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" among eukaryotic anisms alternative splicing is an important process that can generate multipletranscripts from one same precursor messenger rna which greatly increase transcriptome and proteome diversitythis process is carried out by a superprotein complex defined as the spliceosome specifically splicing factor branchpoint binding protein sf1bbp is a single protein that can bind to the intronic branchpoint sequence bpsconnecting the ² and ² splice site binding complexes during early spliceosome assembly the molecular functionof this protein has been extensively investigated in yeast metazoa and mammals however its counterpart inplants has been seldomly reportedresults to this end we conducted a systematic characterization of the sf1 gene family across plant lineages inthis work a total of sequences from plant species were identified phylogenetic relationships of thesesequences were constructed and subsequent bioinformatic analysis suggested that this family likely originatedfrom an ancient gene transposition duplication event most plant species were shown to maintain a single copy ofthis gene furthermore an additional rna binding motif rrm existed in most members of this gene family incomparison to their animal and yeast counterparts indicating that their potential role was preserved in the plantlineage our analysis presents general features of the gene and protein structure of this splicing factor familyand will provide fundamental information for further functional studies in plantskeywords alternative splicing expression profile phylogenetics plants promoter splicing factor correspondence fyzhunjfueducn kailu zhang zhen feng jingfang yang and feng yang contributedequally to this work1coinnovation center for sustainable forestry in southern china college ofbiology and the environment nanjing forestry university nanjing jiangsu province chinafull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0czhang bmc plant biology page of in eukaryotes canonical splicing removes noncoding intronic sequences and assembles the coding elements intomature mrnas while alternative splicing as generatesdifferent multiple transcripts that encode proteins withdistinct structures and functions by differential usage ofexons or splice site [ ] the resulting transcripts ofas greatly contribute to posttranscriptional regulationbiological complexity and proteome diversity in eukaryotes [ ] given that on average there are approximately exons in each transcript in the humantranscriptome and the degenerative nature of corresponding splice sites premrna splicing is sophistically catalysed by the spliceosome spliceosome is amultimegadalton protein complex which consists offive u1 u2 u4 u5 and u6 small nuclear ribonucleoprotein ps snrnps and over spliceosomalproteins furthermore the early assembly of spliceosome complex e or the commitment complex is anatpindependent process and contains u1 snrnps sf1and u2 snrnp auxiliary factors u2af large and u2afsmallthe prespliceosome complex a is formed by replacing sf1 withsf3b155sap155 of u2 snrnps [ ] stepwiseassembly of the following spliceosome during the splicing reaction has been reported as well [ ] however splice site recognition is a critical step during earlyassembly of the spliceosome the current model describes the binding of u1 snrnp and u1 snrna to ashort stretch of nucleotides at the ² splice site of splicing factor sf1mammalian branch point bindingprotein mbbp at the branch point and of u2 snrnpauxiliary factors at the ² splice site these threeciselements are necessary but usually insufficient to define a specific exonintron boundary thus additionalsplicing enhancers or silencers located at exons and introns may allow the recognition of genuine splice sitesduring early spliceosome assembly [ ] subsequentlysubunitsimportantly sf1 preferentially binds to the intronbranch point sequence bps which is adjacent to thebinding site polypyrimidine tract py of u2af largesubunits mammal u2af65 and fission yeast u2af59bridging u1 and u2af to form an intermediate lariatstructure [ ] in particular sf1 is characterized bythe presence of two types of rna binding motifs at thenterminus a k homologyquaking khqua2 domain which originated from the human heterogeneousribonucleoprotein hnrnp k protein [ ] and oneor two zinc knuckle motifs cx2cx4hx4c x represents any amino acid sf1 also contains a prolinerichregion at cterminus [ ] intriguingly the yeast khdomain specifically binds to the bps of premrnas witha glyproarggly motif and the variable loop of thekh domain and is necessary for spliceosomeassembly the first but not the second zinc knuckledomain in yeast has been demonstrated to bind rnawith high affinity moreover the stability of thesf1u2af65rna complex is further affected by thephosphorylation status of several sf1 serine residuesser20 ser80 and ser82 in vitro the prolinerichregion of sf1 interacts with u1 snrnp prp40fbp11 inyeast and human [ ] in regards to its interactionpartner the u2af large subunit the nterminal of sf1interacts with its noncanonical rna recognition motifsrrm or u2af homology motif[ ]whereas the other two rrms of u2af large subunitbind to the py region uhmsaccharomycescerevisiae common fruita previous study in fission yeast schizosaccharomycespombe suggests that the initial corecognition of thebranch site and ² splice site is pivotal for correct splicing of target premrnas because of the importance of splice site recognition for gene expression andprotein diversity sf1 has been demonstrated to play essential roles in a number of eukaryotic species includinghuman homo sapiens mice mus musculus buddingyeastflydrosophila melanogaster and roundworm caenorhabditis elegans [ ] for example in humansmissense mutation of splicing factors which are responsible for splice site recognition such as sf1 has beenlinked to tumourigenesis similarly heterozygoussf1 knockdown mice are susceptible to colontumourigenesis induced by an anotrophic carcinogenazoxymethane and sf1 has been found to associatewith betacatenintcf4 complex suggesting its role incarcinogenesis in contrast knockdown of sf1 suppresses the development of germ cell tumours in mice indicating its tissue dependency in cancer researchfurthermore the molecular function of sf1 has been extensively studied in yeast for instance a sf1 mutantstrain causes frequent exon skipping in fission yeast additionally sf1 has been proposed to recognize suboptimal sequences in specific introns and lead to nuclearaccumulation of premrna with aberrant splicing however increasing evidence indicates that this proteinis a regulator of splice site recognition and does not reduce general splicing specifically during alternative splicing by targeting a subset of genes [ ] thishypothesis is supported by the fact that knockdown ofsf1 in both yeast and human extracts only slightly affects the splicing outcome rnai targeting of thisgene has been demonstrated to not affect the splicingpattern of several splicing marker genes tested in comparison to studies in human and yeast few reports have been published related to plant sf1 genessimilar functions of the arabidopsis sf1 gene were proposed in an early study in this plant sf1homologue is reportedly responsible for the splicing of a 0czhang bmc plant biology page of to maintain itsgroup of transcripts the lossoffunction mutant atsf1 of this gene leads to abnormal development earlyflowering and dwarfism and aba or heat stress sensitivity in arabidopsis [ ] subsequently the domainstructure and its functional relationships have been substantially investigated and the rrm domain is considered crucialfunction in plantsmoreover sf1 may have a different mechanism of ²splice site recognition in plant because the plant sf1 homologs contain a different rrm domain compared withfungal and metazoan counterparts [ ] on the otherhand a study found that atsf1 may be likely to play afunctional role in the cytoplasm because it was found toshuttle between the nucleus and cytoplasm however no related investigations have been conducted onthe phylogenetic analysis of plant sf1 genes and theirregulatory mechanisms although it is a highly conserved family and has conserved functions in eukaryotesplant sf1 genes may have overlapping and distinct rolescompared to the mammalian genes hence studying thephylogenetic relationship and regulatory mechanism ofplant sf1 genes may make us understand the evolutionary history characteristics an expression profile of thisgene family and predict specific functions in plants thiscan lay the foundation for further functional studies inviridiplantae to this end we systematically identified sf1 sequences from plant species ranging fromalgae to higher plants meanwhile the gene and proteinstructure potential regulation at promoter regions andexpression pattern of these genes were further investigated in this study we hypothesize that plant sf1 isstructurally different from its counterparts in animalsand yeast but it is conserved among lower and higherplants indicating its specific role in alternative splicingin branch point recognitionthalianasf1methodssequence acquisition and identification of plant sf1genesthe arabidopsisprotein sequenceat5g51300 was used to search similar sequences inall available plant species from the phytozome v121databasehttpsphytozomejgidoegovpzportalhtml by running the blastp program with an evaluecutoff 1e10 the other parameters were the default settings then the retrieved protein sequences wereexamined and filtered using the hmmer score defaultsettings which contained pf16275 splicing factorkhomology domain kh_1 and pf00076 rna recognition motif rrm_1 finally putative sf1 sequencesfrom plant species were identified detailed information including groups plant species common namesand number of sf1 homologs reported for each planthelixhairpinsf1hhdomainpf00013species for subsequent analysis are listed in table s1subcellular location prediction of identified sf1 proteinswas carried out using wolf psort httpswolfpsorthgcjp construction of molecular phylogenetic tree of plant sf1genesprotein sequences of the aforesaid plant sf1 genes wereextracted from phytozome v121 database for phylogenetic relationship analysis the sequences with the longestcoding sequences were chosen for genes with multipledifferent splicing isoforms then multiple sf1 proteinsequences were aligned with the muscle v38 softwarewith default settings the molecular phylogenetictree of plant sf1 genes was then constructed using themaximum likelihood method ml jtt g i modelvia phyml v30 program with the following parametersinitial tree bionj discrete gamma model yes numberof categories gamma shape parameter proportion of invariant subtree patterns aliasing no figtree v143 was used to visualize and edit the phylogenetic treegene structure protein domain and multiple em for motifelicitation meme analysisrequired genomic cdna and peptide sequences and allsf1 gene structures were downloaded from the phytozome v121 database corresponding intron phases weregenerated using the online program gene structure display server gsds20httpgsdscbipkueducn correlation analysis of sf1 exons were performedby using the piece2 webserver httpwwwbioinfogenomenetpiecesearchphp tdsourcetags_pctim_aiomsg sf1 protein sequences were used to search formatching pfam families using the hmmer websitehttpswwwebiacuktoolshmmer then protein domain patterns were drawn by using tbtools software according to the full pfam resultanttableconserved motifs of plant sf1 cdna sequences andprotein sequences were analysed on the meme onlineprogram httpmemesuitetoolsmeme considering a maximum of the most preserved motifs predicted for each sequence and leaving other settings onthe default parametersmotif prediction in promoter regions of plant sf1 genesthe 15kb ²flanking sequences of plant sf1 geneswere extracted from genomic data available in phytozome database prediction of plant putative ciselementswas performed with the online server plantcarehttpbioinformaticspsbugentbewebtoolsplantcarehtml motifs related to tissuespecific expressioninternal hormones and external environmental stress response were selected for further analysis and discussion 0czhang bmc plant biology page of expression analysis base on microarray datasets and geneexpression experimentsexpression data of arabidopsis s tuberosum g max slycopersicum p trichocarpa and b distachyon includingtissue specificity and stress responses were extracted fromthe efp browser series of the bioanalytic resource forplant biology httpbarutorontoca expressionvalues of selected plant sf1 genes were log transformedlg to generate visualize expression difference heatmapsby using bar heatmapper tool program httpbarutorontocantoolscgibinntools_heatmappercgigene expression experimentstotal rna of samples from different plant tissues wereextracted by rneasy mini kit qiagen usa and subsequently reversed transcribed into cdna by fastkinggdna dispelling rt supermix fastking tiangenchina according to the manufacturers instruction rtpcr amplification were programmed asfollowings °c min °c s °c s °c s cycles °c min sybr premix ex taqtm accuratebiotechnology co ltd hunan china was used forquantitative realtime rtpcr analysis which was conducted on the stepone plus realtime pcr system following optimized program °c s °c s °c s cycles the data were normalized to the expression of internal reference genes table s6 and the transcript abundance was determined by the comparativect value method analysis of proteinprotein interaction network andstructural conservationa proteinprotein interaction network was generated bythe string website httpsstringdb withrepresentative protein sequences from arabidopsis thefollowing basic settings were employed meaning of network edges evidence line colour indicates the type ofinteraction evidence and active interaction sourcesexperimentsthere are three domains in the arabidopsis sf1 protein the phosphorylation and u2af65 binding of thenterminal domain of splicing factor during ² splicesite recognition of homo sapiens pdbid 2m0g identity evalue 7e17 was similar to that of the khomology domain the structure for recognition of theintron branch site rna by splicing factor of homo sapiens pdbid 1k1g identity evalue 9e27 canbe used as the template for the splicing factor helixhairpin domain therefore homology modelling wasperformed with modeller based on two crystalstructures the amino acid conservation scores were calculated using the consurf web server based on the mlmethod input attributes were the 3d model andmultiple sequence alignment figure s4 related figureswere created based on pymol with default settings analysis gene structure evolution with orthologue groupof sf1 genesreconstruction of the evolutionary history of the structure of the plant sf1 family of orthologous genes wascarried out by searching at5g513001 in the piece severhttpwwwbioinfogenomenetpieceindexphpthis provided an exonintron display for orthologousgenes from gene structure data sets linked to the phylogenetic treeresultssequence identification and phylogenetic analysis of theplant sf1 gene familyto identify sf1 gene family members in plants we carried out a blastp search using the arabidopsis atsf1at5g51300 amino acid sequence against the phytozome database v121 after filtering the sequence without sf1 signature or truncated sequences a total of sequences from plant species were retrieved whichwere roughly classified as algae bryophyta basicangiosperm monocots and eudicots table s1specifically the only species with four copies of plantsf1s was eutrema salsugineum salt cress table s1 inparticular three copies of sf1 genes were observed infive species including panicum virgatum switchgrasstriticum aestivum common wheat daucus carotacarrot kalanchoe laxiflora milky widows thrill andsalix purpurea purple osier willow additionally plant species contained two copies and species including the model plant arabidopsis possessed only onecopy of plant sf1s respectively the relatively largernumber of sf1 genes and higher number of plant speciesin this work demonstrated the universality and complexity of the sf1 gene family the retrieved sequences of plant species provided us with more complete information to analyse the phylogenetic relationship of the sf1gene family subsequently a rooted phylogenetic treewas constructed based on the abovementioned protein sequences by using the maximum likelihoodmethod the trees bootstrap threshold was represented by a colour gradient fig in general all sf1protein sequences were clustered into four major cladesincluding alga in yellow other land plants in greenmonocots in pink and eudicots in blue and one species amborella trichopoda belonged to basic angiosperm shown in colourless the phylogenetic tree ofsf1s figs and left panel with clear topology andoverall high bootstrap values was similar to evolutionarytrend from lower plants to higher plants reported inother studies for example the genes of algae in the yellow branch were representative members of the lineage 0czhang bmc plant biology page of fig circular phylogenetic tree of the sf1 gene family available in plants the phylogenetic tree of sf1 genes in plants was constructed basedon maximumlikelihood with jtt g model by using phyml v3037 a total of protein sequences from plant species were chosen tocalculate the phylogenetic relationship for tree construction bootstrap values are labelled at each major branch the corresponding informationof each transcript such as species name common name number of identified transcripts and their transcript id nomenclature are shown intable s1 taxonomies based on apgiv system 0czhang bmc plant biology page of fig see legend on next page 0czhang bmc plant biology page of see figure on previous pagefig gene structure comparisons and conserved motif identification among plant sf1 genes from left panel to right panel verticalphylogenetic tree genomic anization and identified cdna conserved motifs by meme analysis intron phase and are shown on thegene structure the conserved sequence of identified motifs represented by different coloured boxes are listed below some long genes werereduced to onehalf of their original length to fit this picturethat diverged before the evolution of land plants whichwas the basal part of the phylogeny in the blue branchfive sequences from kalanchoe with higher bs valuesformed a subclade showing their closer evolutionary relationships additionally cagra3782 s00261p fromcapsella grandiflora and carubv10025900m from c rubella formed a subclade with the arabidopsis sequencesbecause they all belong to brassicaceae which is consistent with the apg iv system fig and table s1 usually some homologous sf1 sequences from the samespecies were clustered in the same small branch next toeach otherthese species included cashew soybeanapple woodland strawberry quinoa carrot coloradoblue columbine maize common wheat cereal grassmoss and bog moss fig and table s1 in contrastsome other homologous sf1 members from the samespecies were clustered into the different subclades suchas purple osier willow poplar eastern cottonwood saltcress potato diploid kalanchoe milky widows thrillhalls panicgrass switchgrass green algae and volvoxfig and table s1gene structure and conserved motif analysisit is necessary to compare the exonintron anizationand conserved motifs of the plant sf1 gene family toclarify their evolutionary process and potential functionthe gene structure models of sf1 genes were attachedto the phylogenetic tree fig and the correspondingintron phase of each was also displayed fig tables2 figure middle panel shows that the gene lengthand structure of each member of the sf1 family exhibitssignificant differences for example the gene structureof members of sf1 family genes did not containintron sequences this subset accounts for of thetotal number of members fortyeight sequences of sf1genes had exon1 intron anizations accounting for of all genes in particular some genes from algaehad multiple exons including vocar0008 s02941p volvox carteri which contained the most exons exonsmoreover different gene structures were also observedat the same subbranch for instance two sequencesfrom zea mays maize zm00008a037777_p01 exonsand zm00008a007621_p01 exons were observed tohave distinctive gene structures although the dissimilation of gene structure of each member of sf1s was substantial we found that the length of cdss did notsignificantly change fig thus whether it influencesthe differentiation of their gene function needs to befurther investigated further investigation on conservedmotifs by using multiple em for motif elicitationmeme search tool demonstrated that most sf1 genes sequences exhibited similar sequence signatures andthe same order and all contained the analysed motifsexcept one sequence of micromonas pusilla hada different position fig right panel although no obvious differences in identified conserved motifs werefound among basal angiosperm monocots and eudicotssequences from the same species were found to have different motifs fig for example aqcoe5g4069001pand aqcoe7g0393001p from the eudicot aquilegiacoerulea had motifs and motifs respectively thesame situation was found in d carota dcar_006843dcar_008506 and dcar_004968 had motifs motifs and motifs respectively intriguingly the cdslength of dcar_008506 was the longest notably somesequences from algae and moss had fewer conservedmotifs for examplein bryophyta the sequences ofphyscomitrella patens pp3c7_10890v31p and pp3c11_24710v31p sphagnum fallax sphfalx0015s00771pand sphfalx0010s01971p and marchantia polymorphamapoly0009s01891p had nine motifs in algal plantsthe sequences of and from micromonashad only motifs and motifs respectively moreoveralthough the sequences of volvox carteri vocar0007s03451p and vocar0008 s02941p and chlamydomonasandcre09g386731t11 contained multiple exons they had motifs indicating their sequence variation had little influence on function classes further correlation analysisof the sf1 exon regions were carried out to elucidate thegainloss of introns correlations between transcripts ofplant sf1s are shown in fig providing additional information for phylogenetic analysis for example thereis more similarity between pgsc0003dmt400081859and migutd025312 because of multiple exact matchesbetween the exons of the two transcriptscre12g553750t11reinhardtiianalysis of protein domain and conserved motifs inpeptidesthe protein domains were analysed by using the aboveselected peptide sequences from plant species thepeptides annotations were splicing factorrelated andconserved protein motifs were predicted according tothe retrieved peptide sequences by meme analysisfig consequently all sf1s were found having sf1_hh nterminal domain on the nterminal ofthe 0czhang bmc plant biology page of fig analysis of gene structure evolution with orthologue group of sf1 genes exonintron structure and intron phase right panel are linked tothe plant species tree left panel genes with red colour represent the members of the plant sf1 genes different coloured lines mean differentexon comparison results between species 0czhang bmc plant biology page of fig comparisons of protein domains and conserved motif identification among plant sf1 genes protein domain middle panel and identifiedprotein conserved motifs right panel identified by meme analysis are shown against the vertical phylogenetic tree left panel the conservedsequence of identified motifs represented by different coloured boxes are listed below 0czhang bmc plant biology page of peptides followed by a kh domain and a cterminal domain namely an rna recognition motif rrm fig middle panel interestingly in algae peptides from mpusilla v carteri vocar0008 s02941p andc reinhardtii cre09g386731t11 had two rrm domains the amino acid lengths of sf1 proteins rangedfrom aa to aa and most of them possessed to amino acids table s3 consistently most ofthem are approximately to amino acids inlength subcellular location prediction showed that themajority of sf1 proteins were had nuclear localization table s3 moreover proteins of 147m014250 ricinus communis and migutf011911pmimulus guttatus were located in the vacuoles proteins of traes_2dl_6f03f05fa4 t aestivum and m pusilla were predicted to be cytoplasmic proteins of gsmua_achr5p25100_001 musa acuminataand cre09g386731t11 c reinhardtii were located inthe chloroplast and endoplasmic reticulum respectivelymeme analysis for sf1 peptide sequences was used topredict a total of conserved motifs which are presented as coloured boxes and cover most of the proteinfig right panel further analysis showed that peptides had all motifs accounting for approximately of all sf1 protein sequences analysed in the studyinterestingly all sequences from moss have conservedmotifs in the analysis suggesting the conservation ofsf1 proteins in bryophyta furthermore almost all eudicots had conserved motifsexcept anacardium occidentalegrandifloracagra3782 s00261p which lacked motif and malusdomesticavescamrna211921v10hybrid and brassica rapa brarac014811p which lacked motif while most monocots had eight conserved motifs in contrast algal plantsonly possess approximately half of the predicted motifs due to their peptides with integrant protein domainsimplying the least degree of conservation and divergenceof plant sf1 proteins in algae t motifs that all algaeshared were motif motif motif and motif anaoc0018 s04251pmdp0000558834fragariaand canalysis of promoter and tissuespecific expression of sf1genesto further analyse the regulation of plant sf1 genes atthe transcriptional level the 15kb upstream sequencesof plant sf1 genes were obtained from the phytozomedatabase then the ciselements of each promoter wereidentified by using the plantcare program table s4 consequently a total of motifs were predictedgenerally eight ciselements related to tissuespecificexpression among them were selected fig and tables4 including hdzip1 for differentiation of the palisademesophyll cells the ryelement which regulates seedspecific expression the aaca_motif and gcn4_motif waspresentfurther hdzipinvolved in endosperm expression and the catboxccgtccbox doct and oct for meristem expression further analysis showed that there were only promoters of sf1 genes which had tissuespecific regulatory ciselements particularly the catbox and ccgtccbox turned up at the highest frequency and greatestabundance in the promoters of sf1 genes both of themregulate meristemspecific expression and play key rolesduring development and growth of plants consistentlypurple false brome brachypodium distachyon of monocots not only had a catbox and ccgtccbox butwas also highly expressed in young leaves internode adventitious roots and roots fig and figure s2however no motifs were found to link the highexpression of two sf1s of glycine max soybean insam and roottip figure s1 additionally the aaca_motif was only detected in solanum tuberosumpgsc0003dmp400032853 of potato suggesting itsspecific role in regulating endospermspecific negativeexpressioninpodel03g1132001p of populus deltoideseasterncottonwood and spipo17g0046100 of spirodela polyrhiza greater duckweed the ryelement was detectedin the promoter of the dicot model plant arabidopsisand low expression was also reported in dry seed inarabidopsis fig suggesting that the ryelement isinvolved in seedspecific negative expression of arabidopsis moreover expression levels in the same tissuetype showed significant differences during differentgrowth stages for example the expression level in stamen of flower stage of arabidopsis was obviouslyhigher than that of the other flower development stageshowever the expression levels of different growth stagesof solanum lycopersicum were not only similar butlower and no motifs were found in the promoter in tomato figs and s1 furthermore different expressionpatterns were detected in several sf1 genes with multiple copies figs s1 and s6 for instance similar tissue expression profiles were detected in two sf1trichocarpahomologuespotri001g1264001 and potri003g1072001 and themonocotandzm00008a037777_p01 figure s1 and s5 in contrasttwo sf1 genes of s tuberosum showed differential expression patterns similar to in g max figs and s1zm00008a007621_p01dicot populuszea maysfrom theanalysis of promoter and internal and external hormonesexpression of sf1 genesin longterm evolution and development plants havegradually formed mechanisms of adaptation and resistance to adversity to maintain their life and sustaingrowth to understand the regulatory mechanisms of internal and external stimuli on plant sf1s cisacting elements involved in hormone and stress were studied with 0czhang bmc plant biology page of fig see legend on next page 0czhang bmc plant biology page of see figure on previous pagefig analysis of motifs related to tissue specificity in the plant sf1 promoter regions eight cisacting motifs are represented in different colortriangles positions of these identified motifs are labeelled along the kb ²flanking regions of each sf1 gene the line solid and dottedrepresents regions with basic pairs and regions of no sequences or annexed base n respectively symbols on above the line represent the motifsat the plus strand whereas symbols on below the line represent the motifs at the minus strand function of motifs aacamotif involved inendospermspecific negative expression catbox cisacting regulatory element related to meristem expression ccgtccbox cisactingregulatory element related to meristem specific activation doct cisacting regulatory element related to meristem specific activationgcn4_motif cisregulatory element involved in endosperm expression hdzip1 element involved in differentiation of the palisade mesophyllcells ryelement cisacting regulatory element involved in seedspecific regulation the black vertical lines represent break at that particularbranch oct cisacting regulatory element related to meristem specific activationlowtemperatureltr droughtthe plantcare database fig table s4 finally hormone and stressrelated motifs were selected from promoter sequences of plant sf1s there are hormonerelated motifs including abscisic acid abreauxin auxre auxrecore tgabox tgaelementethylene ere gibberellin garemotif pbox tatcbox meja cgtcamotif tgacgmotif and salicylic acid tcaelement and five stressrelated motifsincludingmbswound wunmotif and anoxic are gcmotif motifs almost each sf1 sequence had a great diversity ofciselements in its promoter regions except some sequences such as araha13031 s00021 and traes_2al_3d67296921 which did not contain a single motif dueto the sequences contain n or no promoter suggestingthat multiple hormonesmediated signalling pathwaysare closely related to sf1 plants resistance analysisshowed that more than half of sf1 promoters containedabre cgtcamotif tgacgmotif and are respectively moreover external hormone signals also affect theabundance of sf1 transcripts figure s3 for examplein arabidopsis at5g513001 mj methyl jasmonateinhibited its expression fig and treatment withother hormones like acc a precursor of ethylene iaaauxin | 0 |
"Oral squamous cell carcinoma is one of the most common causes of malignancyassociated death Early diagnosis of oral squamous cell carcinoma OSCC is important in patient treatment and prognostic evaluation Due to the lack of significant therapeutic benefit the year survival rate has not improved Therefore effective novel markers are needed to improve diagnosis To determine novel promising diagnostic biomarkers for OSCC upregulated and downregulated differentially expressed genes were screened from OSCC tissues using an RNA microarray The results suggested that minichromosome maintenance protein MCM5 mRNA was significantly overexpressed in OSCC tissues compared with that in adjacent normal tissues Moreover silencing of MCM5 expression an OSCC cell line SCC significantly impaired proliferation and colony formation Furthermore negative regulation of the mRNA and protein expression of MCM5 and demonstrated that MCM5 served as a cancerpromoting gene modulating OSCC cell proliferation through induced G2M phase arrest In this process the mRNA expression of cyclin E and cyclindependent kinase was downregulated while p21 expression was upregulated These results suggested that MCM5 may be an important pathogenic factor of OSCC High expression levels of MCM5 may serve as a marker for the early diagnosis of OSCC IntroductionOral squamous cell carcinoma OSCC affects individuals worldwide annually Presently the clinical treatment of OSCC is primarily surgery radiotherapy or chemotherapy Over the past decades the overall survival Correspondence to Dr Xiaofeng Wang Department of Stomatology ChinaJapan Union Hospital of Jilin University Xiantai Changchun Jilin PR ChinaEmail wangxiaofengjlueducnContributed equallyKey words minichromosome maintenance protein cell cycle oral squamous cell carcinoma biomarker microarrayOS rate of patients with OSCC has not significantly improved with a year survival rate of Insufficient sensitive and specific biomarkers may lead to the diagnosis of OSCC at advanced stages Therefore it is necessary to identify novel biomarkers for the early diagnosis and treatment of OSCCRecently with the continuous development of sequencing technology researchers can efficiently distinguish differentially expressed genes DEGs by transcriptome sequencing which allows screening of potential tumor markers or therapeutic drug targets For example a number of new potential tumor markers have been found in human malignancies such as breast cancer epithelial ovarian cancer and glioma Minichromosome maintenance protein MCM5 a member of minichromosome maintenance family of proteins plays an important role in cell proliferation and DNA replication Some studies have confirmed that MCM5 is highly expressed in numerous human malignancies such as renal cell carcinoma pancreatic ductal adenocarcinoma cervical cancer and skin cancer Further studies have found that high expression of MCM5 is closely associated with the clinicopathological features of specific cancer types For example overexpression of MCM5 is significantly associated with overall survival rate OS in hepatocellular carcinoma Moreover increased expression of MCM5 is positively correlated with larger tumor size positive lymph node metastasis more advanced clinical stage higher histological grade deeper invasion depth and perineural invasion of OSCC However thus far the expression function and potential mechanisms of MCM5 in OSCC are still unclear Therefore the present study aimed to analyze the DEGs in OSCC using a microarray screen for MCM5 and further evaluate the possible functions of MCM5 in OSCC The present results may provide evidence to support the value of MCM5 as a biomarker or a therapeutic target of OSCC Materials and methodsTissue sampling Pairs of OSCC tissues and adjacent normal tissues were obtained from patients undergoing resection operations at the ChinaJapan Union Hospital of Jilin University Changchun China Clinicopathological data were also collected No patient received preoperative treatment including radiotherapy or chemotherapy No other inclusionexclusion criteria were used Matched normal OSCC 0cHAO MCM5 IS A POTENTIAL EARLY DIAGNOSTIC MARKER FOR ORAL SQUAMOUS CELL CARCINOMAtissues were obtained from a segment of the resected specimens cm away from the tumor Pathological analysis was used to identify surgically resected specimens Pathological analysis was performed by our group with no specific diagnostic guidelines Three paired samples were obtained for transcriptome sequencing Then to confirm the reliability of sequencing data the samples size was increased using the remaining paired tissues and analyzed using quantitative qPCR All comparisons between OSCC tissues and adjacent normal tissues were performed simultaneously The KaplanMeier analysis of OS and survival curves were from the Cancer Genome Atlas database TCGA wwwcancergovThe study was approved by the Ethics Committee of ChinaJapan Union Hospital of Jilin University Written informed consent was obtained from all patients who participated in this studyTranscriptome sequencing and functional annotation analysis Total RNA extraction RNA library construction and transcriptome sequencing were performed at Sangon Biotech Co Ltd The biological relevance of unique genes in expression profiles of DEGs were screened according to the threshold values of log2foldchange¥ and P005 Then the differentially expressed mRNAs were analyzed by Gene Ontology GO whose annotations were downloaded from Gene Ontology httpgeneontology UniProt sparqluniprot and NCBI wwwncbinlmnihgov Significant GO categories were identified using Fisher's exact test with a P005 which indicated that significantly upregulated genes in the set of DEGs were assigned to a specific functional category more often than expected by chance Significant pathways of the DEGs were then analyzed and identified according to the Kyoto Encyclopedia of Genes and Genomes KEGG database wwwkeggjpCell lines The human tongue squamous cell carcinoma SCC and CAL were obtained from the American Type Culture Collection CAL cells were cultured in DMEM medium with fetal bovine serum FBS Gibco Thermo Fisher Scientific Inc Uml penicillin and streptomycin at ËC in a humidified atmosphere containing CO2 SCC cells were cultured in MEM medium with FBS NEAA Uml penicillin and streptomycin at ËC in a humidified atmosphere containing CO2MCM5specific siRNA and transfection Three MCM5 siRNA sequences were synthesized by Suzhou GenePharma Co Ltd The sequences were as follows '' siRNA Forward CCG ACU ACU UGU ACA AGC ATT and reverse UGC UUG UAC AAG UAG UCG GTT siRNA forward CCA AAU GUC UAU GAG GUC ATT and reverse UGA CCU CAU AGA CAU UUG GTT siRNA forward GUC GUC UGU AUU GAC GAG UTT and reverse ACU CGU CAA UAC AGA CGA CTT and scrambled forward UUC UCC GAA CGU GUC ACG UTT and reverse ACG UGA CAC GUU CGG AGA ATT The mock was an untransfected empty vector serving as the controlSCC cells 45x104 cellswell were cultured in well plates overnight at ËC Then cells were transfected with nM negative control siRNA or MCM5 siRNA using Lipofectamine® Transfection Reagent Invitrogen Thermo Fisher Scientific Inc according to the manufacturer's protocol After h transfection cells were collected and then RNA was extracted by TRIzol® regent Invitrogen Thermo Fisher Scientific Inc for further experiments as indicatedReverse transcription RTqPCR RNA extracted from tissues samples were reverse transcribed into cDNA using a GoScript Reverse Transcription System kit Monad httpwwwmonadbiotechcom according to the manufacturer's instructions Relative mRNA expressions were quantified by qPCR using the QuantiTect SYBR Green PCR kit Roche Diagnostics and normalized to GAPDH using primers listed in Table I The cycling parameters were cycles of ËC for sec ËC for sec and ËC for sec Relative mRNA levels were assessed by the comparative ÎÎCq method All analyses of the samples were conducted in triplicateFor association of MCM5 expression levels with clinicopathologic features of OSCC the relative expression levels of MCM5 were evaluated using qPCR as aforementioned Relative mRNA levels of paired samples adjacent vs cancer tissues were assessed by the comparative ÎÎCq method A ratio was considered to have high MCM5 expression whereas a ratio ¤ was considered to have low MCM5 expression Cell proliferation assay To analyze cell proliferation a Cell Counting Kit CCK Dojindo Molecular Technologies Inc was used according to the manufacturer's instructions In total cells were cultured to each well of well plates After h post siRNA transfection cells were incubated for and h CCK reagent was added h prior to detection The OD was measured at nm using a microplate reader BioTek The experiment was performed three timesColony formation assay This assay was performed according to a previous study Briefly cells were cultured in well plates at cellswell followed by culture in complete medium DMEM supplemented with FBS Uml penicillin and streptomycin for weeks The colonies were fixed with methanol for min at room temperature and washed with PBS and stained with crystal violet at room temperature Beyotime Institute of Biotechnology solution for min A cell colony was defied as a group formation of at least cells Finally formed colonies were observed and images were captured under a light microscope at magnification x200 Cell migration analysis using scratching assays SCC cells were cultured in a well plate at 5x105 cellswell overnight at ËC Then the cells were scratched and scraped with fresh DMEM Cells were observed and images were captured under a light microscope magnification x200 at h The width of the scratch was measured and referred to as Wbefore Then the cells were starved with no FBS and returned to the incubator for h at ËC The width of the same scratch was measured and referred to as Wafter Migrating distance was calculated by subtracting Wafter from Wbefore The migration of the control was set as Western blot analysis The protein extractions from the cells were isolated using RIPA Lysis Buffer P0013B Beyotime Institute of Biotechnology Then µg protein was loaded per lane on a gel resolved using SDSPAGE and electroblotted onto 0cONCOLOGY LETTERS Table I Primers used for reverse transcription quantitativePCRmRNA MCM5 P21 CyclinE CDK2 GAPDH MCM5 minichromosome maintenance protein cyclindependent kinase Forward primer '' GATCCTGGCATTTTCTACAG GGAGACTCTCAGGGTCGAAA TTCTTGAGCAACACCCTCTTCTGCAGCC GCTAGCAGACTTTGGACTAGCCAG AGAAGGCTGGGGCTCATTTG Reverse primer ''CCCTGTATTTGAAGGTGAAGGGATTAGGGCTTCCTCTTGGTCGCCATATACCGGTCAAAGAAATCTTGTGCCAGCTCGGTACCACAGGGTCAAGGGGCCATCCACAGTCTTCFigure Volcano plots and KEGG pathway analysis of differentially expressed genes between OSCC cancer tissue group and adjacent normal tissue group A Differences in gene expression profiles between OSCC cancer tissue group and adjacent normal tissue group The horizontal line corresponds to a fold log2 scaled change up or down and the vertical line represents P005 The red points on the plot represent the differentially expressed genes with a fold change upregulation while the green points represent downregulation with P005 B Top KEGG enrichment terms of DEG in OSCC The vertical axis represents the pathway category and the horizontal axis represents the enrichment score [lgPvalue] of the pathway LgP was the logarithm of Pvalue and P005 was considered significant KEGG Kyoto Encyclopedia of Genes and Genomes OSCC oral squamous cell carcinoma DEG different expressed genes OSCC oral squamous cell carcinomaPVDF membranes Roche Diagnostics After blocking at ËC for h nonfat milk in PBS plus Tween the blots were incubated with primary antibodies against antiMCM5 D220960 BBI Life Sciences antip21 Proteintech anticyclin E ProteinTech Group Inc and antiβactin D16H11 CST Biological Reagents Co Ltd at ËC for h Western blots were probed with secondary antibodies and detected using the Odyssey infrared imaging system LICOR BiosciencesCell cycle analysis SCC cells were harvested and fixed with ethanol on ice for min and then washed with PBS to decant the ethanol solution Then the cells were suspended and stained by PI and RNase A treatment Cell cycle analysis was performed using a flow cytometer FACSARIA
¡ BD Biosciences The data was performed using CXP Analysis software Beckman Coulter Inc Statistical analysis All the data analysis was performed using SPSS version SPSS Inc The results are presented as mean SD Associations between MCM5 mRNA expression and clinicopathological factors were analyzed using the Pearson's Ï2 test or Fisher's exact test The differences in MCM5 mRNA expression between carcinoma and adjacent normal tissues were evaluated by a paired ttest Oneway ANOVA followed by Tukey's post hoc test was used to determine the differences between groups and unpaired ttests for the rest of the data The survival rate was calculated by the KaplanMeier method and compared using the logrank test P005 was considered to indicate a statistically significant difference All experiments were performed in triplicateResultsRNA sequencing and functional annotation analysis To explore novel biomarkers for OSCC the RNAs derived from tissue samples by sequencing were detected Three matched primary OSCC tissues and adjacent normal tissues were randomly selected As shown in Fig 1A the aberrant expression of genes was detected in tissue samples To screen 0cHAO MCM5 IS A POTENTIAL EARLY DIAGNOSTIC MARKER FOR ORAL SQUAMOUS CELL CARCINOMATable II Twenty randomly selected differentially expressed genes between oral squamous cell carcinoma and adjacent tissue samples The genes selected randomly instead of listing based on rank or foldchange in expressionGene ID ENSG00000160182 ENSG00000205592 ENSG00000171195 ENSG00000126549 ENSG00000090382 ENSG00000161798 ENSG00000161055 ENSG00000107562 ENSG00000214711 ENSG00000106066 ENSG00000184330 ENSG00000137745 ENSG00000243207 ENSG00000107159 ENSG00000183072 ENSG00000196611 ENSG00000171217 ENSG00000178445 ENSG00000100297 ENSG00000127564 Log2 foldchange Gene nameTFF1MUC19MUC7STATHLYZAQP5SCGB3A1CXCL12CAPN14CPVLS100A7AMMP13PPANP2RY11CA9NKX2MMP1CLDN20GLDCMCM5PKMYT1Pvalue 881x10 468x10 144x10 140x10 209x10 528x10 959x10 604x10 601x10 846x10 234x10 753x10 222x10 161x10 380x10 154x10 705x10 751x10 565x10 240x10 Result Downregulation Downregulation Downregulation Downregulation Downregulation Downregulation Downregulation Downregulation Downregulation Downregulation Upregulation Upregulation Upregulation Upregulation Upregulation Upregulation Upregulation Upregulation Upregulation Upregulation the candidate biomarkers for diagnosing OSCC the DEGs were selected when changes in RNA expression were foldchanges As shown in Fig 1A aberrant RNAs were significantly downregulated while RNAs were upregulated In order to represent all differentially expressed genes twenty randomly selected dysregulated genes between OSCC and adjacent tissue samples are summarized in Table II The Pvalue and log2 foldchanges of all aberrant expression genes are shown in Table SI The DEGs were selected randomly instead of listing based on rank or foldchange in expression To explore the role of differentially expressed RNAs in OSCC KEGG pathway analysis was performed Depending on the Pvalue and enrichment signal pathways associated with OSCC were identified Table SII The top KEGG enrichment terms of DEGs are shown in Fig 1B including cell cycle pathways in cancer cell cycleyeast meiosisyeast and cytokinecytokine receptor interaction Among these it was reported that some genes such as MCM5 cell division cycle related protein kinase and Cyclindependent kinase homolog were primarily enriched in the cell cycle pathway Some genes such as lysozyme C statherin and aquaporin were enriched in the saliva secretion pathway MCM5 which participated in cell cycle regulation and had high expression in OSCC was selected for further study and it was hypothesized that MCM5 might be a candidate tumor marker for OSCCValidation of MCM5 using RTqPCR To further verify the aforementioned expression profile data MCM5 expression levels were investigated using RTqPCR in tumor and Figure Relative expression levels of MCM5 mRNA in paired adjacent normal tissues and oral squamous cell carcinoma tissues using quantitative PCR The relative expression data were analyzed by the ÎÎCq method GAPDH was used as an internal control P005 P001 vs adjacent normal tissue MCM5 minichromosome maintenance proteinadjacent normal tissues As shown in Fig MCM5 mRNA was significantly upregulated in of tumor tissues compared with that in matched normal tissues These results showed that MCM5 was highly expressed in OSCC tissues which was in line with the sequencing data Association of MCM5 expression levels with clinicopathologic features of OSCC and survival analysis The results of the potential association between MCM5 expression and clinicopathological features in patients with OSCC are presented in Table III No significant association with MCM5 expression was found for age sex histological differentiation metastasisrecurrence and survival status P05 0cONCOLOGY LETTERS Value n MCM5 expressionLow n3 High n12 Table III Association between expression of MCM5 and clinicopathologic features of patients with oral squamous cell carcinoma Characteristic Age years ¥ Sex Male Female Histological differentiation Well and Moderate Poor MetastasisRecurrence Yes No Survival status Death Survival PvalueA KaplanMeier analysis of OS is shown in Fig Analysis of clinical data from TCGA showed that high MCM5 expression was no associated with a shorter OS in patients with OSCC logrank P062 These results suggested that MCM5 might not be a prognostic biomarker for OSCC Inhibitory effect of MCM5 in OSCC cell lines To determine the functional role of MCM5 first MCM5 expression was analyzed using RTqPCR in two OSCC cell lines Notably SCC cells expressed significantly higher levels of MCM5 compared with Cal cells P001 Fig 4A Considering that knockdown of MCM5 in the cell line with high MCM5 expression may bring about more significant changes the SCC cell line was selected for further investigation of the functional role of MCM5 Three specific siRNA sequences were designed to inhibit MCM5 expression and transfected in SCC cells and the impact on MCM5 expression was determined using RTqPCR As shown in Fig 4B siRNA1 siRNA2 and siRNA3 transfection decreased MCM5 expression by 651P001 P001 and P001 respectively compared with the negative control Then the efficiencies were confirmed using western blotting Fig 4C The inhibitory effect of siRNA1 and siRNA2 was significant but not found in siRNA3 The results were consistent with those of RNA expression siRNA1 transfection reduced MCM5 expression significantly in SCC cells Therefore siRNA1 was used for subsequent experiments Effect of MCM5 inhibition on proliferation colony formation and migration To determine whether MCM5 regulates cell cycle and modulates cell proliferation in OSCC the effect of inhibiting MCM5 expression on SCC cell proliferation was investigated As shown in Fig 5A the results showed that downregulation of MCM5 had significant antiproliferative Figure Survival curves from The Cancer Genome Atlas datasets n518 containing high and low MCM5 expression levels MCM5 minichromosome maintenance protein HR hazard ratio TPM transcripts per millioneffect compared with the negative control P001 Colony formation assays were performed and the results revealed that compared with the number of colonies in the control group downregulation of MCM5 significantly reduced colony formation P001 Fig 5B and C To estimate the impact of MCM5 on OSCC migration scratching assays were conducted and inhibition of MCM5 showed no significant impact on the migration of SCC cells P005 Fig 5D These results suggested that inhibiting MCM5 expression inhibited cell proliferation and colony formation but had no effect on migration in SCC cells\x0cHAO MCM5 IS A POTENTIAL EARLY DIAGNOSTIC MARKER FOR ORAL SQUAMOUS CELL CARCINOMAFigure Inhibition of MCM5 expression in oral squamous cell lines A MCM5 expression in Cal and SCC cells B Efficiency of siRNAMCM5 was confirmed by reverse transcription quantitativePCR in SCC cells C The efficiency of siRNAMCM5 was confirmed by western blot in SCC cells MCM5 minichromosome maintenance protein si small interfering NC negative controlFigure Effects of MCM5 inhibition on antiproliferation colony formation and migration capacity in SCC cells A Downregulation of MCM5 expression suppressed SCC cell proliferation compared with the corresponding control at different time points B Downregulation of MCM5 expression inhibited SCC cells colony formation compared with the corresponding control C Quantification of MCM5 inhibition on colony formation compared with the corresponding control D Cells scratching wounds observed by microscopy E Downregulation of MCM5 had no effect on the migration capacity of SCC cells P001 vs NC MCM5 minichromosome maintenance protein si small interfering NC negative controlEffect of MCM5 inhibition on cell cycle To determine the potential mechanism for the observed proliferation inhibition of SCC cells by MCM5 inhibition cell cycle analysis was performed using flow cytometry As shown in Fig 6A and B after MCM5 inhibition the number of cells were decreased in the G0G1 phase and the S phase but significantly increased in the G2M phase compared with the negative control These results indicated that MCM5 inhibition significantly induced G2M phase arrest compared with that in the control group\x0cONCOLOGY LETTERS Figure Effects of MCM5 inhibition on cell cycle regulation in SCC cells A Flow cytometry assays were performed to analysis the cell cycle progression when SCC cells transfected with siRNAMCM5 B The bar chart represented the percentage of cells in G0G1 S or G2M phase as indicated C Expression levels of cell cycleregulated genes detected by quantitative PCR and normalized to GAPDH D Expression levels of cell cycleregulated proteins determined using western blotting βactin was used as the loading control Data are presented as mean ± SD P005 P001 vs control group MCM5 minichromosome maintenance protein si small interfering NC negative control CDK2 cyclindependent kinase To elucidate the mechanism underlying this effect the expressions levels of cyclin E cyclindependent kinase CDK2 and p21 related to cell cycle arrest were determined using RTqPCR As shown in Fig 6D MCM5 inhibition decreased both cyclin E and CDK2 mRNA levels but increased the mRNA expression of p21 significantly Then cyclin E and p21 were selected to detect the protein levels using western blotting As shown in Fig 6C cyclin E levels decreased while p21 levels increased in MCM5downregulated SCC cells which was consistent with the RTqPCR results DiscussionDespite notable progress in cancer research and treatment the survival rate of patients with OSCC has not significantly improved in the past few decades To date there are no effective tumorspecific biomarkers for the early detection and prognosis prediction of OSCC Several studies have shown that DEGs serve an important role in the development of tumors in different cancer types However few studies have reported differentially expressed genes in OSCC The present screened genes that regulate the progression of oral cancer by gene expression profiling and found that genes were dysregulated of which DEGs were upregulated and were downregulated DEGs significantly affected GO terms and KEGG pathways MCM5 did not have one of the highest log2 foldchange values and log10 qValues however MCM5 is one of the differentially expressed genes in cell cycle signaling pathway which was the most significant enrichment of differentially expressed genes Therefore MCM5 which regulates the cell cycle was selected for further investigation However previously published studies on biomarkers in OSCC mainly focused on pathological studies The present study not only verified the overexpression of MCM5 in OSCC but also confirmed using cell experiments that MCM5 affects cell proliferation by regulating the cell cycle MCM5 is a member of the MCM family of proteins and is a component of the starting complex for DNA synthesis MCM5 has been identified as a cell cycle biomarker of aberrant proliferation which is associated with the progression of various cancer types Previously MCM5 has been found to be overexpressed in numerous human malignancies such as esophageal thyroid and ovarian cancer For example increased MCM5 levels in urine sediment cells predicts the presence of bladder cancer Inhibition of transcription factor SOX can inhibit the proliferation of skin melanocytes and MCM5 expression is significantly decreased following downregulation of SOX Moreover high expression of MCM5 is associated with poor prognosis and poor malignant status in patients with cervical adenocarcinoma It is well known that immunohistochemistry and western blotting are necessary methods to evaluate protein expression 0cHAO MCM5 IS A POTENTIAL EARLY DIAGNOSTIC MARKER FOR ORAL SQUAMOUS CELL CARCINOMAHowever due to a limited number of tissue samples the present study did not have enough samples for simultaneous qPCR western blotting and immunohistochemistry detection Therefore this is a limitation of the present study However the study did perform qPCR to evaluate the expression of MCM5 at the mRNA level The results demonstrated that of patients with OSCC have high MCM5 expression which is consistent with the results of the aforementioned studies indicating that high MCM5 expression may play an important role in the pathogenesis of OSCC In addition other members of the MCM family of proteins have also served as biological markers of dysplasia and malignancy such as glioma cervical colorectal breast prostate and lung cancer Therefore some researchers even suggested that changes in MCM5 expression may be a sign of cell cycle disorders It is worth noting that some researchers reported that the high expression of MCM family members may be closely related to tumorigenesis and prognosis For example MCM2 MCM4 and MCM6 are overexpressed in breast cancer of high histological grades MCM7 expression is a potent prognostic marker in nonsmall cell lung cancer while MCM5 may be an independent adverse prognostic marker in lung squamous cell carcinoma It is well known that the cell cycle is related to cell proliferation signaling pathways In most tumors an increase in the expression level of genes encoding proteins that regulate cell proliferation is observed The abnormal expression of cellcyclerelated genes is associated with infinite proliferation of tumors and poor prognosis Thus far only Yu reported the relationship between MCM5 and OSCC The study reported that overexpression of MCM5 in patients with OSCC was significantly associated with tumor site tumor size positive lymph node metastasis later clinical stage higher histological grade deeper infiltration depth and peripheral nerve infiltration However in the present study association between high expression of MCM5 with survival metastasis and poor histologic differentiation was not observed A KaplanMeier analysis of the overall survival rate was not significantly changed in patients with high MCM5 expression compared with patients with low MCM5 expression It was speculated that due to the small number of samples that there were large differences between individuals Therefore in future research a larger sample size should be used to clarify the relationship between high expression of MCM5 and prognosis of OSCC Little is known about the role and potential function of MCM5 in OSCC In the present study a lossoffunction analysis was conducted and it was demonstrated that MCM5 participated in regulating cell cycle and cell proliferation in OSCC cells In fact inhibiting the expression of MCM5 in SCC cells resulted in the downregulation of cyclin E and CDK expression and upregulation of p21 expression which ultimately led to G2M phase arrest in oral cancer cells These results further verified that MCM5 is highly expressed in patients with OSCC which promotes the proliferation of OSCC cells and regulates cell cycle In addition it was observed that MCM5 was not only expressed in SCC cells but also expressed in CAL cells Fig 4A Notably according to the ATCC the MCM5 gene had no mutations in either of the two cell lines indicating that the two cell lines selected in this study have similar genetic backgrounds and could be used for the study of MCM5 cell functions Considering MCM5knockdown experiments in SCC cells with high MCM5 expression received more significant results SCC cells were selected for followup studies However analyzing the functional effects of MCM5knockdown in CAL27 cell lines may provide more information In addition both SCC and CAL cells are transformed cell lines In future investigations untransformed cell lines for multiple comparisons should be used to clarify the role of MCM5 in OSCC Surgical resection is currently the main method to treat OSCC However considering the particularity of the oral structure surgical resection will lead to a huge impact on patients' quality of life Therefore it is important to find effective diagnostic biomarkers for early detection or to develop targeted drugs for OSCC In the present study it was reported that MCM5 is overexpressed in OSCC and that MCM5 can affect cell proliferation by regulating cell cycle Therefore the results suggested that MCM5 might be one of the important pathogenic factors of OSCC and is expected to be used as a potential tumor marker for OSCC target drugs The specific mechanism of action of MCM5 is still worthy of further investigationOverall the present study evaluated differentially expressed genes using sequencing patterns in OSCC tumor tissues and further validated MCM5 upregulated expression in OSCC tissues By knocking down MCM5 expression in SCC cells it was revealed that cell proliferation and colony formation was significantly inhibited by inducing G2M phase arrest The results also suggested that during this process cyclin E and cell cyclerelated gene expression levels were decreased while p21 was significantly upregulated Therefore MCM5 may modulate OSCC cell proliferation by regulating the cell cycle MCM5 is an important pathogenic factor and might have important role as a potential diagnostic marker or drug target for OSCCAcknowledgementsNot applicableFunding This study was funded by the National Natural Science Foundation of China grant no the Bethune Project of Jilin University of China grant no 2018B02 the Education Department of Jilin Province grant no JJKH20190074KJ and Department of Science and Technology of Jilin Province grant no 20190103086JH and 20200201398JC Availability of data and materials The datasets used andor analyzed during the present study are available from the corresponding author on reasonable request The other datasets generated andor analyzed during the current study are available in The Cancer Genome Atlas wwwcancergov Gene Ontology httpgeneontology UniProt sparqluniprot NCBI wwwncbinlmnihgov and Kyoto Encyclopedia of Genes and Genomes wwwkeggjp databases\x0cONCOLOGY LETTERS Authors' contributions HW CZ and CL performed the experiments HW MH and XW analyzed the data MH and HW wrote the manuscript MH and XW designed and supervised | 2 |
predominant male sex hormones drive the development andmaintenance of male characteristics by binding to androgen receptor AR As androgensare systemically distributed throughout the whole anism they affect many tissues andcell types in addition to those in male sexual ans It is now clear that the immunesystem is a target of androgen action In the lungs many immune cells express ARs andare responsive to androgens In this review we describe the effects of androgens and ARson lung myeloid immune cellsmonocytes and macrophagesas they relate to healthand disease In particular we highlight the effect of androgens on lung diseases such asasthma chronic obstructive pulmonary disease and lung ï¬brosis We also discuss thetherapeutic use of androgens and how circulating androgens correlate with lung diseaseIn addition to human studies we also discuss how mouse models have helped to uncoverthe effect of androgens on monocytes and macrophages in lung disease Although therole of estrogen and other female hormones has been broadly analyzed in the literaturewe focus on the new perspectives of androgens as modulators of the immune systemthat target myeloid cells during lung ammationEdited byFlavia BazzoniUniversity of Verona School ofMedicine and Surgery ItalyReviewed byPaola ParronchiUniversity of Florence ItalyTim WillingerKarolinska Institutet SwedenSandra O GollnickUniversity at Buffalo United StatesCorrespondenceNicola HellernhellerjhmieduKeywords androgen androgen receptor monocyte macrophage asthma lung sex difference sex hormoneSpecialty sectionThis was submitted toCytokines and Soluble Mediators inImmunitya section of the journalFrontiers in ImmunologyReceived March Accepted June Published August CitationBecerraDiaz M Song M and Heller N Androgen and AndrogenReceptors as Regulators of Monocyteand Macrophage Biology in theHealthy and Diseased LungFront Immunol 103389ï¬mmu202001698INTRODUCTIONThe immune system is essential for maintaining homeostasis within tissues and ans andprotecting them against threats such as harmful pathogens or cancerous transformation Itcomprises both innate and adaptive components The innate immune system is made up of theinnate lymphoid innate lymphoid cells [ILCs] natural killer cells [NKs] and lymphoid tissueinducers [LTi] and innate myeloid subsets The innate immune system consists of a networkof immune cells and molecules that provide rapid ï¬rstline defense against pathogens In contrastthe adaptive immune response made up of B and T lymphocytes takes days or even weeks tobecome established Innate immune cells express pattern recognition receptors that recognizeunique and conserved pathogenassociated molecular patterns such as lipopolysaccharide LPSviral ssRNA and fungal glucan B and T cells have evolved to recognize a ï¬ner repertoireof self and nonselfantigens that facilitate pathogenspeciï¬c actions immunologic memorygeneration and host immune homeostasis regulation To accomplish this the adaptiveimmune response involves a tightly regulated interplay between T and B lymphocytes andFrontiers in Immunology wwwfrontiersinAugust Volume 0cBecerraDiaz et alAndrogenAR in Lung MonocyteMacrophage Biologyantigenpresenting cells of the myeloid lineage such as dendriticcells DCs monocytes and macrophages Myeloid cells arisefrom the bone marrow The type and magnitude of the immuneresponse is uenced by biological sex and age and thereforediï¬ers between males and females Sex diï¬erences in the functionof the immune system arise from both genetic chromosomalsex diï¬erences and diï¬erences mediated by the action of maleand female sex hormones Because the concentration of sexhormones changes over the lifespan and throughout the courseof the menstrual cycle in women the function of the immunesystem also changes during diï¬erent stages of life Innate myeloidimmune cells like other cell types express sex hormone receptorsand are responsive to sex hormones Sex hormones are synthesized from cholesterol through adeï¬ned enzymatic cascade predominately in the gonads and theadrenal glands Sex hormones are also produced in othertissues including the brain placenta mammary glands liver andadipose tissue In addition to driving sexual developmentof egg and sperm production sex hormones are responsiblefor the development of male and female secondary sexualcharacteristics like breast development and growth of facial hairthat occur during puberty Androgens include testosteronedihydrotestosterone DHT androstenedione androstenedioland dehydroepiandrosterone DHEA with DHT being the mostpotent The concentration of androgens in circulation isabout sevenfold higher in adult men than in adult women Estradiol and progesterone are the predominantfemale sex hormones synthesized by the ovaries andadrenal glands Both male and female sex hormones are boundto the plasma proteins albumin and sex hormone bindingglobulin SHBG and only a small percentage exists as freehormone Thus the bioavailability of sex hormones isregulated by their biosynthesis and also the amount of albuminand SHBGImportantly sex hormones mediate not only anatomicdiï¬erences between women and men but also direct sexdiï¬erences in immune responses leading to diï¬erent risks forimmunologic diseases Overall women have a greaterrisk for autoimmune diseases such as systemic sclerosis andsystemic lupus erythematosus whereas men are morelikely to die of infectious and parasitic diseases Moreovermen have a greater risk of nonreproductive cancers Both gender and sex are important mediators of these andother health and disease diï¬erences observed between men andwomen While gender refers to the array of socially constructedroles attitudes personality traits and behaviors sex representsa biological characteristic of an individual includingthe hormonal milieu and chromosome complement Ingeneral estrogens are considered to have proammatoryproperties and androgens are thought to have antiammatoryproperties In the United States and worldwide relevant evidence highlights important epidemiologic sexdiï¬erences in incidence susceptibility and severity of a numberof diseases that aï¬ect the respiratory tract In this reviewwe will focus on how male sex hormones the androgensmodulate the response of myeloid cells in the lung and howthis modulation impacts the outcome of diï¬erent diseases ofthe lungSEX DIFFERENCES IN HUMAN LUNG ANDLUNG DISEASESsex mediates diï¬erencesBiologicalin the incidence andpathophysiology of lung diseases These diï¬erences arise fromsex diï¬erences in the structure and function of the lung itselfand also in the immune cells that populate the lung and arerecruited to it during ammation Before birth the female lunghas several structural advantages over the male lung Surfactantis produced earlier and although the female lung is smaller ithas more alveoli per unit area Neonatal females have higherexpiratory ï¬ow rates than do male neonates when corrected forsize Thus male sex is a major risk factor for the developmentof respiratory distress syndrome bronchopulmonary dysplasia inneonates and asthma in childhood In addition to the contribution of structural diï¬erences ofthe lung between the sexes sex diï¬erences in lung function andlung diseases are also dependent on the action of sex hormonesWe have summarized some broad concepts that deï¬ne howtestosterone and estrogen aï¬ectlung macrophage functionand how this may contribute to the outcome of particularlung diseases in Figure As testosterone rises after pubertythe immunosuppressive eï¬ects of this hormone on protectiveimmune responses to infectious diseases in males can worsenpulmonary disease This would be exempliï¬ed by tuberculosisor uenza Some of these eï¬ects are a result of androgeneï¬ects on critical ammatory macrophage functions althoughthe eï¬ects on the adaptive immune system also have a significantcontribution to the overall outcome Thus testosterone appearsto play a key immunoregulatory role in lung macrophagesTestosterones immunoregulatory properties also appear to bedependent on the amount of cellular expression of AR andon the concentration of the hormone Low concentrations oftestosterone have been noted in patients with asthma COPD andtuberculosis Low testosterone may also be linked to insuï¬cientcontrol of tissuedamaging ammatory responses seen inCOPD and pulmonary ï¬brosis Estrogen tends to promotewound healing responses in macrophages Dysregulation ofwound healing responses and overactive tissue remodelingmacrophages in the lung could be broadly used to describe theTh2 response in allergic asthma which is worse in womenCancer could also be considered an aberrant wound healingresponse driven by M2like tumor associated macrophages Wehave highlighted here how sex hormones contribute to changesin lung macrophage function that contribute to lung diseaseHowever it should be pointed out that not every sex diï¬erencein lung disease is due to direct eï¬ects on macrophages but on thebroader coordinated immune response as a wholeAsthmaBefore puberty the structural diï¬erences in the lung as wellas gender diï¬erences likely account for the higher incidence ofFrontiers in Immunology wwwfrontiersinAugust Volume 0cBecerraDiaz et alAndrogenAR in Lung MonocyteMacrophage BiologyFIGURE Sex differences in lung diseases discussed in this Review and how they may be connected to the effects of androgens and estrogens on ammatorymacrophages in the lungasthma in boys than in girls With the onset of puberty male andfemale sex hormones and their eï¬ects on the structural cells ofthe lung and on the immune system contribute to the incidenceof asthma The incidence and severity of asthma aregreater in adult women than in adult men and greaterin female than in male mice Female sex hormones suchas estrogen appear to worsen asthma although a straightforwardcorrelation between amount of female sex hormone and asthmasymptoms has not been concluded Androgens have multipleimmunoregulatory and bronchodilatory functions and maycontribute to or be biomarkers for better lung function inmen Accordingly serum testosterone is low in men withmoderate to severe asthma In one study each ngdLincrease in serum testosterone correlated with a CI P decrease in the likelihood of having asthma On the other hand high concentrations of testosterone andcyclic AMP in sputum of asthmatic women during the lutealphase of the menstrual cycle were thought to play a role inpremenstrual exacerbations The idea that sex hormonesmay be a causal factor in asthma was significantly strengthenedby a recent study of adults that quantiï¬ed serum sexhormones and asthma outcomes That study showed thatlow testosterone in both women and men was associated with anincreased incidence of asthma The other interesting ï¬nding wasthat higher testosterone was protective against asthma in obesewomen Obesity is a risk factor for asthma Thereforehow high body mass index BMI and circulating sex hormonestogether aï¬ect asthma requires further investigationAnother androgen dehydroepiandrosterone DHEA alsoknown as androstenolone is an endogenous steroid hormoneand one of the most abundant circulating steroids in humansIt is a precursor for the synthesis of both testosterone andestrogen DHEA is sulfated at the C3 position into DHEAS by the action ofthe sulfotransferase enzymes SULT2A1and SULT1E1 in the adrenal glands The amount of DHEAS in the circulation is ¼ times those of DHEADHEA became of interest to the asthma ï¬eld because womenwith severe asthma had very low concentrations of DHEAS and DHEAS concentration correlated with lung function Interestingly DHEAS is suppressed by oral or inhaledglucocorticoids the mainstay therapy for asthma HumanDHEA peaks at around age and then follows an agedependentdecline until they reach prepubertal concentrations Reducedsecretion of DHEA with age has been related to a numberof ageassociated conditions Replacement of DHEA has beenconsidered as a possible therapeutic that could activate protectiveresponses in an aging immune system DHEA is known todownregulate Th2ammatory cytokines while upregulatingIL2 synthesis in concanavalin Astimulated peripheralblood mononuclear cells from adult males with atopic dermatitis Thus it was hypothesized that it would be a usefultreatment for atopic diseases including asthma and the results ofthe clinical trials for DHEA in asthma patients show promiseThe results are discussed in a later section titled Eï¬ects ofandrogen exposure on monocytes macrophages in humans withlung diseaseCOPDSex diï¬erences also have been reported in chronic obstructivepulmonary disease COPD a heterogeneous chronic andprogressive respiratory disorder that includes chronic bronchitisand emphysema Chronic exposure of the airways to insultssuch as cigarette smoke leads to epithelial cell injury destructionof pulmonary capillary vasculature acceleration of epithelial cellsenescence and airway remodeling The loss of lung complianceultimately leads to COPD COPD was previously thoughtto aï¬ect mostly elderly men primarily because of the higherprevalence of smoking in men However as smoking ratesincreased in women the number of COPD cases in womenexceeded that of men These diï¬erences are not only basedon gender as women develop more severe COPD with earlyonset disease years and have greater susceptibility toCOPD with lower tobacco exposure Moreover increasingage in female smokers leads to a faster annual decline inFrontiers in Immunology wwwfrontiersinAugust Volume 0cBecerraDiaz et alAndrogenAR in Lung MonocyteMacrophage Biologyforced expiratory volume in the ï¬rst second when compared tothat of male smokers even when they smoke fewer cigarettes Similarly pulmonary ï¬brosis is another lung disease thatmanifests sex diï¬erences with men being more aï¬ectedthan women It is characterized by destruction of thepulmonary parenchyma and deposition of extracellular matrixwith alterations in phenotype of both ï¬broblasts and alveolarepithelial cells Inï¬uenzaThe lungs are also the target of respiratory viruses such asuenza A ï¬u respiratory syncytial virus and coronavirusessuch as severe acute respiratory syndrome and the MiddleEast respiratory syndrome The viruses infectthe airwayepithelial cells and cause damage to the epithelial barrierby themselves or as a result ofthe immune response tothe viralinfection Sex diï¬erences have been noted in theimmune response to uenza A virus and to the uenzavaccine In general women have a more robust protectiveimmune response to uenza virus and vaccine than do menAlthough this elevated response is helpful in clearing viruswomen of reproductive age also experience higher mortalityand hospitalizations possibly from collateral tissuedamage to the lungs The vigorous immune response in womenalso means that women experience more adverse events aftervaccination Indeed a systems biology approach identiï¬edthat high testosterone was correlated with a blunted responseto the ï¬u vaccine in men As testosterone wanes in elderlymen mortality increases Since the male immune responseto the virus is also less robustthe incidence of seasonalï¬u is generally higher in men than in women in developedcountries according to the World Health anization It is not yet known how ï¬uctuations in sex hormones acrossthe menstrual cycle and lifespan aï¬ect the immune systemsresponse to the uenza virus in humans Mouse studieshave revealed that estrogen is protective at high but notlow concentrations On the other hand testosteronereplacement in gonadectomized or aged male mice enhancedsurvival rates Despite these ï¬ndings in mouse modelsstudies examining the eï¬ect of sex hormones on cellular andmolecular mechanisms in human immune cells during uenzainfection are lackingTuberculosisLike uenza infection tuberculosis TB a lung disease causedby Mycobacterium tuberculosis exhibits notable sex diï¬erencesin the number of cases worldwide with men being almosttwice as frequently aï¬ected than women Both sexand gender diï¬erences impact the incidence of TB AlthoughTB aï¬ects less women than men in adulthood womenin their economically active years years old have ahigher TB incidence compared to women in other age groups This indicates that factors associated with gender such asexposure to the bacteria are important in this disease Howeverbecause male predominance does not occur in children thissuggests that biological factors such as male sex hormones alsoplay a significant role This is supported by a study ofmedically castrated men who experienced a significantly smallerproportion of death from TB compared to in intactmen Understanding how androgens lead to the greatersusceptibility of men to TB is critical as TB is still one ofthe leading fatal infectious diseases worldwide and may alsomay favor the development of other diseases such as lungcancer Lung CancerLung cancer is a very complex disease that depends on anumber of variants such as sex gender race and socioeconomicstatus The development of lung cancer is also related toenvironmental factors such as pollution due to industrializationand urbanization An additional genderassociated riskfactor significantly linked to developing lung cancer is cigarettesmoking Historically more men develop lung cancer andsuï¬er lung cancerassociated deaths compared to women However the incidence of lung cancer has changed notably inboth women and men In men lung cancer incidence startedto increase in the 1920s and started to decrease in the early1990s while in women the mortality rates and incidence beganto rise in the 1960s Changes in smoking habits in the lastseveral decades with a rise in the number of women who smokecorrelate with an increase in the incidence of lung cancer in thisdemographic group Smoking is deï¬nitely a key factor inthe development of lung cancer however recent studies showa higher incidence of lung cancer in young women comparedto young men even when the prevalence of cigarettesmoking among young women has approached but not exceededthat among men This suggests that the higher incidenceof lung cancer in women is not explained simply by genderdiï¬erences in smoking habits a deeper analysis of diï¬erencesmediated by sex such as greater sensitivity to tobacco smoke inwomen is warranted Furthermore men and women develop diï¬erent speciï¬ctypes of lung cancer Malignant mesothelioma is more commonin men while women develop more adenocarcinoma particularly nonsmall cell lung cancer NSCLC Womenhave a superior survival rate for lung cancer compared tomen Tumorassociated macrophages are critical in tumorprogression yet how androgens uence macrophage behaviorin lung cancer and in responses to treatment must be addressedmore deeply to develop better therapies and increase survivalrates in menTHE MYELOID IMMUNE SYSTEM IN LUNGHEALTH AND DISEASEAlveolar MacrophagesThe lungs are a primary interface with the external environmentThe delicate structures needed for gas exchange make themsusceptible to damage from invading pathogens and toxicmolecules Some insults to the lung can lead to the developmentof chronic conditions such as allergic asthma As a protectivemechanism alveolar macrophages clearspace ofinfectious toxic or allergenic ps to maintain homeostasisin the alveoli Thus alveolar macrophages have a dualthe airFrontiers in Immunology wwwfrontiersinAugust Volume 0cBecerraDiaz et alAndrogenAR in Lung MonocyteMacrophage Biologyfunction as ammatory cells phagocytosing and killinginhaled bacteria or viruses and also as controllers oftheammatory immune response minimizing alveolar damageResident alveolar macrophages are seeded embryonically fromyolk sac and fetalliver monocytes In asthma andother lung diseases recruited alveolar macrophages derived fromblood monocytes can turn into pathogenic cells worseningthe condition Mouse alveolar macrophages arecharacterized by high surface expression of Siglec F and produceTGF TGF both supports AM development and theirmaintenance of immune homeostasis by induction of Tregs andsuppression of B and T cell proliferation Another importantfunction of AM is the clearance of surfactant AM from male andfemale mice respond diï¬erently to surfactant protein A SPA SPA acts as an opsonin and is important in clearanceof pathogens Sex diï¬erences in AM responses to surfactantcould aï¬ect bacterial clearance and regulate the production ofproammatory mediators The molecular mechanisms thatmediate these diï¬erences and how sex hormones change thisimportant AM function is an open questionIn the human lung there appears to be more diversity inthe subtypes of lung macrophages compared to mice The maindeterminant of the frequency of subtypes of macrophages inhumans appears to be their anatomicallocation within thelung AM are the predominantimmune cells in the lungairways bronchi and bronchioalveolar space Flow cytometricpanels have employed HLADR CD163 CD169 and CD206to diï¬erentiate between AM IM and monocytes Human AMwere identiï¬ed as large highly autoï¬uorescent CD14 CD16cells that also express CD206 CD169 and MARCO There appear to be two populations of AM distinguished byeither high or low expression of CD163 More recent approachesto characterize the macrophage populationsin the lunginvolve singlecell transcriptomic analysis Althoughmacrophages show a large variation in the transcriptionalphenotype expression of MARCO CCL18 APOC1 APOEPPARG and MRC1 was found in macrophages from healthydonors while CHI3L1 MARCKS IL1RN PLA2G7MMP9 and SPP1 were highly expressed in macrophages frompulmonary ï¬brosis patients Thus a second contributor todiversity is likely the activation state of the cells There are nodata that describe sex diï¬erences in human AM responses andthe eï¬ect of sex hormones on these cells From our mouse andhuman MDM studies we would predict that androgens augmentthe immune homeostatic functions of these cells in the malelung Further work is still needed to standardize characterizationof the diï¬erent subpopulations of human lung macrophagepopulations and their role in maintaining healthy lung functionand in diseaseIMsInterstitial MacrophagesInterstitial macrophagesanother macrophagepopulation found in the lung They are a minor populationof monocytederived macrophages which comprise of lung macrophages and are localized in the lungparenchyma IMs contribute to maintaining homeostasisthrough the spontaneous release of IL10 a cytokine thataredampens ammation IMs can prevent the developmentof aberranttype allergic responses triggered by inhaledallergens and have been related to reduction of asthma Diï¬erent subpopulations of IMs have been foundin the lung however their characterization has not arrived at aconsensus due to diï¬culties in their identiï¬cation and isolationIn the mouse lung diï¬erent subpopulations of IMs have beendescribed based on the expression of surface markers One reportdescribed three diï¬erent subpopulations of IMs based on thediï¬erential expression of proammatory cytokines chemokineligands MHCII CD11c CD206 and Lyve1 other groupidentiï¬ed two subpopulations based on similar markers butincluding CX3CR1 Moreover IMs subpopulations canbe also described based on the diï¬erent anatomic locationsthese cells populate inside the mouse lung parenchyma Further work is needed to better characterize and deï¬ne thediï¬erent IM populations as the diï¬erent subtypes may havediï¬erent functions during the ammatory process Smallerin size than their AM counterparts human IMs express moreof the monocytic marker CD14 than AM perhaps suggestingtheir monocytic origin and have lower expression of CD169than human AM The responses of IM to androgen will dependon their expression of AR which has not been measured Thiswill be a challenge due to diï¬culties in clearly identifying thispopulation and its subpopulations from the monocytic AMand other myeloid populations in the lungMonocytesMonocytes are produced in the bone marrow along with anumber of other myeloid cells Myeloid cells originate fromcommon pluripotent hematopoietic stem cells and representthe major subset of white cells in circulation These cellscomprise basophils neutrophils eosinophils DCs monocytesand macrophages among others Monocytes are releasedinto circulationthen blood monocytes are recruited intoamed tissue and can mature into macrophages or dendriticcells There are two main subsets of mouse monocytesclassical or Ly6Chigh monocytes that originate directly fromLy6C precursors and nonclassical or Ly6Clow monocytesthat derive from Ly6Chigh monocytes The origin ofLy6C low monocytes was demonstrated by Sunderkotteret al by tracking the maturation of DiIlabeled Ly6Chighmonocytes into DiIlabeled Ly6Clow monocytes Thisprocess depends on the transcription factor Nr4a1 whichregulates the development and survival of Ly6Clow monocytes These two monocyte subsets mirror the human CD14classical and CD16 nonclassical monocyte populationsrespectively Ly6Chigh monocytes highly express thechemokine receptor CCchemokine receptor CCR2 whereasLy6Clow monocytes highly express CX3CR1 ImportantlyCCR2 expression is required for Ly6C monocyte egress fromthe bone marrow into the circulation and entry into nonamed and amed tissues from the blood As monocytes migrate into tissue they mature into macrophagesdeveloping unique tissuedependent morphology and functions They lose expression of Ly6C and gain expression ofMHC class II becoming more eï¬cient antigenpresenting cellsFrontiers in Immunology wwwfrontiersinAugust Volume 0cBecerraDiaz et alAndrogenAR in Lung MonocyteMacrophage Biology Some authors have proposed the concept of tissuemonocytes which are monocytes that can enter nonlymphoidans without obligatory diï¬erentiation into macrophagesTherefore monocytes are much more than simply precursorsfor macrophagesIn human lungs monocytes which can be both beneï¬cialand pathogenic in a variety of pulmonary diseases arepresent at steady state Multiplecolor cytometric analysison cells obtained from diï¬erent anatomical locations of the lungof healthy subjects nonsmokers with normal lung function andabsence of disease or infection revealed that while intermediatemonocytes CD14CD16 are more frequent in the airwaysclassical monocytes CD14CD16 are more frequent in blood Moreover the diï¬erent monocyte subsets produced TNFα to diï¬erent degrees upon stimulation with TLR ligands and Thus the anatomic location where samples are obtainedshould be considered and reported when working with humanbronchoscopies as this may alter the type and abundance ofmonocytes and macrophages found Accurate identiï¬cation ofmonocytes in the lung compartments in humans has been achallenge because monocytic contamination from the bloodvessels Overcoming this challenge Desch et alperformed a ï¬ow cytometric phenotyping study and identiï¬edtwo additional lung monocyte populations by analyzing lungsobtained from donors who died of nonpulmonary causes CD14 CD206 CD1c CD1a intravascular monocyteswere similar to CD14 blood monocytes and CD14 CD206CD1c CD1a monocytes were described as tissue monocytesThese studies highlightthe beginningof understanding the complexity of lung monocyte subtypesand their functions depending on the ammatory state ofthe lungthat we are just atOther myeloid populationslike DCs occupy the lungparenchyma at steady state and their relative numbers changeduring ammation We refer readers to previous excellentreviews in this journal that cover the importance of DCs inimmune responses in the lung and how they are aï¬ectedby sex diï¬erences Therefore we will not discuss DCs here Macrophage ActivationPolarization is a very important eï¬ector characteristic observedin monocytes and macrophages Polarization refers to the changein phenotype and function of monocytes and macrophagesas they are exposed to diï¬erentammatory milieus orfactors in the tissue microenvironment To understand theeï¬ects of the diï¬ering ammatory or tissue environments onmonocytemacrophage phenotype and function researchershave used cytokines and other factors in vitro to mimic diï¬erentammatoryand tissue microenvironments Monocytesand macrophages stimulated with interferonγ LPS TNFαinterleukin IL12colonystimulating factor promote a proammatory macrophagephenotype denoted as M1 polarization The activation state wasalso known as classical activation M1polarized macrophagesmediate immunity to intracellular infections such as viruses andand granulocytemacrophagebacteria and they are generally considered tumoricidal M1 macrophages accomplish these functions by inducingproduction of nitric oxide reactive nitrogen intermediatesreactive oxygen species and hydrogen peroxide Incontrast activation of macrophages with IL4 or IL13 as inextracellular parasitic infections and allergic reactionsleadsto M2 polarization or alternative activation of macrophages M2 macrophages produce ammatory mediatorsand chemokines such as chitinaselike proteins IL13 CCL17 CCL18 CCL22 and CCL24 which activateTh2 cells and promote eosinophil ltration into the lungs In allergic asthma a Th2ammatory response to inhaledallergens drives lung macrophages toward an M2 phenotypeIncreased number and percent of M2 macrophages havebeen correlated with asthma severity and a decline in lungfunction in humans and mouse models SimilarlyM2 macrophages are the predominant subset seen in pulmonaryï¬brosis and are responsible for ï¬brogenesis During COPDthe number of macrophages in airwayslung parenchymabronchoalveolar lavage ï¬uid and sputum increases This increase may occur as a result of enhanced monocyterecruitment from circulation in response to chemokines suchas CCL2 and CXCchemokine ligand1 which are increased inthe sputum and bronchoalveolar lavage ï¬uid of patients withCOPD Unlike in allergic asthma and pulmonary ï¬brosismacrophages in COPD are polarized toward an M1 proï¬le In addition to aï¬ecting men and women diï¬erently anothercommonality of COPD is that macrophages both in the alveolarspace and in lung tissue present an altered activation phenotypeDiï¬erent concentrations of cytokines TNFα IL1 IL6 IL IL12 and chemokines CCL2 CCL5 CCL7 CCL13 CCL22IL8 CXCL9 and CXCL10 are found comparing smokers tohealthy subjects Thus the external provoking stimulusuniquely shapes macrophage phenotype and functionWhile the M1M2 designations are useful for in vitro studieswith stimulation with deï¬ned cytokines the in vivo phenotypeof macrophages exists on a spectrum somewhere in betweenthese two welldeï¬ned opposing phenotypes or does not ï¬tthe paradigm at all For example M1 and M2 markers canexist simultaneously within the same cell in some cases The key factors dictating the macrophage phenotypeor activation state are the stage ofthe immune responseand the soluble factors and interactions in a particular tissuemicroenvironment For example the lung environment is richin GMCSF TGF and PPARγ and is critical for developmentof mature AMs after birth in both mice and humans Furthermoreinteractions betweenCD200 on type II alveolar epithelial cells and CD200R on thesurface of the AM deliver regulatory signals to the AM toprevent proammatory signaling and macrophage activation Thus macrophage nomenclature has evolved as ourunderstanding of the phenotypes and functions of diï¬erenttypes of tissue resident macrophages recruited monocytes andmonocytederived macrophages advances Indepth studies ofthe eï¬ects of androgens and other sex hormones on tissuemacrophage plasticity and phenotype have yet to be carried outFrontiers in Immunology wwwfrontiersinAugust Volume 0cBecerraDiaz et alAndrogenAR in Lung MonocyteMacrophage BiologyMECHANISMS OF ANDROGEN SEXSTEROID ACTIONEFFECTS OF ANDROGEN EXPOSURE ONMONOCYTES MACROPHAGES IN VITROBecause androgens are lipophilic steroid hormones they caneasily diï¬use across cell membranes withoutthe need forreceptormediated import Androgens in circulation arefound mostly bound to sex hormonebinding globulin andalbumin Free unbound steroid sex hormones can signalthrough two diï¬erent mechanisms the classical ARlocate | 2 |
"11-years of experience. Surg Endosc23: 556118437482 7 CerfolioRJ BryantAS McCartyTP MinnichDJ (2011) A prospective study to determine the incidence of non-imaged malignant pulmonary nodules in patients who undergo metastasectomy by thoracotomy with lung palpation. Ann Thorac Surg91: 16961700 discussion 17001691.21619965 8 DownsSH BlackN (1998) The feasibility of creating a checklist for the assessment of the methodological quality both of randomised and non-randomised studies of health care interventions. J Epidemiol Community Health52: 3773849764259 9 KondoH OkumuraT OhdeY NakagawaK (2005) Surgical treatment for metastatic malignancies. Pulmonary metastasis: indications and outcomes. Int J Clin Oncol10: 818515864692 10 PorterGA CantorSB WalshGL RuschVW LeungDH et al (2004) Cost-effectiveness of pulmonary resection and systemic chemotherapy in the management of metastatic soft tissue sarcoma: a combined analysis from the University of Texas M. D. Anderson and Memorial Sloan-Kettering Cancer Centers. J Thorac Cardiovasc Surg127: 1366137215115994 11 PetersenRP PhamD BurfeindWR HanishSI TolozaEM et al (2007) Thoracoscopic lobectomy facilitates the delivery of chemotherapy after resection for lung cancer. Ann Thorac Surg83: 12451249 discussion 1250.17383320 12 PaulS AltorkiNK ShengS LeePC HarpoleDH et al (2010) Thoracoscopic lobectomy is associated with lower morbidity than open lobectomy: a propensity-matched analysis from the STS database. J Thorac Cardiovasc Surg139: 36637820106398 13 WhitsonBA GrothSS DuvalSJ SwansonSJ MaddausMA (2008) Surgery for early-stage non-small cell lung cancer: a systematic review of the video-assisted thoracoscopic surgery versus thoracotomy approaches to lobectomy. Ann Thorac Surg86: 20082016 discussion 20162008.19022040 14 EckardtJ LichtPB (2012) Thoracoscopic versus open pulmonary metastasectomy: a prospective sequentially controlled study. Chest142: 1598160222677347 15 AmbrogiV PaciM PompeoE MineoTC (2000) Transxiphoid video-assisted pulmonary metastasectomy: relevance of helical computed tomography occult lesions. Ann Thorac Surg70: 1847185211156082 16 MargaritoraS PorziellaV D'AndrilliA CesarioA GalettaD et al (2002) Pulmonary metastases: can accurate radiological evaluation avoid thoracotomic approach? Eur J Cardiothorac Surg21: 1111111412048094 17 ParsonsAM DetterbeckFC ParkerLA (2004) Accuracy of helical CT in the detection of pulmonary metastases: is intraoperative palpation still necessary? Ann Thorac Surg78: 19101916 discussion 19161918.15561000 18 KaytonML HuvosAG CasherJ AbramsonSJ RosenNS et al (2006) Computed tomographic scan of the chest underestimates the number of metastatic lesions in osteosarcoma. J Pediatr Surg41: 200206 discussion 200206.16410133 19 Long-term results of lung metastasectomy: prognostic analyses based on 5206 cases. The International Registry of Lung Metastases. J Thorac Cardiovasc Surg113: 37499011700 20 MutsaertsEL ZoetmulderFA RutgersEJ (2001) Port site metastasis as a complication of thoracoscopic metastatectomy. Eur J Surg Oncol27: 32732811373113 21 JohnstonePA RohdeDC SwartzSE FetterJE WexnerSD (1996) Port site recurrences after laparoscopic and thoracoscopic procedures in malignancy. J Clin Oncol14: 195019568656265 22 TreasureT (2007) Pulmonary metastasectomy: a common practice based on weak evidence. Ann R Coll Surg Engl89: 74474817999813 23 RothJA PassHI WesleyMN WhiteD PutnamJB et al (1986) Comparison of median sternotomy and thoracotomy for resection of pulmonary metastases in patients with adult soft-tissue sarcomas. Ann Thorac Surg42: 1341383741009 24 FloresRM IhekweazuUN RizkN DycocoJ BainsMS et al (2011) Patterns of recurrence and incidence of second primary tumors after lobectomy by means of video-assisted thoracoscopic surgery (VATS) versus thoracotomy for lung cancer. J Thorac Cardiovasc Surg141: 596421055770 25 SchaeffB PaolucciV ThomopoulosJ (1998) Port site recurrences after laparoscopic surgery. A review. Dig Surg15: 1241349845574 26 ChenYR YeowKM LeeJY SuIH ChuSY et al (2007) CT-guided hook wire localization of subpleural lung lesions for video-assisted thoracoscopic surgery (VATS). J Formos Med Assoc106: 91191818063512 27 MolnarTF GebitekinC TurnaA (2010) What are the considerations in the surgical approach in pulmonary metastasectomy? J Thorac Oncol5: S14014420502249 28 NakajimaJ TakamotoS TanakaM TakeuchiE MurakawaT et al (2001) Thoracoscopic surgery and conventional open thoracotomy in metastatic lung cancer. Surg Endosc15: 84985311443456 29 MutsaertsEL ZoetmulderFA MeijerS BaasP HartAA et al (2002) Long term survival of thoracoscopic metastasectomy vs metastasectomy by thoracotomy in patients with a solitary pulmonary lesion. Eur J Surg Oncol28: 86486812477479 30 NakasA KlimatsidasMN EntwisleJ Martin-UcarAE WallerDA (2009) Video-assisted versus open pulmonary metastasectomy: the surgeon's finger or the radiologist's eye? Eur J Cardiothorac Surg36: 46947419464921 31 CarballoM MaishMS JaroszewskiDE HolmesCE (2009) Video-assisted thoracic surgery (VATS) as a safe alternative for the resection of pulmonary metastases: a retrospective cohort study. J Cardiothorac Surg4: 1319239710 32 GossotD RaduC GirardP Le CesneA BonvalotS et al (2009) Resection of pulmonary metastases from sarcoma: can some patients benefit from a less invasive approach? Ann Thorac Surg87: 23824319101304 33 ChaoYK ChangHC WuYC LiuYH HsiehMJ et al (2012) Management of lung metastases from colorectal cancer: video-assisted thoracoscopic surgery versus thoracotomya case-matched study. Thorac Cardiovasc Surg60: 39840422228090 101150042 30118 Mol Cancer Res Mol. Cancer Res. Molecular cancer research : MCR 1541-7786 1557-3125 24202705 3946989 10.1158/1541-7786.MCR-13-0300 NIHMS538386 NEDD9 Depletion Leads to MMP14 Inactivation by TIMP2 and Prevents Invasion and Metastasis. McLaughlin Sarah L. 5 * Ice Ryan J. 5 * Rajulapati Anuradha 5 Kozyulina Polina Y. 1 Livengood Ryan H. 4 Kozyreva Varvara K. 5 Loskutov Yuriy V. 5 Culp Mark V. 3 Weed Scott A. 2 5 Ivanov Alexey V. 1 5 Pugacheva Elena N. 1 5 # 1Department of Biochemistry West Virginia University School of Medicine Mantown WV 26506 2Department of Neurobiology and Anatomy West Virginia University School of Medicine Mantown WV 26506 3Department of Statistics West Virginia University School of Medicine Mantown WV 26506 4Department of Pathology West Virginia University School of Medicine Mantown WV 26506 5Mary Babb Randolph Cancer Center West Virginia University School of Medicine Mantown WV 26506 #Corresponding author: Elena N. Pugacheva Mailing address: Department of Biochemistry and Mary Babb Randolph Cancer Center PO Box 9142 1 Medical Center Drive West Virginia University School of Medicine Mantown WV 26506. Phone: (304) 293-5295; Fax: (304) 293-4667; epugachevahsc.wvu.edu S.L. McLaughlin* and R. J. Ice* contributed equally to this work. 17 12 2013 07 11 2013 1 2014 01 1 2015 12 1 69 81 The scaffolding protein NEDD9 is an established pro-metastatic marker in several cancers. Nevertheless the molecular mechanisms of NEDD9 driven metastasis in cancers remain ill defined. Here using a comprehensive breast cancer (BCa) tissue microarray it was show that increased levels of NEDD9 protein significantly correlated with the transition from carcinoma in situ to invasive carcinoma. Similarly it was shown that NEDD9 overexpression is a hallmark of highly invasive BCa cells. Moreover NEDD9 expression is crucial for the protease-dependent mesenchymal invasion of cancer cells at the primary site but not at the metastatic site. Depletion of NEDD9 is sufficient to suppress invasion of tumor cells in vitro and in vivo leading to decreased circulating tumor cells (CTCs) and lung metastases" | 1 |
"Sulindac is a ligand of the aryl hydrocarbon receptor (AhR) an xenobiotic-sensing nuclear receptor that can be activated by chemical structures containing planar aromatic hydrocarbons and thus evokes a cellular response that to detoxify xenobiotics. AhR activation leads to transcriptional upregulation of the NQO1 gene [62] [63]. In previous studies [30] [64] [65] sulindac and its two metabolites have been used to treat cancer cells at concentrations of 200 µM 1 mM i.e. the concentrations used in our present study. In addition to reducing the growth of polyps all three increase NQO1 activity and expression in colon cancer cells [28] and might therefore be good candidates to increase the cytotoxic effect of ?-lapachone against lung cancer cells. When two cancer cell lines CL1-1 and CL1-5 with low NQO1 expression and activity were co-incubated with sulindac or its metabolites and ?-lapachone much higher cell death was seen with the CL1-5 cells than the CL1-1 cells ( and 7). These results demonstrated that the effect of sulindac and its metabolites in upregulating NQO1 was greater in CL1-5 cells which has lower NQO1 level and activity than CL1-1 cells showing that sulindac and its metabolites can be used to increase the ?-lapachone sensitivity of cells with lower NQO1 levels. Many other compounds such as toxifolin [32] and resveratrol [66] can increase NQO1 expression or activity but are not FDA-approved. A search is underway for other compounds that can increase the activity or expression of NQO1 using high-throughput library screening and two compounds DMEBP and TRES were recently found to be potent NQO1 inducers with low toxicity [3]. These compounds may also be valuable in increasing ?-lapachone cytotoxicity for cancer cells with low NQO1 expression or activity. The NQO1 Inhibitor Dicoumarol or Transfection with NQO1 siRNA Inhibits the Effect of Sulindac on ?-lapachone Toxicity for Lung Cancer Cells Dicoumarol is widely used as a specific pharmacologic inhibitor of NQO1 and has been shown to inhibit both enzyme activity and expression [45] [67] [68]. NQO1 siRNA designed to specifically target NQO1 mRNA can lower the expression of NQO1 mRNA and protein. In our study both agents blocked the synergistic effect of sulindac or its metabolites and ?-lapachone on decreasing the survival of CL1-1 or CL1-5 cells. Although ?-lapachone is very toxic for many cancer cells cells with lower NQO1 levels are less sensitive. However from the present study we can conclude that sulindac and its metabolites increase NQO1 expression and enzyme activity and thus are potential synergistic drugs that might be used in combination with ?-lapachone to treat cancer cells with high resistance to ?-lapachone cytotoxicity. Supporting Information Figure S1 ?-lapachone causes cell death of CL1-1 and CL1-5 cells by decreasing the mitochondrial membrane potential. (A) Cells were left untreated or were incubated with 5 µM ?-lapachone for the indicated time and then the cell cycle distribution was analyzed using propidium iodide staining and flow cytometry. (B) Cells were incubated with 5 µM ?-lapachone for the indicated time then pro-caspase 3 and caspase 3 levels were analyzed by Western blotting. (C) Cells were incubated with 5 µM ?-lapachone for the indicated time then the mitochondrial membrane potential (MMP) was measured using the dye JC1 (Life Technology) and flow cytometry. (D) Cells were incubated with 5 µM ?-lapachone for the indicated time and then intracellular H2O2 levels were measured. (TIF) Click here for additional data file. Figure S2 zVAD-FMK ALLM and ALLN do not block the cytotoxicity of ?-lapachone. CL1-1 cells (A) or CL1-5 cells (B) were left untreated or were incubated for 1 h with the indicated concentration of the pan caspase inhibitor zVAD (left panels) or the calpain inhibitor ALLM (center panels) or ALLN (right panels) then 5 µM ?-lapachone was added for 12 or 24 h and cell viability measured using the MTT assay and expressed as percentage survival compared to the untreated cells. (TIF) Click here for additional data file. Figure S3 Dicoumarol an NQO1 inhibitor inhibits NQO1 activity and blocks the increase in intracellular calcium levels induced by ?-lapachone. (A) CL1-1 cells (left) or CL1-5 cells (right) were left untreated (CTL) or were incubated with 10 µM dicoumarol for 6 h then NQO1 activity was measured. (B) CL1-1 cells (top panel) or CL1-5 cells (bottom panel) were left untreated or were incubated with 10 µM dicoumarol and/or 5 µM ?-lapachone for 1 h then were stained with Fluo-4 and the intensity of the Fluo-4 fluorescence measured by flow cytometry. (TIF) Click here for additional data file. Figure S4 Sulindac and its metabolites do not affect survival of lung cancer cells. CL1-1 CL1-5 or A549 cells were left untreated or were incubated for 54 h with 100 or 250 µM sulindac (left panel) or sulindac sulfone (center panel) or for 12 h with 100 or 250 µM sulindac sulfide (right panel) then cell survival was measured by the MTT assay and expressed as percentage survival compared to the untreated cells. (TIF) Click here for additional data file. Figure S5 The cytotoxic effect of ?-lapachone on A549 cells is enhanced by sulindac and its metabolites. Two sets of cells were left untreated or were incubated for 6 h with the indicated concentration of sulindac sulindac sulfone or sulindac sulfide then 2 µM ?-lapachone was added to one set and incubation continued for 12 h when cell survival was measured using crystal violet staining and expressed as percentage survival compared to the untreated cells. (TIF) Click here for additional data file. Figure S6 NQO1 siRNA has no effect on cell morphology or cell growth. CL1-1 cells (top) and CL1-5 (bottom) were transfected with negative siRNA or NQO1 siRNA for 1 to 3 days then pictures were taken using a digital camera and phase contrast microscopy. The scale bar represents 50 µm. (TIF) Click here for additional data file. Figure S7 NQO1 RNA levels are decreased by siRNA targeting NQO1. A549 CL1-1 or CL1-5 cells were transfected for 48 h with siRNA targeting NQO1 (siNQO1) or control siRNA (siNeg) and then NQO1 mRNA levels were measured by realtime PCR and expressed as a fold change compared to the value for CL1-5 cells transfected with siNeg. * : p<0.05 compared to the result for the corresponding siNeg-transfected cells. (TIF) Click here for additional data file. Table S1 Primers used in the realtime PCR for actin and NQO1. (TIF) Click here for additional data file. Materials and Methods S1 (DOCX) Click here for additional data file. This work was supported by grants (NSC 101-2320-B-002-020-MY3 NSC 98-2320-B-715-001-MY3 (YPC) and NSC 101-2320-B-002-008) from the National Science Council Taiwan. References 1 PardeeAB LiYZ LiCJ (2002) Cancer therapy with beta-lapachone. Curr Cancer Drug Targets2: 22724212188909 2 TagliarinoC PinkJJ ReinickeKE SimmersSM Wuerzberger-DavisSM et al (2003) Mu-calpain activation in beta-lapachone-mediated apoptosis. Cancer Biol Ther2: 14115212750552 3 TanXL MarquardtG MassimiAB ShiM HanW et al (2012) High-throughput library screening identifies two novel NQO1 inducers in human lung cells. Am J Respir Cell Mol Biol46: 36537122021338 4 MinamiT AdachiM KawamuraR ZhangY ShinomuraY et al (2005) Sulindac enhances the proteasome inhibitor bortezomib-mediated oxidative stress and anticancer activity. Clin Cancer Res11: 5248525616033843 5 TeraiK DongGZ OhET ParkMT GuY et al (2009) Cisplatin enhances the anticancer effect of beta-lapachone by upregulating NQO1. " | 1 |
"Gastric neoplasms containing neuroendocrine carcinoma NEC components are rare malignancieswith highly aggressive behavior and a poor prognosis and include pure NEC and mixed tumors containing NECcomponents We aimed to investigate whether there is a distinct difference in overall survival OS between gastricneoplasms containing NEC components and gastric adenocarcinomaMethods Surgically resected gastric neoplasms containing NEC components n and gastricadenocarcinomas n from January to December at Peking University Cancer Hospital wereretrospectively analysed Patients were categorized into a surgical group and a neoadjuvant group and adjustedusing pr sity score matching In the two groups gastric neoplasms containing NEC components were dividedinto pure NEC and mixed tumors with less than GHMiNEN between and GHMiNEN andmore than GHMiNEN neuroendocrine carcinoma components OS was compared between thesegroups and the gastric adenocarcinoma groupResults The OS of gastric neoplasms containing neuroendocrine NEC components was poorer than that of gastricadenocarcinomas in the surgical group regardless of whether the percentage of neuroendocrine cancercomponents was less than between and more than or Cox multivariable regressionanalysis suggested that tumor category neoplasms containing NEC components or gastric adenocarcinoma wasan independent risk factor for prognosis Interestingly among patients receiving neoadjuvant therapy thedifference was not significantContinued on next page Correspondence buzhaodecjcrcn jijiafuhscpkueducn Jiahui Chen Anqiang Wang and Ke Ji contributed equally to this workDepartment of Gastrointestinal Surgery Key Laboratory of Carcinogenesisand Translational Research Ministry of Education Peking University CancerHospital Institute No Fucheng Road Haidian District Beijing China The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cChen BMC Cancer Page of Continued from previous pageConclusions Gastric neoplasms containing any proportion of NEC components had poorer overall survival thangastric adenocarcinoma in patients treated with surgery directly indicating that these neoplasms are moremalignant than gastric adenocarcinoma Among the patients receiving neoadjuvant therapy the difference inoverall survival was not significant which was in sharp contrast with the results of the surgery group suggestingthat neoadjuvant therapy may have a good effect in the treatment of these neoplasmsKeywords Neuroendocrine carcinoma Gastric adenocarcinoma Overall survivalBackgroundGastric neoplasms containing neuroendocrine carcinomaNEC components are a heterogeneous subgroup ofgastric cancer with highly aggressive behavior and poorprognosis and include pure NECs and mixed tumorscontaining NEC components Every yearthere areapproximately million new cases of gastric cancerworldwide and gastric neoplasms containing NEC components account for approximately of thesecases [ ] Given the low incidence there is little comprehensive basic and clinical research to systematicallyguide the treatment of these gastric neoplasms makingthe prognosis of these tumors unsatisfactory []According to the World Health anizationWHO digestive neuroendocrine tumor classificationneuroendocrine neoplasm NEN can be divided intothree categories based on Ki67 levels and mitotic counts HPF Grade G1 Ki67 mitoses Grade G2 Ki67 mitoses Grade G3Ki67 mitoses [] Meanwhile the AmericanJoint Committee on Cancer AJCC defines highly differentiated NEN as a neuroendocrine tumor NET and thepoorly differentiated NEN as a neuroendocrine carcinoma NEC based on the degree of tumor cell differentiation Generally G1 G2 and rare welldifferentiated G3NENs belong to the NETs while poorly differentiatedG3 NENs belong to NECs[ ] Gastric mixedneuroendocrinenonneuroendocrineneoplasm GMiNEN is a special type of gastric NEN that is definedas containing more than of both neuroendocrineand nonneuroendocrine components [] accountingfor approximately of all GNENs and of gastricneuroendocrine carcinomas GNECs [] For thosemixed tumors with less than or more than neuroendocrine carcinoma components there is no uniform definition Consideringthe heterogeneity ofMiNEN and the malignancy degree of the different components in the tumor La Rosa [ ] proposeddividing MiNEN into three categories highgradeintermediategrade and lowgrade Highgrade MiNENconsists of NEC and carcinomaadenoma intermediategrade MiMEN consists of NET and carcinoma and lowgrade MiNEN consists of NET and adenoma Thereforein this study gastric highgrade mixed neuroendocrinenonneuroendocrine neoplasm GHMiNEN was defined as gastric cancer containing more than of bothneuroendocrineadenocarcinomacomponentscarcinomaandGenerally the prognosis of mixed tumors is largely determined by the most malignant component Kim et al[] found that GNEC has shorter progressionfree survival PFS than gastric adenocarcinoma Huang et al[] found that the prognosis of patients with more than of neuroendocrine cancer components is significantly poorer than that of patients with less than components All of these studies provide evidence thattumors containing neuroendocrine cancer componentsmay contribute to a worse prognosis Therefore wehypothesized that a mixed tumor containing neuroendocrine carcinoma components would have a worse prognosis than pure adenocarcinoma alone We sought tofind studies on the overall survival OS comparison between GHMiNEN and gastric adenocarcinoma butfailed Thus we think that a study of the comparison ofthe OS of GHMiNEN and gastric adenocarcinoma willprovide a valuable supplement to current research on GHMiNEN To overcome the bias caused by the differences between the covariates in the comparison we usedpr sity score matching PSM to match importantfactors such as age gender tumor location tumor sizepathological staging and adjuvant chemotherapy between the two groups making the research results morereliableMethodsPatient selectionWe retrospectively collected patients diagnosed withgastric NENs and underwent radical resection at PekingUniversity Cancer Hospital Beijing from January to December The inclusion criteria were as follows pathologically confirmed pure NEC or tumorcontaining NEC components no other tumors werediagnosed before the operation complete clinicopathological information and survival information thatcould be obtained through followup Patients diagnosedwith cM1 or cT4b before surgery or died from perioperative complications were excluded from the study 0cChen BMC Cancer Page of Patients with gastric adenocarcinomas undergoing radical surgery were randomly selected for PSM analysesperformed The chisquared test and MannWhitney Utest were used to further verify the matching resultsFollowupWe followed the patients at least twice a year Serumtumor markers test gastroscope and computed tomography CT scans were used to reexamine patients aftersurgery Depending on the patients status Magneticresonance imaging MRI and Positron emission tomography computed tomography PETCT were alsoconsidered For patients who cannot regularly visit ourcenter for postoperative examination we use telephonefollowup to obtain survival informationDiagnosis and classificationWe reevaluated the diagnosis and classification of GHMiNEN Mixed tumors with less than or morethan neuroendocrine carcinoma components werealso included in this study which were defined as GHMiNEN and GHMiNENrespectively Atumor consisting of NEC is defined as pure NECAll neuroendocrine tumors were identified diagnosedand classified by two independent pathologists in accordance with the WHO classification of tumors[] Neuroendocrine components were identified byhistological features and immunohistochemical specificity marks such as synaptophysin Syn chromograninA CgA and neuro cell adhesion molecule CD56 orNCAM The tumor staging described in the study wasbased on the AJCC 8th Edition TNM Staging Guidelines[] All possible disagreements were discussed in ourstudy groupDefinition of variables and groupsIn this study patients were divided into a surgical groupand a neoadjuvant group based on whether they had received neoadjuvant therapy before surgery Patients inthe surgery group were assessed by the pTNM stagingsystem while patients in the neoadjuvanttreatmentgroup were assessed by the ypTNM staging system OSrefers to the time from surgery to the last followup thetime of death or the end ofloss offollowup or other cause of deathfollowup egPr sity score matchingTo accurately compare the prognosis of GHMiNENand gastric adenocarcinoma we employed PSM to balance the differences between the two groups PSM wasperformed through the Pamatching plugin in SPSS software Logistic regression models were used toestimate pr sity scores based on gender age tumorlocation tumor size and pathological staging Given a caliper width nearest neighbor matching wasStatistical analysisAll statistical analyses were performed using SPSS statisticalsoftware IBM United States The chisquared test and MannWhitney U test were used forstatistical analysis of categorical variables and continuous variables respectively KaplanMeier method wasused for the comparison of OS The logrank test wasused to compare survival rates Multivariable Cox proportional hazards models were used to identify predictors of survival outcome P was regarded as thethreshold of significanceResultsPatient selection and PSM resultsBetween and among the patients treated atthe Gastrointestinal Cancer Center of Peking UniversityCancer Hospital a total of patients with gastric neoplasms containing NEC components met the inclusioncriteria for the study including cases of pure NECand cases of mixedtype Of these patients a total of patients received neoadjuvant therapy NEC GHMiNEN GHMiNEN GHMiNEN while the remaining patients receivedsurgery directly NEC GHMiNEN GHMiNEN GHMiNEN There were aninsufficient number of patients in group GHMiNEN group to conduct effective statistical analysisso we combined the GHMiNEN group with theNEC group for further analysis We also randomly selected patients with gastric adenocarcinoma whounderwent radical surgery Among them patientsreceived neoadjuvant therapy and the remaining patients were treated with surgery directly Fig Immunohistochemical specificity markers were utilizedto identify the neuroendocrine components Fig 2aSyn was expressed in almost all neoplasms containingNEC components while the positive rates ofCgA and CD56 were much lower and respectively No significant difference in the positiverate of Syn and CgA was observed between pure NEC GHMiNEN GHMiNEN and GHMiNENFig 2b c only the positive rate of CD56 was found tobe higher in the pure NEC group than that in the GHMiNEN group Fig 2dTherefore priorto OS comparison PSM wasperformed to ensure that there were no significant differences in patient gender age tumor location tumorsize pathological staging and adjuvant chemotherapybetween the two groups 0cChen BMC Cancer Page of Fig Flow chart of patient enrolmentComparison of OS between all patients with NECcomponents and patients with gastric adenocarcinoma inthe surgical group and neoadjuvant groupBefore PSM we compared the survival curves between all patients with NEC components and patientswith gastric adenocarcinoma by the KaplanMeiermethod Fig Apparently patients with NEC components had a poorer OS than those with gastricadenocarcinoma Fig 3a p in the surgicalgroup In contrast no significant difference was observed between the patientsreceiving neoadjuvanttherapy Fig 3b p According to the proportion of NEC components patients were classifiedinto pure NEC GHMiNEN GHMiNENand GHMiNEN The OS was also comparedbetween patients with adenocarcinomaand thesegroups and the results were similar to the overallcomparison Fig 3c dFig Illustrations of immunohistochemical staining patterns in gastric neoplasms containing NEC components a An overview of the expressionof Syn CgA and CD56 in tumors containing NEC components b Syn expression in different NEC component groups c CgA expression indifferent NEC component groups d CD56 expression in different NEC component groups CD56 neuro cell adhesion molecule CgAchromogranin A NEC neuroendocrine carcinoma Syn synaptophysin Pvalue 0cChen BMC Cancer Page of Fig See legend on next page 0cChen BMC Cancer Page of See figure on previous pageFig Comparison of OS between gastric neoplasms containing NEC components and gastric adenocarcinoma a OS comparison betweengastric neoplasms containing NEC components and gastric adenocarcinoma before PSM in the surgical group b OS comparison between gastricneoplasms containing NEC components and gastric adenocarcinoma before PSM in the neoadjuvant group c OS comparison between differentNEC content groups pure NEC GHMiNEN GHMiNEN and GHMiNEN and gastric adenocarcinoma before PSM in the surgicalgroup d OS comparison between the different NEC content groups and gastric adenocarcinoma before PSM in the neoadjuvant group e OScomparison for patients in the surgical group after PSM f OS comparison for patients in the neoadjuvant group after PSM NEC neuroendocrinecarcinoma OS overall survival PSM pr sity score matchingBefore PSM significant differences between the baseline characteristics were observed in the surgical groupand the neoadjuvant group Table Table To balance the clinicopathological differences between the twogroups PSM was performed to ensure that there wereno significant differences in patient gender age tumorlocation tumor size pathological staging and adjuvantchemotherapy between the two groups The detailedclinicopathological characteristics before and after PSMare shown in Table and Table As a result patients with NEC components and patients with gastric adenocarcinoma were matchedin the surgical group Table Patients with NEC components also had a poorer OS than those with gastricadenocarcinoma Fig 3e p Multivariable analysis showed that adjuvant therapy tumor category andTNM stage werefactorsTable independent prognosticTo investigate whether neoadjuvant therapy had an effect on OS patients with NEC components and patients with gastric adenocarcinoma were matched inthe neoadjuvant group Table Interestingly KaplanMeier analysis showed that among patients receivingneoadjuvant therapy there was still no significant difference in OS between the two groups Fig 3f p Comparison of OS between patients with differentproportions of NEC components and patients with gastricadenocarcinomaTo investigate whether the level of NEC componentshad an effect on OS in the surgical group GHMiNEN GHMiNEN pure NEC and pure NEC plus GHMiNEN were compared with gastric adenocarcinoma after PSM The results showed that even thegroup with the lowest proportion of NEC componentsthe GHMiNEN group had a poorer OS thanadenocarcinoma Fig 4a P As expected theGHMiNEN pure NEC and pure NEC plus GHMiNEN groups each with relatively high proportionsof NEC components had worse OS than the gastricadenocarcinoma group Fig 4bd P Detailed clinical information after matching isshown in Additional file Tables S1S4PSM was also performed in the neoadjuvant group Incontrast to the results of the surgery group in the pureNEC group containing the highest proportion ofNEC componentstill no significantdifference in OS from gastric adenocarcinoma Fig5d The other three groups with lower NEC contentwere also notfrom gastricadenocarcinoma in terms of OS Fig 5ac Detailedclinicopathologicaland afterPSM are shown in Additional file Tables S5S8characteristics beforethere wassignificantly differentDiscussionAmong gastric neuroendocrine neoplasms the tumorcontaining NEC components is a special type includingpure NEC and mixed tumor containing NEC components The incidence of these tumors is extremely lowbut they are more invasive and have a poorer prognosisthan welldifferentiated GNENs [ ]received neoadjuvantIn previous study Kim found that in patientschemotherapywho had notprogressionfree survivalPFS of pure GNEC waspoorer than that of gastric adenocarcinoma while thePFS of mixedtype tumors was not significantly differentIn Kimsfrom that of gastric adenocarcinoma []study the mixed type was defined as NET mixed withgastric cancer rather than NEC NET is much less malignant than NEC [ ] This may be the reason whythere was no significant difference in OS between mixedtype and gastric adenocarcinomas In addition mixed tumors with less than or more than of NEC components were not included in that study which webelieve was a deficit of the study PFS is an important indicator for evaluating prognosis in many cases it can reflect the trend of OS Based on Kims research resultswe regarded tumors containing NEC components as awhole and found that the OS of these tumors was poorerthan that of adenocarcinoma in the surgical group Inthe comparison of OS between mixed tumors with different proportions of NEC components and gastricadenocarcinoma the results for pure NEC cases wassimilar to Kims While the OS of mixed tumors was alsopoorer than that of gastric adenocarcinoma whether theproportion of neuroendocrine cancer components wasless than between and or more than which was not mentioned in Kims study Cox multivariable regression analysis showed thattumor categoryneoplasm with NEC component or adenocarcinoma 0cChen BMC Cancer Page of Table Comparison of clinicopathological characteristics before and after PSM in surgical groupPatient CharacteristicsUnmatched comparisonPatients with NECcomponents n P valueMatched comparisonPatients with NECcomponents n Age year mean ± SDGender malefemaleBMI mean ± SDAdjuvant therapyYesNoTumor locationUpper thirdMiddle thirdLower thirdEntireTumor size cm¥ cmType of gastrectomyTotal gastrectomyDistal gastrectomy ± ± Proximal gastrectomy Surgical procedure LaparoscopicT stageT1T2T3T4N stageN0N1N2N3M stageM0M1pTNM stageIIIIIIIV Gastricadenocarcinoman ± ± ± ± P value Gastricadenocarcinoman ± ± BMI Body Mass Index MiNEN Mixed neuroendocrinenonneuroendocrine neoplasm NEC neuroendocrine carcinoma PSM Pr sity Score MatchingPatients with NEC components NEC high grade MiNEN high grade MiNEN and high grade MiNEN 0cChen BMC Cancer Table Comparison of clinicopathological characteristics before and after PSM in neoadjuvant groupMatched comparisonPatient CharacteristicsUnmatched comparisonPatients with NECcomponents n Age year mean ± SDGender malefemaleBMI mean ± SDAdjuvant therapyYesNoTumor locationUpper thirdMiddle thirdLower thirdEntireTumor size cm¥ cmType of gastrectomyTotal gastrectomyDistal gastrectomyProximal gastrectomySurgical procedure LaparoscopicT stageT0T1T2T3T4N stageN0N1N2N3M stageM0M1ypTNM stageIIIIIIIV ± ± Gastricadenocarcinoman ± ± P valuePatients with NECcomponents n ± ± Page of P valueGastricadenocarcinoman ± ± BMI Body Mass Index MiNEN Mixed neuroendocrinenonneuroendocrine neoplasm NEC neuroendocrine carcinoma PSM Pr sity Score MatchingPatients with NEC components NEC high grade MiNEN high grade MiNEN and high grade MiNEN 0cChen BMC Cancer Page of Table Univariate and multivariate analyses of survival after PSM in surgical groupPatient CharacteristicsUnivariate analysisHR CIMultivariate analysisHR CIP valueAge yearGendermale vs femaleBMIAdjuvant therapyYes vs NoTumor size¥ cm vs cmTumor categoryCarcinoma with NEC component vsGastric adenocarcinoma vsType of gastrectomyTotal gastrectomyDistal gastrectomyProximal gastrectomySurgical procedureLaparoscopic vs TNM stageIIIIIIIVP value tumor size and TNM staging were independent risk factors for prognosis This suggests that the prognosis ofgastric neoplasms with NEC components is substantiallydifferent from that of gastric adenocarcinoma and evena small percentage of NEC components can alsoimpair prognosis which challenges the current cutoffvalue of The proportion of each component that must theoretically be greater than was set in [] Andsince WHO has also adopted this standard to define MiNEN [] This largely avoids the overdiagnosisof MiNEN in tumors with only focal neuroendocrinemarker expression and no corresponding morphologicalchanges In additionit also prevents clinicians fromdealing with these rare neoplasms too often withoutguidelines [] Nevertheless it is now being questionedby an increasing number of scholars The componentsin mixed tumors are not evenly distributed For large tumorsthe randomness of biopsy and postoperativepathological sampling causes the proportion of eachcomponent to fluctuate greatly making it difficult to describe the proportion of each component precisely []Park compared the OS between tumors with morethan NEC components and gastric adenocarcinomawith or without less than NEC and they found thattumors with an NEC composition of more than hada worse prognosis This suggests that even a small proportion of malignant components can affect prognosis[] While in Parks study for unknown reasons the authors did not compare the prognosis of mixed tumorswith NEC components less than with gastricadenocarcinomas directly nor did they compare allNECcontaining tumors as a whole with gastric adenocarcinoma which we believe was a deficit of the studyIn our study we regarded tumors containing NECcomponents as a whole and found that the OS of thesetumors was poorer than that of adenocarcinoma in thesurgical group In addition we also found that the OS ofmixed tumors with less than between and more than NEC components or pure NEC wasworse than that of gastric adenocarcinoma Analysis ofimmunohistochemical markers show that there was nosignificant difference in the positive rate of Syn and CgAbetween different NEC content groups only the positiverate of CD56 was found to be higher in the pure NECgroup than that in the GHMiNEN group Therole of CD56 in the diagnosis of NEC is still controversial However Syn and CgA are two wellrecognized 0cChen BMC Cancer Page of Fig Comparison of OS between gastric neoplasm with different proportions of NEC and gastric adenocarcinoma in the surgical group aOverall survival comparison between GHMiNEN and gastric adenocarcinoma b Overall survival comparison between GHMiNEN andgastric adenocarcinoma c Overall survival comparison between GHMiNEN plus pure NEC and gastric adenocarcinoma d Overall survivalcomparison between pure NEC alone and gastric adenocarcinomamarkers Therefore from the results of immunohistochemistry we believed that there was no significantlydifference in tumors containing NEC componentsStudies on the molecular mechanism of pathogenesisshow that NEC components and adenocarcinoma components have similar genomic abnormalities similarlosses of heterozygosity LOH and mutations at multiple loci and key oncogenes such as TP53 APC and RBgenes All these results imply that the two componentsin the mixed tumor may have a common origin and acquire biphenotypic differentiation during carcinogenesis[] Moreoverin the WHO definition of mixedneuroendocrine and nonneuroendocrine neoplasms ofother ans ie lung and thyroid [] no minimumpercentage for either ingredient is established Thereforewe believe that mixed tumors containing NEC components are actually of the same origin have similar biological characteristics and are differentfrom gastricadenocarcinoma We propose considering mixed tumorscontaining NEC components as a whole rather than defining them based on the definition for both tumorcomponents which has not been raised by other studiesPreviously many studies have confirmed the efficacyof neoadjuvant chemotherapy in gastric adenocarcinoma[ ] In a retrospective study involving patientsMa et alfound that neoadjuvant chemotherapy improves the survival of patients with NEC and HMiNENof the stomach [] Van der Veen reported that 0cChen BMC Cancer Page of Fig Comparison of OS between gastric neoplasm with different proportions of NEC components and gastric adenocarcinoma in theneoadjuvant group a Overall survival comparison between GHMiNEN and gastric adenocarcinoma b Overall survival comparisonbetween GHMiNEN and gastric adenocarcinoma c Overall survival comparison between GHMiNEN plus pure NEC and gastricadenocarcinoma d Overall survival comparison between pure NEC and gastric adenocarcinomaneoadjuvant chemotherapy could not benefit the survivalof patients with mixed tumors containing NEC components [] However because only eight patients wereincluded in the neoadjuvant group Vans results arequestionable In our study among patients receivingneoadjuvanttherapy no significant difference in OSbetween mixed tumor and gastric adenocarcinoma wasobserved Even for the pure NEC group with the highestNEC contentthere was no significant differencesuggesting that neoadjuvant therapy may have a positiveeffect on these neoplasmsAlthough this is only a singlecenter retrospectivestudy the sample we reported is considerable for thisrare disease which can provide new ideas for clinicaland basic research In addition we proposed treatingall gastric neoplasms containing NEC components asa whole and found that neoadjuvanttherapy mayhave a good effect on these neoplasms In the futurewe will conduct more genomics studies to confirmour ideas This study also has its limitations Due tothe lack of recurrence and detailed chemotherapy information we were unable to compare progressionfree survival and analyse the effects of differentchemotherapy regimens As a retrospective study despite our performing PSM in advance selection biascannot be completely avoided In addition since theexact proportion of each componentin the mixedtumor could not be obtained we could not determine 0cChen BMC Cancer Page of whether there is a cutoff value for the diagnosis ofthe mixed tumor with NEC componentless than so we could only treat all mixed tumors withNEC component as a wholeConclusionsOur study demonstrated that gastric neoplasms withNEC components regardless of the proportion of components have poorer overall survival than gastric adenocarcinomaindicating a higher degree of malignancythan gastric adenocarcinoma Among the patients receiving neoadjuvant therapy the difference in overallsurvival was not significant which was in sharp contrastwith the results of the surgery group suggesting thatneoadjuvant therapy may have a good effect on theprognosis of these malignancies Therefore for this typeof malignancy we should adopt more aggressive andpowerful treatments than those used for gastric adenocarcinoma to improve the prognosis of patients Neoadjuvant chemotherapy may be a good way to improve theefficacy offor these tumors at advancedstagestreatmentSupplementary informationSupplementary information accompanies this paper at httpsdoi101186s12885020072817Additional file Table S1 Comparison of clinicopathologicalcharacteristics before and after PSM of 30GHMiNEN patients insurgical group Table S2 Comparison of clinicopathologicalcharacteristics before and after PSM of GHMiNEN patients in surgicalgroup Table S3 Comparison of clinicopathological characteristics beforeand after PSM of 70GHMiNEN plus pure NEC patients in surgicalgroup Table S4 Comparison of clinicopathological characteristics beforeand after PSM of pure NEC patients in surgical group Table S5 Comparison of clinicopathological characteristics before and after PSM of 30GHMiNEN patients in neoadjuvant group Table S6 Comparison ofclinicopathological characteristics before and after PSM of GHMiNEN patients in neoadjuvant group Table S7 Comparison of clinicopathologicalcharacteristics before and after PSM of 70GHMiNEN plus pure NECpatients in neoadjuvant group Table S8 Comparison of clinicopathological characteristics before and after PSM of pure NEC patients in neoadjuvant groupAbbreviationsAJCC American Joint Committee on cancer CT Computed tomography GHMiNEN Gastric highgrade mixed neuroendocrinenonneuroendocrineneoplasm GNEC Gastric neuroendocrine carcinoma HPF High power fieldMiNEN Mixed neuroendocrinenonneuroendocrine neoplasmNEC Neuroendocrine carcinoma NEN Neuroendocrine neoplasmNET Neuroendocrine tumor MRI Magnetic resonance imaging OS Overallsurvival PETCT Positron emission tomography computed tomographyPFS Progressionfree survival PSM Pr sity score matching WHO WorldHealth anizationAcknowledgmentsThanks to Dr Zhongwu Li of the Department of Pathology Peking UniversityCancer Hospital and his colleagues for their assistance in pathologicaldiagnosis and review Thanks to all colleagues in the Department ofGastrointestinal Surgery of Peking University Cancer Hospital and Dr JiangHong from the Statistics Department for their assistance in this studyAuthors contributionsAll authors contributed to the study conception and design JC performeddata collection and wrote the manuscript AW wrote and t revised hemanuscript KJ helped with statistical analysis and prepared the illustrationsZB edited the manuscript JJ conceived the study and reviewed themanuscript All authors read and approved the final manuscriptFundingThis work was supported by the National Science Foundation for YoungScientists of China Beijing Youth Talent Plan QML20191101 andScience Foundation of Peking University Cancer Hospital Thefunders had no role in study design data collection and analysis decision topublish or preparation of the manuscriptAvailability of data and materialsThe datasets used andor analysed during the current study are availablefrom the corresponding author on reasonable requestEthics approval and consent to participateThe study was approved by the Ethics Committee of Peking UniversityCancer Hospital and the patients written consent was also obtained Writteninformed consent for publication was obtained and stored in PekingUniversity Cancer HospitalConsent for publicationNot applicableCompeting interestsThe authors declare that they have no competing interestsReceived May Accepted August ReferencesBray F Ferlay J Soerjomataram I Siegel RL Torre LA Jemal A Global cancerstatistics GLOBOCAN estimates of incidence and mortality worldwidefor cancers in countries CA Cancer J Clin Matsubayashi H Takagaki S Otsubo T Iiri T Kobayashi Y Yokota T et alAdvanced gastric glandularendocrine cell carcinoma with 1year survivalafter gastrectomy Gastric Cancer Park JY Ryu MH Park YS Park HJ Ryoo BY Kim MG Prognosticsignificance of neuroendocrine components in gastric carcinomas Eur JCancer La Rosa S Inzani F Vanoli A Klersy C Dainese L Rindi G Histologiccharacterization and improved prognostic evaluation of gastricneuroendocrine neoplasms Hum Pathol Ishida M Sekine S Fukagawa T Ohashi M Morita S Taniguchi H et alNeuroendocrine carcinoma of the stomach morphologic andimmunohistochemical characteristics and prognosis Am J Surg PatholRayhan N Sano T Qian ZR Obari AK Hirokawa M Histological andimmunohistochemical study of composite neuroendocrineexocrinecarc | 2 |
"dysregulation of bcl2 is a pathophysiology observed in haematological malignancies forimplementation of available treatmentoptions it is preferred to know the relative quantificationof bcl2 mrna with appropriate reference genes for the choice of reference genesi reference genes were selected by assessing variation of genes from rnaseq datasets of haematological malignancies followed by filtering based on their go biological processannotations and proximity of their chromosomal locations to known disease translocationsselected genes were experimentally validated across various haematological malignancy samples followed by stability comparison using genorm normfinder bestkeeper and reffinderii commonly used reference genes were obtained from literature through extensive systematic review levels of bcl2 mrna was assessed by qpcr normalized either by novel reference genes from this study or gapdh the most cited reference gene in literature andcompared the analysis showed ptcd2 ppp1r3b and fbxw9 to be the most unregulatedgenes across lymphnodes bone marrow and pbmc samples unlike the reference genesused in literature bcl2 mrna level shows a consistent higher expression in haematologicalmalignancy patients when normalized by these novel reference genes as opposed togapdh the most cited reference gene these reference genes should also be applicable inqpcr platforms using taqman probes and other model systems including cell lines and rodentmodels absence of sample from healthynormal individual in diagnostic cases call for carefulselection of reference genes for relative quantification of a biomarker by qpcrbcl2 can beused as molecular diagnostics only if normalized with a set of reference genes with stable yetlow levels of expression across different types of haematological malignanciesintroductionoverexpression of bcl2 bcell lymphoma a mitochondrial membrane protein has beenobserved in several haematological malignancies due to genetic and epigenetic mechanismsa1111111111a1111111111a1111111111a1111111111a1111111111open accesscitation dwivedi n mondal s p k s t ssachdeva k bathula c relativequantification of bcl2 mrna for diagnostic usageneeds stable uncontrolled genes as reference one e0236338 101371 pone0236338editor pedro v baptista universidade nova delisboa portugalreceived may accepted july published august peer review history recognizes thebenefits of transparency in the peer reviewprocess therefore we enable the publication ofall of the content of peer review and authorresponses alongside final published s theeditorial history of this is available here101371 pone0236338copyright dwivedi this is an openaccess distributed under the terms of thecreative commons attribution license whichpermits unrestricted use distribution andreproduction in any medium provided the originalauthor and source are crediteddata availability statement all relevant data arewithin the manuscript and its supportinginformation files one 101371 pone0236338 august one 0cfunding the study is funded by glue grantscheme number btpr23078med2912532017by department of biotechnology httpdbtindiagovin govt of india awarded to sd and md thefunders had no role in study design data collectionand analysis decision to publish or preparation ofthe manuscriptcompeting interests the authors have declaredthat no competing interests existbcl2 molecular diagnostics with novel reference genesresulting in evasion of apoptosis giving the malignant cells a longer life span and survival benefits at times of nutrient deficiency hypoxia and growth factor deprivation [] estimationof level of bcl2 along with other antiapoptotic genes are essential to avail efficient treatmentoptions by rchop regimen of cyclophosphamide doxorubicin vincristine and prednisone and rituximab or venetoclax in different haematological malignancies [ ] byvisualization of chromosomal aberrations using karyotyping or fish fluorescence insituhybridization bcl2 levels can be inferred indirectly detection of expression of bcl2protein by immunohistochemistry a standard pathological testing procedure for dlbcl hasnot been adopted in the clinics for bone marrow tissues of liquid cancers due to sample inconsistency and challenging procedure of capturing low concentrations of biomarkers western blotting for the very nature of the method cannot be adopted for high throughputpathological testing elisa for detection of bcl2 in human plasma remains limited sinceonly one splice isoform of the mitochondrial membrane protein is available in soluble formthus bringing down the effectiveness of the assay bcl2 at the mrna level can be determined without ambiguity by next generation sequencing nanostring and microarray though increasing time and expense of pathological testing in clinical trials relative quantification by qpcr quantitative polymerase chain reactioncan be successfully used due tothe availability of appropriate controls in untreated or normal groups [ ] although beingtime and costeffective it suffers misinterpretation in pathological setting since the relativequantification depends only on the rg reference gene used due to the absence of normalsamplesnormalization with a rg which shows varying expression across samples can often lead towrong s as seen with the use of glyceraldehyde3phosphate dehydrogenasegapdh as rg in gene expression studies of pulmonary tuberculosis and cd8 tcellsunder inactivated or activated condition similarly abl protooncogene abl1 therecommended rg for gene expression studies with leukemic patients was found to haveextremely low expression in neutrophils making it unsuitable as rg for the specific casesuch discrepancies have prompted researchers to analyze gene expression across multiple tissues or pancancer database like tcga to propose normalization factors using multiple rg candidatesthis study through a systematic review of literature in haematological malignancies concluded that mostly conventionally used housekeeping genes are still being deployed s1table and s1 fig despite their varied expression based on cell type developmental stage andexperimental conditions with rare exceptions [ ] none of the genes thus identified could be used to relatively quantify bcl2 as molecular diagnostics since compared to thefpkm fragments per kilobase of transcript per million mapped reads value of the antiapoptotic genes across databases s2 fig most of the rgs from the literature are not onlyhigher but also varied significantly s3 and s4 figs with few exceptions inspired by genomewide search for rgs from publicly available rnaseq or microarray data in human and otheranisms [] we report here a set of novel candidate rgs obtained from an unbiasedsearch of genes in haematological malignancies to be used to normalize bcl2 andother antiapoptotic genes in qpcr as molecular diagnosticsmaterials and methodsethics statementthe study was performed in compliance with ethical practices and was approved by narayanahealth academics ethics committee narayana health hospitals ethics approval numbernhhaeccl2017152a one 101371 pone0236338 august one 0cbcl2 molecular diagnostics with novel reference genessystematic review of commonly used rgsliterature search was carried out in pubmed databasepubmed as detailed in s5 figaccording to prisma preferred reporting items for systematic reviews and metaanalysesguidelines selection of stable genes proteincoding genes identified from publicly available datasets table using ensembldb annotation package within r statistical software were categorised into four quartiles based on their median expression values across all samples geneswith median expression in middle two quartiles q2 and q3 in all datasets were consideredas q1 and q4 representing extreme ends of the expression spectrum are not preferred as rgcandidates for normalization of molecular diagnostic markersto determine the stability of a gene following statistical measures were employedi cv �xsx where �x and Ïx are mean and standard deviation of a variable x respectively and ii normality pvalue as measured by shapirowilks test where a pvalue less than signifies thatthe distribution is away from normal cv although used most frequently isnt a robustmeasure as it is affected by outliers to solve this a third parameter was used mad medianabsolute deviation medianjx 00 xj where x is the median of x after normalization withmedian mad is a better measure for understanding the spread of the distribution as itdepends on medians a parameter less prone to deviations by outlierslow or comparable statistical variation across samples represented by low values of cvand mad and a normal distribution high value of normality pvalue or low values of pvalue are characteristics of an ideal rg therefore genes with median expression values inmiddle quartiles q2 and q3 were shortlisted and clustered based on their cv mad and pvalue normalized to their respective zscores using pam partitioning around medsalgorithm required optimal number of clusters was calculated using silhouette graphicalmethod for each tissue sample the gene cluster with the lowest med value of parameters was selected and the genes at the intersection of the four clusters were shortlisted the list was further filtered by analysing and eliminating genes based on stop words in theirgo gene ontology annotation such as transcription factors nuclear receptor or other nuclearlocalization dna binding activity response to external stimuli translational and transcriptionalactivation since genes with such characteristics regulated by environmental conditions areunsuitable as rg candidates next genes were ranked in ascending order of their mean euclidqffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffifficv þ mad2 þ ð1 00 pþ2ean distance d ¼all parameters replaced by their zscores in thisthreeparameter hyperspace for each dataset average of d across four datasets was taken to calculate the mean euclidean distance �d genes with �d median were selected for furthertable list of rnaseq databasesdatasetdiseasetcgalamlamltargetaml paediatric amlgdcdlbcdlbclmmrfmmmultiplemyeloma� both primary and recurrent tumor only 1st visit recordstissuebloodbonemarrowlymphnodesbonemarrowsamples n sourcedownload location� tcga research networkwwwcancergovtcgaschmitz multiple myeloma researchfoundationgdccancergovaboutdatapublicationsdlbclresearchthemmrf fpkm data for gdcdlbc dataset was available as log2 transformed normalized value which was converted to fpkm101371 pone0236338t001 one 101371 pone0236338 august one 0cbcl2 molecular diagnostics with novel reference genesanalysis locus of genes associated with pathogenic translocations were identified [ ] andcandidate rgs in close proximity of such loci within bands in the same arm of chromosomewere eliminated by an automated method further only genes with nonzero fpkm value in allsamples from four datasets were retained then each gene was given a composite quartile ranking cqr the sum of quartile indices from each dataset and genes with cqr value median expression in 2nd quartile in at least two datasets were shortlisted s6 figdesign of primersbcl2 primers bcl2 has two known splice isoforms membranebound bcl2α and aless studied soluble bcl2β lacking the transmembrane domain at the cterminal most reported primers amplified only bcl2α or larger amplicon s2 table hence new primers were designed table rg primers primers for shortlisted genes were designed table s3 table using primerbank and idt sample detailsrna was isolated from peripheral blood or bone marrow samples from patient or normalindividuals s7 fig with their informed consent ethics approval number nhhaeccltable primers details of rgs and bcl2primeracy1accession nonm_000666ankrd26nm_014915jmjd4nm_001161465ptcd2nm_0247545ppp1r3bnm_024607fbxw9nm_032301nanpnm_1526673plekhm3nm_0010804753tsga10nm_025244nat1nm_001160174ric8bnm_018157gapdhnm_0012897453bcl2nm_0006572sequence fw 'cactgacaaccgctatatccgrv 'ctcatgcagccgttcatcgtfw 'tctcggcaagatccacaaagcrv 'aatgtagagccgtcctgttcafw 'gtctgtcaatgtctgtgggagrv 'caggtgtgtgtcgcagagt3'fw 'tatgggacactgcacatcac3'rv 'ggctgaccatcctcttgttta3'fw 'agaacctcgcatttgagaagac3'rv 'tctgaaccggcataagtgtcc3'fw 'tagggcggtgcgatgattc3'rv 'cggattttggcggactgaga3'fw 'ggtccgcctacttctattaacg3'rv 'tctctgctctccacctacaa3'fw 'gatgatatcagcccagccttag3'rv 'ggacttcctggatcccataaac3'fw 'tactcagcgacaccttgctaa3'rv 'ccagatcattgagggttccac3'fw 'gggagggtatgtttacagcac3'rv 'acatctggtatgagcgtccaa3'fw 'atagtgttcaacagtcagatggc3'rv 'gcaagcgcaagtcaaagca3'fw 'tcgacagtcagccgcatcttcttt3'rv 'gcccaatacgaccaaatccgttga3'fw 'ggaggattgtggccttcttt3'rv 'gcccaatacgaccaaatccgttga3'fw forward primer rv reverse primer101371 pone0236338t002amplicon length bptm Ëcamplification factor one 101371 pone0236338 august one 0cbcl2 molecular diagnostics with novel reference genes2017152a subjects with hepatitis bc or hiv and pregnant or lactating women wereexcluded from the studypbmcbmmc peripheral blood mononuclear cells bone marrow mononuclear cellswere separated by layering of blood or bone marrow diluted to with 1x pbs gibco¢germany above ficollpaque plus histopaque himedia india followed by centrifugation at rcf for mins with brakes off resultant buffy coat was washed twice with 1x pbs andonce with 1x penstrep himedia india before culturing at cell density of to millioncellsml of rpmi himedia india with fbs gibco¢ germany brazil origin and1x penstrep for subculturing the lymphocyte populationrna cdna and qpcrfrom ffpe formalinfixed paraffinembedded blocks curls were deparaffinized inxylene at Ëc followed by proteinase k himedia india treatment prior to rna isolationeither from lymphocytes or from deparaffinized retrospective samples rna was isolatedby trizol¢ ambion us method and quantified with qubit rna br assay kit thermofisher scientific us before converting to cdna using superscript iv ssiv thermo fisherscientific us as per manufacturers instructions with notemplate control ntc qpcrwas performed in triplicates for each sample using kapa sybr green universal reagentssigma aldrich us cdna dilution and primers in a 5μl reaction mix qpcr condition preincubation at Ëc for minutes followed by amplification for cyclesdenaturation at Ëc for sec amplification at Ëc for sec and extension at Ëc for sec inroche lightcycler ii machineoptimization of primersprimers were optimized for qpcr as required by the miqe guidelines all primers wereused at four different final concentrations forwardreverse 200nm200nm 200nm100nm100nm200nm and 100nm100nm with pooled cdna template obtained from six normalhealthy volunteers to yield single amplification product primer efficiency was checked using atwofold fivepoint dilution of the template primer efficiency was obtained from standardcurve using the formula amplication factor ¼ 00� table ��slope 00 stability analysis of candidate rgsmean of cq quantification cycle of ntc were subtracted from cq values of each gene inqpcr experiments to obtain δcq cq samplemean cq ntc and relative expression aseδcq for each replicate where e is the amplification factor of corresponding genestability of expression of the candidate rgs was analysed using three independent algorithmsgenorm normfinder and bestkeeper and the webbased reffindertool that integrates all three algorithms plus the delta ct method algorithm genorm wasrun using the slqpcr r package whereas authorsupplied r package and excel worksheet were used for normfinder and bestkeeper analysis respectively mean cq values foreach gene for all samples were used as input for bestkeeper and reffinder whereas fenorm and normfinder relative expression values were used since normfinder uses amodelbased approach to quantify inter and intragroup variations the malignant and nonneoplastic or healthynormal samples were used as two groups for normfinder analysiscomprehensive stability rank of each gene was calculated as the geometric mean of stabilityrank given by each method one 101371 pone0236338 august one 0cbcl2 molecular diagnostics with novel reference genesexpression analysis of bcl2rq relative quantification of bcl2 expression was calculated either as ratio of relativeexpression of bcl2 with relative expression of gapdh or the normalization factor which isgeometric mean of relative expression of three candidate rgsrq ðgapdhþ ¼ e 00 dcqðbcl2þe 00 dcqðgapdhþrq ðproposedþ ¼ e 00 dcqðbcl2þgeo mean e 00 dcqðptcd2 ppp1r3b fbxw9þresults and discussionquantification by qpcr could be the choice of pathology laboratories for a quick and costeffective platform for singlegene expression level with appropriate rg towards this effort macrae performed a genome wide search and statistical analysis using rnaseq datafrom leukemia patients in a more recent pancancer study publicly available geneexpression data from microarray studies were analysed to identify a few rg candidates thatshowed minimal variation between malignant and normal samples and were validated in droplet digital pcr on bone marrow samples of all patients we have used types of haematological malignancy samples encompassing bone marrow pbmc and ffpe blocks along with nonneoplastic bone marrow and healthy pbmc samples subsequent to using much wider publiclyavailable data from samples in aml dlbcl and multiple myeloma databases furtherwe have employed an improved statistical analysis including clustering technique described inmethods section instead of an ad hoc approach of selection of top few genes from the clusterswe used important biological considerations to further prune the list of candidate rgssystematic review of commonly used rgs from literaturesystematic review of s yielded rgs used in haematological malignancies througha selection of genes by different analysis methods s4 table and b usage of known rgs inqpcr s1 table fpkm values of all these rgs when examined in public databases showedvaried expression among different types of haematological malignancies s3 and s4 figs withmaybe the exception of pggt1b however since other genes selected in the literatureshowed higher expression and correlated extreme variation we could not depend on the assayand proceeded to select novel rgs with an unbiased approachselection of candidate rgsstatistical analysis stepwise filtration of the number of genes from each dataset is summarized in s6 fig and also in graphical abstract fig shows gene clusters plotted in cv normalized mad and 1pvalue hyperspace for four datasets cluster marked in green in eachfigure represents the cluster with least med value s5 table for the three parametersselected clusters in the four datasets had an overlap of genes indicating large number ofgenes involved in housekeeping processes and hence showing lesser intersample variationacross diverse datasets common genes were pruned further to by go biological processterm filtration disease association and cqr to lead to a final of genes s6 table that weretaken through experimental validation melt curve analysis and efficiency check with pooledcdna from six healthy volunteers narrowed it down to genes with stable median expression and single amplification product of expected size for each table primers for geneswhich did not qualify the efficiency check were eliminated as they failed to show single amplification peak after repeated trials with new experimental conditions and even new primersequences s3 table one 101371 pone0236338 august one 0cbcl2 molecular diagnostics with novel reference genesfig statistical analysis of candidate genes genes plotted in the cv normalized mad and pvalue hyperspace for the fourdatasets a tcgalaml b targetaml c gdcdlbc and d mmrfmm cluster shown in green represents the chosencluster with least value of meds101371 pone0236338g001expression of genes with efficient primers were analysed on samples by qpcr usingobserved cq values preliminary stability analysis of the genes were done with online reffinder tool to select top stable genes ptcd2 ppp1r3b fbxw9 nanp ric8b jmjd4plekhm3 nat1 ankrd26 tsga10 as rg candidatessssstability analysis of candidate rgs results of bestkeeper algorithm used independentlyor as part of reffinder were comparable whereas results of genorm or normfinder analysisdiffered as they used different inputs geometric mean of stability ranks assigned in each algorithm was used to create comprehensive stability ranking of all the candidate rgs s7 tableand fig the analysis shows ptcd2 ppp1r3b and fbxw9 to be most stable across all analysed patient samplesptcd2 pentatricopeptide repeatcontaining protein codes for a mitochondrial proteininvolved in rna binding maturation and respiratory chain function though its exact one 101371 pone0236338 august one 0cbcl2 molecular diagnostics with novel reference genesfig stability rank of candidate reference genes101371 pone0236338g002molecular function is not well understood [ ] ppp1r3b protein phosphatase1 regulatorysubunit3b encodes for a catalytic subunit phosphatase regulatory subunit 3b which isinvolved in hepatic glycogen dysregulation in type diabetes [] fbxw9 fboxwdrepeatcontaining protein is a cytosolic protein involved in ubiquitination and proteasomedegradation expression analysis of bcl2accurate determination of bcl2 expression among few antiapoptotic markers in patients withhaematological malignancies is emerging as a critical diagnostic test for clinicians to suggest efficacious therapy options fpkm values of rgs common and novel from the publicly availabledatabases when compared fig with bcl2 indicated the novel rgs to be better normalizationcandidate for bcl2 in qpcr assays in pathology labs due to less and stable expressioncomparison of relative expression of gapdh versus the proposed normalization facteometric mean of relative expression of the three rg candidates clearly show a large variation in gapdh expression across malignant samples fig 4a s8 table granted itspopularity the expression stability of gapdh has been proven to differ in different conditionsdue to its involvement in apoptotic cell death through ubiquitin ligase membrane trafficking upregulation in aml involvement in nonhodgkins bcell lymphomas and inconsistency in several other cancers on the other hand proposed rgs havelesser variation and their expressions are consorted with each other making them better candidate as rg compared to gapdh this behaviour is translated to bcl2 expressionrq in malignant samples when normalized with gapdh fig 4b evidently normalizationwith gapdh underestimates relative quantification of bcl2 compared to normalization withproposed rgs with a statistically significant difference in median values p wilcoxonrank sum test between the two schemes bcl2 quantification in haematological malignanciesby qpcr is overtly reliant on rg since availability of adjacent normal sample is ruled outabove results clearly demonstrate how the quantification may go off limit due to a wrongchoice of rg one 101371 pone0236338 august one 0cbcl2 molecular diagnostics with novel reference genesfig candidate reference genes in hematological malignancy datasets expression values of candidate genes in four datasets a tcgalamlb targetaml c gdcdlbc and d mmrfmm101371 pone0236338g003broader applicability of proposed reference genesthough primary objective of this study is to discover rg candidates for bcl2 diagnostics in aclinical setting the rgs may have broader utility in other experimental platforms or modelsystems in the systematic review we found a number of research s [] that haveused taqman probes instead of sybr green whereas our validation experiment was carriedout using sybr green probes however studies in different contexts such as a tropical oilseedplant or measurement of expression of various adenosine receptors in breast cancer tissue and in experiments using human reference rna sybr green pcr assays wereobserved having fair concordance with taqman pcr from these evidences we believe thatstability of proposed rgs is not likely to differ between sybr green and taqman qpcr assaysto assess variation of these stable rgs in cell lines we analyzed rpkm values of proteincoding genes across cell lines of haematopoietic and lymphoid tissue origin frombroad institute cancer cell encyclopedia and found the proposed rgs presenting muchlesser variations in expression compared to the common rgs gapdh abl1 b2m gusband actb in cell lines as well s8 figboth transgenic and wild type and occasional rat models are widely used in leukemia andlymphoma research [ ] usability of rgs common between clinical and animal studies one 101371 pone0236338 august one 0cbcl2 molecular diagnostics with novel reference genesfig relative expression of chosen reference genes and relative quantification of bcl2 a relative expression of chosen reference genes solidlines and gapdh dashed line across patient samples b relative quantitation of bcl2 expression with respect to the candidate reference genesand gapdh in malignant patient samples101371 pone0236338g004will thus be of immense advantage we find that the proposed rgsptcd2 ppp1r3b andfbxw9 have sequence similarity and identity with corresponding genes in mice andother commonly used rodent models s9 table suggesting the genes playing similar role incellular function thereby displaying stability similar to that in humans hence normalizationfactor derived from the expression of these rgs may be applicable in murine and other rodentmodels as well with suitable design of primers encompassing conserved regionsbeyond detection of gene expression at mrna level it may be worthwhile to explore theapplicability of protein counterpart of the stable rgs in western blot as control for proteindetection by design we have chosen rgs that are of moderate expression level in middlequartiles of expression among other genes and they may not be detectable by western blotunless a larger amount of sample is loaded which is often not feasible with clinical sampleshowever it may be an interesting proposition to predict stable reference proteins for use inwestern blot by statistical analysis of proteomics data and associated systematic review ofliterature one 101371 pone0236338 august one 0cbcl2 molecular diagnostics with novel reference genesour results indicate that genes ptcd2 ppp1r3b and fbxw9 render more reliability toqpcrbased diagnostic test of bcl2 in haematological malignancies the can beextended to other biomarkers in liquid cancer as well as for research with other model systemssuch as cell lines and rodentssupporting informations1 table list of reference genes in literaturedocxs2 table list of bcl2 primers from literaturedocxs3 table list of unqualified primersdocxs4 table literature explaining analysis and selection of reference genedocxs5 table zscore med valuesdocxs6 table list of selected genesdocxs7 table individual and combined stability rank and scores of candidate reference genesdocxs8 table relative expression of gapdh and the proposed normalization factordocxs9 table sequence similarity and identity with corresponding genes in mice rat andguinea pigdocxs1 fig rgs found in literature with more than one citationtiffs2 fig fpkm values of bcl2 family of antiapoptotic genes in the four datasetstiffs3 fig fpkm values of rgs found in relevant literature with more than one citationtiffs4 fig fpkm values of rgs found in relevant literature with a single citationtiffs5 fig workflow according to prisma guidelines for systematic review for commonlyused reference genestiffs6 fig statistical analysis workflowtiffs7 fig patient samples used in the studytiff one 101371 pone0236338 august one 0cbcl2 molecular diagnostics with novel reference geness8 fig variation in stable rgs in cell lines and animal modeltiffs1 graphical abstracttiffacknowledgmentsauthors acknowledge prof joy kuri chair department of electronic science and engineering indian institute of science bangalore for providing the computational resourcesauthor contributionsconceptualization sujan k dhar manjula dasdata curation nehanjali dwivedi sreejeta mondal smitha p k sowmya t kartik sachdeva christopher bathula vishnupriyan kformal analysis sujan k dharfunding acquisition sharat damodar manjula dasinvestigation nehanjali dwivedi sreejeta mondal smitha p k sowmya tmethodology nehanjali dwivedi sreejeta mondal smitha p k sowmya t vishnupriyank manjula dasproject administration manjula dasresources nataraj k s sharat damodarsoftware sujan k dharsupervision manjula dasvalidation nehanjali dwivedi sreejeta mondal smitha p k sowmya t kartik sachdevachristopher bathula vishnupriyan kvisualization manjula daswriting original draft sreejeta mondal sujan k dharwriting review editing nehanjali dwivedi sreejeta mondal smitha p k sujan kdhar manjula dasreferences perini gf ribeiro gn pinto neto jv campos lt hamerschlak n bcl2 as therapeutic target forhematological malignancies vol of hematology and oncology biomed central ltd gratiotdeans j merino r nuñez g turka la bcl2 expression during tcell development early lossand late return occur at specific stages of commitment to differentiation and survival proc natl acad sciu s a oct 101073pnas912210685 pmid merino r ding l veis dj korsmeyer sj nuñez g developmental regulation of the bcl2 protein andsusceptibility to cell death in b lymphocytes embo j feb pmid li l li y que x gao x gao q yu m prognostic significances of overexpression myc andorbcl2 in rchoptreated diffuse large bcell lymphoma a systematic review and metaanalysis scirep 101038s41598017177655 uchida a isobe y asano j uemura y hoshikawa m takagi m targeting bcl2 with venetoclaxis a promising therapeutic strategy for doubleproteinexpression lymphoma with myc and bcl2 one 101371 pone0236338 august one 0cbcl2 molecular diagnostics with novel reference genesrearrangements haematologica jun 103324haematol2018 pmid baro´ c espinet b salido m garcı´a m sa´nchez b florensa l cryptic ighbcl2 rearrangementswith variant fish patterns in follicular lymphoma leuk res feb 101016jleukres201009011 pmid hofman p heeke s alixpanabières c pantel k liquid biopsy in the era of immunooncology is itready for primetime use for cancer patients suppressed immune microenviron repert brain metastases from patients with resected nsclc fatani s h mukhtar m h ali a s correlation between serum antiapoptotic bcl2 level and its immunohistochemical expression in relation to apoptosis in gastric cancer j mol biomark diagn albitar m zijun xy wang y manman d tzankov a visco c myc and bcl2 mrna expressionas determined by ngs predicts survival in dlbcl in gcb but not in abc subgroup blood nov 134supplement_15092 derenzini e rossi a agostinelli c rossi m melle f motta g integration of nanostring profilingand functional characterization of oxidative and replicative stress biomarkers identifies poor prognosis mycbcl2 positive diffuse large bcell lymphoma subsets providing opportunities for precisiontherapies blood nov 132supplement zhang f yang b zhang k hou ml lu xc li yx ccnd1bcl2 gene network a direct target of amifostine in human acute megakaryocytic leukemia cells chem biol drug des may 101111cbdd12889 pmid patel vm balakrishnan k douglas m tibbitts t xu ey kutok jl duvelisib treatment is associated with altered expression of apoptotic regulators that helps in sensitization of chronic lymphocyticleukemia cells to venetoclax abt199 leukemia sep 101038leu2016382 pmid bomben r ferrero s dagaro t dal bo m re a evangelista a a bcell receptorrelated genesignature predicts survival in mantle cell lymphoma results from the fondazione italiana linfomi mcl trial haematologica apr 103324haematol2017184325pmid dheda k huggett jf chang js kim lu | 0 |
"carcinogenesis is a process of somatic evolution previous models of stem and transient amplifying cells in epithelial proliferating units like colonic crypts showed that intermediate numbers of stem cells in a crypt should optimally prevent progression to cancer if a stem cell population is too small it is easy for a mutator mutation to drift to fixation if it is too large it is easy for selection to drive cell fitness enhancing carcinogenic mutations to fixation here we show that a multiscale microsimulation that captures both withincrypt and betweencrypt evolutionary dynamics leads to a different epithelial tissues are metapopulations of crypts we measured time to initiation of a neoplasm implemented as inactivation of both alleles of a tumor suppressor gene in our model time to initiation is dependent on the spread of mutator clones in the crypts the proportion of selectively beneficial and deleterious mutations in somatic cells is unknown and so was explored with a parameter when the majority of nonneutral mutations are deleterious the fitness of mutator clones tends to decline when crypts are maintained by few stem cells intercrypt competition tends to remove crypts with fixed mutators when there are many stem cells within a crypt there is virtually no crypt turnover but mutator clones are suppressed by withincrypt competition if the majority of nonneutral mutations are beneficial to the clone then these results are reversed and intermediatesized crypts provide the most protection against initiation these results highlight the need to understand the dynamics of turnover and the mechanisms that control homeostasis both at the level of stem cells within proliferative units and at the tissue level of competing proliferative units determining the distribution of fitness effects of somatic mutations will also be crucial to understanding the dynamics of tumor initiation and progressionk e y w o r d scancer evolution initiation metapopulation dynamics neoplastic progression simulationmajor findings competition between epithelial units such as colonic crypts tends to suppress initiation of neoplasms by suppressing mutator clones this suppression of initiation is enhanced when crypts have few stem cells and so are likely to go extinct due to stochastic fluctuations in stem cell numbers this is an open access under the terms of the creative commons attribution license which permits use distribution and reproduction in any medium provided the original work is properly cited the authors evolutionary applications published by john wiley sons ltdevolutionary applications wileyonlinelibrarycom eva 2003 2003 0c 2003 2003 2003 2002 2003 2003introductionthe anization of a population into spatially distinct subpopulations can have a dramatic effect on the evolution of that metapopulation hanski gaggiotti this has implications for both the evolution of anisms and for the effect of tissue architecture on somatic evolution and tissue health in multicellular anisms epithelia are typically divided into subpopulations of tissue stem cells along with the transient amplifying cells and differentiated cells that they produce these subpopulations go by different names in different tissues such as crypts in the intestine or more generally epithelial proliferative units cairns first recognized that the division of stem cells into subpopulations such as crypts acts as a tumor suppressor cairns a mutant stem cell with a reproductive or survival advantage may take over a crypt but is generally constrained from expanding beyond that subpopulation unless it breaches the crypt via a process known as crypt fission which tends to duplicate the mutant crypt cell population however by establishing a population size barrier the mutant clone has to overcome the subpopulation structure of the tissue limits the probability that that clone will acquire further carcinogenic mutations yet clones of mutant stem cells can be observed at scales spanning many crypt diameters especially in compromised tissues such as ulcerative colitis and barrett's esophagus maley salk a fundamental question is therefore how the cryptlevel metapopulation dynamics affect the accumulation of somatic mutations during carcinogenesishere we explore the evolutionary dynamics of mutant stem populations that lead to tumor initiation that is the breach of the crypt barrier allowing clonal expansions of crypts across a tissue as well as of mutant stem cells within and out of a crypt while there may be multiple pathways to tumor initiation it has been shown that the inactivation of a single tumor suppressor gene tsg such as the adenomatous polyposis coli apc gene in colon is sufficient to abrogate crypt homeostasis leading to the formation of aberrant crypts and nascent adenomas humphries wright using an agentbased microsimulation model for both stem cell turnover within a crypt and for the cryptpopulation tissuelevel dynamics we study the role of pretumor evolution in tumor initiation this exploration allows for the selection of mutant crypts across the tissue prior to the inactivation of the tumor suppressor genea form of premalignant field cancerizationwhile the stem cell in which the tumor suppressor is inactivated can proliferate beyond the limit of a single crypt due to crypt bifurcationthe evolution of somatic cells is a complex multiscale process depending on the nature of somatic mutations which may either increase or decrease cell fitness stem cell divisions and differentiation or apoptosis as well as subpopulation eg crypt division and extinction rates there is considerable evidence that carcinogenesis involves both an increase in the rate of epigenetic lesions bielas loeb rubin true loeb breivik ji king weisenberger et al and expansions of clones with a relative fitness advantage over their competitor cells cannataro gaffney townsend maley pepper findlay kassen spencer maley vermeulen williams however it continues to be unclear whether the mutator phenotype is a preinitiation phenomenon in carcinogenesis or is more likely to occur during tumor progression in barrett's esophagus another cryptstructured precancer we found evidence that genomic instability precedes genome doubling and transformation martinez the frequency of deleterious versus beneficial mutations in somatic cells is also unknown though the large number of genes in metazoans devoted to differentiation apoptosis and cell cycle control suggests that the frequency of deleterious mutations may be lower in somatic evolution than anismal evolution rajagopalan nowak vogelstein lengauer recent analysis of somatic mutations in cancer found no evidence of purifying selection except in a few essential genes and strong evidence of positive selection with large selective effects williams suggesting that beneficial mutations are more common than deleterious mutations in somatic evolution martincorena although a definitive answer to these questions can only come from further experimental data a theoretical exploration that recognizes the roles of metapopulation dynamics the mutator phenotype and the proportion of deleterious to advantageous mutations in the process of tumor initiation is called for such an exploration will help the identification of factors that drive the tumor initiation processour model integrates previous efforts to characterize the stem cell dynamics within a crypt cannataro mckinley st mary cannataro mckinley st mary frank iwasa nowak komarova komarova cheng loeffler birke winton potten meineke potten loeffler michor frank may iwasa nowak nowak et al pepper sprouffske maley with models of the dynamics of crypt populations cannataro et al chao eck brash maley luebeck kostadinov maley kuhner loeffler bratke paulus li potten totafurno bjerknes cheng mathematical studies of the stem cell population in the crypt niche suggest that epigenetic alterations that increase the rate of genetic lesions mutator mutations and reduce the fitness of stem cells will tend to drift to fixation if the stem cell population is small whereas carcinogenic mutations that increase the proliferation or survival of a stem cell will tend to spread if the stem cell population is large cannataro komarova michor assuming that most nonneutral somatic mutations are deleterious the accumulation of deleterious mutations may lead to senescence of the intestine over time cannataro however competition between crypts of different fitnesses may significantly change the dynamics of the establishment of a mutator clone through a metapopulation dynamic our in silico experiments suggest that there may have been selection at the level of the anism to minimize the number of stem cells within each subpopulation of birtwell 0c 2003 2002 2003 2003its structured epithelium so as to reduce the probability of tumor suppressor gene inactivation and the initiation of carcinogenesisproliferation rate decreased stem cell loss and caused the mutator phenotypethe following equations and assumptions govern the model 2003 2003methodswe implemented a multiscale model of epithelial tissue architecture with stem cells subdivided into crypts under homeostatic control we examined the time required until the two alleles of a tumor suppressor gene tsg were inactivated in at least one stem cell to represent tumor initiation the model was run at least times for every parameter setting crypts were arranged in a flat hexagonal tissue similar to that observed in colon and contained a population of stem cells as well as an implicitly modeled transient amplifying compartment stem cells divided both symmetrically and asymmetrically symmetric division resulted in two daughter stem cells each having the opportunity during the division event synthesis to acquire a mutation asymmetric division did not result in any new stem cells but did provide an opportunity for stem cell mutation stem cell loss due to cell death or differentiation and stem cell gain due to division events were modeled as a stochastic birthdeath process with parameters that were functions of the stem cell fitness and of homeostatic feedback effects in response to deviations of the crypt cell population from its normal target level equations and a flow chart of the model algorithm is shown in figure s1homeostasis operated at two spatial scales within a crypt if the stem cell population dropped below the target level stem cell division rates increased by a parameterized amount equation if the population rose above the target level stem cell loss rates increased equation the level of homeostatic feedback was proportional to the degree of deviation away from the target equilibrium level equations and we also introduced a mechanism for homeostasis on the hexagonal lattice of crypts if all the stem cells in a crypt died the inhibition on stem cell population growth was released from the neighboring crypts when the stem cell population of a neighbor reached twice the equilibrium level we modeled crypt bifurcation by allocating half of its stem cells to a new crypt in the location of the dead neighborwe included both beneficial mutations that increased the division probability or the survival probability of stem cells as well as deleterious mutations that decreased them these can accumulate indefinitely and affect fitness multiplicatively equation we also implemented a genetic instability mutation that increased the clone's mutation rate 100fold bielas herr kennedy knowels schultz preston ji king the frequency of each mutation type those that changed the cell's fitness the proportion of nonneutral mutations that were deleterious as well as the rate of tsg inactivation was set by parameters each mutation affected proliferation survival or mutation rate parameters by a constant factor half of the deleterious mutations decreased stem cell proliferation and half decreased their survival increased cell loss for the beneficial mutations increased the cell's 2003 2003equationsequation time to stem cell losslet t be a random exponential deviate with distribution function fr t and rate parameter r the time to cell loss due to apoptosis or differentiation is the minimum of the time to cell loss due to background cell death or differentiation and the time to cell loss due to crypt feedbacktcell_loss min 01t1 ¼ fbackground_loss t2 ¼ ffeedback_loss 01 equation homeostatic crypt feedback by differentiationwhen the stem cell population within a crypt expands beyond the homeostatic target level kcrypt\\_size the crypt provides homeostatic feedback via a change in the rate of stem cell loss with a rate parameter equal to the base stem cell loss rate multiplied by the crypt feedback multiplier the crypt feedback rate multiplier is used to calculate the time to stem cell loss due to crypt homeostatic feedback the crypt feedback multiplier is equal to raised to the nth power where n is the excess number of stem cells above the crypt size divided by the kcrypt\\_deviation parameter here kcrypt\\_deviation and kcrypt\\_size is a parameter that we varied across experimentsrfeedback\\_cell_loss rbase_cell_loss2max0ncellskcrypt_sizekcrypt_deviationequation homeostatic crypt feedback by proliferationwhen the stem cell population of a crypt drops below kcrypt\\_size the division rate of the remaining stem cells is increased by a factor that depends on the difference between the current number of stem cells ncellsand kcrypt\\_sizerfeedback_division rbase_division2max0kcrypt_sizencellskcrypt_deviationequation fitness mutation effectskï¬tness is a constant factor representing the effect of a single beneficial mutation on fitness as a first approximation we assume that there are many possible mutations that increase and decrease the fitness of a somatic clone by approximately the same amount and so the effect of n beneficial mutations nbeneï¬cial on stem cell fitness is the constant fitness effect raised to the nth power the effect of n deleterious mutations ndeleterious of small effect is just the inverse of kï¬tness raised to the nth power there is a separate mï¬tness calculated for the division probability and the survival probability of a cell because beneficial and deleterious mutations may affect either of those probabilitiesmï¬tness 01kï¬tness 01nbeneï¬cial 03 kï¬tness 03ndeleteriousbirtwell 0c 2003 2003 2003 2002f i g u r e 2003plots of cumulative hazard functions using the kaplanmeier estimator the tissue was x crypts with stem cells per crypt in panels a through d each colored line represents the function for a specific proportion of deleterious mutations a baseline experiment with default parameter values table b mutation rate reduced to 01x of baseline c mutator phenotype reduced to 01x of baseline d each colored line represents a different number of cells per crypt all proportions of deleterious mutations were included 2003 2003assumptionscrypts consist of stem cells and of transient amplifying cellscrypt density is fixed that is the tissue contains a fixed number of crypts arranged on a hexagonal griddrops below the target level the division rate of each stem cell in the crypt is increased when the number of stem cells grows above the target level the cell loss rate of each stem cell in the crypt is increasedcrypts divide to fill vacant slots left by adjacent crypts that have the number of cells in a crypt transient amplifying compartment gone extinct due to loss of the constituent stem cellsis fixedcrypts attempt to maintain a stable population of stem cells through homeostatic feedback when the number of stem cells the extinction of an adjacent crypt suppresses the homeostatic apoptotic signals allowing the stem cell populations in neighboring crypts to expand once that extinct crypt is replaced the normal birtwell 0cta b l e 2003baseline simulation parameterssimulationmaximum simulation duration in daysstem celldivision rate rbase\\_divisionratio of asymmetric divisions to symmetric divisionsstem cell loss rate rbase\\_cell\\_lossmutation rate per stem cell divisionmutation rate maximumtumor suppressor gene mutation ratetumor suppressor gene mutation rate maximumnumber of transient amplifying cells associated with each stem cellcell division minimum time in dayscell loss minimum time in dayscrypttarget equilibrium level of number of stem cells in a crypt kcrypt\\_sizestandard deviation from equilibrium level the crypt uses this value to determine its level of effect on the cell loss and division rates of the stem cells kcrypt\\_deviationbifurcation threshold factor below which a crypt will not bifurcatecrypt cell loss effect multipliercrypt division effect multipliermultiplier to the division effect for each dead neighbor cryptcanceruncontrolled cell proliferation threshold if a crypt has this threshold times the equilibrium number of stem cells it is considered to be experiencing uncontrolled stem cellnumber of tumor suppressor gene hits that mean cancermutation 2003 2002 2003 2003 years x x x also tested x x default varied from percent of nonneutral mutations that are deleteriousthe factor affecting the loss rate of the stem cell from a beneficial mutation kï¬tnessthe factor affecting the division rate of the stem cell from a beneficial mutation kï¬tnessthe factor affecting the cell loss rate of the stem cell from a deleterious mutation kï¬tnessthe factor affecting the division rate of the stem cell from a deleterious mutation kï¬tnessthe factor affecting the mutation rate of the stem cell from a mutator mutation also tested homeostatic controls on stem cell numbers of neighboring crypts are restoredcrypt division is triggered by an expansion of the stem cell population of a crypt to twice its homeostatic level as hypothesized by garcia park novelli and wright as long as there is an empty slot adjacent to the enlarged crypta stochastic birthdeath process governs the scheduling of division and cell loss eventsfitness mutations affect in a multiplicative fashion the rate parameters of the birthdeath processthere is a single mutator phenotype that requires only a single mutator mutation additional mutator mutations have no effect on the mutation ratethe loss of the first allele of the tsg has no effect on stem cell fitness 2003 2003results 2003 2003tsg inactivation depends on the emergence of a mutatorat baseline for comparison our tissue was a 5x5 hexagonal lattice of crypts each crypt having stem cells stem cell loss and symmetric division rates were balanced mutations were acquired stochastically with probabilities defined by proportions starting with deleterious mutations beneficial mutations and mutator mutations and ranging in increments to deleterious beneficial and mutator beneficial versus mutator the incidence of tsg inactivation decreased as the proportion of deleterious mutations increased figure 1a table birtwell 0c 2003 2003 2003 2002reducing the base mutation rate to of baseline we observed a marked decrease in tsg inactivation figure 1b as expected similarly reducing the effect of the mutator mutation to 10x the baseline mutation rate instead of 100x produced a significant decrease in tsg inactivation figure 1c we found that the vast majority of tsg inactivation occurred in stem cells that had previously acquired the mutator phenotype figure s2 this assumes that the mutator phenotype can be caused by a single mutation that is otherwise neutral eg overexpression of dna polymerase beta canitrot or a dominantnegative mutation in p53 de vries though this assumption is easily relaxed 2003 2003proportion of deleterious mutations negatively correlates with tsg inactivationnot surprisingly we found that the proportion of deleterious mutations and the incidence of tsg inactivation were negatively correlated figure cell divisions per time remained roughly constant across all proportions of mutations however as the proportion of deleterious mutations decreased the cost of being a mutator also decreased because it accumulated less mutational burden of deterious mutations this resulted in an increased emergence of crypts with fixed mutator stem cell populations conversely across all experiments we observed progressively less tsg inactivation as the proportion of deleterious mutations approached our maximum of turnover levels we observed a reduction in the average number of stem cells per crypt figure s3 the implemented homeostatic control was unable to maintain the target stem cell population size in the face of high turnover rates essentially there is a lag between depletion of the stem cell pool due to cell death and differentiation and replenishment provided by an increase in stem cell division rates with higher levels of cell loss the simulated crypts spend more time further below the target homeostatic number of stem cells as a result there were fewer total stem cells in the simulation and therefore fewer mutations per time allowing less chance for mutator acquisition and tsg inactivation this may or may not be realistic second as the turnover level increased above our baseline the number of mutator crypts present in the tissue at any given time decreased figure since increased turnover should lead to increased opportunities for mutator mutations to arise the decline in mutator crypts was a surprise however the loss of mutator crypts is due to intercrypt competition as described below increased turnover led to increased stochastic fluctuations in stem cell numbers and thereby increased crypt extinction events the reduction in stem cell numbers and mutator crypts combined to produce a reduction in the overall incidence of tsg inactivation as turnover rates increased above baseline if in reality homeostatic control of stem cell numbers prevents increased crypt extinctions with increased stem cell turnover this result would likely not hold however all things being equal increased cell turnover would be expected to increase stem cell number fluctuations 2003 2003increased stem cell turnover initially increased and then decreased tsg inactivationwe modulated stem cell turnover by varying cell loss and symmetric division rates in unison at lower turnover levels we found that increased cell turnover increased tsg inactivation however at turnover levels 2x and 5x our baseline level the incidence of tsg inactivation declined figure due to two factors first at higher 2003 2003the number of stem cells per crypt had a varying effect on tsg inactivationin general fewer stem cells per crypt reduced the rate of tsg inactivation even though the total number of stem cells in the tissue was held constant figure 1d however there is a tradeoff between tsg inactivation and tissue death at very low stem cells per crypt with only one stem cell per crypt there is no opportunity for homeostatic signals within a crypt to compensate for stem cell loss in f i g u r e 2003this graph represents the proportion of crypts at the end of the run that contained a population of stem cells with the mutator mutation fixed each bar corresponds to a specific proportion of deleterious mutations the proportion of mutator crypts correlates with the risk of tumor initiation as seen in figure birtwell 0cbase cell loss divison rate base cell loss divison rate 2003 2002 2003 2003base cell loss divison rate base cell loss divison rate f i g u r e 2003the effect of changes in stem cell turnover plots of cumulative hazard functions using the kaplanmeier estimator where each colored line represents the function for a specific proportion of deleterious mutations the baseline division and stem cell loss rates were as seen in figure initially turnover correlated positively with the risk of tumor initiation however at higher turnover rates the risk of tumor initiation decreased due to a reduction in the overall number of living stem cells and decreased incidence of mutator cryptsour model tissues with one stem cell per crypt were mostly unviable and died out before tsg inactivation or the predetermined simulation end timeat and cells per crypt we observed a reduction in the incidence of tsg inactivation figure 1d at all but the lowest proportions of deleterious mutations as was predicted by models of the crypt stem cell niche of single crypts komarova michor figure s4 as in other experiments along with a reduction in the incidence of tsg inactivation the frequency of fixed mutator crypts was reduced as well this shows that selection against mutator cells increases as the number of stem cells increases above some threshold in our model as long as the majority of nonneutral mutations are deleterious supporting the s by michor and komarova komarova michor et al when the stem cell populations are large it is very unlikely that a crypt will go extinct and so there is no intercrypt competition in this case the metapopulation dynamics are reduced to the single crypt dynamicsthe increased risk of tsg inactivation associated with increased stem cells per crypt appeared to plateau after approximately stem cells per crypt the total number of cells in the tissue remained constant as did the total stem cell divisions per time figure s5 the average number of mutations per time increased through stem cells per crypt but then reached a temporary plateau figure there was no statistically significant difference between the average number of mutations per time in the and stem cells per crypt cases as the number of stem cells per crypt increased beyond the average mutations per time decreased except when the proportion of deleterious mutations was the incidence of mutator crypts followed a similar trend figure birtwell 0c 2003 2003 2003 2002f i g u r e 2003this graph represents the proportion of crypts at the end of the run that contained a population of stem cells with the mutator mutation fixed as a function of turnover rate each bar corresponds to a specific proportion of deleterious mutations as turnover increased mutator crypts became more rare at higher proportions of deleterious mutationsf i g u r e 2003number of mutations per unit time as a function of cells per crypt and proportion of deleterious mutations where each bar represents a specific proportion of deleterious mutations at most proportions of deleterious mutations mutations per time peaked around stem cells per crypt at deleterious mutations mutations per time reached a minimum at cells per crypt and increased as the cells per crypt grew past we have chosen to explore the case where the total number of stem cells is kept constant assuming that a certain number of selfrenewing tissue stem cells might be required to maintain an epithelial tissue an alternative view is that a fixed number of epithelial units like the crypts might be required to maintain a tissue and that the number of stem cells per crypt could vary by changing the number of differentiated cells produced by each stem cell in this case the risk of tsg inactivation continues to increase with increasing number of stem cells per crypt figure s4 because we held the number of crypts constant but changed the number of cells per crypt in this case the total number of cells in this simulation also changed leading to a large evolving population size of stem cells and thus an increased chance for at least one cell to inactivate both alleles of the tsg 2003 2003partitioning the tissue into crypts imposed a metapopulation dynamicfitness levels measured by the difference between division and loss rates were greater in tissues with smaller crypts even as the total number of stem cells remained constant figure 6a counts of crypt births and crypt life span measurements showed that there was more crypt turnover in smaller crypts figures s6 and s7 this crypt turnover provided an opportunity for fitter phenotypes to spread more easily across the tissue crypt fitness peaked at cells per crypt where there was the most crypt turnover and bottomed out at and cells per crypt after cells per crypt we observed no crypt death and therefore no turnover crypt fitness began to increase again at and cells per crypt through natural selection of stem cells within larger crypts however fitness levels did not increase to the levels seen at cells per cryptthe metapopulation dynamic was observed in the clonal expansion of mutator crypts across the tissue we considered two crypts to have mutator phenotype agreement if they both have a fixed mutator mutation or neither has a fixed mutator we calculated mutator crypt agreement across spatial distance and found that overall agreement decreased as the cells per crypt increased figure 6b further at and cells per crypt closer crypts had increased mutator agreement suggesting clonal expansion of mutator crypts conversely at cells per crypt and above we found that spatial distance correlated less well with mutator agreement indicating that birtwell 0c 2003 2002 2003 2003f i g u r e 2003a crypt fitness measured by the difference between division and cell loss rates across cells per crypt and proportion of deleterious mutations the total number of stem cells remained constant across these experiments the crypt turnover at and cells per crypt allowed fit clones to spread across the tissue resulting in increased overall fitness b mutator agreement by distance class two crypts were in mutator agreement if they both had fixed mutator mutations or neither did overall agreement was higher in the smaller crypts suggesting that mutator clones were able to spread across the tissuewhen there was less crypt turnover mutator crypts arose de novo rather than through cryptlevel clonal expansion 2003 2003discussionin the colon the development of adenomatous polyps frequently involves the inactivation of the apc gatekeeper gene a member of the wntsignaling pathway which represses proliferation and facilitates orderly cell differentiation in the luminal part of the crypts barker goss groden as long as this gatekeeper gene is active mutant stem cell progeny with neoplastic potential is likely eliminated from the crypt and clonal expansion thus averted however when the gatekeeper gene is inactivated the brakes on mutant stem proliferation are removed and mutant cell progeny may undergo focal clonal expansion therefore we asked the question what are the factors that determine the risk of a tsggatekeeper inactivation leading to tumor initiation in a compartmentalized tissue a metapopulationour microsimulations indicate that the base mutation rate figure 1b and the increase in that rate for a mutator clone figure 1c are the main driving forces behind tsg inactivation and thus the initiation of carcinogenesis the proportion of deleterious mutations is important for its effect on selection of the mutator clone we find that if most mutations are assumed to be deleterious then a mutator clone quickly accumulates a large genetic load of deleterious mutations and tends to be driven to extinction in competition with nonmutator cells however if mutations are more likely to be beneficial then a mutator clone can spread more easily which leads to the early inactivation of both alleles of the tumor suppressor gene figure 1a similarly increasing turnover of the stem cells effectively increases the mutation rate and consequently the rate of initiation however when turnover is very high the homeostatic feedback signals cannot maintain the target number of stem cells and so the total stem cell population of the epithelium decreases which in turn decreases the chances for initiation figure since the rate of tsg inactivation is trivially relat | 0 |
"Optimizing Telemedicine Encounters for Oral and Maxillofacial Surgeons During the COVID19 Pandemic Hwi Sean Moon DDS MD1 Tim T Wang BA23 Karthik Rajasekaran MD4 Ryan Brewster BA5 Rabie M Shanti DMD MD46 Neeraj Panchal DDS MD MA7 1Resident Department of Oral Maxillofacial Surgery University of Pennsylvania Philadelphia PA 2DMD Candidate School of Dental Medicine University of Pennsylvania Philadelphia PA 3MPH Candidate Perelman School of Medicine University of Pennsylvania Philadelphia PA 4Assistant Professor of Otorhinolaryngology University of Pennsylvania Philadelphia PA 5MD Candidate Stanford University School of Medicine Stanford University Stanford CA 6Assistant Professor of Oral and Maxillofacial Surgery University of Pennsylvania Philadelphia PA 7Assistant Professor and Section Chief of Oral and Maxillofacial Surgery Philadelphia Veterans Affairs Medical Center Penn Presbyterian Medical Center University of Pennsylvania School of Dental Medicine Philadelphia PA Corresponding Author Neeraj Panchal DDS MD MA Tel Mailing Address N 39th St Philadelphia PA Email Address npanchalupennedu Disclosures None to report Abstract Word Count Manuscript Word Count Number of References Number of Figurestables Number of Supplements 0c The COVID19 pandemic has changed conventional medical practice patterns across all health disciplines including oral and maxillofacial surgery practices The use of telemedicine has rapidly expanded to uphold safety strategies of physical distancing and disease transmission reduction while maintaining uninterrupted care of patients To date there are no specific guidelines to optimize telemedicine encounters in the oral and maxillofacial surgery practice The goal of this paper is to provide best practices for both oral and maxillofacial surgeons and their patients to effectively utilize telemedicine for the duration of COVID19 and beyond Statement of Clinical Relevance The goal of this paper is to provide best practices for both oral and maxillofacial surgeons and their patients to effectively utilize telemedicine for the duration of COVID19 and beyond INTRODUCTION The COVID19 pandemic has disrupted society in a multitude of ways Healthcare is no exception the SARSCoV virus rapid transmission and high hospitalization rate have strained the availability of medical resources including personal protective equipment PPE respiratory ventilators and hospital beds[] The virus also poses a major threat to healthcare personnel whose risk of exposure are compounded by the aforementioned PPE shortages[] In response the American Association of Oral and Maxillofacial Surgeons AAOMS recommended delaying elective surgeries in accordance with the Centers for Disease Control and Preventions calls to 0c postpone elective medical and dental procedures[] Similarly four out of five dental offices have closed for all except emergency procedures[] In the face of these challenges the medical and dental communities have remained steadfast in caring for patients with nonelective health needs and innovating alternate ways to deliver care One of the most important and popular alterations in the delivery of care is the increased utilization of telemedicine which allows surgeons and patients to connect virtually[ ] This has enabled patients to access muchneeded medical care while preserving PPE and minimizing exposure to pathogens Though studies have found telemedicine to decrease costs and save time without compromising patient satisfaction it was not widely used in healthcare before the COVID19 pandemic[ ] Similarly teledentistry was deemed to be in its infancy by the founder of the American Teledentistry Association in [] Nevertheless telemedicine has shown promise and has been incorporated into the workflow of various oral and maxillofacial surgery institutions and practices across the country Virtual visits are particularly useful in triaging patients For example patients with dentoalveolar infections can meet virtually with surgeons and receive prescriptions for appropriate analgesics and antibiotics without going to the emergency department Also patients with oral lesions can take images and show their surgeon before their inperson visit to expedite the diagnosis and treatment planning workflow This enables patients to access timely attention of providers while lightening the load on the healthcare system by reducing the number of inperson visits 0c Associated with the recent rise in telemedicines popularity is a learning curve for both surgeons and patients The incorporation of technology and the shift to virtual visits can be jarring for the patientsurgeon relationship and must be navigated thoughtfully While there have been helpful telehealth guides for surgeons and patients in other surgical specialties we do not know of any such guidelines for oral and maxillofacial surgery[ ] As such we detail best practices for both oral and maxillofacial surgeons OMS and their patients to effectively utilize telemedicine for the duration of COVID19 and beyond GENERAL CONSIDERATIONS FOR TELEMEDICINE In accordance with the AAOMS White Paper on Telehealth and Remote Treatment virtual management of any oral and maxillofacial surgical condition should only be provided by appropriately licensed oral and maxillofacial surgeons as regulated by the state law[] The delivery of patient care through telemedicine must continue to follow evidencebased guidelines to ensure quality and safety for all patients All providers must comply with the latest telehealth requirements outlined by the United State Health and Human Services to protect patient privacy and comply with the Health Insurance Portability and Accountability Act HIPAA[] Furthermore providers are ethically obligated to inform all patients about the potential benefits limitations and risks of telemedicine[] Patients requiring emergency or urgent services must be directed to the nearest hospital 0c SETTING UP FOR TELEMEDICINE While there are several modalities to conduct a telemedicine encounter we strongly recommend a live synchronous twoway interaction between the patient and the OMS incorporating both audio and visual telecommunications tools This can be achieved with a desktop computer laptop or smartphone The United States Census Bureau reports that approximately percent of households have computers or smartphones and percent have broadband internet subscriptions[] Of these options though we recommend using a desktop or a large screen laptop with a highresolution camera over smartphones even if the latter meets the minimum technical requirements Ideally telemedicine visits can offer a clinic experience that closely simulates inperson encounters Trained administrative staff members should call patients beforehand to discuss the virtual setup and basic expectations for the visit Prasad et al created Figure which exemplifies a patient informational handout with graphic illustrations detailing the set up as well as key examination steps that patients may be asked to perform during the encounter [] In the following we will detail key aspects and considerations for both OMS and patients to maximize their telehealth visits 0c Insurance Coverage and Billing In an effort to reduce the burden posted on healthcare entities and facilitate mitigation efforts the Centers for Medicare Medicaid Services CMS broadened access for Telemedicine coverage with private payers following suit CMS expansion included voiceonly visits which is critical for patients without access to a smartphone or computer video capabilities Furthermore CMS allowed for parity of payment for Telemedicine visits and inperson visits so providers can bill Medicaid and Medicare at the same rate as they would for an inperson visit[] This new policy is especially relevant for older patients covered by Medicare for they are generally at higher risk for COVID19 complications[] Therefore before telehealth encounters providers should confirm if the patients insurance plan has Telemedicine coverage and also whether the insurance plan waives all copays for nonCOVID19 related visits to avoid a scenario where a patient receives a bill unknowingly The American Association of Oral and Maxillofacial Surgeons provide additional detailed information about telehealth billing relevant for OMSs including updated links to AMA and ADA billing codes on its website at httpswwwaaomsorgpracticeresourcestelehealthfaqs 0c Professionalism and provider attire The visit should replicate the same level of professionalism as that of inperson appointments Providers should dress professionally as they would in their office or hospitalbased practice Before the patient comes in providers should review the patients relevant medical records and chief complaints to save time and maximize the efficiency of the visit Also in the interest of both time and professionalism OMS and patients should both be mindful to start the visit on time During the visit OMS should communicate with patients to maintain transparency For instance if an OMS needs to document something during the visit they should respectfully inform the patient of the task to prevent any misunderstanding At the end of the visit it is important for OMS to summarize what they accomplished during the visit and provide a clear plan for appropriate next steps Physical background When possible OMS and patients should conduct virtual encounters in welllit spaces Lighting specifically can have a profound effect on video quality As such overhead lights can be helpful while lights behind the person should be avoided Care should also be taken to prevent other sources of potential disruptions such as background noise or visual distractions It may be helpful for OMS to evaluate their surroundings from the perspective of their patients 0c Technological background It is important for OMS to test their video and audio quality before visits to anticipate any potential technical difficulties that may interrupt the encounter A strong WiFi internet connection is preferred over cellular data to ensure a stable signal With the exception of electronic health records OMS should close any unnecessary programs or internet browser tabs to preserve internet bandwidth In addition OMS should be mindful that their patients may have varying internet speeds As such OMS should give approximately seconds of lag time after patients stop speaking to allow all of their words to come through completely Patient and camera position If possible patients should sit upright on a chair in front of a computer placed on top of a desk They should sit close enough to the camera so that their entire head and neck area are within the video frame The camera should be at approximately eyelevel for both OMS and patients to maintain eye contact and remain engaged during the virtual encounter For patients using a smartphone the device can be propped up at a to 90degree angle from the table surface to allow patients to free both of their hands for physical exam tasks Patient clothing Patients must be notified of the appropriate clothing well in advance prior to the appointment Ideally clothing should allow patients entire head and neck regions to be visualized while maintaining patient comfort and professionalism Any hat or scarves should be removed if at all possible maintaining appropriate cultural and religious norms Patient items Prior to the appointment patients should prepare the following items which can help aid OMS in visualization and retraction during the virtual physical exam Most of these items are commonplace inexpensive and available at the patients home 0c Flashlight A flashlight or a penlight can enhance visualization of certain obscure head and neck structures particularly those in the oral cavity The builtin flashlight of a smartphone can also be used Ruler A ruler or a measuring tape can be used to measure the patients maximal mouth opening and mandibular range of motion Napkins A napkin can be used to touch any intraoral landmarks and also to clean up after any inadvertent salivation from the virtual physical exam Spoon A spoon can be used to retract the cheeks or depress the tongue to evaluate structures of the oropharynx such as the soft palate and tonsillar pillars Cheek retractors A fun way for patients to achieve cheek retraction can be to use the plastic props from the board game Speak Out which was created in [] This game provides horseshoeshaped plastic retractors shown in Figure that can be placed along the patients upper and lower lips to lateralize the cheeks thus allowing handsfree visualization of the dentition and oral cavity soft tissue Patient assistant If available patients should ask a family member or friend to accompany them to the telehealth visit The assistant can help patients perform the physical tasks required during the virtual physical exam Assistants can also help position the web camera to improve the providers view In fact these assistants are essentially mandatory for pediatric patients or patients with disabilities Feedback It is a good practice for providers to seek feedback from patients after a visit This will help OMS hone their telehealth skills and ensure that their patients are receiving an appropriate quality of care 0c THE VIRTUAL HISTORY AND PHYSICAL Thorough patient assessment proper medical documentation and appropriate diagnostic testing are critical components of OMS practice that enable proper diagnosis and treatment planning OMS should obtain patients medical histories in a similar manner as they would in their offices New patients must be asked for comprehensive histories that include chief complaint history of present illness past medical history past surgical history dental history medications allergies pertinent family history social history and complete review of systems For patients of record their medical histories should be updated to reflect their current chief complaint All patients should also be screened with an up to date COVID19 questionnaire If an infection is suspected OMS should refer patients to their primary care physicians or local emergency department depending on the severity of symptoms for appropriate workup Vitals should be obtained if the patient has access to a thermometer blood pressure cuff pulse oximeter or weighing scale Even without any of these devices patients can still measure their pulse by applying two fingers on the patients carotid counting the number of beats per minute Also patients can calculate their respiratory rate by observing the number of chest rises in one minute Finally oxygen saturation can be measured with certain mobile health applications though OMS should not solely rely on their results for major medical decisions Finally patients with fever body temperature F warrants further work up in an emergency setting for a differential diagnosis that includes COVID19 infection The virtual physical exam will be limited to a head and neck exam and a cranial nerve exam While inspection and palpation are the basis of a focused physical examination in oral and maxillofacial surgery OMS must learn to work together with patients to achieve the same goals 0c virtually Patients must perform maneuvers on themselves with the OMSs guidance To this end a printed stepbystep schematic as illustrated in Figure can be helpful for patients to receive before the visit During the visit the OMS can reinforce the diagram with clear verbal instructions that avoids medical jargon The exam itself must be conducted systematically with a topdown outsidein approach as is typical in an oral and maxillofacial surgery practice The exam can be further divided into head and neck subsites The OMS should ask for specific symptoms related to each subsite and carefully inspect for any abnormalities while guiding the patient or the patients assistant through the exam The following section offers additional details and considerations for each subsite Head The OMS should ask about any history of head trauma The head is assessed to ensure that it is normocephalic and atraumatic Face The OMS should ask the patient about any facial pain swelling weakness numbness or history of trauma to the region Then OMS can start the facial exam by asking the patient to lean close to the camera First the face is examined for any skin lesions along the forehead eyelids external ears nose malar region vermilion of the lips and the chin Patients left and right sides of the face should be compared for any gross asymmetry or deformities OMS can then guide patients through palpating their own face for any bony discontinuity or soft tissue swelling Patients can also tap their own face with two fingers to reveal any tenderness in the sinuses Regarding the eyes OMS can assess if the pupils are equal The extraocular muscles along with the oculomotor supratrochlear and the abducens nerves can be tested by having the 0c patient look up down left and right without moving the head The sensory portion of the trigeminal nerve can be tested by asking patients to close their eyes and slide both of their index fingers horizontally along the ipsilateral forehead ophthalmic branch cheek maxillary branch lip and chin mandibular branch The branches of the facial nerves can be tested by asking patients to raise their eyebrows close their eyes tightly puff out their cheeks smile widely and show their bottom teeth Temporomandibular joint TMJ The OMS should ask the patient about any facial jaw or ear pain trismus difficulty mastication clicking or locking of the joint Then the TMJ exam begins by asking patients to palpate their mandibular condyles and muscles of mastication to look for any tender spots Providers can ask patients to open and close their mouth while palpating the condyles to feel for any clicks or crepitus Also maximal interincisal opening can be roughly estimated by the number of fingerbreadth or precisely measured using a ruler The mandibular range of motion can be assessed or measured in protrusive and lateral excursive positions Neck The OMS should ask for any difficulty breathing dysphagia sore throat odynophagia hoarseness or new neck swelling The neck exam begins with inspection looking for any asymmetry or tracheal deviation Patients can be asked to turn the head from side to side look upwards and shrug the shoulders to assess the spinal accessory nerves OMS should ask the patients assistant if possible to stand right behind the patient and palpate the patients neck Using their fingertips on both hands the assistant can palpate the neck in a unidirectional manner from superior to inferior and then from lateral to medial Ask them to note any palpable bumps or tender spots It is particularly important to palpate the lateral neck for enlarged lymph nodes 0c Lastly OMS can identify the thyroid by asking patients to swallow while palpating the appropriate area on the neck to rule out thyromegaly Oral cavity and oropharynx The OMS should ask for any oral pain oral swelling or sores tongue numbness difficulty with tongue movement or dry mouth Examination of the oral cavity exam can be challenging because intraoral structures can be difficult to retract and illuminate The aforementioned cheek retractors whether from a board game or makeshift spoons can be helpful for retraction of soft tissue and visualization In addition patients friends and family can help a great deal by adjusting the camera while also properly angling an additional light source For each intraoral structure the OMS must carefully inspect for ulcers raised lesions abnormal white leukoplakic or bright red erythroplakic lesions In general OMS can best visualize structures near or at the level of the maxilla when patients lift their heads up to degrees Likewise structures near or at level of the mandible are best observed with the patient dropping the chin approximately degrees OMS may find it useful to practice these examination techniques on their own cameras before the visit Patients should be recommended to wash their hands or to use gloves before touching any intraoral landmarks The exam begins by sliding the patients index finger along the maxillary and mandibular vestibule to look for any swelling or fluctuance With the cheeks retracted the patient can palpate their buccal and labial mucosa using the thumb and index finger with one 0c finger compressing along the face extraorally When possible palpation should be bidigital Next the patient can use their index figures to palpate the tuberosity retromolar trigone and the hard palate for tenderness or irregularities The tongue is the most common site for oral cancer and must be thoroughly examined The dorsal surface of the tongue should be examined by asking the patient to fully protrude their tongue Providers should also ask patients to move their protruded tongues to the left and right to inspect the lateral tongue and ensure the function of the hypoglossal nerves The ventral tongue and the floor of the mouth can be observed by asking patients to touch the tip of their tongue to their hard palate The tongue can then be palpated for lumps or masses Next the sublingual and submandibular glands can be palpated for symmetry and lack of elevation by the patient with their extended index fingers on the floor of the mouth The examination of the oropharynx is mostly limited in virtual encounters Nevertheless the soft palate tonsils and uvula can be partially visualized with the patients mouth wide open and using a spoon to depress the tongue Although unpleasant the glossopharyngeal and vagus nerves can be tested by gently touching the soft palate using a spoon to induce a gag reflex Dentition If the patient is dentate the provider should ask about dental pain sensitivity loosening of teeth bleeding or sore gums or malocclusion The patients dentition can be evaluated after retracting soft tissue as described previously Dental caries missing teeth periodontal disease gingival lesions or swelling can be readily identified Mobility of teeth can be assessed by using the patients thumb and index finger In edentulous patients the alveolar 0c ridge should be examined for any abnormalities as part of the aforementioned oral soft tissue exam CONCLUSION The COVID19 pandemic has catalyzed an exponential increase in telemedicine usage Telemedicine helps patients maintain access to care conserves limited medical resources and protects both OMS and patients from pathogen exposure Nevertheless there is an expected learning curve that accompanies such a paradigm shift in the delivery of care As such this paper provides a guide of best practices to aid both OMS and patients to navigate this promising electronic tool In addition we provide an accessible schematic handout that can be given to patients before a telehealth appointment to help them prepare for the visit for both setting up and performing physical exam procedures Because telemedicine may have a role in oral and maxillofacial surgical care even after this pandemic we are optimistic that these best practices can be helpful and relevant for the present situation and beyond 0c FIGURE LEGEND Figure Patient informational handout with graphic illustrations detailing the set up as well as key examination steps that patients may be asked to perform during the telemedicine encounter Reprinted from [] 0c Figure Horseshoeshaped plastic lipcheek retractors REFERENCES Li Q Guan X Wu P et al Early Transmission Dynamics in Wuhan China of Novel CoronavirusInfected Pneumonia N Engl J Med httpsdoiorg101056NEJMoa2001316 Feng S Shen C Xia N et al Rational use of face masks in the COVID19 pandemic Lancet Respir Med httpsdoiorg101016S221326002030134X Li R Rivers C Tan Q et al Estimated Demand for US Hospital Inpatient and Intensive Care Unit Beds for Patients With COVID19 Based on Comparisons With Wuhan and Guangzhou China JAMA Netw Open 3e208297e208297 httpsdoiorg101001jamanetworkopen20208297 White DB Lo B A Framework for Rationing Ventilators and Critical Care Beds During the COVID19 Pandemic JAMA httpsdoiorg101001jama20205046 Heinzerling A Stuckey MJ Scheuer T et al Transmission of COVID19 to Health Care Personnel During Exposures to a Hospitalized Patient Solano County California 0c February MMWR Morb Mortal Wkly Rep httpsdoiorg1015585mmwrmm6915e5 Ng K Poon BH Kiat Puar TH et al COVID19 and the Risk to Health Care Workers A Case Report Ann Intern Med httpsdoiorg107326L200175 Halepas S Ferneini EM A Pinch of Prevention is Worth a Pound of Cure Proactive Dentistry in the Wake of COVID19 Journal of Oral and Maxillofacial Surgery httpsdoiorg101016jjoms202003036 AAOMS Member Alert COVID19 Update Healthcare Facilities Preparing for Community Transmission In Centers for Disease Control and Prevention httpswwwcdcgovcoronavirus2019ncovhcpguidancehcfhtml Accessed May CDC Guidance for Providing Dental Care During COVID19 In Centers for Disease Control and Prevention httpswwwcdcgovoralhealthinfectioncontrolstatementCOVIDhtml Accessed May Carey M Second week of HPI polling shows dentists response to COVID19 American Dental Association Hollander JE Carr BG Virtually Perfect Telemedicine for Covid19 New England Journal of Medicine httpsdoiorg101056NEJMp2003539 Prasad A Brewster R Newman JG Rajasekaran K Optimizing your telemedicine visit during the COVID19 pandemic Practice guidelines for patients with head and neck cancer Head Neck na httpsdoiorg101002hed26197 Russo JE McCool RR Davies L VA Telemedicine An Analysis of Cost and Time Savings Telemedicine and eHealth httpsdoiorg101089tmj20150055 Cain SM Moore R Sturm L et al Clinical assessment and management of general surgery patients via synchronous telehealth Journal of Telemedicine and Telecare httpsdoiorg1011771357633X16636245 Teledental Practice and Teledental Encounters An American Association of Teledentistry Position Paper Jampani ND Nutalapati R Dontula BSK Boyapati R Applications of teledentistry A literature review and update J Int Soc Prev Community Dent httpsdoiorg1041032231076297695 Wicklund E Dentists Use Telehealth to Improve Access to Care And Fight a Phobia In mHealthIntelligence httpsmhealthintelligencecomnewsdentistsusetelehealthtoimproveaccesstocareandfightaphobia Accessed May 0c Smith WR Atala AJ Terlecki RP et al Implementation Guide for Rapid Integration of an Outpatient Telemedicine Program during the COVID19 Pandemic Journal of the American College of Surgeons httpsdoiorg101016jjamcollsurg202004030 AAOMS White Paper on Telehealth and Remote Treatment Notification of Enforcement Discretion for Telehealth Remote Communications During the COVID19 Nationwide Public Health Emergency In HHSgov httpswwwhhsgovhipaaforprofessionalsspecialtopicsemergencypreparednessnotificationenforcementdiscretiontelehealthindexhtml Accessed May Ethical Practice in Telemedicine In American Medical Association httpswwwamaassnorgdeliveringcareethicsethicalpracticetelemedicine Accessed May Ryan C Computer and Internet Use in the United States United States Census Bureau Additional BackgroundSweeping Regulatory Changes to Help US Healthcare System Address COVID19 Patient Surge CMS In CMSgov httpswwwcmsgovnewsroomfactsheetsadditionalbackgroundsweepingregulatorychangeshelpushealthcaresystemaddresscovid19patient Accessed Jun Medicaid Learning Network Telehealth Services Hasbro Brings Mouth Piece Challenge to the Masses with New SPEAK OUT Game In Business Wire httpswwwbusinesswirecomnewshome20160624005633enHasbroBringsMouthPieceChallengeMassesNew Accessed May 0c" | 2 |
" triple negative breast cancer tnbc remains recalcitrant to most targeted therapy approaches however recent clinical studies suggest that inducing tumor damage can render tnbc responsive to immunotherapy we therefore tested a strategy for immune sensitization of murine tnbc 4t1 tumors through combination of focused ultrasound fus thermal ablation and a chemotherapy gemcitabine gem known to attenuate myeloid derived suppressor cells mdscsmethods we applied a sparse scan thermally ablative fus regimen at the tumor site in combination with systemically administered gem we used flow cytometry analysis to investigate the roles of monotherapy and combinatorial therapy in mediating local and systemic immunity we also tested this combination in rag1 mice or t cell depleted wild type mice to determine the essentiality of adaptive immunity further we layered programmed cell death protein pd1 blockade onto this combination to evaluate its impact on tumor outgrowth and survivalresults the immune modulatory effect of fus monotherapy was insufficient to promote a robust t cell response against 4t1 consistent with the dominant mdsc driven immunosuppression evident in this model the combination of fusgem significantly constrained primary tnbc tumor outgrowth and extended overall survival of mice tumor control correlated with increased circulating antigen experienced t cells and was entirely dependent on t cell mediated immunity the ability of fusgem to control primary tumor outgrowth was moderately enhanced by either neoadjuvant or adjuvant treatment with anti pd1 thermally ablative fus in combination with gem restricts primary tumor outgrowth improves survival and enhances immunogenicity in a murine metastatic tnbc model this treatment strategy promises a novel option for potentiating the role of fus in immunotherapy of metastatic tnbc and is worthy of future clinical evaluationtrial registration numbers nct03237572 and nct04116320 metastatic breast cancer brca particularly the triple negative breast cancer tnbc phenotype is resistant to most chemical and molecularly targeted therapeutic approaches interestingly tnbc is often infiltrated with immune cells and the presence of these cells has been shown to have a favorable prognosis in patients treated with neoadjuvant chemotherapy1 early studies in the use of immunotherapies targeting the pd1programmed death ligand pd l1 checkpoint inhibitory axis showed some efficacy2 in tnbc compared with other brca subtypes which are generally recalcitrant to checkpoint blockade activity in the tnbc subtype may be related to the relatively high immune infiltration and correlated with the higher mutational burden observed in tnbc greater immunotherapy efficacy in tnbc has been recently observed with the use of antibodies targeting the pd1pd l1 checkpoint inhibitory axis in combination with nab paclitaxel5 this outcome suggests that inducing tumor damage augments antitumor immunity either by promoting antigen availability or disrupting the immunosuppressive tumor microenvironment tme found in tnbcamong the potential networks in tnbc that could constrain the activity of antitumor immunity is the presence of immunosuppressive myeloid cell subsets these have the capacity to impair adaptive immunity and promote tumor growth and metastasis among these cell types myeloid derived suppressor cells mdscs prevail as a heterogeneous population of immature myeloid cells which serve the eponymous role of suppressing the antitumor immune response limiting both t cell activation and effector functions6 increased levels of this cell type have been demonstrated in tumor tissues of patients with primary brca while those with metastatic disease bear the highest abundance of circulating mdscs8 studies have sheybani a0nd et a0al j immunother cancer 20208e001008 101136jitc2020001008 0copen access shown that approaches that either stimulate myeloid cells with inflammatory mediators or eliminate mdsc can improve antitumor immunity9to this end the central premise put forth in this study is that focused ultrasound fusa safe noninvasive and nonionizing strategy for localized acoustic energy deposition into tissuescan synergize with immunotherapy in a murine model of metastatic tnbc fus is capable of rapidly heating tumors to thermally ablative temperatures its extracorporeal application obviates the need for catheterization injection or implantation fus can be targeted with millimeter precision under mri or ultrasound guidance thereby allowing for thermal damage and destruction of tumor tissue without compromising healthy intervening or peripheral tissues the bioeffects of fus hold distinct implications for tumor antigenicity immune cell activation and trafficking13 thermally active fus regimes have elicited antitumor immune responses in implantable models of melanoma15 pancreatic16 prostate17 colon20 kidney21 and brca23 pertaining to the challenge of myeloid cell immunosuppression in tnbc thermally ablative fus has been shown to induce the expression of heat shock proteins24 and proinflammatory cytokines including interleukin il12 interferonÎ ifnÎ and tumor necrosis factorα tnfα from a variety of cancer cell lines and after in vivo treatment of tumors26 whether the ability of fus to induce these inflammatory mediators is sufficient to overcome myeloid suppression in the context of brca is currently under debate with some studies showing activation of antigen presenting cells and t cell recruitment in patients with brca treated with thermally ablative fus28 while others show that additional innate stimuli are needed to support antitumor immunity23 notably some studies have suggested that a sparse scan thermal ablation regimen more effectively recruits and activates dendritic cells dcs and antitumor immunity than total thermal ablation perhaps by limiting thermal denaturation of tumor antigens and innate stimuli31based on the improved myeloid cell maturation that occurs with sparse scan regimens we herein tested the ability of a sparse scan partial thermal ablation fus regimen as a monotherapy to promote antitumor immunity in an aggressive syngeneic model of metastatic murine tnbc with extensive granulocytic mdsc involvement that is recalcitrant to anti pd1 while some activity is evident with the partial ablation approach significantly greater control was achieved by targeting mdsc inhibition in combination with thermally ablative fus this control was completely dependent on the adaptive immune responsemoreover we demonstrate that layering anti pd1 immune checkpoint blockade onto this combinatorial regimen moderately improves tumor growth restriction these data suggest that in disease settings where myeloid allied approaches to attenuate myeloid immunosuppression may be employed to reveal the full immunotherapeutic immunosuppression predominates potential of thermally ablative fus once immunosuppressive myeloid cells are accounted for fus treatment can promote adaptive immunity that in turn potentiates immune checkpoint blockademethodscell line maintenance4t1 and e0771 cell lines were maintained in rpmi l glut or dulbeccos modified eagles medium dmem gl d glucose l glutamine respectively supplemented with fetal bovine serum fbs at °c and co2 thawed cells were cultured for up to three passages and maintained in logarithmic growth phase for all experiments cells tested negative for mycoplasmaeight week old to week old female balbc or c57bl6 mice were obtained from nci charles river nci crl or the jackson laboratory female balbc rag1 mice were obtained from the jackson laboratory 4t1 or e0771 cells were subcutaneously implanted into the right flank of mice mice were housed on a hour12 hour lightdark cycle and supplied food ad libitum tumor outgrowth was monitored via digital caliper measurements tumor volume was calculated as follows volume lengthwidth22 approximately days 4t1 or days e0771 following tumor implantation mice were randomized into groups in a manner that ensured matching mean starting tumor volume across experimental groupsin vivo ultrasoundguided fus partial thermal ablationmice were treated with fus either days 4t1 cohorts or days e0771 postimplantation on treatment day mice were anesthetized with intraperitoneal injection of ketamine mgkg zoetis and dexdomitor mgkg pfizer in sterilized saline mouse flanks were shaved and depilated following which ultrasound guided fus thermal ablation was performed using one of the two systems system and treatment details are provided in online supplementary materials and methods mice that did not receive fus treatment consistently underwent anesthesia and depilation of the flank additionally these mice underwent a sham treatment consisting of exposure to the °c degassed water bath exposure for min following sham or fus treatment all mice were moved to a heating pad and given antisedan for anesthesia reversal and recoverygemcitabine therapygemcitabine gem mgmouse in µl volume mylan diluted in saline and filter sterilized through a µm syringe filter was administered intraperitoneally once a week on the day of fus treatment following which administration was repeated for an additional weeks administration of gem doses was based on existing literature demonstrating the use of gem for inhibition of mdscs in 4t112 the initial dose of gem was administered immediately prior to sham or fus treatment sheybani a0nd et a0al j immunother cancer 20208e001008 101136jitc2020001008 0cmice that did not receive gem received an intraperitoneal injection of vehicle treatment µl of sterile saline at the time points specifiedpd1 blockade therapyfor checkpoint inhibitor therapy the rat anti mouse pd1 antibody αpd1 rmp114 diluted in sterilized saline was administered intraperitoneally every days for a total of five doses µg per mouse treatment was initiated on day early αpd1 or day delayed αpd1t cell depletionst cell depletion antibodiesanti cd8 clone bio x cell and anti cd4 gk15 clone bio x cellwere diluted in sterilized saline and administered intraperitoneally every to days starting at day days post fus for a total of seven doses µg of each antibody for a total µg per mouseimmunohistochemistryon day sham or fus exposed tumors were excised and fixed in neutral buffered formalin sigma fixed tumors were paraffin embedded sectioned and stained for hematoxylin and eosin digital images of stained slides were acquired using the vectra automated quantitative pathology imaging system akoya biosciences whole slide screening and image capture were subsequently performed using phenochart akoya biosciencesflow cytometrymice were bled at days and via tail vein and samples were rbc lysed hybri max sigma and stained for flow cytometry analysis at days post tumor implantation tissues were obtained from euthanized tumor bearing animals for immune response assessment in order to gain resolution into tissue resident versus vascular immune cell populations mice were injected intravenously with rat anti mouse cd45 fitc clone f11 bd biosciences min prior to euthanasia 4t1 tumors spleens cardiac blood axillary and brachial tumor draining lymph nodes tumor dlns pooled and nondraining inguinal lymph nodes were harvested processed and stained for flow cytometry analysis additional details are provided in online supplementary materials and methodssamples were acquired on an attune nxt flow cytometer thermofisher scientific and data were analyzed with flowjo treestar or fcs express de novo software a representative gating strategy for granulocytic myeloid derived suppressor cell g mdsc and cd44 t cells is provided in online supplementary figure statistical analysisall statistical analyses were performed in graphpad prism graphpad software a detailed description of statistical methods for each experiment is provided in the corresponding figure legendopen accessanimal study approvalall animal work was performed under a protocol approved by the animal care and use committee at the university of virginia and conformed to the national institutes of health guidelines for the use of animals in researchresultspartial thermal ablation of established tnbc tumors promotes peripheral dc activation but has limited impact on the presence of t cells and other myeloid cell subsetsto achieve partial thermal ablation of 4t1 tumors we used an ultrasound guided fus system equipped with a single element therapeutic transducer driven at mhz figure 1a online supplementary figure a grid of sonications was overlaid on the ultrasound visible tumor and ablated in a raster pattern under b mode ultrasound guidance figure 1bc the exceptionally small focus of this system rendered a low ablation fraction of total tumor volume immediately following ablation tumors displayed evidence of coagulative necrosis in the ablated zone with surrounding periablative margins figure 1d one week following fus partial thermal ablation tumors and secondary lymphoid organs were excised for immunological characterization by flow cytometry figure 1b fus partial thermal ablation of 4t1 tumors conferred a significant increase fold in the absolute number of cd11c hi dcs within the axillary tumor draining lymph node adln of mice figure 1e while this was accompanied by a nearly threefold elevation in the absolute number of cd86 dcs within the adln figure 1f the percentage of dcs expressing cd86 did not change figure 1g increased numbers of dcsand cd86 dcs in particularsuggest fus is promoting the maturation or trafficking of these cells in the dlns where they could encounter and activate t cells however this did not translate to tumor growth restriction data not shown we also did not observe significant differences in the absolute number of activated t cells in 4t1 tumors figure 1h or dlns data not shown following fus exposure suggesting limitations in the ability of fus activated dc to further drive an antitumor t cell responseimmune profiling by flow cytometry revealed that irrespective of fus exposure of the intratumoral cd45 immune cell population is comprised of cd11b myeloid cells figure 1i similarly approximately of the circulating immune cell population in 4t1 tumor bearing mice is comprised of myeloid cells a striking fold elevation in circulating myeloid burden compared with naive mice online supplementary figure notably ly6g granulocytic myeloid derived suppressor cells g mdscs significantly dominated the immune cell repertoire within 4t1 tumors relative to other myeloid including f480 macrophages ly6c cell subsets monocytic myeloid derived suppressor cells m mdscs and cd11c hi dcs figure 1j fus partial thermal ablation did not significantly alter the absolute number per sheybani a0nd et a0al j immunother cancer 20208e001008 101136jitc2020001008 0copen access figure partial thermal ablation of established tnbc tumors promotes peripheral dc activation but has limited impact on the presence of t cells and other myeloid cell subsets a design overview of a custom ultrasound guided fus system consisting of a mhz single element transducer orthogonally co registered to an mhz linear ultrasound imaging array the tumor bearing flank of each anesthetized mouse was acoustically coupled to ultrasound transducers via degassed water bath maintained at °c sham mice were similarly positioned but did not undergo sonications b schematic illustration of fus partial thermal ablation scheme and study layout for evaluation of immune sequelae in 4t1 tumor bearing mice a grid of sonications was applied in a raster pattern onto the b mode ultrasound visible tumor in total two planes of sonication spaced mm apart were applied to each tumor grid points were spaced mm apart within a single plane one week following thermal ablation tumors and secondary lymphoid organs were excised for sham n6 or fus treated n5 mice and processed for flow cytometry c representative b mode ultrasound images of ectopic 4t1 tumors either before top or during bottom fus exposure sonication grid depicting targets red points is superimposed on b mode image during treatment subsequent to thermal ablation hyperechoic signatures yellow arrow are occasionally observed d representative he staining of either sham 4t1 tumors or those resected immediately following fus partial thermal ablation zoomed insets depict the transition from necrotic to intact tumor tissue within the periablative zone scale bars400 µm and µm on left and right inset respectively e absolute number of cd11c hi dcs in the axillary tumor draining lymph node adln of 4t1 tumor bearing mice p00136 vs sham f absolute number of cd86 cd11c hi dcs in the adln p00063 vs sham g percentage of cd86 subset out of total cd11c hi dcs within adln h absolute number of intratumoral cd44 cd8 and cd44 cd4 t cells and regulatory t cells tregs per gram tumor i percentage of cd11b myeloid cells out of total cd45 immune cells across tumor spleen adln inguinal dln idln and nontumor draining axillary and inguinal lns ndlns p005 vs all other groups irrespective of fus exposure specifically tumor vs spleen p00226 tumor spleen vs all other organs p00001 j absolute number of intratumoral myeloid cells cd11c hi dcs f480 macrophages ly6c monocytic myeloid derived suppressor cells m mdscs ly6g granulocytic myeloid derived suppressor cells g mdscs per gram 4t1 tumor p00001 vs all other cell types irrespective of fus exposure all data represented as mean±sem significance assessed by unpaired t test fh or two way analysis of variance followed by tukey multiple comparison correction ik nsnot significant dcs dendritic cells fus focused ultrasound hifu high intensityfocused ultrasoundsheybani a0nd et a0al j immunother cancer 20208e001008 101136jitc2020001008 0cgram tumor of these myeloid cell subsets these observations led us to formulate the hypothesis that widespread immunosuppressive mechanisms associated with the 4t1 tme must be addressed in order to facilitate the t cell response to fusfus partial thermal ablation in combination with gem constrains primary tnbc tumor outgrowth and extends overall survivalour observation of the overwhelming mdsc burden following 4t1 tumor implantation warranted implementation of an allied therapeutic strategy in order to counter this immunosuppressive barrier to this end we tested a combinatorial paradigm incorporating gem a myelosuppressive chemotherapy demonstrated to inhibit mdscs transiently in the 4t1 model without consequence to t cell phenotype or function12to evaluate the efficacy of fus and gem in combination we used a preclinical ultrasound guided fus system to achieve partial thermal ablation of established 4t1 tumors 14d after tumor implantation average tumor volume of mm3 in combination with the single session of fus thermal ablation we initiated gem therapy mgmouse which was then readministered weekly for a total of three gem doses figure 2a combinatorial therapy synergized to produce significant constraint of 4t1 tumor outgrowth compared with sham and monotherapy groups figure 2bcby termination of treatments at day 4t1 tumors exposed to fusgem combination saw nearly and reductions in average volume compared with sham or gem exposed tumors respectively figure 2b two dimensional tumor projections at day postimplantation saw a nearly fold reduction in area from sham to combinatorial therapy setting figure 2de in a fraction of mice treated with fusgem we observed complete regression of 4t1 tumors although transient figure 2c tumor outgrowth eventually rebounded after termination of treatments 4t1 tumor bearing mice receiving fusgem treatment additionally saw the greatest extension in overall survival with and increases in median survival time compared with sham and gem groups respectively hrs and for fusgem relative to sham and gem groups respectively figure 2f we additionally observed that fusgem significantly constrained outgrowth in a separate c57bl6 metastatic mammary carcinoma model e0771 online supplementary figure to further the clinical relevancy of these findings we applied this combinatorial strategy with the research grade analog of a clinical ultrasound guided fus system theraclion echopulse that is already ce marked for applications in breast fibroadenoma thyroidparathyroid gland and varicose vein ablation and currently in use for multiple clinical trials leveraging fus thermal ablation in combination with cancer immunotherapy we observed that partial thermal ablation using the theraclion visualization and treatment unit mhz in combination open accesswith gem controlled 4t1 tumor outgrowth to a degree comparable with that observed with the custom in house system online supplementary figure these findings lend credence to the notion that the impact of combining gem with fus may be conserved across partial thermal ablation regimens moreover they demonstrate that the efficacy of fus partial thermal ablation in combination with gem can be recapitulated on a system with a larger focus and in line image guidance that is currently in use clinicallycombination of fus partial thermal ablation with gem increases the levels of circulating t cellslymphocytesin particular cd8 and cd4 t cellsplay an important role in responding to tumor antigen and generating a durable antitumor response based on the extended protective effect observed in mice treated with fusgem flow cytometry analysis was performed to evaluate the contribution of t cells in generating systemic and local tumor control we sampled the circulating immune cell repertoire in 4t1 tumor bearing mice via serial tail bleeds days and prior to readministration of gem and a terminal cardiac bleed at the time of spleen harvest day figure 3a combinatorial therapy significantly elevated absolute number of cd8 and cd4 t cells in the circulation at days and figure 3bc and ef moreover a trend threefold to fivefold increase in circulating t cells was noted in the fus group relative to sham figure 3bc and ef from days to systemic cd44 expressing antigen experienced t cell populations both cd8 and cd4 saw a steady significant increase after combinatorial therapy figure 3d and g a similar modest trend was noted for the fus monotherapy group relative to sham and gem figure 3d and g these changes were concordant with a decrease in circulating myeloid cd11b cells in gem recipient groups demonstrating the ability of gem to partially alleviate circulating myeloid burden figure 3hsplenomegaly is a common signature that arises in parallel with the leukemoid reaction to 4t1 tumors that is the expansion of immunosuppressive myeloid cells during tumor progression we observed that combinatorial therapy most significantly reverses splenomegaly online supplementary figure 6ab consistent with this observation immunological characterization of spleens revealed a significant decrease in cd11b myeloid cellsa reduction in fusgem spleens relative to sham or monotherapy figure 3i while there appeared to be a trend toward more cd11b cells in the monotherapy groups compared with the sham this difference was not significant and there was no difference between these groups in terms of absolute cd11b cell numbers within the spleen data not shown the decrease in myeloid cells in the combination treatment group was accompanied by a significant corresponding elevation in lymphocytes in the spleen following fusgem treatment relative to these sham and gem groups combination therapy elevated splenic cd8 t lymphocytes by fold and sheybani a0nd et a0al j immunother cancer 20208e001008 101136jitc2020001008 0copen access figure combination of focused ultrasound fus partial thermal ablation with gemcitabine gem constrains primary triple negative breast cancer outgrowth and extends overall survival a overview of experimental design for evaluation combination of fus with serial gem treatment in murine mammary carcinoma b average 4t1 tumor outgrowth in sham n7 fus monotherapy n5 gem monotherapy n10 and combinatorial fusgem therapy groups n10 data are represented up to select time points corresponding with mouse dropout due to humane endpoints all data represented as mean±sem significance assessed on outgrowth up to day by repeated measures mixed effects model implementing restricted maximum likelihood method followed by tukey multiple comparison correction p005 vs all other groups specifically sham vs fusgem p00001 fus vs fusgem p00001 shamgem vs fusgem p00026 c 4t1 tumor outgrowth from individual mice in sham fus shamgem or fusgem groups data represent outgrowth from initiation of treatments at day up to removal of mouse from study for meeting a humane endpoint d representative images of 4t1 tumors excised at day scale bar1 cm e quantification of 2d tumor areas from images in previous panel f kaplan meier curve depicting overall survival of sham treatment n9 fus monotherapy n6 gem monotherapy n10 and combinatorial fusgem therapy n10 recipient mice significance assessed by log rank mantel cox test p005 vs all other groups specifically sham vs fus p02154 sham vs fusgem p00001 sham vs shamgem p00050 fus vs fusgem p00021 fus vs shamgem p00312 fusgem vs shamgem p00041sheybani a0nd et a0al j immunother cancer 20208e001008 101136jitc2020001008 0copen accessfigure combination of focused ultrasound fus partial thermal ablation with gemcitabine gem increases the levels of circulating t cells a overview of experimental design to understand the impact of fus andor gem treatment on circulating immune cells bc absolute number of circulating cd8 t cells at day b and day c d percentage of circulating cd8 t cells expressing cd44 from days to ef absolute number of circulating cd4 t cells at day e and day f g percentage of circulating cd4 t cells expressing cd44 from days to h percentage of cd11b myeloid cells out of total cd45 immune cell in circulation from days to ik percentage of myeloid cells i cd8 t cells j and cd4 t cells k out of total cd452 immune cells all data represented as mean±sem all data representative of sham n6 fus monotherapy n4 gem monotherapy n9 and combinatorial fusgem therapy n6 groups significance assessed by analysis of variance followed by tukey multiple comparison correction for b c e f or fishers least significant difference lsd without multiple comparisons correction for ik significance for d g and h assessed by repeated measures mixed effects model implementing restricted maximum likelihood method followed by fishers lsd without multiple comparisons correction p005 vs all other groups unless otherwise indicated p001 p0001 vs groups indicatedsheybani a0nd et a0al j immunother cancer 20208e001008 101136jitc2020001008 0copen access fold figure 3j and cd4 t lymphocytes by fold and fold figure 3k these elevations were accompanied by a modest increase in percentage of foxp3 regulatory t cells tregs online supplementary figure 6e additionally increases in percentage of nk and b cells were noted twofold to fivefold online supplementary figure 6cd these findings indicate that combinatorial therapy with fusgem promotes a systemic lymphocyte response that is discrete from the effects of either intervention alone which may account for reduced mortality associated with pulmonary metastasescombinatorial fusgem therapy does not promote robust local antitumor t cell responsesgiven the robust systemic immune signatures within the blood and spleen following fusgem we assayed 4t1 tumors at a time point within the window of tumor growth restriction and subsequent to termination of treatments ie day to interrogate whether tumor control correlates with an increase in the effector functions of the intratumoral t cell response figure 4a approximately hours prior to euthanasia mice received intravenous brefeldin a injection to inhibit cytokine secretion for subsequent intracellular cytokine staining by flow cytometry immune characterization of tumors at days postimplantationthat is days subsequent to final gem administrationrevealed no significant changes in absolute number of antigen experienced cd44 cd8 or cd4 t lymphocytes figure 4bc moreover the polyfunctionality of these t cells as denoted by ifnÎ and granzyme b expression was not significantly altered figure 4de however intratumoral functional changes were noted in the myeloid compartment gem monotherapy modestly increased il 12p40 production by dcs fold but this was not conserved in the combinatorial therapy group figure 4f moreover while fus monotherapy generated a trend in elevated tnfα production by intratumoral g mdscs gem recipient groups saw a significant increase threefold relative to sham figure 4g these findings indicate that changes in the myeloid compartment in response to monotherapy and combination therapy may contribute to tumor control but are unlikely to drive the protective response entirely interestingly intratumoral t cell representation correlates poorly with circulating lymphocytes suggesting a transitory immune response that either cannot be fully characterized at this time point or is hampered by additional modes of immunosuppressionprotection conferred by combination of fus and gem is dependent on adaptive immunitysince our findings revealed no obvious advantage or function of adaptive immunity in the local tme we next investigated the overarching role of the adaptive immune system in protection offered by combinatorial therapy with fusgem to this end we utilized an rag1 model that is deficient in t and b cells to address the hypothesis that mature t andor b cells play a role in the observed response wild type wt or rag1 mice bearing 4t1 tumors were randomized into groups in a manner that preserved similarity in average initial tumor volumes mice were subsequently treated with either gem monotherapy or the combination of fusgem the tumor growth inhibition offered by fusgem was entirely lost in rag1 mice relative to their wt counterparts with average 4t1 tumor volume in rag1 mice being over fivefold higher than that of wt mice on termination of treatments figure 5a of note despite a trend toward loss of protection in rag1 mice tumor outgrowth in response to gem monotherapy did not significantly stratify between wt and rag1 settings figure 5a we also observed a complete loss of fusgem mediated survival benefit over gem monotherapy in the rag1 setting figure 5b while these results demonstrate that an intact adaptive immune response is required for both the overall survival benefit and restriction of primary tumor offered by fusgem therapy they do not delineate the relative roles of t andor b cellsthus to address the hypothesis that the protective effect of fusgem is specifically dependent on cd4 and cd8 t cells we depleted these populations via serial coinjections of cd8 depleting and cd4 depleting antibodies in 4t1 tumor bearing wt mice on a fusgem figure 5c depletions were maintained between day and day and flow cytometry analysis of circulating immune cells at day confirmed that the target t cell populations were effectively depleted in all mice online supplementary figure consistent with the tumor escape observ | 0 |
Construction of a novel prognosticpredicting modelcorrelated to ovarian cancerWeichun Tang12 Jie Li3 Xinxia Chang12 Lizhou Jia1 Qi Tang12 Ying Wang4 Yanli Zheng4 Lizhou Sun5 andZhenqing Feng121National Health Commission Key Laboratory of Antibody Technique Nanjing Medical University Nanjing Peoples Republic of China 2Department of Pathology Nanjing MedicalUniversity Nanjing Peoples Republic of China 3Department of Nursing The Second Afï¬liated Hospital of Nantong University Nantong Peoples Republic of China 4Department ofGynaecology and Obstetrics The Second Afï¬liated Hospital of Nantong University Nantong Peoples Republic of China 5Department of Obstetrics and Gynecology First Afï¬liatedHospital of Nanjing Medical University Nanjing Peoples Republic of ChinaCorrespondence Zhenqing Feng fengzhenqingnjmueducn or Lizhou Sun lizhou sun163comBackground Ovarian cancer OC is one of the most lethal gynecological cancers worldwide The pathogenesis of the disease and outcomes prediction of OC patients remainlargely unclear The present study aimed to explore the key genes and biological pathwaysin ovarian carcinoma development as well as construct a prognostic model to predict patients overall survival OSResults We identified upregulated and downregulated differentially expressedgenes DEGs associated with OC Gene Ontology GO term enrichment showed DEGsmainly correlated with spindle microtubes For Kyoto Encyclopedia of Genes and GenomesKEGG pathways cell cycle was mostly enriched for the DEGs The proteinprotein interaction PPI network yielded nodes and edges Top three modules and ten hub geneswere further filtered and analyzed Three candidiate drugs targeting for therapy were alsoselected Thirteen OSrelated genes were selected and an eightmRNA model was presentto stratify patients into high and lowrisk groups with significantly different survivalConclusions The identified DEGs and biological pathways may provide new perspective onthe pathogenesis and treatments of OC The identified eightmRNA signature has significantclinical implication for outcome prediction and tailored therapy guidance for OC patientsBackgroundOvarian cancer OC is the most lethal malignant disease in the female reproductive system with over new cases and deaths each year worldwide [] Epithelial OC accounts for ofovarian malignancies listed as the most common histological type Since the ovaries locate in the deeppelvis with mere symptoms emerging at the beginning of ovarian morbid change the early detectionfor the malignancy is truly difficult Hence when OC is detected the patient is usually at an advancedstage with invasion and metastasis accompanied [] For patients in the early stage the 5year survivalrate can reach whereas for advancedstage patients the number is mere ¼ [] Thereforeit is imperative to explore the molecular mechanisms of malignant biological behavior of OC cells andto develop more reliable molecular markers for predicting recurrence and evaluating prognosis furtherguiding clinicians for therapyAt present various highthroughput microarrays and nextgeneration sequence genomic datasetswhich were deposited in the Gene Expression Omnibus GEO [] and The Cancer Genome Atlas TCGAdatabases have been widely analyzed for identifying differentially expressed genes DEGs which couldserve as candidate diagnostic or prognostic markers further effectively improving our understanding ofthe disease from genetic perspective Whereas since the existence of tissue or sample heterogeneity inThese authors contributedequally to this workReceived April Revised July Accepted July Accepted Manuscript online July Version of Record published August The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20201261101042BSR20201261each independent experiment as well as the discrepancy of different data processing methods and technology platforms the DEGs identified from a singlecohort study may generate false positives Herein the Robust Rank Aggregation RRA method which analyzes the significant probability of all elements by a probabilistic model is developedto identify statistically significant genes from multiple datasets and provide more accurate and valuable informationfor clinical use far beyond one gene list [] To date a bunch of novel prognostic markers have been discovered topotentially improve the efficacy of diagnosis and prognosis of OC [] However these identified markers were onlyeffective for partial stages or grades and were difficult to apply widely Hence a prognostic model which is basedon signature gene expression level with high discriminating power to effectively assist prognosis prediction for eachpatient is required in clinical practiceIn the present study we downloaded six primary microarray datasets from the GEO database which containeda total of samples with OC samples and normal samples The geneset and relative clinical information on ovary tissues of OC patients and healthy females from TCGA and GTEx portal were also downloadedIntegrated DEGs between cancerous and normal ovarian samples were screened using the limma R package andRRA method Gene Ontology GO and Kyoto Encyclopedia of Genes and Genomes KEGG pathways enrichmentof DEGs were performed for nextstep functional analysis The Search Tool for the Retrieval of Interacting GenesDatabase STRING and the Connectivity Map CMap online database were then used to analyze the association ofDEGs and explore the molecular mechanisms as well as drugs involved in tumorigenesis Through survival analysisprognostic mRNAs were also selected By performing Cox regression analysis we identified an eightmRNA signature model with the ability to predict the prognosis of OC patients and independent from clinical factors Our studyprovides reliable molecular markers and prognostic models for early detection and outcome prediction as well aseffective drug targets for treating OCMethodsData collectionThrough searching on the GEO Repository with ovarian cancer we downloaded the gene expression profiles ofGSE54388 GSE40595 GSE38666 GSE27651 GSE18520 and GSE14407 and the corresponding annotation files fromthe GPL570 [HGU133 Plus ] Affymetrix Human GenomeU133 Plus Array platform GSE54388 contains ovarian tissue samples with normal ovarian surface epithelium and tumor epithelial components from highgradeserous OC patients GSE40595 includes OCassociated stroma and epithelium samples which consist of cancerassociated stroma samples and epithelial tissues from highgrade serous OC patients along with stromal component and ovarian epitheliums from the normal ovary GSE38666 comprises stroma and matchedovarian epitheliums from healthy females as well as cancer stroma and matched cancer epitheliums from OCpatients GSE27651 incorporates normal ovarian surfaces epithelial and serous borderline ovarian tumors lowgrade serous ovarian carcinomas and highgrade serous ovarian carcinomas GSE18520 covers advancedstage highgrade primary OC specimens and normal ovarian surface epithelium tissues GSE14407 involves healthy ovarian surface epithelial samples and paired serous OC epithelium Note that all samples from these GEOdatasets are classified into the cancerous or normal part to be clear the normal stromal and surface epithelium is defined as normal ovarian tissues whereas the borderline tumors as well as cancerous stromal and epithelial tissues areconsidered as malignancies Besides we also downloaded the FPKM format gene expression data and relative clinicalinformation of OC patients samples and normal ovarian tissues from TCGA and GTEx portal respectivelyScreening for DEGs and integration of microarray dataWe used the limma R package [] to integrate the expression profiles from TCGA and GTEx portal standardize the data from the integrated TCGA and GTEx expression matrix as well as six GEO datasets and furtherscreen the DEGs between ovarian cancerous and normal samples The list of DEGs obtained from six GEO microarray datasets by limma analysis was further integrated by the RRA method to get prioritized commonly upor downregulated gene list The final overlapped DEGs for subsequent biological function analysis were the combination of prioritized jointly dysregulated genes from six GEO microarrays and the results from TCGA and GTExdatabases The cutoff criteria were set as FDR and log2fold change FC GO term and KEGG pathway enrichment analysisGO classified the known genes into three main biological progress Molecular Function MF Cellular ComponentCC and Biological Process BP [] KEGG provides researchers highlayer functions of the biological system frommolecular level information [] The Enrichr online tool amppharmmssmeduEnrichr allows for GO term The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20201261101042BSR20201261Figure The expression heatmap of the top signiï¬cantly dysregulated genes in six GEO datasetsHierarchical clustering that shows the expression profiles of mRNAs from A GSE14407 B GSE18520 C GSE27651 DGSE38666 E GSE40595 F GSE54388annotation and KEGG pathway for a cluster of genes [] We explored the biological functions of overlappedDEGs and hub modules from our proteinprotein interaction PPI network using Enrichr website Pvalue was considered as significant enrichment Likewise the functional biological pathways of the top ten hub genes fromPPI network were also analyzed by the FunRich tool version [] and the top five enriched pathways of up anddownregulated genes were displayed as bar charts respectively We set the Pvalue as statistically significant The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20201261101042BSR20201261PPI network construction and analysisPPI networks display the relationships of various proteins according to their physical or biochemical propertiesSTRING is a database that encompasses the interaction information between known proteins and potentially interacted proteins [] In order to explore the correlations between the DEGs we used the STRING database to constructa PPI network and visualize our results by Cytoscape software [] Confidence score was set as significantMolecular Complex Detection MCODE was utilized to select hub modules of PPI networks in Cytoscape [] Weset the degree cutoff node score cutoff kscore and max Depth was set as the criterion Thenthe significant modules were performed by GO and KEGG analyses Top ten genes were defined according to thehigh degree of connectivity in STRING network [] The coexpression analysis of ten hub genes was performed bySTRING eitherValidation of the hub genesWe downloaded the raw geneset of OC patients from TCGA to explore the expression differences of hub genes inlow and highgrade tumor tissues of OC and draw the survival plot using KaplanMeier plotter webtool [] Thegene and protein expression level of graderelated hub genes were then confirmed by Oncomine and The HumanProtein Atlas HPA database [] Meanwhile we explored the genetic alteration information of the selected tenhub genes in OC patients by plugin cBioPortal cBio Cancer Genomics Portal tool which deposits the genomicsprofiles of various cancer types and provides analysis and visualization of the genomics datasets []Identiï¬cation of candidate small molecule drugsThe CMap database was able to predict potential drugs which might reverse or induce the biological state encoded incertain gene expression signatures in OC [] The DEGs from our study were used to query the CMap databaseThe enrichment scores which represent the similarity were calculated ranging from to The positive connectivity score means an inducing influence on the input signature whereas drugs with negative connectivity score presentreversion impact on the characteristic in human cell lines and are considered as candidate therapeutic molecules After sorting all instances the connectivity score of various instances was filtered by Pvalue Next we investigatethe structures of these candidate molecular drugs from the Pubchem database pubchemncbinlmnihgovEstablishment and evaluation of the prognostic modelThe OC patients from the TCGA project were randomly classified into the training cohort n188 and the testingcohort n186 OSrelated genes were determined by performing univariate Cox regression analysis in the trainingcohort with the Survival R package and further selected for the nextstep screening Least Absolute Shrinkage andSelection Operator LASSO is a parameter selection algorithm which shrinks all highdimensional regression coefficients and generates the penalty regularization parameter λ via the crossvalidation routine by glmnet R packageTo select the optimal prognostic mRNAs we adopted LASSO regression among the selected candidate genes and further perform multivariate Cox proportional hazards regression to evaluate their independent prognostic values Theriskscore model for predicting outcomes of OC patients was the sum of each optimal prognostic mRNA expressionlevel multiplying relative regression coefficient weight calculated from the multivariate Cox regression modelRisk score patient cid2Coefficient mRNAi à Expression mRNAiiAll training cohort patients were classified into high and lowrisk groups according to the median risk score TheKaplanMeier curves of two diverse groups were plotted using survfit function and the receiver operating characteristic ROC curve was unfolded for OS prediction to estimate the sensitivity and specificity of the prognostic modelCox multivariate analysis was also performed to examine whether the risk score was independent of the clinical characters such as age tumor stage and grade Next we used the testing group to check the efficacy of the prognostic riskmodel Each individual in the testing cohort was also categorized as high or lowrisk case by comparing the patientsrisk score with the cutoff value calculated from the training cohort KaplanMeier curve analysis timedependentROC analysis and Cox multivariate analysis were performed eitherSearching tumorinï¬ltrating immune cells associated with patientsprognostic signaturesThe TIMER webtool allows for systematical evaluations of the relationship between the six immune cell types inthe tumor microenvironment which are B cell CD4 T cell CD8 T cell neutrophil macrophage as well as dendritic The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20201261101042BSR20201261Figure The volcano plot of all gene expression distribution in six GEO datasetsVolcano plot of differentially expressed mRNAs of A GSE14407 B GSE18520 C GSE27651 D GSE38666 E GSE40595 FGSE54388cell and clinical impact in various cancer types via a novel statistical method [] To further explore the prognosticsignature we used the TIMER online tool to search the most significant tumorinfiltrating immune cells according tothe TCGA OC gene data To be clear the relative gene expression levels of six types of immune cells for each patientin high and lowrisk groups from training and testing cohort were measuredResultsThe DEGs among six GEO microarray datasetsThe top significantly up and downregulated genes from each microarray dataset were displayed in the heatmapsFigure 1AF and the distribution of all gene expression was presented in volcano plots Figure 2AF ThroughRRA analysis of expression microarrays we identified DEGs which consisted of upregulated and downregulated genes and displayed the top dysregulated genes by pheatmap R package in Figure Next weanalyzed the expression profiles of TCGA and GTEx getting dysregulated genes Intriguingly when these DEGs were combined with the DEGs from GEO datasets we found that genes were commonly dysregulatedin these two databases with upregulated Figure 4A and downregulated genes Figure 4BGO term and KEGG pathway enrichment analysis of DEGsTo study the potential biological function of the DEGs we performed biological pathway analysis and identifiedsignificantly enriched pathways via Enrichr web tools In GO term Figure 5A for the BP group the DEGs weremostly enriched in regulation of attachment of spindle microtubes to kinetochore cellular response to laminar fluidshear stress and microtubule cytoskeleton anization involved in mitosis In MF group the dysregulated geneswere highly correlated to microtubulebinding microtubule motor activity and tubulinbinding As for CC group The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20201261101042BSR20201261Figure Heatmap showing the top upregulated genes and top downregulated genes according to PvalueEach row represents one gene and each column indicates one dataset Red indicates upregulation and blue represents downregulation The numbers in the heatmap indicate log FC in each dataset calculated by the limma R package Abbreviation log FClogarithmic fold changethe DEGs were closely related to condensed nuclear chromosome kinetochore and mitotic spindle KEGG pathwayanalysis showed DEGs highly enriched in cell cycle and Alanine aspartate and glutamate metabolism Figure5B The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20201261101042BSR20201261Figure The intersection of up and downregulated DEGs of GEO and TCGA datasetsA upregulated intersected DEGs in both datasets B downregulated intersected DEGs in both GEO and TCGA dataset Theintersected DEGs were defined as the significant DEGsPPI network construction and modules analysisUsing the STRING database and Cytoscape software a total of DEGs were mapped into the PPI network whichincluded nodes and edges Figure 6A The PPI enrichment Pvalue was 10e16 The top three key modules Figure 6CE within PPI network were then selected Module MCODE score Module MCODEscore Module MCODE score and the biological function of Module which consisted of nodesand edges was further analyzed GO analysis indicated that Module1 was mainly associated with regulation ofattachment of spindle microtubules to kinetochore condensed nuclear chromosome kinetochore and microtubulemotor activity KEGG analysis showed that cell cycle and oocyte meiosis were the most highly enriched pathwaysSupplementary Figure S1The screening of Hub genes and their characteristicsThe top ten hub genes with the highest degree of connectivity were CDC45 CDK1 TOP2A CDC20 CCNB1CEP55 UBE2C HMMR FOXM1 and TPX2 Figure 6B The coexpression analysis results of the hub genes demonstrated that these genes actively interacted with each other Supplementary Figure S2 Besides we established theinteraction network of ten hub genes with their related genes and explored the biological role Supplementary Figure S2ACF of the network by FunRich The gene alteration type and frequency as well as the most frequentlyaltered neighbor genes were also exhibited Figure Gene alteration frequency of ten hub genes among TCGAOC samples was beyond with most genes showed amplified and multiple altered Figure 7AB The top threemost frequently altered genes were FOXM1 CDC20 and CCNB1 with FOXM1 and CDC20 largely amplified whileCCNB1 deep deleted Through analysis of OC patients geneset from TCGA we found that CCNB1 UBE2C CDK1CEP55 as well as FOXM1 expressed significantly higher in highgrade tumors and predicted worse outcomes sincepatients overexpressed above genes owned lower overall survival OS and diseasefree survival DFS rates Figure The Oncomine database showed results from various studies were consistent to our finding Supplementary Figure S3 The HPA website also demonstrate that proteins translated by such five hub genes were overexpressed in OCtissues Supplementary Figure S4 HMMR and TPX2 were also negatively correlated to patients prognosis while noexpression difference was observed in diverse tumor grades and CDC20 was positively associated with tumor gradebut not correlated to patients outcomesRelated small molecule drugs screeningIn total DEGs were analyzed in CMap to screen small molecule drugs and the candidate molecules with top tenconnectivity score are listed in Table Five of these molecules showed a negative correlation and suggested potentialin clinical applications Among them Trichostatin A pyrvinium and 8azaguanine showed a significantly negativecorrelation and the stuctures of such candidate molecule drugs was found in the PubChem database and shown inSupplementary Figure S5 The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20201261101042BSR20201261Figure GO and KEGG functional annotation for the signiï¬cant DEGsA The top ten enriched BP of the DEGs B The top ten enriched CC of the DEGs C The top ten enriched MF of the DEGs DThe top ten enriched KEGG pathways of the DEGs The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20201261101042BSR20201261Figure The PPI network and top hub genes as well as top three modules were constructedA The PPI network of the DEGs B The hub genes of the DEGs CE Top three hub modules were identified by Cytoscapeplugin MCODE C module1 D module2 E module3Table The top ten OCrelated small molecules with highly signiï¬cant correlations in results of CMap analysisRankCMap nametrichostatin A8azaguaninepyrviniumisoï¬upredonequinpirolevorinostatgenisteinantimycin AheptaminolmidodrineMeanNEnrichmentP value The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20201261101042BSR20201261Figure The gene mutation overview of ten hub genes in TCGA OC patientsA Ten hub genes are altered in of queried patients B The summary of mutation type of ten hub genes in OC patientsC The network of hub genes and the most frequently altered neighbor genesTable Univariate cox regression identiï¬ed DEGs correlated to patients OSGene IDCCND1SYNE4CCDC80TMC4MCCFOXQ1KRTCAP3CXCR4IL4I1DEFB1CSGALNACT1KLHL14MCUR1HRAbbreviation HR hazard ratioHR95LHR95HPvalueConstruction of prognostic model and evaluation of its predictive abilityUnivariate Cox regression analysis revealed that of DEGs were significantly correlated to patients OS in thetraining cohort Table The OSrelated genes were listed as follows CCND1 SYNE4 CCDC80 TMC4 MCC The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20201261101042BSR20201261Figure The clinical characteristics of CCNB1 CEP55 CDK1 FOXM1 and UBE2C in OC patientsA Five genes were overexpressed in high grade G1 and G2 compared with low grade G3 and G4 in OC BC The OS time Band DFS time C of five genes in OC patients The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20201261101042BSR20201261Figure Identiï¬cation of prognosisrelated mRNAs using LASSO regression modelA LASSO coefficient profiles of the mRNAs associated with the OS of OC B Plots of the crossvalidation error rates Each dotrepresents a λ value along with error bars to give a confidence interval for the crossvalidated error rateTable Multivariate Cox regression selected eight DEGs correlated to patients OSGene IDTMC4KLHL14CXCR4CCDC80KRTCAP3DEFB1SYNE4FOXQ1HRHR95LHR95HPvalue845E08Abbreviation HR hazard ratioFOXQ1 KRTCAP3 CXCR4 IL4I1 DEFB1 CSGALNACT1 KLHL14 and MCUR1 Through LASSO Cox regression we narrowed the number of prognosisassociated genes to according to the minimum criteria Figure Next based on the multivariate Cox model of candidate mRNAs retained their prognostic significance and werefinally selected as independent remarkable prognostic factors which were TMC4 KLHL14 CXCR4 CCDC80 KRTCAP3 DEFB1 SYNE4 and FOXQ1 Table To predict patients outcomes we developed an individuals risk scoremodel as follows risk score à expression value of TMC4 à expression value of KLHL14 à expression value of CXCR4 à expression value of CCDC80 à expression value of KRTCAP3 à expression value of DEFB1 à expression value of SYNE4 à expression value of FOXQ1 On the basis of the median risk score patients were divided into high orlowrisk groups KaplanMeier curve analysis showed that the OS time of the lowrisk group was significantly longerthan the highrisk group P1147e07 Figure 10E ROC curve analysis revealed the area under the ROC curveAUC of the prognostic model was Figure 10D Meanwhile the risk scores Figure 10A of OC patients inthe training group were ranked for displaying their distribution and the survival status Figure 10B was marked onthe dot plot The expression pattern of eight prognostic mRNAs between high and lowrisk groups was also shown inthe heatmap Figure 10C Univariate and multivariate Cox regression analyses concerning the risk score and clinicalfactors showed that the prognostic model was able to serve as an independent prognostic indicator Figure 11ABROC curve analysis also showed that the AUC value of the model was much significantly higher than the tumor stage AUC grade AUC and patients age AUC Figure 11C Interestingly when The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20201261101042BSR20201261Figure Prognostic analysis of the TCGA training modelA The risk score B survival status C expression heatmap D timedependent ROC curves and E KaplanMeier survival ofthe prognostic model for the TCGA OC training cohort The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20201261101042BSR20201261Figure The clinical independency of the risk model in training cohortUnivariate A and multivariate B regression analyses as well as timedependent ROC curve analysis CD of the prognostic valuebetween the training model and OC patients OS status when compared with or combined with clinical factorscombined the risk score with clinical factors the ROC curve of combination model was much higher than each aloneFigure 11DAs for the testing cohort we divided the group into highrisk and lowrisk individuals based on the trainingcohort cutoff risk score The outcomes of low and highrisk groups patients of the testing cohort were also measuredand the OS time of the highrisk group was significantly shorter than the lowerrisk group P1721e02 Figure12E The AUC of the prognostic model was Figure 12D The risk scores distribution Figure 12A and survivalstatus Figure 12B of OC patients as well as the eightprognostic gene expression heatmap Figure 12C in the testinggroup were also present Meanwhile the independency of the prognostic model was confirmed in testing cohort sinceunivariate and multivariate Cox regression analyses showed the model correctly predicted high or lowrisk factroups patients outcomes without relying on any clinical factors Figure 13AB ROC curve analysis showed that theprognostic model exhibited better sensitivity and specificity when compared with tumor stage grade and patientsage for the AUC value of the model was much higher than later Figure 13C In accordance with results from trainingcohort the combination of risk score and clinical factors showed better OS prediction capability Figure 13DThe prognostic signature correlating to immune cells inï¬ltrationThrough TIMER webtool we analyzed the relative gene expression levels of six types of immune cells for each patientand found that genes concerning macrophage fraction were expressing significantly higher in the highrisk groupP005 compared with the lowrisk group in training cohort Figure Interestingly same result was also observed in the testing cohort Figure The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20201261101042BSR20201261Figure Prognostic analysis of the TCGA testing modelA The risk score B survival status C expression heatmap D timedependent ROC curves and E KaplanMeier survival ofthe prognostic model for the TCGA OC testing cohort The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20201261101042BSR20201261Figure The clinical independency of the prognostic risk signature in testing cohortUnivariate A and multivariate B regression analyses as well as timedependent ROC curve analysis CD of the prognostic valuebetween the testing model and OC patients OS status when compared with or combined to clinical factorsDiscussionIn the present study we used the RRA methods to jointly analyze six GEO OC microarrays which contained OC and normal samples identifying DEGs and overlapped them with dysregulated genes of OC cohort fromTCGA and GTEx portal finally getting upregulated and downregulated genes Functional analysis showedthat DEGs were significantly enriched in the cell division cycle to be clear in the process of the mitotic spindleSpindle microtubules have been proved to play crucial role in physiological and pathological processes As for celldivision only when all chromosomes linked to spindle microtubules through kinetochores and the spindle assemblycheckpoint is satisfied this process could step to anaphase [] Suraokar et al found that the mitotic spindle assemblycheckpoint and microtubule network were significantly altered in malignant pleural mesothelioma MPM whileusing epothiloneB a nontaxane small molecule inhibitor targeting the microtubules could greatly decrease theviability of MPM cell lines [] Rogalska et al compared the antiproliferative capacity of epothilone B with paclitaxelon OC cell line SKOV3 found that this effect of Epo B was greater than latter [] The researches above wereconsistent with our study that the mitotic spindle process was dysregulated in OC progression playing importantroles in OC cell proliferation and tumor developmentPPI network construction of DEGs included nodes and edges among which we identified three keymodules Interestingly the top1 module was also highly associated with spindle microtubules and chromosome kinetochore confirming the role of cell cycle in OC pathogenesis The top ten hub genes from the PPI network were alsorecognized which were CDC45 CDK1 TOP2A CDC20 CCNB1 CEP55 UBE2C HMMR FOXM1 and TPX2 The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20201261101042BSR20201261Figure The expression level of immune cells related genes in high and lowrisk groups of the training cohortA Bcell fraction B dendritic fraction C CD4 Tcell fraction D CD8 Tcell fract | 2 |
"dysregulation of bcl2 is a pathophysiology observed in haematological malignancies forimplementation of available treatmentoptions it is preferred to know the relative quantificationof bcl2 mrna with appropriate reference genes for the choice of reference genesi reference genes were selected by assessing variation of genes from rnaseq datasets of haematological malignancies followed by filtering based on their go biological processannotations and proximity of their chromosomal locations to known disease translocationsselected genes were experimentally validated across various haematological malignancy samples followed by stability comparison using genorm normfinder bestkeeper and reffinderii commonly used reference genes were obtained from literature through extensive systematic review levels of bcl2 mrna was assessed by qpcr normalized either by novel reference genes from this study or gapdh the most cited reference gene in literature andcompared the analysis showed ptcd2 ppp1r3b and fbxw9 to be the most unregulatedgenes across lymphnodes bone marrow and pbmc samples unlike the reference genesused in literature bcl2 mrna level shows a consistent higher expression in haematologicalmalignancy patients when normalized by these novel reference genes as opposed togapdh the most cited reference gene these reference genes should also be applicable inqpcr platforms using taqman probes and other model systems including cell lines and rodentmodels absence of sample from healthynormal individual in diagnostic cases call for carefulselection of reference genes for relative quantification of a biomarker by qpcrbcl2 can beused as molecular diagnostics only if normalized with a set of reference genes with stable yetlow levels of expression across different types of haematological malignanciesintroductionoverexpression of bcl2 bcell lymphoma a mitochondrial membrane protein has beenobserved in several haematological malignancies due to genetic and epigenetic mechanismsa1111111111a1111111111a1111111111a1111111111a1111111111open accesscitation dwivedi n mondal s p k s t ssachdeva k bathula c relativequantification of bcl2 mrna for diagnostic usageneeds stable uncontrolled genes as reference one e0236338 101371 pone0236338editor pedro v baptista universidade nova delisboa portugalreceived may accepted july published august peer review history recognizes thebenefits of transparency in the peer reviewprocess therefore we enable the publication ofall of the content of peer review and authorresponses alongside final published s theeditorial history of this is available here101371 pone0236338copyright dwivedi this is an openaccess distributed under the terms of thecreative commons attribution license whichpermits unrestricted use distribution andreproduction in any medium provided the originalauthor and source are crediteddata availability statement all relevant data arewithin the manuscript and its supportinginformation files one 101371 pone0236338 august one 0cfunding the study is funded by glue grantscheme number btpr23078med2912532017by department of biotechnology httpdbtindiagovin govt of india awarded to sd and md thefunders had no role in study design data collectionand analysis decision to publish or preparation ofthe manuscriptcompeting interests the authors have declaredthat no competing interests existbcl2 molecular diagnostics with novel reference genesresulting in evasion of apoptosis giving the malignant cells a longer life span and survival benefits at times of nutrient deficiency hypoxia and growth factor deprivation [] estimationof level of bcl2 along with other antiapoptotic genes are essential to avail efficient treatmentoptions by rchop regimen of cyclophosphamide doxorubicin vincristine and prednisone and rituximab or venetoclax in different haematological malignancies [ ] byvisualization of chromosomal aberrations using karyotyping or fish fluorescence insituhybridization bcl2 levels can be inferred indirectly detection of expression of bcl2protein by immunohistochemistry a standard pathological testing procedure for dlbcl hasnot been adopted in the clinics for bone marrow tissues of liquid cancers due to sample inconsistency and challenging procedure of capturing low concentrations of biomarkers western blotting for the very nature of the method cannot be adopted for high throughputpathological testing elisa for detection of bcl2 in human plasma remains limited sinceonly one splice isoform of the mitochondrial membrane protein is available in soluble formthus bringing down the effectiveness of the assay bcl2 at the mrna level can be determined without ambiguity by next generation sequencing nanostring and microarray though increasing time and expense of pathological testing in clinical trials relative quantification by qpcr quantitative polymerase chain reactioncan be successfully used due tothe availability of appropriate controls in untreated or normal groups [ ] although beingtime and costeffective it suffers misinterpretation in pathological setting since the relativequantification depends only on the rg reference gene used due to the absence of normalsamplesnormalization with a rg which shows varying expression across samples can often lead towrong s as seen with the use of glyceraldehyde3phosphate dehydrogenasegapdh as rg in gene expression studies of pulmonary tuberculosis and cd8 tcellsunder inactivated or activated condition similarly abl protooncogene abl1 therecommended rg for gene expression studies with leukemic patients was found to haveextremely low expression in neutrophils making it unsuitable as rg for the specific casesuch discrepancies have prompted researchers to analyze gene expression across multiple tissues or pancancer database like tcga to propose normalization factors using multiple rg candidatesthis study through a systematic review of literature in haematological malignancies concluded that mostly conventionally used housekeeping genes are still being deployed s1table and s1 fig despite their varied expression based on cell type developmental stage andexperimental conditions with rare exceptions [ ] none of the genes thus identified could be used to relatively quantify bcl2 as molecular diagnostics since compared to thefpkm fragments per kilobase of transcript per million mapped reads value of the antiapoptotic genes across databases s2 fig most of the rgs from the literature are not onlyhigher but also varied significantly s3 and s4 figs with few exceptions inspired by genomewide search for rgs from publicly available rnaseq or microarray data in human and otheranisms [] we report here a set of novel candidate rgs obtained from an unbiasedsearch of genes in haematological malignancies to be used to normalize bcl2 andother antiapoptotic genes in qpcr as molecular diagnosticsmaterials and methodsethics statementthe study was performed in compliance with ethical practices and was approved by narayanahealth academics ethics committee narayana health hospitals ethics approval numbernhhaeccl2017152a one 101371 pone0236338 august one 0cbcl2 molecular diagnostics with novel reference genessystematic review of commonly used rgsliterature search was carried out in pubmed databasepubmed as detailed in s5 figaccording to prisma preferred reporting items for systematic reviews and metaanalysesguidelines selection of stable genes proteincoding genes identified from publicly available datasets table using ensembldb annotation package within r statistical software were categorised into four quartiles based on their median expression values across all samples geneswith median expression in middle two quartiles q2 and q3 in all datasets were consideredas q1 and q4 representing extreme ends of the expression spectrum are not preferred as rgcandidates for normalization of molecular diagnostic markersto determine the stability of a gene following statistical measures were employedi cv �xsx where �x and Ïx are mean and standard deviation of a variable x respectively and ii normality pvalue as measured by shapirowilks test where a pvalue less than signifies thatthe distribution is away from normal cv although used most frequently isnt a robustmeasure as it is affected by outliers to solve this a third parameter was used mad medianabsolute deviation medianjx 00 xj where x is the median of x after normalization withmedian mad is a better measure for understanding the spread of the distribution as itdepends on medians a parameter less prone to deviations by outlierslow or comparable statistical variation across samples represented by low values of cvand mad and a normal distribution high value of normality pvalue or low values of pvalue are characteristics of an ideal rg therefore genes with median expression values inmiddle quartiles q2 and q3 were shortlisted and clustered based on their cv mad and pvalue normalized to their respective zscores using pam partitioning around medsalgorithm required optimal number of clusters was calculated using silhouette graphicalmethod for each tissue sample the gene cluster with the lowest med value of parameters was selected and the genes at the intersection of the four clusters were shortlisted the list was further filtered by analysing and eliminating genes based on stop words in theirgo gene ontology annotation such as transcription factors nuclear receptor or other nuclearlocalization dna binding activity response to external stimuli translational and transcriptionalactivation since genes with such characteristics regulated by environmental conditions areunsuitable as rg candidates next genes were ranked in ascending order of their mean euclidqffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffifficv þ mad2 þ ð1 00 pþ2ean distance d ¼all parameters replaced by their zscores in thisthreeparameter hyperspace for each dataset average of d across four datasets was taken to calculate the mean euclidean distance �d genes with �d median were selected for furthertable list of rnaseq databasesdatasetdiseasetcgalamlamltargetaml paediatric amlgdcdlbcdlbclmmrfmmmultiplemyeloma� both primary and recurrent tumor only 1st visit recordstissuebloodbonemarrowlymphnodesbonemarrowsamples n sourcedownload location� tcga research networkwwwcancergovtcgaschmitz multiple myeloma researchfoundationgdccancergovaboutdatapublicationsdlbclresearchthemmrf fpkm data for gdcdlbc dataset was available as log2 transformed normalized value which was converted to fpkm101371 pone0236338t001 one 101371 pone0236338 august one 0cbcl2 molecular diagnostics with novel reference genesanalysis locus of genes associated with pathogenic translocations were identified [ ] andcandidate rgs in close proximity of such loci within bands in the same arm of chromosomewere eliminated by an automated method further only genes with nonzero fpkm value in allsamples from four datasets were retained then each gene was given a composite quartile ranking cqr the sum of quartile indices from each dataset and genes with cqr value median expression in 2nd quartile in at least two datasets were shortlisted s6 figdesign of primersbcl2 primers bcl2 has two known splice isoforms membranebound bcl2α and aless studied soluble bcl2β lacking the transmembrane domain at the cterminal most reported primers amplified only bcl2α or larger amplicon s2 table hence new primers were designed table rg primers primers for shortlisted genes were designed table s3 table using primerbank and idt sample detailsrna was isolated from peripheral blood or bone marrow samples from patient or normalindividuals s7 fig with their informed consent ethics approval number nhhaeccltable primers details of rgs and bcl2primeracy1accession nonm_000666ankrd26nm_014915jmjd4nm_001161465ptcd2nm_0247545ppp1r3bnm_024607fbxw9nm_032301nanpnm_1526673plekhm3nm_0010804753tsga10nm_025244nat1nm_001160174ric8bnm_018157gapdhnm_0012897453bcl2nm_0006572sequence fw 'cactgacaaccgctatatccgrv 'ctcatgcagccgttcatcgtfw 'tctcggcaagatccacaaagcrv 'aatgtagagccgtcctgttcafw 'gtctgtcaatgtctgtgggagrv 'caggtgtgtgtcgcagagt3'fw 'tatgggacactgcacatcac3'rv 'ggctgaccatcctcttgttta3'fw 'agaacctcgcatttgagaagac3'rv 'tctgaaccggcataagtgtcc3'fw 'tagggcggtgcgatgattc3'rv 'cggattttggcggactgaga3'fw 'ggtccgcctacttctattaacg3'rv 'tctctgctctccacctacaa3'fw 'gatgatatcagcccagccttag3'rv 'ggacttcctggatcccataaac3'fw 'tactcagcgacaccttgctaa3'rv 'ccagatcattgagggttccac3'fw 'gggagggtatgtttacagcac3'rv 'acatctggtatgagcgtccaa3'fw 'atagtgttcaacagtcagatggc3'rv 'gcaagcgcaagtcaaagca3'fw 'tcgacagtcagccgcatcttcttt3'rv 'gcccaatacgaccaaatccgttga3'fw 'ggaggattgtggccttcttt3'rv 'gcccaatacgaccaaatccgttga3'fw forward primer rv reverse primer101371 pone0236338t002amplicon length bptm Ëcamplification factor one 101371 pone0236338 august one 0cbcl2 molecular diagnostics with novel reference genes2017152a subjects with hepatitis bc or hiv and pregnant or lactating women wereexcluded from the studypbmcbmmc peripheral blood mononuclear cells bone marrow mononuclear cellswere separated by layering of blood or bone marrow diluted to with 1x pbs gibco¢germany above ficollpaque plus histopaque himedia india followed by centrifugation at rcf for mins with brakes off resultant buffy coat was washed twice with 1x pbs andonce with 1x penstrep himedia india before culturing at cell density of to millioncellsml of rpmi himedia india with fbs gibco¢ germany brazil origin and1x penstrep for subculturing the lymphocyte populationrna cdna and qpcrfrom ffpe formalinfixed paraffinembedded blocks curls were deparaffinized inxylene at Ëc followed by proteinase k himedia india treatment prior to rna isolationeither from lymphocytes or from deparaffinized retrospective samples rna was isolatedby trizol¢ ambion us method and quantified with qubit rna br assay kit thermofisher scientific us before converting to cdna using superscript iv ssiv thermo fisherscientific us as per manufacturers instructions with notemplate control ntc qpcrwas performed in triplicates for each sample using kapa sybr green universal reagentssigma aldrich us cdna dilution and primers in a 5μl reaction mix qpcr condition preincubation at Ëc for minutes followed by amplification for cyclesdenaturation at Ëc for sec amplification at Ëc for sec and extension at Ëc for sec inroche lightcycler ii machineoptimization of primersprimers were optimized for qpcr as required by the miqe guidelines all primers wereused at four different final concentrations forwardreverse 200nm200nm 200nm100nm100nm200nm and 100nm100nm with pooled cdna template obtained from six normalhealthy volunteers to yield single amplification product primer efficiency was checked using atwofold fivepoint dilution of the template primer efficiency was obtained from standardcurve using the formula amplication factor ¼ 00� table ��slope 00 stability analysis of candidate rgsmean of cq quantification cycle of ntc were subtracted from cq values of each gene inqpcr experiments to obtain δcq cq samplemean cq ntc and relative expression aseδcq for each replicate where e is the amplification factor of corresponding genestability of expression of the candidate rgs was analysed using three independent algorithmsgenorm normfinder and bestkeeper and the webbased reffindertool that integrates all three algorithms plus the delta ct method algorithm genorm wasrun using the slqpcr r package whereas authorsupplied r package and excel worksheet were used for normfinder and bestkeeper analysis respectively mean cq values foreach gene for all samples were used as input for bestkeeper and reffinder whereas fenorm and normfinder relative expression values were used since normfinder uses amodelbased approach to quantify inter and intragroup variations the malignant and nonneoplastic or healthynormal samples were used as two groups for normfinder analysiscomprehensive stability rank of each gene was calculated as the geometric mean of stabilityrank given by each method one 101371 pone0236338 august one 0cbcl2 molecular diagnostics with novel reference genesexpression analysis of bcl2rq relative quantification of bcl2 expression was calculated either as ratio of relativeexpression of bcl2 with relative expression of gapdh or the normalization factor which isgeometric mean of relative expression of three candidate rgsrq ðgapdhþ ¼ e 00 dcqðbcl2þe 00 dcqðgapdhþrq ðproposedþ ¼ e 00 dcqðbcl2þgeo mean e 00 dcqðptcd2 ppp1r3b fbxw9þresults and discussionquantification by qpcr could be the choice of pathology laboratories for a quick and costeffective platform for singlegene expression level with appropriate rg towards this effort macrae performed a genome wide search and statistical analysis using rnaseq datafrom leukemia patients in a more recent pancancer study publicly available geneexpression data from microarray studies were analysed to identify a few rg candidates thatshowed minimal variation between malignant and normal samples and were validated in droplet digital pcr on bone marrow samples of all patients we have used types of haematological malignancy samples encompassing bone marrow pbmc and ffpe blocks along with nonneoplastic bone marrow and healthy pbmc samples subsequent to using much wider publiclyavailable data from samples in aml dlbcl and multiple myeloma databases furtherwe have employed an improved statistical analysis including clustering technique described inmethods section instead of an ad hoc approach of selection of top few genes from the clusterswe used important biological considerations to further prune the list of candidate rgssystematic review of commonly used rgs from literaturesystematic review of s yielded rgs used in haematological malignancies througha selection of genes by different analysis methods s4 table and b usage of known rgs inqpcr s1 table fpkm values of all these rgs when examined in public databases showedvaried expression among different types of haematological malignancies s3 and s4 figs withmaybe the exception of pggt1b however since other genes selected in the literatureshowed higher expression and correlated extreme variation we could not depend on the assayand proceeded to select novel rgs with an unbiased approachselection of candidate rgsstatistical analysis stepwise filtration of the number of genes from each dataset is summarized in s6 fig and also in graphical abstract fig shows gene clusters plotted in cv normalized mad and 1pvalue hyperspace for four datasets cluster marked in green in eachfigure represents the cluster with least med value s5 table for the three parametersselected clusters in the four datasets had an overlap of genes indicating large number ofgenes involved in housekeeping processes and hence showing lesser intersample variationacross diverse datasets common genes were pruned further to by go biological processterm filtration disease association and cqr to lead to a final of genes s6 table that weretaken through experimental validation melt curve analysis and efficiency check with pooledcdna from six healthy volunteers narrowed it down to genes with stable median expression and single amplification product of expected size for each table primers for geneswhich did not qualify the efficiency check were eliminated as they failed to show single amplification peak after repeated trials with new experimental conditions and even new primersequences s3 table one 101371 pone0236338 august one 0cbcl2 molecular diagnostics with novel reference genesfig statistical analysis of candidate genes genes plotted in the cv normalized mad and pvalue hyperspace for the fourdatasets a tcgalaml b targetaml c gdcdlbc and d mmrfmm cluster shown in green represents the chosencluster with least value of meds101371 pone0236338g001expression of genes with efficient primers were analysed on samples by qpcr usingobserved cq values preliminary stability analysis of the genes were done with online reffinder tool to select top stable genes ptcd2 ppp1r3b fbxw9 nanp ric8b jmjd4plekhm3 nat1 ankrd26 tsga10 as rg candidatessssstability analysis of candidate rgs results of bestkeeper algorithm used independentlyor as part of reffinder were comparable whereas results of genorm or normfinder analysisdiffered as they used different inputs geometric mean of stability ranks assigned in each algorithm was used to create comprehensive stability ranking of all the candidate rgs s7 tableand fig the analysis shows ptcd2 ppp1r3b and fbxw9 to be most stable across all analysed patient samplesptcd2 pentatricopeptide repeatcontaining protein codes for a mitochondrial proteininvolved in rna binding maturation and respiratory chain function though its exact one 101371 pone0236338 august one 0cbcl2 molecular diagnostics with novel reference genesfig stability rank of candidate reference genes101371 pone0236338g002molecular function is not well understood [ ] ppp1r3b protein phosphatase1 regulatorysubunit3b encodes for a catalytic subunit phosphatase regulatory subunit 3b which isinvolved in hepatic glycogen dysregulation in type diabetes [] fbxw9 fboxwdrepeatcontaining protein is a cytosolic protein involved in ubiquitination and proteasomedegradation expression analysis of bcl2accurate determination of bcl2 expression among few antiapoptotic markers in patients withhaematological malignancies is emerging as a critical diagnostic test for clinicians to suggest efficacious therapy options fpkm values of rgs common and novel from the publicly availabledatabases when compared fig with bcl2 indicated the novel rgs to be better normalizationcandidate for bcl2 in qpcr assays in pathology labs due to less and stable expressioncomparison of relative expression of gapdh versus the proposed normalization facteometric mean of relative expression of the three rg candidates clearly show a large variation in gapdh expression across malignant samples fig 4a s8 table granted itspopularity the expression stability of gapdh has been proven to differ in different conditionsdue to its involvement in apoptotic cell death through ubiquitin ligase membrane trafficking upregulation in aml involvement in nonhodgkins bcell lymphomas and inconsistency in several other cancers on the other hand proposed rgs havelesser variation and their expressions are consorted with each other making them better candidate as rg compared to gapdh this behaviour is translated to bcl2 expressionrq in malignant samples when normalized with gapdh fig 4b evidently normalizationwith gapdh underestimates relative quantification of bcl2 compared to normalization withproposed rgs with a statistically significant difference in median values p wilcoxonrank sum test between the two schemes bcl2 quantification in haematological malignanciesby qpcr is overtly reliant on rg since availability of adjacent normal sample is ruled outabove results clearly demonstrate how the quantification may go off limit due to a wrongchoice of rg one 101371 pone0236338 august one 0cbcl2 molecular diagnostics with novel reference genesfig candidate reference genes in hematological malignancy datasets expression values of candidate genes in four datasets a tcgalamlb targetaml c gdcdlbc and d mmrfmm101371 pone0236338g003broader applicability of proposed reference genesthough primary objective of this study is to discover rg candidates for bcl2 diagnostics in aclinical setting the rgs may have broader utility in other experimental platforms or modelsystems in the systematic review we found a number of research s [] that haveused taqman probes instead of sybr green whereas our validation experiment was carriedout using sybr green probes however studies in different contexts such as a tropical oilseedplant or measurement of expression of various adenosine receptors in breast cancer tissue and in experiments using human reference rna sybr green pcr assays wereobserved having fair concordance with taqman pcr from these evidences we believe thatstability of proposed rgs is not likely to differ between sybr green and taqman qpcr assaysto assess variation of these stable rgs in cell lines we analyzed rpkm values of proteincoding genes across cell lines of haematopoietic and lymphoid tissue origin frombroad institute cancer cell encyclopedia and found the proposed rgs presenting muchlesser variations in expression compared to the common rgs gapdh abl1 b2m gusband actb in cell lines as well s8 figboth transgenic and wild type and occasional rat models are widely used in leukemia andlymphoma research [ ] usability of rgs common between clinical and animal studies one 101371 pone0236338 august one 0cbcl2 molecular diagnostics with novel reference genesfig relative expression of chosen reference genes and relative quantification of bcl2 a relative expression of chosen reference genes solidlines and gapdh dashed line across patient samples b relative quantitation of bcl2 expression with respect to the candidate reference genesand gapdh in malignant patient samples101371 pone0236338g004will thus be of immense advantage we find that the proposed rgsptcd2 ppp1r3b andfbxw9 have sequence similarity and identity with corresponding genes in mice andother commonly used rodent models s9 table suggesting the genes playing similar role incellular function thereby displaying stability similar to that in humans hence normalizationfactor derived from the expression of these rgs may be applicable in murine and other rodentmodels as well with suitable design of primers encompassing conserved regionsbeyond detection of gene expression at mrna level it may be worthwhile to explore theapplicability of protein counterpart of the stable rgs in western blot as control for proteindetection by design we have chosen rgs that are of moderate expression level in middlequartiles of expression among other genes and they may not be detectable by western blotunless a larger amount of sample is loaded which is often not feasible with clinical sampleshowever it may be an interesting proposition to predict stable reference proteins for use inwestern blot by statistical analysis of proteomics data and associated systematic review ofliterature one 101371 pone0236338 august one 0cbcl2 molecular diagnostics with novel reference genesour results indicate that genes ptcd2 ppp1r3b and fbxw9 render more reliability toqpcrbased diagnostic test of bcl2 in haematological malignancies the can beextended to other biomarkers in liquid cancer as well as for research with other model systemssuch as cell lines and rodentssupporting informations1 table list of reference genes in literaturedocxs2 table list of bcl2 primers from literaturedocxs3 table list of unqualified primersdocxs4 table literature explaining analysis and selection of reference genedocxs5 table zscore med valuesdocxs6 table list of selected genesdocxs7 table individual and combined stability rank and scores of candidate reference genesdocxs8 table relative expression of gapdh and the proposed normalization factordocxs9 table sequence similarity and identity with corresponding genes in mice rat andguinea pigdocxs1 fig rgs found in literature with more than one citationtiffs2 fig fpkm values of bcl2 family of antiapoptotic genes in the four datasetstiffs3 fig fpkm values of rgs found in relevant literature with more than one citationtiffs4 fig fpkm values of rgs found in relevant literature with a single citationtiffs5 fig workflow according to prisma guidelines for systematic review for commonlyused reference genestiffs6 fig statistical analysis workflowtiffs7 fig patient samples used in the studytiff one 101371 pone0236338 august one 0cbcl2 molecular diagnostics with novel reference geness8 fig variation in stable rgs in cell lines and animal modeltiffs1 graphical abstracttiffacknowledgmentsauthors acknowledge prof joy kuri chair department of electronic science and engineering indian institute of science bangalore for providing the computational resourcesauthor contributionsconceptualization sujan k dhar manjula dasdata curation nehanjali dwivedi sreejeta mondal smitha p k sowmya t kartik sachdeva christopher bathula vishnupriyan kformal analysis sujan k dharfunding acquisition sharat damodar manjula dasinvestigation nehanjali dwivedi sreejeta mondal smitha p k sowmya tmethodology nehanjali dwivedi sreejeta mondal smitha p k sowmya t vishnupriyank manjula dasproject administration manjula dasresources nataraj k s sharat damodarsoftware sujan k dharsupervision manjula dasvalidation nehanjali dwivedi sreejeta mondal smitha p k sowmya t kartik sachdevachristopher bathula vishnupriyan kvisualization manjula daswriting original draft sreejeta mondal sujan k dharwriting review editing nehanjali dwivedi sreejeta mondal smitha p k sujan kdhar manjula dasreferences perini gf ribeiro gn pinto neto jv campos lt hamerschlak n bcl2 as therapeutic target forhematological malignancies vol of hematology and oncology biomed central ltd gratiotdeans j merino r nuñez g turka la bcl2 expression during tcell development early lossand late return occur at specific stages of commitment to differentiation and survival proc natl acad sciu s a oct 101073pnas912210685 pmid merino r ding l veis dj korsmeyer sj nuñez g developmental regulation of the bcl2 protein andsusceptibility to cell death in b lymphocytes embo j feb pmid li l li y que x gao x gao q yu m prognostic significances of overexpression myc andorbcl2 in rchoptreated diffuse large bcell lymphoma a systematic review and metaanalysis scirep 101038s41598017177655 uchida a isobe y asano j uemura y hoshikawa m takagi m targeting bcl2 with venetoclaxis a promising therapeutic strategy for doubleproteinexpression lymphoma with myc and bcl2 one 101371 pone0236338 august one 0cbcl2 molecular diagnostics with novel reference genesrearrangements haematologica jun 103324haematol2018 pmid baro´ c espinet b salido m garcı´a m sa´nchez b florensa l cryptic ighbcl2 rearrangementswith variant fish patterns in follicular lymphoma leuk res feb 101016jleukres201009011 pmid hofman p heeke s alixpanabières c pantel k liquid biopsy in the era of immunooncology is itready for primetime use for cancer patients suppressed immune microenviron repert brain metastases from patients with resected nsclc fatani s h mukhtar m h ali a s correlation between serum antiapoptotic bcl2 level and its immunohistochemical expression in relation to apoptosis in gastric cancer j mol biomark diagn albitar m zijun xy wang y manman d tzankov a visco c myc and bcl2 mrna expressionas determined by ngs predicts survival in dlbcl in gcb but not in abc subgroup blood nov 134supplement_15092 derenzini e rossi a agostinelli c rossi m melle f motta g integration of nanostring profilingand functional characterization of oxidative and replicative stress biomarkers identifies poor prognosis mycbcl2 positive diffuse large bcell lymphoma subsets providing opportunities for precisiontherapies blood nov 132supplement zhang f yang b zhang k hou ml lu xc li yx ccnd1bcl2 gene network a direct target of amifostine in human acute megakaryocytic leukemia cells chem biol drug des may 101111cbdd12889 pmid patel vm balakrishnan k douglas m tibbitts t xu ey kutok jl duvelisib treatment is associated with altered expression of apoptotic regulators that helps in sensitization of chronic lymphocyticleukemia cells to venetoclax abt199 leukemia sep 101038leu2016382 pmid bomben r ferrero s dagaro t dal bo m re a evangelista a a bcell receptorrelated genesignature predicts survival in mantle cell lymphoma results from the fondazione italiana linfomi mcl trial haematologica apr 103324haematol2017184325pmid dheda k huggett jf chang js kim lu | 0 |
among synchronous colorectal cancers scrcs reported previously the incidence of quadruple advanced scrcs is very rarewe present the case who underwent laparoscopic twosegment resection of the colon requiring two anastomoses that wasperformed for quadruple advanced cancers and four tumors were curatively removed there were no signs of recurrence at months after surgery laparoscopic surgery provided less invasiveness even for quadruple advanced scrcs in terms of earlyrecovery with an acceptable longterm outcomeintroductionsynchronous colorectal cancers scrcs are characterized bythe simultaneous occurrence of multiple primary tumors inthe same patient synchronous malignancies most commonlyoccur in the colon among other ans [] the occurrence ofadvanced scrcs is rare and may be identified at any locationwithin the large intestine the prevalence of scrcs is reportedto range from to among these however the incidence of quadruple advanced scrcs is extremely rare accounting for of all scrcs surgical resection is considered thestandard treatment for scrcs as a surgical approach laparoscopic surgery has significant advantages in terms of shortterm outcomes including early recovery and no disadvantageouslongterm outcomes according to recent reports laparoscopicsurgery has been used in scrcs but these reports noted thatcontroversy remains concerning operative procedures for multiple segmental resections and for total or subtotal colectomywe report the case who presented with quadruple synchronousadvanced cancers arising from the colon which were successfully treated with laparoscopic twosegment colectomyreceived may accepted june published by oxford university press and jscr publishing ltd all rights reserved the authors this is an open access distributed under the terms of the creative commons attribution noncommercial license httpcreativecommonslicensesbync40 which permits noncommercial reuse distribution and reproduction in any medium provided the original work is properly citedfor commercial reuse please contact spermissionsoupcom 0cj ma figure colonoscopy images showing four tumors a one cauliflowerlike tumor with lumen stenosis is located in the ascending colon b another cauliflowerliketumor is located in the descending colon the third c and fourth d tumors are located in the sigmoid coloncase presentationa 70yearold man who was positive for a blood stool testvisited our hospital colonoscopy computed tomography ctand barium enema indicated quadruple concurrent locallyadvanced cancers the firsttumor with observed lumenstenosis was located in the ascending colon the secondtumor was located in the descending colon and the third andfourth tumors were located in the sigmoid colon fig ctrevealed marked intestinal wall thickness in the ascendingdescending and sigmoid colon fig preoperative precisesimulation using 3d angiography was performed to determineadequate lymph node dissection along the arteries feedingthe tumors and appropriate resection to avoid anastomoticleakageswe planned the appropriate placement of trocars as shownin figure because we wanted to create a single minilaparotomy for specimen retrieval and extracorporeal reconstruction after lymph node dissection and mobilization of thecolonduring the operation laparoscopic exploration confirmed thepresence of known four tumors with no invasion of the serosasubsequently a right hemicolectomy and sigmoid colectomywere performed laparoscopically the right half of the colon wasseparated and a sidetoside anastomosis between the jejunumand transverse colon was performed followed by the sigmoidcolon and a colorectal anastomosis between the descendingcolon and rectum was performed the resected tissue specimensrevealed four tumors fig histological examination showedthat the first tumor in the ascending colon the second tumor inthe descending colon and the third tumor in the sigmoid colonhad invaded up to the subserosa whereas the fourth tumor inthe sigmoid colon had invaded up to the muscularis propriafig according to the american joint committee on cancertumornodemetastasis staging system the pstage was iiia t3n1m0the patient was discharged days after surgery for adjuvantchemotherapy the patient chose to take an oral fluoropyrimidine agent for months fortunately there have been no signsof metastasis or recurrence after the operation at months offollowupdiscussionwe reported a rare case of quadruple scrcs all four tumorswere removed curatively by laparoscopic surgery with d3 lymphnode dissection we planned a strategy for quadruple scrcsbased on preserving the remnant large intestine and sufficientd3 lymph node dissection through a laparoscopic approachwe believe that laparoscopic surgery can be a safe even forquadruple scrcs this is the first case report of laparoscopicsurgery with d3 lymph node dissection for quadruple advancedscrcsthe incidence of malignant scrc with four or five synchronous lesions is extremely rare with a rate of beingreported this is a quite rare case of quadruple synchronous 0cquadruple advanced synchronous colorectal cancersfigure placement of trocars and miniincision in the present casethan the index cancer however all of the scrcs in our patienthad the same histological grade and t staging pstage iiiawith the tumor locations being in the ascending descending andsigmoid colonsurgical management of scrcs needs to be tailored tothe individual based on tumor location invasion status andthe patients health condition some studies have suggestedtotal or subtotal colectomy to remove any potential existingsynchronous tumors or polyps that have not been detected however other studies recommend a more conservativesurgical approach it is thought that the removal of the entirecolon will prevent the development of metachronous tumorsand a previous study indicated that subtotal colectomy mayincrease defecation frequency as the normal colon cannot bepreserved we successfully performed laparoscopic surgerycombining twosegment resection of a right hemicolectomyand sigmoid colectomy with no intra or postoperative adverseevents in our patient we tried preserving as much colonas possible considering the patients quality oflife aftersurgery in addition to performing sufficient d3 dissection ofcourse the meaning of preserving colon in terms of patientpostoperative quality of life needs to be more clearly assessed infuturewe encountered a rare case of advanced quadruple scrcsfor which we achieved a curative resection that required twoanastomoses through a laparoscopic approach we suggest thatlaparoscopic surgery that requires multiple anastomoses foradvanced scrcs can be a safe procedure even if the number ofcolorectal cancers is multiplefigure abdominal ct scan revealing a tumor of the ascending colon aarrowhead a tumor in the descending colon b arrowhead and two tumorsin the sigmoid colon are also visible c d arrowheadadvanced cancer arising from the ascending descending andthe sigmoid colon it was reported that scrcs often occur inthe same or adjacent segment of the large intestine and thatother smaller colorectal cancers in the patients with scrcs wereusually smaller and of lower pathological grade and t stagingconflict of interest statementnone declared 0cj ma figure the surgical specimens of the ascending colon cancer a descending colon cancer b and the two sigmoid colon cancers c and dfigure histopathological examination of the tissue specimens revealed four tumors showing cancerous cells arranged in a tubular pattern 0cfundingnonereferencesjiang x xu c tang d wang d laparoscopic subtotal colectomy for synchronous triple colorectal cancer a case reportoncol lett yang j peng jy chen w synchronous colorectal cancersa review of clinical features diagnosis treatment and prognosis dig surg aky l synchronous colorectal cancer clinical pathological and molecular implications world j gastroenterol fukatsu h kato j nasu ji kawamoto h okada h yamamotoh clinical characteristics of synchronous colorectalcancer are different according to tumour location dig liverdis holubar sd wolff bg poola vp soop m multiple synchronous colonic anastomoses are they safe colorectal disquadruple advanced synchronous colorectal cancers li z wang d wei y liu p xu j clinical outcomes oflaparoscopicassisted synchronous bowel anastomoses forsynchronous colorectal cancer initial clinical experienceoncotarget nosho k kure sirahara n shima k baba yspiegleman d a prospective cohort study showsunique epigenetic genetic and prognostic features ofsynchronous colorectal cancers gastroenterology 20e1 lam ak carmichael r gertraud buettner p gopalan dho yh siu s clinicopathological significance of synchronous carcinoma in colorectal cancer am j surg easson am cotterchio m crosby ja sutherland h dale daronson m a populationbased study of the extent ofsurgical resection of potentially curable colon cancer annsurg oncol tsantilas d ntinas apetrasp zambas n aimogrambi s frangandreas g metachronouscolorectals202adenocarcinomastech coloproctol 0c' | 0 |
"s/Design A parallel group randomized controlled trial is conducted to compare MBSR with TAU. Lung cancer patients who have received or are still under treatment and their partners are recruited. Assessments will take place at baseline post intervention and at three-month follow-up. The primary outcome is psychological distress (i.e. anxiety and depressive symptoms). Secondary outcomes are quality of life (only for patients) caregiver appraisal (only for partners) relationship quality and spirituality. In addition cost-effectiveness ratio (only in patients) and several process variables are assessed. Discussion This trial will provide information about the clinical and cost-effectiveness of MBSR compared to TAU in patients with lung cancer and their partners. Trial registration ClinicalTrials.gov NCT01494883. Mindfulness-based stress reduction Lung cancer patients Partners Psychological distress Randomized controlled trial Background With an estimated 1.4 million deaths per year lung cancer is the leading cause of death by cancer worldwide. Even with the best available treatment five-year survival is merely 16% and about 60 to 70% of patients die within the first year after diagnosis [1]. This poor prognosis is often caused by a late diagnosis as the presentation usually occurs when the lung cancer is advanced. Patients may develop burdensome symptoms like pain dyspnoea fatigue and cough and they may undergo radical treatment including surgery chemo- and radiotherapy. Not surprisingly lung cancer has a major impact on the psychological wellbeing of patients and their family. Akechi and colleagues [2] showed that 19% of patients with advanced lung cancer meets the criteria of psychiatric disorders especially depressive and adjustment disorders. Of patients who had been successfully treated for lung cancer 15% met the criteria for a minor or major depressive disorder [3]. The prevalence rate of depressive and anxiety symptoms among lung cancer patients ranges from 20 to 47% [4-7]. Compared to patients with other cancer diagnoses lung cancer patients report the highest rates of distress (43 to 58%) [89] resulting in a lower quality of life [10]. Family friends and especially partners of patients with lung cancer also have to deal with its psychological impact [11-14]. Partners not only provide emotional and practical support they also have to cope with their own concerns including the uncertainty regarding the course of the illness and the fear of losing their partner [15]. More than 50% of partners of lung cancer patients report negative emotional effects of caregiving [16]. Around 40% of partners of patients with advanced lung cancer report high levels of distress [17]. The relationship between patient and partner can also be affected by the cancer. It has been shown that some partners report a lower quality of their relationship after the diagnosis of lung cancer [18]. Though numerous studies examined the psychological distress of lung cancer patients and their partners [2-22] not much research is done on how to alleviate distress in these groups [23]. In addition the available studies on managing the psychosocial care needs of cancer patients and their families have focused on care at the very end of life (e.g. [24-26]). Recently studies have demonstrated that palliative care initiated early in treatment improves the quality of life and depressive symptoms of lung cancer patients [1027]. This stresses the importance of integrating psychosocial care for lung cancer patients and their partners early in the treatment rather than instigating it once life-prolonging therapies fail. In the past ten years MBSR has become a promising psychosocial intervention for cancer patients. Mindfulness is defined as intentionally paying attention to moment-by-moment experiences in a non-judgmental way [28]. MBSR is an 8-week group-based training consisting of meditation practices such as the bodyscan gentle yoga sitting and walking meditation. By repeatedly bringing attention back to the current experience participants gradually learn to disengage from dysfunctional thoughts and directly experience the emotions and bodily sensations of the present moment. MBSR aims to provide participants with the ability to step back from ruminating about the past or worrying about the future and simply allow experiences to unfold [2829]. A recent meta-analysis [30] of 13 nonrandomized studies and 9 randomized controlled trials (RCT) concluded there is positive evidence for the use of mindfulness-based interventions in reducing psychological distress in cancer patients. Among the RCTs a reduction in symptom severity was found for both anxiety and depression corresponding to moderate pooled controlled effect sizes (Hedgess g = 0.37 and Hedgess g = 0.44 respectively) [30]. Though mindfulness-based interventions seem to be effective the authors note that across studies the majority of participants were women (85%) and diagnosed with breast cancer (77%). Compared to breast cancer patients patients with lung cancer are more often male older and have a poorer prognosis. Furthermore of these 22 studies only one study included the partners of the patients showing that partners also benefit from the MBSR training [31]. This is quite surprising since partners of cancer patients also report high levels of distress [32]. Aims The aim of the Mindfulness for Lung Oncology Nijmegen (MILON) study is to examine the effectiveness of MBSR compared to TAU in reducing psychological distress in patients with lung cancer and their partners. We hypothesize that patients in the MBSR group will report a lower level of psychological distress (i.e. anxiety and depressive symptoms) higher levels of quality of life quality of relationship and spirituality than those in the TAU group. Medical and societal costs will be lower in the MBSR versus TAU group. We expect partners in the MBSR group to report a lower level of psychological distress and higher levels of caregiver appraisal relationship quality and spirituality than their counterparts in the TAU group. With regard to the working mechanisms of the MBSR programme we will examine changes in mindfulness skills self-compassion rumination intrusion avoidance and adherence to MBSR. Methods/Design Study design The design of the MILON study is a parallel group randomized controlled trial with an embedded process study. Participants are randomized between MBSR and TAU. The study protocol has been approved by our ethical review board (CMO Arnhem-Nijmegen) and registered under number 2011519. Participants and procedure Patients and partners are recruited at the outpatient clinic of the Department of Pulmonary Diseases Radboud University Nijmegen Medical Centre (RUNMC) by a nurse practitioner and the attending physician. Patients and partners are invited to participate together but both are welcome to participate on their own if they do not have a partner or their partner is not willing to participate. Patients and/or partners who are interested are provided with an information leaflet. If they are willing to participate they are invited for a research interview in which in- and exclusion criteria are assessed and informed consent is taken. At other participating hospitals (Department of Pulmonary Diseases Canisius-Wilhelmina Hospital Nijmegen; Department of Pulmonary Medicine Rijnstate Arnhem; Department of Oncology Elkerliek Hospital Helmond; Department of Pulmonary Medicine Jeroen Bosch Hospital; Department of Pulmonary Diseases Maas hospital Pantein Boxmeer) patients and their partners will be sent a letter with the invitation to participate in the study. One week later the researcher calls the patients to answer possible questions and asks whether the patient and partner are interested in participation. If so they are invited for a research interview at the RUNMC. Eligibility We include patients and/or partners of patients who are (a) diagnosed with cytologically or histologically proven non-small cell lung cancer or small cell lung cancer and (b) have received or are still under treatment. Exclusion criteria for both patient and partner include: (a) being under 18 years of age (b) not being able to understand or use the Dutch language (c) former participation in MBSR or Mindfulness-Based Cognitive Therapy (MBCT) (d) current and regular treatment by psychologist or psychiatrist (e) current participation in other psychosocial programme and (f) physical or cognitive (<26 on the Mini-Mental State Examination (MMSE)) impairments hampering participation in MBSR training or completion of questionnaires. Baseline Patients and partners are interviewed to obtain demographics and clinical characteristics after which they are screened for cognitive impairments with the MMSE [33]. After that baseline questionnaires including the Distress Thermometer (DT) [3435] are administered followed by randomization. Table 1 shows the assessment instruments and time points at which the questionnaires are administered to patients and partners. Table 1 Measurements and corresponding time points for patient and partner Measure Target T0 T1 T2 pt pr pt pr pt pr MMSE Cognitive impairments x x DT General distress x x HADS Psychological distress x x x x x x QLQ-C30 Quality of life x x x QLQ-LC13 Quality of life x x x SIP Impact of sickness x x x SPPIC Caregiver burden x x x CRA-SE Caregiver self-esteem x x x IMS-S Relationship satisfaction x x x x x x MIS Communication about cancer x x x x x x SAIL Spirituality x x x x x x FFMQ Mindfulness skills x x x x x x SCS Self-compassion x x x x x x RRS-EXT Rumination x x x x x x IES Psychological stress reaction x x x x x x Diary Health care use work absence Monthly during study period for pt Calendar Mindfulness adherence Monthly during study period for pt and pr Note. T0 = Baseline measurement; T1 = Post-intervention measurement; T2= 3-month follow-up measurement; pt = Patient; pr = Partner; MMSE = Mini Mental State Examination; DT = Distress Thermometer; HADS = Hospital Anxiety and Depression Scale; QLQ-C30 = Quality of Life Cancer; QLQ-LC13 = Quality of Life Lung Cancer; SIP = Sickness Impact Profile; SPPIC = Self-Perceived Pressure from Informal Care; CRA-SE = Caregiver Reaction Assessment Care-Derived Self-Esteem; IMS-S = Investment Model Scale-Satisfaction; MIS = Mutuality and Interpersonal Sensitivity; SAIL = Spiritual Attitude and Involvement List; FFMQ = Five Facet Mindfulness Questionnaire; SCS = Self-Compassion Scale; RRS-EXT = Rumination Response Scale Extended Version; IES = Impact of Event Scale. Randomization Randomization is stratified according to setting and minimized for (a) stage of disease (curative versus palliative) (b) baseline level of anxiety and depressive symptoms (anxiety or depression subscale score of Hospital Anxiety and Depression Scale (HADS) <8 versus ?8) (c) treatment during MBSR (no treatment versus chemo- and/or radiotherapy) and (d) participation (patient alone versus partner alone versus patient and partner together). Randomization is computerized using a randomization website specifically designed for this study on which the researcher can fill out the required data. The researcher communicates treatment allocation to the nurse practitioner who informs the patient and/or partner. Follow-up assessments Follow-up assessments take place post intervention and at three-month follow-up. Participants who have access to the internet and have an email address receive the questionnaires online. If not they receive the questionnaires on paper along with a reply envelope. In case of drop-out the researcher tries to contact the participant by phone to complete a minimum set of outcome measures and to identify the main reason for drop-out. Intervention The MBSR curriculum used is primarily based on the Mindfulness-Based Stress Reduction programme as developed by Kabat-Zinn [28] but contains some elements of the MBCT programme by Segal Williams and Teasdale [29] like psycho-education on the interrelatedness of feelings and thoughts. Moreover some modifications have been made to make the intervention more suitable for patients with lung cancer and their partners such as psycho-education about grief [36]. In addition a mindful communication exercise in which partners talk with each other about the cancer was added. The programme consists of 8 weekly 2.5-hour sessions a silent day between session six and seven and home practice assignments of about 45 minutes 6 days per week. Participants receive a set of CDs with guided mindfulness meditation exercises for home practice and a folder with information and home practice instructions for the forthcoming week. Table 2 shows the content of the MBSR programme per session. The MBSR courses are taught by mindfulness teachers with extensive training in MBSR. They all fulfil the advanced criteria of the Center for Mindfulness of the University of Massachusetts Medical School [37] and maintain a regular personal meditation practice. Teachers were trained supervised and assessed to ensure their competency levels met the qualification criteria to instruct the MBSR classes. During the trial teachers will receive weekly supervision and a number of sessions will be videotaped to evaluate competence and adherence with the Mindfulness-Based Interventions Teaching Assessment Criteria [38]. Table 2 Content of MBSR programme per session Theme of session Meditation exercise Didactic teaching Homework 1. Automatic pilot - Bodyscan - Intention of participating - Bodyscan - Raisin exercise - Eating one meal mindfully - Attention for routine activity 2. Mindfulness of the breath - Bodyscan - Imagery exercise to demonstrate relationship between thoughtsand feelings - Bodyscan - Sitting mediation with focus on breath - Attention for breath - Awareness of pleasant events - Attention for routine activity 3. Observing limits - Yoga while lying down - Seeing exercise to demonstrate difference between observation and interpretation - Bodyscan or yoga - 3-min breathing space - Sitting meditation - Awareness of unpleasant events - 3-min breathing space 4. Opening up to distress - Sitting mediation with focus on breath body and sound - Interrelatedness of feelings thoughts and bodily sensations - Bodyscan or yoga - Sitting meditation - 3-min breathing space - Psychoeducation about grief - Awareness of stress reactions - 3-min breathing space 5. Responding to distress - Sitting mediation with focus on breath body sound thoughts difficulty - Reacting versus responding - Meditation by choice - Coping with grief - Awareness of reaction in difficult situation - Walking meditation - Awareness of communication difficulties - 3-min breathing space - 3-min breathing space 6. Mindful communication - Yoga in standing position - Mindful communication exercise about effect of lung cancer with their own partner - Sitting meditation or yoga - 3-min breathing space - Awareness of communication - 3-min breathing space during stress Silent day - Varying meditation exercises - Silent lunch and tea break 7. Taking care of yourself - Sitting meditation ending in choiceless awareness - Exercise on taking care of yourself by examining how to improve balance in life - Meditation without CD - Yoga or walking meditation - Reflect on training - 3-min breathing space 8. The rest of your life - Bodyscan - Reflection on training - Further sources of information - Short sitting meditation - Maintaining practice Outcome measures Primary outcome measure Psychological distress The primary outcome measure is the total score on the HADS [39-41] which is developed to measure psychological distress in somatic patient populations. It consists of a 7-item anxiety (HADS-A) and 7-item depression (HADS-D) subscale. The HADS shows good psychometric properties in the general medical population including oncology patients [42]. Internal consistency as measured with Cronbachs ? varied from .84 to .90 [4042].Test-retest reliability was good as Pearsons r > .80 were obtained [4043]. Though the cut-off scores of the HADS vary among populations [44] in lung cancer patients they have found to be <8 versus ?8 on the HADS-A or HADS-D [45]. The HADS has been shown to be highly correlated with the Beck Depression Inventory [42]. It has previously been used in intervention studies of mindfulness and shown to be sensitive to change (e.g. [46]). Secondary outcome measures Quality of life (only for patients) The European Organisation for Research and Treatment of Cancer (EORTC) Core Quality of Life Questionnaire (QLQ-C30) [47] is included along with the supplemental Lung Cancer questionnaire module (QLQ-LC13) [48]. The QLQ-C30 is designed to use in clinical trials on physical treatments for cancer patients. It incorporates five functional scales (physical role cognitive emotional social) three symptom scales (fatigue pain nausea and vomiting) a global health and quality of life scale and an array of single-item symptom measures. After revisions in the role functioning global health and physical functioning scale internal consistency of the subscales varied between .65 and .94 except for the cognitive functioning scale with ? varying from .56 to .63 [474950]. Test-retest reliability varied from .63 to .86 [51]. The lung cancer questionnaire module is designed to supplement the core questionnaire and comprises specific symptoms associated with lung cancer (coughing haemoptysis dyspnoea pain) and side-effects from conventional chemo- and radiotherapy (hair loss neuropathy sore mouth dysphagia). While the multi-item dyspnoea scale showed high internal consistency the pain subscale did not. When combined with the dyspnoea and pain items of the core questionnaire both the dyspnoea (? = .86) and pain (? = .71) subscale showed high internal consistency. Since the QLQ-C30 and QLQ-LC13 are mainly focused on physical symptoms we added the items Social Interaction and Alertness Behavior of the Sickness Impact Profile (SIP) [52]. Internal consistency was .94 and test-retest reliability was .92. The SIP correlated with self-assessed sickness and dysfunction [52]. Caregiver appraisal (only for partners) We use the 9-item Self-Perceived Pressure from Informal Care (SPPIC) [53] to assess the extent to which caregiving is experienced as burdensome. To also measure positive aspects of caregiving the 9-item subscale Care-Derived Self-Esteem of the Caregiver Reaction Assessment (CRA-SE) [54] is included. Internal consistency of the SPPIC was .79 and of the CRA-SE was .73. The SPPIC and CRA-SE were unrelated to each other [55]. Relationship quality To measure relationship satisfaction we included the 10-item Satisfaction subscale of the Investment Model Scale (IMS-S) [56]. The IMS-S starts with 5 items that measure concrete examplars of satisfaction to enhance the comprehensibility of the global items which are utilized to form the construct. Internal consistency varied from .79 to .95 and the IMS-S was related to the Dyadic Adjustment Scale. Also the Mutual Interpersonal Sensitivity scale (MIS) [57] is included to measure communication between partners about the cancer. It contains 18 items and is divided into two scales: open communication and avoiding negative thoughts about the cancer. Spirituality is measured with the Spiritual Attitude and Involvement List (SAIL) [58] and consists of 26 items divided into the subscales meaningfulness trust acceptance caring for others connectedness with nature transcendent experiences and spiritual activities. The internal consistency varied from .74 to .88 and test-retest reliability varied from .77 to .92. All subscales except for connectedness with nature were related with the Functional Assessment of Chronic Illness Therapy Spiritual Well-Being Scale. Costs (only for patients) The cost-effectiveness evaluation is carried out from a societal perspective considering direct as well as indirect health costs. Data on costs are collected prospectively using a diary in which participants register a) health care utilization: the type of care and its duration and b) cancer-related absence from work. Unit cost estimates are derived from the national manual for cost prices in the health care sector [59]. Costs of reduced ability to work are estimated using the friction costs method which results in a more realistic estimate than the human capital approach [60]. Treatment costs of MBSR are calculated using activity-based-costing methods thus measuring actual resources (time of therapist time of patients facilities) used. All unit cost prices are adjusted to 2013 prices. Unit cost estimates are combined with resource utilization data to obtain a net cost per patient over the entire follow-up period. Process measures Mindfulness skills are examined with the 39-item Five Facet Mindfulness Questionnaire (FFMQ) [6162]. The FFMQ is based on an exploratory factor analysis of five mindfulness measures which allowed items from different instruments to form factors providing an empirical integration of these independent attempts to operationalize mindfulness. This led to the following five subscales: observing describing acting with awareness non-judging of inner experience and non-reactivity to inner experience. Internal consistency varied from .72 to .93 among the different subscales. Most subscales were related to meditation experience Psychological Well-Being scales and psychological symptoms including the Brief Symptom Inventory [61]. FFMQ is sensitive to change in mindfulness-based interventions and is found to mediate the relationship between mindfulness practice and improvements in psychological symptoms (e.g. [63]). Self-compassion is assessed with the Self Compassion Scale (SCS) [6465] which has 26 items and is divided into six subscales: self-kindness v" | 1 |
" microwave ablation mwa is widely used to treat unresectable primary and secondary malignanciesof the liver and a limited number of studies indicate that ablation can cause not only necrosis at the in situ site butalso an immunoreaction of the whole body this study aimed to investigate the effects of mwa on cytokines inpatients who underwent mwa for a hepatic malignancymethods patients admitted to the oncology department in the first affiliated hospital of soochow universitybetween june and february were selected peripheral blood was collected from patients with a hepaticmalignancy treated with mwa the levels of cytokines il2 ifnÎ tnfα il12 p40 il12 p70 il4 il6 il8 il10and vascular endothelial growth factor vegf were detected with a milliplex® map kit the comparison times wereas follows before ablation h after ablation days after ablation and days after ablation data were analyzedusing a paired sample ttests and spearmans correlation analysisresults a total of patients with hepatic malignancies were assessed there were significant differences in il2il12 p40 il12 p70 il1 il8 and tnfα at h after mwa significant increases 2fold vs before ablation wereobserved in il2 il1 il6 il8 il10 and tnfα after mwa elevated il2 and il6 levels after ablation werepositively correlated with energy output during the mwa procedures wa treatment for hepatic malignancies can alter the serum levels of several cytokines such as il2 and il6keywords microwave ablation hepatic malignancy cytokines il2 il6 immunoregulation primary and secondary malignancies of the liver have asubstantial impact on morbidity and mortality worldwidein china hepatocellular carcinoma hcc has the secondhighest mortality rate of malignancies the treatmentof primary and secondary hepatic malignancies via correspondence lengbengsudaeducn jing zhao qiang li and merlin muktiali contributed equally to this work2department of oncology the first affiliated hospital of soochow universitysuzhou china5division of neurosurgery city of hope beckman research institute duartecalifornia usafull list of author information is available at the end of the interventional imaging therapy is undertaken by investigators in the field of interventional radiology and possibly bya smaller group of practitioners known as interventionaloncologists whose major focus is cancer care via minimally invasive approaches [ ] recently percutaneous ablation therapy has been widely accepted as a radicaltreatment method for hcc and its fiveyear survival rateis similar to that of resection microwave ablationmwa is widely used to treat unresectable hcc and recurrent hcc and has the advantages of minimal invasiona good curative effect and no side effects due to radiationor chemotherapy immune checkpoint inhibitors icis the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0czhao bmc cancer page of such as pd1pdl1 and ctla4 antibodies have beenwidely applied in several cancers and studies have indicated that ici treatment could enhance the effect of ablation evidence hasindicated that hyperthermicdestruction causes the release of a large population of heterogeneous tumor antigens and inflammatory cytokinesmay play crucial roles in this process cytokines aremediators that regulate a broad range of processes involved in the pathogenesis of cancer several cytokineswhich can arise from either tumor cells or immunocytes such as tumor necrosis factor tnfα interleukinil1 il6 il8 il10 and vascular endothelial growthfactor vegf have been linked with cancers and can either promote or inhibit tumor development the serumlevels of cytokines differ during cancer development although cytokines have been found to be altered after anticancer treatment such as chemotherapy and radiotherapy[ ] few investigations have focused on cytokines beforeand after mwa it is still unknown whether the above cytokines changed before andor after mwa in patientswith hepatic malignancies in this study we investigatedthe effects of mwa on the serum levels of cytokines inpatients with hepatic malignanciesmethodspatients and samplesthe patient population examined in this study was derivedfrom the first affiliated hospital of soochow universitypatients were admitted to the oncology department between june and february the total number ofpatients was with liver metastases and primaryliver cancers the inclusion criterion was a tumor locatedat a hepatic site either primary or metastases all patients with metastatic hepatic malignances should be givensystematic treatments chemotherapy or target therapyand get at least stable disease sd or partial responsepr for more than days informed consent for blooddraw and the relevant therapy was obtained from all patients the protocol was approved by the human ethicscommittee of the first affiliated hospital of soochowuniversity and was conducted in accordance with thedeclaration of helsinki all written informed consent wasobtained from all participants and clearly stated wholeblood ml was drawn into edta anticoagulant tubeson days to before and h days and days afterablation mostly on the last day of the course for cytometry and cytokine analysesablation procedurethe ablation procedure used in this research was mwathe puncture site and pathway were determined underthe guidance of a computed tomography ct scanlocal infiltration anesthesia was achieved by using lidocaine the placement of microwave ablation probeswas guided by a ct scan or ultrasonic device and allprobes were placed at the maximum diameter layerdouble probes were employed when the maximumdiameter of the tumor was up to cm the power andtime of ablation were designed for each patient in therange of w and min respectively basedon the size number and position of the tumor theboundaries of ablation zones were designed as extended cm upon the tumor sitecytokine detectiona milliplex map kit with human cytokinechemokinepanels that measured ifnÎ il2 il6 il8 il10 il12p40 il12 p70 il1 tnfα and vegf was utilized according to the manufacturers instructions briefly chemically dyed antibodybound beads were mixed withstandard or sample incubated overnight at °c washedand then incubated with a biotinylated detection antibodyafter the beads were washed they were incubated with astreptavidin phycoerythrin complex and the mean fluorescent intensities were quantified on a luminex analyzer luminex corporation all samples were measured in duplicate standard curves of known concentrations of recombinant human cytokineschemokines wereused to convert fluorescence units to cytokine concentration units pgml the minimum detectable concentrations were as follows ifnÎ pgml il2 pgmlil12 p40 pgml il12 p70 pgml il1 pgml il6 pgml il8 pgml il10 pgml tnfα pgml and vegf pgml all resultsbelow the minimum concentrations were processed as theminimum concentrationsstatistical analysisibm spss statistics software was used for the statistical analysis along with graphpad prism for figurecreations normally distributed numerical data areexpressed as the mean ± standard deviation and nonnormally distributed numerical data are expressed as themedian and confidence interval ci cytokinesat different times were compared using a onetailedpaired ttest spearmans correlation analysis was executed to determine the correlation between clinical indexes and cytokine levels p indicates a significantdifferenceresultsclinical characteristics of the enrolled patientsas shown in table a total of patients with tumorslocated on the liver liver metastases primary livercancers were analyzed the patients cytokine levelswere compared according to time before treatment h after treatment days after treatment and daysafter treatment 0czhao bmc cancer page of table clinical characteristics of the patients enrolled n characteristicsexmalefemaleagepathogenesisprimarysecondaryprimary site for metastatic hepatic malignancescolon rectalpancreasstomachebreastothersmaximum tumor length mmablation probe usedablation time minaverage power per probe w ± ± ± ± average energy time à power time à power¼¼ time and power indicate the time and power respectively ofdifferent probes used during the operation ± ifnÎ il12 p40 and il12 p70 were slightly increasedafter mwa treatmentas shown in table and fig the median level ofifnÎ before the mwa treatment was pgml ci pgml at days and days after themwa treatment there was a slight increase comparedto that premwa with median levels of pgml ci pgml and pgml ci pgml respectively the median level of il p40 before the mwa treatment was pgml ci pgml there was a slight increase to pgml ci pgml days postmwathe median il12 p70 level before the mwa treatmentwas pgml ci pgml and increasedto pgml ci pgml days afterthe mwa treatment and to pgml ci pgml days postmwa no significant alteration in the vegf median level was detected after themwa treatmentil2 il1 il6 il8 and il10 were elevated over 2foldafter the mwa treatmentas shown in table fig and fig the median levelof il2 before the mwa treatment was pgml ci pgml there was a significant increase at h postmwa with a median level of pgml ci pgml the median level ofil1 before the mwa treatment was pgml ci pgml and a significantincrease wasnoted days after the mwa treatment pgml ci pgml the median level of il6before the mwa treatment was pgml ci pgml and significantly increased daysafter the mwa treatment pgml ci pgml the median level ofil8 before themwa treatment was pgml ci pgml and increased significantly to pgml ci pgml days after the mwa treatmentthe median level of il10 before the mwa treatmentwas pgml ci pgml and increasedsignificantly days after the mwa treatment pgml ci pgml the median level oftnfα before the mwa treatment was pgml ci pgml and increased significantlyto pgml ci pgml days afterthe mwa treatmentlevelselevated il2 and il6 levels after ablation were positivelycorrelated with energy output during mwato further evaluate the relationship between the increased cytokineand mwa treatment weemployed the concept of energy time à power time à power time and power indicated thetime and power of different probes used in the operation to reflect total hyperthermic damage to hepatictissues during the mwa procedure as shown in table and fig the il2 levels at h postmwa and the il levels at days postmwa illustrated significant correlations with energy the relative indexes were and respectivelydiscussionas technology continues to develop other types of localtherapy such as radiotherapy chemical ablation andhyperthermal ablation for primary and metastatic livercancer are increasingly being used mwa for liver malignances is reserved for patients who cannot undergosurgical removal or for whom other treatments havefailed a consensus guideline was recently developed to address indications for mwa in these patientsthermal ablation is a process that heats the target tissueto a temperature that causes immediate coagulative necrosis usually over °c terminal treatment requiresthat a necrotic area surrounds the target site with anadditional 10mm margins however in the liverhigh tissue perfusion and large blood vessels can cause aheat sink effect around the ablation zone making itdifficult to achieve terminal ablation the heat sink 0czhao bmc cancer page of table median levels of cytokines before and after mwacytokineifnÎil2premwa pgml ci ci ci ci ci ci ci ci ci ci h postmwa pgml ci ci ¼ ci ci ci ci ci ci ci ci il12 p40il12 p70il1il6il8il10tnfαvegf p vs premwa ¼ 2fold vs premwa days postmwa pgml ci ci ci ci ci ¼ ci ¼ ci ¼ ci ¼ ci ¼ ci days postmwa pgml ci ci ci ci ci ci ci ci ci ci effect can lead to sublethal temperatures and the retention of malignant cells thereby increasing the likelihoodof local tumor progression ltp however an incompletely ablated zone containing immune cells andcancer cells as well as functional vessels could establisha serious inflammatory site that may provide tumorspecific antigens cytokines and activated immune cellsin our study significant increases in the secretion ofchemokines il8 proinflammatory cytokines il1il12 ifnÎ and tnfα and antiinflammatory cytokines il10 were observed after mwa il8 is mainlyproduced by macrophages the classical biological activity of il8 is to attract and activate neutrophils whichcan lead to a local inflammatory response however recent studies have indicated that il8 both macrophageand cancer cellderived can recruit myeloidderivedsuppressor cells mdscs into the tumor microenvironment eventually inhibiting antitumor immunity andpromoting cancer progression [ ] il1 is mainlyproduced by macrophages b cells and nk cells couldproduce il1 under certain circumstances generallycells can only synthesize and secrete il1 after beingstimulated by foreign antigens or mitogens il1 couldpromote the th1 response promoting the activation ofdendritic cells dcs and cytotoxic t lymphocytesctls il12 is mainly produced by b cells and macrophages human il12 is a heterodimer with two subunits p40 kd and p35 kd which areinactivated in isolated form in general il12 functionsas a combination of two subunits il12 p70 while p40alone possesses partial functions of il12 p70 its mentionable that il12 p40 and p35 are not expressed inequal proportions so the amounts of il12 p40 and il p70 are different in one cell il12 can stimulate theproliferation of activated t cells and promote the differentiation of th0 cells into th1 cells moreover il12could induce the cytotoxic activity of ctls and nk cellsand promote the secretion of several cytokines such asifnÎ and tnfα previous research indicatedthat tnfα may play a crucial role in mwa in combination with immunotherapy notably our data illustrated that the il12 results were consistent with thoseof ifnÎ after the ablation operation but not with thoseof tnfα this result indicated that upregulation ofifnÎ may be a major effect of the il12 increase aftermwa on the other handan antiinflammatory and immunosuppressive cytokine wasevaluated after mwa il10 is a multicellularderivedmultifunctional cytokine that regulates cell growth anddifferentiation and could participate in inflammatoryand immune responses il10 was reported to increaseafter thermal ablation in the literature [ ] strategiesto inhibit il10induced immunosuppression after thermal ablation treatment would be of interestil10asablation therapy can mediate antitumor immunity astumor tissue necrosis caused by ablation may release various antigens that eventually form a kind of in situ vaccination moreover ablative therapy can not onlydirectly kill cancer cells in situ but also regulate immunecells and promote the immune function of patients withliver cancer [ ] many immunoregulatory cytokineswere released or expressed after thermal ablation notablythe cytokines released after thermal ablation can regulatethe positive and negative aspects of the cancer immunecycle previously researchers demonstrated that proinflammatory cytokines such as il1 il6 il8 il18 andtnfα were increased several hours or days after thermalablation [ ] to our knowledge terminal tumorthermal ablation may not only cause local heat injury intissues surrounding the tumor site but also induce a systemic reaction this systemic reaction would becaused by different mechanisms first interventional operation may result in trauma to the liver although this procedure is very minimally invasive the healing process maycause alteration of some cytokines second heat injurycould cause acute thermal necrosis in liver and tumor 0czhao bmc cancer page of fig levels of cytokines before and after mwa treatment slightly increased ifnÎ il12 p40 and il12 p70 levels after mwa treatment over fold enhancement of il2 h postmwa and of il1 il6 il8 il10 and tnfα d postmwa p 0czhao bmc cancer page of fig trends in cytokines significantly altered after mwa treatment the levels of il2 at h postmwa il1 at d postmwa il6 at dpostmwa il8 at d postmwa and il10 at d postmwa were elevated over 2fold compared to the levels premwatable correlation between the ablation energy and significantly elevated cytokinesenergyvsil2 h postmwaenergyvsil1 d postmwaenergyvsil6 d postmwaenergyvsil8 d postmwaenergyvsil10 d postmwaenergyvstnfα d postmwaspearmans rp value onetailed p 0czhao bmc cancer page of fig correlation between the ablation energy and the serum levels of il2 and il6 the serum levels of il2 at h postmwa and il6 at dpostmwa were positively correlated with energy output during the mwa procedureand nonspecifictissues and release of necrotic tissue fragments into bloodcould cause immunological reactions including nonspecific and specific reactions generally cytokines affectedby wound healingimmunologicalreactions do not last longer than those affected by specificimmunologicalreactions ablation treatmentinducedspecific immunological reactions are more complicatedand could affect more immunocytes [ ] which wouldmake this process last longer than other reactions theseexplanations may be the reason why the cytokine changeslasted different durations moreover cytokines affected bythe second manner would be positively correlated withthe ablation scale which is why we employed the energyindex in our ablation operation design to receive a terminal ablation larger tumor would cost higher energy including higher power and longer duration time terminaltumorthermal ablation would release tumorrelatedneoantigen to blood circulation eventually induce a systemic reaction this reaction is dependent on the scale ofthermal injury and the local immunological microenvironment of the tumor our findings indicated that il2 andil6 were significantly altered after the ablation procedureand positively correlated with mwa energy il2 is commonly derived from activated t cells primarily th1 cellsil2 can stimulate t cells to proliferate and differentiateactivate natural killer nk cells and macrophages and enhance the functions of cytotoxic t lymphocytes ctls our data illustrated that il2 is significantly increased at h after mwa indicating that il2 may induce a nonspecific immune response after mwa but il decreased after h postmwa in our study suggesting that the il2induced immune response may not belong lasting mentionable many cytokines detected il8il1 il12 were mainly derived from macrophagewhich was a widely distributed antigen presenting cellthis result support the theory that mwa could releasefragment of cancer cells into blood as neoantigen macrophages could response to this proceed and cause a systemic immunoreaction additional cytokines alterationsuch as il6 after ablation may be no anspecific inliver evidences indicate that increase of il6 was not onlyoccurred in liver ablation researches focus on lung cancerincluding primary lung cancer and pulmonary metastasesdemonstrated that serum il8 il1 il6 il10 il12and tnfα were significantly raised after radiofrequencythermal ablation moreover joseph found that imageguided thermal ablation of tumors located in lung liver orsoft tissues increases plasma levels of il6 and il10 another question remain unveiled was if our result wascancerspecific we checked literature about cytokinemodulation after thermal ablation in benign diseases andonly got limit evidences based on benign thyroid nodules and adenomyosis according to these literatureil6 levels did not show any significant difference aftertreatment compared with pretreatment values indicatingthat elevation of il6 may be caused by tumour antigenreleased by ablation treatment however the ablationenergy used in thyroid nodules was much lower thanliver and lung which would lead to a false negativein cytokine detection to the research about adenomyosis on the other hand experiment design was determined to followup the il6 at months afterhifu ablation as our data demonstrated mostly cytokines were return to premwa level after monthdetection after months may miss the modulation ofil6 overall few evidences support that some of thecytokines were altered in a cancerspecific mannerwhile no solid results could confirm that further animal experiments were required to make a clarifieddata and answer this question 0czhao bmc cancer page of thetumorassociated immunein recent years ablationinduced systemic effects suchasresponse haveattracted increased attention de baere t first reported two cases of spontaneous regression of multiplepulmonary metastases occurring after radiofrequencyablation of a single lung metastasis although growing evidence suggests that thermal ablation can inducespontaneous regression of the socalled abscopal effecton distant tumors the incidence rate of such an effect israre probably due to uncontested immunological activation caused by one ablation treatment and the lack ofimmuneamplification management in it was described that in situ tumor destruction can provide a useful antigen source forthe induction of antitumorimmunity however clinical studies could not sufficiently utilize such an effect until the development ofimmune checkpoint inhibitors icis [ ] icis suchas pd1pdl1 and ctla4 antibodies are widely applied in several cancers and studies have indicated thatici treatment could enhance the effect of ablation evidence indicates that hyperthermic destruction causesthe release of a large population of heterogeneous tumorantigens and inflammatory cytokines may play crucialroles in this process however opposite evidence indicated that incomplete radiofrequency ablation couldinduce inflammation which may accelerates tumor progression and hinders pd1 immunotherapy suggesting that ablation treatment may promote tumorprogression our data demonstrated that il6 was significantly increased after mwa treatment il6 is derived from monocytes macrophages dcs th2 cells andsometimes cancer cells and it plays a key role in t cellproliferation and survival the role of il6 appearsto be rather complex korn classified il6 as differentiation factor which could involve in differentiation ofth17 cells however il6 does not direct the commitment to the th1 or th2 cell lineage but controls theproliferation and survival of immunocytes cooperatingwith other cytokines such as tgf tnf or il21 for instance il6 activated stat3 pathway in naivecd4 t cells in the presence of the morphogen tgfbpromotes the population expansion of th17 cells recent evidence indicates that il6 plays an indispensable role in t cellinfiltration to the tumor sitewhich could benefit immunomodulatory therapy incontrast il6 can increase mdscs inhibit the development and maturation of dendritic cells dcs and inhibit the polarization of th1 cells eventuallyresulting in negative immunomodulatory effects according to muneeb ahmeds work the adjuvant uses ofa nanop smallinterfering rna sirna can besuccessfully used to target the il6mediated locoregional and systemic effects of thermal ablation il6 knockout via a nanop antiil6 sirna in mice coulddecrease the local vegf level at the ablation site therefore how to utilize the positive effect of il6 whileavoiding the negative effect after mwa needs further investigation preclinical research indicated that il6 andpdl1 blockade combination therapy reduced tumorprogression in animal models [ ] thus an antiil strategy after ablation should be considered whencombined with ici therapy previous studies and ourshave demonstrated that most cytokine levels returned topretreatment levels days after ablation this resultsuggests that h to days after ablation may be optimal timing for additional immunomodulatory therapysour results reported here support the evidence for terminal tumor thermal ablation could cause heat injury totissues surrounding the tumor site and release neoantigento blood circulation eventually induce a systemic reactionthis reaction could lead to a detectable alteration of cytokine levels further investigation is required to revealwhether the cytokines altered by mwa treatment couldaffect cancer progression whether positive or negativeabbreviationsmwa microwave ablation hcc hepatocellular carcinoma icis immunecheckpoint inhibitors tnf tumor necrosis factor il interleukinvegf vascular endothelial growth factor sd stable disease pr partialresponse ct computed tomography ci confidence interval ltp likelihoodof local tumor progression mdscs myeloidderived suppressor cellsctls cytotoxic t lymphocytes nk natural killer sirna small interfering rnaacknowledgementsnot applicableauthors contributionsjz conceptualization data curation writingoriginal draft and writingreview and editing ql conceptualization and writingreview and editingmm conceptualization and writingreview and editing brconceptualization and writingreview and editing and collect samples yhexecute milliplex assay and collect data dpl patient enrollment executemwa ablation and collect samples zl execute mwa ablation and collectsamples dml patient enrollment execute mwa ablation and collectsamples yx execute milliplex assay and collect data mt conceptualizationand writingreview and editing rl conceptualization data curation formalanalysis visualization writingoriginal draft and writingreview and editingall authors have read and approved the manuscriptfundingthis work was supported by the national natural science foundation ofchina the natural science foundation ofjiangsu province of china bk20140295 the jiangsu governmentscholarship for oversea studies js2018179 and the six one projects forhighlevel health personnel in jiangsu province lgy2018077availability of data and materialsthe datasets used andor analysed during the current study are availablefrom the corresponding author on reasonable requestethics approval and consent to participatethe protocol was approved by the human ethics committee of the firstaffiliated hospital of soochow university and was conducted in accordancewith the declaration of helsinki patients were informed that the bloodsamples were stored by the hospital and potentially used for scientific 0czhao bmc cancer page of research and that their privacy would be maintained all written informedconsent was obtained from all participants and clearly statedconsent for publicationnot applicablecompeting intereststhere is no financial or personal relationship with other people oranizations that could inappropriately influence bias this workauthor details1department of radiation oncology the first affiliated hospital of soochowuniversity suzhou china 2department of oncology the first affiliatedhospital of soochow university suzhou china 3department of lymphatichematologic oncology jiangxi cancer hospital nanchang china4department of interventional radiology the first affiliated hospital ofsoochow university suzhou china 5division of neurosurgery city of hopebeckman research institute duarte california usareceived january accepted august referencesfu j wang h precision diagnosis and treatment of liver cancer in chinacancer lett bruix j han kh gores g llovet jm mazzaferro v liver cancer approachinga personalized care j hepatol suppls144rognoni c ciani o sommariva s bargellini i bhoori s cioni r facciorussoa golfieri r gramenzi a mazzaferro v transarterial radioembolizationfor intermediateadvanced hepatocellular carcinoma a budget impactanalysis bmc cancer nault jc sutter o nahon p gannecarrie n seror o percutaneoustreatment of hepatocellular carcinoma state of the art and innovations jhepatol yin j dong j gao w wang y a case report of remarkable response toassociation of radiofrequency ablation with subsequent atezolizumab instage iv nonsmall cell lung cancer medicine baltimore 20189744e13112shi l chen l wu c zhu y xu b zheng x sun m wen w dai x yang m pd1 blockade boosts radiofrequency ablationelicited adaptiveimmune responses against tumor clin cancer res lippitz be cytokine patterns in patients with cancer a systematic reviewlancet oncol 2013146e218jin yb zhang gy lin kr chen xp cui jh wang yj luo w changes ofplasma cytokines and chemokines expression level in nasopharyngealcarcinoma patients after treatment with definitive intensitymodulatedradiotherapy imrt plos one 2017122e0172264kim mj jang jw oh bs kwon jh chung kw jung hs jekarl dw lee schange in inflammatory cytokine profiles after transarterial chemotherapy inpatients with hepatocellular carcinoma cytokine gillams a goldberg n ahmed m bale r breen d callstrom m chen mhchoi bi de baere t dupuy d thermal ablation of colorectal livermetastases a position paper by an international panel of ablation expertsthe interventional oncology sans frontieres meeting eur radiol ahmed m solbiati l brace cl breen dj callstrom mr charboneau jwchen mh choi bi de baere t dodd gd 3rd imageguided tumorablation standardization of terminology and reporting criteriaa 10yearupdate radiology chiang j hynes k brace cl flowdependent vascular heat transfer duringmicrowave thermal ablation conf proc ieee eng med biol soc huang hw influence of blood vessel on the thermal lesion formationduring radiofrequency ablation for liver tumors med phys najjar yg rayman p jia x pavicic pg jr rini bi tannenbaum c ko jhaywood s cohen p hamilton t myeloidderived suppressor cellsubset accumulation in renal cell carcinoma parenchyma is associated withintratumoral expression of il1beta il8 cxcl5 and mip1alpha clin cancerres alfaro c teijeira a onate c perez g sanmamed mf andueza mp alignanid labiano s azpilikueta a rodriguezpaulete a tumorproducedinterleukin8 attracts human myeloidderived suppressor cells and elicitsextrusion of neutrophil extracellular traps nets clin cancer res kundu m roy a pahan k selective neutralization of il12 p40 monomerinduces death in prostate cancer cells via il12ifngamma proc natl acadsci u s a onishi h kuroki h matsumoto k baba e sasaki n kuga h tanaka mkatano m morisaki t monocytederived dendritic cells that capture deadtumor cells secrete il12 and tnfalpha through il12tnfalphanfkappabautocrine loop cancer immunol immunother yu z geng j zhang m zhou y fan q chen j treatment of osteosarcomawith microwave thermal ablation to induce immunogenic cell deathoncotarget yang w wang w liu b zhu b li j xu d ni y bai l liu gimmunomodulation characteristics by thermal ablation therapy in cancerpatients asia pac j clin oncol 2018145e490erinjeri jp thomas ct samoilia a fleisher m gonen m sofocleous ctthornton rh siegelbaum rh covey am brody la imageguidedthermal ablation of tumors increases the plasma level of interleukin6 andinterleukin10 j vasc interv radiol den brok mh sutmuller rp van der voort r bennink ej figdor cg ruerstj adema gj in situ tumor ablation creates an antigen source for thegeneration of antitumor immunity cancer res zerbini a pilli m laccabue d pelosi g molinari a negri e cerioni sfagnoni f soliani p ferrari c radiofrequency thermal ablation forhepatocellular carcinoma stimulates autologous nkcell responsegastroenterology zhang h hou x cai h zhuang x effects of microwave ablation on tcellsubsets and cytokines of patients with hepatocellular carcinoma minim | 0 |
" among eukaryotic anisms alternative splicing is an important process that can generate multipletranscripts from one same precursor messenger rna which greatly increase transcriptome and proteome diversitythis process is carried out by a superprotein complex defined as the spliceosome specifically splicing factor branchpoint binding protein sf1bbp is a single protein that can bind to the intronic branchpoint sequence bpsconnecting the ² and ² splice site binding complexes during early spliceosome assembly the molecular functionof this protein has been extensively investigated in yeast metazoa and mammals however its counterpart inplants has been seldomly reportedresults to this end we conducted a systematic characterization of the sf1 gene family across plant lineages inthis work a total of sequences from plant species were identified phylogenetic relationships of thesesequences were constructed and subsequent bioinformatic analysis suggested that this family likely originatedfrom an ancient gene transposition duplication event most plant species were shown to maintain a single copy ofthis gene furthermore an additional rna binding motif rrm existed in most members of this gene family incomparison to their animal and yeast counterparts indicating that their potential role was preserved in the plantlineage our analysis presents general features of the gene and protein structure of this splicing factor familyand will provide fundamental information for further functional studies in plantskeywords alternative splicing expression profile phylogenetics plants promoter splicing factor correspondence fyzhunjfueducn kailu zhang zhen feng jingfang yang and feng yang contributedequally to this work1coinnovation center for sustainable forestry in southern china college ofbiology and the environment nanjing forestry university nanjing jiangsu province chinafull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0czhang bmc plant biology page of in eukaryotes canonical splicing removes noncoding intronic sequences and assembles the coding elements intomature mrnas while alternative splicing as generatesdifferent multiple transcripts that encode proteins withdistinct structures and functions by differential usage ofexons or splice site [ ] the resulting transcripts ofas greatly contribute to posttranscriptional regulationbiological complexity and proteome diversity in eukaryotes [ ] given that on average there are approximately exons in each transcript in the humantranscriptome and the degenerative nature of corresponding splice sites premrna splicing is sophistically catalysed by the spliceosome spliceosome is amultimegadalton protein complex which consists offive u1 u2 u4 u5 and u6 small nuclear ribonucleoprotein ps snrnps and over spliceosomalproteins furthermore the early assembly of spliceosome complex e or the commitment complex is anatpindependent process and contains u1 snrnps sf1and u2 snrnp auxiliary factors u2af large and u2afsmallthe prespliceosome complex a is formed by replacing sf1 withsf3b155sap155 of u2 snrnps [ ] stepwiseassembly of the following spliceosome during the splicing reaction has been reported as well [ ] however splice site recognition is a critical step during earlyassembly of the spliceosome the current model describes the binding of u1 snrnp and u1 snrna to ashort stretch of nucleotides at the ² splice site of splicing factor sf1mammalian branch point bindingprotein mbbp at the branch point and of u2 snrnpauxiliary factors at the ² splice site these threeciselements are necessary but usually insufficient to define a specific exonintron boundary thus additionalsplicing enhancers or silencers located at exons and introns may allow the recognition of genuine splice sitesduring early spliceosome assembly [ ] subsequentlysubunitsimportantly sf1 preferentially binds to the intronbranch point sequence bps which is adjacent to thebinding site polypyrimidine tract py of u2af largesubunits mammal u2af65 and fission yeast u2af59bridging u1 and u2af to form an intermediate lariatstructure [ ] in particular sf1 is characterized bythe presence of two types of rna binding motifs at thenterminus a k homologyquaking khqua2 domain which originated from the human heterogeneousribonucleoprotein hnrnp k protein [ ] and oneor two zinc knuckle motifs cx2cx4hx4c x represents any amino acid sf1 also contains a prolinerichregion at cterminus [ ] intriguingly the yeast khdomain specifically binds to the bps of premrnas witha glyproarggly motif and the variable loop of thekh domain and is necessary for spliceosomeassembly the first but not the second zinc knuckledomain in yeast has been demonstrated to bind rnawith high affinity moreover the stability of thesf1u2af65rna complex is further affected by thephosphorylation status of several sf1 serine residuesser20 ser80 and ser82 in vitro the prolinerichregion of sf1 interacts with u1 snrnp prp40fbp11 inyeast and human [ ] in regards to its interactionpartner the u2af large subunit the nterminal of sf1interacts with its noncanonical rna recognition motifsrrm or u2af homology motif[ ]whereas the other two rrms of u2af large subunitbind to the py region uhmsaccharomycescerevisiae common fruita previous study in fission yeast schizosaccharomycespombe suggests that the initial corecognition of thebranch site and ² splice site is pivotal for correct splicing of target premrnas because of the importance of splice site recognition for gene expression andprotein diversity sf1 has been demonstrated to play essential roles in a number of eukaryotic species includinghuman homo sapiens mice mus musculus buddingyeastflydrosophila melanogaster and roundworm caenorhabditis elegans [ ] for example in humansmissense mutation of splicing factors which are responsible for splice site recognition such as sf1 has beenlinked to tumourigenesis similarly heterozygoussf1 knockdown mice are susceptible to colontumourigenesis induced by an anotrophic carcinogenazoxymethane and sf1 has been found to associatewith betacatenintcf4 complex suggesting its role incarcinogenesis in contrast knockdown of sf1 suppresses the development of germ cell tumours in mice indicating its tissue dependency in cancer researchfurthermore the molecular function of sf1 has been extensively studied in yeast for instance a sf1 mutantstrain causes frequent exon skipping in fission yeast additionally sf1 has been proposed to recognize suboptimal sequences in specific introns and lead to nuclearaccumulation of premrna with aberrant splicing however increasing evidence indicates that this proteinis a regulator of splice site recognition and does not reduce general splicing specifically during alternative splicing by targeting a subset of genes [ ] thishypothesis is supported by the fact that knockdown ofsf1 in both yeast and human extracts only slightly affects the splicing outcome rnai targeting of thisgene has been demonstrated to not affect the splicingpattern of several splicing marker genes tested in comparison to studies in human and yeast few reports have been published related to plant sf1 genessimilar functions of the arabidopsis sf1 gene were proposed in an early study in this plant sf1homologue is reportedly responsible for the splicing of a 0czhang bmc plant biology page of to maintain itsgroup of transcripts the lossoffunction mutant atsf1 of this gene leads to abnormal development earlyflowering and dwarfism and aba or heat stress sensitivity in arabidopsis [ ] subsequently the domainstructure and its functional relationships have been substantially investigated and the rrm domain is considered crucialfunction in plantsmoreover sf1 may have a different mechanism of ²splice site recognition in plant because the plant sf1 homologs contain a different rrm domain compared withfungal and metazoan counterparts [ ] on the otherhand a study found that atsf1 may be likely to play afunctional role in the cytoplasm because it was found toshuttle between the nucleus and cytoplasm however no related investigations have been conducted onthe phylogenetic analysis of plant sf1 genes and theirregulatory mechanisms although it is a highly conserved family and has conserved functions in eukaryotesplant sf1 genes may have overlapping and distinct rolescompared to the mammalian genes hence studying thephylogenetic relationship and regulatory mechanism ofplant sf1 genes may make us understand the evolutionary history characteristics an expression profile of thisgene family and predict specific functions in plants thiscan lay the foundation for further functional studies inviridiplantae to this end we systematically identified sf1 sequences from plant species ranging fromalgae to higher plants meanwhile the gene and proteinstructure potential regulation at promoter regions andexpression pattern of these genes were further investigated in this study we hypothesize that plant sf1 isstructurally different from its counterparts in animalsand yeast but it is conserved among lower and higherplants indicating its specific role in alternative splicingin branch point recognitionthalianasf1methodssequence acquisition and identification of plant sf1genesthe arabidopsisprotein sequenceat5g51300 was used to search similar sequences inall available plant species from the phytozome v121databasehttpsphytozomejgidoegovpzportalhtml by running the blastp program with an evaluecutoff 1e10 the other parameters were the default settings then the retrieved protein sequences wereexamined and filtered using the hmmer score defaultsettings which contained pf16275 splicing factorkhomology domain kh_1 and pf00076 rna recognition motif rrm_1 finally putative sf1 sequencesfrom plant species were identified detailed information including groups plant species common namesand number of sf1 homologs reported for each planthelixhairpinsf1hhdomainpf00013species for subsequent analysis are listed in table s1subcellular location prediction of identified sf1 proteinswas carried out using wolf psort httpswolfpsorthgcjp construction of molecular phylogenetic tree of plant sf1genesprotein sequences of the aforesaid plant sf1 genes wereextracted from phytozome v121 database for phylogenetic relationship analysis the sequences with the longestcoding sequences were chosen for genes with multipledifferent splicing isoforms then multiple sf1 proteinsequences were aligned with the muscle v38 softwarewith default settings the molecular phylogenetictree of plant sf1 genes was then constructed using themaximum likelihood method ml jtt g i modelvia phyml v30 program with the following parametersinitial tree bionj discrete gamma model yes numberof categories gamma shape parameter proportion of invariant subtree patterns aliasing no figtree v143 was used to visualize and edit the phylogenetic treegene structure protein domain and multiple em for motifelicitation meme analysisrequired genomic cdna and peptide sequences and allsf1 gene structures were downloaded from the phytozome v121 database corresponding intron phases weregenerated using the online program gene structure display server gsds20httpgsdscbipkueducn correlation analysis of sf1 exons were performedby using the piece2 webserver httpwwwbioinfogenomenetpiecesearchphp tdsourcetags_pctim_aiomsg sf1 protein sequences were used to search formatching pfam families using the hmmer websitehttpswwwebiacuktoolshmmer then protein domain patterns were drawn by using tbtools software according to the full pfam resultanttableconserved motifs of plant sf1 cdna sequences andprotein sequences were analysed on the meme onlineprogram httpmemesuitetoolsmeme considering a maximum of the most preserved motifs predicted for each sequence and leaving other settings onthe default parametersmotif prediction in promoter regions of plant sf1 genesthe 15kb ²flanking sequences of plant sf1 geneswere extracted from genomic data available in phytozome database prediction of plant putative ciselementswas performed with the online server plantcarehttpbioinformaticspsbugentbewebtoolsplantcarehtml motifs related to tissuespecific expressioninternal hormones and external environmental stress response were selected for further analysis and discussion 0czhang bmc plant biology page of expression analysis base on microarray datasets and geneexpression experimentsexpression data of arabidopsis s tuberosum g max slycopersicum p trichocarpa and b distachyon includingtissue specificity and stress responses were extracted fromthe efp browser series of the bioanalytic resource forplant biology httpbarutorontoca expressionvalues of selected plant sf1 genes were log transformedlg to generate visualize expression difference heatmapsby using bar heatmapper tool program httpbarutorontocantoolscgibinntools_heatmappercgigene expression experimentstotal rna of samples from different plant tissues wereextracted by rneasy mini kit qiagen usa and subsequently reversed transcribed into cdna by fastkinggdna dispelling rt supermix fastking tiangenchina according to the manufacturers instruction rtpcr amplification were programmed asfollowings °c min °c s °c s °c s cycles °c min sybr premix ex taqtm accuratebiotechnology co ltd hunan china was used forquantitative realtime rtpcr analysis which was conducted on the stepone plus realtime pcr system following optimized program °c s °c s °c s cycles the data were normalized to the expression of internal reference genes table s6 and the transcript abundance was determined by the comparativect value method analysis of proteinprotein interaction network andstructural conservationa proteinprotein interaction network was generated bythe string website httpsstringdb withrepresentative protein sequences from arabidopsis thefollowing basic settings were employed meaning of network edges evidence line colour indicates the type ofinteraction evidence and active interaction sourcesexperimentsthere are three domains in the arabidopsis sf1 protein the phosphorylation and u2af65 binding of thenterminal domain of splicing factor during ² splicesite recognition of homo sapiens pdbid 2m0g identity evalue 7e17 was similar to that of the khomology domain the structure for recognition of theintron branch site rna by splicing factor of homo sapiens pdbid 1k1g identity evalue 9e27 canbe used as the template for the splicing factor helixhairpin domain therefore homology modelling wasperformed with modeller based on two crystalstructures the amino acid conservation scores were calculated using the consurf web server based on the mlmethod input attributes were the 3d model andmultiple sequence alignment figure s4 related figureswere created based on pymol with default settings analysis gene structure evolution with orthologue groupof sf1 genesreconstruction of the evolutionary history of the structure of the plant sf1 family of orthologous genes wascarried out by searching at5g513001 in the piece severhttpwwwbioinfogenomenetpieceindexphpthis provided an exonintron display for orthologousgenes from gene structure data sets linked to the phylogenetic treeresultssequence identification and phylogenetic analysis of theplant sf1 gene familyto identify sf1 gene family members in plants we carried out a blastp search using the arabidopsis atsf1at5g51300 amino acid sequence against the phytozome database v121 after filtering the sequence without sf1 signature or truncated sequences a total of sequences from plant species were retrieved whichwere roughly classified as algae bryophyta basicangiosperm monocots and eudicots table s1specifically the only species with four copies of plantsf1s was eutrema salsugineum salt cress table s1 inparticular three copies of sf1 genes were observed infive species including panicum virgatum switchgrasstriticum aestivum common wheat daucus carotacarrot kalanchoe laxiflora milky widows thrill andsalix purpurea purple osier willow additionally plant species contained two copies and species including the model plant arabidopsis possessed only onecopy of plant sf1s respectively the relatively largernumber of sf1 genes and higher number of plant speciesin this work demonstrated the universality and complexity of the sf1 gene family the retrieved sequences of plant species provided us with more complete information to analyse the phylogenetic relationship of the sf1gene family subsequently a rooted phylogenetic treewas constructed based on the abovementioned protein sequences by using the maximum likelihoodmethod the trees bootstrap threshold was represented by a colour gradient fig in general all sf1protein sequences were clustered into four major cladesincluding alga in yellow other land plants in greenmonocots in pink and eudicots in blue and one species amborella trichopoda belonged to basic angiosperm shown in colourless the phylogenetic tree ofsf1s figs and left panel with clear topology andoverall high bootstrap values was similar to evolutionarytrend from lower plants to higher plants reported inother studies for example the genes of algae in the yellow branch were representative members of the lineage 0czhang bmc plant biology page of fig circular phylogenetic tree of the sf1 gene family available in plants the phylogenetic tree of sf1 genes in plants was constructed basedon maximumlikelihood with jtt g model by using phyml v3037 a total of protein sequences from plant species were chosen tocalculate the phylogenetic relationship for tree construction bootstrap values are labelled at each major branch the corresponding informationof each transcript such as species name common name number of identified transcripts and their transcript id nomenclature are shown intable s1 taxonomies based on apgiv system 0czhang bmc plant biology page of fig see legend on next page 0czhang bmc plant biology page of see figure on previous pagefig gene structure comparisons and conserved motif identification among plant sf1 genes from left panel to right panel verticalphylogenetic tree genomic anization and identified cdna conserved motifs by meme analysis intron phase and are shown on thegene structure the conserved sequence of identified motifs represented by different coloured boxes are listed below some long genes werereduced to onehalf of their original length to fit this picturethat diverged before the evolution of land plants whichwas the basal part of the phylogeny in the blue branchfive sequences from kalanchoe with higher bs valuesformed a subclade showing their closer evolutionary relationships additionally cagra3782 s00261p fromcapsella grandiflora and carubv10025900m from c rubella formed a subclade with the arabidopsis sequencesbecause they all belong to brassicaceae which is consistent with the apg iv system fig and table s1 usually some homologous sf1 sequences from the samespecies were clustered in the same small branch next toeach otherthese species included cashew soybeanapple woodland strawberry quinoa carrot coloradoblue columbine maize common wheat cereal grassmoss and bog moss fig and table s1 in contrastsome other homologous sf1 members from the samespecies were clustered into the different subclades suchas purple osier willow poplar eastern cottonwood saltcress potato diploid kalanchoe milky widows thrillhalls panicgrass switchgrass green algae and volvoxfig and table s1gene structure and conserved motif analysisit is necessary to compare the exonintron anizationand conserved motifs of the plant sf1 gene family toclarify their evolutionary process and potential functionthe gene structure models of sf1 genes were attachedto the phylogenetic tree fig and the correspondingintron phase of each was also displayed fig tables2 figure middle panel shows that the gene lengthand structure of each member of the sf1 family exhibitssignificant differences for example the gene structureof members of sf1 family genes did not containintron sequences this subset accounts for of thetotal number of members fortyeight sequences of sf1genes had exon1 intron anizations accounting for of all genes in particular some genes from algaehad multiple exons including vocar0008 s02941p volvox carteri which contained the most exons exonsmoreover different gene structures were also observedat the same subbranch for instance two sequencesfrom zea mays maize zm00008a037777_p01 exonsand zm00008a007621_p01 exons were observed tohave distinctive gene structures although the dissimilation of gene structure of each member of sf1s was substantial we found that the length of cdss did notsignificantly change fig thus whether it influencesthe differentiation of their gene function needs to befurther investigated further investigation on conservedmotifs by using multiple em for motif elicitationmeme search tool demonstrated that most sf1 genes sequences exhibited similar sequence signatures andthe same order and all contained the analysed motifsexcept one sequence of micromonas pusilla hada different position fig right panel although no obvious differences in identified conserved motifs werefound among basal angiosperm monocots and eudicotssequences from the same species were found to have different motifs fig for example aqcoe5g4069001pand aqcoe7g0393001p from the eudicot aquilegiacoerulea had motifs and motifs respectively thesame situation was found in d carota dcar_006843dcar_008506 and dcar_004968 had motifs motifs and motifs respectively intriguingly the cdslength of dcar_008506 was the longest notably somesequences from algae and moss had fewer conservedmotifs for examplein bryophyta the sequences ofphyscomitrella patens pp3c7_10890v31p and pp3c11_24710v31p sphagnum fallax sphfalx0015s00771pand sphfalx0010s01971p and marchantia polymorphamapoly0009s01891p had nine motifs in algal plantsthe sequences of and from micromonashad only motifs and motifs respectively moreoveralthough the sequences of volvox carteri vocar0007s03451p and vocar0008 s02941p and chlamydomonasandcre09g386731t11 contained multiple exons they had motifs indicating their sequence variation had little influence on function classes further correlation analysisof the sf1 exon regions were carried out to elucidate thegainloss of introns correlations between transcripts ofplant sf1s are shown in fig providing additional information for phylogenetic analysis for example thereis more similarity between pgsc0003dmt400081859and migutd025312 because of multiple exact matchesbetween the exons of the two transcriptscre12g553750t11reinhardtiianalysis of protein domain and conserved motifs inpeptidesthe protein domains were analysed by using the aboveselected peptide sequences from plant species thepeptides annotations were splicing factorrelated andconserved protein motifs were predicted according tothe retrieved peptide sequences by meme analysisfig consequently all sf1s were found having sf1_hh nterminal domain on the nterminal ofthe 0czhang bmc plant biology page of fig analysis of gene structure evolution with orthologue group of sf1 genes exonintron structure and intron phase right panel are linked tothe plant species tree left panel genes with red colour represent the members of the plant sf1 genes different coloured lines mean differentexon comparison results between species 0czhang bmc plant biology page of fig comparisons of protein domains and conserved motif identification among plant sf1 genes protein domain middle panel and identifiedprotein conserved motifs right panel identified by meme analysis are shown against the vertical phylogenetic tree left panel the conservedsequence of identified motifs represented by different coloured boxes are listed below 0czhang bmc plant biology page of peptides followed by a kh domain and a cterminal domain namely an rna recognition motif rrm fig middle panel interestingly in algae peptides from mpusilla v carteri vocar0008 s02941p andc reinhardtii cre09g386731t11 had two rrm domains the amino acid lengths of sf1 proteins rangedfrom aa to aa and most of them possessed to amino acids table s3 consistently most ofthem are approximately to amino acids inlength subcellular location prediction showed that themajority of sf1 proteins were had nuclear localization table s3 moreover proteins of 147m014250 ricinus communis and migutf011911pmimulus guttatus were located in the vacuoles proteins of traes_2dl_6f03f05fa4 t aestivum and m pusilla were predicted to be cytoplasmic proteins of gsmua_achr5p25100_001 musa acuminataand cre09g386731t11 c reinhardtii were located inthe chloroplast and endoplasmic reticulum respectivelymeme analysis for sf1 peptide sequences was used topredict a total of conserved motifs which are presented as coloured boxes and cover most of the proteinfig right panel further analysis showed that peptides had all motifs accounting for approximately of all sf1 protein sequences analysed in the studyinterestingly all sequences from moss have conservedmotifs in the analysis suggesting the conservation ofsf1 proteins in bryophyta furthermore almost all eudicots had conserved motifsexcept anacardium occidentalegrandifloracagra3782 s00261p which lacked motif and malusdomesticavescamrna211921v10hybrid and brassica rapa brarac014811p which lacked motif while most monocots had eight conserved motifs in contrast algal plantsonly possess approximately half of the predicted motifs due to their peptides with integrant protein domainsimplying the least degree of conservation and divergenceof plant sf1 proteins in algae t motifs that all algaeshared were motif motif motif and motif anaoc0018 s04251pmdp0000558834fragariaand canalysis of promoter and tissuespecific expression of sf1genesto further analyse the regulation of plant sf1 genes atthe transcriptional level the 15kb upstream sequencesof plant sf1 genes were obtained from the phytozomedatabase then the ciselements of each promoter wereidentified by using the plantcare program table s4 consequently a total of motifs were predictedgenerally eight ciselements related to tissuespecificexpression among them were selected fig and tables4 including hdzip1 for differentiation of the palisademesophyll cells the ryelement which regulates seedspecific expression the aaca_motif and gcn4_motif waspresentfurther hdzipinvolved in endosperm expression and the catboxccgtccbox doct and oct for meristem expression further analysis showed that there were only promoters of sf1 genes which had tissuespecific regulatory ciselements particularly the catbox and ccgtccbox turned up at the highest frequency and greatestabundance in the promoters of sf1 genes both of themregulate meristemspecific expression and play key rolesduring development and growth of plants consistentlypurple false brome brachypodium distachyon of monocots not only had a catbox and ccgtccbox butwas also highly expressed in young leaves internode adventitious roots and roots fig and figure s2however no motifs were found to link the highexpression of two sf1s of glycine max soybean insam and roottip figure s1 additionally the aaca_motif was only detected in solanum tuberosumpgsc0003dmp400032853 of potato suggesting itsspecific role in regulating endospermspecific negativeexpressioninpodel03g1132001p of populus deltoideseasterncottonwood and spipo17g0046100 of spirodela polyrhiza greater duckweed the ryelement was detectedin the promoter of the dicot model plant arabidopsisand low expression was also reported in dry seed inarabidopsis fig suggesting that the ryelement isinvolved in seedspecific negative expression of arabidopsis moreover expression levels in the same tissuetype showed significant differences during differentgrowth stages for example the expression level in stamen of flower stage of arabidopsis was obviouslyhigher than that of the other flower development stageshowever the expression levels of different growth stagesof solanum lycopersicum were not only similar butlower and no motifs were found in the promoter in tomato figs and s1 furthermore different expressionpatterns were detected in several sf1 genes with multiple copies figs s1 and s6 for instance similar tissue expression profiles were detected in two sf1trichocarpahomologuespotri001g1264001 and potri003g1072001 and themonocotandzm00008a037777_p01 figure s1 and s5 in contrasttwo sf1 genes of s tuberosum showed differential expression patterns similar to in g max figs and s1zm00008a007621_p01dicot populuszea maysfrom theanalysis of promoter and internal and external hormonesexpression of sf1 genesin longterm evolution and development plants havegradually formed mechanisms of adaptation and resistance to adversity to maintain their life and sustaingrowth to understand the regulatory mechanisms of internal and external stimuli on plant sf1s cisacting elements involved in hormone and stress were studied with 0czhang bmc plant biology page of fig see legend on next page 0czhang bmc plant biology page of see figure on previous pagefig analysis of motifs related to tissue specificity in the plant sf1 promoter regions eight cisacting motifs are represented in different colortriangles positions of these identified motifs are labeelled along the kb ²flanking regions of each sf1 gene the line solid and dottedrepresents regions with basic pairs and regions of no sequences or annexed base n respectively symbols on above the line represent the motifsat the plus strand whereas symbols on below the line represent the motifs at the minus strand function of motifs aacamotif involved inendospermspecific negative expression catbox cisacting regulatory element related to meristem expression ccgtccbox cisactingregulatory element related to meristem specific activation doct cisacting regulatory element related to meristem specific activationgcn4_motif cisregulatory element involved in endosperm expression hdzip1 element involved in differentiation of the palisade mesophyllcells ryelement cisacting regulatory element involved in seedspecific regulation the black vertical lines represent break at that particularbranch oct cisacting regulatory element related to meristem specific activationlowtemperatureltr droughtthe plantcare database fig table s4 finally hormone and stressrelated motifs were selected from promoter sequences of plant sf1s there are hormonerelated motifs including abscisic acid abreauxin auxre auxrecore tgabox tgaelementethylene ere gibberellin garemotif pbox tatcbox meja cgtcamotif tgacgmotif and salicylic acid tcaelement and five stressrelated motifsincludingmbswound wunmotif and anoxic are gcmotif motifs almost each sf1 sequence had a great diversity ofciselements in its promoter regions except some sequences such as araha13031 s00021 and traes_2al_3d67296921 which did not contain a single motif dueto the sequences contain n or no promoter suggestingthat multiple hormonesmediated signalling pathwaysare closely related to sf1 plants resistance analysisshowed that more than half of sf1 promoters containedabre cgtcamotif tgacgmotif and are respectively moreover external hormone signals also affect theabundance of sf1 transcripts figure s3 for examplein arabidopsis at5g513001 mj methyl jasmonateinhibited its expression fig and treatment withother hormones like acc a precursor of ethylene iaaauxin | 0 |
as one of the most common gynecological malignant tumors cervical cancer is the fourth leadingcause of cancerrelated death among women worldwide although eï¬orts including periodiccancer screening prompt surgical treatment chemotherapy and radiotherapy have been madeto decrease the mortality of cervical cancer the prognosis of patients is still poor and cervicalcancer remains an important public health issue the pathogenesis of cervical cancer has notbeen clearly illustrated but it is conï¬rmed that the activation of tumorpromoting genes and theinactivation of tumor suppressor genes participate in the progression of cervical cancer toscreen for novel abnormally expressed genes functioning in cervical cancer may provide potentialprognostic markers and therapeutic targets for treatmentedited byihab youniscarnegie mellon university inqatar qatarreviewed byweifeng dingnantong university chinamassimo brogginimario negri pharmacologicalresearch institute irccs italycorrespondencelin xuxulin83njmueducnemei lulem13705179888sinacnbinhui renrobbishren163com these authors have contributedequally to this workspecialty sectionthis was submitted tocancer geneticsa section of the frontiers in oncologyreceived april accepted june published august citationzhu b wu y luo j zhang qhuang j li q xu l lu e and ren b mnx1 promotes malignantprogression of cervical cancer viarepressing the transcription ofp21cip1 front oncol 103389fonc202001307frontiers in oncology wwwfrontiersinaugust volume 0czhu mnx1 enhances progression of cervical cancermnx1 motor neuron and pancreas homeobox also knownas hb9 hlxb9 is a member of homeobox gene family andencodes a nuclear protein the homeobox genes are agroup of genes containing homeobox a base pairs longdna sequence and encode homeodomain proteins that actas transcription factors many homeobox genes have beenproved to be implicated in various human cancers acting asoncogenes or tumor suppressors mnx1 was ï¬rstly foundto be expressed in lymphoid and pancreatic tissues and deï¬nedas a novel human homeobox gene in early studiesshowed that mnx1 was involved invertebrate and pancreaticdevelopment and motor neuronal diï¬erentiation defects in this gene result in currarino syndrome an autosomicdominant congenital malformation in followup studymnx1 was found to be abnormally expressed in several cancertypes including prostate cancer hepatocellular carcinoma andacute myeloid leukemia furthermore recent studiesconï¬rmed that mnx1 played oncogenic roles in colorectalcancer breast cancer and bladder cancer the aim of this study is to identify the expression andfunction of mnx1 in cervical cancer our results revealedthat mnx1 was significantly upregulated in cervical cancerand correlated with poorer prognosis the knockdown oroverexpressed mnx1 inhibited or promoted aggressiveness ofcervical cancer including proliferation migration and invasioncapacities by enhancing or repressing the transcription of p21cip1thus regulating the g2m cell cycle transition these ï¬ndingssuggested that mnx1 might be a potential diagnostic marker andtherapeutic target for cervical cancermaterials and methodsbioinformaticsthe tcga dataset termed tcga_cesc_exp_hiseqv22015 was downloaded from the ucsc cancer browser genomecancerucscedu to evaluate the expression ofmnx1 in cervical cancer and adjacent normal tissues gepiagene expression proï¬ling interactive analysis httpgepiacancerpku cnindexhtml was used to analyze the expressionof mnx1 with disease free survival dfs of cervical cancerpatients the cbioportal website httpwww cbioportal was utilized to obtain highly coexpressed genes with mnx1totally genes highly correlated with mnx1 pearson score table s1 were submitted to david bioinformaticsresources httpdavidabccncifcrfgov for geneontology go kyoto encyclopedia of genes and genomeskegg and reactome pathway analysis and we analyzed thebinding site of mnx1 and p21cip promoters through the jaspardatabase httpjaspardevgeneregnet human cervical cancer cell linesthe human normal cervical celllines hacat and cervicalcancer cell lines hela siha caski and c33a were purchasedfrom american type culture collection atcc usa helasiha c33a and hacat cells were incubated in dmem mediumkeygen nanjing china and caski cells were cultured inrpmi1640 keygen nanjing china medium containing fetal bovine serum gibcobrl invitrogen carlsbad causa and cultured at ¦c in a humidiï¬ed incubator containing co2human cervical cancer tissuesthe pairs of cervical cancer tissues and adjacent tissues wereselected from the aï¬liated cancer hospital of nanjing medicaluniversity and informed consent was obtained from all subjectsall tumors and paired nontumor tissues were conï¬rmed byexperienced pathologists and no patients received chemotherapyor radiotherapy before surgery the mrna expression ofmnx1 and p21cip1 in cervical cancer tissues was detected byqrtpcr collection of human tissue samples was conductedin accordance with the international ethical guidelines forbiomedical research involving human subjects cioms thisstudy was approved by the ethics committee of the nanjingmedical university aï¬liated cancer hospitaltissue microarrayspaired cervical cancer tissue microarrays were obtained fromshanghai outdo biotech co ltd cat no odctrputr03 and odctrputr03006 totally pairs of paraï¬nembedded human cervical cancer sections were analyzed formnx1 expression all tissues were examined by two experiencedpathologists and the tnm stage was conï¬rmed in each patientwith blinded methods the sections were incubated with an antimnx1 primary antibody abcam ab79541 the ihcscores were calculated according to intensity and percentage ofpositive cells the staining intensity was evaluated as the basis offour grades negative staining 1weak staining moderatestaining or strong staining the product percentage ofpositive cells and respective intensity scores was used as the ï¬nalstaining scores a minimum value of and a maximum valueof rna preparation reverse transcriptionand qrtpcrtrizol reagent invitrogen carlsbad ca usa was used toextract total rna from tissue samples or cultured cells accordingto the manufacturers protocol a reverse transcription kittakara cat rr036a keygen was utilized to generate cdnaqrtpcr was performed with sybr select master mix appliedbiosystemscat keygen nanjing china and primersare shown in table s2western blottinglysis buï¬er ripa keygen containing protease inhibitorspmsf keygen was used to extract protein of cells andtissues and protein concentration was detected with a bcakit keygen protein samples µg were loaded into sodium dodecyl sulfate polyacrylamide electrophoresissdspage gels and transferred onto a pvdf membraneafter electrophoresis the membrane was blocked with nonfatmilk for h and incubated overnight with antibodies againstrespective antibodies mnx1 abcam ab79541 p21cip1cell signaling technology pthr161cdk1 cellsignaling technology cdk1 cell signalingfrontiers in oncology wwwfrontiersinaugust volume 0czhu mnx1 enhances progression of cervical cancertechnology p27kip1 cell signaling technology cyclinb1 abcam ab72 cycline1abcam ab3927 cycline1 abcam ab3927 cyclind1santa cruz biotechnology sc246 actinabcam ab15265 sirna and plasmid transfectionthe sirnas targeting mnx1 and p21cip1 were conductedand purchased from ribobio guangzhou china all sirnasequences are shown in table s3 the fulllength cdnaof human mnx1 were pcrampliï¬ed and cloned intothe expression vector pgpu6gfpneo vigene biosciencesshandong china the sirnas and overexpression plasmidswere transfected into cervical cancer cells according to thelipofectamine reagent invitrogen carlsbad ca usaprotocol nonsense rnai sinc and empty plasmids oencwas used as negative controls²analysiscell proliferation assaythe cell proliferation assays were performed h aftertransfection for real timexcelligencesystemrtca cells100 µl were seeded in eplates and theplates were locked into the rtca dp device in the incubator tocalculate the cell index value in colony formation assay a totalof cells were placed in afresh 6wellplate and the cells werestained with crystal violet solution after days for5ethynyl2deoxyuridine edu assay keyfluor488 clickitedu kit ribobio guangzhou china the transfected cells wereplaced in 96wellplates cellswell overnight in a co2incubator then cells were incubated with µlwell of µmedu for h at ¦c and ï¬xed with µl paraformaldehydecontaining pbs for min at room temperature subsequentlythe cells were cultured for min with µl of mgmlglycine and then washed with µl bsa in pbs afterpermeabilization with triton x100 for min the cellswere cultured with à clickit reaction solution for minat room temperature in dark conditions after that cells wereincubated with µlwell of à hoechst solutionsfor min at room temperature in the dark after washing with µl of pbs the cells were then imaged using ï¬uorescencemicroscopy and proliferation cell ratios were counted fromï¬ve random ï¬elds in every well each experiment was repeatedthree times a total of cells in a fresh sixwellplates weremaintained in medium containing fbs the medium wasreplaced every or days after weeks the cells were ï¬xedwith paraformaldehyde and stained with crystal violeteach experiment was repeated three timesmigration and invasion assayfor wound healing assay cells were growing on the 6wellplate then artiï¬cial scratch on a conï¬uent monolayer of cellswas created with a µl pipette tip the medium wasreplaced with the serumfree and cells imaged h later fortranswell and matrigel assay totally transfected cells wereadded to the upper chamber of transwell assay inserts µmpet 24well millicell or a matrigel coated membrane bdbiosciences containing µl serumfree dmem media thelower chambers were ï¬lled with µl dmem media containing fbs after a 24h migration assay or 48h invasionassay incubation the cells were ï¬xed with polyformaldehydestained with crystal violet and imaged migration and invasionwere assessed by counting cell nuclei from ï¬ve random ï¬elds onevery ï¬lter each experiment was repeated three times rtcawas also used to evaluated the ability of migration and invasioncimplates installation and baseline measurement was carriedout according to the instructions add µl of mixed serumfree cell suspension à cells to the upper chamber in cimplates and the plates were locked into the rtca dp device in theincubator to calculate the cell index valuecell cycle analysiscells were digested with trypsinedta and ï¬xed with ethanol for h at ¦c the ethanolsuspended cells werecentrifuged and stained with pi staining solution for minin the dark at ¦c a facscalibur ï¬ow cytometer was usedto detect cell cycle distribution the percentage of the cells ing0g1 s and g2m were counted and comparedchromatin immunoprecipitation chipcells were crosslinked in paraformaldehyde and the reactionwas quenched with glycine after two washes with cold pbscells were added with precooling pbs containing cocktailhalttm protease inhibitor cocktail thermo scientiï¬c and scraped into a centrifuge tube the cells were centrifugedfor min at g at ¦c then added with µl celllysis buï¬er containing µl cocktail and incubated onice for min cells were then centrifuged for min at à g ¦c and cell precipitates were resuspended in µlnucleus lysis buï¬er containing µl cocktail the cellswere sonicated amplitude on ice for min and solublechromatin was obtained by centrifuging for min at g at¦c five micrograms of antimnx1 antibody sigmaaldrichsab2101494 coupled to magnetic beads resin m2 sigmashanghai china was used to immunoprecipitate the dnaprotein complex and the igg antibody was used as a negativecontrol the immunoprecipitation products were washed with µl low salt buï¬er high salt buï¬er lici buï¬er and tebuï¬er successively all for min at ¦c the chip elution buï¬ercontaining proteinase k was used for dna puriï¬cation thebeads were wiped out on a magnetic frame and the dna waseluted with elution buï¬er c from adsorption column chipdna samples were subjected to pcr ampliï¬cation with primersspeciï¬c to p21cip1 promoter region pcr products were then usedfor agarose gel electrophoresis the sequence of primers used areshown in table s4 and gapdh was used as a controlluciferase reporter assaythe p21cip1 cdkn1a promoter region bp wasampliï¬ed and cloned into luciferase reporter plasmid pgl3basic the p21cip1 promoter wildtype plasmids or mutanttype plasmids were cotransfected with cmvmnx1 expressionplasmids in hek293t cells and cmvempty vectors were usedas a negative control relative luciferase activity was corrected forfrontiers in oncology wwwfrontiersinaugust volume 0czhu mnx1 enhances progression of cervical cancerrenilla luciferase activity of pgl3basic and normalized to theactivity of the controlxenograft modelall animal studies were conducted in accordance with nihanimal use guidelines and protocols were approved by nanjingmedical university animal care committee sixteen femalenude mice weeks old were purchased from nanjingmedical university school of medicines accredited animalfacility the mice were randomly divided into two groupsusing random number generator in each group à exponentially growing cervical cancer cells were injected inaxilla subcutaneously before tumor transplantation cells weretransfected with shrnas or overexpression plasmids thetransfection was performed by transient transfection accordingto the speciï¬cation of lipofectamine invitrogen carlsbadca usa the shnc and empty vector pcdna31 were usedas controls and totally µg plasmid vectors were transfectedinto cells for each group the sequences of shrnas are shown intable s5 tumors were harvested at weeks after injection theweight of tumor was measured on the scale and tumor volumewas estimated using calipers [length à width2] and tissueswere immunohistochemically stained with mnx1 abcamab79541 ki67 abcam ab79541 and p21cip1abcam ab109520 western blotting was performed aspreviously described no blinding was done in the animal studiesstatistical analysisresults are presented as the mean ± standard deviation sdstatistical analyses were performed using spss statistics version chicago ill and graphpad prism software graphpadsoftware inc la jolla ca usa p was consideredstatistically significantresultsoverexpression of mnx1 correlates withpoorer prognosis and more aggressiveclinical featuresanalysis of tcga dataset revealed that the mrna expressionof mnx1 was remarkably upregulated in cervical cancer tissuescompared with paratumor tissues p figure 1a ingepia gene expression proï¬ling interactive analysis websitepatients with higher expression of mnx1 bore a worse diseasefree survival nhigh nlow p figure 1b theexpression of mnx1 in cervical cancer tissues were significantlyhigher than adjacent tissues in of cervicalcancer patients p figures 1cd ihc assays based ontissue microarrays tmas were performed to detect the proteinexpression of mnx1 in paired human cervical cancer tissuesand paratumor tissues and results showed that staining scoresof mnx1 were higher in cancer tissues p figure 1ecombined with the patients clinical information the expressionof mnx1 was higher in patients with more advanced tnm stagestage iii vs iiiiv p figure 1f t stage t1 vst2t3 p figure 1g and n stage n0 vs n1 p figure 1h moreover mnx1 staining scores were linkedto higher pathological grade level ii vs iii p figure 1iand larger tumor maximum diameter d vs ¥ cm p figure 1j and ihc images of two patients with diï¬erentclinical stages were presented figure 1kknockdown of mnx1 inhibited progressionof cervical cancer in vitroto evaluate the expression of mnx1 in cell lines qrtpcr andwestern blotting were performed and results showed that mnx1was generally upregulated in cervical cancer cell lines comparedwith normal human cervical cell lines hacat figures 2ab tofurther investigate the biological function of mnx1 in cervicalcancer two speciï¬c sirnas targeting mnx1 were transfectedinto hela and siha cells both two sirnas showed favorablesuppression eï¬ciency in hela figures 2cd and siha cellsfigures 2ef the rtca proliferation assay figure 2g eduassay figure 2h and colony formation assay figure 2ishowed that knockdown of mnx1 inhibited the proliferationability of cervical cancer in hela and siha cells moreover rtcamigration assay figure 2j transwell assay and matrigel assayfigure 2k and wound healing assay figure 2l revealed thatsilencing mnx1 inhibited the ability of cervical cancer cells tomigrate and invade these results suggest that mnx1 plays a vitalrole in the malignant phenotype of cervical cancerectopic expression of mnx1 enhancedaggressiveness of cervical cancer in vitroto further verify the biological role of mnx1 in cervical cancer apcdna31 plasmid to overexpress mnx1 was constructed andtransfected into c33a and hela cells the plasmid eï¬ectivelyupregulated the expression of mnx1 conï¬rmed by qrtpcrand western blotting figures 3ab consistently our resultsshowed that ectopic expression of mnx1 promotes proliferationmigration and invasion figures 3cg of cervical cancer cellssimnx1 induced g2m cell cycle arrestand upregulated the expression of p21cip1two hundred and eight genes highly correlated with mnx1were used for go kegg and reactome pathway analysisresults showed that mnx1 may participate in transcriptionand metabolism pathway figure 4a cell cycle detectionshowed that knockdown of mnx1 induced g2m cell cyclearrest in hela and siha cells figure 4b furthermore weexamined the eï¬ect of mnx1 on the expression of cell cyclekeyrelated genes including p15ink4b p16ink4a p21cip1 p27kip1cdk1 cdk2 cdk4 cyclinb1 cyclind1 and cycline1 bothin hela and siha cells knockdown of mnx1 upregulated theexpression of p21cip1 which has been conï¬rmed as a tumorsuppressor gene in multiple cancers figure 4c and westernblotting results suggested that knockdown of mnx1 increasedthe expression of p21cip1 while decreased the expression ofphosphorylated cdk1 pthr161cdk1 a downstream eï¬ectorof p21cip1 figure 4d consistently with these results ectopicexpression of mnx1 decreased the expression of p21cip1 whileincreased the expression of pthr161cdk1 in c33a and helacells figures 4ef it suggested that mnx1 might exerted itsbiological function via modulating the expression of p21cip1frontiers in oncology wwwfrontiersinaugust volume 0czhu mnx1 enhances progression of cervical cancerfigure mnx1 is upregulated in cc tissues and positively correlates with aggressive clinical characteristics a mnx1 is upregulated in cc tissues compared withadjacent normal tissues in tcga dataset p b patients with high expression of mnx1 have poor disease free survival dfs in cc p cd themrna expression of mnx1 in cervical cancer tissues was significantly higher than that in adjacent normal tissues in patients p e the mnx1staining score was upregulated compared with that in adjacent normal tissues p f the mnx1 staining score was positively correlated with tnm stage p g t stage p h lymph node metastasis p i tumor differentiation p and j local primary tumor diameter p incc patients k representative ihc staining images in tmas were shown error bars represent the mean ± sd values ns no signiï¬cance represents p frontiers in oncology wwwfrontiersinaugust volume 0czhu mnx1 enhances progression of cervical cancerfigure knockdown of mnx1 suppressed the proliferation migration and invasion in cc cells ab mnx1 mrna and protein level are upregulated in cc celllines cf two speciï¬c sirna si1 and si2 of mnx1 were designed and the transfection efï¬ciencies of sirnas in hela and siha cells were analyzed by qrtpcrand western blot gi the proliferation abilities were evaluated by xcelligence system assay edu incorporation assay and colony formation assay were inhibitedafter knockdown of mnx1 in hela and siha cells j the xcelligence system assay k transwell and matrigel assay and l wound healing assay indicated thatmigration and invasion capacities were suppressed after simnx1 in hela and siha cells error bars represent the mean ± sd values of three independentexperiments p p p ns no signiï¬cancefrontiers in oncology wwwfrontiersinaugust volume 0czhu mnx1 enhances progression of cervical cancerfigure ectopic expression of mnx1 enhanced aggressive abilities in c33a and hela cells ab the pcdna31mnx1 was synthesize and the transfectionefï¬ciencies were analyzed by qrtpcr and western blot the proliferation functions were measured by c the xcelligence system assay d colony formationassays and e edu incorporation assays were elevated in oemnx1 c33a and hela cells f the transwell assay and matrigel invasion assay g wound healingassay also showed that oemnx1 strengthened migration and invasion capacities error bars represent the mean ± sd values of three independent experiments p p p ns no signiï¬cancemnx1 suppressed the expression ofp21cip1 via binding to its promoter regionour previous results showed that knockdown or ectopicexpression of mnx1 altered the expression of p21cip1 to furtherverify the mechanism we analyzed the correlation betweenmnx1 and p21cip1 in cases of cc samples and the resultswere shown that mnx1 and p21cip1 had a negative correlationn p figure 5a as transcription factors usuallybind to sequencespeciï¬c dna to regulate transcription weutilized jaspar database to predict the binding site betweenmnx1 and the promoter region upstream bp of codingregion of cdkn1a the gene symbol of p21cip1 it turnedout that mnx1 was predicted to have four binding sites withthe promoter region of cdkn1a of which bpfrontiers in oncology wwwfrontiersinaugust volume 0czhu mnx1 enhances progression of cervical cancerfigure knockdown of mnx1 expression induced g2m phage arrest by regulating the p21cip1 expression a many genes were enriched in regulation oftranscription by go analysis most of the genes were enriched in the metabolic pathways by kegg and reactome pathway analysis b knockdown of mnx1generated g2m stage arrest in hela and siha cells were measured by ï¬ow cytometry cf the p21cip1 mrna levels were upregulated or downregulated after si oroemnx1 in cc cell lines de the protein level of p21cip1 was upregulated or downregulated while the expression of pthr161cdk1 was decreased or increased afterknockdown or ectopic mnx1 of cc cells the expression of cdk1 ccne1 ccnd1 and ccnb1 had no obvious changes error bars represent the mean ± sdvalues of three independent experiments p p p ns no signiï¬canceaacaataaat and bp gcccattaat showedhigher combination scores figure 5b accordingly the wildcdkn1a promoter region and mutant types 226mt and1371mt were generated and cloned into luciferase reportervector pgl3basic figure 5c and in luciferase reporterassay overexpression of mnx1 inhibited the transcriptionalactivity of the wild cdkn1a promoter but not mutant typefigure 5d moreover chip assay also revealed that mnx1bound to the p21cip1 promoter region in hela and sihacells figures 5effrontiers in oncology wwwfrontiersinaugust volume 0czhu mnx1 enhances progression of cervical cancerfigure mnx1 bounds to the p21cip1 promoter region and suppresses p21cip1 transcription a the expression of mnx1 and p21cip1 is negatively correlated in cervical cancer tissues p b the jarspar database indicates that mnx1 has several binding sites with the promoter region of p21cip1 c schematicdiagram shows that the two sites with the highest score of mnx1 on p21cip1 promoter and the mutant p21cip1 promoter were selected d overexpression of mnx1remarkably decreased wild type but not mutant p21cip1 promoter luciferase activity p21cip1226 p p21cip11371 p e chromatinimmunoprecipitation chip assays using normal igg or antimnx1 demonstrated that mnx1 directly binding to p21cip1 promoter region f the results of chippcrproduct electrophoresis were showed that a clear band was observed in the antimnx1 group while almost no band was detected in the igg control groupp p frontiers in oncology wwwfrontiersinaugust volume 0czhu mnx1 enhances progression of cervical cancerfigure downregulation of p21cip1 partially recovered the malignant phenotypes of simnx1 cells a the transfection efï¬ciency of p21cip1 was determined byqrtpcr and si1p21cip1 was chosen to further experiments bd the proliferative abilities were partially rescued after knockdown p21cip1 in simnx1 hela cellswere measured by the xcelligence system assay colony formation assay and edu incorporation assay ef the invasion and migration capacities have also beensignificantly improved after knockdown p21cip1 in simnx1 cells compared with simnx1 alone cells g the protein level of p21cip1 and pthr161cdk1 were partiallyreversed when knockdown of p21cip1 in simnx1 compared with simnx1 alone error bars represent the mean ± sd values of three independent experiments p p p ns no signiï¬cancefrontiers in oncology wwwfrontiersinaugust volume 0czhu mnx1 enhances progression of cervical cancerfigure knockdown or overexpression of mnx1 inhibited or promoted tumor growth in vivo ab the transfection efï¬ciency of shmnx1 was measured byqrtpcr and western blot c a total of eight nude female mice were sacriï¬ced and xenograft tumors were collected after injection with shmnx1 cells weeksde tumor volume and weight were reduced in the shmnx1 group compared with those in the shnc group f the expression of mnx1 and ki67 wasdownregulated and p21cip1 was upregulated in shmnx1 xenograft tumors analyzing by ihc staining g the protein level of mnx1 pthr161cdk1 weredownregulated and p21cip1 was upregulated in shmnx1 mouse xenograft tumors analyzed by western blot h a total of eight nude female mice were sacriï¬ced andxenograft tumors were collected after injection with oemnx1 cells weeks jk tumor volume and weight was increased in the oemnx1 group compared withthose in the oenc group i the expression of mnx1 and ki67 was upregulated and p21cip1 was downregulated in oemnx1 xenograft tumors analyzing by ihcstaining error bars represent the mean ± sd values p p p ns no signiï¬cancesilencing p21cip1 rescued the function ofsimnx1to determine whether the function of mnx1 was relied onp21cip1 we designed three sirnas table s3 to knockdownthe expression of p21cip1in hela cells the si1p21cip1showed the best transfection eï¬ciency figure 6a and it wasused for the following experiment rtca proliferation assaycolony formation assay edu assay transwell assay matrigelassay and would healing assay revealed that silencing p21cip1partially rescued the decreased proliferation migration andinvasion ability of hela cells caused by knockdown of mnx1figures 6bf and western blotting showed that the proteinlevel of p21cip1 and pthr161cdk1 were partially reversed bysilencing p21cip1 figure 6gmnx1 promoted tumor growth of cervicalcancer in vivothe xenograft models were used to explore the function ofmnx1 in vivo the shrnamnx1 shrnanc as control wastransfected into hela cells and the knockdown eï¬ciency wasconï¬rmed by qrtpcr and western blotting figures 7abresults showed that knockdown of mnx1 inhibited tumrowth measured by tumor weight and volume in vivofigures 7ce ihc staining and western blotting of harvestedtumors revealed that knockdown of mnx1 upregulated theprotein level of p21cip1 and downregulated ki67 and pthr161cdk1 in vivo figures 7fg moreover ectopic expression ofmnx1 promoted tumor growth and altered the expression ofp21cip1 and ki67 in vivo figures 7hkfrontiers in oncology wwwfrontiersinaugust volume 0czhu discussionin this study we identiï¬ed mnx1 a transcription factor ofhomeobox family was significantly upregulated and involvedin the progression of cervical cancer the overexpression ofmnx1 correlated with advanced clinical stages and poorerprognosis of cervical cancer patients furthermore mnx1exerted its oncogenic role via modulating the expression ofp21cip1 especially by targeting the promoter region of p21cip1thus to repress its transcriptionin accordance with ourï¬ndings a recent showed that mnx1 had a role in theprogression of cervical cancer partially through upregulating cellcycle regulators ccne1 and ccne2 and mnxas1 theantisense lncrna of mnx1 was also reported to promote theinvasion and metastasis of gastric cancer through repression ofcdkn1a all this results indicated that mnx1 played acritical role in cancer growth and cell cycle progression andmnx1 might serve as a useful diagnostic and treatment targetfor cervical cancermnx1is a member of homeobox gene family which allcontain a homeobox a dna sequence around basepairs long and encode homeodomain protein products astranscription factors this cluster of genes has beenidentiï¬ed to participate in the regulation of development andmorphogenesis in animals fungi and plants for examplecdx1 which is stably expressed in the human intestine playsan important role in embryonic epicardial development and the protagonist of our study mnx1 participates inmotor neuron development and neurodegenerative processesof zebraï¬sh and moreover controls cell fate choice inthe developing endocrine pancreas in recent years moreand more researches uncovered the role of developmentrelatedhomeobox genes in carcinogenesis and these genes show greatapplication prospect in tumor diagnosis and prevention asthe role of carcinoembryonic antigen cea in gastroenterictumors and alpha fetal protein afp in liver cancer for instance pdx1 is a key regulator in pancreatic developmentand cell function and meanwhile dynamically regulatespancreatic ductal adenocarcinoma initiation and maintenance hoxc13 a highly conserved transcription factor involvedin morphogenesis of all multicellular anisms is aberrantlyexpressed and associated with cancer progression in esophagealcancer lung adenocarcinoma and liposarcomas likewise mnx1 has been reported to promote sustainedproliferation in bladder cancer by upregulating ccne12 and to act as a novel oncogene in prostate cancer and in ourstudy mnx1 was also conï¬rmed to be upregulated in cervicalcancer and enhance the progression of cervical cancerin terms of mechanism we found that mnx1 promotedtumor growth of cervical cancer via accelerating the progressionof the cell cycle especially by modulating the expression ofp21cip1 cell cycle is a vital process by which a cellleadsto duplication and disorders of the cell cycle regulation maylead to tumor formation the cell cycle progress isdetermined by two types of regulatory factors cyclins and cyclindependent kinases cdks active cyclincdk complexesphosphorylate proteins to elevate the expression levels of cyclinsmnx1 enhances progression of cervical cancerand enzymes required for dna replication converselythe cell cycle progression can be prevented by inhibitors bybinding to and thus inactivating cyclincdk complexes suchas p21cip1 p27kip1 p16ink4a and so on the p21cip1also known as cyclindependent kinase inhibitor cdkn1ahas been identiï¬ed as a regulator of cell cycle and a tumorsuppressor in multiple kinds of cancers our results provedthat mnx1 repressed the transcription of p21cip1 by directlytargeting its promoter region and furthermore promoted thephosphorylation of downstream cdk1 the mnx1p21cip1pthr161cdk1 axis played crucial roles in the progression ofcervical cancer and meanwhile provided new evidence forthe pathogenesis of cervical cancer moreover the associationbetween cervical cancer and hpv has long been identiï¬ed as asexually transmitted agent hpv are involved in transformationand maintaining of transformed status many studies havereported that hpv can also alter the expression of p21 thus we searched the geo dataset to seek for information aboutthe relationship between mnx1 and hpv viral infection weanalyzed the gse dataset and found that there were nosignificant changes in the expression of mnx1 nm_005515 inhacat cells infected with hpv11e6 or hpv18e6 in gse3292gds1667 hpv positive or negative head and neck squamouscell carcinoma hnscc showed no expression diï¬erences ofmnx1 figure s1 this information suggests that mnx1 mightnot be directly involved in hpv carcinogenesis and furtherinvestigations are needed in the future in addition cervicalcancer i | 0 |
antibioticresistant pathogen strainlevel investigations are beginning to reveal themolecular mechanisms used by vrefm to colonize regions of the human bowelhowever the role of commensal bacteria during vrefm colonizationin particularfollowing antibiotic treatment remains largely unknown we employed amplicon16s rrna gene sequencing and metabolomics in a murine model system to try andinvestigate functional roles of the gut microbiome during vrefm colonization firstorder taxonomic shifts between bacteroidetes and tenericutes within the gut microbial community composition were detected both in response to pretreatment usingceftriaxone and to subsequent vrefm challenge using neural networking approaches to ï¬nd cooccurrence proï¬les of bacteria and metabolites we detected keymetabolome features associated with butyric acid during and after vrefm colonization these metabolite features were associated with bacteroides indicative of a transition toward a preantibiotic naive microbiome this study shows the impacts of antibiotics on the gut ecosystem and the progression of the microbiome in responseto colonization with vrefm our results offer insights toward identifying potentialnonantibiotic alternatives to eliminate vrefm through metabolic reengineering topreferentially select for bacteroidesimportance this study demonstrates the importance and power of linking bacterial composition proï¬ling with metabolomics to ï¬nd the interactions betweencommensal gut bacteria and a speciï¬c pathogen knowledge from this researchwillinform gut microbiome engineering strategies with the aim of translatingobservations from animal models to humanrelevant therapeutic applicationscitation mu a carter gp li l isles ns vrbanacaf morton jt jarmusch ak de souza dpnarayana vk kanojia k nijagal b mcconvillemj knight r howden bp stinear tp microbemetabolite associations linked to therebounding murine gut microbiomepostcolonization with vancomycinresistantenterococcus faecium msystems 5e0045220101128msystems0045220editor manuel liebeke max planck institutefor marine microbiologycopyright mu this is an openaccess distributed under the terms ofthe creative commons attribution international licenseaddress correspondence to andre muandremuunimelbeduaureceived may accepted july published august julyaugust volume issue e0045220msystemsasm 0cmu keywords microbiome multiomics metagenomics metabolomics gutmicrobiome vancomycinresistant enterococci colonization antimicrobial resistanceceftriaxonevancomycinresistant enterococcus faecium vrefm is a significant health careassociated pathogen vrefm infections can be difï¬cult to treat due to their intrinsicand acquired resistance to nearly all classes of antibiotics the world healthanization categorizes vrefm as a high priority bacterial pathogen advocatingresearch to stop the global increase in antibiotic resistance recent studies highlightthe importance of the gut microbiota in modulating the growth and virulence of vrefmin the gastrointestinal ecosystem for instance the depletion of normal gut ï¬ora usingantibiotics exacerbates the severity of vrefm infection whereas transplant ofcommensal species including a consortium of clostridium bolteae blautia productablautia sartorii and parabacteroides distasonis can drive established vrefm colonization to below levels of culture detection speciï¬cally b productaa colonizer ofthe colonreduces vrefm growth in vivo by secreting a lantibiotic these observations raise the intriguing possibility that metabolic traits act in concert betweenpathogen and select gut commensals to confer mutual beneï¬ts during pathogenpersistence these ï¬ndings also highlight the greater risk posed to immunocompromised patients when colonized with vrefm for instance allogeneic hematopoietic celltransplantation patients have gastrointestinal tracts that are dominated by vrefm as aresult of losing a large portion of the intestinal commensal microbiota upon receivingbroadspectrum antibiotics as pretreatment hildebrand discovered longtermecological impacts to the gut microbiome with strong bacterial species turnover afterceftriaxone treatment in humans further mice receiving broadspectrum antibioticscombination of metronidazole neomycin and vancomycin showed markedly increased vrefm colonization of the cecum and colon the compromised intestinal innateimmune defenses in these animals allowed proliferation of vrefm caused by theantibiotic exposure and subsequently reduced the expression of antimicrobial molecules produced by bacteria in the intestinal mucosa the problem with vrefm is further complicated by the fact that enterococci aremembers of the gastrointestinal tract microbiota a key reservoir of antimicrobialresistance amr genes and potentially facilitating gene transfer within the gut microbiome for example the vanb resistance gene was detected in human fecalspecimens that did not contain culturable vre and instead demonstrated that isolatescarrying the resistance transposon are anaerobic commensal bacteria eggerthella lentaand clostridium innocuum colonization of and persistence in the gastrointestinaltract therefore presents as a key mechanism for de novo vre and may lead to severeinvasive diseasethe current study aimed to understand the impact of antibiotics on the murine gutmicrobiota and the subsequent colonization pattern of vrefm to this extent wedesigned a murine model timeseries study that consisted of two main perturbativephases i antibiotic pretreatment with ceftriaxone and ii vrefm challenge our 16srrna gene proï¬ling analyses highlighted a ï¬rstorder shift in bacterial biodiversitycomposition across time a secondorder clustering of samples associated with theexperimental phases and the transition of the postvrefm colonization gut microbiotaand its metabolome toward resembling an asymptomatic carriagelike microbiomephenotype this research provides support for engineering the metabolic potential ofthe gut microbiome using for example prebiotics as a nonantibiotic alternative fortreating multidrugresistant bacterial infectionsresultsexperimental design the following experimental design was developed to address the hypothesis that there are speciï¬c murine gut microbiome factors thatfacilitate vrefm colonization three groups of three c57bl6 mice cocaged wildtypejulyaugust volume issue e0045220msystemsasm 0cgut microbiome and colonization with vretable summary of samples analyzed in this studyday of exptphase of exptannnnnnabxtxabxtxabxwnvreevreevreevreevrelvrelamplicon 16s rrnagene datab«º«º«º«ºmetabolomicsb«º«º«º«º«º«º«º«ºavg no ofobserved sotusc«º«º«º«ºathe key phases of the experiment where n represents naive abxtx represents antibiotic treatment abxwn represents antibiotic weaning vree represents earlyphase postvrefm colonization and vrelrepresents latephase postvrefm colonizationbsymbols sample processed «º data unavailablecthe average number of sotus observed across all mice for each day of the experimentmales were monitored and fecal samples were collected over a 14day period with twointervention time points including i ceftriaxone treatment administered at 05gliter indrinking water across a 2day period and ii colonization via oral gavage with «» vrefm st796 per mouse postantibiotic treatment at a single time point mice werehoused in groups of ï¬ve and samples were collected from the same three mice torepresent technical replicates per cage herein each group of cohoused mice will bereferred to as group a group b and group c the remaining two mice per group werereserved for microbiological assays table highlights samples and data sets collectedamplicon 16s rrna gene sequencing revealed ï¬rstorder shifts in bacterialcommunity composition amplicon 16s rrna gene sequencing was performed tocapture the bacterial community composition in an effort to track changes in responseto antibiotic pretreatment and vrefm colonization bacterial community proï¬les wereassessed in fecal samples from nine mice before during and after the two interventionstable a total of of reads reads passed quality control with reads on average per sample and a total of exact variant sequence typesie features with an average of features per sample and an upper bound of features when rareï¬ed to reads alpha rarefaction analysis demonstratedsufï¬cient sequencing depth to capture microbial diversity to saturation see fig s1 inthe supplemental materialthe biodiversity proï¬les of each sample were compared and showed that keysuboperational taxonomic units sotus were differentially abundant throughout thecourse of the experiment fig there was a shift in the dominance of bacteroidiabacteroidetes light green colored bars during the naive phase of the experiment tomollicutes tenericutes fuschia colored bars in response to ceftriaxone treatment witha return to the predominance of bacteroidia during the late phase of the experimentafter vrefm colonization ie days to of note is the predominance of lactobacillales in mouse to from group a fig the murine gut microbiota responds to antibiotics and microbial communityrichness begins to rebound days after vre colonization principalcoordinateanalysis pcoa of the unweighted unifrac distances was used to assess clusteringof fecal samples based on bacterial composition this assessment showed that the fecalmicrobiota from samples collected from each phase clustered together but were clearlyseparated between phases after exposure to ceftriaxone and challenge with vrefmpostantibiotic treatment fig 2a permutationbased statistical testing demonstratesthe groups are significantly different from one another fig s2 temporal tracking ofjulyaugust volume issue e0045220msystemsasm 0cmu fig biodiversity plot of sotus as relative frequencies at the taxonomic level of class firstorder shifts in microbial communitycomposition as revealed by 16s rrna gene community proï¬ling from a predominance of bacteroidetes to tenericutes and return tobacteroidetes was observed each column displays the relative bacterial community composition in a mouse fecal sample collecteddaily and sorted by the chronology of the experiment ie day of experiment table the columns are further sorted by group iegroup a group b and group c and individual mice within each group mouse mouse and mouse stacked bars are presentedas relative frequencies at the taxonomical level of class however annotations of key taxa are at the phylum level bacteroidetes [green]firmicutes [gray] and tenericutes [fuschia] or order level lactobacillales [yellow]the changing microbiomes against each mouse on the pcoa sample space demonstrated a clear unidirectional trajectory that followed the chronology of the experiment106084m9ï¬gshare12775859 procrustes analyses of weighted andunweighted unifrac distances showed that the same general patterns on the samplespace were preserved meaning that there is congruency in global spatial patternsbetween qualitative and quantitative measures of community dissimilarity fig s3analysis of community diversity faiths phylogenetic diversity index revealed astable and rich microbial community during the naive phase preceding a sharpdecrease following antibiotic treatment and a further decrease immediately followingvrefm colonization fig 2b of note is the responsiveness of the microbiota within h to the removal of antibiotics at the end of day community richness began torebound at approximately days after vrefm colonization ie day with group ademonstrating a higher rate of rebound compared to groups b and c calculating thedistances of dissimilarity unweighted unifrac distances of each mouse microbiotatime point relative to day a proxy for the naive bacterial community phenotyperevealed a small dissimilarity distance for samples collected during the naive phase andan increasing dissimilarity distance following antibiotic treatment day and vrefmcolonization day fig 2c there was a downward trajectory in distance scores daysafter vrefm colonization ie day group a followed a sharper return to a microbiotaresembling day these observations suggest that mice were transitioning toward apersistent carrierlike state and that the rebounding community richness toward levelsrepresentative of the naive phase was by a microbial community structure that resembled the naive phase additional studies where the time frame of postvrefm challengeextends beyond week of monitoring are needed to understand whether the perturbed microbiome will return to resemble an absolute naive state or arrive at a newaltered statemultinomial regression identiï¬es sotus most positively associated with vrefmcolonization multinomial regression using songbird was employed to identify sotusjulyaugust volume issue e0045220msystemsasm 0cgut microbiome and colonization with vrefig diversity analyses a principalcoordinate analysis plot of unweighted unifrac distances data points areprojected onto the sample space and colored by prevrefm colonization red and postvrefm colonization bluenote that circles and ellipses function to highlight the separation of experimental phases and do not indicatestatistical conï¬dence intervals principal coordinate axis explains of the variation observed between thenaive microbiota and those from the postvrefm colonization phase b community richness of the murine gutmicrobiome as measured by faiths phylogenetic diversity in response to ceftriaxone treatment and challengewith vrefm c community dissimilarity distances as calculated by unweighted unifrac of each time point relativeto day naive phasethat were most positively and negatively associated with the postvrefm colonizationphase fig the ï¬ve most positively associated sotus were enterococcus bacteroideserysipelotrichaceae catabacter and lachnospiraceae while the ï¬ve most negativelyassociated were clostridiales adlercreutzia mollicutes peptostreptococcaceae and clostridiales temporal tracking of exact sequence variants esvs demonstrated that theesv feature classiï¬ed as enterococcusand identiï¬ed as the most positively associatedwith the postvrefm colonization phasewas most abundant on days of challengeconï¬rming that this esv likely was the st796 vrefm colonization challenge anismsfig there were a further eight esv features classiï¬ed as enterococcus however theywere absent during the days representing vrefm colonization and lacked positiveassociations with the postvrefm colonization phase suggesting that these featuresrepresent murine gut commensal enterococcijulyaugust volume issue e0045220msystemsasm 0cmu fig multinomial regression multinomial regression identiï¬ed an enterococcus exact sequence variant as the most positivelyassociated with the colonization phase log fold change score of read counts for the enterococcus esv tracked daily acrossthe experiment showing high abundance during the days of vrefm challengemolecular networking identiï¬es differential metabolome proï¬les duplicatefecal samples from key time points throughout the experiment ie days and were analyzed by datadependent tandem mass spectrometry msms performed on a liquid chromatography quadrupole time of ï¬ight lcqtof system tomonitor changes in the murine gut metabolome table polar metabolite analysiswas given preference in an effort to broadly capture primary metabolites that play a keyrole in metabolic handoffs that deï¬ne interspecies interactions analysis of the globalmetabolome proï¬le of each sample was measured based on their overlapping molecules and a pcoa plot using a binary jaccard distance metric through the global naturalproducts social molecular networking gnps platform a separation of metaboliteproï¬les along pcoa1 was observed fig 4a metabolomes from the naive andlate vrefm colonization phase tended to cluster together while samples from thepostantibiotic phases including the early vrefm colonization phase clustered togethersupporting pairwise permutational multivariate analysis of variance permanovatesting fig s4 highlights that naive and early vre samples are significantly differentwhile late vre has a lower distance to naive samples compared to antibiotictreatedantibiotic wean and early vre samplesrandom forest analysis of spectrum proï¬les from lcmsms was used to predictexperimental phase and rank the importance of metabolite association with eachexperimental phase the top metabolite features for each experimental phase arehighlighted in fig 4b unique proï¬les of metabolite features were observed for eachphase of the experiment importantly the late vrefm colonization phase capturesan unknown metabolite feature with a masstocharge ratio mz of and retention time rt of this metabolite is exclusively present during whatrepresents the transition toward resembling the naive microbiome manual curationoffeature in positiveion mode predicts a molecular formula c5h8n4o3with ±10 ppm in mass error the major peaks in the msms spectrum for feature are precursor ion [m«¹h]«¹ assumed precursor ion [h2o] product ion [likely c2h2o] [c2h2o«¹h] plus product ionfurther supporting neutral loss of c2h2o given the summation of results the chemicalstructure of feature is likely to contain a nacetylated hydroxyl group peakquantiï¬cation values indicate its presence during the late phase of vre colonizationfig 4c further manual curation of msms data identiï¬ed ceftriaxone as feature julyaugust volume issue e0045220msystemsasm 0cgut microbiome and colonization with vrefig metabolomic analyses a emperor plot displaying principalcoordinate analysis of binary jaccard distances of metabolomic proï¬les samples are colorcoded and the colors represent the naive orange antibiotic treatment red antibiotic weaning blue early vre colonization green and late vrefmcolonization purple phases b random forest classiï¬er identifying metabolite features spectra for each phase of the experiment the heatmap is color codedfrom low ranking score white ie lowest importance to high ranking score dark blue highest importance metabolite features are labeled by theirmasscharge ratios and retention times for the reason that current databases do not capture their chemical structure andor identiï¬cations abx tx antibiotictreatment c peak quantiï¬cation values for feature mz «½ and rt «½ present in abundance during vre colonization late phase dpeak quantiï¬cation values for ceftriaxone mz «½ and rt «½ tracked across the experiment ceftriaxone values are highest during antibiotictreatment phase and begins to wane during antibiotic weaning phase julyaugust volume issue e0045220msystemsasm 0cmu with an mz of and rt of and mostly abundant during days of antibioticexposure fig 4dbacteroidalesassociated metabolites implicated in latephase postvrefm colonization a distinct proï¬le shift in microbe and metabolite abundances as calculatedby multinomial regression was observed particularly during latephase vrefm colonization fig s6 shallow neural networking analysis with mmvec was used to predictmicrobemetabolite interactions through their cooccurrence probabilities fig sequential biplots captured the shift in experimental phases and highlighted the cooccurrences of microbiota and metabolomic data sets fig 5a to c there was a strongenterococcus effect as indicated by the magnitude of the corresponding arrow and therebounding species during the latephase vrefm colonization are predominantly bacteroidales sotus fig 5c with cooccurring metabolite features mz rt and mz rt metabolite feature mz rt was ranked asbeing highly associated with the postvre colonization phase these results integratemicrobial and metabolite data sets to reveal which microbes may be responsible fordetected metabolites in this instance the metabolite present during the phase representing a transition toward a microbiome approximating the naive state feature mz and rt fig 4b is linked with bacteroidales fig 5adiscussionin this study of the murine gut ecosystem we employed a mouse model ofgastrointestinal tract colonization that replicates the shift in bacterial compositionwhen patients enter the health care system develop an imbalance in their microbiomeas a result of pretreatment eg antibiotic treatment and are subsequently colonizedwith a hospital superbug the resolution of current studies describes a consortiumof commensal microbes that can for example reduce the magnitude of vrefm colonization however understanding the key metabolic shifts relative to the gutmicrobiota remains challenging here we employed amplicon 16s rrna genesequencing and highresolution mass spectrometry metabolomics in an effort towarddetermining microbiotametabolome interactions during vrefm colonization we demonstrated clear changes in the gut microbiome in response to ceftriaxone and vrefmchallengeconceptual and statistical advances in analysis of amplicon 16s rrna gene data whereby otus are clustered at a nucleotide similarity threshold allows for theidentiï¬cation of exact sequence variants esvs query against an errorcorrecteddatabase can detect multiple esvs that may be classiï¬ed to the same taxonomicrank for example our analyses identiï¬ed multiple esvs classiï¬ed as enterococcihowever when the relative abundances were tracked across the chronology of theexperiment only one enterococcus esv was dominant in relative abundances and mostpositively associated with the days of postvrefm challenge fig this highlights theresolving power to differentiate between commensal and pathogenic strains of enterococci when the composition of the microbial community is considered the factthat this was achievable at the level of amplicon 16s rrna gene sequencing alludes tothe possibility of implementing microbiota screenings as routine diagnostics for patients entering health care systems further ï¬rstorder level shifts in microbial community composition was observed in response to ceftriaxone and subsequent vrefmchallenge fig three days after vrefm colonization ie day the microbiomerichness begins to rebound suggesting that mice are transitioning toward a persistentcarrierlike state interestingly the group a cohort exhibited a higher rate of reboundthat may be facilitated by their initially higher microbial community richness andpredominance of lactobacillales on the day of vrefm challenge fig 2b this observation supports the need to prescreen baseline microbiota proï¬les of patients uponadmission into hospital for the reason that it is not necessarily which microbialpopulations are removed postperturbation eg antibiotic pretreatment but insteadwhich populations persist that drives the responding phenotype we can begin toassess patients from across different wards eg intensive care unit oncology neuroljulyaugust volume issue e0045220msystemsasm 0cgut microbiome and colonization with vrefig microbemetabolite vector biplots sequential biplots highlighting the changing metabolitedifferentials across each key phase of the experiment abx tx is the antibiotic treatment phase and abxwean is the period when antibiotics were removed for a 24h period prior to colonization with vrefmeach point on the sample space represents metabolites and arrows represent microbes microbe andcontinued on next pagejulyaugust volume issue e0045220msystemsasm 0cmu ogy and healthy cohorts and build a database of microbiome proï¬les that can be usedas biomarkers to predict i the susceptibility of patients to develop persistent bacterialcolonization and ii propensity to clear the pathogen once colonized the clinicalimplication is that new patients are screened and identiï¬ed via betadiversity metaanalyses by these biomarkers and placed in bedding cohorts accordingly therebyimproving infectious disease management and isolation precautions within healthcareassociated ecosystemsthe shortlist of microbes ranked as most negatively associated with the colonizationphase clostridiales adlercreutzia mollicutes peptostreptococcaceae and clostridialesfig are hypothesized to play a role in maintaining the health of the animals indeedamong the microbes identiï¬ed are known shortchain fatty acid eg butyric acidproducers which supports and expands upon those previously identiï¬ed bycaballero further the use of deblur to identify esvs facilitates the temporaltracking of their relative abundances to inform selection of primary fecal samples thatwill provide the best probability ie highest relative abundance of culturing targettaxa for downstream screening of probiotic potential however translating animalderived observations from experimental animal models to human clinical situationsremains challenging particularly where the key microbes are rodentspeciï¬c microbesone solution may be to integrate metabolomics to reveal shared metabolic capacityamong taxonomically divergent microbes our supervised classifying approaches suggests an altered metabolome composition during the late phase of vrefm challengethat may facilitate the apparent suppression of vrefm to levels below the limit ofdetection by culture despite the caveat of poor resolution in current databases to linkmetabolite features to associated chemical structures microbemetabolite vector analysis linked metabolite feature mz «½ and rt «½ to bacteroidesfig our efforts toward manually identifying feature suggests a chemicalformula of c5h8n4o3 and a structure likely to contain a nacetylated hydroxyl group aputative annotation through pubchem search is 3hydroxy4nitrosocyanamido butyramide butyramide is the amide group of butyric acid a shortchain fatty acid thathas been shown to play a key role in colonization resistance against intestinal pathogens further research to comprehensively characterize interactions betweenmicrobe and metabolites will be critical to address the gaps in our understanding of thebiochemical parameters that deï¬ne interspecies microbiome interactions during antibiotic pretreatment and persistent infectionsthe resolution of our results provides the basis in which to begin to identifynonantibiotic alternatives to engineer the gut microbiome through prebiotic interventions eg butyric acid and translating animal studies to humanrelevant therapeuticapplications by delineating taxonomically diverse microbes with shared metaboliccapacity here achieving integrative omics to link microbemetabolite associations ourï¬ndings add support to the incorporation of microbiome proï¬ling approaches intoroutine clinical microbiology particularly in the context of monitoring the impacts ofantibiotic usematerials and methodsmouse gastrointestinal colonization model sixweekold wildtype c57bl6 male mice were usedto establish an animal model of gastrointestinal colonization with vrefm mice were cohoused and hadfree access to food ordinary chow and water and had environmental enrichment eg fun tunnels chewblocks and tissue paper the lightdark cycle was 12h light12h dark and cages were changed weeklyfig legend continuedmetabolite features are ï¬xed upon the sample space with gradient coloring of metabolites indicating thetransition across key phases of the experiment the distance between each point is indicative ofmetabolite cooccurrence frequency and the angle between arrows indicates microbial cooccurrencethe directionality of the arrows describes the variation in the metabolites explained by the microbesrepresented by the arrows for example metabolite feature mz and rt isdemonstrated to cooccur with bacteroides information about the abundances of these cooccurringfeatures are provided as heatmaps in fig s6 in the supplemental materialjulyaugust volume issue e0045220msystemsasm 0cgut microbiome and colonization with vremice were pretreated with gliter ceftriaxone in drinking water for days followed by an antibioticwean period of h mice were then challenged with «» cfu vrefm st796genomic dna extraction and sequencing wholecommunity genomic dna gdna was extractedfrom mouse fecal samples using the qiagen powersoil dna extraction kit formerly mobio following themanufacturers protocol a preprocessing step of mechanicallysis was incorporated using a bertintechnologies precellysis machine for one round of a 40s cycle at rpm the v4 region of thebacterial 16s rrna gene was ampliï¬ed using small subunit forward golaybarcoded and ssu806reverse primers following the earth microbiome project protocol and sequenced using the illuminamiseq platform v2 cycles illumina inc san diego ca usa further primary derived data egbiom tables used to produce results can be found within qiita study id amplicon 16s rrna gene proï¬ling analyses sequence data were processed within the qiitav010 framework for quality control split libraries v q2191 demultiplexing trimming sequencereads to a length of nucleotides nt and picking suboperational taxonomic units sotus usingdeblur v110 to resolve singlenucleotide community sequencing patterns ie feature identiï¬cation ofsotus [] the output biom ï¬les were further processed using qiime2 v20197 for downstreamstatistical analyses alpha rarefaction curves were generated to determine whether each sample hadbeen sequenced to saturation the feature table was subsequently rareï¬ed to reads per sampletaxonomy was assigned using the sklearn classiï¬er and greengenes otus from 515f806rregion of sequences classiï¬er available from docsqiime220184dataresources furthermorerelative abundances of each taxa were visualized as bar plots using the qiime2 taxa plugin a phylogenetic tree was constructed using fragment insertion qiime fragmentinsertion sepp [] to guidephylogeneticaware statistical analyses generated using the qiime2 plugin q2diversity coremetricsphylogenetic key metrics computed by default include both alphadiversity eg shannons diversityindex faiths phylogenetic diversity and evenness and betadiversity eg braycurtis distance andunweighted unifrac distance metrics the unweighted unifrac distance matrix was used tocompute ï¬rst distances and calculate distances relative to day as the baseline between sequentialstates qiime longitudinal ï¬rstdistances ggplot2 r v360 ggplot2tidyverse was used tovisualize the distance scores as line plots emperor was used to visualize principalcoordinate analysisplots of unweighted unifrac distances permutationbased statistical testing permanova on unweighted unifrac distances was used to determine whether samples grouped by phase of experimentwere significantly different from one another q2betagroupsigniï¬cance songbird githubcommortonjtsongbird was employed to determine the importance ie fold change of each sotu inrelation to a given metadata variable eg vrefm colonization microbial features from all samples weresplit into training and test sets for supervised learning classiï¬er analyses of input samples wereallocated to train the random forest classiï¬er within qiime2 the estimator method used for sampleprediction the different experimental phases were the response variables while the 16s rrna gene datawere the featuresmetabolite extraction and liquid chromatographytandem mass spectrometry analysis duplicate fecal samples as outlined in table were processed for polar metabolite extraction and analysisdays and feces were metabolically arrested by immediate collection into dry ice andstored at °c until further processing metabolite extraction from the fecal samples was undertaken bythe addition of \u242el per sample of methanolwater solution [volvol] containing \u242em [13c]sorbitoland \u242em [13c15n]valine and \u242em [13c]leucine as internal standards fecal samples were homogenizedat rpm for min in a thermomixer maintained at °c mechanically disrupted and incubated fora further min in the thermomixer samples were randomized for metabolite extractionmetabolite analysis of the extracted samples pooled biological quality control pbqc samples and mixtures of authentic standard mixes was performed by liquid chromatographymass spectrometrylcms using hydrophilic interaction column zicphilic and highresolution agilent seriesquadrupole time of ï¬ight mass spectrometry qtof ms as described previously pcoa of binaryjaccard distances of test standard mixes and pbqc samples are presented in fig s5 in the supplementalmaterial ions were analyzed in positive mode with full scan range of to mz and in datadependent tandem ms mode to facilitate downstream metabolite identiï¬cationmetabolomic analyses | 0 |
"adipogenesis is the process through which mesenchymalstem cells mscs commit to the adipose lineage and diï¬erentiate into adipocytes during this process preadipocytescease to proliferate begin to accumulate lipid droplets anddevelop morphologic and biochemical characteristics ofmature adipocytes such as hormoneresponsive lipogenesisand lipolytic programs currently there are mainly twomodels of benign adipocyte diï¬erentiation in vitro one isï¬broid pluripotent stem cells which can diï¬erentiate intonot only adipocytes but also muscle cartilage and othercells there are two kinds of ï¬broid pluripotent stem cellsbone marrow and adipose mesenchymal stem cells anothergroup is ï¬broblastic preadipocytes which have a single direction of diï¬erentiation namely lipid diï¬erentiation including3t3l1 and 3t3f422a cells cancer cells with tumorinitiation ability designated as cancer stem cells cscshave the characteristics of tumorigenesis and the expressionof speciï¬c stem cell markers as well as the longterm selfrenewal proliferation capacity and adipose diï¬erentiationpotential in addition to cscs cancer cells undergoing epithelialmesenchymaltransformation emt havebeen reported to be induced to diï¬erentiate into adipocytes[] lung cancer ncih446 cells can be induced to differentiate into neurons adipocytes and bone cells in vitro the adipogenesis diï¬erentiation treatment is promisingin the p53 gene deletion type of ï¬broblastderived cancer cancer cells with homologous recombination defectssuch as ovarian and breast cancer cells with breast cancersusceptibility genes brca mutations can be inducedto diï¬erentiate by poly adpribose polymerase parp 0cstem cells internationalinhibitors the nuclear receptor peroxisome proliferatoractivated receptor Î pparÎ agonist antidiabetic thiazolidinedione drug can induce growth arrest and adipogenicdiï¬erentiation in human mouse and dog osteosarcoma cells thyroid cancer cells expressing the pparÎ fusion proteinppfp can be induced to diï¬erentiate into adipocytes bypioglitazone adipogenesis can be induced in welldiï¬erentiated liposarcoma wdlps and dediï¬erentiatedliposarcoma ddlps cells by dexamethasone indomethacininsulin and 3isobutyl1methyl xanthine ibmx in this review we highlight some of the crucial transcription factors that induce adipogenesis both in mscs and inincluding the wellstudied pparÎ and ccaatcscsenhancerbinding proteins cebps as well as othercell factors that have been recently shown to have an important role in adipocyte diï¬erentiation we focus on understanding the complex regulatory mechanism of adipocytediï¬erentiation that can contribute to the clinical treatmentof human diseases including those caused by obesity andadipocytes dysfunction especially for the malignant tumorwhich can be transdiï¬erentiated into mature adipocytes adipocyte differentiationcell proliferation and diï¬erentiation are two opposingprocesses and there is a transition between these two processes in the early stages of adipocyte diï¬erentiation theinteraction of cell cycle regulators and diï¬erentiation factors produces a cascade of events which ultimately resultsin the expression of adipocyte phenotype adipogenesishas diï¬erent stages each stage has a speciï¬c gene expression pattern in general adipocyte diï¬erentiation ofpluripotent stem cells is divided into two phases the ï¬rstphase known as determination involves the commitment ofpluripotent stem cells to preadipocytes the preadipocytescannot be distinguished morphologically from their precursor cells but also have lost the potential to diï¬erentiate intoother cell types in the second phase which is known asterminal diï¬erentiation the preadipocytes gradually acquirethe characteristics of mature adipocytes and acquire physiological functions including lipid transport and synthesisinsulin sensitivity and the secretion of adipocytespeciï¬cproteins the diï¬erentiation of precursor adipocytes is also dividedinto four stages proliferation mitotic cloning early diï¬erentiation and terminal diï¬erentiation after the precursors are inoculated into the cell culture plates the cellsgrow exponentially until they converge after reaching contact inhibition the growth rate slows and gradually stagnatesand the proliferation of precursor adipocytes stops which isvery necessary for initiating the diï¬erentiation of precursoradipocytes adipocyte precursors exhibit transient mitosiscalled clonal expansion a process that relies on the actionof induced diï¬erentiation factors some preadipocyte cellsmouse cell lines 3t3l1 3t3f442a undergo one or tworounds of cell division prior to diï¬erentiation whereasother cell lines mouse c3h10t12 diï¬erentiating into adipocyte do not undergo mitosis clonal expansion whether mitotic clonal expansion is required for adiposediï¬erentiation remains controversial however it is certainthat some of the checkpoint proteins for mitosis regulateaspects of adipogenesis [ ] when cells enter the terminaldiï¬erentiation stage the de novo synthesis of fatty acidsincreases significantly the transcription factors and adipocyterelated genes work cooperatively to maintain precursor adipocyte diï¬erentiation into mature adipocytes containing largelipid droplets regulatory pathways inpreadipocytes commitmentadipocyte diï¬erentiation is a complex process in which geneexpression is ï¬nely regulated the most basic regulatory network of adipose diï¬erentiation has not been updated inrecent years but some factors and signaling pathways thatdo aï¬ect adipose diï¬erentiation have been continuouslyreported adipocyte diï¬erentiation is the result of the geneexpression that determines the phenotype of adipocyteswhich is a complex and delicate regulatory process figure wnt signal pathway in adipogenesis wnt signaling isimportant for adipocytes proliferation and diï¬erentiationboth in vitro and in vivo the wnt family of secretedglycoproteins functions through paracrine and autocrinemechanisms to uence cell fate and development wntprotein binding to frizzled receptors initiates signalingthrough catenindependent and independent pathways wnt signaling inhibits adipocyte diï¬erentiation in vitroby blocking the expression of pparÎ and cebpα constitutive wnt10b expression inhibits adipogenesis wnt10b isexpressed in preadipocytes and stromal vascular cells butnot in adipocytes in vivo transgenic expression of wnt10bin adipocytes results in a reduction in white adipose tissuemass and absent brown adipose tissue development wnt10a and wnt6 have also been identiï¬ed as determinantsof brown adipocyte development [ ] wnt5b is transiently induced during adipogenesis and promotes diï¬erentiation indicating that preadipocytes integrate inputs fromseveral competing wnt signals the hedgehog hh signaling pathway mechanismthree vertebrate hh ligands including sonic hedgehogshhindian hedgehog ihh and desert hedgehogdhh have been identiï¬ed and initiated a signaling cascademediated by patched ptch1 and ptch2 receptors [ ]hh signaling had an inhibitory eï¬ect on adipogenesis inmurine cells such as c3h10t12 ks483 calvaria mscslines and mouse adiposederived stromal cells thesecells were visualized by decreased cytoplasmic fat accumulation and the expression of adipocyte marker genes after hhsignaling was inhibited although it is generally agreedthat hh expression has an inhibitory eï¬ect on preadipocytediï¬erentiation the mechanisms linking hh signaling andadipogenesis remain poorly deï¬ned erkmapkppar signal pathway extracellularregulated protein kinase erk is required in the proliferativephase of diï¬erentiation erk activity blockade in 3t3l1 0cstem cells internationaldex insulin demxwnt 10band othersshhpbc smotgfð½p smad3 smad3testosteroneð½catentinarirspi3kaktcrebpkapcrebfoxo1a2tcflef gata23cebpð½mapkg3k3ð½p2cebpð½cebpαppará½»bmpssmad1srebpadipocytegenesfigure regulation pathways in preadipocytes commitment bmp and wnt families are mediators of mscs commitment to producepreadipocytes exposure of growtharrested preadipocytes to diï¬erentiation inducers igf1 glucocorticoid and camp triggers dnareplication leading to adipocyte gene expression due to a transcription factor cascade the dotted line indicates an uncertain molecularregulatory mechanismcells and embryonic stem cells can inhibit adipogenesis inthe terminal diï¬erentiation phase erk1 activity leads topparÎ phosphorylation which inhibits adipocyte diï¬erentiation this implies that erk1 activity must be reduced afteradipocyte proliferation so that diï¬erentiation can proceedthis reduction is mediated in part by mitogenactivatedprotein kinase mapk phosphatase1 mkp1 [ ]these extracellular and intracellular regulation factors causeadipocytespeciï¬c gene expression and eventually lead toadipocyte formation adipocyte differentiationregulatory proteins pparÎ and adipocyte diï¬erentiation pparÎ is a member of the nuclearreceptor superfamily and is both necessaryand suï¬cient for adipogenesis forced expression ofpparÎ is suï¬cient to induce adipocyte diï¬erentiation broblasts indeedthe proadipogenic cebps andkrüppellike factors klfs have all been shown to induceat least one of the two pparÎ promoters in contrast antiadipogenic transcription factor gata functioned in part byrepressing pparÎ expression pparÎ itself has twoisomers the relative roles of pparÎ1 and pparÎ2 in adipogenesis remain an open question pparÎ2 is mainlyexpressed in adipose tissue while pparÎ1 is expressed inmany other tissues although both can promote adipocytediï¬erentiation pparÎ2 could do so eï¬ectively at very lowligand concentration compared with pparÎ1 the twoprotein isoforms are generated by alternative splicing andpromoter usage and both are induced during adipogenesispparÎ1 can also be expressed in cell types other than adipocytes ren et al used engineered zincï¬nger proteins tothe expression ofthe endogenous pparÎ1 andinhibitpparÎ2 promoters in 3t3l1 cells ectopic expression ofpparÎ2 promotes adipogenesis whereas that of pparÎ1does not zhang et al reported that pparÎ2 deï¬ciencyimpairs the development of adipose tissue and insulin sensitivity there are transcriptional cascades between adipocytesgenes including pparÎ and cebpα which are the coreadipocyte diï¬erentiation regulators in the early stage of adipocyte diï¬erentiation the expression of cebp and cebpδincrease which upregulates cebpα expressionfurtheractivate pparÎ pparÎ activating cebpα in turn resultsin a positive feedback pparÎ binding with retinoic acid xreceptor rxr forms diï¬erent heterodimers the variousdimmers can combine with the pparÎ response elementppre and initiate the transcription of downstream genesfor diï¬erentiation into adipocytes cebps participate in adipogenesis and several cebpfamily members are expressed in adipocytesincludingcebpα cebp cebpÎ cebpδ and cebphomologous protein chop the temporal expression of thesefactors during adipocyte diï¬erentiation triggers a cascadewhereby early induction of cebp and cebpδ leads tocebpα expression this notion is further supported by thesequential binding of these transcription factors to severaladipocyte promoters duringadipocyte diï¬erentiationcebp is crucial for adipogenesis in immortalized preadipocyte lines cebp and cebpδ promote adipogenesis atleast in part by inducing cebpα and pparÎ cebpαinduces many adipocyte genes directly and plays an important role in adipose tissue development once cebpα isexpressed its expression is maintained through autoactivation despite the importance of cebps in adipogenesis 0cstem cells internationalthese transcription factors clearly cannot function eï¬cientlyin the absence of pparÎ cebp cannot induce cebpαexpression in the absence of pparÎ which is required torelease histone deacetylase1 hdac1 from the cebpαpromoter furthermore ectopic cebpα expressioncannot induce adipogenesis in pparÎï¬broblasts however cebpα also plays an important role in diï¬erentiated adipocytes overexpression of exogenous pparÎ incebpαdeï¬cient cells showed that although cebpα isnot required for lipid accumulation and the expression ofmany adipocyte genes it is necessary for the acquisition ofinsulin sensitivity [ ] figure human ï¬broblasts withthe ability to diï¬erentiate into adipocytes also do not undergomitotic cloning ampliï¬cation however pparÎ exogenousligands need to be added to promote adipocyte diï¬erentiation therefore it can be inferred that mitotic cloning expansion can produce endogenous ligands of pparÎ bmp and transforming growth factor tgf inadipocyte diï¬erentiation a variety of extracellular factorsaï¬ect the preadipocyte commitment of stem cells includingbone morphogenetic protein bmp transforminggrowth factor tgf insulininsulinlike growthfactor igf1 tumor necrosis factor α and interleukin matrix metalloproteinase ï¬broblast growthfactor fgf and fgf2 bmp and tgf have variedeï¬ects on the diï¬erentiation fate of mesenchymal cells the tgf superfamily members bmps and myostatinregulate the diï¬erentiation of many cell types includingadipocytes tgf inhibitor can promote adipose diï¬erentiation of cancer cells with a mesenchymal phenotypein vitro and transgenic overexpression of tgf impairsadipocyte development inhibition of adipogenesis couldbe obtained through blocking of endogenous tgf with adominantnegative tgf receptor or drosophila mothersagainst decapentaplegic protein smad inhibitionsmad3 binds to cebps and inhibits their transcriptionalactivity including their ability to transactivate the pparÎ2promoter [ ] exposure of multipotent mesenchymalcells to bmp4 commits these cells to the adipocyte lineageallowing them to undergo adipose conversion theeï¬ects of bmp2 are more complex and depend on the presence of other signaling molecules bmp2 alone has little eï¬ecton adipogenesis and it interacts with other factors such astgf and insulin to stimulate adipogenesis of embryonicstem cells bmp2 stimulates adipogenesis of multipotentc3h10t12 cells at low concentrations and can contribute tochondrocyte and osteoblast development at higher concentrations klfs in adipocyte diï¬erentiation during adipocyte differentiation some klf family members are overexpressedsuch as klf4 klf5 klf9 and klf15 while klf16 expression is reduced [ ] klf15 is the ï¬rst klf family members which were identiï¬ed to be involved in adipocytediï¬erentiation its expression increased significantly on thesixth day of 3t3l1 adipocyte diï¬erentiation and peakedon the second day of adipocyte induction in mscs andmouse embryonic ï¬broblasts inhibition of klf15 by sirnaor mutation led to a decrease in pparÎ cebpα fatty acidbinding protein fabp4 and glucose transporter glut4 however overexpression of klf15 in nih3t3cells was found to be associated with lipid accumulation aswell as increases in pparÎ and fabp4 mice with complete absence of klf5 showed embryonal lethality and micewith singlechromosome klf5 knockout showed a significant reduction in white fat in adulthood suggesting thatklf5 plays an important role in adipocyte diï¬erentiationklf5 can be activated by cebp or cebpδ which isinvolved in early adipocyte diï¬erentiation klf5 can beactivated by cebp or cebpδ which is involved in earlyadipocyte diï¬erentiation direct binding of klf5 to thepparÎ2 promoter in combination with cebps inducespparÎ2 expression transfection of klf5 dominantnegative mutants in 3t3l1 cells reduced lipid droplet accumulation and inhibited pparÎ and cebpα expressionwhereas overexpression of wild klf5 significantly promotedadipocyte diï¬erentiation even without exogenous hormonestimulation similar to klf5 klf9 knockdown can inhibitthe expression of a series of adipocyte diï¬erentiation genessuch as pparÎ cebpα and fabp4 hence inhibitingadipocyte diï¬erentiation however klf9 overexpressiondid not upregulate the expression of pparÎ and cebpα in addition klf4 can transactivate cebp by bindingto the region of kb upstream of the cebp promoter and promote lipid diï¬erentiation klf6 can forma complex with histone deacetylase3 hdac3 inhibitingpreadipocyte factor1 pref1 expression and promotinglipid diï¬erentiation klf2 is highly expressed in adiposeprogenitors and its expression decreases during the processof lipid diï¬erentiation overexpressed klf2 can bind to thecaccc region of pparÎ2 proximal promoter and inhibitlipid diï¬erentiation as well as the expression of pparÎcebpα and sterolregulated elementbinding proteinssrebp by inhibiting the promoter activity rnasequence analysisshowed that klfl6 expression wasdecreased on the ï¬rst day of adipocyte diï¬erentiation of3t3l1 cells adipocyte diï¬erentiation was promoted byklf16 knockdown but was inhibited by klf16 overexpression via inhibition of pparÎ promoter activity in addition klf3 and klf7 were also found to play a negativeregulatory role in adipocyte diï¬erentiation [ ] signal transducers and activators of transcriptionstats and adipocyte diï¬erentiation the activated statprotein enters the nucleus as a dimer and binds to the targetgene to regulate gene transcription in the adipocyte diï¬erentiation of mouse 3t3l1 cells the expression of stat1 andstat5 was significantly increased while that of stat3and stat6 was not significantly changed in the adipocyte diï¬erentiation of human subcutaneous adipose precursor cells stat1 expression was significantly decreased while the expression of stat3 and stat5 wasincreased and stat6 expression was unchanged therole of stat1 in adipocyte diï¬erentiation is not clearbecause its expression trend in humans and mice diï¬ersduring the adipocyte diï¬erentiation process early adipocytediï¬erentiation of 3t3l1 cells was inhibited by stat1 0cstem cells internationalklf5srebp1cklf15klf2chopcebpá½»krox20ligandcebpð½cebpð¿gata23ppará½»cebpð¼proadipogenicantiadipogenicgenes of terminaladipocytedifferentiationfigure a cascade of transcription factors that regulate adipogenesis pparÎ is one of the key transcription factors in adipogenesis and thecore of the transcriptional cascade that regulates adipogenesis pparÎ expression is regulated by several proadipogenic blue andantiadipogenic red factors cebpα is regulated through a series of inhibitory proteinprotein interactions some transcription factorfamilies include several members that participate in adipogenesis such as the klfs black lines indicate eï¬ects on gene expression violetlines represent eï¬ects on protein activityagonist interferon Î loss of stat1 in 3t3l1 cells can rescue the inhibition of adipocyte diï¬erentiation caused byprostaglandin factor 2α other studies have found thatstat1 is required for adipose diï¬erentiation and stat1overexpression in c3h10t12 cells can prevent the inhibition of lipid diï¬erentiation caused by bcell lymphoma6knockdown there was no abnormal adipose tissuein stat1 knockout mice stat3 not only aï¬ectsthe proliferation of 3t3l1 cells but also coregulates theiradipocyte diï¬erentiation with high mobility group protein the fabp4 promoter was used to speciï¬callyknock out stat3 in the adipose tissue of mice and theresults showed that mice weight significantly increasedand the adipocyte quantity increased compared with thewildtype mice stat5a and stat5b have diï¬erenteï¬ects on adipocyte diï¬erentiation abnormal adipose tissuewas found in the mice with stat5a or stat5b knockout ordouble knockout and the amount of adipose tissue was onlyoneï¬fth of the original adipose tissue in mice withoutknockdown histone modiï¬cation in adipocyte diï¬erentiation histone deacetylase sirtuin sirt plays an important rolein biological processes such as stress tolerance energymetabolism and cell diï¬erentiation during the adipocyte diï¬erentiation of c3h1012 cells sirt1 expressiondecreased overexpression of sirt1 activated thewnt signal which caused the deacetylation of cateninthe accumulation of catenin in the nucleus could inhibitadipocyte diï¬erentiation sirt1 knockdown resulted inincreased acetylation of the histones h3k9 and h4k16 inthe secreted frizzledrelated protein sfrp and sfrp2 promoters thereby promoting transcription of these genes andpromoting lipid diï¬erentiation forkhead box proteino foxo is a member of the transcription factor foxofamily it can recruit cyclic amp response elementbindingprotein cbphistone acetyltransferase p300 to initiate anacetylation the acetylated foxo1 can be phosphorylatedby phosphorylated protein kinase b pkbakt the phosphorylation of foxo1 by akt inhibits the transcriptionalactivation of foxo1 the acetylation of foxo1 lost the ability of dnabinding aï¬nity and promoted its shuttling fromnuclei to cytoplasm sirt1 and sirt2 can deacetylateand active foxo1 activated foxo1 nonphosphorylatednuclear foxo1 in the nucleus binds to the promoters of target genes encoding p21 p27 and pparÎ and initiates subsequent transcriptions sirt2 inhibits the acetylation andphosphorylation of foxo1 thereby induces the accumulation of activated foxo1 in the nucleus activated foxo1could inhibit adipogenesis via pparÎ [] lysinespeciï¬c histone demethylase lsd1 expression increasedduring the adipocyte diï¬erentiation of 3t3l1 cells lsd1could reduce the dimethylation levels of histone h3k9 andh3k4 in the cebpα promoter region thereby promotingadipocyte diï¬erentiation set domaincontaining setd8 catalyzed the monomethylation of h4k20 andpromoted pparÎ expression the activation of pparÎ transcriptional activity leads to the induction of monomethylatedh4k20 and modiï¬cation of pparÎ and its targets therebypromoting adipogenesis enhancer of zeste homolog ezh2 is a methyltransferase and can bind methyl groupsto histone h3k27 which is also necessary for lipid diï¬erentiation the absence of ezh2 in brown fat precursors results inreduced levels of the wnt promoter histone h3k27me3which is also saved by the ectopic ezh2 expression or theuse of a wntcatenin signal inhibitor in addition histone demethylases such as lysinespeciï¬c histone demethylase lsdkdm kdm6 and histone lysine demethylasephf2 are also involved in adipose diï¬erentiation andkdm2b inhibits transcription factor activator protein 2αpromoter via h3k4me3 and h3k36me2 role of microrna and long noncodingrna in adipogenesismicrorna mir can bind and cut target genes or inhibittarget gene translation endogenous sirna can be producedby the action of dicer enzyme and bind to a speciï¬c proteinto change its cellular location many kinds of mirsare involved in regulating adipocyte diï¬erentiation the 0cstem cells internationalexpression of mir143 increased during the diï¬erentiationof adipose progenitor cells overexpression of mir143promoted gene expression involved in adipose diï¬erentiationand triglyceride accumulation inhibition of mir143 prevented the adipose diï¬erentiation of human fat progenitorcells [ ] additionally mir8 promotes adipocyte diï¬erentiation by inhibiting wnt signaling moreover mir mir103 mir21 mir519d mir210 mir30mir204211 and mir375 also play a certain role in promoting adipocyte diï¬erentiation while mir130 mir448and let7y inhibit lipid diï¬erentiation [ ] in additionto mirs long noncoding rna lncrna is a type of noncoding rna and is important during epigenetic regulationand can form a doublestranded rna complex with mrnacauses protein transcription lncu90926 inhibits adipocytediï¬erentiation by inhibiting the transactivation of pparÎ2 as a novel lncrna hoxaas3 expression increasedduring the adipose diï¬erentiation of mscs and hoxaas3 silencing reduced the marker gene of adipose diï¬erentiation and inhibited the adipose diï¬erentiation zhu et al reported that hoxaas3 interacted with ezh2 toregulate lineage commitment of mscs hoxa as3 canregulate the trimethylation level of h3k27 in the runx2promoter region by binding to ezh2 therefore hoxaas3 is considered to be an epigenetic switch regulating mscslineage speciï¬city adipocyte diï¬erentiationassociatedlncrna can act as a competitive endogenous rna of mir in the process of lipid diï¬erentiation thereby promotingthe expression of sirt1 the target gene of mir204 and thusinhibiting lipid diï¬erentiation the lncrna neat1can also regulate adipocyte diï¬erentiation under the uence of mirna140 other lncrna including lncrnablnc1 and plnc are also involved in regulating adipocytediï¬erentiation [ ] other biochemical response involved inadipocyte differentiation unfolded protein responses in adipocyte diï¬erentiationin the endoplasmic reticulum of eukaryotes unfolded protein response involves three proteinsinositolrequiringenzyme 1α doublestranded rnadependent proteinkinaselike er kinase and activating transcription factoratf 6α knockdown of atf6α aï¬ects the expressionof adipocytes genes and inhibits c3h10t12 adipocyte differentiation the inhibitory eï¬ect of berberine on adipocyte diï¬erentiation of 3t3l1 cells is also due to inducedchop and decorin expressions and this inhibitory eï¬ectis ameliorated by chop knockout in the adipocytediï¬erentiation process of 3t3l1 cells increases in pparÎand cebpα as markers of adipocyte diï¬erentiation wereaccompanied by an increase in the corresponding proteinexpressions of phosphorylated eukaryotic translation initiaeif 2α phosphorylated endoribonucleasetion factorire1α atf4 chop and other unfolded protein responsesendoplasmic reticulum stress inducer or hypoxic endoplasmic reticulum stress can inhibit adipocyte diï¬erentiationadditionally eif2α mutation results in continuous activation or overexpression of chop which also inhibits adipocyte diï¬erentiation after the initiation of adiposediï¬erentiation numerous diï¬erentiationassociated proteinsare synthesized exogenous endoplasmic reticulum stressinducers can lead to excessive endoplasmic reticulumresponse which in turn aï¬ects the synthesis of proteinsrelated to diï¬erentiation and inhibits adipocyte formationfigure role of oxidative stress in adipogenesis during thedirectional diï¬erentiation of mscs mitochondrial complexi and iii and nadph oxidase nox4 are the main sourcesof oxygen species ros production currently it is believedthat ros aï¬ects not only the cell cycle and apoptosis but alsodiï¬erentiation through uencing the signaling pathwaysincluding the wnt hh and foxo signaling cascade duringmscs diï¬erentiation the diï¬erentiation ability ofstem cells is determined by the arrangement of perinuclearmitochondria which speciï¬cally manifests as low atpcellcontents and a high rate of oxygen consumption the lackof these characteristics indicates stem cell diï¬erentiation adipocyte diï¬erentiation is a highly dependent rosactivation factor related to mitosis and cell maturation schroder et al found that exogenous h2o2 could stimulate adipocyte diï¬erentiation of mouse 3t3l1 cells andhuman adipocyte progenitor cells in the absence of insulinh2o2 regulates adipocyte diï¬erentiation of 3t3l1 cells ina dosedependent manner high doses of h2o2 and μm promote adipocyte diï¬erentiation [ ] tormos et al found that ros synthesis increased in humanmscs at the early stage of adipose diï¬erentiation and targeted antioxidants could inhibit lipid diï¬erentiation byknocking down rieske ironsulfur protein and ubiquinonebinding protein ros produced by mitochondrial complexiii was found to be necessary in initiating adipose diï¬erentiation however other studies have shown that theexpression levels of adiponectin and pparÎ were decreasedby using h2o2 mm in 3t3l1 cells free radical nitric oxide no also promotes lipid diï¬erentiationbecause treatment with no inducer hydroxylamine or nosynthase nos substrate arginine can significantly induceadipose diï¬erentiation of rat adipose progenitor cells nosinduced adipose diï¬erentiation mainly via enos rather thaninos ros can induce adipose diï¬erentiation primarily by inhibiting wnt foxo and hh signaling pathwaysthat inhibit lipid diï¬erentiation autophagy in adipocyte diï¬erentiation the increase inautophagosomes during lipid diï¬erentiation indicates thatautophagy may play an important role in lipid diï¬erentiation baerga et al conï¬rmed that the adipocyte diï¬erentiation eï¬ciency was significantly inhibited in mouse embryonic ï¬broblasts lacking autophagyrelated gene atg agene encoding an essential protein required for autophagy knockdown of atg5 in 3t3l1 cells promotesproteasomedependent degradation of pparÎ2therebyinhibiting adipocyte diï¬erentiation zhang reportedthat autophagyrelated gene 7atg7 is also crucial for adipose development atg7deï¬cient mice were slim and onlyhad of white fat compared to wildtype mice and the 0cstem cells internationalcebpð½ geneebf1 geneklf4egr2cebpð½cytosolcebpð¿ genecebpð¿klf5geneppará½» geneklf5nr2f2nfkb11433relasrebf1a2rxrappará½»ppará½»rxra heterodimerppará½»rxracorepressor complexfabp4ligands of ppará½»fam120bthrap3ep300ncoa2ncoa3helz2ncoa1crebbpebf1adipoq geneaidrfcebpð¼ geneznf638znf467cebpð¼ncor1hdac3ncor2 slc2a4 geneglut4 genelep genefabp4 genecdk4ccnd3plin1 genepck1 genefabp4cd36 geneppararxracoactivator complexppará½»fatty acidrxramediatorcoactivator complexangptlgeneppargc1amediator complex consensuslpl genenucleoplasmproteins bind to gene promoterstranscription of genes into proteinsacting on proteins compoundingtgfð½1wnt1wnt10btnf77233adipoqglut4slc2a4 tetramerlepfabp4lipid dropletplin1pck1papa pa4xpalmccd36paangptl4lplfigure regulation of adipocyte diï¬erentiation a regulatory loop exists between pparÎ and cebp activation transcription factor coeebf activates cebpα cebpα activates ebf1 and ebf1 activates pparÎ cebp and cebpδ act directly on the pparÎ gene bybinding its promoter and activating transcription cebpα cebp and cebpδ can activate the ebf1 gene and klf5 the ebf1 and klf5proteins in turn bind the promoter of pparÎ which becomes activated other hormones such as insulin can aï¬ect the expression ofpparÎ and other transcription factors such as srebp1c pparÎ can form a heterodimer with the rxrα in the absence of activatingligands the pparÎrxrα complex recruits transcription repressors such as nuclear receptor corepressor ncor ncor1 andhdac3 upon binding with activating ligands pparÎ causes a rearrangement of adjacent factors corepressors such as ncor2 are lostand coactivators such as transcription intermediary factor tif2 cbp and p300 are recruited which can result in the expression of cyclicampresponsive elementbinding protein creb followed by pparÎ pparÎ expression initiates the expression of downstream genesincluding angiopoietinrelated protein pgar perilipin fabp4 cebpα fatty acid transportrelated proteins carbohydrate metabolismrelated proteins and energy homeostasisrelated proteinslipid metabolism and hormoneinduced lipolysis in the adipocytes were altered autophagy related gene atg4b isactivated by cebp in the process of lipid diï¬erentiationand autophagy activation is necessary for the degradationof klf2 and klf3 two negative regulators of lipid diï¬erentiation these results showed that adipose diï¬erentiation andautophagy are mutually complementary in 3t3l1cells autophagy was inhibited by aspartate ammonia or 0cstem cells internationalmethyladenine at diï¬erent lipid induction periods and days and only autophagy inhibition at days hindered the formation of lipid droplets and the expression of lipid marker genes indicating that autophagy wasvery important in the early stage of lipid diï¬erentiation recent studies showed that lc3 is overexpressed in3t3l1 cells further demonstrating the important role ofautophagy in lipid diï¬erentiation role of alternative splicing in adipogenesis selectivesplicing is uenced by splicing regulators which regulateadipocyte diï¬erentiation by regulating the selective splicingof genes speciï¬c to this process lipin1 is an important regulator in the process of adipocyte diï¬erentiation and includestwo isomers lipin1α and lipin1 which have diï¬erenteï¬ects high expression of lipin1α promotes adipocyte differentiation while that of lipin1 promotes lipid droplet formation in sam68deï¬cient mice the ï¬fth intron ofserinethreonineprotein kinase mtor was retained resulting in unstable and rapid mtor degradation and inhibitionof adipocyte diï¬erentiation furthermore there arefour isomers of pref1 pref1a and pr | 0 |
prevalence of pathogenic variants in DnA damage response and repair genes in patients undergoing cancer risk assessment and reporting a personal history of earlyonset renal cancerTiffiney a0R a0Hartman12 a0Elena a0V a0Demidova345 a0Randy a0W a0Lesh6 a0Lily a0Hoang7 a0Marcy Richardson7 a0Andrea a0Forman8 a0Lisa a0Kessler1 a0Virginia a0Speare7 a0Erica a0A a0Golemis4 a0Michael a0J a0Hall38 a0Mary a0B a0Daly38 Sanjeevani Arora3Pathogenic a0variants a0PVs a0in a0multiple a0genes a0are a0known a0to a0increase a0the a0risk a0of a0earlyonset a0renal a0cancer a0eoRC a0However a0many a0eoRC a0patients a0lack a0PVs a0in a0RCspecific a0genes a0thus a0their a0genetic a0risk a0remains a0undefined a0Here a0we a0determine a0if a0PVs a0in a0DNA a0damage a0response a0and a0repair a0DDRR a0genes a0are a0enriched a0in a0eoRC a0patients a0undergoing a0cancer a0risk a0assessment a0Retrospective a0review a0of a0deidentified a0results a0from a0 a0eoRC a0patients a0undergoing a0testing a0with a0a a0multigene a0panel a0for a0a a0variety a0of a0indications a0by a0Ambry a0Genetics a0PVs a0in a0cancerrisk a0genes a0were a0identified a0in a0 a0of a0patientswith a0 a0in a0RCspecific a0and a0 a0in a0DDRR a0genes a0DDRR a0gene a0PVs a0were a0most a0commonly a0identified a0in a0CHEK2 a0BRCA1 BRCA2 and ATM a0Among a0the a0 a0of a0patients a0with a0a a0BRCA1 or BRCA2 a0PV a0 a0 a0reported a0a a0personal a0history a0of a0hereditary a0breast a0or a0ovarianassociated a0cancer a0No a0association a0between a0age a0of a0RC a0diagnosis a0and a0prevalence a0of a0PVs a0in a0RCspecific a0or a0DDRR a0genes a0was a0observed a0Additionally a0 a0patients a0reported a0at a0least a0one a0additional a0cancer a0breast a0cancer a0being a0the a0most a0common a0 a0of a0females a0 a0of a0males a0Multigene a0testing a0including a0DDRR a0genes a0may a0provide a0a a0more a0comprehensive a0risk a0assessment a0in a0eoRC a0patients a0Further a0validation a0is a0needed a0to a0characterize a0the a0association a0with a0eoRCRenal cancer RC often develops with no signs or symptoms and is referred to as the silent disease While factors including smoking environment obesity and race have been linked to increased risk of RC inherited factors are the most wellvalidated source of increased risk2 Hereditary RC syndromes typically associated with earlyonset disease and a clinically significant family history of cancer result from germline pathogenic variants PV in highpenetrance RCspecific genes including VHL MET FLCN TSC1 TSC2 FH SDH PTEN and BAP15 A previous report of an earlyonset RC eoRC cohort screened with an RCspecific panel found of individuals had a PV in an RCspecific gene7 However for most eoRC patients a PV in an RCspecific gene is not identified leaving many eoRC genetically undefined Thus there is a need to identify additional genes related to eoRC risk Currently there are no National Comprehensive Cancer Network NCCN guidelines for detection prevention or risk reduction in individuals who present with an eoRC but lack a PV in a defined RCspecific gene81Arcadia University Glenside PA USA 2Cancer Biology Program Fox Chase Cancer Center Philadelphia PA USA 3Cancer Prevention and Control Program Fox Chase Cancer Center Cottman Avenue Philadelphia PA USA 4Molecular Therapeutics Program Fox Chase Cancer Center Philadelphia PA USA 5Kazan Federal University Kazan Russian Federation 6Geisinger Commonwealth School of Medicine Scranton PA USA 7Ambry Genetics Konica Minolta Aliso Viejo CA USA 8Department of Clinical Genetics Fox Chase Cancer Center Philadelphia PA USA email SanjeevaniArorafccceduScientific RepoRtS 101038s41598020704495Vol0123456789wwwnaturecomscientificreports 0cDNA damage response and repair genes DDRR play an important role in maintaining genome integrity and when mutated in the germline can increase cancer risk for several types of cancers including breast colorectal ovarian and others9 Although PVs in DDRR genes are associated with increased risk of a variety of cancer types they are not typically considered risk factors for RC However germline PVs in some DDRR genes have been observed in RC including PVs in the DNA mismatch repair Lynch syndrome genes MSH2 and MLH1 in renal urothelial carcinoma and PVs in CHEK2 in advanced renal cell carcinoma10 To address the hypothesis that PVs in additional DDRR genes may contribute to the missing heritability of eoRC we analyzed germline sequencing data from a cohort of individuals with RCMaterials and methodsAmbry a0Genetics a0eoRC a0study a0cohort a0and a0variant a0determination a0 Deidentified data were requested from RC patients that were tested by Ambry Genetics Konica Minolta Aliso Viejo California using germline cancer testing panels Ambry samples were selected for patients with RC and deidentified data was obtained for all RC patients tested with multigene cancer panels n a0years at diagnosis specimens collected between July December All genetic test results from germline testing of individuals diagnosed with RC at during this time period were used in this studyThere is currently no standard definition specifying the age when RC is considered earlyonset Different models have been used to determine a specific age as a trigger for germline testing in patients with RC who lack family history of RC including ages or a0years For this study we selected individuals a0years or younger as the cutoff for our cohort which is substantially below the median age of RC diagnosis of a0years in the general population as reported in SEER2223 but considerably older than other suggested cutoffs We did so because the main hypothesis of the study was that PVs in DDRR genes might be responsible for increased risk of RC Variants in multiple DDRR genes are associated with earlyonset colorectal cancer2425 which typically manifests in patients at a0years or younger We considered that PVs in DDRR genes were most likely to impact repair of DNA damage induced during cell replication leading to genetic instability and cancer given renal cells turn over much less frequently than colon cells we hypothesized that it may take longer for cancers associated with PVs in DDRR genes to manifest in RC causing us to select a cutoff of a0years old for assessmentDeidentified data included family history of cancer genetic test results personal history of cancers apart from RC presence of multifocal tumors and RCsubtypestage The RC patients had been tested with CancerNext versions and CancerNextExpanded versions and Table a0S1 Deidentified patient information was analyzed for genetic test results and personal and family medical histories Classification of variants by Ambry Genetics is based on ACMG recommendations for standards for interpretation and reporting of sequence variations These variants are also regularly deposited in ClinVar by Ambry Genetics Variant classification was updated through March for all data Gene variants were classified as pathogenic variant PVsee below for criteria variant of uncertain significance VUS or inconclusive or negativeindeterminate Ambry Genetics follows strict criteria when classifying variants as PV Variant Likely Pathogenic VLP VUS Variant Likely Benign VLB and Benign for details see wwwambry gencomclini cianourscien tific excel lence varia ntclass ifica tion Variants reported as PV and VLPs were grouped as PVs All test results were used for this study The analysis of VUS which currently lack clinical significance was beyond the scope of this study Given updating of test panels by Ambry Genetics not all patients were tested for all genes Individuals were provided different versions of the panel over the course of the study see below and also see Table a0S1Any deidentified personal or family history information including sex ethnicityrace age of cancer diagnosis tumor histology history of additional personal cancer and history of family cancer and types was reported first as summarized data and later as deidentified individual case reports For analysis comparing outcomes for RCspecific genes versus genes not typically associated with RC we focused our statistical comparison on only those individuals who had CancerNext Expanded panel version testing which analyzes all genes including the RCspecific genes individuals who had the CancerNext Expanded version test were used for this statistical comparison For additional statistical test comparisons that analyzed the correlations between specific genes and categories such as tumor pathology or age any individual who had been tested for that specific gene was includedThe Western IRB issued a regulatory opinion that the Genomic Data Sharing Policy for Ambry Genetics does not involve human subjects based on CFR46102f and associated guidance thus the requirement to obtain written patient informed consent was waived A Data Use Agreement and Materials Transfer Agreement was established between Ambry Genetics and Fox Chase Cancer Center The FCCC Institutional Review Board IRB provided study oversight and approval protocol number Ambry Genetics provided deidentified patient medical and family history where available and genetic results for the patients All methods were performed in accordance with the relevant guidelines and regulation of the approved studyGenetic a0analysis a0with a0Ambry a0CancerNext a0and a0CancerNext a0Expanded a0panels a0Individuals were provided different versions of the panel by their healthcare provider see Table a0S1 The number of genes in the panels ranged from the smallest CancerNext panel Version which include genes APC ATM BARD1 BRIP1 BMPR1A CDH1 CHEK2 EPCAM MLH1 MRE11A MSH2 MSH6 MUTYH NBN PALB2 PMS2 PTEN RAD50 RAD51C SMAD4 STK11 TP53 to the largest CancerNext Expanded Version panel which contained genes APC ATM BAP1 BARD1 BRCA1 BRCA2 BRIP1 BMPR1A CDH1 CDK4 CDKN2A CHEK2 EPCAM FH FLCN GREM1 MAX MEN1 MET MITF MLH1 MRE11A MSH2 MSH6 MUTYH NBN NF1 PALB2 PMS2 POLD1 POLE PTEN RAD50 RAD51C RAD51D RET SDHA SDHAF2 SDHB SDHC SDHD SMAD4 SMARCA4 STK11 TMEM127 TP53 TSC1 TSC2 VHL The DDRRs identified in germline testing of this cohort are bolded26Scientific RepoRtS 101038s41598020704495Vol1234567890wwwnaturecomscientificreports 0cAmbry Genetics sequenced genomic DNA that was obtained from patient blood or saliva samples DNA was evaluated by next generation sequencing NGS of all coding sequences and ± bases into the ² and ² ends of flanking introns and untranslated regions In addition sequencing of the promoter region was performed for the following genes PTEN c to c MLH1 c to c and MSH2 c to c Additional Sanger sequencing was performed for any regions missing or with insufficient depth of coverage for reliable heterozygous variant detection and on potentially homozygous variants variants in regions with complicated pseudogene interference and when variant calls did not meet allele frequency quality thresholds Additional details on specific testing methods are available at wwwambry gencomclini ciangenet ictesti ng28oncol ogycance rnext expan dedControl a0population a0in a0ExAc a0and a0gnomAD a0 To compare the frequency of DDRR gene PVs found in the study to that in the general population our results were compared to the Exome Aggregation Consortium ExAc dataset of largely unrelated whole exome sequencing results and to the Genome Aggregation database gnomAD dataset consisting of exomes and genomes2728 These datasets are the most commonly used genomic data at the populationlevelClinVar a0analysis a0 ClinVar wwwncbinlmnihgovclinv ar a database of medically relevant gene variants was used to investigate all PVs in this study retrieved on February PVs that were not reported in ClinVar were noted as not reported Associated conditions for each PV were categorized into hereditary cancer predisposing syndromes conditions related to renal cancer and any other conditions To further elucidate any PVs related to renal cancer the search term renal cancer was queried and the results were noted as associated with ClinVar search term Renal CancerStatistical a0 analysis a0 To identify potential correlations between PVs and characteristics such as tumor pathology additional primary tumor type and age of diagnosis genes were combined into pathwaysgroups of interest histologys were grouped and cancer types were grouped Each individual was categorized as having a variant in one of the genes within the group or no variant in the group Gene categories were used for comparison of RC diagnosis with a DDRR or an RCspecific geneWe also tested the hypothesis that different gene groups are associated with age at RC diagnosis We used the median age of RC diagnosis in the study cohort a0years and studied PVs in patients a0years or ¥ a0years of age To test the association between the presence of PVs and age of RC diagnosis twosided Fishers exact tests were used and a0pvalues were considered significant Odds ratios OR were calculated to determine the odds that an outcome had occurred given a particular variant compared to the odds of the outcome occurring in the absence of that variant in the population tested Finally we queried the SEER database for patients under a0years old with RC to compare the distribution of their clinical characteristics where available to those in our study cohort22Due to the evolving nature of the panels during the course of this study each version included a different total number of genes and analysis of each gene is based on the number of individuals whose test included that gene Table a0S1 Only data from individuals was considered for comparison of individuals with RCspecific genes compared to those with variants in genes not typically associated with RC as the other individuals did not have all genes analyzed For statistical comparisons analyzing correlations between specific genes with various characteristics all individuals who had been tested for that specific gene were includedTo identify potential correlations between PVs and characteristics such as tumor pathology additional primary tumor type and age of diagnosis RCspecific genes other cancerassociated genes and DDRR genes were combined into groups and histologies were grouped The categories for comparison of PVs and patient characteristics are as follows Known RC genes BAP1 FH FLCN MEN1 MET MITF PTEN SDHA SDHAF2 SDHB SDHC SDHD TSC1 TSC2 and VHL versus genes not typically associated with RC APC ATM BARD1 BRCA1 BRCA2 BRIP1 BMPR1A CDH1 CDK4 CDKN2A CHEK2 EPCAM GREM1 MAX MLH1 MRE11A MSH2 MSH6 MUTYH NBN NF1 PALB2 PMS2 POLD1 POLE RAD50 RAD51C RAD51D RET SMAD4 SMARCA4 STK11 TEMEM127 TP53 versus DDRR genes alone ATM BARD1 BRCA1 BRCA2 BRIP1 CHEK2 MLH1 MRE11A MSH2 MSH6 MUTYH NBN PALB2 PMS2 POLD1 POLE RAD50 RAD51C RAD51D Histology categories combined from the original categories Chromophobe Papillary renal Clear cell Wilms Renal cell likely clear cell but cannot be confirmed Unknown Mixed papillary [clear cell papillary type papillary renalchromophobe renal and sarcomatoidpapillaryclear cell] Mixed chromophobe [chromophobeoncocytoma chromophoberenal cell clear cellchromophobe and clear celloncocytomachromophobe] Oncocytoma Mixed oncocytoma [clear celloncocytoma oncocytomacollecting duct and renal celloncocytoma] and Others [included clear cellsarcomatoid collecting duct mixed epithelial and stromal mucinous tubular and spindle cell multilocular cystic renal neuroendocrine renal cellWilms renal cortical sarcomatoid transitional urothelial and urothelial transitional] Transitional urothelial urothelial and papillary transitional cases were not included in the analysis for counts of pathogenic variants Renal oncocytomas mixed epithelial and stromal tumors are considered benign tumors and were not included in the analysis for counts of pathogenic variants Study a0approval a0 The Western IRB issued a a0regulatory opinion that the Genomic Data Sharing Policy for Ambry Genetics does not involve human subjects based on CFR46102f and associated guidance thus a0the Scientific RepoRtS 101038s41598020704495Vol0123456789wwwnaturecomscientificreports 0crequirement to obtain written patient informed consent was waived A Data Use Agreement and Materials Transfer Agreement was established between Ambry Genetics and Fox Chase Cancer Center The FCCC Institutional Review Board IRB a0provided study oversight and approval protocol number Ambry Genetics a0provided deidentified results for the study All methods were performed in accordance with the relevant guidelines and regulation of the approved studyResultsPatient a0characteristics a0 We first benchmarked the eoRC study cohort to the reported incidence of RC in SEER data for the general US population to provide context In the study cohort of cases were between a0years of age and median age of diagnosis was a0years As expected a higher percentage of RC cases were diagnosed between a0years of age as compared to patients diagnosed with RC in the general US population SEER versus Fig a01A The study cohort was female and male Fig a01B Table a0 versus female and male for the general US population prevalence of RC diagnosed Fig a01B Raceethnicities in study cohort were Caucasian African AmericanBlack Ashkenazi Jewish Hispanic other and unknown Table a0The tumor pathologies reported varied Fig a01C and Table a0 Clear cell constitutes of all RCs in SEER and was the most commonly reported histology in the eoRC cohort Renal cell not defined but likely to predominantly reflect clear cell was also common Fig a01C and Table a0 Papillary and chromophobe histology were each identified in of the individuals and respectively Other histologies were identified rarely but included Wilms tumor and oncocytoma For of patients the RC subtype was unknownHigh a0incidence a0of a0other a0cancers a0in a0study a0cohort a0 n of the cases in the study cohort reported at least one additional primary cancer Fig a01D Table a0 Table a0S2 Each of the primary cancer types is also represented at a higher level in the study cohort than in the general US population as reported by the SEER database Fig a01D For femalespecific cancers of females also had breast cancer in comparison to the breast cancer rate in women in the general population SEER Fig a01D and Table a0S2 The rate of additional primary cancer in the study cohort is much higher than the rate of each cancer type observed in SEER cases with eoRC Fig a01E Finally patients out of reported a family history of cancer and of these patients specifically reported at least one family member with RC Table a0Multigene a0cancer a0panel a0testing a0identifies a0PVs a0in a0DDRR a0genes a0in a0the a0study a0cohort a0 The most common gene with PVs identified in the eoRC patients was the DDRR gene CHEK2 Fig a02A Table a0S3 and S4 consistent with a recent report by Carlo et a0al16 Of patients with CHEK2 PVs n had a common highly damaging variant c1100delC pThr367Metfs that is known to be associated with an increased risk for breast prostate colorectal and thyroid cancers Table a0S434After CHEK2 PVs were most frequently observed in the DDRR genes BRCA2 ATM and BRCA1 Table a0S3 We compared the overall frequency of PVs in CHEK2 BRCA1 BRCA2 and ATM to the control population in ExAc and gnomAD representing individuals sequenced for diseasespecific and population genetic studies2728 Overall PVs in each of these genes were more common in the study cohort versus the control populations Fig a02BC Table a0S5A An outlier was the moderate risk CHEK2 c470TC p I157T PV38 identified in individuals in the study cohort which was higher in the controls gnomAD OR CI ExAc OR CI We compared the prevalence of all PVs in DDRR genes presented in Table a0S4 from cases to controls from gnomAD23 We found 48fold enrichment of PVs in DDRR genes in the study cohort versus the controls in gnomAD vs respectively Table a0S5B each DDRR gene was corrected for number of patients assessedCancer risk with MUTYH DDRR gene has only been defined for individuals with homozygous or compound heterozygous PVs but not for heterozygous carriers39 We identified individuals with MUTYH PVs out of which were heterozygous carriers and only was compound heterozygous Only the individual with compound heterozygous MUTYH PVs was counted in the full study cohort n Table a0S3 and Fig a02A Similar to MUTYH cancer risk from the FH RCspecific gene c1431_1433dupAAA pK477DUP variant is currently considered to be pathogenic only in the compound heterozygous or homozygous state40 We identified RC patients who were heterozygous carriers of this specific FH variant Tables a0S3 and S4The overall gene variation rate in the full study cohort n is presented in Table a0S3 The full study cohort was not tested for all genes The largest panel was tested in the subcohort of cases and consisted of genes which included RCspecific genes and othercancer associated genes including DDRR genes Table a0S1 Here of cases had PVs PVs were identified in one or more of the genes not typically associated with RC in cases n Table a0S6 versus n with a PV in RCspecific genes Fig a02D Table a0S6 Of the genes not typically associated with RC were in DDRR genes n or n Among the patients patients were found to have PVs in two genes One patient had PVs in two DDRR genes BRCA1 and MUTYH het and the other patient in a RCspecific gene and a DDRR gene SDHB and MUTYH het Table a0S4 There was no MUTYH or FH compound heterozygous or homozygous PV in the subcohort of casesDDRR a0genes a0PVs a0are a0similarly a0enriched a0in a0patients a0diagnosed a0with a0eoRC a0alone a0or a0with a0eoRC a0and a0other a0cancers a0Individuals who were tested for all genes n could be further separated into two subcohorts those with eoRC as their only diagnosis n and those with eoRC and one or more additional types of cancer n To test the hypothesis that DDRR gene PVs might be Scientific RepoRtS 101038s41598020704495Vol1234567890wwwnaturecomscientificreports 0cAiega yb ssongad iCsesac AgenrnrWilmsersothal cellnrenrnwonknunramebohpomchroebod hmixepomchroar cellncocytocleodncocytomixeoamalnpillarypillary read pmixeapKey A C D E FSEER cohort n97805Ambry cohort n844Bsesac FemaleMaleSEER cohortAmbry cohortDtear recnac brainstabrectalolorecmiaekuleamonelamnariavoaticcrenapstateproamoarcsyroidthetrialuterinemodneEsesac number of primary cancers reportedFigure a0 Patient characteristics A Age range of individuals diagnosed with RC a0years in SEER cohort compared to the study cohort n of the remaining individuals in the study were diagnosed a0years were diagnosed at a0years and were excluded from the calculations as their age was reported as a wide range of years B Percentage of males and females diagnosed with RC a0years in SEER compared to the study cohort n C The percentages of reported RC histology up to age a0years in the SEER data n compared to the study cohort n not all histological subtypes reported in SEER were reported in the study cohort D The percentage of cancer incidence at a0years in the general SEER population versus the study cohort The SEER data reflect individuals reporting the indicated cancer type not individuals with RC in addition to the indicated cancer type E Percentage of different primary cancers reported a0years in SEER n versus the study cohort n Less than not reported for figure clarityScientific RepoRtS 101038s41598020704495Vol0123456789wwwnaturecomscientificreports 0cCharacteristicSexMaleFemaleEthnicityAfrican AmericanAshkenazi JewishAsianCaucasianHispanicMiddle EasternMixed EthnicityNative AmericanOtherUnknownMedian age of testingHistologyChromophobeMixed chromophobeClear cellOncocytomaMixed oncocytomaPapillary renalMixed papillaryRenal cellWilmsOthersUnknownPersonal cancer historyRenal cancer onlyRenal cancer plus additional cancer typeFamily history of cancerYesNoNot reportedunknownFamily history of renal cancerYesNoTotalNumber of patients in Ambry study cohort Rate in general population from birth to age SEER of renal cancers of renal cancersnrnrnrnrnrnr a0years Table Demographics and clinical characteristics of RC patients in the Ambry Genetics study cohort Demographics and clinical characteristics of the RC cases in the study cohort were compared to those of RC from birth to age in the SEER data Personal and family history of cancer were reported for the cases in the study cohort For family history of renal cancers numbers include only those who reported on cancer history n nr not reported SEER data included types of renal cancer histologies not all were represented in dataset other based on other category from Ambry cohort Family histories as selfreported on the intake formmedical records and have not been validatedassociated with eoRC we first analyzed PVs in eoRC cases with no additional primary cancer diagnosis Among the patients who only presented with eoRC PVs were identified in of cases n Fig a02E which is approximately twice the reported frequency of PVs in RCspecific genes7 Among this n of PVs were in one of DDRR genes ATM BRCA1 BRCA2 BRIP1 CHEK2 MLH1 MRE11A NBN PALB2 RAD51C n were in one of RCspecific genes BAP1 FLCN SDHB VHL and the remaining cases bore PVs in nonDDRR genes associated with cancers other than RC Fig a02ENext we performed similar analysis as described above for patients who presented with eoRC plus one or more additional cancers Among the patients who presented with eoRC and at least one additional cancer Scientific RepoRtS 101038s41598020704495Vol1234567890wwwnaturecomscientificreports 0cPVs were identified in cases Fig a02F Among these of cases PVs in othercancer associated genes including DDRR genes were found in of cases n versus n of cases with PVs in RCspecific genes This population was also enriched for PVs in DDRR genes n ATM BRCA1 BRCA2 CHEK2 MSH6 PALB2 PMS2 versus PVs in RCspecific genes BAP1 FLCN MET MITF PTEN SDHB VHLOverall these data suggest that DDRR gene PVs are enriched similarly in individuals diagnosed with eoRC alone or eoRC plus at least one additional primary cancer but that the frequency of PVs in DDRR genes in either group exceeded that in the control populations tested gnomADExAc Fig a0 Table a0S5A The specific PVs identified were similar in frequency to those identified in the full patient cohort n with CHEK2 the most represented DDRR genes Fig a0 To gain additional insight into the prevalence of these PVs in cancer patients we surveyed ClinVar wwwncbinlmnihgovclinv ar and found that multiple PVs from this study Table a0S4 have been reported in hereditary cancer predisposing syndromes HCPS summarized in Table a0S7 HCPS reflects a pattern of cancers in a family characterized by earlier onset with individuals not necessarily having the same tumor andor having more than one primary tumor and having tumors that are more likely to be multicentricRC a0patients a0with a0BRCA1 or BRCA2 a0PVs a0 Notably of the eoRC cases had a BRCA2 PV and RC cases had a BRCA1 PV Table a0 Table a0S3 This included n Table a0 of the cases who presented with only eoRC Interestingly despite the fact that the cohort was female of the detected BRCA1 and BRCA2 PVs were in males Table a0 Of the RC cases with a BRCA1 or BRCA2 PV had an additional cancer associated with hereditary breast and ovarian cancer HBOC syndrome breast ovarian prostate pancreatic or melanoma had an additional nonHBOC cancer and presented with only eoRC Table a0 Family history was reported for cases and of those indicated that at least one family member had an HBOCassociated cancer Of those with a BRCA2 PV reported that at least one family member had RC Table a0No a0correlation a0between a0age a0of a0RC a0diagnosis a0and a0type a0of a0PV a0in a0RC a0 To determine if identification of specific classes of germline PV correlated with age of diagnosis in this cohort genes were divided into four broad overlapping categories all genes in the panel RCspecific genes nonRC genes including DDRR genes and DDRR genes see Methods The groups were compared to median age at first RC diagnosis of or ¥ a0years Given the invariable earlyonset of Wilms tumor the individuals with this diagnosis were excluded from the analysis Within this eoRC cohort there was no significant association between age at diagnosis of RC and the type of PV for any of the four broad categories above Fig a03ACorrelation a0of a0renal a0histologies a0with a0PVs a0in a0specific a0genes a0 Of the clear cell cases in this cohort had a PV of which were RCassociated PVs Similar findings were observed for the cases described as renal cell carcinoma had a PV of which were RCassociated DDRR gene PVs were found in of clear cell cases and in of renal cell cases Figure a03BC contrast the findings in clear cell and renal cell histology with the other nonclear cell histologiesDiscussionThis study for the first time demonstrates that PVs in multiple DDRR genes occur in patients with eoRC Importantly this study found that DDRR gene PVs were represented both in cases diagnosed with eoRC and additional cancers and also cases diagnosed with eoRC alone Comparison with a large control population indicated that germline PVs in DDRR genes were more common in this study cohort than in the control population although further studies are required to confirm this finding and estimate the penetrance of PVs in DDRR genes for eoRC We also found that germline testing using an RCspecific panel would have identified only of the RC cases with actionable PVs according to the NCCN recommended screening or management guidelines compared to the additional cases identified with the expanded panelsThe most common gene with PVs identified in the patients in this study was the DDRR gene CHEK2 This is consistent with recent reports by Carlo et a0al and Huszno et a0al1516 While evidence is mounting that CHEK2 PVs may increase risk for RC in this study we did not consider CHEK2 as a gene typically associated with RC as it is not currently included on RC panels and would fail to be included in testing in many cases In addition limitations of the previous studies and the analysis reported here together indicate that larger studies with appropriate controls are needed before confirming that CHEK2 indeed confers a risk for RCIdentification of germline DDRR gene PVs can have specific implications for the proband and the family For example of cases diagnosed with eoRC alone had PVs in BRCA1 or BRCA2 but not all of these cases had a family history strongly indicative of HBOC syndrome This is important because identification of a BRCA PV can potentially change medical management for instance PARP inhibitor therapy is effective in tumors with BRCA PVs including nonbreast tumors4142 Also screening and prevention of HBOCsyndrome cancers would likely be increased significantly in the proband and in family members found to have the same PV Further many of the specific PVs identified in this study have been annotated as relevant to various HCPS emphasizing their role in the development of multiple cancer types Our results support broader panel testing as a way to identify unexpected highpenetrant PVs in eoRC patients when there is a personal or family history of additional cancers especially an HBOCsyndrome cancerScientific RepoRtS 101038s41598020704495Vol0123456789wwwnaturecomscientificreports 0cA stinairav cnegohapt lan deifitneditot BKey A D E FDDRR genesother cancer associated genesrenal cancer associated genesMTADRABACRBACRBPIRBKEHCHLMHSMHSMAERMHYTUMNBNBLAPSMPCDARCPAARPMBANKDCFNPTPABNCLFTEMFTIMNETPAHDSBHDSLHVPathogenic DDRR variants in Ambry cohort vs ExAc populationC Pathogenic DDRR variants in Ambry cohort vs GnomAD populationKey B CATM BRCA1BRCA2CHEK229211GA3576GA8655dupT5712dupA68_69delAG2475delC7558CT9294CG7069_7070delCT3847_3848delGT2339CG4284dupT518delG4441GA4441CT470TC1 | 2 |
"pd1pdl1 blockade therapy is a promising cancer treatment strategy which has revolutionized the treatmentlandscape of malignancies over the last decade pd1pdl1 blockade therapy has been trialed in a broad range ofmalignancies and achieved clinical success despite the potentially curelike survival benefit only a minority ofpatients are estimated to experience a positive response to pd1pdl1 blockade therapy and the primary oracquired resistance might eventually lead to cancer progression in patients with clinical responses accordingly theresistance to pd1pdl1 blockade remains a significant challenge hindering its further application to overcomethe limitation in therapy resistance substantial effort has been made to improve or develop novel antipd1pdl1based immunotherapy strategies with better clinical response and reduced immunemediated toxicity in thisreview we provide an overview on the resistance to pd1pdl1 blockade and briefly introduce the mechanismsunderlying therapy resistance moreover we summarize potential predictive factors for the resistance to pd1pdl1blockade furthermore we give an insight into the possible solutions to improve efficacy and clinical response inthe following research combined efforts of basic researchers and clinicians are required to address the limitation oftherapy resistancekeywords pd1pdl1 blockade cancer immunotherapy resistance immunotherapy is a validated and significant cancertreatment strategy which eliminates tumors by normalizing the antitumor immune responses [ ] over thelast decade cancer immunotherapy has revolutionizedthe treatment landscape of malignancies and achievedclinical success especially in immune checkpoint inhibitors correspondence 189whueducn lschrjjs163com jinyu sun and dengke zhang are cofirst authors4department of general surgery the first affiliated hospital of nanjingmedical university nanjing china2key laboratory of imaging diagnosis and minimally invasive interventionresearch lishui hospital of zhejiang university the fifth affiliated hospitalof wenzhou medical university clinical medicine of center hospital of lishuicollege lishui chinafull list of author information is available at the end of the signalsandprogrammed death1 pd1 is a class of receptorexpressed on the t cell surface which could downregulate the immune system by abrogating t cellreceptorinducedantigenmediated t cell activation the interaction betweenpd1 and its ligand programmed deathligand pdl1 plays an essential role in maintaining selftoleranceand avoiding autoimmune diseases however pd1pdl1 could also prevent the activation of t cells in thetumor and thus result in immune resistance preventingpd1pdl1 blockade is a breakthrough in cancerimmunotherapy and it has been trialed in a broadrange of malignancies in the preclinical or clinicalincluding melanoma hodgkins lymphomastage breast cancer [ ] nonsmall celllung cancer as well as hepatocellular carcinomansclc the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0csun biomarker research page of [ ] despite the longterm potentially curelikeclinical benefits therapy resistance remains a significant challenge for the further application of pd1pdl1 blockade therapy only a minority of patientsin general are estimated to experience apositive response to pd1pdl1 blockade therapy[] and the primary or acquired resistance mighteventually lead to cancer progression in patients withclinical response [ ]in this review we provide an overview on the resistance to pd1pdl1 blockade and its underlying mechanisms moreover we summarize potential predictivefactors for the resistance to pd1pdl1 blockade furthermore we give an insight into the possible solutionsto improve efficacy and clinical response of pd1pdl1blockade therapyresistance to pd1pdl1 blockade therapycheckpoint inhibitors targeting pd1 or pdl1 coulddisturb the interaction between pd1 and pdl1 whichwould preserve antitumor properties of t cells withdraw immune escape and normalize their ability to induce tumor cell death currently pd1pdl1 blockadehas shown sustained survival benefits in multiple malignancies and is at the forefront of cancer immunotherapy howeverjust as tumor cells can avoid immuneevasion several cancers may evolve to resist pd1pdl1 blockade therapy clinical evidence indicated thateven for patients with tumors highly positive for pdl1more than of them might not respond to pd1pdl1 blockade due to tumor heterogeneity and manyother reasons clinical responses vary largely across different tumor entities the objective response rate was in melanoma in nsclc in head and neck carcinoma and in kidneycancer besides for most patients experiencing initial clinical response acquired resistance remains another problem which would lead to cancer progressionor relapse after a few years [ ]many studies have demonstrated that antipd1therapy can significantly improve survival outcomes forpatients with metastatic or unresectable melanoma however only a small number of patients approximately could achieve a complete response in arecent phase i trial of atezolizumab antipdl1 involving patients with metastatic melanoma the overall response rate was among efficacy evaluablepatients and the median response duration was months moreover in another study on the longtermoutcomes of melanoma patients receiving antipd1therapy complete responses were only observed in of patients after a median followup of months of patients were alive without additional melanoma therapy additionally in the retreatedpatients after disease progression the response was onlyobserved in retreated patients receiving singleagent pd1 blockade therapy and of patientsescalated to pd1 blockade plus ipilimumab therapy inthis cohort most complete responses were durable withthe treatment failure rate of at three years whilethe response to retreatment remained relatively infrequent with a response rate of for patients withsingleagent pd1 blockade therapy moreover in aphase ii study of pembrolizumab on patients withadvancedobjectiveresponse was observed in of patients with a diseasecontrol rate of after a median followup of months adrenocorticalcarcinomatheinterestingly the response rate of some malignanciesis relatively high in hematological malignancies for example for patients with relapsed or refractory classicalhodgkin lymphoma tislelizumab antipd1 achievedan objective response rate of and a completeresponse of in a phase ii singlearm multicenterstudy similarly the complete response rate of camrelizumab antipd1 was with a partial remission rate of mechanisms underlying the resistance to pd1pdl1 blockadesince therapy resistance remains a significant limitationof pd1pdl1 blockade in clinical practice interest isgrowing in understanding the mechanisms underlyingthe resistance the response to pd1pdl1 blockaderelies on a preexisting immune response and determinants of adaptive immunity currently multiple factorshave been discovered to be involved in the efficacy ofpd1pdl1 blockade therapy such as tumor immunogenicity t celltumormicroenvironment and so forthfunction pdl1 expressionthe lack of tumor antigensthe genetic alterations are centralin the oncogenicprocess which could lead to tumor immunogenicity andprovide an opportunity for cancer immunotherapy tumor immunogenicity is positively associated with theability of the t cell to recognize tumor cells which isessential for the antitumor effect of pd1pdl1 blockade however the lack of tumor antigen will significantlyimpede the recognition ability of t cells and eventuallyresult in the failure of immunotherapymicrosatellites are prone to dna replication errorswhich will usually be repaired in normal cells however in tumors with mismatch repair mmr deficiencythese errors will accumulate which eventually result in alarge number of mutations and lead to microsatellite instability msi importantly high msi positivelycontributes to increased neoantigen production greater 0csun biomarker research page of immunogenicity and a more robust immune response moreoverthe resultant high tumor mutationburden would contribute to tumor immunogenic andenhance the response to pd1pdl1 blockade therapy[ ]multiple studies have demonstrated that the tumormutation burden is positively correlated with neoantigenburden as well as response to immunotherapy [ ]for example in colorectal cancer with mmr deficiencywhich usually exhibits a high tumor mutation burdenantipd1 therapy showed a higher response rate andbetter survival outcome compared to other subtypeswith mmr proficiency [] yarchoan analyzed the objective response rates of pd1pdl1blockade therapy for the corresponding tumor mutationburden in various cancers and their results showed thatthe mutation burden was closely associated with the objective response rate moreover pancreatic cancer generally exhibits a lowermutation load compared with other solid tumors andtherefore pd1pdl1 blockade is usually ineffective forthose patients and fails to improve their survival outcomes nevertheless in pancreatic cancer patients harboring an mmr deficiency they appear to be responsiveto pd1pdl1 blockade therapy mmr deficiency significantly increases the somatic mutation rate whichcould be translated into neoantigens and recognized bythe immune system thus making these patients responsive to pd1pdl1 blockade therapy [ ] accordingly pembrolizumab has been approved for selectedcancer patients with mmr deficiencyt cell dysfunctioneffective pd1pdl1 blockade therapy relies on the tcell function and any disruption in the processes of tcell immune function will result in the failure of pd1pdl1 blockade therapy a recent review by ren has provided an indepth insight into the mechanisms underlying the t cell dysfunctionmediated resistance with a focus on t cell recognition activationdifferentiation infiltration depletion as well as chemotaxis identification byantigen presentation is a critical process for the tumorantigensinitial t cells beta2microglobulin b2m is a significant hla1 moleculewhose mutation will hinder tumor antigen presentationand result in therapy resistance [] zaretsky analyzed biopsy samples from patients with metastatic melanoma receiving pembrolizumab who exhibited disease progression after an initial tumor regressionand they found a truncating mutation in the b2m genein the following research gettinger identifiedacquired homozygous loss or downregulation of b2m inlung patients with resistance to pd1pdl1 blockadeto further explore the role of b2m in mediating resistance they knocked out the b2m gene in immunocompetent lung cancer mice by crispr technology and theloss of b2m resulted in the resistance to pd1pdl1blockade additionally b2m mutationinducedresistance primarily occurred in an environment ofactivated pd1 positive t cellinfiltration whichresistance to pd1pdl1 blockadesuggested thattherapy might be particularly common in patients withhigh pd1 positive t cell for example t cellmoreover t cell activation is another critical processfor pd1pdl1 blockade therapy after blocking pd1pdl1 tumor cells can still counteract the activity ofimmune checkpoints and activate additional inhibitorypathways via expression of other immune checkpointsand their ligands within the tumor immune microenvironmentimmunoglobulinmucin3 tim3 is another type of immune checkpointreceptor expressed on tumorinfiltrating lymphocytes inhuman head and neck squamous cell carcinoma tumorinfiltrating lymphocytes pd1 blockade was demonstrated to upregulate tim3 expression which inhibitedt cells activation and contributed to tim3mediatedescape from pd1 blockade in the tumor microenvironment via pi3kakt pathway pd1 or pdl1physiologicallyinteractions between pd1 and pdl1block t cell activation pathways related to the immuneresponse against specific antigens and the expression ofpd1 or pdl1 has gained importance as a significantplayerin regulating the response to pd1pdl1blockade therapy pd1 and pdl1 are upregulated inthe tumor immune microenvironment of various malignancies which is considered as a strategy to evadeimmunosurveillance and imposes a significant barrier ofthe antitumor immune response importantly pdl1 primarily exhibits two distinct expression patternson tumor cells or on tumorinfiltrating immune cellspdl1 expression on immune cells reflects the adaptiveregulation meditated by ifnγ which is accompanied byincreased effector t cells as well as tumorinfiltratinglymphocytes effector t cells differently the expressionof pdl1 on tumor cells is less prevalent and it indicates the epigenetically dysregulated pdl1 gene whichis correlated with reduced immune infiltration scleroticor desmoplastic stroma as well as mesenchymal molecular features multiple studies have revealed a significantly higherobjective response rate in tumor pdl1 positive patientsthan pdl1 negative subgroups together with an improved progressionfree and overall survival [ ]kowanetz observed that atezolizumab antipdl1 achieved an objective response rate of in 0csun biomarker research page of patients with high pdl1 levels on tumor cells alone andof in those with a high expression on immune cellsalone although these observations indicated that thefunctional importance of pdl1 expression in regulatingpd1pdl1 blockadeinduced t cellthemechanistic significance of pdl1 on tumor cells or immune cells remains vagueresponsenoncoding rnasa large amount of micrornas mirnas and some longnoncoding rnas lncrnas have emerged as players inregulating tumor immunity [] and resistance topd1pdl1 blockade therapy recently huber identified a panel of circulating mirnas mir146a mir155 mir125b mir let7e mir125a mir146b mir99b which wereassociated with phenotypic and functional features ofmyeloidderived suppressor cells mdscs in melanomapatients importantly mdscs are a subclass of immature myeloid cells pathologically associated with cancerand play an inhibitory role against antitumor t cell immunity the transcriptional analysis showed thatthese mirnas could facilitate the conversion of monocytes into mdscs by melanoma extracellular vesiclesand the expression level ofthese mirna was upregulated in circulating cd14 monocytes and tumorsamples which was associated with myeloid cell infiltration and could predict the resistance to pd1 blockadetherapy moreover hu revealed the role of oncogeniclncrna for kinase activation linka in losing antigenicity and evading immune checkpoints and demonstrated lncrnadependent antigenicity downregulationsuppression for patients withand intrinsic tumortriplenegative breast cancer and resistantto pd1blockade therapythey showed upregulated linkalevels and downregulated peptideloading complex components the analysis suggested that linka expressioncould attenuate protein kinase amediated phosphorylation of the e3 ubiquitinprotein ligase trim71 via facilitating the crosstalk between phosphatidylinositol []trisphosphate and inhibitory gproteincoupled receptor pathways consequently linka could contribute to the degradation of the antigen peptideloadingcomplex and upregulate intrinsic tumor suppressors gut microbiomethe gut microbiome is a complex system composed ofmore than trillion microanisms which has beendemonstrated to regulate the efficacy and toxicity ofcancer immunotherapy many studies have reported theinfluence of the gut microbiome on cancer immunotherapy and the therapeutic response of pd1pdl1blockade therapy can be improved or diminished via gutmicrobiome modulationin mice models with distinct microbiome a significantly different response to pd1pdl1 blockade therapy was observed for example melanoma mice with anincreased bifidobacterium species in the gut microbiomeexhibited an effective response to pd1 blockade therapy similarly antibiotic administration was reported toreduce the diversity and aggravate dysbiosis of the gutmicrobiome thus influencing the clinical response topd1pdl1 blockade in tumorbearing mice as well ascancer patients [] compared to patients withoutantibiotic treatment the oral antibiotic application couldsignificantly diminish the clinical benefit of pd1pdl1blockade therapy and decrease progressionfree survivaland overall survival therefore dysbiosis of the gut microbiome is considered as one of the putative mechanisms underlying poorresponse to pd1pdl1 blockade therapy and thedualdirectional modulation of the gut microbiome oncancer immunotherapy is increasingly revealed howeverit is still unclear how gut microbiome regulatestherapy response and whether a specific bacterial taxaor gut microbiome as a whole plays a primary role remains largely unclear further research is required toprovide a more indepth understanding of the underlying mechanismspredictive factors for pd1pdl1 blockadetherapydespite the clinical success achieved in pd1pdl1blockade across multiple cancers the knowledge concerning therapy selection criteria is relatively limitedconsidering the potential adverse events and high costof immune checkpoint inhibitor agents there is a substantial need to identify predictive factors to select patients likely to benefit from this therapy currently apartfrom the functional status of immune cells [] ortumor infiltrating lymphocytes multiple factorshave been identified to predict the response to pd1pdl1 blockade therapy such as pd1pdl1 expression antigen recognition gut microbiome and so forthtable pd1 or pdl1 expressioninhibiting the pd1 pathwaymediated immune suppression is the basis and premise of pd1pdl1 blockadetherapy accumulating research has suggested that pdl1 is a biomarker to predict therapeutic response to pd1pdl1 blockade across multiple tumor types forexample atezolizumab achieved overall survival benefitacross all pdl1 expression subgroups in nsclc patients while those with high pdl1 expression experienced a more substantial survival benefit currently 0csun biomarker research page of table predictive factors for pd1pdl1 blockade therapytumor typenonsmall cell lung canceragentatezolizumabmultiple cancerscolorectal cancerurothelial carcinomaurothelial carcinomaurothelial cancermelanomamelanomamelanomapembrolizumabnivolumabatezolizumabatezolizumabatezolizumabantipd1 therapyantipd1 therapyantipd1 therapymmr mismatch repair msi microsatellite instability tmb tumor mutation burdenpredictive factorpdl1pdl1mmr msitmbtmbtmbgut microbiomegut microbiomegut microbiomereference pdl1 testing is recommended as a predictive test fornsclc urothelial carcinoma or head andneck cancers and so forthott assessed the predictive value of pdl1expression in patients with advanced solid tumors receiving pembrolizumab and the analysis showed that tumors with higher pdl1 expression and tumor mutationburden were significantly associated with higher response rate and more prolonged progressionfree survival heat map analysis revealed a close correlationbetween pdl1 expression and a broader pattern ofcoregulated gene expression which involved cytokine recruitment of t cells t cell activation markers as well asantigen presentation also the regression metaanalysisdemonstrated that pdl1 expression level was positivelyassociated with objective response rate p aswell as progressionfree survival p moreover nct02853305 and nct02807636 evaluated the efficacy of pembrolizumab or atezolizumab asfirstline treatment and the current data showed reduced survival in patients with low expression of pdl1accordingly it is advised that pembrolizumab or atezolizumab should be used for adult patients with a relativelyhigh pdl1 expression pdl1 expression of ¥ foratezolizumab and a combined positive score of ¥ forpembrolizumab however the efficacy of pd1pdl1blockade therapy as firstline therapy for advancedurothelial carcinoma still remains unclear [ ]importantly pdl1 positive only is not a predictivefactor for the response to pd1pdl1 blockade sincemultiple factors are involved in the pd1pdl1 blockade therapy in a study on patients with metastaticmelanoma receiving pembrolizumab preexisting cd8t cells were demonstrated as a prerequisite for thetumor regression after pd1pdl1 blockade therapy besidesin advanced adrenocortical carcinomatumor pdl1 expression status was not associated withtherapy response additionally it was reported thatpdl1 expression on tumor cells was not associatedwith therapy response in resected head and necksquamous cell cancer additionalinvestigation isrequired to illustrate the mechanisms accounting for thedifferenceantigen recognitionantigen recognition plays a vital role in initiating theadaptive immune response while the lack of tumor antigens significantly impedes the response to pd1pdl1blockade therapycurrently the fda has approved pembrolizumab totreat unresectable solid tumors with high msi or mmrdeficiency in a study on recurrent or metastaticcolorectal cancer patients with mmr deficiency or highmsi nivolumab showed an objective response rate of and of the patients had a disease control rateof ¥ weeks which indicated that patients with highmmr deficiency or high msi might exhibit better responses to pd1pdl1 blockade therapy [ ] interestingly the responses of tumors with mmrdeficientare highly variable and approximately half are resistantto pd1pdl1 blockade therapy mandal revealed that msi and the resultant mutation load wereresponsible for the variable response to pd1 blockadetherapy in mmrdeficiency tumors and the responsedegree was significantly correlated with the degree ofinsertiondeletion mutation loadseveral studies have revealed the association betweentumor mutation burden and the response to pd1pdl1blockade therapy [ ] mariathasan examined samples from patients with metastatic urothelial cancer receiving atezolizumab treatment and identified highneoantigen and tumor mutation burden as major determinants of clinical outcome their results showed that thetumor mutation burden was closely correlated with the response in the excluded and inflamed phenotypes 0csun biomarker research page of gut microbiome compositionclinical experiments on the human gut microbiomehave identified several specific bacteria genres that playimportant roles in human immunity and can be used asprognostic biomarkers for clinical response to pd1pdl1 blockade therapy based on the gut microbiome analysis of melanomapatients receiving pd1 blockade gopalakrishnan found that patients with prolonged progressionfree survival showed a higher multiplicity of bacteriaand clostridiales ruminococcaceae and faecalibacterium were abundant in therapy responders moreovermatson evaluated the baseline stool samplesfrom patients with metastatic melanoma before pd1pdl1 blockade treatment and the results showed thatcommensal microbial composition was significantly associated with the clinical response bifidobacteriumlongum collinsella aerofaciensand enterococcusfaecium were more abundant in responders similarlyin patients with epithelial tumors routy revealed that akkermansiacea muciniphila and enterococcus hirae were significantly abundant in those withbetter clinical response progressionfree survival months all these results indicate that gut microbiomecomposition may be a potential determinant of therapyresponse and might be used as a predictive factor inthe following research more studies are needed to validate the predictive value of gut microbiome in largercohorts and explore their efficiency in the context ofvarious types of tumorsstrategies and it hasfuture perspectivesimmunotherapy is one of the most promising cancertreatmentrevolutionized thelandscape of cancer management over the last decadehowever together with the costly and timeconsumingtrialanderror approach the limited therapy responseremains a tricky problem which hinders the furtherapplication of pd1pdl1 blockade to overcome therapy resistance and potential adverse events substantialeffort has been made on developing novel antipd1pdl1 based immunotherapy strategies with better clinical response and limited immunemediated toxicityfigs tobetterclinicallikelyachievesystem issince the interaction between cancer and the immunecomplex and involves multiplefactors strategies in combination with multiple agentsareoutcomescompared with singleagent administration a largenumber ofcombinedtherapy is an effective therapeutic strategy againstcancers for example transforming growth factor βtgfβblocking agents concomitantly with combinedpd1pdl1 blockade combined provides a clinicallyrevealed thatstudies haveexperimentson mice withfeasible strategy to improve efficacy and reduce toxicity mariathasan revealed that metastaticurothelial cancer with upregulated tgfβ signalingbefore treatmentresponded poorly to pd1pdl1blockade therapy the tumors with dense collagenfibrils could trap t cells in the stromal compartmentthus preventing them from playing their functions inpreclinicalimmuneexcluded phenotype they demonstrated that the coadministration of pdl1 blockade and tgfβblockingagents could reduce tgfβ signaling facilitate t cellinfiltration and achieve active antitumor immunityand tumor regression similarly the combination ofpd1pdl1 blockade with tumor necrosisfactorinhibitor [ ] metformin antivegf agents or otherinhibitors egcxcr4 has been demonstrated as a clinicallyfeasible strategy with improved antitumor efficacyand reduced toxicityimmune checkpointinhibitor agentspd1pdl1 blockade usually acts on the whole hostimmune system instead ofsitespecifically targetingtumorspecific immune cells while nanomedicine technology provides a powerful tool to selectively deliverimmune checkpointto tumors orlymphoid ans using drugloaded nanops usually to nm in diameter recent studies suggest that the pd1pdl1 antibody could be conjugatedor modified on the surface of nanops which couldmaintain their stability enhance efficiency and minimizethe toxicity of pd1pdl1 blockade [ ] forexamplein gastric cancer cells the pdl1 blockadeconjugated nanops contributed to significantlyhigher cellular uptake and achieved more effective inhibition of pdl1 expression compared with the controlgroups moreover in patients with metastatic triplenegative breast cancer the coadministration of nabpaclitaxelatezolizumabprolonged progressionfree survival owing to thesuccess in previous research clinical trials on nanoimmunotherapysuch asnct03589339 and nct03684785 these clinical trialsshould provide substantial evidence for the combinationof nanomedicine and pd1pdl1 blockade in the nextfew yearscurrently underwayblockadepdl1plusarethe manipulation offurthermore accumulating evidence has demonstrated that gut microbiome significantly impacts theefficacy of cancer immunotherapy which in turn indithe gut microbiomecates thatcould latently affectthe response to pd1pdl1blockade therapy [] currently antibiotic applicationfecal microbiota transplantation fmt anddiet regulation are considered as practical approachesto manipulate gut microbiome for example fmtfrom patients with a positive response to germfree or 0csun biomarker research page of fig overview on the strategies to improve the resistance to pd1pdl1 blockade therapy multiple strategies have been proposed toimprove the resistance to pd1pdl1 blockade therapy including combined therapy nanoimmunotherapy gut microbiome manipulation andso forthin contrastantibiotictreated mice could improve tumor controlaugment t cell responses and ameliorate the antitumor effects of pd1 blockadethetransplantation from resistant patients did not resultin improvement similarly responses to pdl1blockade are distinctin mice with different commensal microbes and the positive response of micewith advantageous gut microbiome can be transplanted to mice with negative responses by fmt orcohousing conclusionsdespite the success across multiple types of cancersonly a minority of patients are estimated to exhibit apositive response to pd1pdl1 blockade therapy andthe primaryacquired resistance might eventually leadto progression in patients with clinical responses thelimitation in clinical response impairs the efficacy andhinders its further application since the understandingof the mechanisms underlying therapy resistance remains vague only a few therapeutic options areavailable for those patients currently illustrating thedeterminants of response or resistance is significant toaccelerate improving survival outcomes and developingimproved treatment options for cancer patients tobetter realize the therapeutic potential of pd1pdl1blockade therapyit is essential to identify predictivebiomarkers for therapy response develop novel therapeutic strategies and improve therapeutic strategies incombination with other agents in the following research combined efforts of basic researchers and clinicians are required to address the pd1pdl1 blockadetherapy resistanceabbreviationspd1 programmed death1 pdl1 programmed deathligand nsclc nonsmall cell lung cancer mmr mismatch repair msi microsatelliteinstability b2m beta2microglobulin tim3 t cell immunoglobulin mucin3mirnas micrornas lncrnas long noncoding rnas mdscs myeloidderived suppressor cells tgfβ transforming growth factor β fmt fecalmicrobiota transplantationacknowledgmentsnot applicableauthors contributionsjys dk z mx and xz wrote original draft preparation sq w jsj and xjprovided critical revision all authors read and approved the final manuscriptfundingthis study was supported by national key research and developmentprojects intergovernmental cooperation in science and technology of chinano 2018yfe0126900 to jiansong ji the key research and developmentproject of zhejiang province no 2018c03024 to jiansong ji the nationalnatural science foundation of china to xl 0csun biomarker research page of availability of data and materialsnot applicableethics approval and consent to participatenot applicableconsent for publicationnot applicablecompeting intereststhe authors declare that they have no competing interestsauthor details1the first college of clinical medicine the first affiliated hospital of nanjingmedical university nanjing medical university nanjing china 2keylaboratory of imaging diagnosis and minimally invasive interventionresearch lishui hospital of zhejiang university the fifth affiliated hospitalof wenzhou medical university clinical medicine of center hospital of lishuicollege lishui china 3college of medicine lishui college lishui china 4department of general surgery the first affiliated hospitalof nanjing medical university nanjing china 5department of radiologyaffiliated lishui hospital of zhejiang university lishui chinareceived april accepted august referenceshellmann md pazares l bernabe caro r zurawski b kim sw carcerenycosta e nivolumab plus ipilimumab in advanced nonsmallcell lungcancer n engl j med niglio sa jia r ji j ruder s patel vg martini a programmed death1or programmed death ligand1 blockade in patients with platinumresistant metastatic urothelial cancer a systematic review and metaanalysis eur urol sun jy lu xj cancer immunotherapy current applications and challengescancer lett andrews lp yano h vignali daa inhibitory receptors and ligands beyondpd1 pdl1 and ctla4 breakthroughs or backups nat immunol prestipino a zeiser r clinical implications of tumorintrinsic mechanismsregulating pdl1 sci transl med betof warner a palmer js shoushtari an goldman da panageas ks hayessa et | 0 |
" it is well established that retrieved lymph node rln counts were positively correlated with betteroverall survival in gastric cancer gc but little is known about the relationship between rln count and shorttermcomplications after radical surgerymethods a total of consecutive gc patients between january and december at nanjing drumtower hospital were retrospectively analyzed univariate analyses were performed to elucidate the associationbetween rln count and postoperative complications we further identified clinical factors that might affect the rlncountresults among all of the patients postoperative complications occurred in patients the mean rlncount was and patients were diagnosed with lymph node metastasis univariate analyses showedno significant difference between rln count and postoperative complications both overall and stratified by cdcgrade univariate and multivariate analyses further revealed that type of resection tumor invasion and lymph nodemetastasis were associated with rln counts the current study demonstrated that rln count was not associated with postoperative shorttermcomplications following gastrectomy of gc which provided a rationale for the determination of a proper rlncount of curative gastrectomykeywords retrieved lymph nodes postoperative complications gastric cancer there are approximately one million new cases of gastriccancer gc each year worldwide and half of them occurin eastern asia including china japan and south korea despite advances in early screening and comprehensive treatment of gc it remains the third most commoncause of cancerrelated death in the world for advanced gc a consensus has been reached of radical gastrectomy with d2 lymphadenectomy however there correspondence medguanwenxian163com wangmeng001263net feng sun song liu and peng song contributed equally to this workdepartment of gastrointestinal surgery nanjing drum tower hospital theaffiliated hospital of nanjing university medical school nanjing chinais still controversy over the number of retrieved lymphnodes rlns for accurate pathological stagingseveral studies have reported that rln count waspositively correlated with better overall survival in gceven in lymph nodenegative gc [] an rln countof ¥ has been recommended by the 8th edition tnmclassification for gc to guarantee the accurate pn stage moreover okajima suggested an optimal rlncount of ¥ for nodal staging recently by stratumanalysis of patients deng proposed an optimal rln count of ¥ for lymph nodenegative gc and for lymph nodepositive gc these abovestudies are all conducted by comparing the rln count the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0csun world of surgical oncology page of table demographic and clinical features of patientscharacteristicsage yearsgender nn ± malefemalebmi kgm2preoperative comorbidities nprevious abdominalsurgerydiabetes mellitushypertensionpreoperative laboratory dataserum albumin glcrp glasa ¥ mode of surgical approach nlaparoscopicopentype of resection ndistal gastrectomyproximal gastrectomytotal gastrectomyoperation time minblood loss mltumor invasiont1t3tumor sitecardiafundusbodypylorusantrumrln countlymph node metastasispositivenegativelnrloddsptnm stage iiiiiiivlauren subtypeintestinaldiffusemixedunknownpostoperative complicationspositive ± ± ± ± ± ± ± ± table demographic and clinical features of patientscontinuedcharacteristicsnegativepostoperative stay daystotal hospital charges ¥n ± ± bmi body mass index crp creactive protein asa american society ofanesthesiologists rlns retrieved lymph nodes lnr lymph node ratio loddslog odds of positive lymph nodeswith longterm survival but little is known about the relationship between the rln count and shortterm complications after radical surgerypostoperative complications of gc pose a significantimpact on the length of postoperative stay and hospitalcharges which further affect the quality oflife thereforeinvestigating the relationship between rlncount and postoperative shortterm complications wouldprovide more comprehensive evidence for selecting theappropriate rln countmethodspatientsa total of consecutive gc patients between january and december at nanjing drum tower hospital were retrospectively reviewed all patients underwent curative r0 gastrectomy and were histologicallyconfirmed the exclusion criteria were as follows multivisceral resection patients accepting preoperative radiotherapy or chemotherapy patients with previous stomach surgery and patients with incompleteclinical data this study was approved by the ethicscommittee of nanjing drum tower hospitalcharacteristicsfor preoperativedata collectiondataintraoperativeindex and postoperative features were extracted preoperative characteristics included age gender body massindex bmi comorbidities and laboratory data the intraoperative index involved the american society of anesthesiologists asa grade surgical approach type ofresection operation time and blood loss postoperativefeatures included depth of tumor invasion tumor site retrieved lymph node count lymph node metastasis lymphnode ratio lnrlog odds of positive lymph nodeslodds ptnm stage lauren subtype shortterm complications postoperative stay and total hospital chargeslnr was defined as the ratio of positive to retrievedlymph nodes lodds was calculated by log [positivelymph nodes 05total lymph nodes positive lymphnodes ] the postoperative shortterm complications occurring in the hospital or within days werecollected all complications were evaluated according tothe claviendindo classification system 0csun world of surgical oncology page of statistical analysisstatistical analyses were conducted by spss chicago il usa continuous variables were shown asmeans ± sd students t test was applied for normallydistributed data mannwhitney u test was applied fornonnormally distributed data categorical variable datawere presented as numbers and analyzed using the chisquared test or the fisher exact test univariate andmultivariate analyses were performed to analyze the riskfactors associated with the postoperative complicationsor retrieved lymph node count the optimal cutoffvalues of lnr and lodds were determined by receivertable univariate and multivariate analyses of characteristics associated with postoperative complicationscharacteristicsunivariateormultivariateor cipreferenceage ¥ gendermalefemalebmi kgm2preoperative comorbiditiesprevious abdominal surgerydiabetes mellitushypertensionpreoperative laboratory dataserum albumin glcrp ¥ glasa ¥ mode of surgical approachlaparoscopicopentype of resectiontotal gastrectomydistal gastrectomyproximal gastrectomyoperation timeblood losstumor sitecardiafundusbodypylorusantrumtumor invasion t3rlnslymph node metastasislnr lodds ptnm stage ¥ iiilauren subtypeintestinaldiffusemixedunknown cireferencereferencereferencep 0csun world of surgical oncology page of operating characteristic roc analysis all statisticaltests were conducted twosided and statistical differences were termed as p value resultspatient characteristicsthe characteristics of the patients enrolledin this study were presented in table there were gc patients in all including men and women the median age was years with arange from to years a total of patients underwent open gastrectomy while underwent laparoscopic surgery the type of resectionwas distal gastrectomy in patients proximalgastrectomy in and total gastrectomy in the mean operation time was min and themean intraoperative blood loss was ml pathologicalresults were stage iiiiiiiv in patientsrespectively the mean rln count was range and patients were tested with lymphnode metastasis overall postoperative shortterm complications occurred in patients the meanpostoperative stay was days and the mean total hospital charges were à ¥association between perioperative characteristics andpostoperative complicationsas presented in table univariate and multivariate analyses indicated that postoperative shortterm complications were significantly correlated with age gender levelof preoperative serum albumin and operation timestratified analyses by type of resection revealed thatcomplications occurred frequently in proximal gastrectomy compared with total gastrectomy while there wasno significant difference between distal gastrectomy andtotal gastrectomy no significant association was observed between rln count and overall postoperativecomplicationsimpact of rln count on postoperative complicationsof the patients developed complications of encountered a single complication and of encountered multiplecomplications the details of patients with shorttermcomplications based on the claviendindo classification are for grade i for grade ii frade iii for grade iv and for grade vthe rate of major complications cdc grade ¥ iiiwas the median rln count in this study was so we divided all patients into two groups basedon the median rln count univariateanalysesshowed no significant difference between rln countand postoperative complicationsboth overall andstratified by cdc grade table table univariate analyses of postoperative complicationsassociated with rln countcharacteristicsallrln count ¥ overall ngrade i nfever °cemesispainabdominopelvic collectionpleural effusiongrade ii nblood transfusionsearly postoperative bowel obstructiongastroparesisliver function abnormalitieswound infectionpneumoniaintraabdominal infectionsurinary tract infectionenteritisbacteremiagrade iii nanastomotic leakagelymphatic leakagepancreatic fistulabiliary fistulableedingabdominopelvic collectionpleural effusionintraabdominal abscesswound disruptiondelayed wound healinggastroparesisearly postoperative bowel obstructionsplenic necrosisgrade iv nheart failurekidney failurebrain infarctionmodspvaluegrade v ngrade ¥ iii nrlns retrieved lymph nodes mods multiple an dysfunction syndrome 0csun world of surgical oncology page of factors associated with rln countwe further explored the potential factors associated withrln count univariate analyses revealed that preoperative serum albumin type of resection tumor invasionlymph node metastasis and ptnm stage were associatedwith rln count p table stratification bytype of resection showed that rln count in either distalgastrectomy or proximal gastrectomy was significantlyin total gastrectomy multivariatelowerthan thatanalyses further indicated that type of resection tumorinvasion and lymph node metastasis were still significantly associated with rln count p table discussionnodal involvement significantly affected the prognosis ofgc patients because it is the major root of tumor relapse after surgery [ ] thus standardized lymphnode dissection is the basic requirement for curativetable univariate and multivariate analyses of factors associated with rln count ¥ characteristicsunivariateormultivariateor cipreference age ¥ gendermalefemalebmi kgm2preoperative comorbiditiesprevious abdominal surgerydiabetes mellitushypertensionpreoperative laboratory dataserum albumin glcrp ¥ glasa ¥ mode of surgical approachlaparoscopicopentype of resectiontotal gastrectomydistal gastrectomyproximal gastrectomyoperation timeblood losstumor sitecardiafundusbodypylorusantrumtumor invasion t3lymph node metastasisptnm stage ¥ iiilauren subtypeintestinaldiffusemixedunknown cireferencereferencereferencep bmi body mass index crp creactive protein asa american society of anesthesiologists rlns retrieved lymph nodes or odds ratio ci confidence interval 0csun world of surgical oncology page of r0 gastrectomy curative gastrectomy with d2 lymphadenectomy has been considered as the standard fashionfor decades in eastern asia especially in japan [ ]this procedure has been gradually accepted by westerncountries in recent years [ ] as for the rln countthe 8th edition tnm classification for gc recommendeddissecting at least lymph nodes moreover emergingevidence revealed the positive correlations between rlncount and overall survival of gc patients [ ] bycomparing rln count to survival time okajima suggested an optimal rln count of ¥ deng proposed an optimal rln count of ¥ for lymphnodenegative gc and for lymph nodepositive gcby stratum analysis of patients sano reported that rln count preferably achieved or moreby a multicenter study enrolling patients additionally lnr and lodds were also reported to[] thesebe associated with gc prognosisabove studies mainly focused on the relationship between rln count and longterm prognosis howeverlittle is known aboutits effects on postoperativeshortterm complicationsin this study we concentrated on the association betweenrln count and shortterm prognosis univariate analysesshowed no significant difference between rln count andpostoperative complications both overall and stratified bycdc grade therefore more lymph nodes were encouragedto be dissected from the perspective of shortterm prognosisalthough curative gastrectomy with d2 lymphadenectomy is considered a pivotal strategy for advanced gcthere are international and institutional differences in thenumber of rln count [ ] various factors were reported to influence the rln count including the confidence and enthusiasm of doctors both surgeons andpathologists surgical situation and innate lymph nodecount in each patient [ ] in our study we concludedthat rln count was related to the type of resection tumorinvasion and lymph node metastasis of note rln countwas positively correlated with the lymph node metastasisrate which underlined the importance of rln count foraccurate stagingactuallyfor a thorough pathological examinationrlns should be individually divided from a completetissue sample after surgery owing to much time andeffort was required during this procedureit has notbeen widely implemented clinically therefore the examined lymph node count by pathologists might belower than the dissected lymph node count multipleattempts have been conducted to improve the detection rate of lymph nodes [] li elucidatedthat the mean number of rlns could be significantlyelevated by injecting carbon nanops before surgery compared with controls vs markl and colleagues reported a twofold lymph nodepick up rate utilizing methylene blue staining thanunstained groups vs several dye materials were also used to increase the number of lymphnodes dissected during surgery such as fluorescentindocyanine green icg and 5aminolevulinic acid5ala [ ]we acknowledge that this study had some potentialit was a retrospective singlecenterlimitations firststudy so the results might be flawed because of residualconfounding factors second the rln count was closelyrelated to the quality of surgeons and pathologists theperioperative variables might differ in different doctorstherefore multicenter studies are needed to confirmour resultssin the current study demonstrated thatrlns\\ count was not associated with postoperativeshortterm complications following gastrectomy of gctherefore our analysis encouraged more lymph nodesto be dissected for accurate pathologic stagingabbreviationsbmi body mass index crp creactive protein asa american society ofanesthesiologists rlns retrieved lymph nodes lnr lymph node ratiolodds log odds of positive lymph nodesacknowledgementsthe authors gratefully acknowledge all the investigators for theircontributions to the trialauthors contributionsfs worked on the study design collected the data and drafted themanuscript sl contributed to the study design and data collection ps wasinvolved in the data collection and extraction cz helped collect the datawg was involved in the study design and data extraction mw revised themanuscript all authors have read and approved the final manuscriptfundingthere is no funding supporting this workavailability of data and materialsaccess to the data and the calculation method can be obtained from theauthors by email medsunfeng163comethics approval and consent to participatethis retrospective study was approved by the ethics committee of nanjingdrum tower hospital medical school of nanjing university due to theretrospective nature the requirement for informed consent was waived bythe irbs from nanjing drum tower hospital medical school of nanjinguniversityconsent for publicationnot applicablecompeting intereststhe authors declare that they have no competing interestsreceived june accepted august referencesstewart b wild cp world cancer report public health 0csun world of surgical oncology page of degiuli m de manzoni g di leo a ugo dd galasso e marrelli d gastric cancer current status of lymph node dissection world jgastroenterol son t hyung wj lee jh kim ym kim hi an jy clinical implication ofan insufficient number of examined lymph nodes after curative resectionfor gastric cancer cancer li z ao s bu z wu a wu x shan f clinical study of harvestinglymph nodes with carbon nanops in advanced gastric cancer aprospective randomized trial world j surg oncol markl b kerwel tg jahnig hg oruzio d arnholdt hm scholer c methylene blueassisted lymph node dissection in colon specimens aprospective randomized study am j clin pathol aoyama t yoshikawa t morita s shirai j fujikawa h iwasaki k methylene blueassisted technique for harvesting lymph nodes after radicalsurgery for gastric cancer a prospective randomized phase iii study bmccancer he m jiang z wang c hao z an j shen j diagnostic value of nearinfrared or fluorescent indocyanine green guided sentinel lymph nodemapping in gastric cancer a systematic review and metaanalysis j surgoncol koizumi n harada y murayama y harada k beika m yamaoka y detection of metastatic lymph nodes using 5aminolevulinic acid inpatients with gastric cancer ann surg oncol publishers notespringer nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliationsbray f ferlay j soerjomataram i siegel rl torre la jemal a global cancerstatistics globocan estimates of incidence and mortality worldwidefor cancers in countries ca cancer j clin van cutsem e sagaert x topal b haustermans k prenen h gastric cancerlancet zhang w zhangyuan g wang j jin k liu y wang f effect of lymphnodes count in nodepositive gastric cancer j cancer chu x yang zf impact on survival of the number of lymph nodes resectedin patients with lymph nodenegative gastric cancer world j surg oncoljiang l yang kh guan ql zhao p chen y tian jh survival and recurrencefree benefits with different lymphadenectomy for resectable gastric cancera metaanalysis j surg oncol deng j yamashita h seto y liang h increasing the number of examinedlymph nodes is a prerequisite for improvement in the accurate evaluationof overall survival of nodenegative gastric cancer patients ann surg oncolamin mb greene fl edge sb compton cc gershenwald je brookland rk the eighth edition ajcc cancer staging manual continuing to build abridge from a populationbased to a more personalized approach tocancer staging ca cancer j clin okajima w komatsu s ichikawa d kosuga t kubota t okamoto k prognostic impact of the number of retrieved lymph nodes in patients withgastric cancer j gastroenterol hepatol deng j liu j wang w sun z wang z zhou z validation of clinicalsignificance of examined lymph node count for accurate prognosticevaluation of gastric cancer for the eighth edition of the american jointcommittee on cancer ajcc tnm staging system chin j cancer res kim th suh ys huh yj son yg park jh yang jy the comprehensivecomplication index cci is a more sensitive complication index than theconventional claviendindo classification in radical gastric cancer surgerygastric cancer wang j hassett jm dayton mt kulaylat mn the prognostic superiority oflog odds of positive lymph nodes in stage iii colon cancer j gastrointestsurg dindo d demartines n clavien pa classification of surgical complicationsa new proposal with evaluation in a cohort of patients and results of asurvey ann surg hirabayashi s kosugi s isobe y nashimoto a oda i hayashi k development and external validation of a nomogram for overall survivalafter curative resection in serosanegative locally advanced gastric cancerann oncol tóth d bÃró a varga z török m árkosy p comparison of different lymphnode staging systems in prognosis of gastric cancer a biinstitutional studyfrom hungary chin j cancer res de steur wo dikken jl hartgrink hh lymph node dissection in resectableadvanced gastric cancer dig surg maruyama k kaminishi m hayashi ki isobe y honda i katai h gastric cancer treated in in japan data analysis of nationwide registrygastric cancer liang h deng j evaluation of rational extent lymphadenectomy for localadvanced gastric cancer chin j cancer res degiuli m sasako m ponti a vendrame a tomatis m mazza c randomized clinical trial comparing survival after d1 or d2 gastrectomy fastric cancer br j surg sano t coit dg kim hh roviello f kassab p wittekind c proposal ofa new stage grouping of gastric cancer for tnm classification internationalgastric cancer association staging project gastric cancer zhao e zhou c chen s prognostic nomogram based on log odds ofpositive lymph nodes for gastric carcinoma patients after surgical resectionfuture oncol alatengbaolide lin d li y xu h chen j wang b liu c lu p lymph noderatio is an independent prognostic factor in gastric cancer after curativeresection r0 regardless of the examined number of lymph nodes am jclin oncol wang j dang p raut cp pandalai pk maduekwe un rattner dw comparison of a lymph node ratiobased staging system with the 7th ajccsystem for gastric cancer analysis of patients from the seer databaseann surg 0c" | 0 |
"cellular recognition of microbial dna is an evolutionarily conserved mechanism by which the innate immunesystem detects pathogens cyclic gmpamp synthase cgas and its downstream effector stimulator of interferongenes sting are involved in mediating fundamental innate antimicrobial immunity by promoting the release oftype i interferons ifns and other inflammatory cytokines accumulating evidence suggests that the activation ofthe cgassting axis is critical for antitumor immunity the downstream cytokines regulated by cgasstingespecially type i ifns serve as bridges connecting innate immunity with adaptive immunity accordingly a growingnumber of studies have focused on the synthesis and screening of sting pathway agonists however chronicsting activation may lead to a protumor phenotype in certain malignancies hence the cgassting signalingpathway must be orchestrated properly when sting agonists are used alone or in combination in this review wediscuss the dichotomous roles of the cgassting pathway in tumor development and the latest advances in theuse of sting agonistskeywords cgassting innate immunity type i interferon sting agonists antitumor response cancerdevelopmentintroductionthe discovery of phagocytosis in advanced the understanding of innate immunity the first line of host defenses protection againston patternrecognition receptors prrs which recognize microbialproducts coordinate antimicrobial defenses and activateinfection dependsagainstinfection byvarious pathogens correspondence zqliucsueducn juyan zheng and junluan mo contributed equally to this work1department of clinical pharmacology hunan key laboratory ofpharmacogenetics and national clinical research center for geriatricdisorders xiangya hospital central south university changsha peoples republic of china2institute of clinical pharmacology engineering research center for appliedtechnology of pharmacogenomics of ministry of education central southuniversity changsha peoples republic of chinafull list of author information is available at the end of the adaptive immunity abnormal rna or dna rnadna hybridization and cyclic dinucleotides derived frommicrobes are usually considered pathogenassociated molecular patterns pamps [ ] cells associated with innate immunity recognize different microbial pampsthrough specific prrs thereby playing key roles in hostresistance to microbial infection the pathways governing rna recognition such as retinoid acid induciblegene i rigilike receptors have been reviewed elsewhere and will not be covered herein in the case of dnarecognition one of the best known prrs is tolllike receptor tlr9 which senses extracellular cpg hypomethylated dna that has entered the cytosol through thephagosomelysosome system in addition the aim2like receptor aim2 inflammasome can be triggered afterthe entry of doublestranded dna dsdna into the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0czheng molecular cancer page of cytosolic compartment which induces the proteolyticmaturation of proinflammatory cytokines such as il1and il18 and the activation of gasdermin d leading topyroptosis [] nevertheless the most notable prr iscgas a direct cytosolic dsdna sensor which was identified by dr chens group in once cgas bindsto dsdna the cgassting pathway is activated to further induce the expression of type i ifns and other inflammatory cytokinesthus triggering innate immuneresponses mounting evidence suggests that cgassting signaling not only plays pivotal roles in the hostdefense against microbialinfection but also modulatestumorigenesis hence in this review we summarize themechanism of cgassting activation and elaboratefindings regarding its dual effects on tumor developmentcurrent advances in the use of sting agonists as a novelstrategy for antitumor therapy are also reviewedinsights into the cgassting signal transductioncascadecgas is an innate immune sensor that identifies variouscytosolic dsdnaincluding dna with viral bacterialmitochondrial micronuclei and retroelement originswhich can be mainly divided into pathogenderiveddna and selfdna table in the cytoplasm cgas isactivated by interacting with dsdna in a sequence[]independent butstructural and biochemical analyses have revealed thatthe cterminal lobe of cgas contains a conserved zinclengthdependent mannerionbinding module that mediates dna binding andcgas dimerization [ ] dna ligands promotecgas activation primarily by inducing conformationalchanges around the catalytic site and in the dnabinding structures of cgasthe gscontaining loopundergoes conformational change to maintain stabilitywhich is a major mechanism of cgas activation bydna in addition to the primary dnabinding sitementioned above the secondary site located beside theprimary site is a helix formed between strands 78and several surfaceexposed loops the proximity ofthe two dnabinding sites in cgas leads to a cgasdna complex assembly in which two cgas moleculesembrace two molecules of dsdna [ ] the cgasdimers are anized in headtohead alignment nextto the dna and thus form stable ladderlike networks between one long curved dsdna helix or two independent dsdna strands [ ] in this way eachindividual cgasdsdna complex can be cooperativelystabilized and can lead to stronger enzymatic activitywhich may provide a possible explanation for longerdsdna as more likely to activate cgas in additionlong dna is more efficient than short dna in drivingthe liquidliquid phase separation of cgas and the formation ofcriticallydependent on the concentration of cgas and dna inthe cytoplasm cgas and dsdna are spatially concentratedcgasdimerization and activation [] once cgas andcgas liquidlike dropletsin liquiddropletsistofacilitatetable classification of the cytosolic dsdna that activates the cgassting signaling axisclassificationselfdnasource of dsdnamicronucleipossible mechanismsrupture of the micronuclei membrane leads to exposureof chromatin dna that is recognized by cgas whichactivates the cgassting pathwayreferences mitochondrionnuclear rnapathogenderived dnadna virushsv1 hsv2 kshv adenovirus vacciniavirus cytomegalovirus papillomavirusmurine gammaherpesvirus retrovirushiv siv murine leukemia virusrna viruswest nile virus dengue virus vsvsarscov2bacterialisteria monocytogenes mycobacteriumtuberculosis listeria shigella francisellachlamydia and neisseriamitochondrial stress induces mtdna leakage into thecytosol thus activating the sting pathway and inducingproduction of cytokinesfacilitated by endogenous retroelements nuclear rnacan be reversely transcribed into dna that activatescgassting signaling dna viruses invade host cells and release pathogenderiveddna to induce sting activation[]dna intermediates generated from reverse transcription maybe recognized by cgas to stimulate downstream stingsignaling infection with rna viruses might cause cellular damage andcell death which results in the release of cellular dna andfurther activation of the cgassting axis sarscov2 bindingto ace2 can lead to excessive angiotensin ii signaling thatactivates the sting pathway in mice[]bacteria produce cdns such as cyclic digmp and cyclicdiamp which can directly bind to and activate sting[ ]hsv1 herpes simplex virus hsv2 herpes simplex virus kshv kaposi sarcomaassociated herpesvirus hiv human immunodeficiency virus siv simianimmunodeficiency virus vsv vesicular stomatitis virus cdns cyclic dinucleotides and sarscov2 severe acute respiratory syndrome coronavirus 0czheng molecular cancer page of dsdna interacts structural switches rearrange the catalytic pocket to enable cgas to catalyze the synthesis of²²cyclic gmpamp ²²cgamp with atp andgtp as substrates the first step in this process is theformation of a linear dinucleotide ²pppg ²²pawith atp serving as the donor and ²oh on gtp serving as the acceptor then the intermediate product flipsover in the catalytic pocket placing gtp at the donorposition and amp at the acceptor position to form asecond ²² phosphodiester bond [ ] notablyalthough dsrna or singlestrand dna ssdna is ableto bind to cgas neither can rearrange the catalyticpocket which may explain the exclusive activation ofcgas by dsdna ultimately cgamp acts as a secondmessenger to bind to and activate sting a small endoplasmic reticulum erlocated protein kd withfour putative transmembrane domains [ ] normally in a resting state sting is retained in the er byinteracting with the ca2 sensor stromalinteractionmolecule stim1 the cytosolic ligandbindingdomain lbd of sting exists as the most functionalunit capable of integrating with ²² cgamp or cdnscyclic dinucleotides such as cdiamp cdigmp or ²²cgamp from bacteria upon interaction the obviousclosure of the ligand binding pocket in the lbd is observed which is related to the activation of sting next sting transforms into a tetramer through a highorder oligomerization reaction and is translocated fromthe er to the perinuclear area facilitated by cytoplasmiccoat protein complex ii copii and adpribosylationfactor arf gtpases [ ] in the golgi sting ispalmitoylated atcys88 andcys91 a posttranslational modification necessary forsting activation modified sting recruits thekinase tankbinding kinase tbk1 in turn the cterminal domains of sting are phosphorylated bytbk1 and then phosphorylated sting recruits interferon regulatory factor irf3 which is also phosphorylated by tbk1 and dimerizes ultimately dimerizedirf3 enters the nucleus and exerts its function in thetranscription of type i ifns and interferonstimulatedgenes isgs in parallel sting can also bind toand stimulate iκb kinase ikk to mediate the production of nuclear factorκb nfκbdriven inflammatorygenes upon signal transduction termination sting istransferred to endolysosomes for degradation considering that cgamp can be transferred through gapjunctions or delivered in viralexosome packages cgassting signaling may be activated in the cytoplasmwithout dsdna [ ] moreover newly produced typei ifns activate heterodimer interferon receptors ifnar1 and ifnar2 through paracrine signaling and thusinduce the transcription of isgs [ ] in summaryonce virusderived dna and selfdna are located intwo cysteine residuesthe cytoplasm they can be sensed by cgas and a cgasdsdna complex is formed to catalyze the synthesis of ²²cgamp with atp and gtp then ²²cgamp and bacteriaderived cdns induce sting activation and mediate the release of downstream type iifns tnfα and il6 which are prerequisites for antimicrobial defense and antitumor effects the wholeprocess shows that the dsdnacgassting axis canlead to the activation of both innate and adaptive immunity fig the antitumor functions of the cgasstingsignaling pathwayrecent evidence has revealed the close association of thecgassting pathway with cancer development thissignaling pathway is generally regarded as a potent regulator of cancer immunity a stingmediated immunesupportive microenvironment can hamper malignancyoccurrence stressbytumor cell cytosolic dsdna induces sting activationunder normal circumstances dna is strictly unaffiliatedwith the cytoplasm in eukaryotic cells to avoid autoimmunity however dna leaks aberrantly in tumorcells [ ] cancer cells share common features including genome instability tumor suppressor gene mutation or deletion oxidativeand vigorousmetabolism under these intense states nuclear andmitochondrial dna is fragile and easily damaged whichleads to eventual dna leakage in the forms of micronuclei chromatin fragments andor free telomeric dna[ ] chromosomal instability cin is the primary source of cytoplasmic dna in malignant cells andis generally associated with tumor progression distantmetastasis and therapeutic tolerance excessive proliferation of cancer cells results in unstable genomes usuallychromosomal missegregation during mitosis due to defects in segregation lagging chromosomes generate micronuclei in a cellcycledependent manner the vulnerable membraneof micronuclei easily exposes the inner dna to the cytoplasm and activates the cgassting signaling axis exogenous stimuli such as chemotherapy and irradiation can also cause dna damage in addition to leakednuclear dna oxidative stressinduced mitochondrialdna leakage is another crucial initiator of sting pathway activation several anticancer treatments that precisely attack mitochondrial membranes result in effluxand cell death therefore the permeabilization of mitochondria membranes provides a reasonable explanationfor mitochondrial dna escape [ ] other sourcessuch as apoptotic cellderived dna exosomal dnaexodna and transposable elements have also beencharacterized 0czheng molecular cancer page of fig the cgassting dna sensing signaling pathway various dna derived from virus dying tumor cells or nucleus and mitochondria binds toand activates the cytosolic dna sensor cgas cgas catalyzes the synthesis of ²²cgamp in the presence of atp and gtp then ²²cgamp bindsto the er adaptor sting which also can be activated by cdns derived from bacteria upon activation sting translocates from er to golgicompartments where it activates tbk1 and ikk which phosphorylate irf3 and iκbα respectively then irf3 and iκbα dimerize and enter nucleusinitiating the transcription of type i ifn tnf and il6 the primary roles of these cytokines are reflected in host defense inflammation andantitumor immunitydemonstrated to evoke cgassting activation intumor cells [ ]type i ifns mediators of sting and adaptive antitumoreffectscgassting signaling exerts antitumor functions incancer cells both in an autonomous and nonautonomousmanner on the one hand dna damage can provokeacute sting signal transduction and induce cellularsenescence an irreversible cell cycle arrest state whichthwarts the aberrant proliferation of tumor cells throughacquisition ofsecretoryphenotype sasp which is associated with the releaseof abundantinflammatory mediators proteases andgrowth factors [ ] in contrast to undergoingsenescence tumor cells also directly propel apoptosisprocesses by upregulating proapoptosis protein bcl2associated x bax and downregulating the bcl2 apoptosis on the other hand stingsenescenceassociatedtheregulatoractivation in tumor cells not only facilitates the transcription of downstream type i ifns to induce dendriticcell maturation but also recruits supportive immunecells for direct nonspontaneous tumor elimination sting activation in nonmalignant cells causes tumorsuppressive effects as well sting signaling protectsagainst colitisassociated carcinomas cacs induced byazoxymethane aom and dextran sulfate sodiumdss which induce dna damage in intestinal epithelialcells and further trigger sting activation downstreamcytokines of sting signaling such as il1 and il18prevent neoplastic transformation by facilitating woundrepair more importantly sting signaling can also provoke cytotoxic t cell responses to control tumorigenesis necrotic cancer cells are commonly engulfed byantigenpresenting cells especially the basic leucine zippertranscription factor atflike batf3drivenlineage of dendritic cells dcs batf3 dcs take intumorassociated antigens and migrate towardsthe 0czheng molecular cancer page of tumordraining lymph node via the lymphatic systemwhere they crossprime tumorspecific cd8 t cellsthen cd8 t cells undergo activation and clonal expansion in the lymph nodes and are trafficked throughblood vessels to kill tumor cells in turn damaged cancercells release more antigens that are further captured bydcs the whole process forms a positive feedback loopcalled the cancerimmunity cycle tumor eradication can be achieved by multiple processes in thecancerimmunity cycle including tumor antigen captureand presentation and t cell priming and activation withtumor antigenspecific t cell priming and activationrelying on dcs and type i ifn release the involvement of type i ifns in innate immune sensing and adaptive immunity provides a reasonable hypothesis forexploring candidate prr pathways as potential immunomodulators mice lacking tlr9 myeloid differentiationprimary response gene myd88 cytosolic rna sensor mavs or the purinergic receptor p2x7r maintainintact antitumor immunity responses whereas mice deficient in sting or irf3 present with impaired cd8 tcell priming and activation [ ] in fact dying tumorcells can release multiple damageassociated molecularpatterns damps to trigger innate immune responsesin dcs among these released stimuli tumor cellderiveddna is a pivotal inducer in general the phagocytosis ofapoptotic cells causesimmune silence because ofdnasebased degradation nevertheless tumor cellreleased dna can be preserved in the dc endolysosomal compartment through an unknown mechanism cgas recognizes dna invading the cytoplasm andinduces the activation of sting cascades excretion oftype i ifns and expression of isgs additionally undersome physiological conditions such as hypoxia andacidic environments nuclear or mitochondrial dnamight be packaged in exosomes exosomal dnaexodna animates sting signaling once it is absorbedby tumorinfiltrating dcs finallytumor cellderived cgamp can also be transferred to host dcs bythe folate transporter slc19a1 and then directly bindsto sting activating it in dcs a recent study moredirectly demonstrated that cellautonomous sting promoted the maintenance of stem celllike cd8 t cellsand augmented antitumor t cell responses and mechanistically cgasstingmediated type i interferon signaling reinforced the stem celllike cd8 t celldifferentiation program mainly by restraining akt activity immune cellderived type i ifns have crucial functions in antitumor immunity control on the one handtype i ifns boost cross presentation by various mechanisms first they stimulate the maturation of dcs secondthey slow the endosomelysosome acidificationprocess to prevent engulfed tumor antigen clearance andelevate the expression of mhc i molecules on the cellsurface [ ] finally they accelerate dc migrationtowardslymph nodes where they can crossprimetumorspecific cd8 t cells on the other handtype i ifns drive the expression of multiple chemokinessuch as cxcl9 and cxcl10 both of which are necessary for cytotoxic t lymphocyte ctl transfer and infiltration similarly type i ifns restrain the defaultimmune suppressive action of regulatory t treg cellsby downregulating phosphodiesterase pde4 and upregulating cyclic amp camp consequently typei ifns serve as bridges linking the cgassting pathway with cd8 t cellmediated antitumor immunitythe antitumor mechanisms of the cgassting signaling axis are illustrated in fig indeed previous studies revealed that sting activation can stimulate antitumor immune responses inleukemia melanoma glioma and hepatocellular carcinoma [] additionally sting expression is downregulated in a wide variety of tumor tissues and celllines according to a pancancer analysis with a smallproportion of tumors approximately bearing silent sting expression lower sting expressionwas found in hepatic carcinoma and gastric cancer compared with its level in corresponding normal tissues andthis lower expression level was correlated with highertumor stage and poorer prognosis [ ] consistentlycompared with that in the mcfg10a mammary epithelial cell line lower sting expression was detected inmalignant breast cancer cellincluding mcf7hbl100 and t47d cells as well as human melanomacell lines and colorectal adenocarcinoma lines [ ] collectivelythat cgassting signaling might act as a tumor suppressor in certain types of cancersthese findings suggestlinessting pathway agonists as cancer therapeuticsthe immunostimulatory potential of the cgasstingpathway makes it an attractive pharmacological targetsince its activation in the tumor microenvironmenttme can induce efficient crosspriming oftumorspecific antigens and facilitate the infiltration of effectort cells recent drug research has focused on the development of sting agonists because of their potential inanticancer therapy [ ] to date various kinds ofsting agonists have been discovered and they aremainly divided into the following categories cyclic dinucleotides and their derivates dmxaa and its analogsand small molecular agonists in addition some conventional antitumor therapeutics can also indirectly activatesting such as chemotherapy radiotherapy rt andtargeted therapy in addition sting agonists areable to enhance the efficacy of other anticancer therapeutic agents when used in combination sting 0czheng molecular cancer page of fig the antitumor immunity effect of the cgassting pathway dna damage leads to the formation of dsdna in tumor cells upon itsstimulation sting signaling is activated and promotes the release of type i ifn which is crucial for dc maturation sting signaling activation indcs is the core step of the whole cancerimmunity cycle which can be initiated through engulfment of dyingdamaged tumor cells exosometransfer and cgamp gap junctions then dcs migrate towards the tumordraining lymph node and crossprime tumor specific cd8 t cells withthe help of type i ifns finally t cells undergo clonal expansion and traffic through the blood vessel to conduct tumor killingagonists and their synergistic use with other remedies isfurther explored in detail belowcyclic dinucleotides cdnscdns constitute a main type of sting agonist whichmainly originate from bacteria the known naturalcdns consist of exogenous cyclic digmp cdigmpcdiamp ²²cgamp and endogenous ²²cgampamong these cdigmp cdiamp and ²²cgampare synthesized by bacteria and identified as secondarymessengers that mediate sting signal transduction inprokaryotic cells while ²²cgamp functions as theinitiator of sting in mammalian cells the antitumor potential of these natural dinucleotides was firstproven by the finding that cdigmp could inhibit theproliferation of human colon cancer cells in vitro andbasal cell proliferation of human cecal adenocarcinomah508 cells was inhibited with μm cdigmp intraperitoneal ip injection of highdose cdigmpdirectly activated caspase3 and triggered t1 tumoripcell apoptosis in vitro nmol of cdigmp reduced thegrowth of t1 tumor cells in vitro by and nmreduced it by while lowdose cdigmp nmol accelerated the adaptive t cell response by converting a subgroup of myeloidderived suppressor cellsmdscs into immune stimulatory cells producing il12injection of ²²cgamp consistentlymgkg expedited dramatic leukemic elimination in eltcl1 transgenic mice bearing chronic lymphocyticleukemia cll and promoted tumor shrinkage of multiple myeloma in vivo from the perspective of endogenous cdns ²²cgamp mgkg was alsoshown to restrain tumorigenesis and improve the survival rate of mice bearing ct26 colon adenocarcinomain a dosagedependent manner relying on dc activationand t cell crosspriming more recently ohkurit further demonstrated that intratumoral it injection of ²²cgamp μg25 μldose on and days after the injection of tumor cells significantly mitigated tumor growth and prolonged the survival of breast 0czheng molecular cancer page of cancer t1luc squamous cell carcinoma mscc1colon cancer ct26 and melanoma b16f10 mousemodels notably the it injection of ²²cgampinhibited not only tumor growth but also lung metastases in mice bearing b16f10 cellderived tumors suggesting that cgampinduced cd8 tcell priming can drivesystemic antitumor immunity to control local and distant tumor growth termedvaccinestingvaxconsidering the superior properties of sting signaling in activating adaptive immunityit is rational toutilize sting agonists such as cdns as cancer vaccineadjuvants to increase tumor immunogenicity fu investigated the in vivo therapeutic efficacy of acancercomprisinggranulocytemacrophage colonystimulating factor gmcsf and bacteriaderived or synthetic cdns theyobserved that after it injection of stingvax with μg of cdns per vaccine dose the volume of b16melanoma tumors was dramatically reduced in a dosedependent manner compared to mice receiving gmcsf cancer vaccine alone stingvaxtreated mice hadmore infiltrating cd8 ifnγ t cells in the tumormicroenvironment the in vivo antitumor effect of stingvax was also verified in models of colon carcinomact26 pancreatic carcinoma panc02 and upper aerodigestive squamous cell carcinoma sccfvii although natural cdns are able to produce robust antitumor immunity their chemical features might hindertheir future application in the clinical setting first native cdns are easily degraded by enzymes inside the cellor in the bloodstream second their negatively chargedproperty hydrophilicity and phosphate moieties severelyimpede cdns from penetrating cell membranes to activate cytosolic sting leading to low bioavailability andpoor retention of the cdns in specific cells and tissuesthird unintentional toxicities and narrow therapeuticwindows are also unavoidable thus new strategies toimprove therapeutic efficacy and reduce adverse effectsare urgently needed including drug delivery carrier engineering original structural modification and nonnucleotide agonist screening regarding agonistdelivery smith reported that biopolymer implantscodelivering cdigmp μg and chimeric antigen receptor t cart cells resulted in significant tumor regression in mice bearing pancreatic tumors moreoveriv administration of cdigmpysk05lip equivalent to μg of cdigmp aysk05liposome delivery system encapsulating cdigmp led to a tremendous decrease in metastatic lesionsin a b16f10 mouse melanoma model with nearly ofthe injected mice showing resistance against tumor relapsethe adaptive immune responsememory was successfully induced chen alsofound thatliposomalindicating thatinjection ofintravenousintravenousivnanopdelivered cgamp cgampnp could activate the sting axis more effectively than solublecgamp and converted the immunosuppressive tme toa tumoricidal state in a transplanted b16f10 cell melanoma model and in a genetically engineered triplenegative breast cancer model moreover a recentstudy creatively suggested that modified bacteria mightbe exploited as a selective carrier of sting agonistsintroduction of a dinucleotide cyclasecoding gene intothe escherichia coli nissle strain was an attempt at realizing this effect however advancements to the systemare needed tobysnakeapartdigestionresistancecompoundatoms the modifiedfrom improving delivery methods cdnswith superior properties are currently being synthesized and tested for instance to prevent enzymatichydrolysis of cgamp the nonbridging oxygen atomsin cgamp phosphodiester linkages were replaced by²²sulfurcgsasmp showed resistance against degradation byenpp1 a major ²²cgamp hydrolasetherebyleading to a longer halflife and sustained high affinity for human sting hsting syntheticdithio mixedlinkage cdns with both rp rp r rand rp sp r s dithio diastereomers possessed notonlyvenomphosphodiesterase but also enhanced affinityforsting a novel superior modified product ml rrs2 cda also termed adus100 had the potencyto activate all hsting variants and mouse stingmsting adus100 had higher efficiency in activating sting signaling than endogenous or exogenous cdns mainly because of its enhanced stabilityand lipophilicity its powerful tumor elimination effect was extensively demonstrated in multiple murinemodelsincluding b16 melanoma t1 breast cancer and ct26 colon cancer with all treated animalsshowing significant and durable tumorregressionafter itinjection of adus100 three mg doseswhen tumor volumes reached mm3 theremarkableforhsting laid the foundation for its clinical use related clinicaltrials of adus100 are outlined intable in addition to adus100 some other novelsting agonists have been well designed iacs8779and iacs8803 are two highly potent ²²thiophosphate cdn analogs that induced striking systemicantitumorin a b16 melanoma murineinjection μg on and daysmodel after itposttumor implantation compared with adus100or cgamp the characteristics and preclinicalapplications of all these mentioned cnds are summarized in table because of the structural modification and optimization of delivery strategiestheapplication range and efficacy of cdns have beenand high affinityresponsescurativeeffect 0czheng molecular cancer page of table characteristics and preclinical applications of different sting agonistsclassificationcharacteristicsapplicationmodelsnatural cdnagonistscdigmppoor membrane permeabilitysuitable for various codeliverytechnologiescolon cancer h508cells t1 metastaticbreast cancertreatmentinformation μm nmol ip nmol ip nmol ip²²cgamp²²cgamphigher binding affinity formsting than for hstinghigher affinity for hsting thanits lineage isomers binds tovarious sting nucleotidepolymorphisms observed inhumans easily degraded byphosphodiesteraseimpermeable to the cellmembranechronic lymphocyticleukemia mgkg ipmultiple myeloma mgkg ipct26 colonadenocarcinoma mgkgbreast cancer t1lucsquamous cellcarcinomasmscc1 μg25 μldose it μg25 μldose itcolon cancer ct26 μg25 μldose itmelanoma b16f10 μg25 μldose ittherapeutic effects references[ ] [ ]inhibitsproliferation tumorregression tumorregressionaccelerates tcellresponseleukemiaeliminationsuppressesgrowthrestrainstumorigenesisimproves survivalratedelays tumrowthdelays tumrowthdelays tumrowthdelays tumrowthstingvaxsyntheticcdnagonistspotent in vivo antitumor efficacyin multiple therapeutic modelsof established cancercgampnpsbiopolymer scaffolds cdigmp and car t cellscdigmpysk05lip²²cgsasmpadus100iacs8779iacs8803noncdnagonistsfaaliposomal nanops npsdeliver cgamp intracellularlymore effectively than realizedwith soluble cgamperadicates tumors moreeffectively than systemicdeliveryysk05 is a lipid that can efficientlydeliver cdigmp to the cytosolpossesses high fusogenic activitywhich enhances endosomalescapemore resistant to degradation byenpp1 tenfold more potent atinducing ifn secretion potentialuse as a cancer vaccine adjuvantimproves stability and lipophilicityhigher affinity for hsting thannatural cdn agonists capable toactivate all hsting variants andmstingstimulates a superior systemicantitumor response thanadus100 and cgampcauses hemorrhagic necrosisfailed in a phase i clinical trialdue to species specificity μg cdns itreduces tumorvolume b16 melanomacolon carcinomact26pancreaticcarcinoma panc02b16f10 melanomaivtnbccreates atumoricidal state pancreatic cancer μg cdigmptumor regression b16f10 mousemelanoma μg cdigmp ivdecreasesmetastasisthp1 monocytesb16 melanomathree mg doses it t1 breast cancerthree mg doses itmc26 colon cancerthree mg doses itdurable tumorregressiondurable tumorregressiondurable tumorregression b16 melanoma μg on day and posttumor implantationantitumorresponse murine colontumorsextensive tumorrejection[ ]dmxaafirst discovered as a vascularrat mammary mgkg iphigh anticancer[ 0czheng molecular cancer page of table characteristics and preclinical applications of different sting agonists continuedclassificationcharacteristicsapplicationmodelstreatmentinformationinduces proinflammatory cytokinesin a stingdependent mannerhuman fibroblastsantiviral activity selectively induces stingdependentsynthesis and secretion of bioactiveifns no evidence of binding directlyto stingactivates sting in openconformation submicromolarlevels induce sting activationand ifn productionhuman fibroblastsantiviral activity colon tumors mgkg iv of a treatedgroup remainedtumor free faa flavone acetic acid dmxaa 56dimethylxanthenone4acetic acid cma 10carboxymethyl9acrid | 0 |
t he gut is the largest immune an of the humanbody and also an important target an for variouskinds of stress caused by severe insults such as infectiontrauma and shock12 these stresses are considered to haveinstitution at which the work was performed department oftraumatology and acute critical medicine osaka universitygraduate school of medicine yamadaoka suita osaka japancorresponding yasutaka nakahori md division of trauma andsurgical critical care osaka general medical center bandaihigashi sumiyoshi ward osaka japan emailp_olysyahoocojpreceived apr accepted jul funding informationno funding information providedan important role in promoting infectious complications andmultiple an dysfunction syndrome from the viewpointsof deteriorated intestinal epithelium the immune systemand commensal bacteria gut dysfunction is now recognizedas a cause for the promotion of diseases34lipids have been a focus not only as energy sources butalso as immunemodulating substrates5 unlike long andmediumchain fatty acids shortchain fatty acids scfaswhich mainly consist of acetate propionate and butyratewith two to four carbon atoms are principally derived fromthe fermentation of carbohydrates and amino acids by anaerobic microanisms shortchain fatty acids are usedmainly by intestinal epithelial cells as energy substrates andthey uence the motility of the intestinal tract and increaseintestinal blood ï¬ow we previously reported on altered gutï¬ora and fecal anic acids in critically ill patients andshowed that these patients had significantly lower levels of the authors acute medicine surgery published by john wiley sons australia ltd on behalf ofjapanese association for acute medicinethis is an open access under the terms of the creative commons attributionnoncommercial licensewhich permits use distribution and reproduction in any medium provided the original work is properly cited andis not used for commercial purposes of 0c of y nakahori acute medicine surgery 20207e558fecal anic acids especially scfas than healthy volunteers67despite the evidence implicating the importance ofscfas in the gut the effect of scfas on prognosis in critically ill patients has not yet been clariï¬ed in this study wemeasured eight kinds of fecal anic acidsincludingscfas in critically ill patients and investigated the effect ofscfas on their prognosismethodspatientst his retrospective study enrolled patientswho i fulï¬lled the criteria of systemic ammatoryresponse syndrome sirs according to the american college of chest physicians and the society of critical caremedicine8 ii had a serum creactive protein crp levelgreater than mgdl iii were admitted to the departmentof traumatology and acute critical medicine osakauniversity graduate school of medicine osaka japan during the period november january iv were treated in the intensive care unit for more than days we usedcrp ¥ mgdl to focus on cases with severe ammationbecause crp is mainly used as a marker of ammationwe excluded patients from whom we could not collect anyfecal samples during hospitalization continuous enteralfeeding was initiated with kcalday and graduallyincreased depending on the patients condition we usedimpact novartis nutrition minneapolis mn usa as anenteral nutritional product for all patients in our intensivecare unit and used kcalkg ideal body weight as thegoal for the administered dose fourteen healthy volunteersprovided fecal samples as controls for examinations of fecalanic acids concentrations this study was approved bythe institutional review board of osaka university approval no and informed consent was obtained fromeach patients familyfecal samples and determination of fecalanic acid concentrationswe collected fecal samples serially from all patients thefecal samples were transported at 00°c to the yakult central institute tokyo japan we measured eight kinds ofanic acid in the feces by highperformance liquid chromatography feces were homogenized in ml distilledwater the homogenate was then placed in an eppendorftube and centrifuged at g for min at °c a mixture of ml of the resulting supernatant and ml of moll perchloric acid was mixed well in a glass tubeand allowed to stand at °c for h the suspension wasthen passed through a ï¬lter with a pore size of lmnihon millipore tokyo japan the sample was analyzedfor anic acids by highperformance liquid chromatography as previously described9 using a waters conductivity detector waters milford ma usa equipped withtwo columns shodex kc811 showa denko tokyojapan the concentrations of fecal anic acids were calculated with the use of external standards the reproducibilityand stability of these measurements were shown previously10 we retrospectively evaluated the minimum andmaximum value of each fecal anic acid measured duringhospitalization and determined prognostic factors by classiï¬cation and regression tree cart analysissurveillance and deï¬nition of complicationsbacterial infection was diagnosed in accordance with thedeï¬nitions of the centers for disease control and prevention11 body temperature was measured continuouslysurveillance cultures from blood and sputum were undertaken routinely for each patient in cases of suspected infectionand computedtomography scanning were carried out as necessary bacteremia was deï¬ned as a positive blood culturelaboratory testingchest xraystatistical analysiscontinuous variables expressed as the median 25th and 75thpercentiles were compared by the mannwhitney utestcategorical variables expressed as number were comparedby the v2test unless the expected counts in any of the cellswere below in which case the fisher exact test was usedwe used cart analysis which is a binary recursive partitioning using nonparametric approaches to identify keyfecal anic acids and their cutoff values for mortality12we also used multivariate logistic regression analysis toquantitatively evaluate the effect of the covariates that weresuggested by cart analysisthe most important prognostic factors were selected anda predictive model was developed as follows first the minimum and maximum values of each fecal anic acid wereselected from all patients the maximum value was the highest value and the minimum value was the lowest value measured during hospitalization second the mannwhitney utest was applied to discover variables with potential prognostic value and covariates with p were excludedfinally cart was used to create a tree using the minimumand maximum data values of all fecal anic acids theeffects of the minimum and the maximum values of the fecalanic acids that were selected as key prognostic factors by the authors acute medicine surgery published by john wiley sons australia ltd on behalf ofjapanese association for acute medicine 0cacute medicine surgery 20207e558fecal anic acids in critically ill patients of table characteristics of critically ill patients included in this studytotal n survivors n nonsurvivors n pvalueage yearssex maleicu stay daysapache ii score on admissionorigins of sirssepsistraumaburnunknownbacteremianumber of antibiotic typesduration of antibiotic use days categorical values are expressed as number continuous values are expressed as median 25th75th percentiles bold values indicatestatistical signiï¬canceapache acute physiology and chronic health evaluation icu intensive care unit sirs systemic ammatory response syndromecart analysis and age sex and acute physiology andchronic health evaluation apache
± score on admission were analyzed with the multivariable logistic regressionmodelsall reported pvalues were twosided and a pvalue of was considered to indicate statistical signiï¬cance allstatistical analyses were undertaken using ibm spss statisticsversion armonk ny usaresultspatient characteristicsc haracteristics of the patients are shown intable there were survivors and nonsurvivors the two groups did not differ significantly in termsof sex or apache ii score age in the nonsurvivors wassignificantly older than that in the survivors p the principal origin of sirs was sepsis in of the patients in the two groups significantly more types ofantibiotics were used in the nonsurvivors than in the survivors during hospitalization p and the durationof antibiotic use in the nonsurvivors was significantlylonger than that in the survivors p the incidenceof bacteremia was significantly higher in the nonsurvivorsthan that in the survivors vs p anic acid proï¬lestiming of fecal sample collection is shown in table wecollected fecal samples for scfa analysis fecalsamples were collected evenly throughout hospitalization inboth the survivors and the nonsurvivors the minimum andmaximum values of each fecal anic acid and the resultsof univariate analysis are shown in tables and in termsof the minimum values total anic acids acetate propionate and isovaleric acid were significantly decreased in thenonsurvivors in terms of the maximum values lactate formic acid and succinic acid were significantly increased inthe nonsurvivors otherwise isovaleric acid and acetatewere significantly decreased in the nonsurvivorsadditionally cart and multivariate logistic regressionanalyses of the predominant covariates were carried out fortable timing of fecal sample collection in critically illpatientssurvivorsnonsurvivorstotalweek week week week week week week week week week weekstotal the authors acute medicine surgery published by john wiley sons australia ltd on behalf ofjapanese association for acute medicine 0c of y nakahori acute medicine surgery 20207e558table minimum values of fecal anic acids in eachgroup of critically ill patientssurvivorsnonsurvivorspvaluetotal anic acidsacetatepropionatebutyratesuccinic acidlactateformic acidisovaleric acidvaleric acidvalues are expressed as mean 06 se lmolg of feces bold val 06 06 06 06 06 06 06 06 06 06 06 06 06 06 06 06 06 06 ues indicate statistical signiï¬cancetable maximum values of fecal anic acids in eachgroup of critically ill patientssurvivorsnonsurvivorspvalue 06 06 06 06 06 06 06 06 06 total anic acidsacetatepropionatebutyratesuccinic acidlactateformic acidisovaleric acidvaleric acidvalues are expressed as mean 06 se lmolg of feces bold val 06 06 06 06 06 06 06 06 06 ues indicate statistical signiï¬cancethe minimum and maximum values of the fecal anicacids for the minimum values the primary split variablewas determined to be the minimum value of propionate andthe cutoff value was lmolg of feces the secondarysplit variable was determined to be the minimum value ofacetate and the cutoff value was lmolg of feces themortality in each partition and the diagnostic characteristicsof this tree are shown in figure for the maximum values of fecal anic acids the primary split variable was determined to be the maximumvalue of lactate and the cutoff value was lmolg offeces the secondary split variable was determined to bethe maximum value of formic acid and the cutoff valuewas lmolg of feces the mortality in each partitionand the diagnostic characteristics of this tree are shown infigure the diagnostic characteristics of the tree for minimumvalue showed a sensitivity of and speciï¬city of whereas those ofthe tree for maximum valueshowed a sensitivity of and speciï¬city of for the minimum values of fecal anic acids multivariate logistic regression analysis revealed that age and theminimum values of propionate and acetate were significantprognostic factors for mortality table for the maximumvalues age and maximum lactate were identiï¬ed as significant prognostic factors for mortality table discussiont his study provides the ï¬rst in vivo evidence toour knowledge that the altered balance of fecal anicacids is associated with mortality in critically ill patients arecent study has shown that scfas particularly acetate andpropionate bind the gproteincoupled receptor gpr43in adipose tissue stimulation of gpr43 by scfas was necessary for the antiammatory responses because gpr43deï¬cient mice showed exacerbated ammation in modelsof colitis arthritis and asthma the scfagpr43 interactions profoundly affected intestinal and systemic ammatory responses13 the low concentrations of acetate andpropionate observed in nonsurvivors in the present studycould reduce the antiammatory effect fukuda 14showed that acetate produced by protective biï¬dobacteriaimproves intestinal defense mediated by epithelial cells andthereby protects the host against lethal infection furthermore asahara 15 showed a positive correlation betweenfecal acetic acid level and tightjunctionrelated gene expression in the intestinal epithelium in their experiment using aninfected mouse modelthe maximum values of lactate and formic acid were alsoselected as prognostic factors fig our group previouslyshowed that when patients had severely acidic feces fecallactate succinic acid and formic acid were significantlyincreased over those of patients with normal feces we alsoshowed that abnormal fecal ph was associated with the incidence of bacteremia and mortality16 the increase of lactateand formic acid can make feces severely acidic and severelyacidic feces could injure intestinal epithelial cells and worsen the patients condition the present results indicate thatnot only a lack of scfas but also an accumulation of lactateand formic acid might be associated with disruption of thegastrointestinal environmentin critically ill conditionswhich leads to bacterial translocationthere was a significant difference between the two groupsin age number of antibiotic types and duration of antibioticuse in this study previous studies have suggested thatchanges in the composition of bacterial species and genes in the authors acute medicine surgery published by john wiley sons australia ltd on behalf ofjapanese association for acute medicine 0cacute medicine surgery 20207e558fecal anic acids in critically ill patients of fig charts showing mortality in critically ill patients partitioned by the minimum value of fecal anic acids using classiï¬cationand regression tree analysis min minimumfig charts showing mortality in critically ill patients partitioned by the maximum value of fecal anic acids using classiï¬cationand regression tree analysis max maximum the authors acute medicine surgery published by john wiley sons australia ltd on behalf ofjapanese association for acute medicine 0c of y nakahori acute medicine surgery 20207e558table results of multivariate logistic regression analysis in critically ill patients using the minimum value of fecal anic acidscoeff bsepvalueodds ratio conï¬dence intervallower limitupper limitmortalityagesexapache iipropionate minacetate min 00 00 00bold values indicate statistical signiï¬canceapache acute physiology and chronic health evaluation coeff b coefï¬cient min minimum se standard errortable results of multivariate logistic regression analysis in critically ill patients using the maximum value of fecal anic acidscoeff bsepvalueodds ratio conï¬dence intervallower limitupper limitmortalityagesexapache iilactate maxformic acid max 00bold values indicate statistical signiï¬canceapache acute physiology and chronic health evaluation coeff b coefï¬cient max maximum value se standard errorthe intestinal ï¬ora occur with aging17 it is also reported thatthe genes related to scfa production were lost from theintestinal ï¬ora and that glycolytic ability was decreased withaging18 these changes could be one of the reasons whyaging was detected as an important factor related to mortality in the present study there is no doubt that antibiotics areimportant butthe intestinal ï¬orasome systemically administered antibiotics are excreted inthe bile and secreted in the upper gastrointestinal tract andupon reaching the colon they cause serious damage to theintestinal ï¬ora19 shortchain fatty acids could also bereduced with the destruction of the intestinal ï¬ora whichmight have worsened the prognosis20they adversely affectshortchain fatty acids are mainly used by intestinalepithelial cells as energy substrates and some are absorbedinto the portal ï¬ow to the liver and used as systemic energysources they can also uence the motility of the intestinaltract absorption of electrolytes and pancreatic secretion andcan increase intestinal blood ï¬ow in the present study totalanic acids including scfas were significantly decreasedin the critically ill patients compared with those in thehealthy controls data not shown one reason would be thealtered gut ï¬ora that produce scfas the commensal gutï¬ora in humans plays an essential role in homeostasis andprotection from injury in the gut2122 under critically illconditions it is difï¬cult to maintain normal gut ï¬ora6 notonly does the stress derived from diseases such as traumaand burns affect the balance of the gut microbiota but alsothe stress caused by the treatment with various therapeuticagents such as histamine h2 receptor blockers catecholamines and broadspectrum antibiotics can uence the gutï¬ora we reported that critically ill patients had to times fewer total anaerobes biï¬dobacterium andlactobacillus and times greater staphylococcus whencompared with counts in healthy volunteers6 these dataindicate that the balance of gut ï¬ora is significantly disturbed in critically ill patients the concentrations of fecalanic acids decreased significantly in the nonsurvivors asshown in table and we believe thatthis was likelybecause the number of good bacteria biï¬dobacterium andlactobacillus had decreased similarly the cause of thereduction of shortchain fatty acids in the present study may the authors acute medicine surgery published by john wiley sons australia ltd on behalf ofjapanese association for acute medicine 0cacute medicine surgery 20207e558fecal anic acids in critically ill patients of have been the reduction of the scfaproducing bacteriasuch as clostridium eubacterium peptococcusandfusobacterium2324synbiotics are the combination of probiotics and prebiotics the clinical effects of synbiotics have been reported inthe ï¬elds of pediatric surgery abdominal surgery and intensive care25 in our preliminary report critically ill patientstreated with synbiotics maintained the necessary amounts offecal anic acids including scfas and had fewer incidences of enteritis pneumonia and bacteremia than thosewithout synbiotics26 these reports suggest that the maintenance of gut ï¬ora and scfas would be a promising intestinal therapy synbiotics were given to patients in thisstudy in the synbioticstreated group maximum values offecal anic acids acetic acid propionic acid succinicacid lactic acid and formic acid increased significantly butminimum values did not increase only the minimum valueof lactic acid decreased significantly however there wasno ï¬xed protocol for the treatment with synbiotics in thisstudy the treatment start date was variable and the averagehospital day of starting treatment was day so itwas difï¬cult to evaluate the effects of synbiotics in thisstudy evaluating the impact of synbiotics on fecal anicacids and prognosis in critically ill patients will be a futuretaskour study has some limitations this was a retrospectivestudy and thus the timing of feces sampling was differentbetween patients the number of samples was too small tomake conclusions about the temporal patternsin conclusion the minimum values of fecal acetic acidand propionate in the nonsurvivors were significantly lowerthan those in the survivors the altered balance of fecalanic acids was associated with mortality in critically illpatients thus the maintenance of scfas could be a targetfor future intestinal therapyacknowledgementsw e thank all the staff of the department of traumatology and acute critical medicine osakauniversity school of medicinedisclosureapproval of the research protocol this study was approvedby the institutional review board of osaka universityinformed consent informed consent was obtained from eachpatients familyregistry and the registration no of the studytrial naanimal studies naconï¬ict of interest nonereferences meddings j the signiï¬cance of the gut barrier in disease gut macfie j oboyle c mitchell cj buckley pm johnstoned sudworth p gut origin of sepsis a prospective studyinvestigating associations between bacterialtranslocationgastric microï¬ora and septic morbidity gut clark ja coopersmith cm intestinal crosstalk a new paradigm for understanding the gut as the motor of critical illness shock deitch ea bacterial translocation or lymphatic drainage oftoxic products from the gut what is important in humanbeings surgery kreymann kg berger mm deutz ne espen guidelines on enteral nutrition intensive care clin nutr shimizu k ogura h goto m altered gut ï¬ora andenvironment in patients with severe sirs j trauma yamada t shimizu k ogura h rapid and sustainedlongterm decrease of fecal shortchain fatty acids in criticallyill patients with systemic ammatory response syndromejpen j parenter enteral nutr american college of chest physicianssociety of criticalcare medicine consensus conference deï¬nitions for sepsisand an failure and guidelines for the use of innovative therapies in sepsis crit care med sugawara g nagino m nishio h perioperative synbiotic treatment to prevent postoperative infectious complications in biliary cancer surgery a randomized controlled trialann surg kikuchi h yajima t correlation between waterholdingcapacity of different types of cellulose in vitro and gastrointestinal retention time in vivo of rats j sci food agr horan tc andrus m dudeck ma cdcnhsn surveillancedeï¬nition of health careassociated infection and criteria forspeciï¬c types of infections in the acute care setting am jinfect control shimizu k ogura h hamasaki t altered gut ï¬ora areassociated with septic complications and death in critically illpatients with systemic ammatory response syndrome digdis sci maslowski km vieira at ng a regulation of ammatory responses by gut microbiota and chemoattractantreceptor gpr43 nature fukuda s toh h hase k biï¬dobacteria can protectfrom enteropathogenic infection through production of acetate nature asahara t takahashi a yuki n kaji r takahashi tnomoto k protective effect of a synbiotic against multidrug the authors acute medicine surgery published by john wiley sons australia ltd on behalf ofjapanese association for acute medicine 0c of y nakahori acute medicine surgery 20207e558resistant acinetobacter baumanniimodel antimicrob agents chemother in a murine infection guarner f malagelada jr gut ï¬ora in health and diseaselancet osuka a shimizu k ogura h prognostic impact of gill sr pop m deboy rt metagenomic analysis of thefecal ph in critically ill patients crit care r119human distal gut microbiome science otoole pw jeffery ib gut microbiota and aging science macfarlane s macfarlane gt regulation of shortchain fatty rampelli s candela m turroni s functional metagenomic proï¬ling of intestinal microbiome in extreme ageingaging albany ny de gunzburg j ghozlane a ducher a protection ofthe human gut microbiome from antibiotics j infect dis p 13erezcobas ae gosalbes mj friedrichs a gutmicrobiota disturbance during antibiotic therapy a multiomic approach gut acid production proc nutr soc pryde se duncan sh hold gl stewart cs flint hj themicrobiology of butyrate formation in the human colonfems microbiol lett shimizu k ogura h asahara t probioticsynbiotictherapy for treating critically ill patients from a gut microbiotaperspective dig dis sci shimizu k ogura h goto m synbiotics decrease theincidence of septic complications in patients with severesirs a preliminary report dig dis sci the authors acute medicine surgery published by john wiley sons australia ltd on behalf ofjapanese association for acute medicine 0c' | 0 |
"A pulmonary function test showed mild restrictive patterns. The patient underwent bronchoscopy and no endobronchial lesion was found. Bronchoalveolar lavage results were negative for malignant cells. Thick varicose septal veins and intraalveolar macrophages were identified on transbronchial lung biopsy. After seven months a follow-up chest CT scan revealed increased interstitial septal thickening (Fig. 1E F) new peribronchovascular cuffing and small amounts of bilateral pleural effusion (Fig. 1G). Finally the patient underwent video-assisted thoracoscopic surgery wedge resection of the right middle and lower lobes. In the surgical field abnormal hypervascularity was noted on the lung surface (Fig. 1H). Microscopic examination showed proliferation of thinwalled anastomosing vascular spaces lined by a single layer of endothelial cells lacking cytological atypia (Fig. 1I). Theses lesions were located along the lymphatic distribution and were highlighted by D2-40 (1:100 Dako Glostrup Denmark) immunohistochemical stain (Fig. 1J) characteristic of DPL. Presently the patient is alive without any symptoms and being observed without specific treatment such as low fat medium chain fat. DISCUSSION Diffuse pulmonary lymphangiomatosis is a rare condition in which diffuse proliferation of anastomosing lymphatic channels is observed. It manifests equally in both sexes mostly in children and young adults (3). Symptoms include dyspnea nonproductive cough bronchial casts milky sputum fever and recurrent pneumonia. However patients can present with the disease later in adulthood and often have symptoms dating back to their childhood (5). To the best of our knowledge only five cases of DPL in middle-aged patients have been reported in the English literature () (1-5) with the current report being the sixth. Ours is a unique one in the following aspects; although the disease progression revealed on serial follow-up CT required differentiation from lymphangitic metastasis the patient remained asymptomatic and did not need any treatment; also the extent of the disease markedly increased within one year and a neoplastic but possibly benign component was presumed. Making a preoperative diagnosis of DPL in adults is difficult. The most common radiologic finding is increased interstitial markings on chest radiography. CT demonstrates smooth or nodular interlobular septal and peribronchovascular thickenings. Patchy ground glass opacities are also seen. Pleural thickening pleural effusion and diffuse mediastinal soft tissue infiltration can also occur (5). Pulmonary edema pulmonary veno-occlusive disease Erdheim-Chester disease and lymphangiectasis can be considered possible diagnoses when smooth interlobular septal thickening is found. In this patient pulmonary edema was less likely since there was no evidence of congestive heart failure or pleural effusion. Prominent central pulmonary arteries were also not identified making pulmonary veno-occlusive disease less likely (6). The possibility of Erdheim-Chester disease characterized by proliferation of lipid-containing foamy histiocytes in the skeleton and other ans was ruled out by the absence of sclerotic changes in the diaphyses and metaphyses of long bones in the current case (7). The CT appearance of DPL is virtually identical to that of pulmonary lymphangiectasia (8). Pulmonary lymphangiectasia is a rare condition characterized by the diffuse dilatation of pulmonary lymphatics and classified as congenital or secondary (9). In this case there was no evidence of pulmonary hypertension or venous obstruction factors that can cause secondary lymphangiectasia. Congenital lymphangiectasia typically presents shortly after birth and is associated with high neonatal morbidity and mortality. Lymphangiomatosis typically presents in older children and is rarely seen in adults (10). Histopathologically lymphangiomatosis is characterized by an increased number of variable-sized lymphatic spaces. This should be distinguished from the findings of lymphangiectasia in which nonproliferative lymphatic channels are dilated (4). Other diseases with lymphatic distributions such as lymphangitic carcinomatosis sarcoidosis and pulmonary lymphoma can also be considered. In these diseases interlobular septal thickening tends to be nodular. There is no established treatment for DPL. " | 1 |
"ChIP assays were performed with anti-acetyl-H3 (panel b) and anti-Sp1 antibodies (panel c). DNA was extracted for PCR with miR-182 and p21 primers. Data were quantified after three independent experiments (panel d). (F) A549 cells were harvested for DAPA with a biotin-conjugated p21 and miR-182 promoter probes and samples were analyzed by Western blotting using anti-Sp1 antibodies (panel a). Data were quantified after three independent experiments (panel b). (G) Plasmids GFP or GFP-Sp1 were co-transfected with pGL2 pGL2-miR-182 WT or mutation plasmids into H1299 cells for 24 h and then cells were harvested for luciferase activity assays. Data are representative of three independent experiments each of which was performed in triplicate and presented as the mean ± SEM. The level of statistical significance determined by t-test (* p<0.05; ** p<0.01). Because Sp1 is highly expressed in lung cancer we studied the expression of Sp1 and miR-182 in various lung cancer cell lines and patient samples (). Compared with normal human lung cells (BEAS-2B) lung cancer cell lines expressed higher levels of miR-182 (A). We also assessed the correlation between the miR-182 and Sp1 expression patterns. Sp1 levels in clinical lung tissue samples were highly elevated in the tumorous sections of the lung accompanied by increased expression of miR-182 (B). To confirm this result Sp1 and miR-182 levels were measured in 32 lung cancer patients. Sp1 and miR-182 were upregulated by more than 1.3-fold in 59.4% of the lung adenocarcinoma specimens when compared to expression in normal tissue (C). These results indicate that Sp1 expression positively correlates with miR-182 expression (D). The miR-182 level correlates to Sp1 level Total RNA and cell lysates were prepared from indicated cell lines (A) or from clinical lung tissues of lung cancer patients (B). The miR-182 level was determined by stem-loop RT-PCR and Sp1 level was studied by Western blotting with anti-Sp1 antibodies. U6 and tubulin served as the internal control. (C) Total RNA and cell lysates were prepared from 32 paired normal lung tissues and lung adenocarcinoma samples. The miR-182 level was studied by stem-loop RT-PCR and Sp1 levels were studied by RT-PCR. (D) The relationship between Sp1 and the miR-182 level in the 32 lung cancer samples was statistically analyzed using Fisher's exact test. miR-182 increases lung tumor growth The data shown in indicated that Sp1 regulated miR-182 expression during lung tumorigenesis. To identify the specific gene targets of miR-182 we searched public miRNA target prediction databases (miRDB miRWalk and TargetScanHuman) for candidate target genes. By combining the data from these three databases we identified 161 genes potentially regulated by miR-182 (Supplementary Figure S1A). Moreover pathway analysis using Ingenuity software indicated that the cellular growth and proliferation pathway had the highest score when the association of these 161 genes with biological pathways was examined. This suggests that miR-182 may play a functional role in cancer-associated processes (Supplementary Figure S1B). Indeed when miR-182 was knocked down with miRZip-182 shRNA the percentage of cells in G2/M and sub-G1 phases increased suggesting that miR-182 positively regulated cell cycle progression in the lung cancer cells (A). To further elucidate miR-182's effect on the cell cycle cells were synchronized at prometaphase using nocodazole treatment. After removing nocodazole more miRZip-182 than miRZip cells remained in G2/M phase providing further evidence that miR-182 positively regulates cell cycle progression (B). Consistently knockdown of miR-182 expression inhibited cell growth (C). miR-182 increases cancer cell proliferation (A) The miRZip and miRZip-182 stably expressed H1299 cells were fixed with 70% ethanol and stained with propidium iodide for cell cycle analysis by FACS. (B) Mitotic cells were released into growth by removing nocodazole then fixed at indicated time points for cell cycle progression assay by FACS. (C) The growth rates of miRZip and miRZip-182 stably expressed H1299 cells were calculated by cell counting within 5 days. Data are representative of six independent experiments and presented as the mean ± SEM. (D) Bioluminescent imaging was performed on 10 severe combined immunodeficient (SCID) mice implanted with miRZip and miRZip-182 stable expression H1299 cells (106 cells/mouse) at day 14 (panel a) then image signal was analyzed using Living Image software and presented as total flux measurements in photons/second (panel b). (E) Tumors from SCID mice implanted with miRZip and miRZip-182 stable expression H1299 cells for 4 weeks are shown (panel a) and tumor weights were analyzed (panel b). Data are representative of ten independent experiments and are presented as the mean ± SEM. The level of statistical significance determined by t-test (* p<0.05; ** p<0.01; *** p<0.001). To confirm the effect of miR-182 on tumor formation cells stably expressing with miRZip or miRZip-182 were implanted into SCID mice and tumor growth was monitored in vivo (D). The miRZip lentivector contains a copGFP gene and the GFP signal in miRZip-182-expressing cells was lower than that in miRZip control cells (D). Furthermore tumor volume and tumor weight were also lower in miRZip-182-implanted mice than in miRZip-implanted mice (N = 10 per group) (E). These results suggest that miR-182 overexpression facilitates lung tumor growth in vivo. Sp1 inhibits FOXO3 expression by inducing miR-182 expression To investigate the molecular mechanism underlying miR-182-mediated cancer cell proliferation we studied an important miR-182 target gene FOXO3. FOXO3 expression was higher in cells stably expressing miRZip-182 than in control cells (A). Knockdown of miR-182 expression enhanced the luciferase activity of a pGL3 vector containing the 3?-UTR of FOXO3 (B) indicating that miR-182 downregulated FOXO3 expression. Further to determine whether Sp1 downregulated FOXO3 expression through miR-182 GFP-Sp1 was expressed in cells stably expressing miRZip-182 (C). Overexpression of GFP-Sp1 reduced FOXO3 protein expression in miRZip stable cells but increased FOXO3 levels in miRZip-182-expressing cells implying that different effect of Sp1 is existed on the regulation of FOXO3 expression. Regulation of FOXO3 by miR-182 and Sp1 (A) Lenti-miRZip and lenti-miRZip-182 viruses were infected into H1299 for 96 h individually. FOXO3 level was studied by Western blotting with anti-FOXO3 antibodies and miR-182 level was studied by stem-loop RT-PCR. (B) Plasmids pGL3 and pGL3-FOXO3-3'UTR were transfected into miRZip and miRZip-182 stably expressed H1299 cells for 24 h and then cells were harvested for luciferase activity assays. (C) Different doses of GFP-Sp1 adenovirus were infected into the miRZip and miRZip-182 stably expressed H1299 cells for 48 h. FOXO3 level was studied by Western blotting using anti-FOXO3 antibodies (panel a). Quantitative results from three independent experiments are shown (panel b). The level of statistical significance was determined by t-test (* p<0.05; *** p<0.001). Therefore we further investigated the relationship between Sp1 and miR-182 in the context of FOXO3 regulation. The expression of Sp1 and FOXO3 in patients with lung cancer was examined (A). In normal tissue samples Sp1 levels were low and FOXO3 levels were high. In tumor tissue samples two Sp1 expression patterns i.e. high and low Sp1 expression were identified. Samples with higher Sp1 levels exhibited lower FOXO3 levels whereas samples with lower Sp1 levels exhibited higher FOXO3 levels suggesting that there is an inverse correlation between Sp1 and FOXO3 levels in lung specimen (A). The levels of FOXO3 and Sp1 in the lung cancer cell lines A549 H1299 CL 1-0 and CL 1-5 were studied (B). Higher levels of Sp1 expression were accompanied by lower levels of FOXO3 expression in A549 and CL 1-0 cells and lower levels of Sp1 expression were accompanied by higher levels of FOXO3 expression in H1299 and CL 1-5 cells suggesting that there is an inverse correlation between Sp1 and FOXO3 expression in lung tumorigenesis. Overexpression of GFP-Sp1 decreased FOXO3 mRNA and protein levels in a dose-dependent manner (C panel a) whereas knockdown of Sp1 expression increased FOXO3 mRNA and protein levels (C panel b). These results indicate that Sp1 negatively regulates FOXO3 expression. Sp1 negatively regulates FOXO3 expression through regulating miR-182 (A) The Sp1 and FOXO3 levels in clinical lung tissue samples were studied by IHC staining using antibodies against Sp1 and FOXO3 respectively. (B) Cell lysates were harvested from various cell lines for Western blotting using antibodies against FOXO3 and Sp1 and tubulin as an internal control. (C) Adeno-GFP-Sp1 viruses were infected into IMR-90 cells for 48 h and FOXO3 mRNA and protein were studied by RT-PCR and Western blotting respectively. GAPDH served as the internal control (panel a). Scramble and Sp1 shRNAs were transfected into H1299 for 48 h then FOXO3 mRNA and protein levels were studied by RT-PCR and Western blotting (panel b). (D) Scramble and Sp1 shRNAs were transfected into H1299 for 48 h and then cells were harvested at indicated time points following cycloheximide treatment for studying the Sp1 and FOXO3 levels with Western blotting. The levels of FOXO3 protein from three independent experiments were quantified using tubulin as an internal control. (E) Plasmids pGL2 or pGL2-FOXO3 (-1000/+50) were cotransfected with GFP or GFP-Sp1 into H1299 cells for 24 h then cell lysates were harvested for luciferase activity assays. (F) Adeno-GFP-Sp1 viruses were infected into H1299 cells for 24 h and cells were then transfected with pGL3 or pGL3-FOXO3-3'UTR plasmid for 24 h. Cells lysates were harvested for luciferase activity assays. (G) H1299 cells which were infected with GFP-Sp1 adenovirus for 24 h were then transfected with pGL3 or pGL3-FOXO3-3'UTR plasmid for 24 h. Total RNA was extracted at various time points following actinomycin D treatment. The mRNA levels of luciferase were determined by using quantitative RT-PCR and quantified using GAPDH as an internal control. Data are representative of three independent experiments each of which was performed in triplicate and presented as the mean ± SEM. The level of statistical significance determined by t-test (* p<0.05; ** p<0.01). Next we investigated the mechanism by which Sp1 regulates FOXO3 expression. FOXO3 protein half-life was studied after Sp1 knockdown. Knockdown of Sp1 expression did not affect FOXO3 protein stability (D). We then constructed a luciferase reporter construct containing the FOXO3 promoter (-1000/+50) to study the effect of Sp1 on the promoter-mediated transcription of FOXO3 (E). GFP-Sp1 overexpression significantly enhanced the luciferase activity indicating that Sp1 positively regulated FOXO3 transcription (E). However FOXO3 mRNA and protein levels decreased as shown in C. The data shown in indicated that Sp1 increased miR-182 expression which suggests that post-transcriptional processing contributes to the regulation of FOXO3 expression. Thus the 3'-UTR of FOXO3 might play an important role in stabilizing FOXO3 mRNA and in FOXO3 translation. Consequently a luciferase reporter construct containing the 3?-UTR of FOXO3 was generated. GFP-Sp1 overexpression reduced the luciferase activity (F). Furthermore the stability of the luciferase mRNA containing the 3?-UTR sequence of FOXO3 decreased dramatically upon GFP-Sp1 overexpression (G). These results indicate that Sp1 regulates FOXO3 expression through transcriptional and post-transcriptional regulation with a net negative effect on FOXO3 expression. miR-182 inhibits lung cancer metastasis activity The data shown in indicated that miR-182 positively regulated lung cancer cell growth. Therefore the role of miR-182 in lung cancer metastasis was studied (Figure 6). The morphology of miRZip-182 cells was markedly altered: circular structures of actin filaments were absence and pseudopodia were enriched suggesting that miR-182 decreased the cells' migratory ability (Figure 6A). Indeed knockdown of miR-182 expression increased the migration ability of lung cancer cells suggesting that miR-182 inhibits lung cancer migration (Figure 6B). Moreover transwell migration assays showed that knockdown of miR-182 expression enhanced cell's invasive capacity (Figure 6C). In mice injected with miRZip-182-treated cells the knockdown of miR-182 expression also increased the number of nodules in the lung suggesting that miR-182 represses metastatic ability in vivo (Figure 6D). The effects of miR-182 knockdown were partially reversed by knockdown of FOXO3 suggesting that miR-182 functions as a suppressor of lung cancer metastasis by repressing FOXO3 expression (Figure 6E panel a). The endothelial-mesenchymal transition (EMT) marker N-cadherin increased after miR-182 knockdown but this effect was abolished by FOXO3 knockdown. Thus miR-182 might repress lung cancer metastasis by decreasing the expression of N-cadherin (Figure 6E panel b). However the expression of other genes regulated by miR-182 might also play a role in metastasis (Figure 6F and Supplementary Figure S3). Therefore we generated gene expression profiles using microarray analysis. Functional grouping analysis using DAVID bioinformatics resources showed that 19 of the genes differentially regulated by miR-182 knockdown were related to cell migration. The expression of these genes was increased in miR-182-knockdown cells indicating that they are potential targets of miR-182 (Figure 6F). Many metastasis-related genes such as CD44 CDH9 and ADAM9 were upregulated after the knockdown of miR-182 expression (Figure 6F). Figure 6 miR-182 attenuates lung cancer cell metastasis (A) Immunofluorescent staining of Alexa Fluor 568-conjugated phalloidin that is a high-affinity probe for F-actin (red) in miRZip and miRZip-182 stably expressed H1299 cells. DNA was stained with DAPI (blue). Stained cells were photographed under a fluorescence microscope at x 600 magnification. (B) Confluent monolayers of miRZip or miRZip-182 stably expressed H1299 cells were wounded and incubated for an additional 16 h (panel a). Migratory area was calculated for quantification (panel b). (C) The migration activities of H1299 cells (2 x 104) expressing miRZip or miRZip-182 were studied by Transwell chambers. (D) The miRZip or miRZip-182 stably expressed H1299 cells (4 x 106) were suspended in 100 ?l of PBS and injected into the lateral tail vein of SCID mice. After 8 weeks all mice were killed and the number of pulmonary tumor nodules was calculated after fixation of lungs with 4% formaldehyde for 48 h (panel a) and the number of pulmonary metastatic tumor nodules was counted (panel b). (E) FOXO3 and miR-182 in H1299 cells were knockdown by shFOXO3 and miRZip-182 respectively and then migration of cells (3 x 104) was studies by Transwell chambers (panel a). In addition cell lysates were harvested from FOXO3 and miR-182 knockdown cells for Western blotting using antibodies against N-cadherin ?-catenin vimentin FOXO3 and tubulin (panel b) respectively. (F) Heat map of the 19 of genes from miRZip and miRZip-182 microarray data the red color represents genes that are upregulated and the green color represents genes that are downregulated. The level of statistical significance determined by t-test (* p<0.05; ** p<0.01; *** p<0.001). DISCUSSION Our recent studies showed that Sp1 increased the growth of lung cancer cells but inhibits metastatic activity [23 32]. In the present study we found that Sp1 which accumulated in the early stages of cancer positively regulated miR-182 gene expression to silence FOXO3 expression and thereby promote cancer cell growth. In addition decreased levels of Sp1 in the late stages of cancer increased the expression of FOXO3 and N-cadherin leading to cancer metastasis (Figure 7). Figure 7 (A) Clinical samples from lung cancer patients of stage I and IV were used to study the Sp1 level by IHC staining with anti-Sp1 antibodies (B) Schematic diagram illustrates Sp1 regulates miR-182 to silence FOXO3 expression in early and late stages of lung cancer progression. Sp1 functions as a transcriptional activator by recruiting p300 to its target genes and as a repressor by the recruiting HDACs. Because Sp1 accumulates in several types of cancer including lung cancer [33] understanding the Sp1 transcriptional regulatory network may provide novel insights into the molecular origins and treatment of lung cancer. In our previous studies of lung cancer we found that Sp1 was highly upregulated in the early stages of cancer progression but partially down regulated in the late stages. Our previous studies also showed that regulation of Sp1 protein stability by phosphorylation and sumoylation contributed to its expression in the early and late stages of cancer respectively [32]. Kras activation and the Notch pathway might activate ERK1/2 to phosphorylate Sp1 thus stabilizing Sp1 in the early stages of cancer [32 34]. In the late stages Sp1 could be sumoylated leading to recruitment of its E3-ligase RNF4 followed by polyubiquitination and degradation [32]. To clarify the molecular mechanism underlying gene regulation by Sp1 we used microarray analysis to assess gene expression in KrasG12D-induced lung tumor transgenic mice and identified thousands of genes potentially regulated by Sp1 [23]. However some of the genes do not harbor a conserved Sp1 binding motif within their promoter region suggesting that another regulatory mechanism is involved in Sp1-mediated gene regulation. In this study we identified a novel pathway for Sp1-mediated activation wherein miR-182 expression downregulated the expression of FOXO3 a known miR-182 target gene [35]. Sp1 activated miR-182 and FOXO3 at the transcriptional level; however FOXO3 protein expression decreased. These results suggest that post-transcriptional regulation by miRNAs is a powerful mechanism by which to control the final level of protein expression. Many coding genes with Sp1 binding element(s) in their promoters harbor conserved miRNA target sequences in their 3'-UTR. To our knowledge this is first study to demonstrate that Sp1 regulates the expression of a target gene by regulating promoter activity and post-transcriptional processing in parallel. Few studies have characterized the regulation of miRNA by Sp1. Herein using a bioinformatics approach we identified several miRNAs potentially regulated by Sp1 including miR-182. We then showed that Sp1 specifically targets the miR-182 promoter region and activates miR-182 expression. miR-182 reportedly forms a gene cluster with two adjacent miRNAs (miR-96 and miR-183) [35]. The expression of miR-96 and miR-183 also decreased following Sp1 knockdown (Supplementary Figure S2A). Moreover we also investigated the binding of Sp1 to the miR-212 promoter because the latter contains 13 putative Sp1 binding sites (Supplementary Table S1). We found that Sp1 bound to the miR-212 promoter sequence (Supplementary Figure S2B and S2C). Interestingly a recent study showed that FOXO3 is a direct target of miR-212 in the neurons of patients with Alzheimer's disease [36]. miR-182 and miR-212 might cooperate to downregulate FOXO3 expression upon Sp1 overexpression. We cannot rule out this possibility. However depletion of miR-182 was sufficient to impair the Sp1-mediated reduction of FOXO3 expression in our experiments (C) suggesting that miR-182 is the major regulator of FOXO3 in lung cancer cells. Several studies have shown that miR-182 is upregulated in lung cancer. This suggests that miR-182 plays a positive role in lung tumorigenesis. However in two studies of miR-182 function in lung cancer miR-182 inhibited the proliferation of human lung adenocarcinoma cells [37 38]. Our results in this study provide several pieces of evidence to support the notion that miRNA-182 is a positive regulator of lung cancer cell proliferation. Firstly miR-182 was upregulated in the majority of lung cancer clinical samples and lung cancer cell lines examined. Secondly miR-182 knockdown inhibited cell cycle progression and cell growth. Finally miR-182 knockdown reduced lung tumor growth in vivo. Discrepancies in the role of miRNA-182 in lung cancer cell proliferation might derive from the different experimental designs of the studies. For example because miR-182 expression is upregulated in lung cancer we knocked down its expression and examined the effects on cancer cell proliferation. However other studies that described a negative role of miR-182 in lung cancer used miR-182 overexpression to study miR-182's role in cancer cell proliferation. Overexpression conditions can alter the function of many genes [39]. For example Sp1 accumulates in most of cancers; knockdown of Sp1 expression decreases cell proliferation but Sp1 overexpression also attenuates cancer cell growth [40]. Because post-translational modifications affect protein function overexpressed proteins might not be completely processed which could affect their function. Previous studies in melanoma and hepatocellular carcinoma indicated that miR-182 enhanced tumor metastasis [35 41]. However our data as shown in Figure 6 indicated that miR-182 knockdown altered cell morphology and increased migration and invasion activities. In addition miR-182 knockdown increased N-cadherin levels suggesting that miR-182 promotes the mesenchymal to epithelial transition (MET) [42]. Previous studies have shown that TIMP-2 enhances the E-cadherin/?-catenin complex in A549 lung cancer cells [43]. Whether Sp1 or miR-182 regulates TIMP-1 in lung cancer needs to be addressed in future studies. Finally miR-182 levels were lower in CL1-5 cells then in CL1-0 cells resulting in increased metastatic activity in CL1-5. Collectively our data suggest that miR-182 inhibits lung cancer metastasis. Our previous study indicated that Sp1 is down regulated in the late stages of lung cancer progression [32]. Therefore in the late stages of lung tumorigenesis miR-182 expression was down regulated compared with expression in the early stages which led to tumor metastasis through at least in part an increase in FOXO3 expression. It is still not clear why miR-182 has different roles in different types of cancer; this awaits further study. Although we found that FOXO3 is involved in miR-182-mediated lung cancer progression FOXO3 knockdown did not completely abolish the effects of miR-182 knockdown suggesting that other genes regulated by miR-182 contribute to the inhibition of metastasis by miR-182. With this in mind we determined the expression profile of miR-182-regulated genes. Many metastasis-related genes were induced in miR-182-knockdown cells including CD44 ADAM9 and CDH9. CD44 which localizes to the cell membrane is reportedly involved in cell migration in various cancer types [44]." | 1 |
the bacteroides fragilis b fragilis produce biofilm for colonisation in the intestinal tract can cause a series of inflammatory reactions due to b fragilis toxin bft which can lead to chronic intestinal inflammation and tissue injury and play a crucial role leading to colorectal cancer crc the enterotoxigenic b fragilis etbf forms biofilm and produce toxin and play a role in crc whereas the nontoxigenic b fragilis ntbf does not produce toxin the etbf triggers the expression of cyclooxygenase cox2 that releases pge2 for inducing inflammation and control cell proliferation from chronic intestinal inflammation to cancer development it involves signal transducers and activators of transcription stat3 activation stat3 activates by the interaction between epithelial cells and bft thus regulatory tcell tregs will activates and reduce interleukin il2 amount as the level of il2 drops thelper th17 cells are generated leading to increase in il17 levels il17 is implicated in early intestinal inflammation and promotes cancer cell survival and proliferation and consequently triggers il6 production that activate stat3 pathway additionally bft degrades ecadherin hence alteration of signalling pathways can upregulate spermine oxidase leading to cell morphology and promote carcinogenesis and irreversible dna damage patient with familial adenomatous polyposis fap disease displays a high level of tumour load in the colon this disease is caused by germline mutation of the adenomatous polyposis coli apc gene that increases bacterial adherence to the mucosa layer mutatedapc gene genotype with etbf increases the chances of crc development therefore the colonisation of the etbf in the intestinal tract depicts tumour aetiology can result in risk of hostility and effect on human health keywords bacteroides fragilis colon cancer stat3 pathway bacteroides fragilis toxin inflammationintroductionbacteroides species are nonspore forming anaerobe and gramnegative bacteria there are more than different species of bacteroides these bacteria act as normal flora in the intestine to maintain healthy intestinal microflora in humans bacteroides fragilis b fragilis has two classes nontoxigenic b fragilis ntbf and enterotoxigenic b fragilis etbf the differences between ntbf and etbf are the presence of b fragilis toxin bft gene and its ability to produce biofilm bft product is a kda zincdependent metalloprotease toxin also known as fragilysin or bft bft plays an important role in intestinal inflammation and tissue injury by damaging the tight junction and increasing intestinal permeability furthermore it has been proven that tissue inflammation and injury promote cancer formation simultaneously the biofilm produced by b fragilis induces carcinogenesis fortunately only etbf encompasses bft and can produce biofilms hence ntbf does not harm the intestinal tract malays j med sci julaug wwwmjmsusmmy penerbit universiti sains malaysia this work is licensed under the terms of the creative commons attribution cc by httpcreativecommonslicensesby40 0cmalays j med sci julaug in the united states colorectal cancer crc is the third most common cancer in both genders it is also the second most common cancerrelated death especially for older patients who are ¥ years old in the american cancer society stated that there were new cases of crcs that led to the death of people moreover crc is the fourth leading cancer resulting in deaths worldwide inflammatory bowel disease ibd and genetic mutations are factors predisposing an individual towards colon cancer this indicates that crc has a high mortality rate microbes are capable in promoting cancer development through several routes such as activation of chronic inflammation alteration of tumour microenvironment and production of toxins that damage dna when there is chronic etbf colonisation in the intestine it stimulates chronic intestinal inflammation triggering signal transducers and activators of transcription stat3 activation which contributes to interleukin il17 production il17 is involved in colon inflammation bft produced by etbf causes the alteration of signalling pathways and production of reactive oxygen species ros that leads to dna damage and cleavage of ecadherin in the below review we have provided a general information regarding bft produced by etbf triggering crc developmentliterature reviewcolon cancer associated with microbespromote nutrition in the human gastrointestinal tract there are nearly trillion microbes out of which make up normal flora in the intestine meanwhile the normal flora is characterised into beneficial and harmful microbes beneficial microbes including production of vitamins in the intestine and prevent disease formation however harmful microbes produce carcinogenic toxin and substances in the intestine these harmful substances may cause cancer there are many types of bacteria that stimulate a variety of cancer formation through their respective site of inflammation eg bacteria such as enterococcus faecalis e faecalis colibactinproducing escherichia coli e coli and etbf are involved in crc development however the mechanisms between each bacterium in contributing to crc formation are different for instance e faecalis damages the dna through ros colibactinproducing e coli produces colibactin that damages the dna and etbf produces bft that contributes to inflammation and immunecell infiltration intestinal dysbiosis inflammation and colon cancergenetic normal flora is advantageous to a person as it maintains intestinal health and gut homeostasis however as the bacteria such as etbf in the gut undergoes dysbiosis it brings harmful effects to the person according to deng et al a correlation was observed between microbiota imbalance and cancer progression while liu et al claimed is associated with that crc development intestinal microecology disorder imbalance among microbiota leads to bacterial infection that can progress to chronic inflammation one of the main environmental risk factors contributing to crc development is chronic intestinal inflammation chronic inflammation alters cellular microenvironment enhances gene mutation inhibits apoptosis and induces neovascularisation and cell proliferation that causes precancerous conditions eventually leading cancer simultaneously chronic inflammation alterations that directly affect the stat3 pathway and promoting there are three stages involved in tumour development namely initiation promotion and progression during initiation and progression cancer cells and microbes interact both producing genetic inflammatoryimmunological factors that are responsible for their survival and replication in tumour progression tumour cells interact with the inflammatory cells in the tumour microenvironment these tumour cells inflammatoryimmunological factors to attract the inflammatory cells and the stromal cells simultaneously activate both stromal cells start to produce various soluble factors including growth factors and protease these soluble factors play an important role in facilitating the growth differentiation and survival of tumour cells hence it promotes tumour progression and promotion additionally cytokines or microbes promote cancer by changing genetic sequence during gene mutation epithelial cells inflammatory and activated carcinogenesis cytokines chemokines causes and secrete wwwmjmsusmmy 0creplicate rapidly and develop into a hyperplastic epithelium which progresses into adenomas and then towards adenocarcinomas both adenomas and adenocarcinomas affect the growth rate of colonic epithelial cells and improve the cells toleration towards apoptosis and abnormal cells escape from the immune cells furthermore these invade submucosa turning into cancer when the growth of malignant cells continues the tumour continues to spread in the colon thus carcinogenesis becomes more efficientadenocarcinomas begin to ibd is an example of chronic intestinal inflammation that is associated with etbf pathogenic bacteria are capable of stimulating infection inflammation and carcinogenesis whereas the relationship between ibd and crc is well established surprisingly patients with ibd show a high level of immunoglobulin ig g antibodies il6 vascular endothelial growth factor vegf and tumour necrosis factor tnf igg antibodies are responsible for killing bacteria moving into the intestinal lumen simultaneously il6 and vegf are responsible for stat3 activation ibd is also known as ulcerative colitis uc and crohns disease cd this chronic intestinal inflammation increases the risk of colitisassociated crc the probability of which depends on multiple casual factors including severity duration of inflammation in the intestine and gut microbiota imbalance patients with uc or cd have folds higher incidence of crc when compared to healthy individuals it is also stated that patients with uc and cd have and respectively higher risks of crc compared to a normal healthy person this indicates that patients with uc tend to be more susceptible to crc than those with cd furthermore it is evident that the large intestine tends to have a higher risk of crc compared to the small intestine which can be attributed to the higher amount of bacteria simultaneously people with ibd and crc have a higher quantity of etbf in the intestine or stool examination compared to healthy persons additionally etbf are biofilm producers they can reduce or redistribute ecadherin in the colonic epithelial cells trigger the production of il6 by epithelial cells activate stat3 pathway and enhance cells proliferation at the site of crypt epithelial in normal colon mucosa this shows that biofilms are associated with the risk of colon cancer development review article formation of colon cancercox enzymes involved in inflammation carcinogenesis and biomarkerchronic inflammation is a principal factor that contributes to carcinogenesis prostaglandin is a paracrine hormone that plays an important role in inflammation cyclooxygenase cox is the ratelimiting enzyme responsible for producing prostaglandins cox1 and cox2 are the isoforms of cox enzymes that break down arachidonic acid into prostaglandins cox2 plays an important role in maintaining environment for the development of cancer inflammation cox2 is normally expressed in epithelial and stromal cells and the expression level is increased in both inflammation and cancer due to the presence of proinflammatory cytokines additionally bft triggers colonic epithelial cells to express cox2 but not cox1 cox2 releases prostaglandin e2 pge2 that triggers pain and inflammation at the site of tissue injury simultaneously pge2 controls cell proliferation by binding at the cell receptor and activating oncogenic signalling pathways thus it is proven that cox2 plays an important role in carcinogenesis and cancer progression by promoting cell proliferation angiogenesis and cancer stem cell formation inhibiting cell apoptosis and heightening metastatic potential through producing pge2 the in certain studies it is stated that aspirin and nonsteroidal antiinflammatory drugs have the ability to inhibit the activity of cox enzyme which reduces inflammatory response thus it delays crc occurrence fortunately cox1 and cox2 act as biomarkers for screening purposes the biomarker is defined as any substance structure or process that is measurable in the body to determine the incidence of a disease it is commonly detected in circulation and body fluids cox1 is present in most cells thus it is not a specific biomarker however cox2 is only detected when is stimulated by trauma release of cytokines and stimulation of arachidonate metabolism by a toxin such as bft thus cox2 acts as a useful biomarker to detect cox2 is also a useful biomarker for colorectal carcinogenesis screening the level of cox2 biomarker in the blood is dependent upon epithelial cell proliferation apoptosis inhibition and neoangiogenesis patients with crc have high levels of cox2 compared to normal individuals indicating more aggressive growth rate and higher mortality rate this inflammatory responses inflammation the wwwmjmsusmmy 0cmalays j med sci julaug suggests that cox2 expression is correlated to the aggressiveness of growth rate and mortality rate etbf activates stat3immune is activated to eliminate regulation of etbf is associated with ibd due to the abnormal response to bacteria the systemic adaptive immune response foreign antigens in the body this action eventually reduces intestinal mucosal tolerance although immune cells kill foreign antigens neutrophils and th17 cells contribute to inflammation and tumourigenesis transcription factors are known as stat protein family comprising seven members each stat protein responds to its specific cytokines they play an important role in regulating immune responses by controlling th cell types generation for instance the activation and generation of th17 cells require transcription factor stat3 protein the roles of stat3 protein include promotion of cell proliferation cell survival inflammation cellular transformation metastasis of cancer blood vessel formation and tumourpromoting moreover stat3 intrinsic pathway for cancer inflammation it induces genes in tumour cells that are responsible for inflammation within a tumour cell it exhibits an overly expressed stat3 pathway inflammation is a major factors etbf has the ability to activate stat3 rapidly in both colonic epithelial cells and colonic mucosal immune cells through phosphorylation and nuclear translocation however stat3 activation first occurs in colonic mucosal immune cells followed by colonic epithelial cells to activate stat3 in immune cells epithelial cells should respond in the production of cytokines such as il6 il10 and il23 besides cytokines growth including vegf and fibroblast growth factor fgf2 are also involved in activating stat3 when etbf and bft first interact with colonic epithelial cells they stimulate early stat3 activation in colonic mucosal immune cells this stat3 activation continuously rises slowly until it reaches the peak level the peak indicates that etbf activates the immune system due to barrier dysfunction during etbfinduced colitis it activates both stat3 and th17 immune response in the colonic mucosa stat3 activation induces prooncogenic inflammatory pathways and increases the permeability of mucosa although stat3 activation is longterm and lasts for months it highly increases the chance of getting a tumour as a result of chronic inflammation additionally stat3 activation promotes the accumulation of tumour regulatory tcell tregs and blocks the generation of antitumour immune responses which give an adverse effect to the body this abnormal persistent stat3 activation increases the cancer cell tolerance prevents rejection of cancer by the immune system reduces the effectiveness of immunotherapy and enhances the effectiveness of oncogenesis activated stat3 predominantly detected in human cancers is constitutively activated and depicts its association with neoplasms patients with ibd tend to show stat3 activation and a high level of th17 cells and il17 the level of activated stat3 in patients with ibd and dysplasia is different from patients with ibd and without dysplasia patients with ibd and dysplasia show a higher level of activated stat3 compared to those without dysplasia simultaneously the level of activated stat3 increases together with the continuum of dysplasia to colitisassociated cancer it is clear that b fragilis can either be toxigenic or nontoxigenic the latter does not activate stat3 because it does not produce bft therefore ntbf does not contribute to colon cancer development but etbf does are tregs th17 and il17 good or badreduces intestinal il17 this process in a normal healthy condition tregs play an important role in inflammatory responses and immune homeostasis they express high levels of il2 receptor and produce endogenous il2 which inhibits the production of intestinal inflammation and prevents carcinogenesis however when etbf colonises a particular site of the colon it produces a large amount of bft damaging the intestinal mucosa to initiate etbftriggered colitis with the activation of the stat3 pathway this leads to direct contact between tregs and etbf and promotes tregs activation activated tregs lack the ability to produce endogenous il2 instead of producing endogenous il2 tregs consume exogenous il2 for their survival the consumption of exogenous il2 by tregs reduces the levels of exogenous il2 and produces an environment that favours the growth of th17 cells as the levels of il2 drop th17 cells are no longer inhibited and undergo expansion to produce a large quantity of naïve tcells this naïve subset of tcells then differentiates into th17 cells in excess wwwmjmsusmmy 0ccarcinogenesis this shows that colonisation of etbf promotes the accumulation of both tregs and th17 cells th17 cells start to produce large amounts of cytokines including tnf and il17 these cytokines promote cell survival and proliferation during injuries although th17 cells heal an injured site they turn into pathogenic th17 cells when deregulated these pathogenic th17 cells initiate chronic inflammatory condition il17 produced by pathogenic th17 cells are involved in an early inflammatory stage of the injuries it promotes tumour cell survival proliferation tumour neovascularisation and metastasis which allow additionally tumour cells and fibroblasts are stimulated by il17 to produce high amounts of angiogenic factors for angiogenesis il17 can activate stat3 pathway indirectly through il6 when il17 binds to il17 receptorbearing tumour cells it stimulates il6 production that is highly important for stat3 pathway activation as mentioned above this stat3 pathway activation contributes to several characteristics such as cancer proliferation antiapoptosis and angiogenesis that favour carcinogenesis in the colon this shows that there is a relationship between stat3 pathway and tregs in contributing to crc formation when etbf is accumulating in the intestinal tract as shown in figure to some extent stat5 and stat6 have been reported to be involved in inhibiting antitumour immunity when all stat3 and are activated together it highly enhances the tumourigenesis effect cleavage of ecadherin stimulate cell proliferationapart from inflammation bft alters the structure and function of colon epithelial cells by degrading ecadherin ecadherin is a 120kda glycoprotein that is the major structural protein in zonula adherens and is also known to be a tumour suppressor and zonula adherence protein this protein is responsible for the epithelial polarity in normal conditions the expression of ecadherin is linked to cellular functions including apoptosis and homotypic cellcell unfortunately when ecadherin interacts with bft in the intestinal epithelial cells it degrades ecadherin rapidly in an atpindependent manner this cleavage promotes colonic injury inflammation and loss of membraneassociation resulting in morphological enhances cellular metastatic potential it is proven that changes and adhesion review article formation of colon canceretbfbftintestinal lumenepithelial cells il2intestinal immune system th17tnfcell proliferation il17tregsinflammationcarcinogenesisil17 receptorbearing tumour cellsstat3 activationfigure the mechanism through abnormal systemof intestinal carcinogenesis immune factordependent the cleavage of ecadherin correlates with the changes of cell morphologies simultaneously degradation of ecadherin also promotes the binding of nuclear localisation of βcatenin and tcell transcriptional activator this binding promotes gene regulation and transcription additionally βcatenin plays an important role in wingless and int wnt signalling pathway promoting cell proliferation and epithelialmesenchymal transition and enhancing the expression of protooncogene in primary colorectal tumours cells in the centre of the tumour exhibit the presence of βcatenin and ecadherin however when the cells move away from the centre of the tumour they exhibit high amounts of nuclear βcatenin and the junction of ecadherin is lost important role ecadherin plays an in maintaining the morphology of cells there is a relationship with the ecadherin and the apical factin ring of the intestinal epithelial cells secretion when the loss of ecadherin increases the integrity of the apical factin is lost resulting in the increase in cell volume and chloride secretion and cell and epithelial barriers become wwwmjmsusmmy 0cmalays j med sci julaug more permeable this contributes to intestinal inflammation diarrhoea and colon carcinogenesisalteration of the signalling pathway of colorectal cancerbft is involved in many colonic epithelial cell signal transductions when bft disturbs or activates the signalling pathway it brings adverse effects to the body and can lead to colorectal tumourigenesis figure the colonic epithelial cell signal transduction transpires through the nuclear factor kappalightchainenhancer of activated bcells nfκb wnt and mitogenactivated protein kinase mapk signalling pathways bft can stimulate nfκb pathway in the intestinal epithelial cells with the expression of heme oxygenase1 ho1 and cytokines to induce mucosal inflammation this pathway has the ability to enhance the survival of neoplastic cells by preventing them from undergoing apoptosis leading to tumour formation furthermore in figure it shows that when nfκb of intestinal epithelial cells is activated for a long time it induces the activity of nitric oxide synthase that breaks down larginine to produce nitric oxide which can damage cellular dna wnt signalling pathway is important to maintain the structures of the intestinal epithelium however wnt signalling pathway contributes negatively and affects cells which are extremely important for colorectal carcinogenesis and progression as wnt signalling pathway is activated it weakens tight junctions and reduces cellular adhesion this allows the cancer cells to undergo migration and metastasis hence cancerous cells can migrate to another ans spermine oxidase is a catabolic enzyme that increases ros which can be upregulated by bft in normal conditions figure ros acts as an important mediator in multiple cell signalling pathways and immune response that is produced naturally within biological systems it consists of superoxide hydroxyl radical and hydrogen peroxide however as the amount of ros becomes excessive it imparts negative effects in the disruption of redox homeostasis figure this excessive ros induces oxidative stress it oxidises cellular components including dna lipids and proteins within the cells etbfbftbftepithelial cellactivation of signaling pathwaynfκbwntnitric oxide synthase reduce cell adhesion weaken tight junction cancer cell migratenitric oxidedamage cellular dnafigure the role of the signalling pathways when epithelial cells contact bftspermine oxidase smorelative oxygen species ros mediatorimmune responsemultiple cell signalling pathwaysfigure normal condition of the smo and ros that helps in immune response and cell signalling pathwayswwwmjmsusmmy 0conce the cellular components are oxidised it generates irreversible damage to host cells additionally ros plays an important role in the survival of cancer cells enhancing the effectiveness of carcinogenesis and aggravating cancer formed in the body spermine oxidase smo upregulated relative oxygen species ros mediatordnalipidproteinirreversible damagecarcinogenesisfigure the adverse effect of smo contacted bftfamilial adenomatous polyposisfactors contributes the combination of both genetic and environmental to crc formation it is estimated that of crc development is due to genetic predisposition wherein nearly of all crcs are attributed to familial adenomatous polyposis fap fap is an autosomal dominant inherited disorder that describes the development of numerous colorectal adenomatous polyps these polyps are able to develop in the teenagers colon meanwhile the number of polyps formed in the colon depends on the age of a person which means the number of polyps is directly proportional to the age of a person if these polyps are not removed from the colon they may transform from benign to malignant developing crc the source of fap disease is mainly due in the adenomatous to germline mutation polyposis coli apc gene this apc mutation occurs due to frameshifts insertions or deletions that may introduce a premature stop codon during the halfway through the transcription process these earlyintroduced premature stop codons in the gene sequence lead to incompletetruncated apc protein formation thus the normal function of apc protein is lost eventually facilitating carcinogenesis review article formation of colon canceradditionally germline mutations along with somatic mutations of the normal allele or loss of the normal allele lead to inactivation of apc once apc is inactivated it precisely commences carcinogenesis in normal conditions apc pathway acts as a gatekeeper controlling a part of wnt signalling pathway unfortunately when apc is mutated the function of apc pathway is lost or inactivated this inactivation of the apc pathway results in the activation of wnt signalling pathway this characteristic is mainly found in crc moreover apc mutation has the ability to alter bacteriahost epithelial interaction where it allows the bacteria to attach onto the mucosa if a person has the apcmutated gene and is exposed to etbf the chances of developing crc are high concurrently high amount of tumour load is displayed in the persons colon conclusionthe human gastrointestinal tract contains its own bacterial flora that benefit humans daily b fragilis is one of them and consists of two classes namely ntbf and etbf the differences between both the classes is the presence of bft etbf is able to produce bft that can disrupt the intestinal environment and promotes inflammation simultaneously bft degrade ecadherin and causes inflammation ibd is a chronic intestinal inflammation associated with etbf and can induce crc however patients with cd have lower risk of developing crc as compared to those with uc patients with ibd exhibit stat3 activation due to the stimulation of immune response that favours th17 cell generation as the levels of th17 cell increase it brings a huge disadvantage to the intestinal tract due to the production of il17 furthermore il17 stimulates the production of il6 that is required to activate stat3 this indicates that the stat3 pathway activates for a long time longterm stat3 activation blocks antitumour immune response which supports the growth of cancer cells thus stat3 th17 and il17 are highly important in carcinogenesis concurrently the production of proinflammatory cytokines at the site of inflammation triggers the production of cox2 enzymes that release pge2 cox2 is also known for its carcinogenic abilities due to the production of pge2 that controls cell proliferation additionally bft affects signal transductions such as wnt wwwmjmsusmmy 0cmalays j med sci julaug nfκb and mapk signalling pathways and induces tumourigenesis considering that bft induces inflammation activates stat3 and alters signalling pathways it can be concluded that bft produced by etbf plays an important role in colon carcinogenesis boleij a hechenbleikner em goodwin ac badani r stein em lazarev mg et al the bacteroides fragilis toxin gene is prevalent the colon mucosa of colorectal cancer in patients clin infect dis httpsdoi101093cidciu787acknowledgementsnoneconflict of interestnonefundsnoneauthors contributionsconception and design hkkdrafting of the article with supervision cwtcritical revision of the article for important intellectual content fdfinal approval of the article hkk fdcorrespondencedr fabian davamaniphd microbiology university of madrasfaculty of biomedical scienceschool of health sciences international medical university kuala lumpur malaysia tel fax email fabian_davamaniimuedumyreferences snezhkina av krasnov gs lipatova av sadritdinova af kardymon ol fedorova ms et al the dysregulation of polyamine metabolism in colorectal cancer is associated with overexpression of cmyc and cebpβ rather than enterotoxigenic bacteroides fragilis infection oxid med cell longev httpsdoi10115520162353560 sears cl geis al housseau f bacteroides fragilis from carcinogenesis j clin symbiont invest httpsdoi101172jci72334subverts mucosal to biology colon lv y ye t wang h zhao j chen w wang x et al suppression of colorectal tumorigenesis by recombinant bacteroides fragilis enterotoxin2 in vivo world j gastroenterol httpsdoi103748wjgv23i4603 pierce jv bernstein hd genomic diversity of enterotoxigenic strains of bacteroides fragilis plos one 2016116e0158171 httpsdoi 101371journalpone0158171 sun j kato i gut microbiota inflammation and colorectal cancer genes dis httpsdoi101016jgendis201603004 erdrich j zhang x giovannucci e willett w proportion of colon cancer attributable to lifestyle in a cohort of us women cancer causes control httpsdoi101007s105520150619z mughinigras l schaapveld m kramers j mooij s neefjesborst ea van pelt w et al increased colon cancer risk after severe salmonella infection plos one 2018131e0189721 httpsdoi101371journalpone0189721 francescone r hou v grivennikov si microbiome inflammation and cancer cancer j httpsdoi101097ppo0000000000000048 wick ec rabizadeh s albesiano e wu x wu s chan j et al stat3 activation in murine enterotoxigenic bacteroides inflamm bowel dis httpsdoi101097mib0000000000000019induced by fragili | 0 |
colorectal carcinoma crc as one of the most commongastrointestinal malignancies has developed the worldsfourth most deadly cancer with a high rate of incidence andmortality [ ] liver metastasis which is the most commonform for crc metastasis is the leading cause of high mortality for this severe malignancy however few clinical prevention and treatment measures could be available for tumormetastasis therefore it is really urgent to develop new biomarkers and explore the underlying mechanism for crcmetastasis and eventually develop new therapeutic strategiesfor crc patientsduring cancer progression metabolic reprograming withincreasing glucose utilization is termed as warburg aï¬ectwhich is accompanied by altered pyruvate and mitochondrialmetabolism the fate of pyruvate is the core manifestationto distinguish normal cells and tumor cells through metabolism in normal cells pyruvate was used for eï¬cient atpproduction directly into mitochondria however pyruvatewas converted into lactate in cytosol despite of normoxicand hypoxic conditions in cancer cells this may be dueto the impaired process of pyruvate from the cytosol intothe mitochondrial matrix which is a critical metabolic steplinking glycolysis and mitochondrial oxidative phosphorylation the mitochondrial pyruvate carriermpc a 0c of immunology researchmultimeric complex containing two distinct proteins mpc1and mpc2 which is located in the inner mitochondrialmembrane is responsible for eï¬cient mitochondrial pyruvate uptake loss of mpc expression or activity blockspyruvate entry into the tca cycle which results in a metabolism switch to increase glycolysis and the compensatoryusage of glutamine [ ]existed studies have reported that mpc1 was related withimmunoregulation stemness metabolism cellular morphology etc [ ] currently the important role of mpc1was uncovered in several tumors in crc and esophagealsquamous cell carcinoma decreased mpc1 results in accelerated aerobic glycolysis and malignant progression [ ] inlung adenocarcinoma mpc1 deï¬ciency accelerates lung adenocarcinoma progression through the stat3 pathway in prostate cancer mpc1 was reported to be involved instemness and metabolism which regulated by couptfii[ ] in renal cell carcinoma hypoxiainduced loss ofmpc1 enhanced the expression of mmp7 and mmp9 to promote cell invasion collectively these data suggestedthat mpc1 maybe serves as a suppressor to disrupt tumormalignancy however whether mpc1 is involved in crcmetastasis and the underlying mechanisms remain to beillustratedin the present study we ï¬gured out the relationshipbetween the mpc1 expression and crc liver metastasiswe identiï¬ed that decreased mpc1 was closely correlatedwith patients metastasis as well as led to poor prognosisfunctionally mpc1 overexpression could attenuate themigration and invasion capacities of crc cells both in vitroand in vivo mechanically mpc1 suppressed crc metastasisthrough mediating the wntcatenin signaling thus ourï¬nding ï¬rstly revealed a critical role of mpc1 in crc livermetastasis materials and methods data mining seven geo datasets gse21510 gse5206gse20916 gse9348 gse89393 gse67675 and gse4183and tcga were used to analyze the mpc1 expression pattern in crc the primary data for tcga datasets weredownloaded wwwcancergov the primary data feo datasets were downloaded at wwwncbinlmnihgovgeo the oncolnc database httpwwwoncolnc was used to detect the prognostic value of mpc1 inthe tcga cohort patients and clinical specimens in our study a total of patients containing paired crc tissues and adjacentnontumor tissues were enrolled from the department of gastrointestinal surgery renji hospital school of medicineshanghai jiao tong university among them cases ofliver metastases were collected and only cases wereavailable with complete followup data for survival analysisinformed consents were signed by all patients the researchwas approved by the research ethics committee of renjihospital and carried out in accordance with ethical standardsas formulated in the helsinki declarationtable sequences of primers used for realtime pcrprimermmp7 forwardmmp7 reverseecadherin forwardecadherin reversesnail1 forwardsnail1 reversemyc forwardmyc reversegapdh forwardgapdh reversesequence ²²gagtgagctacagtgggaacactatgacgcgggagtttaacatatgagtgtcccccggtatcttcacgagcagagaatcataaggcggtatccagagctgtttggaaacattttcctcccaggccatcacagccctcactcacacagattccacaaggtgcctgggctacactgagcaccaagtggtcgttgagggcaatg cell culture and cell transduction human crc celllines hct116 ht29 sw620 rko sw480 and lovoand mouse crc cell line mc38 were gained from the cellbank of the chinese academy of sciences shanghai chinaall cells were cultured in dmem medium supplemented°with fetal bovine serum and antibiotics at c in ahumidiï¬ed incubator with co2mc38 cells were transfected with lentivirus containing aluciferase reporter plasmid stable transfection cells werescreened with μgml blastisidin for days which termedmc38luc in addition one short hairpin rna shrnasequence against mpc1 was packaged as lentivirus andtransfected into mc38luc cells lovo and sw480 were transfected with lentivirus containing fulllength human mpc1cdna or empty vehicle control stable transfection cells werescreened under μgml puromycin for days and veriï¬edby western blot in those assays all lentiviral transfectionswere performed in the presence of μgml polybrene immunohistochemical ihc the protocol of this assayand quantify the mpc1 protein expression level were performed according to previously reported primary antibodies used as follows mmp7 yt2663 immunoway ecadherin cst snail1 ab53519 abcam and mycyp0861 immunoway cellular immunoï¬uorescence assays were performedaccording to the previous description brieï¬y cells wereincubated with antibodies against catenin ab32572abcam and incubated with alexa 488conjugated secondaryantibody the nuclei were stained with ²6diamidino2phenylindole dapi western blotting wholecell lysates or nuclear proteinwas extracted using a protein extraction buï¬er beyotimeshanghai china or nucleoprotein extraction kit sangonbiotech c500009 respectively proteins were resolved bysdspage and transferred onto nitrocellulose nc membranes using standard methods primary antibodies used asfollows mpc1 ab74871 abcam catenin ab32572abcamlamin ac ab8984 abcam and gapdhab9485 abcam speciesspeciï¬c secondary antibodies used 0c of immunology researchtable related enzyme and carrier of pyruvate analyzed in this studygenepdha1pdhbpdk4pdhxmpc2pdk2pdk1pdk3mpc1pcpdp1pdprpdha2pdp2pklrdescriptionpyruvate dehydrogenase lipoamide alpha pyruvate dehydrogenase lipoamide betapyruvate dehydrogenase kinase isozyme pyruvate dehydrogenase complex component xmitochondrial pyruvate carrier pyruvate dehydrogenase kinase isozyme pyruvate dehydrogenase kinase isozyme pyruvate dehydrogenase kinase isozyme mitochondrial pyruvate carrier pyruvate carboxylasepyruvate dehydrogenase phosphatase catalytic subunit pyruvate dehydrogenase phosphatase regulatory subunitpyruvate dehydrogenase lipoamide alpha pyruvate dehydrogenase phosphatase catalytic subunit pyruvate kinase liver and rbclocationmitochondrion matrixmitochondrion matrixmitochondrion matrixmitochondrion matrixmitochondrion inner membranemitochondrion matrixmitochondrion matrixmitochondrion matrixmitochondrion inner membranemitochondrion matrixmitochondrion matrixmitochondrion matrixmitochondrion matrixmitochondrion matrixmitochondrionas follows irdye goat antimouse igg licor andirdye goat antirabbit igg licorttest was used for comparison between groups p wasconsidered as statistically significantsynthesized using the primescript realtime pcr total rna was extracted using trizoltakara according to the manufacturersinstructionscdna wasreversetranscriptionpolymerasechain reaction rtpcr kittakara the qpcr was performed using sybr green masct method was used to analyze theter mix roche the data and gapdh was used as a loading control the primersequences are listed in table liver metastasis model this study was performed inaccordance with the recommendations in the guide for thecare and use of laboratory animals and relevant chineselaws and regulations the protocol was approved by theinstitutional animal care and use committee iacucof shanghai jiao tong university the mc38 cells transplanting luciferaseexpressing were injected into the spleensof c57bl6n mice n with a concentration of cellsmouse two weeks later the mice were killed andthe liver metastasis tissues were harvested luciferase reporter assay the protocols of this assaywere operated in accordance with previous reported ng top reporter plasmid wntcatenin signalingor ng fop reporter plasmid negative control ofwntcatenin signaling and ng renilla were mixedand transfected into crc cells using lipofectamine dualluciferase reporter assay system promega was usedto detect the ï¬reï¬y and renilla luciferase activities statistical analysesdata are shown as means ± sd spss chicago il usaand graphpad prism software were used to manipulate statistical analyses kaplanmeier method was used to calculatecumulative survival time the chisquare test or students results mpc1 expression was aberrantly decreased in crcpyruvate is a pivotal intermediate in the process of cellmetabolism which connects glycolysis and the tca cycleto determine the potential maladjustment genes involvedin pyruvate metabolism which is located in mitochondriaas shown in table we analyzed the tcga dataset containing crc and their normal counterparts the resultsshowed that multiple genes are significantly upregulated ordownregulated in crc t tissues compared to normal colonn notably mpc1 had a log2 fold change less than figures 1a and 1b as known to us pyruvate translocation from the cytoplasm to the mitochondria is the ï¬rst stepinto the tca cycle which needs mpc1mpc2 heterodimerin the analysis no significant change was found in mpc2therefore mpc1 was selected for further study the expression pattern of mpc1 was further analyzed in ï¬ve independent geo datasetsgse21510 gse5206 gse20916gse9348 and gse4183 consistently we found thatmpc1 was downregulated with statistical diï¬erence in crctissues figure 1c and inï¬ammatory tissue supplementary figure in comparison to their normal counterpartsmeanwhile we found decreased mpc1 expression inhuman crc tissues compared to their normal counterpartsfigure 1d in addition a similar phenomenon wasrevealed in aomdss induced mouse crc modelsfigure 1e then we evaluated the protein level of mpc1used a tissue microarray containing matching cancerand corresponding adjacent nontumortissues whichsubjected ihc staining the expression of mpc1 wasscored as based on the staining area andintensity figure 1f we found that more lower mpc1expression score as and was presented in crc 0c of immunology researcheulavp goltcga t nmpc1mpc2log2 fold changenoisserpxe cpm evitalertcgan normaltumornoisserpxe cpm evitalergeo datasetsgse21510 gse5206 gse20916 gse9348normaltumorabhuman samplecrcnormalaom dssnormalccrcesacesacesacesacdempc1 lower expressionmpc1 higher expressionf egatnecrepromutlamrongfigure expression pattern of mpc1 in crc a volcano plot showed fold changes xaxis and corresponding p values log10 yaxis ofpyruvate metabolismrelated genes located in mitochondria analyzed in the tcga dataset between paired normal and crc samplesstudents ttest b the comparison of mpc1 expression in tumor and matched normal tissues using the tcga dataset students ttest p c expression analysis of mpc1 in tumors and corresponding normal tissue using four independent geo datasetsgse21510 gse5206 gse20916 and gse9348 students ttest p d ihc staining of mpc1 expression in matched crctumor and nontumor tissues scale bar μm e ihc staining of mpc1 expression in the aomdss induced crc mouse models andcontrol animal scale bar μm f representative ihc staining of mpc1 expression in tissue microarray from the ren ji cohortwhich contained crc patients and paired adjacent normal tissue n the tumor tissues were divided into two groups based on theexpression level which scored as scale bar μm g the percentage of tissue displaying diï¬erent expression level ofmpc1 in crc tumor and adjacent nontumor tissues fishers exact test p tissues figure 1g overall these results revealed thatmpc1 was disrupted during crc decreased mpc1 enhanced tumor metastasis capabilityand predicated poor prognosis in crc it is well known thatcrc is one of the most malignant tumors given its strongmetastasis ability so we tried to ï¬gure out whether thempc1 expression was correlated with metastasis we foundthat lower mpc1 expression was closely correlated withmetastasis p lymph node invasion p and tnm stage p which revealed by the analysisbetween the clinical signiï¬cance and mpc1 expression incrc table next to illuminate the expression patternof mpc1 in diï¬erent process of crc data mining was carried out by two independent geo datasets gse21510 andgse89393 which contained normal tissue primary crcand metastatic lesion in the liver mcrc as shown infigures 2a and 2b mpc1 expression was gradually downregulated in patients with an increase in metastasis ability asimilar result was found in mice cells ct26 with high livermetastasis hmct26 or poor liver metastasis pmct26figure 2c which isolated by in vivo selection in an orthotopic mouse model of colon cancer metastasis to the liverfurther analysis showed that mpc1 protein expression was 0c of immunology researchtable clinicopathological correlation of mpc1 expression in theren ji crc cohortclinicopathological featureexpression ofmpc1lowhighp value Ï2testage years¥gendermalefemalemetastasisyesnolymph node invasionyesnotumor size cm¥ cmtnm stageiiiiiiivkras mutationmutationno mutationgradually downregulated in normal tissue primary crc andliver metastasis crc crlm tissues figure 2d furthermore survival analysis showed that patients with lowermpc1 expression had a worse outcome compared to thepatients with higher mpc1 expression using the tcgacohort figure 2e and ren ji cohort figure 2f additionally among patients with metastasis worse prognosiswas emerged in the mpc1 low cases figure 2g while nosignificant association was observed in patients withoutmetastasis figure 2h mpc1 overexpression impaired crc cells motility bothin vitro and in vivo to evaluate the role of mpc1 on themotility of crc cells the transwell assay was performedfirstly we examined low mpc1 protein expression in humanand high mpc1 protein expression in mouse mc38 crccells by western blot figure 3a mpc1overexpressingstable cell lines were established using a lentivirus carrying the mpc1 gene in lovo and sw480 cells and theoverexpression eï¬ciency was conï¬rmed by immunoblotsfigure 3b mpc1 knockdown in lucmc38 cells wereestablished and veriï¬ed by wb figure 3c mpc1 overexpression exhibited significantly weaker migration and invasion ability than the control cells in both lovo figure 3dand sw480 figure 3e cells following the liver metastasismodel of crc was established by spleen orthotopicallyinjecting mc38 cells transplanting luciferaseexpressingwhich would simulate mc38 metastasis to the liver throughsplenic veinportal vein the results revealed that the mpc1knockdown promoted mc38 cells metastasis to the liverthrough detecting the luminescence intensity monitoredby bioluminescence imaging figure 3f notablythenumber of metastatic liver nodules in the mpc1 silencinggroup wasin the control groupfigure 3g histological examination also proved thatmpc1 knockdown decreased the metastatic potential ofcrc in vivo figure 3gthan thatsmaller decreased mpc1 activated the wntcatenin pathwayby promoting nuclear translocation of catenin to furtherexplore the underlying mechanism of mpc1mediated inhibition of crc metastasis the tcga database was used toperform gsea analysis the results indicated that mpc1was involved in the wntcatenin signaling when set themrna expression median as a cutoï¬ figure 4a anddualluciferase reporter gene assay revealed that mpc1 overexpression obviously inhibited the activity of wntcateninpathway figure 4b which conï¬rmed the result abovewe then tested the distribution changes of catenin innuclear and cytoplasmic which was a crucial step inwntcatenin pathway as shown in figure 4c no significant diï¬erence was emerged in the total amount ofcatenin between mpc1 overexpression cells and the control cells figure 4c however obviously decreased distribution of nuclear catenin was presented in mpc1overexpression cells compared to that in the control cellsas revealed by the stronger gray corresponding to cateninfigures 4c and 4d consistently immunoï¬uorescenceif staining showed that mpc1 overexpression weakenednuclear catenin localization in both lovo and sw480 cellswhen compared to control cells figures 4e and 4f asimilar phenomenon was observed in mouse liver metastasistissues by ihc figure 4g following qpcr analyses displayed some downstream target genes of catenin such asmmp7 ecadherin snail1 and myc obviously increasedin ecadherin and decreased in mmp7 snail1 and mycwere observed after the mpc1 overexpression in both lovoand sw480 cells compared to that in the control cellsfigures 4h and 4i meanwhilethe opposite trendwas observed in mc38 cell after mpc1 knockdownfigure 4j and similar results were observed in mouseliver tissue detected by ihc figures 4k4n takentogether the data above indicated that mpc1 mediatedcrc cell metastasis through the wntcatenin pathway discussionaccumulating evidences have shown that reprogrammedenergy metabolism conduces to the tumor malignant propertiesincluding enhanced crc liver metastatic capacity[] mitochondria as the primary site of energy production regulate the pyruvate metabolism under both physiologic and pathologic conditions the ï¬rst step of the tcacycle is mediated by mpc which transports pyruvate into 0cnoisserpxe cpm evitalergse21510normal crc mcrcnoisserpxe cpm evitalernormalacrccrlmesacesac of immunology researchgse67675pmct26 hmct26ctcgan n p hr time daysgse89393normal crcmcrcbnoisserpxe cpm evitaler egatnecreplavivrus llarevolamroncrcmlrclavivrus llarevoren ji cohortn p hr n time monthsfdlavivrus llarevolavivrus llarevocrc with metastasis n n p hr n time monthsmpc1 higher expressionmpc1 lower expressiongecrc without metastasis n n n p hr time monthshfigure decreased mpc1 enhances tumor metastasis and predicts poor prognosis in crc a b data mining showed mpc1 expressionwas gradually decreased in normal tissue primary crc and liver lesion of metastatic crc mcrc from two independent geo datasetsoneway anova a gse21510 p b gse89393 p c data from gse67675 revealed that mpc1 expressionwas lower in high liver metastasis ct26 cells hmct26 than that in poor liver metastasis ct26 cells pmct26 students ttestp d gradually decreased mpc1 expression was presented in normal tissue primary crc and liver metastasis crccrlm tissue scale bar μm n fishers exact test p e kaplanmeier overall survival os curves in the tcgadataset of crc patients according to the mrna expression of mpc1 the lower quartile value of expression was utilized as a cutoï¬logrank test p f kaplanmeier os curve for the mpc1 expression in the ren ji cohort logrank test p gkaplanmeier os curve for the mpc1 expression in patients with metastasis logrank test p h kaplanmeier os curve forthe mpc1 expression in patients without metastasis logrank test p mitochondrial from cytoplasm in the beginning tcgawas used to analyze the relative genes involved in pyruvatemetabolism which is located in mitochondria the resultsrevealed that mpc1 expression was significantly downregulated in crc tissues meanwhile the geo datasets analysisas well as ihc staining on crc patients tissue and mousemodels conï¬rmed this trend these phenomena may indicate that loss of mpc activity enhanced tumorigenic glucoseutilization by blocking mitochondrial pyruvate uptake andoxidation interestingly in the course of data analysis wefound that the expression of mpc1 was decreased at thestage of intestinal inï¬ammation which was not diï¬erentfrom that in tumor tissue mpc1 has been reported to beinvolved in immune regulation of peripheral t cell homeostasis through metabolic regulation and a decrease inmpc1 was found at the earliest stages of crc hence 0c of immunology researchhumanmouselovosw480lovosw480mpc1gapdhmpc1mpc1gapdhmpc1mpc1gapdhwsthwsovolokrcmtchablovorotcevitnelcpmitnelrotcevitnelcpmitneldlefi rep sllec edavnidlefi rep sllec edavnirotcevitnelcpmitneldsw480rotcevitnelcpmitnelcncpmcncpmdlefi rep sllec detargimdlefi rep sllec detargimccncpmcncpmmc38shnc shmpc1¨¯ sp xufl latotcnhscpmhscnhscpmhsradiancepseccm2srfemc38suledon sisatsatemcnhscpmhsgfigure mpc1 overexpression inhibits the motility of crc cells in vitro and in vivo a mpc1 expression in human and mouse crc celllines examined by western blot gapdh serves as loading control b mpc1 overexpression in lovo and sw480 cells c mpc1 silencing by shrnampc1 in mouse mc38 cells d e transwell assays showed that upregulated mpc1 suppressed the invasion and migration ability of lovod and sw480 e cells quantiï¬cation of invaded and migrated cells was performed for ï¬ve randomly selected ï¬elds values are means ± sd frepresentative bioluminescence photograph of mice spleen implanted with luciferaseexpressing mc38 cells treated with shmpc1 or controlvector total ï¬ux was quantiï¬ed by the ivis system to verify the ability of liver metastasis g representative image of liver metastases andquantiï¬ed by the nodules in mice inoculated with mc38 cells treated with shmpc1 or control vector as well as representative images ofhe staining of the liver metastatic lesions scale bar μm students ttest p p p 0c of immunology researchse erocs tnemhcirnewnt signaling pathwaynes p mpc1 highmpc1 lowoitar pof pot evitalerð½cateninlamin acð½cateningapdhmpc1alovosw480csuelcunlatot icanmalnnetacð½ilovoncmpc1bsw480lovosw480ncmpc1dð½catenindapimergeð½catenindapimergeð½catenincpmcnovolnoisserpxe evitaleregnahc doflelovolianscympmmncmpc1nirehdacehcpmcnwsfsw480lianscympmmncmpc1nirehdaceifigure continuedcmcnhscpmhsmc38glianscympmmnirehdaceshncshmpc1jnoisserpxe evitaleregnahc doflnoisserpxe evitaleregnahc dofl 0c of immunology researchmmp7ecadherinsnail1myccmcnhscpmhscmcnhscpmhscmcnhscpmhscmcnhscpmhsklmnfigure decreased mpc1 activates the wntcatenin pathway by promoting nuclear translocation of catenin a gsea analysis ofmpc1 expression in crc using the tcga dataset nes normalized enrichment score b luciferase reporter gene assay of crc cellstreated with mpc1 overexpression or not c the expression of total catenin and nuclear catenin was detected in control and mpc1overexpression crc cells respectively gapdh and lamin ac were used as the loading control of total and nuclear protein respectivelyd the gray value analysis of nuclear catenin in mpc1overexpression cells and control cells e f mpc1 overexpression could inhibitthe nuclear translocation of catenin in crc cells scale bar μm g ihc staining of catenin in mouse liver metastatic lesionsinoculated with mc38 cells treatment with shmpc1 or control vector scale bar μm h i relative mrna expression level of catenin target genes in crc cells with mpc1 overexpression or control vector j relative mrna expression level of catenin targetgenes in mc38 cells with shmpc1 or control vector kn relative protein expression level of catenin target genes in mouse livertissue detected by ihc scale bar μm students ttest p p we suspect that mpc1 is involved in bowel inï¬ammation totumorigenesis and more studies need to be devised to illustrate this processfollowing in the analysis between the clinical signiï¬cance and mpc1 expression in crc we found that mpc1expression was especially correlated with metastasis inspiredby this we detected the mpc1 expression pattern in normaltissue primary crc and metastasis crc by geo datasetsand patients tissue all results revealed that gradually downregulated mpc1 was in patients with an increase in metastasis ability survival analysis indicated that worse outcome waspresented in patients with lower mpc1 expression especiallyin patients with metastasis additionally function assays veriï¬ed that mpc1 overexpression could attenuate the migration and invasion capacities of crc cells in vitro andmpc1 knockdown could enhance the metastasis capacityin vivo and existing studies have revealed that the mpc1was participated in metastatic dissemination of pgc1αtransduced cholangiocarcinoma through elevating reactiveoxygen species ros production besides mpc inhibitor uk5099 treatment could trigger strong invasive capacitythrough blocking pyruvate translocation into the mitochondria so as to attenuate mitochondrial oxidative phosphorylation and trigger aerobic glycolysis these phenomenatogether with our results indicate that mpc1 could act as atumor suppressor through inhibiting tumor metastasis andexisting studies have shown that mpc1 could alter the maintenance and fate of stem cells through regulating cancermetabolism in crc however the relationship betweenthe metabolism and tumor metastasis regulated by mpc1was not being mentioned thus further evidence should bereceived to conï¬rm thissubsequently the underlying mechanism of mpc1 inregulating metastasis was explored for this purpose gseaanalysis was performed the results indicated that mpc1was involved in the wntcatenin signaling the next seriesof experiments also conï¬rmed that mpc1 could mediate thewntcatenin pathway by redistribution of catenin previous reports showed that cytoplasmic catenin phosphorylated in nterminally localized to sites of cellcell contact isassociated with ecadherin and was required for intact cellcell adhesions without any change detected in the levels oftotal catenin [] simultaneously cellcell adhesionbased on cadherin binding with catenin limited wnt signals in addition catenin was reported to interact withusp9x to inhibit the degradation of catenin through thedeubiquitination of catenin in breast cancer a constitutive irs1 and catenin protein interaction activated mycexpression in acute lymphoblastic leukemia cells inhcc catenin was reported to interact with yap1 to leadto rapid tumorigenesis hence it is reasonable to guessthat accumulated cytoplasmic catenin maybe crosstalkwith other genes or involved in other biological processeswhat is more some downstream target genes of cateninsuch as mmp7 ecadherin snail1 and myc were changedin expression as known to us mmp7 is a member of theproteolytic enzyme family which promotes the invasionand metastasis of tumor cells by degrading the basementmembrane and extracellular matrix and previous studies had evidenced for involvement of mmp7 activation incolorectal cancer liver metastases [ ] ecadherin andsnail1 were considered as the epithelialmesenchymal transition emt marker which was involved in metastasis ofmalignant tumor moreover ecadherin was reportedto be involved in cellcell junction to regulate cancer invasionand metastasis [ ] and the gsea analysis also revealedthat mpc1 could aï¬ect the cellcell contacts data notshown as described previously mmp7 could facilitatemorphological transition by cleaving ecadherin thecommunication between the cells is disrupted when ecadherin was shredded leading to destructed cell adhesionand induction of emt followed by increased cell migration inspired by this we assumed that mpc1 could mediatetumor cell motility through aï¬ecting mmp7 activity cellcell 0c of immunology researchcontacts and emt however further studies need to be performed to clarify the detailed underlying mechanisms conclusionsin conclusion we ï¬rstly demonstrated that decreased mpc1was closely correlated with patients metastasis as well as ledto poor outcome moreover mpc1driven nuclear translocation of catenin contributed to crc cell motility thismeans that mpc1 has the potential to be a diagnostic biomarker and therapeutic target for metastasis patientsabbreviationscrc colorectal carcinomampc mitochondrial pyruvate carrieremt epithelialmesenchymal transitiongsea gene set enrichment analysisdata availabilitythe data used to support the ï¬ndings of this study are available from the corresponding author upon requestconflicts of interestall authors declare no conï¬icts of interestauthors contributionsyahui wang and guangang tian conceived and designedthe study chunjie xu and kaixia zhou obtained and anized the data guangang tian analyzed the data zhigangzhang and jianren gu contributed reagentsmaterialsanalysis tools xueli zhang and guangang tian wrote themanuscript guangang tian chunjie xu and kaixiazhou contributed equally to this workacknowledgmentsthis work was supported by the national natural sciencefoundation of china no to zhigang zhangand the natural science foundation of shanghai no18zr1436900 to xuelili zhangsupplementary materialssupplementary figure expression analysis of mpc1 innormal ibd and tumor tissues using the gse4183 datasetoneway anova was used to analyze the statistical diï¬erences between ibd adenoma and crc tissues ns no significance students ttest p p p supplementary materialsreferences h brody colorectal cancer nature vol no r l siegel k d miller s a fedewa et al colorectal cancerstatistics ca a cancer for clinicians vol no pp a j rauckhorst and e b taylor mitochondrial pyruvatecarrier function and cancer metabolism current opinion ingenetics development vol pp m g vander heiden l c cantley and c b thompsonunderstanding the warburg eï¬ect the metabolic requirements of cell proliferation science vol no pp s herzig e raemy s montessuit et al identiï¬cation andfunctional expression of the mitochondrial pyruvate carrierscience vol no pp c yang b ko c t hensley et al glutamine oxidationmaintains the tca cycle and cell survival during impairedmitochondrial pyruvate transport molecular cell vol no pp t bender g pena and j c martinou regulation of mitochondrial pyruvate uptake by alternative pyruvate carrier complexes the embo vol no pp a g ramstead j a wallace sh lee et al mitochondrialpyruvate carrier promotes peripheral t cell homeostasisthrough metabolic regulation of thymic development cellreports vol no pp 2899e6 x zhou z j xiong s m xiao et al overexpression ofmpc1 inhibits the proliferation migration invasion and stemcelllike properties of gastric cancer cells oncotargets andtherapy vol pp d li c wang p ma et al pgc1α promotes cholangiocarcinoma metastasis by upregulating pdha1 and mpc1 expression to reverse the warburg eï¬ect cell death diseasevol no j c schell k a olson l jiang et al a role for the mitochondrial pyruvate carrier as a repressor of the warburg eï¬ectand colon cancer cell growth molecular cell vol no pp y li x li q kan et al mitochondrial pyruvate carrierfunction is negatively linked to warburg phenotype in vitroand malignant features in esophageal squamous cell carcinomas oncotarget vol no pp h zou q chen a zhang et al mpc1 deï¬ciency accelerateslung adenocarcinoma progression through the stat3 pathway cell death disease vol no l wang m xu j qin et al mpc1 a key gene in cancermetabolism is regulated by couptfii in human prostatecancer oncotarget vol no pp y zhong x li d yu et al application of mitochondrialpyruvate carrier blocker uk5099 creates metabolic reprogramand greater stemlike properties in lncap prostate cancer cellsin vitro oncotarget vol no pp x p tang q chen y li et al mitochondrial pyruvatecarrier functions as a tumor suppressor and predicts theprognosis of human renal cell carcinoma laboratory investigation vol no pp g a tian c c zhu x x zhang et al ccbe1 promotesgist developmentthrough enhancing angiogenesis andmediating resistance to imatinib scientiï¬c reports vol no article c xu g tian c jiang et al nptx2 promotes colorectalcancer growth and live | 0 |
ovarian cancer is a silent and largely asymptomatic cancer leading to late diagnosis and worseprognosis the latestage detection and low survival rates makes the study of the spacetime evolution of ovariancancer particularly relevant in addition research of this cancer in small areas like provinces or counties is still scarcemethods the study presented here covers all ovarian cancer deaths for women over years of age in the provincesof spain during the period spatiotemporal models have been fitted to smooth ovarian cancer mortalityrates in age groups [ [ [ and [ borrowing information from spatial and temporal neighboursmodel fitting and inference has been carried out using the integrated nested laplace approximation inla techniqueresults large differences in ovarian cancer mortality among the age groups have been found with higher mortalityrates in the older age groups striking differences are observed between northern and southern spain the globaltemporal trends by age group reveal that the evolution of ovarian cancer over the whole of spain has remainednearly constant since the early 2000s differences in ovarian cancer mortality exist among the spanish provinces years and age groups as theexact causes of ovarian cancer remain unknown spatiotemporal analyses by age groups are essential to discoverinequalities in ovarian cancer mortality women over years of age should be the focus of followup studies as themortality rates remain constant since highmortality provinces should also be monitored to look for specific riskfactorskeywords agespacetime models disease mapping inla ovarian cancer mortality smoothing the number and scientific impact of publications on ovarian cancer are continuously increasing not in vainovarian cancer is the eighth most common cancer inwomen and the 18th most frequent overall with nearly new cases worldwide in around of cases are concentrated in developed countries whereovarian cancer is the most lethal gynecological tumorcorrespondence lolaunavarraes1department of statistics computer science and mathematics publicuniversity of navarre campus de arrosadia pamplona spain2inamat public university of navarre campus de arrosadia pamplonaspainthe highest incidences are found in northern and easterneurope austria the czech republic germany irelandlatvia lithuania the nordic countries slovakia and theuk and the united states whereas in africa andasia this tumor is virtually nonexistent this pattern isattributed mostly to the low birth rates found in developedcountries in the past years the overall number of tumors hasundergone a constant increase in spain this is due notonly to the population growth but also to the increasedlife expectancy and the use of early detection techniquesin the estimated number of new cases of ovarian the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you give appropriatecredit to the original authors and the source provide a link to the creative commons licence and indicate if changes weremade the images or other third party material in this are included in the s creative commons licence unlessindicated otherwise in a credit line to the material if material is not included in the s creative commons licence and yourintended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtain permission directlyfrom the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40 the creativecommons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to the data madeavailable in this unless otherwise stated in a credit line to the data 0ctrandafir bmc public health page of cancer was representing of all female cancersbeing the fifth cause of cancer deaths in women afterlung breast colon and uterine tumors [ ] the agestandardized incidence rate calculated using the directmethod and the world standard population is per women which may be considered high as forits temporal evolution there was a slight decline between and but since then mortality rate starts toincrease slowly but constantly in addition there is a greatdeal of variability among the spanish provinces for example during the period one finds a rate of per women in the canary islands and a rate of per women in cuenca ovarian cancer is a disease affecting mostly older postmenopausal women with more than of cases beingdiagnosed in women over according to medicalexperts it is a silent and mostly asymptomatic cancer acircumstance that leads to late diagnosis and worse prognosis furthermore in the cases where symptoms doappear these may be confused with digestive problemsbloating early satiety abdominal andor pelvic pain orbenign gynecological alterations such as endometriosis orpolycystic ovary syndrome to date no method for earlydetection is available this is reflected in the fact that upto of cases are detected in the advanced stages of thedisease the etiology of ovarian cancer is poorly understoodhowever several factors associated with an increased riskof ovarian cancer have been identified age number ofovulations early menarche infertility low parity theuse of hormone replacement therapy hrt obesity physical inactivity a family history of breast andovarian cancer including brca1 and brca2 gene mutations and past and current smoking associations between exposure to asbestos in the workplace orat home and ovarian cancer have also been found further research is needed to corroborate this finding inspain as most jobs with a high exposure to asbestos arepredominantly maledominated eg mining milling orshipyard work nevertheless a study of asbestos exposure among italian women found that the main factorwas secondhand contact due to occupationally exposedrelatives for example from soiled work clothes broughthome as of this writing however there is no documented registry for asbestos exposure in the workplaceor at home in spain some epidemiological studies havedetected an increased risk of ovarian cancer in womenwith less exposure to sunlight and consequently with lowlevels of vitamin d in particular the higher the latitudethe less overall accumulated sunlight and the higher theincidence of ovarian cancer some protective factors against ovarian cancer have also been identified suchas multiparity oral contraceptives and tubal ligation orhysterectomy the 5year agestandardised relative survival in spain is estimated at less than similar tothe european average of as mentioned aboveovarian tumors are the eighth cause of cancer deathsamong women worldwide with deceases registered in overall among malign tumors in about women died yearly in spain from ovarian cancer representing of all cancer deaths and of all deaths among women the mean age of decease from ovarian cancer inspain is years cabanes analyzedthe ageadjusted mortality trends of ovarian cancer inspain for the period ovarian cancer caused deaths in this period with rates in women over showing a tenfold increase versus those in youngerwomen in women under rates increased peryear until and afterwards started to drop at a rateof per year in the age group mortalityrates significantly increased annually up to andbecame stable thereafter in women and older mortality rates increased annually up to the year anddecreased per year afterthe disproportionate impact on older women togetherwith the aforementioned concerns regarding latestagedetection and low survival rates makes the study of thespacetime evolution of ovarian cancer particularly relevant in addition it is important to mention that agegroups are not equally affected by ovarian cancer mortality and then it is necessary not just to standardize by agebut to analyze the different age groups hence the maingoal in this paper is to study the temporal evolution of thegeographical patterns of ovarian cancer mortality rates infour age groups of women aged years or moremethodsdata sourcethe study presented here covers all ovarian cancer deathscode c56 of the 10th edition of the international classification of diseases for women over years of agein the provinces of spain excluding the autonomouscities of ceuta and melilla recorded throughout theperiod by the spanish statistical officestatistical analysisa bayesian hierarchical spatiotemporal model is used toestimate rates the model is briefly described in whatfollows for better interpretation of resultsspain is divided into s provinces indexed by i s and data are available for t27 time periods corresponding to years labeled as t tfor each age group let nit represent the population at riskfor region i and time period t then conditional on themortality rates rit the number of ovarian cancer deathsoit is assumed to follow a poisson distribution with mean 0ctrandafir bmc public health page of table descriptive statistics of observed cases and crude mortality rates per women disaggregated by age groups provinceand year min minimum q1 first quartile q3 third quartile max maximumobserved casescrude ratesage group[ [ [ [ [ [ [ [ minq1medianmeanq3maxμit nitrit that isoitrit ¼ poissonμit nitritlog μit log nit log ritwhere the lograte is modelled aslog rit α ξi γt δithere α denotes the logarithm of the overall rate ξi andγt are the main spatial and temporal effects respectivelyand δit corresponds to the spacetime interaction effectssince each of these components are supposed to be gaussian markov random fields gmrf the integratednested laplace approximation inla technique hasbeen used for model fitting and inference specifically theleroux car prior distribution has been considered for the spatial random effects and a firstorderrandom walk prior distribution for the temporal randomeffects in addition the four different types of interactionintroduced by knorrheld have been considered forthe spatiotemporal random effects these interactionsallow the spacetime effects to be completely independenttype i interaction structured in time but not in spacetype ii interaction structured in space but not in timetype iii interaction or completely structured in spaceand time type iv interactionall the computations have been done using the interactive web application sstcdapp which can befound at httpsemisstcdappunavarraeslogin thisapplication provides a user interface for the analysis ofspatiotemporal areal count data allowing to fit a widevariety of spacetime models using the inla estimationtechnique in addition the application provides differentmodel selection criteria in this paper the model with the[[[[nemow rept shaed etar edurcyearfig temporal trends by age groups temporal trend of the ovarian cancer mortality crude rates by agegroups 0ctrandafir bmc public health page of lowest value of the deviance information criterion dic has been selected for further details about modelspecification prior distribution of the hyperparametersidentifiability constraints and additional model selectioncriteria see for example adin and the referencesthereinresultsa total of ovarian cancer deaths were registered inthe population of spanish women over years of age during the period since ovarian cancer is mainlyrelated to the onset of menopause the age groups we areconsidering in this paper are [ [ [ and [ a brief summary of observed cases and mortality rates per women by age groups provinceand year is shown in table clear differences areobserved in the mean and median mortality rates amongthe youngest and oldest age groups with values ranging from cases per women up to casesper women approximately respectively figure displays the global temporal trend of crude rates by agegroup here the different behaviour of the age groups iseven more evident a pronounced slope from the lastdecade of the twentieth century to the beginning of thetwentyfirst century is observed in the older age groupsmodel was fitted to smooth spatiotemporal rates ineach age group the interaction considered in the modelwas chosen on the basis of the dic values for each subgroup of ageclass the dic pointed toward a type ivinteraction for age groups [ [ and [ whereas a type ii interaction was selected for the agegroup [ to make the different terms in all themodels comparable a decomposition of the estimated logrates was computed by defining posterior spatial ξi t and spatiotemporal δtemporal γ it patterns see adini γ so that log rit α ξit note thatexpα represents the overall mortality rate for the wholeof spain during the period in order to facilitate interpretation of the results a map of spain showingits provinces is given in fig t δla coruñalugopontevedraorenseasturiasleoncantabriavizcayaguipuzcoaalavanavarrapalenciaburgosriojahuescaleridageronazamoravalladolidsoriazaragozabarcelonasegoviatarragonasalamancaguadalajaraavilamadridcacerestoledocuencabadajozciudad realalbaceteteruelcastellonvalenciaalicantebalearescordobajaenmurciahuelvasevillagranadaalmeriamalagacadizsanta cruz de tenerifepalmaslasfig administrative division of spain map with the administrative division of spain showing provinces source map was generated by the authorsusing the library tmap from the r statistical software version no licenses are required to use or publish 0ctrandafir bmc public health page of figure shows the map with the posterior meanieestimates of provincespecific mortality ratesexpα ξexceedance probabilitiesofrate being greaterthanthe overall spanish rate have also been computedsee fig i posteriorthis provincespecificthe estimated spatial pattern draws attention toasturias as a high ovarian cancer mortality rate provincefor all age groups in the age group [ the highest spatial rates are found in the northwestern provincesasturias lugo and la coruña but also in vizcaya andhuesca over cases per women in age group[ the regions with the highest estimated rates areasturias the balearic islands and valencia with an estimated rate of over cases per women in thethird age group [ the highest rates are located inthe centralnorthern areas with salamanca and asturiasleading the ranking and in the canary islands tenerifeprovince all of them with more than cases per women the oldest age group [ exhibits high rateareas in asturias barcelona gerona and guadalajarawith a rate of over cases per women all ofthese highrate provinces have a rate significantly higherthan the overall spanish rate in their respective age groupssee fig in general northern spain has greater ovarian cancermortality rates compared to the southern regions thelowest rates are found in guipúzcoa for age groups [and [ and in almerÃa for age groups [ and[ the northwestern province of la coruña showsage group [age group [ to to to to to to to to age group [age group [ to to to to to to to to fig provincespecific mortality rates estimates by agegroups posterior mean estimates of provincespecific mortality rates expα ξsource maps were generated by the authors using the library tmap from the r statistical software version no licenses are required touse or publishi 0ctrandafir bmc public health page of age group [age group [[][[[[[][[[[[][[[[age group [age group [[][[[[fig provincespecific posterior exceedance probabilities by agegroups posterior exceedance probabilities of each province in comparison withthe spanish overall rate pexpα ξstatistical software version no licenses are required to use or publish expαo source maps were generated by the authors using the library tmap from the ria surprising behaviour with high rates in the age group[ but one of the lowest rates in the age group [the estimated global temporal pattern expα γ t isvisualized in fig rates seem to have decreased duringthe last few years from to in age group [ but have remained nearly constant in the other threeage groups which experienced a sharp increase in ratesfrom up to the beginning of the twentyfirst centuryapproximatelyfigures and display maps showing the spatiotemporal evolution of ovarian cancer mortality rates foreach spanish province for the period dividedinto intervals of years for age groups [ [ [ and [ respectively specifically these mapsrepresent the posterior mean of rit expα ξi γ t δit the corresponding maps of probabilities showing theprobability that a particular rate in a given province andyear is greater than the spanish rate during that periodare not shown here to conserve space however we haveclassified a province as having a rate significantly greaterthan the spanish rate if this probability is greater than for age group [ we find that the mortality ratefor the period was highest in the northeasternregion with the province of huesca having a significantrate that lasted until in the latter years of the periodstudied only asturias showed a significant rate withinthis age group the percentage of the rates variabilityexplained by the spatiotemporal term was implying that the specific temporal evolution of each provinceis rather high in this age group 0ctrandafir bmc public health page of age group [age group [age group [age group [fig temporal trends estimates by agegroups for the whole of spain posterior mean estimates of yearspecific mortality rates expα γ and credible intervals dotted lines provide the estimated rate in each age group in the whole period in spain expαt to to to to to to fig mortality rates estimates for age group [ posterior mean estimates of mortality rates rit for age group [ source maps weregenerated by the authors using the library tmap from the r statistical software version no licenses are required to use or publish 0ctrandafir bmc public health page of to to to to to to fig mortality rates estimates for age group [ posterior mean estimates of mortality rates rit for age group [ source maps weregenerated by the authors using the library tmap from the r statistical software version no licenses are required to use or publish to to to to to to fig mortality rates estimates for age group [ posterior mean estimates of mortality rates rit for age group [ source maps weregenerated by the authors using the library tmap from the r statistical software version no licenses are required to use or publish 0ctrandafir bmc public health page of to to to to to to fig mortality rates estimates for age group [ posterior mean estimates of mortality rates rit for age group [ source maps weregenerated by the authors using the library tmap from the r statistical software version no licenses are required to use or publishin age group [ rates exhibit a greater variability among provinces between cases per women and about cases per women thehighest rates occurred in the late 1990s and early 2000smainly in the northern half of spain and in the balearicislands asturias lugo salamanca valladolid huescateruel gerona and the balearic islands show significantlyhigh rates at the end of the periodfor women between and years of age the lowest rates are found at the beginning of the study periodwith significantly low rates the highest significant ratesare located mainly in the northern and western parts ofspain and in the canary and balearic islands in the period the provinces with the lowest rates at the endof the period are gerona and madridwomen over years of age show significantly low ratesbetween again asturias shows the highestmortality rate at the end of the period some provinceslocated in southern spain mainly along the coastlineshow significantly low ratesthe temporal evolution of the rates for some selectedprovinces are plotted in fig the colors used in thebands are associated to the posterior exceedance probabilities of each province at year t in comparison withthe temporal pattern for the whole of spain in that yearnamely prit α γ t o in the case of barcelonathe probability that the mortality rate lies above the spanish rate reaches a maximum between for agegroups [ and [ and between forage groups [ and [ madrid behaves similar tospain in all age groups in asturias the probability is quitehigh for all age groups with a significant rising trend forage group [ from onwards la coruña showssignificantly low rates during the whole study period forthe age group [ whereas this provinces behaviourfor women over years of age is similar to the spanishratediscussionthe ovaries are one of the cancer sites where knownrisk factors are not enough to explain all the cases andthus spatiotemporal analyses provide additional important information allowing for the examination of thespatial temporal and spatiotemporal mortality patternsas the age groups are not equally affected by ovarian cancer mortality it is necessary not just to standardize by age but also to analyze the different agegroupsresults show large differences in mortality among theage groups with higher mortality rates in the older agegroups indeed the last age groups women of more than years of age double the rate of women between and more than cases in the last age groups vs casesper women on average in age group [ thiscan be explained by poor survival rates of gynaecologicalcancers in the elderly which in turn is influenced by late 0ctrandafir bmc public health page of barcelonamadridasturiasla coruñabarcelonamadridasturiasla coruñabarcelonamadridasturiasla coruñabarcelonamadridasturiasla coruñafig temporal evolution of ovarian cancer mortality rates estimates for some selected provinces barcelona madrid asturias and la coruñatemporal evolutions of the posterior mean estimates of mortality rates rit for some selected provinces and twosided credible intervals for agegroups [ first row [ second row [ third row and [ fourth row the colors used in the bands are associated to theposterior exceedance probabilities of each province in time t in comparison with the temporal pattern of spain that is prit expα γ orepresented with a red line in the graphstdiagnoses and the failure of treatments due to comorbidity[ ]the global temporal trends by age group reveal thatthe evolution of ovarian cancer over the whole of spainhas remained nearly constant since the early 2000s particularly for women aged years or more after a sharpincrease during the period approximatelythe stabilization of the rates may be due to an increasedconcern among women regarding their personal health inparticular among older women that led them to be testedmore frequently access to better information via massmedia and the internet and the increasing effectiveness of 0ctrandafir bmc public health page of cancer treatments in the nineties access to informationvia the internet was more limited and health care was possibly less advanced which could explain in part the sharpincrease in mortality rates in the first half of the periodmortality in women between and years of age showsa slight decrease since until nearly althoughthis decrease does not seem to be significant with respectto the average mortality in this age group it seems thatin fact during the last two years of the study period ratesare starting to rise slightly but once again this trend is notsignificant as yetthe global geographical patterns show in general thatthe north has higher rates than the south a situation similar to that observed overall in europe the variabilityobserved in all age groups between northern and southernspain remains unknown although in general women inthe south are prone to marry earlier and have more children on average interestingly asturias the provincewith the highest rates in all age groups is one of thespanish provinces with the lowest average number of children the differences observed between the north and thesouth could also be explained taking into account the relationship between exposure to sunlight provitamin dand ovarian cancer although spain is in general a sunnycountry there is a great variability between the northand south in terms of average daily hours of sunlight forexample bilbao in the north receives about hoursof sunlight per year while sevilla in the south receives thus the observed increasing trend with latitude might be at least partly explained by a cumulativeexposure to sunlightheterogeneity among the provinces regarding ovariancancer mortality rates can be elucidated at least in partby a heterogeneous distribution of other risk factorsthere are differences among provinces in the age at whichwomen have their first period menarche the averageage of first childbirth and the total number of children[] the average fecundity rate in spain has beenmarkedly declining in particular the areas registering thelowest fertility rates were the basque country asturiasnavarre and aragón [ ] we should also point outthat the age of first birthing is closely related to socioeconomic development and is steadily increasing navarreand the basque country are the regions of spain wherewomen delayed childbearing the longest additionally hormonal replacement therapy has proven to have animportant influence on the appearance of ovarian cancerin postmenopausal women [] however its use inspain has been very limited another risk factor is the presence of a family history ofthe disease hereditary ovarian cancer syndrome presenting a mutation in brca genes is important between of ovarian cancer cases are linked to bcra mutation women who are carriers of the bcra1 mutationhave a chance of suffering ovarian cancer before age in spain the accumulated risk of developing ovarian cancer before age has been estimated at ci in carriers of a mutation in bcra1 and ci in carriers of a mutation in brca2 the prevalence of brca1 and brca2 mutation in spainis heterogeneous and varies according to geographical origin moreover blay showed that the brca1 andbrca2 spectrum of mutations in asturias was largely different from other areas of spain this could also explainin part the high mortality rates found in this provincediez studying a large group of spanish patientsshowed that there is only a slight difference betweenthe percentages of deleterious mutations in brca1 andbrca2 genes and respectively however somevariation due to geographic origin is present with a higherproportion of brca1 in families from the northwesternpart of spain according to vega the differencesfound in galicia could be due to founder effectsovarian cancer is also linked to lifestyle habits tobaccoand alcohol consumption the smoking habit is a riskfactor for epithelial ovarian cancer with an odds ratio of ci [ ] differences in alcoholand tobacco consumption can be found among spanishprovinces all in all to better understand the etiology of the disease and to better determine the effect of risk factors onthe spanish female population it would have been helpful to have the medical and workplace histories of all thewomen participating in this study this is the main limitation of the current work on the other hand as thereis a lack of scientific studies analyzing the associationbetween ovarian cancer mortality rates and risk factorsin the domains analyzed here age groups provinces andyears spatiotemporal analyses by age groups are essential to discover inequalities in ovarian cancer mortality todetect provinces with high risks in each age group and tokeep track of how the rates are evolving with timesdifferences in ovarian cancer mortality exist amongthe spanish provinces years and age groups as theexact causes of ovarian cancer remain unknown spatiotemporal analyses by age groups are very useful to look forpotential risk factors associated to the observed geographical patterns and to allocate funds among spanish regionsfor future clinical and epidemiological practice we recommend to followup women over years as the mortality rates remain constant since highmortalityprovinces should also be monitored to look more closelyfor specific risk factors some risk factors for ovarian cancer like getting old or having a family history cannot bechanged however women may slightly decrease their riskby avoiding other risk factors for example maintaining 0ctrandafir bmc public health page of a healthy weight avoiding tobacco and alcohol consumption or not receiving hormone replacement therapy aftermenopauseabbreviationsaei spanish research agency brca breast cancer dic deviance informationcriterion ue european union inla integrated nested laplaceapproximations gmrf gaussian random markov field sstcdapp spatialand spatiotemporal count data application uk united kingdomacknowledgementswe acknowledge the spanish statistical institute for providing the dataauthors contributionsstudy conception and design mdu pct acquisition of the data mdu aaanalysis of the data mdu pct aa interpretation of the data mdu aa pctwriting the pct mdu critical revision of the all authorsapproved the final manuscript and the decision to submit the manuscriptfundingthis research has been supported by the spanish ministry of science andinnovation project mtm 201782553r aeifeder ue the content of thispaper is solely the responsibility of the authors and does not represent theofficial views of the spanish ministry of science and innovationavailability of data and materialdata have been provided by the spanish statistical institute at municipalitylevel under a contract and aggregated later up on reasonable request thecorresponding author will make the datasets available mortality data fromcancer and other causes from by sex and province is available in theinteractive epidemiological information system ariadna httpariadnacneisciiiesevindexhtml of the spanish national center for epidemiologyethics approval and consent to participatenot applicableconsent for publicationnot applicablecompeting intereststhe authors declare that they have no competing interestsreceived january accepted july referencesbrüggmann d pulch k klingelhöfer d pearce c groneberg d ovariancancer density equalizing mapping of the global research architectureint j health geogr bray f ferlay j soerjomataram i siegel r torre l jemal a global cancerstatistics globocan estimates of incidence and mortality worldwidefor cancers in countries ca cancer j clin national institute for health and care excellence nice ovarian cancerthe recognition and initial management of ovarian cancer httpswwwniceukguidancecg122resourcesovariancancerrecognitionandinitialmanagementpdf35109446543557 accessed jan dos santos silva i beral v socioeconomic differences in reproductivebehaviour iarc sci publ ferlay j soerjomataram i ervik m dikshit r eser s mathers c rebelom parkin dm forman d bray f globocan v10 cancer incidenceand mortality worldwide iarc cancer base no [internet] lyoninternational agency for research on cancer available from httpglobocaniarcfr accessed jan ferlay j steliarovafoucher e lortettieulent j rosso s coebergh jcomber hea cancer incidence and mortality patterns in europeestimates for countries in eur j cancer forman d bray f brewster dh gombe mbalawa c kohler b piñerosm steliarovafoucher e swaminathan r ferlay j cancer incidence infive continents vol x iarc scientific publication no available from httppublicationsiarcfr_publicationsmediadownload3743e886b2754a75a0f70e190e9b56e5346047319c17pdfaccessed jan ledermann j raja f fotopoulou c gonzalezmartin a colombo n cs european society for medical oncology esmo guidelines workinggroup newly diagnosed and relapsed epithelial ovarian carcinomaesmo clinical practice guidelines for diagnosis treatment and followupann oncol coleman m gatta g verdecchia a esteve j sant m storm h allemanic ciccolallo l santaquilani m berrino f eurocare3 summary cancersurvival in europe at the end of the 20th century ann oncol bouchardy c rapiti e blagojevic s vlastos a vlastos g older femalecancer patients importance causes and consequences of undertreatment j clin oncol meindl a ditsch n kast k rhiem k schmutzler r hereditary breast andovarian cancer new genes new treatments new concepts deutschesärzteblatt | 0 |
"cellular recognition of microbial dna is an evolutionarily conserved mechanism by which the innate immunesystem detects pathogens cyclic gmpamp synthase cgas and its downstream effector stimulator of interferongenes sting are involved in mediating fundamental innate antimicrobial immunity by promoting the release oftype i interferons ifns and other inflammatory cytokines accumulating evidence suggests that the activation ofthe cgassting axis is critical for antitumor immunity the downstream cytokines regulated by cgasstingespecially type i ifns serve as bridges connecting innate immunity with adaptive immunity accordingly a growingnumber of studies have focused on the synthesis and screening of sting pathway agonists however chronicsting activation may lead to a protumor phenotype in certain malignancies hence the cgassting signalingpathway must be orchestrated properly when sting agonists are used alone or in combination in this review wediscuss the dichotomous roles of the cgassting pathway in tumor development and the latest advances in theuse of sting agonistskeywords cgassting innate immunity type i interferon sting agonists antitumor response cancerdevelopmentintroductionthe discovery of phagocytosis in advanced the understanding of innate immunity the first line of host defenses protection againston patternrecognition receptors prrs which recognize microbialproducts coordinate antimicrobial defenses and activateinfection dependsagainstinfection byvarious pathogens correspondence zqliucsueducn juyan zheng and junluan mo contributed equally to this work1department of clinical pharmacology hunan key laboratory ofpharmacogenetics and national clinical research center for geriatricdisorders xiangya hospital central south university changsha peoples republic of china2institute of clinical pharmacology engineering research center for appliedtechnology of pharmacogenomics of ministry of education central southuniversity changsha peoples republic of chinafull list of author information is available at the end of the adaptive immunity abnormal rna or dna rnadna hybridization and cyclic dinucleotides derived frommicrobes are usually considered pathogenassociated molecular patterns pamps [ ] cells associated with innate immunity recognize different microbial pampsthrough specific prrs thereby playing key roles in hostresistance to microbial infection the pathways governing rna recognition such as retinoid acid induciblegene i rigilike receptors have been reviewed elsewhere and will not be covered herein in the case of dnarecognition one of the best known prrs is tolllike receptor tlr9 which senses extracellular cpg hypomethylated dna that has entered the cytosol through thephagosomelysosome system in addition the aim2like receptor aim2 inflammasome can be triggered afterthe entry of doublestranded dna dsdna into the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0czheng molecular cancer page of cytosolic compartment which induces the proteolyticmaturation of proinflammatory cytokines such as il1and il18 and the activation of gasdermin d leading topyroptosis [] nevertheless the most notable prr iscgas a direct cytosolic dsdna sensor which was identified by dr chens group in once cgas bindsto dsdna the cgassting pathway is activated to further induce the expression of type i ifns and other inflammatory cytokinesthus triggering innate immuneresponses mounting evidence suggests that cgassting signaling not only plays pivotal roles in the hostdefense against microbialinfection but also modulatestumorigenesis hence in this review we summarize themechanism of cgassting activation and elaboratefindings regarding its dual effects on tumor developmentcurrent advances in the use of sting agonists as a novelstrategy for antitumor therapy are also reviewedinsights into the cgassting signal transductioncascadecgas is an innate immune sensor that identifies variouscytosolic dsdnaincluding dna with viral bacterialmitochondrial micronuclei and retroelement originswhich can be mainly divided into pathogenderiveddna and selfdna table in the cytoplasm cgas isactivated by interacting with dsdna in a sequence[]independent butstructural and biochemical analyses have revealed thatthe cterminal lobe of cgas contains a conserved zinclengthdependent mannerionbinding module that mediates dna binding andcgas dimerization [ ] dna ligands promotecgas activation primarily by inducing conformationalchanges around the catalytic site and in the dnabinding structures of cgasthe gscontaining loopundergoes conformational change to maintain stabilitywhich is a major mechanism of cgas activation bydna in addition to the primary dnabinding sitementioned above the secondary site located beside theprimary site is a helix formed between strands 78and several surfaceexposed loops the proximity ofthe two dnabinding sites in cgas leads to a cgasdna complex assembly in which two cgas moleculesembrace two molecules of dsdna [ ] the cgasdimers are anized in headtohead alignment nextto the dna and thus form stable ladderlike networks between one long curved dsdna helix or two independent dsdna strands [ ] in this way eachindividual cgasdsdna complex can be cooperativelystabilized and can lead to stronger enzymatic activitywhich may provide a possible explanation for longerdsdna as more likely to activate cgas in additionlong dna is more efficient than short dna in drivingthe liquidliquid phase separation of cgas and the formation ofcriticallydependent on the concentration of cgas and dna inthe cytoplasm cgas and dsdna are spatially concentratedcgasdimerization and activation [] once cgas andcgas liquidlike dropletsin liquiddropletsistofacilitatetable classification of the cytosolic dsdna that activates the cgassting signaling axisclassificationselfdnasource of dsdnamicronucleipossible mechanismsrupture of the micronuclei membrane leads to exposureof chromatin dna that is recognized by cgas whichactivates the cgassting pathwayreferences mitochondrionnuclear rnapathogenderived dnadna virushsv1 hsv2 kshv adenovirus vacciniavirus cytomegalovirus papillomavirusmurine gammaherpesvirus retrovirushiv siv murine leukemia virusrna viruswest nile virus dengue virus vsvsarscov2bacterialisteria monocytogenes mycobacteriumtuberculosis listeria shigella francisellachlamydia and neisseriamitochondrial stress induces mtdna leakage into thecytosol thus activating the sting pathway and inducingproduction of cytokinesfacilitated by endogenous retroelements nuclear rnacan be reversely transcribed into dna that activatescgassting signaling dna viruses invade host cells and release pathogenderiveddna to induce sting activation[]dna intermediates generated from reverse transcription maybe recognized by cgas to stimulate downstream stingsignaling infection with rna viruses might cause cellular damage andcell death which results in the release of cellular dna andfurther activation of the cgassting axis sarscov2 bindingto ace2 can lead to excessive angiotensin ii signaling thatactivates the sting pathway in mice[]bacteria produce cdns such as cyclic digmp and cyclicdiamp which can directly bind to and activate sting[ ]hsv1 herpes simplex virus hsv2 herpes simplex virus kshv kaposi sarcomaassociated herpesvirus hiv human immunodeficiency virus siv simianimmunodeficiency virus vsv vesicular stomatitis virus cdns cyclic dinucleotides and sarscov2 severe acute respiratory syndrome coronavirus 0czheng molecular cancer page of dsdna interacts structural switches rearrange the catalytic pocket to enable cgas to catalyze the synthesis of²²cyclic gmpamp ²²cgamp with atp andgtp as substrates the first step in this process is theformation of a linear dinucleotide ²pppg ²²pawith atp serving as the donor and ²oh on gtp serving as the acceptor then the intermediate product flipsover in the catalytic pocket placing gtp at the donorposition and amp at the acceptor position to form asecond ²² phosphodiester bond [ ] notablyalthough dsrna or singlestrand dna ssdna is ableto bind to cgas neither can rearrange the catalyticpocket which may explain the exclusive activation ofcgas by dsdna ultimately cgamp acts as a secondmessenger to bind to and activate sting a small endoplasmic reticulum erlocated protein kd withfour putative transmembrane domains [ ] normally in a resting state sting is retained in the er byinteracting with the ca2 sensor stromalinteractionmolecule stim1 the cytosolic ligandbindingdomain lbd of sting exists as the most functionalunit capable of integrating with ²² cgamp or cdnscyclic dinucleotides such as cdiamp cdigmp or ²²cgamp from bacteria upon interaction the obviousclosure of the ligand binding pocket in the lbd is observed which is related to the activation of sting next sting transforms into a tetramer through a highorder oligomerization reaction and is translocated fromthe er to the perinuclear area facilitated by cytoplasmiccoat protein complex ii copii and adpribosylationfactor arf gtpases [ ] in the golgi sting ispalmitoylated atcys88 andcys91 a posttranslational modification necessary forsting activation modified sting recruits thekinase tankbinding kinase tbk1 in turn the cterminal domains of sting are phosphorylated bytbk1 and then phosphorylated sting recruits interferon regulatory factor irf3 which is also phosphorylated by tbk1 and dimerizes ultimately dimerizedirf3 enters the nucleus and exerts its function in thetranscription of type i ifns and interferonstimulatedgenes isgs in parallel sting can also bind toand stimulate iκb kinase ikk to mediate the production of nuclear factorκb nfκbdriven inflammatorygenes upon signal transduction termination sting istransferred to endolysosomes for degradation considering that cgamp can be transferred through gapjunctions or delivered in viralexosome packages cgassting signaling may be activated in the cytoplasmwithout dsdna [ ] moreover newly produced typei ifns activate heterodimer interferon receptors ifnar1 and ifnar2 through paracrine signaling and thusinduce the transcription of isgs [ ] in summaryonce virusderived dna and selfdna are located intwo cysteine residuesthe cytoplasm they can be sensed by cgas and a cgasdsdna complex is formed to catalyze the synthesis of ²²cgamp with atp and gtp then ²²cgamp and bacteriaderived cdns induce sting activation and mediate the release of downstream type iifns tnfα and il6 which are prerequisites for antimicrobial defense and antitumor effects the wholeprocess shows that the dsdnacgassting axis canlead to the activation of both innate and adaptive immunity fig the antitumor functions of the cgasstingsignaling pathwayrecent evidence has revealed the close association of thecgassting pathway with cancer development thissignaling pathway is generally regarded as a potent regulator of cancer immunity a stingmediated immunesupportive microenvironment can hamper malignancyoccurrence stressbytumor cell cytosolic dsdna induces sting activationunder normal circumstances dna is strictly unaffiliatedwith the cytoplasm in eukaryotic cells to avoid autoimmunity however dna leaks aberrantly in tumorcells [ ] cancer cells share common features including genome instability tumor suppressor gene mutation or deletion oxidativeand vigorousmetabolism under these intense states nuclear andmitochondrial dna is fragile and easily damaged whichleads to eventual dna leakage in the forms of micronuclei chromatin fragments andor free telomeric dna[ ] chromosomal instability cin is the primary source of cytoplasmic dna in malignant cells andis generally associated with tumor progression distantmetastasis and therapeutic tolerance excessive proliferation of cancer cells results in unstable genomes usuallychromosomal missegregation during mitosis due to defects in segregation lagging chromosomes generate micronuclei in a cellcycledependent manner the vulnerable membraneof micronuclei easily exposes the inner dna to the cytoplasm and activates the cgassting signaling axis exogenous stimuli such as chemotherapy and irradiation can also cause dna damage in addition to leakednuclear dna oxidative stressinduced mitochondrialdna leakage is another crucial initiator of sting pathway activation several anticancer treatments that precisely attack mitochondrial membranes result in effluxand cell death therefore the permeabilization of mitochondria membranes provides a reasonable explanationfor mitochondrial dna escape [ ] other sourcessuch as apoptotic cellderived dna exosomal dnaexodna and transposable elements have also beencharacterized 0czheng molecular cancer page of fig the cgassting dna sensing signaling pathway various dna derived from virus dying tumor cells or nucleus and mitochondria binds toand activates the cytosolic dna sensor cgas cgas catalyzes the synthesis of ²²cgamp in the presence of atp and gtp then ²²cgamp bindsto the er adaptor sting which also can be activated by cdns derived from bacteria upon activation sting translocates from er to golgicompartments where it activates tbk1 and ikk which phosphorylate irf3 and iκbα respectively then irf3 and iκbα dimerize and enter nucleusinitiating the transcription of type i ifn tnf and il6 the primary roles of these cytokines are reflected in host defense inflammation andantitumor immunitydemonstrated to evoke cgassting activation intumor cells [ ]type i ifns mediators of sting and adaptive antitumoreffectscgassting signaling exerts antitumor functions incancer cells both in an autonomous and nonautonomousmanner on the one hand dna damage can provokeacute sting signal transduction and induce cellularsenescence an irreversible cell cycle arrest state whichthwarts the aberrant proliferation of tumor cells throughacquisition ofsecretoryphenotype sasp which is associated with the releaseof abundantinflammatory mediators proteases andgrowth factors [ ] in contrast to undergoingsenescence tumor cells also directly propel apoptosisprocesses by upregulating proapoptosis protein bcl2associated x bax and downregulating the bcl2 apoptosis on the other hand stingsenescenceassociatedtheregulatoractivation in tumor cells not only facilitates the transcription of downstream type i ifns to induce dendriticcell maturation but also recruits supportive immunecells for direct nonspontaneous tumor elimination sting activation in nonmalignant cells causes tumorsuppressive effects as well sting signaling protectsagainst colitisassociated carcinomas cacs induced byazoxymethane aom and dextran sulfate sodiumdss which induce dna damage in intestinal epithelialcells and further trigger sting activation downstreamcytokines of sting signaling such as il1 and il18prevent neoplastic transformation by facilitating woundrepair more importantly sting signaling can also provoke cytotoxic t cell responses to control tumorigenesis necrotic cancer cells are commonly engulfed byantigenpresenting cells especially the basic leucine zippertranscription factor atflike batf3drivenlineage of dendritic cells dcs batf3 dcs take intumorassociated antigens and migrate towardsthe 0czheng molecular cancer page of tumordraining lymph node via the lymphatic systemwhere they crossprime tumorspecific cd8 t cellsthen cd8 t cells undergo activation and clonal expansion in the lymph nodes and are trafficked throughblood vessels to kill tumor cells in turn damaged cancercells release more antigens that are further captured bydcs the whole process forms a positive feedback loopcalled the cancerimmunity cycle tumor eradication can be achieved by multiple processes in thecancerimmunity cycle including tumor antigen captureand presentation and t cell priming and activation withtumor antigenspecific t cell priming and activationrelying on dcs and type i ifn release the involvement of type i ifns in innate immune sensing and adaptive immunity provides a reasonable hypothesis forexploring candidate prr pathways as potential immunomodulators mice lacking tlr9 myeloid differentiationprimary response gene myd88 cytosolic rna sensor mavs or the purinergic receptor p2x7r maintainintact antitumor immunity responses whereas mice deficient in sting or irf3 present with impaired cd8 tcell priming and activation [ ] in fact dying tumorcells can release multiple damageassociated molecularpatterns damps to trigger innate immune responsesin dcs among these released stimuli tumor cellderiveddna is a pivotal inducer in general the phagocytosis ofapoptotic cells causesimmune silence because ofdnasebased degradation nevertheless tumor cellreleased dna can be preserved in the dc endolysosomal compartment through an unknown mechanism cgas recognizes dna invading the cytoplasm andinduces the activation of sting cascades excretion oftype i ifns and expression of isgs additionally undersome physiological conditions such as hypoxia andacidic environments nuclear or mitochondrial dnamight be packaged in exosomes exosomal dnaexodna animates sting signaling once it is absorbedby tumorinfiltrating dcs finallytumor cellderived cgamp can also be transferred to host dcs bythe folate transporter slc19a1 and then directly bindsto sting activating it in dcs a recent study moredirectly demonstrated that cellautonomous sting promoted the maintenance of stem celllike cd8 t cellsand augmented antitumor t cell responses and mechanistically cgasstingmediated type i interferon signaling reinforced the stem celllike cd8 t celldifferentiation program mainly by restraining akt activity immune cellderived type i ifns have crucial functions in antitumor immunity control on the one handtype i ifns boost cross presentation by various mechanisms first they stimulate the maturation of dcs secondthey slow the endosomelysosome acidificationprocess to prevent engulfed tumor antigen clearance andelevate the expression of mhc i molecules on the cellsurface [ ] finally they accelerate dc migrationtowardslymph nodes where they can crossprimetumorspecific cd8 t cells on the other handtype i ifns drive the expression of multiple chemokinessuch as cxcl9 and cxcl10 both of which are necessary for cytotoxic t lymphocyte ctl transfer and infiltration similarly type i ifns restrain the defaultimmune suppressive action of regulatory t treg cellsby downregulating phosphodiesterase pde4 and upregulating cyclic amp camp consequently typei ifns serve as bridges linking the cgassting pathway with cd8 t cellmediated antitumor immunitythe antitumor mechanisms of the cgassting signaling axis are illustrated in fig indeed previous studies revealed that sting activation can stimulate antitumor immune responses inleukemia melanoma glioma and hepatocellular carcinoma [] additionally sting expression is downregulated in a wide variety of tumor tissues and celllines according to a pancancer analysis with a smallproportion of tumors approximately bearing silent sting expression lower sting expressionwas found in hepatic carcinoma and gastric cancer compared with its level in corresponding normal tissues andthis lower expression level was correlated with highertumor stage and poorer prognosis [ ] consistentlycompared with that in the mcfg10a mammary epithelial cell line lower sting expression was detected inmalignant breast cancer cellincluding mcf7hbl100 and t47d cells as well as human melanomacell lines and colorectal adenocarcinoma lines [ ] collectivelythat cgassting signaling might act as a tumor suppressor in certain types of cancersthese findings suggestlinessting pathway agonists as cancer therapeuticsthe immunostimulatory potential of the cgasstingpathway makes it an attractive pharmacological targetsince its activation in the tumor microenvironmenttme can induce efficient crosspriming oftumorspecific antigens and facilitate the infiltration of effectort cells recent drug research has focused on the development of sting agonists because of their potential inanticancer therapy [ ] to date various kinds ofsting agonists have been discovered and they aremainly divided into the following categories cyclic dinucleotides and their derivates dmxaa and its analogsand small molecular agonists in addition some conventional antitumor therapeutics can also indirectly activatesting such as chemotherapy radiotherapy rt andtargeted therapy in addition sting agonists areable to enhance the efficacy of other anticancer therapeutic agents when used in combination sting 0czheng molecular cancer page of fig the antitumor immunity effect of the cgassting pathway dna damage leads to the formation of dsdna in tumor cells upon itsstimulation sting signaling is activated and promotes the release of type i ifn which is crucial for dc maturation sting signaling activation indcs is the core step of the whole cancerimmunity cycle which can be initiated through engulfment of dyingdamaged tumor cells exosometransfer and cgamp gap junctions then dcs migrate towards the tumordraining lymph node and crossprime tumor specific cd8 t cells withthe help of type i ifns finally t cells undergo clonal expansion and traffic through the blood vessel to conduct tumor killingagonists and their synergistic use with other remedies isfurther explored in detail belowcyclic dinucleotides cdnscdns constitute a main type of sting agonist whichmainly originate from bacteria the known naturalcdns consist of exogenous cyclic digmp cdigmpcdiamp ²²cgamp and endogenous ²²cgampamong these cdigmp cdiamp and ²²cgampare synthesized by bacteria and identified as secondarymessengers that mediate sting signal transduction inprokaryotic cells while ²²cgamp functions as theinitiator of sting in mammalian cells the antitumor potential of these natural dinucleotides was firstproven by the finding that cdigmp could inhibit theproliferation of human colon cancer cells in vitro andbasal cell proliferation of human cecal adenocarcinomah508 cells was inhibited with μm cdigmp intraperitoneal ip injection of highdose cdigmpdirectly activated caspase3 and triggered t1 tumoripcell apoptosis in vitro nmol of cdigmp reduced thegrowth of t1 tumor cells in vitro by and nmreduced it by while lowdose cdigmp nmol accelerated the adaptive t cell response by converting a subgroup of myeloidderived suppressor cellsmdscs into immune stimulatory cells producing il12injection of ²²cgamp consistentlymgkg expedited dramatic leukemic elimination in eltcl1 transgenic mice bearing chronic lymphocyticleukemia cll and promoted tumor shrinkage of multiple myeloma in vivo from the perspective of endogenous cdns ²²cgamp mgkg was alsoshown to restrain tumorigenesis and improve the survival rate of mice bearing ct26 colon adenocarcinomain a dosagedependent manner relying on dc activationand t cell crosspriming more recently ohkurit further demonstrated that intratumoral it injection of ²²cgamp μg25 μldose on and days after the injection of tumor cells significantly mitigated tumor growth and prolonged the survival of breast 0czheng molecular cancer page of cancer t1luc squamous cell carcinoma mscc1colon cancer ct26 and melanoma b16f10 mousemodels notably the it injection of ²²cgampinhibited not only tumor growth but also lung metastases in mice bearing b16f10 cellderived tumors suggesting that cgampinduced cd8 tcell priming can drivesystemic antitumor immunity to control local and distant tumor growth termedvaccinestingvaxconsidering the superior properties of sting signaling in activating adaptive immunityit is rational toutilize sting agonists such as cdns as cancer vaccineadjuvants to increase tumor immunogenicity fu investigated the in vivo therapeutic efficacy of acancercomprisinggranulocytemacrophage colonystimulating factor gmcsf and bacteriaderived or synthetic cdns theyobserved that after it injection of stingvax with μg of cdns per vaccine dose the volume of b16melanoma tumors was dramatically reduced in a dosedependent manner compared to mice receiving gmcsf cancer vaccine alone stingvaxtreated mice hadmore infiltrating cd8 ifnγ t cells in the tumormicroenvironment the in vivo antitumor effect of stingvax was also verified in models of colon carcinomact26 pancreatic carcinoma panc02 and upper aerodigestive squamous cell carcinoma sccfvii although natural cdns are able to produce robust antitumor immunity their chemical features might hindertheir future application in the clinical setting first native cdns are easily degraded by enzymes inside the cellor in the bloodstream second their negatively chargedproperty hydrophilicity and phosphate moieties severelyimpede cdns from penetrating cell membranes to activate cytosolic sting leading to low bioavailability andpoor retention of the cdns in specific cells and tissuesthird unintentional toxicities and narrow therapeuticwindows are also unavoidable thus new strategies toimprove therapeutic efficacy and reduce adverse effectsare urgently needed including drug delivery carrier engineering original structural modification and nonnucleotide agonist screening regarding agonistdelivery smith reported that biopolymer implantscodelivering cdigmp μg and chimeric antigen receptor t cart cells resulted in significant tumor regression in mice bearing pancreatic tumors moreoveriv administration of cdigmpysk05lip equivalent to μg of cdigmp aysk05liposome delivery system encapsulating cdigmp led to a tremendous decrease in metastatic lesionsin a b16f10 mouse melanoma model with nearly ofthe injected mice showing resistance against tumor relapsethe adaptive immune responsememory was successfully induced chen alsofound thatliposomalindicating thatinjection ofintravenousintravenousivnanopdelivered cgamp cgampnp could activate the sting axis more effectively than solublecgamp and converted the immunosuppressive tme toa tumoricidal state in a transplanted b16f10 cell melanoma model and in a genetically engineered triplenegative breast cancer model moreover a recentstudy creatively suggested that modified bacteria mightbe exploited as a selective carrier of sting agonistsintroduction of a dinucleotide cyclasecoding gene intothe escherichia coli nissle strain was an attempt at realizing this effect however advancements to the systemare needed tobysnakeapartdigestionresistancecompoundatoms the modifiedfrom improving delivery methods cdnswith superior properties are currently being synthesized and tested for instance to prevent enzymatichydrolysis of cgamp the nonbridging oxygen atomsin cgamp phosphodiester linkages were replaced by²²sulfurcgsasmp showed resistance against degradation byenpp1 a major ²²cgamp hydrolasetherebyleading to a longer halflife and sustained high affinity for human sting hsting syntheticdithio mixedlinkage cdns with both rp rp r rand rp sp r s dithio diastereomers possessed notonlyvenomphosphodiesterase but also enhanced affinityforsting a novel superior modified product ml rrs2 cda also termed adus100 had the potencyto activate all hsting variants and mouse stingmsting adus100 had higher efficiency in activating sting signaling than endogenous or exogenous cdns mainly because of its enhanced stabilityand lipophilicity its powerful tumor elimination effect was extensively demonstrated in multiple murinemodelsincluding b16 melanoma t1 breast cancer and ct26 colon cancer with all treated animalsshowing significant and durable tumorregressionafter itinjection of adus100 three mg doseswhen tumor volumes reached mm3 theremarkableforhsting laid the foundation for its clinical use related clinicaltrials of adus100 are outlined intable in addition to adus100 some other novelsting agonists have been well designed iacs8779and iacs8803 are two highly potent ²²thiophosphate cdn analogs that induced striking systemicantitumorin a b16 melanoma murineinjection μg on and daysmodel after itposttumor implantation compared with adus100or cgamp the characteristics and preclinicalapplications of all these mentioned cnds are summarized in table because of the structural modification and optimization of delivery strategiestheapplication range and efficacy of cdns have beenand high affinityresponsescurativeeffect 0czheng molecular cancer page of table characteristics and preclinical applications of different sting agonistsclassificationcharacteristicsapplicationmodelsnatural cdnagonistscdigmppoor membrane permeabilitysuitable for various codeliverytechnologiescolon cancer h508cells t1 metastaticbreast cancertreatmentinformation μm nmol ip nmol ip nmol ip²²cgamp²²cgamphigher binding affinity formsting than for hstinghigher affinity for hsting thanits lineage isomers binds tovarious sting nucleotidepolymorphisms observed inhumans easily degraded byphosphodiesteraseimpermeable to the cellmembranechronic lymphocyticleukemia mgkg ipmultiple myeloma mgkg ipct26 colonadenocarcinoma mgkgbreast cancer t1lucsquamous cellcarcinomasmscc1 μg25 μldose it μg25 μldose itcolon cancer ct26 μg25 μldose itmelanoma b16f10 μg25 μldose ittherapeutic effects references[ ] [ ]inhibitsproliferation tumorregression tumorregressionaccelerates tcellresponseleukemiaeliminationsuppressesgrowthrestrainstumorigenesisimproves survivalratedelays tumrowthdelays tumrowthdelays tumrowthdelays tumrowthstingvaxsyntheticcdnagonistspotent in vivo antitumor efficacyin multiple therapeutic modelsof established cancercgampnpsbiopolymer scaffolds cdigmp and car t cellscdigmpysk05lip²²cgsasmpadus100iacs8779iacs8803noncdnagonistsfaaliposomal nanops npsdeliver cgamp intracellularlymore effectively than realizedwith soluble cgamperadicates tumors moreeffectively than systemicdeliveryysk05 is a lipid that can efficientlydeliver cdigmp to the cytosolpossesses high fusogenic activitywhich enhances endosomalescapemore resistant to degradation byenpp1 tenfold more potent atinducing ifn secretion potentialuse as a cancer vaccine adjuvantimproves stability and lipophilicityhigher affinity for hsting thannatural cdn agonists capable toactivate all hsting variants andmstingstimulates a superior systemicantitumor response thanadus100 and cgampcauses hemorrhagic necrosisfailed in a phase i clinical trialdue to species specificity μg cdns itreduces tumorvolume b16 melanomacolon carcinomact26pancreaticcarcinoma panc02b16f10 melanomaivtnbccreates atumoricidal state pancreatic cancer μg cdigmptumor regression b16f10 mousemelanoma μg cdigmp ivdecreasesmetastasisthp1 monocytesb16 melanomathree mg doses it t1 breast cancerthree mg doses itmc26 colon cancerthree mg doses itdurable tumorregressiondurable tumorregressiondurable tumorregression b16 melanoma μg on day and posttumor implantationantitumorresponse murine colontumorsextensive tumorrejection[ ]dmxaafirst discovered as a vascularrat mammary mgkg iphigh anticancer[ 0czheng molecular cancer page of table characteristics and preclinical applications of different sting agonists continuedclassificationcharacteristicsapplicationmodelstreatmentinformationinduces proinflammatory cytokinesin a stingdependent mannerhuman fibroblastsantiviral activity selectively induces stingdependentsynthesis and secretion of bioactiveifns no evidence of binding directlyto stingactivates sting in openconformation submicromolarlevels induce sting activationand ifn productionhuman fibroblastsantiviral activity colon tumors mgkg iv of a treatedgroup remainedtumor free faa flavone acetic acid dmxaa 56dimethylxanthenone4acetic acid cma 10carboxymethyl9acrid | 0 |
there is currently a major human pandemic caused by the novelsevere acute respiratory syndrome sars coronavirus2 sarscov2 that leads to coronavirusinduced disease covid191 itis primarily a viralinduced ammatory disease of the airwaysand lungs that causes severe respiratory issues sarscov2 usesthe angiotensin converting enzymeii receptor ace2 to bindand infect cells leading to internalization and proliferation23ammatoryinnate and adaptive immune responses areinduced to clear the virus but also cause host tissue damage45consequent hypoxia leads to systemic involvement particularlyofleads to vasoconstriction reducedperfusion and an failure6 much remains to be understoodof the ammatory and immune responses that are induced bythe infection and how they induce pathogenesis ventilation andoxygen therapy are primary treatments and it is emerging thatthose with severe disease who survive develop lung ï¬brosis7 themost effective pharmacological treatments remain illdeï¬nedwith varying results with hydroxychloroquine8 but more promising results with dexamethasone9the vasculature thatelucidating the mechanisms of pathogenesis will enable theidentiï¬cation of the most effective therapies animal models ofsarscov2 infection and covid19 that recapitulate the hallmarkfeatures of the human disease will undoubtedly be valuable inelucidating pathogenic mechanisms identifying new therapeutictargets and developing and testing new and effective treatmentshuman infection and diseasesarscov2 is a betacoronavirus closely related to sarscovthat caused a relatively small outbreak in the early 2000s210similar to sarscov sarscov2 binds the ace2 receptor andrequires proteases such as serine tmprss2 to cleave the viralspike s protein required for sarscov and sarscov21112 cellentry2 this step may be facilitated by endosomal proteases suchas cathepsinl and enhanced by the protein furin13 the virusthen enters the host cell by endocytosisa critical element of sarscov2 tropism in humans is theabundance of ace2 in the upper respiratory tract urt especiallythe nasopharynx14 the molecular conï¬guration of the sarscov21centre for ammation centenary institute and university of technology sydney faculty of science sydney australia 2zhejiang universityuniversity of edinburgh institutezhejiang university school of medicine zju international campus haining china 3second afï¬liated hospital zhejiang university school of medicine hangzhou china4department of genomes and genetics institut pasteur paris france 5centre for innate immunity and infectious diseases hudson institute of medical research clayton vicaustralia 6department of molecular and translational sciences monash university clayton vic australia 7ritchie centre hudson institute of medical research clayton vic australia 8department of paediatrics monash university clayton vic australia 9monash newborn monash childrens hospital clayton vic australia 10priorityresearch centre for healthy lungs hunter medical research institute and university of newcastle newcastle nsw australia 11school of chemistry and molecular biosciencesand australian infectious diseases research centre the university of queensland brisbane australia 12centenary institute and dermatology the university of sydney andcancer services royal prince alfred hospital sydney nsw australia 13centenary institute and faculty of medicine and health university of sydney sydney australia 14dr johnand anne chong lab for functional genomics charles perkins centre centenary institute and school of life and environmental sciences university of sydney sydney nswaustralia 15charles perkins centre and school of life and environmental sciences university of sydney and faculty of medicine and health concord clinical school anzacresearch institute and centre for education and research on ageing sydney australia 16discipline of infectious diseases and immunology central clinical school faculty ofmedicine and health and the charles perkins centre the university of sydney camperdown sydney australia 17woolcock institute of medical research sydney australia and18centenary institute the university of sydney and department of clinical immunology royal prince alfred hospital sydney nsw australiacorrespondence p m hansbro philiphansbroutseduaureceived july revised july accepted july society for mucosal immunology 0canimal and translational models of sarscov2 infection and covid19md johansen membrane binding component of the s protein binds with greaterafï¬nity to ace2 than does sarscov which likely contributes to thehigher infectivity of the former15 the clinical course commenceswith an incubation period with a median of days with illnesstypically developing by days16 this phase is characterized bymild symptoms with most people remaining asymptomatic andinfection thought to be conï¬ned to the urt although they arecapable of transmitting infection symptoms when they do occur aretypically acute viral respiratory illness with fever cough dyspnoeafatigue anosmia myalgia and confusion17 in of people thecourse remains mild and disease does not extend to the lowerrespiratory tractlrt however develop more severesymptoms with diffuse widespread pneumonia with havingsevere gas exchange problems acute lung injury and progress ontosyndrome ards1819 the clearestacute respiratory distresspredictor of mortality is age with the case fatality rate risingdramatically over years of age20 other predisposing factors forheightened mortality are male sex social deprivation and chronicdisease particularly chronic obstructive pulmonary disease copdcardiovascular disease cvd obesity and diabetes21a key issue is why some individuals progress to more severe lowerrespiratory disease but others do not one factor is the ability of theammatory and immune responses to conï¬ne the infection to theurt ace2 is expressed in the lrt but at lower levels than in thenasopharynx22 also while ciliated airway epithelial cells are readilyinfected and transmit to surrounding cells the reduction in ace2may be a barrier to lrt infectionin those that progress severesystemic ammatory response or cytokine storm develop thepneumonia associated with severe infection bears all the pathological features of ards with diffuse alveolar damageinterstitialpneumonitis and lymphocytic ltrates2324 unique features ofcritical disease are extravascular ï¬brin deposition neutrophiltrapping microvascular thrombosis and large vessel pulmonaryemboli24 widespread thrombosis and microangiopathy in criticalcovid19 occurs at higher rates than in ards associated withuenza and dysregulated coagulation and angiogenesis are alsofeatures25increased and dysregulated th1 and th17 responses werepresent in ards in middle eastern respiratory syndrome merscov and uenza2627 the occurrence of severe lung disease at days postinfection dpi reï¬ects the dual features of spreadof infection to the lrt and coincident development of adaptiveimmune responses with heightened activation of virusspeciï¬c teffector cells this coincides with lymphopenia associated withsevere disease and is a predictive marker earlier in disease withworse outcome28 there is also evidence that tcells aredysfunctional with increased expression of exhaustion moleculesrelated to heightened systemic ammation29 the role ofelevated systemic immune dysregulation is supported by analysisof the transcriptomic responses in people with sarscov2infection demonstrating that impaired interferon ifn responseswere related to persistent viremia and increased systemicil6 and il1030 elevatedammation with elevated tnfαsystemic levels of il17 are also present in critical illness with ardsand heightened ammation and it is plausible that increasedth17 responses drive ongoing ammation31 while interestingthese are observations often performed with limited patientnumbers where ards and disease severity are associated withheightened ammatory and immune activation and a causativerole cannot be established2732 interrogation of representativeanimal models is needed to deï¬ne cause and effect elucidatemechanisms of pathogenesis and test treatmentssmall animal modelsa range of animal models have been used to study covid19 withvarying susceptibility likely dependent on species speciï¬c makeup for ace2 fig fig ace2 protein phylogenetic divergence and in vivo modelseverity left panel fast minimum evolution distance tree for ace2protein sequences using griphin for evolutionary distance unsortedspecies included are gallus gallus chicken anas platyrhynchos duckcavia porcellus guinea pig mustela putoris furo ferret canis lupusfamiliaris dog felis catus cat sus scrofa pig rousettus aegyptiacusfruitbat mesocricetus auratus golden hamster mus musculus mousecallithrixjacchus common marmoset macaca mulatta rhesus macaque macaca fascicularis cynomolgus macaque chlorocebus aethiopsafrican green monkey and homo sapiens macaques are representedas one image due to close divergence severity of disease is colorcoded from refractory to infection blue no virus detected to severered shedding common marmoset and guinea pig have only beenassessed for sarscov all others with sarscov2 scale indicates amino acid divergencemouse modelssmall animal models are widely used to study emerging virusesbut often they need to be genetically modiï¬ed or the virus needsto be adapted for different species to be susceptible and this is thecase for sarscov2 in most studies to date the mouse strainshave been incompletely or incorrectly described and should beincluded in future publicationsgenetically modiï¬ed miceinbred mouse strains were used to model sarscovseveralinfection including balbc33 c57bl634 and 129svev incorrectlyreported name35 as well as factordeï¬cient mice such ascd1 rag1 and stat1 the identiï¬cation of ace2 asthe host receptor for sarscov38 initiated considerable globalinterest in developing murine models that are representative ofhuman disease this led to the generation of transgenic mice thatexpress human hace2 k18hace2 in the epithelia of tissues andans including lungs liver kidney spleen heart and gut k18hace2 mice still express the mouse mace2 however this isprimarily localised in the ileum39 they are highly susceptible tosarscov infection and experience high virallung titressigniï¬cant weight loss and morbidity from dpi39 viral dissemination to the brain was the main cause of death40 similar resultswere obtained in another transgenic strain expressing hace2under the control of the mace2 promoter41mucosal immunology __ 0cwith the mers outbreak in and identiï¬cation of that virusentry receptor dipeptidyl peptidase4 dpp4 cd26 it was foundthat neither wildtype wt nor immunodeï¬cient mice weresusceptible to merscov infection42 transgenic mice expressinghuman hdpp4 in epithelial and endothelial cells in the lungbrain heart liver kidney spleen and intestine were generated43they were highly susceptible to merscov infection withsigniï¬cant weight loss high virallung titres and ammatorycytokine production from dpi and mortality from day inother studies exons of the mdpp4 locus were modiï¬ed toresemble hdpp4 expression44 which led to merscov lungreplication but not disease development4748with the discovery of sarscov2ace2 interactions21249 globalinterest in hace2 mouse models reemerged however due tofalling interest with the resolution of the sars outbreak most labsceased maintaining hace2 mice to satisfy the global demand forhace2 mice the jackson laboratory reanimated k18hace2 micewhich are becoming available the lagtime has slowed theunderstanding of disease mechanisms in covid19 and theidentiï¬cation of effective drug and vaccine candidates to progressto clinical trialsthe ï¬rst live sarscov2 infection model used transgenic miceexpressing hace2 under the control of the mace2 promoter4150mice had signiï¬cant weight loss over a 14day infection periodand high viral lung titres dpi histological lung examinationrevealed moderate interstitial pneumonia ltration of lymphocytes mucus accumulation and desquamation of bronchoepithelial cells from day there were no detectable viral titres orpathology in other tissues or ans except on day in theintestine suggesting that infection is localized almost exclusivelyto the lungssimilarly transgenic mice overexpressing hace2 under thecontrol of hfh4foxj1 lung ciliated epithelial cellspeciï¬cpromoters are also susceptible to sarscov2 infection5152 mostinfected hfh4hace2 mice had minimal weight loss over 7days ofinfection however mice that later became moribund showedsigniï¬cant weight loss from day and signiï¬cant lymphopeniaand neutrophilia in peripheral blood at day which recapitulatessevere human disease2831 lung histology showed initial macrophage and lymphocyte ltration and ï¬brin exudation from dpiwhich steadily progressed to severe pneumonia blockage ofterminal bronchioles extensive ï¬broplasia and alveolar necrosisby day in contrastlung tissuespeciï¬city50 hfh4hace2 infected mice had detectable viral titresin the lung eyes brain and heart suggesting that the virus mayhave additional tissue tropisms following initial lung infection5051reinfection following recovery from initial sarscov2 infectionresulted in reduced weight loss and viral titres and improvedsurvivalindicating the development of protective immunityfollowing initial challengeto previous ï¬ndings ofrecentlythe ï¬rst k18hace2 sarscov2 infection wasexamined53 mice exhibited no clinical symptoms or weight lossuntil dpi by day mice had weight loss with variableclinical presentation ranging from reduced activity to increasedrespiration and lethargy infected mice also had moderate virallung titres suggesting productive infection bronchoalveolarlavage revealed increased ltrating granulocytes monocytesand eosinophils accompanied by alveolar debris and septalthickening on histopathology analysis while these results areencouraging the study only examined ï¬ve mice up until dpi inagreement with these initial ï¬nding53 a recent preprint papersimilarly shows that sarscov2 infected k18hace2 mice lost weight by dpi which steadily decreased to bodyweight by dpi54 they had high virallung titres from dpiaccompanied by modest viral titres in the heart brain kidney andspleen declines in pulmonary function were notable at dpievidenced by reduced inspiratory capacity and increased tissueresistance and elastance rnasequencing analysis of infectedmucosal immunology __animal and translational models of sarscov2 infection and covid19md johansen type i and iiinnate immunelung tissues identiï¬ed the upregulation ofsignatures particularly nfkbdependentifnsignalling and leucocyte activation pathways another preprintpaper shows that sarscov2 infected k18hace2 mice produce arobust th1217 cytokine storm in the lungs and spleens from dpi55 highlighting the relevance of this mouse model thatrecapitulates critical human clinical features of covid19 importantly multiple reports have shown that sarscov2 infection offrom dpi55k18hace2 mice is dosedependently fatalsuggesting that it is similarly lethal as sarscov39 in thesetransgenic miceadenoviral systemsadenovirus vectorbased systems can be used to insert humanreceptors into mouse genomes and are valuable for use withalready factordeï¬cient or transgenic mice replicationdefectiveadenovirus vectors have been used to insert hddp4 into wt micerendering them susceptible to merscov infection58 infectedmice developed pneumonia with extensive pulmonary immunecell ltration and viral clearance by dpi58 but were less affectedthan fully hdpp4 transgenic micesimilarly transduction of balbc mice with adenovirus containing hace2 advhace2 led to stable hace2 expression in thelungs from h posttransfection59 sarscov2 infected advhace2 mice had weight loss over days high viral lungtitres and modest titres in the heart brainliver and spleenextensive neutrophil accumulation in perivascular and alveolarlocations and vascular congestion upon histological examinationadministration of antiifnar1 monoclonal antibodies to transiently inhibit typei ifn signalling resulted in up to weight lossand more severe lung ammation compared to infection alonein this system neutralizing antibodies against sarscov2 sprotein 1b07 were protective against severe disease mice lostless body weight and had lower viral titres in the lung heart andspleen at dpi and reduced expression of proammatorycytokines and chemokines ifnb il6 cxcl10 cxcl1 ccl2 ccl5 andimmune cell ltrates in the lungs at dpicrispr systemsusing crisprcas9 an alternative humanised hace2 mousemodel was developed where the mace2 was disrupted byinserting hace2 linked to the red ï¬uorescent protein tdtomato60hace2 expression is under the control of the mace2 promoter inthe native locus with expression predominantly in the lungs andkidneys similar to mace2 in wt mice sarscov2 infectedhumanized hace2 mice had high viral titres in the brain tracheaand lungs with no differences between young and aged micehowever infected aged mice had lung neutrophilia and extensivealveolarinjury and focal hemorrhagingcompared to young mice intragastric infection resulted in highviral titres in the trachea and lungs suggesting that the oraladministration route can lead to productive pulmonary infectionthickening vascularmouseadapted virusesviruses can be adapted to infect wt mice through serial passagingor targeted mutation their use is advantageous as they reducebiological risks to researchers and may more closely resemblenatural hostpathogen interactions in mice however they arelimited due to mouse adaptation that may result in infection thatdoes not recapitulate all aspects of human diseasethis approach was applied to sarscov which was seriallypassaged in the respiratory tracts of young balbc mice this ledto the generation of a mouseadapted sarscov strain ma15which was lethal to mice from dpi61 infection resulted in highviral titres in the lungs from dpifollowed by viremia anddiffusion to extrapulmonary sites including the brain liver andspleen signiï¬cant lymphopenia and neutrophilia mild and focalpneumonitis and necrotic cellular debris in the airways and alveoli 0canimal and translational models of sarscov2 infection and covid19md johansen another mouseadapted sarscov strain v2163 was producedby serial passage in 6weekold balbc mice62 infection resultedin more severe symptoms and higher mortality rate than withma15 with greaterimmune responses and lung pathologymouseadapted sarscov strains lacking the critical viral envelopei protein induce varying degrees of protection against reinfectionwith virulent strains highlighting the potential for liveattenuatedvaccines6364serial passage of merscov through the lungs of mice withhuman modiï¬ed exons of dpp4 resulted in a virus thatinduced signiï¬cant weight loss and mice became moribund from dpi mice had high stable viral titres in the lungs and bloodammatory cellltrates and oedema necrotic debris andvascular permeabilization in lungs47mouseadapted sarscov2 has been reported in a preprint withmutations in the receptor binding domain rbd of the spike proteinfollowing serial passageinducing productive infection of bothyoung and aged wt balbc mice65 infected mice had high virallung loads up to dpi and displayed mild pneumonia withammatory cell ltration alveolar damage focal exudation andhaemorrhage and endothelial cell denaturation the efï¬cacy of arbdfc based vaccine was examined in this model which inducedthe production of neutralizing antibodies which potently inhibitedthe infection a similar preprint describes the modiï¬cation of therbd of the sarscov2 s protein which facilitated the efï¬cientbinding of the s protein to mace2 for host cell entry66 infectionwith this virus resulted in viral replication in the upper and lowerairways of young and aged balbc mice aged mice had greaterweight loss and pulmonary function decline compared to youngmice reproducing important aspects of human diseasea summary of the different mouse models that are permissiveto sarscov2 infection table and a comparison of featuresbetween mouse models and human covid19 fig are showncovid191875 animal models that combine these risk factorswith infection will be invaluable in elucidating mechanisms ofincreased susceptibility and severity8081chronic respiratory diseasesboth cigarette smoking cs and copd are strong independentrisk factors for severe covid19 with extensive lung damage andincreased mortality82 cs upregulates ace2 expression in theairways which may increase infection risk83 the use of shorttermmurine models thatreplicate csinduced copd has greatlyincreased our understanding of disease and identiï¬ed and testednovel therapeutic interventions808184early data from china showed asthma prevalence in covid19patients were lower than the general population suggesting thatasthma may be protective89 however emerging reports showthat asthma is one ofthe most prevalent comorbidities inhospitalized covid19 patients90 and is associated with higher riskof death especially in severe asthma based on oral corticosteroiduse21 elucidating how severe asthma predisposes to severecovid19 is needed and can be achieved using preclinical animalmodels of severe asthma that recapitulate the human disease91k18hace2 mice and sarscov2 infectionlung ï¬brosis is the most severe sequelae of covid19 in up to of the patients after months94 the mechanisms areunclear but ammation and cytokine storm likely contribute7sarscov2 infection of human alveolar epithelial cells results inaltered proï¬brotic gene expression similar to pulmonary ï¬brosisincluding ace2 tgfβ connective tissue growth factor tissueinhibitor of metalloproteinase3 and ï¬bronectin98 fibrotic sequalae can be assessed in mouse models with long rest periods afterinfection8699 bleomycininduced experimental pulmonary ï¬brosiscould be combined with sarscov2 infection to investigatesequalae8699diversiï¬ed modelsthe immediate aim of producing infectionpermissive mice ormouseadapted viruses is to generate models with high respiratory viral titres and lung lesions resembling those observed inhumans host factors are associated with increased risk of severecomplications including age and obesity67 and comorbidities likecopd cvd and diabetes68 human genetic variantslikelymodulate susceptibility to covid1969in mice the impact of host genetic variations were investigatedin sarscov using the collaborative cross cc a collection ofmouse inbred strains produced by crossing eight founder inbredstrainsincluding ï¬ve classic laboratory strains aj c57bl6j129s1svimj nodshiltj and nzohiltj and three wildderivedcasteij pwkphj and wsbeij strains70 cc strains segregate million polymorphisms and have more genetic diversity thanthe human population71 this resource is ideally suited forinvestigating the role of host genetic variants in the pathophysiology of infectious diseases72 ma15 infected cc mice had abroad range of phenotypes including weight loss and increasedlung viral titres and lung pathology73 susceptibility varied fromabsence of symptoms to mortality with normallungparenchyma and lung viral titres at dpi that varied over logunits genetic analysis of cc strains identiï¬ed trim55 and ticam2as host susceptibility genes7374 this shows that the geneticbackground of mice is important in replicating distinct humanclinical presentations investigating multiple genetic backgroundsis possible using adenovirustransduced hace2 or mouseadaptedvirusespredisposing risk factors and mouse modelschronic diseases like copd asthma lung ï¬brosis cvd diabetesmellitus as well as obesity male sex and old age are associatedincreased susceptibility to sarscov2 infection and severeobesity diabetes and cvdmouse models of genetic or dietary modiï¬cations to inducefeatures of human disease are widely available8687100101 inbredmouse strains such as c57blb6 have genetic susceptibility toobesity and diabetes while male mice and rats are more pronecompared to females101 ace2 is highly expressed in hearttissues and so it is expected that cvd models willincreasedsusceptibility to sarscov2 infection104 the link between type andor diabetes and ace2 expression is unknown withconï¬icting reports on the levels of expression in diabetesprogression and severity105106 there are numerous mousemodels of type and diabetes that are chemically or geneticallyinduced and for type diabetes typically incorporate obesity107sarscov and sarscov2 infection has not yet been explored inthese models but these mice will likely be highly susceptible toinfection with pulmonary decline and increased mortality asobserved in humansagerelated susceptibilityold age is the main risk factor for severe covid19108 which isattributed to comorbidityimmunosenescence malnutritionresidential care and biological aging changes109 ace2 expressionin human lungs may either be unchanged83 or decreased104 withold age while expression is increased in the olfactory epitheliumwhich may contribute to susceptibility to sarscov2 infection110the signiï¬cance of age is exempliï¬ed in murine models whereinfection of young balbc and c57blb6 mice with sarscovresulted in high viral titres in the upper and lower airways butwith no disease or mortality33 however aged balbc mice hadsigniï¬cant weight loss and severe disease with extensive alveolardamage and bronchiolitis111 ace2 levels are decreased in agedcompared to younger mice which may explain differences indisease severity112 itis unknown whether aged mice aresusceptible to sarscov2 infection however evidence frommucosal immunology __ 0cybsaytilatrommrofinuhtiwipdybssolithgewllewsaipdmorfgnulehtnisertitlarivhghiipdinarbdnasetanbrutilasanehtnisertitlarivetaredomdnasgnulnidevresbomrotsienkotycipdmorfipdmorfneepslsgnulehtnisertitlarivhghiipdybssolithgewmorfyticapacyrotaripserdecuderipdmorfneepsldnayendkiinarbtraehehtnisertitlarivtsedomdnasyawhtapesnopserenummietanniroflnoitaugerpuhghiswohseussitgnuldetcefnifoqesanripdlygoohtapotsihufpÃufpÃecnereferemoctuoesaesidsesodsuoitcefninoitasilacoldnanoisserpxeecahretomorpenilesuomlarivhghiderevocerllatubithgewydobtsoliytidbromonsngislacinilcisuovboongnulehtniecimsertitdctiÃotpuyendkidnaenitsetnininoisserpxehghitraehninoisserpxeetaredomsgnulninoisserpxewolretomorpecambhecahsisenegohtapvocsrasienmaxeotdesusledomesuomeballiavafoyrammuselbatdnanoitammaï¬niinarbdnagnulevisnetxeipdybsertitgnullarivhghiipdybssolithgewufpÃotpuinarbninoisserpxeegamadgnulerevesdnaailihportuendahecimdnubiromhghiipdybesaesidcitamotpmysdnassolithgewlygoohtapotsihybdecnedveisgnulnistnuoclygoohtapotsihllecdnayrotammaï¬ninoitammaï¬nidnasertitlarivgnulevisnetxesyendkidnarevilninoisserpxewoljxofdctiÃwoltraehdnarevilyendkineepslenitsetnillamsninoisserpxeetaredomnoocldnasgnulninoisserpxehghiknitarekotycecahkdedidnaithgewydobtsolecimfonoitroporpufpÃinarbdnatugsgnulninoisserpxehghihfhrotcafnoitpircsnartdaehkrofecahhfhanimal and translational models of sarscov2 infection and covid19md johansen gnullarivhghisyadrevoithgewydobtsolytilatromdetroperondetroperecimsertituffninoisserpxesgnulninoisserpxedetropertonseussithghirehtolsurivoagemotycfolortnoctnetepmocnirednuhfhniretomorpecahvdadegagnuldnaaehcartinarbnisertitlarivhghdahisngislacinilcisuovboonithgewydobtsolecimecimufpÃninoisserpxewolyendkidnaneepsltraehdnayravoinarbenitsetnillamssgnulninoisserpxehghiretomorpecamevitanecadesinamuhrotcevlarivonedamucosal immunology __ 0canimal and translational models of sarscov2 infection and covid19md johansen fig comparison of disease features shared between humans with covid19 and mouse models of sarscov2 both humans and micedisplay similar clinical signs such as weight loss and pneumonia severe infections are often associated with increased proammatorycytokine production accompanied by high viral lung titres which correlates with extensive lung damage and signiï¬cant pulmonary declinehumanised ace2 mice shows that aged mice display greaterdisease severity60 aged mice showed a reduced response toexperimental vaccination for sarscov with lowerlevels ofneutralizing antibodies than young mice113 similar to responsesto many vaccines in older people thus studying aged mice islikely to be pivotal for developing treatments and vaccines forolder peoplewas less efï¬cient with low levels of viral antigen detectable innasal washes from two of six ferrets and infection was mild withno changes in body temperature overall the ferret model ofsarscov2 is limited as the infection is mild with little lrtinvolvement and no reported characteristics of severe disease inhumans such as oedema or ards they may provide a model tostudy sarscov2 transmissionferretsferrets have been extensively used to study uenza a virus iavpathogenesis and transmission since their respiratory tract isanatomically comparable to humans114 ace2 is most abundantlyexpressed on typeii alveolar and glandular epithelial cells in thetrachea and bronchi of ferrets115 ferrets are susceptible to sarscov infection with replication in the urt and lrt increased bodytemperature sneezing and alveolar damage115recently ferret ace2 was shown to contain the critical residuesrequired for binding by sarscov2 rbd118 the animals weresusceptible to sarscov2 infection with viral replication in theurt however only low levels of virus were detected inlungs119120 viral loads in the lung and nasal turbinates peakedat dpi with clearance by and dpi respectively whileinfectious virus was not detected outside the respiratory tract viralrna was found in the intestine saliva urine rectal swabs andfaeces up to 8dpi this suggests that sarscov2 preferentiallyreplicates in the urt of ferrets in line with this disease is mildwith reduced activity and occasional cough from days in moststudies elevated body temperatures are observed from dpireturning to baseline by day with no change in bodyweight119121 shi 120 reported only out of ferretsdeveloped fever and loss of appetite following intranasalinoculation with different sarscov2 isolates suggesting isolatevariability and dose may alter disease outcomes limited studieshave examined immune responses to sarscov2 in ferretsintranasal infection induced serum neutralizing antibodies119121another study compared transcriptional responses in cells fromiav and sarscov2infected ferrets122 thenasal washes ofmagnitude of antiviral and ifn responses were higher with iavcompared to sarscov2 infection with unique enrichment forcell death and leucocyte activation in the latter kim 119 usedthe ferret model to study sarscov2 transmission viral rna wasdetectable in nasal washes from ferrets h after direct contactwith intranasally inoculated animals suggesting transmission israpid and can be transmitted prior to the peak of disease dpi allsix ferrets that had direct contact developed elevated bodytemperatures in contrast to direct contact airborne transmissiongolden hamstersyrian golden hamsters have been used to model a broad rangeof viral infections123 and genetically modiï¬ed animals have beengenerated124 hamsters are susceptible to sarscov infectionwith comparable viral replication in the urt and lrt but noclinical signs of disease other than reduce activity111127128in silico structural analysis predicts that sarscov2 s proteinrbd effectively binds hamster ace2129130 infection of hamsterswith sarscov2 resulted in clinical signs such as rufï¬ed fur rapidbreathing and weight loss from day with recovery by dpi129 high levels of infectious virus were detected in the lungand nasal turbinates on days and and signiï¬cant histologicalchanges were observed including proteinrich lung ï¬uid exudatemononuclear cell ltration severe cell death and haemorrhageand alveolar damage transmission studies revealed efï¬cient virusspread via direct contact between cohoused infected hamsters129 infectious virus was detectable in the lungs and nasalturbinates accompanied by lung histology changes cohousedhamsters did not lose signiï¬cant body weight suggesting theinfection was less severe than with intranasally inoculation likelydue to differences in inoculum dose this suggests that sarscov infection of hamsters is not dissimilar to humans and may be amodel to study pathogenesis and transmission and test potentialtherapeuticsnonhuman primates nhpsnhp experiments account for only of all animal researchnevertheless clinical translation of nhp studies is much greaterthan for other animal models as they are closest to humanp | 0 |
gastrointestinal nematodes could release excretorysecretory es proteins into the host environment to ensure their survival these es proteins act as immunomodulators to suppress or subvert the host immune response via the impairment of immune cell functions especially in chronic infections in our preliminary study haemonchus contortus adhesionregulating molecule hcadrm1 was identified from h contortus es proteins hcesps that interacted with host t cells via liquid chromatographytandem mass spectrometry analysis however little is known about hcadrm1 as an es protein which may play a pivotal role at the parasitehost interfacemethods based on bioinformatics approaches multiple amino acid sequence alignment was conducted and the evolutionary relationship of hcadrm1 with adrm1 orthologues was extrapolated employing rtqpcr and immunohistochemistry assays temporal transcriptional and spatial expression profiles of hcadrm1 were investigated using immunostaining approaches integrated with immunological bioassays the immunomodulatory potentials of hcadrm1 on goat t cells were assessedresults we hereby demonstrated that hcadrm1 with immunodiagnostic utility was a mammalian adrm1 orthologue abundantly expressed at all developmental stages of h contortus given the implications of adrm1 proteins in cell growth survival and development we further investigated the immunomodulatory property of hcadrm1 as an individual es protein acting at the parasitehost interface the rhcadrm1 stimuli notably suppressed t cell viability promoted intrinsic and extrinsic t cell apoptosis inhibited t cell proliferation and induced cell cycle arrest at g1 phase simultaneously rhcadrm1 stimuli exerted critical controls on t cell cytokine secretion profiles predominantly by restraining the secretions of interleukin il4 il10 and interferongammas importantly hcadrm1 protein may have prophylactic potential for antih contortus vaccine development together these findings may contribute to the clarification of molecular and immunomodulatory traits of es proteins as well as improvement of our understanding of parasite immune evasion mechanism in h contortushost biologykeywords h contortus excretorysecretory protein adhesionregulating molecule adrm1 immunomodulation immune evasioncorrespondence lixiangruinjaueducn moe joint international research laboratory of animal health and food safety college of veterinary medicine nanjing agricultural university nanjing jiangsu peoples republic of chinafull list of author information is available at the end of the the authors this is licensed under a creative commons attribution international license which permits use sharing adaptation distribution and reproduction in any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the creative commons licence and indicate if changes were made the images or other third party material in this are included in the s creative commons licence unless indicated otherwise in a credit line to the material if material is not included in the s creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtain permission directly from the copyright holder to view a copy of this licence visit httpcreat iveco mmons licen sesby40 the creative commons public domain dedication waiver httpcreat iveco mmons publi cdoma inzero10 applies to the data made available in this unless otherwise stated in a credit line to the data 0clu a0et a0al parasites vectors page of the highly conserved and regulated ubiquitin ub proteasome pathway is the primary mechanism for targeted elimination of most shortlived proteins including misfolded or damaged proteins in eukaryotic cells ub can covalently attach to cellular proteins by ub modification which is an atpdependent process mediated via different classes of ub enzymes alongside three families of shuttling factors rad23 dsk2 and ddi1 three proteasome subunits located in the subcomplex of 26s proteasome rpn1 rpn10 and rpn13 are demonstrated to be ub receptors as well as the proteasomeassociated polyubiquitin receptor rpn13 also termed as adhesionregulating molecule adrm1 is recruited by rpn2 to be assembled into the 19s regulatory p and target protein substrates linked to the small protein ub via its pleckstrinlike receptor [ ] simultaneously the cterminal adaptor domain of adrm1 serves to bind and activate the deubiquitylase uchl5uch37 and enhance its isopeptidase activity revealing a mechanism to accelerate ub chain disassembly []with engagement in the ub proteasome pathway that regulates a broad range of physiological functions adrm1 is implicated in multitudinous cellular processes such as cell growth migration survival and development particularly in cancer cells recent publications reveal that adrm1 transcription is consistently elevated in ovarian colorectal and gastric cancer tissues and knockdown of adrm1 expression in both human colon carcinoma and gastric cancer cell lines suppress cell migration and proliferation and induces cell apoptosis [] meanwhile fejzo et a0 al demonstrated that overexpression of adrm1 in ovarian cancer promoted cell growth and migration whereas blocking its expression caused cell death given the association of amounting adrm1 expression with the onset and progression of cancers adrm1 has been defined as a potential predictive and therapeutic target for clinical therapy additionally comparable expressions of adrm1 have also been observed in several lymphocyte cell lines as well as endothelial cell lines and similar physiological roles of adrm1 are described through its excessive expression in skin endothelial cells that facilitates t lymphocyte adhesion in a previous study we identified haemonchus contortus excretorysecretory es proteins hcesps that interacted with host t cells via liquid chromatography mass spectrometry lcmsms analysis haemonchus contortus adrm1 hcadrm1 protein a mammalian adrm1 homologue was ascertained among these interacting proteins additionally recombinant hcadrm1 rhcadrm1 was recognized by serum samples obtained at day and postinfection derived from experimentally h contortusinfected goats as a result of these observations hcadrm1 with immunodiagnostic utility was fostered as a hallmark of h contortus infection and a serological diagnosis assay with high sensitivity and specificity was developed using hcadrm1 antigen furthermore our preliminary analysis showed that hcesps stimuli notably induced intrinsic and extrinsic apoptosis suppressed t cell proliferation and caused cell cycle arrested hcesps consisted of multitudinous modulatory molecules such as kinases phosphatases hydrolases and proteases where the pleiotropic effects were initiated by a cascade of individual es components importantly the exact molecules that modulated t cell immune response in the parasitehost interaction warrant further investigation given the functional diversity of adrm1 and especially its engagement in cell proliferation and apoptosis hcadrm1 might be one of these dominated proteins that exert critical controls on cellular survival and death of host key effector cells therefore herein we aimed to further investigate the molecular traits of hcadrm1 and address its immunomodulatory roles at the parasitehost interfacemethodsparasite animals and a0cellsthe h contortus strain was propagated via serial passages in nematodefree goats in the animal experimental center faculty of veterinary medicine nanjing china the collection of eggs l3 xl3 male and female adults was performed as previously described [ ] sprague dawley sd rats scxk with a standard packing weight a0 g were obtained from jiangsu experimental animal center nanjing china they were maintained in a microbefree room with access to sterilized food and water ad libitumlocal crossbred and healthy goats monthsold were reared in individually ventilated cages to prevent accidental infection with nematodes alongside ad libitum access to water in pens these goats were given hay and whole shelled corn daily peripheral venous blood samples were obtained by venipuncture as described elsewhere as well as the isolation of goat peripheral blood mononuclear cells pbmcs total t cells in goat pbmcs were sorted using a magneticactivated cell sorting system macs miltenyi biotech inc auburn ca usa as described elsewhere briefly every million pbmcs in a0µl staining buffer were incubated with µl mouse antibovine cd2 primary antibody biorad kidlington uk which crossreact with goat cd2 t cells for a0min after two washes in pbs à of total pbmcs resuspended in µl staining buffer were labeled 0clu a0et a0al parasites vectors page of with µl antifitc microbeads miltenyi biotech at room temperature for min subsequently pbmcs were loaded onto the macs magnetic system miltenyi biotech for positive sorting based on the manufacturers specifications t cells were then adjusted to a density of à cellsml in rpmi gibco grand island ny usa containing heatinactivated fetal calf serum fcs gibco and a0uml penicillin a0mgml streptomycin gibco the viability of t cells was as assessed by the trypan blue exclusion test the purity of separated t cells was validated via flow cytometric determination additional file a0 figure s1 three goats biological replicates were used in every experimentsequence alignment and a0phylogenetic analysisthe cloning and amplification of the complete coding sequence of the hcadrm1 gene were performed as previously described the amplified hcadrm1 fragment was cloned into pet28a vector invitrogen carlsbad ca usa and validated by sequence analysis using blast multiple amino acid sequences of adrm1 orthologs were aligned for comparison by the clustal omega software the evolutionary analysis was conducted and extrapolated by mega x software using jtt matrixbased model and the maximum likelihood method with partial deletion option positions with less than site coverage were excluded prior to phylogenetic analysis based on the optimized evaluation of replicates for bootstrap support the evolutionary tree of adrm1 orthologues was generated with several designated and collapsed branchesexpression and a0purification of a0the a0rhcadrm1 proteinthe expression and purification of rhcadrm1 proteins were conducted as elsewhere described briefly escherichia coli bl21 de3 cells containing the reconstructed pet28ahcadrm1 plasmid were incubated with luriabertini medium containing kanamycin a0 µgml sigmaaldrich st louis mo usa and then stimulated by isopropylβdthiogalactopyranoside sigmaaldrich for the induction of rhcadrm1 expression the rhcadrm1 protein fused with a histidinetag was obtained from the supernatant of cell lysates via histrap hp purification columns ge healthcare piscataway nj usa rhcadrm1 proteins were resolved on sodium dodecyl sulfatepolyacrylamide gel electrophoresis sdspage gels for size and purity validation and the concentration was determined by a bicinchoninic acid bca assay thermo fisher scientific rockford il usa employing the detoxigel affinity pak prepacked columns thermo fisher scientific rhcadrm1 proteins were exempt of lipopolysaccharide contamination as rhcadrm1 protein was dissolved in pbs pbstreated t cells served as the control group a0 µgml in functional assays the purified rhcadrm1 was stored at a0°c until further analysispreparation of a0polyclonal antibody pabto obtain antigenspecific pab rhcadrm1 proteins a0 µg blended with freunds complete adjuvant was administrated subcutaneously into sd rats with a 2week interval booster immunizations with a0 µg of rhcadrm1 proteins emulsified in freunds incomplete adjuvant were administered four times seven days after the final boost rat sera containing antirhcadrm1 pab were harvested and kept at a0 °c for further analysis the sera harvested from h contortusinfected goats antih contortus serum were stored at the veterinary parasitology teaching and research center of nanjing agricultural university nanjing chinaimmunoblot analysisrhcadrm1 and hcesps were resolved on protein gels respectively and transferred onto nitrocellulose membranes the blots were blocked using bsa in trisbuffered saline01 tween tbst for a0 h at room temperature the blots with the rhcadrm1 samples were probed with primary goat antih contortus serum in tbst or normal goat serum control at a0°c overnight while the blots with the hcesps samples were probed with primary rat antirhcadrm1 igg in tbst or normal rat igg control after five washes in tbst the blots were incubated with horseradish peroxidasecoupled rabbit antigoat or antirat igg hl secondary antibody sigmaaldrich in tbst for h at a0 °c the blots were then developed with ²diaminobenzidine dab sigmaaldrich for min and visualized by using a chemidoc imaging system biorad hercules ca usahcadrm1 transcription in a0h contortus lifecycle stagesto detect mrna expression of hcadrm1 in h contortus lifecycle stages total rna of eggs l3 xl3 female and male adults were extracted using trizol invitrogen and the resulting cdnas were synthesized in accordance with the manufacturers specifications employing specific primers for the βtubulin gene endogenous reference and target gene hcadrm1 additional file a0 table a0s1 transcriptional analysis of the hcadrm1 gene was conducted by realtime pcr using the quantstudio system applied biosystems carlsbad ca usa with a standard protocol the specificity of the primers was 0clu a0et a0al parasites vectors page of validated to ensure product purity via generation of a melt curve and the absence of primer dimers the amplification efficiencies and correlation coefficients were verified to be stable and similar based on the δδcq method the relative transcription levels of hcadrm1 were normalized on βtubulin transcription each experiment was run in triplicateimmunohistochemistry assaysfreshly collected female and male adults were washed dehydrated fixed embedded and cut into cryostat sections as previous described to minimize nonspecific binding cryosections were treated with normal goat serum in pbs containing tween pbst for a0h subsequently cryosections were served with primary antirhcadrm1 igg or sham control igg overnight at a0 °c prior to dna staining with 24amidinophenyl6indolecarbamidine dihydrochloride dapi sigmaaldrich cryosections were then incubated with cy3labeled goat antirat igg beyotime biotechnology shanghai china at a0 °c for a0 h subsequently the samples were immersed in antifade medium sigmaaldrich to prevent fluorescence fading for microscopic examination finally the sections were imaged at à magnification using a lsm710 fluorescence microscope zeiss jena germany and zen software zeiss was used for the analysis of digital imagesthe interaction of a0hcadrm1 protein with a0t cells in vitrothe interaction of hcadrm1 with goat t cells was investigated as previously described in brief freshly sorted t cells were cultured with or without a0 µgml rhcadrm1 proteins for a0h at a0°c after three washes paraformaldehydefixed t cells were permeabilized by triton x100 in pbst and blocked with bsa in pbst for a0min subsequently prior to the staining with the cy3coupled secondary antibody t cells were treated by primary antihcadrm1 pab or normal rat igg control in a humidified chamber at a0°c for a0h followed by five pbst washes t cells were subjected to gold antifade mounting solution containing dapi life technologies eugene or usa for nuclear staining immunofluorescencelabeled cells were visualized at à magnification using a lsm780 confocal microscope zeiss jena germany zen software zeiss was employed for the interpretation of digital imagescell viabilitythe modulatory effects of rhcadrm1 on goat t cell viability were determined using the cell counting kit8 assay cck8 dojindo kumamoto japan as previously described fresh sorted goat t cells activated with concanavalin a cona a0µgml were incubated in the presence of various doses of rhcadrm1 proteins and a0µgml at a0°c following a0hstimulation cell culture medium was incorporated with a0µl cck8 solution and incubated at a0 °c in the dark for a0h following incubation optical density was measured at a0nm od450 using a microplate reader biorad hercules california usa three independent tests each in triplicate were performedcell apoptosis assayflow cytometry assays were performed for t cell apoptosis determination using the annexin vpe kit bd biosciences san jose ca usa as previously described in brief freshly sorted t cells were cultured in the presence of tested doses of rhcadrm1 proteins and a0µgml followed by annexin v and 7aminoactinomycin d 7aad staining based on the kits specification the pbsstimulated t cells served as negative controls three individual tests each in triplicate were conductedcell proliferation assaycell proliferation analysis was determined using the alexa fluor clickit plus edu flow cytometry kit thermo fisher scientific via the measurement of dna synthesis directly based on the manufacturers instructions after a0h coincubation the cell culture was incorporated with 5ethynyl2²deoxyuridine edu a0μm for another a0h incubation subsequently t cells were harvested fixed with paraformaldehyde in pbs and permeabilized using the clickit saponinbased reagent followed by clickit reaction to coupled edu with alexa fluor dye after three washes with a0ml of bsa in pbs t cells were treated with 7aad staining solution bd biosciences flow cytometry was used for the determination of edu cells in the population each experiment consisting of three replicates was run in triplicatecell cycle assayflow cytometry assays were conducted for cell cycle determination using pirnase staining buffer bd biosciences according to the manufacturers dna staining protocol following coincubation with rhcadrm1 stimuli a0 µgml for a0 h t cells were harvested washed and fixed with icecold ethanol every a0 h after being frozen at a0°c for more than a0h treatedt cells were washed twice with pbs to remove remaining ethanol and resuspended in pirnase staining buffer for flow cytometry analysis each experiment consisting of three replicates was run in triplicate 0clu a0et a0al parasites vectors page of transcription analysist cells treated with different concentrations of rhcadrm1 and a0µgml for h were harvested for the transcription analysis of the cell apoptosis pathway and t cells treated with a0µgml of rhcadrm1 for a0h were collected for transcription analysis of the cell cycle pathway cells were harvested for total rna extraction and cdna obtained by reversetranscription pcr relative quantification of candidate gene expression was conducted using previously published primers [] of endogenous reference and candidate genes additional file a0 table a0s2 based on the δδcq method the relative levels of target gene transcription were normalized to reference gene expression each experiment consisting of three replicates was run in triplicatedetection of a0cytokine secretionsfor the determination of cytokine secretion levels freshly isolated t cells activated by cona a0µgml were treated with or without rhcadrm1 and a0µgml for a0h cell culture medium was harvested and determined for cytokine secretion detection using goat enzymelinked immunosorbent assay elisa kits mlbio shanghai china according to the manufacturers specifications the limit of quantification dependent upon each analytic kit ranged from between and a0pgml each experiment was run in triplicatestatistical analysisoneway and twoway analysis of variance anova with dunnetts multiple comparison test alongside the students ttest were performed for statistical analysis using graphpad premier software graphpad prism san diego ca usa differences were regarded as statistically significant when pvalues were data were denoted as minimum to maximum all points or mean ± standard deviation sdresultssequence alignment and a0phylogenetic analysisthe entire coding region of the hcadrm1 gene a0bp was amplified from the cdna of adult worms encoding a 361amino acid protein with an estimated molecular mass around a0 kda we then performed a sequence alignment of adrm1 orthologues derived from genbank on zebrafish human mouse caenorhabditis elegans and h contortus using clustal omega software hcadrm1 protein showed a moderate degree of identity to zebrafish human mouse and c elegans orthologs fig a0 1a in addition we conducted an evolutionary analysis of hcadrm1 using the maximum likelihood method involving amino acid sequences phylogenetic analysis clearly showed an evolutionary relationship of hcadrm1 with other adrm1 orthologues revealing that hcadrm1 was closely related to the teladorsagia circumcincta homologue but divergent from vertebrate sequences fig a01bprotein expression and a0immunoblot analysisthe rhcadrm1 protein fused with the histidinetag was successfully obtained in the supernatant of cell lysates after purification rhcadrm1 was visualized by coomassie blue staining as a single band with a molecular weight of a0kda fig a01c lane the specificity of the rhcadrm1 protein was determined by western blot probing with antih contortus serum or normal goat serum a single band a0 kda was observed through the specific recognition of rhcadrm1 protein by antih contortus serum fig a01c lane while no band was identified via healthy goat sera fig a01c lane meanwhile native hcadrm1 protein derived from hcesps was identified by rat antirhcadrm1 igg as a single band of a0kda fig a01c lane while no positive band was observed in the control groups fig a01c lane differential mrna expression in a0h contortus lifecycle stages and a0immunolocalizationtranscription analysis by realtime rtpcr revealed that mrna expression of hcadrm1 were detectable at all of the tested h contortus lifecycle stages the data demonstrated rising expression levels from the freeliving stages eggs and l3 to parasitic stages xl3 female and male adults simultaneously the highest level of hcadrm1 transcription was observed in male adults but not in female adults fig a01d given that the highest mrna expression was detected in adult worms we next investigated the localization of native hcadrm1 proteins within h contortus by checking the cryosections of the adult worms specific red fluorescence resulting from tagging hcadrm1 proteins by rat antirhcadrm1 igg was ubiquitously observed from the intracellular and cytoplasmic localization of somatic cells particularly in the intestinal regions and the internal membrane of cuticle for both male and female adults fig a01e however no cy3fluorescence was observed in the sections treated with normal rat igg fig a01ebinding of a0rhcadrm1 protein to a0goat t cellsbased on our preliminary lcmsms analysis we next conducted immunocytochemistry assays to verify the in vitro interaction of hcadrm1 proteins with goat t cells immunocytochemistry assay showed that intense red 0clu a0et a0al parasites vectors page of fig molecular characterization of hcadrm1 derived from hcesps a alignment of hcadrm1 amino acid sequences with other orthologues the positions of adrm1 family motifs are indicated above the multiple sequence alignment containing zebrafish xp_0213255291 human np_0012683661 mouse np_0627962 c elegans np_4983872 and h contortus w6nb91 adrm1 ortholog sequences retrieved from genbank an asterisk indicates the position with one completely conserved amino acid while period denotes weakly conserved similarity within different groups and colon represents strongly similar conservation between groups b phylogenetic analysis of hcadrm1 with vertebrate and parasite orthologues evolutionary relationships of taxa were inferred using the maximum likelihood method with protein sequences including mus musculus np_0627962 homo sapiens xp_0115268051 danio rerio xp_0213255291 toxocara canis khn872021 c elegans np_4983872 dictyocaulus viviparus kjh490541 t circumcincta pio739301 h contortus w6nb91 oesophagostomum dentatum khj973301 and ancylostoma caninum rcn481761 bootstrap support values are shown for each node the scalebar denotes the number of substitutions per site c acquisition of rhcadrm1 proteins and western blot analysis lanes m protein standard ladder lane rhcadrm1 expressed in the supernatant of cell lysates lane sdspage analysis of purified rhcadrm1 protein lane immunoblot analysis of rhcadrm1 using antih contortus serum as primary antibody lane immunoblot analysis of rhcadrm1 using normal goat serum control as primary antibody lane immunoblot analysis of hcesps using rat antirhcadrm1 igg as primary antibody lane immunoblot analysis of hcesps using normal rat igg control as primary antibody d hcadrm1 expression in h contortus lifecycle stages data are presented as the mean ± sd e immunolocalization of native hcadrm1 protein in male and female adults the immunohistochemistry assays were performed using normal rat igg control or rat antirhcadrm1 igg as primary antibody cy3coupled fluorescence red along with dapi blue was identified for the investigation of hcadrm1 distribution scalebars e µm 0clu a0et a0al parasites vectors page of cy3fluorescence resulting from tagging rhcadrm1 was observed in rhcadrm1treated t cells revealing the cytomembrane and cytoplasmic localization of rhcadrm1 fig a0 2a a1 no red fluorescence was detected in both blank and negative control groups fig a0 2a a2 and a3 the results presented here further validated the positive interactions between hcadrm1 protein and host t cellsrhcadrm1 suppressed cell viability and a0induced cell apoptosisgiven the modulatory potential of adrm proteins on cellular development and survival we next investigated the impact of rhcadrm1 proteins on t cell viability the results of cck8 determination showed that t cell viability was dramatically inhibited by the stimulation of a0 µgml anova f4 p a0 µgml anova f4 p a0µgml anova f4 p and a0 µgml anova f4 p of rhcadrm1 proteins fig a02b based on this finding an annexin vpe7aad double staining kit was employed to evaluate the proapoptotic potential of rhcadrm1 proteins flow cytometry results demonstrated that rhcadrm1 stimuli at the tested concentrations of a0 µgml anova f4 p a0 µgml anova f4 p and a0 µgml anova f4 p remarkably caused t cell apoptosis in comparison to the unstimulated group fig a0 2c d additionally transcriptional analysis of key genes involved in apoptosis signaling pathways further validated the proapoptotic effects of rhcadrm1 proteins on host t cells the treatments with and a0µgml of rhcadrm1 dramatically upregulated mrna transcripts of caspase8 anova f4 p p and p respectively and caspase3 anova f4 p p and p respectively fig a0 2e simultaneously a0 µgml anova f4 p and µgml anova f4 p of rhcadrm1 proteins significantly promoted caspase9 transcription fig a02erhcadrm1 protein restrained the a0proliferation of a0t cells and a0caused cell cycle stallingas apoptosis proliferation and cell cycle were interconnected cellular movements we next explored the modulatory potentials of rhcadrm1 stimuli on t cell proliferation and cell cycle at the tested doses of and a0 µgml flow cytometry data showed that rhcadrm1 stimuli significantly inhibited t cell proliferation in vitro fig a03a as indicated by the decreasing proportion of edu cells compared with control cells anova f4 p p and p respectively fig a0 3b given that the treatments with a0µgml rhcadrm1 had significant biological effects on cell viability apoptosis and proliferation as well as the transcription of certain key genes we next treated t cells with a0µgml of rhcadrm1 for cell cycle determination here flow cytometry analysis with pi staining demonstrated that rhcadrm1 stimuli induced cell cycle arrest in a timedependent manner fig a0 3c as indicated by the increased proportion of t cells in g1 phase at a0h anova f8 p a0h anova f8 p and a0h anova f8 p as well as the decreased proportion of t cells in s phase at a0 h anova f8 p a0 h anova f8 p and a0h anova f8 p fig a0 3d consistent with these findings transcriptional analysis of key genes in g1s checkpoints showed that mrna transcripts of ccne1 ttest t16 p and cdk2 ttest t16 p were significantly downregulated by rhcadrm1 stimuli while no significant transcriptional changes of ccnd1 ttest t16 p cdk4 ttest t16 p and cdk6 ttest t16 p were observed fig a03e given the inhibitory effects of p21 and p27 on cdks in ubmediated cell cycle progression the transcription analysis of p21 and p27 was performed in this study in addition the mrna transcripts of iκbα as the physiological substrate of adrm1 and its downstream inhibitor nfκb were determined importantly transcription of p21 ttest t16 p p27 ttest t16 p and iκbα ttest t16 p was notably enhanced by rhcadrm1 stimuli fig a0 3e whereas the mrna transcript of nfκb ttest t16 p was significantly suppressed fig a03edetermination of a0cytokine secretionsto investigate the modulatory effects of rhcadrm1 on t cell cytokine productions il2 il4 il10 il17a ifnγ and tgfβ1 secretions in the cell culture supernatant were determined via elisa assays the data showed that the exposure of goat t cells to rhcadrm1 proteins led to the alteration of their cytokine production profiles intriguingly at the tested doses of and a0µgml rhcadrm1 stimuli predominantly inhibited secretions of il4 anova f4 p p and p respectively anova f4 p p and p respectively and ifnγ anova f4 p p and p respectively fig a0 4b c e however all the tested doses of rhcadrm1 had no notable effects on secretions of il2 anova f4 p p p and p il10 0clu a0et a0al parasites vectors page of fig rhcadrm1 proteins suppressed cell viability and induced apoptosis via the interaction with goat t cells a determination of the interaction of hcadrm1 protein with goat t cells in vitro t cells treated with a1 or without a2 rhcadrm1 protein were incubated with rat antirhcadrm1 igg as the primary antibody t cells stimulated by rhcadrm1 were incubated with normal rat igg as the primary antibody a3 b rhcadrm1 significantly inhibited t cell viability cell viability was determined via the incorporation with cck8 whereas cell viability index was determined by calculating the od values of the control group as results are presented as the mean ± sd asterisks denote statistically significant differences p p p compared with the control group c flow cytometry analysis of t cell apoptosis in responses to rhcadrm1 stimuli apoptosis determination was performed via 7aad and annexin vpe staining d statis | 0 |
" rectus sheath block rsb is known to attenuate postoperative pain and reduce perioperative opioidconsumption thus a retrospective study was performed to examine the effects of bilateral rectus sheath blockbrsb in cytoreductive surgery crs combined with hyperthermic intraperitoneal chemotherapy hipecmethods a total of patients undergoing crshipec at our hospital were included patient information andanaesthesiarelated indicators were collected from the electronic medical record emr system all subjects weredivided into the following two groups the g group general anaesthesia and the gr group rsb combined withgeneral anaesthesia patients in the gr group received ropivacaine for brsb before surgery the primaryoutcomes included the total amount of remifentanil and rocuronium the total consumption of dezocine aftersurgery the visual analogue scale vas score and the patientcontrolled intravenous analgesia pcia input dose at h t6 h t7 h t8 h t9 and h t10 after surgery other outcomes were also recorded such aspatient demographic data the intraoperative heart rate hr and mean arterial pressure map and postoperativecomplicationsresults compared with the g group the gr group showed a shorter time to tracheal extubation p adecreased total amount of remifentanil and rocuronium p and a reduced vas score pcia input dose andnumber of pcia boluses at h h and h after surgery p however at h and h after surgery therewere no differences in the vas score of pain at rest or during motion between the two groups p moreoverthe incidence of hypertension emergence agitation delayed recovery hypercapnia and nausea and vomiting waslower in the gr group than in the g group p there were no differences in the changes in map and hrduring the surgery between the two groups p no complications associated with nerve block occurred brsb could provide shortterm postoperative analgesia reduce perioperative opioid consumption andreduce the incidence of postoperative complications it is an effective and safe procedure in crshipeckeywords cytoreductive surgery hyperthermic intraperitoneal chemotherapy rectus sheath block generalanaesthesia analgesia correspondence trmzltz126comdepartment of anesthesiology beijing shijitan hospital capital medicaluniversity beijing china the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cwang bmc anesthesiology page of radical cytoreductive surgery crs combined withhyperthermic intraperitoneal chemotherapy hipec isconsidered a standard for the treatment of peritonealcancer such as rectal cancer ovarian cancer peritonealpseudomyxoma and peritoneal mesothelioma thistechnique could prolong the longterm survival of patients with a decreased recurrence rate although thepositive results of this treatment have been proven inprevious studies [] because of the large peritonealsurface area involved in this kind of surgery crshipecis time consuming and complex which presents agreat challenge for the anaesthesiologist in terms of perioperative managementdue to the stable respiratory and circulatory supportgeneral anaesthesia is the preferred choice in this surgery however long periods of general anaesthesia leadto drug accumulation in the body followed by increasedanaesthesiarelated complications including delayed recovery respiratory inhibition and cognitive dysfunction consequently exploring better anaesthesia methodsfor this surgery is still a major concerna new approach called ultrasoundguided bilateral rectus sheath block brsb has been proven to amelioratepostoperative pain and reduce the consumption of morphine [] nonetheless there have been no reportson the application of general anaesthesia combined withbrsb in patients undergoing crshipec based on theinformation presented above this retrospective observational study was conducted to examine the efficacy andsafety of brsb in patients treated with crs and hipecmethodssubjectsall patients who underwent crs and hipec at beijingshijitan hospital between august and december were retrieved from the institutional database inthis study the exclusion criteria were as follows laparoscopic surgery with crshipec intraoperativeblood loss volume greater than ml mechanicventilation required after surgery and use of analgesictechniques apart from brsb and general anaesthesiaaccording to this standard a total of patients wereincluded and divided into the following groups generalanaesthesia g group n and general anaesthesiacombined with posterior rsb gr group n anaesthesia methodgeneral anaesthesia was consistently induced in all patients with intravenous propofol mgkg sufentanil μgkg and rocuronium mgkg invasive arterialpressure and central venous pressure were monitored byradial artery puncture flotracvigileo® edwards lifesciences irvine ca usa and internaljugular veinsitetargeteffectpuncture respectively after anaesthesia induction anaesthesia was maintained with sevoflurane and remifenconcentration ngmltanilkeeping the bispectral index bis between and rocuronium mgkg wasintermittently used tomaintain muscle relaxation in the gr group before anaesthesia induction patients received brsb under ultrasound guidance the puncture site was placed at theouter edge of the bilateral rectus abdominis at the levelof the umbilicus fig a a total of ropivacaine ml was injected into each side the spindleshapedspread of ropivacaine was observed between the posterior sheath of the rectus abdominis and the rectus abdominis itself implying success of the procedure fig b c patientcontrolled intravenous analgesia pciawas applied in both groups after the surgery sufentanil μgkg palonosetron hydrochloride mg was diluted to ml the dose was mlh and asingle dose was mlh with a 15min lockout intervalafter the surgery all patients were sent to the surgicalintensive care unit sicu if the visual analogue scalevas score at rest after surgery was ¥ dezocine mgwas used as a rescue analgesicdata collectionall the indicators we needed were obtained from theemr system the records included patient demographicdata patient medical history american society of anesthesiologists asa grade and new york heart association nyha grade the hr and map were recordedat the time before brsb t1 the time of anaesthesiat2 the time of skin incision t3 the time of peritoneal thermochemotherapy t4 and the end of surgeryt5 in addition the duration of the surgery time totracheal extubation the time after skin closure totalamount of remifentanil and muscle relaxants total fluidvolume urine volume and the total volume of allogeneicerythrocytes and plasma infused during the surgery wereall recorded moreover after surgery the occurrence ofhypertension the systolic blood pressure dropped bymore than of baseline blood pressure beforeanesthesia or the sbp mmhg during surgery nausea and vomiting hypoxemia spo2 or pao2 mmhg hypercapnia paco2 mmhg and emergence agitation during the recovery period were recorded the recovery period was considered as the timefrom switching off inhalation anaesthetics remifentaniland muscle relaxant to recovery of the patients abilitiesto command movement orientation as well as conscious state when the recovery period of patients is beyond min it was considered as delayed recovery thevas score for pain at rest and during motion the pciainput dose and the number of boluses at h t6 ht7 h t8 h t9 and h t10 after surgery 0cwang bmc anesthesiology page of the incomplete datachemotherapy crshipec during the year from to in our hospital one hundred and six patientsreceived brsb seventeen patients were excluded because ofincluding patientsundergoing intraoperative haemorrhage blood ml and other patients received mechanic ventilationbecause of acute respiratory distress syndrome ardsallergic shock and cardiac insufficiency thus patients with brsb were eventually obtained finally patients without brsb were randomly selected to analysis in this study fig statistical analysisspss software was used for statistical analysisnormal distribution data were recorded as the mean ±standard deviation sd and analysed by independentsamples t test for comparison between the two groupsnonnormally distributed data are presented as themedian range and were analysed by kruskalwallistest chisquared test or fishers exact test was used forcategorical data a p value of was considered statistically significantresultscharacteristics of study populationin total patients were included in the study thebaseline demographic and surgical variables of patientsare presented in table there were no significant differences in age sex body mass index bmi basic diseases asa grade nyha grade total surgery time totalfluid volume urine volume total volume of allogeneicerythrocyte infusion or total volume of plasma p however the time to tracheal extubation was shorter inthe gr group than in the g group p the totalamount of both remifentanil and rocuronium used wasless in the gr group than in the g group p thus posterior rsb could reduce the use of remifentaniland rocuronium during surgerychanges in haemodynamic parametersthe changes in hr and map are presented in table there were no significant differences in hr or map atany point in time t1 to t5 between the two groupsp the results suggest that brsb did not affectthe haemodynamics ofthe patient undergoing crshipecpainrelated indicatorstable shows the postoperative vas score the pca input dose and the number of pca boluses at h t6 ht7 h t8 h t9 and h t10 after surgeryas well as the dose of dezocine used as a rescue analgesic from t6 to t8 compared with the g group thegr group showed significantly decreased vas scores offig ultrasoundguided brsbas well as the dose of dezocine used as a rescue analgesic were also recordedin addition brsbrelatedcomplications such as peritoneal punctureinternalan injury and systemic toxicity were all recordeda total of patients underwent cytoreductive surintraperitonealcombined withgeryhyperthermic 0cwang bmc anesthesiology page of fig flow chart showing patient consecutive enrolment and analysis abbreviations crshipec cytoreductive surgery and hyperthermicintraperitoneal chemotherapy ga general anesthesia brsb bilateral rectus sheath block ards acute respiratory distress syndrome vas visualanalogue scalepain at rest and during motion p however at h and h after surgery there were no significant differences in the vas scores of pain at rest and duringmotion between the two groups p from t6 tot10 the pcia input dose and the number of pca boluses were also obviously reduced in the gr group compared with the g group p in addition as arescue analgesic the dose of dezocine after surgery inthe gr group was significantly lower than that in the ggroup p postoperative adverse eventsadverse events that occurred in the sicu are presentedin table after surgery there were cases withhypertension cases of emergence agitation cases ofdelayed recovery cases of hypercapnia and cases ofnausea and vomiting in the g group fewer cases of allof these events occurred in the gr group p there were no differences in the incidence of hypoxemiabetween the two groups p there were no complications associated with nerve block in either group 0cwang bmc anesthesiology page of table demographic and surgical variables mean ± sdage ysex malefemalebmi kgm2medical historydiabetes mellitus n yesnohypertension n yesnocoronary heart disease n yesnoasa grade iiiiiinyha grade iiitotal surgery time mintime to tracheal extubation minremifentanil mgrocuronium mgtotal fluid volume mlurine volume mltotal volume of allogeneic erythrocyte infusion mltotal volume of plasma mlg group n ± ± ± ± ± ± ± ± ± ± gr group n ± ± ± ± ± ± ± ± ± ± p value asa american society of anesthesiologists bmi body mass index calculated as weight in kilograms divided by height in metres squared nyha new york heartassociation g general anaesthesia gr bilateral rectus sheath block combined with general anaesthesia before bilateral rectus sheath block t1 the time ofanaesthesia t2 the time of skin incision t3 the time of peritoneal thermochemotherapy t4 and the end of surgery t5discussionin this retrospective study we examined the efficacy andsafety of brsb combined with general anaesthesia in patients undergoing crshipec regarding efficacy theresults show that ultrasoundguided brsb significantlyreduced the total dose of remifentanil used during thesurgery and shortened the time to tracheal catheter extraction which is consistent with the findings of previous studies of other surgeries [ ] in addition rsbreduced the total dose of rocuronium in this studytable haemodynamic parameters in both groups mean ± sdindextimepointgn ± grn ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± pvaluemmhghrbpmt1t2t3t4t5t1t2t3t4t5map mean arterial pressure hr heart rate g general anaesthesia gr bilateralrectus sheath block combined with general anaesthesiawhich may be associated with the high concentration ofropivacaine used in the studyrsb also effectively relieved postoperative pain in thisstudy we found that the vas scores of pain at rest andduring motion were all lower in the gr group than inthe g group at h after surgery however at h and h after surgery there were no differences in the vasscores of pain at rest and during motion between thetwo groups suggesting that the analgesic effects of asingle brsb remained within h after surgery thisresult may be different from the findings of others cho reported that at h after surgerythere were no differences in the vas scores of painat rest and during motion between the rsb and nonrsb groups the discrepant results may be related todifferences in the concentration of ropivacaine andthe physical constitution of patients a high concentration can prolong the duration of action of a localanaesthetic in this study we selected not ropivacaine additionallythese patientsundergoing crshipec may have been adaptive topain furthermore compared with the control groupthe rsb group showed a reduced totalinfused doseof sufentanil as pcia number of pca boluses within h after surgery and total dose of dezocine used asa rescue analgesic after surgery these results furtherprove the role of rsb in providing shortterm postoperative analgesia 0cwang bmc anesthesiology page of table painrelated indicators in both groups median [range]vas score of pain at rest [median range]t6t7t8t9t10vas score of pain during motion [median range]t6t7t8t9t10total infused dose of pcia [ml median range]t6t7t8t9t10cumulative number of pcia boluses [median range]t6t7t8t9t10total dose of dezocine as a rescue analgesic mggn [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] ± grn [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] ± p value vas visual analogue scale pcia patientcontrolled intravenous analgesia g general anaesthesia gr bilateral rectus sheath block combined with generalanaesthesia the time at h after surgery t6 h after surgery t7 h after surgery t8 h after surgery t9 and h after surgery t10table postoperative adverse events in both groupsadverse eventsn gn grn hypertensionemergence agitation delayed recoveryhypoxemiahypercapnia nausea and vomiting peritoneal punctureinternal an injurysystemic toxicity g general anaesthesia gr bilateral rectus sheath block combined withgeneral anaesthesiawe also examined the safety of rsb during the surgery ultrasoundguided brsb had no significant effectson the haemodynamics of patients during surgery compared with general anaesthesia alone in terms of postoperative adverse events the results show that comparedwith the control group the rsb group showed a reducedincidence of hypertension emergence agitation delayedrecovery hypercapnia and nausea and vomiting whichmight be correlated with the decreased analgesic andmuscle relaxant doses no rsbrelated complications occurred in any patient these data indicate that rsbcould reduce the risk of complications associated withgeneral anaesthesia and is safe for patientsrsb an established technique has regained popularityin clinical applications [] previous studies havedemonstrated that this technique could achieve relaxation of the anterior abdominal wall [ ] bashandyreported that anterior branches of the t7t12 thoracicnerve and the l1 lumbar nerve travelled through thepvalue 0cwang bmc anesthesiology page of plane of the transverse abdominis muscle entered the rectus abdominis sheath and distributed on the surface of theskin the main process of rsb is to inject local anaesthetics between the rectus abdominis and the posteriorsheath of the rectus abdominis therefore rsb exerteda good effect in terms of perioperative analgesia for medianabdominal incisions a midline incision is required inthis kind of surgery thus based on these results rsbcould meet the need for analgesia in these patientsin addition for a long time epidural analgesia eawas thought to be an effective method for abdominalsurgery [] studies have proved that epidural analgesia could maintain a good analgesic effect and reduceperioperative opioid consumption including in this typeof surgery [ ] however the safety of ea in crshipec remains controversial especially regarding effectson coagulation and circulatory function coagulationdysfunction and profound fluid loss are the main characteristics of patients with peritoneal cancer [ ]which might limit the administration of eaalthough epidural catheter is standard of care insolankis guideline in our hospital epidural catheter in not the standard of care we performed generalanesthesia combined with epidural anesthesia in somepatients to reduce the consumption of intravenous drugsand provide perfect analgesia but coagulation dysfunction and profound fluid loss are the main characteristicsof patients with peritoneal cancer in our previous studywe found that the mean arterial pressure of patientsundergoing epidural anesthesia was difficult to be maintained in the surgery besides there were many patientswith coagulation dysfunction before surgery who werenot suitable for the epidural anesthesia these results wefound in clinical were similar with others researcheskajdi and colleagues reported a case of epidural haematoma in their study godden found that the incidence of hypotension in the ea group was obviouslyhigher than that in the rsb group consequentlyrsb could be a better choice than ea in crshipecadditionally there are some limitations to this studyfirst all the data of this study were collected from theemr system as this was a retrospective study the findings are not as persuasive as those of a randomized controlled study we plan to conduct prospective studies toexplore the comprehensive influence of rsb in this surgery second we only examined the application of brsbestablished with a single injection which provides only ashortterm analgesic effect the efficacy of continuousanalgesia with brs catheters in crshipec remains unclear and needs further exploration third in our the results are initially presented according to the different aspects the primary outcome of this study is thetotal consumption of remifentanil during the surgeryother indicators were belonged to second outcomessin brsb could provide good postoperativeanalgesia reduce perioperative opioid consumption andreduce the incidence of postoperative complicationsthis is an easily applicable and safe procedure in crshipecabbreviationsrsb rectus sheath block brsb bilateral rectus sheath blockcrs cytoreductive surgery hipec hyperthermic intraperitonealchemotherapy emr electronic medical record vas visual analogue scalepcia patientcontrolled intravenous analgesia hr heart rate map meanarterial pressure bis bispectral index sicu surgical intensive care unitasa american society of anesthesiologists nyha new york heartassociation spo2 pulse oximetry paco2 partial pressure of carbon dioxidepao2 oxygen partial pressure bmi body mass indexacknowledgementsi would like to express my heartfelt thanks to the staff of the informationdata center of beijing shijitan hospital affiliated to capital medical universityauthors contributionswsh study design data collection writing paper lpf gt data collectionand data analysis gl coordinated the study and manuscript revision ltzstudy design and manuscript revision all authors read and approved thefinal manuscript all authors ensure the accuracy of the manuscript andagree to take personal responsibility for their contributionsfundingno fundingavailability of data and materialsthe datasets used andor analyzed during the current study are availablefrom the corresponding author on reasonable requestethics approval and consent to participatethis study was approved by the ethics committee of beijing shijitan hospitalaffiliated to capital medical university approval code research ethicsno69 this study is retrospective only anonymous data sources were usedand informed consent was not requiredconsent for publicationnot applicablecompeting intereststhe authors declare that they have no competing interestsreceived january accepted july referencesklaver ce musters gd bemelman wa punt cj verwaal vj dijkgraaf mgaalbers ag van der bilt jd boerma d bremers aj adjuvanthyperthermic intraperitoneal chemotherapy hipec in patients with coloncancer at high risk of peritoneal carcinomatosis the colopec randomizedmulticentre trial bmc cancer 201515undefined428van oudheusden tr braam hj nienhuijs sw wiezer mj van ramshorst bluyer md lemmens ve de hingh ih cytoreduction and hyperthermicintraperitoneal chemotherapy a feasible and effective option for colorectalcancer patients after emergency surgery in the presence of peritonealcarcinomatosis ann surg oncol passot g vaudoyer d villeneuve l kepenekian v beaujard ac bakrin ncotte e gilly fn glehen o what made hyperthermic intraperitonealchemotherapy an effective curative treatment for peritoneal surfacemalignancy a 25year experience with procedures j surg oncolarjonasánchez a barrios p boldoroda e camps b carrascocampos jmartÃn vc garcÃafadrique a gutiérrezcalvo a morales r ortegapérez g hipect4 multicentre randomized clinical trial to evaluate safety andefficacy of hyperthermic intraperitoneal chemotherapy hipec with 0cwang bmc anesthesiology page of huepenbecker sp cusworth se kuroki lm lu p samen cd woolfolk cdeterding r wan l helsten dl bottros m continuous epiduralinfusion in gynecologic oncology patients undergoing exploratorylaparotomy the new standard for decreased postoperative pain and opioiduse gynecol oncol teoh da hutton mj else s walker a lee a mack la epidural analgesia aprospective analysis of perioperative coagulation in cytoreductive surgeryand hyperthermic intraperitoneal chemotherapy am j surg schmidt c creutzenberg m piso p hobbhahn j bucher m perioperativeanaesthetic management of cytoreductive surgery with hyperthermicintraperitoneal chemotherapy anaesthesia kajdi me beckschimmer b held u kofmehl r lehmann k ganter mtanaesthesia in patients undergoing cytoreductive surgery withhyperthermic intraperitoneal chemotherapy retrospective analysis of asingle centre threeyear experience world j surg oncol solanki sl mukherjee s agarwal v thota rs balakrishnan k shah sb desain garg r ambulkar rp bhorkar nm society of oncoanaesthesia andperioperative care consensus guidelines for perioperative management ofpatients for cytoreductive surgery and hyperthermic intraperitonealchemotherapy crshipec indian j anaesth godden ar marshall mj grice as daniels ir ultrasonography guidedrectus sheath catheters versus epidural analgesia for open colorectal cancersurgery in a single centre ann r coll surg engl publishers notespringer nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliationsmitomycin c used during surgery for treatment of locally advancedcolorectal carcinoma bmc cancer baratti d kusamura s iusco d gimondi s pietrantonio f milione mguaglio m bonomi s grassi a virzì s hyperthermic intraperitonealchemotherapy hipec at the time of primary curative surgery in patientswith colorectal cancer at high risk for metachronous peritoneal metastasesann surg oncol chua tc robertson g liauw w farrell r yan td morris dl intraoperativehyperthermic intraperitoneal chemotherapy after cytoreductive surgery inovarian cancer peritoneal carcinomatosis systematic review of currentresults j cancer res clin oncol li y zhou yf liang h wang hq hao jh zhu zg wan ds qin lx cui szji jf chinese expert consensus on cytoreductive surgery andhyperthermic intraperitoneal chemotherapy for peritoneal malignanciesworld j gastroenterol willschke h bosenberg a marhofer p johnston s kettner sc wanzel o kaprals ultrasonographyguided rectus sheath block in paediatric anaesthesiaanew approach to an old technique br j anaesth azemati s khosravi mb an assessment of the value of rectus sheath blockfor postlaparoscopic pain in gynecologic surgery j minim invasive gynecol dingeman rs barus lm chung hk clendenin dj lee cs tracy s johnsonvm dennett kv zurakowski d chen c ultrasonographyguided bilateralrectus sheath block vs local anesthetic infiltration after pediatric umbilicalhernia repair a prospective randomized clinical trial jama surg relland lm tobias jd martin d veneziano g beltran rj mckee c bhalla tultrasoundguided rectus sheath block caudal analgesia or surgical siteinfiltration for pediatric umbilical herniorrhaphy a prospective doubleblinded randomized comparison of three regional anesthetic techniques jpain res 201710undefined2629 xu l hu z shen j pm mq efficacy of usguided transversus abdominisplane block and rectus sheath block with ropivacaine anddexmedetomidine in elderly highrisk patients minerva anestesiol cho s kim yj jeong k moon hs ultrasoundguided bilateral rectus sheathblock reduces early postoperative pain after laparoscopic gynecologicsurgery a randomized study j anesth li t ye q wu d li j yu j doseresponse studies of ropivacaine in bloodflow of upper extremity after supraclavicular block a doubleblindrandomized controlled study bmc anesthesiol bell jc rylah bg chambers rw peet h mohamed f moran bjperioperative management of patients undergoing cytoreductive surgerycombined with heated intraperitoneal chemotherapy for peritoneal surfacemalignancy a multiinstitutional experience ann surg oncol landmann a visoiu m malek mm development of a novel technique forbilateral rectus sheath nerve blocks under laparoscopicguidance j pediatrsurg kumar a wilson ga engelhardt te ultrasound guided rectus sheathblockade compared to perioperative local anesthetic infiltration in infantsundergoing supraumbilical pyloromyotomy saudi journal of anaesthesia bashandy gm elkholy ah reducing postoperative opioid consumption byadding an ultrasoundguided rectus sheath block to multimodal analgesiafor abdominal cancer surgery with midline incision anesthesiol pain med201443e18263 dowidar aerm ezz haa shama aae eloraby ma postoperative analgesiaof ultrasound guided rectus sheath catheters versus continuous woundcatheters for colorectal surgery a randomized clinical trial egypt journalof anaesth karaarslan e topal a avci o tuncer uzun s research on the efficacy of therectus sheath block method agri piccioni f casiraghi c fumagalli l kusamura s baratti d deraco m arientif langer m epidural analgesia for cytoreductive surgery withperitonectomy and heated intraperitoneal chemotherapy int j surg 16pt a99 vesterandersen m lundstrøm lh møller mh the association betweenepidural analgesia and mortality in emergency abdominal surgery apopulationbased cohort study acta anaesthesiol scand httpsdoi101111aas13461 0c" | 0 |
Association of variant on the promoter of cluster ofdifferentiation in graves disease and gravesophthalmopathyYuHuei Liu123 ChiouYuan Shen1 and FuuJen Tsai3451Graduate Institute of Integrated Medicine China Medical University Taichung Taiwan 2Drug development center China Medical University Taichung Taiwan 3Department ofMedical Genetics and Medical Research China Medical University Hospital Taichung Taiwan 4Department of Pediatrics China Medical University Hospital Taichung Taiwan5School of Chinese Medicine China Medical University Taichung TaiwanCorrespondence YuHuei Liu yuhueiliumailcmuedutwThe macrophage migration inhibitory factor MIFcluster of differentiation CD74plays a role in immunological functions The present study aims to investigate whethersinglenucleotide polymorphisms SNPs in the MIF and CD74 are risk factors for developing Graves ophthalmopathy GO in patients with Graves disease GD A casecontrolstudy enrolled patients with GD with and without GO and healthy individuals SNPs were discriminated using realtime polymerase chain reaction HardyWeinbergequilibrium as well as frequencies of allele and genotype between GD patients with andwithout GO were estimated using the Chisquare test The effects of CD74 on adipocyteproliferation and differentiation were evaluated using 3T3L1 preadipocytes QuantitativeDNAimmunoprecipitation was used to detect the binding capacity of NR3C1 and FOXP3to AG oligonucleotides The results showed that individuals carrying the GG genotype atrs2569103 in the CD74 had a decreased risk of developing GD P3390 oddsratio OR confidence interval CI however patients with GDcarrying the AG genotype at rs2569103 in the CD74 had an increased risk of developing GOP0009 OR CI The knockdown of CD74 reduced adipocyteproliferation and differentiation NR3C1 had a higher affinity for A whereas FOXP3 had ahigher affinity for G of rs2569103 The results suggested the existence of a link between thegenetic variation of CD74 promoter and the risk for developing GD and GO which shouldbe considered in clinical practiceBackgroundGraves disease GD a complex autoimmune disorder that occurs more often in women is characterized by the presence of autoantibodies and thyroidstimulating immunoglobulins targeting thethyroidstimulating hormone receptor to stimulate both thyroid hormone synthesis and thyroid glandgrowth and results in hyperthyroidism and its accompanying features [] Graves ophthalmopathyGO is one common anspecific complication affecting of patients with GD [] Activation oforbital fibroblasts through proliferation and differentiation into adipocytes and myofibroblasts is thoughtto play a major role in the generation of the extracellular matrix During inflammatory cell infiltrationand edema the activation augments the volume of tissues surrounding the eyes which in turn leads to anincrease in intraocular pressure []Genetic predispositions epigenetic regulations and environmental factors are risk factors for GD andGO [] Representative studies shed new light on the pathogenesis of GD such as thyroid antigensthyroidstimulating hormone receptor and human leukocyte antigen HLA class I and II regions []However the genomewide approaches to determining the relative risks of developing GO are relativelyReceived June Revised July Accepted July Accepted Manuscript online August Version of Record published August The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20202072101042BSR20202072limited [] Candidate gene approaches revealed that polymorphisms of genes involved in immune response andinflammation might be linked to the development of GO []Cluster of differentiation CD74 encoded by CD74 is an HLA class II histocompatibility antigen gamma chainalso known as HLADR antigenassociated invariant chain and a signaltransducing receptor of macrophage migration inhibitory factor MIF that maintains cell proliferation and survival [] The singlenucleotide polymorphisms SNPs in HLA class II and MIF play a role in the development of GD [] Conversely the chromosome5q3133 region where CD74 is located 5q32 may play a pivotal role in the development of GD and could be thesusceptibility region for developing GD [] Results from mRNASeq also reveal CD74 as a novel signature fD However to our knowledge there is no study on the putative impact of CD74 locus variations on the risk ofGD or GO In an attempt to contribute to the understanding of the pathogenic processes underlying GD and GO acasecontrol study was designed to evaluate the association between SNPs in the upstreamdownstream regulatoryregion of the MIFCD74 axis and the risk of developing GD and GOMethodsPatients healthy individuals and DNA isolationThe study followed the Declaration of Helsinki and was approved by the Medical Ethics Committee of China MedicalUniversity Hospital DMR100IRB144 CMUH103REC2071 A total of patients with GD females100males mean age y range y at enrollment from the China Medical University Hospital and patients had GO and did not All participants provided written informed consent Detailed descriptions of theinclusionexclusion criteria blood drawing and handling genomic DNA storage and quality assurance have beendescribed [] SNP data for ethnicitymatched healthy individuals were obtained from the Taiwan biobankSNP selection and genotypingSNPs were selected based on the following criteria i a threshold minor allele frequency MAF in the Asian population of ii primerprobe set passed by the manufacturer criteria to ensure a high genotyping success rate andiii SNP data for healthy individuals could be obtained without imputation from the Taiwan biobank Four SNPsnamely rs476240 and rs507715 in the downstream region of MIF which is also the upstream region of MIF antisense RNA [MIFAS1] as well as rs13175409 and rs2569103 in the upstream region of CD74 were analyzedGenotyping using specific primerprobe sets have been described previously []Cell cultureThe human HEK293 cells and mouse 3T3L1 preadipocytes were obtained from Bioresource Collection and Research Center BCRC Hsinchu Taiwan and maintained in Dulbeccos modified Eagles medium DMEM Thermo Fisher Scientific Waltham MA USA with fetal bovine serum Uml penicillin and μgml streptomycin and mM Lglutamine at ¦C in a humidified atmosphere of CO2CD74 knockdownShort hairpin RNAs shRNAs obtained from the RNAi core Academia Sinica Taipei Taiwan were used in CD74knockdown experiments For CD74 knockdown confluent 3T3L1 preadipocytes in sixwell dishes were incubated inOptiMEM Thermo Fisher Scientific and transfected with either CD74 shRNA or nonspecific shRNA using Lipofectamine Thermo Fisher Scientific according to the manufacturers protocol After h the medium was replacedwith complete DMEM with a differentiation cocktail μM 3isobutyl1methylxanthine μM dexamethasoneand μM insulin to induce differentiation into mature adipocytes day Western blottingEqual amounts of protein lysates were subjected to sodium dodecyl sulfatepolyacrylamide gel electrophoresis andthen transferred to polyvinylidene fluoride membranes After blocking with skim milk the membranes wereincubated with primary antibodies and subsequently with appropriate peroxidaseconjugated secondary antibodiesPrimary antibodies including targets catalog numbers dilutions and suppliers were as follows antibodies specific toCD74 GTX110477 were from GeneTex Hsinchu Taiwan and antibodies specific to actin MAB1501 were from MilliporeSigma St Louis MI USA The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20202072101042BSR20202072Adipocyte differentiationThe 2day postconfluency preadipocytes were cultured in complete DMEM with a differentiation cocktail μM3isobutyl1methylxanthine μM dexamethasone and μM insulin On day of differentiation cells wereswitched to complete DMEM with μM insulin for the remaining duration of differentiationCell counting3T3L1 cells were detached from sixwell plates using trypsin Thermo Fisher Scientific resuspended in complete DMEM and counted using a cell counter Millipore every day from day Oil Red O stainingDifferentiated adipocytes were fixed in formalin and stained for min with Oil Red O MilliporeSigma working solution Oil Red O dye in isopropanol Oil Red O was extracted using isopropanol and theabsorbance was measured at nm using a spectrophotometerCell culture and extraction of nuclear proteins from established NR3C1FOXP3 and CD74 transformantsCells were transfected with the pCMV3CMycNR3C1 pCMV3CMycFOXP3 or pCDNA4CD74 usingthe Lipofectamine kit Thermo Fisher Scientific according to the manufacturers protocol The nuclear proteinswere extracted using NEPER nuclear and cytoplasmic extraction reagents Thermo Fisher Scientific supplementedwith protease inhibitor cocktail and phosphatase inhibitors Roche Basel Switzerland according to the manufacturers protocolQuantitative DNA immunoprecipitation qDNAIP assayqDNAIP assays were performed on nuclear extracts from established FOXP3 and NR3C1 transformantsDNA binding of FOXP3 or NR3C1 was assessed using the annealed double strand oligonucleotides 5cid3biotinlabeled rs2569103A probes 5cid3CCAAATGGCTGGTTTCAGGGCTGGAGATGGGGG3cid3 and 5cid3CCCCCATCTCCAGCCCTGAAACCAGCCATTTGG3cid3 as well as 5cid3biotinlabeled rs2569103G probes 5cid3CCAAATGGCTGGTTTCGGGGCTGGAGATGGGGG3cid3 and 5cid3CCCCCATCTCCAGCCCCGAAACCAGCCATTTGG3cid3 PURIGOBiotechnology Taipei Taiwan For the binding reactions μg of nuclear proteins were incubated with or without labeled oligonucleotides in binding buffer [ mM TrisHCl pH mM NaCl mM MgCl2 mMEDTA mM DTT mgml polydIdC and glycerol] for min at ¦C in a final volume of μl FOXP3 or NR3C1nucleotide complexes were crosslinked with formaldehyde final concentration for min at room temperature followed by immunoprecipitation with antibodies specific to Myc tag GTX115046 GeneTex and Protein AG magnetic beads GE Healthcare Immunoprecipitated DNA was detected usinghorseradish peroxidaseconjugated streptavidin The reaction was developed with the 33cid355cid3tetramethylbenzidinereagent Sigma and read at nm with a Microplate reader BioRad Hercules CA USAStatistical analysesThe statistical analyses were performed using the PASW Statistics software from IBM Armonk NY USAA ttest was used to evaluate the associations between GO and age A Chisquare test was used to evaluate the associations between polymorphisms and GD or GO Screening for linkage disequilibrium LD was performed usingHaploview ver [] A twotailed Pvalue less than with Bonferroni correction was considered statistically significant [] Logistic regression with a confidence interval CI was used to estimate odds ratiosORsResultsDemographic data clinical characteristics and their correlations withGO in patients with GDThe frequency distributions of clinical characteristics such as goiter nodular hyperplasia myxedema vitiligo andage in male and female groups were compared between the patients with GD with or without GO As demonstratedin Table gender and age were significantly associated with GO in patients with GD Even myxedema was associatedwith GO in patients with GD however due to a limited number of cases the association needs further investigation The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20202072101042BSR20202072Table Demographic data and clinical characteristics of graves disease patients with or without graves ophthalmopathyCharacteristicGDnonGO N GDGO N PNumber of patientsFemale genderAge of diagnosis Year Mean SD[Range]Presence of goiterNo1a1bPresence of nodular hyperplasiaPresence of myxedemaPresence of vitiligoWith radioiodine therapy historyWith thyroid surgery historyWith smoke historyFree T3 pgmlFree T4 ngdlT3 ngdlT4 μgdlTSH μIUmlTRAb positive [] [] Abbreviations GD graves disease GO graves ophthalmopathy N numberaFrequencies of genotypes were determined by the chisquare test using or contingency tablesbSigniï¬cance of age were evaluated by t testP005P00010039a 105b 0165a0539a0039a 0743a0273a0227a0527a0900a0692a0146a0310a0479a0482aThese results adhered to other epidemiological results that GO occurred more commonly in the middleaged femalepopulationLD among SNPs of MIF and CD74Four SNPs of the MIF and CD74 were genotyped to determine whether polymorphisms in these genes influencethe development of GO in patients with GD The distribution of the four SNPs fit the HardyWeinberg equilibriumHWE in patients with GD and healthy individuals However the strong r208 LD r2 values calculated for thetwo SNPs at the CD74 in healthy individuals were not observed in patients with GD with or without GO suggestingthat there is more variation in the extent of LD within CD74 in patients with GD Figure Allele and genotype distributions of CD74 contribute to GDGOdevelopmentNo significant association was found in the examined SNPs of MIF nor was a significant association found betweenthe polymorphisms and the clinical features or the indicators of thyroid function including free triiodothyronineT3 free thyroxine T4 thyroid stimulating hormone TSH and thyrotropin receptor antibodies TRAbs in patients with GD However allele frequencies showed that individuals carrying a G allele at rs2569103 in the CD74 hada reduced risk of developing GD P0005 OR CI Table Genotype frequenciesfurther showed that individuals carrying the GG genotype at rs2569103 in the CD74 had a reduced risk of developing GD P3390 OR CI which was consistent with results from allelefrequencies however the patients with GD carrying the AG genotype at rs2569103 in the CD74 had an increasedrisk of developing GO P0009 OR CI Table The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20202072101042BSR20202072Figure Linkage disequilibrium LD values between the two polymorphisms rs13175409 and rs2569103 in the CD74region in a TaiwaneseChinese populationThe color scale reflects the strength of LD between the two single nucleotide polymorphisms SNPs A Healthy individuals BPatients with Graves disease GD with and without Graves ophthalmopathy GO C Patients with GD without GO D Patientswith GD with GOTable Allele distributions of MIF and CD74GenotypesControl N GDnonGO NGDGO N Control vs GDPaControl vs GDOR 95CINonGO vsGO PaNonGO vsGO OR95CIMIF rs476240AGMIF rs507715ACCD74 rs13175409CTCD74 rs2569103AG 0929bAbbreviations CI conï¬dence interval GD graves disease GO graves ophthalmopathy N number OR odds ratiosaFrequencies of genotypes were determined by the chisquare test using or contingency tablesbOdds ratios and CI per genotype were estimated by applying unconditional logistic regressionP005 with Bonferroni correction OR with signiï¬canceKnockdown of the expression of CD74 inhibits 3T3L1 adipocytedifferentiationThe swelling of extraocular orbital fat is one reason that the development of GO is triggered [] To understand thepossible regulation between CD74 and adipocyte differentiation 3T3L1 cells were chosen as an experimental modelThe expression of CD74 in CD74 knockdown CD74KD cells by shRNA was confirmed as compared with those withcontrol of shRNA Figure 2A Cell numbers of CD74KD and control cells were counted every day The knockdownof CD74 decreased cell proliferation from days after induction Figure 2B In addition the degree of Oil Red The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20202072101042BSR20202072Table Genotype distributions of MIF and CD74GenotypesControl N GDnonGO NGDGO N Control vs GDP aControl vs GDOR 95CINonGO vsGO P aNonGO vsGO OR95CIMIF rs476240AAAGGGMIF rs507715AAACCCCD74 rs13175409CCCTTTCD74 rs2569103AAAG2495cGG b0154b2467bAbbreviations CI conï¬dence interval GD graves disease GO graves ophthalmopathy N number OR odds ratiosaFrequencies of genotypes were determined by the chisquare test using or contingency tablesbOR and CI per genotype were estimated by applying unconditional logistic regressioncOR and CI per genotype were estimated by adjusting with gender age and myxedemaP005 with Bonferroni correctionOR with signiï¬canceFigure Changes in adipocyte differentiation and proliferation after knockdown of CD74A Endogenous expression of CD74 protein in 3T3L1 cells was examined and knockdown of CD74 was examined by Westernblotting Actin was used as an internal control B The downregulation of CD74 inhibits cell growth 3T3L1 cells were detachedfrom sixwell plates and counted P001 P0001 CD74 knockdown vs control cells C Cells were stained with Oil Red Oafter inducing differentiation Quantitative analyses were performed by measurement of optical density OD at nm in extractsfrom Oil Red Ostained cells transfected with CD74 short hairpin RNA shRNA and control shRNA P0001 CD74 knockdownvs control cellsO staining was weaker in CD74KD cells than in control cells on day and on day respectively forCD74 shRNA vs control cells Figure 2C The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20202072101042BSR20202072Different binding afï¬nities of NR3C1 and FOXP3 for CD74 promoterdepends on SNP rs2569103The CD74 SNP rs2569103 was located within the upstream region of CD74 and showed the strongest associationwith the disease making it a possible target for transcription factors Indeed the putative transcription factorbindingsites were predicted using PROMO [] At SNP rs2569103 the A allele generates motifs for nuclear receptorsubfamily group C member NR3C1 TCAGG whereas the G allele generates a motif for forkhead box P3FOXP3 GTTTCG Bulk RNAseq analysis of NR3C1 and FOXP3 in thyroid and fat tissues from public datasetsPRJEB4337 were demonstrated Figure 3A To interpret the possible regulatory mechanisms of these moleculespublished mRNA expression results were explored The mRNA expression of NR3C1 only showed a negative correlation with that of CD74 in thymoma samples Pearsons correlation Spearmans correlation Figure3B whereas the mRNA expression of FOXP3 showed a positive correlation with that of CD74 Pearsons correlation Spearmans correlation in thymoma samples welldifferentiated papillary thyroidcarcinoma and welldifferentiated thyroid cancer respectively Figure 3CE The qDNAIP results supported thatNR3C1 tends to bind to probes with promoter sequence containing AA at rs2569103 whereas FOXP3 tends to bindto probes with promoter sequence containing GG at rs2569103 Figure 3F These results suggested that the CD74expression may be orchestrated by complex transcription factor networks The AA genotype may play a role in response to NR3C1induced CD74 downregulation whereas the GG genotype on rs2569103 on the CD74 promotermay play an additional role in response to FOXP3induced CD74 upregulationDiscussionEnvironmental factors and genetic loci have been thought to be associated with immune regulation [] Here weidentified new candidates CD74 alleles and genotypes for the susceptibility of GD and GO in a TaiwaneseChinesepopulation CD74 is involved in adipocyte differentiation through its differential promoter binding affinity for transcription factors To the best of our knowledge this is the first study to demonstrate novel CD74 polymorphisms inassociation with the development of GD and GO Our results support wholegenome screening studies in that thechromosome 5q32 may play a role in generating GD and GO in humansThe thyroid gland of patients with GD revealed marked enlargement of the gland due to autoantibodies Patientswith accompanying GO exhibited enlargement of the retroorbital connective tissue and extraocular muscles inpart due to the inflammatory deposition of glycosaminoglycans collagen and fat [] Indeed genes involved inthe regulation of cell survival DNA transcription and protein synthesis have been considered risk factors for GDand GO [] Overexpression of CD74 plays a crucial role in preventing hyperreactivity between immature antigens and major histocompatibility complex class II as well as cell growth and survival whereas downregulation ofCD74 is often correlated with autoimmunity and cell apoptosis [] Upon expression of surface CD74 the cellsmay transduce survival signaling through extracellular signalregulated kinase or cJun Nterminal kinase JNKmitogenactivated protein kinase MAPK pathways or AKT pathways in a MIFdependent manner thereby improving cell survival and proliferation [] Due to the limitation to find identical cells expressed GG or AA genotypeon rs2569103 current results we did not show the direct impact of these transcription factors to the CD74 expression Further evidence such as RNAseq as secondary data was warranted The results showed that GD patients withor without GO although loss the protective GG genotype most of them hold AG heterogenous genotype insteadsuggested the lossofprotect effect on the disease In the present study cellbased experiments showed that CD74 isinvolved in adipocyte differentiation but the link toward GO development remained to be investigated On the otherhand the GG genotype on rs2569103 with a higher frequency in healthy individuals Table increased the bindingof FOXP3 to the CD74 promoter Figure 3F thereby increasing CD74 upregulation and protecting autoimmuneresponses Conversely the AA genotype on rs2569103 increases the binding of NR3C1 to the CD74 promoter whichdownregulates CD74 and increases autoimmune response and manifestations of GDGO Due to the limitation tofind identical cells expressed GG or AA genotype on rs2569103 current results we did not show the direct impactof these transcription factors to the CD74 expression Further evidence such as RNAseq as secondary data was warranted The results showed that GD patients with or without GO although they lost the protective genotype mostof them hold the AG heterogenous genotype instead suggesting the lossofprotection effect of the disease Furtherstudies on the detailed mechanisms through CD74derived adipocyte differentiation are warrantedConversely the ligand of CD74 MIF has previously been reported to be counterregulatory to glucocorticoid secretion [] The glucocorticoidinduced MIF secretion was noted at min after dexamethasone administration[] In addition nonsteroidal antiinflammatory drugs such as aspirin ibuprofen and naproxen have been used torelieve the pain and inflammation of GO This evidence further supports the crucial role of CD74 in the transduction The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20202072101042BSR20202072Figure Different binding afï¬nities of NR3C1 and FOXP3 for CD74 promoter depends on singlenucleotide polymorphismSNP rs2569103A RNAseq analysis of NR3C1 and FOXP3 in thyroid and fat tissues from public datasets PRJEB4337 BE Bioinformaticanalysis of mRNA expression correlation between NR3C1 and CD74 or FOXP3 and CD74 The mRNA expression of NR3C1 andCD74 in thymoma samples B and the mRNA expression of FOXP3 and CD74 in thymoma samples C welldifferentiated papillarythyroid carcinoma D and welldifferentiated thyroid cancer E F Probe with promoter sequence containing rs2569103 probe Ahas a higher affinity for NR3C1 whereas G at rs2569103 probe G has a higher affinity for FOXP3 as shown by quantitative DNAimmunoprecipitation qDNAIP assay P001 P0001 probe A vs probe G The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20202072101042BSR20202072of MIF signaling However due to the limited population of the minor polymorphism the present study is unable toreach the interactions among cells and molecules in the orbital microenvironment and their association toward thetarget polymorphism due to the inaccessibility of the orbital tissues The current finding may have further implications for understanding the link between the polymorphismexpression of CD74 and current treatments for GOatherapeutic effect issue that might be of value for future treatment strategies targeting MIF or CD74In conclusion the current study identified new SNPs in the CD74 that were found to be associated with GD and GOin a TaiwaneseChinese population Biological studies provide insights into the genetic information that influencesthe development of GD and GO via adipocyte proliferation and differentiationPerspectives¢The impact of genetic factors on the orbital microenvironment cannot be closely monitored due tothe inaccessibility of the orbital tissue Studies on feasible cellbased models may help elucidate howgenetic factors such as CD74 SNPs modulate the target gene expression¢¢The present study combined clinical observations and cell models to investigate how CD74 polymorphisms affect adipocyte proliferation and differentiationThe present clinical observations suggest that the genetic factors of CD74 should be considered inclinical practiceCompeting InterestsThe authors declare that there are no competing interests associated with the manuscriptFundingThis work is supported by Ministry of Science and Technology Taiwan [grant numbers MOST 1042815C039002B and MOST1072320B039032MY3] the peak project and thematic project of Academia Sinica Taiwan the higher education sproutproject by the Ministry of Education MOE Taiwan via Drug Development Center of China Medical University from The FeaturedAreas Research Center Program and China Medical University [grant numbers CMU105S33 and CMU106S46] TaichungTaiwanAuthor ContributionYHL proposed the concept designed the experiment anized the study wrote and reviewed the manuscript CYS performed the experiments FJT coordinated patient enrollment collected the clinical samples and applied official applicationAcknowledgementsWe thank Taiwan Biobank for providing related data all anonymous for our research The sponsorfunding anization had norole in the design or conduct of this researchAbbreviationsCD74 cluster of differentiation CI confidence interval FOXP3 forkhead box P3 GD graves disease GO graves ophthalmopathy HLA human leukocyte antigen HWE HardyWeinberg equilibrium JNK cJun Nterminal kinase LD linkagedisequilibrium MAPK mitogenactivated protein kinase MIF macrophage migration inhibitory factor NR3C1 nuclear receptorsubfamily group C member OR odds ratio PCR polymerase chain reaction qDNAIP quantitative DNA immunoprecipitation SNP singlenucleotide polymorphism T3 triiodothyronine T4 free thyroxine TRAb thyrotropin receptor antibody TSHthyroid stimulating hormoneReferences Smith TJ and Hegedus L Graves Disease N Engl J Med 101056NEJMra1510030 Brent GA Clinical practice Graves disease N Engl J Med 101056NEJMcp0801880 Ginsberg J Diagnosis and management of Graves disease CMAJ Canadian Med Assoc J J de lAssoc Med Canadienne The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative CommonsAttribution License CC BY 0cBioscience Reports BSR20202072101042BSR20202072 McIver B and Morris JC The pathogenesis of Graves disease Endocrinol Metab Clin North Am 101016S0889852905702991 Bednarczuk T Gopinath B Ploski R and Wall JR Susceptibility genes in Graves ophthalmopathy searching for a needle in a haystackClin Endocrinol Oxf 101111j13652265200702854x Anvari M Khalilzadeh O Esteghamati A Esfahani SA Rashidi A Etemadi A et al Genetic susceptibility to Graves ophthalmopathy therole of polymorphisms in proinï¬ammatory cytokine genes Eye Lond 101038eye2009244 Bahn RS Understanding the immunology of Graves ophthalmopathy Is it an autoimmune disease Endocrinol Metab Clin North Am vi Manji N CarrSmith JD Boelaert K Allahabadia A Armitage M Chatterjee VK et al Inï¬uences of age gender smoking and familyhistory on autoimmune thyroid disease phenotype J Clin Endocrinol Metab 101210jc20061402 Stan MN and Bahn RS Risk factors for development or deterioration of Graves ophthalmopathy Thyroid Ofï¬cial J Am Thyroid Assoc 101089thy20101634 Bahn RS and Heufelder AE Pathogenesis of Graves ophthalmopathy N Engl J Med Tomer Y Barbesino G Greenberg DA Concepcion E and Davies TF Mapping the major susceptibility loci for familial Graves andHashimotos diseases evidence for genetic heterogeneity and gene interactions J Clin Endocrinol Metab Tomer Y Ban Y Concepcion E Barbesino G Villanueva R Greenberg DA et al Common and unique susceptibility loci in Graves andHashimoto diseases results of wholegenome screening in a data set of multiplex families Am J Hum Genet 101086378588 Gianoukakis AG and Smith TJ Recent insights into the pathogenesis and management of thyroidassociated ophthalmopathy Curr OpinEndocrinol Diabetes Obesity 101097MED0b013e32830eb8ab Shiina T Ota M Shimizu S Katsuyama Y Hashimoto N Takasu M et al Rapid evolution of major histocompatibility complex class I genesin primates generates new disease alleles in humans via hitchhiking diversity Genetics 101534genetics106057034 Liu YH Chen YJ Wu HH Wang TY and Tsai FJ Single nucleotide polymorphisms at the PRR3 ABCF1 and GNL1 genes in the HLA class Iregion are associated with Graves ophthalmopathy in a genderdependent manner Ophthalmology 101016jophtha201404027 Wang S Sun H Chen HY Zhao ZF Yang Y Zhao YJ et al Intercellular adhesion molecule gene polymorphisms do not contribute toGraves disease in Chinese patients Endocrine 101007s1202000700329 Liu YH Chen RH Chen WC Tsai Y Wan L and Tsai FJ Disease association of the CD103 polymorphisms in Taiwan Chinese Gravesophthalmopathy patients Ophthalmology 101016jophtha200912037 Bednarczuk T Hiromatsu Y Seki N Ploski R Fukutani T Kurylowicz A et al Association of tumor necrosis factor and human leukocyteantigen DRB1 alleles with Graves ophthalmopathy Hum Immunol 101016jhumimm200402033 Khalilzadeh O Anvari M Esteghamati A Mahmoudi M Tahvildari M Rashidi A et al Graves ophthalmopathy and gene polymorphisms ininterleukin1alpha interleukin1beta interleukin1 receptor and interleukin1 receptor antagonist Clin Exp Ophthalmol Siegmund T Usadel KH Donner H Braun J Walï¬sh PG and Badenhoop K Interferongamma gene microsatellite polymorphisms inpatients with Graves disease Thyroid Ofï¬cial J Am Thyroid Assoc 101089thy199881013 Wong KH Rong SS Chong KK Young AL Pang CP and Chen LJ Genetic Associations of Interleukinrelated Genes with GravesOphthalmopathy a Systematic Review and Metaanalysis Sci Rep 101038srep16672 Bucala R and Shachar I The integral role of CD74 in antigen presentation MIF signal transduction and B cell survival and homeostasis MiniRev Med Chem 1021741389557515666150203144111 Leng L Metz CN Fang Y Xu J Donnelly S Baugh J et al MIF signal transduction initiated by binding to CD74 J Exp Med 101084jem20030286 Liu YH Chen CC Yang CM Chen YJ and Tsai FJ Dual effect of a polymorphism in the macrophage migration inhibitory factor gene isassociated with newonset Graves disease in a Taiwanese Chinese population PLoS ONE e92849 101371journalpone0092849 Nakabayashi | 2 |
"immunotherapy is revolutionising cancer treatment and has already emerged as standard treatment for patients with recurrent or metastatic gastric cancer gc recent research has been focused on identifying robust predictive biomarkers for gc treated with immune checkpoint inhibitors icis the expression of programmed cell death protein ligand1 pd l1 is considered a manifestation of immune response evasion and several studies have already reported the potential of pd l1 expression as a predictive parameter for various human malignancies meanwhile based on comprehensive molecular characterisation of gc testing for epstein barr virus and microsatellite instability is a potential predictive biomarker culminating evidence suggests that novel biomarkers such as the tumour mutational burden and gene expression signature could indicate the success of treatment with icis however the exact roles of these biomarkers in gc treated with icis remain unclear therefore this study reviews recent scientific data on current and emerging biomarkers for icis in gc which have potential to improve treatment outcomesintroductionthe prognosis for metastatic or recurrent gastric cancer gc remains very poor making it the third leading cause of cancer related death worldwide1 however the recent development of immune checkpoint inhibitors icis targeting the programmed cell death protein1 pd1 and programmed cell death protein ligand1 pd l1 pathways has produced improved outcomes for gc and already been successfully incorporated into clinical practice2 in particular checkpoint inhibition with anti pd1 monoclonal antibodies such as pembrolizumab and nivolumab has led to durable and significant responses in a minority of gcs3 as a result interest has increased in selecting the right patient population to benefit such icis along with further exploration of immunotherapy notably various tumour related and host related factors with a critical impact on systemic immune functions may influence the response to icis6 moreover a significant proportion of patients do not respond to these therapies and there can also be a threat of unpredictable immune related adverse events aes and even severe toxicity7 therefore identifying more robust predictive biomarkers is critical to optimise treatment with icis while avoiding unnecessary treatment of patients who could develop life threatening or life altering aesgc is a heterogeneous and complex disease9 thus various approaches such as molecular classification have already been proposed for the subhistological exploration of gc as a potential tool for more effective therapeutic strategies the cancer genome atlas research network tcga suggested a comprehensive molecular characterisation of gcs using various platforms and proposed four distinct subtypes epstein barr virus ebv positive microsatellite unstable microsatellite instability msi genomically stable and tumours with chromosomal instability10 more recently the asian cancer research group acrg described four subtypes msi microsatellite stable mssepithelial to mesenchymal transition mssand msstp53 negative11 tp53 positive while the above subtypings show some overlapping molecular aberrations msi was identified as a subtype by both tcga and acrg2 msi is a molecular marker of a defective function of the dna mismatch repair mmr system that recognises and repairs the erroneous insertion deletion and mis incorporation of bases that can arise during dna replication and recombination as well as repairing some forms of dna damage12 it is also well known that msi high msi h tumours exhibit a high tumour mutational burden tmb neoantigen load and immune infiltration making them respond well to icis13 meanwhile the distinct characteristics of ebv positive gc is the overexpression of pd l1 and pd l214 the driving features of pd l1 positivity in ebv positive gc can also be effectively targeted with immunotherapy similar to the msi h subtype interestingly in a recent phase ii trial by kim kang a0bw chau a0i esmo open 20205e000791 101136esmoopen2020000791 0copen accesspembrolizumab showed a promising efficacy in patients with msi h and ebv positive tumours15 accordingly based on a better molecular characterisation of gc this review focuses on the current and emerging biomarkers for icis that would facilitate precision medicineclinical evidences of icis in gcseveral phase ii and iii trials have recently investigated the pd1 and pd l1 blockade in gc as summarised in table pembrolizumab is an igg4 human antibody targeting pd1 thereby interfering with the interaction between pd1 and pd l120 in the phase ib keynote trial patients with pd l1 positive gc or gastro oesophageal junction gej cancer were enrolled in both asian and non asian countries21 the objective response rate orr was and durable responses were seen with a median duration of responses of weeks the subsequent phase ii multicohort keynote059 trial cohort enrolled patients with recurrent or metastatic gc and gej cancer who received two pretreated lines of chemotherapy4 here the orr was and median overall survival os was months following these two trials the randomised phase iii keynote061 trial compared pembrolizumb monotherapy with paclitaxel in patients with pd l1 positive gc that had progressed on first line flouropyrimidine and platinum doublet chemotherapy17 while patients with a pd l1 status were initially unselected pd l1 positive patients with a combined positive score cps of or higher were included in the latter part of the study the cps is the number of pd l1 staining cells including tumour cells lymphocytes and macrophages divided by the total number of viable tumour cells while a tumour proportion score tps is the percentage of viable tumour cells showing partial or complete membrane staining relative to all viable tumour cells present in the sample22 approximately of patients were found to have pd l1 positive tumours using the cps pembrolizumab did not meet its primary end point of a longer os and progression free survival pfs in patients with pd l1 positive tumours notwithstanding it is worth noting that patients who expressed pd l1 cps of or higher exhibited a better benefit from treatment with pembrolizumab in post hoc analyses although these subgroup analyses should be interpreted with cautionsubsequently the keynote062 was a large randomised first line clinical trial of patients with advanced gc or gej cancer who were randomly assigned to one of three arms pembrolizumab at mg every weeks for up to years pembrolizumab plus chemotherapy cisplatin and fluorouracil or capecitabine or placebo plus chemotherapy23 pembrolizumab was non inferior to chemotherapy for os in patients with cps or higher no survival benefit was observed with the addition of pembrolizumab to chemotherapy compared with chemotherapy alone in this studynivolumab also an igg4 antibody is very similar in structure to pembrolizumab except that nivolumab binds to the n terminal loop on the pd1 molecule while pembrolizumab binds to the cd loop24 the phase iii attraction2 ono453812 trial compared nivolumab with a placebo in asian patients with unresectable or recurrent gc that was refractory to or intolerant of at least two previous standard chemotherapy regimens5 the results showed a significantly prolonged os for the nivolumab group with a year os rate of vs more recently the long term survival was reported showing year survival rates of for nivolumab and for placebo25 nivolumab also showed a significant advantage compared with the placebo in terms of pfs and the radiological objective response therefore these results provide randomised evidence that nivolumab is a valid approach to improving the clinical outcomes for patients with gc in a third line and subsequent line setting however the overall clinical value of attraction02 was partially limited by several important issues26 the patient population was only asian and pd l1 positivity reported at a low frequency of as this was assessed as tps rather than cps was not associated with the survival outcomes plus there was no comparative data on quality of life a phase iii checkmate032 study also reported that nivolumab and nivolumab plus ipilimumab provide a durable antitumour activity in heavily pretreated western patients with chemotherapy refractory gc gej cancer and oesophageal cancer19 in particular the clinical benefit of nivolumab monotherapy was consistent with that reported for asian patients in the attraction02 study yet similar to the attraction02 study there was no association of pd l1 positivity according to tps with survival outcomes therefore ongoing randomised controlled trials of icis including pembrolizumab and nivolumab in earlier line treatment need to unify assessment of pd l1 expression and create more accurate profiles of aes in gcavelumab is an igg1 antibody which binds to the front beta sheet of pd l1 and possesses pd1pd l1 blockade activity with antibody dependent cell mediated cytotoxicity adcc28 there are some differences between pd1 inhibition and pd l1 inhibition as pd1 targeting therapeutic antibodies including pembrolizumab and nivolumab block the pd1pd l1 or pd1pd l2 interaction to restore tumor specific t cell reactivity without mediating adcc24 the javelin gastric trial the ï¬rst study to compare avelumab with standard chemotherapy in third line treatment for gc did not achieve its primary end point of improving os or the secondary end points of pfs and orr18 this negative finding may be attributed to the usage of the active comparator in the control arm in addition there are possible reasons including the different drug biding sites heterogeneity of tumour biology and methodology of pd l1 testing notwithstanding fewer patients experienced aes with avelumab than with chemotherapy although researchers found no evidence of clinical beneï¬t compared with commonly used chemotherapy in any of the examined subgroups including the tumour pd l1 expression status kang a0bw chau a0i esmo open 20205e000791 101136esmoopen2020000791 0csecnerefersodehcaer tonsfp rristnopdne yramirp ichpargoegrrinogerlaboglso sfplaboglsorrrrinasainasanretsewsolabogl tneitaprebmunrecnac cirtsagn i srotibhniii tnopkcehc enummi lgnitauave seduts iii idna ii esahpdetcees l l ebatsmra tnemtaertsutats ldpgnittesesahpeman ydutstegratstnegabamuzilorbmepdetceesnul eni ldrihtretal robmuzilorbmepevitisop dnoceslexatilcapenilbamuovnilobecapldetceesnul eni ldrihtretal roiiiiiiii trohoc etonyekdpbamuzilorbmepetonyekdpbamuzilorbmepi notcarttaonodpbamuovnlixopac roxosbamuovnil bamumilipibamuovnilbamuovnil bamumilipibamuovnilretal rodetceesnuleni ltsrifiiiiii notcarttadp trapdetceesnul eni ldrihtiiietamkcehcdpbamuovnlibamuovnlii¡ecohc snacsyhpiibamuevaldetceesnuleni ldrihtiii cirtsagnlevaj i ldpbamuevalevitisop ldp erew stneitapgnoma open access dna s xos etar esnopser rri lavvrus eer fnossergorpi sfp dnagii lnetorphtaed llec demmargorp ldp inetorphtaed llec demmargorp dpi lavvrus llarevo so nitaplil axodna enbacepac iixopacnitaplilaxoskeew yreve iipovni gkg mbamum ilipl i supgkg mbamuovn il skeew yreve povniii gkg m bamumilipl i supgkg mbamuovn illecyc tnemtaert kee w a fohcae dna syadno mg m nacetoniri ro dna syad no mg m lexatilcap¡kang a0bw chau a0i esmo open 20205e000791 101136esmoopen2020000791 0copen accessthese results might support the potential of avelumab for combination or maintenance therapy notwithstanding the recent javelin gastric trial with maintenance avelumab therapy following initial chemotherapy in gc produced disappointing results29 although there was more efficacy seen with avelumab in an exploratory analysis of pd l1 positive patients with a cps ¥ vs months no significant difference with respect to os was observed between the avelumab maintenance and continued chemotherapy groups vs months p0177as discussed above several clinical trials are currently ongoing with various strategies30 the phase iii attraction04 nct02746796 trial is evaluating nivolumab plus standard chemotherapy oxaliplatin plus either s1 or capecitabine versus chemotherapy alone in asian patients16 the phase ii component part of this study showed chemotherapy plus nivolumab led to an orr of and median pfs of months while the subsequent phase iii trial part is evaluating the survival outcomes another phase iii trial checkmate649 nct02872116 has completed recruitment with over patients randomising patients to oxaliplatin based chemotherapy alone or in combination with nivolumab or nivolumab plus ipilimumab32 the third arm of this study terminated recruitment early due to early safety signalantiangiogenic treatment such as ramucirumab and bevacizumab is also generating recent interest as several studies have suggested that simultaneously blocking angiogenesis and pd1 pathways induces synergistic antitumour effects especially involving the control of the tumour microenvironment tme33 researchers including the current authors noted that ramucirumab in combination with pembrolizumab jvdf showed a manageable safety profile with favourable antitumour activity in patients with previously treated gc35 consequently the phase iii nivoram nct02999295 trial has evaluated the safety and tolerability of the addition of ramucirumab to nivolumab in patients with gc as second line therapy7 as an alternative antiangiogenic and multitargeted kinase inhibitor a phase i trial of regorafenib plus nivolumab is also exploring in gc nct0340687136 this trial demonstrated an orr of in gc and icis pretreated patients with gc achieved a partial response pr in patients interestingly of the seven patients who had received prior anti pd1pd l1 treatment three achieved an objective responseanother interesting phase ii trial investigating the role of pembrolizumab plus trastuzumab combined with chemotherapy in patients with previously untreated human epidermal growth factor receptor positive tumours is currently ongoing nct0295453637 preliminary results from this study showed of patients experienced reduction in target lesions after one dose of pembrolizumabtrastuzumab before oxaliplatincapecitabine was introduced in second cycle the high orr of was coupled with median pfs of months this has led to the current phase iii keynote811 study randomising patients to chemotherapy plus trastuzumab with or without pembrolizumb nct0361532638 thus these therapeutic strategies including combination treatment represent a true opportunity in the contemporary treatment of gc and may produce further success when considering integrative genomic databiomarkers for icis in gcthe identification and validation of reliable biomarkers are important to facilitate precise patient selection and increase the clinical benefit from icis an overview of the predictive roles of biomarkers obtained from tissue or blood and their characterisation in the management of gc is briefly summarised in table pdl1 expressiontesting for pd l1 expression by ihc is the current standard in most solid tumours and several studies have already assessed the clinical outcomes according to the pd l1 expression status in gc nevertheless different antibodies are being used for ihc to assess with different performance and different scoring criteria for pd l1 expression a recent multicentre study blueprint pd l1 ihc comparison project attempted to compare the performance of each ihc pd l1 assay in lung cancer39 three assays including 22c3 for pembrolizumab for nivolumab and sp263 for durvalumab were found to be comparable to each other in the staining of tumour tissue whereas sp142 for atezolizumab was found to be less sensitive however further study is required to carefully validate these assays in gcas noted above in the keynote059 trial pd l1 expression was assessed using the pd l1 ihc 22c3 pharmdx assay and measured using a cps4 this trial demonstrated a higher orr vs in patients with high pd l1 expression defined as cps ¥ both pfs and os were also more prolonged in this group40 although pembrolizumab in a second line trial keynote061 did not significantly prolong os greater benefits were seen in tumours with higher pd l1 expression cps ¥ orr25 cps ¥ orr16 cps orr217 however the attraction02 and javelin gastric trials showed no clinical improvement for pd l1 positive tumours as they used tps rather than cps18 these differences may also have been due to the use of different cut off points and scoring systems a lack of standardisation of the assays and testing platforms the heterogeneous nature of pd l1 expression in tumours intratumouralintertumoural heterogeneity and intraobserverinterobserver variability41 for instance the attraction02 study retrospectively evaluated pd l1 expression using a pd l1 ihc pharmdx assay defined as the tps while pd l1 expression was prospectively assessed in tumour cells tumour associated lymphocytes and macrophages using a 22c3 pharmdx assay in keynote06117 as such pd l1 positivity was only reported in about patients using tps with nivolumab whereas it is generally kang a0bw chau a0i esmo open 20205e000791 101136esmoopen2020000791 0ctable overview of candidate biomarkers associating with response to immune checkpoint inhibitors in gastric cancerbiomarkerssample source methodspd l1tumourihctreatmentrecent results in gastric cancerreferencespembrolizumab expression of pd l1 ¥ associated with open accessbetter clinical efficacy orr mrdpembrolizumab expression of pd l1 cps of or higher associated with better clinical efficacy os orrpembrolizumab expression of pd l1 associated with higher response rateebv positivitymsi htumourin situ hybridisationpembrolizumab ebv positivity associated with higher tumour msi testing or ihcnivolumbresponse ratemsi h associated with clinical efficacy orr dcr ospembrolizumab msi h associated with better clinical efficacy orrpembrolizumab msi h associated with better clinical efficacy orr ospembrolizumab msi h associated with better clinical tmbtilstumour or bloodtumourwes or targeted sequencingimage analysing software or manually countedtoripalimabicisefficacy orrtmb associated with better clinical efficacy orr ospresence of tils associated with better clinical efficacy in various solid tumours but very limited data in gcgeptumour multigene profilingpembrolizumab ifn gamma gene signature associated with better clinical efficacy orr pfspembrolizumab t cell inflamed gene signature gut microbiotastoolnlrbloodculture or molecular technique sequencingmetagenomicscomplete blood counticisicisnivolumabassociated with better clinical efficacy orr pfsvarious species associated with enhancement and iraes of icis in various solid tumoursincreased nlr correlated with dcr and osdecreased change of nlr associated with better survivalsixty seven patients had tumours from the stomachcps combined positive score dcr disease control rate ebv epstein barr virus gc gastric cancer gep gene expression profiling icis immune checkpoint inhibitors ifn interferon ihc immunohistochemistry iraes immune related adverse events mrd median response duration msi h microsatellite instability nlr neutrophil to lymphocyte ratio orr overall response rate os overall survival pd l1 programmed cell death protein ligand pfs progression free survival tils tumour infiltrating lymphocytes tmb tumour mutational burdenbetween and using cps ¥ as the cut off more recently using the checkmate032 study data tumours were re scored using cps and there was better correlation between nivolumab treatment and survival even at the cps ¥ level44 thus one of the coprimary patient population in the first line checkmate649 study that has recently completed recruitment is including a subpopulation of patients with pd l1 cps ¥ebv positivityebv status is also emerging as a potential biomarker for personalised treatment strategies in gc14 in situ hybridisation detection of ebv encoded small rna in tumour cells is generally recommended to identify ebv associated gc ebvagc30 the incidence of ebvagc varies from to in different regions with an average of worldwide45 nevertheless this subgroup is associated with better prognosis thus less frequently found in advanced or metastatic setting in particular ebv positive tumours frequently display pd l12 overexpression and occasional immune cell signalling activation10 several research groups found that the level of pd l1 expression ranging from approximately to of ebvagc with kang a0bw chau a0i esmo open 20205e000791 101136esmoopen2020000791 0copen accessvariable results between studies20 pd l1 positivity has also been significantly associated with a poorer prognosis than pd l1 negativity in ebvagc furthermore ebv triggers a significantly higher infiltration of cd8 t cells in tme46 in previous studies of patients with ebv positive cancer the current authors showed that high levels of tumour infiltrating lymphocytes tils were associated with a favourable prognosis while intratumoural pd l1 positivity with a worse prognosis47in the phase i javelin solid tumour trial where avelumab was shown to be beneficial for a patient with metastatic gc it is worth noting that ebv positive tumours with a low mutation burden and msi tumours with a high mutation burden had statistically significantly higher tumour lymphocytic infiltration when compared with mss tumours48 the strength of immune mediated signalling signatures in ebv positive tumours also represents a t cell inflamed tme10 these findings support the concept that icis can be used in patients with gc with ebv by suppressing the pd1 pathway in tumour cells and allowing immune activation a recent phase ii trial by kim demonstrated improved efficacy associated with pembrolizumab in patients with ebv positive tumours15 this study enrolled patients with pretreated gc in a subgroup analysis pembrolizumab monotherapy as salvage treatment showed that all six ebv positive patients with gc attained pr orr100 with a median duration of months however in another study out of patients considered ebv positive were treated with toripalimab50 only one pr was observed with two stable and one progressive diseases patients with pr was also pd l1 positive these contrasting results with pembrolizumab could be due to toripalimab rather than ebvagc as a predictive biomarker for icimicrosatellite instabilityhighmmr deficiency is generally characterised by a failure to repair dna replication associated errors leading to the accumulation of mutations in microsatellite regions of the genome51 these phenomena are known as msi52 currently two different methods have been validated for detecting msi h53 the mmr status is assessed by ihc staining to measure the expression levels of the proteins involved in dna mmr and a polymerase chain reaction based exam also tests the length of repetitive dna that are known as microsatellite in the normal and tumour tissues while there are discrepancies between the ihc of mmr protein expression and msi test results the overall concordance in the two tests is high52 the incidence of msi in gc varies between countries being relatively high in approximately of western patients51 msi h gc is commonly associated with intestinal type female sex older age lack of lymph node metastases and onset in the distal stomach54 to date multiple retrospective studies and limited prospective studies have reported on a positive association between msi h and a better prognosis in resectable gc55 for example the magic study reported that patients with msi h tumours have superior survival compared with patients with mssmsi low msi l tumours when treated with surgery alone and conversely have inferior survival to patients with mssmsi l tumours when treated with perioperative chemotherapy plus surgery56 however similar to ebv status patients with msi h had better prognosis thus only of patients with metastatic gc would be mmr deficient the prognostic and predictive values of the msi status on the survival of patients with metastatic gc remain a subject of debate52theoretically in the presence of mmr deficiency undetected dna replication errors leading to a tumour with a high mutational burden reproduce various neoantigens that stimulate t cell activation and tumour infiltration by immune cells keynote012 trial reported that msi h tumours showed a partial response in two out of four patients regardless of pd l1 expression21 a subgroup analysis of keynote059 revealed an orr of for patients with msi h gc4 in keynote061 and more recently reported keynote062 there was a substantially enhanced survival benefit in patients treated with pembrolizumab compared with chemotherapy17 similar to the results for ebv positive tumours the clinical study by kim also showed that msi h tumours responded particularly well to pembrolizumab monotherapy orr85715 in the checkmate032 trial that assessed the efficacy of another pd1 monoclonal antibody nivolumab the orr was for the msi h group vs for the msi l group or mss group19 therefore this evidence highlights the potential of msi h as a predictor of the response to icis in gcof note whereas msi hmmr deficiency is the most consistent predictor of efficacy to icis in gc a substantial portion of msi h gc still has unsatisfactory outcomes even with icis the degree of msi and resultant mutation load in part might explain the variable response to pd1 blockade in mmr deficient tumours57 tumours sensitive to pd1 antibodies showed a loss or a reduction in tumour allele frequency of missense non synonymous single nucleotide variant and indel mutations after pd1 treatment suggestive of immune editing of tumour cellstmb and neoantigentmb may be a potential biomarker of outcomes with icis in multiple solid tumour types41 generally cells have a number of repair pathways to maintain their genome stability13 the mutational load acquired by defective dna repair pathways frequently alters protein function and expression resulting in the formation of neoantigens that serve a source of antitumour immune response therefore it is reasonable to hypothesise that tumours with a high mutational load are more likely to produce neoantigens and increase immunogenicity58 in turn this course of reaction induces a more intensified immune response resulting in tumours becoming more sensitive to treatment with icis41 although tumour specific neoantigens with high clonality are more predictable and beneficial for the response to icis accurate measurement kang a0bw chau a0i esmo open 20205e000791 101136esmoopen2020000791 0cof these neoantigens is known to be expensive and time consuming59 in this situation tmb could be a good approach for indirectly evaluating the neoantigen load tmb is defined by the total number of somatic non synonymous mutations per coding area of the tumour dna58 several studies have already demonstrated the predictive impact of tmb in lung cancer and melanoma one early study by yarchoan observed a significant correlation between tmb and orr for anti pd1 or anti pd l1 therapy60 rizvi also reported that patients with tmb 50th percentile exhibited an improved durable clinical benefit rate and pfs versus those with lower tmb61 this benefit was also seen in the checkmate227 study that included patients with advanced non small cell lung cancer nsclc who received a combination of nivolumab and ipilimumab as the first line metastatic setting62 a significantly prolonged pfs was reported for the patients with higher tmb treated with the combination treatment irrespective of the expression of pd l1 likewise a large scale study across multiple cancer types found a significant association between tmb and the clinical outcome63 these findings can also provide a novel strategy for subgroups with high tmb considering that the measurement of the mutational load is a critical factor for therapeutic successhowever for patients with gc there is still insufficient evaluation and conflicting results on the utility of tmb as a biomarker of the response to icis65 interestingly wang performed a tmb analysis of patients with chemorefractory gc who were treated with toripalimab as a monotherapy in a prospective phase ibii clinical trial50 in this study tmb high tmb h patients responded signiï¬cantly better than the tmb low patients orr vs p0017 a survival benefit has also been demonstrated for patients with high tmb os vs months p0038 similar correlation was also found between tmb h according to circulating tumour dna and better survival when treated with pembrolizumab in gc15 in light of recent approaches this close relationship between tmb and clinical outcomes also points to the possibility of tmb as a predictive biomarker in patients with gc however in the study with regorafenib plus nivolumab due to small sample size tmb did not correlate with response or pfs to this combination36despite its identiï¬ed significant predictive role there are still many challenges in precisely estimating tmb first it is difficult to apply the protocol including whole exome sequencing or targeted sequencing panels using next generation sequencing to clinical practice due to various problems such as the sample amount cost sensitivity coverage and analysis time8 second a standardised cut off value for tmb has not yet been clearly established since many studies have reported a wide range of cutoffs for different tumour types58 thus given the variety of tmb cutoffs assays related with tmb in clinical studies should be interpreted cautiously moreover the availability of tumour sampling to detect tmb is commonly limited and tmb may present temporal open accessvariability a novel blood based tmb approach could be considered as an alternative method as the advantages of repeating sampling during treatment could provide information of a dynamic immune reaction8 plus this approach is less invasive and enables investigators to document the evolution of tmb interestingly in a study by gandara et al blood based tmb was correlated with tissue based tmb and showed a longer pfs in patients with metastatic nsclc who received treatment with atezolizumab68 in summary tmb could be a novel and independent biomarker that reflects the therapeutic effects of icis in gc however its accuracy in predicting the efficacy of icis varies among studies and still needs to be explored for gctumour infiltrating lymphocytesthe immune microenvironment of tumours is now recognised as an important determinant for understanding the relationship between a patients immune system and their cancer informing prognosis and guiding immunotherapy like icis69 tumour cells are typically surrounded by infiltrating inflammatory cells such as cytotoxic t cells helper t cell subsets regulatory t cells tumor associated macrophages dendritic cells and myeloid lineage leucocytes70 among these differentiated lymphocytes referred to as tils are considered a manifestation of the host immune response against tumour cells and seem to play an important role in various human malignancies71 several studies have already reported the potential of ti | 0 |
" preproof 0c highlights f0b7 propolis made by bees from bioactive plant resins has antiviral activity f0b7 propolis potentially can interfere with host cell invasion by sarscov2 f0b7 propolis blocks proinflammatory pak1 a kinase highly expressed in covid19 patients propolis a resinous material produced by honey bees from plant exudates has long been used in traditional herbal medicine and is widely consumed as a health aid and immune system booster the covid19 pandemic has renewed interest in propolis products worldwide fortunately various aspects of the sarscov2 infection mechanism are potential targets for propolis compounds sarscov2 entry into host cells is characterized by viral spike protein f0b7 propolis is a safe widely consumed functional food with medicinal properties f0b7 standardized propolis has consistent properties for lab and clinical research preprooffactor in advanced covid19 disease propolis has also shown promise as an aid in the treatment of various of the comorbidities that are particularly dangerous in covid19 patients including induced lung inflammation fibrosis and immune system suppression propolis components have inhibitory effects on the ace2 tmprss2 and pak1 signaling pathways in addition antiviral activity has been proven in vitro and in vivo in preclinical studies propolis promoted α this immunoregulation involves monocytes and macrophages as well as jak2stat3 nfkb and inflammasome pathways reducing the risk of cytokine storm syndrome a major mortality interaction with cellular angiotensinconverting enzyme ace2 and serine protease tmprss2 this mechanism involves pak1 overexpression which is a kinase that mediates coronavirusimmunoregulation of proinflammatory cytokines including reduction in il6 il1 beta and tnfrespiratory diseases hypertension diabetes and cancer standardized propolis products with consistent bioactive properties are now available given the current emergency caused by the covid19 pandemic and limited therapeutic options propolis is presented as a promising and 0crelevant therapeutic option that is safe easy to administrate orally and is readily available as a natural supplement and functional food keywords propolis sarscov2 covid19 antiviral antiinflammatory pak1 blocker introduction the covid19 pandemic is of grave concern due its impact on human health and on the deadly disease most require more robust data in clinical trials before they can be widely and safely used isolation and stayathome measures do not effectively protect essential workers economy it is much more deadly than influenza and other types of diseases that recently have had worldwide impact forcing countries to take unusual measures such as limiting travel closing schools businesses and other locations where many people can come into contact with each other various public healthcare strategies have been adopted in an attempt to reduce the impact of the disease but with limited effectiveness as the virus continues to spread often through asymptomatic patients unfortunately tests to determine if people are infectious or were previously infected are not widely available often are costly and frequently do not provide timely and accurate results various therapeutic alternatives have been proposed and tested however preproofnonessential professions return to the workplace they become more at risk for infection in this scenario any options that could help ameliorate disease progression and its consequences even marginally would be useful the world needs safe alternatives to help reduce the impact of this especially health care personnel who have become infected and are dying at alarming rates economic and other necessities limit how well and how long these isolation measures can be maintained especially in poor countries and in poor communities such as slums and favelas [ ] as populations gradually try to get back to normalcy reducing social distancing and people in natural products which have historically been widely used to help avoid and alleviate diseases are among the options being considered as an adjuvant treatment for sarscov2 infection because they are generally inexpensive widely available and rarely have undesirable side effects some have proven antiviral activity an important advantage of using natural remedies is that people who have other health problems or who have mild flurelated 0c infection infection by sarscov2 the virus that causes covid19 is characterized by binding symptoms but do not have the means or courage to visit an already overcrowded medical facility could take simple and inexpensive measures to help reduce the impact of infection with sarscov2 considering the large number of deaths and other types of damage that the covid19 pandemic is causing there is an urgent need to find treatments that have been approved as safe and potentially able to inhibit the new coronavirus reduce its infectivity andor alleviate the symptoms of infection [ ] along this line propolis and its components emerge as potential candidate materials that could help to reduce the pathophysiological consequences of sarscovfactors increased pak1 levels also suppress the adaptive immune response facilitating viral replication [ ] sarscov2 infection is associated with increased levels of chemokines and activated proinflammatory cytokines that lead to the development of atypical pneumonia with rapid respiratory impairment and pulmonary failure immunologicalinflammatory between viral spike proteins and angiotensinconverting enzyme ace2 activation of the spike protein is mediated through proteases such as tmprss2 which play important roles in the viral infection after entry followed by endocytosis coronavirus infection causes pak1 upregulation a kinase that mediates lung inflammation lung fibrosis and other critical mortality preproofphenomena such as cytokine release syndrome have been shown to be important in the spectrum of sarscov2 infection these mechanisms are associated with an dysfunction more than the viral load per se along this line a retrospective observational study found higher serum levels of proinflammatory cytokines such as il6 il1 and tnfα in patients with severe inflammatory diseases by affecting various metabolic cycles recently several studies have shown that propolis extract and some of its components act against several important targets in the pathophysiological context of the disease caused by sarscov2 such as reducing tmprss2 expression and reducing ace2 anchorage which would otherwise facilitate entry of covid19 compared to individuals with mild disease there is considerable evidence that propolis can reduce and alleviate the symptoms of the virus into the cell this is in addition to immunomodulation of monocytes macrophages reducing production of and eliminating il1 beta and il6 reduction of the transcription factors 0cviral replication and antiinflammatory action [ ] propolis and its properties are particularly relevant to sarscov2 infection such as immune system fortification reduced nfkb and jak2 stat3 and blocking pak1 which determine inflammatory activities and fibrosis caused by covid19 various comorbidities have been associated with severe covid19 symptoms and a greater chance of patients requiring intensive care these include hypertension and diabetes also mortality rates of covid19 patients are much higher in those with cardiovascular disease chronic respiratory disease and diabetes [ ] there is considerable evidence that these conditions could be alleviated by treatment with propolis this includes research in animal models of diabetes [ ] hypertension [ ] and cardiovascular disease [ ] propolis has properties that preproofthese plant products to make propolis which they use to protect the colony the production and use of propolis by honey bees evolved to the point that these social bees have considerably fewer immune genes than solitary insect species bees in colonies that produce more propolis are healthier and live longer and propolis consumption by the bees augments their immune [ ] as this variability can affect their medicinal properties standardized propolis products have been developed to help meet the need for a product that does not vary in the main bioactive components and is safe with minimal interaction with pharmaceutical drugs and proven efficacy in clinical trials in recent decades it has been shown to have antimicrobial including the composition of propolis varies according to the plant species available in each region propolis is a product derived from resins and plant exudates plants defend themselves from pathogens mainly by producing phytochemicals many of which have been extracted and used in medicine plant defense substances collected by bees include phenols and terpenoids phytochemical compounds that show promise for the inhibition of coronavirus in humans include quercetin myricetin and caffeic acid all components of propolis honey bees and many other species of social bees recognize these antimicrobial properties and selectively collect and process response to bacterial challenge antiviral antiinflammatory immunomodulatory antioxidant and anticancer properties propolis has historically been widely used to alleviate various diseases [ ] it also has been considered among other natural alternatives as an adjuvant treatment for sarscov2 infection 0ccompared to the propolis that the bees make from these plant materials because it is generally inexpensive widely available and rarely causes undesirable side effects some types of propolis that are highly valued for their medicinal properties such as brazilian green propolis are mainly produced by bees from materials they collect from specific plants in this case baccharis dracunculifolia after the botanical origin of the propolis has been identified extracts of the plant can be made to develop useful products such as a medicinal mouthwash however the medicinal properties of these plant extracts are often inferior can be safely consumed these propolis products and the raw material for their manufacture are extensively exported by brazilian companies especially to asian countries including japan south among natural medicine alternatives propolis has been widely studied and is already extensively consumed in many countries [ ] for example propolis products such as throat sprays and extracts are produced by hundreds of companies in brazil and are sold as a health aid in nearly every pharmacy throughout the country demonstrating on a practical basis that they preproofin fact propolis has a wide spectrum of pharmacological properties and is a dietary supplement that is commonly consumed by both healthy and sick people as a preventative precaution and for treatment it is also used in veterinary medicine due its antibacterial antifungal antiviral korea and china [ ] the importance of propolis in chinese japanese russian and korean medicine is reflected in the number of patents for propolis containing products registered by including about by china and each for japan russia and korea since about new propolisrelated patents were applied for in the us patent office it is a key ingredient in traditional chinese medicine japanese scientists have isolated and patented various brazilian propolis components for cancer treatment demonstrating their usefulness why propolis may be a good fit for dealing with covid19 antiparasitic hepatoprotective and immunomodulatory activities in the wake of the coronavirus outbreak south korea has seen a boon in the use of functional foods according to their ministry of food and drug safety health functional foods are nutrients that have been proven to be beneficial to health in march of this year in response to the coronavirus pandemic the ministry eased regulations for propolis which is considered a 0ccould help fight against the covid19 pandemic active site of this enzyme is a relevant target for drug discovery functional food and allowed new oral formulations however despite considerable evidence that propolis can reduce and alleviate disease symptoms its acceptance as a healthpromoting supplement in human medicine has been limited in many countries such as the usa because of a relevant criticism that propolis products are not standardized and vary in their components and biological activity in part this is because propolis varies with the species of plants available in each region from which the bees collect resins to produce it [ ] however standardized propolis products have recently become available to help fill the need for a product that does not vary in the main bioactive components and effectiveness [ ] one such option a standardized brazilian propolis extract blend has been tested for safety and effectiveness in clinical trials for treating kidney disease and diabetes denture stomatitis and burn patients therefore propolis as a nutraceutical or functional food should be considered as a resource that preproofcaffeic acid phenethyl ester cape galangin chrysin and caffeic acid substances found in several different types of propolis around the world appeared as potential drugs against this viral target table specifically cape was predicted to interact with sarscov2 mpro in a potential targets in order to inactive the virus and reduce the damage that it causes the main protease of coronavirus sarscov2 mpro 3chymotrypsinlike cysteine enzyme is essential similar study therefore although it will be necessary to run in vitro assays to evaluate the potential antisarscov2 effects of propolis andor its constituents these in silico results are along this line hashem evaluated various natural compounds with an in silico approach molecular docking to try to find useful options for treating sarscov2 infection curiously for coronavirus processing of polyproteins and for its life cycle and therefore inhibition of the some propolis compounds can potentially interact with sarscov2 mpro the research community has examined the genetic code of coronavirus and the mechanisms underlying the damages caused by sarscov2 to help search for drugs andor well boding propolis can interact with ace and tmprss2 potentially blocking or reducing sarscov2 invasion of the host cell 0c sarscov2 strongly binds to angiotensinconverting enzyme ace2 using this enzyme as a receptor for invasion and replication in the host cell [ ] causing damage and increasing interpersonal transmission [ ] consequently ace inhibitors have been considered as useful drug alternatives however potential deleterious effects on users of angiotensinconverting enzyme ace inhibitors and angiotensin receptor blockers arbs have tool against potential cardiovascular events inhibition of ace2 enzyme is an important target for treatment against sarscov2 myricetin caffeic acid phenethyl ester hesperetin and pinocembrin rutin interacts with zinc fingers of the active sites of ace2 a metalloprotease that presents the same zinc finger in ace1 emerged as a concern for treatment of covid19 patients an observational study involving patients did not confirm this suspicion and therefore these classes of drugs remain an important infection [ ] güler et al prepared an alcoholic extract of propolis and identified some hydroxycinnamic acids caffeic acid pcoumaric acid tcinnamic acid and cape the flavanons rutin and myricetin and the flavones hesperidin chrysin and pinocembrin using molecular docking evaluations they found that rutin had the highest binding energy to ace2 followed by preproofvarious characteristics including inhibition of ace they found strong inhibition for most of the propolis types they studied with higher than ace inhibition the best results were found in addition to the in silico evidence osés et al evaluated several types of propolis for with the propolis components catechin and pcoumaric acid ace2 and tmprss2 transmembrane serine protease on the surface of host cells are used by sarscov2 via interaction with spike glycoproteins in order to proceed with invasion and replication vardhan sahoo studied several molecules commonly found in medicinal herbs using molecular docking procedures with relevant targets such as rnadependent rna polymerase rdrp ace2 and spike glycoproteins and compared the resulting scores with those of hydroxychloroquine limonin was the most active compound however quercetin and kaempferol also propolis compounds gave high docking scores kaempferol was studied in prostate cancer models and the expression of tmprss2 was reduced showing a potential mechanism of action for an antitumoral effect kaempferol could be an important propolis component for use against covid19 since it is involved in the inhibition of tmprss2 0c potentially interacting with ace2 rdrp and spike glycoprotein sgp besides its antiviral activity table propolis blocks pak1 potentially avoiding lung fibrosis and restoring a normal immune response among the possible targets for controlling covid19 damage the major pathogenic contributes to their suppression pak1 inhibitors can both help combat the virus and restore a normal immune response kinase pak1 is key it is an essential component in malaria and viral infections but it is also involved in a wide variety of other diseases and disease conditions including cancer inflammation and immunosuppression when abnormally activated consequences of pak1 activation include lung fibrosis which is an aggravating factor in covid19 pak1 is activated by rac xu et al demonstrated that caffeic acid and its ester cape components of propolis can inactivate rac consequently inhibiting pak1 the inactivation of pak1 directly or upstream can potentially attenuate coronavirus pathogenesis bcells and tcells are lymphocytes that produce specific antibodies against viruses and other intruders and pak1 preproofimprove the immune response its components increase neutralizing antibody titers activate phagocytosis and increase ifnγ levels and the number of lymphocytes an increase in ifnγ levels was also detected by shimizu et al who evaluated the mechanisms involved in the cape caffeic acid phenethyl ester is a potent inhibitor of activation of nfkb in myelomonocytic cells anse et al demonstrated that propolis cape quercetin hesperidin and some other propolis flavonoids can inhibit the cytokine production of th1 and th2 type t cells while increasing tgfbeta an important antiinflammatory cytokine moreover cape can attenuate oxidative stress and inflammation through downregulation of jak2stat3 signaling propolis from europe and temperate asia usually made by bees from resins collected from poplar trees has predominantly flavonoid compounds while green propolis from baccharis dracunculifolia a propolis exclusively found in brazil has various kinds of flavonoids and prenylated phenylpropanoids such as artepellin c baccharin and drupanin these and all other types of propolis can inactivate pak1 artepillin c selectively inhibits pak1 table some studies have shown that propolis can act as an immunostimulant with the ability to effects of some types of propolis in a herpes simplex animal model 0c as well as having an immunomodulatory effect in which cape inhibits il6 phosphorylation and stat3 which are important for proinflammatory th17 development besides the antiinflammatory effect of cape and kaempferol paulino et al evaluated the antiinflammatory effect of artepellin c in rat paw edema and in cell cultures demonstrating that the activity was at least in part mediated by prostaglandin e2 and no inhibition through nfkb modulation artepillin c is an important biomarker of brazilian green propolis botanical source baccharis dracunculifolia immune modulation is desirable since coronavirus infection dysregulates the immune response in the initial phases of infection which facilitates viral replication however in later stages of covid19 the body develops an exaggerated inflammatory response which can greatly damage the lungs and other ans propolis different from typical immunosuppressants can help avoid immunosuppression during the initial phases of disease and in later stages reduce an exaggerated host inflammatory response inhibiting excess il6 il2 and jak signaling cape a propolis component is also known as an immunemodulating agent and should be considered as an alternative to help reduce an exaggerated inflammatory response in a mouse model propolis had immunomodulatory action in vivo on tolllike receptor expression and on preproofreplication and infectivity potentially decreasing lung inflammation due to antiinflammatory properties while promoting immune system fortification these are useful properties that could there is ample evidence for interference of propolis andor its components with viral propolis has been tested against various viral disease anisms initial successes have prompted research to determine the most useful components which may be modified to produce more active and specific pharmaceuticals viruses that were controlled by propolis in animal models with suggestion for control in humans include influenza [ ] herpes simplex virus type and hiv [ ] shimizu et al evaluated three different types of propolis in ethanol extracts using a murine model of herpes simplex virus type despite the chemical differences proinflammatory cytokine production help minimize the symptoms and deleterious effects of covid19 figure figure propolis as an antiviral substance 0cdue to the different plant origins of the resins the bees used to produce the propolis baccharis dracunculifolia baccharis eriodata and myrceugenia euosma all three propolis extracts not only had direct antihsv1 effects but also stimulated immunological activity against intradermal hsv1 infection in mice antiviral activity of propolis has been reported for dna and rna viruses poliovirus herpes simplex virus and adenovirus in an in vitro model cultured cells the best results were obtained against poliovirus and herpes virus with inhibition of the latter at a propolis concentration of ugml the propolis components chrysine and kaempferol caused a concentrationdependent reduction of intracellular replication of herpesvirus strains when host cell monolayers were infected and subsequently cultured in a drugcontaining medium quercetin another propolis component had the same effect but only at the highest concentrations tested ugml against various human herpes simplex virus strains with a intracellular replication reduction of approximately while it reduced the infectivity of bovine herpes virus human adenovirus human coronavirus and bovine coronavirus about the reduction was in the preproofimpairment and pulmonary failure inflammatory response to infection sarscov2 infection is associated with increased levels of chemokines and activated proinflammatory cytokines that lead to the development of atypical pneumonia with rapid respiratory immunologicalinflammatory phenomena such as cytokine release syndrome have been shown to be important mortality factors in sarscov2 infection higher serum levels of proinflammatory cytokines such as il6 il1 and tnfα are found in patients with severe covid compared to those of individuals with mild disease molecular mechanisms involved in this immune process are the targets of various synthetic medicines being tested in patients including ciclesonide hydroxy chloroquine ivermectin and ketorolac which are pak1 blockers pak1 raccdc42activated kinase is overexpressed in the lung in response to sarscov2 infection and is a critical mediator of the cytokine storm that frequently results in mortality the most critical cases of covid19 which require ventilatorassisted intensive care and often result in prolonged ventilator dependency and death are a result of an exaggerated case of rotavirus antiinflammatory and immunomodulatory properties of propolis 0cin hospitalized patients fortuitously propolis components are effective pak1 blockers table there is considerable evidence that propolis can reduce and alleviate the symptoms of inflammatory diseases and has immunomodulatory properties [ ] however these properties can vary according to the plant origin of the propolis as well as the extraction processsolvent used and the inflammatory protocol cell culture animal models induction by lipopolysaccharides when the propolis extracts are tested tests with animal models have shown that propolis can reduce the levels of il6 and tnfalpha which are key proinflammatory mediators and increase the levels of the regulatory cytokine il10 kaempferol a propolis component reduces il6 tnfalpha and vegf vascular endothelial growth factor via the preproofifnγ in serum fernandes et al found that propolis exerted a positive adjuvant effect on vaccines that were developed against canine coronavirus they assayed ifnγ which is an effective way to measure the cellular response induced by a vaccine in a mouse model propolis added as an adjuvant to inactivated swine herpesvirus type vaccine stimulated increased cellular propolis is considered a safe immunostimulant and a potent vaccine adjuvant propolis has been widely tested as a vaccine adjuvant because it induces an earlier immune response and provides a longer protection period it is also included as an adjuvant ingredient in traditional chinese medicine propolis flavonoids have potential as adjuvants enhancing igg il4 and propolis has potential as a vaccine adjuvant erknfkbcmycp21 pathway table tests on macrophage cell cultures also demonstrated that propolis inhibits the production of il1 beta an important component of the inflammasome inflammatory pathway in diseases such as rheumatoid arthritis lupus and other autoimmune diseases although the mechanisms of action are not well elucidated these propolis components have potential as complementary supplements in the preventive treatment of chronic inflammatory diseases and humoral responses increasing ifnγ [ ] propolis enhanced the immune response to inactivated porcine parvovirus vaccine in guinea pigs when added to a trichomonas vaginalis protein vaccine propolis increased the igg antibody response times in mice 0ccancer is considered a relevant comorbidity factor for covid19 cancer patients have a patients cancer patients compared to the protein alone propolis was also effective as an adjuvant in the immunization of cattle with bovine herpesvirus it improved the humoral and cellular responses in mice inoculated with inactivated virus vaccines propolis as an adjuvant gave a similar immune response increasing ifnγ levels to alum and freunds adjuvant in mice vaccinated with an hiv1 polytope vaccine candidate with less risk of undesirable side effects comorbidities and evidence of how propolis can help reduce their impact in covid19 to risk a visit to a clinic or hospital to determine if they have cancer alternative therapies could help retard cancer or reduce the impact of cancer and cancer treatment in covid19 times higher risk of progressing to severe covid19 disease than patients without comorbidities also the hospital environment during the coronavirus pandemic can interfere with or delay the treatment that cancer patients should receive patients with symptoms may choose not preproofacid cape quercetin and chrysin propolis and its components normally have little impact on normal cells displaying differential cytotoxicity in liver cancer melanoma and breast cell carcinoma cell lines [ ] propolis enhances the activity of tumor various types including bladder blood brain breast colon head and neck kidney liver pancreas prostate and skin cancers propolis could help prevent cancer progression in various parts of the world it is considered an alternative therapy for cancer treatment propolis extracts have been found to inhibit tumor cell growth both in vitro and in vivo including inhibition of angiogenesis demonstrating potential for the development of new anticancer drugs various components of propolis have been shown to inhibit cancer cell growth including cinnamic propolis has potential as a complementary therapy for cancer it has shown efficacy against necrosis factor related apoptosis inducing ligand trail in cancer cells hypertension and cardiovascular disease 0c hypertension and cardiovascular disease are considered relevant comorbidities for covid19 propolis has demonstrated antihypertensive effects in rat models [ ] in cameroon it is used in popular medicine to treat various ailments including high blood pressure propolis has been widely used as a dietary supplement for its health benefits including cardiovascular protective effects [ ] in a human trial consumption of propolis improved critical blood parameters including hdl gsh and tbars levels demonstrating that obesity a natural antiobesity agent it could contribute to a reduced risk for cardiovascular disease obesity is a major comorbidity and predictor of increased mortality in covd19 patients obesity and sarscov2 both induce an inflammatory process exacerbating sarscov2 infection in the obese propolis reduced inflammation and prevented hyperlipidemia and metabolic syndromes in highly caloric diet induced obesity in mice body weight gain visceral adipose tissue liver and serum triglycerides cholesterol and nonesterified fatty acids were all reduced in the propolis fed mice [ ] caffeic acid phenethyl ester a propolis component is preproofplatelet aggregation was reduced by propolis in tests on human blood in vitro and in other in vitro tests caffeic acid phenethyl ester cape a wellstudied bioactive propolis component inhibits collagen induced platelet activation thromboembolism thrombosis and microthrombosis microthrombosis disseminated intravascular coagulation and consequent multian failure are common in severely affected covid19 patients with associated high mortality rates anticoagulants are sometimes prescribed to such patients because they can reduce mortality tang et al an elevated level of plasminogen activator inhibitor1 pai1 is a biomarker and risk factor for thrombosis and atherosclerosis [ ] various types of evidence demonstrate that propolis can reduce platelet aggregation and other thrombosisrelated parameters propolis decreased thrombotic tendencies in mice by suppressing lipopolysaccharideinduced increases in pai1 levels [ ] propolis downregulated plateletderived growth factor and platelet endothelial cell adhesion molecules in lowdensity lipoprotein knockout mice 0c old age the elderly are more often affected by chronic inflammation characterized by systemically increased levels of proinflammatory cytokines which can contribute to development of a cytokine storm a major cause of covid19 mortality propolis has antioxidant properties which could help retard o | 0 |
coronaviruses covs belong to a family of envelopedviruses with a positive sense singlestranded rna genomecovs cause illness ranging from upper respiratory tractinfections urtis resembling the common cold to lowerrespiratory tract infections lrtis such as bronchitis pneumonia and even severe acute respiratory syndrome sarswith most serious disease outcomes in the elderly immunocompromised patients and infants [ ] hcovoc43oc43 hcov229e 229e hcovnl63 nl63 andhcovhku1 hku1 were the ï¬rst documented humancovs hcovs which usually cause urtis and less frequently are associated with ltri diseases in the lastdecades two human coronaviruses created great concernfor the world medical community due to significant diseaseand mortality [ ] in severe acute respiratorysyndromecoronavirus sarscov was characterized byacute atypical pneumonia and diï¬use alveolar damagedad in roughly patients and with almost deathsrepresenting a nearly mortality rate more recentlyin a new human coronavirus designated as middle eastrespiratory syndromecoronavirus merscov was identiï¬ed and the global ongoing outbreak of mers with over oï¬cial cases and deaths represented approximately case fatality rate to date in humans over the last few months a new strain of human coronavirus sarscov2 also known as 2019ncov hascaught the worlds seven continents attention with its rapidglobal spread aï¬ecting at least countries and territoriesinfecting more than and claiming more than lives worldwide the coronavirus pandemichas promoted isolation and uncertainly fear and panicworldwide in additionlikely lead to changes inpolitical and economic power in ways that can be determined only later it willit is important to note that there are many similaritiesamong diï¬erent coronavirus species but not in all aspects 0coxidative medicine and cellular longevitydepending on the molecular mechanism of viral inhibitionpromoted by an antiviral agent the analysis of the data andcomparison between animal and human covs must be donevery carefully in fact it is important to note that there arediï¬erences between human and animal cov receptorswhich will likely result in diï¬erent aï¬nities or unlikely interactions of an antiviral agent with the diï¬erent cov receptors howeverif the antiviral agent interferes with thereplication andor assembly of the covs there is a higherprobability of obtaining similar antiviral activity results inhuman cov tests [ ] following this line our searchin specialized literature was focused mainly on studies thatinvestigated the anticoronavirus eï¬ects of natural antioxidants by inhibiting proteases for viral replication materials and methodsthe present study was carried out based on a search of the literature of natural antioxidants and coronavirus the searchperformed in the pubmed database included studies published until march and used the following keywordscoronavirusstressmerscov sarscov 229e nl63 oc43 hku1merscov virus infection and middle east respiratorysyndrome virus the scientiï¬c publications were selectedfrom studies published in the english languageantioxidants ï¬avonoids oxidative pathogenic mechanism of coronavirusinduced cell damagethe high mortality rate associated with the three pathogenichcovs has been mainly attributed to the development ofdigestive and respiratory tract injuries observed followinginfection acute atypical pneumonia and diï¬use alveolardamage that progress to deposition of ï¬brous tissue denudedairways haemorrhage and elevated macrophage ltrationare sometimes accompanied by watery diarrhoea dehydration and vomiting [ ]despite the molecular mechanisms of coronavirusinduced intestine and lung pathogenesis not fully elucidatedand still unclear studies have suggested that lateterm diseaseprogression is unrelated to viremia it is now believed morelikely to be associated with the immunopathological mechanism [ ] viral clearance and subsequent recovery frominfection require activation of an eï¬ective host immuneresponse however many immune eï¬ector cells may alsocause injury to host tissues together with ammatoryand immune response signaling the presence of oxidativecompounds such as reactive oxygen species ros playsimportant roles in the pathogenic mechanism of cell damageinduced by covs through oxidative stress oxidative stress is deï¬ned as an interruption andorderegulation of the signaling and redox system that can becaused by an imbalance in the production of oxidant andantioxidant species among the main oxidant agentsros and reactive nitrogen species rns stand out in orderto counterbalance the oxidant species there is an antioxidantsystem formed by enzymes and nonenzymatic molecules however during pathological events such as viral infections there may be an increase in the production of oxidantspecies not neutralized by the antioxidant system resultingin oxidative stress that promotes cellular damage throughprotein denaturation changes in the functions of nucleicacids lipid peroxidation and cell death []in addition during viral infection oxidative stress contributes to viral pathogenesis through stimulating ammation loss of immune function and increased viral replicationthat may occur due to the activation of the nuclear factorkappa b nfκb transcription pathway [] currentevidence suggests that cytokine dysregulationalso calledcytokine stormcontributes to severe disease caused by thepathogenic covs [ ] the exact mechanisms are notclear yet but research on uenza a virus shows that infection causes a rapid ux of ammatory cells this isfollowed by an increase in reactive oxygen species productionand cytokine expression and release which ultimately leadsto acute lung injury in general rna viruses promotechanges in the bodys antioxidant defense system aï¬ectingenzymes such as superoxide dismutase sod and catalasecat in addition to reducing the levels of antioxidantmolecules such as ascorbic acid carotenoids and reducedglutathione gsh [] wu reported that glucose6phosphate dehydrogenasean important antioxidantenzyme that produces nadph knockdown cells were moresusceptible to infection by hcov229e than normal cells interestingly ye and colleagues have reported that theinhibition of ros production alleviates ammation causedby uenza a virus infections in an experimental model of sarsinduced acute lunginjury in mice it was noted that phospholipid oxidationdue to oxidative stress is one of the main triggering factorsof acute lung injury this happens through the activation ofthe innate immune response culminating in the activationof pulmonary macrophages via tlr4triftraf6nfκbsignaling furthermore hypoxia caused by acute lunginjury can cause myocardial injury due to the production ofros aggravating infections caused by coronavirus disease covid19 mitochondria have an essential function in energy generation and for this reason their function and integrity arestrictly regulated in order to respond to varying energyrequirements and environmental conditions mitochondria are known to function as the control point in apoptoticpathways releasing proapoptotic factors mainly ros whichfunction as a signaling molecule that may result in cell death[ ] some studies have shown a relationship betweencoronavirus infection and dysfunctional or damaged mitochondria leading to the release of ros and other proapoptotic substances [ ] in a recent study xu reportedthat ros and p53 play key roles in regulating many kindsof the cell process during coronavirus infection in vero cellsaccording to the authors coronavirus infection appears toinduce a timedependent ros accumulation which in turnis linked to regulatory mechanisms of p53 activation andapoptosis in infected cells antioxidant substances promote improvement in casesof disease caused by coronaviruses such as apolipoproteinda lipocalin that promoted a neuroprotective eï¬ect against 0coxidative medicine and cellular longevityencephalitis induced by human coronavirus oc43in micethis protective eï¬ect occurred through the reduction of oxidative stress cerebral lipid peroxidation and regulation ofammation [ ] also the treatment with antioxidantssuch as pyrrolidine dithiocarbamate or nacetylcysteine significantly inhibits moreover melatonin promotes downregulation of acute lungoxidative injury due to its antiammatory and antioxidantactions making it a possible compound in the treatment ofcovid19 based on these studies compounds thathave antioxidant actions can be helpful in the treatment ofinfections promoted by coronaviruscoronavirusinduced apoptosisin general antioxidant properties of polyphenolic compounds such as some ï¬avonoids have been associated withthe presence of aromatic phenolic rings that promote theelectron donation and hydrogen atom transfer to free radicals acting as free radical scavengers reducing agents andquenchers of single oxygen formation thus the aimof this study was to investigate the antioxidant capacity andantiviral activity of natural antioxidants against coronavirusthe compounds are illustrated in figure occurrence and antioxidant properties ofanticoronavirus compoundsquercetin can be found in plants such as rubus fruticosus land lagerstroemia speciosa l pers [ ] also quercetinshows antioxidant activity at a concentration of μmoll inhepg2 cells inhibiting oxidative stress promoted by h2o2 promotes an increase in sod cat and glutathioneperoxidase gpx and reduces lipid peroxidation in rats withchronic prostatitischronic pelvic pain syndrome moreover quercetin improves sepsisinduced acute lung injury inrats by reducing lipid peroxidation and ammation andincreasing sod and cat levels in addition quercetin glycosides with antioxidant activity such as quercetin 3βglucoside have already been isolated from plants such as passiï¬ora subpeltata ortega andchamomilla suaveolens pursh rydb [ ] the administration of quercetin 3βglucoside mgkg poinstreptozotocininduced diabetic rats promotes an increasein the levels of antioxidant enzymes sod cat andgpx and nonenzymatic antioxidants vitamins c and eand gsh and a reduction of lipid peroxidation quercetin 3βgalactoside hyperoside is found mainly in plantsof the hypericum genus such as hypericum perforatum l[ ] moreover it showed cardioprotective activity inhigh glucoseinduced injury of myocardial cells throughdecreased apoptosis and ros production and increasedsod levels quercetin 7ramnoside is also found inplants of the hypericum genus such as hypericum japonicum thunb ex murray this ï¬avonoid shows hepatoprotective activity against carbon tetrachloride in mice bydecreasing lipid peroxidation and increasing cat andgsh levels in addition to presenting values of μmand22diphenyl1picrylhydrazyldpph and ²azinobis3ethylbenzthiazoline6sulphonic acid abts assays respectively μm intheepigallocatechin gallate is present in parkia roxburghii gdon and is one of the main metabolites found in green teaand liubao tea camellia sinensis lo kuntze also gallocatechin gallate can be found in this plant [] literaturedata reveal that the administration ip of mg100 g ofepigallocatechin gallate in rats with streptozotocininduceddiabetes mellitus promotes a reduction in oxidative stressthrough reductions in parameters such as indirect nitricoxide synthesis and status total oxidative as well as anincrease in levels of cat and total antioxidant capacity ofplasma furthermore it promotes cardioprotection byantioxidant mechanisms green tea has a high antioxidant capacity due to the highlevels of catechins present he and collaborators compared the antioxidant activities of catechins and reported thatepigallocatechin gallate has greater antioxidant activity viaradical scavenging activity μm with values of ± ± and ± compared to its epimergallocatechin gallate with values of ± ± and ± in the dpph abts and ferric reducingantioxidant power frap respectively amentoï¬avone is a biï¬avonoid present in leaves ofginkgo biloba l garcinia brasiliensis l and nandinadomestica l [] this biï¬avonoid has a high antioxidantcapacity demonstrated in scavenging tests ofdpph abts superoxide and hydroxyl radicals moreover amentoï¬avone prevents acute lung injury induced bysepsis in rats by decreasing thiobarbituric acid reactive substance tbars levels and by increasing levels of sod andgsh apigenin is mainly present in ï¬owers and leaves beingabundantly found in apium graveolens l petroselinum crispum mill fuss and matricaria chamomilla l sánchezmarzo and collaborators evaluated the antioxidantcapacity of apigenin using the trolox equivalent antioxidantcapacity teac oxygen radical absorbance capacityorac and frap assays the results show that apigeninhas good antioxidant activity with values of ± μmol teammol ± μmol teammol and ± μmol fe2mmol respectively in addition oraladministration of apigenin mgkgday for days in anexperimental model of cardiotoxicity induced by doxorubicin in rats promoted cardioprotection by reducing levels ofmalondialdehyde mda increasing sod levels and preventing cardiomyocyte apoptosis luteolin is present in foods such as carrot cabbage teaand apple and is found in ugni molinae turcz [ ] datashow that luteolin μgml increases the levels of gsh theexpression of gsh synthetase and the activity of sod andcat in human colon cancer cells ht29 furthermoreluteolin attenuates the sepsisinduced acute lunginjury in mice by reducing lipid peroxidation and increasingsod and cat activity in addition to suppressing the nfκbpathway herbacetin is ubiquitous in plants of the genus rhodiolasuch as rhodiola rosea l herbacetin glycosides are alsopresent in the roots of r sachalinensis a bor and show antioxidant activity veeramani reported that theadministration of herbacetin mgkg po in mice with 0coxidative medicine and cellular longevityohohohohhoohooohohhooohoh oquercetinohohoooohohohquercetin 3ð½galactosideohohooohoohoohohhoohohohoh oquercetin 7ramnosideohooohohoohoohoohohohquercetin 3ð½glucosidehooohooohohohohohohepigallocatechin gallateohgallocatechin gallateohhooohoh ohooluteolinhooohoooohoohohoohrhoifolinhohohoohohohohohohoohoohooh oamentoflavoneohhooohohohoherbacetinhohoooohooohohohoapigeninooohooohhopectolinarinooopsoralidinhooohohohohcatechinohhoohoooh ohelichrysetinohohomyricetinohohohhohoohoohoisobavachalconeohhooohscutellareinohresveratrolfigure chemical structures of bioactive antioxidants against coronavirusobesityassociated insulin resistance promotes an increasein the activity of the enzyme glucose6phosphate dehydrogenase which is directly related to the production ofnadph pectolinarin is present in plants of the genus cirsiumsuch as cirsium setidens nakai and cirsium japonicum dcthe administration of pectolinarin and mgkg pofor two weeks in rats promotes antioxidant eï¬ects in hepatic 0coxidative medicine and cellular longevitytable antioxidant properties of natural inhibitors of coronaviruscompoundtestedassaysexperimentalconcentration doseantioxidant eï¬ectreferencetype of cells μmoll mgkg po mgkg poinhibiting oxidative stress promoted byh2o2promoted an increase in sod cat andgpx and reduced lipid peroxidationreduces lipid peroxidation and increasessod and cat levelsincreases levels of sod cat gpx mgkg povitamins c and e and gsh and reduces nmoll mgkgic50 μm dpphec50 μm abtsrats with streptozotocininduced diabetes mellitus mg100 g iprats with streptozotocinnicotinamideinduceddiabetes mellitus mgkg po μm mgkg μgml ± μmol teammol ± μmol teammol and ± μmol fe2mmolrespectively mgkg pomodelshepg2 cellsrats with chronicprostatitischronic pelvicpain syndromesepsisinduced acute lunginjury in ratsstreptozotocininduceddiabetic ratshigh glucoseinducedinjury of myocardial cellsccl4induced liver damagemodel in micedpphabtsquercetinquercetin 3βglucosidequercetin 3βgalactosidequercetin ramnosideepigallocatechingallateepigallocatechingallatedpphabtsfrapgallocatechingallateamentoï¬avoneacute lung injury inducedby sepsis in ratsdpph abts superoxideand hydroxyl radicalsteacoracfrapcardiotoxicity induced bydoxorubicin in ratshuman colon cancer cellsht29acute lung injury inducedby sepsis in micemice with obesityassociated insulinresistance inductionhepatic injury induced bydgalactosamine in ratsoracapigeninluteolinherbacetinpectolinarinrhoifolincatechinlipid peroxidationdecreases apoptosis and ros productionand increases sod levelsdecreases lipid peroxidation and increasescat and gsh levelsscavenging of free radicalsreduces indirect nitric oxide synthesis andtotal oxidative statusincreased levels of cat and totalantioxidant capacity of plasmaincreased levels of cat sod and gshreduced levels of superoxide and proteincarbonyl pco and prevented dnadamage ± ± and ± ± and ± respectively ± respectivelydecreases tbars levels and increaseslevels of sod and gshscavenging of free radicalsscavenging of free radicalsreduces levels of mda increases sodlevels and prevents cardiomyocyteapoptosis μgmlincreases levels of gsh expression of gshsynthetase and the activity of sod and mgkg ip mgkg po and mgkg poapproximately troloxequivalents μmcatreduces lipid peroxidation increases theactivity of sod and cat and suppressesthe nfκb pathwayincreases the activity of glucose6phosphate dehydrogenaseincreases levels of sod gsh glutathionereductase and glutathione stransferasescavenging of free radicalsscavenging of free radicals 0coxidative medicine and cellular longevitytable continuedtype of cellscompoundtestedassaysexperimentalconcentration doseantioxidant eï¬ectreferencemodelsabtsfrap ± mol troloxequivalentsmol ± mol trolox equivalentsmoldihydrorhodamine oxidation assayandic50 ± μmisobavachalconedpph sc50frapabts sc50psoralidinelectron spin resonancemyricetinhelichrysetinscutellareinresveratroldpphchinese hamster lungï¬broblast cells v794treated with h2o2oracdpphabtssuperoxide radicalsdpph sc50r2rats with obstructive lungdiseasehypercholesterolemicapoeko mouserespectively μm ± mmic50 μmequivalent to feso4 mm respectively·7h2o and μgml and μgml μgml ± trolox equivalents ± μm ± μm and ± μm respectivelyscavenging of free radicalsscavenging of free radicalsscavenging of free radicals and respectivelyprevents dna damage and lipidperoxidationincreases the activity of sod cat andgpxscavenging of free radicalsscavenging of free radicals μmoldmscavenging of free radicals mgkg mgkgincreases sod activity and reduces mdalevelsinhibits the activity and expression ofnadph oxidasesincreases sod gpx and cat levels injury induced by dgalactosamine by increasing levels ofsod gsh glutathione reductase and glutathione stransferase [ ]rhoifolin is found in citrus fruits such as citrus limettarisso studies have indicated that its radical peroxyl scavenging capacity is higher than trolox in orac assays approximately trolox equivalents μm [ ]meanwhile the catechin is a ï¬avonoid present inleaves of green tea wine and fruits [ ] grzesik investigated the antioxidant action of catechinthrough the abts scavenging activity and frap teststhe results show values of ± mol troloxequivalentsmol and ± mol trolox equivalentsmol respectively in addition catechin shows greaterprotective properties in the dihydrorhodamine oxida ± μm than gsh and ascorbiction assay ic50acid and μm respectively psoralidin is a prenylated coumestan which is found inplants of the fabaceae family such as psoralea corylifolia lxiao and collaborators investigating the antioxidant potential of compounds isolated from p corylifolia observed thatpsoralidin shows the best antioxidant activity by the methodof electron spin resonance spectroscopy with an ic50 value of μm the compound isobavachalcone has been isolated fromplants of the fabaceae and moraceae families [ ] isobavachalcone shows a strong antioxidant activity in dpphsc50 frap and abts sc50 assays with values of μm ± mm equivalent to feso4·7h2o and mm respectively in addition the compound has beenreported to inhibit the nfκb pathway in sephadexinducedlung injury in rats [ ]helichrysetin is a chalcone that is found in plants of thehelichrysum genus such as helichrysum odoratissimum l in a study investigating the antioxidant activity of natural and prenylated chalcones vogel found that helichrysetin is the substance that shows the highest antioxidantactivity in the orac test with values of ± troloxequivalents myricetin is widely found in the plant families myricaceae and anacardiaceae and is widely used as health foodsupplement due to its antioxidant properties [ ]bennett demonstrated that myricetin reacts withoxygencentered galvinoxyl radicals more than timeshigher than vitamin e dalphatocopherol furthermoremyricetin was able to scavenge and on the dpphassay μgml and μgml respectively interestinglythe compound prevents dna damage by lipid 0coxidative medicine and cellular longevity2h2o2 2gsh2o2h2h2o2vit evit cgshnadphnonenzymatic antioxidantsgpxsodcatsemyzne tnadixoitna2h2o gssgo2 h2o2o2 h2omdapcotbarsbio m arkers of oxidative stressgeneration of rosoxidative stresscell damagenaturalantioxidants sesadixohpdan2o2 nadphnadp 2o2 hfigure the main antioxidant mechanisms of natural compounds reported in this review dashed line inhibition full line activationperoxidation and increasing the activity of sod cat andgpx in chinese hamster lung ï¬broblast cells v794treated with h2o2 scutellarein is found in scutellaria barbata d don andpolygonum viscosum buchham [ ] liu investigated the antioxidant activity of scutellarein through thedpph abts and superoxide scavenging assays they notedthat the compound shows good antioxidant activity withvalues of ± μm ± μm and ± μmrespectively while the trolox a standard antioxidant compound presented ± μm ± μm and ± μm respectively resveratrol is found in grapes peanuts and blueberriesand can be isolated from veratrum grandiï¬orum o loes literature shows that resveratrol has good antioxidantvalues of μmoldmactivity with dpph sc50r2moreover it is able to reduce the production of ros by inhibiting the activity and expression of nadph oxidases byeliminating oxidant agentsincluding radical hydroxylsuperoxide hydrogen peroxide and peroxynitrite the treatment of resveratrol mgkg po in ratsreduces oxidative stress in obstructive lung disease byincreasing sod activity and reducing mda levels indicatinga decrease in lipid peroxidation table shows themain actions of natural antioxidants discussed in this studyand figure illustrates these activities effect of natural antioxidants incoronavirus infectionsthis review focused on studies reporting on the anticoronavirus activity of natural antioxidants based on exclusioncriteria data from nineteen compounds were discussedthe oxidative stress pathway could potentially be a keyelement in coronavirusinduced apoptosis and pathogenesis for this reason it is interesting to investigate the useof antioxidants as potential therapeutic toolseither as analternative or as an adjuvant to conventional therapiesinthe treatment of coronavirus infections among the antioxidant compounds evaluated as for coronavirus infectionsare the ï¬avonoids which are compounds widely found infruits vegetables and certain beverages in fact researchgroups have reported that antioxidant ï¬avonoids includingcatechin luteolin apigenin quercetin and quercetin rhamnosideinhibit ros accumulation and apoptosis ofcells infected with diï¬erent coronavirus including porcineepidemic diarrhoea coronavirus pedv and transmissiblegastroenteritis coronavirus tgev []as shown with the recent covid19 pandemic thesearch for alternative or new antiviral therapies for theremainstreatment of coronavirus diseasesimportantbased on the literature antioxidanttherapies oï¬er anattractive option 0coxidative medicine and cellular longevitytable natural antioxidants tested in in vitro coronavirus infection models and their main results and mechanism of actionantioxidanttype of cells testedconcentrationic50antiviral eï¬ectmechanism of actionreferencecatechintgevinfected st cellscatechin μminhibition of tgevinduced apoptosissuppression of the tgevinducedbcl2 reduction baxredistribution cytochrome crelease and caspase3 activation resveratrolmersinfected vero e6cellsresveratrol μmquercetinepigallocatechingallategallocatechingallate gcgquercetin rhamnosideq7rrecombinant 3clprowas expressed in pichiapastoris gs115quercetin μmepigallocatechingallate μmgallocatechingallate μmpedvinfectedvero cellsq7r μminhibition ofmersinducedreduction of the expression ofinfectionapoptosis andnucleocapsid n protein essential prolonged cellular survivalfor merscov replicationafter virus infectioninhibition of coronavirusreplicationreduction of the formationof a visible cytopathiceï¬ect cpe without dnafragmentationgcg displayed a binding energyof kcal mol1 to the active siteof 3clpro and the galloyl moietyat 3oh position was required for3clpro inhibition activitynot speciï¬city amentoï¬avoneapigeninluteolinquercetinquercetin3βgalactosideherbacetinrhoifolinpectolinarinsarscov 3clproinhibition usingï¬uorescence resonanceenergy transfer analysismolecular dockingsprfretbasedbioassays andmutagenesistryptophanbasedï¬uorescence methodherbacetinisobavachalconequercetin3βdglucosidehelichrysetintryptophanbasedï¬uorescence methodamentoï¬avone3βgalactoside μmapigenin μmluteolin μmquercetin μmquercetin μmherbacetin μmrhoifolin μmpectolinarin μmherbacetin μmisobavachalcone μmquercetin3βdglucoside μmhelichrysetin μminhibition of sarscovreplicationflavonoids exhibited sarscov3clpro inhibitory activity[ ]inhibition of merscovreplicationflavonoids exhibited merscov3clpro inhibitory activity isobavachalconepsoralidinlineweaverburk anddixon plotsmyricetinscutellareinsprfretbasedbioassaysisobavachalcone μmpsoralidin μmmyricetin μmscutellarein μminhibition of sarscovreplicationisobavachalcone and psoralidinexhibited sarscov papainlikeprotease inhibitory activitymyricetin and scutellareininhibition of sarscovpotently inhibit the sarscovreplicationhelicase protein in vitro byaï¬ecting the atpase activity the high number of deaths and clinical complicationsobserved in sars and merscov epidemics motivatedthe search for eï¬ective therapeutic agents this was necessitated when many of the tested conventional drugs andtherapies proved ineï¬ective in treating sarsantiviralcov infections for exampletreatment ofthe initial 0coxidative medicine and cellular longevitycell culture coronavirusnaturalantioxidants suppress bcl2 reductionsuppress bax redistributionsuppress cytochorome c releasesuppress caspase3 activationreduce formation of a visiblecytopathic effect without dnafragmentationreduce nucleocapsid n protein expressioninhibit 3clike proteaseinhibit 3clike proteaseinhibit papainlike proteaseinhibit helicase protein by affectedatpase activitytgevpedvmerscovsarscovfigure inhibitory actions of natural antioxidants against coronavirussarscov with antiviral agents such as ribavirin and corticosteroids did not achieve very satisfactory resultsmainly because corticosteroids exertimmunosuppressoreï¬ects on the humoral and cellular immune systems[ ] other drugs such as pentoxifylline were considered for the treatment of sars due to its interestingtherapeutic propertiesantiammatoryantiviral immunomodulatory and bronchodilatory eï¬ectshowever it too was not successful in the clinical treatment of sarscov infection includethatinhibition ofmany antioxidant compounds show antiviral activityagainst sarscov the antiviral activity has been mainlyattributed to thethe 3clike protease3clpro of sarscov a vital enzyme for sarscov replication as an example multiples studies havereported that quercetin and quercetinderived compoundssuch as quercetin 3βgalactoside display potent 3clproinhibitory e5ï¬ect and consequent reduction of sarscovreplication other antioxidants such as epigallocatechin gallate gallocatechin gallate amentoï¬avone apigeninluteolin herbacetin rhoifolin and pectolinarin are alsofound to eï¬ciently block the enzymatic activity of sarscov 3clpro [ ]moreover some natural antioxidants exhibit promisingantiviral activity against sarscov infection by interferingwith diï¬erent targets involved in sarscov replication inparticular the sarscov papainlike protease plpro andsarscov helicase protein kim reported that isobavachalcone and psoralidin inhibit plpro in a dosedependentmanner with ic50 ranging between and μm previously yu reported that myricetin and scutellareinpotently inhibit the sarscov helicase protein in vitro byaï¬ecting the atpase activity merscov is another zoonotic coronavirus transmitted between animals and human beings that causes severemorbidity and mortality no antiviral medicines with satisfactory eï¬cacy for the treatment of merscovinfectedpatients have been identiï¬ed to date similar to sarscov natural antioxidant libraries have been probed forpotentialinhibitory compounds against merscov 3clike protease jo showed that herbacetin isobavachalcone quercetin 3βdglucoside and helichrysetinfourcompounds with recognized antioxidant activity can blockthe enzymatic activity of merscov 3clpro using atryptophanbased ï¬uorescence method furthermore theexperimental and computational studies show that ï¬avonoland chalcone are favourite scaï¬olds to bind with the catalytic site of merscov 3clpro in a study performed by lin the antiviral activitiesof resveratrol were investigated in mersinfected vero e6cells the authors reported a significantinhibition ofmerscov infection and prolonged host cell survival aftervirus infection which they speculate was promoted by resveratrol in addition they also found that the expression ofthe nucleocapsid n protein which is essential for merscov replicationis decreased after resveratrol treatment it is important to mention that in vitro models of coronavirus infection also show antiviral activity of ï¬avonoidsextracted from ï¬owering cherry cultivars and black tea[ ] finally antioxidants such as resveratrol alsoare able to block infection produced by herpesvirus the discovery of antiviral compounds from a bioactivecompound against other viruses is an interesting strategy forobtaining new antiviral drugs table shows the mainactions of the natural antioxidants against the coronavirusand figure summarizes these activities 0c conclusionsin conclusion this review shows that antioxidant compounds prominently ï¬avonoids exhibit antiviral action inmodels of coronavirus infections in general the antiviralactivity might be attributed at least in part to the inhibitoryeï¬ect on the enzymatic activity of targets involved in coronavirus replication including sarscov 3clpro sarscovpapainlike protease plpro sarscov helicase proteinand merscov 3clpro in addition some studies provideevidence that the reduction of ros accumulation retardsthe coronavirusactivated apoptotic signaling thereforethe mechanisms of oxidative stress could be the key elementto be studied in coronavirus infectionsincluding thoserelated to ammatory processes arising from the action ofthis virus obviously further investigations are needed toelucidate other pharmacological mechanisms by which natural antioxidants play an antiviral eï¬ect despite the ï¬ndingsreported in this reviewthey cannot be generalized tocovid19 however the data provided support to the investigation of natural antioxidants as a potential therapeuticapproach in the treatment for covid19 and its severe clinical complications either as an alternative or as an adjuvantto conventional therapies and contribute to the search fornew prototypes in the development of drugs against coronavirus infectionsabbreviations229e3clproabtshuman coronavirus229e3clike protease²azinobis3ethylbenzthiazoline6sulphonic acidcoronavirusescatalasediï¬use alveolar damage22diphenyl1picrylhydrazylferric reducing antioxidant powerreduced glutathioneglutathione peroxidasehuman coronaviruseshuman coronavirushku1lower respiratory tract infectionsmalondialdehydecovscovid19 coronavirus disease catdaddpphfrapgshgpxhcovshku1lrtismdamerscov middle east respiratory syndromecoronavirusnfκbnuclear factor kappa bhuman coronavirusnl63nl63human coronavirusoc43oc43oxygen radical absorbance capacityoracprotein carbonylpcoporcine epidemic diarrhoea coronaviruspedvplpropapainlike proteasereactive nitrogen speciesrnsreactive oxygenated speciesrossarssevere acute respiratory syndromesarscov severe acute respirator | 0 |
"peroxisome proliferatoractivated receptorsppars asligandactivated transcription factors belong to the steroidreceptor superfamily which includes three isoforms pparalpha ppar betadelta and ppar gamma ppars formheterodimers with retinoic x receptors and regulate theexpression of various genes upon ligand binding ppars alsointeract with corepressors or coactivators to modulate thetranscription of its downstream target genes ppars asimportant transcriptional regulators have been suggested tobe involved in lipid metabolism and multiple cellular functions for instance ppar alpha also functions in fatty acidbetaoxidation and vascular ammation ppar gammaacts as a regulator in adipocyte diï¬erentiation and type diabetes ppar betadelta is a key player in cardiac energyproduction angiogenesis and particularly in cancer progression ppar alpha and ppar gamma exert predominantly anantiangiogenic eï¬ect [] but there still exist conï¬ictingstudies showing opposite results [ ] on the contraryppar betadelta produces more obviously proangiogeniceï¬ects [] in this review we will focus on the promotingrole of ppar betadelta in angiogenesis especially in tumorangiogenesis the network of interplay between ppar betadelta and its various downstream signal molecules and alsobetween those key molecules will be further discussed andestablished remarkably diverse important signal moleculesinvolved in tumor angiogenesis and progression and cancercell metabolism have been identiï¬ed as direct ppar betadelta target genes angiogenesisangiogenesis is the physiological process through which anew capillary network forms from the preexisting vasculature[ ] whereas vasculogenesis denotes de novo bloodvessel formation mostly during embryogenesis in whichendothelial progenitor cells epc migrate to sites of vascularization then diï¬erentiate into endothelial cells ec andcoalesce into the initial vascular plexus [ ] besides theinteraction between proangiogenic factors and antiangiogenic factors angiogenesis is also a multiple step biologicalprocess during which a variety of molecules cooperateincluding cell adhesion molecules matrix metalloproteinases 0cppar researchmmps extracellular matrix ecm and basement membrane componentsangiogenesis is a physiological and vital process in development and growth an imbalance of proangiogenic andantiangiogenic factors causes angiogenesis in pathologicalconditions such as diabetic retinopathy and tumor growththus when the imbalance comes to a point at which angiogenesis is triggered by tumor cells then an angiogenicswitch of tumor cells is turned on during tumor progression the angiogenic switch is often activated and remainson [] inducing angiogenesis is known as a hallmarkof cancer and angiogenesis is also a fundamental stepby which most benign tumors transition into malignant ones tumor angiogenesis tumor needs to sprout new vesselsand further develop a vascular network in order to supplynutrients and oxygen remove waste products support a continually high proliferative rate and ultimately expand neoplastic growth [ ] hence angiogenesis is essential forhelping sustain tumor growth and facilitate tumor progression besides being a requirement for angiogenesis an abnormal vasculature also helps to promote tumor progression andmetastasis the tumor vascular wall is imperfect and prone toleakage so it is much easier for tumor cells to directly penetrate into the blood vessels or lymphatic vessels and then proliferate at another distant site to form metastasis due to intensive abnormal neovascularization in tumortissues most malignant tumors grow rapidly and acquirethe ability to spread to adjacent and distant ans whichmakes them more malignant and even life threateningtherefore angiogenesis indeed plays an important role intumor progression and metastasis and to intervene with thisprocess would obviously prevent tumor development andspread thus this has been regarded as a critical target forantitumor therapy ppar alpha and angiogenesisit was reported ï¬rstly that a selective ppar alpha agonistwy14643 did not show any eï¬ect on angiogenesis or ec proliferation but some subsequent studies showed that theactivation of ppar alpha inhibited angiogenesis in vitro byusing fenoï¬brate a clinically used ppar alpha agonist moreover fenoï¬brate suppressed ec proliferation migration and tube formation through inhibition of protein kinaseb akt and disruption of the cytoskeleton furthermore ppar alpha activation was shown to inhibit vascularendothelial growth factor vegf induced ec migrationand basic ï¬broblast growth factor bfgffgf2 inducedcorneal angiogenesis in vitro and in vivo especiallyin vivo reduced tumor growth and microvessel numberswere observed in mice implanted with melanoma lewis lungcarcinoma llc ï¬brosarcoma and glioblastoma due to asystemic treatment of ppar alpha ligand and the antiangiogenic state induced through activation of ppar alpha withelevated thrombospondin1 tsp1 and endostatin expression howeverit was demonstrated inanother observation that activation of ppar alpha stimuin that same yearlated neovascularization in vivo with increased phosphorylation of endothelial nitric oxide synthase enos and akt via avegfdependent manner furthermore zhang andward also suggested that ppar alpha activation inducedproangiogenic responses in human ocular cells inanother study it was shown that a new ppar alpha agonistrk13675 had no eï¬ect on angiogenesis recentlyppar alpha activation is further shown to have antineovascularization eï¬ects with downregulation of vegf and angiopoietin expression in a rat alkali burn model in summary the role of ppar alpha in angiogenesis isstill controversial some observations showed that ligandactivation of ppar alpha had antiangiogenic eï¬ects mediated either through upregulation of antiangiogenic factorssuch as tsp1 and endostatin or downregulation of proangiogenic factors including vegf fgf2 akt and angiopoietins others also reported opposite results showing aproangiogenic role upon ppar alpha activation thus thespeciï¬c molecular mechanism is still unclear and needs tobe further studied ppar gamma and angiogenesisligand activation of ppar gamma was previously shown toinhibit human umbilical vein endothelial cell huvec tubeformation in collagen gels and vegfinduced choroidalneovascularization in vitro and in vivo another studyalso demonstrated that ec apoptosis was induced throughtreatment with the ppar gamma ligand 15dpgj2 furthermore rosiglitazone a potent ppar gamma agonist wasshown to inhibit primary tumor growth and metastasisthrough both direct and indirect antiangiogenic eï¬ectsin vitro and bfgfinduced corneal neovascularizationin vivo moreover a similar observation also displayedthe inhibition of vegfinduced angiogenesis in a chickchorioallantonic membrane model in a mouse modelwith ischemiainduced retinopathy pioglitazone a ppargamma agonist also showed a protective eï¬ect against pathological neoangiogenesis through upregulation of antiammatory adipokine adiponectin additionally theppar gamma antagonist gw9662 was shown to reverseomega3 polyunsaturated fatty acidinduced reduction ofeselectin angiopoietin2 vascular cell adhesion molecule and intracellular adhesion molecule1 implicatingan antiangiogenic potential of ppar gamma itself howeveropposite results also showed that pioglitazone enhanced neovascularization and inhibited apoptosis of epc in vitro andin vivo via a phosphoinositide3kinase pi3k dependentmanner nadra observed that ppar gammanull embryosdisplayed a vascular structural defect at e95 moreover disanized placental layers and an altered placental microvasculature were observed in pregnant wildtype mice treatedwith the ppar gamma agonist rosiglitazone as well asreduced expression of proangiogenic factorsincludingvegf proliferin and plateletendothelial cell adhesionmolecule1 pecam1cd31 suggesting a crucial roleof ppar gamma in placental vascular development the 0cppar researchmajor antiangiogenic properties on ppar gamma activationwere also reviewed here notablyin most cancersthe canonical wntbetacatenin pathway is upregulated while on the contrary ppargamma is downregulated interestingly in numerous tissuesthe activation of ppar gamma inhibits the betacateninpathway whereascanonicalwntbetacatenin signal cascade also inactivates ppargamma implicating a negative regulatory role of ppargamma in carcinogenesis where tumor angiogenesis mightbe a fundamental stepstimulation ofthethein summary ppar gamma predominantly displays anantiangiogenic eï¬ect that may be mediated through the inhibition of vegf or bfgfinduced neovascularization andreduction of the expression level of some proangiogenicfactors ppar betadelta and angiogenesisunlike ppar alpha and ppar gamma on the contrarymany studies have explicitly shown the proangiogenic eï¬ectsof ppar betadelta on physiological and pathological angiogenesis the ï¬rst evidence provided in a study is that activation of ppar betadelta with gw501516 a highly selectiveppar betadelta agonistinduces huvec proliferationand an increased expression of vegf and its receptorvegfr1 flt1 besides inducing ec proliferationppar betadelta activation by itsligand prostacyclinpgi2 also stimulates upregulation of alpha expression an antiapoptotic and antiammatory protein whichthereby protects ecs from h2o2induced apoptosis and oxidant injury moreover a subsequent study further provides evidence that activation of ppar betadelta withgw501516 induces angiogenesis during which vegfrelease is considered as a major trigger factor ï¬rstlysuggesting the promotion for angiogenesis upon pparbetadelta activationmüllerbrüsselbach show that ppar betadelta mice implanted with llc and b16 melanoma exhibit diminished blood ï¬ow and immature microvascular structurescompared with wildtype mice moreover reexpression ofppar betadelta into the matrigelinvading cells triggersmicrovessel maturation and restores normal vascularization indicating a crucial role of ppar betadelta in tumorvascularization additionally another study also observedreduced levels of calcium intracellular channel protein clic4 but it observed enhanced expression of cellular retinol binding protein crbp1 in migrating ecs from pparbetadeltanull mice both of which play a role in tumorvascularization [ ] it was reported that ppar betadeltawas required for placentation and most of the pparbetadeltanull mutant embryos died at e95 to e105 due toabnormal celltocell communication atthe placentaldecidual interface however in these studies [] adefect in angiogenesis was not observed during normal development in ppar betadeltaknockout micesome observations also show the important role of pparbetadelta in physiological angiogenesis for instance skeletal musclespeciï¬c ppar betadelta overexpression leads toppar betadeltaan increase in the number of oxidative muscle ï¬bers andrunning endurance in adult mice [] moreover pparbetadelta activation promotes a rapid muscle remodelingvia a calcineurindependent manner and induces muscleangiogenesis in highly selective ppar betadelta agonistgw0742treated animals furthermore in the heartpharmacologicalstimulation withgw0742 induces rapid cardiac growth and cardiac angiogenesis through direct transcriptional activation of calcineurin interestingly the same cardiac phenotype wasalso observed after treatment with the ppar betadelta agonist gw501516implicating a response speciï¬city forppar betadelta stimulation calcineurin activationfurther leads to the stimulation of nuclear factoractivatedt cell c3 nfatc3 and an enhanced expression of hypoxiainducible factor alpha hif1alpha and cyclindependentkinase cdk9 overall the remodeling in skeletalmuscle and heart is perfectly the same as the phenotypeobserved with exercise and both of them are mediatedthrough activation of calcineurinppar betadelta may act as a key regulator in mediatingpathological angiogenesis for instance ppar betadelta wasshown to regulate retinal angiogenesis in vitro and in vivoand its inhibition reduced preretinal neovascularization possibly via an angiopoietinlike protein angptl4 dependent manner implicating the potential of pparbetadelta in modulating pathological ocular angiogenesisrecently an observation reported that ppar betadeltaknockdown in both retinal pigment epithelial and choroidalendothelial cells caused an antiangiogenic phenotype andppar betadelta promoted laserinduced choroidal neovascular cnv lesions in ppar betadelta mice moreover pharmacological inhibition of ppar betadelta with theantagonist gsk0660 also resulted in a signiï¬cantly decreasedcnv lesion size in vivo suggesting a functional role of pparbetadelta in the development of cnv lesions this indicates that ppar betadelta has an important association withpathological angiogenesisangiotensin ii ang ii the biologically active peptide ofthe reninangiotensin system ras is a major blood pressure and cardiovascular homeostasis regulator and is alsorecognized as a potent mitogen angiotensinconvertingenzyme inhibitors were introduced approximately yearsago as antihypertensive agents and have since become asuccessful therapeutic approach for high blood pressurecongestive heart failure and postmyocardial infarction inexperimental systemsthe antitumor eï¬ects of diverseace inhibitors show that these inhibit cell proliferationand possessand antiammatory eï¬ects [] it has been shown recentlythat activation of ppar betadelta inhibits ang iistimulated protein synthesis in a concentrationdependentmanner and suppresses ang iiinduced generation of reactive oxygen species ros in vascular smooth muscle cells ppar betadelta was further shown to inhibit angiimediated atherosclerosis however it is not clearuntil now if ppar betadelta activation can be consideredis foras an ace inhibitormimicking approach as itexample the case for ppar gamma activators antiangiogenicantimetastatic 0cppar researchthe relevance offurthermorethis hypothetical pparbetadelta feature might be limited for tumor angiogenesiswhere vascular smooth muscle hypertrophy and atherosclerosis do not contribute to the major pathologybesides inducing angiogenesis it has been demonstratedthat ppar betadelta directly acts on early epc through activation of the akt pathway and induces an enhanced vasculogenesis similarlythe ppar betadeltamediatedprovasculogenic eï¬ects are also observed on late epc he showed that ppar betadelta activation withgw501516 induced epc proliferation and tube formationwhereas epc treated with an inhibitor of cyclooxygenasecox or pgi2 synthase or with ppar betadeltaspeciï¬csirna also displayed an opposite eï¬ect furthermoreit has been demonstrated that ppar betadelta inducesangiogenesis and skeletal muscle regeneration throughmatrix metalloproteinase mmp 9mediated insulinlikegrowth factor1 paracrine networks upon epc activation han also observed that ppar betadelta activationpromoted a rapid wound healing with enhanced angiogenesis in a mouse model with skin punch wound overallin addition to ec ppar betadelta is also a key regulator ofepc or even may act as an initiator of activation of epc tofurther induce vasculogenesis ppar betadelta and tumor angiogenesislinked to tumor microenvironmentppar betadelta expression is often upregulated and promotes cancer progression in many major human cancerslung breast and gastric cancers []such as colonwhich suggests a crucial role of ppar betadelta in cancercells even though there exist some conï¬icting studies indicating that the functional role of ppar betadelta in tumorigenesis or carcinogenesis still remains highly controversial [] and dependent on speciï¬c tumor or cancer cell typesthus here we discuss the promotion of ppar betadelta intumor progression through facilitating tumor angiogenesisppar betadelta has been suggested as a critical hubnode transcriptional factor which governs a tumor angiogenic switch [ ] in the transcriptional networkanalysis it was reported that tumor growth and tumor angiogenesis were markedly inhibited in ppar betadeltanullmice in comparison with wildtype mice moreoverthe elevated ppar betadelta expression level was also considered to be highly correlated to pathologically advancedtumor stage and increased cancer risk for recurrence and distant metastasis in patients with pancreatic cancer indicating the crucial association of ppar betadelta withtumor angiogenesis progression and cancer invasivenessppar betadelta may indirectly facilitate tumor angiogenesis and progression through its function on the tumormicroenvironment tme where tumor angiogenesis is fostered moreover a tumor also releases some extracellular signals to closely communicate and constantly collaborate withtme to facilitate tumor angiogenesis in order to furtherenable tumor growth and progression for instance it wasshown that colon cancer cells with ppar betadelta knockoutfailed to stimulate ec vascularization in response to hypoxicstress whereas wildtype cells exposed to hypoxia were ableto induce angiogenesis [ ] suggesting that ppar betadelta is required for the promotion of angiogenesis in hypoxic stressmediated tme moreover in the tme tumorltrating myeloid cells are considered as the most important cells for fostering tumor angiogenesis among the multiple diï¬erent kinds of stromal cells besides stimulatingtumor angiogenesis tumor myeloid cells also support tumrowth by suppressing tumor immunity and promotingtumor metastasis to distinct sites interestingly it hasbeen demonstrated that ppar betadelta activation intumorltrating myeloid cells stimulates cancer cell invasion and facilitates tumor angiogenesis via an interleukin il10 dependent manner moreover impairedtumor growth and angiogenesis were observed in pparbetadelta ko bmt mice due to ppar betadelta deï¬ciencyin tumor myeloid cells suggesting that ppar betadeltaplays a key role in tumor angiogenesis and progression intumor myeloid cells of tmefurthermore the endoplasmic reticulum er an essential anelle involved in many cellular functions is implicated in tme in cancer stressors like hypoxia nutrientdeprivation and acidosis disrupt er function and lead toaccumulation of unfolded proteins in er a condition knownas er stress cells adapt to er stress by activating an integrated signal transduction pathway called the unfolded protein response upr upr represents a survival response bythe cells to restore er homeostasis and has both survivaland cell death eï¬ects the mechanisms that determine cellfate during er stress are not well understood for instanceshort exposure to er stress initially increases akt signalingbut longterm er stress suppresses akt signaling ppar betadelta activation has been shown to reduce endoplasmic reticulum er stressassociated ammation inskeletal muscle through an ampkdependent mechanism and to reduce ammation in response to chronic erstress in cardiac cells furthermore it has been nicelyshown that ppar betadelta can repress rasoncogeneinduced er stress to promote senescence in tumors thisis mediated through the decrease of pakt activity promoting cellular senescence through upregulation of p53 and p27expression it would be interesting to investigate thedirect eï¬ects of ppar betadelta on senescence of tumorendothelial cells in an in vivo setting we recently showedthat senescent endothelial cells are indispensable for ahealthy lifespan and that removal of senescent endotheliumdisrupts vascular function leading to diminished vessel densities and ï¬brotic lesions if ppar betadelta mediatessenescence of tumor endothelium thereby protecting vesselintegrity this might explain the enhanced tumor growthand vascularization upon ppar betadelta activationobserved by us and others [ ]most recently zuo demonstrated that pparbetadelta in cancer cells regulates tumor angiogenesisin vivo and in vitro by promoting the secretion of proangiogenic factors including vegf and interleukin il8 most importantly in our recent works it has beenshown that conditionalinducible vascular endotheliumspeciï¬c ppar betadelta overexpression in vivo leads to 0cppar researchenhanced tumor angiogenesis tumor growth and metastasis formationfurther indicating a vascular ecspeciï¬cppar betadelta action mechanism in tumor progressionindependent of some controversial observations of pparbetadelta in speciï¬c tumor or cancer cell types wagner also ï¬rstly reported the mouse model in whichrapid induction of cardiac angiogenesis and cardiac hypertrophy were observed [ ] crosstalk between ppar betadelta and signalmolecules ppar betadelta activation or overexpressionmay upregulate the expression of its various downstream signal molecules involved in tumor angiogenesis includingproangiogenic factors such as vegf pdgf and fgfproinvasive matrixdegrading enzymes such as mmp9proammatory mediators such as cox2 and cytokinesand chemokines such as il1 and cxcl8 even some ofwhich have been further identiï¬ed as ppar betadelta directtarget genes besides a leading role of ppar betadelta amongthe signal molecules ppar betadelta may function in tmelinked to diverse kinds of cells through direct or indirectmodulation of its downstream molecules interplay between ppar betadelta and ammatoryangiogenesis ammatory angiogenesis is a crucial processin tumor progression for instance the proammatorymediator cyclooxygenase2 cox2 is considered as a keyregulator of angiogenesis and tumor growth through multiple downstream proangiogenic mechanisms such as production of vegf and induction of mmps moreover selectiveinhibition of cox2 has also been shown to suppress angiogenesis in vivo and in vitro it is well known that vegfaplays a critical role in both angiogenesis and vasculogenesis and it leads the directional migration of tip cells andstalk cell proliferation in microtubule branches [ ] ithas also been demonstrated that mmp9 triggers the angiogenic switch during carcinogenesis and enhances the availability of vegf to its receptors furthermore it hasbeen reported that ammatory cell mmp9 initiates theonset of tumor neovascularization during which there existsfunctionalincludingmmp9 leptin is shown to mediate angiogenesisin vivo and in vitro through induction of ec proliferationand expression of mmp2 and mmp9 and to furtherpromote ec diï¬erentiation and directional migrationthrough enhancement of cox2 activity leptin couldalso induce angiogenesis via transactivation of vegfr inecs additionally besides inducing angiogenesisppar betadelta also functions in chronic ammationfacilitating tumorigenesis through induction of cox2 andits product prostaglandin e2 pge2 in vivo [ ]interestingly cox2 vegf mmp9 and leptin have beenidentiï¬ed as ppar betadelta target genes via a direct transcriptional activation mechanism in hepatocellular carcinoma cells colorectal cancer cells [ ] epcs[ ] and liposarcoma cells respectivelylinks between vegf and mmpsin tme tumorltrating ammatory cells also helpto induce and sustain tumor angiogenesis and further tofacilitate tissue invasion and tumor metastatic spread byreleasing some signal molecules such as proinvasive mmp9and ammatory chemokines [] chemotaxis is alsoa crucial process for inducing angiogenesis in tumors eitherdirectly by attracting ecs towards tumor cells to form newvessels or indirectly by mediating immune ammatorycells to ltrate eventually promoting tumor angiogenesis chemotaxis of tumor cells and stromal cells in tmeis also required for tumor dissemination during tumor progression and metastasis [ ]cxc chemokines such as cxcl8 encoding il8 andcxcl5 are also involved in cox2associated angiogenesisto contribute to nonsmallcelllung cancer progression[ ] it is further shown that il8 directly regulatesangiogenesis via recruitment of neutrophils whichfurther drives vegf activation moreoveril8responding neutrophils are considered as the major sourceof angiogenesisinducing mmp9 [ ] chemokine cc motif ligand ccl2 in addition to the promotionof angiogenesis [ ] also enhances tumor metastasis furthermore myeloid monocytic cellssuch asmyeloidderivedtumormdscsassociated macrophages tams and dendritic cells arerecruited to the tumor site mainly by ccl2 and producemany proangiogenic factorssuch as vegf cxcl8plateletderived growth factor pdgf and transforminggrowth factor beta tgf beta [] in fact bothtgf beta and hypoxia are potentinducers of vegfexpression in tumor cells and collaborate with tme toprovide the foundation of tumor angiogenesis and cancercell invasion importantly il8 has been reported asa key target gene of ppar betadelta to promote angiogenesis in vivo and in vitro and ccl2 expression isalso signiï¬cantly upregulated upon vascular ppar betadelta overexpression in vivo suppressorcellscox2 also mediates il1 betainduced angiogenesisin vitro and in vivo [ ] il1 beta supports neovascularization through the regulation of the expression of vegfand its receptor vegfr2 flk1kdr on ecs il1 actsas an upstream proammatory mediator that initiates anddisseminates the ammatory state by inducing a localinteractive network and increasing adhesion moleculeexpression on ecs and leukocytes which facilitates tumorassociated angiogenesis in tme ammatory il1beta recruits myeloid cells from bone marrow and activatesthem to produce proangiogenic factors such as vegf vegffurther activates ecs and myeloid cells promoting tumorinvasiveness and fostering tumor angiogenesis in addition il6 also stimulates angiogenesis and vasculogenesis[ ] however gopinathan observed an il6induced newly forming vascular structure with defectivepericyte pc coverage ex vivo thus facilitating cancercell ltration and tumor metastasis through vascular leakage interestingly il1 and il6 expression levels are signiï¬cantly upregulated in the ppar betadelta overexpressionmouse model reported recently in summary ppar betadelta seems to act as a key leaderin ammatory mediatordriven tumor angiogenesis linkedto tme in which many proammatory mediators chemokines and proangiogenic factors closely communicate with 0cppar researcheach other and also associate with tumorltrating myeloid cells such as neutrophils tams and mdscs other key ppar betadeltamediated proangiogenicfactors it has been demonstrated that wilms tumor suppressor wt1 is a major regulator of tumor neovascularization andtumor progression e26 avian leukemia oncogene ets1 also plays a key role in regulating vascular development and haemopoiesis particularly in angiogenesis in addition ets1 promotes cancer cell invasion throughupregulation of mmps consistent with this silencingof ets1 in highly invasive breast cancer cells also reducesthe expression of mmp9 and mmp1 ets1 also acts as a key regulator of mmps such asmmp1 mmp3 and mmp9 in human cancerassociatedï¬broblasts cafs [ ] cafs support tumor growthby secreting growth factors such as vegf fgf pdgf andchemokines to stimulate angiogenesis and thereby promotecancer cell invasion and metastasis formation [ ]cafs as metastatic tumor stroma are a crucial componentin tumor progression through the remodeling of the ecmstructure thus helping a tumor to acquire an aggressive phenotype [ ] ppar betadelta in cafs also exhibits aprotumorigenic eï¬ect it was reported that ablation of pparbetadelta in cafs attenuated tumor growth by altering theredox balance in tme suggesting that ppar betadeltain cafs is also an important player in tumor developmentets1 induces the expression of vegf vegfr1 andvegfr2 in ecs [] in turn vegf is also a majorinducer of ets1 in ecs through the activation of either thepi3kakt pathway or the mekerk12 signal cascade[ ] wt1 is also reported to regulate tumor angiogenesis via direct transactivation of ets1 sryrelated hmgbox sox18 has also beenreported previously to induce angiogenesis during tissuerepair and wound healing and cancer progression and most recently it was further shown that speciï¬cecderived endovascular progenitors initiated a vasculogenic process and diï¬erentiated into more mature endothelial phenotypes within the core of the growing tumorsthrough reactivation of sox18 interestingly theseimportant proangiogenic molecules including wt1 ets1and sox18 are also signiï¬cantly upregulated in the vascularppar betadelta overexpression model in vivo andwt1 is also identiï¬ed as a target gene of ppar betadeltain melanoma cells ppar betadelta may facilitate cancer progression atdiverse cellular levels in tme ppar betadelta activationis shown to induce colonic cancer stem cell csc expansionand to promote the liver metastasis of colorectal cancerin vivo via direct transactivation of the nanog gene nanog as a key transcriptional factor governs the selfrenewal and pluripotency of stem cells and cancer cellsexpressing nanog also often exhibit stem cell properties protooncogene ckitcd117 is known as the maststem cell factor receptor and receptor tyrosine kinase andits activation in cscs may regulate the stemness to controltumor progression and drug resistance to tyrosine kinaseinhibitors moreover ckit has been identiï¬ed as a potentialmarker of the cancer stemlike cells in addition ckitnot only functions on ecs [ ] but also belongs to thetumor angiogenesispromoting molecule [] studiesalso suggested that activation of ckit enhances the expression of vegf that can be suppressed by imatinib an inhibitor of ckit in gastrointestinal stromal tumor cells whichthereby has an impact on tumor angiogenesis [ ] ckit is also involved in pathological ocular neovascularization and is regulated transcriptionally by wt1 and ppar betadelta pdgfb and its receptor pdgfr beta also known asangiogenic factors are suggested to enhance angiogenesisand vasculogenesis via their function in ecs [] andepcs and to regulate vascular permeability and vesselmaturation through recruitment of pericytes pcs and smooth muscle cells smcs in newly formingvessels moreover pdgfb and pdgfr beta also interactwith other proangiogenic factors such as fgf2 [ ]vegfa and its receptor vegfr2 furthermorepdgfb and pdgfr beta may also aï¬ect cancer growthand progression by directly acting on tme besides thecrosstalk with cafs [] pdgfr beta in stromalï¬broblasts may mediate pdgfbinduced tam recruitment thus implicating a role of pdgfr beta in tumorstroma to facilitate tumor progression most recently it wasfurther shown that speciï¬c targeting of pdgfr beta kinaseactivity in tme inhibited cancer growth and vascularizationin cancers with high pdgfb expression such as llc therefore this indicates the diverse role of pdgfb andpdgfr beta in facilitating tumor angiogenesis and progression at diï¬erent cellular levels in tme pdgfr beta is demonstrated as a target of telomeric repeat binding factor trf2 that is further activated transcriptionally by wt1 pdgfb and pdgfr beta have further been identiï¬edas critical targets of ppar betadelta via a direct transactivation mechanism in vivo in conclusion a variety of key signal molecules involvedin tumor angiogenesis and tumor progression and metastasishave either been identiï¬ed as ppar betadelta direct targetsor largely upregulated in the vascular ppar betadelta overexpression model in vivo reported recently thus pparbetadelta activation seems to give rise to a highly angiogenicphenotype and even plays a hallmark role in promotingtumor angiogenesis and progression interestingly it appearsthat there could also exist a widely interactive networkbetween the downstream protumorangiogenic moleculesas described above therefore the crosstalk network is established between ppar betadelta and the various signal molecules and also between those molecules figure 1amoreover in addition to cancer cells ppar betadeltamay also produce pleiotropic eï¬ects in tme by modulatingdownstream key molecules to act on ecs epcs pcs smcscscs cafs and tumorltrating ammatory cells indirectly facilitating tumor angiogenesis and further promotingcancer development figure 1b other ppar betadelta target genes ppar betadeltaregulates the transcription oftarget genes via a direct 0cppar researchil1 betacox2leptinppar betadeltavegfvegfrpdgfr betatrf2wt1ets1mmp9pdgfbckitcxcl8il8il10ccl2cxcl8il8pdgfr betaappar betadeltaets1mmp9pdgfr betananogckittumorassociatedmacrophage tamdendritic cellneutrophilmyeloidderivedsuppressor cell mdscvegfmmp9pdgfbckitsox18pdgfr betaendothelial cell ece | 0 |
edited byfang zhoucas lamvac biotech co ltd chinareviewed bybart evertsleiden university medicalcenter netherlandsmaria rosa bonouniversity of chile chileandrew john staggqueen mary university of londonunited kingdomcorrespondencedipayan rudradipayanrudragmailcomrudradpostechackrsinhyeog imiimshpostechackr present addresshyunja kokobiolabs inc seoul south koreasungwook hongdepartment of microbiology andimmunology centre for immunologyuniversity of minnesota medicalschool minneapolis mnunited states¡these authors have contributedequally to this work§deceasedspecialty sectionthis was submitted toimmunological tolerance andregulationa section of the frontiers in immunologyreceived april accepted july published august citationko hj hong sw verma r jung jlee m kim n kim d surh cdkim ks rudra d and im sh dietary glucose consumptionpromotes raldh activity in smallintestinal cd103cd11b dendriticcells front immunol 103389ï¬mmu202001897intestinal dendritic cells dcs are critical for the initiation and regulation of innate and adaptiveimmunity by delivering self or foreign antigens to t cells the intestine is spontaneouslyexposed to innumerable antigens comprising of intestinal microbes as well as dietarycomponents to maintain immune homeostasis intestinal dcs regulate the balance betweenthe tolerogenic immune response by inducing cd4foxp3 regulatory t cells treg cells and the protective immune responses by inducing eï¬ector t cells dysregulation of thisbalance by harmful pathogens or dietary intake results in ammatory disorders such asammatory bowel disease ibd celiac disease and food allergy frontiers in immunology wwwfrontiersinaugust volume 0cko glucose promotes silpdc raldh activityintestinal dcs are located in the peyers patches ppsmesenteric lymph nodes mlns and lamina propria lp andcomprise cellular subsets that have diï¬erent origins and functions among these dc subtypes intestinal cd103 dcshave the unique function that metabolizes vitamin a to retinoicacid ra through the activation of aldehyde dehydrogenase member a2 [aldh1a2 also called retinaldehyde dehydrogenaseraldh2] enzyme the ra produced by intestinaldcs play an important role in orchestrating immune responsesimprinting guthoming speciï¬city on t cells b cells andinnate lymphoid cells ilcs inducing igaproducing b cellspromoting tgfdependent diï¬erentiation of induced tregcells suppressing the diï¬erentiation of th17 cells enhancing il production by Îδ t cells and ilcs as well as inducing eï¬ectorfunctions in t cells while vitamin a derived from dietary intake can induceraldh enzymatic activity the ra produced from intestinalepithelial cells iecs by raldh1 and stroma cells in lp andmln by raldh2 in a trans activating mechanism is alsocapable of inducing raldh expression in intestinal dcs furthermore recent data suggest that ra is also involvedin the development of a gut homing precursor for intestinaldcs in the bone marrow as well as is required for theirtranscriptional programming and maturation severalendogenous factors that regulate raldh expression in lpdcs are also reported cytokines such as il4 and granulocytemacrophage colonystimulating factor gmcsf induce orenhance the expression of raldh enzymes in lpdcs whileprostaglandin e2 pge2 negatively regulates raldh activitythrough the induction of inducible cyclic amp early repressoricer despite these ï¬ndings whether additionalcomponents in diet can induce raldh activity in the intestineand promote immune tolerance remains unknown in this studywe uncover a hitherto unknown role of dietary glucose in shapingup intestinal immunological tolerance by facilitating raldhexpression speciï¬cally in intestinal lpdcsresultsmice administered antigen free diet havedefects in development and raldhactivity in cd103cd11b silpdcsto investigate the uence of commensal microbiota and foodcomponents on intestinal immunity we utilized the previouslyestablished antigen free af mice model where germfreegf mice are raised on welldeï¬ned elemental diet [termedantigen free diet afd] devoid of macromolecules such asproteins and starches when dcs in small intestine wereassessed we observed comparable frequencies of cd11cmhcii silpdcs in speciï¬c pathogen free spf gf and af micefigures 1ab however indepth analyses revealed alterationin the frequencies of tolerogenic dc subtypes the frequencyof cd103cd11b silpdcs a subset known to be a majortolerogenic dc population was slightly but significantly lowerin af when compared to spf and gf mice figure 1c acompensatory increase on the other hand was observed inthe cd103cd11b silpdc compartment more interestinglywhile the expression of the characteristic dc surface markerslargely remained comparable in all three groups figure s1athe expression of all three representative genes tested namelyaldh1a2 indoleaminepyrrole 23dioxygenase ido1 andtransforming growth factor beta tgfb1 that are functionallyimplicated in tolerogenic phenotype of cd103cd11b dcswere dramatically reduced in af mice figure 1d interestinglythe expression of aldh1a2 was found to be speciï¬cally reduced inmice raised under afd a phenomenon that was not observedin gf mice figure 1d left panel on the other hand theabsence of gut microbiota appeared to partially uence theexpression of ido1 and tgfb1 which was further enhanced byafd figure 1d middle and right panel these results indicatedthat certain dietary components otherwise absent or underrepresented in afd have most speciï¬c and the largest uenceon the expression of aldh1a2 for this study we therefore focusedon the uence of normal diet on raldh activity in silpdcsraldh is an enzyme that irreversibly metabolizes vitamin ato ra which in turn acts as a key modulator of mucosal immuneresponses to determine whether in concert to itsreduced expression the function of raldh in lpdcs fromaf mice was also negatively aï¬ected we next examined raldhenzyme activity in lpdcs from spf gf and af mice usingthe aldefluor assay in this assay which has been previouslyemployed in the context of cd103 lpdcs and mlndcs the raldh enzyme activity is measured in individual cellsby ï¬ow cytometry with a ï¬uorescent substrate based assay system in agreement with the results obtained by realtime pcranalysis cd103 silpdcs both cd11b and cd11b subsetsfrom af mice displayed significantly lower enzyme activity whencompared with spf and gf mice figures 1ef figure s1b ofnote the characteristic frequencies of the aforementioned silpdc subtypes in spf gf and af mice remained unaltered evenafter performing this assay suggesting that this enzyme assay didnot interfere with the phenotype of silpdcs figure s1cin order to further understand the role of dietary componentson raldh activity we next analyzed silpdc raldh activityin mice at diï¬erent stages of their lives after subjecting them tospeciï¬c dietary conditions we observed that raldh activityin preweaned gf mice weeks of age was significantlylower than in adult gf mice and comparable to af mice thiswas dramatically restored to the level equivalent to adult gfmice within a week after weaning figure 2a furthermorewhen mice raised in af condition were fed with normal chowdiet ncd raldh activity in cd103cd11b silpdcs waspromptly recovered within a week figure 2b mirroring thisan opposite phenomenon was observed when ncd in gf micewas replaced with afd figure 2c these results suggested thatdietary components in ncd absent in afd is required as theinitial trigger for raldh gene expression after mice are weanedthereby promoting enzyme activity as well as homeostasis ofcd103cd11b silpdcsthe above results also implied that supplementing af micewith ra the ï¬nal product of the enzymatic reaction and a knownfeedback inducer of raldh activity would be suï¬cient indriving raldh activity in these mice indeed when adult affrontiers in immunology wwwfrontiersinaugust volume 0cko glucose promotes silpdc raldh activityfigure dietary intake affects differentiation and raldh activity in cd103 silpdcs cell suspensions were prepared from silp harvested from agematchedadult ¼12weekold spf gf and af mice and phenotypic and raldh activity analyses of silpdcs were carried out a representative ï¬uorescenceactivatedcontinuedfrontiers in immunology wwwfrontiersinaugust volume 0cko glucose promotes silpdc raldh activityfigure cell sorting facs plots of silpdc subpopulations gated on lin cd3b220 cells b statistical quantiï¬cation of percentage left and total numbersright of cd11cmhcii cells in mice from indicated experimental groups c graph displays percentage of silpdc subpopulations in cd11cmhcii cells bcdata are combined from four independent experiments d realtime analyses of mrna expression of indicated gene products normalized against hprt mrna levelsef representative facs plots left and quantiï¬cation right of relative mean ï¬uorescence intensity mfi of aldeï¬uor in cd103cd11b e and cd103cd11bf silpdc subpopulations from indicated groups deab is a raldh inhibitor 01mfi is calculated by subtracting background deab mfi from aldeï¬uor mfi relative 01mfi indicates ratio of 01mfi in experimental samples vs control data are combined from six independent experiments mean ± sem are indicated statisticalsigniï¬cance was determined by oneway anova bef and twoway anova c with turkeys multiple comparison tests p ns not statisticallysignificantmice were supplemented in their diet with ra it resulted incomplete recovery of raldh activity figure 2d interestinglyin all the cases changes in raldh activity also correlatedwith the frequencies of cd103cd11b silpdcs suggestingits role in diï¬erentiation as well as function of these cells ofnote while the above results were obtained in a gf setting thebasic ï¬ndings from these experiments could also be recapitulatedin mice raised in spf conditions thereby conï¬rming thatmice with normal repertoire of gut ï¬ora are equally aï¬ectedby dietary components with respect to raldh activity insilpdcs figures s2acraldh activities in different intestinalraproducing cells are differentiallyaffected by dietwe next sorted to understand whether the uence of dieton raldh activity is an lpdc speciï¬c phenomenon orwhether other raldh expressing cells are also aï¬ected itis wellestablished that within the gut associated lymphoidtissues besides lpdcs ra converting enzymes are alsoexpressed in lp associated stroma cells lpscs small intestineepithelial cells iecs as well as mlndcs the ra producedfrom these cellsis known as a localsource of ra for inducing the raldh expression in cd103silpdcs in particular iecsthe nonhematopoietic scs in secondary lymph nodescomprise three diï¬erent cell types based on the expressionof surface markers lymphatic stroma cells [lscs also calledï¬broblast reticular cells frc]lymphatic endothelial cellslecs and blood endothelial cells becs amonglpscs cd45epcam in smallintestine the lscs thatexpressed podoplanin pdpn and are cd31 were foundto be capable of activating raldh enzymes figure 3a aspreviously reported interestingly unlike lpdcstheraldh activity in lpscs remained comparable between gfand af mice figure 3b in contrast when iecs were analyzedthe expression of aldh1a1 raldh1 the major gene encodingfor raldh enzyme in these cells was found to be reducedin af mice figure 3c of note while the iecs are wellestablished to have raldh activity the baseline ofthis activity in these cells is known to be significantly lower thanlpdcs therefore our attempt to measure raldhactivity in iecs was unsuccessful due to lower sensitivity ofthe aldefluor assay however albeit comparatively lowerraldh activity on a per cell basis the cumulative contributionof iecs in ra production is understandably of larger signiï¬cancesince numerically there are many more iecs than the other celltypes in the intestinefinally when mlndcs were analyzed the raldh activityin particular within the cd103cd11b dc population in afmice was found to be significantly albeit to a lesser extentlower than that of gf mice figure 3d taken together theseresults suggested that dietary components diï¬erentially uenceraldh activity in diï¬erentregulatory dc populationswhereas mlndcs and iec are aï¬ected lpscs appeared toremain unaï¬ected from dietary contributions these results alsoimplied that the overall reduction of ra synthesis cumulativelyamong these cell types eventually contributed to the reducedraldh activity in the lpdcs in af miceproteins starches and minerals in diet donot uence raldh activity incd103cd11b silpdcssince for this study we focused on raldh activity insilpdcs we next wished to deï¬ne which dietary factorswere required to trigger the initial raldh activity in thesecells while in our initial ï¬ndings we observed both thesubtypes cd103cd11b and cd103cd11b silpdcs tohave reduced raldh activity in af mice figures 1ef thecell recovery of cd103cd11b silpdcs from spf and gfmice were low and the level of enzyme activity in this celltype showed variability among individual mice figures 1cftherefore henceforth in this study we focused on the raldhactivity in cd103cd11b silpdcswe ï¬rst conï¬rmed that the reduction in raldh activityin cd103cd11b silpdcs was not a consequence of lowvitamin a contentin af diet thereby compromising theprecursor for the assayed reaction based on information offood compositions from the suppliers and from our perviousreport ï¬nal consumption of vitamin a per day by gf andaf mice were comparable [table s1 and ] nonethelessthere remained a possibility that albeit equal consumptionthe absorption of vitamin a into the small intestine may becompromised in af mice however when gf mice were weanedon af diet supplemented with times more vitamin a inthe usual form of oil mix or were administrated additionaloil mix by oral gavage failed to recover the reduction inraldh activity in cd103cd11b silpdcs figure s3we thus concluded that mere unavailability of the precursorvitamin a was not a cause of reduced raldh activity inthese cellsto this end we modiï¬ed the compositions of puriï¬eddiet by removing individual food components table s2 withfrontiers in immunology wwwfrontiersinaugust volume 0cko glucose promotes silpdc raldh activityfigure dietary components in normal chow readily trigger and maintain raldh activity in silpdcs in mice after weaning cell suspensions prepared from silpwere subjected to aldefluor assays and raldh activity in cd103cd11b silpdcs and percentage of silpdc subpopulations in cd11cmhcii cells wereanalyzed by ï¬ow cytometry a gf mice 3weeks old before weaning preweaned gf mice weaned onto ncd for days and adult gf mice were analyzed braldh activity and frequencies of silpdc subpopulations in cd11cmhcii cells in adult af mice ¼12weekold after feeding ncd for and days c raldh activity and frequencies of silpdc subpopulations in cd11cmhcii cells in adult gf mice ¼12weekold after feeding afd for and weeks d raldh activity and frequencies of silpdc subpopulations in cd11cmhcii cells in adult gf af or af mice that were administered intraperitoneal injection of alltrans ra µg per mouse in soybean oil every other day for days data are combined from two to three independent experimentsmean ± sem are indicated bc statistical signiï¬cance was determined by oneway anova with turkeys multiple comparison test p p p ns not statistically significantthe presumption that taking out or adding back individualcomponents in otherwise welldeï¬ned diet may lead us towardidentifying the dietary component required to trigger raldhactivity to obtain relatively accurate results under in vivosettings the modiï¬ed diets were designed to contain similaramount of vitamin a as in ncd tables s1 s2 and thefrontiers in immunology wwwfrontiersinaugust volume 0cko glucose promotes silpdc raldh activityfigure raldh activity in intestinal raproducing cells is differentially regulated by diet ac cell suspensions from mln and silp harvested from agematchedadult ¼12weekold gf and af mice were subjected to aldefluor assays and raldh activity of lpscs and mlndcs were analyzed by ï¬ow cytometry arepresentative facs plot of lpsc distinguished by cd31 and pdpn from cd45epcam cells red dots in facs plot indicate aldeï¬uor positive cells histogramsdepict the mfi of aldeï¬uor in lpsc subtypes b graph displays the level of raldh activity in lscs data is combined from two independent experiments c iecswere isolated from small intestine si by stripping with edta and analyzed for expression of aldh1a1 raldh1 by realtime pcr upon normalization to hprt mrnalevels fold change indicates ratio target gene in experimentcontrol data are combined from four independent experiments d representative facs plots ofmlndc subpopulations distinguished by cd103 and cd11b from cd11cmhciilin cells and the raldh activity in cd103cd11b mlndcs andcd103cd11b mlndcs data are combined from four independent experiments mean ± sem are indicated statistical signiï¬cance was determined bytwotailed unpaired ttest p p ns not statistically significantexperiments were performed primarily in gf condition in orderto eliminate the uence of microbiota in addition to avoidany uence from ncd during the preweaned period neonateaf mice were utilized and these mice were weaned onto eachmodiï¬ed diet for weeksas a starting point we took advantage of two commerciallyavailable diets with welldeï¬ned dietary compositions in the socalled amino acid deï¬ned diet aad like the afd employed sofar proteins were replaced with amino acids however there wereseveral diï¬erences between their compositions table s2 whilefrontiers in immunology wwwfrontiersinaugust volume 0cko glucose promotes silpdc raldh activityfigure depletion of macromolecules from puriï¬ed diet do not alter the raldh activity in cd103cd11b silpdcs af mice ¼4weekold were weaned ontospeciï¬c diets for ¼ weeks following which the indicated analyses were carried on a a cartoon depicting experimental scheme left panel aad is a sterilized formcontinuedfrontiers in immunology wwwfrontiersinaugust volume 0cko glucose promotes silpdc raldh activityfigure of amino aciddeï¬ned diet aad that contains three times more vitamin a than aad where protein macromolecules are replaced with amino acidsaad_stf indicates aad from which cornstarch and maltodextrin are removed the level of raldh activity in cd103cd11b silpdcs in the indicated groups arepresented by relative aldeï¬uor 01mfi right panel b representative facs plots left panel frequencies middle panel and absolute numbers right panel of totalcd4foxp3treg cell and cd4foxp3nrp1lo peripheral treg ptreg cell populations in silp of mice subjected to the indicated diet regimes c experimentalscheme left panel and relative aldeï¬uor 01mfi of silpdcs in the indicated experimental groups right panel min_mix afd indicates afd mixing with the mineral mixpowder td94049 data are combined from four independent experiments mean ± sem are indicated statistical signiï¬cance was determined by oneway anovawith turkeys multiple comparison test p p p ns not statistically significantafd is a liquid diet where the ï¬bers are provided as cellulosebedding aad diet are edible solid pellets with cellulose mixedwith the food components unlike afd aad diet containedstarches in the form of maltodextrin and corn starch and whilethe source of sugar in afd was glucose that in aad was sucrose furthermore in terms of mineral compositionthere are substantial diï¬erences between the groups the secondcommercially available diet is aad_stf which was largelysimilar to aad but was devoid of starches note the inaad indicates a sterilizable form of the diet which is otherwisesimilar to its traditional form aad but with three times morevitamin a to account for presumed losses during sterilization byirradiation surprisingly the mice groups weaned in both aadand aad_stf showed a complete recovery of raldh activityin cd103cd11b silpdcs to an extent similar to the controlncd fed group figure 4a these results were independentof microbiota since the characteristic drop in raldh activityof cd103cd11b silpdcs in mice raised in spf conditionscould also be recovered by aad figure s2d taken togetherthese ï¬ndings led to two important conclusions first starchesare not involved second antigens in the form of peptidesderived from proteins are also dispensable as far as raldhactivity in cd103cd11b silpdcs is concerned to this endwe also considered a possibility that an artifact arising fromunaccounted protein contamination in the amino acid deï¬neddiets may be responsible for the observed recovery of raldhactivity we therefore quantiï¬ed the generation of peripheralregulatory t ptreg also referred to as itreg when inducedin vitro cell population in the silp of these mice ptreg cells area type of treg cells that are extrathymically generated primarilyat mucosal sites and are distinguished from their thymic ttregcounterparts by the lack of expression of the membrane boundcoreceptor neuropilin1 nrp1 notably in a previousreport we have demonstrated that diet derived proteins are theprimary cause for the generation of cd4foxp3nrp1 ptregcells in the small intestine and af mice display dramaticallyreduced ptreg population in silp indeed we found that whiletotal foxp3 treg populations comprising of ttreg and ptregcells remained comparable the frequencies and numbers offoxp3nrp1 ptreg cells among total treg population couldonly be recovered in mice fed with ncd and not aadand aad_stf figure 4b therefore the recovery of raldhactivity in aad and aad_stf groups were not due to anyprotein contamination in the aad dietwe next wished to exclude the possibility that diï¬erencesin minor food components such as minerals between afdand puriï¬ed diet table s2 was responsible for diï¬erences inraldh activity for that we prepared afd by supplementingwith mineral mix powder td94049 derived from aadmin_mixafd as shown in figure 4c min_mixafd failedto induce raldh activity taken togetherthese resultsconcluded that proteins starches and minerals in diet werenot responsible for the induction of raldh activity incd103cd11b silpdcsoptimum glucose level in diet is requiredto induce raldh activity incd103cd11b silpdcsif the macromolecules and micromolecules in diet were notinvolved lack of which factors in afd compromise raldhactivity in cd103cd11b silpdcs to investigate furtherwe compared the food compositions between aad_stf andafd table s2 which were the closest among the four typesof diets tested in case of aad_stf this diet does not containproteins and starches as in afd but does contain unknowndietary factors responsible for the induction of raldh activityin silpdcs there are few diï¬erences between the compositionof these two diets the diet forms pellet vs liquid thecarbohydrate sources sucrose vs glucose the amount ofcarbohydrate sucrose ¼ vs glucose ¼ based on thisobservation as well as in order to minimize the diï¬erencesin diet forms we ï¬rst generated a liquid form of afd with of sucrose afd_s500 or glucose afd_g500germ free diet gfd or aad_stf were used as positivecontrols afd with usual glucose designated as afd_g220here was used as negative control the neonate af micewere weaned onto each diet for weeks and were analyzedfor raldh activity in cd103cd11b silpdcs figure 5aleft panel indeed afd supplemented with sucroseresulted in significantly enhanced raldh activity in these cellsmore interestingly we observed a similar increase in raldhactivity in cd103cd11b silpdcs even when the source ofcarbohydrate was changed to glucose instead of sucrosefigure 5a middle panel these eï¬ects were found to be speciï¬cfor cd103cd11b silpdcs since raldh1 expression iniecs remained unaltered upon carbohydrate supplementationfigure 5a right panel these results suggested that regardlessofits optimum concentrationis important and glucose being a monosaccharide unit ofcarbohydrate is suï¬cient for the initial triggering of raldhactivity in silpdcsthe source of carbohydratein order to further understand whether the positive eï¬ectof dietary carbohydrate supplementation is speciï¬c for raldhactivity or if it can also aï¬ect diï¬erentiation of other immunecells we determined the frequencies of th1 th2 and ptregfrontiers in immunology wwwfrontiersinaugust volume 0cko glucose promotes silpdc raldh activityfigure supplementing glucose in af diet restores raldh activity in cd103cd11b silpdcs a cartoon depicting experimental scheme left panelneonate af mice were weaned onto speciï¬c diets for ¼ weeks afd contains of glucose afd_g220 afd_g500 indicates of glucose in afd andafd_s500 indicates of sucrose in afd aad_stf was utilized as a positive control of raldh activity in lpdcs aldefluor assays were performed on cellsuspensions from silp and raldh activity of silpdc was analyzed by ï¬ow cytometry the level of raldh activity in cd103cd11b silpdcs is represented asrelative 01mfi of aldeï¬uor middle panel aldh1a1 expression was also determined in sorted iecs relative to hprt control right panel data are combined from twoindependent experiments bd graphs display the percentage of tbet in cd4 t cells th1 b gata3 in cd4 t cells th2 c and nrp1lo populations amongin cd4foxp3 treg cells ptreg d data are combined from at least two independent experiments mean ± sem are indicated statistical signiï¬cance wasdetermined by oneway anova with turkeys multiple comparison test p p p ns not statistically significantcells in these mice supplementation of afd with additionalsucrose or glucose did not aï¬ect tbet th1 or gata3 th2 cellsfigures 5bc neither recovered the characteristically reducedptreg population in silp figure 5dwe next directly investigated the eï¬ect of glucose on raldhactivity by employing in vitro culture conditions spldcsmlndcs and cd11c silpdcs were magnetically puriï¬edand cultured either without glucose in commercially availableglucosefree media without glucose but in the presence of rawith glucose or in the presence of glucose and ra for hand then measured for raldh activity in terms of baselineraldh activity as expected spldcs displayed the least whichwas significantly increased in the presence of ra glucose onthe other hand had minimal eï¬ect figure 6a although mlndcs had the highest baseline raldh activity among the threegroups tested neither ra nor glucose had an impact figure 6bin contrast silpdcs derived from 2weeks old neonatal spfmice which had low basal raldh activity at steady state wassignificantly increased in the presence of glucose alone while raitself induced slight increase of this enzyme activity the highestactivity was observed when both ra and glucose was presentfigure 6c moreover this raldh activity promoting eï¬ect ofglucose was also observed when silp mixed lymphocytes derivedfrom 8weeks old adult mice were cultured with glucose for hunder the culture conditions tested ra by itself was found tohave minimal eï¬ect on the already high raldh activity whereasthe addition of glucose in the media was able to further boost thisactivity figure 6dfinally in order to ascertain functional relevance of theseï¬ndings we sorted to determine whether supplementationfrontiers in immunology wwwfrontiersinaugust volume 0cko glucose promotes silpdc raldh activityfigure glucose induces raldh activity speciï¬cally in silpdcs mlndcs and spleen dcs spldc from adult spf mice or silpdcs from 2weeks oldneonatal spf mice were either puriï¬ed ac or total single cell suspensions were isolated from adult spf mice si d cells were cultured for h in glucosefreecontinuedfrontiers in immunology wwwfrontiersinaugust volume 0cko glucose promotes silpdc raldh activityfigure media with or without treatments following which aldefluor assay was performed fluorescence intensities of aldeï¬uor in cd8cd11b spldcsa cd103 mlndcs b and cd103cd11b silpdcs cd were analyzed by ï¬ow cytometry raldh activity depicted in overlaid histograms and bar graphsare from the cells cultured with glucose mm or ra nm or both the line graphs indicate raldh activity from the cells cultured with different concentrations ofglucose and mm in the presence or absence of ra nm data shown is representative of at least three independent experiments mean ± sem areindicated statistical signiï¬cance was determined by oneway anova with turkeys multiple comparison test p p p ns notstatistically significantof glucose presumably through the generation of ra canfacilitate the generation of itreg cells for this we performedan in vitro itreg induction assay with magnetically puriï¬edcd11chi dcs with high cd11c expression that were pretreated with glucose and incubated with naïve t cells undersuboptimal itreg inducing condition we observed significantincrease in itreg induction when silpdcs were pretreatedwith glucose compared to mock this eï¬ect of glucose pretreatment was speciï¬c for silpdcs and was not observedwhen mlndcs were used in the assay figures 7ab andfigure s4 these results in accordance to the results presentedin figure 6b suggested that the silp dcs are particularlymore susceptible to glucose treatment and thereby presumablythrough enhanced raldh activity acquire superior itreg cellinduction capacity compared to mln dcs it is to be notedhowever that while the puriï¬ed cd11chimhcii dcs usedin this assay are presentthere aresome site speciï¬c diï¬erences with regard to the expressionof mhcii and cd11c in mln and silp compared to silpmln has significantly higher proportion of cd11cmhciiand cd11cintmchii with intermediate cd11c expressionpopulations figure s4 left panels furthermore the expressionof mhcii in puriï¬ed mln dcs is lower than that of silpdcs figure s4 right panels these observations raised theformal possibility that enhanced mhcii expression in silprather than glucose mediated enhanced raldh activity maybe responsible for increased itreg conversion however pretreatment with ra either alone or in the presence of glucoseresulted in equally eï¬cient itreg induction irrespective of thesource of the dcs suggesting that the beneï¬t of glucose pretreatment is indeed primarily due to enhanced ra productionfigures 7ab lastly when itreg induction was carried outin vitro in a dc independent manner supplementation ofthe media with excess glucose had little eï¬ect thereby furthersubstantiating the role of dc derived raldh in this processfigure s5in similar frequenciesdiscussionthere is accumulating evidence suggesting that vitamin a and itsmetabolites play a pivotal role in maintaining various biologicalprocesses dietary supplementation of ra in thecontext of cutaneous t cell lymphoma and acute promyelocyticleukemia have been shown to have beneï¬cial outcome furthermore many studies highlight antiammatoryactivities of ra at mucosal sites and tissues such as intestinalmucosa airways lung central nervous system and skin in addition to the eï¬ect of ra on cancer and ammatorydiseases vitamin a or its metabolites play an important roleto suppress dietinduced obesity and insulin resistance therefore a better understanding of the cellular and molecularparameters responsible for vitamin a metabolism is of greatbiological relevance in this study by identifying the role of dietand the importance of glucose consumption for establishingraldh activity early in life in small intestine dcs we makesubstantial contribution to our knowledge related to the role ofnutritional components in establishing immunological toleranceat mucosal sitesby feeding af diet to mice raised under germ free conditionwe found that small intestine cd103cd11b lpdcs require adietary component as an initial trigger for raldh activity sincevitamin a in diet is known to be essential to activate raldhand generate ra in lpdcs lpscs mlndcs mlnscs andiecs one explanation of this observation could bethe possibility that at steady state af mice consume less vitamina compared to gf mice raised on ncd however our furtherexperiments in conjunction with a previous report stronglyindicate that the availability of vitamin a and the way it is feddoes not account for the low raldh activity in af m | 0 |
"PDGF-BB and VEGF-C expression were detected in 109 primary NSCLC tissues while the lymphatic micro-vessel density (LMVD) was counted. Results Of 109 cases PDGF-BB and VEGF-C overexpression was 66.97% (73/109) and 65.14% (71/109) respectively. 52 (47.7%) had overexpression of both PDGF-BB and VEGF-C (P?+?V+) 21 (19.3%) overexpression of PDGF-BB but low expression of VEGF-C (P?+?V-) 19(17.4%) overexpression of VEGF-C but low expression of PDGF-BB (P-V+) and 17(15.6%) low expression of both PDGF-BB and VEGF-C (P-V-). PDGF-BB expression was positively related to that of VEGF-C (r?=?0.451 p?=?0.034). LMVD in cases with P?+?V?+?was much higher than those with P-V- (p?=?0.004). In addition the patients with P?+?V?+?were younger and also had larger tumor size more likely lymph node metastasis and worse histological differentiation than those with P-V-. Moreover the overall survival (OS) of patients with P?+?V?+?was shorter than those with P-V- (p?=?0.015). Conclusion Coexpression of both PDGF-BB and VEGF-C was associated with lymphangiogenesis and poor prognosis in NSCLC and might play a critical role in NSCLC progression. Virtual Slides The virtual slide(s) for this can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/2261801312571320 Platelet-derived growth factor-BB Vascular endothelial growth factor-C Lymphatic micro-vessel density Non-small cell lung cancer Background Lung cancer is the leading cause of tumor-related mortality throughout the world of which 80% are non-small cell lung cancer (NSCLC). In 2008 lung cancer replaced liver cancer as the first cause of death among people with malignant tumors in China [1]. Despite all efforts in the field of early diagnosis and adjuvant therapy the morbidity and mortality of NSCLC trend to ascend straightly [2]. One of the most important factors with direct impact on prognosis and therapeutic strategy in NSCLC is lymphatic metastasis [34]. Lymphangiogenesis the formation of new lymphatic vessels is considered to be an important process in the development of lymphatic metastasis [5]. The status of lymphangiogenesis and lymphatic vessel remodeling has been estimated by lymphatic micro-vessel density (LMVD) [6]. D2-40 is the preferred lymphatic endothelium-specific monoclonal antibody (mAb) for investigating intra-tumoral and peri-tumoral lymphatic micro-vessels [7]. Increased amount of LMVD provides more opportunities for tumor cells to disseminate to the lymph nodes. The correlation between LMVD and prognosis was confirmed in a variety of human cancer including breast cancer melanoma and NSCLC [8-11]. The family of VEGFs is composed of VEGF-A VEGF-B VEGF-C VEGF-D VEGF-E VEGF-F and placental growth factor (PlGF). VEGF-A is directly linked to angiogenesis while VEGF-C is considered as a prime mediator of lymphangiogenesis and has been implicated in carcinogenesis and metastasis. VEGF-C is a ligand for the VEGF receptor (VEGFR)-3 a tyrosine kinase receptor that is expressed predominantly on lymphatic endothelial cells (LECs) [1213]. It is demonstrated that VEGF-C induces lymphangiogenesis by VEGFR-3 signaling [14]. Studies showed that VEGF-C expression is associated with lymphatic invasion LMVD lymph node metastasis and prognosis in some human tumors such as breast cancer gastric cancer and NSCLC [15-18]. Recent studies show that platelet-derived growth factors (PDGFs) also enable the process of functional lymphangiogenesis. They can connect the receptors on LECs to promote LECs proliferation migration and the formation of tubular structures which induce lymphangiogenesis [19]. PDGF family consists of five isoforms -AA -AB -BB -CC and DD [20]. PDGF-BB is a direct lymphangiogenic factor [21]. Emerging evidences indicate that the tight communication between vascular endothelial cells and mural cells by platelet-derived growth factor (PDGF)-BB is essential for capillary stabilization during the angiogenic process [22]. It was reported that the expression of PDGF-BB was correlated with tumor growth lymph node metastasis and lymphatic invasion in human esophageal squmaous cell carcinomas and NSCLC [2324]. Based on these data PDGF-BB and VEGF-C may play an important role in the process of tumor growth and lymphangiogenesis. However it is still unknown about the significance of combination of PDGF-BB and VEGF-C i.e. expression of both PDGF-BB and VEGF-C compared with only PDGF-BB or VEGF-C expression in NSCLC. In this study we examined the expression of PDGF-BB and VEGF-C in primary NSCLC tissues and investigated the clinicopathological significance of their coexpression and association with lymphangiogenesis. Methods Patients characteristics Tumor specimens were obtained from 109 patients with primary NSCLC who underwent surgery at the Jinan Central Hospital Affiliated to Shandong University China during the period from October 2008 to September 2010. They did not receive radiation therapy or chemotherapy before biopsy or surgical resection. There were 78 men (72%) and 31 women (28%) with median age of 58 years (interquartile range: 50?~?65 years) at the time of diagnosis. We determined the cell differentiation degree according to the classification amended in 1999 [25] and found 81 cases of well and moderately differentiated cells and 28 cases of poorly differentiated cells. The tumors were staged according to the USA Cancer Union Guidelines [26]. 38 patients were diagnosed with early NSCLC (I-IIa) and 71 with advanced NSCLC (IIb-III). Other clinical features are summarized in Table 1. All patients were followed up for at least 3 years after surgery. The median follow-up period was 47 months (interquartile range: 42?~?50 months). Overall survival (OS) was calculated from the date of surgery to the last follow up. The work was conducted in accordance with the Declaration of Helsinki. Informed consent was obtained from all the patients in this study. All patients signed the informed consent for use of specimens and the study was approved by the Institutional Review Board (Medical Ethics Committee of Jinan Central Hospital). Correlations of both PDGF-BB and VEGF-C coexpression with clinicopathological factors in primary human NSCLC Factors P?+?V+ P-V- P1 P?+?V - P2 P-V+ P3 Gender Male 35 13 0.476 16 0.716 14 0.847 Female 17 4 5 5 Age >60 years 23 11 0.047 13 0.859 11 0.676 ?60 years 29 6 8 8 Histology SQC 28 6 0.184 6 0.539 5 0.559 ADC 24 11 15 14 Tumor size >5 cm 24 3 0.037 5 0.950 6 0.563 ?5 cm 28 14 16 13 differentiation WD MD 35 17 0.017 17 0.757 12 0.027 PD 17 0 4 7 TNM stage I-IIa 12 8 0.113 10 0.973 8 0.765 IIb-III 40 9 11 11 Nodal status Positive 29 3 0.006 5 0.471 7 0.362 Negative 23 14 14 12 Note: P1 P value between P?+?V?+?and P-V-; P2 P value between P?+?V- and P-V-; P3 P value between P-V?+?and P-V-. Abbreviations: WD well differentiated MD moderately differentiated PD poorly differentiated ADC adenocarcinoma SQC squamous cell carcinoma. Main reagents The main reagents were anti-podoplanin mouse monoclonal antibody D2-40 (Dako Co. Denmark) anti-PDGF-BB rabbit polyclonal antibody (abcam Cambridge UK) Anti-VEGF-C rabbit monoclonal antibody (Beijing Zhongshan Goldenbrige Biotechnology China) immunohistochemical SP reagent box and DAB colour reagent (Fuzhou Maixin Co. China.P.R). Immunohistochemistry Immunohistochemical staining was carried out using the DAKO Envision detection kit (Dako Carpinteria CA USA). In brief paraffin-embedded tissue blocks were sectioned (4 ?m-thick) dried deparaffinized and rehydrated. Antigen retrieval was performed in a microwave oven for 15 min in 10 mM citrate buffer (pH 6.0). For all samples endogenous peroxidase activity was blocked with a 3% H2O2-methanol solution. The slides were blocked with 10% normal goat serum for 10 min and incubated with an appropriately diluted primary antibody mouse monoclonal antibody D2-40 (diluted 1:50) anti-PDGF-BB rabbit polyclonal antibody (diluted 1:200) or anti-VEGF-C rabbit polyclonal antibody (diluted 1:100) overnight at 4°C. The slides were then probed with an HRP-labeled polymer conjugated to an appropriate secondary antibody for 30 min. Each step was followed by washing with PBS. Each batch of staining was accompanied by positive and negative control slides. Primary human NSCLC tissues which are demonstrated to exhibit high levels of PDGF-BB and VEGF-C protein were used as positive controls. Normal mouse IgG substituted for primary antibody was a negative control. Quantitation of immunohistochemistry Clinicopathological findings were evaluated simultaneously using a double-headed light microscope by two independent examiners in a blinded fashion and mean values were calculated. The percentage of stained cells was recorded in at least 5 fields at 400-fold magnification in randomly selected tumor areas. In tumor specimens analysis of staining was exclusively restricted to the NSCLC cell reactions. Staining of stromal cells was not considered. Because cancer cells showed heterogeneous staining the dominant pattern was used for scoring. A combined scoring method that accounts for the intensity of staining as well as the percentage of cells stained was used as described previously [27]. The intensity of staining was graded from 0 to 3 with strong moderate weak and negative staining intensities as grade 321 and 0 respectively. The scores indicating percentage of positive cancer cells and staining intensity were multiplied to get a weighted score for each sample. For example a sample with 10% weak staining 10% moderate staining and 80% strong staining would be assigned a score of 270 (10?Ã?1?+?10?Ã?2?+?80?Ã?3?=?270) out of a possible score of 300. For statistical analyses samples with weighted scores 0100 were defined as negative otherwise as positive. LMVD was performed according to a modification of Weidners method [28]. The immunostained sections were scanned by light-microscopy at low magnification (40Ã) and the areas of tissue with the greatest number of distinctly highlighted microvessels (hot spots) were selected. LMVD was then determined by counting all immunostained vessels at a total magnification of (200Ã) from five areas for each case. Determination of the staining reaction was strictly confined to the hot spots and the mean number of the vessels in each case was evaluated. Statistical analysis Data were analyzed according to the Statistical Package for Social Sciences (SPSS. 18.0 Chicago IL USA). Spearmans coefficient of correlation Chi-squared test and two-tailed Student t test were used as appropriate. " | 1 |
"Early detection of capecitabineresistance could largely increase overall survival of colorectal cancerCRC patients Previous studies suggested examination of immune cells in peripheral blood would help to predictefficacy of chemotherapyMethods We examined the immunological characteristics of peripheral blood in CRC patients with capecitabinetreatment We analyzed the relationships between the abnormal immune cell population in capecitabineresistancepatients and major clinical features Furthermore RNA sequencing analyses of cell surface marker expression andthe correlations with other major immune cell populations were performed using this population to explore thepossible function of these cellsResults The expression level of CD16 on neutrophils was downregulated in capecitabineresistant CRC patientsPatients with CD16lowneutrophils after capecitabine therapy had adverse clinical features Whats important thechange of CD16 expression level on neutrophils appeared much earlier than CT scan RNA sequencing revealedthat CD16lowneutrophils in capecitabineresistant patients had lower expression level of neutrophilrelated genescompared to CD16neutrophils in capecitabinesensitive patients suggesting this CD16lowpopulation might beimmature neutrophils Furthermore the expression level of CD16 on neutrophils in patients with capecitabinetreatment was positively correlated with the number of antitumor immune cell subsets such as CD8T cell CD4Tcell NK cell and monocyteConclusions Our findings indicated that CD16 expression on neutrophils in peripheral blood was a goodprognostic marker for predicting efficacy of capecitabine in CRC patientsKeywords CD16 Neutrophils Capecitabineresistance Colorectal cancer Correspondence drzhongming1966163com gaoweiqiangsjtueducnyanzhsjtueducnYu Lu Yizhou Huang and Lei Huang share first authorship2Department of Gastrointestinal Surgery Renji Hospital School of MedicineShanghai Jiaotong University Shanghai China1State Key Laboratory of Oncogenes and Related Genes RenjiMed X StemCell Research Center Renji Hospital School of Medicine Shanghai JiaotongUniversity Shanghai ChinaFull list of author information is available at the end of the The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cLu BMC Immunology Page of BackgroundColorectal cancer CRC is one of the leading cause ofdeath worldwide More than million patients are diagnosed with CRC every year [] Whats more this lifethreaten disease kills nearly million people annually []In north America and Europe the morbidity and mortalityremain at high level [] despite developments of cancerscreening and endoscopy [ ] In China CRC becomesthe 5th most diagnosed cancer and 5th most deadly cancer[] Nearly million new cases are diagnosed andabout million people die from the disease every year []Postoperative adjuvant chemotherapy is firstline treatment for CRC patients [ ] Capecitabine a carbamatederivative of fluoropyrimidine is the backbone of CRCchemotherapy [ ] Asthe oral prodrug of fluorouracil 5FU it is widely used for postoperative adjuvant chemotherapy due to its long stable durationlower toxicity and convenient dosing compared to infusional 5FU [ ] However this chemotherapeutic drughas only modest efficacy the response rates of 5FU foradvanced CRC is only for single treatment and for combined chemotherapy [ ] The chemoresistance is recognized as a principal obstacle for cancer therapy [] leading to tumor recurrence or metastasisespecially liver and lung metastasis and cause over ofCRC mortality [] Intense researches on the mechanisms underlying the resistance revealed that changes oftumor cells themselves cause resistance although thesefindings are mainly restricted to tumor specimen examinewhich is not that suitable for posttreatment surveillanceWhats more CT computed tomography scan and colonoscopy are insensitive to micro metastasis despite theirgoodrecurrenceCapecitabineresistant patients could only be diagnosedwith cancer recurrence by CT scan or colonoscopy about years after capecitabine therapy [] when tumorsare big enough to be discovered Thus good prognosticmarkers are indispensable for predicting capecitabineresistance in the early stage after capecitabine therapydetection ofaccuracytheforCancer cells and their microenvironment could interactwith each other Immune cells could dynamically reflectcancer status and display multifaceted functions in cancerdevelopment [] Myeloid cells including monocytesmacrophages granulocytes neutrophils eosinophils basophils and mast cells play critical roles in cancer progression [] Myeloidderived suppressor cells MDSCs aheterogeneous population of myeloid cells remain at different stages of differentiation are immature counterparts ofmyeloid cells in cancer MDSCs acquire immunosuppressive features and mainly inhibit lymphocytes including Tcells and NK cells [] Recent studies report that chemotherapeutic agents like 5FU could interact with myeloid cells and influence antitumor efficacy []Vincent J reported that 5FU selectively inducedMDSC apoptotic cell death and increase IFNÎ productionby tumorspecific CD8T cells [] Other researchersshowed that activation of NLRP3 inflammasome and increased amount of HSP70 exosomes on MDSC by 5FUlead to MDSC activation [ ] Yuan Y found thattumorassociated macrophages secret IL6 to induce 5FUchemoresistance []ImportantlyIn this study we discovered that the expression ofCD16 on CD11bmyeloid cells was dramatically decreased in capecitabineresistant CRC patients after capecitabine adjuvanttherapy The expression level ofCD16 was closely related to poor prognosis after capecitabine therapythe downregulation ofCD16 on CD11bmyeloid cells appeared as early as month after capecitabine therapy in patients who werediagnosed with capecitabineresistance by CT scansabout years after the treatment The cutoff value ofCD16 expression would be helpful for the prediction of capecitabine chemoresistance Further analysisdemonstrated that these CD11bCD16lowmyeloid cellswere mainly immature neutrophils and expression levelof CD16 on neutrophils had a positive relationship withfrequencies of antitumor immune cell populations suchas CD8T cells and NK cellsResultsCD16 expression levels on CD11bmyeloid cells inperipheral blood of capecitabineresistant CRC patientsare different from capecitabinesensitive CRC patientsafter capecitabine therapyTo explore if myeloid cells in peripheral blood could predict the treatment efficacy of capecitabine we chose CRC patients with capecitabine adjuvant treatment whoseimmune cells populations in peripheral blood were examined by flow cytometry before and about months afterthe treatment Patients were divided into capecitabinesensitive and capecitabineresistant groups based on thediagnosis of recurrence by CT scan in about years aftercapecitabine treatment Table Additional file Fig S1ENo significant change was observed in major myeloid cellsubsets such as monocytes CD11bCD14CD15 neutrophils CD11bCD15CD14 or CD11bCD66bCD14and MDSCsbetweencapecitabinesensitive patients and capecitabineresistantpatients Additional file S1A B C and D But we foundthat the frequency of CD11bCD16myeloid cells was decreased in capecitabineresistant patients after capecitabinetreatment compared to that before the treatment Fig 1aWhats important a dramatic lower expression level ofCD16incapecitabineresistant patients compared to that of drugsensitive patients Patient and patient are representative patientsgroup andcapecitabineresistant group respectively Fig 1b TheCD11bHLADR\\lowCD33from capecitabinesensitiveon CD11bmyeloidcells wasobserved 0cLu BMC Immunology Page of Table Baseline characteristics of CRC patients in Fig GroupNumber of PatientsAgeSexTNM StageLocationCEA ngmlCA199 ngml Diagnosis of Recurrence AfterCapecitabinesensitiveCapecitabineresistantMMMMMFFFMFMFMMMFMMFFFMFMFMMMMFMMMFFMIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIRectumRectumColonColonRectumRectumRectumColonRectumColonColonColonRectumColonRectumRectumRectumRectumColonColonRectumRectumRectumRectumColonRectumRectumColonColonRectumRectumRectumRectumRectumRectumColonCapecitabine TreatmentNoNoNoNoNoNoNoNoNoNoNoNoNoNoNoNoNoNoNoNoNoNoNoNoNoNoYesYesYesYesYesYesYesYesYesYesdiagnosis of capecitabine resistance was determined by CTscan Additional file Fig S1E However when we analyzed these CD11bCD16myeloid cells in healthy donorsHDs and CRC patients before capecitabine therapy wefound no difference between these two cohorts Additionalfile Fig S1F and G This indicated that change of CD16expression on CD11bCD16myeloid cells was particular inCRC patients who were resistant to capecitabine therapyDecreased CD16 expression is correlated with poorpathological features in CRC patients after capecitabinetherapyTo determine whether the expression level of CD16 onCD11b myeloid cells is related to treatment efficacy of capecitabine we collected peripheral venous blood of CRCpatients months after capecitabine treatment and divided these patients into two groups CD16 group and 0cLu BMC Immunology Page of Fig CD16 expression of peripheral blood myeloid cells were differential in CRC patients after capecitabine therapy Peripheral venous bloodfrom CRC patients received singleagent oral capecitabine adjuvant therapy was collected before the therapy and months after the therapyand analyzed for myeloid cellrelated markers Attention Blood were collected months after capecitabine treatment unless particularlynoted a Frequencies of CD11bCD16myeloid cells were compared before and after capecitabine therapy in capecitabinesensitive andcapecitabineresistant patients n in sensitive group and n in resistant group respectively b Representative images of CD16 expressionon CD11bmyeloid cells before and after capecitabine therapy in two CRC patients from capecitabinesensitive group or capecitabine resistantgroup respectively Diagnosis of drugresistance was proved by CT scan during the followup in Fig S1e Mean ± SEM P005 by t tests aCD16low group Firstly Kmean clustering algorithm wasused to determineto divideCD11bCD16myeloid cells into CD11bCD16highcells andthe boundaryvalueCD11bCD16lowcells based on mean fluorescent intensityMFI of CD16 on CD11bCD16myeloid cells in peripheralblood after capecitabine therapy Additional file Fig S2A 0cLu BMC Immunology Page of ROCanalysisThe boundary value of CD16 MFIfor division ofCD11bCD16high cells and CD11bCD16low cells was à Next we analyzed frequency of CD11bCD16high cellsin peripheral blood after capecitabine therapy Additional file Fig S2B and determined the cutoff value for CD16 expression on CD11bmyeloid cells by receiver operating characteristicand Youden Index valuesAdditional file Fig S2C and S2D The cutoff value was Patients of CD16 group or CD16low group were determined if their frequencies of CD11bCD16highcells werehigher or lower than the cutoff value Additional file FigS2B S2C and S2D Then we assessed correlations betweenthe expression level of CD16 and CRC clinicopathologicalcharacteristics by Ï2 test The data revealed that patients inCD16low group had more cancer recurrence P and high level of carcinoembryonic antigen CEA P as well as carbohydrate antigen CA199 P compared to patients in CD16 group Table There were CRC patients developing recurrenttumor in CD16low group whereas only cases were observed in CD16 group Among CRC patientswith high CEA level patients belonged toCD16low group while only patients wereCD16 And patients with high CA199level were found in CD16low group compared with cases in that of CD16 However no significant difference was observed between these twogroups on age gendertumor sizeand Tumor Node Metastasis TNM stage Table tumor locationTo further confirm these results we divided CRCpatients after capecitabine treatment into two groupsbased on the level of CEA or CA199 and compared theexpression level of CD16 on CD11bCD16myeloid cellsbetween CEAhigh CEA ng and CEAlow CEA ¤ Table Relationship between CD16 expression on CD11bmyeloid cells after capecitabine therapy and clinicopathologiccharacteristicsCharacteristicsCD16low after therapy n nAll patients n nCD16 after therapy n nAge years¥GenderMaleFemaleTumor locationRectumColonTumor Size¥ cm cmCEA level after therapy¤ ngml ngmlCA199 level after therapy¤ ngml ngmlTNM stage AJCCStage IIStage IIILocation of recurrenceLocoregionalliver lungliverlungperitoneumPvalue 0cLu BMC Immunology Page of ng groups or between CA199high CA199 ngand CA199low CA199 ¤ ng groups The boundaryvalue of CEA and CA199 were decided by clinical guidelines The results showed that the expression level ofCD16 was dramatically decreased in either CEAhigh orCA199high groups compared to CEAlow or CA199low groups Fig 2a and b suggesting that the decreasedexpression level of CD16 on CD11bmyeloid cells aftercapecitabine treatment was related to the poor pathological features In conclusion low level of CD16 expression was related to poor pathological features such astumor recurrence CEA and CA199in CRC patientswith capecitabine therapyCD16 serves as a prognostic marker for CRC patientsreceived capecitabine adjuvant chemotherapyTo further explore the prognostic significance of CD16expression on CD11bmyeloid cells in predicting thetreatment efficacy of capecitabine chemotherapy wecompared the differences of overall survival OS anddisease free survival DFS between CD16 group andCD16low group The survival curves revealed that therewere significant association between the expression levelof CD16 and OS P 00006Fig 3a or DFS P 00023Fig 3b suggesting that low expression level ofCD16 was associated with shorter survival Next weused univariate analysis to further elucidate the significance of CD16 expression in predicting prognosis ofCRC patients receiving capecitabine The result demonP HR strated that CD16 expression level was prognostic factor for OS Table Whatsimportant Cox multivariate analysis also demonstratedthat expression level of CD16 P HR wasindependent predictors of OS Table Thesestillresults demonstrated that the expression level of CD16on CD11bmyeloid cells may serve as a good prognosticmarker for overall survival in CRC patients with capecitabine adjuvant chemotherapy[] Next we wondered ifDownregulation of CD16 expression on CD11bmyeloidcells appears earlier than diagnosis of capecitabine byimaging testsAs we know adjuvant chemotherapy remains the firstline therapy for CRC patients Capecitabine the oralprodrug of 5fluorouracil is one of the primary drugsfor the treatment A number of CRC patients becomeinsensitive to the therapy and suffer from cancer recurrence In clinic capecitabineresistance is mainlydiagnosed by cancer recurrence discovered throughcolonoscopy or CT scan in about years after capecitabine treatmentthechange of CD16 expression level on CD11bmyeloidcells appeared earlier than CTshowed recurrence Weselected CRC patients with capecitabine treatmentwhose blood samples were examined before and aftercapecitabine treatment Table The results showedin patients in capecitabineresistant groupthefrequency of CD11bCD16myeloid cells was decreased months after treatment compared to thatbeforecapecitabineresistance was diagnosed by CT scan about yearsafter the treatmentfile Fig S1E Whats important in a resistant patient decreased expression level of CD16 was found as earlyas month after capecitabine treatment Fig 4a Thefrequency of CD11bCD16high cell population waslargely lower than the cutoff value Neverthelesstumor monthsTable and Additional1a whiletreatmentFigafterthecapecitabinetherapyFig CD16 expression of CD11bCD16myeloid cells related to pathological features of CRC patients with capecitabine therapy CRC patientsreceiving capecitabine therapy were divided into different groups according to their CEA or CA199 level n in CEAhigh CEA ng groupand n in CEAlow CEA ¤ ng group n in CA199high CA199 ng group and n in CA199low CA199 ¤ ng group CD16MFI of CD11bCD16myeloid cells in CRC patients acquired from flow cytometry analysis was compared between different groups Mean ± SEMP001 P0001 by t tests a b 0cLu BMC Immunology Page of Fig CD16 high expression on CD11bmyeloid cells was good prognostic marker for CRC patients survival KaplanMeier analysis of overallsurvival OS and disease free survival DFS was performed in CD16 group and CD16low group p values were calculated by logrank test n in CD16 group and n in CD16low grouprecurrence was found in the liver from CT scan Fig 4bThese data suggested that downregulation of CD16on CD11bmyeloid cells served as a more sensitiveexamine than CT in CRC patientstreated withcapecitabineCD11bCD16lowmyeloid cells are mainly immatureneutrophils after capecitabine therapyTo further characterize the population of CD11bCD16lowmyeloid cells we isolated CD11bCD16myeloid cells fromcapecitabinesensitive patients and CD11bCD16myeloidcells from capecitabineresistant patients after capecitabinetherapy Fig 5a The data from flow cytometry revealed thatthese two populations were mainly neutrophils provedby their CD15 and CD66b expression Additional file Fig S3A To further verify these CD11bCD16myeloid cells and CD11bCD16myeloid cells were bothneutrophils we sorted these cells from capecitabineresistant patients and capecitabinesensitive patientsrespectively Characteristics ofthese patients werelisted in Additionalfile Table S1 We comparedour data of RNA sequencing with published data ofneutrophils from Jiang K [] using gene set enrichment analysis GSEA The results revealed thatin gene sets of neutrophil signature the expressionpattern of these cells was similar to that of the neutrophils provided by other group Additionalfile Fig S3B Additionalfile Table S2 Neverthelessthe decline of CD15 and CD66b expression combinewith the elevation of hematopoietic progenitorrelatedmarkers especially CD33 and CD117 suggested thatthese CD11bCD16myeloid cellsin capecitabineresistant patients became more immature after thetherapy compared with CD11bCD16myeloid cells fromcapecitabinesensitive patients Fig 5b The data of RNA sesomequencing also revealed declined expression ofTable Univariate and multivariate analyses for survival in CRC patients after capecitabine therapyPrognosticparameterUnivariate analysisHRCD16 expressionGenderAgeTumor locationTumor sizeCEACA199TNMRecurrenceHR Hazard ratio CI Confident interval95CIp valueMultivariate analysisHR95CIp value 0cLu BMC Immunology Page of Fig Analysis of CD16 expression was more sensitive than CT scan after capecitabine therapy a Peripheral venous blood from CRC patientsreceiving singleagent oral capecitabine adjuvant therapy was collected at different time before capecitabine therapy month and years afterthe therapy Frequencies of CD11bCD16highmyeloid cells were analyzed by flow cytometry b CT scan was performed during followup afteradjuvant chemotherapy in same patients as that of a respectively Sensitive patient normal operation site with no recurrence Resistant patientresectable metachronous liver metastases red arrowsand ATP wereneutrophilrelated genes in CD11bCD16myeloid cells fromcapecitabineresistant patients after capecitabine therapywhich implied immature status of these neutrophils Fig 5cIn addition active metabolism of nitrogen species purinenucleosidetheseCD11bCD16myeloid cells which are tightly related toimmunosuppressive role of MDSC [ ] Fig 5d To verify the immunosuppressive role of these CD11bCD16myeloid cells we sorted peripheral blood CD11bCD16myeloidcellsandCD11bCD16myeloid cellsfrom capecitabinesensitiveCRC patients or HDs and autologous T cells as well Aftercoculture T cells with these myeloid cells in the presence offrom capecitabineresistant CRC patientsinalsofoundleukocyte activators proliferation of T cell was significantlydeclined in resistant CRC patients group compared withsingle T cell group HD group and sensitive CRC patientsgroup Fig 5e ThetheseCD11bCD16myeloid cells in capecitabineresistant patientsmight exert immature cell status and play immunosuppressive role like MDSCsuggested thatresultsThe low expression level of CD16 on neutrophils isrelated to protumor status in CRC patients aftercapecitabine therapyAs we know immature myeloid cells are usually MDSCswhich could exert powerfulimmunosuppressive role 0cLu BMC Immunology Page of Fig CD11bCD16myeloid cells became immature neutrophils after therapy in capecitabineresistant patients a Peripheral venous blood fromcapecitabineresistant and capecitabinesensitive CRC patients was collected after the treatment in months CD11bCD16myeloid cells insensitive patients and that of CD11bCD16 in resistant patients were sorted for further analysis in b c and d b Expression of myeloidassociated and hematopoietic progenitorassociated markers on CD11bCD16myeloid cells in sensitive patients and on CD11bCD16myeloidcells in resistant patients was analyzed by flow cytometry c Peripheral blood CD11bCD16myeloid cells in sensitive patients andCD11bCD16myeloid cells in resistant patients were sorted and analyzed by RNA sequencing Expression of neutrophilrelated and monocyterelated genes derived from the results of RNA sequencing was shown in the heatmap d GO enrichment terms of differentially expressed MDSCrelated immunosuppressive biological processes derived from RNA sequencing e Autologous T cells were cultured alone cocultured withperipheral blood CD11bCD16myeloid cells from HDs and sensitive CRC patients or CD11bCD16myeloid cells from resistant CRC patientsfor h respectively Proliferation of T cells were analyzed by flow cytometry after incubation n for each group CD16N HD CD11bCD16myeloid cells from HDs CD16N CRC S CD11bCD16myeloid cells from sensitive CRC patients CD16N CRC R CD11bCD16myeloid cells from resistant CRC patients Mean ± SEM P005 P001 by t tests epatientscapecitabinesensitiveespecially in inhibiting T cells and NK cells [ ]As our results showed that CD11bCD16myeloid cellsfromandCD11bCD16myeloid cells from capecitabineresistantpatients were mainly neutrophils we tried to find out therelationship between the expression level of CD16 on neutrophils and other major immune cell subsets We collected peripheral venous blood from colorectal cancerpatients months after capecitabine therapy and analyzed frequencies of immune cells by flow cytometry Therelationships between expression level of CD16 on neutrophils and frequencies ofimmune cell subsets wereanalyzed by Pearsons correlation test The results showedthat CD16 expression was positively related to CD8T cellCD4T cell monocyte and NK cell frequencies Fig 6a bc and d but not that of cDC and pDC in patients aftercapecitabine therapy Fig 6e and f suggesting thatCD16lowneutrophils might have immunosuppressive activity as MDSCsDiscussionOver the past few decades numerous researchers haveattempted to improve the efficacy of capecitabine adjuvant therapy to ameliorate prognosis of CRC patients 0cLu BMC Immunology Page of Fig CD16 low expression on neutrophils predicted protumor immune status in CRC patients with capecitabine therapy Peripheral venousblood from CRC patients received singleagent oral capecitabine adjuvant therapy was collected months after the therapy and analyzed fordifferent immune cell subsets by flow cytometry CD16 MFI of peripheral blood neutrophils was calculated by flow cytometry analysis and thecorrelations between CD16 MFI of neutrophils and frequencies of CD8 T cells a CD4 T cells b monocytes c NK cells d cDCs e and pDCsf among total peripheral blood leukocytes were analyzed by Pearsons correlation testHoweverit remains one of the principal obstacle forcancer therapy at present In this study we demonstrated that the expression level of CD16 was downregulated in capecitabineresistant patients and lower expression level of CD16 on neutrophils in peripheralblood was correlated with poor prognosis in CRC patients with capecitabine adjuvant therapy Importantlydownregulation of CD16 was observed as early as month after capecitabine treatment which was moresensitive than CT scan indicating its great value in clinical application We determined the cutoff value ofCD16 expression on neutrophils for the prediction of capecitabine chemoresistance which would behelpful for clinical application and further researchesAnalyzationincapecitabineresistant patients revealed their immaturestatus and the expression of CD16 on neutrophils waspositively correlated with frequencies of antitumor immune cell populationsCD16lowneutrophilstheseofrecurrence which is vitalTo this day coloscopy and CT scan are still themain examines to supervise CRC progression and discoverfor capecitabineresistance diagnosis Unfortunately these two methodscould only provide evidence untiltumors are bigenough to be discovered patients wont have enoughtime to adjustthe treatment CEA and CA199 arewidely used to CRC surveillance as well especiallyCEA [] However CEA and CA199 cannot predictcancer progression so precisely and the false positivelead to anxiety and excessiveor negative results willtherapy Whats more some clinicaltrial also suggested that combining CEA and CT got no advantagecompared with single examine [] In this study ourresults showed that CD16 expression could serve as agood prognostic marker for poor CRC progressionafter capecitabine therapy Analyzation of CD16 expression hasthe downregulation of CD16 expression on neutrophils couldbe observed atcapecitabineresistance after the treatment Fig Previous studieshave demonstrated that CRC patients had primary resistance to 5FU single treatment[ ]thus the marker is essential for the drugselection inthese patients Second this marker is quite accuratefor predicting capecitabineresistance after the therapy In our study we collected totally CRC patients with capecitabinetheexpression level of CD16 on neutrophils Among patients who werecapecitabineresistance patients were observed to have downadvantages Firstto examinediagnosedtherapyasgreattheearlystage of 0cLu BMC Immunology Page of regulation of CD16 in months after capecitabinetreatment Table Third the examination of CD16expression only takes about ml peripheral bloodand it is noninvasive and has nearly no effect on patients healthCapecitabine the oral form of 5FU which is widelyused in CRC therapy has only modest efficacy due tothe chemoresistance Great efforts have been taken tofind out the mechanism Previous studies mainly concentrated on tumor cells themselves such as expressionof specific genes or generation of particular tumor cells[ ] In this research we worked on the correlationbetween changes on immune system and capecitabinechemoresistance and illustrated the conversion fromneutrophilsto immunosuppressive PMNMDSClikeneutrophils in these capecitabine insensitive patients byRNA sequencing and flow cytometry Our conclusioncould also be supported by other studies that 5FUcould promote MDSC protumor function The study byBruchard M found that 5FU could activate NLRP3inflammasome in MDSC and promote tumor growth[] Gobbo J also discovered that 5FU facilitatedproduction of tumorderived HSP70 exosomes whichfavored MDSC activation [] Thus prevention ofMDSC function after capecitabine or 5FU therapyholds great promise for improving drug efficacyreceptorResearchers have revealed that CD16myeloid cellswere tightly related to CRC development[ ]Giulio S found that CD16myeloid cell infiltration in CRC tumor tissue represented favorable prognosis [] and by using in vitro studies these studiesalso demonstrated that colon cancer infiltrate neutrophils enhance the responsiveness of CD8 T cells byTcelltriggering [] Our work differedfrom theirs in some ways Firstly our study focusedon CRC patients who received capecitabine adjuvanttreatment after surgery while Giulio Spagnoli groupfocused on all CRC patients and some healthy donorsSecondly biopsies from different positions were analyzed Peripheral blood was used in our study whileGiulio Spagnoli group mainly focused on tumor biopsies Exceptthese differences some of our resultswere also consistent with studies from Giulio Spagnoligroup Firstly both our data and Giulio Spagnoligroups data found that phenotype of peripheral bloodCD11bCD16myeloid cells had no difference betweenhealthy donors and CRC patients without capecitabinetherapy Fig S1F and G Secondly our work indicated that CD16 highpositive expression after capecitabine therapy predicted sensitivity to the therapyand good prognosis These results were consistentwith the work from Giulio Spagnoli groupthatCD16myeloid cells related to good prognosis of CRCpatientsMDSCs are a heterogeneous population of myeloidcells stay at different stages of differentiation PMNMDSCs are a great part of MDSCs that could be considered as counterparts of immature granulocytes chieflyimmature neutrophils []In this study we founddownregulation of CD16 expression on myeloid cells incapecitabineinsensitive CRC patients after capecitabinetreatment These CD16lowmyeloid cells after the therapy were mainly immature neutrophils CD16 is a lowaffinity FcÎ receptor which could activate antibodydependent process like phagocytosis in neutrophils andother phagocytes [] It is expressed on neutrophilsduring the maturation Researchers also revealed thatCD16 is typically associated with PMN activation andphagocytosis and its expression will change in differentmaturation status [ ] MDSCs could exert protumor roles mainly through inhibition of effective Tcells and NK cells [ ] Our study demonstrated thatlow expression of CD16 on neutrophils after the therapywas related to decreased frequencies of antitumor immune cells like CD8T cells and NK cells suggestingthatthey may have immunosuppressive activity asMDSCs The mechanism underlying the changes induced by capecitabine would be investigated further andit could be a good target to compete against capecitabinechemoresistanceConclusionsIn conclusion CD16 seems to be a promising target forCRC progression surveillance after capecitabine therapyStudies of CD16 expression on neutrophils may light thepath for not only predicting prognosis but also solvingcapecitabine resistance in CRC patientsMethodsPatients and peripheral bloodPeripheral venous blood of CRC patients in Departmentof Gastrointestinal Surgery Renji Hospital ShanghaiChina from January to December was gottenbefore capecitabine adjuvant treatment and at differenttime after the treatment as indicated in figure legendPeripheral venous blood of healthy donors was gotten inRenji Hospital The pathological information of patients was retrieved from the Pathology Department ofRenji Hospital These peripheral blood was used for flowcytometric analysis All the patients were provided withwritten informed consent before enrolment and thestudy was approved by the Research Ethics Committeeof Shanghai Jiao Tong University School of MedicineRenji Hospital Approval No Renji [] N013 Noneof patients had received radiotherapy or chemotherapybefore surgery All patients were followedup until deathor until the final followup May 0cLu e | 2 |
Growing evidence has demonstrated that glutathione peroxidases GPXs family genes play critical roles in onset and progression of human cancer However a systematic study regarding expression diagnostic and prognostic values and function of GPXs family genes in breast cancer remains absentMaterials and methods Several databases were employed to perform in silico analyses for GPXs family genes qRTPCR western blot and immunohistochemistry staining were introduced to validate GPX3 expression in breast cancer The functions of GPX3 in breast cancer cells were successively determinedResults By combination of receiver operating characteristic ROC curve analysis survival analysis and expression analysis GPX3 was considered as a potential tumor suppressor and a promising diagnosticprognostic biomarker in breast cancer Next low expression of GPX3 was confirmed in breast cancer cells and tissues when compared with corresponding normal controls Overexpression of GPX3 markedly suppressed proliferation colony formation migration and invasion of breast cancer in vitro Moreover two potential mechanisms responsible for GPX3 downregulation in breast cancer including hypermethylation of GPX3 promoter and release of hsamiR3245p inhibitionConclusions Collectively we demonstrate that GPX3 is markedly downregulated in breast cancer possesses significant diagnostic and prognostic values and attenuated in vitro growth and metastasis of breast cancerKeywords Glutathione peroxidase GPX3 Breast cancer Diagnosis Prognosis BiomarkerBackgroundBreast cancer is the most common diagnosed womens malignant tumor and also the second leading cause of cancerrelated deaths in women worldwide [ ] Despite a variety of advancements have been achieved in diagnosis and therapy the total outcome of patients with breast cancer remains unsatisfactory Thus developing effective therapeutic targets and promising biomarkers for Correspondence 11718264zjueducn Peifen_Fu163comDepartment of Breast Surgery First Affiliated Hospital College of Medicine Zhejiang University QingChun Road Hangzhou Zhejiang Chinadiagnosis and prognosis prediction is very meaningful to improve prognosis of breast cancerGlutathione peroxidases GPXs consisting of eight members GPX18 are ubiquitously expressed proteins that catalyze the reduction of hydrogen peroxides and anic hydroperoxides by glutathione [] GPX family members have been well demonstrated to be frequently aberrantly expressed and are also closely linked to progression of diverse types of human cancer including kidney cancer [] pancreatic cancer [] hepatocellular carcinoma [] cervical cancer [] and gastric cancer [] However a comprehensive study about expression The Authors This is licensed under a Creative Commons Attribution International License which permits use sharing adaptation distribution and reproduction in any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate if changes were made The images or other third party material in this are included in the s Creative Commons licence unless indicated otherwise in a credit line to the material If material is not included in the s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtain permission directly from the copyright holder To view a copy of this licence visit httpcreat iveco mmons licen sesby40 The Creative Commons Public Domain Dedication waiver httpcreat iveco mmons publi cdoma inzero10 applies to the data made available in this unless otherwise stated in a credit line to the data 0cLou a0et a0al Cancer Cell Int Page of function diagnostic and prognostic values of GPXs family in breast cancer remain absentIn this study we first assessed the roles of GPXs family genes in predicting diagnosis and prognosis of breast cancer and then determined the mRNA and protein expression of GPXs family genes in breast cancer using bioinformatic analysis Next the low expression of GPX3 was detected in breast cancer cells and tissues Subsequently the function of GPX3 in breast cancer cell growth and metastasis was also investigated Finally we explored the potential detailed mechanisms responsible for GPX3 downregulation in breast cancerMaterials and a0methodsROC curve analysisUsing TCGA breast cancer and normal breast expression data the diagnostic values of GPXs family genes were evaluated by ROC curve as we previously described [] Pvalue was considered as statistically significantKaplanMeierplotter database analysisKaplanMeierplotter database httpkmplo tcomanaly sis which is capable to access the effect of genes on survival in cancer types including breast cancer was employed to perform survival analysis for GPXs family genes and miRNAs in breast cancer [] Logrank Pvalue was considered as significantGEPIA database analysisGEPIA database httpgepia cance rpkucnindex html a newly developed interactive web server for analyzing the RNA sequencing expression data of tumors and normal samples from the TCGA and GTEx projects was used to determine mRNA expression profile of GPXs family genes in breast cancer [] Pvalue was considered as statistical significanceOncomine database analysisOncomine database wwwoncom ine which is a cancer microarray database and integrated datamining platform was also utilized to analyze mRNA expression of GPXs family genes in breast cancer [ ] Fold change FC Pvalue and a gene rank in top were set as the thresholds for selecting the included datasetsUALCAN database analysisThe protein expression levels of GPXs family genes in breast cancer were assessed using UALCAN database httpualca npathuabeduindex html which is a comprehensive userfriendly and interactive web resource for analyzing cancer OMICS data [] UALCAN database was also introduced to determine the promoter methylation level of GPX3 in breast cancer Pvalue of statistical analysis was considered to have significant differencesstarBase database analysisstarBase database tarb asesysueducnindex php an source platform for investigating miRNAassociated studies was used to predict the upstream binding miRNAs of GPX3 [ ] The correlation of GPX3 with miRNA in breast cancer and miRNA expression level in breast cancer were also assessed by starBase database Pvalue was considered as statistical significanceCell lines and a0clinical tissuesThe human breast cancer cell lines MCF7 and MDAMB231 and normal breast cell line MCF10A were purchased from Shanghai Institute of Biological Science Chinese Academy of Sciences Shanghai China breast cancer tissues and matched normal tissues were obtained from patients with breast cancer who received surgical resection in the First Affiliated Hospital of Zhejiang University College of Medicine Hangzhou China This study was approved by the ethics committee Table Correlation of a0 GPX3 expression with a0 various clinicopathological features in a0breast cancerFeaturesCasesBreast cancerLow expressionHigh expressionPvalueAge Tumor size Lymph node metastasis Present AbsentHistopathological grade III IIIER status Positive NegativePR status Positive NegativeHER2 status Positive Negative 0cLou a0et a0al Cancer Cell Int Page of of the First Affiliated Hospital of Zhejiang University College of MedicineRNA isolation and a0qRTPCRTotal RNA was isolated from breast cancer cells and tissues by Trizol reagent Invitrogen USA qRTPCR was employed to detect GPX3 mRNA expression in breast cancer as we previously described [] GPX3 expression was normalized to GAPDH by the method of ddCt The sequences of primers used in this study GPX3 forward primer ²GAG CTT GCA CCA TTC GGT CT3² GPX3 reverse primer ²GGG TAG GAA GGA TCT CTG AGTTC3² GAPDH forward primer ²AAT GGA CAA CTG GTC GTG GAC3² GAPDH reverse primer ²CCC TCC AGG GGA TCT GTT TG3²Protein extraction and a0western blotProtein of breast cancer cells was extracted using RIPA buffer Beyotime China supplemented with protease and phosphatase inhibitors Thermo Scientific USA Western blot was performed as previously described [] The primary antibodies of GPX3 and GAPDH were purchased from Abcam and antirabbit peroxidase conjugated secondary antibody was purchased from Sigma GPX3 band density was normalized to GAPDH and quantified by ImageJ softwareImmunohistochemistry IHC analysisIHC was utilized to analyze the protein expression of GPX3 in breast cancer tissues and matched normal breast tissues as we previously reported []Establishment of a0stablyoverexpressed cellFull length of GPX3 was first amplified after which the PCR product was cloned into pcDNA31PURO vector digested with BamH1 and XhoI GPX3overexpressed Lipofectamine¢ Invitrogen USA according to the plasmid was transfected into breast cancer cells using manufactures instruction Then stablyoverexpressed cell was screened using puromycin a0μgmLCCK assay stablyoverexpressed cells were seeded into 96well plates and cultured for varied period and a0h At the culture end of each time point a0μl CCK8 solution was added into each well and incubated for another a0 h at a0 °C Finally the optical density OD value at a0nm of each well was determined by a microplate readerColony formation assay stablyoverexpressed cells were seeded into sixwell plates and cultured for a0weeks At the end of culture the plates were washed using phosphate buffered saline PBS for two times Next the plates were fixed in methanol for a0min and stained with crystal violet solution for another a0 min Finally the visible colonies of each well were countedWound healing assayWound healing assay was introduced to detect the migrated ability of breast cancer cells à stablyoverexpressed cells were seeded into sixwell plates When the cells were grown to confluence a wound cross was made using a micropipette tip Photographs were then taken through a microscopy immediately or a0h after woundingTranswell invasion assayCell invasion was determined by Transwell invasion assay Briefly transwell inserts were firstly coated with Matrigel BD USA Then à stablyoverexpressed cells suspended in a0 mL serumfree medium were added into inserts And a0mL medium containing FBS was added to the lower compartment as a chemoattractant After culturing for a0h the cells on the upper membrane were carefully removed using a cotton bud and cells on the lower surface were fixed with methanol for a0 min and successively stained with crystal violet solution for a0min Photographs were then taken through a microscopyStatistical analysisStatistical analysis of bioinformatic analysis was performed by online databases as mentioned above The results of experimental data were shown as mean ± SD Students ttest was used to assess differences between two groups The diagnostic value was determined by ROC curve analysis A twotailed value of P was considered as statistically significantResultsThe diagnostic and a0prognostic values of a0GPXs family genes in a0breast cancerTo explore if the expression of GPXs family genes possesses significant diagnostic values in patients with breast cancer receiver operating characteristic ROC curve analysis was employed based on breast cancer data from TCGA database Fig a0 As shown in Fig a0 four GPXs family genes had the significant ability to distinguish breast cancer tissues from normal breast tissues including GPX2 GPX3 GPX4 and GPX8 However the other four GPXs family genes GPX1 GPX5 GPX6 and GPX7 showed no statistical diagnostic values in breast cancer Notably these findings suggested that GPX3 was the most potential diagnostic biomarker for patients 0cLou a0et a0al Cancer Cell Int Page of Fig The diagnostic values of GPXs family genes in breast cancer using ROC curve analysis a GPX1 b GPX2 c GPX3 d GPX4 e GPX5 f GPX6 g GPX7 h GPX8with breast cancer with the Area Under Curve AUC value being equal to Next we investigated the prognostic values of GPXs family genes in breast cancer using KaplanMeierplotter database Fig a0 Increased expression of GPX1 Fig a02a indicated poor prognosis of breast cancer Breast cancer patients with higher expression of GPX2 Fig a02b GPX3 Fig a02c or GPX5 Fig a02e had better prognosis GPX4 GPX6 and GPX7 had no significant predictive values for prognosis of breast cancer All these findings together indicated that only GPX2 and Fig The prognostic values of GPXs family genes in breast cancer determined by KaplanMeier plotter database a GPX1 b GPX2 c GPX3 d GPX4 e GPX5 f GPX6 g GPX7 h GPX8 0cLou a0et a0al Cancer Cell Int Page of GPX3 possessed significant diagnostic and prognostic values for breast cancerThe expression levels of a0GPXs family genes in a0breast cancerNext we further studied the expression levels of GPXs family genes in breast cancer First of all TCGA and GTEx databases were introduced to mine the mRNA expression of GPXs family genes in breast cancer The mRNA expression profile of GPXs family was shown in Fig a03a TCGA tumor tissues compared with TCGA normal tissues and Fig a03b TCGA tumor tissues compared with TCGA normal tissues and GTEx normal tissues We found that GPX2 and GPX3 were significantly downregulated in breast cancer Fig a0 3cf Next Oncomine database was used to further analyze mRNA expression of GPXs family genes in breast cancer Fig a04a We performed metaanalysis for included studies about GPX3 and found that GPX3 mRNA expression was markedly decreased in breast cancer Fig a04b The downregulation of GPX3 mRNA expression in breast cancer of the GPX3associated studies was presented in Fig a04cq However we found that GPX2 was not significantly downregulated in breast cancer Subsequently CPTAC database was utilized to assess the protein expression of GPXs family genes in breast cancer Fig a0 The results revealed that GPX1 GPX2 GPX3 and GPX4 protein levels were markedly decreased in breast cancer when compared with normal controls GPX7 protein expression in breast cancer was significantly increased GPX8 showed no statistical difference between breast cancer tissues and normal tissues And GPX5 and GPX6 were not found in CPTAC Taken together GPX3 was the most potential one among all GPXs family genes in breast cancer and was selected for following research Fig a0The expression level of a0GPX3 was a0confirmed in a0breast cancer and a0negatively correlated with a0tumor progressionTo further validate the results from in silico analysis we detected the mRNA and protein expression levels of GPX3 in breast cancer cells and tissues As presented in Fig a0 7a b GPX3 mRNA and protein were significantly downregulated in two breast cancer cells MCF7 and MDAMB231 when compared with normal cell MCF10A We also found that GPX3 mRNA expression in breast cancer tissues was much lower than that in adjacent matched normal tissues Fig a07c The protein expression of GPX3 was also detected using immunohistochemistry IHC analysis The results showed that GPX3 protein expression was significantly decreased in breast cancer tissues Fig a07d Collectively GPX3 mRNA and protein expression levels were significantly downregulated in breast cancer which was identical with the bioinformatic analytic results Furthermore Chi square test revealed that low expression of GPX3 was significantly negatively correlated with ERPR expression and positively linked to tumor size histopathological grade and lymph node metastasis Table a0 All these findings showed that GPX3 was negatively correlated with progression of breast cancer and might function as a tumor suppressor in breast cancerGPX3 overexpression suppressed proliferation and a0colony formation of a0breast cancer cellsGiven the low expression of GPX3 in breast cancer overexpression technology was used to study GPX3²s functions We then constructed the overexpressed plasmid of GPX3 After transfection of GPX3overexpressed plasmid GPX3 mRNA and protein expression levels were significantly upregulated in breast cancer cells Fig a0 8a b Firstly we explored the effect of GPX3 on growth of breast cancer cells CCK8 assay demonstrated that overexpression of GPX3 markedly suppressed in a0vitro proliferation of breast cancer cells MCF7 and MDAMB231 Fig a0 8c d Furthermore colony formation assay also revealed that GPX3 upregulation led to the inhibition of clonogenic capacity of breast cancer cells Fig a08e f These findings indicated that GPX3 overexpression significantly suppressed in a0 vitro proliferation and colony formation of breast cancer cellsGPX3 overexpression inhibited migration and a0invasion of a0breast cancer cellsMetastasis is another hallmark of malignant tumors including breast cancer We intended to ascertain if GPX3 affects metastasis of breast cancer Wound healing assay was first employed to investigate GPX3²s function in controlling migration of breast cancer cells and the result demonstrated that overexpression of GPX3 obviously attenuated the migrated ability of breast cancer cells Fig a09a b Moreover increased expression of GPX3 could also suppressed invasion of breast cancer cells which was detected by transwell invasion assay Fig a09cf Taken together overexpression of GPX3 suppressed in a0vitro migration and invasion of breast cancer cellsThe potential mechanisms responsible for a0GPX3 downregulation in a0breast cancerFinally we preliminarily probed the possible molecular mechanisms that accounted for GPX3 downregulation in breast cancer Promoter hypermethylation may be responsible for expression suppression of tumor suppressors Intriguingly we found that the promoter methylation level of GPX3 was significantly upregulated in breast cancer tissues compared with normal controls Fig a010a Gene expression was also frequently negatively regulated by miRNAs at posttranscriptional 0cLou a0et a0al Cancer Cell Int Page of Fig The mRNA expression of GPXs family genes in breast cancer determined by GEPIA database a The mRNA expression profile of GPXs family genes in breast cancer tissues compared with TCGA normal breast tissues b The mRNA expression profile of GPXs family genes in breast cancer tissues compared with TCGA and GTEx normal breast tissues c d GPX2 was significantly downregulated in breast cancer e f GPX3 was significantly downregulated in breast cancer P level The miRNAs that potentially bind to GPX3 were predicted by starBase database and miRNAs were finally found For better visualization miRNAGPX3 network was established Fig a0 10b Based on the action mechanism of miRNA there should be negative correlation between miRNA and target gene We performed expression correlation analysis for miRNAGPX3 pairs As listed in Table a0 four potential miRNAs hsamiR3245p hsamiR3283p hsalet7a5p and hsamiR449b5p which were inversely associated 0cLou a0et a0al Cancer Cell Int Page of Fig The mRNA expression of GPXs family genes in breast cancer determined by Oncomine database a The mRNA expression of GPXs family genes in breast cancer b Metaanalysis for the included GPX3associated datasets in breast cancer cq The mRNA expression of GPX3 was markedly downregulated in breast cancer in included GPX3assocaited datasets 0cLou a0et a0al Cancer Cell Int Page of Fig The protein expression of GPXs family genes in breast cancer detected by UALCAN database a GPX1 b GPX2 c GPX3 d GPX4 e GPX7 f GPX8 P 0cLou a0et a0al Cancer Cell Int Page of Fig The visual flowprocess diagram of this studywith GPX3 expression in breast cancer were identified The prognostic values of the four miRNAs in breast cancer were also evaluated by KaplanMeierplotter database Fig a0 10c d Survival analysis revealed that among the four miRNAs only high expression of hsamiR3245p indicated poor prognosis for patients with breast cancer Fig a0 10c The expression levels of four miRNAs in breast cancer was subsequently determined by starBase Fig a010gj and showed that miR3245p and hsamiR449b5p were significantly upregulated whereas hsamiR3283p and hsalet7a5p were markedly downregulated in breast cancer compared with normal controls By combination of survival and expression analysis miR3245p was considered as the most potential upstream miRNA of GPX3 in breast cancer The above results implied that promoter hypermethylation and miR3245pmediated suppression were two potential mechanisms that may be responsible for GPX3 downregulation in breast cancer Fig a010lDiscussionBreast cancer is the most common cancer type in women The molecular mechanism of carcinogenesis of breast cancer is still unclear and need to be further investigated Increasing findings have showed that GPXs are critical regulators in onset and progression of human cancer However the knowledge of GPXs in breast cancer is still limitedROC curve and survival analysis for GPXs family revealed that some of them might serve as promising diagnostic and prognostic biomarkers for breast cancer especially GPX2 and GPX3 Expression analysis demonstrated the significant low expression of GPX3 in breast cancer GPX3 was reported to act as a tumor suppressor 0cLou a0et a0al Cancer Cell Int Page of Fig The expression levels of GPX3 in breast cancer cells and tissues The mRNA a and protein b expression of GPX3 in breast cancer cells was significantly lower than that in normal breast cell c The mRNA expression of GPX3 was markedly decreased in breast cancer tissues compared with matched normal breast tissues d IHC analysis of GPX3 expression levels in normal breast tissues and breast cancer tissues Bar scale um P in human cancer For example Cai et a0al indicated that GPX3 prevented migration and invasion of gastric cancer by targeting NFkBWnt5aJNK signaling [] Lee et a0al suggested that GPX3 arrested cell cycle and functioned as a tumor suppressor in lung cancer [] Hua et a0 al showed that silencing GPX3 expression promoted tumor metastasis in human thyroid cancer [] Caitlyn et a0 al revealed that plasma GPX3 limited the development of colitis associated carcinoma [] However the function and mechanism of GPX3 in breast cancer have not been reported and need to be further elucidatedNext we confirmed the low expression of GPX3 in breast cancer cells and tissues using qRTPCR western blot and IHC which supported the results of bioinformatic analysis Functional experiments revealed that overexpression of GPX3 significantly inhibited in a0 vitro proliferation colony formation migration and invasion of breast cancer cellsPrevious studies have showed the effect of promoter methylation level in regulating gene expression [] Thus we preliminarily evaluated the promoter methylation level of GPX3 in breast cancer and found that it was significantly upregulated in breast cancer compared with normal breast tissues Moreover Mohamed et a0 al also demonstrated the link between promoter hypermethylation of GPX3 and inflammatory breast carcinogenesis [] The report together with our finding revealed that hypermethylation of GPX3 promoter might be a potential mechanism responsible for GPX3 downregulation in breast cancermiRNAs are involved in multiple biological processes by suppressing gene expression [ ] We also explored the upstream regulatory miRNAs of GPX3 By combination of correlation analysis survival analysis and expression analysis for these miRNAs miR3245p was regarded as the most potential miRNA which was overexpressed negatively correlated with GPX3 expression and possessed poor prognosis in breast cancer Numerous studies have demonstrated that miR3245p served as an oncogenic miRNA in human cancer For example miR3245p promoted progression of papillary thyroid carcinoma via microenvironment alteration [] miR3245p facilitated progression of colon cancer by activating Wntbetacatenin pathway [] Moreover the 0cLou a0et a0al Cancer Cell Int Page of Fig Overexpression of GPX3 inhibited proliferation and colony formation of breast cancer cells in vitro ab The overexpression effect of GPX3overexpressed plasmid in breast cancer cells cd Overexpression of GPX3 inhibited proliferation of MCF7 and MDAMB231 cells ef Overexpression of GPX3 inhibited colony formation of MCF7 and MDAMB231 cells P relationship between GPX3 and miR3245p has already been reported in lung cancer [] Thus overexpressed miR3244p might be another mechanism that accounted for GPX3 downregulation in breast cancer In the future the oncogenic roles of miR3245p need to be further investigated by in a0vitro and in a0vivo assaysConclusionsIn summary our current findings indicate that GPX3 is markedly downregulated in breast cancer promotes in a0 vitro growth and metastasis of breast cancer cells and servers as a promising diagnostic or prognostic biomarker for patients with breast cancer Moreover we also elucidate that promoter hypermethylation and miR3245pmediated suppression may be two 0cLou a0et a0al Cancer Cell Int Page of Fig Overexpression of GPX3 suppressed migration and invasion of breast cancer cells in vitro a b Increased expression of GPX3 attenuated migration of MCF7 and MDAMB231 cells c d Increased expression of GPX3 attenuated invasion of MCF7 cell e f Increased expression of GPX3 attenuated invasion of MDAMB231 cell Bar scale um P See figure on next pageFig The potential mechanisms responsible for GPX3 downregulation in breast cancer a The promoter methylation level of GPX3 was increased in breast cancer compared with normal controls b The miRNAGPX3 network cf The prognostic values of four miRNAs in breast cancer gj The expression levels of four miRNAs in breast cancer k The intersection analysis of survival analysis and expression analysis l The model of GPX3²s function and dysregulated mechanism in breast cancer P was considered as statistically significant 0cLou a0et a0al Cancer Cell Int Page of 0cLou a0et a0al Cancer Cell Int Page of Table The expression correlation of a0GPX3 with a0predicted miRNAs using TCGA breast cancer datamiRNAhsamiR3245phsamiR3283phsalet7a5phsamiR449b5phsamiR6295phsamiR47565phsamiR642a5phsalet7d5phsamiR449ahsamiR5895phsamiR181d5phsamiR21145phsamiR34a5phsamiR449c5phsamiR23a3phsamiR3150a3phsamiR47315phsamiR23b3phsamiR4915phsamiR4739hsamiR181c5phsamiR3612hsamiR5823phsamiR650hsalet7b5phsamiR3383phsamiR2278hsamiR1225phsamiR181a5phsamiR181b5phsamiR3139hsamiR4644hsamiR5013phsamiR26825phsamiR4306hsamiR95phsamiR620hsamiR1321hsamiR985phsalet7e5phsamiR1855phsamiR1270hsalet7 g5phsamiR2055phsamiR371a5phsamiR8735phsamiR34b5phsamiR5325phsamiR2963pR PvalueTable continuedmiRNAhsamiR7085phsamiR35295phsamiR5745phsamiR8765phsamiR2965phsamiR5023phsamiR1365phsamiR285phsamiR3615phsamiR520 hhsamiR6683phsamiR520 g3phsamiR376b3phsamiR6753phsalet7f5phsamiR34c5phsamiR665hsamiR1385phsamiR146a5phsamiR376a3phsamiR223phsamiR2995phsalet7i5phsamiR4953phsamiR1433phsamiR8893phsamiR146b5phsamiR3795phsamiR2233phsalet7c5pRPvaluepotential mechanisms responsible for GPX3 downregulation in breast cancer These results provide key clues for developing effective therapeutic targets and biomarkers for breast cancerAcknowledgementsNot applicableAuthors contributionsWL and PF designed this work performed experiments analyzed data and draft the manuscript BD performed some experiments SW revised the manuscript All authors read and approved the final manuscriptFundingNot applicableAvailability of data and materialsThe data in this work are available from the corresponding author on reasonable request 0cLou a0et a0al Cancer Cell Int Page of Lou W Ding B Fan W High expression of pseudogene PTTG3P indicates a poor prognosis in human breast cancer Mol Therapy Oncolytics Lou W Liu J Ding B Jin L Xu L Li X Chen J Fan W Five miRNAsmediated PIEZO2 downregulation accompanied with activation of Hedgehog signaling pathway predicts poor prognosis of breast cancer Aging Chen D Si W Shen J Du C Lou W Bao C Zheng H Pan J Zhong G Xu L et al miR27b3p inhibits proliferation and potentially reverses multichemoresistance by targeting CBLBGRB2 in breast cancer cells Cell Death Dis Cai M Sikong Y Wang Q Zhu S Pang F Cui X Gpx3 prevents migration and invasion in gastric cancer by targeting NFsmall ka CyrillicBWnt5aJNK signaling Int J Clin Exp Pathol An BC Choi YD Oh IJ GPx3mediated redox signaling arrests the cell cycle and acts as a tumor suppressor in lung cancer cell lines Plos One 2018139e0204170 Zhao H Li J Li X Han C Zhang Y Zheng L Guo M Silencing GPX3 expression promotes tumor metastasis in human thyroid cancer Curr Prot Peptide Sci Barrett CW Ning W Chen X Smith JJ Washington MK Hill KE Coburn LA Peek RM Chaturvedi R Wilson KT et al Tumor suppressor function of the plasma glutathione peroxidase gpx3 in colitisassociated carcinoma Cancer Res Kulis M Esteller M DNA methylation and cancer Adv Genet Mohamed MM Sabet S Peng DF Nouh MA ElShinawi M Promoter hypermethylation and suppression of glutathione peroxidase are associated with inflammatory breast carcinogenesis Oxid Med Cell Lou W Liu J Ding B Chen D Xu L Ding J Jiang D Zhou L Zheng S Fan W Identification of potential miRNAmRNA regulatory network contributing to pathogenesis of HBVrelated HCC J Transl Med Lou W Liu J Gao Y Zhong G Chen D Shen J Bao C Xu L Pan J Cheng J et al MicroRNAs in cancer metastasis and angiogenesis Oncotarget Lou W Liu J Gao Y Zhong G Ding B Xu L Fan W MicroRNA regulation of liver cancer stem cells Am J Cancer Res Yang Y Xia S Zhang L Wang W Chen L Zhan W MiR3245pPTPRDCEBPD axis promotes papillary thyroid carcinoma progression via microenvironment alteration Cancer Biol Therapy Yan D Liu W Liu Y Luo M LINC00261 suppresses human colon cancer progression via sponging miR3243p and inactivating the Wntbetacatenin pathway J Cell Physiol Lin MH Chen YZ Lee MY Weng KP Chang HT Yu SY Dong BJ Kuo FR Hung LT Liu LF et al Comprehensive identification of microRNA arm selection preference in lung cancer miR3245p and3p serve oncogenic functions in lung cancer Oncol Lett Publishers NoteSpringer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliationsEthics approval and consent to participateThis study was approved by the ethics committee of the First Affiliated Hospital of Zhejiang University College of MedicineConsent for publicationNot applicableCompeting interestsThe authors state that they have no conflicts of interestReceived June Accepted July References Bray F Ferlay J Soerjomataram I Siegel RL Torre LA Jemal A Global cancer statistics GLOBOCAN estimates of incidence and mortality worldwide for cancers in countries CA Cancer J Clin Lou W Liu J Ding B Xu L Fan W Identification of chemoresistanceassociated miRNAs in breast cancer Cancer Manag Res Matouskova P Hanouskova B Skalova L MicroRNAs as potential regulators of glutathione peroxidases expression and their role in obesity and related pathologie | 2 |
pulmonary disease COPD is due to structural changes and narrowing of small airways and parenchymaldestruction loss of the alveolar attachment as a result of pulmonary emphysema which all lead to airï¬ow limitation Extracorporeal shock waves ESW increase cell proliferation and diï¬erentiation of connective tissue ï¬broblasts To date no studiesare available on ESW treatment of human bronchial ï¬broblasts and epithelial cells from COPD and control subjects We obtainedprimary bronchial ï¬broblasts from bronchial biopsies of patients with mildmoderate COPD and control smokers with normallung function 16HBE cells were also studied Cells were treated with a piezoelectric shock wave generator at low energy mJmm2 pulses After treatment viability was evaluated and cells were recultured and followed up for and h Cellgrowth WST1 test was assessed and proliferation markers were analyzed by qRTPCR in cell lysates and by ELISA tests in cellsupernatants and cell lysates After ESW treatment we observed a significant increase of cell proliferation in all cell types CKitCD117 mRNA was significantly increased in 16HBE cells at h Protein levels were significantly increased for cKit CD117 at h in 16HBE p and at h in COPDï¬broblasts p � for PCNA at h in 16HBE p � for y1 CD90 at and h in CSï¬broblasts p � and p � for TGF1 at h in CSï¬broblasts p � for procollagen1 at h inCOPDï¬broblasts p � and for NFκBp65 at and h in 16HBE p � and p � In the peripheral lung tissueof a representative COPD patient alveolar type II epithelial cells TTF1 coexpressing cKit CD117 and PCNA were occasionally observed ese data show an increase of cell proliferation induced by a low dosage of extracorporeal shock waves in16HBE cells and primary bronchial ï¬broblasts of COPD and control smoking subjects Backgrounde progressive chronic airï¬ow limitation in chronic obstructive pulmonary disease COPD is due to two majorpathological processes i remodeling and narrowing ofsmall airways and ii destruction of the lung parenchymawith loss of the alveolar attachments as a result of pulmonaryemphysema [] Chronic ammation in the lung plays a 0cCanadian Respiratory Journaltherapycentral role in both the small airway remodeling and thepulmonary emphysema [] Lung volume reductionsurgery and lung transplantation while possible in endstageCOPD are restricted to just a few selected patients []httpwwwgoldcopdcom RegenerativeforCOPD includes mesenchymal stromal cell MSC or tissueengineering therapies But while bone marrow MSC oradipose tissue MSC treatments showed promising results inmice with induced emphysema [] clinical trials performedin COPD patients have been discouraging [ ] ere are alarge number of animal studies in which lung regenerationhas been successfully stimulated For instance in a rat modelof elastaseinduced emphysema administration of alltransRA ATRA stimulated alveolar regeneration [] keratinocyte growth factor KGF FGF7 administered afterpneumonectomy augmented alveolarization [] administration of HGF stimulated alveolar regeneration enhancedlung vascularization and improved exercise tolerance andgas exchange [] intratracheal administration of bFGF torats and dogs with elastaseinduced emphysema improvedalveolar dimensions and lung microvessel density [] andVEGF administration enhanced postpneumonectomy alveolar growth in mice [] But again the attempts tostimulate lung regeneration in COPD patients with emphysema with orally administered ATRA yielded no differences in computed tomography CT lung function orquality of life scores between treatment groups [ ] andRARc selective agonist administration also showed nodiï¬erences in CT scores or lung function in treated vsnontreated COPD patients [ ] However the therapeutic potential of regenerative pharmacology is still at thebeginning of its development And many authors haveshown that the human lung also in adulthood retains asignificant regenerative potential from the large to the smallairways and in terminal and respiratory bronchioles [] andthat tissue regeneration is achieved in two ways by proliferation of common diï¬erentiated cells andor by deployment of specialized stemprogenitor cells [ ]Extracorporeal shock wave therapy ESWT is applied inmany musculoskeletal diseases and in regenerative medicinebased on its capability to induce neoangiogenesis osteogenesis regeneration and remodeling through stem cellstimulation [] ESW in combination with tenogenicmedium improved the diï¬erentiation of human adiposederived stem cells hASCs into tenoblastlike cells []ESW combined with osteogenic medium increased the osteogenic diï¬erentiation of treated hASCs [] while stemcell diï¬erentiation into myoï¬broblasts was partially reducedby ESW treatment [] But to our knowledge no data areavailable on ESW treatment of primary bronchial ï¬broblastsof patients with COPD and control healthy smokers orbronchial epithelial cells 16HBEMarkers of cell proliferation include CD117 cKit orSCFR a receptor tyrosine kinase protein that binds to stemcell factor SCF expressed on hematopoietic stem cells Itcan also be expressed by mast cells melanocytes in the skininterstitial cells of Cajal in the digestive and urogenital tract[] cardiac pericytes [] amniotic ï¬uid stem cells []stemprogenitor cells in conducting airway epithelium ofporcine lung [] and dendritic cells in the lung []Another marker of cell proliferation is proliferating cellnuclear antigen PCNA It is expressed in the nuclei of cellsand is involved in DNA replication DNA repair andchromatin remodeling [ ] In the lung of COPD patients alveolar type II epithelial cells and endothelial cells[] and small airway bronchiolar epithelium [] expressdecreased PCNA levels compared with related nonCOPDcontrol groups A third marker of cell proliferation is CD90y1thymocyte diï¬erentiation antigen1 a glycophosphatidylinositol cell surface protein expressed by thymocytes CD34 cells mesenchymal stem cells endothelialcells and cardiac ï¬broblasts It is also considered a marker ofmultipotent mesenchymal stem cells when expressed inassociation with other markers CD29 CD44 CD73CD105 [ ]We aimed in this study to analyze the proliferative eï¬ectof shock waves when applied as an external challenge toprimary bronchial ï¬broblasts of COPD patients and controlsmokers and to immortalized bronchial epithelial cells16HBE To this end we investigated cell markers expression related to this proliferative stimulus Methods Ethics Statement Collection and processing of bronchialbiopsies at the Institute of Veruno NO and collection andprocessing of the peripheral lung tissues at the UniversityHospital of Orbassano during lung resection for a solitaryperipheral neoplasm were approved by the ethics andtechnical committees ofthe Istituti Clinici Scientiï¬ciMaugeri CTS p102 and San Luigi Hospital OrbassanoTO CE N Italy the study complied withthe Declaration of Helsinki and written informed consentwas obtained from each participant Cell Culture and Treatments We used the SV40 large Tantigentransformed 16HBE cellline which retains thediï¬erentiated morphology and function of normal humanbronchial epithelial cells NHBE [] and primary humanbronchial ï¬broblasts obtained from bronchial biopsies ofpatients with COPD n � and control smoking subjectsn � with normal lung function Primary bronchial ï¬broblasts were obtained from bronchial biopsies obtainedfor diï¬erent protocol studies [] Bronchial biopsies weretreated with type II collagenase min at °C InvitrogenGIBCA and cultured in DMEM until conï¬uentprimary ï¬broblasts were obtained 16HBE cells and primarybronchial ï¬broblasts were maintained in Dulbeccos modiï¬ed minimum essential medium DMEM supplementedwith vv fetal bovine serum FBS IUmL penicillin μgmL streptomycin 1x nonessential amino acids mMsodium pyruvate and mM glutamine °C CO2 []When cells were conï¬uent the complete mediumwas replaced with DMEM with FBS for starvation time h e shockwave generator utilized for the in vitroexperiments was a piezoelectric device Piezoson Richard Wolf Knittlingen Germany designed for clinical 0cCanadian Respiratory Journaluse in orthopedics and traumatology Aliquots of mL ofcell suspension adjusted to à cellsmL were placed in mm polypropylene tubes completely ï¬lled with culturemedium e shock wave unit was kept in contact with thecellcontaining tube by means of a waterï¬lled cushionCommon ultrasound gel was used as a contact mediumbetween the cushion and tube ESW treatment was as follows energy ï¬ux density EFD � mJmm2 pulsesfrequency � shockss is EFD is a mediumhigh energywe already used for previous in vitro diï¬erentiation studiesin tendons [] After treatment cell viability was evaluatedby trypan blue exclusion and primary ï¬broblasts werepassaged in DMEM complete for hours 16HBEcells were cultured for and h because of their lowerresistance to starvation T0 corresponds to hours post ESWtreatment for all experiments reported Nontreated ï¬broblasts or 16HBE cells were used as controls Cell growth wasevaluated by the colorimetric test WST1 All experimentswere performed in quadruplicate ie four independentexperiments for each type of treatment ESW or noESWand each time exposure Extraction and Quantiï¬cation of RNA and qRTPCRfrom Primary Bronchial Fibroblasts and 16HBE Total RNAfrom treated and nontreated cells was puriï¬ed and isolatedusing an RNAspin Mini RNA Isolation kit GE HealthcareLife Sciences Pittsburgh USA following the manufacturersinstructions Total RNA was resuspended in μL nucleasefree water RNA concentration was determined using aUVvisible spectrophotometer λ260280 nm EppendorfBioPhotometer plus and stored at °CQiagenQT00000728e expression of genes of interest was measured usingSYBR Green Qiagen UK for qPCR in a Corbett RotorGene Corbett Cambridge UK system Onestep realtime PCR was carried out by amplifying mRNA using theQuantiFast¢ SYBR Green RTPCR kitITaccording to the manufacturers instructions and the genespeciï¬c primers Qiagen IT We detected the expression ofcKit or SCFR CD117 Cat QT01844549 Qiagen PCNACat QT00024633 y1 CD90Cat QT00023569TGF1CatProcollagenIQT01005725 and NFκBp65 Cat QT01007370 Weperformed independent experiments and quantitative PCRmeasurements in quadruplicate for each type of treatmentESW or noESW and each time exposure Brieï¬y the PCRreaction mix prepared in a total volume of μL was run onthe Rotor Gene Q Qiagen IT and the following PCR runprotocol was used °C for min reverse transcription°C for min PCR initial activation step ampliï¬cationcycles of °C for s denaturation and °C for scombined annealingextension followed by melting curveanalysis to ensure the speciï¬city of PCR ampliï¬cationGlyceraldehyde phosphate dehydrogenase GAPDHQT01192646 Qiagen was used as the reference gene forevery target gene per sample and the data were normalizedagainst the respective GAPDH signaling Cycle thresholdCT values were determined using the Rotor Gene Qsoftware RotorGene Q Series Software eCatexpression levels of all genes studied were normalized toGAPDH levels in each sample to determine the expressionbetween treated and nontreated cells using the ÎCt method[] for primary bronchial ï¬broblasts and the ÎÎCt for16HBE cells [] ELISA Tests in the Supernatants or Cell Lysates of ESWTreated and Nontreated Cells Protein extraction andquantiï¬cation in the supernatants or cell lysates of ESWtreated and nontreated cells were performed as reported inTable Suppliers Cat Numbers dilution conditions anddetection limits of the ELISA kits used are also reported eELISA kits WST1 cell proliferation kit and MPERmammalian protein extraction kit were used according tothe manufacturers instructions Table CKit CD117PCNA and NFκBp65 were quantiï¬ed in cell lysates CD90TGF1 and procollagen1 were quantiï¬ed in the cellsupernatants Immunohistochemistry of the Lung Parenchyma of Patients with COPD Samples were frozen in liquid nitrogenprecooled is tane after embedding in OCT and used forcryostat sectioning and immunostaining of some cellproliferationrelated molecules Single immunostainings offrozen sections were performed with mouse antithyroidtranscription factor1 TTF1 sc53136 Santa Cruz rabbitanticKit CD117 ARG51826 ARGBIO and rabbit antiPCNA PAS27214 ermo Fisher primary antibodiesAntibody binding was demonstrated with secondary antibodies antimouse Vector BA and antirabbitVector BA followed by ABC kit AP AK5000VECTASTAIN and FastRed Substrate red color Doublestainings were performed using also ABC kit Elite PK6100VECTASTAIN and diaminobenzidine substrate browncolor for identiï¬cation of TTF1 positive alveolar type IIepithelial cells [] coexpressing cKit CD117 or PCNAantigens Slides were included in each staining run usinghuman tonsil nasal polyp or breast cancer as positivecontrols For the negative control slides normal nonspeciï¬cmouse or rabbit immunoglobulins Santa Cruz Biotechnology Santa Cruz CA USA were used at the same proteinconcentration as the primary antibodiesmean± standardthe unpaired ttest Probability values of p were Statistical Analysis Group data were expressed asorinterquartile range IQR for morphologichistologic dataDiï¬erences between treatment groups were analyzed usingor mediandeviationrangeconsidered significant Data analysis was performed usingthe Stat View SE Graphics program Abacus Concepts IncBerkeley CA USA Results ESW Eï¬ects on Cell Proliferation ESW treatment at adosage of mJmm2 pulses frequency � shockssof primary bronchial ï¬broblasts from COPD patients n � 0cCanadian Respiratory JournalPackyearsExsmokercurrent smokerTable Clinical characteristics of chronic obstructive pulmonary disease COPD patients and control smokers who provided bronchialï¬broblasts for in vitro experimentsSubjectsCOPD1COPD2COPD3± Mean± SD± Mean± SDIndividual and mean± standard deviation SD data M male F female FEV1 forced expiratory volume in s BD bronchodilator FVC forced vitalAge years MFMMMMMM± ± ± ± ± ± ± CurrentCurrentCurrentFEV1 postBDFEV1 preBDCurrentCurrentNDNDNDFEV1FVCCS1CS2CS3Excapacity ND not determined Patients were classiï¬ed according to the Global Initiative for Chronic Obstructive Lung disease httpgoldcopdorg levels ofseverity for COPD For COPD patients FEV1FVC are postbronchodilator values ANOVA test FEV1 p � FEV1FVC p � No significantdiï¬erences were observed for age p � and packyears p � smokedTable List of ELISA tests cell proliferation and protein extraction kits used For ELISA tests dilution of the supernatants or cell lysatesamples used and detection limits are also reportedTargetcKit or SCFR CD117PCNAy1 CD90TGF1Procollagen1NFκBp65WST1 cell proliferationMPER mammalian protein extraction ermo Scientiï¬c ngmL ngmL ngmL ngmL pgmL pgmL pgmL pgmL pgmL pgmL17pgmL pgmLCloudClone CorpCloudClone CorpCloudClone CorpCloudClone CorpCloudClone CorpSEA121 HuSEA591MiSEB404 HuSEA124 HuSEA957 HuKHO0371KA1384Dilution PBS PBSDetection limit range diluent buï¬erInvitrogenAbnovaNo dilNo dilNo dilSupplierCat ashowed a significantly increased proliferation index at and h after treatment compared with nontreated bronchial ï¬broblasts Figure 1a ESWtreated primary bronchial ï¬broblasts from control smokers with normal lungfunction n � also showed a significant increase of theproliferation index at and h aftertreatmentFigure 1b Treated bronchial epithelial cells 16HBEshowed significantly increased proliferation index values at and h after treatment when compared with nontreated16HBE cells Figure 1c ESW Eï¬ects on mRNA and Protein Levels of Cell Proliferation and Cell Remodeling Markers Primary bronchialï¬broblasts from COPD patients n � control smokersn � and human bronchial epithelial cells 16HBE werestimulated with extracorporeal shock waves at a dosage of mJmm2 pulses and compared with paired nonstimulated primary bronchial ï¬broblasts and 16HBE cellsCKit mRNA was significantly increased in ESWtreated16HBE cells at h p � and decreased in CSï¬broblasts at h p � compared with nontreated cellsFigures 2b and 2c Furthermore a tendency to increased cKit mRNA levels was observed after ESW treatment for COPDï¬broblasts Figure 2a CKit protein wassignificantly increased in the cell lysates at h after ESWtreatment in primary bronchial ï¬broblasts of COPD patientsp � Figure 2d and in 16HBE cells p at h after ESW treatment Figure 2f No significantchanges were observed for cKit protein in ESWtreatedprimary bronchial ï¬broblasts from control smokers CSbronchialï¬broblastswith normal lung function Figure 2e PCNA mRNAlevels were not significantly changed in ESWtreated ï¬broblasts and 16HBE cells when compared with nontreatedcells Figures 3a3c PCNA protein in the cell lysatesshowed a tendency to be increased in primary bronchialï¬broblasts of CS p � at h after ESW treatmentFigure 3e and a significant increase was observed at hT0 in 16HBE cells p � after ESW treatmentFigure 3f No significant changes were observed inprimaryof COPD patientsFigure 3d y1 CD90 mRNA levels were not significantly diï¬erent in ESWtreated ï¬broblasts and 16HBE cellscompared with nontreated cells Figures 4a4c y1CD90 protein in the cell supernatants was significantlyincreased in primary bronchial ï¬broblasts of CS at hp � after ESW treatment Figure 4e No significant changes were observed in primary bronchial ï¬broblastsof COPD patients or in 16HBE cells Figures 4d and 4fTGF1 mRNA levels were not significantly changed in ESWtreated ï¬broblasts and 16HBE cells when compared withnontreated cells Figures 5a5c TGF1 protein in thecell supernatants was significantly increased in primarybronchial ï¬broblasts of CS at h p � after ESWtreatment Figure 5e No significant changes were observed in primary bronchial ï¬broblasts of COPD patients orin 16HBE cells Figures 5d and 5f Procollagen1 mRNAlevels were not significantly diï¬erent in ESWtreated ï¬broblasts and 16HBE cells compared with nontreated cellsFigures 6a6c Procollagen1 protein in the cellsupernatants wasincreased in primarysignificantly 0cCanadian Respiratory JournalabIncreased cell proliferation was observed in all cellular types studied after challenge with ESW Ttestp andp Figure WST1 test for evaluation of cell proliferation after extracorporeal shock wave ESW stimulation of primary bronchial ï¬broblastsof COPD patients n � a primary bronchial ï¬broblasts of control smokers n � b and bronchial epithelial cells 16HBE ccbronchial ï¬broblasts of COPD patients at h p � after ESW treatment Figure 6d No significant changeswere observed in primary bronchial ï¬broblasts of CS or in16HBE cells Figures 6e and 6f NFκBp65 mRNAlevels were not significantly changed in ESWtreated ï¬broblasts and 16HBE cells when compared with nontreatedcells Figures 7a7c NFκBp65 protein in the cell lysates was decreased in primary bronchial ï¬broblasts ofCOPD patients at h p � after ESW treatmentFigure 7d and increased in 16HBE cells at h p � and h p � after ESW treatment Figure 7f Nosignificant changes were observed in primary bronchial ï¬broblasts of CS Figure 7e Immunohistochemistry in the Lung Parenchyma of COPDPatients of Alveolar Type II Epithelial Cells Expressing cKitand PCNA In the lung parenchyma of COPD patientsalveolar type II epithelial cells were identiï¬ed by the use ofantithyroid transcription factor1 TTF1antibodyImmunopositivity for cKit CD117 and PCNA was alsooccasionally observed in alveolar septa Figure Doublestaining used for identiï¬cation of TTF1 cells coexpressingcKitFigures 9a and 9b and PCNAFigures 9c and 9d showed that alveolar type II epithelial cells coexpressing cKit and PCNA were present eventhough rarely observedCD117 Discussionis study shows that extracorporeal shock waves induce cellproliferation of bronchial epithelial cells 16HBE and primary bronchial ï¬broblasts of COPD patients and controlsmokers As far as markers of cell proliferation are concerned cKit CD117 was increased in bronchial epitheliumat both mRNA and protein levels h after ESW treatmentand it was also increased in primary bronchial ï¬broblasts at h after ESW challenge Other markers indicative of cellproliferation were also increased PCNA protein increased inCOPDWST1NO ESWESWT24T48T72T0012345CST0T24T48T72NO ESWESWWST101234516HBET0T24T48NO ESWESWWST1012345 0cCanadian Respiratory JournaladbecfFigure CKit CD117 mRNA a b c and protein d e f expression after ESW treatment in primary bronchial ï¬broblasts of COPDpatients a d primary bronchial ï¬broblasts of control smokers b e and bronchial epithelial cells c f In bronchial epithelium 16HBEcKit increased at mRNA c and protein f levels In primary bronchial ï¬broblasts of COPD patients cKit increased at protein level d Ttest was used for comparative purposes and p values are reported in the graphsadbecfFigure Proliferating cell nuclear antigen PCNA mRNA a b c and protein d e f expression after ESW treatment in primary bronchialï¬broblasts of COPD patients a d primary bronchial ï¬broblasts of control smokers b e and bronchial epithelial cells c f In bronchialepithelium 16HBE PCNA increased at protein f level Ttest was used for comparative purposes and p values are reported in the graphsT0T24T48T7201234102030COPD CNCOPD ESWcKit CD117 Ct0180021103960767CS CNCS ESWcKit CD117 CtT0T24T48T7202461020304050002016HBE CN16HBE ESWcKit CD117 CtT0T24T480246204060800435041800320010020030006839038680037305624COPD CNCOPD ESWcKit CD117 ngmLT0 CNT0 SWT24 CNT24 SWT48 CNT48 SWT72 CNT72 SWCS CNCS ESW000500100015002000250006727038680877507636cKit CD117 ngmLT0 CNT0 SWT24 CNT24 SWT48 CNT48 SWT72 CNT72 SW16HBE CN16HBE ESW020406080P 000010791501364cKit CD117 ngmLT0 CNT0 SWT24 CNT24 SWT48 CNT48 SWT0T24T48T72PCNA Ct0005101520COPD CNCOPD ESWT0T24T48T7205101520PCNA CtCS CNCS ESWT0T24T48000510152025PCNA Ct16HBE CN16HBE ESW01450027520139305288COPD CNCOPD ESWT0 CNT0 SWT24 CNT24 SWT48 CNT48 SWT72 CNT72 SW00102030PCNA ngmL00002004006008010000512084270367304489PCNA ngmLCS CNCS ESWT0 CNT0 SWT24 CNT24 SWT48 CNT48 SWT72 CNT72 SW0123004620190109820PCNA ngmL16HBE CN16HBE ESWT0 CNT0 SWT24 CNT24 SWT48 CNT48 SW 0cCanadian Respiratory JournalacebdfFigure y1 CD90 mRNA a b c and protein d e f expression after ESW treatment in primary bronchial ï¬broblasts of COPDpatients a d primary bronchial ï¬broblasts of control smokers b e and bronchial epithelial cells c f In primary bronchial ï¬broblasts ofcontrol smokers y1 increased at protein level at and h e Ttest was used for comparative purposes and p values are reported in thegraphsbronchial epithelial cells at h after ESW challenge y1CD90 protein increased in CSprimary bronchial ï¬broblasts at and h after ESW treatment molecules morerelated to remodeling such as TGF1 protein were increased in CSprimary bronchial ï¬broblasts at h afterESW treatment and procollagen1 protein increased at hfollowed by a decrease at h in COPDprimary bronchialï¬broblasts after ESW treatment A marker of ammationtranscription factor NFκBp65 protein was decreased inCOPDprimary bronchial ï¬broblasts at h after ESWtreatment but it was increased in CSprimary bronchialï¬broblasts and in bronchial epithelial cells after ESWtreatment Markers of cell proliferation such as cKit andPCNA were observed in the peripherallung of COPDT0T24T48T72COPD CNCOPD ESWThy1 CD90 Ct0005101520CS CNCS ESWThy1 CD90 CtT0T24T48T72012316HBE CN16HBE ESWThy1 CD90 CtT0T24T4801020304009523087570853209221COPD CNCOPD ESWT0 CNT0 SWT24 CNT24 SWT48 CNT48 SWT72 CNT72 SW00200040006000800010000Thy1 CD90 pgmLCS CNCS ESW01500003150239300410T0 CNT0 SWT24 CNT24 SWT48 CNT48 SWT72 CNT72 SW000200004000060000Thy1 CD90 pgmL16HBE CN16HBE ESW035960811001447T0 CNT0 SWT24 CNT24 SWT48 CNT48 SW010203040Thy1 CD90 pgmL 0cCanadian Respiratory JournaladbecfFigure TGF1 mRNA a b c and protein d e f expression after ESW treatment in primary bronchial ï¬broblasts of COPD patients ad primary bronchial ï¬broblasts of control smokers b e and bronchial epithelial cells c f In primary bronchial ï¬broblasts of controlsmokers TGF1 increased at protein level at h e Ttest was used for comparative purposes and p values are reported in the graphsadbecfFigure Procollagen1 mRNA a b c and protein d e f expression after ESW treatment in primary bronchial ï¬broblasts of COPDpatients a d primary bronchial ï¬broblasts of control smokers b e and bronchial epithelial cells c f In primary bronchial ï¬broblasts ofCOPD patients procollagen1 increased at protein level d at h T0 followed by a decrease at h panel d Ttest was used forcomparative purposes and p values are reported in the graphsT0T24T48T72TGF 1 Ct0005101520COPD CNCOPD ESWT0T24T48T72TGF 1 Ct00051015CS CNCS ESWT0T24T48TGF 1 Ct0005101516HBE CN16HBE ESW07196046450373903445T0 CNT0 SWT24 CNT24 SWT48 CNT48 SWT72 CNT72 SWCOPD CNCOPD ESWTGF 1 pgmL005010015004487044930863500385T0 CNT0 SWT24 CNT24 SWT48 CNT48 SWT72 CNT72 SWCS CNCS ESWTGF 1 pgmL0005001000150004757089490102101199T0 CNT0 SWT24 CNT24 SWT48 CNT48 SW16HBE CN16HBE ESWTGF 1 pgmL010203040T0T24T48T72Procollanen1 Ct000510152025COPD CNCOPD ESWT0T24T48T72Procollagen1 Ct000510152025CS CNCS ESWT0T24T48Procollagen1 Ct00051015202516HBE CN16HBE ESW00220057350024202359T0 CNT0 SWT24 CNT24 SWT48 CNT48 SWT72 CNT72 SWCOPD CNCOPD ESW00100020003000Procollagen1 pgmL00541053750944605958T0 CNT0 SWT24 CNT24 SWT48 CNT48 SWT72 CNT72 SW000100002000030000Procollagen1 pgmLCS CNCS ESW010340898407490T0 CNT0 SWT24 CNT24 SWT48 CNT48 SW050100150200Procollagen1 pgmL16HBE CN16HBE ESW 0cCanadian Respiratory JournaladbecfFigure NFκBp65 mRNA a b c and protein d e f expression after ESW treatment in primary bronchial ï¬broblasts of COPD patientsa d primary bronchial ï¬broblasts of control smokers b e and bronchial epithelial cells c f In bronchial epithelium 16HBE NFκBp65 increased at protein panel f level at and h of exposure In primary bronchial ï¬broblasts of COPD patients NFκBp65 decreased atprotein level d at h In primary bronchial ï¬broblasts of control smokers NFκBp65 increased at protein level e at h Ttest wasused for comparative purposes and p values are reported in the graphspatients and both these markers were occasionally coexpressed by alveolar epithelial type II cells TTF1 in thesepatientsExtracorporeal shock wave therapy is applied in regenerative medicine since it is capable of inducing neoangiogenesis osteogenesis and remodeling through stemcell stimulation [] On the other hand while regenerativetherapy applied to mice with induced emphysema has shownpromising results [] clinical trials performed in COPDpatients were discouraging [ ] Since the human lung alsoin adulthood maintains a significant regenerative potential[] due to proliferation of diï¬erentiated of stemprogenitor cells andor by their stimulation [ ] we hereinvestigated the proliferative action of ESW at low dosage inbronchial epithelial cells and in primary bronchial ï¬broblasts of control smokers CS and patients with COPD Ourdata show that all the cell types studied significantly increased their proliferation index WST1 test after ESWtreatment in agreement with data previously obtained formuscle cells or tendon ï¬broblasts [] Interestingly thecKit CD117 receptor tyrosine kinase protein and mRNAwere increased in 16HBE cells and cKit protein also increased in primary bronchial ï¬broblasts of COPD patientsafter ESW stimulation It is not clear however if this cellresponse represents an intermediate dediï¬erentiation step ora simple proproliferative stimulus for stimulated 16HBEcells and COPDprimary bronchial ï¬broblasts Since weexposed welldiï¬erentiated cells we believe that this transitory increment may be interpreted as a proproliferativestimulus induced by ESW exposureIn bronchial epithelial cells 16HBE proliferating cellnuclear antigen PCNA considered a marker of cellproliferation was increased after ESW stimulation conï¬rming again the proproliferative role of ESW exposurefor these lung structure cells is ï¬nding in view of thedecreased PCNA levels reported in the lung of COPDpatients [ ] compared with control subjects is particularly relevant since ESW stimulation may contrastthese lower PCNA levels characterizing the damaged lungof these patientse increased y1 CD90 protein level shown afterESW exposure in CSprimary bronchial ï¬broblasts was notobserved in ESWtreated COPDprimary bronchial ï¬broblasts or in 16HBE treated cells PCNA protein alsotended to be higher in CSprimary bronchial ï¬broblastsafter ESW treatment but not in COPDprimary bronchialï¬broblasts ese diï¬erences in the response to ESWchallenge of COPD and CSprimary bronchial ï¬broblastsmay in part be due to the reduced proliferation capacity ofthese cells derived from COPD lungs as previously reported [ ] In our welldiï¬erentiated ESWexposedï¬broblasts we interpret the increment of y1 protein NFκBp65 Ct012345T24T48T72T0COPD CNCOPD ESW NFκBp65 Ct01234T24T48T72T0CS CNCS ESW NFκBp65 Ct000510152025T24T48T016HBE CN16HBE ESW00805026820020907045NFκBp65 pgmL0050000100000150000T72 CNT72 SWT24 SWT48 CNT48 SWT0 SWT24 CNT0 CNCOPD CNCOPD ESWNFκBp65 pgmL036510427702233003830020000400006000080000100000T0 SWT24 CNT24 SWT48 CNT48 SWT0 CNT72 SWT72 CNCS CNCS ESWNFκBp65 pgmL0015500002062865000100001500002004006008001000T0 SWT24 CNT24 SWT48 CNT48 SWT0 CN16HBE CN16HBE ESW 0cCanadian Respiratory JournalFigure Photomicrographs showing thyroid transcription factor1 TTF1 expression panels a b cKit CD117 c d and proliferatingcell nuclear antigen PCNA e f in the peripheral lung tissue of a representative patient with chronic obstructive pulmonary diseaseCOPD Arrows indicate positively stained cells mainly located in the alveolar septa Bars � micronsafter ESW treatmentlike that of cKitas a proproliferative stimulus induced by the treatmentWe found increased levels of secreted TGF1 inCSprimary bronchial ï¬broblasts h after ESW stimulation TGF signaling pathways are involved in the regulationof many cell functions and in the maintenance of cellularhomeostasis [] We recently reported a decrease of TGF1and TGF3 in bronchiolar epithelium and alveolar macrophages of COPD patients compared with CS [] and thisdecrease may favor the increase of autoimmunity responsesin these patients [] We speculate that the inductionthrough ESW challenge of an increase of TGF in bronchialï¬broblasts may play a role in the TGF repositioning andgain in homeostatic function of this important protein in thelungs of COPD patientsTGF induced extracellular matrix and procollagen1production has been reported in pulmonary ï¬broblasts[] even though it was also reported that the increase ofproï¬brotic markersincluding procollagen1 in humanlung ï¬broblasts may be NLRP3 ammasome dependentand TGF independent [] and associated with increasedammation ofthe lung [] We here observed aTTF a0Lung COPDCD a0Lung COPDPCNA a0Lung COPDabcdef 0cCanadian Respiratory JournalFigure Photomicrographs showing alveolar type II epithelial cells TTF1 cells red color coexpressing cKit CD117 brown color ab and PCNA brown color c d in the peripheral lung tissue of a representative patient with COPD Positive doublestained cells can berecognized in the alveolar septa even though their presence was only rarely observed Arrows indicate positively stained cells located in thealveolar septa Bars � micronstransitory increase of procollagen1 protein in COPDprimary bronchial ï¬broblasts at h after ESW | 2 |
cancer is still one of the most prevalent and highmortality diseases summing more than million deaths in This has motivated researchers to study the application of machine learningbased solutionsfor cancer detection to accelerate its diagnosis and help its prevention Among several approaches one is toautomatically classify tumor samples through their gene expression analysisMethodsstomach breast and lung To do so we have adopted a previously described methodology with which we comparethe performance of different autoencoders AEs used as a deep neural network weight initialization technique Ourexperiments consist in assessing two different approaches when training the classification model fixing theweights after pretraining the AEs or allowing finetuning of the entire network and two different strategies forembedding the AEs into the classification network namely by only importing the encoding layers or by inserting thecomplete AE We then study how varying the number of layers in the first strategy the AEs latent vector dimensionand the imputation technique in the data preprocessing step impacts the networks overall classification performanceFinally with the goal of assessing how well does this pipeline generalize we apply the same methodology to twoadditional datasets that include features extracted from images of malaria thin blood smears and breast masses cellnuclei We also discard the possibility of overfitting by using heldout test sets in the images datasetsResults The methodology attained good overall results for both RNASeq and image extracted data Weoutperformed the established baseline for all the considered datasets achieving an average F1 score of Continued on next pageCorrespondence mafaldafferreirafeuppt1Faculty of Engineering University of Porto Rua Dr Roberto Frias sn Porto Portugal2INESC TEC Institute for Systems and Computer Engineering Technology andScience Porto Portugal The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you give appropriatecredit to the original authors and the source provide a link to the Creative Commons licence and indicate if changes weremade The images or other third party material in this are included in the s Creative Commons licence unlessindicated otherwise in a credit line to the material If material is not included in the s Creative Commons licence and yourintended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtain permission directlyfrom the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40 The CreativeCommons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to the data madeavailable in this unless otherwise stated in a credit line to the data 0cFalc£o Ferreira BMC Medical Informatics and Decision Making 20Suppl Page of Continued from previous pageand and an MCC of and for the RNASeq when detecting thyroid cancer the Malaria and theWisconsin Breast Cancer data respectivelyConclusions We observed that the approach of finetuning the weights of the top layers imported from the AEreached higher results for all the presented experiences and all the considered datasets We outperformed all theprevious reported results when comparing to the established baselinesKeywords Cancer Classification Deep learning Autoencoders Gene expression analysisBackgroundCancer is a label for a group of diseases that is characterized by abnormal and continuous cell growth with thepotential to spread through its surrounding tissues andother body parts [] During cancer was the secondleading cause of death globally accountable for milliondeaths where around were in developing countries[] Throughout the years and given the evolution of techniques technology and treatments in medicine cancersurvival rates have been improving [] However there arestill some types that have survival rates of under suchas pancreatic esophagus and liver cancers Its prevalencemakes it more crucial to correctly and accurately classify such diseases For tackling this need many researchgroups have been trying to help on accelerating cancerdiagnosis by experimenting and studying the applicationof machine learning algorithms to this problem []When automatically classifying tumor samples oneapproach is to analyze the samples derived molecularinformation which is its gene expression signatures Geneexpression is the phenotypic manifestation of a gene enes by the processes of genetic transcription and translation [] By studying it this gene map can help to betterunderstand cancers molecular basis which can have adirect influence on this diseases life cycle prognosis diagnosis and treatment There are two main cancer genomicsprojects The Cancer Genome Atlas TCGA [] andThe International Cancer Genome Consortium ICGC[] that aim to translate gene expression systematizing thousands of samples across different types of cancersWith this elevated number of features each representing a particular gene one may find genomewide geneexpression assays datasets in these projects However thistype of data presents some challenges because of alow number of samples an unbalanced class distribution with few examples of healthy samples and a high potential of underlying noise and errors due toeventual technical and biological covariates [] This difficulty in gathering data accurately is underlying for everydataset creation The equipment used to collect the datahas intrinsic errors associated mechanical of acquisitionand others hence the dataset will reflect these errorsSeveral authors have chosen the previously mentionedapproach of analyzing the gene expression of tumor samples Many of the developed methodologies in this scopeuse straightforward supervised training especially whenusing deep neural networks DNNs relying on theirdepth to produce the best results Gao [] proposedDeepCC a supervised deep cancer subtype classificationframework based on deep learning of functional spectra quantifying activities of biological pathways robust tomissing data The authors conducted two studies eachwith a different cancer detection colorectal and breastcancer data The authors claimed that the describedmethod achieved overall higher sensitivity specificity andaccuracy compared with other classical machine learningmethods widely used for this kind of task namely randomforests support vector machine SVM gradient boostingmachine and multinomial logistic regression algorithmswith an accuracy higher than Sun [] proposed Genome Deep Learning GDLa methodology aiming to study the relationship betweengenomic variations and traits based on DNNs Thisstudy analyzed over six thousand samples of Whole ExonSequencing WES mutations files from different cancer types from TCGA and nearly two thousand healthyWES samples from the one thousand genomes projectsThe main goal of GDL was to distinguish cancerous fromhealthy samples The authors built models to identify each type of cancer separately a totalspecific modelable to detect healthy and cancerous samples and a mixedmodel to distinguish between all types of cancerbasedon GDL All the experiments were evaluated througha three performance metrics accuracy sensitivityand specificity and b Receiver Operating Characteristic curves with the respective Area Under the CurveROCAUC This methodology achieved a mean accuracy of on the specific models on mixturemodels and on total specific models for canceridentificationIn [] Kim compared the performances of a neural network a linear SVM a radial basisfunctionkernel SVM a knearest neighbors and arandom forest when identifying types of cancers and 0cFalc£o Ferreira BMC Medical Informatics and Decision Making 20Suppl Page of healthy tissues The classifiers were trained with RNAseq and scRNAseq data from TCGA where they selectedup to the most significant genes expressed for eachof the cancer variations To determine the optimal number of genes for each classifiers binary classification taskthe methods mentioned above were trained with different sizes of gene expression datasets from to genes When learning with genes the neural network the linear SVM and the radial basis functionkernelSVM models achieved their best performance with a witha Matthews Correlation Coefficient MCC of and respectively The knearest neighbors and random forest models achieved an MCC of and accordingly when using genes Furthermore theauthors identified classes with an accuracy of over and achieved a mean MCC of and a mean accuracy of with the neural network classifierHowever many DNNs besides the known challenges regarding their training setting [] have a highertendency to overfit which one can detect when applying the same architecture to unseen data or to a heldouttest Thus our motivation focuses on exploring unsupervised pretraining methods based on a lowerdimensionallatent representation with the usage of an autoencoderAE This approach is grounded in the hypothesis thata there is unessential information in high dimensionality datasets and b the acquisition and processing errorspotentially present in the dataset are discarded contributing to a lower probability of overfitting [] Furthermorepretraining AEs and using the learned weights as priorsof the supervised classification task not just improves themodel initialization but also often leads to better generalization and performance [] This may be one of thereasons why AEs are found to be the most predominantstrategy when analyzing RNASeq data []To support our motivation and choices we presentsome works that include unsupervised training in theirmethodologies In [] the authors designed a solution by combining a Multilayer Perceptron and StackedDenoising Autoencoder MLPSAE aiming to predicthow good genetic variants can be a factor in gene expression changes This model is composed of layers inputtwo hidden layers from the AEs and output and trained itto minimize the chosen loss function the Mean SquaredError MSE The authors started by training the AEs witha stochastic gradient descent algorithm to later use themon the multilayer perceptron training phase as weight initialization crossvalidation was used to select the bestmodel The performance of the chosen model was compared with the Lasso and Random Forest methods andevaluated on predicting gene expression values for a different dataset The authors concluded that their approach outperformed both the Lasso and Random Forest algorithms with an MSE of versus and respectively and was able to capture the change ingene expression quantificationThe authors in [] described a study of four different methods of unsupervised feature learning PrincipalComponent Analysis PCA Kernel Principal Component Analysis KPCA Denoising AE DAE and StackedDenoising AE combined with distinct sampling methods when tackling a classification task The authorsfocused on assessing how influential the input nodes areon the reconstructed data of the AEs output when feeding these combinations to a shallow artificial networktrained to distinguish papillary thyroid carcinoma fromhealthy samples The authors highlighted two differentresults in their 5fold cross validation experiment thecombination of a SMOTE [] with Tomek links and aKPCA was the one with the best overall performancewith a mean F1 score of while the usage of a DAEachieved a mean F1 score of In [] presented a stacked sparse autoencoder SSAEsemisupervised deep learning pipeline applied to cancer detection using RNASeq data By employing layerwise pretraining and a sparsity penalty this approachhelps to capture more significant information from theknown high dimensionality of RNASeq datasets usingthe filtered information to the sequent classification taskThe SSAE model was tested on three different TCGARNASeq datasets corresponding to lung stomach andbreast cancers with healthy and cancerous samplesand compared it to four others classification methodsan SVM a Random Forest a neural network supervisedlearning only and a vanilla AE The authors performed5fold cross validation and evaluated the models performance through four metrics accuracy precision recalland F1 score The results show that the semisuperviseddeep learning approach achieved superior performanceover the other considered methods with an average F1score of across the three used datasetsThe authors in [] developed a methodology for detecting papillary thyroid carcinoma They analyzed how theusage of AEs as a weight initialization method affectedthe performance of a DNN Six types of AEs were considered Basic AE Denoising AE Sparse AE DenoisingSparse AE Deep AE and Deep Sparse Denoising AEBefore being integrated into the classifier architecture allAEs were trained to minimize the reconstruction errorSubsequently they were used to initialize the weights ofthe first layers of classification neural network meaningthat the AE layers become the top layers of the wholeclassification architecture using two different strategieswhen importing the weights just the encoding layersand all the pretrained AE Moreover in the training phase the authors studied two different approacheswhen building the classifier a fixing the weights ofthe AE and b allowing subsequent finetuning of all 0cFalc£o Ferreira BMC Medical Informatics and Decision Making 20Suppl Page of the networks weights The authors used stratified 5foldcrossvalidation and evaluated the model through distinct metrics Loss Accuracy Precision Recall and F1score The authors reported that the overall best resultwas achieved through a combination of Denoising AEfollowed by its complete import into the classification network and by allowing subsequent finetuning throughsupervised training yielding an F1 score of in which the main goalIn [] the authors present a transfer learning methodologyis to explore whetherleveraging the information extracted from a large RNASeq data repository with multiple cancer types leadsto extract important latent features that can help complex and specific prediction tasks such as identifyingbreast cancer neoplasia The authors used the TCGAPanCancer dataset which is composed of approximately RNASeq gene expression examples of distincttumor types This data was split into two sets breast cancer and nonbreast cancer data The nonbreast data isfirstly used to train the three selected architectures forthis study a sparse AE a deep sparse AE and a deepsparse denoising AE models Then the breast data isused to finetune the resulting AEs After pretrainingthese models the authors aim to predict the breast tumorintrinsicsubtypes which is given by the PAM50 subtype information included in the clinical data included inthe PanCancer data The extracted features from the AEbased architectures are then fed as input to three differentmachine learning classifiers namely Logistic RegressionSupport Vector Machine and a shallow Neural NetworkTo assess the deep AEs performance as feature extractionmethods the authors compared them to other classical feature extraction methods combining them with theclassification algorithms previously mentioned ANOVAMutual Information ChiSquared and PCA A 10foldcross validation was performed and all the combinationswere compared through the accuracy metric The resultsshowed the deep sparse denoising AE performs best whenusing the AE extracted features where the combinationwith a shallow neural network leads to the best overall of ±In [] Ferreira used the same methodologydescribed in [] to discriminate different types of cancerinstead of distinguishing cancerous samples from healthyones In this case they aimed to identify thyroid skin andstomach cancer correctly Given that a Denoising AE wasthe AE that lead to the best results in previous studiesthe authors chose to single it out instead of the original The rest of the experiments remained the same strategies for importing the pretrained AE into the top layersof the classifier two approaches when training the classifier to detect different types of cancer same evaluation ofthe obtained results Although in a different domain thebest outcome was reached with a combination of the samestrategy and the same approach in the previous work []with an F1 score of when identifying thyroid cancerMethodsWe extend the previously described work in [] byassembling three different types of experiments dividedinto two main parts where we use three different AEsand five types of cancer samples In the first one we analyze the performance of a deep neural network DNNusing the same pipeline to identify different types of cancer In the second part we choose one of the used AEsto assess how the variance of its latent vector dimension impacts the essential information capture and therefore possibly influencing the classifiers performance and different data imputation strategies can influence theoverall performance in the classification task Moreoverwe study if the network architecture is correlated withits overall performance and how the model reacts whentraining with a different data type dataset We built thispipeline in Python using the Numpy [] and Pandas []packages for the data preprocessing step the Keras deeplearning library [] running on top of TensorFlow andthe ScikitLearn [] package to train and evaluate themodels and the Matplotlib [] library for visualizationAdditionally we used an NVIDIA GeForce RTX TiGPU on a Ubuntu operating systemThis section is anized as follows The data subsection describes the used data and its inherent preprocessing Autoencoders subsection overviews the AEs considered to this study Methodology subsection outlinesthe pipeline for each of the referred experiments Evaluation subsection details how we evaluate the results toprovide statistical evidence Finally Baseline subsectionpresents the established baseline results for all the useddatasetsThe dataIn our experiments we use two different types of datawhich are described in the subsections that followRNASeq dataWe used five different RNASeq datasets from The Cancer Genomes Atlas TCGA [] each representing a typeTable Five instances of the thyroid RNASeq dataset we have usedTPTEP1 AKR1C6PUBE2Q2P2 HMGB1P1 LOC155060 ZZZ3 NANANANANANANANANANA NA NA NA NA NAThe first line the header contains the genes names and the column valuesrepresent its expression samplewise except for the first column which is thesample ID NA stands for missing value for a particular gene and sample 0cFalc£o Ferreira BMC Medical Informatics and Decision Making 20Suppl Page of of cancer thyroid skin stomach breast and lung Onecan find a sample of the described data in Table The datasets were downloaded from the cBioPortal []which gathers cancerrelated data from different projectsincluding TCGA To train DNNs we need as many data aswe can get Ergo our first criterion was to choose cancertypes that had the highest number of examples Additionally we decided to gice priority to cancer types withhigh mortality and high incidence rates We use the samethyroid skin and stomach datasets presented in []alongside the lung and breast datasets The data filteringprocess in the cBioPortal comprised searching with thekeywords PanCancer sorting the obtained results fromhighest to lowest RNASeq examples and finally selectingthe thyroid skin stomach breast and lung datasetsAll five datasets are composed of approximately thousand features Each column feature in these datasetsrepresents a specific gene and the cell values for each column are the expression of that gene in a particular sampleAll the RNASeq data were normalized according to thedistribution based on all samples The expression distribution of a gene is estimated by calculating the mean andvariance of all samples with expression values and discarding zeros and nonnumeric values such as NA Nullor NaN which are substituted by NA [] With the fivedatasets we gathered examples of thyroid cancer of skin cancer of stomach cancer of breast cancer and of lung cancer We would like to emphasizethat this dataset is only a toy dataset since the data doesnot fairly reflect the immense difficulty associated withidentifying cancer in a real scenarioThe preprocessing pipeline was executed for each RNASeq dataset separately Firstly we removed the columnsthat had only one value throughout all samples Whena value is constant for all the examples there is noentropic value with no value variation one cannot inferany information In total and columns were removed on the thyroid skin stomach breast and lung datasets respectively By default weattributed the remaining missing values represented byNA in the dataset as observable in Table with the meanvalue of the column where the missing value is [] Further normalization was not applied in the data Finally weadded the Label column to link the instances to their typeof cancer when training the classifierSince we aim to distinguish several cancer variations wetest all cancers against each other assigning the positivevalue one to the class of interest and zero to the remainingones When detecting thyroid cancer all thyroid examplesare labeled as one and the skin stomach breast and lunginstances as zero and henceforwardAfter processing all the datasets it is improbable thatthe preprocessing phase removed the same columns in allof them To guarantee the same features describe all thesamples we intersect all the datasets and use the resultas our final dataset Also given that the breast cancerdatasets had almost the double of instances we applydownsampling and randomly select breast cancerexamples to keep the final dataset as evenly distributedfor all the cancers as possible In the end the resultingdataset has approximately instances and more than thousand genesData of features extracted from imagesWe use two datasets of two different diseases composedof features extracted from images malaria and breast cancer Since we aim to evaluate how well this methodologygeneralizes by using distinct types of data we are nowable to gather evidence supporting this premiseThe malaria dataset was created by the FraunhoferAICOS institution through the MalariaScope project[] Their main goal is to develop lowcost solutions thatcan provide fast reliable and accurate results on detecting such disease particularly in developing countries In[] the authors thoroughly describe the feature extraction process from thin blood smear images exclusivelyacquired with smartphones The resulting dataset is composed of samples and features These featureswere normalized between [ ] via scaling and groupedinto three main groups geometry color and textureFrom all the examples approximately contain malariaparasites Due to the high unbalance between Malariaand NonMalaria labels we performed downsampling onthe NonMalaria class where we randomly selected examples We decided to choose instead of dueto a wide variety of nonparasite artifacts Once the samples were selected and similarly to the preprocessing stepof the RNASeq data we verify if there are features withconstant values and remove them if that is the case Ourworking malaria dataset has instances negativeand positive and feature columnsThe Wisconsin Breast Cancer dataset [] from the UCIMachine Learning Repository is composed of examples and features These features are computed from afine needle aspirate digitized image of a breast mass anddescribe the cell nuclei characteristics present in thoseimages such as texture area concavity and symmetryFrom the examples approximately are benignsamples and are malign ones No under or oversampling techniques were applied since we do not find it to beneeded As performed in the malaria data we checked ifthere were columns with constant values for which therewere not The data was used as is with the proportionsand characteristics described aboveAutoencodersAn autoencoder AE [] is an unsupervised featurelearning neural network that aims to copy its input based 0cFalc£o Ferreira BMC Medical Informatics and Decision Making 20Suppl Page of on a lower dimensional representation This type of architecture is able to extract features by reducing the dimension of its hidden layer [] which helps the AE to focuson capturing the essential features that best represent thedataLet the encoding and decoding functions of the AE be fand g parameterized on θe and θd respectively where θ θeª θd L being the loss function and J the cost function tobe minimized When learning the AE aims to find value θthatargminθJθ X LX gθdfθe Xpenalizing the reconstruction of the input given by ËX fθe X the more distinct ËX is the bigger the appliedgθdpenalty When training an AE we use Mean Squared ErrorMSE as the loss function and the Rectified Linear Unitsactivation function ReLU [] for all its layers Currentlyusing ReLU as activation is the default recommendationwhen training neural networks [] Similarly using MSEas the loss function is a fairly common practice present inthe literature when training AEs [ ]We use the AEs as a weight initialization technique[] since evidence supports that using unsupervised pretraining guides the learning towards basins of attractionof minima that support better generalization from thetraining dataset [] Thus we pretrained them beforeimporting the encoding part or all their layers to theclassification neural networkBasic autoencoder AEThe simplest AE has only one hidden layer This type ofAE learns through the optimization cost function presented in Eq With the combination of linear activationsReLU and the MSE loss function these AEs behave similarly to the Principle Component Analysis PCA method when trained with an MSE an AE learns the principalsubspace of the training data consequentially []Denoising autoencoder DAEA Denoising AE DAE [] aims not just to reproduce theinput but also to keep its information intact to undo theeffect of an intentional corruption process applied to theoriginal data Its cost function can be described byFig Overall pipeline of our experiments This figure illustrates the chosen metodology for our work Firstly we pretrain the autoencoders AEsbefore embedding them to the top layers of the classification network fullfilling either Strategy import only the encoding layers from the AE orStrategy import the complete AE Each of the full assembled architectures is then trained to detect one of the cancer types in the input dataThe training process can follow two different approaches regarding the imported weights of the AEs A fixing them or B allowing subsequentfinetune I represents the input layer E the encoding layerËI the output layer of the AE at the classification region of the network D represents thefully connected layer and O the output of the classifer 0cFalc£o Ferreira BMC Medical Informatics and Decision Making 20Suppl Page of θfθe ËXJθ X LX gθdargminwhere ËX is a copy of the input X intentionally corruptedby a sort of noise [] To simulate a form of BernoulliNoise [] we apply a Dropout layer immediately after theinput layer where of the connections are randomly cutSparse autoencoderSimilarly to a DAE a Sparse AE SAE learning processalso has two main goals minimizing the reconstruction error when aiming to copy the input data and applying a sparsity penatly represented by 01 to theparameters involved in the encoding partJθ X LX gθdfθe X λ · 01θeargminθAlthough it also tries to reproduce X an SAE canaddress unique statistical features of the dataset it hasbeen trained on [ ] To deliver that sparsity elementwe use an L1 penalty with a λ of MethodologyWe have adopted the methodology described in []which was also used in [] Our experiments consist ofan analysis of the performance of a DNN trained to classify different cancer types studying how three differentfactors may impact the network performance The top layers where we use three different AEs asweight initialization The dimension of the latent vector of the AEs thatmeans the encoding layer size The imputation technique to replace missing datawhen preprocessing the datasetsBesides the top layers imported from the AE the classification part of the full architecture is composed of aBatch Normalization layer [] followed by two FullyConnected layers with a ReLU [] activation Since weaim to detect one type of cancer at the time the last layer the predictive one is a single neuron layer with aSigmoid nonlinearity [] This activation considers thatif the probability of the classification is lower than the sample is classified as negative that is not having thedisease otherwise the sample is classified as positiveTo assess the following experiments we decided to onlyuse the AE that achieved the best results in the firstexperiments For points and we try three different dimensions and For the data imputationstudy we use three strategies replacing the data witha the mean column value used as default a constantvalue in this case zero and b with the most frequentvalueFurthermore we want to study if when using Strategy importing the complete AE into the classification network the model yields better results just because it hasone more layer and therefore more parameters to trainTo observe if the classifier is better only by being deeperwe pretrained the AE and at the embedding step forStrategy we add a decoder layer with all its weightsrandomized guaranteeing that there are no discrepanciesconcerning the networks topological complexity for bothstrategiesFinally we want to assess how the pipeline behaveswhen dealing with different data types besides RNAseq entries Hence we apply the same methodologyto the image extracted features datasets described inThe data section to assess if the model can adapt andgeneralize well to these data characteristicsFor all these we follow the same pipeline see Fig Foreach experience we start by pretraining a different AE tominimize the reconstruction error before importing theminto the top of the classification architecture When doingso we choose one of the two strategies considered for thisstudy add just the encoding layers or add all thepretrained AE After the embedding of the AE to the toplayers we consider two different approaches in the training process A fixing the imported weights of the AElayers and B by allowing them to be finetuned duringthe model training for the classification taskWith the complete architectures AE as the top part ofthe classification network assembled we train each oneto distinguish¢ The RNASeq input data as one of cancers namely¢ The malaria input data as Malaria or NonMalaria¢ The breast masses input data as Malign or Benignthyroid skin stomach breast and lungEvaluationWe use stratified 10fold crossvalidation to ensure andprovide statistical evidence The AEs are trained during epochs and the classifier during with a batchsize of The classification model is trained with thebinary crossentropy loss function [] and with an Adamoptimizer [] Furthermore we assess the overall performance of the model in the training and validation setsby analyzing five more metrics Accuracy Matthews Correlation Coefficient MCC [] Precision Recall and F1score and provide the Receiving Operator Curve with therespective Area Under the Curve ROCAUC and thePrecisionRecall CurveFurthermore to study how the model generalizes tounseen data during the training phase we evaluate theperformance of the best architecture combination on aheldout test set for the Malaria and the Wisconsin BreastCancer datasets For both and separately we use a ratio ofone third to create two new splits Therefore 0cFalc£o Ferreira BMC Medical Informatics and Decision Making 20Suppl Page of Table Baseline results for cancer detection using a Fully Connected Neural Network the classification architecture without the AEas top layersThyroidSkinStom | 2 |
"At present the relationship between hypothyroidism and the risk of breast cancer is still inconclusive Thismetaanalysis was used to systematically assess the relationship between hypothyroidism and breast cancer riskand to assess whether thyroid hormone replacement therapy can increase breast cancer riskMethods The relevant s about hypothyroidism and the risk of breast cancer were obtained on the electronicdatabase platform Relevant data were extracted and odd ratios OR with corresponding confidence intervalsCI were merged using Stata SE softwareResults A total of related studies were included in the metaanalysis including cohort studies and casecontrol studies The results show that hypothyroidism was not related to the risk of breast cancer odd ratios CI In the European subgroup we observed that patients with hypothyroidism have a lower risk ofbreast cancerodd ratios CI Furthermore no significant correlation was observed betweenthyroid hormone replacement therapy and the risk of breast cancer odd ratios CI Conclusion Hypothyroidism may reduce the risk of breast cancer in the European population and no significantcorrelation was observed between hypothyroidism and breast cancer risk in nonEuropean populations Due to thelimited number of studies included more largescale highquality longterm prospective cohort studies areneededKeywords Hypothyroidism Thyroid hormone replacement therapy Breast cancer MetaanalysisBackgroundAs a global public health problem breast cancer has anincreasing incidence on a global scale [] According tothe US cancer statistics breast cancer has becomethe most common malignant tumour in women withabout new cases each year accounting for of new malignant tumours in women [] Therefore theidentification of risk factors for breast cancer and the Correspondence Yanhuangdr163com Ruobaolidr163com2Department of Oncology Affiliated Hospital of Weifang Medical UniversityWeifang China3School of Basic Medicine Weifang Medical University Weifang ChinaFull list of author information is available at the end of the adoption of effective early prevention and interventionmeasures are of great significance for patients withbreast cancerThe physiology and pathology of the breast are closelyrelated to the endocrine of the body [] As the largestendocrine an in the human body the thyroid glandhas specific regulation effects on various hormone levelsand cell growth and development in the body whichbrings new enlightenment to the research of breast cancer [] In Kapdi et alfirst proposed thathypothyroidism maybe increase the risk of breast cancer[] Since then many scholars have studied the relationship between hypothyroidism and the risk of breast The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cWang BMC Cancer Page of []cancer However the relationship between the two diseases remains controversial [] Some studies haveshown that hypothyroidism increases the risk of breastcancerthathypothyroidism reduces the risk of breast cancer []Besides some studies have found no correlation betweenthyroid disease and breast cancer risk [] Thereforewhether hypothyroidism can increase the risk of breastcancer is worthy of further studystudiesshownSomehaveTwo metaanalyses have previously been studied forhypothyroidism and breast cancer risk [ ] Based onprevious research we have included more prospectivestudies and Asian population studies to assess the relationship between hypothyroidism and breast cancer risksystematically Besides the impact of thyroid hormonereplacement therapy on breast cancer risk was exploredin this metaanalysisMethodsSearch strategyRelevant clinical literature was extracted by systematicretrieval of PubMed Medline EMBASE Springer Webof Science and Cochrane Library electronic databasesup to date to October Our search strategy includedorhypothyroidism or HT and thyroid diseases orbreast cancer or BC or breast neoplasms or mammarmy cancer and risk orincidence At the sametime we manually screened out the relevant potentialliterature in the references extracteddysfunctionthyroidtermsforInclusion and exclusion criteria The inclusion criteria Types of studies Published studies exploring therelationship between hypothyroidism and breastcancer risk Subject Female Exposure factors Primary hypothyroidism thediagnosis needs to be based on the detection ofthyroid function Outcome indicators the occurrence of primarybreast cancerThe exclusion criteria Nonprimary hypothyroidism due to other causes Non observational studiesInsufficient information was provided or no fulltext Unable to obtain full text or quality assessment ofthe literature Studies were repeated or publications overlappedData extraction and quality assessmentTwo researchers separately conducted literature screening data extraction and literature quality evaluationand any differences could be resolved through discussionor a third inspector Information secured from the enrolled literature included first authors surname year ofpublication country ofthe population sample sizefollowup time and data on the relationship betweenhypothyroidism and the risk of breast cancerThe NewcastleOttawa Scale NOS was used to assessthe quality of the study from three aspects cohort selection cohort comparability and outcome evaluation []NOS scores of at least six were considered highqualityliterature Higher NOS scores showed higher literaturequalityStatistical analysisAll data analysis was performed using Stata120 softwareMetaanalysis was performed according to the PRISMAguidelines The OR and 95CI from included studieswere treated with the combined effect size After thatthe heterogeneity test was conducted When P ¥ orI2 was performed it mean that there was no apparent heterogeneity and the fixedeffect model shouldbe applied for a merger When P or I2 ¥ indicated high heterogeneity the randomeffect model wasapplied Combined effect size if OR indicates thathypothyroidism is an unfavorable factor for breast cancer If OR is the opposite Publication bias Begg funnel plot and Egger test linear regression test were usedto research publication bias detection of the literatureincluded If P indicates obvious publication biasResultsProcess of study selection and description of qualifiedstudiesA total of studies were identified on our online databases After exclusion of duplicate references129 s were considered After screening the andtitle s were excluded After careful review ofthe full texts studies have been excluded because ofthem did not provide relevant data and s didnot have fulltext Nineteen s published between and met the inclusion criteria Fig A total of samples from studies involvingwere enrolled in this metaanalysis [ ] Sixcohort studies and casecontrol studies were includedin the study Twelve s were studied in the European population five in the North American populationand two in the Asian population All s are of highquality because of NOS score no less than The chiefcharacteristics of the enrolled materials are detailed inTable 0cWang BMC Cancer Page of Fig Flow chart of search strategy and study selectionRelationship between hypothyroidism and breast cancerriskThere were studies reported the relationship betweenhypothyroidism and breast cancer risk With obviousheterogeneity I p among these studies so a random effect model was used for assessmentThe pooled analysis suggested that was not related tothe risk of breast cancer OR CI P 0001Fig explorethefurtherrelationshipSubgroup analysis of hypothyroidism and risk of breastcancerTobetweenhypothyroidism and breast cancer risk subgroup analysis was conducted from three aspects study typepopulation distribution and followup time The resultsof subgroup analysis were shown in Table In theEuropean subgroup we observed that patients withhypothyroidism have a lower risk of breast cancer OR CI P In the subgroup witha followup date of more than four years patients withhypothyroidism can reduce the risk of breast cancerwith borderline significance OR CI In otherP found thathypothyroidism was not related to the risk of breastcancersubgroups weRelationship between thyroid hormone replacementtherapy and breast cancer riskA total of studies reported the relationship betweenthe use of thyroid hormone replacement therapy and therisk of breast cancer [ ] Asobvious heterogeneity observed the fixedeffect modelwas usedI p The result suggestedthat patients who received thyroid hormone replacementtherapy was not related to the risk of breast cancerOR CI 109P Fig Publication biasFigure 4a shows the results of publication bias for the relationship between hypothyroidism and breast cancerrisk which were evaluated by funnel plots and Eggerstest The Begg test Pr and the Egger testP were used to detecting publication bias showedthat there was no possibility of publication bias Asshown in Fig 4b there were no publication biases in the 0cWang BMC Cancer Table Main characteristics of the included studies in ouranalysisStudySampleYearRegionAdamiKalacheHoffmanBrintonMosesonSmythSheringTalaminiSimonTurkenKuijpensCristofanilliSandhuHellevikDitschGraniSøgaardWengKimSwedenUKSwedenUSACanadaIrelandIrelandItalyUSAPragueNetherlandsUSACanadaNorwegianGermanyItalyDanishUSAKoreaMedianMean ageyearsNANA ± NANA ± ± ¥ ± ¥Page of NOSFollowupyearsNANANAStudydesignCasecontrolCasecontrolCohortCasecontrolCasecontrolCasecontrolCasecontrolCasecontrolCasecontrolCasecontrolCohortCasecontrolCohortCohortCasecontrolCasecontrolCohortCasecontrolCohortStudyIDAdami Kalache Hoffman Brinton Moseson Smyth Shering Talamini Simon Turken Kuijpens Cristofanilli Sandhu Hellevik Ditsch Grani Sogaard Weng Kim Overall Isquared p ES CIES CI WeightWeightNOTE Weights are from random effects analysisFig Relationship between hypothyroidism and breast cancer risk 0cWang BMC Cancer Table Stratiedanalysis of the relationship between hypothyroidism and breast cancer riskVariableOR95CINoofstudiesPHeterogeneityI2RegionEur orth AmericaAsiaStudy designCasecontrolCohortFollowup date ¤ Page of ModelusedFixedRandomedFixedRandomedFixedFixedRandomedPhStudyIDHoffman Kuijpens Sandhu Ditsch Cristofanilli Simon Moseson Brinton Adami Weng ES CIES CI WeightWeightOverall Isquared p NOTE Weights are from random effects analysisFig Relationship between thyroid hormone replacement therapy and breast cancer risk 0cWang BMC Cancer Page of A ]rr[golB ]rh[golBegg's funnel plot with pseudo confidence limitsEgger's publication bias plotse of log[rr]Begg's funnel plot with pseudo confidence limitstceffe idezdradnatstceffi edezdradnatsprecisionEgger's publication bias plotse of log[hr]precisionFig Publication bias assessment a hypothyroidism b thyroid hormone replacement therapy Metaanalysis estimates given named study is omitted Lower CI Limit Estimate Upper CI Limit Adami Kalache Hoffman Brinton Moseson Smyth Shering Talamini Simon Turken Kuijpens Cristofanilli Sandhu Hellevik Ditsch Grani Sogaard Weng Kim Fig Sensitivity analysis for relationship between hypothyroidism and breast cancer risk 0cWang BMC Cancer Page of s on the study of thyroid hormone replacementtherapy The Egger test was P and the Begg testwas Pr Sensitivity analysisThe results of sensitivity analysis are generally stableand the primary source of heterogeneity is in the research of Cristofanilli []Fig So we excludedthe literature of Cristofanilli and analyzed the otherstudies The results revealed that the hypothyroidismcould reduce the risk of breast cancer was borderlineOR096 95CI092 P andsignificantthere was no heterogeneityI2 P cohortstudy ofDiscussionMore than years ago Beatson used thyroid extracts to treat patients with metastatic advanced breastcancer The condition was significantly alleviated sparkinginterest in exploring the relationship between thyroid andbreast cancer [] Subsequently a prospective study enrolled women and women with earlier diagnosisof hypothyroidism observed the occurrence of breast cancer during followup showed that low serum free thyroxine levels increased the risk of breast cancer [] In aprospective women withhypothyroidism and hyperthyroidism found thathypothyroidism slightly reduced the risk of breast cancer[] However a prospective cohort study of women with autoimmune hypothyroidism and women with normal thyroid function indicated that autoimmune hypothyroidism was not associated with breastcancer risk [] Besides some animal experiments alsoreflect the relationship between the two [ ] Animalexperiments by López Fontanafound thathypothyroidism mice inhibit the development of breastcancer and promote the apoptosis of breast cancer cellsdue to the low expression of βchain protein and activation of the apoptotic pathway on the tumour cell membrane [] Due to the inconsistency ofthe aboveconclusions we performed a metaanalysis to evaluate therelationship between hypothyroidism and breast cancerrisketalA total of studies were included in this metaanalysis and the results showed that patients withhypothyroidism not related to the risk of breast cancerHowever there was significant heterogeneity among theincluded studies After subgroup analysis and sensitivityanalysis we found that Cristofanillis research may causeheterogeneity [] Cristofanillis research is a retrospective study and the diagnosis of hypothyroidism patientswas based on the information recorded in the medicalrecords which may lead to the bias risk of misclassification and have a positive impact on the positive results ofthis study [] After excluding Cristofanillis researchwe found that patients with hypothyroidism had a lowerrisk of breast cancer with borderline significance [] Theresults of the metaanalysis are inconsistent with the findings of Hardefeldt and Angelousi [ ] Perhaps because our study included more prospective studiesand Asian population cohort study In addition we evaluated the risk of breast cancer in thyroid hormone replacement therapy and show that patients who received thyroidhormone replacement therapy was not related to the riskof breast cancerIn the analysis of the European population the resultsshow that hypothyroidism may reduce the risk of breastcancer We also found that patients with hypothyroidismcan reduce the risk of breast cancer was borderline significance in the subgroup with more longer followupdate However the relationship between the two was notobserved in North American and Asian populationsThe possible reasons for these disparities may be as follows First followup time may be the main contributorsto this difference A longer followup is required to demonstrate the relationship between hypothyroidism andbreast cancer risk In the metaanalysis five studies provided North American population data and two reported Asian population data However only one ofseven nonEuropean studies followup time for morethan years Second the differences may be attributedto different ethnicities sharing different genegene andgeneenvironmental backgrounds Third social and environmental factors are another critical cause for thisdifference With these in mind our findings suggest thathypothyroidism may reduce the risk of breast canceronly in the European population and more largescalehighqualitylongterm prospective cohort studies arestill needed to study on different human populationsThe following may explain the potential relationshipbetween hypothyroidism and the risk of breast cancerHealthy mammary epithelial cells can express a largenumber of T3 receptors and breast cancer cells have asimilar ability to bind to T3 [] T3 has an estrogenlike effect that promotes the growth of mammary glandlobes and stimulates normal breast tissue differentiation[ ] Therefore T3 can mimic the effect of estrogenon the proliferation of breast cancer cells When theconcentration of T3 is low in vivo it may inhibit theproliferation of breast cancer cells Hypothyroidism mayreduce the risk of breast cancer by affecting T3concentrationSome basic experiments support this theory In GonzalezSancho studied the relationship betweenT3 and breast cancer [] It was found that there is anoverexpressed T1 gene in human breast cancer cellsand T3 inhibits the proliferation of mammary epithelialcells by inhibiting the expression of cyclin D1 and T1thereby inhibiting the proliferation of breast cancer cells 0cWang BMC Cancer Page of Author details1School of Clinical Medicine Weifang Medical University Weifang China 2Department of Oncology Affiliated Hospital of Weifang MedicalUniversity Weifang China 3School of Basic Medicine WeifangMedical University Weifang ChinaReceived December Accepted July foundthat MartinezIglesias[] Afterthathypothyroidism can inhibit the growth of breast cancercells [] In Tosovic conducted a prospectivestudy of T3 levels associated with breast cancer risk andfound that T3 levels in postmenopausal women werepositively correlated with breast cancer risk in a doseresponse mannerthathypothyroidism through lower levels of T3 could reducethe incidence of breast cancer Our metaanalysis resultsalso confirm the above conjecture[] Therefore we suspectHowever this conclusion needs to be taken with caution as this study has several limitations First the studies that have been included do not adjust for importantrisk factors for breast cancer Second in subgroup analysis for example there are only two s in Asianstudies and we should be cautious about the results ofAsian analysis Third the results of this metaanalysis indicate that there is a large heterogeneity between studiesFourth followup time at different endpoints cannot beuniform Finally publication bias cannot be avoidedentirelyConclusionHypothyroidism may reduce the risk of breast cancer inthe European population and no significant correlationwas observed between hypothyroidism and breast cancerrisk in nonEuropean populations Furthermore therewas no obvious correlation between thyroid hormone replacement therapy and breast cancer risk It is necessaryto conduct a large sample size strictly controlled prospective study of hypothyroidism patients further todemonstrate the relationship between hypothyroidismand breast cancer riskAbbreviationsOR Odd ratios CI Confidence intervals NOS NewcastleOttawa ScaleAcknowledgementsNot applicableAuthors contributionsStudy design BW ZL RLYH and TL Data extraction BW ZL TL and YH Dataanalysis BW ZL RLand YH Manuscript writing BW and RL Manuscriptedition RL and YH All authors have read and approved the manuscriptFundingNo sources of funding were used to conduct this study or prepare this letterAvailability of data and materialsAll the published s and data were available onlineEthics approval and consent to participateNot applicableConsent for publicationNot applicableCompeting interestsNoneReferencesSiegel RL Miller KD Jemal A Cancer statistics CA Cancer J Clin httpsdoi103322caac21442Praestegaard C Kjaer SK Andersson M StedingJensen M Frederiksen KMellemkjaer L Risk of skin cancer following tamoxifen treatment in morethan breast cancer patients a cohort study Breast cancer httpsdoi101007s1228201506605 Mittra I Hayward JL Hypothalamicpituitarythyroid axis in breast cancerLancet httpsdoi101016s0140673674903444Adami HO Rimsten A Thoren L Vegelius J Wide L Thyroid disease andfunction in breast cancer patients and nonhospitalized controls evaluatedby determination of TSH T3 rT3 and T4 levels in serum Acta Chir ScandDargent M Berger M Lahneche B Thyroid function in patients with Cancerof the breast Acta Mustacchi P Greenspan F Thyroid supplementation for hypothyroidism Anlatrogenic cause of breast cancer JAMA Kapdi CC Wolfe JN Breast cancer Relationship to thyroid supplements forhypothyroidism JAMA httpsdoi101001jamaKuijpens JL Nyklictek I Louwman MW Weetman TA Pop VJ Coebergh JWHypothyroidism might be related to breast cancer in postmenopausalwomen Thyroid httpsdoi101089thy200515 Weng CH Chen YH Lin CH Luo X Lin TH Thyroid disorders and breastcancer risk in Asian population a nationwide populationbased casecontrolstudy in Taiwan BMJ 201883e020194 httpsdoi101136bmj 2017020194Sogaard M Farkas DK Ehrenstein V Jensen JO Dekkers OM SorensenHT Hypothyroidism and hyperthyroidism and breast cancer risk anationwide cohort study Eur J Endocrinol httpsdoi101530EJE150989 Angelousi AG Anagnostou VK Stamatakos MK Geiopoulos GAKontzoglou KC Mechanisms in endocrinology primary HT and risk forbreast cancer a systematic review and metaanalysis Eur J Endocrinol httpsdoi101530EJE110838 Hardefeldt PJ Eslick GD Edirimanne S Benign thyroid disease is associatedwith breast cancer a metaanalysis Breast Cancer Res Treat httpsdoi101007s1054901220193Stang A Critical evaluation of the NewcastleOttawa scale for theassessment of the quality of nonrandomized studies in metaanalyses Eur JEpidemiol httpsdoi101007s106540109491zKalache A Vessey MP McPherson K Thyroid disease and breast cancerfindings in a large casecontrol study Br J Surg httpsdoi101002bjs1800690731 Hoffman DA McConahey WM Brinton LA Fraumeni JF Jr Breast cancer inhypothyroid women using thyroid supplements JAMA Brinton LA Hoffman DA Hoover R Fraumeni JF Jr Relationship of thyroiddisease and use of thyroid supplements to breast cancer risk J Chronic Dis httpsdoi1010160021968184900626 Moseson M Koenig KL Shore RE Pasternack BS The influence of medicalconditions associated with hormones on the risk of breast cancer Int JEpidemiol httpsdoi101093ije2261000Shering SG Zbar AP Moriarty M McDermott EW O'Higgins NJ Smyth PPThyroid disorders and breast cancer Eur J Cancer Prevent Smyth PP Smith DF McDermott EW Murray MJ Geraghty JG O'Higgins NJA direct relationship between thyroid enlargement and breast cancer J ClinEndocrinol Metab httpsdoi101210jcem813Talamini R Franceschi S Favero A Negri E Parazzini F La Vecchia CSelected medical conditions and risk of breast cancer Br J Cancer httpsdoi101038bjc1997289 0cWang BMC Cancer Page of Simon MS Tang MT Bernstein L Norman SA Weiss L Burkman RT DalingJR Deapen D Folger SG Malone K Marchbanks PA McDonald JA Strom BLWilson HG Spirtas R Do thyroid disorders increase the risk of breast cancerCancer Epidemiol Biomarkers Prevent Turken O NarIn Y DemIrbas S Onde ME Sayan O KandemIr EG Yaylac IMOzturk A Breast cancer in association with thyroid disorders Breast CancerRes 200355R110 httpsdoi101186bcr609 Cristofanilli M Yamamura Y Kau SW Bevers T Strom S Patangan M Hsu LKrishnamurthy S Theriault RL Hortobagyi GN Thyroid hormone and breastcarcinoma Primary hypothyroidism is associated with a reduced incidenceof primary breast carcinoma Cancer httpsdoi101002cncr20881 Hellevik LR Vierendeels J Kiserud T Stergiopulos N Irgens F Dick ERiemslagh K Verdonck P An assessment of ductus venosus tapering andwave transmission from the fetal heart Biomech Model Mechanobiol httpsdoi101007s1023700901554Sandhu MK BrezdenMasley C Lipscombe LL Zagorski B Booth GLAutoimmune hypothyroidism and breast cancer in the elderly BreastCancer Res Treat httpsdoi101007s10549008 Ditsch N Liebhardt S Von Koch F Lenhard M Vogeser M Spitzweg CGallwas J Toth B Thyroid function in breast cancer patients Anticancer Res Grani G Dicorato P Dainelli M Coletta I Calvanese A Del Sordo M DeCesare A Di Matteo FM D'Andrea V Fumarola A Thyroid diseases inwomen with breast cancer La Clin Terapeut 20121636e401Kim EY Chang Y Lee KH Yun JS Park YL Park CH Ahn J Shin H Ryu SSerum concentration of thyroid hormones in abnormal and euthyroidranges and breast cancer risk a cohort study Int J Cancer httpsdoi101002ijc32283 Beatson GT On The Treatment Of Inoperable Cases Of Carcinoma Of TheMamma Suggestions For A New Method Of Treatment With IllustrativeCases1 Lancet Lopez Fontana CM Zyla LE Santiano FE Sasso CV CuelloCarrion FDPistone Creydt V Fanelli MA Caron RW Hypothyroidism reduces mammarytumor progression via Betacateninactivated intrinsic apoptotic pathway inrats Histochem Cell Biol httpsdoi101007s004180171544x MartinezIglesias O GarciaSilva S Regadera J Aranda A Hypothyroidismenhances tumor invasiveness and metastasis development PLoS One 47e6428 httpsdoi101371journalpone0006428 Nogueira CR Brentani MM Triiodothyronine mimics the effects of estrogenin breast cancer cell lines J Steroid Biochem Mol Biol httpsdoi101016s0960076096001173 Alyusuf RH Matouq JA Taha S Wazir JF The pattern of expression and roleof triiodothyronine T3 receptors and type I ²deiodinase in breastcarcinomas benign breast diseases lactational change and normal breastepithelium Appl Immunohistochem Mol Morphol httpsdoi101097PAI0b013e3182a20917 Pereira B Rosa LF Safi DA Bechara EJ Curi R Control of superoxidedismutase catalase and glutathione peroxidase activities in rat lymphoidans by thyroid hormones J Endocrinol httpsdoi101677joe01400073 GonzalezSancho JM Figueroa A LopezBarahona M Lopez E Beug HMunoz A Inhibition of proliferation and expression of T1 and cyclin D1genes by thyroid hormone in mammary epithelial cells Mol Carcinog httpsdoi101002mc10046Tosovic A Bondeson AG Bondeson L Ericsson UB Malm J Manjer JProspectively measured triiodothyronine levels are positively associatedwith breast cancer risk in postmenopausal women Breast Cancer Res 123R33 httpsdoi101186bcr2587Publishers NoteSpringer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations 0c" | 2 |
"Accumulating evidence has revealed the critical role of long noncoding RNAs lncRNAs in cellularprocesses during tumor progression As documented in cancerrelated literatures LINC00992 expression isassociated with cancer progression whereas its function in tumors including prostate cancer has not beencharacterized yetMethods Data from GEPIA database suggested LINC00992 expression in prostate cancer tissues The expressionlevels of RNAs were monitored via qRTPCR Western blot evaluated the levels of proteins The proliferationapoptosis and migration of prostate cancer cells were assessed by CCK8 EdU TUNEL Transwell and woundhealing assays Luciferase reporter RNA pull down and RIP assays were applied to detect the interplays amongLINC00992 miR3935 and GOLM1Results Elevated levels of LINC00992 and GOLM1 were detected in prostate cancer tissues and cells LINC00992exerted facilitating functions in prostate cancer cell proliferation and migration Mechanically LINC00992 interactedwith and negatively regulated miR3935 to elevate GOLM1 expression in prostate cancer cells In addition thein vitro suppressive effect of silenced LINC00992 on prostate cancer cell proliferation and migration was reversed byGOLM1 upregulation Likewise LINC00992 depletion restrained tumor growth in vivo was offset by enhancedGOLM1 expressionConclusions LINC00992 competitively bound with miR3935 to elevate GOLM1 expression and therefore facilitatethe oncogenic phenotypes of prostate cancer cells implying a potential LINC00992targeted therapy for prostatecancerKeywords INC00992 miR3935 GOLM1 Prostate cancer Correspondence engineyangsinacom5Department of Urology the Second Affiliated Hospital of Bengbu MedicalCollege Hongye Road Bengbu Anhui ChinaFull list of author information is available at the end of the The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cChen BMC Cancer Page of BackgroundClinically prostate cancer manifests as a dominatingcause of malerelated death worldwide and is characterized as the most continually occurred tumor amongmen in the United States [] The biggest challenge isthe detectable bone metastases in roughly advancedprostate cancer [] Virtually all prostate cancer patientsduring years androgen deprivation treatment inevitably undergo castrationresistance which contributes tothe poor clinical consequences in prostate cancer []However the mechanism underlaid prostate cancer remains mostly unknownThe widely studied long noncoding RNAs lncRNAsare transcribed from nonproteincoding human genomeand have more than nt in length [] LncRNAs are increasingly functionally identified and experimentally consolidated to be related to tumor neoplasia and progressionin diverse cancers [] Additionally lncRNAs with dysregulation can functionally modulate tumor developmentfrom multiple pathological aspects such as cell proliferation drugresistance and metastasis [] For examplelncRNA A1BGAS1 inhibits cell proliferation and invasionin hepatocellular carcinoma via targeting miR216a5p []LncRNA LOC730100 sponges miR760 from FOXA1 toaccelerate cell proliferation and invasion in glioma []LncRNA SNHG16 functions as an oncogene in hepatocellular carcinoma [] Long intergenic nonprotein codingRNA LINC00992 is a novel lncRNA that has beenpreviously revealed to be elevated in tumors and substantiated as a master regulator for chemoresistance [] Besides LINC00992 has been uncovered as an elevatedlncRNA in prostate cancer [] which is consistent withthe detection from GEPIA database Despite that no previous study has given a comprehensive explanation aboutthe precise function or detailed mechanism of LINC00992in prostate cancerIn past decades the fact that lncRNAs function in tumors depending on their secondary or tertiary structureshas been reported in many cancerlinked studies For instance in the nucleus lncRNAs are entitled to work asmolecular scaffolds or alternative splicing assistants [] On the contrary lncRNAs dispersing in cytoplasm influence downstream mRNA translation or degradationthrough serving as miRNA sponges [ ] For exampleTNFαinduced lncRNA LOC105374902 promotes themalignant behaviors of cervical cancer cells by acting as asponge of miR12853p [] LncRNA TTNAS1 promotes papillary thyroid cancer tumorigenesis by regulatingmiR1533pZNRF2 axis[] Nevertheless whetherLINC00992 could exert its functions in prostate cancer viaits sponging role of certain miRNA remains unknownWe conducted this research aiming to explore thefunction or probable mechanism of LINC00992 in prostate tumor which might enrich the understanding interms of prostate tumor pathology and contribute to awider applied scopeMethodsTissue samplesThe prostate cancer tissue samples and matched peritumor tissue samples were collected from patientsdiagnosed with prostate cancer under the approval ofthe Ethics Committee of the First Affiliated Hospital ofKunming Medical University Each participant did notreceive radiotherapy and chemotherapy prior to tissuecollection and signed the written informed consentsbefore this study All samples were snapfrozen in liquidnitrogen and then stored at °C until required forfurther analysisCell cultureThe prostate epithelial cell line RWPE1 CRL11609and prostate cancer cellsincluding PC3 CRL1435LNCaP CRL1740 C4 CRL3314 and DU145HTB81 were all purchased from American TypeCulture Collection ATCC Manassas VA USAinOctober All cells were cultured as recommendedin Dulbeccos modified Eagles medium containing FBS GIBCO MA USA under the condition of a cellincubator with CO2 at °C Before using in thisstudy all cell lines were authenticated by STR profilingand tested for mycoplasma contamination in June Cell transfectionLINC00992 shRNA or negative control shRNA andpcDNA31LINC00992 pcDNA31GOLM1 or its emptycontrol pcDNA31 plasmid were chemically synthesizedand provided by Gene Pharma Shanghai China MiR mimics miR3935 inhibitor and theirrelatednegative controls NCmimics NCinhibitor were allpurchased for upregulating or downregulating miR3935from Ribobio Guangzhou China In line with the directions of LipofectamineTM RNAiMAX TransfectionReagent Thermo Fisher Scientific transfection of theseplasmids into DU145 PC3 and RWPE1 cells wasconducted and qRTPCR checked the transfection efficiency The sequences were as follows shNC ²CCGGTAGTAATTGACAACCATTATACTCGAGTATAATGGTTGTCAATTACTATTTTTG3²shLINC009921²CCGGATTATCCAAGAGTATTAACATCTCGAGATGTTAATACTCTTGGATAATTTTTTG3² shLINC0 ²CCGGTGTTAGATGATCATTGAGGTGCTCGAGCACCTCAATGATCATCTAACATTTTTG3² s²CCGGTTACCTAATCAGTAGAThLINC009923GCAGCTCGAGCTGCATCTACTGATTAGGTAATTTTTG3² NCmimics ²UCAGGUAGGGCUCAAACCAACC3² miR3935 mimics ²UGUAGAUACGAGCACCAGCCAC3² NCinhibitor²CUGGCUUUAG 0cChen BMC Cancer Page of GGUGCCACUUAG3² miR3935 inhibitor ²GUGGCUGGUGCUCGUAUCUACA3²Quantitative realtime PCR qRTPCROn the basis of the instructions of Trizol reagent Invitrogen USA RNA extraction was executed in prostatecancer cells After the examination of RNA purity withspectrophotometry cDNA was obtained from aboveRNA with reverse transcription kit ThermoFisher Scientific shanghai China qRTPCR analysiswas devised with the aid of a BioRad CFX96 system andSYBR green was applied for investigating the RNA levelsThe internal reference for LINC00992 and mRNAs wasGAPDH whereas that for miRNAs expression was U6Relative expression was assessed based on the method ofÎÎCtab97779Western blotProtein content in cells was determined by western blotanalysis RIPA lysis buffer Beyotime Shanghai Chinawas adopted for cell lysing followed by the evaluation ofthe protein concentration with BCA Protein Assay KitP0011 Beyotime Tech SDSPAGE gel was applied for separating proteins μg protein per sampleand then proteins were transferred onto μm PVDFmembranes BioRad Hercules CA USA Antibodiesincluding antiGOLM1 ab109628 AbcamCambridge UK antiPCNA ab92552 AbcamantiCDK2 ab32147 Abcam antiCyclin D1ab40754 Abcam antiBax ab32503 Abcam antiBcl2 ab32124 Abcam antiMMP2antiMMP9ab38898 Abcam antipSrc ab40660 Abcam antiSrc ab47405 Abcam antipFAKab81298 Abcam antiFAK ab131435 Abcam antiGAPDH ab8245 Abcam andantiTubulin ab7291 Abcam were applied toprobe the membranes overnight at °C After that themembranes were further incubated for h with HRPconjugated secondary antibody Santa Cruz Co LtdSant Cruz CA USA atroom temperature ECLSubstrates Millipore Billerica MA USA was utilizedfor the visualization of signals followed by exposure toXfilm Kodak Rochester NY USA The quantificationof immunoblots was conducted with the aid of imageJsoftware National Institute of Health Bethesda MDUSA with GAPDH or Tubulin as the normalizer asneeded AbcamLuciferase reporter assayFragments of fulllength LINC00992 with wildtype ormutant binding sites for miR3935 and sequences ofGOLM1 UTR containing wildtype or mutated miR binding sites were inserted into the pmirGLOvectors Promega Madison WI USA for the construction of reporters LINC00992WT LINC00992MUTGOLM1WT GOLM1MUT Then the four reportersand miR3935 mimics or miR3935 inhibitor GenePharma were cotransfected into DU145 and PC3 cellsapplying lipofectamine2000 Invitrogen as neededFortyeight hours later DualLuciferase Reporter AssaySystem Promega was employed for the examination ofthe luciferase activity GloMax® Discover Multimode Microplate Reader Promega assessed the ratio of FireflyRenilla luciferase activity and the activity of Renilla wasthe normalized controlRNA immunoprecipitation RIP assayAccording to the direction for usage of Magna RIP¢RNA Binding Protein Immunoprecipitation Kit Millipore RIP assay was strictly performed RIP lysisbuffer was firstly applied to treat the transfected DU145and PC3 cells Afterwards the obtained cell lysates wereprocessed with magnetic beads integrated with humanantiAgo2 antibodies ab32381 Abcam MA USA orantiIgG AP162KC Millipore Following the recoveryof antibody by the protein AG beads qRTPCR detected the levels of LINC00992 miR3935 and GOLM1mRNA in the precipitates IgG worked as the negativecontrol for the normalization of RNAIPsRNA isolation of nuclear and cytoplasmic fractionsThe dispersion of LINC00992 in the prostate cancercells was assayed as described previously [] The isolation of cytosolic and nuclear sections was executed followingAM1921Invitrogen RNA levels of U1 nuclear control GAPDHcytoplasmic control and LINC00992 were all estimatedby qRTPCR analysisPARIS¢ KittheprotocolofFluorescence in situ hybridization FISH assayIn line with the recommendation of Ribo¢ FISH KitC10910 Ribobio Guangzhou China FISH analysiswas implemented for testing the presence of LINC00992in prostate tumor cells Ribobio Company synthesizedthe LINC00992 probes labeled by Cy3 fluorescent dyeFollowing the fixation by paraformaldehyde and Triton X100 permeabilization DU145 and PC3 cellswere subsequently blocked in prehybridization bufferblocking solution Then incubation of cells with probehybridization buffer was later performed Next day afterrinsing and Hoechst staining the fluorescence was measured under a confocal laser scanning microscope ZeissGermanyCell counting kit8 CCK8 assayFor the viability assessment in DU145 PC3 and RWPE cells CCK8 assay was implemented as described 0cChen BMC Cancer Page of previously [] Cell viability was monitored at and h In short after being seeded onto 96well platesand cultured for indicated times cells were processedwith μl of CCK8 solution Then a microplate readerexamined the absorbance values at the wavelength of nm²ethynyl2²deoxyuridine EdU incorporation assayCell proliferation was examined through EdU assay asdescribed previously [] by using ClickiT EdU AlexaFluor Imaging Kit C10086 Invitrogen After hoftransfection EdU staining was carried out asinstructed The observation and calculation of EdUpositive cells was proceeded under the fluorescencemicroscopyTransferasemediated dUTP nick end labeling TUNELstainingTUNEL assay was carried out as described previously[] for probing DU145 and PC3 cell apoptosis with theassistance of an In Situ Cell Death Detection Kit Roche Mannheim Germany TUNELpositivecells were recorded under a light microscope à from visual fields which were chosen at randomTranswell migration assayThe application of transwell chambers with pore size μm Corning Costar Cambridge MA USA was aimedfor detecting cell migration in strict line with the instructions Cells that were previously suspended inserumfree RPMI1640 media were seeded into theupper chamber RPMI1640 medium containing FBS was supplemented in lower chamber as a chemoattractant Cells in the filters following h incubationwere immobilized in methanol and went through crystal violet staining The images of cells migratedthrough the filters were obtained and counted under themicroscopeWound healing assayThe DU145 PC3 and RWPE1 cells à cellswellwere prepared on glass culture dishes and cultivated at °C for a whole night to allow cells adhered to theplates followed by the straight scratch made with a plastic pipette tip after cell samples reached confluenceLater cells were rinsed in PBS to clear the detachedcells Finally the wounds at and h were imaged viaa light microscopy Olympus Tokyo JapanIn vivo experimentSixteen sixweekold male BALBC athymic nude micewere commercially available from the National Laboratory Animal Center Beijing China and maintained inSPFgrade animal lab All animalrelated protocols wereapproved by the Animal Research Ethics Committee ofthe First Affiliated Hospital of Kunming Medical University The in vivo experiment was undertaken via subcutaneous injection of à DU145 cells into the nudemice while the DU145 cells injected into indicated fourshgroups of mice were transfected with shNCLINC009921shLINC009921 pcDNA31 orshLINC009921 pcDNA31GOLM1 Tumor volume wasmonitored every days 28day after injection nudemice were sacrificed via cervical dislocation and thentumor samples were carefully dissected for weight assessment and hematoxylin and eosin HE stainingImmunohistochemistry IHCThe tumor samples collected from in vivo experimentswere treated with PFA dehydrated and embedded inparaffin Afterwardsthe paraffinembedded sections μm were prepared for IHC assay as described previously [] by use of the antiKi67 and antiPCNA antibodies AbcamStatistical analysisSPSS statistical software SPSS Armonk NY USAwas employed in the processing of data from threebiological replicates and data were expressed as mean ±SD Significance of difference within two groups wasdetermined using Students ttest while that among noless than two groups was tested via oneway or twowayANOVA P was considered as the threshold ofsignificanceResultsLINC00992 is overexpressed in prostate cancer andregulates cell proliferation apoptosis and migrationLINC00992 expression pattern in prostate cancer wasacquired from online GEPIA database As a resultLINC00992 was considerably upregulated in PRADprostate adenocarcinomatissues relative to normalones Fig 1a After detecting LINC00992 expression intissue samples obtained from patients with prostate cancer we observed that LINC00992 expression was higherin prostate cancer tissues than that in peritumor tissuesFigure S1A Moreover clinical data showed that higherexpression of LINC00992 in prostate cancer patientswas associated with lower survival rate Figure S1BFurthermore LINC00992 expression in the prostate cancer cells and RWPE1 cells was evaluated by qRTPCRConsequently higher level of LINC00992 was exhibitedin prostate cancer cells than that in RWPE1 cells Fig1b which was completely consistent with the result presented in previous discovery [] Particularly DU145and PC3 cells expressed the highest level of LINC00992and was thereby chosen for the later assays For silencingLINC00992 special shRNAs targeting LINC00992 was 0cChen BMC Cancer Page of Fig LINC00992 was overexpressed in prostate cancer and regulates cell proliferation apoptosis and migration a GEPIA database demonstratedthe overexpression of LINC00992 in tumor tissues in contrast to adjacent normal ones b LINC00992 expression was detected by qRTPCR in fourprostate cancer cell lines and control RWPE1 cells c LINC00992 expression was monitored by qRTPCR in DU145 and PC3 cells after transfectionwith shRNAs targeting LINC00992 shNC was used as the negative control d The viability of DU145 and PC3 cells was estimated through CCK8assay following LINC00992 depletion e The proliferation of DU145 and PC3 cells was investigated after LINC00992 depletion via EdU assay Scalebar μm f The apoptosis of DU145 and PC3 cells transfected with shLINC0099212 or shNC was estimated via TUNEL assay Scale bar μm g Western blot analysis was applied to examine the expression of apoptosisrelated proteins hi The migration of DU145 and PC3 cellswas analyzed via Transwell migration assay scale bar μm and wound healing assay scale bar μm after inhibiting LINC00992expression The fulllength images for blots in Fig 1g were presented in Supplementary figure P p transfected into DU145 and PC3 cells and the efficiencywas corroborated in qRTPCR Fig 1c And then thedata from CCK8 assay revealed that LINC00992 depletion suppressed the proliferation of DU145 and PC3cells Fig 1d As expected a declined proportion ofEdU positive cells was observed after knocking downLINC00992 Fig 1e suggesting the suppressive effect ofLINC00992 deficiency on prostate cancer cell proliferation Additionally the expression levels of proliferationrelated proteins PCNA CDK2 and Cyclin D1 were allreduced by silenced LINC00992 Figure S1C On thecontrary TUNEL assay uncovered that LINC00992knockdown facilitated cell apoptosis Fig 1f Meanwhile western blot analysis revealed that LINC00992knockdown promoted the apoptosis of DU145 and PC3cells as Bax protein level was increased whereas Bcl2protein level was decreased after LINC00992 was silenced in these two cells Figs 1g Figure S1D FurtherTranswell and wound healing assays indicated that themigration of DU145 and PC3 cells was retarded byLINC00992 depletion Fig 1hi Likewise the expression of migrationrelated molecular markers MMP2MMP9 pSrc and pFAK was decreased by LINC00992inhibition Figure S1E To further verify the biological 0cChen BMC Cancer Page of role of LINC00992 in prostate cancer we carried outgainoffunction assays in RWPE1 cells After overexpressing LINC00992 in RWPE1 cells Figure S2A cellproliferation was promoted Figure S2BC As expectedthe expression of PCNA CDK2 and Cyclin D1 wasdecreased by upregulation of LINC00992 Figure S2DSimilarly LINC00992cellIn addition upregulatingmigration Figure S2EFLINC00992 resulted in the elevated protein levels ofMMP2 MMP9 pSrc and pFAK Figure S2G All thesedata elucidated that LINC00992 could facilitate cell proliferation and migration whereas suppress cell apoptosisin prostate cancerupregulationfacilitatedMiR3935 is targeted by LINC00992Given the high correlation of the sublocalization ofLINC00992 with its functional mechanism the predication of LINC00992 presence in cells was performed viaLncLocatorhttpwwwcsbiosjtueducnbioinflncLocator Result predicted that LINC00992 located mainlyin cytoplasm Fig 2a Likewise FISH assay and RNAisolation of nuclear and cytoplasmic fractions furtherverified the abundance of LINC00992 in the cytoplasmof prostate cancer cells Fig 2bc highlighting a posttranscriptional control of LINC00992 in such cellsHence we speculated that LINC00992 might act as aceRNA in prostate cancer regulation According toDIANAlncBase the top three potential miRNAs possessing the binding capacity with LINC00992 were listedFig 2d To targetthe highlymatched miRNA toLINC00992 qRTPCR analysis was conducted to testthe expression changes of these miRNAs following eitherLINC00992 depletion or augmentation The resultsdemonstrated that only miR3935 expression was increased by LINC00992 depletion Fig 2e but reducedby LINC00992 overexpression in the meantime Fig 2fThus miR3935 was chosen for further analysis Afterwards RNA pull down assay was implemented and theresult depicted that LINC00992 was pulled down byBiomiR3935WT Fig 2g which indicated the bindingof LINC00992 and miR3935 Later we observed thesatisfactory efficiency of miR3935 overexpression andmiR3935 inhibition through qRTPCR analysis Fig2h Thereafter RIP assay applying antiAgo2 was executed Results illustrated that LINC00992 and miR3935were highly enriched in antiAgo2 group in comparisonwith control antibody Fig 2i certifying the associationof LINC00992 with miR3935 in the RNAinduced silencing complexes RISCs To further explore the interaction between LINC00992 and miR3935 the bindingsites between LINC00992 and miR3935 were predictedat first and then data from luciferase reporter assayrevealed that miR3935 upregulation decreased theluciferase activity of LINC00992WT reporter whereasmiR3935 inhibition increased the luciferase activity ofLINC00992WT reporter Fig 2j Altogether LINC0 combined with miR3935 to act as a miRNA decoyin prostate cancerLINC00992 regulates the expression of GOLM1 a targetof miR3935Present evidence has suggested that miRNAs can bindwith downstream target genes to inhibit their expressionHerein we searched the miR3935 target genes andeight mRNAs were found out Subsequently we detectedtheir expression in prostate cancer cells and normalcells Interestingly we found that only Golgi membraneprotein GOLM1 was highly expressed in four prostate cancer cell lines relative to normal controls Fig 3aFurther we discovered that GOLM1 expression wasmarkedly upregulated in prostate cancer tissues according to data from GEPIA database Fig 3b SimilarlyGOLM1 expression was much higher in prostate cancertissue samples than in peritumor samples Figure S3AIn addition the mRNA and protein levels of GOLM1were overexpressed in prostate cancer cells in contrastto RWPE1 cells Fig 3c Figure S3B Besides GOLM1has been previously revealed as a prostate cancer facilitator and was metastasisrelated in prostate tumor [] Thus we hypothesized that GOLM1 might act asthe downstream of LINC00992miR3935 signaling inprostate cancer Through TargetScan httpwwwtargetscanvert_72 the binding site between GOLM1and miR3935 was predicted Fig 3d After conductingluciferase reporter assay in DU145 and PC3 cells weobserved that upregulation of miR3935 specifically decreased the luciferase activity of GOLM1WT reporterFig 3e confirming the interaction between miR3935and GOLM1 relied on the putative binding sites Thenwe unveiled that GOLM1 mRNA and protein levels wereboth reduced by LINC00992 inhibition or miR3935upregulation according to qRTPCR and western blotanalyses Fig 3fg Figure S3C Moreover data fromRIP assay unveiled the binding of miR3935 to GOLM1in the RISCs Fig 3h Further we demonstrated thatthe decreased mRNA and protein levels of GOLM1 induced by LINC00992 depletion could be restored afterinhibiting miR3935 expression Fig 3ij Figure S3DAll the results showed that LINC00992 upregulatedGOLM1 expression via directly binding to miR3935LINC00992 promotes prostate cancer cell proliferationand migration via elevating GOLM1 expressionTo test whether LINC00992 affected prostate cancer cellproliferation apoptosis and migration via regulatingmiR3935targeted GOLM1 we executed the rescue experiments with the upregulation of GOLM1 To beginwiththe efficiency of overexpressing GOLM1 was 0cChen BMC Cancer Page of Fig MiR3935 was targeted by LINC00992 a LncLocator predicted LINC00992 subcellular location b FISH analysis of LINC00992 distribution inprostate cancer cells Scale bar μm c RNA isolation of nuclear and cytoplasmic fractions assayed the subcellular distribution of LIN00992 inprostate cancer cells d Top three miRNAs which might interact with LINC00992 were predicted by DIANAlncBase e After transfection ofLINC00992silencing plasmids the expression of miR31575p miR11783p and miR3935 was examined via qRTPCR f Following LINC00992upregulation qRTPCR tested the levels of miR31575p miR11783p and miR3935 in DU145 and PC3 cells g RNA pull down assay wasimplemented to testify the binding capacity between LINC00992 and miR3935 h miR3935 overexpression efficiency and inhibition efficiencywere examined by qRTPCR i RIP assay disclosed the binding of miR3935 to LINC00992 in the antiAgo2 group j The potential binding sitebetween LINC00992 and miR3935 was shown And the luciferase activity of LINC00992WT or LINC00992MUT reporter was assessed vialuciferase reporter assay in DU145 and PC3 cells after transfection with miR3935mimics miR3935inhibitor NCinhibitor or NCmimics P p p analyzed through qRTPCR and western blot analysesand the outcome turned out to be satisfactory Fig 4abFigure S3E Then we observed that overexpression ofGOLM1 could significantly elevate the mRNA and protein expression of GOLM1 in shLINC009921transfected cells Figure S3F Afterwards data from CCK8revealed that the viability of DU145 cells was firstly hindered due to LINC00992 depletion while subsequentGOLM1 elevation reversed the inhibitory trend onDU145 cell viability Fig 4c Results from EdU assayalso exposed similar trends that GOLM1 upregulationimpactposedthesuppressivecountervailedbyLINC00992 downregulation on DU145 cell proliferationFig 4d Similarly the restraining effect of silencedLINC00992 on the expression of proliferationrelatedproteins could be reversed by GOLM1 upregulationFigure S3G Later TUNEL assay revealed that cellapoptosis rate was elevated by LINC00992 depletion andthen overexpressing GOLM1 reduced the increasedapoptosis rate of LINC00992depleted cells Fig 4eLikewise western blot analysis uncovered that overexpressing GOLM1 could offset the effect of LINC00992 0cChen BMC Cancer Page of Fig LINC00992 regulated the expression of GOLM1 a target of miR3935 a The expression of eight mRNAs in four prostate cancer cell linesand RWPE1 cells was detected by qRTPCR b GOLM1 was overexpressed in prostate cancer tissues according to GEPIA database c The mRNAand protein levels of GOLM1 were evaluated in prostate cancer cell lines and RWPE1 cell line by qRTPCR and western blot respectively d Thebinding sites between GOLM1 and miR3935 were predicted via TargetScan e Luciferase reporter assay presented the inhibited luciferase activityof GOLM1WT reporter in the presence of miR3935 mimics not NCmimics fg GOLM1 expression in transfected cells was tested by qRTPCR andwestern blot analyses h The combination of GOLM1 with miR3935 in the antiAgo2 group was validated by RIP assay ij The mRNA and proteinlevels of GOLM1 in different groups were examined via qRTPCR and western blot The fulllength gels for western blot data in Fig 3c g and jwere presented in Supplementary Figure P p p downregulation on the expression of apoptosisrelatedproteins Fig 4f Figure S3H Moreover Transwellmigration and wound healing assays illuminated thatthe retarding influence of silenced LINC00992 on cellmigration could be rescued by GOLM1 overexpression Fig 4gh As expected the inhibitory effect ofLINC00992 depletion on the expression of migrationrelated molecular markers MMP2 MMP9 pSrc andpFAK could be countervailed by GOLM1 overexpression Figure S3I Collectively GOLM1 was requiredcancercellular processesLINC00992regulatedinprostateLINC00992 contributes to tumor growth via upregulatingGOLM1 expressionAfter the in vitro exploration of LINC00992 performance in prostate cancer we applied the in vivo assays tofurther validate above findings As shown in Fig 5a tumors derived from LINC00992silenced DU145 cellswere smaller with the growth rate quite slower thanthose from control cells more importantly such blockage on tumor growth was obviously countervailed afterGOLM1 overexpression Besides elevating GOLM1 expression could recover the lessened tumor volume anddeclined tumor weight induced by LINC00992 deficiency 0cChen BMC Cancer Page of Fig LINC00992 promoted prostate cancer cell proliferation and migration via elevating GOLM1 expression ab GOLM1 mRNA and proteinlevels in DU145 cells transfected with pcDNA31 or pcDNA31GOLM1 were detected via qRTPCR and western blot pcDNA31 served as thenegative control c The viability of DU145 cells was determined via CCK8 following transfection of different plasmids d The proliferation oftransfected cells was evaluated via EdU assay e The apoptosis of transfected cells was monitored via TUNEL assay Scale bar μm f Theprotein levels of Bax and Bcl2 in different groups were detected via western blot gh The migration of transfected cells was measured viaTranswell migration assay scale bar μm and wound healing assay scale bar μm The fulllength gels for western blot data in Fig 4band f were presented in Supplementary Figure P p Fig 5bc Of note we discovered decreased level ofLINC00992 and enhanced level of miR3935 in tumorsfrom latter three groups compared to control group whilethe lowered expression of GOLM1 in tumors withLINC00992 inhibition was normalized under GOLM1overexpression Fig 5d In addition the inhibitory impactof silenced LINC00992 on the positivity of proliferationassociated proteins PCNA and Ki67 could be reversedby upregulation of GOLM1 Fig 5e Taken togetherLINC00992 promoted the tumorigenesis of prostate cancer through upregulating GOLM1 expressionDiscussionAs documented the aberrant regulation of lncRNAs is afrequent event in diversified tumor types Besides thecorrelation between abnormallncRNA expression andprostate cancer oncogenesis has also been extensivelyexplored For example lncRNA SNHG7 facilitates prostate cancer carcinogenesis via cyclin D1 by spongingmiR503 [] LncRNA SChLAP1 aggravates prostatecancer cell proliferation and metastasis by targetingmiR198 [] LncRNA PCAT1 contributes to prostatecancer tumorigenesis through modulating FSCN1 andsponging miR1455p [] In our work LINC00992 wasrevealed to be highly expressed in prostate cancer tissuesand cells but unlike former investigations our studygave a precise explanation about its role in prostate cancer Our study unveiled that LINC00992 promoted cellproliferation and migration whereas suppressed cellapoptosis in prostate cancer Abovementioned data 0cChen BMC Cancer Page of Fig LINC00992 contributes to tumor growth via upregulating GOLM1 expression a Representative images and the growth curves of tumorsfrom indicated groups bc The volume and weight of tumors from above groups d The expression of LINC00992 miR3935 and GOLM1 intumors from different groups was detected via qRTPCR analysis e The staining of PCNA and Ki67 in different groups was measured via IHCScale bar μm p 0cChen BMC Cancer Page of validated that LINC00992 elicited a tumorpromotingfunction in prostate cancerPresently accumulating evidence has indicated thatcytoplasmic lncRNAs assisted the expression of downstream miRNAtargeted mRNAs via sponging the specific miRNAs Before exploring LINC00992mediatedmechanism in prostate cancer herein we firstly discovered its subcellular distribution in prostate cancer cellswith both aids from online prediction tool LncLocatorand experimental data FISH and RNA isolation of nuclear and cytoplasmic fractions Our study for the firsttime uncovered that LINC00992 located mainly in thecytoplasm of prostate cancer cells Besides our studyalso completed LINC00992modulated mechanism bydisclosing the downstream target miR3935 The directinteraction between LINC00992 and miR3935 | 2 |
" according to the who most chronic diseases including cancer can be prevented by identifyingtheir risk factors such as unhealthy diet smoking and physical inactivity this research examined the effectiveness ofa theorybased educational intervention on colorectal cancerrelated preventive nutritional behaviors among asample of anizational staffmethods in this interventional study employees of shahid beheshti university of medical sciences wererandomly divided into two groups intervention and control with cluster sampling the data gathering tool was aresearchermade questionnaire containing two parts of 10dimensional information and health belief modelconstructs the educational intervention was conducted for month and in four sessions in the form of classroomlecture pamphlet educational text messages via mobile phones and educational pamphlets through the officeautomation system two groups were evaluated in two stages pretest and posttest data were analyzed usingspss18 software analysis of covariance ancova and independent ttest intergroup comparisonsresults two groups were evaluated for variables such as age sex education level and family history of colorectalcancer and there was no significant difference between the two groups p after the months sinceintervention except for the mean score of perceived barriers which was not significant after intervention the meanscores of knowledge perceived susceptibility perceived severity perceived benefits perceived selfefficacybehavioral intention and preventive behaviors were significantly increased after the intervention in the interventiongroup compared to the control group p implementation of educational intervention based on health belief model was effective for thepersonnel and can enhance the preventative nutritional behaviors related to colorectal cancerkeywords educational intervention health belief model nutritional behavior colorectal cancer correspondence mohtashamghaffarisbmuacir1environmental and occupational hazards control research center schoolof public health and safety shahid beheshti university of medical sciencestehran iranfull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0crakhshanderou bmc medical education page of nearly million new cases of colorectal cancer arediagnosed every year worldwide with nearly half of theaffected patients losing their lives due to the disease approximately of men in and of women in are diagnosed with crc during their life time the incidence of colorectal cancer in iran ranges from to per annually with a death rate of about per hundred thousand and it accounts for approximately of all gastrointestinal cancerrelated deaths according to the latest cancer record in iran colonand rectum cancer ranked third in female cancers andfifth in male cancers the global incidence of crc is predicted to increase by to more than million newcases leading to million cancer deaths by therisk of colon cancer increases with age and is higher inmen than in women various factors are involved inthe development of various types of cancerincludingcolorectal cancer which can be attributed to geneticenvironmental and dietary factors among the riskfactors of colorectal cancer nutritionalfactors areconsidered to be the most important and preventableones so that to of cases can be prevented byproper nutrition [ ] colorectal cancer is also morecommon in iran than in other asian countries [ ]therefore the need to educate people about the nutritionalbehaviors associated with colorectal cancer is becomingmore and more evident theories and models identifyfactors that influence health and behavior which meansthat they can be used to develop programs the most effective training programs are based on the theorydrivenapproaches which are rooted in behaviorchanging modelsalso selecting appropriate model or theory is the first stepin the process of planning a training program [ ] asone of the most widely applied theories of health behaviorthe health belief model hbm posits that six constructspredict health behavior perceived susceptibility perceivedseverity perceived benefits perceived barriers perceivedselfefficacy and cues to action fig the hbmposits that when an individual perceives a serious threatalong with a way to reduce the threat they will be morelikely to take action to reduce the threat the hbmhas been applied to predict a wide variety of healthrelatedbehaviors such as being screened for the early detection ofasymptomatic diseases the model has been applied tounderstand patients responses to symptoms of disease lifestyle behaviors and behaviors related to chronicillnesses which may require longterm behaviormaintenance in addition to initial behavior change the research hypotheses are an intervention based onthe hbm can significantly promote colorectal cancer preventive behaviors the score for each and every constructof the hbm eg perceived awareness and susceptibilityperceived severity perceived benefitsbarriers and perceivedselfefficacy is increased significantly after the interventionin the experimental group as compared to the controlmethodsstudy design and samplingthis interventional study was conducted at shahidbeheshti university of medical sciences tehran iranfrom october to june fig health belief models components and links 0crakhshanderou bmc medical education page of in thisstudy using the sample size formula ¾ z¾2δ2d2 in which δ2 α n ¼ °zd and with an attrition rate of finally women subjects in the experimental and in thecontrol group were considered the random samplingmethod clustering and simple random sampling wasused in this study in order to choose from four facultiesfaculties of shahid beheshti university of medicalsciences four faculties were randomly selected and fromthese four faculties two faculties were assigned as intervention group and were considered as control grouprandom sampling method was used to select samplesfrom each clusterinclusion exclusion criteriabeing under years of age having satisfaction to participate in the study and not having serious diseases including gastrointestinal diseases were the inclusion criteriaalso not willing to continue with the study not completing the questionnaire in full and not attending in morethan two educational sessions were the exclusion criteriameasuresthe researchermade questionnaire was used for datacollection in this study three sources of existed toolsliterature review and expert view were used for itemgeneration this instrument consisted of two main partsas followpart one demographic questions about age gendereducational level and economic statuspart two constructs of the health belief model whichincludes knowledge perceived susceptibility perceivedseverity perceived benefits perceived barriersperceived selfefficacy behavioral intention andbehavior table validity and reliabilityface and content validities were applied for validationphase reliability was confirmed based on methods oftestretest and internal consistency cronbachs alphafor face validity a survey was done on employeesabout the difficulty in understanding the words andphrases the probability of misunderstanding the phrasesand lack of clarity in the meaning of the words somemodifications were made to the tools questions todetermine the content validity of the questionnaire twogastroenterologistsfive health education and healthpromotion specialists and one related expert were askedto complete the questionnaire the initial questionnairehad questions theconstructs of knowledgeperceived susceptibility perceived severity perceivedbenefits perceived barriers perceived selfefficacyintention and behavior had and questions respectively internal consistency was used todetermine the reliability of hbm structures the cronbachs alpha coefficient was for all structures andwas statistically acceptable the retest was used to ensure the reliability of the awareness variable in this way employees completed the questionnaire twice and theicc was obtained also construct validity wasperformed by exploratory analysis method the kmovalue was and bartletts research showed thetable description of study instrumentconstruct knowledge refers to a theoretical or practical understanding of asubject perceived susceptibility refers to subjective assessment of risk ofdeveloping a health problem perceived severity perceived severity refers to the subjectiveassessment of severity of a health problem and its potentialconsequences perceived benefits healthrelated behaviors are also influenced bythe perceived benefits of taking an actionno of items format items truefalsedont know items 5point likert scalestrongly disagree to stronglyagree items5point likert scalestrongly disagree to stronglyagree items5point likert scalestrongly disagree to stronglyagreescoring rangecorrect response dont knowresponse incorrect response strongly disagree disagree noidea agree strongly agree strongly disagree disagree noidea agree strongly agree strongly disagree disagree noidea agree strongly agree perceived barriers healthrelated behaviors are also a function ofperceived barriers to taking action perceived selfefficacy refers to an individuals perception of his orher competence to successfully perform a behavior behavioral intention refers to a persons perceived probability orsubjective probability that he or she will engage in a given behavior items5 point likert scalestrongly disagree strongly agree items5 point likert scalestrongly disagree strongly agree items5point likert scalestrongly disagree to stronglyagreestrongly disagree disagree noidea agree strongly agree strongly disagree disagree noidea agree strongly agree strongly disagree disagree noidea agree strongly agree behavior refers preventative behaviors associated with colorectalcancer items5point likert scalealways to neveralways often sometimes rarely never 0crakhshanderou bmc medical education page of significant correlations among the items Ï2 df p therefore the data were suitable forconducting factor analysisinterventionboth intervention and control groups were pretestedusing the questionnaire the analysis of educational needsdetermined the educational methods educational package and the number of educational sessions was obtainedby the pretestreadabilitycomprehensibility and not complexity of educational contents for participants was obtained by pretesting materialssuch as pamphlets messages etc in a sample of employees who were not included in main researchresults assurance abouteducational intervention based on educational textmassagesover the course of days ten text messages were sentto the employees in the intervention group at am mostof which had been prepared according to the educationalobjectives ofthe constructs of knowledge perceivedsusceptibility perceived benefits perceived barriers andperceived selfefficacycounseling there waseducational pamphletstwo pamphlets were given to the employees during twoseparate sessions along with simultaneous provision ofindividuala possibility ofquestioning and answering any ambiguity regarding thecontent of pamphlets the first pamphlet containedsections on the signs and symptoms of colorectal cancerand the risk factors of this cancer and the secondpamphlet contained sections on methods of preventingthis cancereducational packages in the office automation systemeducational packages were uploaded on the staff automation system for days and the employees were askedto study it during the working hoursthe intervention was conducted month and followup months after the intervention the educationalcontents were taken from the trusted sources of theministry of health complemented by what the staffneeded to know about promoting nutritional behaviorsrelated to the prevention of colorectal cancer the education varied in form across the model constructs forperceived susceptibility the facts and figures of the incident rate of colorectal cancer were presented in theclass and for perceived severityimages of colorectalcancer problems were used also for perceived barrierseducational materials were used to somehow incite theindividuals to analyze the cost of optimal behavioragainst the costs of risks time etc involved in unhealthybehavior the educational content used for perceivedbenefits intended to raise awareness on the usefulness ofhealth promoting behaviors to reduce the risk of illnessor to understand the benefits of healthy behaviors infig the research process is presented in generalethical considerationsat first a permission was obtained from the universityto conduct the study and attend the healthcare centerthe samples were assured about the confidentiality oftheir specifications and information they were also toldthat their information will only be used for the purposeof this study and the data collection the participantswere allowed to enter and leave the study at any timesuitable conditions were provided for a proper understanding of questions and responses for the subjectsafter the end of the intervention period the controlgroup was also trained using the slides that were used totrain the intervention group an informed consent wasobtained from the participants the study on whichthese data analyses are based was approved by theethical board committee of shahid beheshti universityof medical sciencesdata analysisdata were analyzed by spss software kolmogorov smirnovtest was used to check the normality of the data to assessthe effectiveness of intervention on variables of knowledgeperceived susceptibility perceived severity perceived benefits perceived barriers perceived selfefficacy behavioralintention and behavior in the intervention and controlgroups two groups were evaluated in two stages pretestand posttest data were analyzed using spss18 softwareanalysis of covariance ancova and independent ttestintergroup comparisonsthe confidence level of and the significance level of were consideredin this studyresultsthe findings of this study showed no drop out until theend of study the questionnaire was completed in bothgroups in a complete and precise manner homogenizationwas done in the two groups by controlling variables such asage sex level of education and related family history theresults showed no significant relationship within thesevariables p table effectiveness of the educationalintervention in improving knowledge perceived susceptibility perceivedseverity perceived benefits perceived selfefficacybehavioral intention and behavior once age gender andlevel of education factors were adjusted was checkedthrough ancova the results revealed that the intervention was successful in improving constructs of thehealth belief model significantly in participants table the mean score ofintention and behavior in the 0crakhshanderou bmc medical education page of fig schematic diagram of designed interventions for colorectal cancer preventionexperimental and control groups before and after theintervention is presented in fig discussionthe purpose of this study was to investigate the effectsof educationalinterventions on the promotion ofcolorectal cancer prevention nutritional behaviors thekmo and bartletts test p results confirmed the suitability of the model for conducting factoranalysis the kmo is in the range if the value ofthe inedex is near to one the data are suitable for factoranalysis kaiser at least kmo to determinestable demographic and variables in intervention and control groups before the interventionvariablegroupintervention group n n control group n n agegenderlevel of educationfemalemalediplomaassociate degreeundergraduate degreeand higherhistory of specialdiet compliancefamily history of cancerchisquareyesnoyesnop value 0crakhshanderou bmc medical education page of table comparison of intervention and control groups in terms of health belief model constructs before and after the interventionp valueconstructsgroupsbefore interventionmean ± sd ± after interventionmean ± sd ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± meandifference ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± knowledgeinterventioncontrolperceived susceptibilityinterventionperceived severityperceived benefitsperceived barriersperceived self efficacybehavioral intentionbehavioranalysis of covariance ancovacontrolinterventioncontrolinterventioncontrolinterventioncontrolinterventioncontrolinterventioncontrolinterventioncontrol also bartlett test was used to confirm adequacy ofthe samples in the present study the mean score of behavioralconstruct increased after the intervention in the intervention group and there was significant differencebetween the two groups after the intervention in thisregard the results of this study are consistent with thefindings of abood hart roozitalabi alidoosti and davoodi studies behavioral intention is the thought of doing abehavior and is considered as the immediate determinant of that behavior the mean score in this construct aswell increased in the intervention group after the intervention and there was significant difference between thetwo groups after the intervention in the study of braun and gimeno the results were similar tothe results of present study selfefficacy is a keyprerequisite for behavior change there was significantdifference between mean score of perceived selfefficacyconstruct in the two groups after the intervention in thisfig mean scores of intention and behavior in the experimental and control groups before and after the intervention 0crakhshanderou bmc medical education page of regard the results of the study by braun alidoosti and hart are consistent with thisfinding perceived selfefficacy is considered as a strongmotivational source and in fact is an indicator of theability of individuals to anize themselves in pursuit ofcertain goals studies show that individuals with ahigh level of perceived selfefficacy have a greatercommitmentto engage in activities at a time ofchallenges and difficulties and spent more time andeffort on such activities such individuals are morelikely to contribute to maintaining healthy behaviors andretrieve them even after failure and they have strongerintention and motivation this not only improves thetarget adjustment but also ensures achievement andsustainability in pursuit of the goals another important factor is knowledge that can be pointed to itsrole in healthy behaviors this study showed a significant difference in the two group in terms of the meanscore of knowledge after the educationalinterventionthese results are consistent with the findings of roozitalab ho and gimeno studiesalso there was no significant difference in the controlgroup before and afterthe intervention althoughincreasing knowledge is an important step in changingattitudes and behaviors it is not a major contributor tocrc prevention achieving the intention to behave isinfluenced by individual and environmental factors so inaddition to enhancing individual aspects overcomingthe structural and environmental barriers of the healthsystem regarding the use of cancer prevention nutritional behaviors is also vital in the present study themean score of perceived susceptibility and perceived severity constructs showed a significant difference betweenthe intervention and control group after the educationalintervention studies by kolutek wang cengiz and donadiki reportedthe role of beliefs regarding public health threats perceived susceptibility and perceived severity in the healthpromotion behaviors becker believed that onesintention to selfcare is influenced by his or her perception of vulnerability and the severity of disease outcomes therefore the need for interventions to increasethe perception of society about the irreparable complications of diseases caused by unhealthy behaviors malnutrition habits seems necessary in this study there was asignificant difference between the two groups in terms ofthe constructs of perceived benefits after the educationalintervention this result is consistent with the findings ofgrace alidoosti and abood studies also in the present study the mean score of perceived barrier construct decreased after the interventionthis was a good result but it was not statistically significant in the present study the mean score of perceivedbarrier construct decreased after the intervention which isnot consistent with the results of studies by moatari grace and gimeno the study ofrajabi identified some of the most important causes of barriers to nutrition in preventionof cancer such as the difficulty of preventativemeasuresinappropriate economic status and fear ofcancer information therefore strategies that overcome the individual and environmental barriers thataffect nutritional behaviors should be addressed byplanners and policymakerslimitationsthe limitations of this study which could have had a relative effect on its findings include the short duration ofintervention the sample size the inability to follow thelong term effect of the intervention and the selfreportingof the subjects in responding to questions however theuse of this method in such studies is inevitable and maylead to a bias of the researcherdesired report in thisstudy anonymous questionnaire was used to minimizethis biasthe findings of this study confirmed the effectiveness ofhealth belief modelbased education in improvement ofcolorectal cancerrelated preventive behaviors on theother hands interventions based on hbm concepts couldpromote nutritional behaviors related to colorectal cancerprevention consequently offering educational programsincluding public information campaigns workshopsvideos websites exhibitions etc should be used to informpeople about crc symptoms and risk factors alsomodelbased education will have a greater effect on nutritional behaviors improvement by focusing on perceptionsand enhancing beliefs aboutthe applicability oftheprogram and understanding the benefits and barriersabbreviationscrc colorectal cancer hbm health belief modelacknowledgementsthis is a part of an msc dissertation in health education approved by theshahid beheshti university of medical sciences the authors of this paperwould like to express their gratitude and appreciation to all the contributorswho have somehow collaborated on the design guidance andimplementation of this projectauthors contributionsmgh sr as and mm designed the study mm and mgh wrote the firstdraft sr and asm conducted the analyses all authors contributed towriting revising and approved the final manuscriptfundingthis study is sponsored by shahid beheshti university of medical sciences intehran the funding agencies had no role in the design of study datacollection and analysis or presentation of the resultsavailability of data and materialsthe datasets used and analyzed during the current study are available fromthe corresponding author on reasonable request 0crakhshanderou bmc medical education page of ethics approval and consent to participatethe study on which these data analyses are based was approved by theethical board committee of shahid beheshti university of medical sciencesparticipants were provided information about the study and verbalconsented by proceeding to take the survey this implied verbal consent wasapproved by the ethical board committee of shahid beheshti university ofmedical sciencesconsent for publicationnot applicablecompeting intereststhe authors have no conflict of interestsauthor details1environmental and occupational hazards control research center schoolof public health and safety shahid beheshti university of medical sciencestehran iran 2school of public health and safety shahid beheshti universityof medical sciences tehran iranreceived december accepted august screening in general practice in central england j epidemiol communityhealth roozitalab m moatari m gholamzadeh s saberifiroozi m zare n the effectof health belief on participation of the official administrative personnel incolorectal cancer screening programs in shiraz university of medicalsciences govaresh alidosti m sharifirad g hemate z delaram m najimi a tavassoli e theeffect of education based on health belief model of nutritional behaviorsassociated with gastric cancer in housewives of isfahan city daneshvarmed davodi a anoosheh m memarian r the effect of selfcare education onquality of life in patients with esophageal cancer following esophagectomyzums j braun kl fong m kaanoi me kamaka ml gotay cc testing a culturallyappropriate theorybased intervention to improve colorectal cancerscreening among native hawaiians prev med gimenogarca az quintero e nicol¡sp©rez d parrablanco a jim©nezsosa a impact of an educational videobased strategy on the behaviorprocess associated with colorectal cancer screening a randomizedcontrolled study cancer epidemiol bandura a social cognitive theory handbook of social psychologicaltheories london sage bandura a social cognitive theory an agentic perspective annu revpsychol luszczynska a guti©rrezdo±a b schwarzer r general selfefficacy invarious domains of human functioning evidence from five countries int jpsychol ho tv effects of an educational intervention on breast cancer screeningand early detection in vietnamese american women oncol nurs forumkolutek r avci ia sevig u the effects of scheduled observation at homeon health beliefs related to breast and cervical cancer screening andattitudes of married women eur j oncol nurs 201418s25 wang wl hsu sd wang jh huang lc hsu wl survey of breast cancermammography screening behaviors in eastern taiwan based on a healthbelief model kaohsiung j med sci cengiz b bahar z use of the health belief model in screening methodsfor colorectal cancer eur j oncol nurs 201418s27 donadiki e jim©nezgarca r hern¡ndezbarrera v sourtzi p carrascogarrido p de andr©s al jimeneztrujillo i velonakis e health belief modelapplied to noncompliance with hpv vaccine among female universitystudents public health becker mh drachman rh kirscht jp a new approach to explaining sickrole behavior in lowincome populations am j public health ma gx shive s tan y gao w rhee j park m kim j toubbeh jicommunitybased colorectal cancer intervention in underserved koreanamericans cancer epidemiol moatari m roozitalab m saber f zare m gholamzadeh s effect ofeducation on health beliefs on knowledge and participation j res med rajabi r sharifi a shamsi m almasi a dejam s investigating the effectof package theorybased training in the prevention of gastrointestinal cancers publishers notespringer nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliationsreferencesarnold m sierra ms laversanne m soerjomataram i jemal a bray f globalpatterns and trends in colorectal cancer incidence and mortality gut american cancer society colorectal cancer facts and figures available at httpswwwcancercontentdamcancerresearchcancerfactsandstatisticscolorectalcancerfactsandfigures 20172019pdf[accessed ]ansari r amjadi h norozbeigi n zamani f mirnasseri s khaleghnejad amalekzadeh r survival analysis of colorectal cancer in patients underwentsurgical operation in shariati and mehr hospitaltehran in a retrospectivestudy govaresh centers for disease control and prevention cdc colorectal cancer risk byage available at httpwwwcdcgovcancercolorectalstatisticsagehtm[accessed apr ] malekzadeh r bishehsari f mahdavinia m ansari r epidemiology andmolecular genetics of colorectal cancer in iran a review kz aa saadat a jalalian hr esmaeili m epidemiology and survival analysisof colorectal cancer and its related factors trauma monthly winter239ghaffari m mehrabi y rakhshanderou s safarimoradabadi a jafarian szeffectiveness of a health intervention based on who food safety manual iniran bmc public health hosseini sv izadpanah a yarmohammadi h epidemiological changes incolorectal cancer in shiraz iran anz j surg yazdizadeh b jarrahi a mortazavi h mohagheghi ma tahmasebi s nahvijoa time trends in the occurrence of major gi cancers in iran asian pac jcancer prev glanz k rimer bk viswanath k health behavior and health educationtheory research and practice john wiley sons ghaffari m rakhshanderou s safarimoradabadi a torabi s oral and dentalhealth care during pregnancy evaluating a theorydriven intervention oraldis becker mh the health belief model and sick role behavior health educmonogr janz n champion v strecher vj the health belief model k glanz bk rimerjanz nk becker mh the health belief model a decade later health educ qlp o review of translation and cultural adaptation process ofquestionnaires kellar sp kelvin ea munro's statistical methods for health care researchwolters kluwer healthlippincott williams wilkins abood da black dr feral d nutrition education worksite intervention foruniversity staff application of the health belief model j nutr educ behav hart ar barone tl gay sp inglis a griffin l tallon ca mayberry jf theeffect on compliance of a health education leaflet in colorectal cancer 0c" | 0 |
" patients who have undergone radical cystectomy for urinary bladder cancer are not sufficientlyphysically active and therefore may suffer complications leading to readmissions a physical rehabilitationprogramme early postoperatively might prevent or at least alleviate these potential complications and improvephysical function the main aim of the canmore trial is to evaluate the impact of a standardised and individuallyadapted exercise intervention in primary health care to improve physical function primary outcome and habitualphysical activity healthrelated quality of life fatigue psychological wellbeing and readmissions due tocomplications in patients undergoing roboticassisted radical cystectomy for urinary bladder cancermethods in total patients will be included and assigned to either intervention or control arm of the study allpatients will receive preoperative information on the importance of early mobilisation and during the hospital staythey will follow a standard protocol for enhanced mobilisation the intervention group will be given a referral to aphysiotherapist in primary health care close to their home within the third week after discharge the interventiongroup will begin weeks of biweekly exercise the exercise programme includes aerobic and strengtheningexercises the control group will receive oral and written information about a homebased exercise programmephysical function will serve as the primary outcome and will be measured using the sixminute walk test secondaryoutcomes are gait speed handgrip strength leg strength habitual physical activity healthrelated quality of lifefatigue psychological wellbeing and readmissions due to complications the measurements will be conducted atdischarge ie baseline postintervention and year after surgery to evaluate the effects of the intervention mixedor linear regression models according to the intention to treat procedure will be usedcontinued on next page correspondence andreaporserudkise1department of neurobiology care sciences and society division ofphysiotherapy karolinska institutet stockholm sweden2allied health professionals function medical unit occupational therapy andphysiotherapy karolinska university hospital stockholm swedenfull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cporserud bmc cancer page of continued from previous pagediscussion this proposed randomised controlled trial has the potential to provide new knowledge withinrehabilitation after radical cystectomy for urinary bladder cancer the programme should be easy to apply to otherpatient groups undergoing abdominal surgery for cancer and has the potential to change the health care chain forthese patientstrial registration clinicaltrialsgov clinical trial registration number nct03998579 first posted june keywords abdominal surgery behaviour bladder neoplasm complications exercise physical activity primaryhealth care process evaluation the most common treatment for solid cancer tumoursis surgery often in combination with chemotherapy orradiotherapy or both minimising postoperative complications is important in health care for the individual patient and in reducing health care costs for society earlymobilisation at the ward and physical activity at homeafter discharge are important activities to reduce complications common complications after abdominal surgery are postoperative pulmonary complications andvenous thrombosis [ ] which generally are thought tobe partially avoidable with early mobilisationafter radical cystectomy for urinary bladder cancerthere is a high risk for postoperative complications thecomplications could be directly related to the patientshigh age to a high degree of comorbidity or both the major risk factor for developing urinary bladdercancer is smoking most of the patients are men andthe median age of undergoing a radical cystectomy is years [ ] as much as of patients are at severe nutritional risk before a radical cystectomy afterroboticassisted radical cystectomy rarc for urinarybladder cancer ofthe patients need to bereadmitted to hospital after discharge because of complications [ ]there is strong evidence that aerobic physical activityhas a positive impact on health survival and quality oflife qol patients diagnosed with cancer shouldfollow the general recommendations on physical activityand exercise for health [ ] yet most patients areinsufficiently active consequently with an increasing number of cancer survivors the importance of supporting high physical function and qol increases research has shown that exercise has a positive effect onhealthrelated qol hrqol in patients who have completed active cancer treatment moreoverin patients living with or beyond a diagnosis of cancerbehaviouraleg goalsetting andgraded tasks are important components in the exerciseinterventions with high adherence and positive physicaloutcomes support methodsin a recent study we evaluated the activity board®phystec sweden as a method to enhance mobilisationand recovery after abdominal surgery for cancer theactivity board is a tool based on techniques to supportbehaviour change [ ] the evaluation showed thatthe activity board resulted in a higher level of mobilisation in the group with the activity board compared withthe group who received standard treatment although evidence for exercise after abdominal surgery isscarce a few studies have evaluated exercise programmes for patients postoperatively atthe hospitalward with promising results [ ]despite the lack of exercise interventions after surgeryit has been shown that functional performance after aradical cystectomy for urinary bladder cancer correlatesto overall survival a large proportion of patientswith urinary bladder cancer do not achieve the recommendations on physical activity and exercise it isalso common that patients who undergo radical cystectomy have not performed physical exercise for a longtime before surgery finally after surgery patientsreport a low level of physical exercise recently tworeviews have been published on physical and psychological interventions to improve healthrelated outcomesin this patient group [ ] both reviews include thesame two postoperative exercise studies [ ] onelarger rct showed that early physical exercise and enhanced mobilisation after radical cystectomy positivelyaffected some domains of hrqol in addition in apilot study we tested a model for physical rehabilitationafter radical cystectomy the model consisted of weeks of individually tailored exercise after dischargefrom the hospital the exercise programme conductedat the hospital showed both short and longterm effectson physical function and hrqolconsequently the few studies within the field raiseseveral research questions for future exercise interventions in patients with urinary bladder cancer undergoingradical cystectomy current recommendations proposethe following areas supervised exercise after dischargethe optimal type of exercise fidelity and adherence ofthe intervention if shortterm outcomes are sustainedclinical relevance longterm outcomes and readmissionsto hospital [ ] we also need to understand thekinds of support that are optimal use behaviour change 0cporserud bmc cancer page of screened for eligibility in medical records by the responsible researcher rr and given written information by a registered nurse at a preoperative meetingafter days the rr will phone the patient provideoral information and then ask about participation informed consent will be signed before surgery the rrkeeps a protocol for enrolment the patient flowchartis depicted in fig eligibility criteriainclusion criteria will be patients who are planned for ararc for urinary bladder cancer the patients shouldbe able to talk and understand swedish without an interpreter be mobile with or without a walking aid and livein the stockholm regionstrategies and implement the intervention as a part ofthe patients clinical pathway through the healthcare system [ ]in summary patients who have been treated forurinary bladder cancer are not sufficiently physicallyactive and suffer from readmissions to hospital due tocomplications therefore there is a need for developing a physical rehabilitation programme to supportpatients who have a radical cystectomy in the earlypostoperative period in this paper we present a studyprotocol for the canmore trial a physical rehabilitation programme after rarc for urinary bladdercancerimpact ofamethodsdesignmain objectiveis to evaluatethe main aim of the canmore trialthestandardised and individuallyadapted exercise intervention in primary health carephcfunction primary outcome and habitual physical activity hrqol fatiguepsychological wellbeing and readmissions due tocomplications in patients undergoing rarc for urinary bladder cancerto improve physicalhypothesiswe hypothesise that the canmore programme is morebeneficial than homebased exercises in increasing physical function primary outcometrial designthe canmore trialis a randomised controlled trialrct with a singleblinded design the interventiongroup will receive a 12week h twice a week standardised and individually adapted exercise intervention inphc and behavioural support for daily physical activitythe control group will receive a homebased exerciseprogramme as well as recommendations on daily physical activity based on general guidelines we will followthe spirit standard protocol items recommendationsfor interventional trials statement and guidelinesfor reporting the study protocol the clinical trial registration number for this trial is nct03998579study settingthe study will be conducted in two settings a universityhospital and a phc context in region stockholmrecruitment and screeningparticipants will be recruited through theme canceratthe karolinska university hospital solna andscreened for eligibility recruitment will be performedconsecutively based on power analysis patientswill be included potential participants will initially befig patient flowchart 0cporserud bmc cancer page of exclusion criteriapatients with planned palliative surgery or cognitive impairment identified by screening of medical records willnot be includedrandomisation assignment of interventionpatients who fulfil the criteria for inclusion will afterhaving given their oral and written consent be randomised in the alea system operated by the clinical trials office cto atthe centre for clinical cancerstudies theme cancer karolinska university hospitalsolna swedenrandomisation will be conducted in blocks of patients stratified by sex and age ¥ years a confirmation email will be sent to the entering investigatorand an enrolment log will be filed at the centre the patient will receive the next consecutive code number inthe trial and treatment arm according to the randomisation schemeislogic model for the canmore programmeit is recommended that programme design should bebased on a theory to improve evidence synthesis the theoretical framework underpinning the canmoreprogrammethe movement continuum theorymct and the evidencebased calore taxonomy forbehavioural change techniques the mct posits that anindividual has three stages of movement capability amaximum a current and a preferred the canmoreprogramme identifies the patients current physical function capability and intervenes regarding the patientsneed for function several models and theories are supporting a behaviour change which results in increasedphysical activity however research has shown thatit is most often not necesssary for a complete theorybut instead the different components in the theory thatsupport the behaviour to consider the patients need forsupport the calore taxonomy for behavioural changetechniques has been added the taxonomy is recommended to be used to improve the specification of interventions behavioural techniques proven effective tosupport behaviour change are goalsetting graded tasksselfmonitoring feedback and reward all of these areused in the canmore programmea conceptual framework visualising the inputs theoryintervention components and its intermediate and possible longterm outcomes is depicted in a logic modelfig interventionall patients receive preoperative information on the importance of early mobilisation and postoperative individual physiotherapy during the hospital stay the activityboard is used for enhanced mobilisation before discharge from the hospital the patients receive standardised information about avoiding the lifting of heavyobjects and the importance of physical activity the patients are then randomised to either the intervention orthe control groupintervention grouppatients in the intervention group get a referral to aphysiotherapist in phc close to where they live the patients can choose from phc settings spread throughout the stockholm region within the third week afterdischarge the patients begin weeks of biweekly exercise the patients pay for their primary care visits physiotherapists in the targeted primary care units receive afig logic model of the intervention 0cporserud bmc cancer page of leaflet and a short education before starting comprisinginformation about rarc restrictions potential adverseevents the trial process and the exercise programmethe physical exercise is individually targeted but basedon international recommendations for persons with cancer disease the exercise programme includes aerobicexercise aiming at moderate intensity minsessionand strengthening exercises comprising endurance training with à repetitions see additional file theprogramme is gradually increased based on the patientscapability the programme also includes exercises forabdominal muscles including pelvic floor exercises tominimise the risk of developing stoma hernia however to avoid heavy strain on the surgery wounds restrictions regarding abdominal muscles are followed weeks postoperatively the exercise programme hasbeen approved by the responsible medical surgeons inaddition to the structured exercise sessions the patientsare advised to take daily walks in their neighborhoodthe number of recommended steps per day is set together with the physiotherapist based on the patientscapability to support the patient individual goalsettingfeedback and selfmonitoring of daily steps are usedsimilarly to those of the activity board at the end ofthe exercise periodthe physiotherapist recommendscontinued physical activity according to their clinicalroutinesanasapplicationactivitydiarypedometeroraphoneoutcomesthe measurements will be conducted using validated instruments at discharge ie baseline postintervention months and year after surgery table with thepurpose to receive information on the patients healthand physical function before surgery eg to adjust forin the analysis measurements will also be conductedbefore surgery all measurements will be conducted byexperienced physiotherapist blinded to the interventiona protocol for the measurements has been developedand the physiotherapist will receive specialised trainingby the research staffprimary outcomephysical function the primary outcome will be measured using the validated sixminute walk test 6mwt[ ] the test reproduces activity of daily living at asubmaximal level which is particularly applicable to elderly patients the patients are asked to walk as faras possible for min the number of meters m oxygensaturation and heart rate measured with a pulse oximeter will be recorded at the end of the test according tostandard procedures the primary outcome variablewill be walking distance in meterscontrol groupthe control group will receive oral and written information of a gradually increased homebased exerciseprogramme that includes daily walks and sittostandexercises they will also receive information on supportive techniques to improve physical activity suchsecondary outcomesgait speed will be assessed with the 10m walk test the test is used to determine walking speed in metersper second ms over a short distance the test is performed as three 10m walks without assistance one testwalk one in preferred walking speed and one in thepreop testingxbaselinexposttestingx1year followupxxxxxxxxxxxxxxxxxxxxxxxxxxxxxtable outcome measures and test occasionsvariablephysical functionmeasure6min walk testgait speedleg strength10m walk testchair stand testhandgrip strengthjamar hand dynamometerhabitual physical activityprevious physical activity levelahealthrelated quality of lifefatiguepsychological wellbeingpainactivpal3 microstanford brief activity surveyeortc qlqc30eortc qlqblm30piper fatigue scalehadsnrslength of hospital stay dayscomplicationsmedical recordsmedical recordsxxxxxxxxreadmissionsaprevious physical activity level is only used for adjustment purposes in the analysismedical records 0cporserud bmc cancer page of fastest speed possible time is measured for the intermediate m to allow for acceleration and decelerationthe outcome variable is msgrip strength will be assessed with the validated jamarhydraulic hand dynamometer the patients will sitin a chair and hold the dynamometer the test is performed three times for each hand and a mean value foreach hand is calculated the outcome variable is the gripstrength reading in kgleg strength will be assessed with the 30s sec chairstand test the patient is asked to rise from a chairas many times as possible for s the outcome variableis the number of sittostand transitionshabitual physical activity will be measured usingthe activpal3 micro activity monitor pal technologies ltd glasgow uk [ ] the activpal is asmall device which when attached to the thigh provides information based on position and accelerationof the body the information is then transferred tobody posture the transition between postures stepping and stepping speed the activity monitor is attached to the anterior midline ofthe thigh withdressing and does not provide feedback to the patientthe monitor will be worn for seven consecutive daysafter discharge from hospital and after the intervention period outcome variables will be time spentsittinglying standing stepping numbers of stepcounts and sittostand transitionsselfreported previous physical activity level will bemeasured using the 2item stanford brief activitysurvey sbas the sbas assesses the usualamount and intensity of physical activity during thepast year that a person performs the first item describes five patterns of work activity ranging frommostly sedentary to hard physical labour the seconditem describes five patterns ofleisuretime physicalactivity ranging from sedentary to regular vigorousintensity aerobic activities for respondents who areretired and have no job or regular work they wouldselect the response not applicable for both itemsthe outcome variable was categorical and rated on a5point scalehrqol will be assessed using the eortc qlqc30 with addition of the eortc qlqblm30 questionnaire the eortc qlqblm30 is explicitlydeveloped for patients with muscleinvasive urinarybladder cancerfrom theworst to the best for functional health statusand from the best to the worst for symptoms outcome variables will range from to fordifferent domains scoring rangesfatigue will be assessed using the piper fatigue scale the questionnaire consists of items and scoringranges from noneto severe the score ispresented in four domains plus a total fatigue scoreoutcome variables will range from to psychological wellbeing will be assessed using the hospital anxiety and depression scale hads thescale consists of questions with each scored on ascale from to where represents more symptomsthe questions are equally divided into the domains anxiety or depression each domain can result in a maximum score of outcome variables will range from to pain will be assessed using the numeric rating scalenrs the nrs is an eleven point scale and scoring ranges from no pain to worst pain the nrsis verbally delivereddata on length of stay at the hospital and frequency ofreadmission to hospital due to complications will be extracted from patient medical records readmissions willbe extracted as and days after surgery and complications will be registered using the claviendindo classification [ ]ethicsthe project is approved by the regional board of ethicsin stockholm dnr and the swedishethical review authority dnr the new model for rehabilitation is compared withsimilar care the patients are given in todays care delivery yet the control group will receive less attentionthan the intervention group we find it unethical to askthe patients to come to phc pay their visit and only receive for example stretching exercises in addition itwill be challenging to motivate the physiotherapists inphc to offer such treatmentsample sizefunction evaluatedthe primary outcome is physicalwith the validated 6mwt based on data from our pilotstudy we calculate an increase in m in theintervention group m in the control group and astandard deviation of m to obtain a statistical powerof with a type error set at patients areneeded in each group however as the test is highlycorrelated with sex and age we will stratify the analysis and increase the sample sizefor the secondary outcome readmissions due to complications we will estimate readmissions in theintervention group compared with the proportion of patients being readmitted today which is in the control group to obtain a statistical power of with atype error of patients are needed in eachgroup taken all this into account and guard againstdropout patients in each group will be includedin the study 0cporserud bmc cancer page of data management and study databasedata will be entered using an electronic system pheedit which is based on the sas system provided and operated by the cto at the centre for clinical cancerstudies theme cancer karolinska university hospitalsolna sweden a data management plan is delivered bycto documenting the database and all procedures fordata management the investigator verifies that all dataentries in the case report forms crfs are accurate andcorrect if certain assessments according to the protocolare not performed for any reason or if certain information is not available not applicable or unknown this willbe indicated in the crf by the investigator the investigator is required to sign off all reported datasource datain this study physicaltests movement sensor datapatientreported outcome measures and medical recordswill be regarded as source datacodingin the study database patients will be identified onlythrough the unique randomisation number all data willbe stored with coded identification and no access to thepatients id patient identification will not be revealed intext files logbooks with identification numbers and therespective codes will be stored in a locked environmentat the local centre that is not accessible to personnel notinvolved in conducting the trialstatistical analysisdescriptive statistics will be performed to ensure comparability between data at baseline to evaluate the effectof the intervention mixed models or linear regressionmodels spss inc chicago il usa according to theintention to treat procedure and with an alpha level of will be used significance of main or interaction effects will be explored using the bonferroni posthoc multiple comparison test in the case of skewed distributionlogarithmic transformations or corresponding nonparametric statistics will be used to assess the effect of theinterventionimplementation processbecause the intervention design is intricate we will inaddition to testing effects of the canmore programmeon patientlevel outcome measures also observe andgather information on factors that might have influencedthe implementation of the programme the evaluation of the implementation process will be based on themedical research council guide for process evaluationof complex interventions the knowledge gainedcan also be used to offer recommendations on whichstrategies to use when implementing the canmoreprogramme in other clinical settings and on a largescalethe initial strategies for the process evaluation will include meetings with surgeons the head of the surgicalward and phc clinics discussions and involvementwith physiotherapists and nurses at the surgical wardand physiotherapists at phc clinics and education ofthe canmore programme and outcome measures tophysiotherapists who will be involved in the intervention a leaflet for the patients an extended educationalleaflet and a short education for the physiotherapistshave been developedto study what is delivered measures of fidelity doseadaptation and reach will be assessed fidelity relative tothe canmore programme will be evaluated as the extentto which the programme was delivered as expecteddose will be assessed as the quantity of the interventionthe canmore programme and the education of physiotherapists in phc implemented adaptation such aschanges done to fit different phc settings will be reported in a questionnaire reach will be assessed regarding how many eligible patients signed an informedconsent form and how many in the intervention groupfulfilled the canmore programme in addition adverseevents will be registerediethe extentcontext includes external factors that may act as abarrier or facilitator to both implementation itself andthe patient level effect assessing barriers and facilitators to programme implementation will also involveevaluating programme feasibilitytowhich patients and health care staff regard the canmore as satisfactory in terms of content and complexitydifficulty we plan to conduct an interviewstudy on patients experiences of the intervention inaddition we plan to collect information on possiblebarriers that might have influenced the implementation ofthe programme and facilitators that mighthave supported it at the various clinical sites bothqualitative structured observationfocus groups andindividual semistructured interviews and quantitativequestionnaires and enrolment files methods will beused to assess how the intervention was delivered aswell as experiences of the different stakeholders patients physiotherapists and other health care staff aswell as managersby using several sources for data collection triangulation can be achieved which supports the trustworthinessof the study the consolidated framework for implementation research cfir will be used in the currentstudy to guide the investigation of context ie potentialbarriers and facilitators of the implementation processconstructs that we believe specifically impact the implementation outcomes in the present study and guidelinesforand observation protocolsinterview questions 0cporserud bmc cancer page of published by cifr will be followed httpcfirguidetoolshtmldiscussionto our knowledge this is the first study to examine theeffects of individually targeted exercise in phc compared with traditional advice on home exercise trainingafter rarc moreover the study will include a processevaluation of factors thought to influence the implementation of the programmealthough there is evidence that exercise is beneficialto improve physical function physical activity hrqolreduce fatigue and perhaps reduce complications it isessential to design feasible and easy to implement interventions in a health care setting our intervention is individually targeted and designed based on currentglobal guidelines for physical activity and exercise strategies for behavioural support and adapted to fitwithin the phc structure the length of the interventionis based on exercise principles and what is feasibleto conduct within phc yet this study does not tell uswhether the length of the intervention is the optimallength for best health benefits nor if a booster sessionafter the intervention period is needed the dose of exercise has been previously tested in a pilot study andshowed a positive effect on physical function was safeand had no adverse events after the pilot study werevised the exercise programme to be individually basedbut still include aerobic and musclestrengthening exerto ourcises the addition of behaviourprogrammeselfmonitoring and feedback has been shown to be associated with increased physical activity behaviour setting graded taskseg goalsupportat discharge from the hospital the standard care forpatients includes information on the importance ofphysical activity in this study the control group receivea light intervention ie recommended daily walks andlegstrengthening exercises instead of the standard carethe exercise recommendation is low dose and not specific but can still affectthe results by producing asmaller difference between the groups because there isstrong evidence for the effect of physical activity we findit unethical not to give the control group any advice onphysical activity at discharge many patients are feeblebecause of surgery and the postoperative period at hospital which can also result in fear of movement thesepatients require supervised physical exercise as in theintervention group in this studythe process evaluation using measures offidelitydose adaption adherence and reach as well as patientperspectives and experiences of the programme can helpexplain the results in additionit can speed up theprocess of translating findings from research settings toclinically representative settings the programme fitswell within the healthcare system and the exercises aregeneric to those recommended for cancer survivors andthe tools for motivational support are generic for thewhole population if the programme is proven effectiveit should be generalisable to other patient groupsthatis notthere are some limitations first due to the difficultiesof doubleblinding we have a singleblind design inwhich a physiotherapistinvolved in theprogramme is conducting the measurements secondwe foresee a long recruitment period that can lead to achange in health care staffphysiotherapists in phc toensure quality we plan to have continued contact withthe clinics and a new educational structure for trainingif neededcystectomyin summary this proposed rct has the potential toprovide new knowledge within rehabilitation after radicalcancer theprogramme should be readily applied to other patientgroups undergoing abdominal surgery for cancer andhas the potential to change the health care chain forthese patientsfor urinary bladdersupplementary informationsupplementary information accompanies this paper at httpsdoi101186s12885020071405additional file exercise programmeabbreviationscanmore cancer mobilisation rehabilitation cfir consolidated frameworkfor implementation research crf case report form cto clinical trials officehads hospital anxiety depression scale hrqol healthrelated quality of lifemct movement continuum theory ms meters per second phc primaryhealth care qol quality of life rarc roboticassisted radical cystectomyrct randomised controlled trial rr responsible researcher sbas stanfordbrief activity survey sec seconds spirit standard protocol itemsrecommendations for interventional trials 6mwt sixminute walk testacknowledgementsnot applicableauthors contributionsmh and ap conceived the idea for this study and designed it along with theother authors all authors ap pk er ma lh mnb and mh were involvedin drafting and revising the manuscript all authors will be involved in datacollection analysis or manuscript preparation as the study proceeds allauthors read and approved the final manuscri | 0 |
specialty sectionthis was submitted tomolecular and cellular oncologya section of the frontiers in cell and developmentalbiologyreceived april accepted july published august citationchen w yang j fang h li l andsun j relevance functionof lincror in the pathogenesisof cancerfront cell dev biol 103389fcell202000696cancer is a serious disease that aï¬ects human health being one of the main causes of death all overthe worldwide according to research in of new tumor cases and of the cancerassociated deaths occurred in lowincome and developing countries kumar and sharawat noorolyai owing to a shortage in eï¬ective screening methods and lack of identiï¬cationof early symptoms most patients were already in advanced stages when they were diagnosedwith cancer bray koo additionally some clinical studies have shownthat polarity and adhesion of cancer cells was decreased leading to heir increased mobility andinvasion which is a key step in the development of cancer yan therefore studies haveshown that the high mobility of cancer cells is the main factor leading to high mortality rates inpatients with cancer currently there are many ways employed in the treatment of cancer includingsurgery radiotherapy chemotherapy biotherapy and targeted therapy nie howeverin the past years the survival rate of patients with cancers remains dismal nakashima abbreviations bc breast cancer cenas competing endogenous rna emt epithelialmesenchymal transition hcchepatocellular cancer ipscs induced pluripotent stem cells lincror long intergenic nonprotein coding rna regulatorof reprogramming lncrnas long noncoding rnas pc pancreatic cancerfrontiers in cell and developmental biology wwwfrontiersinaugust volume 0cchen relevance function of lincrorin the process of developing human antitumorthereforestrategiesto ï¬nd new earlyitbiomarkers and thus identify potential regulatory mechanisms toimprove the survival rate of patients with cancersis particularly importantover the past decades ncrnas constitute more than of the rnas made from the human genome but mostof the known noncoding rnas ncrnas havebeen discovered and remain largely unstudied bhan slack and chinnaiyan transfer rna trna and ribosomal rna rrna constitute themajority of ncrnasfollowed in abundance by messengerrnas mrnas thus the remaining ncrnas includingcircular rna circrna small nuclear rna snrna smallnucleolar rna snorna microrna mirna and long nonfor ¼ ofcoding rna lncrna together accounttotalncrna despite their low abundancethese ncrnas havebeen reported to play critical roles in transcription posttranscriptional processing and translation such as epigeneticsposttranscriptional regulation chromatin modiï¬cation andregulation of the cell cycle huarte kondo peng 2017b additionally because ncrnas can be packagedinto extracellular vesicles ev including exosomes meldolesi they have been shown to provide a mechanism forintercellular communication through the transfer of mirnaand lncrna to recipient cells both locally and systemicallysun it is important to note that the expressionof ncrnas their posttranscriptional modiï¬cation particularlylncrnas and their subcellular distribution have been shownto be important to when assigning their potential functionpalazzo and lee recently nextgeneration sequencingand bioinformatics technology have revealed that circrnasplay crucial role in diagnosis and prognosis of various diseasespamudurti brieï¬y circrnas are singlestrandedtranscripts generated by backsplicing jeck and sharpless with covalently linked headtotail closed loop structures withneither 5cid483cid48 polarity nor a polyadenylated tail memczak that range in length from a few hundred to thousandsof nucleotides and are widely expressed in mammals therebyshowing higher stability compared to that in linear rnas chenj and exhibiting a celltype or developmentalstagespeciï¬c expression pattern barrett and salzman wang j j many functions of circrnas have alsobeen identiï¬ed including their role as mirna sponges bindingto rnabinding proteins and protein decoys and functioningas regulators of transcription hansen du yang y interestingly many circrnas havebeen shown to be dysregulated in pathophysiological processesand circrnas are known to regulate the expression of geneby acting as mirna sponges in a mechanism that is termedas competitive endogenous rna cerna mechanism zheng wang j j for example circmto1have been demonstrated to harbor conventional mirna bindingsites and has been identiï¬ed as an inhibitor of mirna9 inhepatocellular carcinoma hcc han additionallyour previous study has demonstrated that mirna plays arole in limiting the development of liver ï¬brosis by markedlyblocking the activation and proliferation of hepatic stellatecells hscs suggesting that mirnas might be involved inthe development and progression of several forms of cancersyang j yang of notelncrnaswhich are mainly transcribed by rna polymerase ii are anew kind of ncrna that are longer than nucleotidesma owing to the lack of open reading frameslncrnas have extremely limited or no protein coding capacityruan li j these new regulatorswere initially regarded as transcriptional noise with no speciï¬cbiologicalfunctions kim and sung recently ourlaboratory found that epigenetic silencing of lncrna anrilpromoted the progression of liver ï¬brosis thereby indicating thatlncrnas were associated with the progression of cancers yang interestingly increasing evidence have shown thatcellular events including diï¬erentiation proliferation invasionapoptosis and migration have all been associated with lncrnasguttman additionally there has been new evidencesuggesting that lncrnas may regulate a variety of biologicaland disease processes from gene transcription and translationto posttranslational modiï¬cations davalos and esteller pang more importantlylncrnas have beenreported to be used as tumor suppressor genes or oncogenesthus aï¬ecting the proliferation and metastasis of various typesof tumors during tumorigenesis chen q n lu subsequent studies have demonstrated thatlncrnas may serve as cernas for mirnas and in chromatinremodeling during the development of cancers huang wang c j figure illustrates the functionsof lncrnas at the molecular level regarding certain cancerassociated human lncrnasit was demonstrated that lincror was demonstrated to be predominantly upregulated intumors peng 2017a the abnormal expression of lincror in tumors has been suggested to be one of the mainleading factors driving the development the main ways torevert this eï¬ect would be to aï¬ect cell growth migrationand invasionthus leading to the inhibition of epithelialmesenchymal transition emt enhancement of the sensitivityto chemotherapy etc chen 2016a zhao forexample the expression level of lincror in hcc tissues wasinhibited compared with the adjacent tissues at the same timethe downregulation of lincror was linked to the aggressiveprocess of the disease in patients with hcc furthermore theability of migration and invasion of hcc cells may be delayedby the low expression level of lincror in this review weattempted to introduce the latest research on the biologicaleï¬ects potential clinical applications and molecular mechanismsof lincror in human tumors and discuss its prognostic andtherapeutic valuesoverview of lincrorlincror isamong lncrnasand importantcarcinogenic kb lncrna located in chromosome which was initially identiï¬ed as a highly expressed transcriptof pluripotent and embryonic stem cells chen 2016bstudies found that the octamerbinding transcription factor a novelfrontiers in cell and developmental biology wwwfrontiersinaugust volume 0cchen relevance function of lincrorfigure paradigms for the function of long ncrnas recent studies have identiï¬ed a variety of regulatory paradigms for the mechanism by which long ncrnasfunction many of which are highlighted here transcription from an upstream noncoding promoter pink can negatively or positively affect the expression ofthe downstream gene purple by inhibiting the recruitment of rna polymerase ii or inducing chromatin remodeling respectively an antisense transcript orangeis able to hybridize to the overlapping sense transcript purple and block the recognition of the splice sites by the spliceosome thus resulting in an alternativelyspliced transcript alternatively hybridization of the sense and antisense transcripts can allow dicer to generate endogenous sirnas by binding to speciï¬cprotein partners a noncoding transcript blue can modulate the activity of the protein serve as a structural component that allows the formation of a largerrnaprotein complex or alter where the protein localizes in the cell long ncrnas green can be processed to yield small rnas such as mirnaspirnas and other less wellcharacterized classes of small transcriptsoct4 srybox transcription factor sox2 and nanoghomeobox nanog key pluripotency factors could regulatelincror wang howeverlincror was alsofound to be expressed in several ans including lungliver breast and colon since its discovery research in thisï¬eld has been extensively expanded during the past yearsrevealing the important role of lincror in tumorigenesislincror has been suggestedadditionally upregulation ofto mediate the reexpression offetal and cardiomyocytehypertrophyrelated genes wang li inmany reportsrecent years have revealed thatlincror is positively correlated with the clinicopathologicalcharacteristics and poor prognosis of tumorsincluding thestages of advanced tumor node metastasis tnm positivelymph node metastasis lnm and lower survival rate buthigher recurrence rateregarding lincror and tumorigenesisthe upregulation ofcurrent evidence have strongly indicated thatlincrormay exert an impact on a variety of cancers pan furthermore both tumorigenesis and metastasis havebeen shown to be induced by lincror via activation of theemt in various cancers hou huang zhan for example lincror was demonstratedto be upregulated thereby promoting emt in hcc li j besides it was also reported that selfrenewal anddiï¬erentiation of glioma stem cells was signiï¬cantly aï¬ectedby lincror zhang feng moreimportantly the chemoresistance of pancreatic cancer pcand breast cancer bc li as well as radioresistance of colorectal cancer crc cells were observed to beelevated by lincror yang p moreover lincror has also been shown to exert a signiï¬cantly eï¬ect onthe stem celllike characteristics and tumorigenic potential ofpc recentlyit was also reported that lincror could beused as a biomarker in the ï¬eld of diagnosis and prognosisof bc and oral cancer arunkumar zhao notably increasing studies showed that lincror couldbe used as a cerna thus exerting its impact in the posttranscriptional network of tumor pathogenesis for examplein triplenegative bc lincror has been shown to serve asa cerna therefore promoting the migration and invasion ofbc cells signal overall lincror is a typicallncrna that plays important regulatory roles in interactionwith mirnas and maintenance of stem cell pluripotencytriggering the emt as well furthermore lincror has alsobeen involved in various key roles under hypoxia and in thepromotion of tumorigenesis figure therefore lincrormay be considered as an oncogene aï¬ecting the progressionof tumor and a promising predictor for the poor prognosis inpatients with cancer the transition of lincror from basicresearch to clinical application requires further investigations asearly as possiblefrontiers in cell and developmental biology wwwfrontiersinaugust volume 0cchen relevance function of lincrorfigure lincror is a typical lncrna that plays important regulatory roles in interacting with mirnas and maintaining stem cell pluripotency as well astriggering the emt as well lincror is also involved in various key roles under various stresses and in epigenetic regulationregulatory role of lincror invarious types of cancerincreasing evidence has shown that the lincror was abnormalexpression in many cancers and its dysregulation was associatedwith cellular functions fu spinelli additionally studies found that the expression level of lincror was substantially upregulated in samples of papillarythyroid carcinomas ptcs and ptcs cell lines as well as inmetastatic ptcs samples and ptcs cell lines zhang simultaneously cell migration and invasion could be regulatedby lincror via aï¬ecting emt pastushenko and blanpain more importantly studies demonstrated that lincrorwas abnormally expressed in several cancers and led to elevatedthe invasion and metastasis of cancer cells to promoting theprogression of tumors hashemian li 2020bcthis review summarizes the status of lincror research invarious human cancers and discusses its mechanism and clinicalsigniï¬cance in the development and progression of tumor theexpression pattern functional role and regulatory mechanism oflincror are summarized in table and depicted in figure breast cancerbc which accounts for a quarter of all female cancer casesis the most commonly diagnosed cancer and the leading causeof cancerassociated deaths among women worldwide li z in it was estimated that there would be million or so newly diagnosed cases of female bc bray hannafon the main risk factors forbc which is the diï¬cult to change due to prolonged exposureto endogenous hormones is diï¬cult to control rudel bray however comprehensive treatmentapproaches have resulted in relatively good clinical outcomesfor some patients with bc rudel goel kumler nevertheless it has been reported that aboutonethird of the patients with bc have the potential for cellmetastasis chemotherapy resistance and even recurrence goel kumler hence there is an urgent need todevelop new therapies targeting various molecular mechanismsof tumorigenesis for the treatment of bcthe level ofthe level ofhou the expression ofinvestigated the expression level of lincrorin patients hou their results revealedlincror was increased in bc tissuesthatmoreoverlincror in theperipheral blood ofthe patients with bc was shown tobe closely related to tnm phase and lnm in additionthe woundhealing assay showed that overexpression of lincror increased bc cells mcf10a mobility transwell assayrevealed that lincror overexpression remarkably increased themigration ability hou more importantly theyfound that ectopic expression of lincror induced an emtprogram in mcf10a cells fluorescence activated cell sorteranalysis demonstrated that the subpopulation of the stem cellphenotype cd44highcd24low was elevated in mcf10a cellstransfected with lincror plasmid mechanistically the resultsof bioinformatic analysis and rna immunoprecipitation analysisfrom hou demonstrated thatlincror functions asa cerna to regulate mir205 activity toward prevention ofthe degradation of transcripts of mir205 target genes suchas zeb1 and zeb2 from degradation additionallyit wasshown that the expression levels of mir205 members weredecreased upon lincror overexpression in mcf10a cellshou more importantly zhao recentlydemonstrated that crisprcas9generated brca1knockdownadiposederived stem cells stimulated a more aggressive behaviorfrontiers in cell and developmental biology wwwfrontiersinaugust volume 0cchen relevance function of lincrorlateillateoaglatenahzlatenehclatecillateihzlateilnusdnanaylatenapalategnahzlateuohsecnereferbezrmrorcniilmkppbtprmrorcniilgonanrmrorcniilnirehdacehzerorcnilsosfdmnlegatsmnttmeinosavnitmeinosavninoitargminoitarefilopicnegocnoldetaugerpuamoncraciraulllecotapehllseuceomyrotaugerllnoitaerroclacniilcleorlanoitcnufleorinosserpxesepytrecnacsrecnacnamuhnirorcnlielbatirmrorcnilevruccormnlegatsmntnoitargmiissotpopanoitarefilorpicnegocnoldetaugerpurecnactsaerbtmeinosavnipbezrorcnilliavvrusroopmnlegatsmntnoitargminoitarefilorphtwicnegocnoldetaugerpurecnaccitaercnapmxofprmrorcniilncsfrmrorcniprmrorcniililirmrorcnilecnatsserigurdtmeinosavnitmelecycllecinosavniliavvrusroopmnlegatsmntnoitargmiissotpopanoitarefilorpicnegocnoldetaugerpurecnacdoryhtisosfdmnlegatsmntinoitargmecyclllecissotpopaicnegocnoldetaugerpurecnacgnulecnatsseriyparehtodaritmeinosavnisosfdmnlegatsmntnoitargmiissotpopanoitarefilorpicnegocnoldetaugerpurecnaclatcerooclin bc cells than wildtype adiposederived stem cells zhao therefore we believe that crisprcas9 may beused to in the treatment of bc by inhibiting the expressionof lincror during the progression of bc conclusively thelincrormirnas axis has been reported to closely aï¬ect theoccurrence and development of bcpancreatic cancerpc is the fourth most common cause of cancerrelated mortalityworldwide leading to approximately deaths annuallysiegel sabater the year relativesurvival of patients with pc remained at approximately for siegel hence pc has been proposedto be one of the top two cancers in terms of fatalities in thenext decade rahib surgical resection remainsthe exclusive potential curative treatment xiong however approximately half ofthe patients present withmetastasis at the time of diagnosis missing the opportunityfor an eï¬ective treatment vincent xiong a growing body of literature has demonstrated that bothmetastasis and limited eï¬ective biomarker for the diagnosis andtreatment are the main obstacles for the eï¬cient medical therapyof pc vincent boj basuroy thus it is an absolute necessity to identify potential biomarkersand therapeutic targets in pczhan have highlighted the oncogenic eï¬ectsof lincror in the initiation and progression of pc theirstudy demonstrated that the level of lincror was signiï¬cantlyelevated in pc tissues zhan moreoverthewoundhealing assay and boydens chamber assay results showedthat lincror silencing reduced the migratory capability andmetastasis of pc cells zhan another study bychen showed that the proliferation rates of shrorcellsin which the level of lincror was suppressed were evidentlylower than those of shnccells this result was conï¬rmed bycolony formation assay suggesting that lincror accelerated thegrowth of pc cells chen 2016a interestingly silencingof lincror was shown to result in increased levels of theepithelial markers ecadherin and αcatenin and decreased levelsof mesenchymal markers ncadherin and vimentin indicatingthat lincror plays an important role in the regulation ofemt in pc cells zhan chen moreimportantly microarray analysis identiï¬ed zeb1 as potentialtarget gene of lincror further the expression of lincrorand zeb1 were observed to be negatively correlated with thatof p53 suggesting that lincror might mediate migration andmetastasis in pc cells may partly via activation of zeb1 throughthe inhibition of the expression of p53 zhan interestingly the ï¬uorescence in situ hybridization and luciferasereporter assay results showed that the expression of lincrorwas demonstrated to be negatively correlated to that of mir145mir145 can induce posttranscriptional silencing of its targetedgenes by binding to the mrna utr or lincror speciï¬csites indicating that lincror can act as a cerna to decreasemir145 in pc cells thereby activating expression of nanogthus leading to the proliferation of pancreatic cancer stem cellspcscs gao additionally li furtherfrontiers in cell and developmental biology wwwfrontiersinaugust volume 0cchen relevance function of lincrorfigure underlying molecular mechanisms of lincror in multiple cancers a lincror binds to mir205 to upregulate zeb2 while it also regulated theexpression of emt markers b lincror could interact with mir145 to inhibit fscn1 and upregulated emtassociated proteins while it decreases g0g1 arrestand facilitated drug resistance c lincror facilitated emt through upregulate ezhz and regulated zeb2 by competitively binding to mir145 and zeb2overexpression leads to increased emt in addition lincror binds to mir8765p to upregulate foxm1 d lincror downregulated through emt production andrepress the expression of mir145 e lincror binds to mir68333p while also regulating the process of emt f lincror upregulated zeb1 to attenuate theexpression of p53 while also decreasing the expression of mir145 to increase the level of nanog and reduce that of mir124 to suppress pkm2 detailedmechanisms of lincror in other cancers are provided in the reviewproved that the impact of lincror can be partly reversed byoverexpression of mir124 consistently lincror was observedto exhibited a negative correlation with the expression of mir li hence a lincrormir124ptbp1pkm2axis was identiï¬ed in pc shedding new light on the lncrnabased diagnosis and therapeutic approaches in pc li 2020b notably recent studies showed that pc cellderivedevs could be used as eï¬ective carriers of paclitaxel to theirparental cells thereby bringing the drug into cells through anendocytic pathway and increasing its cytotoxicity saari additionally it was demonstrated that vesiclecontainingncrnas could serve as evassociated pc detection markersworst thus the presence of lincrors in evs frompatients with pc could serve as a potential diagnostic biomarkerand a novel target for the therapy of patients with pc this isworthy of further and wider research attentionhepatocellular carcinomaas the sixth most international commonly occurring cancer in hcc has become the fourth cause of cancerassociateddeaths worldwide it has been estimated that new casesand deaths will occur each year bray brieï¬yhcc has been reported to account for of all the livercancer cases half of which have been detected in china omata bray as such hcc poses a huge threatto the worldwide health especially that of the chinese peopleomata about of the patients is expected torecrudescent within years after hepatectomy and of thepatients will die from this tumor vigano thereforeon the basis of studying the pathogenesis of hcc it is apparent tolook for more eï¬ective molecular markers and therapeutic targetsfor the management of hccli c and chen reported that theexpression level of lincror was obviously elevated in hcctissues and four cell lines compared to the corresponding nontumor tissues and normal liver cell lines respectively suggestingthat lincror might be critical regulator in the progression ofhcc furthermore biological function assay demonstrated thatlincror could play promoting role in regulating migration andinvasion of hcc chen moreover downregulationof lincror could result in a signiï¬cant increase in g1g0phase and an obvious decrease in s phase li c more importantly silencing of lincror could lead to theincreased expression of ecadherin and decreased expressionlevel of ncadherin in hcc cell lines li c furtherconï¬rmed that lincror could bind to the zeste homolog ezh2 thereby aï¬ecting the expression of ecadherin furtherindicating that lincror could regulate the progression of emtmoreover lincror was further determined to be associatedwith dna repair currently mounting studies have identiï¬edreliable indicators of dna damage such as phosphorylatedhistone h2ax γh2ax chen uncover thatfrontiers in cell and developmental biology wwwfrontiersinaugust volume 0cchen relevance function of lincrorlincror could obviously decrease theoverexpression ofexpression level of γh2ax illuminating the suppressive eï¬ectsof the overexpression of lincror on dna repair in hccfurther research demonstrated that lincror could interactwith mir and dramatically downregulate the expression ofmir in hcc cells li c the abovementionedresults revealed that lincror might play a promoting role inthe proliferation migration invasion and emt of the hcc cellwhich was contrary to the inï¬uence of mir enrichmentli c it suggested that overexpressed mircould eï¬ectively reverse the promotion of hcc tumorigenesisinduced by the overexpression of lincror li c liand his colleagues proposed a mechanistic model that lincrorpromotes hcc tumorigenesis and autophagy partly throughnegatively regulating the expression of mir aside fromthat it has been reported that mir145 represses emt tumormigration and invasion by directly targeting the utrs ofzeb2 in the tumor the decrease in mir and increase inzeb2 can obviously reversed the inhibition of cell migrationand invasion mediated by the lincror knockdown thereforeit was suggested that targeting the lincrormir145zeb2axis might represent a novel therapeutic application in hccli c similarly zhi similarly showedthat the migration and invasion of cells was reduced by theknockdown of lincror moreover they further conï¬rmedthat foxm1mediated activation of lincror contributes tothe poor sensitivity of hcc cells to sorafenib via partiallyregulating the mir8765pfoxm1 axis which forms a positivefeedback loop further evaluation of the regulatory mechanisminvolving this axis may provide new insights for exploringa potential therapeutic strategy for the management of hcczhi consequently these studies may oï¬er newinsights regarding the pathology of hcc and provide potentialstrategies for lncrnadirected treatment however both thein vivo inï¬uence and other underlying mechanisms of lincrorstill remain to be determined and clariï¬ed in the future researchthe prognosiscolorectal cancerthere are approximately million new crc cases and crcrelated deaths worldwide each year thus making crc thethird most common cancer in the world torre although the treatment of crc has signiï¬cantly improvedin recent decadesremains unsatisfactoryespecially in case of advanced tumors with distant metastasesbogousslavsky torre current studiesresults showed that approximately of cases with crchave synchronous liver metastases during the time of diagnosiskawaguchi these patients have inherently lowsurvival rates of less than within years with an even worseprognosis hu kawaguchi thus there isan urgent need to better understand the progression of crc andto identify novel and sensitive biomarkers for the diagnosis andtreatment of patients with crcyang detected the expression of lincror in crctissues compared to normal tissues by using qrtpcr theyfound that the expression of lincror was remarkably increasedin crc tissues compared with normaltissues similarlylincror was shown to be overexpressed in ï¬ve crc cell linesyan and sun li 2020a then they also performeda series of functional assays to clarify the biological eï¬ects ofthe aberrant expression of lincror on proliferation viabilityapoptosis migration and invasion of crc cells knockdownof lincror was shown to eï¬ectively inhibit the proliferationof crc cells whereas its overexpression obviously increasedthe proliferative capacity of crc cells accordingly silencing oflincror strongly inhibited the migratory and invasive abilitiesof crc cells compared with that in the control cells li 2020a in contrast the migratory and invasive ability of cells wasactivated following the overexpression of lincror the mts345dimethylthiazol2yl53carboxymethoxyphenyl24sulfophenyl2htetrazolium assay results showed thatthelincror could enhance the viability ofoverexpression ofcrc cells furthermore the ï¬ow cytometric analysis resultsrevealed that the percentage of apoptotic cells in lincroroverexpression group was reduced by ± indicatingthat the overexpression of lincror could inhibit apoptosisin the crc cell lines li 2020a more importantly arecent study revealed the role of lincror in the emt it wasrevealed that the upregulation of lincror could increase theexpression of ncadherin and vimentin as well as decrease thelevel of ecadherin leading to the promotion of the progressionof emt zhou yan and sun meanwhilethe high expression of lincror in crc was also conï¬rmedby hu mechanistically li 2020a provedthat that lincror could bind to mir68333p which wasdetermined to be signiï¬cantly downregulated in crc tissuesadditionally a negative correlation was exhibited between theexpression of lincror and mir68333p in bc tissues antiago2 rna immunoprecipitation assay further conï¬rmed theseresults li 2020a besides rescue assays demonstratedthat downregulation of mir68333p could partly reversed theinhibition of tumorigenesis induced by lincror knockdown inbc cells li and his colleagues uncovered that lincror exertedits oncogenic role through negatively regulating the expressionof mir68333p during the progression of crc which mightgive new insights into molecular diagnosis and treatment li 2020a in addition li 2020a further uncoveredthat lincror could mediate the expression level of smc bysponging mir68333p in crc cells thus promoting crcprogression as for the eï¬ects of lincror on radiotherapyresistance yan and sun showed that overexpression oflincror increased the ability of crc cells for radiotherapyresistance collectively these ï¬ndings indicated that lincrormight be engaged in the metastatic process of crc cells andcould promote the development of crc through a variety ofmolecular mechanismslung cancerlung cancer is the leading cause of cancerrelated deathsworldwide bray nonsmall celllung cancernsclc accounts for about of the lung cancer typesincluding squamous cell carcinomalung cancerand lung adenocarcinoma herbertz bray brainard and farver although there are variousapproaches for its diagnosis and treatments the 5year overallsurvival os rate for patients with advanced lung cancer is lesslarge cellfrontiers in cell and developmental biology wwwfrontiersinaugust volume 0cchen relevance function of lincrorthan zhou therefore in order to carry outan early diagnosis and treatment of lung cancer the search forvaluable and eï¬cient tumor markers is very urgentin recent yearslincror has appeared as an importantregulator oflung cancer research from qu demonstrated that the expression of lincror was increasedin nsclc tissues compared to that in the normal tissuesthe overexpression of lincror was shown to be closely relatedto the poor prognosis of lnm histological grade and stageof tnm pan qu another study bypan demonstrated that the decreased expressionof lincror could obviously impair the proliferative capacityof lung adenocarcinoma lad cells cause a g0g1 phasearrest and increase the ratio of apoptotic lad cells moreoverdownregulation of lincror substantially inhibited the invasiveand metastatic ability of lad cells meanwhile forced expressionof lincror was observed to reduce the expression of ecadherinand βcatenin which are the characteristic biomarkers ofepithelial cells whereas it increased the expression of ncadherinand vimentinthus displaying a mesenchymal phenotypeconversely downregulation of lincror was demonstrated toresult in increased the expression of epithelial markers anddecreased the expression of mesenchymal markers this resultuncovered the fact that the prometastatic eï¬ects of lincrorwere induced by the regulation of the expression of a number ofgenes involved in cell metastasis and emt progress meanwhileoverexpression of lincror was shown to enhance the resistanceof lad to docetaxel dtx pan suggestingthat lincrorinduced resistance of lad cells to dtx andemt through regulation the expression of mir145 whichwas predicted to interact with lincror more importantlyfurther research conï¬rmed that mir145 could bind to lincror and its downregulation could partly inhibit the resistanceof lad cells to dtx and emt aside from that pan andhis colleagues discovered that a decrease in the expression ofmir145 and increase in the expression fscn1 could obviouslyreverse the inhibition of cell proliferation chemoresistanceand emt mediated by lincror knockdown they identiï¬edthat dysregulation of the lincrormir145fscn1 axis wasassociated with the therapeutic resistance and emt transition inlad cells thereby providing potential therapeutic strategies formanaging drug resistance in patients with lad pan taken together these studies collectively suggested that lincrorcould activate the malignant phenotype of nsclc cells with theguidance of a mechanism involving mirnas hence more eï¬orts | 0 |
Purpose Pancreatic ductal adenocarcinomas exhibit a high degree of desmoplasia due to extensive extracellular matrix deposition Encasement of mesenteric vessels by stroma in locally advanced pancreatic cancer LAPC prevents surgical resection This study sought to determine if the addition of a monoclonal antibody to connective tissue growth factor pamrevlumab to neoadjuvant chemotherapy would be safe and lead to improved resectability in this surgically adverse patient populationMethods In this phase III trial patients with LAPC were randomised to gemcitabinenab paclitaxel plus Arm A n24 or minus Arm B n13 pamrevlumab Those who completed six cycles of treatment were assessed for surgical eligibility by protocol defined criteria Resection rates progression free and overall survival were evaluatedResults Eighteen patients in Arm A and seven in Arm B completed six cycles of therapy with similar toxicity patterns In Arms A and B carbohydrate antigen response as defined by ¥ decline from baseline occurred in and respectively Sixteen per cent of patients were radiographically downstaged by National Comprehensive Cancer Network criteria in Arm A and in Arm B Positron emission tomography normalised in vs of patients in Arm A vs Arm B respectively and correlated with surgical exploration Eligibility for surgical exploration was vs p00019 and resection was achieved in vs of patients in Arm A vs Arm B p01193 respectively Postoperative complication rates were not different between armsConclusions Neoadjuvant chemotherapy with pamrevlumab holds promise for enhancing resection rates in patients with LAPC without added toxicity This combination merits evaluation in a larger patient cohortIntRoduCtIonPancreatic cancer is currently the third leading cause of cancer death in the USA1 and by it will likely become the second leading cause of cancer related death after Key questionsWhat is already known about this subject º Pamrevlumab is anti CTGF1 inhibitor highly safe when given to patients with pancreatic cancer and potentially adds to the activity of gemcitabine and erlotinib when given in metastatic diseaseWhat does this study add º This study examines a the safety and activity of pamrevlumab when added to gemcitabine and nab paclitaxel in locally advanced pancreatic cancer b the impact of this regimen and surgical resection and postoperative safety c explores the utility of a novel study design for locally advanced pancreatic cancerHow might this impact clinical practice º This study has the potential to lead to practice changing activity in locally advanced pancreatic cancer via the eventual approval of pamrevlumab for use in this situation the promulgation of a new study design for locally advanced pancreatic cancer and increased potential for surgical resection and thus prolonged OS curability in locally advanced pancreatic cancerlung cancer surpassing breast and colon cancer2 Surgical resection is generally necessary for treatment with curative intent or to extend life expectancy3 However only of patients have disease amenable to upfront curative resection at the time of diagnosis4 Approximately of patients are diagnosed with locally advanced disease5 determined surgically unresectable per National Comprehensive Cancer Network NCCN guidelines6 Patients with locally advanced pancreatic cancer LAPC have a prognosis similar to those with metastatic disease with a historical median overall survival OS of Picozzi a0V et a0al ESMO 20205e000668 101136esmo 2019000668 0c access months with recent trials demonstrating median OS of months7 Recent single institution retrospective studies have reported the potential for resection of LAPC with neoadjuvant therapy irrespective of imaging findings with promising results8 However these are limited by significant selection bias lack of strict radiographic classification and chemotherapy standardisation Current prospective trials have documented resection rates of LAPC in the range of to therefore novel approaches are needed to improve patient outcomesThe tumour biology inherent to pancreatic ductal adenocarcinoma PDAC significantly contributes to the poor outcomes seen in this disease Notably PDAC exhibits a high degree of desmoplasia often in association with elevated connective tissue growth factor CTGF expression12 CTGF appears to play a central role in the biology of pancreatic cancer affecting both its cellular biology and its extracellular matrix composition This leads to many simultaneous biological effects that promote pancreatic cancer growth including increased cellular proliferation and differentiation increased cellular adherence and migration antiapoptosis vascular permeability angiogenesis and suppression of tumour immunological responses13 This stroma may also contribute to the radiographic imaging findings of mesenteric vessel involvement or encasement that is used to determine resectability of pancreatic tumours Executing a pharmacological intervention on the pancreatic cancer stromal environment is therefore a major goal of the development of novel pancreatic cancer therapeuticsPamrevlumab is a human monoclonal antibody that targets CTGF Preclinical studies showed that CTGF overexpression is associated with both desmoplasia and gemcitabine resistance in the KPC pancreatic cancer mouse model14 When pamrevlumab was used in combination with gemcitabine sensitivity to gemcitabine was enhanced which correlated with inhibition of XIAP an antiapoptotic protein15 When tested in patients with advanced pancreatic cancer Stage IV and locally advanced Stage III treated with gemcitabine and erlotinib in a phase III study n75 pamrevlumab displayed multiple favourable outcomes16We hypothesised that through inhibition of the downstream effects of CTGF overexpression on tissue adhesion and other mechanisms pamrevlumab may influence resectability of PDAC tumours With this in mind this novel phase III randomised multicentre trial was designed to explore the safety and efficacy of pamrevlumab in combination with gemcitabinenab paclitaxel in LAPC with special emphasis on surgical eligibility and safetyMetHodsstudy designThis was a phase III randomised trial of safety and efficacy in patients with LAPC who received gemcitabine and nab paclitaxel with or without pamrevlumab as neoadjuvant therapy The randomisation was preplanned and blinded to the investigator The study was approved by individual institutional review boards at nine US institutions and conducted according to the Declaration of Helsinki The trial was registered at clinicaltrials gov as NCT eligibilityKey protocol eligibility requirements included biopsy proven diagnosis of PDAC radiographic staging consistent with locally advanced unresectable disease as defined NCCN guidelines V2 clinical stage confirmed by diagnostic laparoscopy radiographically measurable disease per Response Evaluation Criteria in Solid Tumors RECIST V11 Eastern Cooperative Oncology Group ECOG performance status of or adequate haematological renal and hepatic function no prior therapy for PDAC and no concomitant cancer diagnosis within the past yearsstudy schemaEligible patients were randomised to Arm A or Arm B to receive a total of six treatment cycles weeks of therapy figure Patients in Arm A received pamrevlumab mgkg by intravenous infusion on Days and of each day cycle with an additional dose given on Day in the first cycle Patients in both Arms A and B received gemcitabine mgm2 by intravenous infusion on Days and of each day treatment cycle nab paclitaxel mgm2 by intravenous infusion on Days and of each day cycle Doses for gemcitabine and nab paclitaxel were modified for haematological and non haematological toxicity as per standard of care SOC15 Patients remained on therapy for six treatment cycles weeks unless they had disease progression an intolerable adverse event AE or toxicity withdrew consent or were withdrawn at the investigators discretion All patients were followed for drug toxicity until days after the last drug dose Patients undergoing surgery were followed for days following hospital discharge for surgical complications CTGF levels were obtained prior to treatment from all patients Plasma samples for pamrevlumab level determination were obtained from all patients receiving this drug After all protocol specified therapy was completed patients were followed for disease progression survival and additional oncological therapy Postoperative complications including day readmissions and day mortality were notedResponse assessmentPatients were evaluated for response by the following measures carbohydrate antigen CA measured at baseline first day of each cycle and end of treatment EOT RECIST V11 read based on full body CT imaging high resolution dual phase fine cut CT imaging at baseline and every weeks thereafter fluorodeoxyglucose FDG positron emission tomography PET imaging and NCCN V2 resectability criteria at baseline and EOTPicozzi a0V et a0al ESMO 20205e000668 101136esmo 2019000668 0c accessFigure Patient flow and surgery outcomes In Arm A four of the eligible subjects had their surgeries cancelled 1portal vein thrombosis 3medical issues precluding surgery In Arm A four eligible subjects underwent surgery but resection was not achieved 3metastatic disease discovered 1extensive SMA encasement In Arm B one eligible subject underwent surgery but resection was not achieved 1extensive vascular encasement SMA superior mesenteric arterysurgical assessmentSubjects who finished six cycles of combination chemotherapy were evaluated for eligibility for surgical exploration per protocol PP defined criteria Given that patients included in the trial were determined to be initially unresectable by radiographic imaging and NCCN criteria objective criteria were developed to standardise attempts at surgical resectionPatients were deemed eligible for surgery if one or more of the following criteria were met reduction in plasma CA level by ¥ at EOT compared with baseline reduction in FDG PET maximum standardised uptake value SUVmax by ¥ at EOT compared with baseline radiological tumour response per RECIST of partial response PR or complete response CR at EOT or met the definition of resectable or borderline resectable per NCCN guidelines Subjects were classified as ineligible for surgical exploration if any of the following occurred development of distant metastases or local progression on CT scan tumour anatomy precluding vascular reconstruction unreconstructible local complications preventing surgery eg portal vein PVsplenic vein thrombosis pancreatitis or decline in performance status to a Karnofsky score ¤ or Picozzi a0V et a0al ESMO 20205e000668 101136esmo 2019000668absolute contraindication to surgery from comorbidity eg recovery from myocardial infarction or uncontrolled diabetes The final decision regarding whether resection was to be performed was made by the treating surgeonendpointsSafety endpoints included serious adverse events SAE during neoadjuvant therapy and surgical complications postresection The efficacy endpoints included surgical eligibility R0 resection R0R1 resection median OS progression free survival PFS and year survival rate All patients were followed and data analysis was stratified by PP population and intention to treat ITT cohortstatistical considerationsThe comparison between selected clinical characteristics toxicity profiles and eligibility for surgical exploration or completed surgical resection was performed using the ϲ test Exact CIs for the point estimates as well as the treatment difference were obtained from the SAS PROC FREQ procedure with the EXACT option The two treatment arms were compared using the Cochran Mantel Haentzel test controlling for baseline factors TNM stage ECOG CA PET SUVmax 0c accesssuperior mesenteric artery SMA involvement coeliac abutment and so on as prespecified in the protocol All cause mortality was used in determining OS which was analysed by the Kaplan Meier method Survival status was updated within month before the data cut off date Data from patients who were alive at the cut off date were censored for survival analysis All statistical tests were performed at the significance level of α005 using two sided testsResultsPatient characteristics and dispositionThirty seven patients were randomised to study treatment to Arm A pamrevlumabgemcitabinenab paclitaxel and to Arm B gemcitabinenab paclitaxel alone Patient characteristics at baseline are summarised in table All patients enrolled were unresectable by NCCN criteria patients had tumour arterial involvement SMA encasement ° coeliac abutment Table Patient characteristicsBaseline demographics years years ¥ years Median Male FemaleAge group Sex BMI kgm2 Mean SD Median Min maxECOG Grade Grade TNM stage T3 N0 M0 T3 N1 M0 T4 N0 M0 T4 N1 M0 T4 NX M0Location of the tumour in the pancreas Non resectability per NCCN criterion Head Body Tail Median tumour size mm ° SMA encasement Any coeliac abutment Inferior vena cava invasion or encasement Unreconstructible SMVportal occlusion Aortic invasion and encasementArm AGNPPN24 Arm BGNPN13 TotalN37 to to to · OK as isNot mutually exclusiveBMI body mass index ECOG Eastern Cooperative Oncology Group G gemcitabine n number of subjects NCCN National Comprehensive Cancer Network NP nab paclitaxel P pamrevlumab PV portal vein SMA superior mesenteric artery SMV superior mesenteric veinPicozzi a0V et a0al ESMO 20205e000668 101136esmo 2019000668 0c accesswithout gastroduodenal artery involvement or aortic involvement inferior vena cava invasion and unreconstructible PVsuperior mesenteric vein SMV occlusion A higher percentage of patients with SMA encasement ° were randomised to Arm A vs Arm B Patient disposition is summarised in figure Twenty four patients in Arm A received gemcitabinenab paclitaxel and pamrevlumab patients completed six treatment cycles Six patients discontinued treatment early due to progressive disease three patients AEs two patients or physician decision one patient Thirteen patients in Arm B received gemcitabinenab paclitaxel patients completed six treatment cycles Six patients discontinued treatment early due to progressive disease two patients AEs two patients or patientphysician decision two patientssafetySAEs are summarised in table Forty one per cent of patients had a treatment emergent SAE Arm A Arm B No individual toxicity category occurred with frequency except systemic infection patients There was no demonstrable increase in any toxicity with the addition of pamrevlumab to gemcitabinenab paclitaxel chemotherapyTable Summary of treatment emergent serious adverse eventsSystem organ classpreferred term Ascites Nausea Pancreatitis Vomiting Device occlusion Drug withdrawal syndrome FeverNo of patients with any treatment emergent SAEBlood and lymphatic disorders Haemolytic uremic syndrome LymphadenopathyCardiac disorders Cardiac failure Supraventricular tachycardiaGastrointestinal disorders General disorders and administrative site conditions Hepatobiliary disorders Infections Sepsis Cellulitis Urinary tract infectionInjury poisoning and procedural complications Respiratory thoracic and mediastinal disorders Skin and subcutaneous disorders Cholangitis Hyperbilirubinaemia Craniocerebral injury Pneumonitis Pulmonary embolism RashArm An24n Arm Bn13n Overalln37n · OK as isPicozzi a0V et a0al ESMO 20205e000668 101136esmo 2019000668 0c accessResponse to therapyIn Arm A had ¥ CA decline at EOT response by RECIST PR ¥ decline in PET SUVmax and were radiographically downstaged by NCCN criteria During the treatment period the median CA decline was patients were non secretors Seven out of patients had best objective RECIST response CRPR Some patients had exceptional responses defined as normalisation or ¥ decline of CA patients or normalisation PET SUVmax in In Arm B had ¥ CA decline at EOT response by RECIST PR ¥ decline in PET SUVmax and were radiographically downstaged by NCCN criteria Four out of patients had best objective RECIST response CR PR In Arm B of patients had an exceptional CA response and had an exceptional PET response as defined by either ¥ normalized Ca response normalized SUV max andorradiographic downstaging post therapy completion surgical evaluationOverall of the total study patients were eligible for surgical exploration using protocol defined criteria Arm A Arm B p00019 Resection was completed in of the patients Arm A Arm B p01193 Details of the nine resected patients are shown in table In Arm A of the patients were eligible for surgical exploration in the ITT population and of the patients were eligible in the PP population patients who completed six cycles of treatment In Arm A out of eligible patients ultimately underwent surgical exploration for resection five operations were cancelled four due to drug toxicitymedical comorbidity sepsis gallbladder perforation declined performance status and fever and one patient declined Eight out of patients in Arm A were resected R0 R1 The remaining four patients who were explored were not resected due to progression or unresectable disease intraoperatively In Arm B of the patients were eligible for surgical exploration in the ITT population and were eligible in the PP population Of the two subjects found to be surgically eligible only one was resected as the other patient had local progressionPredictors of resectionHigh CA response ¥ decline andor normalisation was contributive to surgical eligibility vs p03 Normalisation versus non normalisation of PET SUVmax was predictive of surgical eligibility vs p0013 and successful resection vs p0002 Combining these two criteria was highly predictive for surgical eligibility vs p0003 and completed vs p0002 resection All nine successful resections were identified by one or both of these criteria Table Summary of resected patientsSitesubject IDTreatmentarmResponse to treatmentNCCNbaselineNCCNend of treatmentResection statusAAAAAAAAB UnresectablecoeliacUnresectableSMA SMVUnresectablecoeliacUnresectablecoeliacUnresectableSMVUnresectableSMAUnresectableSMA SMV coeliacUnresectableSMAUnresectablecoeliacUnresectablecoeliacUnresectableSMA SMVUnresectablecoeliacBorderline resectableUnresectableSMVUnresectableSMAUnresectablecoeliacUnresectableSMAUnresectablecoeliacR0R1R0R0R1R1R1R0R0Protocol defined criteria CA decrease FDG PET SUVmax decrease ¥ RECIST V11 response PR or CR NCCN resectable or borderline resectable criteriaCA carbohydrate antigen CR complete response FDG fluorodeoxyglucose NCCN National Comprehensive Cancer Network PET positron emission tomography PR partial response RECIST Response Evaluation Criteria in Solid Tumors SMA superior mesenteric artery SMV superior mesenteric vein SUVmax maximum standardised uptake valuePicozzi a0V et a0al ESMO 20205e000668 101136esmo 2019000668 0cConversely radiographic features of response did not correlate with operative potential Neither RECIST response nor radiographic downstaging per NCCN criteria statistically correlated with completed resectionsurgical complicationsPostoperative complications were summarised according to the Clavien Dindo classification posthoc analysis Ischaemic gastritis and ulceration and right lower lung lobe collapse were reported for one patient in Arm A Grade II There was one episode of clinically significant pancreatic leak in each arm Grade IIIA no reoperations and no day or day surgical mortality were noted One patient in Arm B had a delayed gastric perforation following a distal pancreatectomy with coeliac axis resection likely due to thermal injury and was treated non operatively Grade IIIB No wound complications or superficial site infections were noted in either group Four out of patients and out of patients in Arm A and B respectively were readmitted within days and the difference between treatment arms was not clinically or statistically significantsurvivalAs of the data cut off date of evaluable patients were known to be alive with a median length of follow up at approximately months PFS was months CI to and months CI to in Arm A and Arm B respectively One year survival and median OS were and months CI accessto in Arm A and and months CI NR in Arm B The median OS for all patients who were eligible for surgical exploration Arm A Arm B vs ineligible Arm A Arm B was months CI NR vs months CI to p00766 The median OS for resected Arm A Arm B vs non resected patients Arm A Arm B was not reached CI NR vs months CI to p00141 figure dIsCussIonThe treatment of LAPC with neoadjuvant therapy remains challenging and there is no established SOC Several high volume centres have reported their single centre experiences with varying neoadjuvant chemotherapy and chemoradiation strategies8 The combination of more active regimens delivered over an extended period and surgeons comfort with resecting and reconstructing major mesenteric vessels has led to an increase in resection rates A meta analysis of studies using FOLFIRINOX has demonstrated resection rates ranging from to in LAPC17 One of the larger studies including patients with LAPC reported a resection rate of that was more dependent on duration of therapy months than chemotherapy regimen FOLFIRINOX or gemcitabine based18 Recently a single institution and single arm prospective study of neoadjuvant FOLFIRINOX and losartan with selective use of radiation in patients with LAPC reported an R0 resection rate of Figure Overall survival Resected vs Non resected patientsPicozzi a0V et a0al ESMO 20205e000668 101136esmo 2019000668 0c access However the lack of randomisation makes it difficult to determine what aspect of therapy was responsible for this effect and only of resected patients needed any type of vascular reconstruction perhaps suggesting more favourable outcome than usually seen in locally advanced disease These retrospective studies contain significant interpretative challenges including selection bias treatment variables including radiation and the use of different definitions of LAPCthe anti CTGF mechanism of action With respect to gemcitabine based therapy a recent large scale prospective trial of patients with LAPC treated with induction gemcitabine with or without erlotinib followed by radiation only patients were able to undergo resection10 Furthermore the addition of erlotinib to gemcitabine did not improve any downstaging capacity and there was no difference in survival with the addition of radiation More recently the LA PACT trial examining the role of induction gemcitabine nab paclitaxel found that only out of patients with LAPC were able to undergo surgery following neoadjuvant therapy with combination gemcitabine and nab paclitaxel for six cycles by investigators choice11 Last although FOLFIRINOX has been the most studied induction combination chemotherapy regimen in this population recent randomised data from European patients who received neoadjuvant FOLFIRINOX versus gemcitabinenab paclitaxel prior to resection20 showed no clear difference with respect to R0R1 to resection rate vs p0135 or OS vs months p0268Given for pamrevlumab its use in the neoadjuvant setting has the potential to impact tumour regression and modulate the desmoplastic niche and possibly affect tumour margins allowing for improved resection rates Previous studies have looked at the ability of gemcitabinenab paclitaxel to reduce cancer associated fibroblasts resulting in a softening of tumours by endoscopic ultrasound elastography21 This stromal depletion also translated into a decrease of SUV uptake on PET22 In the study reported herein we combined gemcitabine and nab paclitaxel with pamrevlumab to explore its effect in terms of therapeutic response the impact on eligibility for surgical exploration and improved resection rates in locally advanced patientsThe protocol specified therapeutic response criteria CA PET SUVmax RECIST and NCCN criteria were used as criteria to determine eligibility for surgical exploration in LAPC This is a novel approach specific to the protocol and allows participating patients to be explored for resection when otherwise they may not qualify by current treatment standards NCCN criteria For example by NCCN conversion alone ie converted from unresectable to borderline resectable only of patients in Arm A would have been eligible for surgical exploration However by protocol criteria of patients in Arm A were eligible for surgical exploration A higher percentage of patients were eligible for surgical exploration by the above criteria in Arm A vs Arm B vs respectivelyOverall the rate of successful resections in the pamrevlumab treated group was higher than in the control group but this did not reach statistical significance most probably due to small sample size Of the nine subjects that were successfully resected in this trial only one was converted by NCCN criteria to borderline resectable prior to surgical exploration Despite this phenomenon the data support the hypothesis that pamrevlumab a human monoclonal antibody with anti CTGF mechanism of action could alter tumour characteristics allowing resection in otherwise unresectable patients This hypothesis needs to be confirmed and patients should be stratified by coeliac andor SMA involvementThe most common predictive factors for eligibility for surgical exploration and resection were CA decline and PET SUV max response which are indicators of tumour response to treatment The combination of these two factors proved to be a highly sensitive objective readout for prediction of potential surgical success Both the ability of CA response and the inability of radiographic response RECIST and NCCN criteria of resect ability to predict surgical outcome has been observed by others3 and these observations deserve further examination in subsequent clinical trials In the MPACT study both CA and PET response correlated to improved survival in metastatic patients treated with gemcitabine and nab paclitaxel23 Recent surgical series of patients with borderline resectable and LAPC have also corroborated their impact in the localised setting25 Correlation of clinical response with plasma levels of endogenous CTGF and pamrevlumab exposure as shown in the prior study by Picozzi et al16 may provide added prognostic and predictive insightWith regard to safety no major incremental toxicity in any category was noted with the addition of pamrevlumab to gemcitabinenab paclitaxel In addition a higher number of patients were able to complete six cycles of the three drug combination including pamrevlumab when compared with gemcitabinenab paclitaxel alone Pamrevlumab is well tolerated and considered safe compared with the SOC drugs for patients with PDAC These observations represent a very favourable attribute when considering potential neoadjuvant chemotherapy in a patient population with the frequency of medical problems typically seen in LAPC In addition there were no signals of increased surgical morbidity or wound healing problems with CTGF blockade by pamrevlumab In fact there were only two clinically significant pancreatic leaks one in each arm which is comparable to national outcome data from high volume pancreatic surgery centres Similarly readmissions following resection were comparable between arms and reflected the complexity of this challenging patient populationFinally while survival data are not yet mature both patients who were eligible for surgery and those that Picozzi a0V et a0al ESMO 20205e000668 101136esmo 2019000668 0cwere ultimately resected had longer PFS and OS highlighting the importance of surgical resection of the tumour Therefore more investigation into newer agents targeting LAPC and increased consideration of candidacy for surgery in those patients who do not progress on therapy or suffer toxicity should be of utmost importance to improve outcomes in this diseaseIn conclusion this is the first prospective randomised multicentre trial examining the role of neoadjuvant therapy in LAPC with prespecified criteria for surgical exploration The use of pamrevlumab in combination with gemcitabine and nab paclitaxel showed a potential to enhance tumour response and increase resection rates Further evaluation of this drug combination in the neoadjuvant treatment setting for LAPC is warranted and a larger phase III trial with resection and survival endpoints is ongoingContributors FibroGen Inc was the study sponsor that designed the study in consultation with the Principal Investigator VP and surgical co investigator FGR All authors except those of the sponsor contributed patients to the study FibroGen was responsible for data collection and analysis All authors reviewed the manuscript and signed off on its accuracyFunding The study was funded by FibroGen Inc San Francisco CAdisclaimer The corresponding author has the right to grant on behalf of all authors and does grant on behalf of all authors an exclusive licence or non exclusive for government employees on a worldwide basis to the BMJ Publishing Group Ltd and its Licensees to permit this article if accepted to be published in ESMO editions and any other BMJPGL products to exploit all subsidiary rights as set out in our licenceCompeting interests MC MZ SP EK and EC are employees of FibroGen and hold stock andor stock options | 2 |
"Cardiac arrhythmias Atrial ï¬brillation Sudden cardiac death Long QT syndrome Torsade des pointes Ventricular tachycardia Ventricular ï¬brillation As the coronavirus COVID19 pandemic marches unrelentingly more patients with cardiac arrhyth mias are emerging due to the effects of the virus on the respiratory and cardiovascular CV systems and the systemic ammation that it incurs and also as a result of the proarrhythmic effects of COVID19 pharmacotherapies and other drug interactions and the associated autonomic imbalance that enhance ar rhythmogenicity The most worrisome of all arrhythmogenic mechanisms is the QT prolonging effect of various antiCOVID pharmacotherapies that can lead to polymorphic ventricular tachycardia in the form of torsade des pointes and sudden cardiac death It is therefore imperative to monitor the QT interval dur ing treatment however conventional approaches to such monitoring increase the transmission risk for the staff and strain the health system Hence there is dire need for contactless monitoring and teleme try for inpatients especially those admitted to the intensive care unit as well as for outpatients needing continued management In this context recent technological advances have ushered in a new era in im plementing digital health monitoring tools that circumvent these obstacles All these issues are herein discussed and a large body of recent relevant data are reviewed Elsevier Inc All rights reserved Introduction The ongoing pandemic of coronavirus disease COVID19 has created a global tumult [] According to current data of patients with COVID19 infection are aï¬icted by acute my ocardial injury with an attendant higher mortality rate compared with those without cardiac injury commensurate with the degree of cardiac troponin cTn elevation [] Furthermore of patients develop cardiac arrhythmias Table including malig nant ventricular arrhythmias VAs [ ] with a higher prevalence noted in patients admitted to the intensive care unit ICU [] Importantly clinically stable patients may have a low preva Abbreviations AAD antiarrhythmic drug AF atrial ï¬brillation APCs atrial pre mature complexes AZM azithromycin COVID19 coronavirus CQ chloro quine cTn cardiac troponin CV cardiovascular CYP cytochrome P450 ECG elec trocardiogram HCQ hydroxychloroquine ICU intensive care unit LQTS long QT syndrome NSVT nonsustained ventricular tachycardia OOHCA outofhospital cardiac arrest SCD sudden cardiac death TdP torsade des pointes VAs ventricular arrhythmias VF ventricular ï¬brillation VPCs ventricular premature complexes VT ventricular tachycardia Corresponding author Email address asmotenetgr AS Manolis lence of arrhythmias [] however critically ill patients are at much higher risk for cardiac arrhythmias [] Cardiac arrhythmias including lifethreatening VAs may be the consequence of direct effects of COVID19 infection but also of the deleterious effects of systemic illness and the adverse reactions to drugs employed in the treatment of this pandemic Table Fig [ ] A recent study indicated that among patients with ± years men African Ameri COVID19 mean age can receiving care in the ICU there were cardiac arrests incident atrial ï¬brillation AF episodes bradyarrhythmias and nonsustained ventricular tachycardias NSVTs [] Arrhythmias occurring in patients admitted to the ICU included cardiac arrests all events of cardiac arrest occurred in this group AF odds ratioOR vs those not in the ICU and NSVT OR Car diac arrests were associated with acute inhospital mortality Among patients with conï¬rmed COVID19 ex hibited myocardial injury as indicated by elevated cardiac troponin T cTnT levels [] During hospitalization patients de veloped ventricular tachycardia VTventricular ï¬brillation VF patients with elevated cTnT levels had more frequent VAs vs p compared with those with normal cTnT levels A recent singleday snapshot survey of stable patients with 101016jtcm202008002 Elsevier Inc All rights reserved Please cite this as AS Manolis AA Manolis and TA Manolis COVID19 infection and cardiac arrhythmias Trends in Cardiovascular Medicine 101016jtcm202008002 0cJID TCM IN PRESS [m5G August ] AS Manolis AA Manolis and TA Manolis Trends in Cardiovascular Medicine xxx xxxx xxx Table Cardiac Arrhythmias Occurring in Patients with COVID19 Infection Sinus tachycardia Sinus bradycardia Conduction disturbances AVBBBB Atrial premature complexes Atrial ï¬brillation Supraventricular tachycardia Ventricular premature complexes Nonsustained ventricular tachycardia Polymorphic ventricular tachycardia Torsade des pointes Sustained ventricular tachycardia Ventricular ï¬brillation Pulseless electrical activity AVB atrioventricular block BBB bundle branch block Table Mechanisms of Arrhythmogenicity in Patients with COVID19 Infection Acute myocardial injury Myocarditis Hypoxia Systemic ammation Autonomic imbalance SNS overactivity virusinduced vagal nerve injury Electrolyte abnormalities QT prolonging drugs antiCOVID pharmacotherapies AADs other agents Drugdrug interactions Cardiovascular comorbidities hypertension coronary artery disease cardiomyopathy AADs antiarrhythmic drugs SNS In this review we present current data about the whole spec trum of cardiac arrhythmias encountered in patients with COVID infection either attributable to the effect of the virus itself on the cardiovascular CV and the respiratory system andor to the effects of the treatments that these patients receive in combina tion with autonomic imbalance that is incurred by this unrelenting pandemic Acute myocardial injury and arrhythmias As mentioned in patients with evidence of acute myocardial injury the prevalence of cardiac arrhythmias is higher compared to patients without myocardial injury [] In a recent retrospec tive cohort study among patients with severe COVID19 had a cTnI level measured upon hospital admission of whom had positive results showing cardiac injury [] In patients with cardiac injury mortality was higher compared to patients without cardiac injury vs p Arrhythmias were found in of the patients with cardiac injury includ ing patients with VT or VF all of whom died [] A recent meta analysis of studies including COVID19 patients showed that patients with cardiac injury and newly occurring arrhythmias were at higher risk of developing severe disease or requiring ICU admission relative riskRR p [] sympathetic nervous system Sinus tachycardia Sinus tachycardia is the most common rhythm disturbance in patients with COVID19 infection due to multiple reasons such as fever respiratory insuï¬ciencyhypoxemia hemodynamic compro mise fearanxiety pain and several other physical and emotional symptoms [] Bradycardiaconduction disturbances According to a retrospective series of patients transient si nus bradycardia lasting days is a possible manifestation of COVID19 hence another reason for close monitoring [] There may be many reasons for bradycardia but severe hypoxia am matory injury of the sinus node by circulating cytokines and exag gerated response to medications are possible triggers Interestingly bradycardia has been suggested as a warning sign of the onset of a serious cytokine storm Fig The schema illustrates the various arrhythmias encountered in patients with COVID19 infection as a consequence of the virus infection affecting the heart and lung andor producing systemic ammation the adverse proarrhythmic effects of COVID therapies and the drugdrug interactions that may occur see text for long QT discussion AF interval PEA pulseless electrical activity SB sud sinus tachycardia sympathetic nervous system STach den cardiac death SNS ventricular arrhythmias VF TdP ventricular ï¬bril lation VT atrioventricular block LQT torsade des pointes VAs sinus bradycardia SCD atrial ï¬brillation AVB ventricular tachycardia COVID19 showed a incidence of arrhythmias limited to AF in and supraventricular tachycardia SVT in patients [] A Heart Rhythm Society HRS online survey of electrophysiology professionals n indicated that AF was the most commonly reported tachyarrhythmia whereas severe sinus bradycardia and complete heart block were the most common brad yarrhythmias in hospitalized patients with COVID19 [] Ven tricular tachycardiaVF arrest and pulseless electrical activity were reported by and of respondents respectively A meta analysis of retrospective cohort studies comprising pa tients with COVID19 showed that the pooled incidence was for cardiac arrhythmia for cardiac arrest [] p According to a retrospective cohort study of COVID19 pa tients multivariable logistic regression indicated that among other ECG abnormalities the presence of one or more atrial premature contractions APCs odds ratio OR a right bun dle branch block RBBB or intraventricular block IVB OR p increased the odds of death [] Another study analyzing the ECGs of COVID19 patients showed that abnormal PR interval behavior paradoxical prolonga tion or lack of shortening with increasing heart rate was associ and need ated with increased risk of death vs p for endotracheal intubation vs p compared to patients with PR interval shortening [] Atrial ï¬brillation According to a recent survey of electrophysiology professionals atrial ï¬brillation AF was the most commonly encountered car diac arrhythmia observed in patients with COVID19 infection [] Several mechanisms could be involved in the pathogenesis of AF in these patients virusinduced cardiac injury that could lead to perimyocarditis hypoxemia frequently occurring in these patients systemic infection common occurrence of the COVID19 infection Please cite this as AS Manolis AA Manolis and TA Manolis COVID19 infection and cardiac arrhythmias Trends in Cardiovascular Medicine 101016jtcm202008002 0cJID TCM IN PRESS [m5G August ] AS Manolis AA Manolis and TA Manolis Trends in Cardiovascular Medicine xxx xxxx xxx in older patients who are already susceptible to AF and sympa thetic nervous system overactivity could all account for such a high incidence of this arrhythmia in this particular population [ ] Guidance on acute management of AF In cases of AF associated hemodynamic compromise as done in all cases of hemodynamically unstable arrhythmias synchronized direct cur rent cardioversion should be used to restore sinus rhythm [] In all other cases one needs to initially proceed with a rate control strategy with use of a betablocker when there is no contraindi cation eg bronchospasm acute heart failure a calcium channel blocker in the absence of heart failure andor digoxin In cases of heart failure digoxin andor amiodarone may be used to achieve rate control For newonset AF within the last hours restoring sinus rhythm is the next target This can be achieved with use of class IA IC or III antiarrhythmic drugs AADs with the selection of the appropriate agent made as based on the presence where only amiodarone seems to be relatively safe or absence of under lying structural heart disease where all other options are available with the caveats being detailed in the discussion that follows be low also taking into account drug interactions with COVID phar macotherapies that are in use A major concern in using speciï¬c AADs relates to the baseline measurement of the QT interval and the coadministration of QTprolonging drugs see discussion be low Most importantly all AF patients should be receiving prophy lactic anticoagulation therapy with intravenous heparin Impact of national lockdown on newonset AF diagnosis An other aspect of the impact of national COVID19 lockdowns on the diagnosis of AF has been recently reported by a Danish study [] Using Danish registries the number of patients receiving a new onset AF diagnosis during the ï¬rst months of and was compared A lower incidence of newonset AF during the weeks of lockdown was noted compared with the corresponding weeks in incidence rate ratios RRs for the weeks and There was a drop in total numbers vs [] Patients diagnosed during lockdown were younger and with a lower CHA2DS2VASc score while history of cancer heart fail ure and vascular disease were more prevalent During lockdown patients with newonset AF suffered an ischemic stroke and died compared with and pa tients during the corresponding period respectively The au thors concluded that following a national lockdown in Denmark a drop in registered newonset AF cases was observed indicat ing that the risk of undiagnosed AF patients developing complica tions could potentially translate into poorer outcomes in patients with AF during the COVID19 pandemic Ventricular arrhythmias In the setting of acute myocardial injury and acute myocarditis in patients with COVID19 infection various and serious ventricu lar arrhythmias VAs may occur [] Other important triggers in clude the severe respiratory insuï¬ciency and the systemic am mation incurred by COVID19 infection as well as the proarrhyth mic effects of COVID therapies and other drug interactions and also the autonomic imbalance superimposed in patients aï¬icted by the disease [ ] Furthermore hypoxemia which is common in these patients and electrolyte disturbances occurring for various reasons in this group of patients may aggravate arrhythmogenic ity Depending on preexisting or currently emerging CV disease various VAs may be encountered including ventricular premature complexes VPCs nonsustained VT NSVT and sustained VTVF Special attention is required for the development of polymorphic VT in the form of torsade des pointes TdP in the setting of QT prolongation either preexisting or acquired and induced by drugs especially when combination therapies are employed that are po tentially proarrhythmic [] Acute myocardial injury noted in of COVID19 patients can be the inciting factor for various VAs [ ] Among pa tients with conï¬rmed COVID19 malignant VAs VTVF developed in patients during hospitalization patients with ele vated cTn levels had more frequent malignant arrhythmias vs [] A recent retrospective cohort study of patients with severe COVID19 indicated that among having a cTn level measured on admission with showing cardiac in jury arrhythmias developed in of the patients in cluding patients with VT or VF all of whom died [] Critically ill COVID19 patients often have comorbidities that can increase the risk for malignant VAs These include electrolyte abnormalities hypokalemia hypomagnesemia fever an am matory state and most importantly COVID19 pharmacotherapies that are potentially proarrhythmic as they prolong the QT interval and may thus trigger TdP and sudden cardiac death SCD [] On the other hand the acute myocardial injury induced by the virus could also independently prolong the QT interval According to a recent report of a Kawasakilike syndrome temporally associated with COVID19 infection in children among whom myocardi tis was diagnosed in patients left ventricular ejection fractionLVEF range of these patients displayed im portant ECG changes that included QT interval prolongation and occasional VAs not attributable to any QTprolonging drug [] Inhospital cardiac arrest As mentioned among patients hospitalized with COVID19 infection all cardiac arrests occurred among patients admitted to the ICU [] In a retrospective cohort study inhospital VTVF occurred in of patients with cardiac injury all of whom died [] Outofhospital cardiac arrest OOHCA A recent Italian study compared all the consecutive outofhospital cardiac arrests OOHCA in the months following the ï¬rst documented case of COVID19 in the region with those which occurred in the same time frame in [] The cumulative incidence of COVID19 from February to April in the study territory was COVID19100 inhabitants and the cumulative incidence of OOHCA was cases100 inhabitants with a increase as compared with OOHCAs in vs in p The authors concluded that the increase in OOHCAs in is signiï¬cantly correlated to the COVID19 pandemic and is coupled with a reduction in shortterm outcome A French study comparing the OOHCAs of the pandemic period to the mean of the total over weeks in the non pandemic period indicated that the maximum weekly OOHCA in cidence increased from to per million inhabitants p before returning to normal in the ï¬nal weeks of the pandemic period [] There was a higher rate of OOHCA at home less bystander cardiopulmonary resus vs p citation vs p and shockable rhythm vs and longer delays to intervention median p min vs min p The proportion of OOHCA patients ad mitted alive decreased from to p in the pan demic period After adjustment for confounders the pandemic pe riod remained signiï¬cantly associated with lower survival rate at hospital admission odds ratio p COVID19 infection accounted for about one third of the increase in OOHCA incidence during the pandemic Druginduced prolongation of QTc interval and torsade des pointes Several agents employed for treating COVID19 infection may prolong the QT interval and lead to polymorphic VT in the form of TdP Table Chloroquinehydroxychloroquine and azithromycin which have been recently used for potential prophylaxis or treat ment for COVID19 infection are listed as deï¬nite causes of TdP Please cite this as AS Manolis AA Manolis and TA Manolis COVID19 infection and cardiac arrhythmias Trends in Cardiovascular Medicine 101016jtcm202008002 0cJID TCM IN PRESS [m5G August ] AS Manolis AA Manolis and TA Manolis Trends in Cardiovascular Medicine xxx xxxx xxx Table QTProlonging Drugs in COVID19 Infection Antibiotics Antiviral agents Anesthetics Antiemetics Antiarrhythmics Antipsychotics ChloroquineHydroxychloroquine Macrolides Azithromycin Quinolones LopinavirRitonavir Favipiravir Tocilizumab Fingolimod Propofol Domperidone Class IA Class III Haloperidol at crediblemeds [] According with the FDA azithromycin other macrolides and ï¬uoroquinolones can cause lethal arrhyth mias as a potential consequence of QTinterval prolongation [] Chloroquine hydroxychloroquine Chloroquine CQ and hydroxychloroquine HCQ have been used for treatment and prophylaxis of malaria while they have also been employed for treatment of amebiasis that is occurring out side the gastrointestinal tract rheumatoid arthritis and lupus ery thematosus These agents were also found to have antiviral effects and have been proposed for the treatment of COVID19 infection [] However both these agents can be proarrhythmic by pro longing the QT interval and potentially initiating lifethreatening VAs including TdP they can also cause QRS widening Chloroquine interacts with multiple cardiac ion channels including the human etheragogorelated gene hERG potassium channel a reduction in hERG channel potassium current is the main cause of acquired druginduced long QT syndrome Recent experimental data indi cated that HCQ markedly increases the action potential dispersion and results in the development of repolarization alternans and ini tiates polymorphic VT [] Preliminary ï¬ndings from a recent study suggested that the QTc prolonging effect of CQ is dosedependent [] Among pa tients enrolled with COVID19 infection were allocated to highdosage group ie mg CQ bid for days and to lowdosage group ie mg bid on day and qd for days Lethality until day was in the highdosage group of and in the lowdosage group of The highdosage group presented more instance of QTc interval ms compared with the lowdosage group Respiratory secre tion at day was negative in only of patients The authors suggested that the higher CQ dosage should not be rec ommended for critically ill patients with COVID19 because of its potential safety hazards especially when taken concurrently with azithromycin and oseltamivir A recent disproportionality analysis of HCQassociated CV ad verse reactions using the FDA adverse event reporting system FAERS database of datasets and patient records indicated that HCQ was associated with higher reporting odds ratios ROR of TdP ROR CI to complete atrioventricular AV block ROR CI to and QT prolongation ROR CI to [] QT prolongation and TdP are more frequent with high doses for a comparatively short period and represent the most common HCQassociated side effects A systematic review of data on COVID19 patients showed that of COVID19 patients treated with CQHCQ developed QT prolongation [] Ventricular arrhythmias developed in COVID patients from a group of treated with highdose CQ The authors suggest daily ECG monitoring and other risk mitigation strategies to be adopted in order to prevent possible arrhythmic sideeffects Macrolide antibiotics Azithromycin AZM also can cause modest QT interval pro longation but not through potent hERG channel blockade rather when used chronically through an increase in peak and late cardiac sodium current to cause potential loading of cardiomyocytes with sodium and calcium to produce calcium overload Advanced age and female gender are considered risk factors [] Azithromycin can also provoke nonpausedependent polymorphic VT in the ab sence of QT prolongation [ ] After reviewing the data of AZM regarding risk of QT prolonga tion and associated TdP the FDA revised AZM product labels ad vising against its use in patients with known risk factors such as QTinterval prolongation hypokalemia hypomagnesemia bradycar dia or use of certain QTprolonging antiarrhythmic agents includ ing class IA eg quinidine and procainamide and class III eg dofetilide amiodarone and sotalol agents [] Antiviral agents The combined antiviral regimen of ritonavirlopinavir approved for human immunodeï¬ciency virus HIV infection was also con sidered to be able to suppress SARSCoV2 replication [ ] Lopinavir is metabolized by the hepatic cytochrome P450 sys tem CYP3A [] it also inhibits drug transporters such as Pglycoprotein Pgp [] Thus ritonavirlopinavir may increase plasma concentrations of drugs primarily metabolized by CYP3A or substrates of these drug transporters Ritonavirlopinavir may require dose reductions or avoidance of CYP3Amediated drugs such as rivaroxaban and apixaban Ritonavirlopinavir has also been shown to cause QT and PR interval prolongation or occasionally second or thirddegree AV block particularly in patients with un derlying structural heart disease and preexisting conduction sys tem abnormalities [] Due to its competitive inhibition of the RNAdependent RNA polymerase favipiravir is being evaluated in treating patients with COVID19 alone or in combination therapies the risk for QT inter val prolongation by favipiravir is considered to be low [] Other agents Fingolimod is an immunomodulator and immuno suppressant which reduces lymphocyte migration and is used in the treatment of multiple sclerosis [] it has been proposed as a potential adjuvant therapeutic agent against COVID19 [] Fin golimod has Ltype calcium channel blockade effect causing pro longation of PR RR and QT interval It also activates acetylcholine dependent potassium channels IKach in sinoatrial node causing dosedependent bradycardia [] Thus ï¬ngolimod increases the risk of bradycardia and heart block through Ltype calcium channel and IKach blockade [] Combined therapies Treatments employed for COVID19 may increase arrhythmia risk particularly the risk for VAs through drug interactions Drug combinations can lead to greater prolongation of cellular action potential duration analogous to QT prolongation compared with single drug therapies [] The combination effect can result from both pharmacokinetic and pharmacodynamic drug interactions Importantly females with preexisting CV disease seem to be more susceptible to druginduced arrhythmias compared to males with CV disease or healthy persons of either gender Please cite this as AS Manolis AA Manolis and TA Manolis COVID19 infection and cardiac arrhythmias Trends in Cardiovascular Medicine 101016jtcm202008002 0cJID TCM IN PRESS [m5G August ] AS Manolis AA Manolis and TA Manolis Trends in Cardiovascular Medicine xxx xxxx xxx Table Measures to Prevent Arrhythmias in Patients with COVID19 Infection ¢ Withhold QT prolonging drugs in patients with baseline QTc ¢ Withdraw QTprolonging drugs when QTc increases to ¢ Do not use chloroquinehydroxychloroquine azithromycin other macrolides ï¬uoroquinolones lopinavirritonavir or favipiravir in patients with known risk factors such as prolonged QTc hypokalemia hypomagnesemia bradycardia or concomitant use of certain QTprolonging antiarrhythmic drugs including class IA eg quinidine and procainamide and class III eg dofetilide amiodarone and sotalol agents ¢ Maintain K ¢ Monitor QTc via ECG or preferably via telemetry monitor or smart phone measurements ms compared to baseline measurement ms or if QTc is prolonged by ms or with known LQTS mEqL and Mg level to level to mgdL An online survey of electrophysiology professionals revealed that of respondents reported having to discontinue therapy with HCQ AZM due to signiï¬cant QTc prolongation and reported cases of TdP in patients on HCQCQ and AZM [] Amiodarone was the most common antiarrhythmic drug used for VA management Among COVID19 positive suspected patients stud ± years male received AZM HCQ ied age ± and received both drugs [] Baseline mean QTc was ± ms p with medications Sig ms and increased to ± ms vs niï¬cant prolongation was observed only in men ± ms in women p of patients reached critical ms or QTc QTc prolongation maximum QTc ¥ ms Changes in ¥ ms if QRS QTc were highest with the combination compared to either drug ± with much greater prolongation with combination vs AZM vs ¥ ms or QTc increase of No patients manifested TdP ¥ ms if QRS ± ms p ± ms p ± ms vs Another recent cohort study of patients treated for COVID with CQHCQ reported that patients received CQ received HCQ and also received AZM [] Although the maximum QTc during treatment was signiï¬ cantly longer in the combination group vs the monotherapy group TdP was not ob served in the entire population and there were no arrhythmogenic deaths reported A study of COVID patients receiving combined HCQAZM therapy indicated longer QTcinterval than before ther ¥ ms had apy vs ms p higher values of transaminases p compared with those with ms [] At h Holter ECG monitoring COVID19 QTc patient and no control had No patients showed R on T VPCs Analysis of h QTc dynamics revealed that COVID19 patients had higher QTc values than controls with no signiï¬cant hourly variability Therapy with HCQ and AZM pro longs QTc interval in patients with COVID19 particularly in those with high levels of transaminases ¥ run of NSVT p patients with a QTc Interestingly in nonCOVID patients a retrospective cohort study identiï¬ed only two SCDVA events among com bination users CQHCQ plus AZM [] However the doses were lower in this study compared to doses used in COVIDpatients drugs were not used acutely in a hospital setting as currently done for COVID patients fewer cardiac patients received the drugs all suggesting an attenuated risk for cardiac arrhythmias in this par ticular cohort Nevertheless when all measures and precautions are taken Table the incidence of QT prolongation and the TdPevent rate may remain low In a recent study of patients with COVID ± years male HCQAZM was infection mean age ¤ 480ms and potassium level initiated only if baseline QTc was mmolL [] Two patients were not eligible for drug ± ms initiation QTc ± ms after h of combined therapy The and increased to treatment had to be stopped because of signiï¬cant QTc prolonga tion in patients No druginduced TdP nor death was ob served In this speciï¬c population HCQAZM could not be initiated or had to be interrupted in ¥ ms Baseline average QTc was of the cases QTc monitoring Congenital long QT syndrome LQTS with a prevalence of in the general population may often be asymptomatic and if an ECG has not been recorded it will remain unknown to the affected person and the ï¬rst manifestation may be SCD usually triggered by a drug [] Furthermore silent genetic variants or forme fruste of congenital LQTS encountered in of people may render a person vulnerable to QT prolongation TdP and SCD [] Therefore a large number of healthy individuals will be at an increased risk of a druginduced LQTS Data suggest that in African Americans may be at a higher risk of druginduced TdP during the COVID19 pandemic due to clustering of intrinsic genetic susceptibility ie exclusive oc currence of the proarrhythmic ion channel variant pSer1103Tyr SCN5A acquired risk factors eg electrolyte disturbances and QTcprolonging drug use and COVID19speciï¬c risk factors eg profound hypoxemia and cytokine storm [] A heart ratecorrected QT QTc interval is measured with use of various formulas among which the Bazetts correction formula is most commonly used QTc QT RRsec QTc is deï¬ned as pro longed when it exceeds ms in males and ms in females as measured preferably in lead II or V on a standard 12lead ECG [] A prolonged QTc predisposes to polymorphic VT in the form of TdP that may degenerate into VF and SCD For the wideQRS adjusted QTc methods that have been suggested include the JT ad justment obtained as QTcQRS [] or subtracting of the QRS duration from the measured QT [] For patients receiving QTprolonging drugs it is imperative to monitor the QTc interval during treatment Table Traditionally this can be accomplished by obtaining a 12lead ECG however in the era of the COVID19 pandemic this poses a certain risk and puts considerable strain on medical personnel and the health sys tem [] Many telemetry systems are equipped with features of real time QTc monitoring and could be used in hospitalized pa tients and those managed in the ICU setting In addition smart phone heart monitors are also capable of providing remote accu rate QTc measurements [] In this context AliveCor has recently received clearance from the FDA to market the KardiaMobile6L device a previously FDAapproved device for AF detection for QTc monitoring of COVID19 patients treated with QT prolonging drugs such as CQHCQ [] Similarly the Apple Watch ECG an F | 2 |
" medical practice variation in caesarean section rates is the most studied type of practice variation inthe field of obstetrics and gynaecology this has not resulted in increased homogeneity of treatment betweengeographic areas or healthcare providers our study aim was to evaluate whether current study designs on medicalpractice variation of caesarean section rates were optimized to identify the unwarranted share of practice variationand could contribute to the reduction of unwarranted practice variation by meeting criteria for audit and feedbackmethods we searched pubmed embase ebscocinahl and wileycochrane library from inception to march24th studies that compared the rate of caesarean sections between individuals institutions or geographicareas were included study design was assessed on selection procedure of study population data source casemixcorrection patient preference aggregation level of analysis maternal and neonatal outcome and determinantsprofessional and anizational characteristicsresults a total of studies were included most studies measured the caesarean section rate in the entirestudy population instead of using a sample national databases were most often used as information source casemix correction was performed in studies the robson classification was used in of thestudies following its endorsement by the who in the most common levels of aggregation were hospitallevel and grouped hospitals eg private versus public the percentage of studies that assessed therelationship between variation in caesarean section rates and maternal outcome was neonatal outcome determinants professional and anizational characteristics and patient preference s study designs of practice variation in caesarean sections varied considerably raising questions abouttheir appropriateness studies focused on measuring practice variation rather than contributing to the reduction ofunwarranted practice variation future studies should correct for differences in patient characteristics casemix andpatient preference to identify unwarranted practice variation practice variation studies could be used for audit andfeedback if results are presented at lower levels of aggregation and appeal to intrinsic motivation of physicians forexample by including the health effects on mother and childkeywords caesarean section medical practice variation study design characteristics correspondence mdhvinkvunl mdhvinkgmailcom1department health sciences faculty of science talma institute vrijeuniversiteit de boelelaan hv amsterdam the netherlands2department of obstetrics and gynaecology university medical centergroningen groningen the netherlandsfull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cvink bmc pregnancy and childbirth page of the caesarean section has been the most performed surgical procedure worldwide for many decades it hasbeen extensively studied both to optimize treatment and to understand why deviations from optimal treatment occur this longterm popularity has not resulted in evidence based homogeneoustreatmentbetween geographic areas or healthcare providers [ ]the magnitude of the variation has raised questionsabout regional differences in quality of healthcare especially in countries with similar resources moreover years of study on practice variation shows no trend ofincreasing regional l one worldwide conformity variation in medical practice can be divided in warranted and unwarranted practice variation variationis warranted when it results from variation in need forexample due to varying rates of extreme premature deliveries extremely preterm deliveries are centralized atinstitutions with the highest expertise of neonatal careas it yields the most optimal outcome these institutions may deviate from the national average as theyserve a highrisk populationmedical practice variation is unwarranted if it cannotbe explained by patient characteristics or patient preference [ ] to identify unwarranted practice variationstudies should compare study groups that are comparable in terms of relevant patient characteristics or makethem comparable through careful casemix correction patient preference is important when both modesof delivery vaginal delivery and caesarean section arean acceptable option variation of caesarean sectionrates is desirable to allow for differences in patient preference across healthcare providers and random or unmeasured differences in need of having a caesareansection when a reported rate deviates more from an acceptable range differences may less likely be attributableto differences in patient preference as both over andundertreatment of caesarean sections harms mother andchild it is therefore likely that quality of healthcare formother and child can be improved by reducing unwarranted practice variation of caesarean sections sufficientevidence on risks and benefits of caesarean sections mayhelp to reduce variation higher quality evidence will result in better guidance on the optimal caesarean sectionrate for specific obstetric conditions subsequentlyuptodate clinical guidelines and clinical support systems may facilitate clinicians to implement recent evidence finally shared decision making should beincorporated in daily clinical practice to empower patients to decide what suits them best improving the process of generating evidence implementing clinical guidelines and incorporating shareddecision making requires healthcare professionals tochange their clinical behavior which is complex auditand feedback is a nonclinical intervention that supportschange of clinical behavior literature shows thathealthcare professionals may be encouraged with information relevant to them one example is performancefeedback on a low level of aggregation ie group or individual level another is to tap into the intrinsic motivation to do well for patients evidence shows thatunnecessary caesarean sections cause an increase in maternal death rates and may affect infant health negatively monitoring and reporting mother and child healthmay motivate change to reduce unnecessary caesareansections audit and feedback has been put forwardas a way through which research can contribute to thereduction of practice variation but it is unclearwhether current research designs of studies on medicalpractice variation of caesarean section rates can be usedfor this purposemedical practice variation in caesarean section rateshas been extensively studied studies started as earlyas and the interest for the topic has remainedstrong however unwarranted medical practicevariation of caesarean section rates has not been reduced the objective of this scoping review is to evaluatewhether studies on medical practice variation of caesarean section rates have used an optimal study design toidentify unwarranted practice variation and when identified can also be used for audit and feedback to contributereduction of unwarranted practicevariationto themethodsto evaluate the characteristics of all caesarean sectionvariation studies we opted for a scoping review wefollowed the prisma statement the researchprotocol was not publishedsearch strategywe searched studies that compared caesarean sectionrates between individual healthcare professionals hospitals groups of hospitals or geographic areas a comprehensive search was performed in collaboration with amedical librarian in the bibliographic databases pubmedembase ebscocinahl and wileycochrane libraryfrom inception up to march 24th the followingterms were used including synonyms and closely relatedwords as index terms or freetext words practice patterns caesarean section the freetext term caesarean section was only used in titles the meshtermpractice patterns includes several descriptions of practice variation the search strategy was performed without date orsearchlanguage restriction the full 0cvink bmc pregnancy and childbirth page of strategies for all databases can be found in additional filestudy selectionall studies that reported on any variation in caesareansection rates between individual healthcare professionals hospitals groups of hospitals or geographicareas were included we included any type of study designie crosssectional study designs and both prospective and retrospective longitudinal studiesexclusion criteriawe excluded studies that were not published in englishand studies that did not publish data on variation of caesarean section rates between healthcare professionalshospitals groups of hospitals or geographic areasprocess of study identification and selectiontitles and abstracts were downloaded and entered inendnote version x81 duplicates were removed tworesearchers mv and pdb screened titles and abstractsthe researchers independently decided whether to include the for full text screening disagreementwas resolved by consensus if no consensus could beachieved a third researcher made the decision evdhfull text screening was performed by two researcherswho decided independently whether to include the or not mv and pdb in case of disagreement thethird researcher evdh decided whether the metthe predefined inclusion criteria data on the designcharacteristics of the studies were extracted by one researcher mv these data were randomly crosscheckedby a second researcher pdbdata extractionto describe the variation of caesarean section rates wescored the minimum and maximum caesarean sectionrate when caesarean section rates of multiple yearswere reported the rate of the most recent year was usedas an indicator for the risk of selection bias we scoredthe selection of study population we differentiated between the use of a study sample or the entire studypopulation to estimate the caesarean section rate theuse of a study sample was considered as a high risk ofselection bias if the study lacked a description of thesampling frame the assessment of the caesarean sectionrate of the entire study population was considered as alow risk of selection bias to indicate the risk of information bias we differentiated between the use of electronic patientepf a national database orquestionnaires the use of epf and databases were considered as low risk of bias as the information was collected by attending healthcare professionalsfilesidentification of unwarranted practice variationto identify whether studies distinguished between warranted and unwarranted practice variation we scoredwhether casemix correction was performed and if patient preference was taken into accountno restriction was imposed on the method of casemix correction examples of casemix corrections include calculating an adjusted or expected caesarean section rate reporting stratified odds ratios by patientcharacteristics or using logistic regression to adjust forpatient characteristics we extracted which variableswere used for casemix correction and whether the robson classification was used the latter is the system proposed by the world health anisation who andthe international federation of obstetrics and gynaecology figo to classify caesarean section casemix no restriction was imposed on how patient preferencewas measured measuring patient preference requiresadditional data collection this could be unfeasible forlarge cohort studies unless a truly random sample of sufficient size is used we assessed whether all studies tookpatient preference into account registered the cohortsize and noted how patient preference was measured ifpatient preference was measured we assessed whether asample was used and whether it was randomusefulness for audit and feedbackto evaluate whether the studies were able to providehealthcare professionals feedback on their clinical behavior in order to reduce unwarranted practice variationwe extracted the aggregation level of analysis that wasused and differentiated betweenindividual physiciangroup of physicians hospital hospital category regionor country similarly we scored whether maternal andneonatal outcomes were measured as outcome reporting informs healthcare professionals on their clinicalperformance we extracted all reported variablesin addition we extracted several explanatory factorsthat might contribute to unwarranted practice variationincluding anizational characteristics ie profitstatusor teachingstatus of the hospital and physician characteristics ie physician gender and age furthermore wescored whether studies analysed financial consequencesof unwarranted practice variation of caesarean sectionratesresultsthe process of study identification and selection is schematically presented according to the prisma statementin fig a total of records were identified from pubmed from embase from cochraneand from cinahl after duplication we screened abstracts for eligibility in total s were 0cvink bmc pregnancy and childbirth page of fig prisma flowchartfig average of the reported minimum and maximum caesarean section rate per year is not presented in fig as only studies areonly included until 24th of march 0cvink bmc pregnancy and childbirth page of selected for fulltext screening we excluded studiesthe reasons for exclusion are presented in fig intotal studies met the inclusion criteria and were included the included studies and their design characteristics and reported variation in caesarean section ratesare listed in additional file the included studies were published between and the cohorts that were studied varied between and more than million women most studies analyzed variation of caesarean section rates in the unitedstates studies followed by brazil studies andaustralia studies the number of studies per country are shown in additional file a wide variation incaesarean section rates is reported some subsaharanregions perform caesarean sections in less than ofthe deliveries by contrast the reported caesarean section rate of some municipalities in brazil reached the variation of caesarean section rates did not decrease over time figure shows the average reportedminimum and maximum caesarean section rates peryear the outlier in is one study that reported variation between four hospitals in rio de janeiro in oneprivate hospital more than of the women deliveredby a caesarean section in a similar situation occurred only two studies were included both reportingon variation in indiain studies a sample of the study populationwas used to estimate the caesarean section rate the majority of the studies studies measured themode of delivery of the entire study population bothmethods were used in eight studies and the selection frame was unclear in nine studies an exampleof a study in which both methods were used was theanalysis of variation in caesarean section rates betweencountries some country estimates were based on a sample of their population while others were based on registries ofin the samplebasedstudies studies defined a selection frame designed to select a representative samplethe entire populationthe majority of studies used data from registries suchas national databases studies or electronicpatient files studies questionnaires were usedin studies eg the demographic and healthsurvey dhs that was used in studies suchquestionnaires were sent to a sample of households inorder to collect information on live births of the pastyears in studies multiple data sources wereused or the data source was not describedidentification of unwarranted practice variationcasemix correction was performed in studies the variables that were used for casemix correction areshown in additional file baseline patient characteristics were observed in studies some studies didnot describe patient characteristics per cohort but didperform a correction for maternal or neonatal characteristics many different maternal and obstetric variables were used as baseline characteristic or for casemix correction age parity gestational age birthweight and maternal education level are the characteristics that were used most often morethan half of the variables were only used in one or twostudiesto reduce this heterogeneity and to increase the quality of casemix correction the who in recommended to use the robson classification as the standardfor casemix correction for studies on caesarean sectionrates in the period out of studiesused the robson classification four of these studies did perform additional casemix correction byusing specific patient characteristics the who notesthat the robson classification should especially be usedwhen comparing caesarean section rates between healthcare facilities or within healthcare facilities over time in the period studies compared caesarean section rates between healthcare providers individuallevel andhospital category of which used the robson classification within the same period of the studiesthat compared caesarean section rates between geographic areas used the robson classification thepercentage of studies that corrected for casemix did notchange over time figure shows the number of studiesthat corrected for casemix by yearlevel group of physicians hospitalthe effect of casemix correction is shown in fig of the studies that corrected for casemix studies calculated an adjusted caesarean sectionrate figure shows these rates per study categorizedper aggregation level the remaining studies calculated an expected caesarean section rate reportedstratified odds ratios by patient characteristics or usedlogistic regression to adjust for patient characteristicsat provider level individual physician group of physicians and hospital level of the studies and at geographic level regional or national of the studiescorrected for casemix figure shows that at the provider level casemix correction had a substantial impacton the provider caesarean section rate at the geographic level the impact of casemix correction wascomparatively smallsix studies took patient preference into accountthese studies did not assess variation of caesarean section rates for a specific obstetric condition for instancepatients with a history of caesarean section in which acaesarean section and vaginal delivery are both an acceptable option all studies measured patientpreference by questionnairesthat were handed tomothers that gave birth between one day and years 0cvink bmc pregnancy and childbirth page of fig number of studies per year with and without casemix correction is not presented in fig as only studies are only included until24th of march rates was least often studied at the level of the individualphysician and group of physicians no clear timetrend was observed in the choice of aggregation level ortrend in observed variation based on the level of aggregation the largest variation in caesarean section rates is reported on both the lowest ie individual level and thehighest ie international level of aggregationneonatal outcomes were captured in andmaternal outcomes in of the studies all variables that were used to measure these outcomes arelisted in additional file many different variables wereused to measure neonatal and maternal outcomes neonatal mortality apgar score maternal mortalityandhaemorrhage are outcomes that were measured mostoften half of the outcome variables were used in justone single study table shows the numbers of studiesper aggregation level that took neonatal and maternaloutcome into account neonatal and maternal outcomeswere most often reported if variation of caesarean nicu admissionprior to the survey patient preference was assessed byposing a variety of questions on the reason of the caesarean section and perceived influence on decision makingno studies reported the quality of decision making in of the studies a sample of the study populationwas used to measure patient preference samples variedbetween and women and three studies used apredefined sampling frameusefulness for audit and feedbacktable shows the study characteristics by aggregation levelthe majority of the studies used one aggregationlevel a minority used two and only three studies used three aggregation levels healthcare providersindividual physicians group of physicians hospitals or hospital categories were compared in studies andgeographic areas national or regional in studies hospitals and grouped hospitals eg private vspublic hospitals were most often used as aggregation levelof analysis medical practice variation of caesarean sectionfig the effect of casemix correction on different aggregation levels 0cvink bmc pregnancy and childbirth page of table study characteristics by aggregation levelnumber of studiesaverage cohort sizemedian cohort sizeentire population measured instead of samplereported variation in caesarean section rate average ofstudiesreported variation in caesarean section rate median ofstudiescasemix correctionmedical outcomeindividuallevelgroup ofphysicians hospitallevelhospitalcategoryregionnational sections was studied atphysiciansthe level ofthe individualdeterminants and financial consequencesa limited number of studies explored determinants toexplain medical practice variation of caesarean sectionrates in an additional analysis hospital characteristics orphysician characteristics were used in studies to explain differences in caesarean section rates thevariables that were used are listed in additional file financial consequences of unwarranted variation of caesarean section rates were calculated in six studiesdiscussionalmost four decades have past and studies werepublished following opits first report on variation ofcaesarean section rates between geographic areas inaustralia clearly the issue raised by the first studies hasnot lost its sense of urgency among researchers nor forthe funders of research because the magnitude of unwarranted variation was considered problematic andremained stable over time while previous reviewsnarrowed their focus on measuring variation betweengeographic areas [ ] or studied the difference between public and private hospitals the focus of thisreview was on the presence of study characteristics thatmay help to reduce unwarranted variation of caesareansection ratesstrengths and limitationsa strength of our review was the systematic search andselection procedure which allowed us to identify almost all studies on medical practice variation that compared caesarean section rates this resulted in a largenumber of included studies a second strength is thehigh level of detail of our analysis the selection of theindividual variables that were used for casemix correction outcomes or determinants enabled us to present anindepth overview of study characteristicsseverallimitations should be addressed first weaimed to describe study characteristics of all relevantpublished studies ie irrespective of the quality in theenglish language and therefore did not perform a qualityassessment of the included studies second we were unable to retrieve all publications that were selected forfulltext screening in order to limit the number of missing s we contacted the authors of missing sby email or through researchgate however this wasnot successful for of the studiesinterpretationfirst we appraised whether studies were designed to distinguish between warranted and unwarranted practicevariation by performing casemix correction and analysing patient preference casemix correction is an essential aspect of the quality of studies on practice variationbecause without casemix correction it remains unclearwhat share of the variation is attributable to health differences between populations and thus to what extentthe variation is warranted our results show that only of the studies that compared caesarean section ratesbetween healthcare providers performed casemix correction casemix correction was performed by calculating adjusted rates expected rates stratified odds ratiosor logistic regression analysis over time we observed noimprovement in the performance of casemix correctionpatient preference another important aspect to identify unwarranted practice variation was only measured insix studies without the assessment of patient preference it remains unclear whether practice variation canbe explained by differences in the outcome of shared decision making this is especially important when bothmodes of delivery vaginal delivery and caesarean section are an acceptable option patients with a historyof caesarean section breech or twin delivery are examples in which information on patient preference is necessary to differentiate between warranted and unwarrantedmedical practice variation [] our results show 0cvink bmc pregnancy and childbirth page of that none of studies that assessed variation in these specific obstetric situations patient preference was analysedto improve the identification of unwarranted practicevariation future studies should not only measure patientpreference but should focus on the implementation ofshared decision making recent literature shows thatseveral shared decision making measures are availablewhich could be included in the study design of medicalpractice variation studies to improve comparability both within healthcare facilities and between them robson proposed what latercame to be known as the robson classification systemfor assessing monitoring and comparing caesarean section rates in the who and the figo jointly endorsed this classification as the international standardfor casemix correction [ ] our data show that following the publication of this guideline by the who in the robson classification was only used in of all studies comparing providers and in studies comparing regions literature shows that casemixcorrection can be improved even more if additional patient characteristics are considered only of thestudies that were published between and andused the robson classification did perform additionalcasemix correction ie age adjusted caesarean sectionrates within robson groups studies that performedcasemix correction with or without robson classification used a wide variation of maternal and obstetriccharacteristics to identify unwarranted practice variation we advise to at least use the robson classificationand to standardise variables for additional casemix correction a delphi procedure can help obstetricians andmidwives to reach consensus on which variables to use the necessity to which casemix correction is neededdepends on the level of aggregation the lower the levelof aggregation the more case mix correction contributesto a valid description of clinical performance health care providers often operate in a network andtreat a subset of the entire population that subset ismore likely to differ between providers as they get morespecialized and the casemix differences may justify differences in caesarean section rates this requiresthat studies aimed at lower levels of aggregation placemore emphasis on casemix correction reporting standards and appropriate small number statistics once casemix has been appropriately controlled forfuture studies should aim to decompose regional unwarranted variation into lower levels of aggregation thisdecomposition allows regional variation to be attributedto health care providers however disaggregation ofcontributions to lower levels of aggregation is not without major risk on its own as groups of physicians getsmaller their clientbase may diverge more from theregional averagefor example due to specializationreporting differences or chance within providers eghospitals further disaggregation to the physician levelcould help identify individual contributions while thelower number of observations may harm both validityand reliability they may also provide a stronger stimulusfor change if the information is interpreted intelligentlyand presented in a motivating environmentstimulus for change might be further enhanced whenoutcome reporting becomes integrated in future studydesigns healthcare professionals are intrinsically motivated to deliver the best healthcare for their patients reporting outcomes in casemix corrected feedbackinformation directly stimulates the intrinsic motivationto improve outcome for patients if it becomes visiblethat increased caesarean section rates do not yield improved maternal and neonatal outcomes professionalsmight be encouraged to adapt clinical behavior our results show that the wide variation in outcome variablesdemands consensus and standardization studies will become more comparable and better interpretable when astandard set of outcomes is used for maternal infectiousmorbidity outcomes after caesarean delivery a core outcome set cos is developed we encourage to develop a cos for neonatal outcomes after caesareansectionforty years of research on caesarean section rates havebeen unable to reduce unwarranted practice variationour study shows that most studies do not meet the criteria to identify unwarranted practice variation and cannot be used for audit and feedback to contribute to thereduction of unwarranted practice variation future studies should correct for differences in patient characteristics and patient preference present results at low levelsof aggregation and appeal to intrinsic motivation by including the health effects on mother and childsupplementary informationsupplementary information accompanies this paper at httpsdoi101186s12884020031693additional file search strategies additional file contains the searchstrategies that were used for the databases pubmed embase ebscocinahl and wileycochrane libraryadditional file list of included studies additional file contains a listof all studies that were included in this scoping review per study theresults are summarized we used the following definitions for theindependent variables used in the table year year of publication author first author of study included title title of study included study period years from which caesarean section rates were reported inexample if a researcher performed a questionnaire survey in andincluded deliveries from y prior to the survey we reported study period caesarean section rate unadjusted caesarean section rate ofmost recent year reported cohort size the cohort size from which thecaesarean section rate is calculated if caesareans section rates from 0cvink bmc pregnancy and childbirth page of multiple years were reported we noted specifically the cohort size of thecohort that was used to calculate the most recent caesarean section rate data source data source that was used by the authors to calculate thereported caesarean section rate it is reported as other if data source isunknown or multiple data sources are used casemix correction thestudy reported an adjusted caesarean section rate expected caesareansection rate reported stratified odds ratios by patient characteristics orused logistic regression to adjust for patient characteristics yn aggregation level aggregation level of analysis outcome outcomes maternal or neonatal were noted yn determinants anisational orphysician characteristics were used to explain reported difference in caesarean section rates between healthcare professionals hospitals groupsof hospitals or geographic areas ynadditional file studies per country additional file describes thenumber of studies on medical practice variation of caesarean sectionrates that were conducted in each count | 0 |
" laparoscopic tumorspecific mesorectal excision tsme preserving the left colic artery and superiorrectal artery is still a technically challenging procedure we conducted this study to demonstrate the feasibility ofthis procedure for upper rectal cancermethods a total of patients with upper rectal cancer were retrospectively analyzed in our cancer centerbetween april and april these patients were treated with either laparoscopic tsme n orlaparoscopic total mesorectal excision tme n in the tsme group the left colonic artery and superior rectalartery were preserved while they were not in the tme groupresults the operation time in the tsme group was longer than that in the tme group ± min vs ± min p furthermore the number of resected lymph nodes in the tsme group was greaterthan that in the tme group ± vs ± p the blood loss between the tsme and tmegroups was not significant no mortality occurred in either the tsme or tme groups one patient in the tme groupunderwent conversion to laparotomy the total postoperative complication rates in the tsme and tme groups were and respectively there was no difference in severe complications between the two groupsanastomotic leakage and stenosiss laparoscopic tsme preserving the left colic artery and superior rectal artery can be safely conductedfor upper rectal cancerkeywords laparoscopic surgery rectal cancer tumorspecific mesorectal excision superior rectal artery leftcolonic artery tme correspondence 237721898qqcom 250537471qqcomhuxiang_zc1978sinacom chi zhang haotang wei wenqing hu and yueming sun contributedequally as joint first authors7department of general surgery yizhen peoples hospital clinical medicalcollege yangzhou university yangzhou jiangsu province china3department of gastrointestinal surgery changzhi peoples hospital theaffiliated hospital of changzhi medical college changzhi shanxi provincechina1department of gastrointestinal surgery the first affiliated hospital of dalianmedical university dalian liaoning province chinafull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0czhang world of surgical oncology page of introductiontotal mesorectal excision tme is an important surgical technique to prevent the local recurrence of rectal cancer on the other hand tme may not besuitable for every case of rectal cancer such as rectosigmoid junction and upper rectal cancers the resection range of tme reaches cm below the inferiorborder of the tumor and has acquired an adequatecure rate reported in previous studies for patientswith rectosigmoid junction and upper rectal cancers this tumorspecific resection according to thetumorsite or t staging is called tumorspecificmesorectal excision tsme itsudecks critical point at the rectosigmoid junction isdescribed as the point of origin of the last sigmoid arterial branch originating from the inferior mesenteric artery ima the anastomosis between the lastsigmoidal artery and superior rectal artery sra is absent in some people to avoid the risk of postoperativeischemic necrosis anastomotic leakage colitis and delayed stricturetosudecks point for cases where anastomosis may be absent or insufficiently present in addition the rate ofis desirable to ligate proximalabsence of the left colic artery lca is which maybe associated with a risk of anastomotic leakage due toinsufficient vascularization of the proximal colonic conduit this study introduces the procedure and technicalpoints of laparoscopic tsme with preservation of thelca and sra the operation is still a technically challenging procedure we conducted this study to demonstrate the feasibility of this procedure for upper rectalcancer and shortterm prognosismethodspatientslaparoscopic tsme preserving the lca and sra wasperformed on patients with upper rectal cancer fromapril to april in the same period patients with upper rectal cancer underwent standardtme surgery this study was conducted in accordancewith approved guidelines this study was approved bythe institutional review board of the first affiliatedhospital of dalian medical university written informedconsent was obtained from all patientsfig ima 3d cta 0czhang world of surgical oncology page of equipmentangled ° 10mm diameter 3d laparoscope insufflation equipment and bipolar electrosurgical deviceaesculap german harmonic vascular closure systemjohnson usa 10mm and 5mm port trocars teleflexmedical usa laparoscopic linear staplers mm inlength covidien usa hemolock polymer lockingsurgical clips teleflex medical usa and a circularstapler ethicon endosurgery usa were used in thisstudypreoperative preparationinferior mesenteric artery ima 3d cta examinationshould be performed before the operation to assess themesenteric vascular vessel types fig intestinal preparation was performed days before the operation andprophylactic intravenous antibiotics were used beforethe operation for min central venous catheterizationwas performed after general anesthesia the surgicalposture was the starboard lithotomy position with thehead lower and feet higherthe operating surgeon and camera assistant stood onthe patients right side and the first assistant stood atthe patients left foot side the laparoscopic monitor wasplaced on the patients right foot side the trocar for thelaparoscope was inserted from the right paraumbilicalside and four ports were used as working ports fig surgical techniquesthis surgical technique was characterized by thoroughlymph node dissection based on neurovascular preservation and dissection of the left colon and sigmoid andfig position of the trocarupper rectal vessels along the inferior mesenteric vesselsthe region of operation was the superficial layer of thenerve sheath on the vascular surface the left colonicand superior rectal vessels needed to be preserved andthe vascular branch from the sigmoid vessels and theblood vessels from the superior rectal vessels to the intestinal wall were selected and severed according to thetumor positionfirst we adopted a lateral approach by opening themonks white line along the descending and sigmoidcolon reaching the splenic flexure as the cephalad dissection point the correct plane of dissection wasachieved by toldts fascia we usually used bipolar electrosurgical devices and bipolar scissors to separate thiscorrect plane with gentle blunt and sharp dissectionthe ureter and other retroperitoneal structures weresafely protected by staying in this plane we continuedto dissect along the plane to the root of the ima thehypogastric nerves were visible the nerves were carefully protectedthen the dissection began at the position of the sacralpromontory the junction of the sigmoid mesentery andretroperitoneum from the previous dissection plane in thefirst step ideally we dissected the presacral space belowthe sra from the left side across the midline to the rightside attentively protecting the hypogastric nerves whileusing a bipolar electrosurgical device fig 3a the distaldissection endpoint was approximately cm below thetumor we needed to open the peritoneal reflection anddissect the lateral ligament of the rectum by protectingthe neurovascular bundle nvb using a harmonic vascular closure system in some patients we placed the dissected colon and mesocolon to the right celiac side andthoroughly revealed the left side of the mesocolon wecarefully employed dissection in the correct plane on thevessels to avoid tissue damage for the realization of enbloc resection the technique in this step is to identify therelationship between the left colic artery inferior mesenteric vein imv to the ima and sra and the branch ofthe arteriae sigmoideae fig 3b this vascular bundle canbe traced from the origin of the ima to the rectal segmentapproximately cm below the inferior border of thetumor fig 3cthe second step was performed using a medial approach this step involved thorough lymph node dissection based on neurovascular preservation the leftcolonic and superior rectal vessels need to be preservedand the sigmoid vessels and vessel branch from the superior rectal vessels to the intestinal wall were selectedand severed according to the tumor positiondissection at the correct presacral space and cephaladdissection to the ima could be employed our generalmedial approach was to begin at the presacral space andobtain a connection with the plane ofthe lateral 0czhang world of surgical oncology page of fig a dissection the presacral space below the superior rectal artery sra approached from the left side across the midline to the right sideattentively protected hypogastric nerves while using a bipolar electrosurgical device b identification of the relationship between left colic arteryimv to the ima and sra and the branch of the arteriae sigmoideae c tracing this vascular bundle from the origin of the ima to the rectumsegment approximately cm below the inferior border of the tumor d ligation of arteriae sigmoideae and vascular branch from sra eligation of arteriae sigmoideae and preserving left colonic vasculature f excision of the mesorectum just underneath the rectal wall about cm and avoiding injury to the rectal wall and sra g tsme preserving left colic artery and superior rectal arteryapproach pelvic dissection was performed from the entrance of the pelvic cavity down to the pelvic floor wecould identify both the hypogastric nerve fibers and pelvicnerve by using highdefinition 3d laparoscopy and preservethem the imvleft colic artery bundle was then carefullytraced to the junction position from the ima and lymphnode no253 was dissected the pelvic nerves and ureterwere already carefully insulated and the circumference ofthe ima could be revealed the mesocolon could be freedfrom the retroperitoneal position by anterior dissection bygently applying a bipolar electrosurgical device we dissected the sra and blood vessels from the sra to the intestinal wall and dissected lymph nodes no252 andno251 at this point we had completed lymph node dissection and completely clarified the relationship betweenthe lca imv ima sra and arteriae sigmoideae finallywe ligated the arteriae sigmoideae and vascular branchfrom the sra into the intestinal wall fig 3d while preserving the left colonic vasculature fig 3e energy devicesand hemolocks were used widely in this step 0czhang world of surgical oncology page of after the above procedure was completed we separated the rectal wall from the mesorectum with an adequate distance from the tumor in accordance with thet stage and position of the tumor using a harmonic vascular closure system in order to provide enough spaceto insert an endoscopic linear stapler we excised themesorectum about cm just underneath the rectalwall fig 3f careful surgery was performed to avoid injury to the rectal wall and sra then the endoscopic linear stapler was fixed the rectum was transected andsatisfactory tsme preservation of the left colic and superior rectal arteries was shown fig 3glastly a small 5cm incision was made at the leftlower abdomen and the specimen was taken outside ofthe abdomen and transected intraabdominal presacralanastomosis was performed by double stapling techniques after inserting the anvil head of a 28mm circularstapler into the oral side of the sigmoid colon doubledrains were placed and no diverting stoma wasperformedin the tme group the inferior mesenteric artery wassevered at the root the colon was severed cm awayand digestive tract reconstruction methods were similarto the tsme groupstatisticsspss190 version was used for statistical analysis categorical variables were compared using a Ï2 test continuous variables were presented as the mean standarddeviation or median range these variables were compared using a mannwhitney u test p values of were considered statistically significantresultsthe general characteristics of the included patients arelisted in table there were men and women in the tsme group and men and women in the tme group the meanage was ± years and ± years in thetsme and tme groups respectively there were no significant differences in preoperative comorbidity tumorsize depth of invasion and lymph node metastasis between groups the average distance between the tumorand anus of the tsme group was ± cm andthe distal margin was ± cm the pathologicalstages of the patients for the tsme group were as follows stage i stage iia stage iib stage iic stage iiia and stage iiib theproportion of patients with normal preoperative carcinoembryonic antigen cea was approximately of patients had cea levels between and ngml of patients had cea levels between and ngml and only patients had cea levels ngmltable clinicopathological features between the tsme andtme groupsfactorsage yearstme n ± tsme n ± p valuegendermalefemalebmi kgm2comorbidity ± ± cardiovascular disease respiratory diseasediabetes mellitushistological type differentiated typeundifferentiated type tumor size mm ± ± t categoryt1t2t3t4n categoryn0n1n2 conversion to open surgery operation time min ± ± blood loss ml ± ± lymph node dissection ± ± the operation time in the tsme group was longerthan that in the tme group ± vs ± p table furthermore the number ofresected lymph nodes in the tsme group was greaterthan that in the tme group ± vs ± p table the blood loss between groupswas not significantly different table the averagehospital stay in the tsme group was a little shorter thanthat in the tme group ± days vs ± days table no mortality occurred in either group one patient inthe tme group underwent conversion to laparotomy dueto bowel ischemia in the distal colon table the totalpostoperative complication rates in the tsme and tmegroups were and respectively table forsevere complications between the two groups anastomotic leakage and stenosis the severity of complicationswas claviendindo classification grades and therewas no significant difference between groups 0czhang world of surgical oncology page of tsme n tme n p value ± table postoperative complicationsfactorspostoperative hospital stay days ± mortalitymorbidityabsentpresentanastomotic leakagebleedingabdominal abscessileuswound infectionanastomotic stenosisurinary tract infectionascitesurinary retentionpneumoniacardiacrelated complications discussionin the british surgeon heald proposed tme for rectalcancer and pointed out that the anatomical level of tmewas clear so that the operative quality can be assessed the main concerns were a higher anastomotic leakage ratelonger operative time and higher blood loss after tme lopezkostner pointed out that tme was the standard operation performed for lower rectal cancers tme isnot necessary for cancers of the upper rectum therefore the tsme technique was introduced to achieve satisfactory local control and low morbidity partial mesorectalexcision is applied in tsme according to willians report in only of patients had distal intraluminal diffusion cm pollett and nicholls observed that there were no differencesin the local recurrence rate of rectal cancer between distal margins cm cm and cm a randomized prospective study of nsabb the national surgicaladjustburst and bowel project showed that the localrecurrence rate was not significantly different betweendistal rectal margins cm cm and cm according to the practice parameters for the management of rectal cancer edition a 2cm distal margin is more acceptable than cm but a 5cm distalmargin is still recommended total mesorectal resectiontme should be used for tumors located in the middleand lowerregardless oftwothirds ofwhether itis performed with low anterior resectionlar or combined abdominal and perineal resectionapr for tumors in the upper onethird of the rectumresection of the mesentery can be carried out accordingto the tumor situation and the distance between thethe rectumdistal margin and tumor should be cm the recommended grade was 1a tme was performed according to the distance between the distal margin of the rectal tumor and anus cm while tsme was performed for patients with adistance between the distal end of the rectal tumor andanus of cm in the authors medical departmentoncological outcomes after surgery can be divided intotwo aspects longterm survival and local recurrence ratelaw reviewed patients the 5year local recurrence rate for tme and partial mesorectal excisionpme for proximal cancer was and respectively the disease stage was associated with a higher riskof local recurrence there was no difference in the localrecurrence rates of tme and pme the 5year cancerspecific survival rates with and without tme were similarat and respectively kim reportedthat the 5year cancerspecific survival rate was andthe local recurrence rate was with cases of rectalcancer after tsme with pathologic stages iiii the riskfactors affecting cancerspecific survival rate were the ptstage pn stage positive distal resection margin and positive circumferential resection margin the risk factors affecting local recurrence were the pn stage positive distalresection margin and positive circumferential resectionmargin another study from a korean reviewed experience in patients with rectal cancer showed that theoverall local recurrence rate was the 5year local recurrence rates were and in stages i iiand iii respectively the 5year cancerspecific survivalrates were and in stages i ii and iiirespectively the risk factors were the pn stage and circumferential resection margin zakir performed an analysis with years of experience in rectal cancer patients who underwent laparoscopic andopen tsme surgery the 5year local recurrence rate was the overall 5year and cancerspecific survival rateswere and respectively there was no difference in the local recurrence rate between laparoscopic oropen resection the overall and cancerspecific survivalrates were and in the laparoscopic surgerygroup and and in the open surgery group respectively the results showed that laparoscopic surgerywas better than open surgery in overall and cancerspecific survival there was no difference in survival in patients with stage i however the survival rates in patientswith stages ii and iii among the laparoscopic surgerygroup were better than those in the open surgery groupwhich shows the superiority of laparoscopic tsme surgery for the longterm prognosis of rectal cancerkorean scholars conducted a study on the safety andprognosis of tsme after neoadjuvant chemotherapy forrectal cancer patients received 5fu with leucovorinchemotherapy and radiotherapy cgy for cycles 0czhang world of surgical oncology page of leadership was tsme was performed weeks later the resultsshowed that the overall complication rate was empiricalinternal construction was the 5year survival rate was and the 5yeardiseasefree survival was at present chinasouth korea and the usa have formulated similarguidelines for preoperative radiotherapy and chemotherapy for middle and low rectal cancer but there is nospecific reference data for preoperative radiotherapy andchemotherapy for upper rectal cancer the purpose ofthis paper is to introduce a new method of tsme anddiscuss the safety of the operation longterm survivaland local recurrence have not been discussedtsme surgery based on tme is now accepted as astandard for rectal cancer surgery and laparoscopic rectal cancer resection is accepted widely in the world eventhough it is a challenging procedure for surgery bloodloss in the laparoscopic group is well shown with anaverage of to ml the average blood loss inour study was ml lower than that reported in the literature we can identify neurovascular lesions usinghighdefinition 3d laparoscopy to preserve them and weuse a bipolar electrosurgical device to reduce injurywhich is beneficial for accurate operationthe overall complication rate in laparoscopic tsmeoperation was lower than that in the open operationgroup the rate of anastomotic leak showed no statistical difference between the two operation methods theaverage leak rate for rectal cancers was zakir reported that the overall complicationrate was in tsme for rectal cancer patients therate of anastomotic leakage was in the open tsmegroup and in the laparoscopic tsme group therewas no statistical difference between groups in our studythe incidence rate of postoperative anastomotic leakagewas three patients had complications after surgeryand the overall complication rate was the threecomplications were wound infection fluid collection andurinary retention with a claviendindo grading of yoo evaluated the optimal duration of urinarycatheterization after tsme for rectal cancer logistic regression analysis was performed to determine the risk factors for urinary retention the variables including age sexasa grade surgical procedure tnm stage tumor position preoperative radiotherapy duration of urinarycatheterization and time of surgery were not significantrisk factors for urinary retentionat present a 3d laparoscopic system aesculapgerman is used in laparoscopic surgery in our department single and reduced portlaparoscopic surgeryrobot operations and tatme operations are not usedfor tsme the surgeons who performed tsme had morethan years of experience in gastroenterostomy and hadexperience with open tsme the difficulty of the tsmeoperation is the management of the mesorectum seiji has reported on the management of the mesorectumin the narrow pelvis which our treatment method is basedon first the right part of the mesorectum is lifted fromthe right side of the sigmoid mesocolon to expose the inferior mesenteric artery and vein left colonic vessels sigmoid colonic vessels and superior rectum vessels theassistant lifts the left mesentery of the sigmoid colon exposes the above vessels expands the sigmoid mesocolonagain penetrates the mesentery from the right side andexposes the surrounding vessels expansion of the pelviccavity along the vessels is continued and the mesorectumis repaired from the left to the right side cm above thetumor according to the location ofthebranches of the severed vessels are determined and cm of the intestinal wall is repaired the rectum is dissected using an endogia staplerthe tumorlaparoscopic tsme has been used for rectal cancer andcan obtain satisfactory functional results compared to openresection and tme we do not think that the reduction inthe hospital stay is due to the acceleration of the intervention as per enhanced recovery after surgery eras butis due to an increase in the doctors confidence in reducingthe risk of postoperative complications after vascular preservation threedimensional cta examination is importantfor the preoperative evaluation of sigmoid colonvascular classification and intraoperative management ofthe sigmoid and left colon vessels however preoperativeexamination could not obtain information on the trafficbranch the biggest advantage of this operation is themaintenance of the blood supply of the proximal and distalintestines and the sufficient length of the intestine so thereis no need for temporary defunctioning stoma temporarydefunctioning stoma only increases the complexity of theoperation and closure of the temporary stoma increasesthe risk of complications in addition the results of the statistical analysis showed that the number of lymph nodes inthe tsme group was greater than that in the tme groupit cannot be concluded that tsme was significantly betterthan tme for lymph node dissection suggesting thattsme was not inferior to tmeslaparoscopic tsme with preserved left colic and superior rectal arteries is a technically challenging procedureintact visceral pelvic fibro is protected with even greateraccuracy than other techniques by 3d laparoscopywhich offers an optimal vision tsme with preserved leftcolic and superior rectum arteries did not increase therisk of operation compared with tme but increased thesurgeon s confidence in patient outcomes thereforelaparoscopic tsme with preserved left colic and superior rectal arteries can be safely performed for rectal cancer patients as an alternative to tme 0czhang world of surgical oncology page of abbreviationstme total mesorectal excision tsme tumorspecific mesorectal excisionima inferior mesenteric artery imv inferior mesenteric vein sra superiorrectal artery nvb neurovascular bundle pme partial mesorectal excisionacknowledgementsnoneauthors contributionslz yx and xh designed the study cz yx hw qz zd and whcollected and analyzed the data ma lz ys and xh interpreted the datalz and cz drafted the manuscript lz ma yx and xh revised themanuscript the authors read and approved the final manuscriptfundingthis study was supported by the jiangsu natural science foundationbk20180274 this funding supported the collection analysis andinterpretation of the dataavailability of data and materialsall experimental data used to support these findings are included in theethics approval and consent to participatethis study was approved by the institutional review board of the firstaffiliated hospital of dalian medical university written informed consent forpublication was obtained from all patientsconsent for publicationwritten informed consent was obtained from the patients and legalguardian for the publication of these patientscompeting intereststhe authors declare that they have no conflicts of interestauthor details1department of gastrointestinal surgery the first affiliated hospital of dalianmedical university dalian liaoning province china 2department ofgastrointestinal surgery the third affiliated hospital of guangxi medicaluniversity nanning guangxi province china 3department ofgastrointestinal surgery changzhi peoples hospital the affiliated hospital ofchangzhi medical college changzhi shanxi province china 4department ofcolorectal surgery the first affiliated hospital of nanjing medical universitynanjing china 5department of gastrointestinal surgery the first peopleshospital of dali city dali yunnan province china 6department ofgastrointestinal surgery graduate school of medicine university of tokyotokyo japan 7department of general surgery yizhen peoples hospitalclinical medical college yangzhou university yangzhou jiangsu provincechinareceived february accepted august heald rj husband em ryall rd the mesorectum in rectal cancer surgerythe clue to pelvic recurrence br j surg macfarlane jk ryall rd heald rj mesorectal excision for rectal cancerlancet lee ky factors influencing oncologic outcomes after tumorspecificmesorectal excision for rectal cancer j korean soc coloproctol williams ns dixon mf johnston d reapppraisal of the centimetre rule ofdistal excision for carcinoma of the rectum a study of distal intramuralspread and of patients survival br j durg pollett wg nicholls rj the relationship between the extent of distalclearance and survival and local recurrence rates after curative anteriorresection for carcinoma of the rectum ann surg wolmark n fisher b wieand hs the prognostic value of the modificationsof the dukes c class of colorectal cancer an analysis of the nsabp clinicaltrials ann surg monson jrt weiser mr buie wd chang gj rafferty jf prepared by thestandards practice task force of the american society of colon and rectalsurgeons practice parameters for the management of rectal cancerrevised dis colon rectum law wl chu kw anterior resection for rectal cancer with mesorectalexcision a prospective evaluation of patients ann surg kim sh bae kb kim jm oncologic outcomes and risk factors forrecurrence after tumorspecific mesorectal excision of rectal cancer cases j korean soc coloproctol kim nk min bs kim js hur h lee ky sohn sk oncologic outcomesand safety after tumorspecific mesorectal excision for resectable rectalcancer a single institutions experience with patients with rectalcancer j korean soc coloproctol zakir k mohamed wai lun law outcome of tumorspecific mesorectalexcision for rectal cancer the impact of laparoscopic resection world jsurg kim nk baik sh seong js oncologic outcomes after neoadjuvantchemoradiation followed by curative resection with tumorspecificmesorectal excision for fixed locally advanced rectal cancer impact ofpostirradiated pathologic down staging on local recurrence and survivalann surg poon jt law wl laparoscopic resection for rectal cancer a review annsurg oncol yoo be kye bh kim hj early removal of the urinary catheter aftertotal or tumorspecific mesorectal excision for rectal cancer is safe discolon rectum seiji o takashi t kazuki s a new laparoscopic surgical procedure toachieve sufficient mesorectal excision in upper rectal cancer int j surgoncol httpsdoi1011552011708439publishers notespringer nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliationsreferencesenker w e thaler h t cranor m l polyak t total mesorectal excision inthe operative treatment of carcinoma of the rectum j am coll surg lopezkostner i lavery c hool gr rybicki la fazio vw total mesorectalexcision is not necessary for cancer of the upper rectum surgery zaheer s pemberton jh farouk r dozois rr wolff bg ilstrup d surgicaltreatment of adenocarcinoma of the rectum ann surg sudeck p ueber die gefässversung des mastdarmes in hinsicht auf dieoperative gangrän muenchen med wschr van tonder jj boon jm becker jhr anatomical considerations onsudecks critical point and its relevance to colorectal surgery clin anatcirocchi r randolph j cheruiyot i systematic review and metaanalysis of the anatomical variants of the left colic artery color dis httpsdoi101111codi14891 0c" | 0 |
"objective endoscopic full thickness resection eftr has shown efficacy and safety in the colorectum the aim of this analysis was to investigate whether eftr is cost effective in comparison with surgical and endoscopic treatment alternativesdesign real data from the study cohort of the prospective single arm wall resect study were used a simulated comparison arm was created based on a survey that included suggested treatment alternatives to eftr of the respective lesions treatment costs and reimbursement were calculated in euro according to the coding rules of and eftr r0 resection rate was used as a measure of effectiveness to assess cost effectiveness the average cost effectiveness ratio acer and the incremental cost effectiveness ratio icer were determined calculations were made both from the perspective of the care provider as well as of the payerresults the cost per case was ¬ for the eftr group ¬ for the standard endoscopic resection ser group ¬ for the surgical resection group and ¬ for the pooled alternative treatment to eftr from the perspective of the care provider the acer mean cost per r0 resection was ¬ for eftr ¬ for ser ¬ for surgical treatment and ¬ for all pooled and weighted alternatives to eftr the icer additional cost per r0 resection compared with eftr was ¬ for ser ¬ for surgical resection and ¬ for the pooled rate of alternatives results from the perspective of the payer were similar eftr is cost effective in comparison with surgical and endoscopic treatment alternatives in the colorectumintroductioncolorectal cancer is the third most common type of cancer and the second most common cause of cancer related deaths worldwide1 screening programmes for early detection of premalignant and malignant lesions led summary boxwhat is already known about this subject º endoscopic full thickness resection eftr has shown clinical efficacy and safety in difficult to treat lesions in the colorectum º the cost of the full thickness resection device is higher than the cost of standard endoscopic resection ser devices but lower than surgical devices º cost effectiveness analyses on treatment with eftr compared with treatment alternatives do not existwhat are the new findings º eftr leads to an almost reduction in cost per r0 resection average cost effectiveness ratio compared with surgery º to achieve an additional r0 resection by surgical treatment compared with eftr incremental cost effectiveness ratio an additional cost of ¬ is necessary º these findings are consistent both from the perspective of the care provider as well as the payerhow might it impact on clinical practice in the foreseeable future º in terms of cost effectiveness eftr should be considered first before patients with difficult to treat lesions in the colorectum are sent to surgical treatmentto a significant reduction in cancer related mortality2 with more intense screening more lesions are detected which automatically creates the need for removal standard endoscopic resections ser such as endoscopic mucosal resection emr and endoscopic submucosal dissection esd are well established and sufficient for the vast majority of lesions however ser of non lifting lesions and lesions located at difficult anatomical to cite kuellmer a0a behn a0j beyna a0t et a0al endoscopic full thickness resection and its treatment alternatives in difficult to treat lesions of the lower gastrointestinal tract a cost effectiveness analysis bmj open gastro 20207e000449 101136bmjgast2020000449received may revised july accepted july authors or their employers re use permitted under cc by nc no commercial re use see rights and permissions published by bmjfor numbered affiliations see end of correspondence toarthur schmidt arthur schmidt uniklinik freiburg dekuellmer a0a et a0al bmj open gastro 20207e000449 101136bmjgast2020000449 0copen access locations eg appendiceal orifice is associated with increased complication rates or incomplete resection3 these types of lesions are therefore often referred to surgery which is associated with significant morbidity and mortality and higher costs6 given the high number of polypectomies performed worldwide this is not only an issue of morbidity and mortality but also a huge economic challengethe efficacy and safety of endoscopic full thickness resection eftr of non lifting and other difficult to treat lesions have been demonstrated in multiple retrospective studies and in one prospective study7 the cost of the device is markedly higher than ser devices but lower than surgical treatment the aim of the present analysis was to evaluate whether eftr is cost effective in comparison with ser as well as surgical treatmentmethodsstudy populationto calculate the cost of eftr we analysed the study cohort of the wall resect trial nct02362126 in this single arm multicentre prospective study patients with difficult to treat adenomas eg non lifting andor challenging anatomical location early adenocarcinomas and subepithelial tumours in the colorectum were treated with eftr the primary endpoints of the study en bloc and r0 resection rate were achieved in and respectively7 written informed consent was obtained from each patient included in the studysimulation second study arm based on a survey of endoscopistswith the wall resect study being single armed a second study arm was created based on treatment simulation in order to compare different treatment modalities a case report form crf was created and sent to each participating centre of the wall resect trial endoscopists at the respective location reviewed the endoscopic images and their case relevant data and decided which treatment modality they would have chosen if eftr were not available treatment alternatives included ser such as emr thermal methods and esd as well as surgical resection the crf was filled out in a pseudonymised fashioninformed consent had already been obtained within the wall resect studydetermination of case costs and reimbursementa certified online it tool the diagnosis related group drg web grouper was used to determine the reimbursement rate for each patient http drg uni muenster de index php therefore the code of the international classification of diseases icd10 and the specific code for the procedure performed operationen und prozedurenschl¼ssel ops code in each patient in both groups were put into the web grouper together with the predefined mean length of hospital stay mittlere grenzverweildauer the drg which accounts for reimbursement was calculatedin the comparison arm the drg codes of were used because this was the year the wall resect study was performed for the eftr arm the drg codes of were used as reimbursement for eftr was increased that yearto calculate the cost per case another certified online it tool g drg report browser was used www g drg de g drg system_ abschlussbericht_ zur_ weiterentwicklung_ des_ g drg systems_ und_ report_ browser this was done by filling in the respective drg icd10 and ops code into the browser the data of the g drg report browser derive from the data that were sent in to inek authority managing the german drg system by certified hospitals kalkulationshuser in grouping was performed following the rules of g drg version the main and secondary diagnoses are shown according to icd10 german modification gm version and the procedures according to ops version g drg report browser inek gmbhas reimbursement for the eftr group was taken from the cost per patient case would ideally also have been calculated from unfortunately these data will be first published by inek in to overcome this problem costs for eftr cases from the university of freiburg between and which were reported to inek were used for the analysiswith the cost of each patient case the mean cost for each treatment modality emr esd laparoscopic surgery transanal endoscopic microsurgery tem eftr could be calculated in the next step the mean cost for each treatment path ser surgical treatment and casemix alternative was determined this was done in the following fashion for ser the mean costs of emr and esd were used for calculation of the surgical treatment laparoscopic surgery and tem were taken together the mean costs of endoscopic and surgical treatments were subsumed as the casemix alternativedetermination of effectivenessthe r0 resection rate was defined as the efficacy parameter to determine cost effectiveness the r0 rate of eftr in the wall resect trial was to determine the efficacy of the therapeutic alternatives to eftr a selective literature review was performed in pubmed and cochrane databases identifying the largest studies comparing resection techniques and r0 rates the respective rates regarding ser found in the literature were for emr and for esd9 for the surgical oncological resection treatment as the gold standard a r0 resection rate was assumed for the tem a rate of had been reported10in order to compare all ser methods emresd all surgical resection methods laparoscopic surgerytem and all alternative methods endoscopic and surgical kuellmer a0a et a0al bmj open gastro 20207e000449 101136bmjgast2020000449 0ctable alternative treatment strategies to eftr with their respective efficacy based on literature review and calculationtreatmentefficacy n n180surgical oncological resection laparoscopictememresdsurgical treatment laparoscopic and temser emresdcasemix alternative assumed arezzo et al fujiya et al arezzo et al calculated calculated calculatedthe combined effectiveness of surgical treatment ser and casemix alternative was calculated by multiplication of the number of patients in each modality eg emr cases for ser with the respective r0 resection rate as the first step in the second step this result would be summed up to the result of the other modalities eg esdemr result for the ser methods and divided by the number of patients in this group of resection method eg patients in the ser group overall efficacy of surgical treatment and casemix alternative was performed in the same mannereftr endoscopic full thickness resection emr endoscopic mucosal resection esd endoscopic submucosal dissection ser standard endoscopic resection tem transanal endoscopic microsurgerycasemix alternative with the eftr procedure a combined effectiveness of each treatment group was calculated this was done by multiplication of the number of patients in each modality eg emr cases with the respective r0 resection rate as the first step in the second step this result would be summed up to the result of the other modalities eg esdemr result for the ser methods and divided by the number of patients in this group of resection method eg patients in the ser group using this approach the overall efficacy in the ser group was calculated as overall efficacy of surgical treatment and casemix alternative was performed in the same manner and was calculated as and respectively the respective r0 rates are shown in table calculation of costeffectivenessfor assessment of cost effectiveness the average cost effectiveness ratio acer and the incremental cost effectiveness ratio icer were calculated acer expresses the mean costs for the investigated outcome11 in our study acer describes the mean costs per successful r0 resection in the different treatment modalitiesacer is calculated with the following computational formula acer mean costseffect open accessicer expresses the additional costs of a treatment alternative for improvement in the investigated outcome12 in our study these are the incremental costs for the alternative treatment to eftr required to achieve an r0 resectionicer is calculated with the following computational formula icer mean costs interventionmean costs controleffect intervention effect control the mean costs were the total costs of the respective treatment modality divided by the number of patients in each group for the calculation of cost effectiveness the ser methods emr and esd as well as the surgical resection methods laparoscopic resection and tem were taken together furthermore cost effectiveness was calculated for the casemix alternative to compare eftr with all alternativesresultscomparative study armendoscopist surveyfrom patients of the study cohort responses were included for further analysis in one patient the investigator recommended solely thermal ablation as alternative treatment of choice thus the primary endpoint r0 resection could not be evaluated from the remaining patients the endoscopists recommended surgical treatment in of of cases thereof of were laparoscopic resections and of tem in of of cases an endoscopic resection was proposed thereof of were emr and of were esdcosts from the perspective of the care providercosts per case were derived from the drg report browser which represent costs of the respective treatment alternative for the year the costs for the eftr treatment were derived from university hospital freiburg and represent the mean costs from years to mid2019 the mean cost for eftr was ¬ the cost per surgical treatment laparoscopic surgery and tem was ¬ and for ser ¬ all alternative treatment strategies casemix alternative op laparscopic surgery tem esd and emr were calculated as ¬ per case the results are shown in figure costs from the perspective of the thirdpartyaccording to the german drg system reimbursement for eftr is ¬ for surgical treatment ¬ was calculated the cost per case for ser is ¬ the cost for the casemix alternative is ¬ the results are shown in figure costeffectiveness analysis care provider viewpointaverage costeffectiveness ratiothe mean cost per r0 resection is ¬ in the eftr group and ¬ in the surgical group in the ser group the cost per r0 resection is ¬ in the casemix alternative group including all treatment kuellmer a0a et a0al bmj open gastro 20207e000449 101136bmjgast2020000449 0copen access figure case costs ¬ for the different treatment modalities are shown costs from the perspective of the third party payer reimbursement are shown in black while actual case costs from the perspective of the care provider are shown in grey surgery mean costs for tem and laparoscopic surgical oncological resection ser mean costs for esd and emr casemix mean costs for esd emr tem and laparoscopic surgery eftr endoscopic full thickness resection emr endoscopic mucosal resection esd endoscopic submucosal dissection ser standard endoscopic resection tem transanal endoscopic microsurgeryfigure incremental cost effectiveness ratio for the different treatment modalities compared with eftr is shown costs from the perspective of the third party payer reimbursement are shown in black while actual case costs from the perspective of the care provider are shown in grey surgery mean costs for tem and laparoscopic surgical oncological resection ser mean costs for esd and emr casemix mean costs for esd emr tem and laparoscopic surgery eftr endoscopic full thickness resection emr endoscopic mucosal resection esd endoscopic submucosal dissection ser standard endoscopic resection tem transanal endoscopic microsurgeryalternatives except eftr the mean cost per r0 resection is ¬ the results are shown in figure the casemix alternative ¬ the results are shown in figure incremental costeffectiveness ratioin comparison with eftr the incremental cost for an additional r0 resection is ¬ if ser is performed the cost for the surgical approach is ¬ and for figure average cost effectiveness ratio ¬ for the different treatment modalities is shown costs from the perspective of the third party payer reimbursement are shown in black while actual case costs from the perspective of the care provider are shown in grey surgery mean costs for tem and laparoscopic surgical oncological resection ser mean costs for esd and emr casemix mean costs for esd emr tem and laparoscopic surgery eftr endoscopic full thickness resection emr endoscopic mucosal resection esd endoscopic submucosal dissection ser standard endoscopic resection tem transanal endoscopic microsurgerycosteffectiveness analysis health insurance reimbursement viewpointaverage costeffectiveness ratiofrom the perspective of the health insurance the cost per r0 resection is ¬ in the eftr group in the ser group the cost is ¬ and in the surgical treatment group ¬ in the casemix alternative the cost per r0 resection is ¬ the results are shown in figure incremental costeffectiveness ratiothe icer of ser in comparison with eftr is ¬ the surgical approach costs an additional ¬ for the casemix alternative ¬ is necessary for an additional r0 resection the results are shown in figure discussionwith technical endoscopic progress patient care has constantly improved over the years however as with any technical innovation this is associated with higher costs therefore the efficacy of new methods and devices needs to be evaluated in relation to their costs13 to our knowledge this is the first cost effectiveness analysis cea for eftr our results demonstrate that eftr for difficult to treat lesions in the colorectum is cost effective in comparison with ser as well as surgical therapy furthermore the results are consistent when analysed from the perspective of the care provider as well as of the payerkuellmer a0a et a0al bmj open gastro 20207e000449 101136bmjgast2020000449 0cfor our analysis a simulated control arm was created this was necessary as to date no randomised controlled trial rct investigating eftr versus alternative treatments has been published in our survey endoscopists proposed surgical treatment as the likely alternative to eftr in the majority of cases as opposed to ser via emr or esd all lesions within the wall resect trial were difficult to resect lesions eg non lifting adenomas exhibiting a high risk of perforation or incomplete resection when treated with ser therefore it may be surprising that ser was suggested in of cases however the suggestions were made by expert endoscopists who might have decided towards an advanced endoscopic procedure more generouslyregarding the costs for each treatment modality it was not surprising that the cost of eftr is above ser ¬ vs ¬ this is due to the cost of the device in germany ¬ plus value added tax however the cost of eftr was roughly one third of the cost of surgery ¬ vs ¬ this reflects the minimally invasive nature of eftr compared with laparoscopic or open surgical operationswhile costs for endoscopic resection and surgical therapy were taken from official and certified tools web grouper and drg report browser the factual costs of the eftr procedure for the year that are determined in a representative cross section of hospitals have not been published yet by inek the administrator of the drg system reimbursement of the procedure changed in thus these data should have been used for calculation to overcome this problem the mean case costs for eftr per case in our home institution university hospital of freiburg germany in the time between and were used as a surrogate in an economic analysis of the cost of eftr in germany presented at the annual conference of the german society for digestive diseases obtained from different endoscopic centres reported ¬ per case as this is only above our number and therefore in the same range our calculated ¬ seems to be a realistic number14in our analysis we chose the r0 rate as a means to detemine effectiveness as this is the most objective parameter to assess curative resection and treatment success furthermore the r0 rate can be compared with the treatment alternatives of eftr as high quality meta analyses and therapeutic success rates exist for those procedures10 the r0 rate for surgical colonic resection was assumed to be however the patient cohorts of these studies are not equal the wall resect study included only difficult to resect lesions mainly non lifting while the studies mentioned above included primarily treatment naive lesions larger studies on ser on non lifting lesions do not exist hence it is reasonable to assume that in these indications real r0 rates of ser would be lower and therefore cost effectiveness would be even worsefor measuring cost effectiveness acer and icer were determined the analysis was performed both open accessfrom the perspective of the care provider hospital as well as the reimbursement authority health insurance acer expresses the mean cost per r0 resection for both investigated perspectives our results reveal that costs are much lower for eftr compared with the surgical alternative although the effectiveness of the surgical approach in terms of radicality can be considered to be higher eftr is cost effective an r0 resection by eftr leads to nearly reduction in costs for the care provider ¬¬ and for the health insurance ¬¬ compared with ser eftr leads to marginally higher costs per r0 resection as explained above comparing eftr with ser has limitations as the investigated difficult lesions in the wall resect study are not well studied for emr and esd however in comparison with all treatment alternatives casemix alternative we calculated and reduction in costs similar to the surgical alternatives figure icer expresses the additional costs for an additional increase in the designated outcome in our analysis it expresses the additional costs that are necessary for an additional r0 resection as shown in figure all alternatives to eftr result in additional costs while ser results in a modest increase ¬ and ¬ additional ¬ and ¬ per r0 resection are required in the surgical group in the casemix alternative group additional costs were ¬ and ¬ respectivelyan absolute threshold at which an icer is thought to be cost effective does not exist16 in the literature the willingness to pay threshold ranges from to and is highly subjective to the investigated outcome and the healthcare system for which the cea is made17 for our analysis we assume that a more invasive treatment that produces at least ¬ more costs for an additional r0 resection cannot be regarded as being cost effectivefor our analysis we did not include costs of follow up endoscopies or further treatment arising from recurrency or from adverse events this was done due to the following reasons first reliable recurrence rates and long term follow up after eftr do not exist follow up in the wall resect trial was only weeks second the lesions of our patient cohort were heterogeneous including adenomas carcinomas and neuroendocrine tumours with different biological features and recurrent rates third treatment of recurrent lesions is not standardised and ranges from re eftr to snare polypectomy to removal with a biopsy forceps leading to highly variable costs fourth management of severe complications and consecutive morbidity differs in every patient and depends on severity of complication patients comorbidities and local expertise we do not have reliable data on costs for such treatment and a hypothetical model would have been highly speculative moreover in the wall resect trial of patients required consecutive surgery due to complications this rate is slightly kuellmer a0a et a0al bmj open gastro 20207e000449 101136bmjgast2020000449 0copen access higher but still grossly comparable with the complication rates of emr and esd on the other hand complications after surgical resection eg anastomotic leakage are much more frequent up to and usually lead to higher morbidity hence even if costs related to complications were added icer is still likely to favour eftr compared with the group of treatment alternativesit is difficult to compare our results with other ceas as this is the first one for this indication the only previous cea on ser compared emr and esd in laterally spreading lesions irrespective of location or lifting sign in most analyses as in the study by bahin and colleagues19 a decision tree model was created to compare different outcome scenarios after each treatment path was filled with probabilities of occurrence costs per predefined outcome were calculated a potential bias of this approach is that the data for the probabilities of occurrence which influence the costs most are taken totally or at least in part from different studies19 22this harbours the risk of resulting in a very heterogeneous study population with uncontrolled confounders this risk can be minimised by deriving data from rcts with well balanced patient cohorts as recently published17 for our analysis we used a different approach than a decision tree factual variables and outcome data derived from the only prospective study on eftr treatment and not from assumptions the simulation of the control arm had to be performed due to the lack of rcts in this setting the strength of our study is that the very same clinician who actually performed the respective eftr could review the different lesions and decide on a solid basis which treatment alternative he or she would have used instead of eftr in our view this approach reflects the clinical situation more precisely than a decision tree modelin most ceas the costs per quality adjusted life years are calculated and taken for healthcare decisions neither survival nor quality of life measurements were part of the wall resect trial in line with most of the recently published cea we calculated costs per defined outcome as the primary endpoint17 our study has several limitations first the comparison arm of the study is based on simulation so there is always a risk of a bias second our analysis is specific to the german healthcare system and may therefore not be fully comparable with different healthcare systems in the world third the estimated r0 rate for the ser methods is very low and likely due to the piecemeal resection in the respective study if efficacy would have been measured as freedom of recurrence efficacy would be higher as proven in the australian colonic endoscopic study24 nonetheless we used the published r0 rate because of the possibility to match this with the endpoint of the wall resect study furthermore as described above an endpoint such as freedom of recurrence cannot be determined reliably as such data do not exist for eftr fourth costs of complications and follow up were not included this is mainly due to lack of an rct and the short follow up period in an ideal cea all treatment related and hospital stay related costs would have been calculatedin our data indicate that eftr for difficult to treat lesions of the colorectum is cost effective compared with surgical and endoscopic treatment alternatives the results are consistent both from a care provider as well as from a third party payer perspective rcts and long term follow up are needed to further assess the cost effectiveness of eftrauthor affiliations1department of medicine ii medical center university of freiburg faculty of medicine university of freiburg freiburg germany2department of gastroenterology klinikum ludwigsburg ludwigsburg baden w¼rttemberg germany3department of gastroenterology evangelisches krankenhaus d¼sseldorf dusseldorf nordrhein westfalen germany4department of internal medicine and gastroenterology elisabeth hospital essen nordrhein westfalen germany5department of medicine ii interventional and experimental endoscopy inexen university hospital wurzburg wurzburg bayern germany6department of gastroenterology university hospital augsburg augsburg bayern germany7department of gastroenterology university hospital ulm ulm baden w¼rttemberg germany8department of gastroenterology klinikum dortmund dortmund nordrhein westfalen germany9department of gastroenterology helios klinikum krefeld krefeld nordrhein westfalen germany10department of gastroenterologyoncology klinikum sindelfingen b¶blingen sindelfingen baden w¼rttemberg germanycontributors as and kc invented and planned the present study and also the underlying wall resect study as assisted with data acquisition and analysis and revised the manuscript jb was responsible for data research and acquisition ak was responsible for data analysis and writing the manuscript kc tb bs am hm hn da mb ap mf tf mg and rt took part in the online survey to create the simulation comparison arm of the study furthermore they carefully read and revised the manuscriptfunding the authors have not declared a specific grant for this research from any funding agency in the public commercial or not for profit sectorscompeting interests as and kc received lecture fees and study grants from ovesco endoscopy t¼bingen germany ak jb tb bs am hm hn da mb ap mf tf mg and rt have no conflicts of interest or financial ties to disclosepatient consent for publication not requiredethics approval the wall resect study was approved by the ethical board on january the study protocol conforms to the ethical guidelines of the declaration of helsinki as reflected in a prior approval by the institution's human research committee for the present study an additional approval by the institutional review board was not necessary since no additional personal data were collectedprovenance and peer review not commissioned externally peer revieweddata availability statement all data relevant to the study are included in the or uploaded as supplementary information data were derived from the wall resect trial nct02362126open access this is an open access distributed in accordance with the creative commons attribution non commercial cc by nc license which permits others to distribute remix adapt build upon this work non commercially and license their derivative works on different terms provided the original work is properly cited appropriate credit is given any changes made indicated and the use is non commercial see a0http creativecommons org licenses by nc orcid idarmin a0kuellmer http orcid org kuellmer a0a et a0al bmj open gastro 20207e000449 101136bmjgast2020000449 0creferences who colorectal cancer fact sheet the global cancer observatory available http gco iarc fr today data factsheets cancers 10_ 8_ colorectum fact sheet pdf [accessed sep ] zauber ag winawer sj o'brien mj et a0al colonoscopic polypectomy and long term prevention of colorectal cancer deaths n engl j med hong sn byeon js lee b i et a0al prediction model and risk score for perforation in patients undergoing colorectal endoscopic submucosal dissection gastrointest endosc org mizushima t kato m iwanaga i et a0al technical difficulty according to location and risk factors for perforation in endoscopic submucosal dissection of colorectal tumors surg endosc agapov m dvoinikova e factors predicting clinical outcomes of endoscopic submucosal dissection in the rectum and sigmoid colon during the learning curve endosc int open 20142e235 baum p diers j lichthardt s et a0al sterblichkeit und komplikationen nach viszeralchirurgischen operationen dtsch arztebl int schmidt a beyna t schumacher b et a0al colonoscopic full thickness resection using an over the scope device a prospective multicentre study in various indications gut aepli p criblez d baumeler s et a0al endoscopic full | 0 |
"unrestricted use distribution and reproduction in any medium provided the original work is properly citedPurpose The present study was aimed at determining the serum levels of actinin4 ACTN4 in cervical cancer CC andinvestigating the diagnostic and prognostic value of serum ACTN4 in CC Materials and Methods We included CC patients cervical intraepithelial neoplasia CIN patients and healthy women Serum ACTN4 levels were assessed using an ELISAmethod A receiver operating characteristic ROC curve was performed to evaluate the diagnostic value of serum ACTN4 Thesurvival curves were used to display the overall survival distributions Results Serum ACTN4 levels in CC patients were ± pgmL which is significantly higher than those in CIN patients ± pgmL P and those in healthycontrols ± pgmL P The ROC analysis demonstrated that the area under the curve AUC of ACTN4 was 95CI with sensitivity of and speciï¬city of Serum ACTN4 levels were associated with theFIGO stage lymph node metastasis and lymphovascular space invasion of CC all P The survival curve suggested thathigh serum ACTN4 levels were related to poor prognosis Conclusion Our ï¬ndings suggest that serum ACTN4 levels may bevaluable diagnostic and prognostic biomarkers for CC IntroductionCervical cancer CC is the second most common femalemalignancy globally and it is the most common femalemalignancy in developing countries which has high morbidity and mortality rates [] In recent years the incidence ofCC has increased greatly in young women under the age of [] Despite great advances in surgical and adjuvant therapy the overall survival of CC patients especially that ofadvanced patients is still very poor [] At present a Papsmear combined with an HPV test has been used for the earlyscreening of cervicalthe screeningmethods are invasive and costly leading to lower screeningcoverage in China [] Previous studies have reported thatthe human papillomavirus HPV screening results have arelatively high falsepositive rate and a relatively low speciï¬city [ ] In addition the results of TCT interpretation byï¬lmreading doctors are uneven which might cause somelesions Howevermisleadingness in the choices of prevention measures andtreatment for CC [] Noteworthily when applying the sametreatment plan to patients with similar pathological types theeï¬cacy and prognosis are quite diï¬erent Therefore it is necessary to identify new biomarkers directly related to the progression and prognosis of CCAlphaactinins ACTNs are actinbinding proteins inthe spectrin gene superfamily [] which are known to becrosslinked with ï¬lamentous actin Factin to maintainthe integrity of cytoskeleton and to control cell motility []The ACTN family has four members numbered ACTN1which are present in humans and other mammals []ACTN4 is encoded by the ACTN4 gene and is widelyexpressed in many tissues especially in glomerular podocytes[] ACTN4 has an actinbinding domain at the Nterminus and ACTN4 monomers can form a homodimer throughreverse binding forming a dumbbellshaped structure []As an actinbinding protein ACTN4 is closely related to 0cDisease Markersenhancing cell viability and tumor invasion and metastasis[] Recent researches have reported that the expression ofACTN4 is significantly elevated in multiple cancers including breast cancer [] pancreatic cancer [] ovarian cancer[] and lung cancer [] In addition the ACTN4 levels aremarkedly associated with the poor prognosis of lung cancer[ ] thyroid cancer [] and salivary gland carcinoma[] An [] have found that the expression level ofACTN4 in human cervical tumors is dramatically higherthan that in normal cervical tissues Their ï¬nding demonstratedepithelialtomesenchymal transition and tumorigenesis by regulatingSnail expression and the Akt pathway in CC [] Thereforethe expression of ACTN4 in cervical tissues may be used inthe clinical diagnosis and prognosis prediction of CCthat ACTN4promotestheHowever up to now the signiï¬cance of the serumACTN4 levels in CC has not been evaluated Hence in thecurrent study the serum levels of ACTN4 in patients withCC were measured In addition we estimated the potentialdiagnostic and prognostic value of serum ACTN4 expressionin CC Materials and Methods Study Population A retrospective study was designed toevaluate serum actinin4 as a biomarker for CC Between July and June newly diagnosed female CC patientsand newly diagnosed female cervical intraepithelial neoplasia CIN patients who received treatment at HuaianMaternal and Child Health Care Hospital Huaian JiangsuChina were recruited The diagnoses of all patients were veriï¬ed by the histopathological examination The patients withother types of tumor or autoimmune atherosclerotic andhematologic diseases were excluded The mean age of CCpatients was years with a range of years Meanwhile healthy women with no evidence of neoplasmsand other serious diseases were enrolled from the physicalexamination center in the same hospital There was no significant diï¬erence in age among the CC CIN and healthy control groups This study was consistent with the Helsinkideclaration and was authorized by the Ethics Committee ofHuaian Maternal and Child Health Care Hospital approvalnumber H20130504 All participantssigned writteninformed consent Clinicopathologic Feature Collection and FollowUp Byreviewing the medical records we collected the clinicopathologic characteristics of the patients including age at diagnosis pathological type FIGO stage tumor diï¬erentiationpelvic lymph node metastasis tumor size and lymphovascular space invasion The CC patients were classiï¬ed based onthe revised FIGO staging system for CC in The tumorsize was the maximum tumor diameter determined by agynecologic oncologist during pelvic examination Thepatients in stage 1A1 received hysterectomy the patients instages IB1 and IIB received radical hysterectomy and pelviclymph node dissection the patients with ¥stage IIB receivedradiotherapy or radiotherapy combined with chemotherapyA regular telephone followup was conducted after treatmentto obtain the overall survival OS time of CC patients andthe OS was deï¬ned as the time from diagnosis to death orthe last followup The followup was in accordance withthe FIGO guidelines Blood Sample Collection and Detection of Serum Actinin and SCCA A mL peripheral blood sample from eachpatient was collected before receiving any treatment Afterstanding at room temperature for minutes the blood samples were centrifugated at gmin for min and then°the supernatant was stored at C until further usageThe serum actinin4 concentration was measured by a quantitativeELISAmethod Uscn Life Science Inc Wuhan China The levelsof SCCA in serum were determined using an ELISA kitRD Systems Minneapolis MN The detection of all samples was strictly in accordance with the instructions providedby the manufacturer and was performed in duplicatesenzymelinked immunosorbentassay Statistical Analysis All statistical analyses were conducted by using SPSS and GraphPad Prism The continuous data following normal distribution were expressed asthe mean ± standard deviation°SD A ttest was used tocompare serum ACTN4 levels between the two subgroupsof each clinicopathological parameters and the serumACTN4 levels of CC patients CIN patients and healthy controls were compared by the SNKq test Receiver operatingcharacteristic ROC curves were performed to assess thediagnostic value of serum ACTN4 levels for diï¬erentiatingCC patients from CIN patients and healthy controls TheKaplanMeier method and logrank test were used to plotsurvival curves The Cox proportional hazards models in univariate and multivariate analyses were used for evaluating theprognostic value of serum ACTN4 expression A twotailed Pvalue was considered to be statistically significant Results Serum ACTN4 Levels Are Higher in Patients with CCSerum concentrations of ACTN4 were detected to rangefrom to pgmL with a mean ±SD of ± pgmL for CC patientsto range from to ngmL with a mean ±SD of ± pgmL forCIN patients and to range from to ngmL witha mean ±SD of ± pgmL for healthy controlsSerum ACTN4 levels in CC patients were significantly higherthan those in CIN patients and healthy controls P However no significant diï¬erence in serum ACTN4 wasfound between CIN patientscontrolsP as shown in Figure and healthy The Diagnostic Value of Serum ACTN4 Levels for CC Wenext used ROC curve analysis to estimate the diagnostic valueof serum ACTN4 expression for CC The ROC curve showedthat the serum levels of ACTN4 were robust for discriminating CC patients from benign and healthy control subjectswith an area under the curve AUC value of 95CI as demonstrated in Figure index we usedAccordingto maximum Youdens 0cDisease Markerslymph node metastasis were the independent prognostic factors for CC all P Table Lmgp NTCAnsCCCINCON DiscussionCervical cancer is a heterogeneous disease with complicatedetiology Genetic and environmental factors play a crucialrole in the pathogenesis of CC [] Although the diagnosisand prognosis of CC have improved greatly over the pastfew decades it is necessary to improve early detection andscreening methods to determine additional promising circulating biomarkers for better patient selection and more personalized treatments [] As far as we know this studyrepresented the ï¬rst eï¬ort to evaluate the serum expressionof ACTN4 as a new biomarker for CCAs an actinbinding protein ACTN4 can participate inregulating cell migration invasion and metastasis via regulating the actin ï¬lament ï¬exibility at the leading edge ofinvading cancer cells [ ] ACTN4overexpressing cancercells have the potential to metastasize because the overexpression of ACTN4 protein in cancer cells can stimulate thedynamic reconstruction of the actin cytoskeleton [] Upto now numerous studies have reported the associationbetween ACTN4 and multiple cancers Okamoto []observed that ACTN4 is expressed in smallcell lung cancerNSCLC and it had a significant correlation with invasionand distant metastasis Additionally ACTN4 was reportedto be a potential predictive biomarker for the eï¬cacy of adjuvant chemotherapy in patients with NSCLC [] Watabe [] revealed that the copy number increase of ACTN4is a novel indicator for poor overall survival of patients withsalivary gland carcinoma and the copy number variationwould aï¬ect the expression of protein A recent study demonstrated that serum ACTN4 levels were dramatically elevated in patients with breast cancer when compared tohealthy controls and serum ACTN4 may be an eï¬ective clinical indicator for diagnosing or predicting the clinical outcomes of breast cancer patients [] In addition ACTN4was proven to be associated with the pathogenesis of CCAn [] proposed a novel mechanism for epithelialtomesenchymal transition and tumorigenesis in CC whichcould be induced by ACTN4 through regulating Snail expression and βcatenin stabilization Hence it is significant toinvestigate the role of serum ACTN4 in CCIn the current study we observed that serum levels ofACTN4 in CC patients were statistically higher than thosein CIN patients and those in healthy controls Howeverserum ACTN4 levels were not significantly diï¬erent betweenthe CIN group and the control group It was shown thatserum ACTN4 expression could strongly diï¬erentiate CCpatients from CIN patients and healthy controls The ROCanalysis demonstrated that the AUC of ACTN4 was and at the optimal cutoï¬ of pgmL the sensitivity andspeciï¬city were respectively and suggestingthat serum ACTN4 might be a potential diagnostic biomarker for CC In a recent study which included Chinesewomen Hu [] reported that the sensitivity and speciï¬city of HPV screening in the diagnosis of CC were and The sensitivity of the HPV test was a litter higherFigure The serum ACTN4 levels in CC patients CIN patientsand healthy controls P pgmL as the cutoï¬ value and the sensitivity and speciï¬city were and respectively Association between Serum ACTN4 Levels andClinicopathological Parameters of CC Patients We furtherinvestigated the correlations between serum levels of ACTN4and clinical pathological data of CC patients and theresults are demonstrated in Table We observed that serumACTN4 levels were related to the FIGO stage lymph nodemetastasis and lymphovascular space invasion all P Nevertheless no significant association was found betweenserum ACTN4 levels and age pathological type diï¬erentiation degree and tumor size in CC patients all P Survival Analysis of Serum ACTN4 Levels in CC Duringthe followup period nine CC patients were lost and thefollowed up rate is Finally the prognostic value ofserum ACTN4 was assessed in patients The patients werefollowed up to December The range of followup timewas to months with the median time of months andmean time of months According to the median serumlevels of ACTN4 in CC patients pgmL the CCpatients were divided into the high ACTN4 level group pgmL N and low ACTN4 level group¥ pgmL N The estimated 5year OS of patientswith high serum ACTN4 levels and low serum ACTN4 levelswere and respectively The KaplanMeier survival curve and logrank test indicated that CC patients withhigh serum ACTN4 levels had a worse prognosis than thosewith low serum ACTN4 levels P Figure Univariate Cox regression analyses showed that theserum ACTN4 levels P FIGO stage P diï¬erentiation degree P lymph node metastasisP and lymphovascular space invasion P had significant prognostic value for OS Multivariate analysiswas further performed to evaluate the prognostic value ofserum ACTN4 as an independent factor for CC All the statistically significant factors from univariate analyses wereincluded and the results indicated that the FIGO stage and 0cDisease MarkersytivitisneS specificityFigure ROC curve analysis assessed the diagnostic performance of serum ACTN4 in CC The AUC was P Table Serum ACTN4 levels in CC patients according toclinicopathological parametersParametersAge years¤Pathological typeSquamous cell carcinomaAdenocarcinomaFIGO stageIA1IB1¥IB2Diï¬erentiationWell and moderatelydiï¬erentiatedPoorly diï¬erentiatedLymph node involvementNegativePositiveTumor size¤Lymphovascular space invasionNegativePositiveN ACTN4pgmLP ± ± ± ± ± ± ± ± ± ± ± ± ± ± than that of serum ACTN4 detection though the speciï¬cityof serum ACTN4 detection was well above that of the HPVtest Hence comparing with the HPV test in diagnosingCC detecting serum ACTN4 has some advantages Furthermore serum ACTN4 levels have been indicated to be a greatbiomarker for diagnosing multiple cancers Fang [] intheir study reported that serum ACTN4 was a promisingindicator for diagnosing breast cancer with the AUC of Wang [] used ACTN4 expression in peripheralblood to diï¬erentiate NSCLC patients from healthy individuals in two groups of participants and they obtained bothsatisfactory eï¬ects Furthermore we investigated the correlation between serum ACTN4 and clinical characteristics ofCC patients The serum ACTN4 levels were significantlyassociated with the FIGO stage lymph node metastasis andlymphovascular space invasion of CC which suggests thatACTN4 could contribute to the development invasion andmetastasis of CC In addition our results indicated that highACTN4 levels were associated with the poor survival of CCpatients In the multivariate analysis although ACTN4 levelsdid not reach the statistical signiï¬cance it still seems to beable to inï¬uence the OSHowever several limitations in the present study should betaken into consideration First the sample size was relativelysmall which was likely to reduce the statistical power of ourresults Second we only explored the relationship betweenserum ACTN4 and OS and other prognostic indicators werenot examined due to the incomplete data which needs to beimproved in the future Third this study was a primary studyto determine the clinical signiï¬cance of serum ACTN4 levelsfor the diagnosis and prognosis of CC but the speciï¬c molecular mechanisms remain unclear Hence further experimentsshould be conducted to elucidate the mechanismsIn conclusion our study showed that serum ACTN4levels were increased in CC patients and were related to the 0cDisease Markers lavivrus muCLog rank P Overall survival monthsLow ACTN4 groupHigh ACTN4 groupFigure KaplanMeier curve compared OS of CC patients with high serum ACTN4 levels versus those with low serum ACTN4 levelsTable Univariate and multivariate Cox regression analysis of OS in CC patientsUnivariate CIVariablesAge vs ¤ yearsPathological type squamous cell carcinoma vs adenocarcinomaFIGO stage ¥IB2 vs IA1IB1Diï¬erentiation poorly diï¬erentiated vs well and moderately diï¬erentiated Lymph node involvement positive vs negativeTumor size vs ¤ cmLymphovascular space invasion positive vs negativeSerum ACTN4 levels high vs low levelsHR PMultivariate CIPHRFIGO stage lymph node metastasis and lymphovascularspace invasion of CC patients In addition serum levels ofACTN4 have great diagnostic and prognostic value in CCNevertheless further studies with a larger sample size shouldbe carried out to conï¬rm our resultsAcknowledgmentsWe thank all the patients and blood donors who participatedin our study This study was funded by grants from the Science and Technology Project of Traditional Chinese Medicine Bureau of Jiangsu province China YB2015128Data AvailabilityReferencesThe datasets used andor analyzed during the present studyare available from the corresponding author on reasonablerequestConflicts of InterestAll authors declare that they have no conï¬icts of interestAuthors ContributionsXigui Ma and Huiying Xue contributed equally to this workand should be considered as coï¬rst authors[] M H Forouzanfar K J Foreman A M Delossantos et alBreast and cervical cancer in countries between and a systematic analysis The Lancet vol no pp [] E Pelkofski J Stine N A Wages P A Gehrig K H Kimand L A Cantrell Cervical cancer in women aged yearsand younger Clinical Therapeutics vol no pp [] Y Zhou W Wang R Wei Serum bradykinin levels as adiagnostic marker in cervical cancer with a potential mechanism to promote VEGF expression via BDKRB2 International Journal of Oncology vol pp [] Y J Hu H P Zhang B Zhu H Y Chen L H Ma andY Wang The role of FH detection combined with HPV 0cDisease Markers[] N Miura M Kamita T Kakuya Eï¬cacy of adjuvantchemotherapy for nonsmall cell lung cancer assessed by metastatic potential associated with ACTN4 Oncotarget vol no pp [] N Tanaka T Yamashita S Yamamoto Histologicalgrowth pattern of and alphaactinin4 expression in thyroidcancer Anticancer Research vol no pp [] Y Watabe T Mori S Yoshimoto Copy numberincrease of ACTN4 is a prognostic indicator in salivary glandcarcinoma Cancer Medicine vol no pp [] HT An S Yoo and J Ko αActinin4 induces theepithelialtomesenchymal transition and tumorigenesis viaregulation of Snail expression and βcatenin stabilization incervical cancer Oncogene vol no pp [] F Niu T Wang J Li The impact of genetic variants inIL1R2 on cervical cancer risk among Uygur females fromChina a casecontrol study Molecular Genetics GenomicMedicine vol no article e00516 [] W Li Y Zhao L Ren and X Wu Serum human kallikrein represents a new marker for cervical cancer Medical Oncology vol no p [] H Shao J HC Wang M R Pollak and A Wells αActinin4 is essential for maintaining the spreading motility andcontractility of ï¬broblasts PLoS One vol no articlee13921 [] K Honda T Yamada Y Hayashida Actinin4 increasescell motility and promotes lymph node metastasis of colorectalcancer Gastroenterology vol no pp [] D G Thomas and D N Robinson The ï¬fth sense mechanosensory regulation of alphaactinin4 and its relevance forcancer metastasis Seminars in Cell Developmental Biologyvol pp screening on the diagnostic signiï¬cance of cervical cancer andprecancerous lesions European Review for Medical and Pharmacological Sciences vol no pp [] KH Wang C J Lin C J Liu Global methylationsilencing of clustered protocadherin genes in cervical cancerserving as diagnostic markers comparable to HPV CancerMedicine vol no pp [] T Li Y Li G X Yang Diagnostic value of combination of HPV testing and cytology as compared to isolatedcytology in screening cervical cancer a metaanalysis Journal of Cancer Research and Therapeutics vol no pp [] K Honda T Yamada R Endo Actinin4 a novel actinbundling protein associated with cell motility and cancer invasion The Journal of Cell Biology vol no pp [] E de Almeida Ribeiro N Pinotsis A Ghisleni Thestructure and regulation of human muscle αactinin Cellvol no pp [] D Wang X W Li X Wang Alphaactinin4 is a possible target protein for aristolochic acid I in human kidneycellsin vitro The American Journal of Chinese Medicinevol no pp [] I V Ogneva N S Biryukov T A Leinsoo and I M Larina Possible role of nonmuscle alphaactinins in musclecell mechanosensitivity PLoS One vol no articlee96395 [] K Honda The biological role of actinin4 ACTN4 in malignant phenotypes of cancer Cell Bioscience vol no p [] X Zhao K S Hsu and J H Lim αActinin potentiatesnuclear factor κlightchainenhancer of activated BcellNFκB activity in podocytes independent of its cytoplasmic actin binding function The Journal of BiologicalChemistry vol no pp [] H Shams J Golji K Garakani and M R Mofrad DynamicRegulation of α Actinin's Calponin Homology Domains on FActin Biophysical Journal vol no pp [] C Fang J J Li T Deng B H Li P L Geng and X TZeng Actinin4 as a diagnostic biomarker in serum ofbreast cancer patients Medical Science Monitor vol pp [] T Watanabe H Ueno Y Watabe ACTN4 copynumber increase as a predictive biomarker for chemoradiotherapy of locally advanced pancreatic cancer British Journal of Cancer vol no pp [] S Yamamoto H Tsuda K Honda ACTN4 gene ampliï¬cation and actinin4 protein overexpression drive tumourdevelopment and histological progression in a highgrade subset of ovarian clearcell adenocarcinomas Histopathologyvol no pp [] M C Wang Y H Chang C C Wu Alphaactinin is associated with cancer cell motility and is a potential biomarker in nonsmall cell lung cancer Journal of ThoracicOncology vol no pp [] N Okamoto H Suzuki K Kawahara The alternativelyspliced actinin4 variant as a prognostic marker for metastasisin smallcell lung cancer Anticancer Research vol no pp 0c" | 2 |
"The posttherapy ADC value was higher than pretherapy ADC value (P < 0.001) (). When all tumors were divided into the PR and SD groups according to the mentioned criterion it was noticed that the changes of tumor diameters on T2 images had significant difference between the SD and PR groups (P = 0.007 for longest diameter; P = 0.045 for shortest diameter) (). The pretherapy ADC value had no significant difference between the PR group and SD group (P = 0.517) (). The change of ADC value was statistically significantly higher in PR group compared with that in SD group ( ). The logistic regression model and receiver operator characteristic curve analysis showed that combination of the change of longest diameter and ADC value had a higher area under curve than any other parameter alone for evaluating treatment response in lung cancer (P < 0.01 ). When we used the change of ADC value for differentiating the PR lesion from the SD lesion the best cutoff value was 0.41 10?3?mm2/s the overall sensitivity specificity positive predictive value and negative predictive value were 100%0.64710.5714 and 100% respectively and the area under the receiver operator characteristic curve was 0.827. 4. Discussion The chemotherapy response was usually observed after 2 cycles of chemotherapy according to tumor size change by radiographies CT or standard MR rarely by functional imaging dynamic or diffusion weighted MR imaging or PET-CT for example [3621 24]. This study investigated whether the change of ADC value and diameters after chemotherapy could be used to evaluate early treatment response in lung cancer. The ADC value had significant increase after 1 cycle of chemotherapy compared with baseline especially in the PR group (). This result was in agreement with the results of previous studies of both lung cancer and other cancers [17 2427]. However the current result contradicted with the result of rectal cancer research on the decrease trend of ADC values 24 weeks after chemotherapy. Chemotherapy-induced fibrosis might be a contributor to decrease of ADC value [28]. The difference was probably caused by disparity in fibrosis appearance and progression. The increase of ADC value was related to necrosis and reduced cell density histologically [29] while the decrease of ADC value was relevant to cytotoxic edema and fibrosis on histology [28]. From the current results the changes of ADC value were significant between the PR and SD groups () which was in agreement with the previous DWI study of lung cancer treatment response evaluation after chemotherapy [24 26] even when a different b value was used in the previous study [27]. Therefore the noninvasive DWI could be potentially used to early predict and monitor lung cancer response to chemotherapy (). This study further demonstrated that the combination of longest diameter and ADC value change had a higher diagnostic ability than any other parameter alone for evaluating treatment response in lung cancer (P < 0.01 ). The receiver operator characteristic curve showed that the combination of longest diameter change and ADC value change adds additional value for a single parameter alone to predict treatment response. The cutoff value of ADC change could predict response to chemotherapy in lung cancer with 100% sensitivity 64.71% specificity 57.14% positive predictive value 100% negative predictive value and 82.7% accuracy. Comparing with CT the MR imaging had two benefits to evaluate tumor response: first the DWI had the potential to evaluate early treatment response from the tumor inner structure change before the morphological change; second by combining the change of ADC value and tumor diameter the treatment response could be predicted with a high sensitivity and moderate specificity by using MR imaging only. For the small cell lung cancer the tumor diameter change may be significant even in the early stage of chemotherapy. But for the less sensitive non-small cell lung cancer especially on target therapy there may not be apparent anatomical changes initially even if the chemotherapeutic regime was appropriate [29]. Therefore tumor size evaluation alone had a smaller area under the curve than the combination of functional and anatomical assessment. The change of ADC value might have the potential to monitor and early predict lung cancer treatment response to chemotherapy. Furthermore the diagnostic ability increased when combined with the change of ADC value and longest diameter. In our study MR images at baseline and the end of 1 cycle of chemotherapy not only showed the tumor volume change on T2-weighted images but also provided the change of ADC value on DWI. Even through MR imaging could not be regarded as the most appropriate and a one stop examination method to predict treatment response; it indeed provided precious information to CT. There were several limitations of this study. Firstly the sample number in our study was relatively small especially the pathological subtypes. Secondly the tumor volume pathologic types and chemotherapy regimens were nonuniform which may affect the treatment response of tumor. At last the interval between the start of chemotherapy and treatment response evaluation by DWI was relatively long. One week interval may make the advantage of ADC value change prominent. 5. Conclusions Our data suggested that the change of ADC value may be a sensitive indicator to predict early response to chemotherapy in lung cancer. Prediction ability could be improved by combining the change of ADC value and longest diameter. Acknowledgments The authors thank Rongchao Zhou for collecting part of patients. They appreciate the statistical suggestion from Ningnannan Zhang. This study was supported by Tianjin Medical University Graduate Innovation Fund. Conflict of Interests The authors declared that there was no conflict of interests regarding the publication of this paper. 1 Patz EF Jr. Pinsky P Gatsonis C Overdiagnosis in low-dose computed tomography screening for lung cancer JAMA Internal Medicine 2013 2013 6 pages 12738 2 Smith SM Campbell NC MacLeod U Factors contributing to the time taken to consult with symptoms of lung cancer: a cross-sectional study Thorax 2009 64 6 523 531 2-s2.0-67249109028 19052045 3 Gazala S Pelletier JS Storie D Johnson JA Kutsogiannis DJ Bedard EL A systematic review and meta-analysis to assess patient-peported putcomes after lung cancer surgery Scientific World Journal 2013 2013 789625 4 Sokucu SN Kocaturk C Urer N Evaluation of six patients with pulmonary carcinosarcoma with a literature review Scientific World Journal 2012 2012 167317 5 Pignon J-P Tribodet H Scagliotti GV Lung adjuvant cisplatin evaluation: a pooled analysis by the LACE collaborative group Journal of Clinical Oncology" | 1 |
case of a 14year old boy with tumorassociated refractory epilepsy Positron emission tomography imaging demonstrated a region with heterogeneous high 11Cmethionine uptake and a region with homogenous low 18Ffluorodeoxyglucose uptake within the tumor Histopathological and genomic analyses confirmed the tumor as BRAF V600Emutated polymorphous lowgrade neuroepithelial tumor of the young PLNTY Within the highmethionineuptake region we observed increased protein levels of Ltype amino acid transporter LAT1 a major transporter of methionine cMyc and constituents of the mitogenactivated protein kinase MAPK pathway We also found that LAT1 expression was linked to the BRAF V600E mutation and subsequent activation of MAPK signaling and cMyc Pharmacological and genetic inhibition of the MAPK pathway suppressed cMyc and LAT1 expression in BRAF V600Emutated PLNTY and glioblastoma cells The BRAF inhibitor dabrafenib moderately suppressed cell viability in PLNTY Collectively our results indicate that BRAF V600E mutationactivated MAPK signaling and downstream cMyc induces specific metabolic alterations in PLNTY and may represent an attractive target in the treatment of the diseaseKeywords PLNTY BRAF V600E mutation Methionine PET LAT1IntroductionPediatric lowgrade neuroepithelial tumors PLGNTs encompass a group of central nervous system neoplasms that longterm epilepsyassociated tumors LEATs such as ganglioglioma and dysembryoplastic neuroepithelial tumor DNT PLGNTs have different characteristics than their adult counterparts and includes Correspondence ktate12yokohamacuacjp Department of Neurosurgery Graduate School of Medicine Yokohama City University Fukuura Kanazawa Yokohama JapanFull list of author information is available at the end of the are commonly driven by genomic alterations in the Rasmitogenactivated protein kinase MAPK pathway such as mutations in BRAF and NF1 [ ] Recent largescale genomic studies and genomewide methylation analyses allowed a thorough characterization of PLGNTs [] and cIMPACTNOW the Consortium to Inform Molecular and Practical Approaches to CNS Tumor Taxonomy currently classifies PLGNTs as distinct disease entities [ ] In Huse et a0al described ten cases of polymorphous lowgrade neuroepithelial tumor of the young PLNTY which were histologically characterized by oligodendrogliomalike cellular components with intense CD34 immunopositivity According to previous publications PLNTYs are indolent tumors The Authors Access This is licensed under a Creative Commons Attribution International License which permits use sharing adaptation distribution and reproduction in any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate if changes were made The images or other third party material in this are included in the s Creative Commons licence unless indicated otherwise in a credit line to the material If material is not included in the s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtain permission directly from the copyright holder To view a copy of this licence visit httpcreat iveco mmons licen sesby40 The Creative Commons Public Domain Dedication waiver httpcreat iveco mmons publi cdoma inzero10 applies to the data made available in this unless otherwise stated in a credit line to the data 0cTateishi a0et a0al acta neuropathol commun Page of that generally exhibit a benign clinical course and harbor either a BRAF V600E mutation or FGFR2FGFR3 fusion [] Based on its histological and genomic profiles cIMPACTNOW Update recommends PLNTY as a possible future classification for pediatrictype glialglioneuronal tumors However because of their rare etiology only a few PLNTYs have been described to date [ ] and it is unclear how genomic alterations promote the pathogenesis of the disease Herein we present a case of PLNTY with unique metabolic imaging features Using positron emission tomography PET we found regions of heterogeneous high 11Cmethionine uptake and homogenous low 18Ffluorodeoxyglucose FDG uptake within the tumor Activation of the MAPK pathway cMyc and expression of Ltype amino acid transporter LAT1 were increased in the highmethionineuptake area compared with the surrounding cortex lowmethionineuptake Glycolytic metabolites were expressed only weakly in tumor cells Pharmacological and genetic inhibition of the MAPK pathway suppressed cMyc and LAT1 and inhibited tumor cell viability suggesting that MAPK signaling and downstream cMyc activates methionine metabolism and inhibition of this pathway induces therapeutic vulnerability in PLNTYMaterials and a0methodsCell viability analysisAM38 and normal human astrocytes was purchased from JCRB Cell Bank and ScienCell Research Laboratories respectively Tumorsphere lines were cultured in serumfree neural stem cell medium as previously described [] Normal human astrocytes were cultured with astrocyte medium ScienCell To assess cell viability primary cultured cells were dissociated into single cells and seeded into 96well plates at a density of cellswell After a0h dabrafenib Selleck and trametinib Selleck were serially diluted and added to the wells Cell viability was measured using the CellTiterGlo Promega assay at day and the results were indicated as viability of the DMSO controlshRNA cell line generationTo knockdown BRAF 293T cells were transfected with lentiviral vector packaging plasmid DNA containing a0 μg of Human BRAF shRNA TRCN0000381693 GP and a0μg of a0pVSVgRev a0with Lipofectamine¢ TRCN0000196844 Sigma Aldrich a0μg of a0pHIVThermo Fisher Scientific YMG62 and AM38 cells were infected with lentivirus in polybrene a0μgmL for a0h Two days later the cells were selected with puromycin a0μgmL for a0days and used for experiments GIPZ nonsilencing lentiviral shRNA Control RHS4348 Horizon Discovery was used as a nonsilencing NS controlImmunohistochemistryTumor tissue specimens were fixed in neutral buffered formalin and embedded in paraffin Hematoxylin and eosin staining was performed using standard procedures For immunohistochemical analysis 5µmthick sections were deparaffinized treated with H2O2 in methanol rehydrated and heated for a0min for antigen retrieval After blocking with serum tissue sections were incubated with primary antibodies against CD34 Novus Biologicals LAT1 Cell Signaling Technology phosphoMEK Cell Signaling Technology phosphoERK Bethyl Laboratories and cMyc Cell Signaling Technology at a0°C overnight The next day sections were washed with PBS incubated with biotinylated secondary antibody for a0 min at room temperature and then incubated with ABC solution PK6101 PK6102 Vector laboratories for a0 min at room temperature Finally the sections were incubated with DAB Dako and counterstained with hematoxylinWestern blottingcOmplete¢ Mini EDTAfree Protease Inhibitor Cocktail Cells were lysed in RIPA buffer SigmaAldrich with a Roche Fifty micrograms of protein was separated by SDSPAGE gel and transferred to polyvinylidene difluoride membranes Millipore by electroblotting After blocking with or nonfat dry milk in TBST a0mM Tris [pH ] a0 mM NaCl Tween20 membranes were incubated at a0 °C overnight with primary antibodies After washing and incubation with horseradish peroxidaseconjugated secondary antibodies Cell Signaling Technology blots were washed and signals were visualized with chemiluminescent HRP substrate Millipore Primary antibodies against BRAF Gene Tex cMyc Cell Signaling Technology GAPDH Gene Tex LAT1 Cell Signaling Technology phosphoMEK Cell Signaling Technology a0phosphoERK Bethyl Laboratories and Vinculin Novus Biologicals were used for western blottingCase presentationThis study was performed in accordance with declaration of Helsinki and was approved by the Institutional Review Board Yokohama City University [YCU Yokohama Japan] IRB numbers A1711300006 and B190600002 Written informed consent was obtained from the patient and parents A 14year old boy presented with chronic medial temporal lobe epilepsy for a year Magnetic resonance imaging MRI indicated 0cTateishi a0et a0al acta neuropathol commun Page of See figure on next pageFig Characteristics of a patient with PLNTY a T2weighted left T1weighted middle and contrastenhanced right MR images b Computed tomography CT left 18FfluorodeoxyglucosePETCT middle and 11CmethioninePETCT right images c Video electroencephalography indicating ictal onset in the left temporal lobe with spread to the contralateral temporal lobe d PETCT and MRI merged intraoperative navigation image left and surgical image right showing the highmethionineuptake region and surrounding abnormal lesion on MRIhypointensity on T2weighted images and hyperintensity on T1weighted images with a cystic component in the left temporal lobe Contrastenhanced MRI showed no significant enhancement in the lesion Fig a01a while computed tomography revealed heavy calcification FDGPET showed lower FDG uptake in the tumor while 11CmethioninePET demonstrated increased methionine uptake in the same lesion SUVmax tumornormal tissue ratio Fig a01b Videoelectroencephalographic EEG monitoring indicated ictal onset in the left temporal lobe with subsequent spread to the contralateral temporal lobe Fig a01c We speculated that this abnormal lesion was a LEAT Since we considered this tumor to be completely resectable the patient underwent craniotomy and resection of the neoplasm including the highmethionineuptake region Fig a01d To achieve epileptic control electrocorticography was performed intraoperatively After removal of the highmethionineuptake and T2 hyperintense lesions the surrounding tissue was resected until interictal epileptiform discharge could no longer be detected by electrocorticography The patient became epilepsyfree after lesion removal and MRI indicated complete remission a0months after the surgeryTissue samples of the highmethionineuptake region and surrounding cortex low methionine uptake were collected Hematoxylin and eosin staining indicated diffusely infiltrating growth patterns and presence of oligodendroglialike cellular components Fig a02a Astrocytic and highgrade features were absent with a Ki67 index of less than Chicken wirelike branching capillaries and microcalcification were also found in region Despite lower cellularity oligodendroglialike cells were present in the surrounding tissue Immunohistochemistry revealed extensive CD34 expression and peripherally associated ramified neural elements in the tumor cells Fig a0 2a Targeted DNA sequencing identified a BRAF V600E mutation in the tumor without recurrent mutations in IDH1 IDH2 TERT promoter FGFR1 H3F3A or HIST3H1B Fig a0 2b Chromosome 1p19q codeletion was absent Fig a02c The above histological and genetic features fulfilled the diagnostic criteria for PLNTYTo assess the mechanisms underlying the methionineFDG uptake mismatch indicated by PET we compared the expression of LAT1 glucose transporter GLUT and hexokinase2 HK2 between tissue regions and Notably LAT1 which is a major methionine transporter was more highly expressed in than in Fig a0 3a In contrast GLUT1 and HK2 which is correlated with FDG uptake and lactate dehydrogenase A LDHA expression were weak in either region Additional file a0 Fig a0 S1 LAT1 expression is mediated by cMyc activation and BRAF V600E mutation activates the MAPK pathway and downstream cMyc [ ] Therefore we hypothesized that BRAF V600E mutation promotes LAT1 expression through MAPK signaling and consequent cMyc activation a0 in PLNTY Levels of phosphoMEK phosphoERK and cMyc were higher in tissue region than in Fig a03a suggesting activation of the MAPK pathway and cMyc within the highmethionineuptake lesion To verify whether the BRAF V600E mutation can induce the expression of LAT1 we exposed primary cultured YMG83 PLNTY cells to a BRAF inhibitor dabrafenib As expected the expression of phosphoMEK phosphoERK cMyc and LAT1 was suppressed after dabrafenib treatment in YMG83 cells Fig a0 3b Notably BRAF inhibitor dabrafenibtreated YMG83 cells had lower cell viability compared to normal human astrocytes Fig a03c To confirm the reproducibility of these molecular features we used patientderived YMG62 cells epithelioid glioblastoma with the BRAF V600E mutation which exhibited high 11Cmethionine uptake by PET imaging Additional file a0 Fig a0 S2 and AM38 glioblastoma cells BRAF V600E mutant We found that dabrafenib and a MEK inhibitor trametinib inhibited the expression of proteins in the MAPK pathway as well as cMyc and LAT1 Fig a03d and 3e Similarly BRAF knockdown suppressed the expression of proteins in the MAPK pathway as well as cMyc and LAT1 Fig a03f Collectively these findings indicated that activation of the MAPK pathway by the BRAF V600E mutation deregulates cMyc and promotes LAT1 expression This oncogenic signaling pathway increases methionine metabolism and tumor maintenance in PLNTYDiscussionThirty cases of PLNTY have been described to date with the first ten reported by Huse et a0al in [ ] BRAF V600E mutation was seen in of the patients and BRAF fusion in patient These BRAF alterations were mutually exclusive with other genomic events including FGFR3TACC3 fusion FGFR3 amplification FGFR2CTNNA3 fusion FGFR2INA fusion 0cTateishi a0et a0al acta neuropathol commun Page of 0cTateishi a0et a0al acta neuropathol commun Page of Fig Histopathologic and genomic features of a patient with PLNTY a Hematoxylin and eosin HE staining top and CD34 immunohistochemistry bottom in the highmethionineuptake and lowmethionineuptake region within tumor tissue Bars μm b Sanger sequencing for detection of BRAF V600E arrow left and IDH1 R132H arrow right mutations c Fluorescence in situ hybridization for detection of 1p311q25 left and 19q1319p13 right chromosomal deletionsFGFR2 KIAA1598 fusion FGFR2 rearrangement and NTRK2 disruption suggesting that the vast majority of PLNTYs are induced by BRAF mutation or FGFR fusion and subsequent MAPK activation Therefore targeting MAPK signaling may become a potential therapeutic strategy in PLNTY Indeed BRAF V600Emutated PLNTY cells were relatively vulnerable to dabrafenib and trametinib in the present study Thus targeted molecular therapy for the MAPK pathway may be particularly useful in PLNTY located in surgically unresectable regions In addition Koh et a0 al reported that the BRAF V600E mutation contributes to the intrinsic epileptogenicity in pediatric brain tumors and that inhibition of BRAF suppressed epileptic seizures [] Thus BRAFMEK inhibitors could exert antiepileptic as well as antitumor effects in PLNTYPET imaging revealed a region with increased methionine uptake and low FDG uptake within tumor tissue in our patient Consistent with this finding previous case reports demonstrated increased methionine uptake but only mild FDG uptake in patients with BRAF V600Emutated PLNTY [ ] Thus excessive 0cTateishi a0et a0al acta neuropathol commun Page of Fig Activating the MAPK pathway induces LAT1 expression in a patient with PLNTY a Immunohistochemistry of indicated proteins in the highmethionineuptake and lowmethionineuptake regions within tumor tissue Bars μm b Western blot analysis of phosphoMEK phosphoERK cMyc and LAT1 proteins in YMG83 PLNTY left cells treated with DMSO and μM BRAF inhibitor BRAFi dabrafenib for h GAPDH loading control c Relative cell viability of dabrafenibtreated left and trametinibtreated right YMG83 cells and immortalized normal human astrocytes NHA P DMSO versus dabrafenib left and trametinib right d Western blot analysis for indicated proteins in YMG62 epithelioid glioblastoma left and AM38 glioblastoma right cells treated with DMSO μM BRAF inhibitor BRAFi dabrafenib and μM MEK inhibitor MEKi trametinib for h GAPDH loading control e Western blot analysis of BRAF phosphoMEK phosphoERK cMyc and LAT1 proteins in YMG62 left and AM38 right cells treated with DMSO and dabrafenib at indicated concentrations Vinculin loading control f Western blot analysis for indicated proteins in nonsilencing NS and BRAF and transduced YMG62 and AM38 cells GAPDH loading controlmethionine uptake and low FDG uptake may be imaging features specific to PLNTY A preclinical study has demonstrated that high uptake of 18FFDG was correlated with increased Glut1 and HK2 expression in human cancers [] Although the diagnostic accuracy is insufficient FDGPET imaging is useful to differentiate highgrade from lowgrade gliomas [] In the present case low FDG uptake and weak expression of Glut1 HK2 and LDHA were observed in tumor tissue suggesting low glycolytic activity in PLNTY On the other hand due to a high signaltonoise ratio 11Cmethionine PET imaging is practical for brain tumors [ ] Several PET imaging studies have demonstrated that methionine uptake was higher in 0cTateishi a0et a0al acta neuropathol commun Page of highgrade adult gliomas than in lowergrade gliomas [ ] In epileptogenic brain tumors however all gangliogliomas and of DNT had increased methionine uptake although these tumors are classified as WHO grade I [ ] implying that methionine uptake may be irrespective of tumor grade in LEATsPrevious studies have reported that methionine uptake was correlated with LAT1 in gliomas [ ] LAT1 plays a major role in the transport of neutral essential amino acids including methionine and is driven by several cancerrelated genes such as MYC [] It has been demonstrated that cMyc which is partly mediated by the MAPK pathway regulates LAT1 expression and MEK inhibitor suppresses LAT1 SLC7A5 transcription [ ] thereby indicating a role of the MAPK pathway and cMyc in the regulation of LAT1 Since RASMAPK pathwayassociated genomic alterations are common in LEATs [] and that the BRAF V600E mutation has been identified in and of gangliogliomas and DNTs respectively [ ] there is a possibility that the BRAF V600E mutation and MAPK pathwayrelated genomic alterations may activate methionine metabolism in LEATs To investigate this hypothesis we evaluated the protein expression of LAT1 and the molecules that are involved in the MAPK pathway As expected levels of phosphoMEK phosphoERK cMyc and LAT1 were higher in the highmethionineuptake area than in the lowmethionineuptake area We also found that genetic andor pharmacological BRAF inhibition suppressed MAPK pathway activation and attenuated LAT1 expression in BRAF V600EmutatedPLNTY cells and glioblastoma cell lines These findings support the hypothesis that the BRAF V600E mutation may upregulate LAT1 and methionine metabolism through cMyc activation for cell survival In addition to LAT1 methionine uptake was correlated with microvascular density MVD in gliomas [] PLNTYs are considered benign brain neoplasms proposed as WHO grade I however in the present case a chicken wirelike MVD which is one of the histopathological characteristics of oligodendroglioma was also observed in the highmethionineuptake tissue region Intriguingly methionine uptake has been reported to be relatively higher in oligodendrogliomas than in astrocytomas [] Thus PLNTY which has an oligodendrogliomalike microvascular structure might show unique metabolic imaging features Further studies are warranted to validate this hypothesis Nonetheless our data indicated that the BRAF V600E mutation induced MAPK pathway activation and downstream cMyc promoted LAT1 expression and methionine metabolism with little effect on glycolytic pathway activation These findings may explain the unique metabolic imaging features of FDGmethionine mismatch in PLNTYSupplementary informationSupplementary information accompanies this paper at https doi101186s4047 Additional file a0 a0Figure S1 Low glycolysis activation in a patient with PLNTY Immunohistochemistry for glucose transporter hexokinase and lactate dehydrogenase A in the highmethionineuptake upper and lowmethionineuptake lower region within tumor tissue A Bars μm Figure S2 Images of the patients glioblastoma with the BRAF V600E mutation Contrastenhanced magnetic resonance left and 11Cmethionine positron emission tomography right images of the YMG62 patientAcknowledgementsWe thank Mrs Emi Hirata and Yasuko Tanaka YCU for technical and administrative assistance We also would like to thank Editage wwweditagecom for English language editingAuthors contributionsKT led the study collected samples designed experiments performed experiments interpreted data and wrote the manuscript JS TH and YM performed experiments NI HM provided tumor samples and associated clinical details TO RM and DU interrupted PET and MRI studies NU and SY performed the histological classification of tumor samples TY designed experiments and interpreted data All authors read and approved the final manuscriptFundingThis work was supported by GrantAid for Scientific Research 19K09488 Princess Takamatsu Cancer Research Fund Takeda Science Foundation SGH Cancer foundation Yokohama Foundation for Advancement of Medical Science and BristolMyers Squibb FoundationCompeting interestsThe authors declare that they have no competing interestsAuthor details Department of Neurosurgery Graduate School of Medicine Yokohama City University Fukuura Kanazawa Yokohama Japan Department of Pathology Yokohama City University Hospital Yokohama Japan Department of Radiology Graduate School of Medicine Yokohama City University Yokohama Japan Departmento of Radiology Division of Nuclear Medicine National Center for Global Health and Medicine Tokyo Japan Received June Accepted August References Borbely K Nyary I Toth M Ericson K Gulyas B Optimization of semiquantification in metabolic PET studies with 18Ffluorodeoxyglucose and 11Cmethionine in the determination of malignancy of gliomas J Neurol Sci https doi101016jjns200602015 Chappe C Padovani L Scavarda D Forest F NanniMetellus I Loundou A Mercurio S Fina F Lena G Colin C et al Dysembryoplastic neuroepithelial tumors share with pleomorphic xanthoastrocytomas and gangliogliomas BRAFV600E mutation and expression Brain Pathol https doi101111bpa12048 Chen Y Tian T Guo X Zhang F Fan M Jin H Liu D Polymorphous lowgrade neuroepithelial tumor of the young case report and review focus on the radiological features and genetic alterations BMC Neurol https doi101186s1288 Ellison DW Hawkins C Jones DTW OnarThomas A Pfister SM Reifenberger G Louis DN cIMPACTNOW update diffuse gliomas 0cTateishi a0et a0al acta neuropathol commun Page of characterized by MYB MYBL1 or FGFR1 alterations or BRAFV600E mutation Acta Neuropathol https doi101007s0040 Gupta VR Giller C Kolhe R Forseen SE Sharma S Polymorphous lowgrade neuroepithelial tumor of the young a case report with genomic findings World Neurosurg https doi101016jwneu201908221 Hafliger P Graff J Rubin M Stooss A Dettmer MS Altmann KH Gertsch J Charles RP The LAT1 inhibitor JPH203 reduces growth of thyroid carcinoma in a fully immunocompetent mouse model J Exp Clin Cancer Res https doi101186s1304 60180907z Hatakeyama T Kawai N Nishiyama Y Yamamoto Y Sasakawa Y Ichikawa T Tamiya T 11Cmethionine MET and 18Ffluorothymidine FLT PET in patients with newly diagnosed glioma Eur J Nucl Med Mol Imaging https doi101007s0025 Hayashi K Jutabha P Endou H Anzai N cMyc is crucial for the expression of LAT1 in MIA Paca2 human pancreatic cancer cells Oncol Rep https doi103892or20121878 Huse JT Snuderl M Jones DT Brathwaite CD Altman N Lavi E Saffery R SextonOates A Blumcke I Capper D et al Polymorphous lowgrade neuroepithelial tumor of the young PLNTY an epileptogenic neoplasm with oligodendrogliomalike components aberrant CD34 expression and genetic alterations involving the MAP kinase pathway Acta Neuropathol https doi101007s0040 Johnson DR Giannini C Jenkins RB Kim DK Kaufmann TJ Plenty of calcification imaging characterization of polymorphous lowgrade neuroepithelial tumor of the young Neuroradiology https doi101007s0023 y Kato T Shinoda J Oka N Miwa K Nakayama N Yano H Maruyama T Muragaki Y Iwama T Analysis of 11Cmethionine uptake in lowgrade gliomas and correlation with proliferative activity AJNR Am J Neuroradiol https doi103174ajnrA1242 Katsanos AH Alexiou GA Fotopoulos AD Jabbour P Kyritsis AP Sioka C Performance of 18FFDG 11Cmethionine and 18FFET PET for glioma grading a metaanalysis Clin Nucl Med https doi101097RLU00000 dysembryoplastic neuroepithelial tumors and other epileptogenic brain neoplasms with [11C]methionine PET Neuro Oncol https doi101093neuon cnou02 Riva G Cima L Villanova M Ghimenton C Sina S Riccioni L Munari G Fassan M Giangaspero F Eccher A Lowgrade neuroepithelial tumor unusual presentation in an adult without history of seizures Neuropathology https doi101111neup12504 Rosenberg DS Demarquay G Jouvet A Le Bars D Streichenberger N Sindou M Kopp N Mauguiere F Ryvlin P [11C]Methionine PET dysembryoplastic neuroepithelial tumours compared with other epileptogenic brain neoplasms J Neurol Neurosurg Psychiatry https doi101136jnnp200405160 Ryall S Tabori U Hawkins C Pediatric lowgrade glioma in the era of molecular diagnostics Acta Neuropathol Commun https doi101186s4047 z Ryall S Zapotocky M Fukuoka K Nobre L Guerreiro Stucklin A Bennett J Siddaway R Li C Pajovic S Arnoldo A et al Integrated molecular and clinical analysis of pediatric lowgrade gliomas Cancer Cell 583e565 https doi101016jccell Salisbury TB Arthur S The regulation and function of the ltype amino acid transporter LAT1 in cancer Int J Mol Sci https doi103390ijms1 Schindler G Capper D Meyer J Janzarik W Omran H HeroldMende C Schmieder K Wesseling P Mawrin C Hasselblatt M et al Analysis of BRAF V600E mutation in nervous system tumors reveals high mutation frequencies in pleomorphic xanthoastrocytoma ganglioglioma and extracerebellar pilocytic astrocytoma Acta Neuropathol https doi101007s0040 Sumdani H Shahbuddin Z Harper G Hamilton L Case report of rarely described polymorphous lowgrade neuroepithelial tumor of the young and comparison with oligodendroglioma World Neurosurg https doi101016jwneu201903181 Surrey LF Jain P Zhang B Straka J Zhao X Harding BN Resnick AC Storm PB Buccoliero AM Genitori L et al Genomic analysis of dysembryoplastic neuroepithelial tumor spectrum reveals a diversity of molecular alterations dysregulating the MAPK and PI3KmTOR pathways J Neuropathol Exp Neurol https doi101093jnennlz10 Kobayashi K Ohnishi A Promsuk J Shimizu S Kanai Y Shiokawa Y Nagane Tateishi K Nakamura T Yamamoto T Molecular genetics and M Enhanced tumor growth elicited by Ltype amino acid transporter in human malignant glioma cells Neurosurgery discussion https doi10122701neu00003 Koh HY Kim SH Jang J Kim H Han S Lim JS Son G Choi J Park BO Heo WD et al BRAF somatic mutation contributes to intrinsic epileptogenicity in pediatric brain tumors Nat Med https doi101038s4159 10180172x Kracht LW Friese M Herholz K Schroeder R Bauer B Jacobs A Heiss WD Methyl[11C] lmethionine uptake as measured by positron emission tomography correlates to microvessel density in patients with glioma Eur J Nucl Med Mol Imaging https doi101007s0025 Lelotte J Duprez T Raftopoulos C Michotte A Polymorphous lowgrade neuroepithelial tumor of the young case report of a newly described histopathological entity Acta Neurol Belg https doi101007s1376 Louis DN Wesseling P Aldape K Brat DJ Capper D Cree IA Eberhart C FigarellaBranger D Fouladi M Fuller GNet al cIMPACTNOW update new entity and diagnostic principle recommendations of the cIMPACTUtrecht meeting on future CNS tumor classification and grading Brain Pathol https doi101111bpa12832 Okubo S Zhen HN Kawai N Nishiyama Y Haba R Tamiya T Correlation of Lmethyl11Cmethionine MET uptake with Ltype amino acid transporter in human gliomas J Neurooncol https doi101007s1106 Ong LC Jin Y Song IC Yu S Zhang K Chow PK [18F]2deoxydglucose FDG uptake in human tumor cells is related to the expression of GLUT1 and hexokinase II Acta Radiol https doi10108002841 Rheims S Rubi S Bouvard S Bernard E Streichenberger N Guenot M Le Bars D Hammers A Ryvlin P Accuracy of distinguishing between therapeutic targets of pediatric lowgrade gliomas Brain Tumor Pathol https doi101007s1001 Tateishi K Tateishi U Nakanowatari S Ohtake M Minamimoto R Suenaga J Murata H Kubota K Inoue T Kawahara N 62Cudiacetylbis N4methylthiosemicarbazone PET in human gliomas comparative study with [18F]fluorodeoxyglucose and Lmethyl[11C]methionine PET AJNR Am J Neuroradiol https doi103174ajnrA3679 Wakimoto H Kesari S Farrell CJ Curry WT Jr Zaupa C Aghi M Kuroda T StemmerRachamimov A Shah K Liu TC et al Human glioblastomaderived cancer stem cells establishment of invasive glioma models and treatment with oncolytic herpes simplex virus vectors Cancer Res https doi10115800085472CAN083886 Yue M Jiang J Gao P Liu H Qing G Oncogenic MYC activates a feedforward regulatory loop promoting essential amino acid metabolism and tumorigenesis Cell Rep https doi101016jcelre p201712002 Zhang W Liu HT MAPK signal pathways in the regulation of cell proliferation in mammalian cells Cell Res https doi101038sjcr72901 Zhao C Zhang Y Wang J A metaanalysis on the diagnostic performance of 18FFDG and 11Cmethionine PET for differentiating brain tumors AJNR Am J Neuroradiol https doi103174ajnrA3718 Publishers NoteSpringer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations 0c' | 2 |
"cellular recognition of microbial dna is an evolutionarily conserved mechanism by which the innate immunesystem detects pathogens cyclic gmpamp synthase cgas and its downstream effector stimulator of interferongenes sting are involved in mediating fundamental innate antimicrobial immunity by promoting the release oftype i interferons ifns and other inflammatory cytokines accumulating evidence suggests that the activation ofthe cgassting axis is critical for antitumor immunity the downstream cytokines regulated by cgasstingespecially type i ifns serve as bridges connecting innate immunity with adaptive immunity accordingly a growingnumber of studies have focused on the synthesis and screening of sting pathway agonists however chronicsting activation may lead to a protumor phenotype in certain malignancies hence the cgassting signalingpathway must be orchestrated properly when sting agonists are used alone or in combination in this review wediscuss the dichotomous roles of the cgassting pathway in tumor development and the latest advances in theuse of sting agonistskeywords cgassting innate immunity type i interferon sting agonists antitumor response cancerdevelopmentintroductionthe discovery of phagocytosis in advanced the understanding of innate immunity the first line of host defenses protection againston patternrecognition receptors prrs which recognize microbialproducts coordinate antimicrobial defenses and activateinfection dependsagainstinfection byvarious pathogens correspondence zqliucsueducn juyan zheng and junluan mo contributed equally to this work1department of clinical pharmacology hunan key laboratory ofpharmacogenetics and national clinical research center for geriatricdisorders xiangya hospital central south university changsha peoples republic of china2institute of clinical pharmacology engineering research center for appliedtechnology of pharmacogenomics of ministry of education central southuniversity changsha peoples republic of chinafull list of author information is available at the end of the adaptive immunity abnormal rna or dna rnadna hybridization and cyclic dinucleotides derived frommicrobes are usually considered pathogenassociated molecular patterns pamps [ ] cells associated with innate immunity recognize different microbial pampsthrough specific prrs thereby playing key roles in hostresistance to microbial infection the pathways governing rna recognition such as retinoid acid induciblegene i rigilike receptors have been reviewed elsewhere and will not be covered herein in the case of dnarecognition one of the best known prrs is tolllike receptor tlr9 which senses extracellular cpg hypomethylated dna that has entered the cytosol through thephagosomelysosome system in addition the aim2like receptor aim2 inflammasome can be triggered afterthe entry of doublestranded dna dsdna into the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0czheng molecular cancer page of cytosolic compartment which induces the proteolyticmaturation of proinflammatory cytokines such as il1and il18 and the activation of gasdermin d leading topyroptosis [] nevertheless the most notable prr iscgas a direct cytosolic dsdna sensor which was identified by dr chens group in once cgas bindsto dsdna the cgassting pathway is activated to further induce the expression of type i ifns and other inflammatory cytokinesthus triggering innate immuneresponses mounting evidence suggests that cgassting signaling not only plays pivotal roles in the hostdefense against microbialinfection but also modulatestumorigenesis hence in this review we summarize themechanism of cgassting activation and elaboratefindings regarding its dual effects on tumor developmentcurrent advances in the use of sting agonists as a novelstrategy for antitumor therapy are also reviewedinsights into the cgassting signal transductioncascadecgas is an innate immune sensor that identifies variouscytosolic dsdnaincluding dna with viral bacterialmitochondrial micronuclei and retroelement originswhich can be mainly divided into pathogenderiveddna and selfdna table in the cytoplasm cgas isactivated by interacting with dsdna in a sequence[]independent butstructural and biochemical analyses have revealed thatthe cterminal lobe of cgas contains a conserved zinclengthdependent mannerionbinding module that mediates dna binding andcgas dimerization [ ] dna ligands promotecgas activation primarily by inducing conformationalchanges around the catalytic site and in the dnabinding structures of cgasthe gscontaining loopundergoes conformational change to maintain stabilitywhich is a major mechanism of cgas activation bydna in addition to the primary dnabinding sitementioned above the secondary site located beside theprimary site is a helix formed between strands 78and several surfaceexposed loops the proximity ofthe two dnabinding sites in cgas leads to a cgasdna complex assembly in which two cgas moleculesembrace two molecules of dsdna [ ] the cgasdimers are anized in headtohead alignment nextto the dna and thus form stable ladderlike networks between one long curved dsdna helix or two independent dsdna strands [ ] in this way eachindividual cgasdsdna complex can be cooperativelystabilized and can lead to stronger enzymatic activitywhich may provide a possible explanation for longerdsdna as more likely to activate cgas in additionlong dna is more efficient than short dna in drivingthe liquidliquid phase separation of cgas and the formation ofcriticallydependent on the concentration of cgas and dna inthe cytoplasm cgas and dsdna are spatially concentratedcgasdimerization and activation [] once cgas andcgas liquidlike dropletsin liquiddropletsistofacilitatetable classification of the cytosolic dsdna that activates the cgassting signaling axisclassificationselfdnasource of dsdnamicronucleipossible mechanismsrupture of the micronuclei membrane leads to exposureof chromatin dna that is recognized by cgas whichactivates the cgassting pathwayreferences mitochondrionnuclear rnapathogenderived dnadna virushsv1 hsv2 kshv adenovirus vacciniavirus cytomegalovirus papillomavirusmurine gammaherpesvirus retrovirushiv siv murine leukemia virusrna viruswest nile virus dengue virus vsvsarscov2bacterialisteria monocytogenes mycobacteriumtuberculosis listeria shigella francisellachlamydia and neisseriamitochondrial stress induces mtdna leakage into thecytosol thus activating the sting pathway and inducingproduction of cytokinesfacilitated by endogenous retroelements nuclear rnacan be reversely transcribed into dna that activatescgassting signaling dna viruses invade host cells and release pathogenderiveddna to induce sting activation[]dna intermediates generated from reverse transcription maybe recognized by cgas to stimulate downstream stingsignaling infection with rna viruses might cause cellular damage andcell death which results in the release of cellular dna andfurther activation of the cgassting axis sarscov2 bindingto ace2 can lead to excessive angiotensin ii signaling thatactivates the sting pathway in mice[]bacteria produce cdns such as cyclic digmp and cyclicdiamp which can directly bind to and activate sting[ ]hsv1 herpes simplex virus hsv2 herpes simplex virus kshv kaposi sarcomaassociated herpesvirus hiv human immunodeficiency virus siv simianimmunodeficiency virus vsv vesicular stomatitis virus cdns cyclic dinucleotides and sarscov2 severe acute respiratory syndrome coronavirus 0czheng molecular cancer page of dsdna interacts structural switches rearrange the catalytic pocket to enable cgas to catalyze the synthesis of²²cyclic gmpamp ²²cgamp with atp andgtp as substrates the first step in this process is theformation of a linear dinucleotide ²pppg ²²pawith atp serving as the donor and ²oh on gtp serving as the acceptor then the intermediate product flipsover in the catalytic pocket placing gtp at the donorposition and amp at the acceptor position to form asecond ²² phosphodiester bond [ ] notablyalthough dsrna or singlestrand dna ssdna is ableto bind to cgas neither can rearrange the catalyticpocket which may explain the exclusive activation ofcgas by dsdna ultimately cgamp acts as a secondmessenger to bind to and activate sting a small endoplasmic reticulum erlocated protein kd withfour putative transmembrane domains [ ] normally in a resting state sting is retained in the er byinteracting with the ca2 sensor stromalinteractionmolecule stim1 the cytosolic ligandbindingdomain lbd of sting exists as the most functionalunit capable of integrating with ²² cgamp or cdnscyclic dinucleotides such as cdiamp cdigmp or ²²cgamp from bacteria upon interaction the obviousclosure of the ligand binding pocket in the lbd is observed which is related to the activation of sting next sting transforms into a tetramer through a highorder oligomerization reaction and is translocated fromthe er to the perinuclear area facilitated by cytoplasmiccoat protein complex ii copii and adpribosylationfactor arf gtpases [ ] in the golgi sting ispalmitoylated atcys88 andcys91 a posttranslational modification necessary forsting activation modified sting recruits thekinase tankbinding kinase tbk1 in turn the cterminal domains of sting are phosphorylated bytbk1 and then phosphorylated sting recruits interferon regulatory factor irf3 which is also phosphorylated by tbk1 and dimerizes ultimately dimerizedirf3 enters the nucleus and exerts its function in thetranscription of type i ifns and interferonstimulatedgenes isgs in parallel sting can also bind toand stimulate iκb kinase ikk to mediate the production of nuclear factorκb nfκbdriven inflammatorygenes upon signal transduction termination sting istransferred to endolysosomes for degradation considering that cgamp can be transferred through gapjunctions or delivered in viralexosome packages cgassting signaling may be activated in the cytoplasmwithout dsdna [ ] moreover newly produced typei ifns activate heterodimer interferon receptors ifnar1 and ifnar2 through paracrine signaling and thusinduce the transcription of isgs [ ] in summaryonce virusderived dna and selfdna are located intwo cysteine residuesthe cytoplasm they can be sensed by cgas and a cgasdsdna complex is formed to catalyze the synthesis of ²²cgamp with atp and gtp then ²²cgamp and bacteriaderived cdns induce sting activation and mediate the release of downstream type iifns tnfα and il6 which are prerequisites for antimicrobial defense and antitumor effects the wholeprocess shows that the dsdnacgassting axis canlead to the activation of both innate and adaptive immunity fig the antitumor functions of the cgasstingsignaling pathwayrecent evidence has revealed the close association of thecgassting pathway with cancer development thissignaling pathway is generally regarded as a potent regulator of cancer immunity a stingmediated immunesupportive microenvironment can hamper malignancyoccurrence stressbytumor cell cytosolic dsdna induces sting activationunder normal circumstances dna is strictly unaffiliatedwith the cytoplasm in eukaryotic cells to avoid autoimmunity however dna leaks aberrantly in tumorcells [ ] cancer cells share common features including genome instability tumor suppressor gene mutation or deletion oxidativeand vigorousmetabolism under these intense states nuclear andmitochondrial dna is fragile and easily damaged whichleads to eventual dna leakage in the forms of micronuclei chromatin fragments andor free telomeric dna[ ] chromosomal instability cin is the primary source of cytoplasmic dna in malignant cells andis generally associated with tumor progression distantmetastasis and therapeutic tolerance excessive proliferation of cancer cells results in unstable genomes usuallychromosomal missegregation during mitosis due to defects in segregation lagging chromosomes generate micronuclei in a cellcycledependent manner the vulnerable membraneof micronuclei easily exposes the inner dna to the cytoplasm and activates the cgassting signaling axis exogenous stimuli such as chemotherapy and irradiation can also cause dna damage in addition to leakednuclear dna oxidative stressinduced mitochondrialdna leakage is another crucial initiator of sting pathway activation several anticancer treatments that precisely attack mitochondrial membranes result in effluxand cell death therefore the permeabilization of mitochondria membranes provides a reasonable explanationfor mitochondrial dna escape [ ] other sourcessuch as apoptotic cellderived dna exosomal dnaexodna and transposable elements have also beencharacterized 0czheng molecular cancer page of fig the cgassting dna sensing signaling pathway various dna derived from virus dying tumor cells or nucleus and mitochondria binds toand activates the cytosolic dna sensor cgas cgas catalyzes the synthesis of ²²cgamp in the presence of atp and gtp then ²²cgamp bindsto the er adaptor sting which also can be activated by cdns derived from bacteria upon activation sting translocates from er to golgicompartments where it activates tbk1 and ikk which phosphorylate irf3 and iκbα respectively then irf3 and iκbα dimerize and enter nucleusinitiating the transcription of type i ifn tnf and il6 the primary roles of these cytokines are reflected in host defense inflammation andantitumor immunitydemonstrated to evoke cgassting activation intumor cells [ ]type i ifns mediators of sting and adaptive antitumoreffectscgassting signaling exerts antitumor functions incancer cells both in an autonomous and nonautonomousmanner on the one hand dna damage can provokeacute sting signal transduction and induce cellularsenescence an irreversible cell cycle arrest state whichthwarts the aberrant proliferation of tumor cells throughacquisition ofsecretoryphenotype sasp which is associated with the releaseof abundantinflammatory mediators proteases andgrowth factors [ ] in contrast to undergoingsenescence tumor cells also directly propel apoptosisprocesses by upregulating proapoptosis protein bcl2associated x bax and downregulating the bcl2 apoptosis on the other hand stingsenescenceassociatedtheregulatoractivation in tumor cells not only facilitates the transcription of downstream type i ifns to induce dendriticcell maturation but also recruits supportive immunecells for direct nonspontaneous tumor elimination sting activation in nonmalignant cells causes tumorsuppressive effects as well sting signaling protectsagainst colitisassociated carcinomas cacs induced byazoxymethane aom and dextran sulfate sodiumdss which induce dna damage in intestinal epithelialcells and further trigger sting activation downstreamcytokines of sting signaling such as il1 and il18prevent neoplastic transformation by facilitating woundrepair more importantly sting signaling can also provoke cytotoxic t cell responses to control tumorigenesis necrotic cancer cells are commonly engulfed byantigenpresenting cells especially the basic leucine zippertranscription factor atflike batf3drivenlineage of dendritic cells dcs batf3 dcs take intumorassociated antigens and migrate towardsthe 0czheng molecular cancer page of tumordraining lymph node via the lymphatic systemwhere they crossprime tumorspecific cd8 t cellsthen cd8 t cells undergo activation and clonal expansion in the lymph nodes and are trafficked throughblood vessels to kill tumor cells in turn damaged cancercells release more antigens that are further captured bydcs the whole process forms a positive feedback loopcalled the cancerimmunity cycle tumor eradication can be achieved by multiple processes in thecancerimmunity cycle including tumor antigen captureand presentation and t cell priming and activation withtumor antigenspecific t cell priming and activationrelying on dcs and type i ifn release the involvement of type i ifns in innate immune sensing and adaptive immunity provides a reasonable hypothesis forexploring candidate prr pathways as potential immunomodulators mice lacking tlr9 myeloid differentiationprimary response gene myd88 cytosolic rna sensor mavs or the purinergic receptor p2x7r maintainintact antitumor immunity responses whereas mice deficient in sting or irf3 present with impaired cd8 tcell priming and activation [ ] in fact dying tumorcells can release multiple damageassociated molecularpatterns damps to trigger innate immune responsesin dcs among these released stimuli tumor cellderiveddna is a pivotal inducer in general the phagocytosis ofapoptotic cells causesimmune silence because ofdnasebased degradation nevertheless tumor cellreleased dna can be preserved in the dc endolysosomal compartment through an unknown mechanism cgas recognizes dna invading the cytoplasm andinduces the activation of sting cascades excretion oftype i ifns and expression of isgs additionally undersome physiological conditions such as hypoxia andacidic environments nuclear or mitochondrial dnamight be packaged in exosomes exosomal dnaexodna animates sting signaling once it is absorbedby tumorinfiltrating dcs finallytumor cellderived cgamp can also be transferred to host dcs bythe folate transporter slc19a1 and then directly bindsto sting activating it in dcs a recent study moredirectly demonstrated that cellautonomous sting promoted the maintenance of stem celllike cd8 t cellsand augmented antitumor t cell responses and mechanistically cgasstingmediated type i interferon signaling reinforced the stem celllike cd8 t celldifferentiation program mainly by restraining akt activity immune cellderived type i ifns have crucial functions in antitumor immunity control on the one handtype i ifns boost cross presentation by various mechanisms first they stimulate the maturation of dcs secondthey slow the endosomelysosome acidificationprocess to prevent engulfed tumor antigen clearance andelevate the expression of mhc i molecules on the cellsurface [ ] finally they accelerate dc migrationtowardslymph nodes where they can crossprimetumorspecific cd8 t cells on the other handtype i ifns drive the expression of multiple chemokinessuch as cxcl9 and cxcl10 both of which are necessary for cytotoxic t lymphocyte ctl transfer and infiltration similarly type i ifns restrain the defaultimmune suppressive action of regulatory t treg cellsby downregulating phosphodiesterase pde4 and upregulating cyclic amp camp consequently typei ifns serve as bridges linking the cgassting pathway with cd8 t cellmediated antitumor immunitythe antitumor mechanisms of the cgassting signaling axis are illustrated in fig indeed previous studies revealed that sting activation can stimulate antitumor immune responses inleukemia melanoma glioma and hepatocellular carcinoma [] additionally sting expression is downregulated in a wide variety of tumor tissues and celllines according to a pancancer analysis with a smallproportion of tumors approximately bearing silent sting expression lower sting expressionwas found in hepatic carcinoma and gastric cancer compared with its level in corresponding normal tissues andthis lower expression level was correlated with highertumor stage and poorer prognosis [ ] consistentlycompared with that in the mcfg10a mammary epithelial cell line lower sting expression was detected inmalignant breast cancer cellincluding mcf7hbl100 and t47d cells as well as human melanomacell lines and colorectal adenocarcinoma lines [ ] collectivelythat cgassting signaling might act as a tumor suppressor in certain types of cancersthese findings suggestlinessting pathway agonists as cancer therapeuticsthe immunostimulatory potential of the cgasstingpathway makes it an attractive pharmacological targetsince its activation in the tumor microenvironmenttme can induce efficient crosspriming oftumorspecific antigens and facilitate the infiltration of effectort cells recent drug research has focused on the development of sting agonists because of their potential inanticancer therapy [ ] to date various kinds ofsting agonists have been discovered and they aremainly divided into the following categories cyclic dinucleotides and their derivates dmxaa and its analogsand small molecular agonists in addition some conventional antitumor therapeutics can also indirectly activatesting such as chemotherapy radiotherapy rt andtargeted therapy in addition sting agonists areable to enhance the efficacy of other anticancer therapeutic agents when used in combination sting 0czheng molecular cancer page of fig the antitumor immunity effect of the cgassting pathway dna damage leads to the formation of dsdna in tumor cells upon itsstimulation sting signaling is activated and promotes the release of type i ifn which is crucial for dc maturation sting signaling activation indcs is the core step of the whole cancerimmunity cycle which can be initiated through engulfment of dyingdamaged tumor cells exosometransfer and cgamp gap junctions then dcs migrate towards the tumordraining lymph node and crossprime tumor specific cd8 t cells withthe help of type i ifns finally t cells undergo clonal expansion and traffic through the blood vessel to conduct tumor killingagonists and their synergistic use with other remedies isfurther explored in detail belowcyclic dinucleotides cdnscdns constitute a main type of sting agonist whichmainly originate from bacteria the known naturalcdns consist of exogenous cyclic digmp cdigmpcdiamp ²²cgamp and endogenous ²²cgampamong these cdigmp cdiamp and ²²cgampare synthesized by bacteria and identified as secondarymessengers that mediate sting signal transduction inprokaryotic cells while ²²cgamp functions as theinitiator of sting in mammalian cells the antitumor potential of these natural dinucleotides was firstproven by the finding that cdigmp could inhibit theproliferation of human colon cancer cells in vitro andbasal cell proliferation of human cecal adenocarcinomah508 cells was inhibited with μm cdigmp intraperitoneal ip injection of highdose cdigmpdirectly activated caspase3 and triggered t1 tumoripcell apoptosis in vitro nmol of cdigmp reduced thegrowth of t1 tumor cells in vitro by and nmreduced it by while lowdose cdigmp nmol accelerated the adaptive t cell response by converting a subgroup of myeloidderived suppressor cellsmdscs into immune stimulatory cells producing il12injection of ²²cgamp consistentlymgkg expedited dramatic leukemic elimination in eltcl1 transgenic mice bearing chronic lymphocyticleukemia cll and promoted tumor shrinkage of multiple myeloma in vivo from the perspective of endogenous cdns ²²cgamp mgkg was alsoshown to restrain tumorigenesis and improve the survival rate of mice bearing ct26 colon adenocarcinomain a dosagedependent manner relying on dc activationand t cell crosspriming more recently ohkurit further demonstrated that intratumoral it injection of ²²cgamp μg25 μldose on and days after the injection of tumor cells significantly mitigated tumor growth and prolonged the survival of breast 0czheng molecular cancer page of cancer t1luc squamous cell carcinoma mscc1colon cancer ct26 and melanoma b16f10 mousemodels notably the it injection of ²²cgampinhibited not only tumor growth but also lung metastases in mice bearing b16f10 cellderived tumors suggesting that cgampinduced cd8 tcell priming can drivesystemic antitumor immunity to control local and distant tumor growth termedvaccinestingvaxconsidering the superior properties of sting signaling in activating adaptive immunityit is rational toutilize sting agonists such as cdns as cancer vaccineadjuvants to increase tumor immunogenicity fu investigated the in vivo therapeutic efficacy of acancercomprisinggranulocytemacrophage colonystimulating factor gmcsf and bacteriaderived or synthetic cdns theyobserved that after it injection of stingvax with μg of cdns per vaccine dose the volume of b16melanoma tumors was dramatically reduced in a dosedependent manner compared to mice receiving gmcsf cancer vaccine alone stingvaxtreated mice hadmore infiltrating cd8 ifnγ t cells in the tumormicroenvironment the in vivo antitumor effect of stingvax was also verified in models of colon carcinomact26 pancreatic carcinoma panc02 and upper aerodigestive squamous cell carcinoma sccfvii although natural cdns are able to produce robust antitumor immunity their chemical features might hindertheir future application in the clinical setting first native cdns are easily degraded by enzymes inside the cellor in the bloodstream second their negatively chargedproperty hydrophilicity and phosphate moieties severelyimpede cdns from penetrating cell membranes to activate cytosolic sting leading to low bioavailability andpoor retention of the cdns in specific cells and tissuesthird unintentional toxicities and narrow therapeuticwindows are also unavoidable thus new strategies toimprove therapeutic efficacy and reduce adverse effectsare urgently needed including drug delivery carrier engineering original structural modification and nonnucleotide agonist screening regarding agonistdelivery smith reported that biopolymer implantscodelivering cdigmp μg and chimeric antigen receptor t cart cells resulted in significant tumor regression in mice bearing pancreatic tumors moreoveriv administration of cdigmpysk05lip equivalent to μg of cdigmp aysk05liposome delivery system encapsulating cdigmp led to a tremendous decrease in metastatic lesionsin a b16f10 mouse melanoma model with nearly ofthe injected mice showing resistance against tumor relapsethe adaptive immune responsememory was successfully induced chen alsofound thatliposomalindicating thatinjection ofintravenousintravenousivnanopdelivered cgamp cgampnp could activate the sting axis more effectively than solublecgamp and converted the immunosuppressive tme toa tumoricidal state in a transplanted b16f10 cell melanoma model and in a genetically engineered triplenegative breast cancer model moreover a recentstudy creatively suggested that modified bacteria mightbe exploited as a selective carrier of sting agonistsintroduction of a dinucleotide cyclasecoding gene intothe escherichia coli nissle strain was an attempt at realizing this effect however advancements to the systemare needed tobysnakeapartdigestionresistancecompoundatoms the modifiedfrom improving delivery methods cdnswith superior properties are currently being synthesized and tested for instance to prevent enzymatichydrolysis of cgamp the nonbridging oxygen atomsin cgamp phosphodiester linkages were replaced by²²sulfurcgsasmp showed resistance against degradation byenpp1 a major ²²cgamp hydrolasetherebyleading to a longer halflife and sustained high affinity for human sting hsting syntheticdithio mixedlinkage cdns with both rp rp r rand rp sp r s dithio diastereomers possessed notonlyvenomphosphodiesterase but also enhanced affinityforsting a novel superior modified product ml rrs2 cda also termed adus100 had the potencyto activate all hsting variants and mouse stingmsting adus100 had higher efficiency in activating sting signaling than endogenous or exogenous cdns mainly because of its enhanced stabilityand lipophilicity its powerful tumor elimination effect was extensively demonstrated in multiple murinemodelsincluding b16 melanoma t1 breast cancer and ct26 colon cancer with all treated animalsshowing significant and durable tumorregressionafter itinjection of adus100 three mg doseswhen tumor volumes reached mm3 theremarkableforhsting laid the foundation for its clinical use related clinicaltrials of adus100 are outlined intable in addition to adus100 some other novelsting agonists have been well designed iacs8779and iacs8803 are two highly potent ²²thiophosphate cdn analogs that induced striking systemicantitumorin a b16 melanoma murineinjection μg on and daysmodel after itposttumor implantation compared with adus100or cgamp the characteristics and preclinicalapplications of all these mentioned cnds are summarized in table because of the structural modification and optimization of delivery strategiestheapplication range and efficacy of cdns have beenand high affinityresponsescurativeeffect 0czheng molecular cancer page of table characteristics and preclinical applications of different sting agonistsclassificationcharacteristicsapplicationmodelsnatural cdnagonistscdigmppoor membrane permeabilitysuitable for various codeliverytechnologiescolon cancer h508cells t1 metastaticbreast cancertreatmentinformation μm nmol ip nmol ip nmol ip²²cgamp²²cgamphigher binding affinity formsting than for hstinghigher affinity for hsting thanits lineage isomers binds tovarious sting nucleotidepolymorphisms observed inhumans easily degraded byphosphodiesteraseimpermeable to the cellmembranechronic lymphocyticleukemia mgkg ipmultiple myeloma mgkg ipct26 colonadenocarcinoma mgkgbreast cancer t1lucsquamous cellcarcinomasmscc1 μg25 μldose it μg25 μldose itcolon cancer ct26 μg25 μldose itmelanoma b16f10 μg25 μldose ittherapeutic effects references[ ] [ ]inhibitsproliferation tumorregression tumorregressionaccelerates tcellresponseleukemiaeliminationsuppressesgrowthrestrainstumorigenesisimproves survivalratedelays tumrowthdelays tumrowthdelays tumrowthdelays tumrowthstingvaxsyntheticcdnagonistspotent in vivo antitumor efficacyin multiple therapeutic modelsof established cancercgampnpsbiopolymer scaffolds cdigmp and car t cellscdigmpysk05lip²²cgsasmpadus100iacs8779iacs8803noncdnagonistsfaaliposomal nanops npsdeliver cgamp intracellularlymore effectively than realizedwith soluble cgamperadicates tumors moreeffectively than systemicdeliveryysk05 is a lipid that can efficientlydeliver cdigmp to the cytosolpossesses high fusogenic activitywhich enhances endosomalescapemore resistant to degradation byenpp1 tenfold more potent atinducing ifn secretion potentialuse as a cancer vaccine adjuvantimproves stability and lipophilicityhigher affinity for hsting thannatural cdn agonists capable toactivate all hsting variants andmstingstimulates a superior systemicantitumor response thanadus100 and cgampcauses hemorrhagic necrosisfailed in a phase i clinical trialdue to species specificity μg cdns itreduces tumorvolume b16 melanomacolon carcinomact26pancreaticcarcinoma panc02b16f10 melanomaivtnbccreates atumoricidal state pancreatic cancer μg cdigmptumor regression b16f10 mousemelanoma μg cdigmp ivdecreasesmetastasisthp1 monocytesb16 melanomathree mg doses it t1 breast cancerthree mg doses itmc26 colon cancerthree mg doses itdurable tumorregressiondurable tumorregressiondurable tumorregression b16 melanoma μg on day and posttumor implantationantitumorresponse murine colontumorsextensive tumorrejection[ ]dmxaafirst discovered as a vascularrat mammary mgkg iphigh anticancer[ 0czheng molecular cancer page of table characteristics and preclinical applications of different sting agonists continuedclassificationcharacteristicsapplicationmodelstreatmentinformationinduces proinflammatory cytokinesin a stingdependent mannerhuman fibroblastsantiviral activity selectively induces stingdependentsynthesis and secretion of bioactiveifns no evidence of binding directlyto stingactivates sting in openconformation submicromolarlevels induce sting activationand ifn productionhuman fibroblastsantiviral activity colon tumors mgkg iv of a treatedgroup remainedtumor free faa flavone acetic acid dmxaa 56dimethylxanthenone4acetic acid cma 10carboxymethyl9acrid | 0 |
" laparoscopic tumorspecific mesorectal excision tsme preserving the left colic artery and superiorrectal artery is still a technically challenging procedure we conducted this study to demonstrate the feasibility ofthis procedure for upper rectal cancermethods a total of patients with upper rectal cancer were retrospectively analyzed in our cancer centerbetween april and april these patients were treated with either laparoscopic tsme n orlaparoscopic total mesorectal excision tme n in the tsme group the left colonic artery and superior rectalartery were preserved while they were not in the tme groupresults the operation time in the tsme group was longer than that in the tme group ± min vs ± min p furthermore the number of resected lymph nodes in the tsme group was greaterthan that in the tme group ± vs ± p the blood loss between the tsme and tmegroups was not significant no mortality occurred in either the tsme or tme groups one patient in the tme groupunderwent conversion to laparotomy the total postoperative complication rates in the tsme and tme groups were and respectively there was no difference in severe complications between the two groupsanastomotic leakage and stenosiss laparoscopic tsme preserving the left colic artery and superior rectal artery can be safely conductedfor upper rectal cancerkeywords laparoscopic surgery rectal cancer tumorspecific mesorectal excision superior rectal artery leftcolonic artery tme correspondence 237721898qqcom 250537471qqcomhuxiang_zc1978sinacom chi zhang haotang wei wenqing hu and yueming sun contributedequally as joint first authors7department of general surgery yizhen peoples hospital clinical medicalcollege yangzhou university yangzhou jiangsu province china3department of gastrointestinal surgery changzhi peoples hospital theaffiliated hospital of changzhi medical college changzhi shanxi provincechina1department of gastrointestinal surgery the first affiliated hospital of dalianmedical university dalian liaoning province chinafull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0czhang world of surgical oncology page of introductiontotal mesorectal excision tme is an important surgical technique to prevent the local recurrence of rectal cancer on the other hand tme may not besuitable for every case of rectal cancer such as rectosigmoid junction and upper rectal cancers the resection range of tme reaches cm below the inferiorborder of the tumor and has acquired an adequatecure rate reported in previous studies for patientswith rectosigmoid junction and upper rectal cancers this tumorspecific resection according to thetumorsite or t staging is called tumorspecificmesorectal excision tsme itsudecks critical point at the rectosigmoid junction isdescribed as the point of origin of the last sigmoid arterial branch originating from the inferior mesenteric artery ima the anastomosis between the lastsigmoidal artery and superior rectal artery sra is absent in some people to avoid the risk of postoperativeischemic necrosis anastomotic leakage colitis and delayed stricturetosudecks point for cases where anastomosis may be absent or insufficiently present in addition the rate ofis desirable to ligate proximalabsence of the left colic artery lca is which maybe associated with a risk of anastomotic leakage due toinsufficient vascularization of the proximal colonic conduit this study introduces the procedure and technicalpoints of laparoscopic tsme with preservation of thelca and sra the operation is still a technically challenging procedure we conducted this study to demonstrate the feasibility of this procedure for upper rectalcancer and shortterm prognosismethodspatientslaparoscopic tsme preserving the lca and sra wasperformed on patients with upper rectal cancer fromapril to april in the same period patients with upper rectal cancer underwent standardtme surgery this study was conducted in accordancewith approved guidelines this study was approved bythe institutional review board of the first affiliatedhospital of dalian medical university written informedconsent was obtained from all patientsfig ima 3d cta 0czhang world of surgical oncology page of equipmentangled ° 10mm diameter 3d laparoscope insufflation equipment and bipolar electrosurgical deviceaesculap german harmonic vascular closure systemjohnson usa 10mm and 5mm port trocars teleflexmedical usa laparoscopic linear staplers mm inlength covidien usa hemolock polymer lockingsurgical clips teleflex medical usa and a circularstapler ethicon endosurgery usa were used in thisstudypreoperative preparationinferior mesenteric artery ima 3d cta examinationshould be performed before the operation to assess themesenteric vascular vessel types fig intestinal preparation was performed days before the operation andprophylactic intravenous antibiotics were used beforethe operation for min central venous catheterizationwas performed after general anesthesia the surgicalposture was the starboard lithotomy position with thehead lower and feet higherthe operating surgeon and camera assistant stood onthe patients right side and the first assistant stood atthe patients left foot side the laparoscopic monitor wasplaced on the patients right foot side the trocar for thelaparoscope was inserted from the right paraumbilicalside and four ports were used as working ports fig surgical techniquesthis surgical technique was characterized by thoroughlymph node dissection based on neurovascular preservation and dissection of the left colon and sigmoid andfig position of the trocarupper rectal vessels along the inferior mesenteric vesselsthe region of operation was the superficial layer of thenerve sheath on the vascular surface the left colonicand superior rectal vessels needed to be preserved andthe vascular branch from the sigmoid vessels and theblood vessels from the superior rectal vessels to the intestinal wall were selected and severed according to thetumor positionfirst we adopted a lateral approach by opening themonks white line along the descending and sigmoidcolon reaching the splenic flexure as the cephalad dissection point the correct plane of dissection wasachieved by toldts fascia we usually used bipolar electrosurgical devices and bipolar scissors to separate thiscorrect plane with gentle blunt and sharp dissectionthe ureter and other retroperitoneal structures weresafely protected by staying in this plane we continuedto dissect along the plane to the root of the ima thehypogastric nerves were visible the nerves were carefully protectedthen the dissection began at the position of the sacralpromontory the junction of the sigmoid mesentery andretroperitoneum from the previous dissection plane in thefirst step ideally we dissected the presacral space belowthe sra from the left side across the midline to the rightside attentively protecting the hypogastric nerves whileusing a bipolar electrosurgical device fig 3a the distaldissection endpoint was approximately cm below thetumor we needed to open the peritoneal reflection anddissect the lateral ligament of the rectum by protectingthe neurovascular bundle nvb using a harmonic vascular closure system in some patients we placed the dissected colon and mesocolon to the right celiac side andthoroughly revealed the left side of the mesocolon wecarefully employed dissection in the correct plane on thevessels to avoid tissue damage for the realization of enbloc resection the technique in this step is to identify therelationship between the left colic artery inferior mesenteric vein imv to the ima and sra and the branch ofthe arteriae sigmoideae fig 3b this vascular bundle canbe traced from the origin of the ima to the rectal segmentapproximately cm below the inferior border of thetumor fig 3cthe second step was performed using a medial approach this step involved thorough lymph node dissection based on neurovascular preservation the leftcolonic and superior rectal vessels need to be preservedand the sigmoid vessels and vessel branch from the superior rectal vessels to the intestinal wall were selectedand severed according to the tumor positiondissection at the correct presacral space and cephaladdissection to the ima could be employed our generalmedial approach was to begin at the presacral space andobtain a connection with the plane ofthe lateral 0czhang world of surgical oncology page of fig a dissection the presacral space below the superior rectal artery sra approached from the left side across the midline to the right sideattentively protected hypogastric nerves while using a bipolar electrosurgical device b identification of the relationship between left colic arteryimv to the ima and sra and the branch of the arteriae sigmoideae c tracing this vascular bundle from the origin of the ima to the rectumsegment approximately cm below the inferior border of the tumor d ligation of arteriae sigmoideae and vascular branch from sra eligation of arteriae sigmoideae and preserving left colonic vasculature f excision of the mesorectum just underneath the rectal wall about cm and avoiding injury to the rectal wall and sra g tsme preserving left colic artery and superior rectal arteryapproach pelvic dissection was performed from the entrance of the pelvic cavity down to the pelvic floor wecould identify both the hypogastric nerve fibers and pelvicnerve by using highdefinition 3d laparoscopy and preservethem the imvleft colic artery bundle was then carefullytraced to the junction position from the ima and lymphnode no253 was dissected the pelvic nerves and ureterwere already carefully insulated and the circumference ofthe ima could be revealed the mesocolon could be freedfrom the retroperitoneal position by anterior dissection bygently applying a bipolar electrosurgical device we dissected the sra and blood vessels from the sra to the intestinal wall and dissected lymph nodes no252 andno251 at this point we had completed lymph node dissection and completely clarified the relationship betweenthe lca imv ima sra and arteriae sigmoideae finallywe ligated the arteriae sigmoideae and vascular branchfrom the sra into the intestinal wall fig 3d while preserving the left colonic vasculature fig 3e energy devicesand hemolocks were used widely in this step 0czhang world of surgical oncology page of after the above procedure was completed we separated the rectal wall from the mesorectum with an adequate distance from the tumor in accordance with thet stage and position of the tumor using a harmonic vascular closure system in order to provide enough spaceto insert an endoscopic linear stapler we excised themesorectum about cm just underneath the rectalwall fig 3f careful surgery was performed to avoid injury to the rectal wall and sra then the endoscopic linear stapler was fixed the rectum was transected andsatisfactory tsme preservation of the left colic and superior rectal arteries was shown fig 3glastly a small 5cm incision was made at the leftlower abdomen and the specimen was taken outside ofthe abdomen and transected intraabdominal presacralanastomosis was performed by double stapling techniques after inserting the anvil head of a 28mm circularstapler into the oral side of the sigmoid colon doubledrains were placed and no diverting stoma wasperformedin the tme group the inferior mesenteric artery wassevered at the root the colon was severed cm awayand digestive tract reconstruction methods were similarto the tsme groupstatisticsspss190 version was used for statistical analysis categorical variables were compared using a Ï2 test continuous variables were presented as the mean standarddeviation or median range these variables were compared using a mannwhitney u test p values of were considered statistically significantresultsthe general characteristics of the included patients arelisted in table there were men and women in the tsme group and men and women in the tme group the meanage was ± years and ± years in thetsme and tme groups respectively there were no significant differences in preoperative comorbidity tumorsize depth of invasion and lymph node metastasis between groups the average distance between the tumorand anus of the tsme group was ± cm andthe distal margin was ± cm the pathologicalstages of the patients for the tsme group were as follows stage i stage iia stage iib stage iic stage iiia and stage iiib theproportion of patients with normal preoperative carcinoembryonic antigen cea was approximately of patients had cea levels between and ngml of patients had cea levels between and ngml and only patients had cea levels ngmltable clinicopathological features between the tsme andtme groupsfactorsage yearstme n ± tsme n ± p valuegendermalefemalebmi kgm2comorbidity ± ± cardiovascular disease respiratory diseasediabetes mellitushistological type differentiated typeundifferentiated type tumor size mm ± ± t categoryt1t2t3t4n categoryn0n1n2 conversion to open surgery operation time min ± ± blood loss ml ± ± lymph node dissection ± ± the operation time in the tsme group was longerthan that in the tme group ± vs ± p table furthermore the number ofresected lymph nodes in the tsme group was greaterthan that in the tme group ± vs ± p table the blood loss between groupswas not significantly different table the averagehospital stay in the tsme group was a little shorter thanthat in the tme group ± days vs ± days table no mortality occurred in either group one patient inthe tme group underwent conversion to laparotomy dueto bowel ischemia in the distal colon table the totalpostoperative complication rates in the tsme and tmegroups were and respectively table forsevere complications between the two groups anastomotic leakage and stenosis the severity of complicationswas claviendindo classification grades and therewas no significant difference between groups 0czhang world of surgical oncology page of tsme n tme n p value ± table postoperative complicationsfactorspostoperative hospital stay days ± mortalitymorbidityabsentpresentanastomotic leakagebleedingabdominal abscessileuswound infectionanastomotic stenosisurinary tract infectionascitesurinary retentionpneumoniacardiacrelated complications discussionin the british surgeon heald proposed tme for rectalcancer and pointed out that the anatomical level of tmewas clear so that the operative quality can be assessed the main concerns were a higher anastomotic leakage ratelonger operative time and higher blood loss after tme lopezkostner pointed out that tme was the standard operation performed for lower rectal cancers tme isnot necessary for cancers of the upper rectum therefore the tsme technique was introduced to achieve satisfactory local control and low morbidity partial mesorectalexcision is applied in tsme according to willians report in only of patients had distal intraluminal diffusion cm pollett and nicholls observed that there were no differencesin the local recurrence rate of rectal cancer between distal margins cm cm and cm a randomized prospective study of nsabb the national surgicaladjustburst and bowel project showed that the localrecurrence rate was not significantly different betweendistal rectal margins cm cm and cm according to the practice parameters for the management of rectal cancer edition a 2cm distal margin is more acceptable than cm but a 5cm distalmargin is still recommended total mesorectal resectiontme should be used for tumors located in the middleand lowerregardless oftwothirds ofwhether itis performed with low anterior resectionlar or combined abdominal and perineal resectionapr for tumors in the upper onethird of the rectumresection of the mesentery can be carried out accordingto the tumor situation and the distance between thethe rectumdistal margin and tumor should be cm the recommended grade was 1a tme was performed according to the distance between the distal margin of the rectal tumor and anus cm while tsme was performed for patients with adistance between the distal end of the rectal tumor andanus of cm in the authors medical departmentoncological outcomes after surgery can be divided intotwo aspects longterm survival and local recurrence ratelaw reviewed patients the 5year local recurrence rate for tme and partial mesorectal excisionpme for proximal cancer was and respectively the disease stage was associated with a higher riskof local recurrence there was no difference in the localrecurrence rates of tme and pme the 5year cancerspecific survival rates with and without tme were similarat and respectively kim reportedthat the 5year cancerspecific survival rate was andthe local recurrence rate was with cases of rectalcancer after tsme with pathologic stages iiii the riskfactors affecting cancerspecific survival rate were the ptstage pn stage positive distal resection margin and positive circumferential resection margin the risk factors affecting local recurrence were the pn stage positive distalresection margin and positive circumferential resectionmargin another study from a korean reviewed experience in patients with rectal cancer showed that theoverall local recurrence rate was the 5year local recurrence rates were and in stages i iiand iii respectively the 5year cancerspecific survivalrates were and in stages i ii and iiirespectively the risk factors were the pn stage and circumferential resection margin zakir performed an analysis with years of experience in rectal cancer patients who underwent laparoscopic andopen tsme surgery the 5year local recurrence rate was the overall 5year and cancerspecific survival rateswere and respectively there was no difference in the local recurrence rate between laparoscopic oropen resection the overall and cancerspecific survivalrates were and in the laparoscopic surgerygroup and and in the open surgery group respectively the results showed that laparoscopic surgerywas better than open surgery in overall and cancerspecific survival there was no difference in survival in patients with stage i however the survival rates in patientswith stages ii and iii among the laparoscopic surgerygroup were better than those in the open surgery groupwhich shows the superiority of laparoscopic tsme surgery for the longterm prognosis of rectal cancerkorean scholars conducted a study on the safety andprognosis of tsme after neoadjuvant chemotherapy forrectal cancer patients received 5fu with leucovorinchemotherapy and radiotherapy cgy for cycles 0czhang world of surgical oncology page of leadership was tsme was performed weeks later the resultsshowed that the overall complication rate was empiricalinternal construction was the 5year survival rate was and the 5yeardiseasefree survival was at present chinasouth korea and the usa have formulated similarguidelines for preoperative radiotherapy and chemotherapy for middle and low rectal cancer but there is nospecific reference data for preoperative radiotherapy andchemotherapy for upper rectal cancer the purpose ofthis paper is to introduce a new method of tsme anddiscuss the safety of the operation longterm survivaland local recurrence have not been discussedtsme surgery based on tme is now accepted as astandard for rectal cancer surgery and laparoscopic rectal cancer resection is accepted widely in the world eventhough it is a challenging procedure for surgery bloodloss in the laparoscopic group is well shown with anaverage of to ml the average blood loss inour study was ml lower than that reported in the literature we can identify neurovascular lesions usinghighdefinition 3d laparoscopy to preserve them and weuse a bipolar electrosurgical device to reduce injurywhich is beneficial for accurate operationthe overall complication rate in laparoscopic tsmeoperation was lower than that in the open operationgroup the rate of anastomotic leak showed no statistical difference between the two operation methods theaverage leak rate for rectal cancers was zakir reported that the overall complicationrate was in tsme for rectal cancer patients therate of anastomotic leakage was in the open tsmegroup and in the laparoscopic tsme group therewas no statistical difference between groups in our studythe incidence rate of postoperative anastomotic leakagewas three patients had complications after surgeryand the overall complication rate was the threecomplications were wound infection fluid collection andurinary retention with a claviendindo grading of yoo evaluated the optimal duration of urinarycatheterization after tsme for rectal cancer logistic regression analysis was performed to determine the risk factors for urinary retention the variables including age sexasa grade surgical procedure tnm stage tumor position preoperative radiotherapy duration of urinarycatheterization and time of surgery were not significantrisk factors for urinary retentionat present a 3d laparoscopic system aesculapgerman is used in laparoscopic surgery in our department single and reduced portlaparoscopic surgeryrobot operations and tatme operations are not usedfor tsme the surgeons who performed tsme had morethan years of experience in gastroenterostomy and hadexperience with open tsme the difficulty of the tsmeoperation is the management of the mesorectum seiji has reported on the management of the mesorectumin the narrow pelvis which our treatment method is basedon first the right part of the mesorectum is lifted fromthe right side of the sigmoid mesocolon to expose the inferior mesenteric artery and vein left colonic vessels sigmoid colonic vessels and superior rectum vessels theassistant lifts the left mesentery of the sigmoid colon exposes the above vessels expands the sigmoid mesocolonagain penetrates the mesentery from the right side andexposes the surrounding vessels expansion of the pelviccavity along the vessels is continued and the mesorectumis repaired from the left to the right side cm above thetumor according to the location ofthebranches of the severed vessels are determined and cm of the intestinal wall is repaired the rectum is dissected using an endogia staplerthe tumorlaparoscopic tsme has been used for rectal cancer andcan obtain satisfactory functional results compared to openresection and tme we do not think that the reduction inthe hospital stay is due to the acceleration of the intervention as per enhanced recovery after surgery eras butis due to an increase in the doctors confidence in reducingthe risk of postoperative complications after vascular preservation threedimensional cta examination is importantfor the preoperative evaluation of sigmoid colonvascular classification and intraoperative management ofthe sigmoid and left colon vessels however preoperativeexamination could not obtain information on the trafficbranch the biggest advantage of this operation is themaintenance of the blood supply of the proximal and distalintestines and the sufficient length of the intestine so thereis no need for temporary defunctioning stoma temporarydefunctioning stoma only increases the complexity of theoperation and closure of the temporary stoma increasesthe risk of complications in addition the results of the statistical analysis showed that the number of lymph nodes inthe tsme group was greater than that in the tme groupit cannot be concluded that tsme was significantly betterthan tme for lymph node dissection suggesting thattsme was not inferior to tmeslaparoscopic tsme with preserved left colic and superior rectal arteries is a technically challenging procedureintact visceral pelvic fibro is protected with even greateraccuracy than other techniques by 3d laparoscopywhich offers an optimal vision tsme with preserved leftcolic and superior rectum arteries did not increase therisk of operation compared with tme but increased thesurgeon s confidence in patient outcomes thereforelaparoscopic tsme with preserved left colic and superior rectal arteries can be safely performed for rectal cancer patients as an alternative to tme 0czhang world of surgical oncology page of abbreviationstme total mesorectal excision tsme tumorspecific mesorectal excisionima inferior mesenteric artery imv inferior mesenteric vein sra superiorrectal artery nvb neurovascular bundle pme partial mesorectal excisionacknowledgementsnoneauthors contributionslz yx and xh designed the study cz yx hw qz zd and whcollected and analyzed the data ma lz ys and xh interpreted the datalz and cz drafted the manuscript lz ma yx and xh revised themanuscript the authors read and approved the final manuscriptfundingthis study was supported by the jiangsu natural science foundationbk20180274 this funding supported the collection analysis andinterpretation of the dataavailability of data and materialsall experimental data used to support these findings are included in theethics approval and consent to participatethis study was approved by the institutional review board of the firstaffiliated hospital of dalian medical university written informed consent forpublication was obtained from all patientsconsent for publicationwritten informed consent was obtained from the patients and legalguardian for the publication of these patientscompeting intereststhe authors declare that they have no conflicts of interestauthor details1department of gastrointestinal surgery the first affiliated hospital of dalianmedical university dalian liaoning province china 2department ofgastrointestinal surgery the third affiliated hospital of guangxi medicaluniversity nanning guangxi province china 3department ofgastrointestinal surgery changzhi peoples hospital the affiliated hospital ofchangzhi medical college changzhi shanxi province china 4department ofcolorectal surgery the first affiliated hospital of nanjing medical universitynanjing china 5department of gastrointestinal surgery the first peopleshospital of dali city dali yunnan province china 6department ofgastrointestinal surgery graduate school of medicine university of tokyotokyo japan 7department of general surgery yizhen peoples hospitalclinical medical college yangzhou university yangzhou jiangsu provincechinareceived february accepted august heald rj husband em ryall rd the mesorectum in rectal cancer surgerythe clue to pelvic recurrence br j surg macfarlane jk ryall rd heald rj mesorectal excision for rectal cancerlancet lee ky factors influencing oncologic outcomes after tumorspecificmesorectal excision for rectal cancer j korean soc coloproctol williams ns dixon mf johnston d reapppraisal of the centimetre rule ofdistal excision for carcinoma of the rectum a study of distal intramuralspread and of patients survival br j durg pollett wg nicholls rj the relationship between the extent of distalclearance and survival and local recurrence rates after curative anteriorresection for carcinoma of the rectum ann surg wolmark n fisher b wieand hs the prognostic value of the modificationsof the dukes c class of colorectal cancer an analysis of the nsabp clinicaltrials ann surg monson jrt weiser mr buie wd chang gj rafferty jf prepared by thestandards practice task force of the american society of colon and rectalsurgeons practice parameters for the management of rectal cancerrevised dis colon rectum law wl chu kw anterior resection for rectal cancer with mesorectalexcision a prospective evaluation of patients ann surg kim sh bae kb kim jm oncologic outcomes and risk factors forrecurrence after tumorspecific mesorectal excision of rectal cancer cases j korean soc coloproctol kim nk min bs kim js hur h lee ky sohn sk oncologic outcomesand safety after tumorspecific mesorectal excision for resectable rectalcancer a single institutions experience with patients with rectalcancer j korean soc coloproctol zakir k mohamed wai lun law outcome of tumorspecific mesorectalexcision for rectal cancer the impact of laparoscopic resection world jsurg kim nk baik sh seong js oncologic outcomes after neoadjuvantchemoradiation followed by curative resection with tumorspecificmesorectal excision for fixed locally advanced rectal cancer impact ofpostirradiated pathologic down staging on local recurrence and survivalann surg poon jt law wl laparoscopic resection for rectal cancer a review annsurg oncol yoo be kye bh kim hj early removal of the urinary catheter aftertotal or tumorspecific mesorectal excision for rectal cancer is safe discolon rectum seiji o takashi t kazuki s a new laparoscopic surgical procedure toachieve sufficient mesorectal excision in upper rectal cancer int j surgoncol httpsdoi1011552011708439publishers notespringer nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliationsreferencesenker w e thaler h t cranor m l polyak t total mesorectal excision inthe operative treatment of carcinoma of the rectum j am coll surg lopezkostner i lavery c hool gr rybicki la fazio vw total mesorectalexcision is not necessary for cancer of the upper rectum surgery zaheer s pemberton jh farouk r dozois rr wolff bg ilstrup d surgicaltreatment of adenocarcinoma of the rectum ann surg sudeck p ueber die gefässversung des mastdarmes in hinsicht auf dieoperative gangrän muenchen med wschr van tonder jj boon jm becker jhr anatomical considerations onsudecks critical point and its relevance to colorectal surgery clin anatcirocchi r randolph j cheruiyot i systematic review and metaanalysis of the anatomical variants of the left colic artery color dis httpsdoi101111codi14891 0c" | 0 |
"colorectal cancer crc remains the third most prevalent cancer type and leading cause of cancerrelated deaths with million cases and deaths worldwide during the occurrence and progression of crc result from a wide array of cellular transformation processes which include genetic and epigenetic mutations that drive uncontrolled cell proliferation and escape from apoptosis2 chemotherapy and surgery remain the major therapeutic treatment for crc patients5 fluoropyrimidinebased chemotherapy eg 5fluorouracil has been used as the firstline systemic chemotherapy of treating advanced crc for over a half century6 however most patients receiving chemotherapy finally develop drug resistance which is considered to be the major reason for crc therapy failure7 furthermore even though chemotherapy has significant antitumor activity the side effects can affect the quality of a patient's life which makes the new therapeutic approaches urgentdrug design development and therapy sun this work is published and licensed by dove medical press limited the full terms of this license are available at wwwdovepresscomtermsphp and incorporate the creative commons attribution non commercial unported v30 license httpcreativecommonslicensesbync30 by accessing the work you hereby accept the terms noncommercial uses of the work are permitted without any further permission from dove medical press limited provided the work is properly attributed for permission for commercial use of this work please see paragraphs and of our terms wwwdovepresscomtermsphp 0csun dovepresstraditional chinese medicines such as dendrobium have been shown to exert anticancer activity in many kinds of cancers89 erianin 2methoxy5[2345trimethoxy phenylethyl]phenol figure 1a a natural compound derived from dendrobium candidum shows various pharmacological activities and therapeutic potential to inhibit multiple cancers in vivo and in vitro10 li demonstrated that erianin inhibited the proliferation of acute promyelocytic leukemia hl60 cells by regulating the expression of bcl2 and bax10 in addition erianin caused moderate growth delay in xenografted human hepatoma bel7402 and melanoma a37511 furthermore erianin induced cell cycle g2mphase arrest and apoptosis via the jnk signalling pathway in osteosarcoma and bladder cancer1213 erianin can also inhibit cell invasion metastasis and angiogenesis in lung cancer and breast cancer by the figure erianin inhibited crc cells growth a chemical structure of erianin b and c sw480 and hct116 cells were treated with indicated concentration b and time c of erianin cell viability was assessed by cck8 assay p Ë p Ë d and e ncm460 cells were treated with indicated concentration d and time e of erianin cell viability was assessed by cck8 assay f sw480 and hct116 cells were performed colony formation assay after being treated with indicated concentration of erianinsubmit your manuscript wwwdovepresscom dovepress drug design development and therapy 0cdovepress sun regulation of ido mpp and timp expressions1415 interestingly besides the function on cell growth apoptosis and migration erianin was found to strongly affect the serum levels of cytokines and immune response in liver cancer16 more importantly in addition to the anticancer effects previous a study also suggested that erianin had no major anrelated toxicities12however to the best of our knowledge neither the mechanism nor the effect of erianin on colorectal cancer has been reported hence in this study we evaluate the antitumor potential and molecular mechanisms of erianin in human colorectal cancer sw480 and hct116 cells and provide a theoretical basis of erianin application for colorectal cancer therapymaterials and methodsmaterialsantibodies against cleaved parp cat bak cat bax cat bcl2 cat bclxl cat catenin cat cyclin d1 cat cmyc cat hdac2 cat and gapdh cat were purchased from cell signaling technologies danvers ma usa antibody against αtubulin cat t6199 was purchased from sigma aldrich co st louis mo usaerianin was purchased from shanghai yuanye bio technology co ltd china and dissolved in dmso wntcatenin signaling inhibitor wnt974 was purchased from medchemexpress monmouth junction nj usa and dissolved in dmsocell culturethe human colorectal cancer cell lines sw480 and hct116 were purchased from american type culture collection atcc manassas va usa cells were maintained in rpmi1640 medium supplemented with fbs thermo fisher scientific waltham ma usa uml penicillin and µgml streptomycin thermo fisher scientific and cells were cultured at °c with co2cell viability and colony formation assaycell viability was assessed with the cell counting kit cck8 dojindo japan according to the manufactorers instructionsfor the colony formation assay crc cells cells well were seeded in a sixwell plate and maintained in medium for days subsequently the colonies were fixed with paraformaldehyde and stained with crystal violet and the number of clones was counted using an inverted microscopekit quantitative realtime pcr qrtpcrtotal rna from crc cells was isolated using rna isolation kit omega norcross ga usa according to the manufacturers protocol total rna µg was used as the template for cdna synthesis by using iscripttm reverse transcription super mix biorad laboratories inc hercules ca usa before the samples were analyzed using sybr green master mix on a realtime pcr system biorad laboratories inc the primer sequences used were as follows cmyc forward 5ʹ aaacacaaacttgaaca gctac3ʹ reverse 5ʹ atttgaggcagtttacatt atgg3ʹ cyclin d1 forward 5ʹaggcggatgagaac aagcaga3ʹ reverse 5ʹcaggcttgactccagaag gg3ʹ cd47 forward 5ʹggcaatgacgaaggaggt ta3ʹ reverse 5ʹatccggtggtatggatgaga3ʹ and gapdh forward 5ʹcacccactcctccacctttg3ʹ and reverse 5ʹccaccaccctgttgctgtag3ʹ the 2δδcq method was used to calculate the relative expression levelswestern blottingfor western blotting μg cellular protein extracts were separated in sdspage gel and were then transferred to nitrocellulose membranes emd millipore burlington ma usa the membrane was blocked with nonfat milk and incubated with primary antibodies overnight at ° then the membranes were incubated with secondary antibody and the proteins were visualized using super signal west pico chemiluminescent substrate thermo fisher scientifictransit transfectionplasmid pegfpn1betacatenin was purchased from addgene watertown ma usa lipofectamine thermo fisher scientific carlsbad ca usa was used for transit transfection according to the instructionscatenin sirna was purchased from sigmaaldrich co lipofectamine rnaimax thermo fisher scientific was used for transfection according to the instructiondrug design development and therapy submit your manuscript wwwdovepresscom dovepress 0csun dovepresscell cycle analysisafter treated with vehicle or indicated drugs crc cells were harvested by trypsinization fixed with ethanol and retained at °c overnight after cells were centrifuged and washed with pbs they were resuspended in propidium iodide pi solution containing rnase μgml in the dark at room temperature for min and then studied in a flow cytometercaspase37 activity assayapoone¢ homogeneous caspase37 assay promega corporation madison wi usa was used to measure caspase37 activity briefly apoone® homogeneous caspase37 reagent μlwell was added to a 96well plate and the plate was then placed on a shaker for five minutes rpm before incubating for h at room temperature the reading of each well was measured by spectrofluorometerapoptosis assay by annexin vannexin vfitc staining was used to detect the extent of apoptosis induced by erianin briefly crc cells were treated with erianin for h and were then collected and resuspended in μl annexin vbinding buffer and μl pi for minat room temperature in the dark then the cells were finally analyzed by the flow cytometry bd facs calibur with an emission filter of nm for pi red and nm for fitc greenapoptosis assay by dapithe effect of erianin on apoptosis induction was evaluated by dapi staining assay crc cells à were seeded in a 96well plate after treatment the cells were washed three times with pbs and paraformaldehyde was added to each well for fixation after permeabilization with triton x100 solution dapi solution was added the cells with condensed and fragmented chromatin were analyzed by echo fluorescence microscopycellular thermal shift assayfor cellular thermal shift assay crc cells were pretreated with μm mg132 for one hour and then incubated with erianin for four hours after washing with icecold pbs cells were aliquot into pcr tubes μl each and incubated at different temperatures for four minutes after being frozen and thawed twice using liquid nitrogen cells were centrifuged and proteins were analyzed by western blottingtopfop luciferase reporter assaythe transcriptional activity of catenin was assessed using the topfop dualluciferase reporter system dual glo¢ luciferase assay system promega the renilla luciferase plasmid prltk promega which controls for transfection efficiency was cotransfected with catenin responsive firefly luciferase reporter plasmid topflash emd millipore or the negative control fopflash emd millipore using the lipofectamine thermo fisher scientific cells were harvested after h in culture and the luciferase activity was determined by the luciferase assay system promega using a microplate luminometer berthold bad wildbad germanyflow cytometry analysiserianin treated crc cells were washed and resuspended in μl facs buffer and stained with fitcconjugated anticd47 bd biosciences san jose ca usa antibodies all samples were incubated for minutes at °c and then washed twice with facs buffer flow cytometry analyses were performed on bd facs canto iiin vitro phagocytosis assayfor phagocytosis assay thp1 derived macrophages were seeded in a sixwell tissue culture plate erianintreated crc cells were washed and labeled with μm of carboxyfluorescein succinimidyl ester cfse thermo fisher scientific after incubating macrophages in serum free medium for two hours cfselabeled crc cells were added to the macrophages for another two hours at °c macrophages were then washed and imaged with an inverted microscope the phagocytosis efficiency was calculated as the number of macrophages containing cfse labeled crc cells per macrophageschromatin immunoprecipitation chip assaychip assays were performed using the simplechip® enzymatic chromatin ip kit cell signaling technologies according to manufacturer's instructions using the antibodies against h3k27ac immunoprecipitated dna was analyzed by qrtpcr using the following primers cd47 promoter fragment f ²aggatgaatgatgtggcctgt3² and r ² caaacaggcattagcagcgt3² fragment f submit your manuscript wwwdovepresscom dovepress drug design development and therapy 0cdovepress sun ²ggggatgtgttggatacgct3² and r ² ctctg cgttcggctcgtcta3² fragment f ²agggaag agcagagcgagta3² and r ² ttgctttcactcc caccctc3² fragment f ²agagagaggacag tggggc3² and r ² ccagtcgcaggctccaga3² fragment f ² gccgcgtcaacagca3² and r ² aaaggcatcattcttggaaattgt3²with ¨° sw480 cells per mouse suspended in vivo xenograftnodscid shanghai slac laboratory animal co ltd china mice were injected subcutaneously in right flank in µl pbs and mixed with an equal volume of matrigel animals with tumors volume mm3 were divided into two groups n6 and treated with either placebo or mgkg erianin for continuously three weeks by intraperitoneal injection tumor size were measured at the indicated times all the animalrelated procedures were approved by the animal care and use committee of the changchun university of chinese medicine all animal experiments were conducted according to the nih guide for the care and use of laboratory animalsstatistical analysisdata were presented as mean ±sd from three independent experiments p value was determined using paired students ttest and a p value Ë was deemed to indicate statistical significanceresultserianin inhibited crc cell growthfigure 1a illustrates the chemical structure of erianin to investigate the inhibitory effect of erianin on crc cell viability we treated two crc cell lines sw480 and hct116 with different concentrations of erianin and nm for and h as shown in figure 1b and c erianin treatment significantly inhibited the viability of crc cells in a dose and timedependent manner importantly erianin did not show cytotoxic effects on normal human colon mucosal epithelial cell line ncm460 figure 1d and e in addition consistent with the shortterm growth assay our colony forming unit assay also showed that erianin inhibited the colony formation ability of sw480 and hct116 cells figure 1ferianin elevated cell cycle arrest and apoptosisto verify the causal relation of cell viability inhibition the cell cycle distribution was analyzed erianin increased cell number at g2m phase but decreased cell number at s and g0g1 phases after 24h incubation with indicated concentration in sw480 and hct116 cells figure 2a and b to explore the effect of erianin on apoptosis we examined the activity of caspase the protein level of cleaved parp bax bak bcl and bclxl as shown in figure 2ce the activity of caspase protein level of cleaved parp bak and bax pro apoptosis increased as the concentration of erianin increased in contrast the protein level of bcl2 and bclxl anti apoptotic decreased after erianin treatment figure 2e annexin v flow cytometry and dapi staining further confirmed that erianin could induce cell apoptosis figure 2f and gerianin inhibited catenin translocationincreasing evidence revealed that the wntcatenin pathway plays critical role in colorectal cancer tumorigenesis we hypothesized that erianin might have effect in modulating the wntcatenin pathway first we investigated the effect of erianin on catenin phosphorylation as shown in figure 3a no obvious change was observed on catenin phosphorylation level we then evaluated the effect of erianin on catenin translocation as shown in figure 3be catenin expression in cytoplasm was increased whereas expression in the nucleus was decreased with the treatment of erianin in a dose and timedependent manner to further explore the effect of erianin on catenin transcription activity we performed topfop dual luciferase assay we found that topfop relative luciferase activity was significantly decreased after erianin treatment both in sw480 and hct116 cells figure 3f and gerianin bound catenin directlysince erianin inhibited catenin translocation to the nuclear without changing its phosphorylation level we hypothesized that erianin might bind catenin directly to determine whether erianin physically binds catenin we performed a cellular thermal shift assay the results from this experiment indicated that erianin treatment increased the thermal stability of catenin when cells were pretreated with the proteasome inhibitor mg132 for one hour figure 4a and b in contrast erianin treatment had no effect on the thermal stability of gapdh a loading control figure 4a and b these results strongly suggested a specific physical interaction between erianin and catenindrug design development and therapy submit your manuscript wwwdovepresscom dovepress 0csun dovepressfigure erianin elevated cell cycle arrest and apoptosis a and b sw480 and hct116 cells were treated with erianin for h and then analyzed by pi staining to determine cell cycle phase distribution c sw480 and hct116 cells were treated with erianin for h the relative caspase37 activity was measured using apoone¢ homogenous caspase37 assay p Ë p Ë d and e the protein level of cleaved parp1 bak bax bcl2 and bclxl were analyzed by western blotting after treated with indicated concentration of erianin f and g sw480 and hct116 cells were treated with erianin for h apoptosis was assessed using annexinv flow cytometry analysis f or dapi staining gsubmit your manuscript wwwdovepresscom dovepress drug design development and therapy 0cdovepress sun figure erianin inhibited catenin translocation a the protein level of indicated proteins was analyzed by western blotting after being treated with indicated concentration of erianin for h be the protein level of catenin in cytosol and nucleus was analyzed by western blotting after treated with erianin for indicated concentration b and c and time d and e f and g sw480 and hct116 cells were treated with erianin for indicated concentration f and time g the transcriptional activity of catenin was assessed by topfop luciferase reporter assay p Ë p Ëerianin inhibited the expression of cmyc and cyclin d1as cmyc and cyclin d1 are the direct targets of the wnt catenin pathway we then evaluated the mrna and protein level of cmyc and cyclin d1 unsurprisingly both mrna and protein level of these two proteins were significantly decreased after erianin figure 5ac interestingly no synergetic effect was observed when combining erianin with wntcatenin signaling inhibitor wnt974 which indicated that erianin regulates cmyc treatment drug design development and therapy submit your manuscript wwwdovepresscom dovepress 0csun dovepressfigure erianin interacted with catenin a and b sw480 a and hct116 b cells were treated with μm mg132 for one hour followed by four hours incubation with nm erianin before performing thermal shift assay the lower panel shows the charts of percentages of nondenatured protein fractionand cyclin d1 via wntcatenin signaling figure 5d furthermore the inhibitory effect of erianin on cmyc and cyclin d1 expression and cell viability could be reversed by catenin overexpression figure 5e and f which indicated that erianin regulates crc cell growth via catenincd47 mediated phagocytosis we used an in vitro assay by coculturing thp1 derived macrophage with crc cell lines sw480 or hct116 as shown in figure 6g and h treatment of erianin markedly promote colorectal cancer cell phagocytosis by macrophages these results suggest that erianin treatment can attenuate cd47 expression and ultimately promote phagocytosis of crc cellserianin decreased cd47 expression and increased phagocytosisthe immune checkpoint protein cd47 is included in the list of wntcatenin target molecules with a role in immunity escape17 since catenin depletion by sirna inhibited the expression of cd47 figure 6a we then sought to know whether erianin regulates the expression of cd47 first we explored the effects of erianin on cd47 mrna protein and cell surface level in both sw480 and hct116 cells erianin treatment significantly decreased the mrna protein and cell surface level of cd47 figure 6bd promoter analysis by ucsc genome browser demonstrates that h3k27 acetyl marks are enriched in cd47 promoter regions figure 6e next our chip assay demonstrated that h3k27ac enrichment specifically near promoter region f3f5 was significantly decreased with erianin treatment figure 6f to investigate the effect of erianin on erianin inhibited tumor growth in vivoto investigate the possibility of erianin as a potential therapy in crc we tested the function of erianin on tumor growth in a mouse model the mouse model was established by s c injection of sw480 cells into nodscid mice after three weeks treatment we analyzed the tumor size and weight as shown in figure 7ac the tumor size and weight from the erianin treatment group were significantly lower than that from the control group in addition after days of bearing tumor the weight of the mice had no significant change figure 7dto examine the impact of therapy on catenin and its downstream signaling localization of catenin protein level of cd47 cmyc bcl2 and bax three representative tumors from each group were analyzed using western blotting as shown in figure 7e and f catenin expression in cytoplasm was increased whereas expression in nucleus was decreased with the treatment of erianin the submit your manuscript wwwdovepresscom dovepress drug design development and therapy 0cdovepress sun figure erianin inhibited the expression of cmyc and cyclin d1 ac after treated with indicated concentration and time of erianin mrna and protein level of cmyc and cyclin d1 were analyzed by qrtpcr and western blotting p Ë d sw480 cells were treated with erianin orand wnt974 for h protein levels of cmyc and cyclin d1 were analyzed by western blotting e and f sw480 cells were treated with erianin for h followed by overexpression with catenin plasmid for h protein levels of cmyc and cyclin d1 were analyzed by western blotting e and cell viability was assessed by cck8 assay f p Ëprotein level of cd47 cmyc and bcl2 decreased while bax increased after erianin treatment these data indicated that erianin inhibited tumor growth via catenin in vivodiscussioncrc is one of the most malignant and commonly diagnosed solid tumors all around the world18 although crc incidence rates have declined somewhat chemotherapies are inefficient in most crc patients due to resistance2122 thus the development of acquired therapeutic drugs researching novel and safe treatment strategies is essential for improving the prognosis of crc patients in recent years natural medicinal plants are receiving more and more attention and considered to be important sources of treatment23 novel dendrobium is considered as one of the most important herbs in the orchidaceae family and shows diverse pharmacological functions including anticancer neuroprotective antidiabetic and immunemodulating activities24 erianin derived from dendrobium is one of the most for cancer drug design development and therapy submit your manuscript wwwdovepresscom dovepress 0csun dovepressfigure erianin decreased cd47 expression and increased phagocytosis a sw480 cells were transfected with nontarget nt or catenin sirna for h protein levels of indicated protein weres measured by western blotting bd sw480 and hct116 cells were treated with erianin for indicated dose the mrna level b protein level c and cell surface cd47 d were detected by qrtpcr and flow e the ucsc genome browser revealed the enrichment of h3k27ac on cd47 promoter f the enrichment of h3k27ac on cd47 promoter f1f6 was detected by chip assay g and h sw480 and hct116 were treated with indicated concentration of erianin for h representative images showed the effect of erianin on phagocytosis g and bar graphs showed quantitative analysis of phagocytosis h p Ë p Ësubmit your manuscript wwwdovepresscom dovepress drug design development and therapy 0cdovepress sun figure erianin inhibited tumor growth in vivo a typical photos of tumors from the control and erianin treated groups b and c erianin decreased tumor volume and weight p Ë d mice body weight of control and erianin treated groups was measure at indicated time e the protein level of catenin in cytosol and nucleus in three representative tumors from mouse to mouse of each group were analyzed by western blotting f the protein level of indicated protein in three representative tumors from mouse to mouse of each group were analyzed by western blottingdrug design development and therapy submit your manuscript wwwdovepresscom dovepress 0csun dovepressnoteworthy constituents that have been used as an antipyretic and an analgesic in traditional chinese medicine25 recently several studies have proved that erianin shows significant antitumour activity in a variety of human cancer cells10 consistent with literature in this study we found that erianin had a significant antiproliferative effect against crc cells the inhibitory effect caused by erianin may result from induction of apoptosis and arrest of cell cycle at g2m since the effect of erianin on crc cells has never been studied before we further confirm its antitumor activity in a mouse model which indicated that erianin significantly inhibited tumor growth in vivoseveral signaling pathways including egfrmapk pi3kakt or wntcatenin have been linked to crc genesis and progression26 as the aberrant activation is present in almost all crc cases wntcatenin signaling is prominent among these pathways27 inactivated mutations in the apc gene leads to stabilization and ensuing nuclear translocation of catenin to facilitate tcflef dependent transcription of wntcatenin signaling target genes such as cmyc and cyclin d1 to drive cell proliferation survival and metastasis28 to understand the mechanisms of action of erianin we assessed the effect of erianin on wntcatenin pathway interestingly we found that erianin treatment had no effect on catenin phosphorylation but inhibited the translocation of catenin in the nucleus which suggested to us that erianin physically interacts with catenin our cellular thermal shift assay confirmed this hypothesis the thermal stability of catenin increased after erianin treatment as catenin downstream targets the expression level of cmyc and cyclin d1 significantly decreased after erianin treatmentcd47 a transmembrane glycoprotein expresses ubiquitously and mediates a selfdonoteatme signal on normal cells however cd47 is often upregulated in tumor cells to evade innate immunity31 anticd47 antibodies which block cd47 sirpα interactions and promote macrophage mediated phagocytosis of tumor cells has shown promise in several solid tumors31 in colorectal cancer cd47 promotes colon cancer cell migration and metastasis34 in addition upregulated immuneescape pathways such as cd47 sirpα are responsible for immune escape and survival in circulating tumor cells of colorectal cancer35 myc an oncogene identified as a wntcatenin target gene was reported to control cd47 transcription therefore mutations in components of the wntcatenin signaling pathway which induced | 0 |
"Accumulating evidence has revealed the critical role of long noncoding RNAs lncRNAs in cellularprocesses during tumor progression As documented in cancerrelated literatures LINC00992 expression isassociated with cancer progression whereas its function in tumors including prostate cancer has not beencharacterized yetMethods Data from GEPIA database suggested LINC00992 expression in prostate cancer tissues The expressionlevels of RNAs were monitored via qRTPCR Western blot evaluated the levels of proteins The proliferationapoptosis and migration of prostate cancer cells were assessed by CCK8 EdU TUNEL Transwell and woundhealing assays Luciferase reporter RNA pull down and RIP assays were applied to detect the interplays amongLINC00992 miR3935 and GOLM1Results Elevated levels of LINC00992 and GOLM1 were detected in prostate cancer tissues and cells LINC00992exerted facilitating functions in prostate cancer cell proliferation and migration Mechanically LINC00992 interactedwith and negatively regulated miR3935 to elevate GOLM1 expression in prostate cancer cells In addition thein vitro suppressive effect of silenced LINC00992 on prostate cancer cell proliferation and migration was reversed byGOLM1 upregulation Likewise LINC00992 depletion restrained tumor growth in vivo was offset by enhancedGOLM1 expressionConclusions LINC00992 competitively bound with miR3935 to elevate GOLM1 expression and therefore facilitatethe oncogenic phenotypes of prostate cancer cells implying a potential LINC00992targeted therapy for prostatecancerKeywords INC00992 miR3935 GOLM1 Prostate cancer Correspondence engineyangsinacom5Department of Urology the Second Affiliated Hospital of Bengbu MedicalCollege Hongye Road Bengbu Anhui ChinaFull list of author information is available at the end of the The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cChen BMC Cancer Page of BackgroundClinically prostate cancer manifests as a dominatingcause of malerelated death worldwide and is characterized as the most continually occurred tumor amongmen in the United States [] The biggest challenge isthe detectable bone metastases in roughly advancedprostate cancer [] Virtually all prostate cancer patientsduring years androgen deprivation treatment inevitably undergo castrationresistance which contributes tothe poor clinical consequences in prostate cancer []However the mechanism underlaid prostate cancer remains mostly unknownThe widely studied long noncoding RNAs lncRNAsare transcribed from nonproteincoding human genomeand have more than nt in length [] LncRNAs are increasingly functionally identified and experimentally consolidated to be related to tumor neoplasia and progressionin diverse cancers [] Additionally lncRNAs with dysregulation can functionally modulate tumor developmentfrom multiple pathological aspects such as cell proliferation drugresistance and metastasis [] For examplelncRNA A1BGAS1 inhibits cell proliferation and invasionin hepatocellular carcinoma via targeting miR216a5p []LncRNA LOC730100 sponges miR760 from FOXA1 toaccelerate cell proliferation and invasion in glioma []LncRNA SNHG16 functions as an oncogene in hepatocellular carcinoma [] Long intergenic nonprotein codingRNA LINC00992 is a novel lncRNA that has beenpreviously revealed to be elevated in tumors and substantiated as a master regulator for chemoresistance [] Besides LINC00992 has been uncovered as an elevatedlncRNA in prostate cancer [] which is consistent withthe detection from GEPIA database Despite that no previous study has given a comprehensive explanation aboutthe precise function or detailed mechanism of LINC00992in prostate cancerIn past decades the fact that lncRNAs function in tumors depending on their secondary or tertiary structureshas been reported in many cancerlinked studies For instance in the nucleus lncRNAs are entitled to work asmolecular scaffolds or alternative splicing assistants [] On the contrary lncRNAs dispersing in cytoplasm influence downstream mRNA translation or degradationthrough serving as miRNA sponges [ ] For exampleTNFαinduced lncRNA LOC105374902 promotes themalignant behaviors of cervical cancer cells by acting as asponge of miR12853p [] LncRNA TTNAS1 promotes papillary thyroid cancer tumorigenesis by regulatingmiR1533pZNRF2 axis[] Nevertheless whetherLINC00992 could exert its functions in prostate cancer viaits sponging role of certain miRNA remains unknownWe conducted this research aiming to explore thefunction or probable mechanism of LINC00992 in prostate tumor which might enrich the understanding interms of prostate tumor pathology and contribute to awider applied scopeMethodsTissue samplesThe prostate cancer tissue samples and matched peritumor tissue samples were collected from patientsdiagnosed with prostate cancer under the approval ofthe Ethics Committee of the First Affiliated Hospital ofKunming Medical University Each participant did notreceive radiotherapy and chemotherapy prior to tissuecollection and signed the written informed consentsbefore this study All samples were snapfrozen in liquidnitrogen and then stored at °C until required forfurther analysisCell cultureThe prostate epithelial cell line RWPE1 CRL11609and prostate cancer cellsincluding PC3 CRL1435LNCaP CRL1740 C4 CRL3314 and DU145HTB81 were all purchased from American TypeCulture Collection ATCC Manassas VA USAinOctober All cells were cultured as recommendedin Dulbeccos modified Eagles medium containing FBS GIBCO MA USA under the condition of a cellincubator with CO2 at °C Before using in thisstudy all cell lines were authenticated by STR profilingand tested for mycoplasma contamination in June Cell transfectionLINC00992 shRNA or negative control shRNA andpcDNA31LINC00992 pcDNA31GOLM1 or its emptycontrol pcDNA31 plasmid were chemically synthesizedand provided by Gene Pharma Shanghai China MiR mimics miR3935 inhibitor and theirrelatednegative controls NCmimics NCinhibitor were allpurchased for upregulating or downregulating miR3935from Ribobio Guangzhou China In line with the directions of LipofectamineTM RNAiMAX TransfectionReagent Thermo Fisher Scientific transfection of theseplasmids into DU145 PC3 and RWPE1 cells wasconducted and qRTPCR checked the transfection efficiency The sequences were as follows shNC ²CCGGTAGTAATTGACAACCATTATACTCGAGTATAATGGTTGTCAATTACTATTTTTG3²shLINC009921²CCGGATTATCCAAGAGTATTAACATCTCGAGATGTTAATACTCTTGGATAATTTTTTG3² shLINC0 ²CCGGTGTTAGATGATCATTGAGGTGCTCGAGCACCTCAATGATCATCTAACATTTTTG3² s²CCGGTTACCTAATCAGTAGAThLINC009923GCAGCTCGAGCTGCATCTACTGATTAGGTAATTTTTG3² NCmimics ²UCAGGUAGGGCUCAAACCAACC3² miR3935 mimics ²UGUAGAUACGAGCACCAGCCAC3² NCinhibitor²CUGGCUUUAG 0cChen BMC Cancer Page of GGUGCCACUUAG3² miR3935 inhibitor ²GUGGCUGGUGCUCGUAUCUACA3²Quantitative realtime PCR qRTPCROn the basis of the instructions of Trizol reagent Invitrogen USA RNA extraction was executed in prostatecancer cells After the examination of RNA purity withspectrophotometry cDNA was obtained from aboveRNA with reverse transcription kit ThermoFisher Scientific shanghai China qRTPCR analysiswas devised with the aid of a BioRad CFX96 system andSYBR green was applied for investigating the RNA levelsThe internal reference for LINC00992 and mRNAs wasGAPDH whereas that for miRNAs expression was U6Relative expression was assessed based on the method ofÎÎCtab97779Western blotProtein content in cells was determined by western blotanalysis RIPA lysis buffer Beyotime Shanghai Chinawas adopted for cell lysing followed by the evaluation ofthe protein concentration with BCA Protein Assay KitP0011 Beyotime Tech SDSPAGE gel was applied for separating proteins μg protein per sampleand then proteins were transferred onto μm PVDFmembranes BioRad Hercules CA USA Antibodiesincluding antiGOLM1 ab109628 AbcamCambridge UK antiPCNA ab92552 AbcamantiCDK2 ab32147 Abcam antiCyclin D1ab40754 Abcam antiBax ab32503 Abcam antiBcl2 ab32124 Abcam antiMMP2antiMMP9ab38898 Abcam antipSrc ab40660 Abcam antiSrc ab47405 Abcam antipFAKab81298 Abcam antiFAK ab131435 Abcam antiGAPDH ab8245 Abcam andantiTubulin ab7291 Abcam were applied toprobe the membranes overnight at °C After that themembranes were further incubated for h with HRPconjugated secondary antibody Santa Cruz Co LtdSant Cruz CA USA atroom temperature ECLSubstrates Millipore Billerica MA USA was utilizedfor the visualization of signals followed by exposure toXfilm Kodak Rochester NY USA The quantificationof immunoblots was conducted with the aid of imageJsoftware National Institute of Health Bethesda MDUSA with GAPDH or Tubulin as the normalizer asneeded AbcamLuciferase reporter assayFragments of fulllength LINC00992 with wildtype ormutant binding sites for miR3935 and sequences ofGOLM1 UTR containing wildtype or mutated miR binding sites were inserted into the pmirGLOvectors Promega Madison WI USA for the construction of reporters LINC00992WT LINC00992MUTGOLM1WT GOLM1MUT Then the four reportersand miR3935 mimics or miR3935 inhibitor GenePharma were cotransfected into DU145 and PC3 cellsapplying lipofectamine2000 Invitrogen as neededFortyeight hours later DualLuciferase Reporter AssaySystem Promega was employed for the examination ofthe luciferase activity GloMax® Discover Multimode Microplate Reader Promega assessed the ratio of FireflyRenilla luciferase activity and the activity of Renilla wasthe normalized controlRNA immunoprecipitation RIP assayAccording to the direction for usage of Magna RIP¢RNA Binding Protein Immunoprecipitation Kit Millipore RIP assay was strictly performed RIP lysisbuffer was firstly applied to treat the transfected DU145and PC3 cells Afterwards the obtained cell lysates wereprocessed with magnetic beads integrated with humanantiAgo2 antibodies ab32381 Abcam MA USA orantiIgG AP162KC Millipore Following the recoveryof antibody by the protein AG beads qRTPCR detected the levels of LINC00992 miR3935 and GOLM1mRNA in the precipitates IgG worked as the negativecontrol for the normalization of RNAIPsRNA isolation of nuclear and cytoplasmic fractionsThe dispersion of LINC00992 in the prostate cancercells was assayed as described previously [] The isolation of cytosolic and nuclear sections was executed followingAM1921Invitrogen RNA levels of U1 nuclear control GAPDHcytoplasmic control and LINC00992 were all estimatedby qRTPCR analysisPARIS¢ KittheprotocolofFluorescence in situ hybridization FISH assayIn line with the recommendation of Ribo¢ FISH KitC10910 Ribobio Guangzhou China FISH analysiswas implemented for testing the presence of LINC00992in prostate tumor cells Ribobio Company synthesizedthe LINC00992 probes labeled by Cy3 fluorescent dyeFollowing the fixation by paraformaldehyde and Triton X100 permeabilization DU145 and PC3 cellswere subsequently blocked in prehybridization bufferblocking solution Then incubation of cells with probehybridization buffer was later performed Next day afterrinsing and Hoechst staining the fluorescence was measured under a confocal laser scanning microscope ZeissGermanyCell counting kit8 CCK8 assayFor the viability assessment in DU145 PC3 and RWPE cells CCK8 assay was implemented as described 0cChen BMC Cancer Page of previously [] Cell viability was monitored at and h In short after being seeded onto 96well platesand cultured for indicated times cells were processedwith μl of CCK8 solution Then a microplate readerexamined the absorbance values at the wavelength of nm²ethynyl2²deoxyuridine EdU incorporation assayCell proliferation was examined through EdU assay asdescribed previously [] by using ClickiT EdU AlexaFluor Imaging Kit C10086 Invitrogen After hoftransfection EdU staining was carried out asinstructed The observation and calculation of EdUpositive cells was proceeded under the fluorescencemicroscopyTransferasemediated dUTP nick end labeling TUNELstainingTUNEL assay was carried out as described previously[] for probing DU145 and PC3 cell apoptosis with theassistance of an In Situ Cell Death Detection Kit Roche Mannheim Germany TUNELpositivecells were recorded under a light microscope à from visual fields which were chosen at randomTranswell migration assayThe application of transwell chambers with pore size μm Corning Costar Cambridge MA USA was aimedfor detecting cell migration in strict line with the instructions Cells that were previously suspended inserumfree RPMI1640 media were seeded into theupper chamber RPMI1640 medium containing FBS was supplemented in lower chamber as a chemoattractant Cells in the filters following h incubationwere immobilized in methanol and went through crystal violet staining The images of cells migratedthrough the filters were obtained and counted under themicroscopeWound healing assayThe DU145 PC3 and RWPE1 cells à cellswellwere prepared on glass culture dishes and cultivated at °C for a whole night to allow cells adhered to theplates followed by the straight scratch made with a plastic pipette tip after cell samples reached confluenceLater cells were rinsed in PBS to clear the detachedcells Finally the wounds at and h were imaged viaa light microscopy Olympus Tokyo JapanIn vivo experimentSixteen sixweekold male BALBC athymic nude micewere commercially available from the National Laboratory Animal Center Beijing China and maintained inSPFgrade animal lab All animalrelated protocols wereapproved by the Animal Research Ethics Committee ofthe First Affiliated Hospital of Kunming Medical University The in vivo experiment was undertaken via subcutaneous injection of à DU145 cells into the nudemice while the DU145 cells injected into indicated fourshgroups of mice were transfected with shNCLINC009921shLINC009921 pcDNA31 orshLINC009921 pcDNA31GOLM1 Tumor volume wasmonitored every days 28day after injection nudemice were sacrificed via cervical dislocation and thentumor samples were carefully dissected for weight assessment and hematoxylin and eosin HE stainingImmunohistochemistry IHCThe tumor samples collected from in vivo experimentswere treated with PFA dehydrated and embedded inparaffin Afterwardsthe paraffinembedded sections μm were prepared for IHC assay as described previously [] by use of the antiKi67 and antiPCNA antibodies AbcamStatistical analysisSPSS statistical software SPSS Armonk NY USAwas employed in the processing of data from threebiological replicates and data were expressed as mean ±SD Significance of difference within two groups wasdetermined using Students ttest while that among noless than two groups was tested via oneway or twowayANOVA P was considered as the threshold ofsignificanceResultsLINC00992 is overexpressed in prostate cancer andregulates cell proliferation apoptosis and migrationLINC00992 expression pattern in prostate cancer wasacquired from online GEPIA database As a resultLINC00992 was considerably upregulated in PRADprostate adenocarcinomatissues relative to normalones Fig 1a After detecting LINC00992 expression intissue samples obtained from patients with prostate cancer we observed that LINC00992 expression was higherin prostate cancer tissues than that in peritumor tissuesFigure S1A Moreover clinical data showed that higherexpression of LINC00992 in prostate cancer patientswas associated with lower survival rate Figure S1BFurthermore LINC00992 expression in the prostate cancer cells and RWPE1 cells was evaluated by qRTPCRConsequently higher level of LINC00992 was exhibitedin prostate cancer cells than that in RWPE1 cells Fig1b which was completely consistent with the result presented in previous discovery [] Particularly DU145and PC3 cells expressed the highest level of LINC00992and was thereby chosen for the later assays For silencingLINC00992 special shRNAs targeting LINC00992 was 0cChen BMC Cancer Page of Fig LINC00992 was overexpressed in prostate cancer and regulates cell proliferation apoptosis and migration a GEPIA database demonstratedthe overexpression of LINC00992 in tumor tissues in contrast to adjacent normal ones b LINC00992 expression was detected by qRTPCR in fourprostate cancer cell lines and control RWPE1 cells c LINC00992 expression was monitored by qRTPCR in DU145 and PC3 cells after transfectionwith shRNAs targeting LINC00992 shNC was used as the negative control d The viability of DU145 and PC3 cells was estimated through CCK8assay following LINC00992 depletion e The proliferation of DU145 and PC3 cells was investigated after LINC00992 depletion via EdU assay Scalebar μm f The apoptosis of DU145 and PC3 cells transfected with shLINC0099212 or shNC was estimated via TUNEL assay Scale bar μm g Western blot analysis was applied to examine the expression of apoptosisrelated proteins hi The migration of DU145 and PC3 cellswas analyzed via Transwell migration assay scale bar μm and wound healing assay scale bar μm after inhibiting LINC00992expression The fulllength images for blots in Fig 1g were presented in Supplementary figure P p transfected into DU145 and PC3 cells and the efficiencywas corroborated in qRTPCR Fig 1c And then thedata from CCK8 assay revealed that LINC00992 depletion suppressed the proliferation of DU145 and PC3cells Fig 1d As expected a declined proportion ofEdU positive cells was observed after knocking downLINC00992 Fig 1e suggesting the suppressive effect ofLINC00992 deficiency on prostate cancer cell proliferation Additionally the expression levels of proliferationrelated proteins PCNA CDK2 and Cyclin D1 were allreduced by silenced LINC00992 Figure S1C On thecontrary TUNEL assay uncovered that LINC00992knockdown facilitated cell apoptosis Fig 1f Meanwhile western blot analysis revealed that LINC00992knockdown promoted the apoptosis of DU145 and PC3cells as Bax protein level was increased whereas Bcl2protein level was decreased after LINC00992 was silenced in these two cells Figs 1g Figure S1D FurtherTranswell and wound healing assays indicated that themigration of DU145 and PC3 cells was retarded byLINC00992 depletion Fig 1hi Likewise the expression of migrationrelated molecular markers MMP2MMP9 pSrc and pFAK was decreased by LINC00992inhibition Figure S1E To further verify the biological 0cChen BMC Cancer Page of role of LINC00992 in prostate cancer we carried outgainoffunction assays in RWPE1 cells After overexpressing LINC00992 in RWPE1 cells Figure S2A cellproliferation was promoted Figure S2BC As expectedthe expression of PCNA CDK2 and Cyclin D1 wasdecreased by upregulation of LINC00992 Figure S2DSimilarly LINC00992cellIn addition upregulatingmigration Figure S2EFLINC00992 resulted in the elevated protein levels ofMMP2 MMP9 pSrc and pFAK Figure S2G All thesedata elucidated that LINC00992 could facilitate cell proliferation and migration whereas suppress cell apoptosisin prostate cancerupregulationfacilitatedMiR3935 is targeted by LINC00992Given the high correlation of the sublocalization ofLINC00992 with its functional mechanism the predication of LINC00992 presence in cells was performed viaLncLocatorhttpwwwcsbiosjtueducnbioinflncLocator Result predicted that LINC00992 located mainlyin cytoplasm Fig 2a Likewise FISH assay and RNAisolation of nuclear and cytoplasmic fractions furtherverified the abundance of LINC00992 in the cytoplasmof prostate cancer cells Fig 2bc highlighting a posttranscriptional control of LINC00992 in such cellsHence we speculated that LINC00992 might act as aceRNA in prostate cancer regulation According toDIANAlncBase the top three potential miRNAs possessing the binding capacity with LINC00992 were listedFig 2d To targetthe highlymatched miRNA toLINC00992 qRTPCR analysis was conducted to testthe expression changes of these miRNAs following eitherLINC00992 depletion or augmentation The resultsdemonstrated that only miR3935 expression was increased by LINC00992 depletion Fig 2e but reducedby LINC00992 overexpression in the meantime Fig 2fThus miR3935 was chosen for further analysis Afterwards RNA pull down assay was implemented and theresult depicted that LINC00992 was pulled down byBiomiR3935WT Fig 2g which indicated the bindingof LINC00992 and miR3935 Later we observed thesatisfactory efficiency of miR3935 overexpression andmiR3935 inhibition through qRTPCR analysis Fig2h Thereafter RIP assay applying antiAgo2 was executed Results illustrated that LINC00992 and miR3935were highly enriched in antiAgo2 group in comparisonwith control antibody Fig 2i certifying the associationof LINC00992 with miR3935 in the RNAinduced silencing complexes RISCs To further explore the interaction between LINC00992 and miR3935 the bindingsites between LINC00992 and miR3935 were predictedat first and then data from luciferase reporter assayrevealed that miR3935 upregulation decreased theluciferase activity of LINC00992WT reporter whereasmiR3935 inhibition increased the luciferase activity ofLINC00992WT reporter Fig 2j Altogether LINC0 combined with miR3935 to act as a miRNA decoyin prostate cancerLINC00992 regulates the expression of GOLM1 a targetof miR3935Present evidence has suggested that miRNAs can bindwith downstream target genes to inhibit their expressionHerein we searched the miR3935 target genes andeight mRNAs were found out Subsequently we detectedtheir expression in prostate cancer cells and normalcells Interestingly we found that only Golgi membraneprotein GOLM1 was highly expressed in four prostate cancer cell lines relative to normal controls Fig 3aFurther we discovered that GOLM1 expression wasmarkedly upregulated in prostate cancer tissues according to data from GEPIA database Fig 3b SimilarlyGOLM1 expression was much higher in prostate cancertissue samples than in peritumor samples Figure S3AIn addition the mRNA and protein levels of GOLM1were overexpressed in prostate cancer cells in contrastto RWPE1 cells Fig 3c Figure S3B Besides GOLM1has been previously revealed as a prostate cancer facilitator and was metastasisrelated in prostate tumor [] Thus we hypothesized that GOLM1 might act asthe downstream of LINC00992miR3935 signaling inprostate cancer Through TargetScan httpwwwtargetscanvert_72 the binding site between GOLM1and miR3935 was predicted Fig 3d After conductingluciferase reporter assay in DU145 and PC3 cells weobserved that upregulation of miR3935 specifically decreased the luciferase activity of GOLM1WT reporterFig 3e confirming the interaction between miR3935and GOLM1 relied on the putative binding sites Thenwe unveiled that GOLM1 mRNA and protein levels wereboth reduced by LINC00992 inhibition or miR3935upregulation according to qRTPCR and western blotanalyses Fig 3fg Figure S3C Moreover data fromRIP assay unveiled the binding of miR3935 to GOLM1in the RISCs Fig 3h Further we demonstrated thatthe decreased mRNA and protein levels of GOLM1 induced by LINC00992 depletion could be restored afterinhibiting miR3935 expression Fig 3ij Figure S3DAll the results showed that LINC00992 upregulatedGOLM1 expression via directly binding to miR3935LINC00992 promotes prostate cancer cell proliferationand migration via elevating GOLM1 expressionTo test whether LINC00992 affected prostate cancer cellproliferation apoptosis and migration via regulatingmiR3935targeted GOLM1 we executed the rescue experiments with the upregulation of GOLM1 To beginwiththe efficiency of overexpressing GOLM1 was 0cChen BMC Cancer Page of Fig MiR3935 was targeted by LINC00992 a LncLocator predicted LINC00992 subcellular location b FISH analysis of LINC00992 distribution inprostate cancer cells Scale bar μm c RNA isolation of nuclear and cytoplasmic fractions assayed the subcellular distribution of LIN00992 inprostate cancer cells d Top three miRNAs which might interact with LINC00992 were predicted by DIANAlncBase e After transfection ofLINC00992silencing plasmids the expression of miR31575p miR11783p and miR3935 was examined via qRTPCR f Following LINC00992upregulation qRTPCR tested the levels of miR31575p miR11783p and miR3935 in DU145 and PC3 cells g RNA pull down assay wasimplemented to testify the binding capacity between LINC00992 and miR3935 h miR3935 overexpression efficiency and inhibition efficiencywere examined by qRTPCR i RIP assay disclosed the binding of miR3935 to LINC00992 in the antiAgo2 group j The potential binding sitebetween LINC00992 and miR3935 was shown And the luciferase activity of LINC00992WT or LINC00992MUT reporter was assessed vialuciferase reporter assay in DU145 and PC3 cells after transfection with miR3935mimics miR3935inhibitor NCinhibitor or NCmimics P p p analyzed through qRTPCR and western blot analysesand the outcome turned out to be satisfactory Fig 4abFigure S3E Then we observed that overexpression ofGOLM1 could significantly elevate the mRNA and protein expression of GOLM1 in shLINC009921transfected cells Figure S3F Afterwards data from CCK8revealed that the viability of DU145 cells was firstly hindered due to LINC00992 depletion while subsequentGOLM1 elevation reversed the inhibitory trend onDU145 cell viability Fig 4c Results from EdU assayalso exposed similar trends that GOLM1 upregulationimpactposedthesuppressivecountervailedbyLINC00992 downregulation on DU145 cell proliferationFig 4d Similarly the restraining effect of silencedLINC00992 on the expression of proliferationrelatedproteins could be reversed by GOLM1 upregulationFigure S3G Later TUNEL assay revealed that cellapoptosis rate was elevated by LINC00992 depletion andthen overexpressing GOLM1 reduced the increasedapoptosis rate of LINC00992depleted cells Fig 4eLikewise western blot analysis uncovered that overexpressing GOLM1 could offset the effect of LINC00992 0cChen BMC Cancer Page of Fig LINC00992 regulated the expression of GOLM1 a target of miR3935 a The expression of eight mRNAs in four prostate cancer cell linesand RWPE1 cells was detected by qRTPCR b GOLM1 was overexpressed in prostate cancer tissues according to GEPIA database c The mRNAand protein levels of GOLM1 were evaluated in prostate cancer cell lines and RWPE1 cell line by qRTPCR and western blot respectively d Thebinding sites between GOLM1 and miR3935 were predicted via TargetScan e Luciferase reporter assay presented the inhibited luciferase activityof GOLM1WT reporter in the presence of miR3935 mimics not NCmimics fg GOLM1 expression in transfected cells was tested by qRTPCR andwestern blot analyses h The combination of GOLM1 with miR3935 in the antiAgo2 group was validated by RIP assay ij The mRNA and proteinlevels of GOLM1 in different groups were examined via qRTPCR and western blot The fulllength gels for western blot data in Fig 3c g and jwere presented in Supplementary Figure P p p downregulation on the expression of apoptosisrelatedproteins Fig 4f Figure S3H Moreover Transwellmigration and wound healing assays illuminated thatthe retarding influence of silenced LINC00992 on cellmigration could be rescued by GOLM1 overexpression Fig 4gh As expected the inhibitory effect ofLINC00992 depletion on the expression of migrationrelated molecular markers MMP2 MMP9 pSrc andpFAK could be countervailed by GOLM1 overexpression Figure S3I Collectively GOLM1 was requiredcancercellular processesLINC00992regulatedinprostateLINC00992 contributes to tumor growth via upregulatingGOLM1 expressionAfter the in vitro exploration of LINC00992 performance in prostate cancer we applied the in vivo assays tofurther validate above findings As shown in Fig 5a tumors derived from LINC00992silenced DU145 cellswere smaller with the growth rate quite slower thanthose from control cells more importantly such blockage on tumor growth was obviously countervailed afterGOLM1 overexpression Besides elevating GOLM1 expression could recover the lessened tumor volume anddeclined tumor weight induced by LINC00992 deficiency 0cChen BMC Cancer Page of Fig LINC00992 promoted prostate cancer cell proliferation and migration via elevating GOLM1 expression ab GOLM1 mRNA and proteinlevels in DU145 cells transfected with pcDNA31 or pcDNA31GOLM1 were detected via qRTPCR and western blot pcDNA31 served as thenegative control c The viability of DU145 cells was determined via CCK8 following transfection of different plasmids d The proliferation oftransfected cells was evaluated via EdU assay e The apoptosis of transfected cells was monitored via TUNEL assay Scale bar μm f Theprotein levels of Bax and Bcl2 in different groups were detected via western blot gh The migration of transfected cells was measured viaTranswell migration assay scale bar μm and wound healing assay scale bar μm The fulllength gels for western blot data in Fig 4band f were presented in Supplementary Figure P p Fig 5bc Of note we discovered decreased level ofLINC00992 and enhanced level of miR3935 in tumorsfrom latter three groups compared to control group whilethe lowered expression of GOLM1 in tumors withLINC00992 inhibition was normalized under GOLM1overexpression Fig 5d In addition the inhibitory impactof silenced LINC00992 on the positivity of proliferationassociated proteins PCNA and Ki67 could be reversedby upregulation of GOLM1 Fig 5e Taken togetherLINC00992 promoted the tumorigenesis of prostate cancer through upregulating GOLM1 expressionDiscussionAs documented the aberrant regulation of lncRNAs is afrequent event in diversified tumor types Besides thecorrelation between abnormallncRNA expression andprostate cancer oncogenesis has also been extensivelyexplored For example lncRNA SNHG7 facilitates prostate cancer carcinogenesis via cyclin D1 by spongingmiR503 [] LncRNA SChLAP1 aggravates prostatecancer cell proliferation and metastasis by targetingmiR198 [] LncRNA PCAT1 contributes to prostatecancer tumorigenesis through modulating FSCN1 andsponging miR1455p [] In our work LINC00992 wasrevealed to be highly expressed in prostate cancer tissuesand cells but unlike former investigations our studygave a precise explanation about its role in prostate cancer Our study unveiled that LINC00992 promoted cellproliferation and migration whereas suppressed cellapoptosis in prostate cancer Abovementioned data 0cChen BMC Cancer Page of Fig LINC00992 contributes to tumor growth via upregulating GOLM1 expression a Representative images and the growth curves of tumorsfrom indicated groups bc The volume and weight of tumors from above groups d The expression of LINC00992 miR3935 and GOLM1 intumors from different groups was detected via qRTPCR analysis e The staining of PCNA and Ki67 in different groups was measured via IHCScale bar μm p 0cChen BMC Cancer Page of validated that LINC00992 elicited a tumorpromotingfunction in prostate cancerPresently accumulating evidence has indicated thatcytoplasmic lncRNAs assisted the expression of downstream miRNAtargeted mRNAs via sponging the specific miRNAs Before exploring LINC00992mediatedmechanism in prostate cancer herein we firstly discovered its subcellular distribution in prostate cancer cellswith both aids from online prediction tool LncLocatorand experimental data FISH and RNA isolation of nuclear and cytoplasmic fractions Our study for the firsttime uncovered that LINC00992 located mainly in thecytoplasm of prostate cancer cells Besides our studyalso completed LINC00992modulated mechanism bydisclosing the downstream target miR3935 The directinteraction between LINC00992 and miR3935 | 2 |
EpidemiologyEPIDEMIOLOGICAL SCIENCERisk factors for hospital admissions related to COVID19 in patients with autoimmune inflammatory rheumatic diseasesDalifer D Freites Nu±ez1 Leticia Leon Arkaitz Mucientes1 Luis Rodriguez Rodriguez Judit Font Urgelles3 Alfredo Madrid Garca1 Jose I Colomer1 Juan A Jover34 Benjamn Fernandez Gutierrez3 Lydia Abasolo1Handling editor Josef S Smolen1Rheumatology Department and IDISSC La Fundacion para la Investigacion Biomedica del Hospital Clinico San Carlos Madrid Spain2Department of Health and Education Universidad Camilo Jose Cela Villafranca del Castillo Madrid Spain3Rheumatology Department Hospital Clinico San Carlos Madrid Spain4Medicine Department Universidad Complutense de Madrid Madrid Comunidad de Madrid SpainCorrespondence toDr Leticia Leon IdISSC and Rheumatology La Fundacion para la Investigacion Biomedica del Hospital Clinico San Carlos Madrid Spain lleon hcsc salud madrid Received May Revised July Accepted July Objectives To describe patients with autoimmune inflammatory rheumatic diseases AIRD who had COVID19 disease to compare patients who required hospital admission with those who did not and assess risk factors for hospital admission related to COVID19Methods An observational longitudinal study was conducted during the pandemic peak of severe acute respiratory syndrome coronavirus March to April All patients attended at the rheumatology outpatient clinic of a tertiary hospital in Madrid Spain with a medical diagnosis of AIRD and with symptomatic COVID19 were included The main outcome was hospital admission related to COVID19 The covariates were sociodemographic clinical and treatments We ran a multivariable logistic regression model to assess risk factors for the hospital admissionResults The study population included patients with AIRD and COVID19 Of these patients required hospital admission related to COVID19 The mean age on admission was years and the median time from onset of symptoms to hospital admission was days The median length of stay was days A total of patients died during admission Compared with outpatients the factors independently associated with hospital admission were older age OR p000 and autoimmune systemic condition vs chronic inflammatory arthritis OR p001 No statistically significant findings for exposure to disease modifying antirheumatic drugs were found in the final modelConclusion Our results suggest that age and having a systemic autoimmune condition increased the risk of hospital admission whereas disease modifying antirheumatic drugs were not associated with hospital admission Authors or their employers No commercial re use See rights and permissions Published by BMJTo cite Freites Nu±ez a0DD Leon a0L Mucientes a0A et a0al Ann Rheum Dis Epub ahead of print [please include Day Month Year] 101136annrheumdis2020217984INTRODUCTIONSevere acute respiratory syndrome coronavirus SARS CoV2 causes a myriad of clinical signs and symptoms together with typical laboratory abnormalities that manifest as the disease COVID191Since the confirmation of the first patient infected with SARS CoV2 in Spain in January the current COVID19 outbreak has had a considerable impact especially in the Madrid region where the highest incidence of COVID19 cases has been Key messagesWhat is already known about this subject º The epidemiological scenario is changing daily There is little evidence for risk factors of poor outcome with COVID19 specific to autoimmune inflammatory rheumatic diseasesWhat does this study add º Patients with an autoimmune systemic condition have a higher risk of hospital admission related to COVID19 compared with those with chronic inflammatory arthritis º Disease modifying agents were not associated with a higher risk of hospital admission related to COVID19How might this impact on clinical practice or future developments º Our data show that in a real world setting a high percentage of patients with autoimmune inflammatory rheumatic diseases and COVID19 required hospital admission The patients were mainly elderly with comorbidities and a systemic autoimmune conditionrecorded with more than patients admitted to the hospital until the first week of May2The incidence and severity of COVID19 disease seem to be higher in patients with risk factors such as advanced age and associated comorbidities mainly hypertension diabetes heart disease and previous respiratory diseases3 It is not clear whether patients with rheumatic diseases are more susceptible to SARS CoV2 infection or when they are infected whether they have more severe disease or a poorer outcome Previous outbreaks caused by coronaviruses did not yield overwhelming evidence that patients with rheumatic diseases are at an increased risk4 although some patients are candidates for a higher number of infections owing to their rheumatic disease predominantly systemic or the treatment they are receiving for rheumatic diseases5 Preliminary experiences in patients with COVID19 show that those with chronic arthritis treated with synthetic conventional or targeted syntheticbiologic disease modifying antirheumatic Freites Nu±ez DD et a0al Ann Rheum Dis 101136annrheumdis2020217984 0cEpidemiologydrugs DMARDs do not seem to be at a greater risk of respiratory or life threatening complications from SARS CoV2 than the general population6 The epidemiological scenario is changing and evidence on the risk factors of poor outcome with COVID19 specific to inflammatory rheumatic disease is scarce In addition there are little data on how the hospital admissions of these patients with severe COVID19 infection have evolved8The aim of our study was to describe patients with autoimmune inflammatory rheumatic diseases AIRD who had COVID19 during the pandemic peak We also explored possible risk factors associated with hospital admission related to COVID19 disease in patients with AIRD from a tertiary hospital in Madrid SpainMETHODSSetting study design and patientsThe study was performed in a public tertiary hospital Hospital Clnico San Carlos HCSC in Madrid Spain The catchment area is home to almost peopleWe performed a prospective observational study from March when our health area had the first hospital admission related to COVID19 to April We preselected all patients attended at the rheumatology outpatient clinic of our centre during the study period whose data were recorded in the electronic clinical history of our department HCR Penelope The inclusion criteria were age years a medical diagnosis according to International Classification of Diseases ICD10 of inflammatory rheumatic disease and symptomatic COVID19 disease assessed by medical diagnosis or confirmed with a positive SARS CoV2 PCR diagnostic testPatient data were obtained during routine clinical practice The study was conducted in accordance with the Declaration of Helsinki and the principles of Good Clinical Practice and was approved by the HCSC Ethics Committee approval number E BSVariablesThe primary outcome was admission to hospital with a medical diagnosis of COVID19 andor a positive PCR result between March and April compared with outpatients with symptomatic COVID19 diseaseThe covariables recorded were as follows sociodemographic baseline characteristics including sex age and rheumatic disease duration Type of AIRD including systemic autoimmune conditions polymyalgia rheumatica mixed connective tissue disease systemic sclerosis Sjogrens syndrome vasculitis Raynaud phenomenon polymyositis polychondritis sarcoidosis antiphospholipid syndrome autoinflammatory syndromes and systemic lupus erythematosus and chronic inflammatory arthritis rheumatoid arthritis inflammatory polyarthritis juvenile idiopathic arthritis psoriatic arthritis axial spondyloarthritis uveitis and inflammatory bowel disease Baseline comorbid conditions including hypertension dyslipidaemia depression diabetes mellitus smoking habit kidney disease chronic liver disease respiratory diseases chronic obstructive pulmonary disease and interstitial lung disease thyroid disease heart disease valve disease arrythmias cardiomyopathy heart failure and pericarditis ischaemic vascular disease stroke cardiovascular and peripheral vascular disease venous thrombosislung embolism and cancer Treatment for inflammatory rheumatic disease a glucocorticoids b non steroidal anti inflammatory drugs NSAIDs c conventional synthetic disease modifying antirheumatic drugs csDMARDs antimalarials hydroxychloroquine and chloroquine azathioprine cyclophosphamide cyclosporine colchicine leflunomide methotrexate mycophenolate mofetilmycophenolic acid and sulfasalazine d targeted syntheticbiologic DMARDs tsbDMARDs including antitumour necrosis factor TNF alpha drugs infliximab adalimumab etanercept certolizumab and golimumab other biologics anti interleukin IL6 tocilizumab and sarilumab rituximab abatacept belimumab anti IL1723 anti IL17 ustekinumab ixekizumab and secukinumab Janus kinase JAK inhibitors tofacitinib and baricitinibTreatment had to start at least month before the beginning of the study and continue during the study period until the end of the study or hospital admission for antimalarial therapy glucocorticoids sulfasalazine NSAIDs or colchicine Regarding csDMARDs and tsbDMARDs treatment had to start at least month before the beginning of the study and continue until at least 21st March the end of the study or hospital admission In the case of rituximab the last infusion had to be at least in JanuaryData sourcesPatient sociodemographic clinical laboratory and data on treatment of rheumatic disease were obtained through HCR PenelopePatients with COVID19 were detected by warning calls to our rheumatologists or nurses or via routine telephone consultation Other infected patients were detected through their sick leave forms for COVID19 The results of SARS CoV2 PCR diagnostic assays were obtained from the microbiologyinfectious service of HCSC In addition our Hospital Central Services registered all medical admissions to HCSC This information was provided from March to AprilThe researchers carried out an exhaustive review of the clinical histories of admitted patients to identify COVID19 cases and rule out patients admitted for other reasons Once the COVID19 cases were identified we collected clinical laboratory and treatment data during admission until the end of admission either discharge or death in order to describe the progress of the disease The review was performed until 24th April in order to include follow up data from patients admitted to the hospital with COVID19Statistical analysisPatient characteristics are expressed as mean and SD or median and IQR for continuous variables categorical variables are expressed as percentages Statistical tests were performed to compare characteristics between patients admitted with COVID19 and those without hospital admissions Continuous variables were analysed using the Mann Whitney test or t test and discrete variables were analysed using the Ï2 or Fisher exact test Univariable logistic regression analyses were performed to assess differences between hospital admissions related to COVID19 risk and covariates Multivariable logistic regression models adjusted for age sex and comorbidity were run in a stepwise manner to examine the possible effect of sociodemographic clinical and therapeutic factors on hospital admissions related to COVID19 The model also included csDMARDs and all other variables with a p02 from the simple regression analysis The results were expressed as the OR with its respective CIAll analyses were performed in Stata V13 statistical software Stata Corp A two tailed p value was considered to indicate statistical significanceFreites Nu±ez DD et a0al Ann Rheum Dis 101136annrheumdis2020217984 0cRESULTSA total of patients with AIRD with symptomatic COVID19 disease were included in the study table The tests were performed as an exploratory measure of the association between a variable and the outcomeMost of the patients were women with a mean age of years and a mean time since diagnosis of years The main diagnosis was rheumatoid arthritis followed by axial spondyloarthritis Many patients had at least one baseline comorbid condition the most prevalent being hypertension dyslipidaemia and lung disease Most patients were taking csDMARDs Half of the patients were taking glucocorticoids a quarter were taking NSAIDs and were taking tsbDMARDs of which adalimumab was the most frequently prescribed followed by rituximab Only one patient was taking a JAK inhibitor Interestingly of the patients taking tsbDMARDs were taking the drug in combination with a csDMARDA total of patients had to be admitted to the hospital because of COVID19 Of these were evaluated in the HCSC Emergency Department were admitted to HCSC and were transferred to the Institucion Ferial de Madrid IFEMA support hospital owing to the lack of capacity in our hospital at that time The remaining three patients were evaluated and admitted to other hospitals in the Autonomous Community of Madrid Table presents data for the patients admitted to HCSCOf the patients admitted to our hospital were women with a mean age at admission of years and median lag time from the onset of symptoms to the admission of days The median length of stay was days table At admission the median haemoglobin was gdL and the median total lymphocyte count was ngmL The median D dimer value was ngmL In of patients median interleukin IL6 levels were pgmL Patients received various antibiotics mainly azithromycin levofloxacin and third generation cephalosporinsMost patients were treated with hydroxychloroquine during admission About half received glucocorticoids Eighteen were treated with lopinavirritonavir and received the anti IL 6R antibody tocilizumab table 2FEDERA total of patients developed relevant complications during admission the most frequent being myocarditis thrombosis and kidney failure Only two patients were admitted to the intensive care unit during admission The first was a patient in 50s with mixed connective tissue disease and associated comorbidities who developed acute respiratory insufficiency and bilateral pneumonia The patient was treated with antibiotic therapy lopinavirritonavir hydroxychloroquine and βinterferon Finally the patient was extubated days later and is recovering The other was a young adult patient with systemic lupus erythematosus treated with methotrexate rituximab hydroxychloroquine and glucocorticoids who days after being diagnosed with COVID19 PCR developed an erythematous rash and generalised urticaria requiring hospitalisation in the intensive care unit owing to general clinical and laboratory worsening elevated D dimer values The patient was treated with methylprednisolone heparin and a cephalosporin A few days later the patients condition improved and he recovered completely at dischargeOf the patients admitted to HCSC were sent to another care centre converted hotel hospitalIFEMA support hospital when their condition improved A further patients Epidemiologywere discharged home to continue self isolation after improvement At the end of the study five patients remained in hospital A total of patients died during admission men and women with a median age of years Of the patients who died had relevant comorbidity diabetes mellitus pulmonary disease ischaemic vascular disease hypertension venous thrombosislung embolism lung disease and or liver disease The main diagnoses were rheumatoid arthritis followed by spondyloarthritis polymyalgia rheumatica vasculitis and Sjogrens syndrome The results of the univariable analysis are shown in table Older age systemic autoimmune conditions vs chronic inflammatory arthritis OR CI p0014 hypertension diabetes mellitus lung disease heart disease and glucocorticoids were associated with statistically significant greater risk of admission to the hospital Female sex NSAIDs and anti TNF drugs vs non use were associated with a statistically significant lower risk The differences reported for the remaining variables did not reach statistical significanceThe multivariable analysis was adjusted for gender age and comorbidities related to COVID19 These comorbidities were diabetes mellitus pulmonary disease ischaemic vascular disease hypertension venous thrombosislung embolism lung disease andor liver disease table Age and systemic autoimmune conditions had more probability of hospital admissions regardless of other factors Differences in exposure to glucocorticoids were not statistically significant The type of exposed DMARDs did not reach statistical significance in the multivariate model In fact long term treatment with antimalarials OR CI p066 other csDMARDs including methotrexate leflunomide and azathioprine OR CI p09 and NSAIDs OR CI p05 dropped from the final model The variable tsbDMARDs was also eliminated from the final model anti TNF vs none OR CI p016 and non anti TNF vs none OR CI p03DISCUSSIONOur study aims to shed light on rheumatologists concerns regarding their patients We found that in a real world setting of patients with AIRD and COVID19 required hospital admission These were mainly elderly patients with more comorbidities and systemic autoimmune conditions Our data show that patients exposed to disease modifying agents do not seem to be at higher risk of hospital admission related to COVID19Of the patients included in the study with COVID19 required hospital admission Comparison of the characteristics of patients admitted to hospital because of COVID19 and those who did not require hospital admission were as follows admitted patients had a median age close to years that is more than years older than patients who were not admitted Moreover those who were admitted more frequently had baseline comorbidities and systemic autoimmune conditions As for therapy admitted patients were less frequently exposed to antimalarial and anti TNF alpha agentsThe median lag time from onset of symptoms to admission was days and almost of patients had pneumonia at admission The baseline laboratory results for admitted patients in our study are consistent with those published elsewhere9 and are characterised by lymphopenia and elevated acute phase reactants In fact of the patients had elevated D dimers normal and elevated IL6 normal pgmL Treatment during admission varied widely as the disease proved Freites Nu±ez DD et a0al Ann Rheum Dis 101136annrheumdis2020217984 0cEpidemiologyTable Baseline demographic and clinical characteristics of patients with AIRD and with COVID19 admitted vs no admitted at the hospitalAIRDCOVID19 patientsAIRDCOVID non admitted patientsAIRDCOVID admitted patientsVariableN123N69N54P value Positive Negative Not performed Women n Age years mean SDTime since diagnosis years mean SDPCR test n Smoking habit active vs noneDiagnosis AIRD n Rheumatoid arthritis Axial spondyloarthritis Polymyalgia rheumatica Psoriatic arthritis Systemic lupus erythematosus Mixed connective tissue disease Sjogrens syndrome Vasculitis Uveitis Systemic sclerosis Inflammatory polyarthritis Polychondritis Polymyositis Raynaud phenomenon OtherComorbidities n NSAIDs n Glucocorticoids n csDMARDs n TsbDMARDs n JAKi n Others inflammatory bowel disease antiphospholipid syndrome juvenile idiopathic arthritis autoinflammatory syndromes and sarcoidosis Heart disease arrhythmiasvalve disease cardiomyopathy and heart failure Ischaemic vascular disease stroke cardiovascular and peripheral vascular diseaseAIRD autoimmune inflammatory rheumatic disease Anti TNF tumour necrosis factor alpha COPD chronic obstructive pulmonary disease csDMARD conventional synthetic disease modifying antirheumatic drug ILD interstitial lung disease JAKi JAK inhibitor tsbDMARDs target syntheticbiologic disease modifying antirheumatic drug Hypertension Dyslipidaemia Depression Diabetes mellitus Heart disease Vascular disease Liver disease Kidney disease Lung disease ILDCOPD Cancer Venous thrombosislung embolism Thyroid disease Anti TNF alpha agent Other biologics Abatacept Tocilizumab Belimumab Rituximab Methotrexateleflunomideazathioprine Sulfasalazine AntimalarialsFreites Nu±ez DD et a0al Ann Rheum Dis 101136annrheumdis2020217984 0cTable Hospital admissions related to COVID19 among patients with AIRDVariableValueTable OR of hospital admission related to COVID19 in patients with AIRD univariable analysisVariable CIORPEpidemiology Haemoglobin gdL D dimer ngmL Neutrophil count 109L Lymphocyte count 109L CRP mgdL LDH UL Platelet count 109L Creatinine mgdL Ferritine ngmLAdmissions nLag time from onset of symptoms to admission days median IQRPneumonia at admission n Systemic autoimmune conditions n Laboratory data at admission median IQR COVID19 related treatments during admission n Admitted by intensive care unit during hospital admission Length of stay days median IQRDischarge reason n Azithromycin Other antibiotics Glucocorticoids Lopinavirritonavir Remdesivir Darunavircobicistat Tocilizumab Interferon HCQ Immunoglobulin Improvement home isolation Other care centre medicalised hotelIFEMA hospital Death End of study no discharge No Yes Data for patients patients were treated in other support centres after referral or admission in other centresCRP C reactive protein HCQ hydroxychloroquine LDH lactate dehydrogenase challenging for specialists who prescribed various combinations of drugs based on little published evidence In this sense the anti IL 6R antibody tocilizumab has proven to be beneficial in patients with COVID1912 Treatment may also be successful in the early stages of cytokine release syndrome if it can effectively block the signal transduction pathway of IL6 therefore tocilizumab and sarilumab are likely to emerge as effective drugs for patients with moderate to severe COVID1913 In our study almost of the patients were treated with tocilizumabThe patients who eventually died had a median age of years This finding is in line with data for the general population where over of deaths occurred in persons years and more than of all deaths were in people aged ¥ years7The multivariable regression model showed that only age increasing by per year and systemic autoimmune conditions continued to be risk factors for hospital admission related to COVID19 Rheumatoid arthritis Inflammatory polyarthritis Systemic lupus erythematosus Psoriatic arthritis Spondyloarthritis MTCD Sjogren syndrome Hypertension Dyslipidaemia Depression Diabetes mellitus Heart disease Vascular disease Liver disease Kidney disease Lung disease ILDCOPD Cancer Venous thrombosislung embolism Thyroid diseaseGender womenAge yearsDiagnosis AIRD one category vs the rest Disease durationSmoking habit active vs noneComorbidities yes NSAIDsGlucocorticoidscsDMARDSs TsbDMARDs JAKisOther biologics anti IL6 tocilizumab sarilumab rituximab Rtx anti IL1723 anti IL17Othercategories could not be represented polymalgia rheumatica systemicsclerosis vasculitis Raynaud phenomenon polychondritis Beh§et diseasepolymyositis uveitis inflammatory boweldisease antiphospholipid syndrome juvenile idiopathic arthritis autoinflammatorysyndromes and sarcoidosisAIRD autoimmune inflammatory rheumatic disease anti TNF tumour necrosis factor csDMARD Conventional synthetic disease modifying antirheumatic drug IL6 interleukin6 JAKi JAK inhibitors tsbDMARDs target syntheticbiologic disease modifying antirheumatic drug Methotrexateleflunomideazathioprine Sulfasalazine Antimalarial agents None Anti TNF agents Other biologicsAs for the association between sex and risk of hospital admission we did not find a higher risk of admission in women despite the fact that rheumatic diseases are more prevalent in this group The type of diagnosis seems to play an important role in the probability of hospital admission and patients with systemic autoimmune conditions seem to have the highest risk compared with chronic inflammatory arthritisAs it has been reported elsewhere comorbidities play an important role in the risk of hospital admission15 Clinical outcomes are poorer in patients with COVID19 with a comorbid condition than in those without and a greater number of comorbidities correlates with poorer clinical outcomes16 Diabetes is a major comorbidity in COVID19 and patients history of diabetes is an independent risk factor for morbidity and mortality in this condition17 Diabetes has been associated with admissions to Freites Nu±ez DD et a0al Ann Rheum Dis 101136annrheumdis2020217984 0cEpidemiologyTable Multivariable analysis risk factors for hospital admission related to COVID19 in patients with AIRDVariableOR CIP valueGender womenAge yearsAIRD systemic autoimmune conditions vs chronic inflammatory arthritisCOVID comorbidities yesGlucocorticoidsSystemic autoimmune conditions polymyalgia rheumatica mixed connective tissue disease systemic sclerosis Sjogrens syndrome vasculitis Raynaud polymyositis polychondritis sarcoidosis antiphospholipid syndrome autoinflammatory syndromes and systemic lupus erythematosus vs chronic inflammatory arthritis rheumatoid arthritis inflammatory polyarthritis juvenile idiopathic arthritis psoriatic arthritis axial spondyloarthritis uvetis and inflammatory bowel disease Comorbidities including the presence of at least one of the follows hypertension heart disease vascular disease diabetes mellitus venous thrombosislung embolism chronic kidney disease liver disease and lung disease ILDCOPDAIRD autoimmune inflammatory rheumatic disease COPD chronic obstructive pulmonary disease ILD interstitial lung diseasethe intensive care unit due to COVID19 in recent series19 and has been shown to increase mortality6 Therefore we adjusted for comorbidity in the multivariable analysisTreatment with glucocorticoids lost its statistical significance in the multiple regression model However the dose was not reported in our data and in the case of these agents the risk could be dose dependent In a recent publication from a European registry the authors found that exposure to mgday was associated with a greater probability of hospitalisation21The exposure to DMARDs regardless of whether they were biological or synthetic does not seem to be associated with a higher hospital admission related to COVID19 Although we have to consider the limited number of patients in our study our results are in concordance with data reported elsewhere8 Our results should be interpreted taking into account other limitations First patients were included from a single centre Second of all the patients with COVID19 who did not require admission one third contacted the rheumatology service to report the disease and the remainder were detected through the COVID19 discharge reports sent to their primary care physician Elderly persons and homemakers who did not contact us can be considered missing Consequently there may be some selection bias between those admitted and those not admitted although this problem was addressed by adjusting for confounders in the multivariable analysis Third while it is acknowledged that clinical suspicion must be confirmed by PCR assay almost of patients admitted did not undergo PCR owing to the lack of tests or the extreme healthcare overload Nevertheless all cases included were clinically compatible and managed as COVID19The key strength of our study is that it was performed in real life conditions during then pandemic peak with access to complete sociodemographic and clinical data from our rheumatology electronic clinical history including thorough hospital admission data such as laboratory abnormalities and COVID19 treatment data from the hospital computer services Consequently this has allowed us to analyse the risk of hospital admission related to COVID19 adjusted for confounders thus avoiding possible biasAlthough we are unable to modify the factors reported here knowing them can help rheumatologists to treat and advise their patients during this new and challenging period Results provided by our study are preliminary and should be corroborated with other real life studies but we consider our findings helpful to increase the knowledge in the management of patients with AIRD and COVID19Twitter Benjamn Fernandez Gutierrez Fergutbe2001Acknowledgements The authors would like to thank Ana M Perez for her help with data collection They would like to say a special thanks to all the rheumatologists and nurses who contributed to the care of the patients in such an innovative and conscientious wayContributors BF G LL JAJ LR R and LA conceived and designed the study DDFN JFU AMG JIC and LL collected data LA and LL performed the data analysis and interpreted the data All of the authors were involved in the drafting andor revising of the manuscriptFunding This work was supported by the Instituto de Salud Carlos III ISCIII Ministry of Health Spain CP1600916 PI1801188 and RD1600120014 and cofunded by el Fondo Europeo de Desarrollo Regional FEDER The funders had no role in study design data collection analysis manuscript preparation or decision to publishCompeting interests None declaredPatient and public involvement Patients andor the public were not involved in the design or conduct or reporting or dissemination plans of this researchPatient consent for publication Not requiredEthics approval The study was approved by the Hospital Clnico San Carlos institutional ethics committee approval number E BS This study was conducted according to the principles of the Declaration of HelsinkiProvenance and peer review Not commissioned externally peer reviewedData availability statement Data are available upon reasonable requestThis article is made freely available for use in accordance with BMJs website terms and conditions for the duration of the covid19 pandemic or until otherwise determined by BMJ You may use download and print the article for any lawful non commercial purpose including text and data mining provided that all copyright notices and trade marks are retainedORCID iDsLeticia a0Leon http orcid Luis a0Rodriguez Rodriguez http orcid REFERENCES Fernandez Gutierrez B COVID19 with pulmonary involvement An autoimmune disease of known cause Reumatol Clin COVID19 Situaci³n actual en La Comunidad de Madrid Informe de situaci³n del de Mayo Available httpswww comunidad madrid sites default files doc sanidad 200508_ cam_ covid19 pdf [Accessed May ] Chen N Zhou M Dong X et a0al Epidemiological and clinical characteristics of cases of novel coronavirus pneumonia in Wuhan China a descriptive study Lancet Figueroa Parra G Aguirre Garcia GM Gamboa Alonso CM et a0al Are my patients with rheumatic diseases at higher risk of COVID19 Ann Rheum Dis Favalli EG Ingegnoli F De Lucia O et a0al COVID19 infection and rheumatoid arthritis Faraway so close Autoimmun Rev Zhou F Yu T Du R et a0al Clinical course and risk factors for mortality of adult inpatients with COVID19 in Wuhan China a retrospective cohort study Lancet Monti S Balduzzi S Delvino P et a0al Clinical course of COVID19 in a series of patients with chronic art | 2 |
Radial shock waves prevent growth retardation caused by the clinically used drug vismodegib in ex vivo cultured bonesSowmya Ramesh123 Lars Svendahl245 Vrisha Madhuri135 farasat Zaman2In childhood medulloblastoma patients the hedgehog antagonist vismodegib is an effective anticancer treatment but unfortunately induces irreversible growth arrests and growth impairment limiting its use in skeletally immature patients We hypothesized that radial shock wave treatment rSWT may protect druginduced growth impairment owing to its osteogenic effects Fetal rat metatarsal bones were exposed to vismodegib day nM andor rSWT single session other bones from day were continuously exposed to a Gli1 antagonist GANT61 µM andor rSWT single session Control bones were untreated The bone length was measured at intervals histomorphometric analysis and immunostaining for PCNA Gli1 and Ihh were performed on the sectioned bones Bones treated with vismodegib showed impaired bone growth reduced height of the restingproliferative zone and reduced hypertrophic cell size compared to control In vismodegib treated bones a single session of rSWT partially rescued bone growth increased the growth velocity hypertrophic cell size and restored growth plate morphology Bones exposed to GANT61 showed impaired bone growth and disanized growth plate while when combined with rSWT these effects were partially prevented Locally applied rSWT had a chondroprotective effect in rat metatarsal bones and suggest a novel strategy to prevent growth impairment caused by vismodegibHedgehog Hh proteins are wellknown to be overexpressed in paediatric medulloblastoma1 Mutations that occur in the family of Hhpathway genes such as patched1 suppressor of fuse and smoothened leads to an increased level of the gliomaassociated oncogene Gli1 a downstream transcription factor of Hh2 In the clinic hedgehog inhibitors are used to decrease the Hhactivity and thereby impede tumor progression3 However stable expression of the Hhgene is essential to maintain chondrocyte proliferation and hypertrophy during bone growth6 A recent study reported that prolonged exposure to vismodegib a Hhantagonist in children with medulloblastoma resulted in irreversible growth plate fusion causing growth arrest of long bones78 Preclinical studies in young mice exposed to a Hhantagonist also showed growth arrests and bone growth defects9 Mechanistic studies revealed that even brief exposure to a Hhinhibitor was enough to damage the growth plates by diminishing the numbers of reserve and proliferative chondrocytes9 These findings further imply that it may not be possible to arrive at a dose that selectively targets tumor growth with no sideeffects on bone development Therefore in children a protective strategy for growth plate shielding without interfering with the desired anticancer effects of vismodegib in the neural tissue is highly desiredIn vitro studies using cultured rat metatarsal bones10 and in a0vivo studies in rabbits11 and humans12 have shown that radial shock wave treatment rSWT a noninvasive modality used in the clinic have stimulatory effects on bone growth10 Furthermore the observed stimulation of chondrocyte proliferation and hypertrophy induced by rSWT was partially linked to local upregulation of Gli1 in cultured metatarsal bones10 Interestingly previous in a0vitro studies revealed that highenergy shock wave treatment increased the uptake of chemotherapy agents13 1Department of Paediatric Orthopaedics Christian Medical College Vellore India 2Division of Paediatric Endocrinology Department of Womens and Childrens Health Karolinska Institutet Solna Sweden 3Centre for Stem Cell Research A Unit of inStem Bengaluru Christian Medical College Bagayam Vellore India 4Paediatric Endocrinology and Metabolism Astrid Lindgren Children²s Hospital Karolinska University Hospital Solna Sweden 5These authors contributed equally Lars Svendahl and Vrisha Madhuri email sowmyarameshkiseScientific RepoRtS 101038s41598020699040Vol0123456789wwwnaturecomscientificreports 0cFigure a0 Rescuing effects of rSWT on vismodegib treated bones a Fetal rat metatarsal bones cultured exvivo were treated with the Hhinhibitor vismodegib a0nM for a0days n dotted line a single session of highenergy rSWT a0Hz a0mJ n or both n and thereafter followed for a0days The graph shows increases in bone length over time a0from day All error bars indicate SD Twoway ANOVA was applied b Graph shows the increase in the growth velocity on day of control vismodegib rSWT and vismodegib rSWT treated bones All error bars indicate SD Representative images of metatarsal bones stained with Alcian blue c untreated control d vismodegib e rSWT and f vismodegib rSWT Magnification 10x g height measurements of R P zone and h hypertrophic cell size Quantification of immunostaining for i Gli1 and j Ihh using the ImageJ software p p p thereby lowering the dose of the drug when applied to cell lines derived from human osteosarcoma14 human colorectal adenocarcinoma15 and anaplastic thyroid cancer16 The effect was mediated by a transient increase in cell membrane permeability allowing passage of a higher concentration of the drug16A recent report suggested that locally applied rSWT can promote longitudinal bone growth of rat metatarsal bones cultured exvivo in the absence of serumgrowth factors10 Based on these findings we hypothesized that rSWT may also have the capacity to prevent growth failure caused by Hhinhibitors We aimed to investigate the potential for rSWT to prevent growth retardation caused by two different Hhantagonists vismodegib and the Gli1 antagonist GANT61 in a wellestablished model of cultured fetal rat metatarsal bonesResultsEffect of vismodegib on bone growth and rescuing effects of rSWT To allow transient inhibition of Hhactivity cultured fetal rat metatarsal bones were treated with vismodegib for a0days whereafter growth was monitored for another a0days without any treatment Bones treated with vismodegib grew less than untreated controls and the difference was significant when measured on day ± vs ± bone length increase from day respectively p Fig a01aTo address the primary objective of this study if rSWT can prevent growth failure caused by hedgehog inhibition we first studied if a single exposure to rSWT can rescue metatarsal bone growth after transient exposure to a0days treatment with vismodegib Indeed bones treated with rSWT single exposure on day in combination with vismodegib day grew significantly better than bones treated with vismodegib alone when measured Scientific RepoRtS 101038s41598020699040Vol1234567890wwwnaturecomscientificreports 0cFigure a0 Rescuing effects of rSWT on GANT61 treated bones a Fetal rat metatarsal bones cultured exvivo treated with the Hhinhibitor GANT61 a0µM n a single session of rSWT a0Hz a0mJ n or both n and followed for a0days The graph shows increases in bone length a0from day All error bars indicate SD Twoway ANOVA was applied Representative images of metatarsal growth plates stained with Alcian blue b untreated control c GANT61 d rSWT and e GANT61 rSWT Magnification 10x f Height measurements of R P zone and g hypertrophic cell size Quantification of immunostaining for h PCNA and i Gli1 using the ImageJ software p p on day ± vs ± respectively p Fig a01a Also the growth velocity tended to be higher in rSWT vismodegib compared to vismodegib alone Fig a01bGrowth plate morphology Histological evaluation revealed severe disruption in the growth plate morphology in the bones treated with vismodegib compared to control Fig a01cd The disrupted growth plate morphology in vismodegib treated bones was found to be more normal in bones receiving rSWT alone Fig a01e and rSWT vismodegib Fig a01f When comparing vismodegib treated bones to controls we found reduced height of the resting proliferative R P zone ± a0µm vs ± a0µm respectively p Fig a01g The height of the R P zone tended to be increased in bones exposed to rSWT vismodegib when compared to vismodegib alone ± a0µm vs ± a0µm respectively p ns Fig a01g Histomorphometric analysis showed decreased size of hypertrophic chondrocytes in the bones treated with vismodegib compared to control ± a0µm vs ± a0µm respectively p Fig a01h In bones treated with rSWT vismodegib the hypertrophic chondrocytes were significantly larger compared to vismodegib alone and similar in size as in untreated control bones ± a0µm ± a0µm and ± a0µm respectively p vs vismodegib p ns vs control Fig a01h Immunostaining for Gli Fig a01i and Ihh Fig a01j did not show any significant effects of vismodegib andor rSWTShock wave treatment prevents bone growth retardation caused by GANT61 To study the effects of continuous inhibition of Hhactivity fetal rat metatarsal bones were treated with the Gli1 antagonist GANT61 from day of culture until the termination of the experiment on day Already on day GANT61 induced a pronounced suppression of the bone growth ± ± p vs control which remained at endpoint on day ± ± p vs control Fig a02aNext we asked if a single exposure to rSWT can rescue from growth retardation caused by continuous Hhinhibition induced by GANT61 Indeed metatarsal bones exposed to rSWT on day were rescued from GANT61induced growth retardation already from day of culture ± ± p vs GANT61 alone which remained until endpoint ± ± p vs GANT61 alone Fig a02aGrowth plate morphology Compared to control in the bones continuously treated with GANT61 growth plate morphology was found to be disturbed with disanized chondrocyte columns on day Fig a02b c Bones treated with rSWT alone showed anized growth plate morphology Fig a02d The growth rescuing Scientific RepoRtS 101038s41598020699040Vol0123456789wwwnaturecomscientificreports 0ceffect of rSWT in GANT61 treated bones was accompanied by a normalization of growth plate morphology including anized chondrocyte columns Fig a02eHistomorphometric analyses showed no significant differences in the height of the R P zone between GANT61 treated bones with or without rSWT ± a0µm ± a0µm p ns Fig a02f There was an increased size of hypertrophic chondrocytes in rSWT GANT61 group when compared to GANT61 alone ± a0µm ± a0µm p Fig a02g Furthermore the number of proliferative chondrocytes tended to be lower in GANT61 treated bones when compared to control ± cells ± cells p ns Fig a02h The number of proliferative PCNA positive cells tended to be higher in the rSWT GANT61 group compared to GANT61 alone ± cells ± cells p ns Fig a02h Immunoexpression of Gli1 tended to be reduced in GANT61 treated bones p ns vs control whereas bones treated with rSWT GANT61 tended to have higher Gli1 expression compared to GANT61 alone p ns Fig a02iDiscussionWe aimed to investigate the potential for locally applied rSWT to prevent bone growth impairment caused by the hedgehog inhibitor vismodegib a therapeutic investigational drug using a wellestablished model of ex a0vivo cultured fetal rat metatarsal bones Herein we report that a single session of rSWT partially prevented growth retardation caused by both transient and continuous Hhinhibition induced by vismodegib and GANT61 respectively The growth rescuing effects by rSWT were accompanied by preservation of growth plate morphology disrupted by the Hhinhibitors Altogether our data suggest that rSWT has the potential to noninvasively protect bones from growth retardation caused by vismodegibBone growth is majorly dependent on the preservation of a unique anization of chondrocytes in the growth plate17 Recent reports have demonstrated that long term exposure to vismodegib the first Hhantagonist approved in the US by FDA led to permanent growth impairment in children with medulloblastoma78 To date no successful strategy that targets tumor cells with no adverse effect on longitudinal bone growth has been described Previous reports have shown that rSWT a treatment modality that is already used in children for musculoskeletal indications18 can stimulate longitudinal bone growth locally in exvivo cultured metatarsal bones even in the absence of any systemic growth factors10 Proinflammatory cytokines are also known to impair bone growth19 and interestingly shockwave treatment has been shown to reduce inflammation and apoptosis while stimulating the regeneration of various tissues2021 These findings encouraged us to expand this knowledge and further investigate the potential for rSWT to prevent bone growth impairment caused by HhinhibitorsVismodegib at a0nM concentration has shown to impair bone growth in exvivo cultured metatarsal bones22 and decrease proliferation of the precursors of a0cerebellar granule neurons23 while in a0vivo studies in a model of medulloblastoma have also shown that vismodegib inhibits Gli1 at a IC50 of a0nM24 a similar range of concentration as used in the present study In young mice transient inhibition of the Hh pathway has been reported to cause permanent defects in bone and growth plate structure9 Similar to the previous in a0vivo observations in young mice9 our histomorphometric growth plate data suggest that partial loss of Hhactivity may result in the breakdown of chondrocyte columnar anization and reduced size of hypertrophic chondrocytes Also the disrupted growth plate ultrastructure caused by Hhinhibition explains the observed growth deficit in our study model system Besides undesired apoptosis of stemlike cells within the growth plate is another wellknown contributing factor linked to growth retardation caused by anticancer drugs2526Our key finding is that a single administration of rSWT not only prevented bone growth retardation caused by transient exposure to the Hhinhibitor vismodegib but also rescued bone growth under a condition of continuous Hhinhibition induced by another Hhinhibitor GANT61 Furthermore rSWT also improved growth velocity and restored growth plate morphology in bones exposed to vismodegib or GANT61 Thus our findings highlight the potential for shock wave technology to be developed as a new and safe treatment strategy to minimize deleterious effects of Hhinhibitors selectively in the growth plates of treated childrenHedgehog signaling drives chondrocyte proliferation and hypertrophy in the growth plate cartilage6 From in a0vitro studies we know that Hhinhibitors decrease the expression of Gli1 and induce cell cycle arrest in prostate cancer cells27 Despite rSWT rescued bones from Hhinhibitor impaired bone growth we did not see significant alterations in the expression of Gli1 and Ihh suggesting a crosstalk between hedgehog signaling and other pathways28 We speculate that the bone rescuing effect of rSWT is more evident if there is any ongoing disturbance within growth plate chondrocytes Indeed it was interesting to note that despite continuous exposure to a Hhinhibitor a single session of rSWT could partially rescue the bone growthOur study has several limitations Firstly the bone growth rescuing effects of rSWT were documented in an exvivo bone culture model and we do not know if this will be applicable under in a0vivo conditions Nevertheless in a0vivo studies in rats or mice are of limited value when it comes to exploring the potential for rSWT to rescue from vismodegib induced bone growth impairment as their growth plates do not normally fuse29 Secondly we only applied a single dose of rSWT while multiple sessions could potentially be even more efficient when it comes to preventing growth impairment caused by Hhinhibitors Thirdly the concentration of vismodegib used in the present study is different from the plasma concentration a0µM achieved in children30 Nevertheless mimicking a gradual decline in bone growth in order to test the rescuing effect of rSWT is more important in our experimental setting We therefore claim a protective effect and not a clinical effect which will require more rigorous testing Consequently our proof of concept finding s up a window of opportunity to explore the potential for locally applied rSWT to prevent bone growth impairment caused by vismodegib as it may not be possible to extrapolate the doses used for preclinical studies to a clinical setting3132In summary we here present a novel treatment strategy based on clinically used rSWT to locally prevent bone growth impairment caused by vismodegib a promising anticancer drug used in children with medulloblastoma Scientific RepoRtS 101038s41598020699040Vol1234567890wwwnaturecomscientificreports 0cFigure a0 A hypothetical schematic diagram showing how extracorporeal radial shockwave treatment rSWT may be administered to lowhigh growth velocity areas a0encircled selectively to restore vismodegibinduced bone growth impairment in children with medulloblastomaFig a0 Before any clinical studies our promising ex a0vivo findings need to be validated in an in a0vivo animal model like the rabbit where growth plate fusion normally occurs just like in humansMethodsThe experiments were approved by the local institutional review board Min No and the institutional animal ethics committee Min No at Christian Medical College Vellore Animal care compiled with the Guide for the Care and Use of Laboratory Animals A a0radial shock wave machine from Radialspec Medispec Gaithersburg MD USA was used for the studyBone an culture system Metatarsal bones were microdissected from the hind limbs of Sprague Dawley rat fetuses sacrificed on day of gestation Ex a0vivo cultures were performed as previously reported33 Briefly each bone was transferred to a 24well plate and cultured in medium containing DMEMF12 a0mM betaglycerophosphate ascorbic acid a0µgml and gentamycin The medium was replenished every two days Figure a0 shows the experimental overviewExposure to Vismodegib Vismodegib was purchased from Selleck Chemicals Houston Texas USA The experimental setup consisted of four groups Metatarsal bones were cultured for a0days and were either a treated with vismodegib a0nM n from day to b a single exposure to rSWT impulses a0Hz a0mJ n on day c vismodegib rSWT n or d were left untreated control n The bone length was measured on days and Exposure to GANT61 GANT61 was purchased from Sigma Aldrich St Louis Missouri USA Metatarsal bones were treated with the a Hhinhibitor GANT61 a0µM from day to n b a single exposure Scientific RepoRtS 101038s41598020699040Vol0123456789wwwnaturecomscientificreports 0cFigure a0 Experimental overview Middlethree metatarsals from the hind limb of embryonic rat at a 1920th day of gestation were dissected and b cultured exvivo c Bones were either exposed to vismodegibGANT61 a single session of highenergy radial shock wave treatment rSWT vismodegibGANT61 with rSWT or left untreated During this time d total bone length measurements at different intervals are performedto rSWT on day impulses a0Hz a0mJ n c GANT61 rSWT or d were left untreated control n The bone length was measured on days and Bone length measurement Digital images were captured for bone length measurements using an inverted microscope Leica Microsystems All measurements were performed by one of the investigators blinded to the nature of the group using an inbuilt measurement tool Bone growth is expressed as percent bone length increase from day Bowed bones were measured in two parts added togetherQuantitative histology and immunostaining After termination of the culture the metatarsal bones were fixed with paraformaldehyde embedded in paraffin and fivemicrometer sections were cut along the longitudinal axis proximal to distal followed by staining with SafraninO and Alcian blue The microscopic description of the growth plate morphology included an assessment of the anization of the chondrocyte column Histomorphometric analysis was performed to measure the height of the restingproliferating zone at five different regions of the growth plate and the size of hypertrophic chondrocytes Hypertrophic cells were defined by a height along the longitudinal axis greater than a0µm Eight hypertrophic chondrocytes from the proximal and distal growth plate were measuredImmunostaining was performed as previously described34 Antigen retrieval was performed in citrate buffer at ° Celsius and endogenous peroxidase activity was quenched with H2O2 in methanol for a0min followed by a wash with PBS For immunostaining sections were blocked with bovine serum albumin for a0h incubated with primary antibodies dilution Gli1 Abcam Cambridge MA USA and Ihh mouse monoclonal antibody Santa Cruz Biotechnology Dallas TX USA overnight at ° Celcius After incubation for a0h with secondary polyclonal antimouse or antirabbit biotinylated antibody DakoCytomation Glostrup Denmark dilution sections were incubated with ABC solution and developed with diaminobenzidine Sections were counterstained with Alcian blue Nonimmune immunoglobin G IgG of the same species as the primary antibodies were used as negative controls Three to five bones per group were analyzed To quantify immunostaining the ImageJ software National Institutes of Mental Health Bethesda MD USA was used and the percentage of DAB positivity was calculated digitally using a plugin IHC profilerStatistical analysis All statistics were carried out using GraphPad Prism GraphPad Software Inc La Jolla CA USA Data were summarized using means ± SD for the bone length measurements and histomorphometric assessments A twoway ANOVA with Dunnett multiple comparisons test35 was performed to examine the change in bone length in terms of treatment and days Pairwise comparisons were done corrected for the alpha levels Margin plots with SD were presented to visualize the change in bone length KruskalWallis and ManWhitney U test were performed when the data were not normally disturbed A p value of was considered to indicate a significant differenceData availabilityAll data generated or analysed during this study are included in this manuscriptScientific RepoRtS 101038s41598020699040Vol1234567890wwwnaturecomscientificreports 0cReceived March Accepted July References Gerber N et al Recent developments and current concepts in medulloblastoma Cancer Treat Rev Gorlin R J Neurocutaneous Disorders Phakomatoses and Hamartoneoplastic Syndromes Springer Berlin Gajjar A J Robinson G W Medulloblastomatranslating discoveries from the bench to the bedside Nat Rev Clin Oncol Kool M et al Molecular subgroups of medulloblastoma an international metaanalysis of transcriptome genetic aberrations and clinical data of WNT SHH Group and Group medulloblastomas Acta Neuropathol Kieran M W Targeted treatment for sonic hedgehogdependent medulloblastoma Neurooncology Ohba S Hedgehog signaling in endochondral ossification J Dev Biol Robinson G W et al Irreversible growth plate fusions in children with medulloblastoma treated with a targeted hedgehog pathway inhibitor Oncotarget Kieran M W et al Phase I study of oral sonidegib LDE225 in pediatric brain and solid tumors and a phase II study in children and adults with relapsed medulloblastoma Neurooncology Kimura H Ng J M Curran T Transient inhibition of the Hedgehog pathway in young mice causes permanent defects in bone structure Cancer Cell Ramesh S Zaman F Madhuri V Svendahl L Radial extracorporeal shock wave treatment promotes bone growth and chondrogenesis in cultured fetal rat metatarsal bones Clin Orthop RelatRes Gollwitzer H et al Radial extracorporeal shock wave therapy rESWT induces new bone formation in a0vivo results of an animal study in rabbits Ultrasound Med Biol Kertzman P Cs¡sz¡r N B Furia J P Schmitz C Radial extracorporeal shock wave therapy is efficient and safe in the treatment of fracture nonunions of superficial bones a retrospective case series J Orthop Surg Res Sansone V et al Shockwave Medicine vol Karger Publishers Basel Palmero A et al High energy shock waves enhance the cytotoxic effect of doxorubicin and methotrexate to human osteosarcoma cell lines Oncol Rep Anticancer Res Canaparo R et al High energy shock waves HESW for sonodynamic therapy effects on HT29 human colon cancer cells Gambihler S Delius M In a0vitro interaction of lithotripter shock waves and cytotoxic drugs Br J Cancer Pines M Hurwitz S The role of the growth plate in longitudinal bone growth Poult Sci Speed C A systematic review of shockwave therapies in soft tissue conditions focusing on the evidence Br J Sports Med FernandezVojvodich P Zaman F Svendahl L Interleukin6 acts locally on the growth plate to impair bone growth Ann Ciampa A R et al Nitric oxide mediates antiinflammatory action of extracorporeal shock waves FEBS Lett Rheum Dis e24e24 Chen YL et al Extracorporeal shock wave therapy effectively prevented diabetic neuropathy Am J Transl Res Zaman F et al Humanin is a novel regulator of Hedgehog signaling and prevents glucocorticoidinduced bone growth impairment FASEB J Sharpe H J et al Genomic analysis of smoothened inhibitor resistance in basal cell carcinoma Cancer Cell Wong H et al Pharmacokineticpharmacodynamic analysis of vismodegib in preclinical models of mutational and liganddependent Hedgehog pathway activation Clin Cancer Res Eriksson E et al Bortezomib is cytotoxic to the human growth plate and permanently impairs bone growth in young mice PLoS Zaman F Fadeel B Svendahl L Proteasome inhibition therapies in childhood cancer Leukemia Gonnissen A et al The hedgehog inhibitor GANT61 sensitizes prostate cancer cells to ionizing radiation both in a0vitro and in a0vivo ONE e50523 Oncotarget Zhong L Huang X Karperien M Post J N The regulatory role of signaling crosstalk in hypertrophy of MSCs and human articular chondrocytes Int J Mol Sci Roach H I Mehta G Oreffo R O Clarke N M Cooper C Temporal analysis of rat growth plates cessation of growth with age despite presence of a physis J Histochem Cytochem Gajjar A et al Phase I study of vismodegib in children with recurrent or refractory medulloblastoma a pediatric brain tumor consortium study Clin Cancer Res Singer T Appropriate Dose SelectionHow to Optimize Clinical Drug Development Springer Berlin ReaganShaw S Nihal M Ahmad N Dose translation from animal to human studies revisited FASEB J Chagin A S Chrysis D Takigawa M Ritzen E Svendahl L Locally produced estrogen promotes fetal rat metatarsal bone growth an effect mediated through increased chondrocyte proliferation and decreased apoptosis J Endocrinol Bov©e J V van den Broek L J CletonJansen AM Hogendoorn P C Upregulation of PTHrP and Bcl2 expression characterizes the progression of osteochondroma towards peripheral chondrosarcoma and is a late event in central chondrosarcoma Lab Investig Vincent K et al Aging of mouse intervertebral disc and association with back pain Bone AcknowledgementSR would like to personally acknowledge Dr Sumith K Mathew Assistant Professor Clinical Pharmacology Christian Medical College for his input on calculating for clinically relevant dose This study was supported by internal grants received from Christian Medical College Vellore Centre for Stem Cell Research Swedish Research Council Swedish Childhood Cancer Foundation HKH Kronprinsessan Lovisas f¶rening Ake Wibergs Stiftelse and Karolinska Institutet Sweden access funding provided by Karolinska InstituteAuthor contributionsSR LS VM FZ designed the study SR carried out data collection and statistical analysis SR LS VM FZ analysed data SR wrote the initial draft of the manuscript FZ VM and LS contributed to the revision of the manuscript All authors contributed to the interpretation of resultsScientific RepoRtS 101038s41598020699040Vol0123456789wwwnaturecomscientificreports 0ccompeting interests The authors declare no competing interestsAdditional informationCorrespondence and requests for materials should be addressed to SRReprints and permissions information is available at wwwnaturecomreprintsPublishers note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations Access This article is licensed under a Creative Commons Attribution International License which permits use sharing adaptation distribution and reproduction in any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the Creative Commons license and indicate if changes were made The images or other third party material in this article are included in the articles Creative Commons license unless indicated otherwise in a credit line to the material If material is not included in the articles Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtain permission directly from the copyright holder To view a copy of this license visit httpcreat iveco mmons licen sesby40 The Authors Scientific RepoRtS 101038s41598020699040Vol1234567890wwwnaturecomscientificreports 0c' | 2 |
"The cancer tissue used in this study was received from patients that had surgical resection of both the primary tumor and related metastases. None of the patients had received chemo- or radiotherapy before the resection of the primary tumor. Medical charts and pathology reports were reviewed to record clinical and pathological data. Glass slides were reviewed to determine the histological type according to the WHO classification [30]. Follow-up information including the patient outcome and the time interval between the date of surgical resection and death was collected. The cases lost to follow-up and deaths from causes other than CRC were considered censored data for the survival analysis. The median follow-up period was 37.9 months (range 0.8104.6 months). Ethical statement All human specimens were obtained from the files of surgically resected cases examined at the Department of Pathology Seoul National University Bundang Hospital for the pathologic diagnosis. The retrospective study was performed using the stored samples after the pathologic diagnosis and all of the samples were anonymized before the study. The participants did not provide written informed consent in this study. The study was approved by the Institutional Review Board of Seoul National University Bundang Hospital under the condition of anonymization (reference: B-1109/136-302). Tissue array methods To evaluate the regional stromal differences samples were taken from each patient from four areas: the center and periphery of the primary cancer distant metastases and lymph node metastases. The distant metastatic sites for the tissue arrays were as follows: liver in 83 cases (45.9%) lung in 38 cases (21.0%) seedings in 38 cases (21.0%) distant lymph nodes in 6 cases (3.3%) and ovary in 16 cases (8.3%). The representative core tissue specimens (2 mm in diameter) were taken from individual paraffin blocks and rearranged in new tissue array blocks using a trephine apparatus (Superbiochips Laboratories Seoul South Korea) [31]. Immunohistochemistry Array slides were labelled by immunohistochemistry using antibodies for CD31 (1?100 DAKO Glostrup Denmark) D2-40 (1?100 DAKO) SMA (1?1000 Neomarkers Fremont CA USA) desmin (1?300 DAKO) and PTEN (1?80 Epitomics Burlingame CA USA) after a microwave antigen retrieval procedure except SMA. Non-reactive sites were blocked using 1% horse serum in Tris-buffered saline (pH 6.0) for 3 min. Primary antibodies were applied and antibody binding was detected with diaminobenzidine (DAB). Sections were counterstained with hematoxylin. The reactivity of PTEN in each tissue section was scored as negative faint or strong and the percentage of PTEN-positive fibroblasts was quantified. For the statistical analysis the sample was deemed PTEN-positive if 5% or more CAFs were scored as strong positives. Calculation of LVD MVD and CAFs using digital pathology Slides were concurrently evaluated by two pathologists (H.E.L and H.S.L) using light microscopy to improve the accuracy of the results (Fig. 1). CRC cells were considered as internal negative controls. Medium- to large-sized vessels were considered as internal positive controls for CD31 and D2-40. Intestinal muscular layer or medium- to large-sized vessels were considered as internal positive controls for desmin and SMA. Samples showing inappropriate staining in internal negative or positive controls were considered non-informative and were excluded from the analysis. Slides were scanned using an Aperio ScanScope® CS instrument (Aperio Technologies Inc. Vista CA) at 20 magnification. Subsequently they were analyzed in ImageScope¢ using the Microvessel Analysis v1 algorithm (Aperio Technologies) and MVD and LVD were calculated. Because desmin-positive muscularis mucosa and propria are positive for SMA immunostaining the area of CAFs (mm2) was calculated by subtracting the areas of desmin staining from that of SMA staining (SMA - desmin). .0091811.g001 Representative sections from four tumor locations stained with CD31 D2-40 SMA or desmin antibodies (100). Statistical analysis A chi-squared test or Fisher's exact test (2-sided) for non-continuous variables and Mann-Whitney or Kruskal-Wallis analysis for continuous variables were used to compare each parameter with respect to the CRC site and according to its clinicopathologic features. The correlation between continuous variables was analyzed using the Pearson correlation coefficient. To determine the best cut-offs of continuous variables for predicting patient survival the maximal chi-squared method was performed using R program (http://cran.r-project./). The overall survival curves were plotted using the Kaplan-Meier product-limit method and the significance of the differences between these curves was determined using the log-rank test. A univariate and multivariate regression analysis was performed using the Cox's proportional hazards model to determine hazard ratios (HRs). P-values of less than 0.05 were considered statistically significant. All statistical analysis excluding the maximal chi-squared test was performed with the IBM SPSS statistics 20 (Armonk NY USA). Results 1. Heterogeneity of cancer-associated stroma according to examined tumor locations The clinicopathological characteristics of the advanced CRC patients are described in . The CRC patients with synchronous metastases had aggressive features including larger tumor size more advanced pT and pN stage and the presence of perineural and venous invasion than the patients with metachronous metastasis (p<0.05). .0091811.t001 Clinicopathologic characteristics of advanced colorectal cancers. Parameters Total Metachronous Synchronous P value (n?=?181) (n?=?57) (n?=?124) Age (median range) 60.00 (2893) 62.00 (3679) 60.00 (2893) 0.241 Sex 0.007 Male 97 39 (68.4%) 58 (46.8%) Female 84 18 (31.6%) 66 (53.2%) Location 0.055 Right colon 37 6 (10.5%) 31 (25.0%) Left colon 75 29 (50.9%) 46 (37.1%) Rectum 69 22 (38.6%) 47 (37.9%) Size of primary tumor 5.30 (2.013.0) 4.20 (29) 5.50 (2.513) <0.001 Histologic grade 0.227 Low grade 157 52 (91.2%) 105 (84.7%) High grade 24 5 (8.8%) 19 (15.3%) T stage <0.001 T1 0 0 0 T2 5 3 (5.3%) 2 (1.6%) T3 107 45 (78.9%) 62 (50.0%) T4 69 9 (15.8%) 60 (48.4%) N stage <0.001 N0 35 23 (40.4%) 12 (9.7%) N1 58 23 (40.4%) 35 (28.2%) N2 88 11 (19.3%) 77 (62.1%) Perineural invasion 0.011 Absent 89 36 (63.2%) 53 (42.7%) Present 92 21 (36.8%) 71 (57.3%) Venous invasion 0.028 Absent 126 46 (80.7%) 80 (64.5%) Present 55 11 (19.3%) 44 (35.5%) The heterogeneous values for LVD MVD and CAF area are shown in Fig 2. LVD was the highest in center of the primary cancers (median interquartile range (IQR); 37.00 10.5081.00) than any other site (5.00 1.0023.75 at the periphery; 2.50 1.0015.00 in lymph node metastases; 3.00 1.0020.00 in distant metastases). MVD was lower in distant metastases (median IQR; 641.50 428.001006.75) than at the periphery of the primary cancer (731.00 508.251049.75) and lymph node metastases (893.50 520.251275.25). The area occupied by CAFs was the lowest in the distant metastases (median IQR; 0.91 0.681.18) than any other site (1.12 0.881.41 in the center; 1.22 0.961.54 in the periphery1.4 1.001.71 in lymph node metastases). In addition the stromal characteristics varied in relation to the metastatic an examined. MVD and LVD were the higher in lung metastases than those in the liver peritoneum or lymph nodes (p<0.001; Fig. 3). However the amounts of CAFs were consistent among the different metastatic ans (p?=?0.180). .0091811.g002 Heterogeneity of lymphatic vessel density (LVD) microvessel density (MVD) and amount of cancer-associated fibroblasts (CAFs) with respect to tumor location. The LVD (A) MVD (B) and CAF area (C) was significantly different according to each tumor location. .0091811.g003 LVD MVD and CAF area at different distant metastasis sites. The characteristics of cancer-associated stroma differed with respect to the metastatic site. LVD (A) and MVD (B) were greater in the metastatic tumor samples collected from the lung than in samples collected from other metastatic sites (p<0.001). However the amount of CAFs was not significant different between metastatic sites (C). Despite the heterogeneity of stromal characteristics CRC cases with higher LVD MVD and CAFs in center of the primary cancers had a tendency of higher LVD MVD and CAFs in periphery (p<0.05; Table S1). However LVD in center and periphery of primary cancer were not correlated with LVD in related distant metastasis (Table S1). In addition the amount of microvasculature was significantly correlated with the amount of CAFs (Table S2). 2. Clinical significance of cancer-associated stroma in advanced CRCs The MVD LVD and amount of CAFs present at each tumor location were compared according to their clinicopathologic features (Table 2). High grade CRCs were associated with lower CAFs in samples taken from the central cancer site (p?=?0.041). When compared with synchronous metastases the patients with metachronous metastases had higher LVD in center and periphery of the primary cancer and had higher MVD in lymph node metastases. Most patients with metachronous metastases were treated by adjuvant chemotherapy before metastasectomy. LVD and MVD in the distant metastases were significantly higher in the patients who had received chemotherapy before metastasectomy than those who did not (p?=?0.011 and 0.048 respectively). .0091811.t002 Table 2 Clinicopathologic factor and LVD MVD and CAFs. Center (median) Periphery (median) LN metastasis (median) Distant metastasis (median) LVD MVD CAFs LVD MVD CAFs LVD MVD CAFs LVD MVD CAFs Total 39 717 1.13 5 740 1.22 3 888 1.42 3 648 0.91 Histologic grade Low grade 40 717 1.15* 5 741 1.23 3 895 1.43 3 665 0.92 High grade 34 683.5 0.94* 6 643.5 1.18 2 656 1.32 6 498 0.82 pT stage pT2 34 758 1.15 16 870 1.48 6 772 0.73 pT3 47 737 1.19 5 803 1.22 2.5 884 1.43 3 724 0.92 pT4 33 639 1.09 4 630 1.22 3 895 1.41 3 520 0.93 LN metastasis Absent 49 602 1.15 8 712 1.42 4 772 0.94 Present 39 737.5 1.12 4 740 1.21 3 884 1.41 3 617 0.91 Perineural invasion Absent 41 738 1.12 6 772 1.32 5.5 931.5 1.42 4 687 0.94 Present 39 672 1.13 4 702 1.2 2 796 1.39 3 548.5 0.86 Metastasis Synchronous 34* 717.5 1.11 3.0* 741 1.21 3 797* 1.39 3 617 0.93 Metachronous 55* 716 1.21 8.0* 712 1.23 2 1117* 1.63 5 698 0.91 Chemotherapy Not done 2.0* 597.5* 0.93 Done 10.0* 684* 0.91 * p<0.05; ** p<0.01; chemotherapy prior to metastatectomy of distant metastasis. 3. Expression loss of PTEN in CAFs PTEN was expressed in cytoplasm and sometimes the nucleus of both cancer and non-neoplastic cells when examined using immunohistochemistry. Expression of PTEN was lost in 8 cases in the center 2 cases in the periphery 4 cases in lymph node metastases and 11 cases in distant metastases (Table S3). In all 11 distant metastases with PTEN loss PTEN expression was intact in both the center and periphery of primary cancer (data not shown). PTEN loss in distant metastasis was correlated with synchronous metastasis (p?=?0.018). 4. Cancer-associated stroma and patient prognosis By using the obtained cut-offs lower LVD MVD and CAFs in the center LVD and CAFs in the periphery and MVD and CAFs in distant metastases were all significantly correlated with lower survival (p<0.05; Fig. S1). Among other clinicopathologic features synchronous metastasis old age larger size high histologic grade advanced pT and pN stage and presence of perineural invasion were associated with a worse prognosis (Table 3). By multivariate Cox regression analysis the hazard ratio of synchronous versus metachronous was the highest (4.029) with the lowest p value (p<0.001). CAFs in distant metastasis LVD and MVD in the center LVD in the periphery age and perineural invasion also independently predicted patient survival. In addition loss of PTEN expression in CAFs in distant metastases was associated with a worse prognosis (p?=?0.042; Fig S2) but not in primary cancer or lymph node metastasis. .0091811.t003 Table 3 Univariate and multivariate survival analysis according to clinicopathologic features. Univariate survival analysis Multivariate survival analysis Factors HR (95% CI) P value HR (95% CI) P value Synchronous vs. Metachronous 4.617 (2.4728.624) <0.001 3.762 (1.8387.701) <0.001 Age 1.023 (1.0041.044) 0.020 1.033 (1.0111.056) 0.003 Sex (female vs. male) 1.428 (0.9202.218) 0.113 Location (left vs. right) 0.503 (0.3140.806) 0.004 0.700 (0.4131.188) NS (0.186) Size 1.073 (1.0051.146) 0.036 1.040 (0.9031.198) NS (0.584) Histologic grade (high vs. low) 1.862 (1.0613.269) 0.030 1.491 (0.7632.912) NS (0.243) pT stage (pT4 vs. pT2/3) 2.341 (1.5033.645) <0.001 1.137 (0.6741.921) NS (0.630) pN stage (pN1/2 vs. pN0) 3.848 (1.7608.411) 0.001 1.773 (0.7584.146) NS (0.186) Perineural invasion 2.628 (1.6404.211) <0.001 2.108 (1.2653.513) 0.004 Venous invasion 1.217 (0.7571.956) 0.418 Center LVD (high vs. low) 0.364 (0.1580.836) 0.017 0.298 (0.1180.753) 0.010 Center MVD (high vs. low) 0.391 (0.2330.655) <0.001 0.437 (0.2380.801) 0.007 Center CAFs (high vs. low) 0.579 (0.3520.954) 0.032 1.038 (0.6071.773) NS (0.892) Periphery LVD (high vs. low) 0.235 (0.0860.644) 0.005 0.279 (0.0960.809) 0.019 Periphery MVD (high vs. low) 1.456 (0.9112.327) 0.117 Periphery CAFs (high vs. low) 0.524 (0.3360.817) 0.004 0.813 (0.4991.326) NS (0.406) LN LVD (high vs. low) 1.646 (0.8743.100) 0.123 LN MVD (high vs. low) 0.597 (0.2941.213) 0.154 LN CAFs (high vs. low) 0.717 (0.4231.217) 0.218 Metastasis LVD (high vs. low) 0.569 (0.3141.032) 0.063 Metastasis MVD (high vs. low) 0.579 (0.3640.921) 0.021 1.262 (0.7202.211) NS (0.417) Metastasis CAFs (high vs. low) 0.492 (0.2710.894) 0.020 0.290 (0.1440.582) 0.001 Metastasis PTEN (intact vs. loss) 0.454 (0.2080.993) 0.048 0.575 (0.2391.383) NS (0.217) Discussion Carcinoma cells in different tissue areas have distinct characteristics [32]. In central areas of the tumor carcinoma cells maintain an epithelial cell phenotype but carcinoma cells in the invasive front acquire a more malignant and mesenchymal phenotype and are thought to have an increased migratory capacity and contribute to metastatic diseases. These metastatic cells may restore the epithelial phenotype at metastatic sites [33]. In addition to carcinoma cells themselves microenvironment is suggested to be uneven within a given tumor because tumor formation and progression involve the co-evolution of cancer cells and microenvironments [34]. The present study demonstrated that the cancer-associated microenvironment also had distinct characteristics in different areas. Of the sites examined LVD was highest in the center of the primary cancer. MVD was slightly higher in center than at the periphery of the primary cancer but this difference was not statistically significant. Interestingly the amount of CAFs in distant metastases was significantly lower than in center and periphery of the primary cancer. We show that the stromal microenvironment has regional heterogeneity both within the primary tumor and between the primary site and its related metastases. Furthermore our data suggests that the stromal heterogeneity might be attributable to tumor heterogeneity. Therefore it would be beneficial to consider both stromal and tumor cell heterogeneity in order to manage CRC patients better. We evaluated the MVD LVD and amount of CAFs in metastatic tissues of various ans including the liver lung peritoneal seeding distant lymph nodes and ovary. Of the metastatic ans we examined both LVD and MVD were the highest in lung. In our previous study the KRAS discordance rate was also significantly higher in matched lung metastases than in other matched metastatic ans [35]. The underlying mechanism is not known. It could be that primary CRCs with high LVD and MVD have a tendency to produce lung metastases; however our results indicated that LVD and MVD in the center and at the periphery of the primary cancers were lower in the patients with lung metastases (data not shown). Alternatively it may be due to the physiological characteristics of metastatic ans interactions between cancer cells and microenvironment within the metastatic an or the characteristics of the cancer cell clones prone to lung metastasis. However technical or sampling errors also may be possible thus further large-scale studies are required. Although numerous studies have attempted to demonstrate an association between tumor microenvironment characteristics and survival the prognostic impacts of MVD and LVD are still controversial. Some studies have been presented that active angiogenesis and lymphangiogenesis represented by high MVD and LVD are associated with poor prognosis and aggressive clinicopathologic factors [36] [37]. Recent meta-analysis has demonstrated that LVD was significantly associated with disease-free survival but not overall survival [38]. Other studies have reported no statistical significance of MVD and LVD on survival [39]. Prall et al. has reported that high MVD and LVD are related with better survival in a consecutive series and liver metastases [40]. Our results were based on patients with advanced disease with distant metastasis and we showed that high MVD and LVD were related with improved survival. This might be because all the patients in this study had confirmed to have distant metastasis and microvasculatures could influence even delivery of the chemotherapeutic drug into the tumor. However our study had some limitations in terms of the survival analysis. We enrolled the CRC patients with available surgically resected cancer tissues from both primary tumors and corresponding metastatic tumors. Not all advanced CRC patients with metastatic diseases were included and far advanced cases were not enrolled because of their inoperability. Therefore unrecognized biases might have influenced our survival results. Some studies have demonstrated an anti-tumorigenic effect of fibroblasts [20] [21]. However it has become clear that CAFs contribute to the progression of cancer and their prognostic significance in various cancers also has been raised [41] and furthermore several studies have observed genetic alterations in CAFs [26] [27]. PTEN loss of CAFs has been observed in breast cancer and prognostic association of it has been suggested [27] [28]. " | 1 |
" the ampactivated protein kinase ampk is an evolutionarily conserved regulator of cellular energyhomeostasis as a nexus for transducing metabolic signals ampk cooperates with other energysensing pathwaysto modulate cellular responses to metabolic stressors with metabolic reprogramming being a hallmark of cancerthe utility of agents targeting ampk has received continued scrutiny and results have demonstrated conflictingeffects of ampk activation in tumorigenesis harnessing multiomics datasets from human tumors we seek toevaluate the seemingly pleiotropic tissuespecific dependencies of ampk signaling dysregulationmethods we interrogated copy number variation and differential transcript expression of ampk pathway genesacross diverse cancers involving over patients cox proportional hazards regression and receiver operatingcharacteristic analyses were used to evaluate the prognostic significance of ampk dysregulation on patientoutcomesresults a total of and seven ampk pathway genes were identified as having loss or gainoffunction featuresthese genes exhibited tissuetype dependencies where survival outcomes in glioma patients were most influencedby ampk inactivation cox regression and logrank tests revealed that the 24ampkgene set could successfullystratify patients into high and lowrisk groups in glioma sarcoma breast and stomach cancers the 24ampkgeneset could not only discriminate tumor from nontumor samples as confirmed by multidimensional scaling analysesbut is also independent of tumor node and metastasis staging ampk inactivation is accompanied by the activationof multiple oncogenic pathways associated with cell adhesion calcium signaling and extracellular matrixanization anomalous ampk signaling converged on similar groups of transcriptional targets where a commonset of transcription factors were identified to regulate these targets we also demonstrated crosstalk between procatabolic ampk signaling and two proanabolic pathways mammalian target of rapamycin and peroxisomeproliferatoractivated receptors where they act synergistically to influence tumor progression significantlycontinued on next page correspondence alvinalaiuclacukinstitute of health informatics university college london euston roadlondon nw1 2da uk the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cchang and lai bmc cancer page of continued from previous page genetic and transcriptional aberrations in ampk signaling have tissuedependent pro or antitumorimpacts pancancer investigations on molecular changes of this pathway could uncover novel therapeutic targetsand support risk stratification of patients in prospective trialskeywords ampk glioma lossoffunction tumor metabolism pancancer the ampactivated protein kinase ampk is an evolutionary conserved key player responsible for energy sensing and homeostasis orthologous copies of ampkprevail universally as heterotrimeric complexes wherethe human genome encodes two genes for the α catalyticsubunit two regulatory subunit genes and three Πsubunit genes historically ampk was discovered as a crucial regulator of lipid metabolism since then ampkis implicated in a wide variety of fundamental metabolicprocesses as well as in metabolic diseases such as cancerand diabetes the first link between ampk and cancer was identified through the tumorsuppressive function of lkb1 which is upstream of the mtor pathway theroles of ampk werepharmacologically demonstrated by the application ofmetabolic inhibitors such as the antidiabetic metforminand the mimetic of amp aicar [] numerousstudies have since compellingly established the promiscuous nature of these pharmacological agents wherebythe inhibition of cancer cell proliferation occurs throughnonspecific ampkindependent avenues [ ]tumorsuppressivestressorssuchasin contrastof metabolicto the tumorsuppressive results frompharmacological studies genetic experiments on cancercells have credibly demonstrated that ampk activationis crucial for tumor progression and survival [] amyriadoxygendeprivation nutrient starvation and oxidative stress exists within the tumor microenvironment metabolic reprogramming during carcinogenesis would thus triggerampk activation to enable cells to survive under conditions of stress typically found in the tumor microenvironment hence conferring an overall tumorpromotingeffect ampk is also shown to support cancer growthand migration through crosstalk with other prooncogenicpathways for instance overexpression of oncogenes mycand src or the loss of the tumor suppressor folliculincould lead to ampk activation []genetic and pharmacological studies have paved theway for our understanding of the function of ampk incancer however anti and proneoplastic features ofampk remain controversial potentially due to the oversimplification of ampkmodulated processes in in vitroand nonhuman invivo models the genetic and clinicallandscape of ampk signaling has not been systematically investigated thus our study aims to address anunmet need to rigorously investigate the role of ampkin diverse cellular context using multiomics data fromactual tumors where we examined somatic copy numberalterations transcriptional and clinical profiles of tumorsfrom cancer types our analyses of clinical samples atscale would complement evidence from pharmacologicaland genetic studies to better elucidate the multifacetedand cellspecific nature of ampk signaling on tumorprogressionmethodsampk pathway genes and cancer cohortsninetytwo ampk pathway genes were retrieved fromthe kyoto encyclopedia of genes and genomes keggdatabase additional file clinical genomic and transcriptomic datasets of cancers involving patients were downloaded from the cancer genome atlastcga copy number variation differential expressionmultidimensional scaling and survival analysesdetailed methods of the above analyses were previouslypublished and thus will not be repeated here as per the guidelines [] to summarize discrete amplification and deletion indicators for copy number variation analyses were obtained from gistic geneleveltables gistic values of and were annotatedas shallow amplification and shallow deletion heterozygous events respectively gistic values of and were annotated as deep amplification and deep homozygous deletion events respectively multidimensionalscaling analyses and permutational multivariate analysisof variance permanova were performed using the rvegan package survival analyses were performed usingcox proportional hazards regression and the logranktest sensitivity and specificity of the 24ampkgene setwere assessed using receiver operating characteristicanalyses differential expression analyses were performed on patients stratified into high 4th quartileand low 1st quartile expressing groups using the genesetto determine the transcriptional effects ofanomalous ampk signalingpathway and transcription factor analysesgenes that were differentially expressed degs betweenthe 4th and 1st quartile patient groups were mapped to 0cchang and lai bmc cancer page of kegg gene ontology and reactome databases using gprofiler to ascertain biological processes and signaling pathways that were enriched the enrichr tool was used to map degs to the chea and encodetranscription factor tf databases to identify tfs thatwere significantly enriched as regulators of the degsirs2pik3r1sirt1 tbc1d1calculating the 24ampkgene score peroxisomeproliferatoractivated receptors ppar score andmammalian target of rapamycin mtor scoreampk scores were calculated from the mean expressionthe following genes slc2a4 foxo3 ppp2cbofpik3cd cab39l ccna1 fbp1 fbp2 foxo1 hmgcrppargc1appp2r2c mlycd pfkfb3 ppp2r2b prkaa2 leprcab39 irs1 and pfkfb1 ppar scores for each patientwere calculated by taking the mean expression of pparsignature genes plin5 pparg acadm gk cpt2scp2 acaa1 apoa1 ppara acox2 angptl4fabp3 plin2 aqp7 acsl1 fabp5 acadl andpck2 mtorpi3kakt scores for each patientwere calculated using the following equation mtorpi3kakt score akt mtor gsk3 s6k s6 pten all figures were generated using r version andadobe illustrator version cs6resultspancancer genomic and transcriptional alterations ofampk pathway genesfocusing on the genomic and transcriptomic landscapeof genes associated with ampk signaling retrievedfrom kegg across cancer types involving patients additional file we interrogated somatic copynumber alterations scna and mrna expression seeadditional file for a flowchart illustrating the study design to determine the effects of genomic alterations inampk pathway genes we classified genes as havinghighlevel amplifications gains lowlevel amplificationsdeep homozygous deletions and shallow heterozygousdeletions to evaluate pancancer patterns of scnaswe considered genes that were gained or lost in at least of samples within a cancer type and in at least onethird of cancer types ie at least seven cancer types atotal of genes were recurrently amplified while genes were recurrently lost fig additional file ampk is the central regulator of cellular energy levelswhich controls a number of downstream targets an example being the nuclear receptor hnf4a remarkablyhnf4a was found to be the most amplified gene identified as being recurrently amplified in of samplesin all cancers fig additionalfile this isfollowed by cftr cancer types and four other genesthat were amplified in cancer types adipor2 lepfile fig additionalprkag2 and rhebincontrast ppp2r2a was the most deleted gene found in of samples across cancers followed by the deletion of slc2a4 in cancers and five additional genesfoxo3 ppp2cb ppp2r2d ppp2r5c and ppp2r5ein cancer types fig additional file among allcancer types the highest number of amplified ampkpathway genes was observed in esophageal carcinomaesca genes followed by bladder cancer blca genes and lung cancer genes in both lung squamouscell carcinoma [lusc] and adenocarcinoma [luad]fig glioma tumors gbmlggin contrast hadonly five genes that were recurrently amplified fig in terms of somatic deletions lusc and esca both had genes deleted while no recurrent deletions were observed in papillary renal cell carcinoma kirp fig lossoffunctionevents differentialwe reasoned that scnas associated with transcriptional alterations could be considered as putative gainorexpressionanalyses between tumor and nontumor samples in eachcancer revealed that and genes were significantlyupregulated and downregulated in at least seven cancertypes respectively additional file of these differentially expressed genes seven and genes were alsorecurrently amplified and deleted respectively venn diagram in fig both gene sets were mutually exclusiveie the genes either had gainorfunction or lossoffunction features but not bothmolecular underpinnings of patient survival involvingputative lossoffunction ampk pathway componentswe next investigated the impact of transcriptional dysregulation ofthe putative gain and lossoffunctiongenes identified previously on patient survival outcomesacross all cancer types employing cox proportional hazards regression we observed that all genes sevengainoffunction and lossoffunction genes wereprognostic in at least one cancer type fig 2a thehighest number of prognostic genes was observed inglioma gbmlgg tumors genes while none ofthe genes were significantly associated with overallsurvival outcomes in esca and cholangiocarcinomachol fig 2a intriguingly although esca had thehighest number of scnas fig none of the genesharbored prognostic information suggesting that alterations in ampk signaling components have minimalroles in driving tumor progression and patient outcomes fbp1 was significantly associated with overallsurvival outcomes in cancers while ppp2r2c andppp2r2b in cancers fig 2a fbp2 is the least prognostic gene in only one cancer type cervical squamouscell carcinoma and endocervical adenocarcinoma cescfig 2a 0cchang and lai bmc cancer page of fig the landscape of somatic copy number alterations of ampk pathway genes heatmaps depict a fraction of samples within each cancertype that harbor somatic deletions and b somatic amplifications fortynine genes are recurrently deleted in at least of tumors within eachcancer and in at least seven cancer types fortysix genes are recurrently amplified in at least of tumors within each cancer and in at leastseven cancer types stacked bar charts on the yaxes illustrate the fraction of samples that possess copy number variation of a gene underconsideration grouped by shallow and deep deletions or amplifications stacked bar charts on the xaxes illustrate the fraction of samples withineach cancer type that contain shallow and deep deletions or amplifications the bar charts on the right of each heatmap depict the number ofcancer types with at least of samples affected by gene deletions and amplifications the venn diagrams demonstrate the identification of putative loss and seven gainoffunction genes from gene sets that are somatically altered and differentially expressed cancer cohorts analyzedwith corresponding tcga abbreviations are listed in parentheses bladder urothelial carcinoma blca breast invasive carcinoma brca cervicalsquamous cell carcinoma and endocervical adenocarcinoma cesc cholangiocarcinoma chol colon adenocarcinoma coad esophagealcarcinoma esca glioblastoma multiforme gbm glioma gbmlgg head and neck squamous cell carcinoma hnsc kidney chromophobekich pankidney cohort kipan kidney renal clear cell carcinoma kirc kidney renal papillary cell carcinoma kirp liver hepatocellularcarcinoma lihc lung adenocarcinoma luad lung squamous cell carcinoma lusc pancreatic adenocarcinoma paad sarcoma sarcstomach adenocarcinoma stad stomach and esophageal carcinoma stes and uterine corpus endometrial carcinoma ucec number ofsamples for each cancer type are indicated in parentheses blca brca cesc chol coad esca gbm gbmlgg hnsc kich kipan kirc kirp lihc luad lusc paad sarc stad stes and ucec given the prevalence of lossoffunction phenotypes indetermining clinical outcomes fig 2a we proceeded toexamine the combined impact of all lossoffunctiongenes on patient survival and oncogenic dysregulationto determine the extent of ampk pathway variationacross the cancers we calculated pathway scores foreach of the tumor samples by taking the meantranscript expression values of the genes slc2a4foxo3 ppp2cb pik3cd cab39l ccna1 fbp1fbp2 foxo1 hmgcr irs2 pik3r1 sirt1 tbc1d1ppargc1a ppp2r2c mlycd pfkfb3 ppp2r2bprkaa2 lepr cab39 irs1 and pfkfb1 we observedinteresting patterns when cancers were ranked from lowto high based on their median pathway scores fig 2bgbmlgg had the highest median pathway score whileblca and cesc were found at the lower end of thespectrum fig 2b as expected kaplanmeier analysisrevealed a significant difference in overall survival between glioma patients p stratified by low andhigh 24gene pathway scores fig 2c interestingly thecontribution of ampk signaling in cancer prognostication is cancertype dependent as in gliomalogranktests revealed that patients with high 24gene scores hadsignificantly improved survival outcomes in breast cancer p and sarcoma p fig 2c incontrast high expression of the genes was associated 0cchang and lai bmc cancer page of fig prognostic significance of ampk loss and gainoffunction genes a heatmap illustrates significant hazard ratio values from coxproportional hazards regression analyses on the lossoffunction and seven gainoffunction genes across all cancers b the distributions of ampkgene scores in each cancer are illustrated in the boxplot cancers are sorted from low to high median scores refer to fig legend forcancer abbreviations c kaplanmeier analyses and logrank tests revealed the prognostic significance of the 24ampkgene set in four cancertypes patients are stratified into q1 1st quartile and q4 4th quartile groups based on their 24gene scores for logrank tests dmultidimensional scaling analyses of the 24gene set depicted in 2dimensional space significance differences in the distribution between tumorand nontumor samples are confirmed by permanovawith increased mortality rates in stomach adenocarcinoma p fig 2c these results were in agreement when independently validated using the coxregression approach breast hazard ratio [hr] p glioma hr p sarcomahr p and stomach hr p cancers additionalfile since the 24genescore could be used to stratify patients into high andlowrisk groups we predict that when considered together gene expression values could discriminate tumorfrom nontumor samples although analysis could notbe performed on sarcoma this dataset only had twonontumor samples multidimensional scaling analysesand permanova tests of breast p gliomap and stomach p cancers revealed significantseparation between tumor and nontumor 0cchang and lai bmc cancer page of samples in twodimensional space fig 2d overall thissuggests that the 24gene set could be harnessed as adiagnostic biomarker for early cancer detectionconsistent with our previous observation that highpathway scores were associated with good prognosis insarcoma fig 2cto determine the independence of the 24gene setfrom other clinicopathologicalfeatures we employedmultivariate cox regression and observed that the gene set is independent of tumor node and metastasistnm staging where available in breast hr p and stomach cancers hr p additional file similarly kaplanmeier analyses andlogrank tests confirmed that the 24gene set allowedfurther risk stratification of patients with tumors of thesame tnm stage breast p and stomach p fig 3a furthermore we observed that within ahistological subtype of sarcomaleiomyosarcoma patients with elevated ampk signaling had significantlybetter3asurvival outcomesp figexploredthepredictivewe nextperformancesensitivity versus specificity of the 24gene set in allfour cancer types using receiver operating characteristicanalysis the area under the roc curve auc is an indication of how well the gene set could predict patientsurvival which ranges from to we found that thecombined model uniting both 24gene set and tnm staging outperformed the 24gene set when considered onits own in breast cancer patients auc vs fig 3b for stomach cancer the 24gene set onlycontributed to a marginally higher auc when used incombination with tnm staging when compared to the24gene set alone auc vs fig 3baucs of the 24gene set in glioma and sarcoma werefig the 24ampkgene set is independent of tumor stage and histological subtype a kaplanmeier analyses of patients grouped by tumornode and metastasis tnm stage breast and stomach cancers or by the histological subtype of leiomyosarcoma and the 24gene score forleiomyosarcoma the logrank test reveals a significant difference in survival rates between 1st and 4th quartile patients b receiveroperatingcharacteristic roc analyses on the 5year predictive performance of the 24gene set roc curves generated by the 24gene set are compared tocurves generated from both 24gene set and tnm staging where available or histological subtype auc area under the curve 0cchang and lai bmc cancer page of and respectively fig 3b within the leiomyosarcoma histological subtype auc was even higherat fig 3boncogenic transcriptional alterations associated withampk pathway inactivationampk pathway inactivation was associated with alteredsurvival outcomes in patients figs and we predictthat this could be due to broad transcriptional dysregulation arising from abnormal ampk signaling to investigate this phenomenon we performed differentialexpression analyses between patients stratified by the24gene set into high 4th quartile and low 1st quartileexpression groups and found that an outstanding number of common genes that were significantly differentially expressed in all four cancer types fig 4a thehighest number of differentially expressed genes degswas observed in stomach cancer genes followedby sarcoma genes glioma genes and breastcancer genes fig 4a additionalfile thedegs were mapped to kegg gene ontology andreactome databases to determine whether they were associated with any functionally enriched pathways intriguingly all four cancer types share similar patterns offunctional enrichments fig 4b and c for instance biological processes associated with cell communicationsignal transduction cell differentiation cell signalingcell adhesion and cell morphogenesis were enriched inall four cancers fig 4c in terms of specific signalingpathways calcium signaling camp signaling and processes associated with extracellular matrix anizationwere among the most enriched fig 4cto further identify potential transcriptional regulatorsof the degs we mapped the degs to encode andchea transcription factor tf binding databases remarkably we identified common tfs shared across allfour cancers that were implicated as direct binding partners of the degs fig 4c five tfs suz12 smad4rest ezh2 and nfe2l2 were found to be enriched inall four cancers suggesting that transcriptional dysregulation of tumors with aberrant ampk signaling involveddirect physical associations of these tfs with targetdegs fig 4c curiously foxm1 and e2f4 wereenriched only in glioma tumors which deserves furtherexploration in the next section overall our analysesdemonstrated that impaired ampk signaling resulted incommon patterns of oncogenesis which affect the severity of cancer and consequently mortality ratesinpatientsdownstream targets of ezh2 nfe2l2 rest smad4 andsuz12 were associated with survival outcomespathways modulating energy homeostatic may transducesignals to influence other cognate signaling modulesezh2 nfe2l2 rest smad4 and suz12 were all implicated as common transcriptional regulators of degsin glioma sarcoma breast and stomach cancers suggesting that altered ampk signaling converged on similargroups of transcriptional targets of all the target degsof the aforementioned tfs and geneswere found to be common targets of ezh2 nfe2l2rest smad4 and suz12 respectively in all four cancers fig 5a concatenating all five gene sets yielded unique genesie genes that were binding targets ofmore than one tf were considered only once to gainfurther insights into how ampk inactivation influencestumor progression we performed cox regression analyses to determine the association between each of the genes and survival outcomes the highest number ofprognostic genes was observed in glioma genes good prognoses and five adverse prognoses fig 5b incontrast out of genes were associated with adverseprognosis in stomach cancer fig 5b these observations were consistent with the 24ampkgene set beingpositive and negative prognostic factors in glioma andstomach cancer respectively fig which mirrored thebehavior of degs identified as a result of aberrantampk signaling fig 4c of the genes only andten were significantly associated with survival outcomesin sarcoma and breast cancer respectively fig 5b collectively our results suggest that the ampk pathwayand its interaction with other signaling modules are keydeterminants of patient outcomes in multiple cancertypesprognostic significance of joint ampk pathway activityand transcriptional levels of five oncogenic tfs inpatients with gliomahaving discovered the importance of the 24ampk geneset we sought to explore the crosstalk between ampksignaling and tf activity in glioma as previously mentioned glioma had the highest 24ampkgene scorefig 2b with a vast majority of the genes conferringprognostic information fig 2a moreover of the transcriptional targets of the five common tfs identifiedin patients with altered ampk signaling were significantly associated with survival outcomes in glioma fig5b additionally tfs foxm1 and e2f4 were identifiedto be enriched only in glioma tumors fig 4c thus wepredict that a joint model uniting ampk and tf expression profiles would allow further delineation of patientsinto additional risk groups and if so allowing combinedtargeting of ampk and candidate tfs for therapeuticaction as done previously we calculated ampk scoresfor each patient based on the mean expression of the genes interestingly we found that ampk scores weresignificantly negatively correlated with tf expressionlevels in glioma e2f4 rho p ezh2 0cchang and lai bmc cancer page of fig see legend on next page 0cchang and lai bmc cancer page of see figure on previous pagefig ampk inactivation drives oncogenic transcriptional alterations in diverse biological processes and signaling modules a venn diagramillustrates the number of differentially expressed genes degs between 1st and 4th quartile patients as stratified using the 24ampkgene set infour cancer types a total of degs were common in all four cancers b dot plots depict the number of significantly enriched pathways andbiological processes upon the mapping of degs to kegg gene ontology and reactome databases each dot represents an enriched event contologies that exhibit similar patterns of enrichment across four cancers are shown degs are also mapped to encode and chea transcriptionfactor tf databases to determine enriched tf binding associated with degsrho p foxm1 rho p smad4 rho p and suz12rho p fig 6a we subsequentlycategorized patients into four groups using the mediancutoff of the ampk scores and tf expression values lowlow highhigh low ampk score and high tfexpression and high ampk score and low tf expression logrank tests revealed that patients stratified intothe four groups had survival rates that were significantlydifferent e2f4 p ezh2 p foxm1p smad4 p and suz12 p fig 6b for e2f4 ezh2 foxm1 and suz12patients with low ampk scores and high tf expressionperformed the worst e2f4 hr p ezh2 hr p foxm1 hr p and suz12 hr p fig6c for smad4 patients within the lowlow categoryhad the highest mortality rates hr p fig 6ccrosstalk between ampk and other anabolicrelatedpathways ppar and mtorampks antianabolic and procatabolic activities maywork in concert with other metabolic pathways toinvestigate the synergistic effects of ampk and two proanabolic pathways peroxisome proliferatoractivated receptors ppar and mammalian target of rapamycinmtor signaling in tumor progression we calculatedppar and mtor pathway scores detailed in themethods section for each glioma tumor low ampkscores were associated with poor outcomes in gliomafig to evaluate ampk and ppar or mtor ascombined models patients were separated into fourgroups using the median cutoff as mentioned previously interestingly when ampk and ppar scores werecollectively used for patient stratification we found thatpatients with low ampk and high ppar scores had thehighest death rates hr p confirmingthat ppar hyperactivation is associated with poor outcomes in glioma tumors with low ampk activity fig in contrast when considering mtor activitypatients with low ampk and low mtor scores performed the worst hr p fig theresults overall suggest that the ampk pathway could actsynergistically with ppar and mtor signaling to influence cancer progression significantlydiscussionwhile the role of ampk in energysensing is wellunderstood its full potential in metabolic diseases suchas cancer remains an open topic of debate despite extensive efforts spent on elucidating the role of ampksignaling [ ] there remains no consensus onwhether ampk promotes or suppresses tumor progression exploiting a rich reservoir of pancancer datasetsafforded to us by tcga we performed a thoroughexamination of genomic and transcriptomic profiles of ampk pathway genes in diverse cancer types ourcurrent understanding of ampk signaling is fueled bygenetic studies in cell lines and animal models although useful in determining causal relationships resultsfrom in vitro cell lines and animal models may have limited translational relevance as they do not accurately reflect human pathology animal models may offeradditional mechanistic insights but limitations in ethicsand costs remain moreover the complexity of humancancers is not accurately modeled in animals less than of results from animal models are translated to clinical trials despite analyses on tumor genetic datasets providing mostly correlative outcomes they remainvaluable in understanding diseasespecific molecularpathology when interrogated at scale on large patientgroups [] and when results are considered in relation to those obtained from celllines and animalmodelsemploying pancancer population data our studyidentified conserved and unique patterns of ampk signaling across diverse cancer types analyses at two molecular levels genetic and transcriptional yielded amore comprehensive depiction of ampk signalingwhere we identified genes that were both somatically altered and differentially expressed these putative lossor gainoffunction genes are more likely to impacttumor progression as they are altered at both macromolecular levels as reported in other studies we confirmedthat ampk signaling could either be oncogenic ortumor suppressive depending on the cellular context intuitively since ampk is antianabolic its function maynot be fitting for tumor growth and proliferation this isconsistent with reports demonstrating ampks tumorsuppressive activity [ ] a study on lymphoma demonstratesthewarburg effect and hypoxia signaling in mice that ampk downregulation induces 0cchang and lai bmc cancer page of fig see legend on next page 0cchang and lai bmc cancer page of see figure on previous pagefig prognostic significance of degs targeted by enriched tfs a venn diagrams illustrate the extent of overlap between degs targeted byezh2 nfe2l2 rest smad4 and suz12 across four cancers b forest plots depict degs that are significantly associated with overall survivaloutcomes hazard ratios are denoted as purple squares while pink bars represent the confidence intervals significant wald test p values areindicated in blueits loss ofampk is proposed to act as a metabolic gatekeeper tolimit cancer cell division hencefunctionwould contribute to tumor aggression because of theloss in metabolic checkpoints [ ] ampk regulatesthe tumorsuppressive function of the serinethreoninekinase lkb1 ablation of lkb1 results in enhanced riskof developing gastrointestinal lung and skin squamouscell cancers [ ] moreover ampk is shown to inhibit pi3kaktmtor signaling which is activated inmany cancers [ ] also metabolic inhibitors such asmetformin which indirectly activates ampk could suppress tumor growth via autophagy induction and mtorinhibition [ ] metformin is shown to inhibit theproliferation of estrogen receptor α erα negative andpositive breast cancer cell lines through ampk stimulation however when tested in mice models metformin contributes to enhanced tumor progression andincreased angiogenesis providing us with a glimpse ofpotential proneoplastic effects of ampk activation in our study we observed that high levels of ampkpathway activity were associated with better outcomes inglioma breast cancer and sarcoma fig corroboratingprevious results on the tumorsuppressive function ofthe opposite is true in stomachampk converselyfig prognostic relevance of candidate tfs and the 24ampkgene set in glioma a scatter plots illustrate significant negative correlationsbetween ampk scores and tf expression levels in glioma patients are separated and colorcoded into four categories based on median ampkand tf scores density plots appended to the y and xaxes demonstrate the distribution of ampk and tf scores b logrank tests are performedon the four patient grou | 0 |
continuously increasing with development of the economy and the environment [] the prognosis for hcc patients remains extremely poor although significant progress has been achieved strategies for early diagnosis are urgently needed because the majority of patients with hcc are diagnosed in very late stages however the molecular mechanism of hcc has not been clearly defined circular rnas circrnas are a new class of rna molecules that have functions as regulators of parental gene transcription in alternative splicing and as mirna sponges through use of rna deep sequencing gtechnology numerous circrnas have been identified as the predominant regulatory elements in diseases moreover accumulating evidence shows that circrnas play pivotal roles in many diseases in particular abnormally expressed circrnas are involved in tumor progression including cell proliferation migration and invasion [] in addition some research indicates that circrnas level are closely correlated wit specific phenotypes and tumorigenesis in hcc [] nevertheless the research concerning circrnas is frankly in its infancy which greatly hinders the application of circrnas as biomarkers for diagnosis of hcc in clinicsrelated research shows that circrnas possess great potential to be used for diagnosis of hcc recent studies have found that hsa_circ_0067934 plays oncogenic roles by accelerating cell proliferation and metastasis in glioblastoma gbm circsmarca5 was significantly elevated and thereby suppressed cell apoptosis and arrested cell cycle in prostate cancer in addition previous studies have shown that downregulation of hsa_circ_0005986 facilitated cell proliferation by promoting the g0g1 to s phase transition in hcc similarly alteration in expression of circrnas correlated with development and metastasis of malignant tumors these data suggest that circrnas may be of greater benefit in clinical diagnosis of hcc however reliable circrna biomarkers for hcc are still lacking therefore this review synthetically integrates available data on the role of circrna in hcc progression and attempts to provide crucial clues for investigating the molecular mechanism regarding hccoverview of circrnacircrnas are a category of singlestranded closedcircle molecules which take part in multifaceted biological regulation recently research has verified that the majority of circrnas are synthesized by backspliced exons and that others are formed from intron intergenic and untranslated regions utr therefore biogenesis of circrnas can be divided into eicirnas exonintron circrnas ecircrnas circular exonic rnas and cirnas circular intronic rnas meanwhile over circrnas have been identified and this type of transcript has been considered a new form of gene expression generally the structure of the transcription is inverted and the order of genomic exons is altered and these exons are spliced over time the biological functions of circrnas gradually have been recognized including roles in embryonic development maintainenance of homeostasis and promotion of tumor progression figure properties of circrnascircrnas recently have attracted great attention related to their pathological role in disease development compared with linear rnas circrnas have special properties including biological roles and clinical use circrnas are mainly enriched in certain body fluids comprising blood saliva and urine they are covalently closed loop structures degradation of most rna is highly dependent on rna exonuclease or rnase hence circrnas remain highly stable based on their high resistance to enzyme degradation moreover studies have shown that expression of circrnas is tissuespecific and correlated with different phases of development and they exhibit different expression patterns at different developmental stages roles of circrnasaccumulating evidence shows that circrnas play a crucial role in the pathogenesis of diseases as a result of their complex biological functions generally the molecular functions of circrnas mainly include being sponges of mirna acting as rnabinding proteins performing alternative splicing of premrnas regulating transcription and translation and potentially encoding proteins these properties are described in detail belowsponges of mirnathe different types of circrnas have different mirna binding sites some circrnas negatively regulate mirnas by absorbing and specifically binding to mirnas then decreasing mirna activity and elevating expression of mirnarelated target genes researchers have shown that cirs7 inhibits mir7 function and positively mediates mir7 target genes acting as a molecular sponge in addition functional analyses have indicated that circrnas constitute an entire molecular regulatory network which specifically regulates degradation of mirnas as mirna sponges this work is licensed under creative common attributionnoncommercialnoderivatives international cc byncnd e9238322indexed in [current contentsclinical medicine] [sci expanded] [isi alerting system] [isi s master list] [index medicusmedline] [embaseexcerpta medica] [chemical scas]review s 0czou rc research progress on circrnas in hcc med sci monit e923832premrnae1e2abcbasepairinge3dguriche4ecrichpretrnarna bindingproteinsrbpgnicilpskcablariat splicingaecircrnaelcirnacirnatrnatricrnafigure1 biogenesis of circular rnas a lariatdriven circularization the hydroxyl of the upstream exon reacts with the phosphate of the downstream exon to form a covalent linkage then producing a lariat including exons and introns the hydroxyl of the intron interacts with the phosphate of the intron to form an ecircrna following an interaction between the hydroxyl of the exon and the phosphate of the exon b rnabinding protein rbpdriven circularization rbps accelerate interaction of the downstream intron and upstream intron thereby promoting formation of ecircrna c basepairingdriven circularization the downstream introns and upstream introns are paired depends on inverserepeatingcomplementary sequences formation of ecircrnaeicirna was derived from the introns are removedretained d biosynthesis of cirna formation of cirnas mainly based on a 7nt gurich element and an 11nt crich element to escape debranching and exonucleolytic degradation e formation of tricrna trna splicing enzymes divide pretrna into two parts tricrnas are generated by a phosphodiester bond and the other part generates trnascircrnasbinding proteinsrna binding proteins rbps are a broad class of proteins involved in gene transcription translation and interaction studies suggest that distribution of rbps is widespread in many tissue types furthermore rbps participate in development of disorders by regulating posttranscriptional regulation of rnas rbps assemble ribonucleoprotein complexes to bind rna sequences thereby affecting the function of the target rnas previous research has shown that circrnas serve as protein decoys to harbor binding sites of specific proteins and block protein activity circfoxo3 induces cell cycle arrest resulting in defective cdk2 gene function by binding to p21 and cdk2 moreover circrna ciacgas binds to cgas protein and suppresses enzymatic activity of cgas thereby preventing cgas from recognizing selfdna e9238323indexed in [current contentsclinical medicine] [sci expanded] [isi alerting system] [isi s master list] [index medicusmedline] [embaseexcerpta medica] [chemical scas]review sthis work is licensed under creative common attributionnoncommercialnoderivatives international cc byncnd 0czou rc research progress on circrnas in hcc med sci monit e923832circrnas regulate alternative splicing transcription and translationcellular localization of most circrnas is cytoplasmic which is the basis for the biological function of mirna and protein decoys several studies have suggested that circrnas participate in rna splicing assembly and biosynthesis recently research has shown that circrnas may play pivotal roles in regulating alternative splicing transcription and translation in addition the exon of the splicing factor may form a circrna by affecting formation of linear rna eicirnas interact with the u1 small nuclear ribonucleoproteinsnrnp thereby regulating parental gene transcription by binding to rna polymerase ii interestingly translation of circrnas is mediated by ires and n6methyladenosine m6a and translation efficiency of circrna is regulated by the level of m6a modification moreover circfbxw7 effectively inhibits glioma proliferation and cell cycle progression by antagonizing usp28induced cmyc stabilization potential to encode proteinscircrnas are implicated in numerous physiological processes and pathogenesis of diseases strong evidence indicates that circrnas can encode proteins by mimicking dna rolling circle amplification related studies indicate that circrna circppp1r12a plays a key molecular role by encoding a functional protein circppp1r12a73aa which promotes proliferation migration and invasion of colon cancer circanril interacts with pescadillo zebrafish homolog pes1 to mediate ribosome biogenesis and prerrna processing in vascular macrophages and smooth muscle cells these studies have significantly increased the knowledge base about the biological functions of circrnascircrnas in diseasescircrnas are involved in processes that lead to development of various disorders such as neuronal and cardiovascular diseases and cancers circrnas participate in regulating gene transcription and protein expression and are indirectly and directly associated with time and regionspecific variations as mentioned previously abnormal expression of circrnas is implicated in neurological disorders atherosclerosis and ribosomal rna maturation reportedly are regulated by circanril simultaneously some studies have suggested that circrnas upregulation significantly affects sprouting and proliferation of vascular endothelial cells and elicits vascular dysfunction recently several experiments have implicated circrnas in pathogenesis of cancer via activation of a series of cascade reactions however the underlying mechanism for the effect of circrnas in initiation and progression of tumors has not been fully clarified to date related studies have revealed that certain circrnas are highly expressed in tumor tissues and overexpression of circrnas promotes tumor proliferation and deterioration an investigation revealed that hsa_circ_002059 was downregulated in gastric cancer while hsa_circ_0004018 was upregulated in hcc meanwhile tumorspecific circrnas candidates were screened in lung adenocarcinoma tissue by microarrays and circrnas were identified downregulated and upregulated of the circrnas hsa_circ_0013958 clearly was positive correlated with lymph node metastasis and tnm stage these findings indicate that circrnas have important roles in tumor progression and may have potential for broad applicatoins in medicine scienceoverview of hcchcc is one of the most prevalent tumors worldwide with diagnoses and approximately deaths annually epidemiological survey data indicate that morbidity and mortality from hcc are gradually increasing risk factors for hcc include diabetes mellitus obesity smoking alcohol consumption older age male sex chronic hbv liver cirrhosis and chronic hepatitis c virus hcv the primary risk factors include liver cirrhosis viral hepatitis alcohol intake and obesity worldwide approximately hcc patients are infected with hepatitis b virus hbv or hcv in addition alcohol abuse is a crucial factor for onset of hcc [] obesity hypertension and diabetes are closely linked with development of hcc but specific correlations remain unknown moreover regular screening has been widely applied for early detection and to ensure effective treatment of hcc most commonly good results have been achieved with regular screening with ultrasonography blood alphafetoprotein content testing mri and ct generally surgical resection and chemotherapy are mainstays of therapy in patients with hcc yet some tumors cannot be fully removed which results in tumor growth invasion and metastasis local and systemic metastases are the main reasons for the unsatisfactory prognosis in patients with hcc therefore more effective therapeutic approaches need to be developedroles of circrnas in hccnumerous studies have documented the important role that circrnas play in tumorigenesis metastasis and invasion research has shown that circrnas are localized in the nucleus and interfere with transcription and promote alternative this work is licensed under creative common attributionnoncommercialnoderivatives international cc byncnd e9238324indexed in [current contentsclinical medicine] [sci expanded] [isi alerting system] [isi s master list] [index medicusmedline] [embaseexcerpta medica] [chemical scas]review s 0czou rc research progress on circrnas in hcc med sci monit e923832hsa_circ_0001649circzkscan1circitchwntbetacatenincircmto1mir9p21hsa_circ_00059836mir1295phmgb1 ragenfκbmir7hsa_circ_101368hsa_circ_001569cdr1ashsa_circ_0000673figure the function of circrnas in hcc carcinogenesis this graph demonstrates the role of circrnas in hcc carcinogenesis including positive and negative effects respectivelytable brief summary of circrnas as biomarkers for hccnamediseaseconclusiondoicirs7hsa_circ_0003570hsa_circ_0005075hepatocellular carcinomahepatocellular carcinomahepatocellular carcinomacirs7 was one of the independent factors and may be a promising biomarker for hepatic mvi and a novel therapy target for restraining mvi101007s0043201622567hsa_circ_0003570 expression levels were associated with hcc clinicopathological characteristics101002jcla22239hsa_circ_0005075 promotes proliferation migration and invasiveness of hcc via mir431 regulation101016jbiopha201801150splicing circpvt1 is overexpressed in gastric cancer tissues compared with nontumor tissues and circpvt1 acts as an oncogene to mediate expression of mir4975p however studies concerning the role of circrnas in development and progression of hcc remain in their infancytumor inhibitioncurrently circrnas are considered promising diagnostic biomarkers and ideal therapeutic targets for hcc studies have revealed that circitch inhibits tumor proliferation by suppressing the wntbetacatenin pathway expression of circitch has been positively correlated with good survival outcome in patients with hcc analysis of the circrnas expression profile in human hcc tissues showed that circmto1 was markedly decreased in hcc tissues and that expression of circmto1 was positively correlated with survival rate circmto1 reportedly inhibits hcc progress by sponging mir9 and thereby increasing p21 expression meanwhile overexpression of hsa_circ_0001649 negatively affects invasion and proliferation and promotes apoptosis of hcc cells downregulation of zkscan1 and circzkscan1 enhances cell proliferation and promotes progression of hcc tumor promotionin patients with hcc cdr1was more abundant in tumor specimens than in adjacent normal tissues cdr1as effectively suppresses the invasion and proliferation of hcc cells by targeting mir7 some reports have shown that hsa_circ_0000673 is significantly upregulated in hcc tissues and hsa_circ_0000673 downregulation markedly inhibits proliferation and invasion of hcc cells in vitro meanwhile a positive correlation was found between circ_001569 expression level and tumor size advanced tnm stages and unfavorable prognosis in patients with hcc circrna101368 was abundantly expressed in hcc tissue which correlated with poorer prognosis in addition circrna101368 inhibited cell migration by reducing protein levels in nfkb rage and hmgb1 figure e9238325indexed in [current contentsclinical medicine] [sci expanded] [isi alerting system] [isi s master list] [index medicusmedline] [embaseexcerpta medica] [chemical scas]review sthis work is licensed under creative common attributionnoncommercialnoderivatives international cc byncnd 0czou rc research progress on circrnas in hcc med sci monit e923832biomarkerconclusionsprevious studies have shown that circrnas are closely related to development of tumors clinicopathological features in patients with hcc are correlated to with levels of expression of cirs7 and its targeted mrnas global circrna expression profile analysis showed that hsa_circ_0005075 exhibited significant differences in tumor tissue versus adjacent tissues in patients with hcc expression of hsa_circ_0005075 also was related to tumor proliferation and metastasis therefore an increasing number of circrnas have been identified as diagnostic markers as summarized in table given the high incidence and mortality fo hcc worldwide it is one of the most serious diseases threatening human health increasing attention is being paid due to this serious situation evidence is increasing to support the close association between circrnas progression of hcc circrnas may play an important role in the occurrence and development of tumors however the molecular mechanism underlying the relationship between circrnas and hcc has not been fully elucidated therefore indepth research is needed on the potential regulatory relationships and to uncover regulatory patterns between circrnas and hcc so that new diagnostic markers for hcc can be developedreferences bray f ferlay j soerjomataram i global cancer statistics globocan estimates of incidence and mortality worldwide for cancers in countries cancer j clin feng rm zong yn cao sm xu rh current cancer situation in china good or bad news from the global cancer statistics cancer commun lond jemal a bray f center mm global cancer statistics cancer j clin li r jiang j shi h circrna a rising star in gastric cancer cell mol life sci salzman j gawad c wang pl circular rnas are the predominant transcript isoform from hundreds of human genes in diverse cell types plos one e30733 lukiw wj circular rna circrna in alzheimers disease ad front genet liu y yang y wang z insights into the regulatory role of circrna in angiogenesis and clinical implications atherosclerosis zhao y alexandrov pn jaber v lukiw wj deficiency in the ubiquitin conjugating enzyme ube2a in alzheimers disease ad is linked to deficits in a natural circular mirna7 sponge circrna cirs7 genes basel shen f liu p xu z circrna_001569 promotes cell proliferation through absorbing mir in gastric cancer j biochem song t xu a zhang z circrna hsa_circrna_101996 increases cervical cancer proliferation and invasion through activating tpx2 expression by restraining mir8075 j cell physiol min l wang h zeng y circrna_104916 regulates migration apoptosis and epithelialmesenchymal transition in colon cancer cells front biosci landmark ed verduci l strano s yarden y blandino g the circrnamicrorna code emerging implications for cancer diagnosis and treatment mol oncol wei j wei w xu h circular rna hsa_circrna_102958 may serve as a diagnostic marker for gastric cancer cancer biomark li p chen s chen h using circular rna as a novel type of biomarker in the screening of gastric cancer clin chim acta xin j zhang xy sun dk upregulated circular rna hsa_circ_0067934 contributes to glioblastoma progression through activating pi3kakt pathway eur rev med pharmacol sci kong z wan x zhang y androgenresponsive circular rna circsmarca5 is upregulated and promotes cell proliferation in prostate cancer biochem biophys res commun fu l chen q yao t hsa_circ_0005986 inhibits carcinogenesis by acting as a mir1295p sponge and is used as a novel biomarker for hepatocellular carcinoma oncotarget zhu x wang x wei s hsa_circ_0013958 a circular rna and potential novel biomarker for lung adenocarcinoma febs j zhang q wang w zhou q roles of circrnas in the tumour microenvironment mol cancer qu z jiang c wu j ding y exosomes as potent regulators of hcc malignancy and potential biotools in clinical application int j clin exp med memczak s jens m elefsinioti a circular rnas are a large class of animal rnas with regulatory potency nature cocquerelle c mascrez b hetuin d bailleul b missplicing yields circular rna molecules faseb j zhao x cai y xu j circular rnas biogenesis mechanism and function in human cancers int j mol sci qu s yang x li x circular rna a new star of noncoding rnas cancer lett bahn jh zhang q li f the landscape of microrna piwiinteracting rna and circular rna in human saliva clin chem hsu mt cocaprados m electron microscopic evidence for the circular form of rna in the cytoplasm of eukaryotic cells nature yu x odenthal m fries jw exosomes as mirna carriers formationfunctionfuture int j mol sci hanan m soreq h kadener s circrnas in the brain rna biol constantin l circular rnas and neuronal development adv exp med biol van rossum d verheijen bm pasterkamp rj circular rnas novel regulators of neuronal development front mol neurosci ebert ms sharp pa microrna sponges progress and possibilities rna ebert ms neilson jr sharp pa microrna sponges competitive inhibitors of small rnas in mammalian cells nat methods hansen tb jensen ti clausen bh natural rna circles function as efficient microrna sponges nature hsiao ky lin yc gupta sk noncoding effects of circular rna ccdc66 promote colon cancer growth and metastasis cancer res janga sc mittal n construction structure and dynamics of posttranscriptional regulatory network directed by rnabinding proteins adv exp med biol du ww yang w liu e foxo3 circular rna retards cell cycle progression via forming ternary complexes with p21 and cdk2 nucleic acids res xia p wang s ye b a circular rna protects dormant hematopoietic stem cells from dna sensor cgasmediated exhaustion immunity 701e7 li z huang c bao c exonintron circular rnas regulate transcription in the nucleus nat struct mol biol this work is licensed under creative common attributionnoncommercialnoderivatives international cc byncnd e9238326indexed in [current contentsclinical medicine] [sci expanded] [isi alerting system] [isi s master list] [index medicusmedline] [embaseexcerpta medica] [chemical scas]review s 0czou rc research progress on circrnas in hcc med sci monit e923832 yang y fan x mao m extensive translation of circular rnas driven by n6methyladenosine cell res yang y gao x zhang m novel role of fbxw7 circular rna in repressing glioma tumorigenesis j natl cancer inst abe n hiroshima m maruyama h rolling circle amplification in a prokaryotic translation system using small circular rna angew chem int ed engl zheng x chen l zhou y a novel protein encoded by a circular rna circppp1r12a promotes tumor pathogenesis and metastasis of colon cancer via hippoyap signaling mol cancer holdt lm stahringer a sass k circular noncoding rna anril modulates ribosomal rna maturation and atherosclerosis in humans nat commun gokul s rajanikant gk circular rnas in brain physiology and disease adv exp med biol idda ml munk r abdelmohsen k gorospe m noncoding rnas in alzheimers disease wiley interdiscip rev rna luo q chen y long noncoding rnas and alzheimers disease clin interv aging li cy ma l yu b circular rna hsa_circ_0003575 regulates oxldl induced vascular endothelial cells proliferation and angiogenesis biomed pharmacother chen j cui l yuan j circular rna wdr77 target fgf2 to regulate vascular smooth muscle cells proliferation and migration by sponging mir biochem biophys res commun kristensen ls hansen tb veno mt kjems j circular rnas in cancer opportunities and challenges in the field oncogene fu l yao t chen q screening differential circular rna expression profiles reveals hsa_circ_0004018 is associated with hepatocellular carcinoma oncotarget massarweh nn elserag hb epidemiology of hepatocellular carcinoma and intrahepatic cholangiocarcinoma cancer control salem r gilbertsen m butt z increased quality of life among hepatocellular carcinoma patients treated with radioembolization compared with chemoembolization clin gastroenterol hepatol 65e1 ozer ed suna n boyacioglu as management of hepatocellular carcinoma prevention surveillance diagnosis and staging exp clin transplant 15suppl lou w liu j ding b identification of potential mirnamrna regulatory network contributing to pathogenesis of hbvrelated hcc j transl med yang t hu ly li zl liver resection for hepatocellular carcinoma in nonalcoholic fatty liver disease a multicenter propensity matching analysis with hbvhcc j gastrointest surg nishibatake km minami t tateishi r impact of directacting antivirals on early recurrence of hcvrelated hcc comparison with interferonbased therapy j hepatol toyoda h kumada t tada t the impact of hcv eradication by directacting antivirals on the transition of precancerous hepatic nodules to hcc a prospective observational study liver int zhao j oneil m vittal a prmt1dependent macrophage il6 production is required for alcoholinduced hcc progression gene expr vandenbulcke h moreno c colle i alcohol intake increases the risk of hcc in hepatitis c virusrelated compensated cirrhosis a prospective study j hepatol fabris c toniutto p falleti e mthfr c677t polymorphism and risk of hcc in patients with liver cirrhosis role of male gender and alcohol consumption alcohol clin exp res vernon g baranova a younossi zm systematic review the epidemiology and natural history of nonalcoholic fatty liver disease and nonalcoholic steatohepatitis in adults aliment pharmacol ther bruix j reig m sherman m evidencebased diagnosis staging and treatment of patients with hepatocellular carcinoma gastroenterology zhang bh yang bh tang zy randomized controlled trial of screening for hepatocellular carcinoma j cancer res clin oncol verduci l ferraiuolo m sacconi a the oncogenic role of circpvt1 in head and neck squamous cell carcinoma is mediated through the mutant p53yaptead transcriptioncompetent complex genome biol yu j xu qg wang zg circular rna csmarca5 inhibits growth and metastasis in hepatocellular carcinoma j hepatol wang m yu f li p circular rnas characteristics function and clinical significance in hepatocellular carcinoma cancers basel guo w zhang j zhang d et al polymorphisms and expression pattern of circular rna circitch contributes to the carcinogenesis of hepatocellular carcinoma oncotarget han d li j wang h circular rna circmto1 acts as the sponge of microrna9 to suppress hepatocellular carcinoma progression hepatology qin m liu g huo x hsa_circ_0001649 a circular rna and potential novel biomarker for hepatocellular carcinoma cancer biomark yao z luo j hu k zkscan1 gene and its related circular rna circzkscan1 both inhibit hepatocellular carcinoma cell growth migration and invasion but through different signaling pathways mol oncol xu l zhang m zheng x the circular rna cirs7 cdr1as acts as a risk factor of hepatic microvascular invasion in hepatocellular carcinoma j cancer res clin oncol yu l gong x sun l the circular rna cdr1as act as an oncogene in hepatocellular carcinoma through targeting mir7 expression plos one e0158347 jiang w wen d gong l circular rna hsa_circ_0000673 promotes hepatocellular carcinoma malignance by decreasing mir7673p targeting set biochem biophys res commun liu h xue l song c overexpression of circular rna circ_001569 indicates poor prognosis in hepatocellular carcinoma and promotes cell growth and metastasis by sponging mir4115p and mir4325p biochem biophys res commun li s gu h huang y circular rna 101368mir200a axis modulates the migration of hepatocellular carcinoma through hmgb1rage signaling cell cycle shang x li g liu h comprehensive circular rna profiling reveals that hsa_circ_0005075 a new circular rna biomarker is involved in hepatocellular carcinoma development medicine baltimore e3811 yao t chen q shao z circular rna as a new biomarker for hepatocellular carcinoma metastasis j clin lab anal e22572e9238327indexed in [current contentsclinical medicine] [sci expanded] [isi alerting system] [isi s master list] [index medicusmedline] [embaseexcerpta medica] [chemical scas]review sthis work is licensed under creative common attributionnoncommercialnoderivatives international cc byncnd 0c' | 0 |
gastrointestinal nematodes could release excretorysecretory es proteins into the host environment to ensure their survival these es proteins act as immunomodulators to suppress or subvert the host immune response via the impairment of immune cell functions especially in chronic infections in our preliminary study haemonchus contortus adhesionregulating molecule hcadrm1 was identified from h contortus es proteins hcesps that interacted with host t cells via liquid chromatographytandem mass spectrometry analysis however little is known about hcadrm1 as an es protein which may play a pivotal role at the parasitehost interfacemethods based on bioinformatics approaches multiple amino acid sequence alignment was conducted and the evolutionary relationship of hcadrm1 with adrm1 orthologues was extrapolated employing rtqpcr and immunohistochemistry assays temporal transcriptional and spatial expression profiles of hcadrm1 were investigated using immunostaining approaches integrated with immunological bioassays the immunomodulatory potentials of hcadrm1 on goat t cells were assessedresults we hereby demonstrated that hcadrm1 with immunodiagnostic utility was a mammalian adrm1 orthologue abundantly expressed at all developmental stages of h contortus given the implications of adrm1 proteins in cell growth survival and development we further investigated the immunomodulatory property of hcadrm1 as an individual es protein acting at the parasitehost interface the rhcadrm1 stimuli notably suppressed t cell viability promoted intrinsic and extrinsic t cell apoptosis inhibited t cell proliferation and induced cell cycle arrest at g1 phase simultaneously rhcadrm1 stimuli exerted critical controls on t cell cytokine secretion profiles predominantly by restraining the secretions of interleukin il4 il10 and interferongammas importantly hcadrm1 protein may have prophylactic potential for antih contortus vaccine development together these findings may contribute to the clarification of molecular and immunomodulatory traits of es proteins as well as improvement of our understanding of parasite immune evasion mechanism in h contortushost biologykeywords h contortus excretorysecretory protein adhesionregulating molecule adrm1 immunomodulation immune evasioncorrespondence lixiangruinjaueducn moe joint international research laboratory of animal health and food safety college of veterinary medicine nanjing agricultural university nanjing jiangsu peoples republic of chinafull list of author information is available at the end of the the authors this is licensed under a creative commons attribution international license which permits use sharing adaptation distribution and reproduction in any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the creative commons licence and indicate if changes were made the images or other third party material in this are included in the s creative commons licence unless indicated otherwise in a credit line to the material if material is not included in the s creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtain permission directly from the copyright holder to view a copy of this licence visit httpcreat iveco mmons licen sesby40 the creative commons public domain dedication waiver httpcreat iveco mmons publi cdoma inzero10 applies to the data made available in this unless otherwise stated in a credit line to the data 0clu a0et a0al parasites vectors page of the highly conserved and regulated ubiquitin ub proteasome pathway is the primary mechanism for targeted elimination of most shortlived proteins including misfolded or damaged proteins in eukaryotic cells ub can covalently attach to cellular proteins by ub modification which is an atpdependent process mediated via different classes of ub enzymes alongside three families of shuttling factors rad23 dsk2 and ddi1 three proteasome subunits located in the subcomplex of 26s proteasome rpn1 rpn10 and rpn13 are demonstrated to be ub receptors as well as the proteasomeassociated polyubiquitin receptor rpn13 also termed as adhesionregulating molecule adrm1 is recruited by rpn2 to be assembled into the 19s regulatory p and target protein substrates linked to the small protein ub via its pleckstrinlike receptor [ ] simultaneously the cterminal adaptor domain of adrm1 serves to bind and activate the deubiquitylase uchl5uch37 and enhance its isopeptidase activity revealing a mechanism to accelerate ub chain disassembly []with engagement in the ub proteasome pathway that regulates a broad range of physiological functions adrm1 is implicated in multitudinous cellular processes such as cell growth migration survival and development particularly in cancer cells recent publications reveal that adrm1 transcription is consistently elevated in ovarian colorectal and gastric cancer tissues and knockdown of adrm1 expression in both human colon carcinoma and gastric cancer cell lines suppress cell migration and proliferation and induces cell apoptosis [] meanwhile fejzo et a0 al demonstrated that overexpression of adrm1 in ovarian cancer promoted cell growth and migration whereas blocking its expression caused cell death given the association of amounting adrm1 expression with the onset and progression of cancers adrm1 has been defined as a potential predictive and therapeutic target for clinical therapy additionally comparable expressions of adrm1 have also been observed in several lymphocyte cell lines as well as endothelial cell lines and similar physiological roles of adrm1 are described through its excessive expression in skin endothelial cells that facilitates t lymphocyte adhesion in a previous study we identified haemonchus contortus excretorysecretory es proteins hcesps that interacted with host t cells via liquid chromatography mass spectrometry lcmsms analysis haemonchus contortus adrm1 hcadrm1 protein a mammalian adrm1 homologue was ascertained among these interacting proteins additionally recombinant hcadrm1 rhcadrm1 was recognized by serum samples obtained at day and postinfection derived from experimentally h contortusinfected goats as a result of these observations hcadrm1 with immunodiagnostic utility was fostered as a hallmark of h contortus infection and a serological diagnosis assay with high sensitivity and specificity was developed using hcadrm1 antigen furthermore our preliminary analysis showed that hcesps stimuli notably induced intrinsic and extrinsic apoptosis suppressed t cell proliferation and caused cell cycle arrested hcesps consisted of multitudinous modulatory molecules such as kinases phosphatases hydrolases and proteases where the pleiotropic effects were initiated by a cascade of individual es components importantly the exact molecules that modulated t cell immune response in the parasitehost interaction warrant further investigation given the functional diversity of adrm1 and especially its engagement in cell proliferation and apoptosis hcadrm1 might be one of these dominated proteins that exert critical controls on cellular survival and death of host key effector cells therefore herein we aimed to further investigate the molecular traits of hcadrm1 and address its immunomodulatory roles at the parasitehost interfacemethodsparasite animals and a0cellsthe h contortus strain was propagated via serial passages in nematodefree goats in the animal experimental center faculty of veterinary medicine nanjing china the collection of eggs l3 xl3 male and female adults was performed as previously described [ ] sprague dawley sd rats scxk with a standard packing weight a0 g were obtained from jiangsu experimental animal center nanjing china they were maintained in a microbefree room with access to sterilized food and water ad libitumlocal crossbred and healthy goats monthsold were reared in individually ventilated cages to prevent accidental infection with nematodes alongside ad libitum access to water in pens these goats were given hay and whole shelled corn daily peripheral venous blood samples were obtained by venipuncture as described elsewhere as well as the isolation of goat peripheral blood mononuclear cells pbmcs total t cells in goat pbmcs were sorted using a magneticactivated cell sorting system macs miltenyi biotech inc auburn ca usa as described elsewhere briefly every million pbmcs in a0µl staining buffer were incubated with µl mouse antibovine cd2 primary antibody biorad kidlington uk which crossreact with goat cd2 t cells for a0min after two washes in pbs à of total pbmcs resuspended in µl staining buffer were labeled 0clu a0et a0al parasites vectors page of with µl antifitc microbeads miltenyi biotech at room temperature for min subsequently pbmcs were loaded onto the macs magnetic system miltenyi biotech for positive sorting based on the manufacturers specifications t cells were then adjusted to a density of à cellsml in rpmi gibco grand island ny usa containing heatinactivated fetal calf serum fcs gibco and a0uml penicillin a0mgml streptomycin gibco the viability of t cells was as assessed by the trypan blue exclusion test the purity of separated t cells was validated via flow cytometric determination additional file a0 figure s1 three goats biological replicates were used in every experimentsequence alignment and a0phylogenetic analysisthe cloning and amplification of the complete coding sequence of the hcadrm1 gene were performed as previously described the amplified hcadrm1 fragment was cloned into pet28a vector invitrogen carlsbad ca usa and validated by sequence analysis using blast multiple amino acid sequences of adrm1 orthologs were aligned for comparison by the clustal omega software the evolutionary analysis was conducted and extrapolated by mega x software using jtt matrixbased model and the maximum likelihood method with partial deletion option positions with less than site coverage were excluded prior to phylogenetic analysis based on the optimized evaluation of replicates for bootstrap support the evolutionary tree of adrm1 orthologues was generated with several designated and collapsed branchesexpression and a0purification of a0the a0rhcadrm1 proteinthe expression and purification of rhcadrm1 proteins were conducted as elsewhere described briefly escherichia coli bl21 de3 cells containing the reconstructed pet28ahcadrm1 plasmid were incubated with luriabertini medium containing kanamycin a0 µgml sigmaaldrich st louis mo usa and then stimulated by isopropylβdthiogalactopyranoside sigmaaldrich for the induction of rhcadrm1 expression the rhcadrm1 protein fused with a histidinetag was obtained from the supernatant of cell lysates via histrap hp purification columns ge healthcare piscataway nj usa rhcadrm1 proteins were resolved on sodium dodecyl sulfatepolyacrylamide gel electrophoresis sdspage gels for size and purity validation and the concentration was determined by a bicinchoninic acid bca assay thermo fisher scientific rockford il usa employing the detoxigel affinity pak prepacked columns thermo fisher scientific rhcadrm1 proteins were exempt of lipopolysaccharide contamination as rhcadrm1 protein was dissolved in pbs pbstreated t cells served as the control group a0 µgml in functional assays the purified rhcadrm1 was stored at a0°c until further analysispreparation of a0polyclonal antibody pabto obtain antigenspecific pab rhcadrm1 proteins a0 µg blended with freunds complete adjuvant was administrated subcutaneously into sd rats with a 2week interval booster immunizations with a0 µg of rhcadrm1 proteins emulsified in freunds incomplete adjuvant were administered four times seven days after the final boost rat sera containing antirhcadrm1 pab were harvested and kept at a0 °c for further analysis the sera harvested from h contortusinfected goats antih contortus serum were stored at the veterinary parasitology teaching and research center of nanjing agricultural university nanjing chinaimmunoblot analysisrhcadrm1 and hcesps were resolved on protein gels respectively and transferred onto nitrocellulose membranes the blots were blocked using bsa in trisbuffered saline01 tween tbst for a0 h at room temperature the blots with the rhcadrm1 samples were probed with primary goat antih contortus serum in tbst or normal goat serum control at a0°c overnight while the blots with the hcesps samples were probed with primary rat antirhcadrm1 igg in tbst or normal rat igg control after five washes in tbst the blots were incubated with horseradish peroxidasecoupled rabbit antigoat or antirat igg hl secondary antibody sigmaaldrich in tbst for h at a0 °c the blots were then developed with ²diaminobenzidine dab sigmaaldrich for min and visualized by using a chemidoc imaging system biorad hercules ca usahcadrm1 transcription in a0h contortus lifecycle stagesto detect mrna expression of hcadrm1 in h contortus lifecycle stages total rna of eggs l3 xl3 female and male adults were extracted using trizol invitrogen and the resulting cdnas were synthesized in accordance with the manufacturers specifications employing specific primers for the βtubulin gene endogenous reference and target gene hcadrm1 additional file a0 table a0s1 transcriptional analysis of the hcadrm1 gene was conducted by realtime pcr using the quantstudio system applied biosystems carlsbad ca usa with a standard protocol the specificity of the primers was 0clu a0et a0al parasites vectors page of validated to ensure product purity via generation of a melt curve and the absence of primer dimers the amplification efficiencies and correlation coefficients were verified to be stable and similar based on the δδcq method the relative transcription levels of hcadrm1 were normalized on βtubulin transcription each experiment was run in triplicateimmunohistochemistry assaysfreshly collected female and male adults were washed dehydrated fixed embedded and cut into cryostat sections as previous described to minimize nonspecific binding cryosections were treated with normal goat serum in pbs containing tween pbst for a0h subsequently cryosections were served with primary antirhcadrm1 igg or sham control igg overnight at a0 °c prior to dna staining with 24amidinophenyl6indolecarbamidine dihydrochloride dapi sigmaaldrich cryosections were then incubated with cy3labeled goat antirat igg beyotime biotechnology shanghai china at a0 °c for a0 h subsequently the samples were immersed in antifade medium sigmaaldrich to prevent fluorescence fading for microscopic examination finally the sections were imaged at à magnification using a lsm710 fluorescence microscope zeiss jena germany and zen software zeiss was used for the analysis of digital imagesthe interaction of a0hcadrm1 protein with a0t cells in vitrothe interaction of hcadrm1 with goat t cells was investigated as previously described in brief freshly sorted t cells were cultured with or without a0 µgml rhcadrm1 proteins for a0h at a0°c after three washes paraformaldehydefixed t cells were permeabilized by triton x100 in pbst and blocked with bsa in pbst for a0min subsequently prior to the staining with the cy3coupled secondary antibody t cells were treated by primary antihcadrm1 pab or normal rat igg control in a humidified chamber at a0°c for a0h followed by five pbst washes t cells were subjected to gold antifade mounting solution containing dapi life technologies eugene or usa for nuclear staining immunofluorescencelabeled cells were visualized at à magnification using a lsm780 confocal microscope zeiss jena germany zen software zeiss was employed for the interpretation of digital imagescell viabilitythe modulatory effects of rhcadrm1 on goat t cell viability were determined using the cell counting kit8 assay cck8 dojindo kumamoto japan as previously described fresh sorted goat t cells activated with concanavalin a cona a0µgml were incubated in the presence of various doses of rhcadrm1 proteins and a0µgml at a0°c following a0hstimulation cell culture medium was incorporated with a0µl cck8 solution and incubated at a0 °c in the dark for a0h following incubation optical density was measured at a0nm od450 using a microplate reader biorad hercules california usa three independent tests each in triplicate were performedcell apoptosis assayflow cytometry assays were performed for t cell apoptosis determination using the annexin vpe kit bd biosciences san jose ca usa as previously described in brief freshly sorted t cells were cultured in the presence of tested doses of rhcadrm1 proteins and a0µgml followed by annexin v and 7aminoactinomycin d 7aad staining based on the kits specification the pbsstimulated t cells served as negative controls three individual tests each in triplicate were conductedcell proliferation assaycell proliferation analysis was determined using the alexa fluor clickit plus edu flow cytometry kit thermo fisher scientific via the measurement of dna synthesis directly based on the manufacturers instructions after a0h coincubation the cell culture was incorporated with 5ethynyl2²deoxyuridine edu a0μm for another a0h incubation subsequently t cells were harvested fixed with paraformaldehyde in pbs and permeabilized using the clickit saponinbased reagent followed by clickit reaction to coupled edu with alexa fluor dye after three washes with a0ml of bsa in pbs t cells were treated with 7aad staining solution bd biosciences flow cytometry was used for the determination of edu cells in the population each experiment consisting of three replicates was run in triplicatecell cycle assayflow cytometry assays were conducted for cell cycle determination using pirnase staining buffer bd biosciences according to the manufacturers dna staining protocol following coincubation with rhcadrm1 stimuli a0 µgml for a0 h t cells were harvested washed and fixed with icecold ethanol every a0 h after being frozen at a0°c for more than a0h treatedt cells were washed twice with pbs to remove remaining ethanol and resuspended in pirnase staining buffer for flow cytometry analysis each experiment consisting of three replicates was run in triplicate 0clu a0et a0al parasites vectors page of transcription analysist cells treated with different concentrations of rhcadrm1 and a0µgml for h were harvested for the transcription analysis of the cell apoptosis pathway and t cells treated with a0µgml of rhcadrm1 for a0h were collected for transcription analysis of the cell cycle pathway cells were harvested for total rna extraction and cdna obtained by reversetranscription pcr relative quantification of candidate gene expression was conducted using previously published primers [] of endogenous reference and candidate genes additional file a0 table a0s2 based on the δδcq method the relative levels of target gene transcription were normalized to reference gene expression each experiment consisting of three replicates was run in triplicatedetection of a0cytokine secretionsfor the determination of cytokine secretion levels freshly isolated t cells activated by cona a0µgml were treated with or without rhcadrm1 and a0µgml for a0h cell culture medium was harvested and determined for cytokine secretion detection using goat enzymelinked immunosorbent assay elisa kits mlbio shanghai china according to the manufacturers specifications the limit of quantification dependent upon each analytic kit ranged from between and a0pgml each experiment was run in triplicatestatistical analysisoneway and twoway analysis of variance anova with dunnetts multiple comparison test alongside the students ttest were performed for statistical analysis using graphpad premier software graphpad prism san diego ca usa differences were regarded as statistically significant when pvalues were data were denoted as minimum to maximum all points or mean ± standard deviation sdresultssequence alignment and a0phylogenetic analysisthe entire coding region of the hcadrm1 gene a0bp was amplified from the cdna of adult worms encoding a 361amino acid protein with an estimated molecular mass around a0 kda we then performed a sequence alignment of adrm1 orthologues derived from genbank on zebrafish human mouse caenorhabditis elegans and h contortus using clustal omega software hcadrm1 protein showed a moderate degree of identity to zebrafish human mouse and c elegans orthologs fig a0 1a in addition we conducted an evolutionary analysis of hcadrm1 using the maximum likelihood method involving amino acid sequences phylogenetic analysis clearly showed an evolutionary relationship of hcadrm1 with other adrm1 orthologues revealing that hcadrm1 was closely related to the teladorsagia circumcincta homologue but divergent from vertebrate sequences fig a01bprotein expression and a0immunoblot analysisthe rhcadrm1 protein fused with the histidinetag was successfully obtained in the supernatant of cell lysates after purification rhcadrm1 was visualized by coomassie blue staining as a single band with a molecular weight of a0kda fig a01c lane the specificity of the rhcadrm1 protein was determined by western blot probing with antih contortus serum or normal goat serum a single band a0 kda was observed through the specific recognition of rhcadrm1 protein by antih contortus serum fig a01c lane while no band was identified via healthy goat sera fig a01c lane meanwhile native hcadrm1 protein derived from hcesps was identified by rat antirhcadrm1 igg as a single band of a0kda fig a01c lane while no positive band was observed in the control groups fig a01c lane differential mrna expression in a0h contortus lifecycle stages and a0immunolocalizationtranscription analysis by realtime rtpcr revealed that mrna expression of hcadrm1 were detectable at all of the tested h contortus lifecycle stages the data demonstrated rising expression levels from the freeliving stages eggs and l3 to parasitic stages xl3 female and male adults simultaneously the highest level of hcadrm1 transcription was observed in male adults but not in female adults fig a01d given that the highest mrna expression was detected in adult worms we next investigated the localization of native hcadrm1 proteins within h contortus by checking the cryosections of the adult worms specific red fluorescence resulting from tagging hcadrm1 proteins by rat antirhcadrm1 igg was ubiquitously observed from the intracellular and cytoplasmic localization of somatic cells particularly in the intestinal regions and the internal membrane of cuticle for both male and female adults fig a01e however no cy3fluorescence was observed in the sections treated with normal rat igg fig a01ebinding of a0rhcadrm1 protein to a0goat t cellsbased on our preliminary lcmsms analysis we next conducted immunocytochemistry assays to verify the in vitro interaction of hcadrm1 proteins with goat t cells immunocytochemistry assay showed that intense red 0clu a0et a0al parasites vectors page of fig molecular characterization of hcadrm1 derived from hcesps a alignment of hcadrm1 amino acid sequences with other orthologues the positions of adrm1 family motifs are indicated above the multiple sequence alignment containing zebrafish xp_0213255291 human np_0012683661 mouse np_0627962 c elegans np_4983872 and h contortus w6nb91 adrm1 ortholog sequences retrieved from genbank an asterisk indicates the position with one completely conserved amino acid while period denotes weakly conserved similarity within different groups and colon represents strongly similar conservation between groups b phylogenetic analysis of hcadrm1 with vertebrate and parasite orthologues evolutionary relationships of taxa were inferred using the maximum likelihood method with protein sequences including mus musculus np_0627962 homo sapiens xp_0115268051 danio rerio xp_0213255291 toxocara canis khn872021 c elegans np_4983872 dictyocaulus viviparus kjh490541 t circumcincta pio739301 h contortus w6nb91 oesophagostomum dentatum khj973301 and ancylostoma caninum rcn481761 bootstrap support values are shown for each node the scalebar denotes the number of substitutions per site c acquisition of rhcadrm1 proteins and western blot analysis lanes m protein standard ladder lane rhcadrm1 expressed in the supernatant of cell lysates lane sdspage analysis of purified rhcadrm1 protein lane immunoblot analysis of rhcadrm1 using antih contortus serum as primary antibody lane immunoblot analysis of rhcadrm1 using normal goat serum control as primary antibody lane immunoblot analysis of hcesps using rat antirhcadrm1 igg as primary antibody lane immunoblot analysis of hcesps using normal rat igg control as primary antibody d hcadrm1 expression in h contortus lifecycle stages data are presented as the mean ± sd e immunolocalization of native hcadrm1 protein in male and female adults the immunohistochemistry assays were performed using normal rat igg control or rat antirhcadrm1 igg as primary antibody cy3coupled fluorescence red along with dapi blue was identified for the investigation of hcadrm1 distribution scalebars e µm 0clu a0et a0al parasites vectors page of cy3fluorescence resulting from tagging rhcadrm1 was observed in rhcadrm1treated t cells revealing the cytomembrane and cytoplasmic localization of rhcadrm1 fig a0 2a a1 no red fluorescence was detected in both blank and negative control groups fig a0 2a a2 and a3 the results presented here further validated the positive interactions between hcadrm1 protein and host t cellsrhcadrm1 suppressed cell viability and a0induced cell apoptosisgiven the modulatory potential of adrm proteins on cellular development and survival we next investigated the impact of rhcadrm1 proteins on t cell viability the results of cck8 determination showed that t cell viability was dramatically inhibited by the stimulation of a0 µgml anova f4 p a0 µgml anova f4 p a0µgml anova f4 p and a0 µgml anova f4 p of rhcadrm1 proteins fig a02b based on this finding an annexin vpe7aad double staining kit was employed to evaluate the proapoptotic potential of rhcadrm1 proteins flow cytometry results demonstrated that rhcadrm1 stimuli at the tested concentrations of a0 µgml anova f4 p a0 µgml anova f4 p and a0 µgml anova f4 p remarkably caused t cell apoptosis in comparison to the unstimulated group fig a0 2c d additionally transcriptional analysis of key genes involved in apoptosis signaling pathways further validated the proapoptotic effects of rhcadrm1 proteins on host t cells the treatments with and a0µgml of rhcadrm1 dramatically upregulated mrna transcripts of caspase8 anova f4 p p and p respectively and caspase3 anova f4 p p and p respectively fig a0 2e simultaneously a0 µgml anova f4 p and µgml anova f4 p of rhcadrm1 proteins significantly promoted caspase9 transcription fig a02erhcadrm1 protein restrained the a0proliferation of a0t cells and a0caused cell cycle stallingas apoptosis proliferation and cell cycle were interconnected cellular movements we next explored the modulatory potentials of rhcadrm1 stimuli on t cell proliferation and cell cycle at the tested doses of and a0 µgml flow cytometry data showed that rhcadrm1 stimuli significantly inhibited t cell proliferation in vitro fig a03a as indicated by the decreasing proportion of edu cells compared with control cells anova f4 p p and p respectively fig a0 3b given that the treatments with a0µgml rhcadrm1 had significant biological effects on cell viability apoptosis and proliferation as well as the transcription of certain key genes we next treated t cells with a0µgml of rhcadrm1 for cell cycle determination here flow cytometry analysis with pi staining demonstrated that rhcadrm1 stimuli induced cell cycle arrest in a timedependent manner fig a0 3c as indicated by the increased proportion of t cells in g1 phase at a0h anova f8 p a0h anova f8 p and a0h anova f8 p as well as the decreased proportion of t cells in s phase at a0 h anova f8 p a0 h anova f8 p and a0h anova f8 p fig a0 3d consistent with these findings transcriptional analysis of key genes in g1s checkpoints showed that mrna transcripts of ccne1 ttest t16 p and cdk2 ttest t16 p were significantly downregulated by rhcadrm1 stimuli while no significant transcriptional changes of ccnd1 ttest t16 p cdk4 ttest t16 p and cdk6 ttest t16 p were observed fig a03e given the inhibitory effects of p21 and p27 on cdks in ubmediated cell cycle progression the transcription analysis of p21 and p27 was performed in this study in addition the mrna transcripts of iκbα as the physiological substrate of adrm1 and its downstream inhibitor nfκb were determined importantly transcription of p21 ttest t16 p p27 ttest t16 p and iκbα ttest t16 p was notably enhanced by rhcadrm1 stimuli fig a0 3e whereas the mrna transcript of nfκb ttest t16 p was significantly suppressed fig a03edetermination of a0cytokine secretionsto investigate the modulatory effects of rhcadrm1 on t cell cytokine productions il2 il4 il10 il17a ifnγ and tgfβ1 secretions in the cell culture supernatant were determined via elisa assays the data showed that the exposure of goat t cells to rhcadrm1 proteins led to the alteration of their cytokine production profiles intriguingly at the tested doses of and a0µgml rhcadrm1 stimuli predominantly inhibited secretions of il4 anova f4 p p and p respectively anova f4 p p and p respectively and ifnγ anova f4 p p and p respectively fig a0 4b c e however all the tested doses of rhcadrm1 had no notable effects on secretions of il2 anova f4 p p p and p il10 0clu a0et a0al parasites vectors page of fig rhcadrm1 proteins suppressed cell viability and induced apoptosis via the interaction with goat t cells a determination of the interaction of hcadrm1 protein with goat t cells in vitro t cells treated with a1 or without a2 rhcadrm1 protein were incubated with rat antirhcadrm1 igg as the primary antibody t cells stimulated by rhcadrm1 were incubated with normal rat igg as the primary antibody a3 b rhcadrm1 significantly inhibited t cell viability cell viability was determined via the incorporation with cck8 whereas cell viability index was determined by calculating the od values of the control group as results are presented as the mean ± sd asterisks denote statistically significant differences p p p compared with the control group c flow cytometry analysis of t cell apoptosis in responses to rhcadrm1 stimuli apoptosis determination was performed via 7aad and annexin vpe staining d statis | 0 |
"30 These findings prompted us to investigate the possible presence of SETDB1 gene amplification and its associated overexpression in lung cancer cells and primary tumors. Extra copies of an oncogene may give tumor cells a growth advantage as well as being a mechanism associated with different sensitivity to therapies.31 The identification of amplified target genes is of great importance for cancer diagnosis and prognosis and ultimately for designing targeted therapies ERBB2/HER-2 in breast cancer being the best example.32 Thus we examined whether the SETDB1 gene amplification occurs in lung cancer cell lines and primary tumors and studied its impact on mRNA and protein expression levels its functional role in lung cancer growth and its potential value as a biomarker for predicting the response to particular chemotherapeutic agents in lung tumors. Results and Discussion We first screened a collection of 15 human lung cancer cell lines for SETDB1 gene copy-number alterations. These included seven non-small (A549 NCI-H1299 NCI-H1975 NCI-H1993 NCI-H2170 NCI-H1437 and NCI-H1395) and eight small (HCC-33 N417 NCI-H446 NCI-H1048 NCI-H1963 NCI-H2029 DMS-114 and DMS-273) cell lung cancer types. The lung cancer cell lines were purchased from the American Type Culture Collection (Rockville MD USA) and were grown and maintained in 10% fetal bovine serum in Roswell Park Memorial Institute medium. Primary normal tissues such as lung epithelium and leukocytes were used as normal SETDB1 copy-number control samples. Using a quantitative genomic PCR approach we observed that two non-small (NCI-H1437 and NCI-H1395) and one small (DMS-273) cell lung cancer lines had a greater than four-fold change in SETDB1 gene copy number (a). This increase was particularly important in the small lung cancer cell line DMS-273 (a). The remaining eleven lung cancer cell lines did not present any evident change in SETDB1 gene copy number (that is amplification half gene dosage or homozygous deletion). We performed fluorescence in situ hybridization analyses to confirm the presence of SETDB1 gene amplification suggested to occur in the three lung cancer cell lines by the competitive genomic PCR approach (b). The fluorescence in situ hybridization technique confirmed the presence of SETDB1 gene amplification in NCI-H1437 NCI-H1395 and DMS-273 (b). The applied fluorescence in situ hybridization technique was validated by the observation of the normal copy-number of the SETDB1 gene in lymphocytes (b). Using reported copy-number variation data33 we have determined the size of the amplicon that contains SETDB1 in the three studied lung cancer cell lines: DMS-273 (1?038?210?bp) NCI-H1437 (4?539?342?bp) and NCI-H1395 (4?623?419?bp). SETDB1 is genomically located in the middle of all three amplicons even in the case of the smallest one (DMS-273; Supplementary Figure S1). In melanoma the only other tumor type where SETDB1 genetic amplification has been reported21 all the melanoma cell lines with amplification at this genomic locus (1q21) contain the SETDB1 gene according to the copy-number variation data.33 In addition in the smallest amplicon detected in the melanoma setting (cell line COLO-679 2?797?611?bp) SETDB1 is also located right in the middle (Supplementary Figure S1). Thus SETDB1 is within the smallest identified region of recurrent amplification. We next considered the possible existence of an association between extra copies of the SETDB1 gene and overexpression of the corresponding RNA transcript and protein using quantitative reverse transcriptionPCR and western blot approaches respectively (Figures 1c and d). We found that the expression of SETDB1 for both mRNA and protein was enhanced in cancer cell lines harboring the SETDB1 gene amplification event relative to non-amplified cancer cells (Figures 1c and d). In the smallest SETDB1 amplicon (present in DMS-273 cells) thirty-four genes are also co-amplified (Supplementary Figure S1). Using reported microarray expression data we have observed that in addition to SETDB1 only 7 of these other 34 genes (21%) are overexpressed in DMS-273 NCI-H1437 and NCI-H1395 in comparison with unamplified lung cancer cells (NCI-H1299 and A549; Supplementary Figure S1). In this regard we have confirmed by quantitative reverse transcriptionPCR that six of these seven genes are overexpressed in the SETDB1-amplified lung cell lines (Supplementary Figure S1). Once we had demonstrated the presence of SETDB1 gene amplification and its associated overexpression in the lung cancer cell lines we examined its contribution to the tumorigenic phenotype in vitro and in vivo. We first analyzed the effect of SETDB1 depletion in lung cancer cells harboring its gene amplification and its associated overexpression. Supplementary Table S1 includes all the used short hairpin RNA (shRNA) sequences. Three SETDB1 shRNA-depleted clones were established for DMS-273 cells (A30 A31 and B32-63) and two clones for NCI-H1437 (A56-B and B49-6). Experiments for each clone were performed in triplicate. We observed that the reduction of SETDB1 expression in the gene-amplified cells had cancer growth inhibitory features (). Upon stable transfection of shRNAs against SETDB1 in the gene-amplified DMS-273 and NCI-H1437 lung cancer cell lines and efficient depletion of the SETDB1 protein (a) the cells proved less viable in the 3-(45-dimethyl-2-thiazolyl)-25-diphenyl-2H-tetrazolium bromide (MTT) assay (b and Supplementary Figure S2) and had a markedly reduced percentage colony-formation density in the assay developed on plastic plate (c and Supplementary Figure S2). Transfection of the scramble shRNA did not reduce cell viability (b and Supplementary Figure S2) and had no impact on the colony formation assay (c and Supplementary Figure S2). Confocal microscopy experiments in the NCI-H1437 lung cancer cell line confirmed the expected nuclear staining of the SETDB1 protein (4'6-diamidino-2-phenylindole colocalization; Supplementary Figure S3) and the disappearance of the nuclear signal upon SETDB1 shRNA-mediated depletion (Supplementary Figure S3). SETDB1 knockdown by the shRNA approach in a lung cancer cell line without gene amplification (NCI-H1299) did not cause a significant effect in the cell growth determined by the MTT experiments or the colony formation assay (Supplementary Figure S3) suggesting the SETDB1 dependence for cell growth only in the amplified cancer cells. We next tested the ability of SETDB1 shRNA-transfected DMS-273 and -NCI-H1437 cells to form tumors in nude mice compared with scramble shRNA-transfected cells (d). DMS-273- and NCI-H1437-scramble shRNA-transfected cells formed tumors rapidly but cells with shRNA-mediated depletion of SETDB1 had much lower tumorigenicity (d). " | 1 |
"The estimate of the OR of each individual trial corresponds to the middle of the squares and horizontal line gives the 95% CI.On each linethe numbers of events as a fraction of the total number randomized are shown for both treatment groups.For each subgroupthe sum of the statistics along with the summary OR is represented by the middle of the solid diamonds.A test of heterogeneity between the trials within a subgroup is given below the summary statistics. Four studies compared the 1-year disease free survival rate (OR?=?1.31; 95% CI 0.792.19; p?=?0.30)finding no significant heterogeneity among these studies (x2?=?1.82P?=?0.61I2?=?0%) (Fig 5) and four studies compared the 3-year disease free survival rate (OR?=?0.59; 95% CI0.380.91; p?=?0.02) finding no significant heterogeneity (x2?=?1.82P?=?0.61I2?=?0%) between the patients who underwent VATS and those who underwent open thoracotomy (Fig 6). Because of the heterogeneity in the sample size sensitivity analyses were conducted using larger sample size studies; however there was no difference between the two surgical methods with an OR of 1.71 (95% CI1.022.89) and with heterogeneity (?2?=? 3.07P ?=?0.22 I2?=?35%). There were significant 3-year disease free survival rate benefits with open thoracotomy. We attempted to evaluate the 5-year disease free survival rate.Only two studies reported these ratesand the published data were not sufficient for the combined analysis. .0085329.g005 1-year disease-free survival rate. Forest plot of the Odds Ratio(OR) of the 1-year disease free survival rate following VATS versus open thoracotomy for metastatic lung cancer.The estimate of the OR of each individual trial corresponds to the middle of the squares and horizontal line gives the 95% CI.On each linethe numbers of events as a fraction of the total number randomized are shown for both treatment groups.For each subgroupthe sum of the statistics along with the summary OR is represented by the middle of the solid diamonds.A test of heterogeneity between the trials within a subgroup is given below the summary statistics. .0085329.g006 3-year disease-free survival rate. Forest plot of the Odds Ratio(OR) of the 3-year survival rate following VATS versus open thoracotomy for metastatic lung cancer.The estimate of the OR of each individual trial corresponds to the middle of the squares and horizontal line gives the 95% CI.On each linethe numbers of events as a fraction of the total number randomized are shown for both treatment groups.For each subgroupthe sum of the statistics along with the summary OR is represented by the middle of the solid diamonds.A test of heterogeneity between the trials within a subgroup is given below the summary statistics. Publication bias Publication bias might exist when nonsignificant findings remain unpublishedthus artificially inflating the apparent magnitude of an effect.The funnel plots of the study are shown in .The funnel plots of the 1-year survival rate following VATS and thoracotomy for the treatment of metastatic lung cancer showed asymmetry which suggested that there was some publication bias. .0085329.g007 Funnel plot of the outcome of 1-year survival rate. Discussion Many tumors can metastasize to the lungand colorectal and breast tumors are the most common primary tumors[9].Pulmonary resection has been shown to be beneficial for patients with resectable and isolated pulmonary metastases[10]. Traditional open thoracotomy and VATS are two principally different surgical methods for pulmonary metastasectomy.The selection of an approach depends more on the theoretical knowledge and personal experience of the surgeon than on the evidence. Over the past two decades several studies have demonstrated the benefits of VATS that included less postoperative pain shorter hospital stays a smaller degree of immunosuppression and enhanced recovery and the ability to tolerate adjuvant therapy[11][13]. Whether the long-term advantages are comparable to those of open thoracotomy is not well documented. The major deficiency of the VATS approach is that nodules might be undetected by VATS that might be detected by manual palpation during thoracotomy; such missing nodules are not imaged on a preoperative CT scan. The VATS approach has long been controversial because VATS does not consistently detect all the metastases and it is recognized that complete resection remains a major determining factor of survival [14].The detection rate of HRCT for pulmonary metastases is 7884%[15][17].Kayton[18]found that 35% of the pathologically verified metastases were missed by CT. In the International Registry of Lung Metastases study of 5206 patients the 5-year survival rate was 36% for complete resection compared with 13% incomplete resectoin[19]. It is not certain whether the nodule imaged on a CT scan and resected by VATS is the correct one [14]. Those who disagree with the use of VATS hypothesize that VATS-related recurrence is commonly observed including port-site recurrence and resection stump recurrence[20]. Johnstone reported 23 cases of port-site chest wall recurrence related to VATS[21]. They hypothesized that the thoracoscopic approach should only be used in patients with a solitary lesion and when resection is requried for diagnostic purposes. The surgeons who favor the VATS approach advocate that VATS minimizes pain and trauma to the patients and that the VATS group might have an improved tolerance of chemotherapy which would likely ensure delivery of planned post-resection adjuvant therapy without a reduction in dosage or delay. The standard surgical procedure for pulmonary metastases is wedge resection that usually does not require manipulation of the pulmonary hilum which is appropriate for the VATS approach.They hypothesiezd that a lesion overlooked by CT but detected by palpation might not result in a survival gain[22] [23] and may be partially compensated for by carefully follow-up.Flores[24] hypothesized that the VATS group might demonstrate a great number of metachronous tumors over time;however the metachronous lesions in each group was similar. Our work suggests that thoracoscopic resection of metastatic lung cancer is a safe and curative procedure with 13 and 5-year survival rates comparable to those of thoracotomy. Patients with metastatic lung cancer are likely to relapse in the lung and after lung metastasectomy by VATS patients might benefit from a second metastasectomy. We hypothesize that earlier chemotherapy and radiation are essential to maximizing survival. Our study might be subject to pretreatment selection bias because most of the patients selected for open thoracotomy had multiple lesions and high risk and were not suitable for treatment with VATS.The missing lesions perhaps skewed the data more toward VATS as an equivalent procedure. We were also interested in the recurrence of cancerand the disease-free survival rates were evaluated. This study demonstrates a similar 1-year disease-free survival rate;however the 3-year disease-free survival rate is inferior for three reasons. First unrelated cancer deaths were included in our analysis of the 13 and 5-year overall survival which might account for VATS having a comparable overall survival rate but an inferior disease-free survival rate. Secondthe patients in the VATS group might have lesions that are missed and there are more likely to relapse in the lung leading to the inferior 3-year disease-free survival rate.Third some of our included studies were in the early period of VATS development when the technology was immature and some of the complications can now be prevented with more experience. Schaeff[25] reported 23 cases of port-site recurrence associated with VATS that occurred before 1998.The number of cases studied was small and the observation period was limitied. Spiral computed tomography has a far higher detection rate today than it did 20 years ago;so small lesions can be accurately localized before surgery[26] which ensures the success of VATS. With advances in imaging technology palpaiton during open thoracotomy is becoming less important.The latest VATS technology has a high-definition resolution and the flexible-tip thoracoscope enables complete inspection of the pleural cavity.These advancements ensure that VATS is an ideal method for patients with a solitary and relatively small peripheral lesions.Tamas[27] hypothesizes that palpation is necessary in a therapeutic metastasectomy as opposed to a diagnostic procedure.Whether patients with multiple lesions should be treated with open thoracotomy or VATS is controversial. This study is the first meta-analysis of the oncological outcome of thoracoscopic surgery for the treatment of metastatic lung cancer. In our work we observed that VATS might be a promising treatment for metastatic lung cancer. No randomized trials existing to guide doctors in the field of metastatic lung cancer currently. A prospective randomized study of the different surgical strategies is needed. Limitation No randomized controlled trials existing to comparing VATS with thoracotomy have been conducted. Heterogeneity was observed between the sample size and the years covered. Most studies are limited to small observational studies and single-institution case series. For these reasonsthere are only a total of 546 patients were included in the two groups for a study period spans more than a decade. Two of the studies comprise almost 65% of the patients and one study has only 20 patients; there are potential sources of bias in our work.Additional randomized controlled trials in the studies we accessed would have increased the strength of our results.There is a bias for the English language. Conclusion In our meta-analysis we found that for patients with metastatic lung cancer comparing VATS with thoracotomy showed almost equivalent survival rates. The VATS can not replace open thoracotomy completely. Further study is neededand a large multicenter randomized trial comparing VATS and thoracotomy would be ideal. Supporting Information Checklist S1 PRISMA Checklist. (DOC) Click here for additional data file. References 1 RuschVW (2010) Pulmonary metastasectomy: a moving target. J Thorac Oncol5: S13013120502246 2 CassonAG PutnamJB NatarajanG JohnstonDA MountainC et al (1992) Five-year survival after pulmonary metastasectomy for adult soft tissue sarcoma. Cancer69: 6626681730117 3 van HalterenHK van GeelAN HartAA ZoetmulderFA (1995) Pulmonary resection for metastases of colorectal origin. Chest107: 152615317781341 4 KandiolerD KromerE TuchlerH EndA MullerMR et al (1998) Long-term results after repeated surgical removal of pulmonary metastases. Ann Thorac Surg65: 9099129564899 5 McCormackPM BainsMS BeggCB BurtME DowneyRJ et al (1996) Role of video-assisted thoracic surgery in the treatment of pulmonary metastases: results of a prospective trial. Ann Thorac Surg62: 213216 discussion 216217.8678645 6 SaishoS NakataM SawadaS YamashitaM SaekiH et al (2009) Evaluation of video-assisted thoracoscopic surgery for pulmonary metastases" | 1 |
" gastric neoplasms containing neuroendocrine carcinoma nec components are rare malignancieswith highly aggressive behavior and a poor prognosis and include pure nec and mixed tumors containing neccomponents we aimed to investigate whether there is a distinct difference in overall survival os between gastricneoplasms containing nec components and gastric adenocarcinomamethods surgically resected gastric neoplasms containing nec components n and gastricadenocarcinomas n from january to december at peking university cancer hospital wereretrospectively analysed patients were categorized into a surgical group and a neoadjuvant group and adjustedusing propensity score matching in the two groups gastric neoplasms containing nec components were dividedinto pure nec and mixed tumors with less than ghminen between and ghminen andmore than ghminen neuroendocrine carcinoma components os was compared between thesegroups and the gastric adenocarcinoma groupresults the os of gastric neoplasms containing neuroendocrine nec components was poorer than that of gastricadenocarcinomas in the surgical group regardless of whether the percentage of neuroendocrine cancercomponents was less than between and more than or cox multivariable regressionanalysis suggested that tumor category neoplasms containing nec components or gastric adenocarcinoma wasan independent risk factor for prognosis interestingly among patients receiving neoadjuvant therapy thedifference was not significantcontinued on next page correspondence buzhaodecjcrcn jijiafuhscpkueducn jiahui chen anqiang wang and ke ji contributed equally to this workdepartment of gastrointestinal surgery key laboratory of carcinogenesisand translational research ministry of education peking university cancerhospital institute no fucheng road haidian district beijing china the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cchen bmc cancer page of continued from previous pages gastric neoplasms containing any proportion of nec components had poorer overall survival thangastric adenocarcinoma in patients treated with surgery directly indicating that these neoplasms are moremalignant than gastric adenocarcinoma among the patients receiving neoadjuvant therapy the difference inoverall survival was not significant which was in sharp contrast with the results of the surgery group suggestingthat neoadjuvant therapy may have a good effect in the treatment of these neoplasmskeywords neuroendocrine carcinoma gastric adenocarcinoma overall survival gastric neoplasms containing neuroendocrine carcinomanec components are a heterogeneous subgroup ofgastric cancer with highly aggressive behavior and poorprognosis and include pure necs and mixed tumorscontaining nec components every yearthere areapproximately million new cases of gastric cancerworldwide and gastric neoplasms containing nec components account for approximately of thesecases [ ] given the low incidence there is little comprehensive basic and clinical research to systematicallyguide the treatment of these gastric neoplasms makingthe prognosis of these tumors unsatisfactory []according to the world health anizationwho digestive neuroendocrine tumor classificationneuroendocrine neoplasm nen can be divided intothree categories based on ki67 levels and mitotic counts hpf grade g1 ki67 mitoses grade g2 ki67 mitoses grade g3ki67 mitoses meanwhile the americanjoint committee on cancer ajcc defines highly differentiated nen as a neuroendocrine tumor net and thepoorly differentiated nen as a neuroendocrine carcinoma nec based on the degree of tumor cell differentiation generally g1 g2 and rare welldifferentiated g3nens belong to the nets while poorly differentiatedg3 nens belong to necs[ ] gastric mixedneuroendocrinenonneuroendocrineneoplasm gminen is a special type of gastric nen that is definedas containing more than of both neuroendocrineand nonneuroendocrine components accountingfor approximately of all gnens and of gastricneuroendocrine carcinomas gnecs [] for thosemixed tumors with less than or more than neuroendocrine carcinoma components there is no uniform definition consideringthe heterogeneity ofminen and the malignancy degree of the different components in the tumor la rosa [ ] proposeddividing minen into three categories highgradeintermediategrade and lowgrade highgrade minenconsists of nec and carcinomaadenoma intermediategrade mimen consists of net and carcinoma and lowgrade minen consists of net and adenoma thereforein this study gastric highgrade mixed neuroendocrinenonneuroendocrine neoplasm ghminen was defined as gastric cancer containing more than of bothneuroendocrineadenocarcinomacomponentscarcinomaandgenerally the prognosis of mixed tumors is largely determined by the most malignant component kim found that gnec has shorter progressionfree survival pfs than gastric adenocarcinoma huang found that the prognosis of patients with more than of neuroendocrine cancer components is significantly poorer than that of patients with less than components all of these studies provide evidence thattumors containing neuroendocrine cancer componentsmay contribute to a worse prognosis therefore wehypothesized that a mixed tumor containing neuroendocrine carcinoma components would have a worse prognosis than pure adenocarcinoma alone we sought tofind studies on the overall survival os comparison between ghminen and gastric adenocarcinoma butfailed thus we think that a study of the comparison ofthe os of ghminen and gastric adenocarcinoma willprovide a valuable supplement to current research on ghminen to overcome the bias caused by the differences between the covariates in the comparison we usedpropensity score matching psm to match importantfactors such as age gender tumor location tumor sizepathological staging and adjuvant chemotherapy between the two groups making the research results morereliablemethodspatient selectionwe retrospectively collected patients diagnosed withgastric nens and underwent radical resection at pekinguniversity cancer hospital beijing from january to december the inclusion criteria were as follows pathologically confirmed pure nec or tumorcontaining nec components no other tumors werediagnosed before the operation complete clinicopathological information and survival information thatcould be obtained through followup patients diagnosedwith cm1 or ct4b before surgery or died from perioperative complications were excluded from the study 0cchen bmc cancer page of patients with gastric adenocarcinomas undergoing radical surgery were randomly selected for psm analysesperformed the chisquared test and mannwhitney utest were used to further verify the matching resultsfollowupwe followed the patients at least twice a year serumtumor markers test gastroscope and computed tomography ct scans were used to reexamine patients aftersurgery depending on the patients status magneticresonance imaging mri and positron emission tomography computed tomography petct were alsoconsidered for patients who cannot regularly visit ourcenter for postoperative examination we use telephonefollowup to obtain survival informationdiagnosis and classificationwe reevaluated the diagnosis and classification of ghminen mixed tumors with less than or morethan neuroendocrine carcinoma components werealso included in this study which were defined as ghminen and ghminenrespectively atumor consisting of nec is defined as pure necall neuroendocrine tumors were identified diagnosedand classified by two independent pathologists in accordance with the who classification of tumors neuroendocrine components were identified byhistological features and immunohistochemical specificity marks such as synaptophysin syn chromogranina cga and neuro cell adhesion molecule cd56 orncam the tumor staging described in the study wasbased on the ajcc 8th edition tnm staging guidelines all possible disagreements were discussed in ourstudy groupdefinition of variables and groupsin this study patients were divided into a surgical groupand a neoadjuvant group based on whether they had received neoadjuvant therapy before surgery patients inthe surgery group were assessed by the ptnm stagingsystem while patients in the neoadjuvanttreatmentgroup were assessed by the yptnm staging system osrefers to the time from surgery to the last followup thetime of death or the end ofloss offollowup or other cause of deathfollowup egpropensity score matchingto accurately compare the prognosis of ghminenand gastric adenocarcinoma we employed psm to balance the differences between the two groups psm wasperformed through the pamatching plugin in spss software logistic regression models were used toestimate propensity scores based on gender age tumorlocation tumor size and pathological staging given a caliper width nearest neighbor matching wasstatistical analysisall statistical analyses were performed using spss statisticalsoftware ibm united states the chisquared test and mannwhitney u test were used forstatistical analysis of categorical variables and continuous variables respectively kaplanmeier method wasused for the comparison of os the logrank test wasused to compare survival rates multivariable cox proportional hazards models were used to identify predictors of survival outcome p was regarded as thethreshold of significanceresultspatient selection and psm resultsbetween and among the patients treated atthe gastrointestinal cancer center of peking universitycancer hospital a total of patients with gastric neoplasms containing nec components met the inclusioncriteria for the study including cases of pure necand cases of mixedtype of these patients a total of patients received neoadjuvant therapy nec ghminen ghminen ghminen while the remaining patients receivedsurgery directly nec ghminen ghminen ghminen there were aninsufficient number of patients in group ghminen group to conduct effective statistical analysisso we combined the ghminen group with thenec group for further analysis we also randomly selected patients with gastric adenocarcinoma whounderwent radical surgery among them patientsreceived neoadjuvant therapy and the remaining patients were treated with surgery directly fig immunohistochemical specificity markers were utilizedto identify the neuroendocrine components fig 2asyn was expressed in almost all neoplasms containingnec components while the positive rates ofcga and cd56 were much lower and respectively no significant difference in the positiverate of syn and cga was observed between pure nec ghminen ghminen and ghminenfig 2b c only the positive rate of cd56 was found tobe higher in the pure nec group than that in the ghminen group fig 2dtherefore priorto os comparison psm wasperformed to ensure that there were no significant differences in patient gender age tumor location tumorsize pathological staging and adjuvant chemotherapybetween the two groups 0cchen bmc cancer page of fig flow chart of patient enrolmentcomparison of os between all patients with neccomponents and patients with gastric adenocarcinoma inthe surgical group and neoadjuvant groupbefore psm we compared the survival curves between all patients with nec components and patientswith gastric adenocarcinoma by the kaplanmeiermethod fig apparently patients with nec components had a poorer os than those with gastricadenocarcinoma fig 3a p in the surgicalgroup in contrast no significant difference was observed between the patientsreceiving neoadjuvanttherapy fig 3b p according to the proportion of nec components patients were classifiedinto pure nec ghminen ghminenand ghminen the os was also comparedbetween patients with adenocarcinomaand thesegroups and the results were similar to the overallcomparison fig 3c dfig illustrations of immunohistochemical staining patterns in gastric neoplasms containing nec components a an overview of the expressionof syn cga and cd56 in tumors containing nec components b syn expression in different nec component groups c cga expression indifferent nec component groups d cd56 expression in different nec component groups cd56 neuro cell adhesion molecule cgachromogranin a nec neuroendocrine carcinoma syn synaptophysin pvalue 0cchen bmc cancer page of fig see legend on next page 0cchen bmc cancer page of see figure on previous pagefig comparison of os between gastric neoplasms containing nec components and gastric adenocarcinoma a os comparison betweengastric neoplasms containing nec components and gastric adenocarcinoma before psm in the surgical group b os comparison between gastricneoplasms containing nec components and gastric adenocarcinoma before psm in the neoadjuvant group c os comparison between differentnec content groups pure nec ghminen ghminen and ghminen and gastric adenocarcinoma before psm in the surgicalgroup d os comparison between the different nec content groups and gastric adenocarcinoma before psm in the neoadjuvant group e oscomparison for patients in the surgical group after psm f os comparison for patients in the neoadjuvant group after psm nec neuroendocrinecarcinoma os overall survival psm propensity score matchingbefore psm significant differences between the baseline characteristics were observed in the surgical groupand the neoadjuvant group table table to balance the clinicopathological differences between the twogroups psm was performed to ensure that there wereno significant differences in patient gender age tumorlocation tumor size pathological staging and adjuvantchemotherapy between the two groups the detailedclinicopathological characteristics before and after psmare shown in table and table as a result patients with nec components and patients with gastric adenocarcinoma were matchedin the surgical group table patients with nec components also had a poorer os than those with gastricadenocarcinoma fig 3e p multivariable analysis showed that adjuvant therapy tumor category andtnm stage werefactorstable independent prognosticto investigate whether neoadjuvant therapy had an effect on os patients with nec components and patients with gastric adenocarcinoma were matched inthe neoadjuvant group table interestingly kaplanmeier analysis showed that among patients receivingneoadjuvant therapy there was still no significant difference in os between the two groups fig 3f p comparison of os between patients with differentproportions of nec components and patients with gastricadenocarcinomato investigate whether the level of nec componentshad an effect on os in the surgical group ghminen ghminen pure nec and pure nec plus ghminen were compared with gastric adenocarcinoma after psm the results showed that even thegroup with the lowest proportion of nec componentsthe ghminen group had a poorer os thanadenocarcinoma fig 4a p as expected theghminen pure nec and pure nec plus ghminen groups each with relatively high proportionsof nec components had worse os than the gastricadenocarcinoma group fig 4bd p detailed clinical information after matching isshown in additional file tables s1s4psm was also performed in the neoadjuvant group incontrast to the results of the surgery group in the purenec group containing the highest proportion ofnec componentstill no significantdifference in os from gastric adenocarcinoma fig5d the other three groups with lower nec contentwere also notfrom gastricadenocarcinoma in terms of os fig 5ac detailedclinicopathologicaland afterpsm are shown in additional file tables s5s8characteristics beforethere wassignificantly differentdiscussionamong gastric neuroendocrine neoplasms the tumorcontaining nec components is a special type includingpure nec and mixed tumor containing nec components the incidence of these tumors is extremely lowbut they are more invasive and have a poorer prognosisthan welldifferentiated gnens [ ]received neoadjuvantin previous study kim found that in patientschemotherapywho had notprogressionfree survivalpfs of pure gnec waspoorer than that of gastric adenocarcinoma while thepfs of mixedtype tumors was not significantly differentin kimsfrom that of gastric adenocarcinoma study the mixed type was defined as net mixed withgastric cancer rather than nec net is much less malignant than nec [ ] this may be the reason whythere was no significant difference in os between mixedtype and gastric adenocarcinomas in addition mixed tumors with less than or more than of nec components were not included in that study which webelieve was a deficit of the study pfs is an important indicator for evaluating prognosis in many cases it can reflect the trend of os based on kims research resultswe regarded tumors containing nec components as awhole and found that the os of these tumors was poorerthan that of adenocarcinoma in the surgical group inthe comparison of os between mixed tumors with different proportions of nec components and gastricadenocarcinoma the results for pure nec cases wassimilar to kims while the os of mixed tumors was alsopoorer than that of gastric adenocarcinoma whether theproportion of neuroendocrine cancer components wasless than between and or more than which was not mentioned in kims study cox multivariable regression analysis showed thattumor categoryneoplasm with nec component or adenocarcinoma 0cchen bmc cancer page of table comparison of clinicopathological characteristics before and after psm in surgical grouppatient characteristicsunmatched comparisonpatients with neccomponents n p valuematched comparisonpatients with neccomponents n age year mean ± sdgender malefemalebmi mean ± sdadjuvant therapyyesnotumor locationupper thirdmiddle thirdlower thirdentiretumor size cm¥ cmtype of gastrectomytotal gastrectomydistal gastrectomy ± ± proximal gastrectomy surgical procedureopenlaparoscopict staget1t2t3t4n stagen0n1n2n3m stagem0m1ptnm stageiiiiiiiv gastricadenocarcinoman ± ± ± ± p value gastricadenocarcinoman ± ± bmi body mass index minen mixed neuroendocrinenonneuroendocrine neoplasm nec neuroendocrine carcinoma psm propensity score matchingpatients with nec components nec high grade minen high grade minen and high grade minen 0cchen bmc cancer table comparison of clinicopathological characteristics before and after psm in neoadjuvant groupmatched comparisonpatient characteristicsunmatched comparisonpatients with neccomponents n age year mean ± sdgender malefemalebmi mean ± sdadjuvant therapyyesnotumor locationupper thirdmiddle thirdlower thirdentiretumor size cm¥ cmtype of gastrectomytotal gastrectomydistal gastrectomyproximal gastrectomysurgical procedureopenlaparoscopict staget0t1t2t3t4n stagen0n1n2n3m stagem0m1yptnm stageiiiiiiiv ± ± gastricadenocarcinoman ± ± p valuepatients with neccomponents n ± ± page of p valuegastricadenocarcinoman ± ± bmi body mass index minen mixed neuroendocrinenonneuroendocrine neoplasm nec neuroendocrine carcinoma psm propensity score matchingpatients with nec components nec high grade minen high grade minen and high grade minen 0cchen bmc cancer page of table univariate and multivariate analyses of survival after psm in surgical grouppatient characteristicsunivariate analysishr cimultivariate analysishr cip valueage yeargendermale vs femalebmiadjuvant therapyyes vs notumor size¥ cm vs cmtumor categorycarcinoma with nec component vsgastric adenocarcinoma vstype of gastrectomytotal gastrectomydistal gastrectomyproximal gastrectomysurgical procedurelaparoscopic vs opentnm stageiiiiiiivp value tumor size and tnm staging were independent risk factors for prognosis this suggests that the prognosis ofgastric neoplasms with nec components is substantiallydifferent from that of gastric adenocarcinoma and evena small percentage of nec components can alsoimpair prognosis which challenges the current cutoffvalue of the proportion of each component that must theoretically be greater than was set in andsince who has also adopted this standard to define minen this largely avoids the overdiagnosisof minen in tumors with only focal neuroendocrinemarker expression and no corresponding morphologicalchanges in additionit also prevents clinicians fromdealing with these rare neoplasms too often withoutguidelines nevertheless it is now being questionedby an increasing number of scholars the componentsin mixed tumors are not evenly distributed for large tumorsthe randomness of biopsy and postoperativepathological sampling causes the proportion of eachcomponent to fluctuate greatly making it difficult to describe the proportion of each component precisely park compared the os between tumors with morethan nec components and gastric adenocarcinomawith or without less than nec and they found thattumors with an nec composition of more than hada worse prognosis this suggests that even a small proportion of malignant components can affect prognosis while in parks study for unknown reasons the authors did not compare the prognosis of mixed tumorswith nec components less than with gastricadenocarcinomas directly nor did they compare allneccontaining tumors as a whole with gastric adenocarcinoma which we believe was a deficit of the studyin our study we regarded tumors containing neccomponents as a whole and found that the os of thesetumors was poorer than that of adenocarcinoma in thesurgical group in addition we also found that the os ofmixed tumors with less than between and more than nec components or pure nec wasworse than that of gastric adenocarcinoma analysis ofimmunohistochemical markers show that there was nosignificant difference in the positive rate of syn and cgabetween different nec content groups only the positiverate of cd56 was found to be higher in the pure necgroup than that in the ghminen group therole of cd56 in the diagnosis of nec is still controversial however syn and cga are two wellrecognized 0cchen bmc cancer page of fig comparison of os between gastric neoplasm with different proportions of nec and gastric adenocarcinoma in the surgical group aoverall survival comparison between ghminen and gastric adenocarcinoma b overall survival comparison between ghminen andgastric adenocarcinoma c overall survival comparison between ghminen plus pure nec and gastric adenocarcinoma d overall survivalcomparison between pure nec alone and gastric adenocarcinomamarkers therefore from the results of immunohistochemistry we believed that there was no significantlydifference in tumors containing nec componentsstudies on the molecular mechanism of pathogenesisshow that nec components and adenocarcinoma components have similar genomic abnormalities similarlosses of heterozygosity loh and mutations at multiple loci and key oncogenes such as tp53 apc and rbgenes all these results imply that the two componentsin the mixed tumor may have a common origin and acquire biphenotypic differentiation during carcinogenesis[] moreoverin the who definition of mixedneuroendocrine and nonneuroendocrine neoplasms ofother ans ie lung and thyroid no minimumpercentage for either ingredient is established thereforewe believe that mixed tumors containing nec components are actually of the same origin have similar biological characteristics and are differentfrom gastricadenocarcinoma we propose considering mixed tumorscontaining nec components as a whole rather than defining them based on the definition for both tumorcomponents which has not been raised by other studiespreviously many studies have confirmed the efficacyof neoadjuvant chemotherapy in gastric adenocarcinoma[ ] in a retrospective study involving patientsma found that neoadjuvant chemotherapy improves the survival of patients with nec and hminenof the stomach van der veen reported that 0cchen bmc cancer page of fig comparison of os between gastric neoplasm with different proportions of nec components and gastric adenocarcinoma in theneoadjuvant group a overall survival comparison between ghminen and gastric adenocarcinoma b overall survival comparisonbetween ghminen and gastric adenocarcinoma c overall survival comparison between ghminen plus pure nec and gastricadenocarcinoma d overall survival comparison between pure nec and gastric adenocarcinomaneoadjuvant chemotherapy could not benefit the survivalof patients with mixed tumors containing nec components however because only eight patients wereincluded in the neoadjuvant group vans results arequestionable in our study among patients receivingneoadjuvanttherapy no significant difference in osbetween mixed tumor and gastric adenocarcinoma wasobserved even for the pure nec group with the highestnec contentthere was no significant differencesuggesting that neoadjuvant therapy may have a positiveeffect on these neoplasmsalthough this is only a singlecenter retrospectivestudy the sample we reported is considerable for thisrare disease which can provide new ideas for clinicaland basic research in addition we proposed treatingall gastric neoplasms containing nec components asa whole and found that neoadjuvanttherapy mayhave a good effect on these neoplasms in the futurewe will conduct more genomics studies to confirmour ideas this study also has its limitations due tothe lack of recurrence and detailed chemotherapy information we were unable to compare progressionfree survival and analyse the effects of differentchemotherapy regimens as a retrospective study despite our performing psm in advance selection biascannot be completely avoided in addition since theexact proportion of each componentin the mixedtumor could not be obtained we could not determine 0cchen bmc cancer page of whether there is a cutoff value for the diagnosis ofthe mixed tumor with nec componentless than so we could only treat all mixed tumors withnec component as a wholesour study demonstrated that gastric neoplasms withnec components regardless of the proportion of components have poorer overall survival than gastric adenocarcinomaindicating a higher degree of malignancythan gastric adenocarcinoma among the patients receiving neoadjuvant therapy the difference in overallsurvival was not significant which was in sharp contrastwith the results of the surgery group suggesting thatneoadjuvant therapy may have a good effect on theprognosis of these malignancies therefore for this typeof malignancy we should adopt more aggressive andpowerful treatments than those used for gastric adenocarcinoma to improve the prognosis of patients neoadjuvant chemotherapy may be a good way to improve theefficacy offor these tumors at advancedstagestreatmentsupplementary informationsupplementary information accompanies this paper at httpsdoi101186s12885020072817additional file table s1 comparison of clinicopathologicalcharacteristics before and after psm of 30ghminen patients insurgical group table s2 comparison of clinicopathologicalcharacteristics before and after psm of ghminen patients in surgicalgroup table s3 comparison of clinicopathological characteristics beforeand after psm of 70ghminen plus pure nec patients in surgicalgroup table s4 comparison of clinicopathological characteristics beforeand after psm of pure nec patients in surgical group table s5 comparison of clinicopathological characteristics before and after psm of 30ghminen patients in neoadjuvant group table s6 comparison ofclinicopathological characteristics before and after psm of ghminen patients in neoadjuvant group table s7 comparison of clinicopathologicalcharacteristics before and after psm of 70ghminen plus pure necpatients in neoadjuvant group table s8 comparison of clinicopathological characteristics before and after psm of pure nec patients in neoadjuvant groupabbreviationsajcc american joint committee on cancer ct computed tomography ghminen gastric highgrade mixed neuroendocrinenonneuroendocrineneoplasm gnec gastric neuroendocrine carcinoma hpf high power fieldminen mixed neuroendocrinenonneuroendocrine neoplasmnec neuroendocrine carcinoma nen neuroendocrine neoplasmnet neuroendocrine tumor mri magnetic resonance imaging os overallsurvival petct positron emission tomography computed tomographypfs progressionfree survival psm propensity score matching who worldhealth anizationacknowledgmentsthanks to dr zhongwu li of the department of pathology peking universitycancer hospital and his colleagues for their assistance in pathologicaldiagnosis and review thanks to all colleagues in the department ofgastrointestinal surgery of peking university cancer hospital and dr jianghong from the statistics department for their assistance in this studyauthors contributionsall authors contributed to the study conception and design jc performeddata collection and wrote the manuscript aw wrote and t revised hemanuscript kj helped with statistical analysis and prepared the illustrationszb edited the manuscript jj conceived the study and reviewed themanuscript all authors read and approved the final manuscriptfundingthis work was supported by the national science foundation for youngscientists of china beijing youth talent plan qml20191101 andscience foundation of peking university cancer hospital thefunders had no role in study design data collection and analysis decision topublish or preparation of the manuscriptavailability of data and materialsthe datasets used andor analysed during the current study are availablefrom the corresponding author on reasonable requestethics approval and consent to participatethe study was approved by the ethics committee of peking universitycancer hospital and the patients written consent was also obtained writteninformed consent for publication was obtained and stored in pekinguniversity cancer hospitalconsent for publicationnot applicablecompeting intereststhe authors declare that they have no competing interestsreceived may accepted august referencesbray f ferlay j soerjomataram i siegel rl torre la jemal a global cancerstatistics globocan estimates of incidence and mortality worldwidefor cancers in countries ca cancer j clin matsubayashi h takagaki s otsubo t iiri t kobayashi y yokota t advanced gastric glandularendocrine cell carcinoma with 1year survivalafter gastrectomy gastric cancer park jy ryu mh park ys park hj ryoo by kim mg prognosticsignificance of neuroendocrine components in gastric carcinomas eur jcancer la rosa s inzani f vanoli a klersy c dainese l rindi g histologiccharacterization and improved prognostic evaluation of gastricneuroendocrine neoplasms hum pathol ishida m sekine s fukagawa t ohashi m morita s taniguchi h neuroendocrine carcinoma of the stomach morphologic andimmunohistochemical characteristics and prognosis am j surg patholrayhan n sano t qian zr obari ak hirokawa m histological andimmunohistochemical study of composite neuroendocrineexocrinecarcinomas of the stomach j med investig jiang sx mikami t umezawa a saegusa m kameya t okayasu i gastriclarge cell neuroendocrine carcinomas a distinct clinicopathologic entityam j surg pathol ohike nan la rosa s who classification of tumours of endocrineans 4th ed lyon iarc press amin mb edge sb ajcc cancer s | 0 |
" Innovation Primary liver cancer PLC is a fatal disease that affects millions of livesworldwide PLC is the leading cause of cancerrelated deaths and theincidence rate is predicted to rise in the coming decades PLC can becategorized into three major histological subtypes hepatocellular carcinoma HCCintrahepatic cholangiocarcinoma ICC and combinedHCCICC These subtypes are distinct with respect to epidemiology clinicopathological features genetic alterations and clinical managementswhich are thoroughly summarized in this review The state of treatmentstrategies for each subtype including the currently approved drugs andthe potential novel therapies are also discussedKEYWORDS PRIMARY LIVER CANCER HEPATOCELLULAR CARCINOMA INTRAHEPATIC CHOLANGIOCARCINOMA COMBINED HCCICC PLC THERAPYIntroductionPrimary liver cancer PLC is a deadly malignancy with significant histological and biological heterogeneity and ranks as the fourth leading cause ofcancerrelated death worldwide12 Therefore it has become a major publichealthy challenge Over the past decades the morbidity and mortality associated with PLC have steadily risen According to Globocan's latest GlobalCancer Statistics Report cases of liver cancer were reported worldwide in accounting for of the total cancer cases in the sameperiod while deaths totaled accounting for of total cancerdeaths3 On the basis of annual projections the World Health anization estimates that patients will die from liver cancer in Incidenceand mortality of PLC differ widely between regions The highest incidenceof PLC was observed in East Asia and in subSaharan Africa4 In particularChina experiences the highest number of cases of PLC with a high incidencerate cases100000 inhabitants5PLC manifests as three subtypes hepatocellular carcinoma HCC intrahepatic cholangiocarcinoma ICC and combined HCCICC cHCCICCwhich differ notably in epidemiology clinicopathological morphology geneticalteration and appropriate therapeutic responses HCCs are primarily relatedto viral infection alcohol abuse and metabolic syndrome6 whereas ICCs aremainly associated with chronic liver ammation and biliary tract diseases78 Risk factors for development of cHCCICC include overweightobsess nonalcoholic steatohepatitis and liver cirrhosis910 HCCs show asolid and trabecular pattern with local invasion restricted to the liver11whereas ICCs are ductular papillary or solid tumor structures with highmetastasis to distal ans14 cHCCICCs are the combination of theHCC and ICC phenotypes present in liver tissue and are classiï¬ed into separate combined and mixed cHCCICC subclasses which are more aggressiveand have a poorer prognosis217The three PLC subtypes have distinct genetic alterations and molecularpatterns HCCs are associated with genetic alterations in speciï¬c chromosomal regions and genes including TERT promoter mutation TP53 deletionand WNT signaling CTNNB1 and AXIN1 activation22 ICCs show aunique mutational landscape with recurrent mutations with the genetic alterations in TP53 KRAS isocitrate dehydrogenase IDH and ï¬broblastgrowth factor receptor FGFR gene fusions30 Combined cHCCICCsshow strong ICClike features whereas mixed cHCCICCs show HCClikefeatures3637 Understanding the molecular alterations that initiate variousPLC subtypes is of great importance for us to decipher the mechanisms oftumorigenesis Genetic alterations can be transformed into biomarkersthat may represent new therapeutic targets affectthe treatmentdecisions and ultimately improve the treatment of liver cancer patientsHCCs mainly respond to targeted therapy immunotherapy and antiviralagents while ICC patients beneï¬t from classical chemotherapy targetedtherapy and immunotherapy Based on the pathological classiï¬cation andthe molecular features of cHCCICCs combined cHCCICCs should betreated with similar therapies to ICCs whereas mixed cHCCICCs are treatedmore like HCCs In this review we systematically summarize the epidemiology pathogenesis genetic alteration and treatment for each subtype andcomprehensively describe current therapy drugs and the potential novel therapies for PLCEpidemiology and Risk Factors HCC HCC represents the major histologic subtype accounting for approximately of all cases of PLC The riskfactors for HCC includes hepatitis B virus HBVhepatitis C virus HCV infection aï¬atoxin B1 alcoholic abuse and nonalcoholic metabolic symptomssuch as diabetes and obesity6 According to the Global Burden of Diseasefrom to HBV and HCV accounted for liver cancer deaths alcohol for and other causes for deathsIn particular of all HCC cases worldwide are reported from China38 dueto the locally high prevalence of HBV infectionICC As the second most common liver carcinoma following HCC ICCaccounts for around of PLC cases with a high incidence of per population worldwide annually39 The most common risk factorsfor ICC are biliary tract diseasesincluding choledochal cysts cholelithiasis choledocholithiasisliver ï¬ukes viral hepatitis metabolic syndromeand other risk factors including tobacco and alcohol use and cirrhosis7Recently the incidence of ICC has been increasing more rapidly owing torisk factors8 including increasing chronic liver disease and environmentaltoxins and is found more often due to improved diagnostic tools andimagingcHCCICC cHCCICC presents as a heterogeneous tumor showing both hepatocyte and cholangiocyte differentiation and has a poor prognosis40cHCCICC is a rather rare tumor with an incidence rate less than Thepoor prognosis associated with cHCCICC is due to the limited treatment options and difï¬culty of diagnosis To date the largest cohort analysis whichincluded patients diagnosed with cHCCICC between and across registries41 reported that the incidence of cHCCICC in men andwomen was and per per year respectively with the averageage of years at diagnosis One and 5year causespeciï¬c survival rates forcHCCICC were and respectively with the median survival of months Among racial groups cHCCICCs are most common in Asianraces and Paciï¬c Islanders Obesity nonalcoholic steatohepatitis and livercirrhosis were observed in some cHCCICC cohorts910 and are potentialrisk factors for cHCCICCClinicopathological Features HCC HCC shows a solid trabecular andpseudoglandular pattern with a high density of tumor cells It has three subtypes welldifferentiated HCC moderately differentiated HCC and poorlyllThe Innovation August 0cnoitavonnIehTReviewdifferentiated HCC11 Welldifferentiated HCCs are often small less than cm in diameter and are composed of cells with a higher nuclear to cytoplasmic ratio arranged in a thin trabecular pattern with rare pseudoglandularstructures Moderately differentiated HCCs are usually larger tumors largerthan cm showing polygonal tumor cells in a thick trabecular arrangementwith a frequent pseudoglandular pattern Poorly differentiated HCCs arecomposed of pleomorphic tumor cells in a solid or compact growth patternICC ICC can be divided into two subtypes a small duct type that originatesfrom small intrahepatic ductules with no or minimal mucin production and alarge bile duct type that arises from large intrahepatic ducts proximal to thebifurcation of the right and left hepatic ducts with high mucin production ability14 Further ICC shows three different growth patterns mass formingMF periductal ltrating PI and intraductal growth IG42 MF ICC is aï¬rm multilobulated unencapsulated whitegray tumor owing to its extensivedesmoplastic stroma The PI subtype shows extensive ltration along theintrahepatic hilum structure and the IG subtype is usually restricted to tubeswith papillary structures MF ICC is the most common type associated with apoor prognosis while IG type is rare but has a favorable prognosis17cHCCICC Though the phenomenon of HCC and ICC being present in thesame liver was ï¬rst described in cHCCICC was not systematicallydescribed until when it was classiï¬ed into three subtypes dependingon the location of HCC and ICC type A separate type has separate nodulesof hepatocellular and bile duct carcinoma type B combined type showscontiguity with intermingling but with clearly deï¬ned areas type C mixedtype presents as intimate association without clear boundaries18 In another classiï¬cation system with three subtypes was established type Icollision tumors is the simultaneous occurrence of both HCC and ICC inthe same patient type II transitional tumors is an identiï¬able intermediatetransition between HCC and ICC type III ï¬brolamellar tumors resemblesthe ï¬brolamellar variant of HCC but also contains mucinproducing pseudoglands19 Presently the World Health anization WHO classiï¬cationis commonly used in which cHCCICC is classiï¬ed into two main types theclassic type and the stem cell SC type subtype with SC features with theSC type subdivided into three subtypes including the typical subtype intermediate subtype INT and cholangiocellular type43The lack of a uniï¬ed classiï¬cation system greatly adds to the difï¬culty forcHCCICC research and the clinicopathological characteristics of cHCCICCremain illdeï¬ned cHCCICC can exhibit stemprogenitor cell phenotypesconsisting of small cells with scant cytoplasm hyperchromatic nucleiembedded within a thick desmoplastic stroma a high nuclearcytoplasmicratio and increased mitotic activity1 In addition the immunohistochemistryhas identiï¬ed stemnessrelated markers KRT19 CD56 EpCAM CD117CD113 OV6120 cHCCICC clinicopathologic characteristics include morefrequent multifocallesions more microvascular emboli and portal veinand lymph node invasion all of which indicate a poor prognosis21Genetic Alterations HCC Widescale genomic studies have revealedthat hundreds of somatic DNA alterations accrue in HCC including chromosome aberrations and mutations Highlevel DNA ampliï¬cations are enrichedin chromosome locations 6p21 and 11q13 in HCC44 which occur in of cases Recently some oncogenic genes were identiï¬ed in the regions offrequent DNA gain For example LINC01138 is an oncogenic long intergenicnoncoding RNA located in this region which has been identiï¬ed as a driver ofHCC45 VEGFA and CCND1FGF19 have also identiï¬ed in these regions andare potential therapeutic targets46 Loss of heterozygosity on chromosome8p is a frequent event in HCC47 These DNA alterations are often associatedwith cancer progression due to the deletion of tumor suppressor genesIntriguingly in these regions a variety of vulnerability genes have recentlybeen identiï¬ed For example TSLNC8 was characterized as a tumor suppressor gene on chromosome 8p12 the region that shows allelic loss in HCC andwas shown to inhibit the proliferation and metastasis of HCC48 The geneticmutations of HCC have been well studied Mutations in the TERT promoteroccur in approximately of cases and cause recurrent viral insertion ofHBV49 Deletion mutations in TP53 are the most frequent genetic alterationsaccounting for about of cases22 and are thought to be the initiatingevent driving the formation of precursor lesions Mutated genes in WNTsignaling CTNNB1 and AXIN1 and chromatin remodeling ARID1A accountfor approximately of cases22 Accumulation of activating mutations in oncogenes including activation of AKT or mTOR and of the oxidativestress pathway activation occurs throughout tumor progression and couldbe potentially targeted with molecular therapies in the futureICC ICC shows a unique mutational landscape with recurrent mutationscompared with HCC It harbors the genetic alterations in TP53 KRASARID1A BAP1 IDH1 IDH2 PIK3CA SMARCB1 EPHA2 SMAD4 GNAS andPBRM1 as well as FGFR gene fusions30 Gain of function of IDH1 andIDH2 mutation on R132 and R172 two hotpot codons was observed in of ICC cases32 Fusions ampliï¬cations translocations and rearrangements of FGFR genes are found in ICC and are closely related to theinitiation and progression of ICC50 The activating mutation of KRAS is another frequent genomic alteration in ICC315152 The KRAS mutationoften exists concurrently with FGFR2 fusions and IDH mutations suggestinga possible cooperative role in ICC pathogenesis5354 In addition recentstudies have shown that BRAF and Notch are considerably more prevalentin ICC and function in ICC pathogenesis55cHCCICC cHCCICCs are genetically complex tumors The combined subtype of cHCCICC shows strong ICClike features with the high expression ofEPCAM KRT19 PRDM5 and KRAS The mixed subtype of cHCCICC showsHCClike features with the high expression levels of AFP GPC3 APOE SALL4and AFP8136The most frequent mutation observed in cHCCICCs is TP53 with a strikingly high mutation frequency much higher than that in HCC and ICC Interestingly several studies have foundthat the disruption of Trp53 alone in livers of mice can induce the formationof cHCCICC3757 which further implies that TP53 may be the driver gene incHCCICC It is notable that Nestin a type VI intermediate ï¬lament IF proteinthat is commonly used as a neuroectodermal SC marker is highly expressedin cHCCICC and is strongly associated with poorer prognosis36 Hence Nestin may be a promising biomarker for cHCCICCChallenges and Limitations of Current Treatment Strategies ResectionTransplantation Local and Regional Therapies HCC The commonlyused staging system for HCC is the Barcelona Clinic Liver Cancer staging system Figure HCCs in the very early stage or intermediate stage can betreated with local regional therapies which include radiofrequency ablationRFA resection da Vinci surgery laparoscopic surgery or traditional surgery transplantation orthotopic liver transplantation piggyback transplantation split liver transplantation auxiliary liver transplantation percutaneousethanoltranscatheter arterial chemoembolizationTACE58injections PEI orICC Surgery is currently the only curative treatment for ICCs but only aminority of patients in early stages are considered candidates for resectionIn surgery ICC is usually treated with hepatic resection to achieve negativeresection margins59 For patients with locally unresectable ICC tumor ablation such as RFA or hepatic arterybased therapies like yttrium90 radioembolization appear promising59cHCCICC An accurate diagnosis is of paramount importance for thetreatment of cHCCICC Currently major hepatectomy is the optimal management for cHCCICC65 The rarity of this cancer as well as the lack of biomarkers have made this cancer difï¬cult to diagnosis and manage Surgicalresection remains the only curative option for patients with cHCCICCThe treatment options for cHCCICC are similar to those for HCC and ICCand include surgery radiation yttrium90 radioembolization chemotherapycombined radiation and chemotherapy combined surgery and chemotherapy and triple therapy surgery radiation and chemotherapy4166 Arecently retrospective analysis from to of PLC patientsincluding cHCCICC HCC and ICC patients who underwentresection found that although cHCCICC is more poorly differentiated thanHCC and ICC it had a similar 5year survival rate and respectively and 3year recurrence rate respectively70Systemic Chemotherapy HCC Systemic chemotherapy has limited efï¬cacy on HCC several clinicaltrials of chemotherapy have shownlow response rates and worse toxicity without a significant improvement inThe Innovation August wwwcellcomtheinnovation\x0cReviewFigure Barcelona Clinic Liver Cancer Staging Systemand Corresponding Treatment Options The schematic diagram illustrates therapeutic choice by which a treatmenttheoretically recommended for a different stage is the besttreatment option 1L ï¬rstline 2L secondline ECOGEastern Cooperative Oncology Group M metastasis stageN nodal stage PEI percutaneous ethanolinjection PSperformance status T tumor stage TACE transarterialchemoembolization TARE transarterialradioembolizationY90 Y90 radioembolizationTheInnovation[5FU]including gemcitabine and doxorubicinbasedthe overall survival OStreatment FOLFOX 5ï¬uorouracilleucovorin oxaliplatin andPIAF cisplatininterferon alpha2bdoxorubicin5FU71 This suggestsa limited role for traditional chemotherapy in the treatment of advanced HCCICC Current ï¬rstline standard of treatment for ICC is the combination ofgemcitabine and platinumderived chemotherapy Figure 2B With the poorprognosis the median survival of advanced ICC patients is less than one yearVery limited effective treatments are available for patients who progress onï¬rstline chemotherapy so there is a high medical demandFirstLine Treatment Effective molecular targeted therapy and immunotherapy is lacking so chemotherapy with gemcitabine platinum compoundsand ï¬uoropyrimidines is still the mainstream of standard treatment for unresectable ICCThe primary chemotherapy for ICC is gemcitabine which was establishedas the ï¬rstline therapy for advanced biliary tract cancer BTC in In the randomized controlled ABC02 phase III clinical trial comparedthe beneï¬t of gemcitabine plus cisplatin CisGem chemotherapy with thesingle agent gemcitabine75 This study showed an advantage for CisGemin OS months versus months hazard ratio [HR] conï¬dence intervalPFS months versus months p This effectiveness wasconï¬rmed in a Japanese randomized phase II study BT22 median OS months versus months HR Based on these promising results CisGem is currently regarded as the standard of care in the ï¬rstlinetreatment for advanced cholangiocarcinoma[CI] and progressionfree survivalOther than cisplatin gemcitabine plus other agents such as oxaliplatin S1capecitabine bevacizumab and Nabpaclitaxel have also been considered asthe ï¬rstline choices for advanced cholangiocarcinoma based on the promising outcomes from several phase II or III trials77A recent multicenter randomized phase III clinical trial NCT01470443showed that XELOX has the comparable efï¬cacious effect to GEMOX interms of tumor response survival rate OS and PFS and safety Also XELOXhas an advantage of low hospital visits compared with GEMOX Thus XELOXcould be an alternative for cholangiocarcinoma therapiesSecondLine Treatment There is no established standard secondlinechemotherapy for advanced cholangiocarcinoma and all regimens haveshown limited efï¬cacy with a median PFS of around months and medianOS of about months92FOLFOX Lfolinic acid 5FU and oxaliplatin is an optional secondlinetreatment option based on the randomized phase III multicenter labelABC06 study NCT01926236 FOLFOX showed increased beneï¬t for median OS months and months and OS rate months and compared with months and for the control groupactive symptom control [ASC] arm92cHCCICC In contrastCurrently several phase II and III chemotherapy clinical trials are under wayTable Combined therapy with chemotherapy shows promise in the treatment of cholangiocarcinoma selective internal radiotherapy SIRT pluschemotherapy or hepatic arterialinfusion plus systemic chemotherapyboth had antitumor activity and are promising for the treatment of ICC9394to surgerybased treatments for resectablecHCCICC systemic therapy is the nonstandard option for advanced and unresectable cHCCICC based on the standard treatment strategy for the unresectable HCC or ICC Chemotherapy for advanced or unresectable cHCCICCis largely understudied with only a few case reports and some retrospectivestudies having been published91095 Recently a multicenter retrospectiveanalysis has been conducted by Kobayashi and colleagues10According to dividedgroup treatment with gemcitabine plus cisplatinn 5FU plus cisplatin n sorafenib monotherapy n others n they found that patients with platinumcontaining treatment had longer OS time than those treated by sorafenib monotherapyshowing OS of months CI months CI months CI and months CI respectivelyA similar conclusion was drawn in another retrospective study of cHCCICC patients with receiving gemcitabinebased therapygemcitabine platinum or gemcitabine 5FU or targeted agents sorafenib9 Median PFS favored gemcitabineplatinum and gemcitabine5FU and months respectively over sorafenib monotherapy monthsllThe Innovation August 0cnoitavonnIehTReviewABFigure Treatment Strategy for Advanced HCC and ICC The schematic illustration represents FDAapproved drugs for treatment of advanced HCC and ICC Firstlinedrugs for HCC include sorafenib lenvatinib atezolizumab plus bevacizumab tremelimumab plus durvalumab and donafenib whereas for ICC the combination ofgemcitabine and cisplatin is currently proposed as ï¬rst line The bottom row represents corresponding secondline therapies that come in when patients are not suitable fortheir ï¬rstline therapyMolecular Targeted Therapy HCC FirstLine Drugs Sorafenib Sorafenib was the ï¬rst US Food and Drug Administration FDAapproved ï¬rstline systemic targeted drug for advanced HCC It is an oral smallmoleculemultikinase inhibitor targeting VEGFR1 VEGFR2 VEGFR3 PDGFRb andRaf Two large international multicenter clinical trials SHARP and AsianPaciï¬c have proved that sorafenib can suppress tumor progression and prolong OS in patients with advanced HCC102103 These trials showed that sorafenib can increase PFS and OS by months in patients with advancedHCC in Western countries As the ï¬rst generation of targeted drugs forHCC sorafenib has been used for over a decade During this time many patients have beneï¬tedthough others quickly developed resistance tosorafenib104Lenvatinib Lenvatinib is becoming available for HCC patients whodevelop sorafenib resistance Lenvatinib is an oral tyrosine kinase inhibitorinhibiting VEGFR1 FGFR1 PDGFR RET and KIT In August theFDA approved lenvatinib for ï¬rstline treatment of patients with unresectableHCC after lenvatinib was proved to be noninferior to sorafenib in the phase REFLECT trial105Median OS in the lenvatinib arm and sorafenib arm was months and months HR CI respectively The adverse effectswere hypertension diarrhea and decreased appetite withlenvatinib and palmarplantar erythrodysesthesia diarrhea decreased weight hypertension and decreased appetite with sorafenibDonafenib Similar to sorafenib donafenib is a novel multikinase inhibitortargeting RAF kinase and various receptor tyrosine kinases RTKs includingVEGF receptor VEGFR and BRAF106 According to the report from International Conference of the American Society of Clinical Oncology CSCOdonafenib significantly improves OS over sorafenib versus monthswith fewer side effects and higher patient tolerance for advanced HCC patients in its phase IIIII label trial107 The grade and above adverse reaction rates for donafenib and sorafenib were and respectivelyThus donafenib was recommended as the ï¬rstline therapy in the CSCOguidelines for HCCSecondLine Drugs Regorafenib Regorafenib an oral multikinase inhibitor inhibits the activity of protein kinases involved in multiple biological processes such as tumorigenesis tumor angiogenesis distant metastasisand tumor immune escape These kinases include VEGFR TIE2RAF1 KIT RET RAF BRAF PDGFR FGFR and CSF1R The randomized doubleblind multicenter phase III clinical trial RESORCE showed that regorafenib significantly improves the OS of patients as compared with the placebofrom to months HR p Grade adverse eventswere reported in of patients receiving regorafenib and of patientsreceiving the placebo In regorafenib received FDA approval as the secondline drug for the treatment of patients with advanced HCC who fail torespond to the sorafenib treatmentCabozantinib Cabozantinib is an oral inhibitor and targets multiple kinasesincluding VEGFR2 cMET RET ROS1 TYRO3 MER KIT TRKBFLT3 TIE2 as well as the GAS6 receptor AXL109110 It was originallyapproved for medullary thyroid cancer in and advanced renal carcinoma in According to the randomized doubleblind multicenter phase clinical trial conducted across centers in countries median OS was months for patients receiving cabozantinib and months for patientstreated with placebo HR p Median PFS was monthsand months respectively Grade or adverse events occurred in of patients in the cabozantinib arm and in the placebo arm Theobserved hepatotoxicity can be mostly controlled through dose modiï¬cations Based on the encouraging results of prolonged OS and PFS cabozantinib received its FDA approval for HCC in Ramucirumab Ramucirumab is a completely human monoclonalantibody that can speciï¬cally inhibit VEGFR2112 For patients with alphafetoprotein R400 ngmL and who have been previously treated with sorafenib ramucirumab was approved as a monotherapy by the FDA on May The Innovation August wwwcellcomtheinnovation\x0cTable Systemic Therapies Currently or Promising Approved for Advanced HCC and ICCReviewTargetTherapy LineApproved YearTrialDrugsHCCSorafenib NexavarLenvatinib LenvimaRegorafenib StivargaNivolumab OpdivoVEGFR2 VEGFR3 PDGFRb RAF kinasesFGFR VEGFR PDGFRa RET KITTie2 VEGFR PDGFR FGFRPD1Cabozantinib CabometyxcMet VEGFR2 AXL RETPembrolizumab KeytrudaRamucirumab CYRAMZAPD1VEGFR2Nivolumab ipilimumab Opdivo YervoyPD1 CTLA4Atezolizumab bevacizumabTremelimumab durvalumabDonafenibApatinibICCGemcitabine cisplatinPemigatinib PemazyreIvosidenibPDL1VEGFPD1 CTLA4VEGFR BRAFVEGFR2chemotherapyFGFR1IDH12TheInnovationpromisingpromisingpromisingpromisingSHARP AsianPaciï¬cREFLECTRESORCECHECKMATE040CELESTIALKEYNOTE224REACH2Cohort of CHECKMATE040IMbravel50NCT02519348NCT02645981NCT02329860ABC02FIGHT202promisingClarlDHyApproval was based on REACH NCT02435433 a randomized doubleblind multicenter phase III study of patients with AFP R400 ngmL whohad disease progression after sorafenib or were intolerant to sorafenib113More recently a study further conï¬rmed the efï¬cacy of ramucirumab inelderly patients with HCC and elevated AFP after sorafenib in REACH andREACH2 with a survival beneï¬t observed across all age subgroups and atolerable safety proï¬le supporting its value irrespective of age including forpatients R75 years114Apatinib Apatinib a tyrosine kinase inhibitor targeting VEGFR2 significantly prolonged OS and PFS in Chinese patients with advanced HCC whohad previously been treated with sorafenib andor chemotherapy accordingto the results of a randomized placebocontrolled phase III trial conducted in sites in China115 Median OS was almost months longer for patients whoreceived apatinib compared with patients receiving the placebo monthsversus months and median PFS was more than months longer months versus months115 The most common grade or worseadverse events occurred at a rate of in the apatinib arm and inthe placebo arm With the significantly prolonged OS and PFS and a manageable safety proï¬le apatinib has potential to become a new secondline therapy for liver cancerNovel Therapeutic Targets Even with all these available treatments Table the median PFS for HCC patients remains less than a year Thus noveltreatment is still a critical unmet need for treatment of HCC Based on thegenomic proï¬le and biomarkers reported in HCC several clinical trials targeting various pathways are currently ongoing Table Recently a ï¬rstinhuman phase I study NCT02508467 of ï¬sogatinib BLU554 an orally bioavailable inhibitor of human FGFR4 demonstrated its antitumor activity in HCCand further validated that the aberrant FGF19FGFR4 signaling pathwaymay be a driver event116 In addition the TGFb1 receptor type I inhibitor galunisertib also showed an acceptable safety and prolonged OS outcome in combination with sorafenib in a phase II trial NCT01246986117118 Other potential candidatesincluding the cyclindependent kinase CDK inhibitorsregulating the cell cycle pathways ribociclib palbociclib119120 abemacicliband milciclib as well as the cMET inhibitors tepotinib121 and tivantinib122are being evaluated in HCC clinical trialsICC Moleculartargeted therapy controls tumor cell proliferationapoptosis adhesion and movement by inhibiting the surface molecules oftumor cell membranes and thereby inhibiting intracellular signaling pathways ICC genetic alterations primarily include FGFR IDH epidermal growthfactor EGFR and breast cancer type susceptible protein associated protein1 BAP1123 Genetic alterations of these genes all have implicationsfor therapy At present a variety of molecular targeted drugs are in the clinicalresearch stage Table some of which have made progress in the treatment of ICC Table FGFR Inhibitors The most promising target therapy for cholangiocarcinoma identiï¬ed in recent years is the inhibitor of the ï¬broblast growth factorFGF signaling pathway which consists of members labeled FGF1FGF15 FGF19 called FGF1519 and four interacting transmembrane receptors FGFR1 FGF signals regulate cell proliferation in which FGFR2fusions occurred in of ICC patients and are considered as a promising therapeutic target3351127128 Currently several FGFR inhibitors are being evaluated in clinical trials for cholangiocarcinomas with FGFR geneticaberrationsPemigatinib INCB054828 Pemigatinib is the ï¬rst and only targeted therapy so far approved in by the FDA for the treatment of this rare cancerIt is a selective potent oral inhibitor of FGFR and Approval wasbased on ï¬ndings from the phase II FIGHT202 trial NCT02924376 whichenrolled patients with locally advanced or metastatic cholangiocarcinoma with FGFR2 fusions or rearrangements cohort A other FGFFGFR genetic alterations cohort B or no FGFFGFR genetic alterations cohort CFor those in cohort A treatment with pemigatinib resulted in a median OSof months and median PFS of months The FIGHT202 study suggests that locally advanced or metastatic cholangiocarcinoma patientswith FGFR2 fusions or rearrangements may beneï¬t from potent oralFGFR1 and inhibitor treatment Median PFS was months for patientswith FGFR2 alterations months for patients with other FGFFGFR alterations and months for those with no alterations in these genes MedianOS was months months and months for the respective cohorts130 With the promising results of phase II the phase III clinical trial ofpemigatinib is currently underway NCT03656536llThe Innovation August 0cnoitavonnIehTDrugTargeted TherapyCabozantinibLenvatinibDonafenibMilciclibPalbociclibRibociclibGalunisertib versus LY2157299 sorafenib versus placebo sorafenibImmunotherapyVEGFRVEGFRVEGFRCDK2CDK46CDK46TGFbToripalimab versus placeboNivolumab versus placeboNivolumab versus sorafenibPD1PD1PD1Hospices Civils de Lyonrecruitingphase Eisai Pharmaceuticals IndiaPvt Ltdnot yetrecruitingphase NCT03963206NCT04297254completedphase phase NCT02645981Suzhou ZelgenBiopharmaceuticalsTiziana LifeSciencesPï¬zeractive notrecruitingactive notrecruitingphase phase Texas Universityrecruitingphase Eli Lillyactive notrecruitingphase NCT03109886NCT01356628NCT02524119NCT02178358NCT03412773NCT03859128NCT03383458ReviewTable Selected Ongoing Systemic Therapy Clinical Trials for Advanced HCCTargetSponsorStatusPhaseEnrollmentTrial Identiï¬erTislelizumab versus sorafenibPD1BeiGeneactive notrecruitingphase Shanghai Junshi Biosciencerecruitingphase phase BristolMyers Squibbrecruitingphase BristolMyers Squibbactive notrecruitingphase NCT02576509Pembrolizumab versus placeboPD1Merck Sharp Dohmerecruitingphase AvelumabPDL1Seoul National UniversityHospitalactive notrecruitingphase Combined TherapyLenvatinib pembrolizumabversus lenvatinib placeboCS1003 lenvatinib versusplacebo lenvatinibVGFR PD1Merck Sharp Dohmeactive notrecruitingphase VGFR PD1CStone Pharmaceuticalsrecruitingphase Tislelizumab regorafenibversus placebo regorafenibVEGF PD1National Taiwan UniversityHospi | 2 |
" laparoscopic tumorspecific mesorectal excision tsme preserving the left colic artery and superiorrectal artery is still a technically challenging procedure we conducted this study to demonstrate the feasibility ofthis procedure for upper rectal cancermethods a total of patients with upper rectal cancer were retrospectively analyzed in our cancer centerbetween april and april these patients were treated with either laparoscopic tsme n orlaparoscopic total mesorectal excision tme n in the tsme group the left colonic artery and superior rectalartery were preserved while they were not in the tme groupresults the operation time in the tsme group was longer than that in the tme group ± min vs ± min p furthermore the number of resected lymph nodes in the tsme group was greaterthan that in the tme group ± vs ± p the blood loss between the tsme and tmegroups was not significant no mortality occurred in either the tsme or tme groups one patient in the tme groupunderwent conversion to laparotomy the total postoperative complication rates in the tsme and tme groups were and respectively there was no difference in severe complications between the two groupsanastomotic leakage and stenosiss laparoscopic tsme preserving the left colic artery and superior rectal artery can be safely conductedfor upper rectal cancerkeywords laparoscopic surgery rectal cancer tumorspecific mesorectal excision superior rectal artery leftcolonic artery tme correspondence 237721898qqcom 250537471qqcomhuxiang_zc1978sinacom chi zhang haotang wei wenqing hu and yueming sun contributedequally as joint first authors7department of general surgery yizhen peoples hospital clinical medicalcollege yangzhou university yangzhou jiangsu province china3department of gastrointestinal surgery changzhi peoples hospital theaffiliated hospital of changzhi medical college changzhi shanxi provincechina1department of gastrointestinal surgery the first affiliated hospital of dalianmedical university dalian liaoning province chinafull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0czhang world of surgical oncology page of introductiontotal mesorectal excision tme is an important surgical technique to prevent the local recurrence of rectal cancer on the other hand tme may not besuitable for every case of rectal cancer such as rectosigmoid junction and upper rectal cancers the resection range of tme reaches cm below the inferiorborder of the tumor and has acquired an adequatecure rate reported in previous studies for patientswith rectosigmoid junction and upper rectal cancers this tumorspecific resection according to thetumorsite or t staging is called tumorspecificmesorectal excision tsme itsudecks critical point at the rectosigmoid junction isdescribed as the point of origin of the last sigmoid arterial branch originating from the inferior mesenteric artery ima the anastomosis between the lastsigmoidal artery and superior rectal artery sra is absent in some people to avoid the risk of postoperativeischemic necrosis anastomotic leakage colitis and delayed stricturetosudecks point for cases where anastomosis may be absent or insufficiently present in addition the rate ofis desirable to ligate proximalabsence of the left colic artery lca is which maybe associated with a risk of anastomotic leakage due toinsufficient vascularization of the proximal colonic conduit this study introduces the procedure and technicalpoints of laparoscopic tsme with preservation of thelca and sra the operation is still a technically challenging procedure we conducted this study to demonstrate the feasibility of this procedure for upper rectalcancer and shortterm prognosismethodspatientslaparoscopic tsme preserving the lca and sra wasperformed on patients with upper rectal cancer fromapril to april in the same period patients with upper rectal cancer underwent standardtme surgery this study was conducted in accordancewith approved guidelines this study was approved bythe institutional review board of the first affiliatedhospital of dalian medical university written informedconsent was obtained from all patientsfig ima 3d cta 0czhang world of surgical oncology page of equipmentangled ° 10mm diameter 3d laparoscope insufflation equipment and bipolar electrosurgical deviceaesculap german harmonic vascular closure systemjohnson usa 10mm and 5mm port trocars teleflexmedical usa laparoscopic linear staplers mm inlength covidien usa hemolock polymer lockingsurgical clips teleflex medical usa and a circularstapler ethicon endosurgery usa were used in thisstudypreoperative preparationinferior mesenteric artery ima 3d cta examinationshould be performed before the operation to assess themesenteric vascular vessel types fig intestinal preparation was performed days before the operation andprophylactic intravenous antibiotics were used beforethe operation for min central venous catheterizationwas performed after general anesthesia the surgicalposture was the starboard lithotomy position with thehead lower and feet higherthe operating surgeon and camera assistant stood onthe patients right side and the first assistant stood atthe patients left foot side the laparoscopic monitor wasplaced on the patients right foot side the trocar for thelaparoscope was inserted from the right paraumbilicalside and four ports were used as working ports fig surgical techniquesthis surgical technique was characterized by thoroughlymph node dissection based on neurovascular preservation and dissection of the left colon and sigmoid andfig position of the trocarupper rectal vessels along the inferior mesenteric vesselsthe region of operation was the superficial layer of thenerve sheath on the vascular surface the left colonicand superior rectal vessels needed to be preserved andthe vascular branch from the sigmoid vessels and theblood vessels from the superior rectal vessels to the intestinal wall were selected and severed according to thetumor positionfirst we adopted a lateral approach by opening themonks white line along the descending and sigmoidcolon reaching the splenic flexure as the cephalad dissection point the correct plane of dissection wasachieved by toldts fascia we usually used bipolar electrosurgical devices and bipolar scissors to separate thiscorrect plane with gentle blunt and sharp dissectionthe ureter and other retroperitoneal structures weresafely protected by staying in this plane we continuedto dissect along the plane to the root of the ima thehypogastric nerves were visible the nerves were carefully protectedthen the dissection began at the position of the sacralpromontory the junction of the sigmoid mesentery andretroperitoneum from the previous dissection plane in thefirst step ideally we dissected the presacral space belowthe sra from the left side across the midline to the rightside attentively protecting the hypogastric nerves whileusing a bipolar electrosurgical device fig 3a the distaldissection endpoint was approximately cm below thetumor we needed to open the peritoneal reflection anddissect the lateral ligament of the rectum by protectingthe neurovascular bundle nvb using a harmonic vascular closure system in some patients we placed the dissected colon and mesocolon to the right celiac side andthoroughly revealed the left side of the mesocolon wecarefully employed dissection in the correct plane on thevessels to avoid tissue damage for the realization of enbloc resection the technique in this step is to identify therelationship between the left colic artery inferior mesenteric vein imv to the ima and sra and the branch ofthe arteriae sigmoideae fig 3b this vascular bundle canbe traced from the origin of the ima to the rectal segmentapproximately cm below the inferior border of thetumor fig 3cthe second step was performed using a medial approach this step involved thorough lymph node dissection based on neurovascular preservation the leftcolonic and superior rectal vessels need to be preservedand the sigmoid vessels and vessel branch from the superior rectal vessels to the intestinal wall were selectedand severed according to the tumor positiondissection at the correct presacral space and cephaladdissection to the ima could be employed our generalmedial approach was to begin at the presacral space andobtain a connection with the plane ofthe lateral 0czhang world of surgical oncology page of fig a dissection the presacral space below the superior rectal artery sra approached from the left side across the midline to the right sideattentively protected hypogastric nerves while using a bipolar electrosurgical device b identification of the relationship between left colic arteryimv to the ima and sra and the branch of the arteriae sigmoideae c tracing this vascular bundle from the origin of the ima to the rectumsegment approximately cm below the inferior border of the tumor d ligation of arteriae sigmoideae and vascular branch from sra eligation of arteriae sigmoideae and preserving left colonic vasculature f excision of the mesorectum just underneath the rectal wall about cm and avoiding injury to the rectal wall and sra g tsme preserving left colic artery and superior rectal arteryapproach pelvic dissection was performed from the entrance of the pelvic cavity down to the pelvic floor wecould identify both the hypogastric nerve fibers and pelvicnerve by using highdefinition 3d laparoscopy and preservethem the imvleft colic artery bundle was then carefullytraced to the junction position from the ima and lymphnode no253 was dissected the pelvic nerves and ureterwere already carefully insulated and the circumference ofthe ima could be revealed the mesocolon could be freedfrom the retroperitoneal position by anterior dissection bygently applying a bipolar electrosurgical device we dissected the sra and blood vessels from the sra to the intestinal wall and dissected lymph nodes no252 andno251 at this point we had completed lymph node dissection and completely clarified the relationship betweenthe lca imv ima sra and arteriae sigmoideae finallywe ligated the arteriae sigmoideae and vascular branchfrom the sra into the intestinal wall fig 3d while preserving the left colonic vasculature fig 3e energy devicesand hemolocks were used widely in this step 0czhang world of surgical oncology page of after the above procedure was completed we separated the rectal wall from the mesorectum with an adequate distance from the tumor in accordance with thet stage and position of the tumor using a harmonic vascular closure system in order to provide enough spaceto insert an endoscopic linear stapler we excised themesorectum about cm just underneath the rectalwall fig 3f careful surgery was performed to avoid injury to the rectal wall and sra then the endoscopic linear stapler was fixed the rectum was transected andsatisfactory tsme preservation of the left colic and superior rectal arteries was shown fig 3glastly a small 5cm incision was made at the leftlower abdomen and the specimen was taken outside ofthe abdomen and transected intraabdominal presacralanastomosis was performed by double stapling techniques after inserting the anvil head of a 28mm circularstapler into the oral side of the sigmoid colon doubledrains were placed and no diverting stoma wasperformedin the tme group the inferior mesenteric artery wassevered at the root the colon was severed cm awayand digestive tract reconstruction methods were similarto the tsme groupstatisticsspss190 version was used for statistical analysis categorical variables were compared using a Ï2 test continuous variables were presented as the mean standarddeviation or median range these variables were compared using a mannwhitney u test p values of were considered statistically significantresultsthe general characteristics of the included patients arelisted in table there were men and women in the tsme group and men and women in the tme group the meanage was ± years and ± years in thetsme and tme groups respectively there were no significant differences in preoperative comorbidity tumorsize depth of invasion and lymph node metastasis between groups the average distance between the tumorand anus of the tsme group was ± cm andthe distal margin was ± cm the pathologicalstages of the patients for the tsme group were as follows stage i stage iia stage iib stage iic stage iiia and stage iiib theproportion of patients with normal preoperative carcinoembryonic antigen cea was approximately of patients had cea levels between and ngml of patients had cea levels between and ngml and only patients had cea levels ngmltable clinicopathological features between the tsme andtme groupsfactorsage yearstme n ± tsme n ± p valuegendermalefemalebmi kgm2comorbidity ± ± cardiovascular disease respiratory diseasediabetes mellitushistological type differentiated typeundifferentiated type tumor size mm ± ± t categoryt1t2t3t4n categoryn0n1n2 conversion to open surgery operation time min ± ± blood loss ml ± ± lymph node dissection ± ± the operation time in the tsme group was longerthan that in the tme group ± vs ± p table furthermore the number ofresected lymph nodes in the tsme group was greaterthan that in the tme group ± vs ± p table the blood loss between groupswas not significantly different table the averagehospital stay in the tsme group was a little shorter thanthat in the tme group ± days vs ± days table no mortality occurred in either group one patient inthe tme group underwent conversion to laparotomy dueto bowel ischemia in the distal colon table the totalpostoperative complication rates in the tsme and tmegroups were and respectively table forsevere complications between the two groups anastomotic leakage and stenosis the severity of complicationswas claviendindo classification grades and therewas no significant difference between groups 0czhang world of surgical oncology page of tsme n tme n p value ± table postoperative complicationsfactorspostoperative hospital stay days ± mortalitymorbidityabsentpresentanastomotic leakagebleedingabdominal abscessileuswound infectionanastomotic stenosisurinary tract infectionascitesurinary retentionpneumoniacardiacrelated complications discussionin the british surgeon heald proposed tme for rectalcancer and pointed out that the anatomical level of tmewas clear so that the operative quality can be assessed the main concerns were a higher anastomotic leakage ratelonger operative time and higher blood loss after tme lopezkostner pointed out that tme was the standard operation performed for lower rectal cancers tme isnot necessary for cancers of the upper rectum therefore the tsme technique was introduced to achieve satisfactory local control and low morbidity partial mesorectalexcision is applied in tsme according to willians report in only of patients had distal intraluminal diffusion cm pollett and nicholls observed that there were no differencesin the local recurrence rate of rectal cancer between distal margins cm cm and cm a randomized prospective study of nsabb the national surgicaladjustburst and bowel project showed that the localrecurrence rate was not significantly different betweendistal rectal margins cm cm and cm according to the practice parameters for the management of rectal cancer edition a 2cm distal margin is more acceptable than cm but a 5cm distalmargin is still recommended total mesorectal resectiontme should be used for tumors located in the middleand lowerregardless oftwothirds ofwhether itis performed with low anterior resectionlar or combined abdominal and perineal resectionapr for tumors in the upper onethird of the rectumresection of the mesentery can be carried out accordingto the tumor situation and the distance between thethe rectumdistal margin and tumor should be cm the recommended grade was 1a tme was performed according to the distance between the distal margin of the rectal tumor and anus cm while tsme was performed for patients with adistance between the distal end of the rectal tumor andanus of cm in the authors medical departmentoncological outcomes after surgery can be divided intotwo aspects longterm survival and local recurrence ratelaw reviewed patients the 5year local recurrence rate for tme and partial mesorectal excisionpme for proximal cancer was and respectively the disease stage was associated with a higher riskof local recurrence there was no difference in the localrecurrence rates of tme and pme the 5year cancerspecific survival rates with and without tme were similarat and respectively kim reportedthat the 5year cancerspecific survival rate was andthe local recurrence rate was with cases of rectalcancer after tsme with pathologic stages iiii the riskfactors affecting cancerspecific survival rate were the ptstage pn stage positive distal resection margin and positive circumferential resection margin the risk factors affecting local recurrence were the pn stage positive distalresection margin and positive circumferential resectionmargin another study from a korean reviewed experience in patients with rectal cancer showed that theoverall local recurrence rate was the 5year local recurrence rates were and in stages i iiand iii respectively the 5year cancerspecific survivalrates were and in stages i ii and iiirespectively the risk factors were the pn stage and circumferential resection margin zakir performed an analysis with years of experience in rectal cancer patients who underwent laparoscopic andopen tsme surgery the 5year local recurrence rate was the overall 5year and cancerspecific survival rateswere and respectively there was no difference in the local recurrence rate between laparoscopic oropen resection the overall and cancerspecific survivalrates were and in the laparoscopic surgerygroup and and in the open surgery group respectively the results showed that laparoscopic surgerywas better than open surgery in overall and cancerspecific survival there was no difference in survival in patients with stage i however the survival rates in patientswith stages ii and iii among the laparoscopic surgerygroup were better than those in the open surgery groupwhich shows the superiority of laparoscopic tsme surgery for the longterm prognosis of rectal cancerkorean scholars conducted a study on the safety andprognosis of tsme after neoadjuvant chemotherapy forrectal cancer patients received 5fu with leucovorinchemotherapy and radiotherapy cgy for cycles 0czhang world of surgical oncology page of leadership was tsme was performed weeks later the resultsshowed that the overall complication rate was empiricalinternal construction was the 5year survival rate was and the 5yeardiseasefree survival was at present chinasouth korea and the usa have formulated similarguidelines for preoperative radiotherapy and chemotherapy for middle and low rectal cancer but there is nospecific reference data for preoperative radiotherapy andchemotherapy for upper rectal cancer the purpose ofthis paper is to introduce a new method of tsme anddiscuss the safety of the operation longterm survivaland local recurrence have not been discussedtsme surgery based on tme is now accepted as astandard for rectal cancer surgery and laparoscopic rectal cancer resection is accepted widely in the world eventhough it is a challenging procedure for surgery bloodloss in the laparoscopic group is well shown with anaverage of to ml the average blood loss inour study was ml lower than that reported in the literature we can identify neurovascular lesions usinghighdefinition 3d laparoscopy to preserve them and weuse a bipolar electrosurgical device to reduce injurywhich is beneficial for accurate operationthe overall complication rate in laparoscopic tsmeoperation was lower than that in the open operationgroup the rate of anastomotic leak showed no statistical difference between the two operation methods theaverage leak rate for rectal cancers was zakir reported that the overall complicationrate was in tsme for rectal cancer patients therate of anastomotic leakage was in the open tsmegroup and in the laparoscopic tsme group therewas no statistical difference between groups in our studythe incidence rate of postoperative anastomotic leakagewas three patients had complications after surgeryand the overall complication rate was the threecomplications were wound infection fluid collection andurinary retention with a claviendindo grading of yoo evaluated the optimal duration of urinarycatheterization after tsme for rectal cancer logistic regression analysis was performed to determine the risk factors for urinary retention the variables including age sexasa grade surgical procedure tnm stage tumor position preoperative radiotherapy duration of urinarycatheterization and time of surgery were not significantrisk factors for urinary retentionat present a 3d laparoscopic system aesculapgerman is used in laparoscopic surgery in our department single and reduced portlaparoscopic surgeryrobot operations and tatme operations are not usedfor tsme the surgeons who performed tsme had morethan years of experience in gastroenterostomy and hadexperience with open tsme the difficulty of the tsmeoperation is the management of the mesorectum seiji has reported on the management of the mesorectumin the narrow pelvis which our treatment method is basedon first the right part of the mesorectum is lifted fromthe right side of the sigmoid mesocolon to expose the inferior mesenteric artery and vein left colonic vessels sigmoid colonic vessels and superior rectum vessels theassistant lifts the left mesentery of the sigmoid colon exposes the above vessels expands the sigmoid mesocolonagain penetrates the mesentery from the right side andexposes the surrounding vessels expansion of the pelviccavity along the vessels is continued and the mesorectumis repaired from the left to the right side cm above thetumor according to the location ofthebranches of the severed vessels are determined and cm of the intestinal wall is repaired the rectum is dissected using an endogia staplerthe tumorlaparoscopic tsme has been used for rectal cancer andcan obtain satisfactory functional results compared to openresection and tme we do not think that the reduction inthe hospital stay is due to the acceleration of the intervention as per enhanced recovery after surgery eras butis due to an increase in the doctors confidence in reducingthe risk of postoperative complications after vascular preservation threedimensional cta examination is importantfor the preoperative evaluation of sigmoid colonvascular classification and intraoperative management ofthe sigmoid and left colon vessels however preoperativeexamination could not obtain information on the trafficbranch the biggest advantage of this operation is themaintenance of the blood supply of the proximal and distalintestines and the sufficient length of the intestine so thereis no need for temporary defunctioning stoma temporarydefunctioning stoma only increases the complexity of theoperation and closure of the temporary stoma increasesthe risk of complications in addition the results of the statistical analysis showed that the number of lymph nodes inthe tsme group was greater than that in the tme groupit cannot be concluded that tsme was significantly betterthan tme for lymph node dissection suggesting thattsme was not inferior to tmeslaparoscopic tsme with preserved left colic and superior rectal arteries is a technically challenging procedureintact visceral pelvic fibro is protected with even greateraccuracy than other techniques by 3d laparoscopywhich offers an optimal vision tsme with preserved leftcolic and superior rectum arteries did not increase therisk of operation compared with tme but increased thesurgeon s confidence in patient outcomes thereforelaparoscopic tsme with preserved left colic and superior rectal arteries can be safely performed for rectal cancer patients as an alternative to tme 0czhang world of surgical oncology page of abbreviationstme total mesorectal excision tsme tumorspecific mesorectal excisionima inferior mesenteric artery imv inferior mesenteric vein sra superiorrectal artery nvb neurovascular bundle pme partial mesorectal excisionacknowledgementsnoneauthors contributionslz yx and xh designed the study cz yx hw qz zd and whcollected and analyzed the data ma lz ys and xh interpreted the datalz and cz drafted the manuscript lz ma yx and xh revised themanuscript the authors read and approved the final manuscriptfundingthis study was supported by the jiangsu natural science foundationbk20180274 this funding supported the collection analysis andinterpretation of the dataavailability of data and materialsall experimental data used to support these findings are included in theethics approval and consent to participatethis study was approved by the institutional review board of the firstaffiliated hospital of dalian medical university written informed consent forpublication was obtained from all patientsconsent for publicationwritten informed consent was obtained from the patients and legalguardian for the publication of these patientscompeting intereststhe authors declare that they have no conflicts of interestauthor details1department of gastrointestinal surgery the first affiliated hospital of dalianmedical university dalian liaoning province china 2department ofgastrointestinal surgery the third affiliated hospital of guangxi medicaluniversity nanning guangxi province china 3department ofgastrointestinal surgery changzhi peoples hospital the affiliated hospital ofchangzhi medical college changzhi shanxi province china 4department ofcolorectal surgery the first affiliated hospital of nanjing medical universitynanjing china 5department of gastrointestinal surgery the first peopleshospital of dali city dali yunnan province china 6department ofgastrointestinal surgery graduate school of medicine university of tokyotokyo japan 7department of general surgery yizhen peoples hospitalclinical medical college yangzhou university yangzhou jiangsu provincechinareceived february accepted august heald rj husband em ryall rd the mesorectum in rectal cancer surgerythe clue to pelvic recurrence br j surg macfarlane jk ryall rd heald rj mesorectal excision for rectal cancerlancet lee ky factors influencing oncologic outcomes after tumorspecificmesorectal excision for rectal cancer j korean soc coloproctol williams ns dixon mf johnston d reapppraisal of the centimetre rule ofdistal excision for carcinoma of the rectum a study of distal intramuralspread and of patients survival br j durg pollett wg nicholls rj the relationship between the extent of distalclearance and survival and local recurrence rates after curative anteriorresection for carcinoma of the rectum ann surg wolmark n fisher b wieand hs the prognostic value of the modificationsof the dukes c class of colorectal cancer an analysis of the nsabp clinicaltrials ann surg monson jrt weiser mr buie wd chang gj rafferty jf prepared by thestandards practice task force of the american society of colon and rectalsurgeons practice parameters for the management of rectal cancerrevised dis colon rectum law wl chu kw anterior resection for rectal cancer with mesorectalexcision a prospective evaluation of patients ann surg kim sh bae kb kim jm oncologic outcomes and risk factors forrecurrence after tumorspecific mesorectal excision of rectal cancer cases j korean soc coloproctol kim nk min bs kim js hur h lee ky sohn sk oncologic outcomesand safety after tumorspecific mesorectal excision for resectable rectalcancer a single institutions experience with patients with rectalcancer j korean soc coloproctol zakir k mohamed wai lun law outcome of tumorspecific mesorectalexcision for rectal cancer the impact of laparoscopic resection world jsurg kim nk baik sh seong js oncologic outcomes after neoadjuvantchemoradiation followed by curative resection with tumorspecificmesorectal excision for fixed locally advanced rectal cancer impact ofpostirradiated pathologic down staging on local recurrence and survivalann surg poon jt law wl laparoscopic resection for rectal cancer a review annsurg oncol yoo be kye bh kim hj early removal of the urinary catheter aftertotal or tumorspecific mesorectal excision for rectal cancer is safe discolon rectum seiji o takashi t kazuki s a new laparoscopic surgical procedure toachieve sufficient mesorectal excision in upper rectal cancer int j surgoncol httpsdoi1011552011708439publishers notespringer nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliationsreferencesenker w e thaler h t cranor m l polyak t total mesorectal excision inthe operative treatment of carcinoma of the rectum j am coll surg lopezkostner i lavery c hool gr rybicki la fazio vw total mesorectalexcision is not necessary for cancer of the upper rectum surgery zaheer s pemberton jh farouk r dozois rr wolff bg ilstrup d surgicaltreatment of adenocarcinoma of the rectum ann surg sudeck p ueber die gefässversung des mastdarmes in hinsicht auf dieoperative gangrän muenchen med wschr van tonder jj boon jm becker jhr anatomical considerations onsudecks critical point and its relevance to colorectal surgery clin anatcirocchi r randolph j cheruiyot i systematic review and metaanalysis of the anatomical variants of the left colic artery color dis httpsdoi101111codi14891 0c" | 0 |
" laparoscopic tumorspecific mesorectal excision tsme preserving the left colic artery and superiorrectal artery is still a technically challenging procedure we conducted this study to demonstrate the feasibility ofthis procedure for upper rectal cancermethods a total of patients with upper rectal cancer were retrospectively analyzed in our cancer centerbetween april and april these patients were treated with either laparoscopic tsme n orlaparoscopic total mesorectal excision tme n in the tsme group the left colonic artery and superior rectalartery were preserved while they were not in the tme groupresults the operation time in the tsme group was longer than that in the tme group ± min vs ± min p furthermore the number of resected lymph nodes in the tsme group was greaterthan that in the tme group ± vs ± p the blood loss between the tsme and tmegroups was not significant no mortality occurred in either the tsme or tme groups one patient in the tme groupunderwent conversion to laparotomy the total postoperative complication rates in the tsme and tme groups were and respectively there was no difference in severe complications between the two groupsanastomotic leakage and stenosiss laparoscopic tsme preserving the left colic artery and superior rectal artery can be safely conductedfor upper rectal cancerkeywords laparoscopic surgery rectal cancer tumorspecific mesorectal excision superior rectal artery leftcolonic artery tme correspondence 237721898qqcom 250537471qqcomhuxiang_zc1978sinacom chi zhang haotang wei wenqing hu and yueming sun contributedequally as joint first authors7department of general surgery yizhen peoples hospital clinical medicalcollege yangzhou university yangzhou jiangsu province china3department of gastrointestinal surgery changzhi peoples hospital theaffiliated hospital of changzhi medical college changzhi shanxi provincechina1department of gastrointestinal surgery the first affiliated hospital of dalianmedical university dalian liaoning province chinafull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0czhang world of surgical oncology page of introductiontotal mesorectal excision tme is an important surgical technique to prevent the local recurrence of rectal cancer on the other hand tme may not besuitable for every case of rectal cancer such as rectosigmoid junction and upper rectal cancers the resection range of tme reaches cm below the inferiorborder of the tumor and has acquired an adequatecure rate reported in previous studies for patientswith rectosigmoid junction and upper rectal cancers this tumorspecific resection according to thetumorsite or t staging is called tumorspecificmesorectal excision tsme itsudecks critical point at the rectosigmoid junction isdescribed as the point of origin of the last sigmoid arterial branch originating from the inferior mesenteric artery ima the anastomosis between the lastsigmoidal artery and superior rectal artery sra is absent in some people to avoid the risk of postoperativeischemic necrosis anastomotic leakage colitis and delayed stricturetosudecks point for cases where anastomosis may be absent or insufficiently present in addition the rate ofis desirable to ligate proximalabsence of the left colic artery lca is which maybe associated with a risk of anastomotic leakage due toinsufficient vascularization of the proximal colonic conduit this study introduces the procedure and technicalpoints of laparoscopic tsme with preservation of thelca and sra the operation is still a technically challenging procedure we conducted this study to demonstrate the feasibility of this procedure for upper rectalcancer and shortterm prognosismethodspatientslaparoscopic tsme preserving the lca and sra wasperformed on patients with upper rectal cancer fromapril to april in the same period patients with upper rectal cancer underwent standardtme surgery this study was conducted in accordancewith approved guidelines this study was approved bythe institutional review board of the first affiliatedhospital of dalian medical university written informedconsent was obtained from all patientsfig ima 3d cta 0czhang world of surgical oncology page of equipmentangled ° 10mm diameter 3d laparoscope insufflation equipment and bipolar electrosurgical deviceaesculap german harmonic vascular closure systemjohnson usa 10mm and 5mm port trocars teleflexmedical usa laparoscopic linear staplers mm inlength covidien usa hemolock polymer lockingsurgical clips teleflex medical usa and a circularstapler ethicon endosurgery usa were used in thisstudypreoperative preparationinferior mesenteric artery ima 3d cta examinationshould be performed before the operation to assess themesenteric vascular vessel types fig intestinal preparation was performed days before the operation andprophylactic intravenous antibiotics were used beforethe operation for min central venous catheterizationwas performed after general anesthesia the surgicalposture was the starboard lithotomy position with thehead lower and feet higherthe operating surgeon and camera assistant stood onthe patients right side and the first assistant stood atthe patients left foot side the laparoscopic monitor wasplaced on the patients right foot side the trocar for thelaparoscope was inserted from the right paraumbilicalside and four ports were used as working ports fig surgical techniquesthis surgical technique was characterized by thoroughlymph node dissection based on neurovascular preservation and dissection of the left colon and sigmoid andfig position of the trocarupper rectal vessels along the inferior mesenteric vesselsthe region of operation was the superficial layer of thenerve sheath on the vascular surface the left colonicand superior rectal vessels needed to be preserved andthe vascular branch from the sigmoid vessels and theblood vessels from the superior rectal vessels to the intestinal wall were selected and severed according to thetumor positionfirst we adopted a lateral approach by opening themonks white line along the descending and sigmoidcolon reaching the splenic flexure as the cephalad dissection point the correct plane of dissection wasachieved by toldts fascia we usually used bipolar electrosurgical devices and bipolar scissors to separate thiscorrect plane with gentle blunt and sharp dissectionthe ureter and other retroperitoneal structures weresafely protected by staying in this plane we continuedto dissect along the plane to the root of the ima thehypogastric nerves were visible the nerves were carefully protectedthen the dissection began at the position of the sacralpromontory the junction of the sigmoid mesentery andretroperitoneum from the previous dissection plane in thefirst step ideally we dissected the presacral space belowthe sra from the left side across the midline to the rightside attentively protecting the hypogastric nerves whileusing a bipolar electrosurgical device fig 3a the distaldissection endpoint was approximately cm below thetumor we needed to open the peritoneal reflection anddissect the lateral ligament of the rectum by protectingthe neurovascular bundle nvb using a harmonic vascular closure system in some patients we placed the dissected colon and mesocolon to the right celiac side andthoroughly revealed the left side of the mesocolon wecarefully employed dissection in the correct plane on thevessels to avoid tissue damage for the realization of enbloc resection the technique in this step is to identify therelationship between the left colic artery inferior mesenteric vein imv to the ima and sra and the branch ofthe arteriae sigmoideae fig 3b this vascular bundle canbe traced from the origin of the ima to the rectal segmentapproximately cm below the inferior border of thetumor fig 3cthe second step was performed using a medial approach this step involved thorough lymph node dissection based on neurovascular preservation the leftcolonic and superior rectal vessels need to be preservedand the sigmoid vessels and vessel branch from the superior rectal vessels to the intestinal wall were selectedand severed according to the tumor positiondissection at the correct presacral space and cephaladdissection to the ima could be employed our generalmedial approach was to begin at the presacral space andobtain a connection with the plane ofthe lateral 0czhang world of surgical oncology page of fig a dissection the presacral space below the superior rectal artery sra approached from the left side across the midline to the right sideattentively protected hypogastric nerves while using a bipolar electrosurgical device b identification of the relationship between left colic arteryimv to the ima and sra and the branch of the arteriae sigmoideae c tracing this vascular bundle from the origin of the ima to the rectumsegment approximately cm below the inferior border of the tumor d ligation of arteriae sigmoideae and vascular branch from sra eligation of arteriae sigmoideae and preserving left colonic vasculature f excision of the mesorectum just underneath the rectal wall about cm and avoiding injury to the rectal wall and sra g tsme preserving left colic artery and superior rectal arteryapproach pelvic dissection was performed from the entrance of the pelvic cavity down to the pelvic floor wecould identify both the hypogastric nerve fibers and pelvicnerve by using highdefinition 3d laparoscopy and preservethem the imvleft colic artery bundle was then carefullytraced to the junction position from the ima and lymphnode no253 was dissected the pelvic nerves and ureterwere already carefully insulated and the circumference ofthe ima could be revealed the mesocolon could be freedfrom the retroperitoneal position by anterior dissection bygently applying a bipolar electrosurgical device we dissected the sra and blood vessels from the sra to the intestinal wall and dissected lymph nodes no252 andno251 at this point we had completed lymph node dissection and completely clarified the relationship betweenthe lca imv ima sra and arteriae sigmoideae finallywe ligated the arteriae sigmoideae and vascular branchfrom the sra into the intestinal wall fig 3d while preserving the left colonic vasculature fig 3e energy devicesand hemolocks were used widely in this step 0czhang world of surgical oncology page of after the above procedure was completed we separated the rectal wall from the mesorectum with an adequate distance from the tumor in accordance with thet stage and position of the tumor using a harmonic vascular closure system in order to provide enough spaceto insert an endoscopic linear stapler we excised themesorectum about cm just underneath the rectalwall fig 3f careful surgery was performed to avoid injury to the rectal wall and sra then the endoscopic linear stapler was fixed the rectum was transected andsatisfactory tsme preservation of the left colic and superior rectal arteries was shown fig 3glastly a small 5cm incision was made at the leftlower abdomen and the specimen was taken outside ofthe abdomen and transected intraabdominal presacralanastomosis was performed by double stapling techniques after inserting the anvil head of a 28mm circularstapler into the oral side of the sigmoid colon doubledrains were placed and no diverting stoma wasperformedin the tme group the inferior mesenteric artery wassevered at the root the colon was severed cm awayand digestive tract reconstruction methods were similarto the tsme groupstatisticsspss190 version was used for statistical analysis categorical variables were compared using a Ï2 test continuous variables were presented as the mean standarddeviation or median range these variables were compared using a mannwhitney u test p values of were considered statistically significantresultsthe general characteristics of the included patients arelisted in table there were men and women in the tsme group and men and women in the tme group the meanage was ± years and ± years in thetsme and tme groups respectively there were no significant differences in preoperative comorbidity tumorsize depth of invasion and lymph node metastasis between groups the average distance between the tumorand anus of the tsme group was ± cm andthe distal margin was ± cm the pathologicalstages of the patients for the tsme group were as follows stage i stage iia stage iib stage iic stage iiia and stage iiib theproportion of patients with normal preoperative carcinoembryonic antigen cea was approximately of patients had cea levels between and ngml of patients had cea levels between and ngml and only patients had cea levels ngmltable clinicopathological features between the tsme andtme groupsfactorsage yearstme n ± tsme n ± p valuegendermalefemalebmi kgm2comorbidity ± ± cardiovascular disease respiratory diseasediabetes mellitushistological type differentiated typeundifferentiated type tumor size mm ± ± t categoryt1t2t3t4n categoryn0n1n2 conversion to open surgery operation time min ± ± blood loss ml ± ± lymph node dissection ± ± the operation time in the tsme group was longerthan that in the tme group ± vs ± p table furthermore the number ofresected lymph nodes in the tsme group was greaterthan that in the tme group ± vs ± p table the blood loss between groupswas not significantly different table the averagehospital stay in the tsme group was a little shorter thanthat in the tme group ± days vs ± days table no mortality occurred in either group one patient inthe tme group underwent conversion to laparotomy dueto bowel ischemia in the distal colon table the totalpostoperative complication rates in the tsme and tmegroups were and respectively table forsevere complications between the two groups anastomotic leakage and stenosis the severity of complicationswas claviendindo classification grades and therewas no significant difference between groups 0czhang world of surgical oncology page of tsme n tme n p value ± table postoperative complicationsfactorspostoperative hospital stay days ± mortalitymorbidityabsentpresentanastomotic leakagebleedingabdominal abscessileuswound infectionanastomotic stenosisurinary tract infectionascitesurinary retentionpneumoniacardiacrelated complications discussionin the british surgeon heald proposed tme for rectalcancer and pointed out that the anatomical level of tmewas clear so that the operative quality can be assessed the main concerns were a higher anastomotic leakage ratelonger operative time and higher blood loss after tme lopezkostner pointed out that tme was the standard operation performed for lower rectal cancers tme isnot necessary for cancers of the upper rectum therefore the tsme technique was introduced to achieve satisfactory local control and low morbidity partial mesorectalexcision is applied in tsme according to willians report in only of patients had distal intraluminal diffusion cm pollett and nicholls observed that there were no differencesin the local recurrence rate of rectal cancer between distal margins cm cm and cm a randomized prospective study of nsabb the national surgicaladjustburst and bowel project showed that the localrecurrence rate was not significantly different betweendistal rectal margins cm cm and cm according to the practice parameters for the management of rectal cancer edition a 2cm distal margin is more acceptable than cm but a 5cm distalmargin is still recommended total mesorectal resectiontme should be used for tumors located in the middleand lowerregardless oftwothirds ofwhether itis performed with low anterior resectionlar or combined abdominal and perineal resectionapr for tumors in the upper onethird of the rectumresection of the mesentery can be carried out accordingto the tumor situation and the distance between thethe rectumdistal margin and tumor should be cm the recommended grade was 1a tme was performed according to the distance between the distal margin of the rectal tumor and anus cm while tsme was performed for patients with adistance between the distal end of the rectal tumor andanus of cm in the authors medical departmentoncological outcomes after surgery can be divided intotwo aspects longterm survival and local recurrence ratelaw reviewed patients the 5year local recurrence rate for tme and partial mesorectal excisionpme for proximal cancer was and respectively the disease stage was associated with a higher riskof local recurrence there was no difference in the localrecurrence rates of tme and pme the 5year cancerspecific survival rates with and without tme were similarat and respectively kim reportedthat the 5year cancerspecific survival rate was andthe local recurrence rate was with cases of rectalcancer after tsme with pathologic stages iiii the riskfactors affecting cancerspecific survival rate were the ptstage pn stage positive distal resection margin and positive circumferential resection margin the risk factors affecting local recurrence were the pn stage positive distalresection margin and positive circumferential resectionmargin another study from a korean reviewed experience in patients with rectal cancer showed that theoverall local recurrence rate was the 5year local recurrence rates were and in stages i iiand iii respectively the 5year cancerspecific survivalrates were and in stages i ii and iiirespectively the risk factors were the pn stage and circumferential resection margin zakir performed an analysis with years of experience in rectal cancer patients who underwent laparoscopic andopen tsme surgery the 5year local recurrence rate was the overall 5year and cancerspecific survival rateswere and respectively there was no difference in the local recurrence rate between laparoscopic oropen resection the overall and cancerspecific survivalrates were and in the laparoscopic surgerygroup and and in the open surgery group respectively the results showed that laparoscopic surgerywas better than open surgery in overall and cancerspecific survival there was no difference in survival in patients with stage i however the survival rates in patientswith stages ii and iii among the laparoscopic surgerygroup were better than those in the open surgery groupwhich shows the superiority of laparoscopic tsme surgery for the longterm prognosis of rectal cancerkorean scholars conducted a study on the safety andprognosis of tsme after neoadjuvant chemotherapy forrectal cancer patients received 5fu with leucovorinchemotherapy and radiotherapy cgy for cycles 0czhang world of surgical oncology page of leadership was tsme was performed weeks later the resultsshowed that the overall complication rate was empiricalinternal construction was the 5year survival rate was and the 5yeardiseasefree survival was at present chinasouth korea and the usa have formulated similarguidelines for preoperative radiotherapy and chemotherapy for middle and low rectal cancer but there is nospecific reference data for preoperative radiotherapy andchemotherapy for upper rectal cancer the purpose ofthis paper is to introduce a new method of tsme anddiscuss the safety of the operation longterm survivaland local recurrence have not been discussedtsme surgery based on tme is now accepted as astandard for rectal cancer surgery and laparoscopic rectal cancer resection is accepted widely in the world eventhough it is a challenging procedure for surgery bloodloss in the laparoscopic group is well shown with anaverage of to ml the average blood loss inour study was ml lower than that reported in the literature we can identify neurovascular lesions usinghighdefinition 3d laparoscopy to preserve them and weuse a bipolar electrosurgical device to reduce injurywhich is beneficial for accurate operationthe overall complication rate in laparoscopic tsmeoperation was lower than that in the open operationgroup the rate of anastomotic leak showed no statistical difference between the two operation methods theaverage leak rate for rectal cancers was zakir reported that the overall complicationrate was in tsme for rectal cancer patients therate of anastomotic leakage was in the open tsmegroup and in the laparoscopic tsme group therewas no statistical difference between groups in our studythe incidence rate of postoperative anastomotic leakagewas three patients had complications after surgeryand the overall complication rate was the threecomplications were wound infection fluid collection andurinary retention with a claviendindo grading of yoo evaluated the optimal duration of urinarycatheterization after tsme for rectal cancer logistic regression analysis was performed to determine the risk factors for urinary retention the variables including age sexasa grade surgical procedure tnm stage tumor position preoperative radiotherapy duration of urinarycatheterization and time of surgery were not significantrisk factors for urinary retentionat present a 3d laparoscopic system aesculapgerman is used in laparoscopic surgery in our department single and reduced portlaparoscopic surgeryrobot operations and tatme operations are not usedfor tsme the surgeons who performed tsme had morethan years of experience in gastroenterostomy and hadexperience with open tsme the difficulty of the tsmeoperation is the management of the mesorectum seiji has reported on the management of the mesorectumin the narrow pelvis which our treatment method is basedon first the right part of the mesorectum is lifted fromthe right side of the sigmoid mesocolon to expose the inferior mesenteric artery and vein left colonic vessels sigmoid colonic vessels and superior rectum vessels theassistant lifts the left mesentery of the sigmoid colon exposes the above vessels expands the sigmoid mesocolonagain penetrates the mesentery from the right side andexposes the surrounding vessels expansion of the pelviccavity along the vessels is continued and the mesorectumis repaired from the left to the right side cm above thetumor according to the location ofthebranches of the severed vessels are determined and cm of the intestinal wall is repaired the rectum is dissected using an endogia staplerthe tumorlaparoscopic tsme has been used for rectal cancer andcan obtain satisfactory functional results compared to openresection and tme we do not think that the reduction inthe hospital stay is due to the acceleration of the intervention as per enhanced recovery after surgery eras butis due to an increase in the doctors confidence in reducingthe risk of postoperative complications after vascular preservation threedimensional cta examination is importantfor the preoperative evaluation of sigmoid colonvascular classification and intraoperative management ofthe sigmoid and left colon vessels however preoperativeexamination could not obtain information on the trafficbranch the biggest advantage of this operation is themaintenance of the blood supply of the proximal and distalintestines and the sufficient length of the intestine so thereis no need for temporary defunctioning stoma temporarydefunctioning stoma only increases the complexity of theoperation and closure of the temporary stoma increasesthe risk of complications in addition the results of the statistical analysis showed that the number of lymph nodes inthe tsme group was greater than that in the tme groupit cannot be concluded that tsme was significantly betterthan tme for lymph node dissection suggesting thattsme was not inferior to tmeslaparoscopic tsme with preserved left colic and superior rectal arteries is a technically challenging procedureintact visceral pelvic fibro is protected with even greateraccuracy than other techniques by 3d laparoscopywhich offers an optimal vision tsme with preserved leftcolic and superior rectum arteries did not increase therisk of operation compared with tme but increased thesurgeon s confidence in patient outcomes thereforelaparoscopic tsme with preserved left colic and superior rectal arteries can be safely performed for rectal cancer patients as an alternative to tme 0czhang world of surgical oncology page of abbreviationstme total mesorectal excision tsme tumorspecific mesorectal excisionima inferior mesenteric artery imv inferior mesenteric vein sra superiorrectal artery nvb neurovascular bundle pme partial mesorectal excisionacknowledgementsnoneauthors contributionslz yx and xh designed the study cz yx hw qz zd and whcollected and analyzed the data ma lz ys and xh interpreted the datalz and cz drafted the manuscript lz ma yx and xh revised themanuscript the authors read and approved the final manuscriptfundingthis study was supported by the jiangsu natural science foundationbk20180274 this funding supported the collection analysis andinterpretation of the dataavailability of data and materialsall experimental data used to support these findings are included in theethics approval and consent to participatethis study was approved by the institutional review board of the firstaffiliated hospital of dalian medical university written informed consent forpublication was obtained from all patientsconsent for publicationwritten informed consent was obtained from the patients and legalguardian for the publication of these patientscompeting intereststhe authors declare that they have no conflicts of interestauthor details1department of gastrointestinal surgery the first affiliated hospital of dalianmedical university dalian liaoning province china 2department ofgastrointestinal surgery the third affiliated hospital of guangxi medicaluniversity nanning guangxi province china 3department ofgastrointestinal surgery changzhi peoples hospital the affiliated hospital ofchangzhi medical college changzhi shanxi province china 4department ofcolorectal surgery the first affiliated hospital of nanjing medical universitynanjing china 5department of gastrointestinal surgery the first peopleshospital of dali city dali yunnan province china 6department ofgastrointestinal surgery graduate school of medicine university of tokyotokyo japan 7department of general surgery yizhen peoples hospitalclinical medical college yangzhou university yangzhou jiangsu provincechinareceived february accepted august heald rj husband em ryall rd the mesorectum in rectal cancer surgerythe clue to pelvic recurrence br j surg macfarlane jk ryall rd heald rj mesorectal excision for rectal cancerlancet lee ky factors influencing oncologic outcomes after tumorspecificmesorectal excision for rectal cancer j korean soc coloproctol williams ns dixon mf johnston d reapppraisal of the centimetre rule ofdistal excision for carcinoma of the rectum a study of distal intramuralspread and of patients survival br j durg pollett wg nicholls rj the relationship between the extent of distalclearance and survival and local recurrence rates after curative anteriorresection for carcinoma of the rectum ann surg wolmark n fisher b wieand hs the prognostic value of the modificationsof the dukes c class of colorectal cancer an analysis of the nsabp clinicaltrials ann surg monson jrt weiser mr buie wd chang gj rafferty jf prepared by thestandards practice task force of the american society of colon and rectalsurgeons practice parameters for the management of rectal cancerrevised dis colon rectum law wl chu kw anterior resection for rectal cancer with mesorectalexcision a prospective evaluation of patients ann surg kim sh bae kb kim jm oncologic outcomes and risk factors forrecurrence after tumorspecific mesorectal excision of rectal cancer cases j korean soc coloproctol kim nk min bs kim js hur h lee ky sohn sk oncologic outcomesand safety after tumorspecific mesorectal excision for resectable rectalcancer a single institutions experience with patients with rectalcancer j korean soc coloproctol zakir k mohamed wai lun law outcome of tumorspecific mesorectalexcision for rectal cancer the impact of laparoscopic resection world jsurg kim nk baik sh seong js oncologic outcomes after neoadjuvantchemoradiation followed by curative resection with tumorspecificmesorectal excision for fixed locally advanced rectal cancer impact ofpostirradiated pathologic down staging on local recurrence and survivalann surg poon jt law wl laparoscopic resection for rectal cancer a review annsurg oncol yoo be kye bh kim hj early removal of the urinary catheter aftertotal or tumorspecific mesorectal excision for rectal cancer is safe discolon rectum seiji o takashi t kazuki s a new laparoscopic surgical procedure toachieve sufficient mesorectal excision in upper rectal cancer int j surgoncol httpsdoi1011552011708439publishers notespringer nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliationsreferencesenker w e thaler h t cranor m l polyak t total mesorectal excision inthe operative treatment of carcinoma of the rectum j am coll surg lopezkostner i lavery c hool gr rybicki la fazio vw total mesorectalexcision is not necessary for cancer of the upper rectum surgery zaheer s pemberton jh farouk r dozois rr wolff bg ilstrup d surgicaltreatment of adenocarcinoma of the rectum ann surg sudeck p ueber die gefässversung des mastdarmes in hinsicht auf dieoperative gangrän muenchen med wschr van tonder jj boon jm becker jhr anatomical considerations onsudecks critical point and its relevance to colorectal surgery clin anatcirocchi r randolph j cheruiyot i systematic review and metaanalysis of the anatomical variants of the left colic artery color dis httpsdoi101111codi14891 0c" | 0 |
" fasassociated factor faf1 has been implicated in parkinsons disease pd and activates the celldeath machinery in the cytosol however the presence of extracellular faf1 has not been studiedmethods serumfree conditioned medium cm from faf1transfected shsy5y cells was concentrated andanalyzed by western blotting exosomes were isolated from cm by ultracentrifugation and analyzed by westernblotting electron microscopy and nanop tracking analysis soluble faf1 from cm was immunodepleted usingantifaf1 antibody transmission of secreted faf1 was examined by transwell assay under a confocal microscopecminduced cell death was determined by measuring propidium iodide pi uptake using a flow cytometerresults faf1 was secreted from shsy5y cells via exocytosis and brefeldin a bfaresistant secretory pathwaysfurthermore faf1 was secreted as a vesiclefree form and a genuine exosome cargo in the lumen of exosomes inaddition faf1 increased the number of exosomes suggesting a regulatory role in exosome biogenesis extracellularfaf1 was transmitted via endocytosis to neighboring cells where it induced cell death through apoptotic andnecrotic pathwayss this study presents a novel route by which faf1 induces neuronal death through celltocelltransmissionkeywords faf1 secretion exosome vesiclefree form celltocell transmission cell death cells secrete proteins harboring signal peptides throughthe classical secretory pathway via the endoplasmicreticulum ergolgi complex from which vesicles withcargo proteins move toward and fuse with the plasmamembrane and subsequently export cargos to the extracellular space however proteins lacking signal peptides are secreted via alternative nonclassical secretorypathways these pathways are classified as vesicularand nonvesicular secretory pathways some proteins correspondence eunheecnuackr gyeongrin park and bokseok kim are considered cofirst authorsdepartment of biological sciences chungnam national university daehakro yuseonggu daejeon south koreaare secreted via extracellular vesicles including exosomesand other vesicles of various sizes [ ] alternativelyother proteins are secreted through membrane poresand atpbinding cassette abc transporters althoughthe exact mechanisms of nonvesicular secretory pathways are elusive [ ]exosomes which are nanosized membrane vesicles nm in diameter secreted into the extracellular environment by various cell types are associated with intercellular communication with neighboring cells and play arole in a myriad of pathological functions in diseases including cancer cardiovascular and neurodegenerative diseasestheextracellular matrix and promote metastasis angiogenesisin particular exosomes[]remodel the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cpark cell communication and signaling page of thrombosis and tumor cell proliferation in cancer exosomes in cardiovascular disease display proangiogenesis procoagulant and proinflammatory effects on thevessel walls in neurodegenerative diseases exosomesare potential sources of key pathogenic proteins such astau amyloid prion and αsynuclein []inflammation triggersin addition to their vesiclemediated secretion manyproteins are secreted through nonvesicular mechanismsfor instance the proteins such as tau and fibroblastgrowth factor are recruited to the plasma membrane toform lipidic pores and are subsequently secreted [ ]in additionthe secretion ofinterleukin1 and transglutaminase through pores fibroblast growth factor translocates across the plasmamembrane via poorly understood mechanisms includingthe membrane release complex upon stress proteinssuch as hydrophilic acylated surface protein b are secretedby abc transporters proteins secreted via nonvesicular secretory pathways are advantageous over cargo proteins in vesicles as immunotherapeutic targets because ofthe antibody accessibility of the extracellular spacefasassociated factor faf1 is involved in diverse biochemical processes including cell death inflammation cellproliferation and proteostasis [] consistent with itsdiverse functions faf1 has been implicated in certain diseases first faf1 plays a tumor suppressive rolethrough activation of the apoptotic machinery and nfκbsuppression [ ] faf1 also suppresses tumor metastasis via tgf signaling moreover faf1 expressionis downregulated in various cancers including lung colonliver prostate brain ovarian and breast cancers second faf1 is involved in parkinsons disease pd as it iscolocalizes with αoverexpressed in pd patientssynuclein and acts as a substrate of parkin furthermorefaf1 mediates the degeneration of dopaminergic neuronsthrough apoptosis and parthanatos []faf1 is an intracellular protein present mainly in thecytosol to date faf1 secretion has not yet been reportedherein we uncovered for the first time that faf1 is secreted via an ergolgiindependent pathway specificallyfaf1 is secreted through exosomal and nonvesicular pathways in shsy5y cells in addition faf1 augments thenumber of exosomes suggesting the involvement of faf1in exosome biogenesis furthermore extracellular faf1moves into neighboring cells via pinocytosis and clathrinmediated endocytosis transmitted faf1 induces cell deathvia apoptosis and necrosis collectivelythese resultspresent a novel measure by which faf1 propagates itsdeath function through celltocell transmissionmethodsreagents and antibodiesthe following reagents and antibodies used in this studywere purchased commercially tnfα from abfrontierseoul south korea zietdfmk caspase8 inhibitormouse antiha antibody and rabbit antigm130 antibody from abcam cambridge uk ²²dichlorofluorescin diacetate dcfhda hydrogen peroxide h2o21methyl4phenylpyridinium mpp propidium iodidepi polydlysine brefeldin a bfa gw4869 monensin cycloheximide chx necrostatin1 nec1 dpqproteinase k pk dynasore heparin heparinase iiimouse antiactin and mouse antiflag antibody fromsigmaaldrich saint louis mo usa ²6diamidino2phenylindole dapi horseradish peroxidase hrpconjugated antimouse antibody and hrpconjugatedantirabbit antibody from thermo fisher scientific incrockford il usa mouse antiflotillin1 from bd biosciences san jose ca usa bafilomycin a1 mouseantialix antibody mouse antigalactosidase antibody401a rabbit anticalregulin antibody mouse anticd63 antibody mouse antiparkin antibody and mouseantifaf1 antibody from santa cruz biotechnologydallas tx usa mouse antihsc70 antibody andmouse antihsp90 antibody from enzo life sciencesfarmingdale ny usa and zvadfmk zvad fromcalbiochem darmstadt germanycell culture and transfectionshsy5y mef hek293 raw2647 hela panc1mia paca2 and mcf7 cells were maintained in dulbeccos modified eagles medium dmem welgenedaegu korea containing fetal bovine serum fbsatlas biologicals fort collins co usa and antibioticantimycotic gibco brl grand island neusa unless otherwise specified cells were transfectedwith the indicated plasmids using bio t manvillescientific inc manville nj usa following the manufacturers protocol rat midbrain cultures derived frompostnatal day were prepared using standard procedures briefly material dissected form the ventral portion of the midbrain was cleaned free of meningealtissue minced and enzymatically dissociated in a mixture of papaindnase sigmaaldrich dissociated cellswere plated onto aminecoated 6well plates bd biosciences cells were maintained in neurobasal mediumgibco with b27 serumfree supplements gibco mm lglutamine after days of culture the cells wereinfected using aav1 adenoassociated virus 1hfaf1viral vectors moi of and sitedirected mutagenesisthe sirnaresistant parkin construct was generatedusing quikchange sitedirected mutagenesis kit stratagene la jolla ca usa the primers were as follows²gagctgagaaacgactggactgtgcagaattgtg3²²gggaaggagctgagaaacgattgcactgtgcaga ² 0cpark cell communication and signaling page of rna interferenceall small interfering rnas sirnas against parkin andscrambled rna scrna were purchased from bioneerdaejeon south korea the sequences of the sirnasused in this study were as follows sirna against parkin²ugaggaaugggacugu3² thescrna orsirna were transfected into shsy5y cells using lipofectamine rnai max thermo fisher scientific according to the manufacturers instructionspreparation of conditioned mediumto prepare conditioned medium cm cells transfectedwith the indicated plasmids were cultured in mmdiameter dishes in dmem containing fbs after h the cells were switched to serumfree dmem forthe indicated times here we used serumfree mediumto avoid interference from albuminenriched fbs thenthe cm was collected and centrifuged at ¨¯g for min and ¨¯g for min to remove cellular debrisfor western blot analysis the cm was concentratedusing kda or kda cutoff amicon ultra filtersmillipore billerica ma usa at ¨¯g for min or minwestern blot analysiscells were harvested washed twice with pbs and lysedwith mammalian lysis buffer [ mm triscl ph mm nacl mm edta nonidet p40 mmphenylmethylsulfonylfluoride] then the protein concentrations were quantified by using a biorad proteinassay kit biorad hercules ca usa after quantification samples were boiled in à protein sample buffer[ mm triscl ph glycerol sds mercaptoethanol] then samples were electrophoresedby sdspage and transferred to nitrocellulose membranes ge healthcare maidstone uk the membranes were blocked with skim milk in pbs with tween20 pbst and incubated with the indicatedprimary antibodies overnight after their washing withpbst the membranes were incubated with secondaryantibodies immunoblot signals were measured by usingchemiluminescent detection lab frontier anyangkoreachx chase assayshsy5y cells were transiently transfected with the indicated plasmids for h after transfection the cells wereswitched to serumfree dmem containing chx sigmaaldrich μgml for the indicated of times subsequently the medium was concentrated with kda cutoff amicon ultra filters millipore and analyzed bywestern blot analysispurification of exosomeswe followed a previously described protocol with somemodifications briefly cells were transfected withthe indicated plasmids for h and incubated in dmemcontaining exosomedepleted fbs system biosciences palo alto ca usa for h the cm was thencollected and subjected to sequential centrifugation at¨¯g for min ¨¯g for min and ¨¯g for min at °c to remove cellular debris using a beckmancoulter optima l90 k ultracentrifuge with a type 41tirotor the supernatant was then spun down at ¨¯gfor min the pellet was resuspended in pbs and thenspun again at ¨¯g for min at °c finally thepellet was resuspended in pbs or radioimmunoprecipitation assay ripa buffer sigmaaldrichin addition to their isolation via ultracentrifugationwe isolated exosomes using exoquicktc system biosciences according to the manufacturers instructionstreatment of vesicles with na2co3to separate integral membrane proteins and luminalproteins purified exosomes were treated with mmna2co3 ph for min at °c as previously described after centrifugation at ¨¯g for minintegral proteins remained in the pellet fraction whileluminal proteins remained in the supernatant fractionthe pellet fractions were resuspended in ripa buffersigmaaldrich and the supernatants were collected ina separate tube for western blot analysisproteinase k digestionpk sigmaaldrich was added to the samples at a finalconcentration of μgml then the samples were incubated at °c for min and mm phenylmethylsulfonyl fluoride was added to inhibit the activation of pkfollowed by the addition of protein sample bufferimmunodepletion of cmfifty microliters of protein gconjugated dynabeadsthermofisher scientific was incubated overnight withmouse monoclonal antibody against faf1 final concentration of or μgml before its addition to cm theantibodydynabeads complex was incubated with mlof cm at °c overnight after the complex was removedusing a magnet the immunodepleted cm was concentrated using a kda cutoff amicon ultra filter andused for western blot analysis for flow cytometryunconcentrated immunodepleted cm was applied to recipient cellsflow cytometryto evaluate cell death we measured pipositive cellstaining by using a guava easycyte flow cytometermillipore briefly cells were switched to serumfree 0cpark cell communication and signaling page of dmem or neurobasal medium for the indicated timeadditionally to measure recipient cell death shsy5yand rat primary neuronal cells were treated with cmfrom donor cells for the indicated times the cells were μgml and evaluatedharvested stained with piusing a guava easycyte flow cytometer following whichthe results were quantified using incyte softwaremilliporeconcentrated cm treatmentshsy5y cells donor cells were plated on mm tissueculture dishes and transfected with gfpvector or gfpfaf1 plasmid at h after transfection the cells wereincubated in serumfree medium for h after the cmwas concentrated by using kda cutoff amicon ultrafiltersthe concentrated cm was dissolved in newserumfree medium that was applied to recipient cellson polyllysinecoated coverslips in 12well plates for hpropagation assayshsy5y cells donor cells were plated on mm tissueculture dishes donor cells were transfected with gfpvector or gfpfaf1 plasmids at h after transfectionthe cells were collected and replated in cell culture inserts polycarbonate membrane μm pore size corning kennebunk me usa at a density of ¨¯ cellsshsy5y cells recipient cells were plated at a densityof ¨¯ cells on polydlysinecoated coverslips in well plates after h the cultures were combined suchthat the donor cells were in the insert and separatedfrom recipient cells plated on a coverslipconfocal microscopycells were fixed with paraformaldehyde for minthe cisgolgi were stained with gm130 after the nucleiwere stained with dapi for min the coverslips weremounted onto microscope slides using fluorescencemounting medium dako carpinteria ca usa andanalyzed using a zeiss lsm laser scanning confocalmicroscope carl zeiss oberkochen germanyelectron microscopyfor transmission electron microscopy tem sampleswere prepared using the exosometemeasy kit containing a formvarcarboncoated em mesh gridwash buffer and em solution bio mountain viewca usa the pellets from the ml of cm vector orfaf1transfected cells obtained by ultracentrifugationwere resuspended in μl of pbs μl of which was applied to the grid all samples were prepared followingthe manufacturers instructions for immunoem thepellets were first fixed with μl of paraformaldehyde and glutaraldehyde sigmaaldrich overnightat °c then the fixed exosome solution was transferredto grids and subsequently treated with m glycinefor min to quench free aldehyde groups after blocking with pbs containing bsa for min the gridswere incubated for h with the indicated antibodies diluted in pbs containing bsa atroomtemperature after three washes with pbs containing bsa the grids were incubated for h with the secondary antibody antimouse igg conjugated to nmgoldroomtemperature three washes to eliminate secondary antibody were followed by incubation with em solution anda wash step samples were viewed under a talos f200xtransmission electron microscope fei hillsboro orusa operated at kv and images were capturedwith a ceta m pixel cmos camera feisigmaaldrichatpsnanop tracking analysisfollowing isolation by differential ultracentrifugation orexoquicktc system biosciences the exosome pelletswere resuspended in μl of pbs then μl of theexosome solution was diluted in pbs to a total volumeof ml the samples were analyzed by nanoptracking analysis using a nanosight ns300 malvernpanalytical ltd malvern uk equipped with a nmlaser to accurately identify the vesicles the detectionthreshold was set at the number of vesicles in eachsample represents the number of ps per ml ofmedium cells were counted using a muse count viability kit millipore on a muse cell analyzer milliporecaspase3 activity assayshsy5y cells were treated with cm from faf1transfected cells plus caspase8 inhibitor or tnfα abfrontier plus chx sigmaaldrich at the indicated concentrations for the indicated times then caspase3 activity was measured by using a caspase3 colorimetricassay kit biovision milpitas ca usa in accordancewith the manufacturers protocol the absorbance at nm was measured with the use of a victor microplate reader perkinelmer norwalk ct usasignalp41we investigated the presence of a signal peptide in faf1using signalp41 httpwwwcbsdtudkservicessignalp41 with secretogranin1 used as a positive controlas it contains a signal peptidestatistical analysesexperiments were independently carried out three timesn all the data are expressed as the mean ± standarddeviation sd statistical comparisons were performedusing students ttest or oneway analysis of varianceanova followed by tukeys hsd post hoc analysis 0cpark cell communication and signaling page of using spss software statistics version ibm incchicago il usa statistical significance was established when the pvalue was lower than resultsfaf1 is secreted via nonclassical exocytosisaccording to the csf proteome resource faf1 is detected in the cerebrospinal fluid and plasma additionalfile this study aimed to determine whether faf1 isalso secreted at the cellular level and investigate faf1secretion in shsy5y human neuroblastoma cellsbecause faf1 is a deathpromoting protein sublethal experimental faf1 transfection conditions wereused to exclude cellular debris due to death additionalfile fig s1a and b the faf1 transfection conditionsin shsy5y cells under which faf1 secretion but notcell death occurs were determined by pi stainingfollowed by flow cytometry cells were transfected with3xflagfaf1 plasmid at a sublethal dose for h andsubsequently moved to serumfree medium faf1 wasdetected in the serumfree medium at h and accumulated henceforth indicating that faf1 is secreted in atimedependent manner fig 1a this finding suggeststhat faf1 is constitutively released from shsy5y cellsto exclude a tagging artifact another epitope taghemagglutinin ha was used hafaf1 was alsodetected in the cm conditioned medium in a dosedependent manner additional file fig s1c furthermore we examined the faf1 secretion with rat primaryneurons using aav1 adenoassociated virus 1mediated faf1 overexpression faf1 overexpression in ratprimary neuronal cells demonstrated consistent resultsin shsy5y cells fig 1b moreover with thatgalactosidase was not detected in the cm of galactosidasetransfected cells demonstrating that therelease of faf1 is not a transfection artifact fig 1c next a pulsechase experiment using chx to inhibit de novo protein synthesis was performed consistently faf1 was secreted and accumulated over timefurther confirming that faf1 is constitutively secretedfrom shsy5y cells fig 1d next we examinedwhether faf1 is also secreted by other cells faf1 wasdetected in the extracellular space of mefs and hek293cells furthermore faf1 was present in the cm of eachof a number of various cancer cell types mcf7 helapanc1 and mia paca2 cells this shows that faf1secretion is notadditional file fig s2afspecific to shsy5y cellsbecause faf1 has been implicated in pd pathogenesisfaf1transfected shsy5y cells were treated with thepdassociated stressors such as mpp h2o2 bafilomycin a1 and αsynuclein overexpression at sublethal dosesadditional file fig s3 there were no significantchanges of faf1 expression in clslysatescelldependent on various pdassociated stressortypeshowever faf1 secretion increased in cms upon allstressors used in this study implying that pdassociatedstressors positively regulate faf1 secretion fig 1eto elucidate the mechanism by which faf1 is secreted two sets of experiments were performed as follows first we examined whether faf1 is released viaexocytosis or passive diffusion as exocytosis is affectedby temperature faf1transfected shsy5y cellswere incubated at either °c or °c faf1 secretionat °c was significantly reduced compared to that at °cindicating that faf1 is released via exocytosisfig 1f second we examined whether faf1 is secretedvia the classical ergolgidependent secretory pathwayusing bfa which generates ros and disassembles golgithrough ergolgi pathway inhibition bfa did not affectfaf1 release fig 1g additional file fig s1d furthermore a signal peptide was not found in faf1when its sequence was analyzed using singalp41 furtherexcluding the possibility of ergolgimediated secretionof faf1 additionalfile fig s4 taken togetherthese data demonstrate that faf1 is released via nonclassical exocytosisfaf1 is secreted via exosomal and nonvesicular pathwaysto determine the mechanism by which faf1 is secreted exosomes were isolated from cm using a differential ultracentrifugation procedure as previouslydescribed and exoquicktc following the manufacturers protocol western blot analysis of exosomesisolated by ultracentrifugation revealed the presenceofthe exosome markers alix cd63 hsc70 andhsp90 but not calregulin an er resident protein a negative exosome control fig 2a exosomes isolated from shsy5y cells by exoquicktc showed anexosome marker profile consistent with that of exosomes purified by ultracentrifugation additional file fig s5a moreover nanop tracking analysisand tem data further confirmed that our purifiedexosomes exhibited a typical size distribution with adiameter ranging from to nm and a typicalmorphology of exosomes fig 2b and cendogenous as well as overexpressed faf1 proteinswere detected in the exosomal fractions isolated by bothmethods indicating that faf1 is secreted as a genuineexosomal cargo fig 2a similarly faf1 was also foundin exosomes isolated from the various indicated cell linesby exoquicktc additional file fig s5bh next weinvestigated whether faf1 is present on the membraneor in the lumen of exosomes exosomes were treatedwith mm na2co3 ph to distinguish betweenthe exosomal membrane and the lumen both alix andflotillin1 were present in the exosomal membrane positive controls whereas faf1 was mainly present in the 0cpark cell communication and signaling page of fig see legend on next page 0cpark cell communication and signaling page of see figure on previous pagefig faf1 is secreted via nonclassical exocytosis a shsy5y cells were transfected with vector control vc or 3xflagfaf1 plasmid at h aftertransfection the culture medium was replaced with serumfree medium for the indicated times se short exposure le long exposure b ratprimary neuronal cells were transduced with aav1hfaf1 viral vectors at days after transduction the culture medium was replaced withserumfree neurobasal medium for h c cells were transfected with lacz or 3xflagfaf1 plasmid at h after transfection the culture mediumwas replaced with serumfree medium and cells were cultured for h d cells were transfected with vc or 3xflagfaf1 plasmid at h aftertransfection the culture medium was replaced with serumfree medium containing chx μgml for the indicated times e left panel cellswere transfected with vc or 3xflagfaf1 plus αsyn plasmid at h after transfection the culture medium was replaced with serumfreemedium containing dmso vehicle mpp mm h2o2 μm or baf a1 nm for h right panel the graph shows the densitometricanalysis of immunoblotting of faf1 in conditioned medium cm shown in the left panel n f upper panel cells were transfected with3xflagfaf1 plasmid at h after transfection the culture medium was replaced with serumfree medium and the cells were cultured at °cor °c for h lower panel the graph shows the result of densitometric analysis of faf1 immunoblotting in cm shown in the upper paneln g upper panel cells were transfected with 3xflagfaf1 plasmid at h after transfection the culture medium was replaced with serumfree medium containing bfa μgml for h lower panel the graph shows the result of densitometric analysis of faf1 immunoblotting incm shown in the upper panel n cell lysate cl and concentrated cm were analyzed by western blotting with the indicated antibodies alllanes were loaded with the same amount of total protein the data are expressed as the mean ± sd of three independent experimentsstatistical comparisons were performed using using anova followed by tukeys hsd post hoc analysis e and students ttest f and g p p p and ns not significanttheexosomeabolishedstructureslumen of the exosomes fig 2d the topology of exosomal faf1 was further examined using pk a nonspecificprotease to digest proteins outside of the exosomesexosomal faf1 but not cytosolic faf1 was protectedfrom pk treatment in the absence of triton x100 txfig 2e tx in contrast pk treatment with tx disintegratingtheexosomemediated protection of faf1 fig 2e txour data demonstrated the presence of faf1 in thelumen of exosomes furthermore the presence of faf1in theconfirmed with theimmunogoldlabeled faf1 under anvisualization ofelectron microscope as shown in fig 2f immunogoldlabeled cd63 was mainly present outside exosomeswhereas immunogoldlabeled faf1 was present in thelumen of exosomes collectively these results consistently showed that faf1 is located in the lumen ofexosomeslumen wasexosomalbecause certain exosomal cargo proteins are alsosecreted via the nonvesicular secretory pathway [] the possibility that faf1 is also secreted viaa nonvesicular route was investigated to this endthe cm was fractionated into exosomal pellet andnonexosomal supernatant fractions by ultracentrifugation faf1 from the cm was present in nonexosomal as well as exosomalfractions fig 2gimplying the presence of a soluble form of faf1 tofurther investigate this cm from faf1transfectedshsy5y cells was treated with antifaf1 antibodyone microgram of antifaf1 antibody almost eliminated faf1 from the cmindicating that faf1 ispredominantly secreted in a soluble form fig 2htaken together these results demonstrate that faf1is concurrently released as a bona fide cargo of exosomes and in a soluble form this new finding addsfaf1 to the list of proteins secreted by nonvesicularas well as exosomal routesrespectivelytransfection offaf1 positively regulates exosome numberthe exosomal cargos such as hsp20 hsp90 andstat3 increase exosome number [] here weexamined whether faf1 also participates in regulating exosome number the exosome markers hsc70alix and cd63 were increased by ± 009fold ± 013fold and ± 022foldinthe cm of faf1transfected shsy5y cells compared with control cells fig 3a additional file fig s6a next we further studied exosome numberchanges using sirnamediated depletion of parkina e3ubiquitin ligase of faf1 siparkin treatment elevated secretion as well as expression of faf1 inshsy5y cells moreoversirnaresistant parkin construct reverted the increased secretion and expression of faf1 by siparkin in shsy5y cells the expression levels of alix and cd63in the cm of shsy5y cells in which parkin hadbeen depleted were elevated by ± and ± 040fold respectively fig 3b collectivelythese data imply that faf1 positively controls exosome number for the direct quantification of exosome number nanop tracking analysis wasapplied the vesicle size distribution profile showinga diverse range of sizes showed that exosomes werepresent predominantly fig 3c the exosome numbers were normalized by cell number additionalfile fig s6b the exosomes of faf1transfectedcells were increased by 25fold compared to thoseof control cells hence these data robustly demonstrate that faf1 augments exosome numberinaddition gw4869 an exosome release inhibitor interfered with the ability of faf1 to increase exosome number while monensin an exosome releasepromoter enhanced this ability implying that faf1functions upstream of the exosome release processfig 3a [ ] 0cpark cell communication and signaling page of fig see legend on next page 0cpark cell communication and signaling page of see figure on previous pagefig faf1 is released to the extracellular space via both exosomal and nonvesicular pathways a shsy5y cells plated on mm dishes weretransfected with vc or 3xflagfaf1 plasmid at h after transfection the culture medium was replaced with exosomedepleted medium andthe cells were cultured for h then exosomes were isolated from the cm by ultracentrifugation cl and isolated exosomes exos wereanalyzed by western blotting with the indicated antibodies calr calregulin b purified exosomes were characterized using nanop trackinganalysis c representative tem images of exosomes are shown scale bar nm left or nm right d the purified exosomes were treatedwith na2co3 after centrifugation at ¨¯g the integral membrane proteins were pelleted memb and nonintegral and luminal proteinsremained in the supernatant sol these fractions were analyzed by western blotting with the indicated antibodies e the purified exosomeswere incubated with pk μgml in the absence or presence of triton x100 tx tx with tx tx without tx f immunogold labelingof purified exosomes with anticd63 antibodies left and antifaf1 antibodies from vctransfected middle or 3xflagfaf1transfected rightcells scale bar nm g cells were transfected with vc or 3xflagfaf1 plasmid at h after transfection the culture medium was replacedwith serumfree medium and the cells were cultured for h after the cm was isolated by ultracentrifugation the supernatant sup and pelletexo were analyzed by western blot with the indicated antibodies h after immunoprecipitation of faf1 from cm with antifaf1 monoclonalantibody the immunodepleted cm was concentrated and analyzed by western blotting with the indicated antibodies all lanes were loaded withthe same amount of total proteinsecreted faf1 is transmitted to adjacent cellswe wondered whether extracellular faf1 is internalizedby neighboring cells donor cells were transfected withgfp or gfpfaf1 plasmid for h subsequently thecm which contained gfp or gfpfaf1 was concentrated and nontransfected recipient cells were treatedwith the concentrated cm for h confocal microscopic images showed the presence of gfpfaf1 in thecytoplasm of recipient cells indicating that gfpfaf1had moved from donor cells to recipient cells in contrast gfp from the cm of donor cells was not detectedin recipient cells excluding the effect of tagging ontransmission fig 4a similarly donor cells were transfected with 3xflagfaf1 plasmid as described abovedonor cellderived faf1 also moved into recipient cellsas shown by western blotting fig 4b corroborating theimmunofluorescence results in fig 4a in these data consistently demonstrate that secreted faf1can be internalized by neighboring cellsto further validate the celltocell transfer of faf1 anin vitro coculture system in which donor cells expressinggfpfaf1 were incubated in upper transwellinsertswhile nontransfected recipient cells were incubated inlower compartments was used consistent with theabove data confocal microscopy of recipient cells revealed the presence of gfpfaf1 indicating that gfpfaf1 moved from cells in the upper transwell inserts tocells in the lower compartments fig 4c consequentlythese results show that extracellular faf1 was transferred to neighboring cells without celltocell contactwe investigated the type of extracellular faf1 thatmoves into recipient cells to this end donor cells weretreated with gw4869 an exosome release inhibitorafter which recipient cells were analyzed by confocal microscopy gw4869 failed to inhibit faf1 transmissionto recipient cells to eliminate vesiclefree faf1 cm ofdonor cells was immunodepleted wit | 0 |
the european commission asked efsa for a scientiï¬c opinion on the risks for animal and humanhealth related to the presence of glycoalkaloids gas in feed and food this risk assessment coversedible parts of potato plants and other food plants containing gastomato andaubergine in humans acute toxic effects of potato gas asolanine and achaconine includegastrointestinal symptoms such as nausea vomiting and diarrhoea for these effects the contampanel identiï¬ed a lowestobservedadverseeffect level of mg total potato gaskg body weight bwper day as a reference point for the risk characterisation following acute exposure in humans noevidence of health problems associated with repeated or longterm intake of gas via potatoes hasbeen identiï¬ed no reference point for chronic exposure could be identiï¬ed from the experimentalanimal studies occurrence data were available only for asolanine and achaconine mostly forpotatoes the acute dietary exposure to potato gas was estimated using a probabilistic approach andapplying processing factors for food due to the limited data available a margin of exposure moeapproach was applied the moes for the younger age groups indicate a health concern for the foodconsumption surveys with the highest mean exposure as well as for the p95 exposure in all surveysfor adult age groups the moes indicate a health concern only for the food consumption surveys withthe highest p95 exposures for tomato and aubergine gas the risk to human health could not becharacterised due to the lack of occurrence data and the limited toxicity data for horses farm andcompanion animals no risk characterisation for potato gas could be performed due to insufï¬cient dataon occurrence in feed and on potential adverse effects of gas in these species european food safety authority efsa published by john wiley and sons ltd on behalfof european food safety authoritykeywords glycoalkaloids gas solanine chaconine potato margin of exposure moe food feedrequestor european commissionquestion number efsaq201600811correspondence contamefsaeuropaeu leon brimer was a member of the working group on glycoalkaloids in food and feed until august wwwefsaeuropaeuefsa efsa 0cglycoalkaloids in feed and foodpanel members margherita bignami laurent bodin james kevin chipman jes 13us del mazo bettinagraslkraupp christer hogstrand laurentius ron hoogenboom jeancharles leblanc carlo stefanonebbia elsa nielsen evangelia ntzani annette petersen salomon sand dieter schrenk tanjaschwerdtle christiane vleminckx and heather wallaceacknowledgements the panel wishes to thank the following for the support provided to thisscientiï¬c output kelly niermans the panel wishes to acknowledge all european competentinstitutions member state bodies and other anisations that provided consumption and occurrencedata for this scientiï¬c outputsuggested citation efsa contam panel efsa panel on contaminants in the food chain schrenk dbignami m bodin l chipman jk del mazo j hogstrand c hoogenboom lr leblanc jc nebbia csnielsen e ntzani e petersen a sand s schwerdtle t vleminckx c wallace h brimer l cottrill bdusemund b mulder p vollmer g binaglia m ramos bordajandi l riolo f rold 13antorres r and graslkraupp b scientiï¬c opinion risk assessment of glycoalkaloids in feed and food in particular inpotatoes and potatoderived products efsa pp 102903jefsa20206222issn european food safety authority efsa published by john wiley and sons ltd on behalfof european food safety authoritythis is an open access under the terms of the creative commons attributionnoderivs licensewhich permits use and distribution in any medium provided the original work is properly cited and nomodiï¬cations or adaptations are madereproduction of the images listed below is prohibited and permission must be sought directly from thecopyright holderfigure elsevier figure springer figure american chemical society springerthe efsa is a publication of the european foodsafety authority an agency of the european unionwwwefsaeuropaeuefsa efsa 0cglycoalkaloids in feed and foodsummarythe european commission asked efsa for a scientiï¬c opinion on the risks for animal and humanhealth related to the presence of glycoalkaloids gas in feed and food in particular in potatoes andpotatoderived products this risk assessment covers edible parts of potato plants and other foodplants containing gas in particular tomato and aubergine nonedible parts of ga containing plantshave not been considered with the exception of potato sprouts the panel developed the draftscientiï¬c opinion which underwent a public consultation from february to april thecomments received and how they were taken into account when ï¬nalising the scientiï¬c opinion werepublished in an efsa technical report efsa gas are present in many plants of the family of solanaceae and contribute to plant resistanceagainst pests and pathogens gas are composed of a steroidal aglycone and an oligosaccharide sidechain in commercial potato cultivars s tuberosum the main gas are achaconine and asolanineconsisting of the aglycone solanidine and chacotriose and solatriose as oligosaccharide side chainsrespectively the aubergine fruit s melongena contains primarily the gas asolamargine and asolasonine composed of the aglycone solasodine and chacotriose and solatriose respectively inlycopersicum atomatine and adehydrotomatine are the major gas withtomato fruitlycotetraose coupled to the aglycones tomatidine and tomatidenol respectivelyshuman risk assessmentin experimental animals the potato gas asolanine and achaconine show a relatively low oralbioavailability with differences between species hamsters exhibit higher absorption and slowerexcretion rates for both substances when compared to rats due to the limited information themetabolic proï¬les of potato gas in experimental animals could not be characterisedin humans asolanine and achaconine are systemically absorbed following ingestion for bothsubstances relatively long serum halflives were reported suggesting a possible accumulation the bloodclearance of the respective aglycone solanidine appears to be slow accordingly levels of solanidine wereregularly detected in the blood of human volunteers in several studies suggesting hydrolysis of gas nofurther information is available on metabolism and excretion of potato gas in humansthere are no toxicokinetic data on tomato and aubergine gas and their aglycones in experimentalanimals and humansin acute oral toxicity studies no adverse effects of asolanine were observed at doses of mgkgbody weight bw per day in rats and mgkg bw per day in mice reliable data on other potatogas or tomato and aubergine gas and their aglycones are missingin repeated oral dose studies on potato gas rodents showed nonspeciï¬c effects such as reducedbody weight and relative liver weight with indication of similar potencies of asolanine and achaconine hamsters exhibited these symptoms after a 5day treatment with mg of asolanine ora chaconinekg bw per day while mice showed these effects after one week of daily treatments with mg of asolanine or mg of achaconinekg bw solanidine however increased the absoluteand relative liver weight at mgkg bw per day in mice suggesting a different effect of theaglycone compared to the gasthe tomato ga atomatine and its aglycone tomatidine exerted no effects in rats when applied at mgkg bw per day for a period of day at higher doses atomatine reduced the cholesterol uptakeand increased fecal sterol and coprostanol excretion in hamsters and rats in mice a to 2weektreatment with the aubergine ga asolasonine increased the body weight gain at mgkg bw perday while its aglycone solasodine decreased body weight gain and caused gastric gland degenerationand liver toxicity at mgkg bw per daydevelopmental studies have been performed mainly in hamsters treated with potato gas and theiraglycones for only one day or for a short very restricted time period during gestation outcomes weremainly analysed in late gestational embryos and comprised effects in the central nervous systempredominantly exencephaly encephalocele and anophthalmia these malformations occurred at dosesof mgkg bw per day and above for gas and of mgkg bw per day and above for theaglycones no noobservedadverseeffectlevelloael could be identiï¬ed from these studies reduced postnatal survival of pups due to insufï¬cientmilk production was reported when pregnant holtzman rats had been exposed to mg of asolaninekg bw per day studies on the male fertility in dogs have been performed only with theaubergine aglycone solasodine decreased epididymal weight and cauda epididymal epithelial heightand also an epididymal lumen depleted of sperm occurred in dogs after mgkg bw per day givenlowestobservedadverseeffectnoael orlevelwwwefsaeuropaeuefsa efsa 0cglycoalkaloids in feed and foodfor month similar effects were observed in rhesus monkeys exposed to mgkg bw per day for monthsfrom the limited number of studies available there was no evidence for genotoxicity of the potatogas asolanine and achaconine and the aglycone solanidine as well as for the aubergine ga asolamargine however there is not sufï¬cient information to conclude on the genotoxic potential ofthese gasno longterm chronic toxicitycarcinogencity study for potato tomato or aubergine gas or for therespective aglycones could be identiï¬edin humans acute toxic effects following ingestion of potato gas include gastrointestinal symptomsof varying severity such as vomiting diarrhoea and abdominal pain which may occur from a totalpotato gas potato tga intake of mgkg bw or more further symptoms including drowsinessapathy confusion weakness vision disturbances rapid and weak pulse and low blood pressure maybe the consequence of dehydration following vomiting and diarrhoeain severe cases paralysis respiratory insufï¬ciency cardiac failure coma and death have beenreported doses in the range of mg potato tgaskg bw are considered to be potentially lethal forhumans results from limited volunteer studies suggest possible differences in the human populationwith respect to the individual susceptibility towards adverse effects associated with the intake ofpotato gasregarding the mode of action adverse effects of gas may be due to their ability to complex withmembrane 3bhydroxy sterols thereby causing disruption and loss of integrity of cell membranesafter oral exposure these effects may affect the mucosa of the gastrointestinal tract and cause thesymptoms observed in intoxicated humans such as nausea vomiting and diarrhoeagas inhibit acetylcholinesterase ache and serum butyrylcholinesterase buche by a reversiblecompetitive mode of action the relative potency of inhibition of asolanine and achaconine appearsto be similar the aglycones exert weak or no inhibitory effects the excess of acetylcholine at theneuronal and neuromuscular junctions upon inhibition of the enzymes might also contribute to thesymptoms described for intoxications with gasat high doses atomatine may form a nonabsorbable complex with cholesterol and other sterols inthe enteral lumen which may impair the absorption of cholesterol as a consequence blood cholesterollevels were lowered in rodentsthe contam panel considered that the use of rodent data on acute toxicity was not appropriate toestablish a reference point for acute exposure to potato gas in humans the contam panel selectedthe loael of mg potato tgakg bw per day as the reference point for acute risk characterisationbased on human data from case reports outbreaks and studies in volunteers the available data onacute toxicity were considered insufï¬cient to establish a healthbased guidance value instead thepanel used the margin of exposure moe approach to assess a possible health concern from acuteexposure to potato tgas via foodassuming the main symptoms to be mainly due to localirritation of the gastrointestinal mucosarather than inhibition of ache activity the panel considered that the possible interindividual variabilityin toxicodynamics is more relevant than the interindividual variability in toxicokinetics accordingly anmoe higher than indicates that there is no health concern this moe of takes into account theextrapolation from a loael to a noael a factor of and the interindividual variability intoxicodynamics a factor of the experimental data available for repeated dose toxicity are not sufï¬cient to identify a referencepoint for chronic exposure to potato gas in humans no evidence of health problems associated withrepeated or longterm intake of gas via potatoes has been identiï¬edregarding gas or aglycones occurring in edible parts of food plants other than s tuberosum nosuitable study for determining a reference point for tomato or aubergine gas or aglycones wasidentiï¬edoccurrence data were only available for asolanine and achaconine and mostly for maincroppotatoes and new potatoes few data were available for processed food no data on the occurrenceof tomato and aubergine gas and their aglycones were submitted to efsasince the occurrence data on potato gas did not cover all the food categories containing potatoesin the consumption database it was decided that the best approach for the exposure assessmentwould be to use the occurrence data in the raw primary commodities rpc maincrop potatoes andnew potatoes and the rpc consumption database the panel decided to combine the occurrence ofnew potatoes with that of maincrop potatoes and the mean upper bound ub occurrence sum ofwwwefsaeuropaeuefsa efsa 0cglycoalkaloids in feed and foodasolanine and achaconine for these two groups was mgkg and the p95 occurrence was mgkg the minimum and maximum reported concentrations were and mgkg respectivelythe acute dietary exposure to potato tgas was estimated using a probabilistic approach includingonly days in which there was consumption of maincrop potatoes as no occurrence data wereavailable for gas in tomato and aubergine these foods were not included in the exposure assessmentprocessing of potatoes has been reported to reduce the content of gas in the ï¬nal processedproduct in general and according to the literature the peeling of potatoes reduced the ga contentby boiling in water and blanching of peeled potatoes by and frying in oil of peeledpotatoes by microwave and oven baking of unpeeled potatoes may cause a reduction in thega content by and by respectively no information has been found about thechemical nature of the ga degradation products for the exposure assessment processing factors forthe major food processing steps comprising peeling and heat processing boiling frying bakingwere applied to the occurrence data as follows processing factors between and wereattributed to the peeling of potatoes between and for frying and deep frying and between and for all other cooking methodsinformation about the peeling of potatoes was not available in the consumption database but itwas assumed that of the potatoes are consumed as peeled where information of the cookingmethod was not available a cooking method was randomly attributed to the eating event based onthe relative frequency of cooking methods reportedthe mean ub exposure to potato tgas across surveys ranged from lgkg bw per day inadults to lgkg bw per day in toddlers the 95th percentile exposure ranged from lgkgbw per day in adults to lgkg bw per day in toddlers up to lgkg bw per day in theupper limit of the conï¬dence intervalcomparing the loael for potato tgas of mgkg bw per day with the acute exposure estimatesthe moes for the younger age groups indicate a health concern for the food consumption surveys withthe highest mean exposure as well as for the p95 exposure in all surveys for adult age groups themoes indicate a health concern only for the food consumption surveys with the highest p95exposuresthe contam panel calculated the mean percentage of days with potato consumption acrosssurveys per age group on which the potato tga intake may be below the moe of the highestnumber of survey days with intake of potatoes below the moe of was estimated for toddlers followed by children for the other age groups the estimated tga intake was below the moeof in up to of the survey daysfor tomato and aubergine gas the risk to human health could not be characterised due to the lackof occurrence data in food and the limited information on the adverse effects in experimental animalsand humansthe contam panel considered that the impact of the uncertainties on the risk assessment of acuteexposure to potato gas in food is moderate and that overall the identiï¬ed uncertainties may eithercause an over or underestimation of the riskfarm animals horses and companion animals risk assessmentinformation on the toxicokinetics of gas was limited to ruminants for which the data suggest anextensive conversion of asolanine and achaconine to aglycones in rumen and a low potential ofsolanidine to transfer into cows milkno data on the potential adverse effects of potato gas in horses companion animals cats anddogs or fur animals were identiï¬ed due to an insufï¬cient database on the adverse effects of gas inruminants pigs poultry rabbits and ï¬sh an acute reference dose could not be derivedpotatoes are not grown speciï¬cally as feed for livestock but when supply exceeds marketrequirements for human consumption whole raw potatoes may be used as feed for ruminants andpigs some byproducts of potato processing and starch extraction are used as feeds for farmedlivestock principally nonruminants and for companion animalsdata on potato gas in feed were insufï¬cient to perform an exposure assessmentthus no risk characterisation could be performed due to insufï¬cient occurrence data of gas forfeed and the lack of or limited data on the adverse effects of gas in farm animals horses orcompanion animalswwwefsaeuropaeuefsa efsa 0cglycoalkaloids in feed and foodrecommendationsthe following needs have been identiï¬ed to improve the risk assessment for humans and reducethe uncertaintiescid129research on the occurrence of gas and their aglycones and other potentially toxicologicallyrelevant secondary plant metabolites in the potato cultivars available on the market and onnew potato cultivars resulting from breeding experimentscid129 occurrence data on gas and their aglycones in potato processed products including foods forinfantscid129 occurrence data on gas and their aglycones in tomato and aubergine and products thereofcid129 data on the toxicokinetics of potato tomato and aubergine gas and aglycones in experimentalanimals and humanscid129 data on repeated dose toxicity including reproductive and developmental toxicity of potatotomato and aubergine gas and aglycones in experimental animalsstudies in humans linking dietary exposure biomarkers of exposure and adverse effectscid129the following needs have been identiï¬ed to improve the risk assessment for farm animals horsesand companion animals and reduce the uncertaintiescid129 occurrence data on potato gas and their aglycones in feedcid129studies on the kinetics and the potential adverse effects from feed material containing gas ofpotato gas in farm animals horses and companion animalswwwefsaeuropaeuefsa efsa 0cglycoalkaloids in feed and foodtable of contentsabstractsummaryintroduction background and terms of reference as provided by the requestor interpretation of the terms of reference supporting information for the assessment chemistry analytical methods sources potatoes tomatoes aubergine previous risk assessments legislation and other standards data and methodologies methodology for data collection selection of evidence and study appraisal food and feed occurrence data submitted to efsa data collection and validation data analysis food and feed consumption data food consumption data feed consumption data food classiï¬cation methodology for exposure assessment methodology for risk characterisation assessment hazard identiï¬cation and characterisation toxicokinetics experimental animals asolanine achaconine humans mixtures of asolanine and achaconine solanidine biomarkers of exposure farm animals horses and companion animals summary on toxicokinetics toxicity in experimental animals acute toxicity studies gas from edible parts of s tuberosum gas from edible parts of food plants other than s tuberosum summary on acute toxicity studies repeated dose toxicity studies gas and aglycones from edible parts of s tuberosum gas and aglycones from edible parts of food plants other than s tuberosum developmental and reproductive toxicity studies developmental effects reproductive effects immunotoxicity studies studies on cardiovascular effects neurotoxicity studies genotoxicity gas from edible parts of s tuberosum gas from edible parts of food plants other than s tuberosum carcinogenicity studies studies on metabolic effects gas from edible parts of s tuberosum gas from edible parts of food plants other than s tuberosum observations in humans wwwefsaeuropaeuefsa efsa 0cglycoalkaloids in feed and food gas from s tuberosum reports on intoxications studies in human volunteers epidemiological studies summary gas from food plants other than s tuberosum case reports adverse effects in farm animals horses and companion animals ruminants pigs poultry rabbits fish horses companion animals cats and dogs fur animals reports on intoxications mode of action membrane effects with implications for the gastrointestinal tract inhibition of cholinesterases ches comparative determination of inhibition of ches in vitro determination of inhibitory constants ki for gas on inhibition of ches in vitro inhibition of ches in vivo developmental and reproductive effects of gas and their aglycones inhibition of cholinesterases and effects in the immune system interference with metabolism considerations of critical effects and doseresponse analysis for the human risk assessment gas from edible parts of s tuberosum considerations of critical effects and doseresponse analysis derivation of a healthbased guidance value hbgv or margin of exposure moe approach gas from edible parts of food plants other than s tuberosum considerations of critical effects and doseresponse analysis consideration of critical effects and doseresponse analysis for the farm animal horses andcompanion animals risk assessment occurrence data occurrence data submitted to efsa previously reported occurrence data in the open literature literature on occurrence data on food occurrence data on gas in potatoes occurrence data on gas in tomatoes occurrence data on gas in aubergines occurrence data on gas in other food products literature occurrence data in feed inï¬uence of storage and processing on the content of gas gas from s tuberosum storage of potatoes processing of potatoes for food consumption processing of potatoes for feed gas from food plants other than s tuberosum summary on the inï¬uence of storage and processing on the levels of gas exposure assessment current acute dietary exposure assessment for humans previously reported dietary exposure assessments current dietary exposure assessment for farm animals horses and companion animals risk characterisation human health risk characterisation ga from edible parts of s tuberosum gas from edible parts of food plants other than s tuberosum farm animals horses and companion animal risk characterisation uncertainty analysis assessment objectives exposure scenarioexposure model wwwefsaeuropaeuefsa efsa 0cglycoalkaloids in feed and foodhazard identiï¬cation and characterisation summary of uncertainties conclusions hazard identiï¬cation and characterisation toxicokinetics toxicity in experimental animals observations in humans adverse effects in farm animals horses and companion animals mode of action margin of exposure moe approach occurrence and exposure food feed risk characterisation human health risk characterisation farm animals horses and companion animal health risk characterisation recommendations documentation provided to efsa references abbreviations appendix a major glycoalkaloids and their aglycones present in solanum species appendix b identiï¬cation and selection of evidence relevant for the risk assessment of glycoalkaloids infeed and food appendix c details of the study design of the toxicokinetic studies appendix d comparison of developmental toxicity of single dose studies appendix e inhibition of cholinesterases by gas appendix f rapid alert system for food and feed rasff reports on the presence of solanum nigrum infood products appendix g studies on the toxicity of glycoalkaloids not considered in the risk assessment appendix h additional scenario for the human risk characterisation annex a occurrence data in food and feed submitted to efsa and dietary exposure assessment forhumans wwwefsaeuropaeuefsa efsa 0cglycoalkaloids in feed and foodintroductionbackground and terms of reference as provided by the requestorbackgroundmany plants in the family solanaceae contain glycoalkaloids and they are considered to be naturaltoxins the plant glycoalkaloids are toxic steroidal glycosides and the commonest types found in foodplants are asolanine and achaconine their natural function is probably to serve as stress metabolitesor phytoalexins for the protection of the plant when attacked by insects fungi etcamongst the most widely cultivated food crops aubergines tomatoes and potatoes are in thesolanaceae family but the levels of glycoalkaloids in tomatoes and aubergines are generally quite lowthe glycoalkaloids of most relevance to food safety are those occurring in the potato thepredominant toxic steroidal glycosides in potato are asolanine and achaconine they occur in potatotubers peel sprouts berries leaves and blossoms and their concentration in tubers depends on anumber offactors concentrations ofglycoalkaloids are times greater in the peel than in the ï¬esh there is considerable variation inglycoalkaloid content among potato cultivars storage conditions especially light and temperature aremainly responsible for increases in solanine although the glycoalkaloid content can increase in thedark the rate of formation is only about the rate of formation in light increases of solanine inthe potato peel are closely associated with greening synthesis of chlorophyll of the peel thesebiochemical processes are independent of each other but are both activated by lightsuch as cultivar maturity and environmentalfactorsbitter or burning sensation in the mouth are sensory impressions which may accompanyglycoalkaloid poisoning symptoms from potatoes that include ï¬ulike symptoms such as nauseavomiting stomach and abdominal cramps and diarrhoea more severe cases of glycoalkaloid poisoningmay be accompanied by a variety of neurological effects ie drowsiness apathy restlessnessshaking confusion weakness and disturbed vision there are a few reports of deaths beingattributed to glycoalkaloid exposure from the consumption of potatoes potato leaves and potatoberriespotatoes and potatoderived products are listed in the catalogue of feed materials1terms of referencein accordance with art of regulation ec no the european commission asks theeuropean food safety authority for a scientiï¬c opinion on the risks for animal and human healthrelated to the presence of glycoalkaloids in feed and food in particular in potatoes and potatoderivedproductsinterpretation of the terms of referencethe contam panel considered that the opinion should cover edible parts of potato plants and alsoof other food plants containing glycoalkaloids gas eg tomato and aubergine nonedible parts ofga containing plants have not been considered with the exception of potato sprouts in particular thecontam panel concluded this opinion should comprise thea evaluation of the toxicity of gas in feed and food in particular in potatoes and potatoderivedproducts for farm and companion animals and humans considering all relevant toxicologicalend pointsb evaluation of the alkaloid proï¬le ie composition of the alkaloids and their concentration ofthe food and feed samples submitted to efsac estimation of the dietary exposure of the european population to gas in food in particular inpotatoes and potatoderived products including the consumption patterns of speciï¬c groupsof the population if appropriated estimation of the dietary exposure offarm and companion animals to gas in feedinparticular in potatoes and potatoderived productse assessment of the human health risks for the european population including speciï¬c groupsof the population if appropriate as the consequence of the estimated dietary exposure commission regulation eu no of january on the catalogue of feed materials ojl p wwwefsaeuropaeuefsa efsa 0cglycoalkaloids in feed and foodf assessment of the farm and companion animal health risks in europe as the consequence ofthe estimated dietary exposure exposure to gas from weeds containing ga is only addressedin this opinion in the context of accidental intake by farm animalswhen referring to gas in potatoes the term total gas tga refers to a material comprising asolanineand achaconine as major fraction with no speciï¬cation on the occurrence of minor gas as well as band cforms of solanine and chaconine similarly when referring to tomato and aubergine the termtga refers to the gas from the corresponding species and forms thereofsupporting information for the assessment chemistrysolanine is one of the ï¬rst alkaloids that has been isolated from nature by desfosses in friedman et al in zwenger and kind reported that solanine contains a glycoside sidechain zwenger and kind only in it was shown that solanine extracted from potato is infact a mixture of two glycoalkaloids gas asolanine and achaconine that share the same solanidineaglycone kuhn and l¬ow since then at least different gas have been isolated and fullystructurally elucidated from over species of the solanaceae family s 13anchezmata et al alsinani and eltayeb the chemical structures and some physical properties of the most importantones are listed in appendix agas are composed of a steroidal aglycone and an oligosaccharide sidechain attached to the 3bhydroxy group of the aglycone see figure friedman et al friedman milner et al the gas of relevance can be divided into the i solanidane group with solanidine as thesteroid backbone and the ii spirosolane group with either the solasodine or the tomatidenoltomatidine backbone gas often contain a double bond between c5 and c6 but the corresponding 5a6hydrogenated forms are also common and in some species eg tomato they constitute the majorcomponents the stereochemistry at carbons c22 and c25 is well deï¬nedtheconï¬guration is 22r 25stheitconï¬guration is 22s 25s friedman et al in solanidineis 22r 25r and in tomatidenoltomatidinein solasodinefurther diversiï¬cation is generated by the composition of the glycoside sidechain most gascontain either a trisaccharide chacotriose or solatriose or a tetrasaccharide lycotetraose ascarbohydrate in commercial potato cultivars solanum tuberosum mostly achaconine and asolaninecomposed ofthe solanidine aglycone and chacotriose and solatriose respectively are presentfigure wild s tuberosum varieties may contain a much wider range of gas friedman et al distl and wink the aubergine fruit derived from s melongena contains primarily asolamargine and asolasonine composed of the solasodine aglycone and chacotriose and solatrioselycopersicum varieties atomatine and arespectivelydehydrotomatine are the major compounds composed of the aglycones tomatidine and tomatidenolrespectively coupled to lycotetraose friedman derived from sin tomato fruitthe preï¬x alpha a refers to the intact glycoside while the preï¬xes beta b gamma c anddelta d refer to the corresponding gas with progressively truncated carbohydrate sidechains due tothe action of enzymatic or acidic hydrolysis friedman milner et al wwwefsaeuropaeuefsa efsa 0cglycoalkaloids in feed and foodohoooohohoohohooohohohsolatrioseohohooohohohsolatrioseohoohoohhh22rnhsolanidine25shhhohsolanine22r 25rnhohsolasodinehhhhooohsolasonineohohoohohohoooohoohoohohoohchacotrioseohohohchacotrioseoohoohohohoooohohohoooohohohohlycotetraoseoohohhhhoohoohoohohhohohoooohoh25sohoh22snhohtomatidinelycotetraoseoohohoohoohooohhhhhhnhsolanidinechaconinehnhohsolasodinehhhsolamarginehhhhnhohtomatidenoltomatinedehydrotomatinefigure s | 0 |
"The extent of lung parenchymal change depends on the time interval. Acute lung injury after the percutaneous injection of lipiodol or methylene blue was reported in animal studies (28 29); however there are no clinical results that show the adverse effect of acute lung injury in human lungs. Injection material (such as barium) can potentially complicate the pathologic diagnosis of the target lesion due to acute inflammation (29 30). To our knowledge no study has indicated that lipiodol or methylene blue hinders the histopathologic diagnosis of target lesions in human lungs. The small amount of material injection in human lungs might not create a significant parenchymal change or disrupt underlying lung disease. It is necessary to avoid directly injecting materials into the target lesion in human lungs in order to avoid the adverse effect of injection materials on underlying lung disease (especially ground glass opacity nodule or potential benign lesion). There were several limitations in our study. First we included only a small number of subjects. Second the overall localization success rate was low and the complication rate was high (compared to the results of previous studies) due to the difficulty in an accurate percutaneous injection at the desired location and depth in the small sized rabbit lung. Third we used a 1 mL syringe with manual administration to inject materials in the lung parenchyma and there were possible individual difference in the administering volume of materials. Fourth we could not evaluate complications such as intractable pain material related anaphylaxis or embolism. Fifth we could not evaluate if the histopathologic changes had any effect on underlying lung disease because the lung parenchyma of the experimental rabbits were normal. Finally we did not evaluate a successful localization for the true target lesion in lung parenchyma. The criteria for appropriate staining and radio-opacity were subjective. We expect that further clinical studies might provide an answer to if MLM can be a useful percutaneous injection material for localization in the human lung. In MLM is available for percutaneous injection for the pulmonary localization. The results of this study showed that MLM provides superior ability for appropriate localization than that of methylene blue. Further research on human lungs can clarify the availability of MLM as a CT guided percutaneous injection material. This study was supported by grant from the Seoul National University College of Medicine Research Fund 2012 (800-20120036). We have no potential conflicts of interest or commercial involvement to disclose. 1 Nakashima S Watanabe A Obama T Yamada G Takahashi H Higami T Need for preoperative computed tomography-guided localization in video-assisted thoracoscopic surgery pulmonary resections of metastatic pulmonary nodules Ann Thorac Surg 2010 89 212 218 20103238 2 Chen S Zhou J Zhang J Hu H Luo X Zhang Y Chen H Video-assisted thoracoscopic solitary pulmonary nodule resection after CT-guided hookwire localization: 43 cases report and literature review Surg Endosc 2011 25 1723 1729 21181200 3 Ciriaco P Negri G Puglisi A Nicoletti R Del Maschio A Zannini P Video-assisted thoracoscopic surgery for pulmonary nodules: rationale for preoperative computed tomography-guided hookwire localization Eur J Cardiothorac Surg 2004 25 429 433 15019673 4 Suzuki K Nagai K Yoshida J Ohmatsu H Takahashi K Nishimura M Nishiwaki Y Video-assisted thoracoscopic surgery for small indeterminate pulmonary nodules: indications for preoperative marking Chest 1999 115 563 568 10027460 5 Seo JM Lee HY Kim HK Choi YS Kim J Shim YM Lee KS Factors determining successful computed tomography-guided localization of lung nodules J Thorac Cardiovasc Surg 2012 143 809 814 22104686 6 Gossot D Miaux Y Guermazi A Celerier M Friga J The hook-wire technique for localization of pulmonary nodules during thoracoscopic resection Chest 1994 105 1467 1469 8181339 7 Pittet O Christodoulou M Pezzetta E Schmidt S Schnyder P Ris HB Video-assisted thoracoscopic resection of a small pulmonary nodule after computed tomography-guided localization with a hook-wire system: experience in 45 consecutive patients World J Surg 2007 31 575 578 17318707 8 Chen W Chen L Yang S Chen Z Qian G Zhang S Jing J A novel technique for localization of small pulmonary nodules Chest 2007 131 1526 1531 17494801 9 Bernard A Resection of pulmonary nodules using video-assisted thoracic surgery: the Thorax Group Ann Thorac Surg 1996 61 202 204 8561553 10 Martin AE Chen JY Muratore CS Mayo-Smith WW Luks FI Dual localization technique for thoracoscopic resection of lung lesions in children J Laparoendosc Adv Surg Tech A 2009 19 S161 S164 18999984 11 Kawanaka K Nomori H Mori T Ikeda K Ikeda O Tomiguchi S Yamashita Y Marking of small pulmonary nodules before thoracoscopic resection: injection of lipiodol under CT-fluoroscopic guidance Acad Radiol 2009 16 39 45 19064210 12 Yamagami T Miura H Yoshimatsu R Tanaka O Ono S Iehara T Hosoi H Nishimura T Experience of fluoroscopy-aided thoracoscopic resection of pulmonary nodule localised with Lipiodol in a child J Med Imaging Radiat Oncol 2011 55 401 403 21843175 13 Iwasaki Y Nagata K Yuba T Hosogi S Kohno K Ohsugi S Kuwahara H Takemura Y Yokomura I Fluoroscopy-guided barium marking for localizing small pulmonary lesions before video-assisted thoracic surgery Respir Med 2005 99 285 289 15733503 14 Yoshida J Nagai K Nishimura M Takahashi K Computed tomography-fluoroscopy guided injection of cyanoacrylate to mark a pulmonary nodule for thoracoscopic resection Jpn J Thorac Cardiovasc Surg 1999 47 210 213 10402768 15 Nomori H Horio H Colored collagen is a long-lasting point marker for small pulmonary nodules " | 1 |
" adenovirus serotype ad5 is a commonly used viral vector for transient delivery of transgenes primarily for vaccination against pathogen and tumor antigens however endemic infections with ad5 produce virus specific neutralizing antibodies nabs that limit transgene delivery and constrain target directed immunity following exposure to ad5 based vaccines indeed clinical trials have revealed the limitations that virus specific nabs impose on the efficacy of ad5 based vaccines in that context the emerging focus on immunological approaches targeting cancer self antigens or neoepitopes underscores the unmet therapeutic need for more efficacious vaccine vectorsmethods here we evaluated the ability of a chimeric adenoviral vector ad5f35 derived from the capsid of ad5 and fiber of the rare adenovirus serotype ad35 to induce immune responses to the tumor associated antigen guanylyl cyclase c gucy2cresults in the absence of pre existing immunity to ad5 gucy2c specific t cell responses and antitumor efficacy induced by ad5f35 were comparable to ad5 in a mouse model of metastatic colorectal cancer furthermore like ad5 ad5f35 vector expressing gucy2c was safe and produced no toxicity in tissues with or without gucy2c expression importantly this chimeric vector resisted neutralization in ad5 immunized mice and by sera collected from patients with colorectal cancer naturally exposed to ad5s these data suggest that ad5f35 based vaccines targeting gucy2c or other tumor or pathogen antigens may produce clinically relevant immune responses in more ¥ patients compared with ad5 based vaccines inhibitor introductiontherapies immune checkpoint have revolutionized cancer treatment and cancer drug development by engaging the immune system to target various cancers1 despite this success many tumors are immunologically cold characterized by a dearth of immunogenic neoepitopes3 and lack of tumor infiltrating lymphocytes4 and remain refractory to checkpoint inhibitors6 one emerging strategy to modify a cold tumor into one responsive to immunotherapy is through combination with cancer vaccines8 the goal of this strategy is to use cancer vaccines to create a pool of tumor reactive t cells with antitumor activity alone andor in combination with checkpoint therapies however this approach is significantly limited by the paucity of effective vaccine platforms to safely deliver tumor specificassociated antigens to elicit beneficial antitumor immunitythe ability of adenovirus serotype ad5 to mediate gene transfer and induce potent immune responses has made it a popular vector for experimental vaccines infectious diseases10 against cancer and indeed there have been more than clinical trials using the ad5 vector with most trials focused on developing cancer treatments10 however on natural infection the host immune system develops neutralizing antibodies nabs to the ad5 capsid limiting viral spread and blocking reinfection because ad5 infections are endemic in many human populations pre existing nabs present in of the worldwide population limit ad5 based vaccine strategies12 these considerations highlight the need for improved vectors for use in vaccines targeting cancer and pathogen associated antigens that can create therapeutic immune responses in the greatest number of patients importantly while the adenovirus capsid is composed of hexon penton and fiber proteins nabs elicited by natural ad5 infection in humans are directed primarily to the ad5 fiber15 suggesting that strategies to flickinger jr a0jc et a0al j immunother cancer 20208e001046 101136jitc2020001046 0copen access circumvent pre existing immunity to this element may improve ad5 based vaccineshere we sought to overcome pre existing ad5 nabs by replacing the ad5 fiber with that of a rare adenovirus serotype ad35 international seroprevalence to improve antitumor immunity in mouse models expressing the gastrointestinal gi cancer antigen guanylyl cyclase c gucy2c preclinical models demonstrated that an ad5 based gucy2c directed vaccine ad5 gucy2c s1 elicited cd8 t cell and antibody responses without autoimmunity17 further ad5 gucy2c s1 vaccination of mice induced long term t cell mediated protection against metastatic colorectal cancer in lung and liver19 moreover those results were recapitulated in a recent first in human phase i clinical trial nct01972737 demonstrating that a humanized version of the vaccine ad5 gucy2c padre safely induced gucy2c specific cd8 t cell responses in patients with colorectal cancer following conventional therapies21 however patients possessing high pre existing titers of nabs against ad5 failed to generate gucy2c specific immunity following ad5 gucy2c padre vaccination21 to overcome ad5 nabs we generated a chimeric ad5 vector possessing the fiber of ad35 ad5f35 with equivalent safety and antitumor activity to ad5 and resistance to ad5 nabs in mice and humans this chimeric vaccine can be translated to patients with gi cancer to safely induce gucy2c specific immunity not only in those patients with low ad5 immunity but also in those with high pre existing ad5 nabsmaterials and methodsadenovirus vectorsadenovirus containing mouse extracellular domain gucy2c1429 with the influenza ha107119 cd4 t cell epitope known as site s1 was described previously ad5 gucy2c s120 here gucy2c s1 was cloned into pshuttle and subcloned into the e1 region of previously generated replication deficient chimeric adenovirus ad5f35 in which the ad5 fiber was replaced by the ad35 fiber22 to generate ad5f35 gucy2c s1 all adenovirus vaccines used in this study were produced in hek293 cells and purified by cesium chloride ultracentrifugation under good laboratory practices by the baylor college of medicine in the cell and gene therapy vector development lab and certified to be negative for replication competent adenovirus mycoplasma and host cell dna contamination in vitro gucy2c expression experiments doseresponse and timecourse were carried out in a549 american type culture collection atcc cells virus was added to the cultures at the indicated doses and culture supernatants were collected at the indicated time points relative gucy2c levels were quantified in supernatants by western blot using μgml ms7 mouse anti gucy2c monoclonal antibody23 and μgml horseradish peroxidase conjugated goat antimouse secondary antibody jackson immunomice and immunizationseight week old male and female balbcj mice were purchased from the jackson laboratory for experiments animal protocols were approved by the thomas jefferson university institutional animal care and use committee protocol for immunizations mice received or vp of ad5 gucy2c s1 ad5f35 gucy2c s1 or ad5f35 gfp control administered as two μl intramuscular injections one in each hind limb using a ml insulin syringequantifying tcell responses by elispotelispot assays were performed using a mouse interferonÎ ifnÎ single color elispot kit cellular technology according to the manufacturers protocol26 briefly well plates were coated with ifnÎ capture antibody overnight at °c the next day plates were washed with phosphate buffered saline pbs and splenocytes from immunized mice were plated at cellswell with no peptide or μgml gucy2c254262 peptide in dimethyl sulfoxide dmso in ctl test medium cellular technology for hours at °c for t cell avidity studies splenocytes were plated at cellswell with decreasing concentrations of gucy2c254262 peptide μgml to pgml normalized to cellswell26 after incubation cells were removed and development reagents were added to detect ifnÎproducing spot forming cells the number of spot forming cells per well was determined using the smartcount and autogate functions of an immunospot s6 universal analyzer cellular technology gucy2c specific responses were calculated by subtracting mean spot counts of dmso wells from peptide stimulated wells26 tumor studiesgucy2c expressing mouse balbc ct26 colorectal cancer cells were used for in vivo tumor studies17 luciferase expressing cells were generated by transduction with lentiviral supernatants produced by 293ft cells invitrogen with plenti4 v5 gw luciferase28 for tumor experiments balbcj mice were immunized with vp of ad5 gucy2c s1 ad5f35 gucy2c s1 or pbs control days before delivering à ct26 cells into tail veins tumor burden was quantified weekly by subcutaneous injection of mg of d luciferin potassium salt gold biotechnologies in pbs followed by an min incubation and imaging with a s exposure using a caliper ivis lumina xr imaging station perkinelmer total radiance photonssecond was measured using living image in vivo imaging software perkinelmerantibody neutralization assayserum samples were obtained previously from patients before ad5 gucy2c padre nct01972737 approved by the thomas jefferson university institutional review board21 neutralizing antibody titers against ad5 and ad5f35 vectors were quantified as immunization with flickinger jr a0jc et a0al j immunother cancer 20208e001046 101136jitc2020001046 0cdescribed21 briefly dilutions of heat inactivated serum samples were added to well tissue culture plates containing a549 cells atcc and infected with vp of gfp expressing ad5 or ad5f35 virus ad5 cmv egfp or ad5f35 cmv egfp respectively baylor vector development lab following a hour incubation at °c egfp fluorescence nm excitation nm emission was quantified using a polarstar optimate plate reader bmg labtech sample fluorescence was normalized to control wells containing cells and virus neutralization or wells containing cells alone neutralization titers were quantified using non linear regression as the serum dilution producing neutralization prism v8 graphpad softwaread5 neutralizing immunity studiesto induce anti ad5 immunity mice were exposed intranasally to ad5 gfp once or twice at a week interval thirty days after the last exposure ad5 nabs were quantified in sera as described above and mice were immunized intramuscularly with vp of ad5 gucy2c s1 or ad5f35 gucy2c s1biodistribution and toxicology studybalbcj mice were immunized intramuscularly with a single dose of vp of ad5f35 gucy2c s1 three doses of vp of ad5f35 gucy2c s1 at day intervals or pbs control animals were monitored for adverse events once daily with additional evaluations on the day of dosing min hour and hours after dosing on days and designated animals were sacrificed and brain salivary glands stomach small intestine colon heart lungs kidneys liver and injection site were harvested and weighed for histopathological analysis by a blinded pathologist pathology evaluation was performed by idexx bioanalytics and detection of viral dna by quantitative pcr qpcr using the previously described assay for the gucy2c transgene19 also spleens were collected for histopathological analysis and detection of viral dna as described above as well as quantification of gucy2c specific t cell responses by ifnÎ elispot as described abovestatistical analysisstatistical analyses were conducted using graphpad prism software v8 statistical significance was considered as follows nsp p p p and p cohort sizes were powered based on prior studies with β02 and α005 for multiple comparisons of survival outcomes significance thresholds were corrected using the bonferroni method to identify vaccine induced t cell responders and non responders a previously described21 modified distribution free resampling approach was employed and positive t cell responses were defined as à compared with dmso and specific spots106 cells to determine the impact of gender and number of vaccinations on responses log transformed vaccine response magnitude was compared in mice of different genders cohorts and treatment regimens for up to three way interactions with stepwise backward variable selection by akaike information criterion using r29 package mass30open accessresultsad5gucy2cs1 and ad5f35gucy2cs1 vectorswhile ad5 seroprevalence worldwide exceeds in some regions ad35 is and associated with lower titers figure 1a12 thus we constructed a chimeric adenovirus ad5f35 composed of ad5 in which the fiber was replaced by the ad35 fiber and evaluated its ability to induce gucy2c specific immunity and resist ad5 specific immunity in humans and mice ad5 gucy2c s1 is a replication deficient human ad5 expressing the mouse gucy2c extracellular domain fused to the i ed restricted cd4 epitope known as site at its c terminus20 to generate ad5f35 gucy2c s1 the ad5 fiber l5 was replaced with the ad35 fiber figure 1b replication deficient ad5 gucy2c s1 and ad5f35 gucy2c s1 generated in hek293 cells produced dose dependent figure 1c and time dependent figure 1d expression of gucy2c s1 protein in a549 human alveolar basal epithelial cells in vitroad5f35gucy2cs1 induces gucy2cspecific antitumor immunityfollowing in vitro validation of gucy2c expression by ad5f35 gucy2c s1 we confirmed its ability to induce gucy2c specific immune responses after vaccination in vivo balbc mice immunized intramuscularly with vp of ad5f35 gucy2c s1 produced lower gucy2c specific cd8 t cell responses figure 2a and no gucy2c specific antibody responses figure 2b compared with ad5 gucy2c s1 importantly ad5 and ad5f35 vaccines produced gucy2c specific cd8 t cells of comparable avidity figure 2c a critical determinant of the antitumor efficacy of gucy2c targeted vaccines26 in contrast gucy2c specific antibody responses have no detectable antitumor activity20 similarly ad5 and ad5f35 vaccines produced comparable s1 specific cd4 t cell responses figure 2dluciferase this model previous studies revealed that ad5 gucy2c vaccines induced protective antitumor cd8 t cell responses in murine models of metastatic colorectal cancer17 thus balbc mice were immunized with ad5 or ad5f35 expressing gucy2c s1 and challenged days later with ct26 colorectal cancer cells expressing gucy2c and firefly specifically emulates secondary prevention of metastatic disease the clinical setting for which the gucy2c vaccine is being developed21 as previously demonstrated ad5 vaccination nearly eliminated metastatic tumor burden figure 3ab delayed disease progression figure 3c and improved survival figure 3d similarly ad5f35 also reduced tumor burden figure 3ab disease progression figure 3c and prolonged survival figure 3d importantly the efficacy of ad5 based and ad5f35 based gucy2c vaccines in flickinger jr a0jc et a0al j immunother cancer 20208e001046 101136jitc2020001046 0copen access figure construction of ad5f35 gucy2c s1 and antigen expression a reported international seroprevalence of ad5 and ad3512 b the l5 gene encoding the fiber protein from ad5 was replaced with the l5 gene from ad35 producing the chimeric adenoviral vector ad5f35 recombinant ad5f35 gucy2c s1 was produced by inserting mouse gucy2c s1 into the e1 region of e1e3 deleted ad5f35 c and d the human alveolar basal epithelial cell line a549 was transduced in duplicate with ad5f35 gucy2c s1 at a multiplicity of infection moi from to for hours c or at an moi of for and hours d supernatants from infected cells were analyzed for gucy2c s1 protein expression by immunoblot protein expression was quantified by densitometry and plotted relative to uninfected cells error bars indicate mean±sem ad5 adenovirus serotype reducing tumor burden opposing disease progression and promoting survival was identical figure 3adad5f35 resists ad5directed immunity in mice and humansnabs against ad5 correlated with poor gucy2c specific immune responses in patients receiving ad5 gucy2c padre vaccination and prior exposure of mice to ad5 similarly blunted vaccine induced immunity21 ad5f35 based vaccine resistance to pre existing ad5 immunity was quantified in a model of respiratory pre exposure to ad5 the natural route of infection in patients33 followed by vaccination and quantification of gucy2c specific t cell responses control mice not pre exposed to ad5 naive and those that were pre exposed once à or twice à to intranasal ad5 were vaccinated after weeks with intramuscular ad5 or ad5f35 expressing gucy2c s1 and immune responses were quantified weeks later immunogenicity of ad5 gucy2c s1 and ad5f35 gucy2c s1 ad balbc mice n4 micegroup were figure immunized intramuscularly with control or vp of ad5 gucy2c s1 or ad5f35 gucy2c s1 and serum and splenocytes were collected days later gucy2c specific cd8 t cell responses were quantified by interferon gamma ifnÎ elispot a and antibodies were quantified by elisa b c gucy2c specific t cell avidity measurements were analyzed by elispot using non linear regression logagonist versus normalized response with comparisons made using the extra sum of squares f test avidity plots depict the regression line solid with cis dashed d s1 specific cd4 t cell responses were measured by ifnÎ elispot t cell responses in a and d were analyzed by one way analysis of variance values in a b and d indicate individual animals and bars in a and d indicate means tcr t cell receptor ad5 adenovirus serotype flickinger jr a0jc et a0al j immunother cancer 20208e001046 101136jitc2020001046 0copen accessfigure antitumor efficacy of ad5 gucy2c s1 and ad5f35 gucy2c s1 ad balbc mice n10 micegroup were immunized intramuscularly with control or vp of ad5 gucy2c s1 or ad5f35 gucy2c s1 and challenged days later with a mouse colorectal cancer cell line ct26 expressing gucy2c and luciferase on days and following challenge mice were injected with d luciferin and imaged a to quantify tumor burden day b mice were weighed twice weekly c and monitored for survival d tumor burden b was analyzed by one way analysis of variance and survival comparisons d were analyzed by the mantel cox log rank test in b and d asterisks indicate comparisons of gucy2c vaccines to the control and brackets ] indicate comparisons between ad5 and ad5f35 vaccines ns not significant ad5 adenovirus serotype figure 4a as expected one ad5 pre exposure induced moderate ad5 nabs online supplementary figure s1 and reduced gucy2c specific t cell responses while two pre exposures induced high ad5 nabs online supplementary figure s1 and reduced gucy2c specific t cell responses following ad5 vaccination figure 4b in contrast gucy2c specific t cell responses were reduced only à pre exposure and à pre exposure following ad5f35 vaccination figure 4b importantly ad5f35 produced t cell responses in a substantially greater fraction of the population cohort responses compared with ad5 cohort responses following serial pre exposures to ad5 figure 4cthese observations in mice were recapitulated using sera from patients with colorectal cancer in the ad5 gucy2c padre phase i trial nct0197273721 here nab titers against ad5 and ad5f35 were quantified using an established ad5ad5f35 reporter virus inhibition bioassay in serum samples collected prior to vaccination with ad5 gucy2c padre21 in these patients ad5f35 specific nab titers were substantially lower than ad5 specific titers figure 4d most importantly of patients possessed low ad5 nabs titers figure 4de which closely correlated with a gucy2c specific response rate21 in striking contrast had low ad5f35 nab titers suggesting that the vast majority of patients immunized with ad5f35 based vaccines could produce gucy2c specific responses figure 4e collectively these observations suggest that pre existing viral immunity induced by repeated environmental exposures which neutralizes ad5 delivery platforms may be overcome by the chimeric ad5f35 vector to enhance fractional population vaccine responsessafety biodistribution and toxicity of ad5f35gucy2cs1food and drug administration ind investigational new drug enabling studies quantified the toxicity biodistribution and immunogenicity of ad5f35 gucy2c s1 in balbc mice employing three schemes to examine acute and chronic effects figure 5a cohorts balanced for sex received ad5f35 gucy2c s1 either as a single intramuscular injection or as three intramuscular injections spaced weeks apart monitored daily and sacrificed on day or for analysis as indicated figure 5a there were no signs of acute or chronic toxicity in the in life phase by observation weight changes or survival figure 5bd similarly there were no clinically significant differences in organ weights online supplementary figure s2 or histopathology not shown at necropsy small statistical differences in organ weights were considered clinically insignificant and were unrelated to vaccine exposure dose time online supplementary figure s2 biodistribution quantified by qpcr detected ad5f35 gucy2c s1 at the injection site and in the spleen but not appreciably in other organs after acute and chronic exposures online supplementary figure s3 moreover robust cd8 t cell responses were quantified at day that persisted through day in of mice after a single administration figure 5eg as expected cd8 t cell responses were greater and persisted in more mice at days after three vaccinations figure 5egdiscussionthrough decades of gene therapy trials ad5 has remained a popular vector while high ad5 seroprevalence remains a barrier to universal vaccination33 natural respiratory infection can generate long lived antibodies that neutralize ad5 based vaccines eliminating transgene delivery and potential therapeutic benefit in that context ad5 seroprevalence is across multiple countries12 highlighting an unmet need for alternative vectors here we demonstrate that the chimeric ad5f35 resists pre existing ad5 immunity and induces transgene specific antitumor immunity indeed ad5f35 is less susceptible to neutralization associated with ad5 exposure in mice and humans and generates a substantially higher proportion of vaccine responders in mice pre exposed to ad5 these observations support the suggestion that ad5f35 flickinger jr a0jc et a0al j immunother cancer 20208e001046 101136jitc2020001046 0copen access figure ad5f35 resists neutralization associated with pre existing anti ad5 immunity in mice and humans ac to generate pre existing immunity to ad5 balbc mice n10 micegroup were exposed intranasally once or twice to vp of ad5 gfp at week intervals four weeks after the final ad5 gfp exposure ad5 exposed and naive mice were immunized intramuscularly with vp of ad5 gucy2c s1 or ad5f35 gucy2c s1 b two weeks after immunization gucy2c specific cd8 t cell responses in each group were quantified by interferon gamma ifnÎ elispot and calculated as the of mean responses in naive mice values indicate individual animals and bars indicate means ad5 and ad5f35 were compared by two way analysis of variance c the fraction of animals producing a detectable gucy2c specific cd8 t cell response filled regions in naive à and à ad5 exposed mice was determined from b d and e sera from patients with colorectal cancer collected prior to ad5gucy2c padre vaccination were tested for the ability to neutralize ad5 and ad5f35 vectors and titers were quantified d analyzed by paired t test the dotted line indicates a titer of the threshold for high neutralizing antibody nab titers21 e while subjects had high nab titers against ad5 only had high titers to ad5f35 vector filled regions binomial test ad5 adenovirus serotype will produce a higher proportion of vaccine responders in patient populationsthe extent to which nabs to the ad5 fiber limit reinfection is controversial in some studies replacing the ad5 fiber with that of another serotype circumvents pre existing ad5 immunity34 in contrast other studies suggest that these chimeric adenoviruses do not evade pre existing ad5 nabs suggesting the hexon as the major target of antibody neutralization35 in contrast to those previous studies which generated pre existing ad5 immunity by intramuscular35 or intravenous administration36 here ad5 immunity was induced by intranasal exposure in mice recapitulating natural infection33 moreover natural human respiratory pre existing ad5 nabs in patients with colorectal cancer uniformly produced by repeated respiratory infections33 similarly were overcome by the ad5f35 vector importantly the quality of antibody responses following adenovirus infection is dependent on the route of exposure indeed respiratory infections elicit fiber specific nabs while intramuscular exposure induce capsid specific nabs15 these qualitative differences in nab responses reflecting varying routes of immunization may contribute to observational discrepancies between laboratories the present studies using relevant animal models confirmed and validated with patient samples support the suggestion that ad5f35 based vaccines should produce clinically relevant immune responses in a substantial proportion of patientsflickinger jr a0jc et a0al j immunother cancer 20208e001046 101136jitc2020001046 0copen accessfigure safety and immunogenicity of multiple ad5f35 gucy2c s1 administrations ag balbc mice n10 micegroup were immunized intramuscularly with one or three administrations of vp ad5f35 gucy2c s1 or control at week intervals following immunization body weights b female and c male were recorded weekly and mice were monitored for survival d at days and following first immunization mice were euthanized to quantify organ pathology by weight online supplementary figure s2 biodistribution by quantitative pcr online supplementary figure s3 and gucy2c specific cd8 t cell responses by interferon gamma ifnÎ elispot eg g pie charts indicate proportion of responding animals ad5 adenovirus serotype recognizing the pervasive limitations imposed by endemic ad5 immunity in global populations12 there is an emerging interest in alternative serotypes and chimeric constructs as a tractable strategy in vaccine development ad26 ad35 and ad48 vectors have been advanced into phase i clinical trials37 in that regard a comparison of ad5 ad26 ad35 and ad48 immunity among healthy patients revealed that endemic ad35 seropositivity was lowest across global populations12 reinforcing chimeric strategies employed herein similarly the first hexon chimeric adenovirus comprising ad5 and ad48 components was safe and immunogenic in patients39 interestingly ad5 ad35 chimeric vectors more efficiently transduce a variety of human cell types in vitro compared with either parental vector40 these observations underscore the future potential of intelligently designed chimeric adenoviruses strategically constructed to deliver transgenes for replacement therapy or vaccination and targeted precisely to the cellular or disease context40while antitumor efficacy was equivalent cd8 t cell responses were lower and antibody responses were absent for ad5f35 gucy2c s1 compared with ad5 gucy2c s1 however the antitumor efficacy of gucy2c directed immunotherapy is driven primarily by t cell avidity rather than effector t cell quantity26 in that context the functional avidity of gucy2c specific cd8 t cells following ad5 and ad5f35 immunizations were equivalent consistent with their comparable antitumor efficacy quantitative differences in transgene specific immunity between vectors may reflect a variety of factors thus the quantity and persistence of gucy2c s1 transgene following ad5f35 immunization is lower compared with ad519 consistent with prior observations that ad5 transduction efficiency in vivo may be several fold higher than ad5f3541 moreover the ad5 fiber binds to cxadr coxsackievirus and adenovirus receptor42 while the ad35 fiber binds to cd4643 suggesting the two viruses may infect distinct cell typesflickinger jr a0jc et a0al j immunother cancer 20208e001046 101136jitc2020001046 0copen access while checkpoint inhibitors have generated practice shifting results in the clinic and defined immunotherapy as an effective strategy for the treatment of several malignancies they have not been universally successful in that context the dearth of neoepitopes in many cancer types including microsatellite stable colorectal and pancreatic second and third leading causes of cancer mortality respectively makes them insensitive to checkpoint blockade7 indeed examination of neoepitopes presented on the surface of five colorectal cancer specimens revealed a total of three neoepitopes3 thus vaccines targeting cancer associated self antigens have re emerged alone and in combination with checkpoint inhibitors as a strategy to prevent and treat metastases from these cold tumors44 checkpoint inhibitors have become first line therapy in the metastatic setting for some cancers46 while chimeric antigen receptor expressing t cells car t cells are being deployed in patients with metastatic and refractory disease47 in contrast few cancer immunotherapies have been developed for early stage cancer patients with no evidence of disease ned following conventional surgicalradiochemotherapies who are at significant risk of disease recurrence indeed25 of stage ii and of stage iii patients with colorectal cancer recur following surgery and chemotherapy49 while of patients with resectable pancreatic cancer experience recurrence50 vaccines targeting tumor associated antigens such as ad5f35 gucy2c padre may provide safe and effective immunotherapies for the secondary prevention of metastatic disease in patients with ned who are otherwise ineligible to receive checkpoint inhibitors or car t cellsthe present studies suggest that the chimeric adenoviral vector ad5f35 may be preferable to the widely used ad5 vector and warrants further investigation indeed they suggest that ongoing clinical investigations of gucy2c directed immunotherapy in patients with gucy2c expressing cancers including colorectal pancreatic gastric and esophageal could benefit from using the ad5f35 rather than the ad5 vector in that context an upcoming clinical trial will examine the safety immunogenicity and resistance to pre existing immunity of ad5f35 gucy2c padre in patients with gi cancer nct04111172 safe generation of gucy2c targeted immunity in a high proportion of patients will lead to efficacy trials to establish the ability of ad5f35 gucy2c padre to prevent recurrence following standard therapy in patients with gi cancer who represent of all cancer deaths51 and for whom established immunotherapies are ineffectivetwitter adam e snook adamsnookphdacknowledgements the authors thank adrian p gee phd zhuyong mei md deborah lyon and malcolm brenner md phd center for cell and gene therapy baylor college of medicine for assistance in vaccine manufacturingcontributors jcf js bb saw and aes designed the studies jcf js rc el trb jb ec ap jar and jr carried out the studies tz carried out data analysis and statistical analysis in discussion with aes jcf and aes wrote the manuscript and all authors critically reviewed and approved the final version of the manuscriptfunding this work was supported by the national institutes of health nih r01 ca204881 r01 ca206026 and p30 ca56036 the defense congressionally directed medical research program w81xwh17 prcrp ttsa and targeted diagnostic therapeutics to saw aes received a research starter grant in translational medicine and therapeutics from the phrma foundation a degregorio family foundation award and was supported by the defense congressionally directed medical research programs nos w81xwh1710299 w81xw | 0 |
"Pluripotent stem cellsDirected differentiationIn vitro disease modellingLungAirwayMechanical cuesContentsChronic lung diseases remain major healthcare burdens for which the only curative treatment is lung transplantation In vitro human models are promising platforms for identifying and testing novel compounds to potentiallydecrease this burden Directed differentiation of pluripotent stem cells is an important strategy to generate lungcells to create such models Current lung directed differentiation protocols are limited as they do not recapitulate the diversity of respiratory epithelium generate consistent or sufï¬cient cell numbers for drug discoveryplatforms and establish the histologic tissuelevel anization critical for modeling lung function In this review we describe how lung development has formed the basis for directed differentiation protocols and discussthe utility of available protocols for lung epithelial cell generation and drug development We further highlighttissue engineering strategies for manipulating biophysical signals during directed differentiation such that futureprotocols can recapitulate both chemical and physical cues present during lung development Elsevier BV All rights reservedOverview of key developmental stages Lung anogenesis molecularly deï¬ning lung fate in the embryo Branching morphogenesis and other mechanical cues generated during lung development Introduction Human embryology as a blueprint for lung directed differentiation Directed differentiation of lung epithelia inspired by embryology Mouse embryonic stem cell derived lung epithelia Modeling airway and lung diseases for drug discovery Opportunities to exploit mechanical cues for improving directed differentiation protocols in the future Micropatterning in 2D Stem cell behaviour on substrate topographies Micropatterning in 3D anoid systemsSubstrate textureHuman pluripotent stem cellderived lung epithelia Creation of human proximal lung epithelia Comparisons of proximal airway directed differentiation protocols Creation of human distal lung epitheliaComparisons of distal lung directed differentiation protocols Limitations of current directed differentiation protocols Correspondence to G Karoubi Latner Thoracic Surgery Research Laboratories Toronto General Hospital College St Toronto ON M5G 1L7 Canada Correspondence to A P McGuigan Institute for Biomaterials and Biomedical Engineering University of Toronto College Street Toronto ON M5S 3G9 CanadaEmail addresses golnazkaroubiuhnresearchca G Karoubi alisonmcguiganutorontoca AP McGuigan101016jaddr2020080050169409X Elsevier BV All rights reservedPlease cite this as R Varma JP Soleas TK Waddell Current strategies and opportunities to manufacture cells for modelinghuman lungs Adv Drug Deliv Rev 101016jaddr202008005 0cR Varma Advanced Drug Delivery Reviews xxx xxxAcknowledgementsReferencesFuture outlook IntroductionEndstage lung disease is the third leading cause of morbidity andmortality worldwide [] and produces a significant burden onhealthcare systems due to extensive resource expenditures for diseasemanagement and as lung transplantation is the only curative treatmentoption Such diseases include acute respiratory distress syndromechronic obstructive pulmonary disease cystic ï¬brosis and pulmonaryï¬brosis Chronic pulmonary diseases result in million global deathsper year [] Patients who receive transplants face continued complications associated with chronic immunosuppression and graft rejectionwith the transplant survival rates at and years being and respectively [] Furthermore since lungs function as an important barrier between the internal and the external environments they are a critical site for bacterial and viral infections and disease transmissionparticularly relevant given the current COVID19 pandemic There istherefore a critical need to better elucidate the mechanisms of infectiondisease progression host response and cellular repair in the lung to enable the development of novel targeted therapeutics for lung diseaseTissueengineered models have emerged as a technology to addressthis challenge and shown some success in drug identiï¬cation and toxicology studies For example commercially available airway epithelialmodels such as EpiAirwayTM MatTek Life Sciences serve as convenientplatforms with airliquid interface culture capabilities for assessing theeffect of chemical and physical stimuli [] Other examples includethe Alveolus LungChip and Airway LungChip systems Emulate Incoriginally developed in the Ingber laboratory which mimic theepithelialendothelial interface of the airway and provide a more dynamic platform for testing new antiammatory compounds inasthma [] and new small molecule targets to decrease cancerassociated pulmonary edema [] More complex models have alsobeen reported which involve selfassembly of heterogeneous progenitor cells into 3D structures termed anoids [] These anoidmodels can recapitulate aspects of human lung development in termsof tissue structures and protein expression and therefore present apromising opportunity for drug screening []A challenge in developing such human in vitro lung models to screenfor drugs however is the requirement for large batches of similarhuman cells as a starting population for tissue manufacturing to ensureminimal heterogeneity between test wells [] Achieving this is especially challenging when using primary human lung cells which exhibitconsiderable heterogeneity across donors and have a limited ability togrow and differentiate reliably [] Furthermore primary cells areoften extracted from diseased donors which is not ideal for conductingcontrolled studies due to the wide range of therapeutic and environmental factors these cells have already been exposed to Directed differentiation of pluripotent populations has the potential to create vastnumbers of cells from either healthy or diseased patients It allows introduction of speciï¬c diseaseassociated mutations via CRISPRCas9gene editing to recapitulate and understand pathologies in a controlledmanner As such directed differentiation enables the generation of anattractive cell source for drug screening platforms and personalized disease models that may provide insight into tissue regeneration mechanisms []Directed differentiation protocols to manufacture speciï¬c cell populations from pluripotent stem cells PSCs have been developed to meetthe need for a homogeneous human cell source Older lung directed differentiation protocols from the late 2000s have been proven inefï¬cientdue to the nonstandardized methods through which they derive lungendoderm from embryoid bodies [] A series of more standardizedstepwise protocols have since emerged in the last decade that provideavenues for developing airway and lung epithelia albeit with variableefï¬ciencies [] The ï¬rst uential directed differentiation protocol to produce lung epithelia used human PSCs in [] whichwas further supported by two prominent studies conducted usingmouse PSCs in [] These protocols have continued to be enhanced through adaptations related to the selection of growth factorsand small molecules the chronology of morphogen delivery as wellas innovations in enabling platforms such as cell sorting 3D cultureand singlecell analyses to efï¬ciently derive normal and diseased lungepithelia from human PSCs [] Despite such advancements limitations pertaining to heterogeneity in the resultingpopulations still exist which are likely attributed to variability across directed differentiation trials PSC cell lines or the persistence of contaminating cell populations belonging to other lineages While protocolshave progressed to some degree in differentiating proximal airwayand distal alveolar epithelia they remain limited Overall many unanswered questions remain with regards to the identity maturity andfunctionality of resulting cell types as well as their utility for tissue engineering and drug testing approaches Therefore these protocols mustbe optimized further to reliably produce large numbers of spatially relevant and functional lung and airway epithelial cells that appropriatelyrespond to both chemical and mechanical stimuli in the context of disease modeling and drug discoveryIn this review we discuss the directed differentiation protocols thatattempt to recapitulate lung development and disease and highlightpossible opportunities to enhance these protocols in the future Weï¬rst describe development of native lung tissue and the patterningevents that occur that differentiation models attempt to mimic andhighlight how human lung embryology has served as the blueprint tocreate the common pathway of lung directed differentiation protocolsWe then discuss the evolution of directed differentiation protocols toï¬nd opportunities for creating speciï¬c populations of airway and lungepithelia through targeted manipulation of key signaling pathways in2D and 3D models We further describe how these models have beenused to recapitulate different airway and lung diseases Finally we discuss how tissue engineering and biophysical cues using biomaterialscan be utilized during lung directed differentiation to mimic patterningcues present in development to augment current differentiationprotocols Human embryology as a blueprintdifferentiationforlung directed Overview of key developmental stagesDirected differentiation protocols have been designed to mimicin vivo human lung development [] Indeed in vitro models of lungdevelopment have provided unique insight into human lung development [] As human lung development has been described at greatlength in earlier reviews [] we provide a brief overview as followsschematically represented in Fig During early embryogenesis at days post fertilization a process called gastrulation begins with the appearance of a structure called primitive streak through which cells migrate to form the primary embryonic germ layers deï¬nitive endodermmesoderm and ectoderm [] Deï¬nitive endoderm expandsthereby forming the primitive gut tube comprised of three endodermalregions foregut midgut and hindgut [] This is when lungPlease cite this as R Varma JP Soleas TK Waddell Current strategies and opportunities to manufacture cells for modelinghuman lungs Adv Drug Deliv Rev 101016jaddr202008005 0cR Varma Advanced Drug Delivery Reviews xxx xxxFig Schematic of human lung development from an epithelial perspectivedevelopment begins at approximately four weeks into embryonic lifewith the outgrowth of foregut endoderm [] and continues througheight years of postnatal life [] There are ï¬ve stages to lungdevelopment Embryonic weeks The future lung buds emerge from the ventral side of the primitive foregut endoderm into the surroundingmesenchyme and develop into embryonic lung buds with early trachea and bronchi [] Pseudoglandular weeks Branching of the airway continuesleading to formation of conducting and terminal bronchioles whilethe proximal airway epithelium begins to develop [] Canalicular weeks Development of the respiratory or gasexchanging airways is initiated primitive alveoli form and the future distal epithelium begins to thin as distal epithelial markers areexpressed [] Saccular weeks Emergence of sacshaped distal airwayswhich develop crests with muscle and elastin to create indentationsThese distal airways extend to form alveoli by weeks [] The developing epithelium and vasculature within the future alveolus continue to merge closer together to facilitate future gas exchange andfurther differentiation of alveolar epithelial cells AEC I and II takesplace Alveolar periods week years True alveoli are seen in week and the majority of alveolarization takes place through sacculeseptation a process by which the sacshaped distal airways changetheir internal architecture and create thin walls intraluminallySeptation leads to an increase in surface area of the gas exchangingportion of the developing lung and prepares the fetus to breath airduring this stage [] Lung anogenesis molecularly deï¬ning lung fate in the embryoDuring the embryonic period early lung is genetically deï¬ned by theexpression of transcription factor NK2 Homeobox NKX21 and Srybox SOX2 [] During human lung development it has beenfound that the lung buds and branches given off during thepseudoglandular period are mostly SOX2SOX9 [] BothSOX2 and SOX9 are individual markers of the early proximal or distal lineage respectively [] Over the course of the canalicular and saccular periods of development weeks these double positivepopulations downregulate one SOX protein and maintain expressionof the other as these cells mature towards proximal or distal lineages[] The proximal airway closer to the mouth is comprised of apseudostratiï¬ed columnar epithelium that is responsible for theconducting airway function debris and pathogen removal ciliatedcells mucus production goblet cells prevention of airway ammation club cells and humidiï¬cation of air as it passes through to the distal lung compartment [] The squamous distal epitheliumcomposed of alveolar epithelial cells AEC I and II facilitates the respiratory function of the lung as air in the epithelial compartment is broughtinto close apposition to blood from the pulmonary vasculature it alsosecretes surfactants which play an immunologic role and decrease thesurface tension present at the airliquid interface thereby preventing alveolar collapse [] In humans a number of cell types are found in theproximal airway each identiï¬ed with speciï¬c markers Table This includes basal cells tumor protein p63P63 keratinKRT5 nerve growthfactor receptorNGFR integrin α6ITGA6 integrin β4ITGB4 ciliatedcells Forkhead BoxJ1FOXJ1 acetylated tubulinAcTUB goblet cellsmucin 5ACMUC5AC mucin 5BMUC5B club cells club cell secretoryproteinCCSP or SCGB1A1 and pulmonary neuroendocrine cellsPNECs synaptophysinSYP chromogranin ACHGA On the otherhand homeodomainonly protein HOPX identiï¬es the distal lungalong with AEC I cells T1α podoplaninPDPN aquaporin 5AQP5while AEC II cells are recognized via surfactant protein B SPC prosurfactant protein C proSPC or SPC and HT2280 []One mechanism by which lung epithelia begin to mature is based onchemokine secretions from the surrounding mesenchyme and the developing heart ï¬eld which are well reviewed here [] Key players including ï¬broblast growth factors FGFs [] WNTs []and bone morphogenetic proteins BMPs [] are known to inducethe differentiation of early lung progenitors in a controlled manner Forexample in mouse it has been found that FGF10 plays a role in bud outgrowth [] and drives lung progenitors towards a distal fate []through canonical WNT signaling [] Proximal epithelia developbecause they are located further away from distally located FGF reservoirs in the mesenchyme in a mechanism that appears dependent onconcentration gradients [] BMP4 plays a key role in lung bud formation from foregut endoderm and establishment of both dorsoventralback to front and proximodistal top to bottom patterning in the nascent lung [] BMP4 is also present at high levels in distal bud tips andepithelia including AEC II cells [] however its inhibition promotesa proximal fate and along with BMP2 inhibition ciliated cell development [] Branching morphogenesis and other mechanical cues generated duringlung developmentWhile the cell fate of early proximal and distal lineages is directedthrough chemical signals the lung epithelium itself undergoes markedPlease cite this as R Varma JP Soleas TK Waddell Current strategies and opportunities to manufacture cells for modelinghuman lungs Adv Drug Deliv Rev 101016jaddr202008005 0cR Varma Advanced Drug Delivery Reviews xxx xxxTable Epithelial populations in native human airways and lungsRegionProximal AirwayDistal LungCell TypeAssociated Markers for Cell CharacterizationCiliated CellGoblet CellClub CellBasal CellAlveolar epithelial cell type I AEC IAlveolar epithelial cell type II AEC IIFOXJ1 AcTUBMUC5AC MUC5BCCSP SCGB1A1 SCGB3A2P63 KRT5 NGFR ITGA6 ITGB4HOPX PDPN AQP5SPB SPC HT2280Cell Proportions in Native Lung [] [] [] [] [] []changes in architecture a process known as branching morphogenesis[] From the simple tube of the anterior foregut endoderm to thecomplex tubular structure of the adult a highly stereotyped mechanismof branching morphogenesis facilitates the outgrowth division placement and structure of lung airway [] Branching morphogenesis ofthe lung is driven by three simple and iteratively used processes domain branching planar and orthogonal bifurcation [] The ï¬rst formof branching is domain branching along a primary branch buds formin a linear and sequential fashion from proximal to distal The nextform of branching is planar bifurcation in which the tip of the formingtube bifurcates to create two new tips which subsequently elongateand bifurcate again creating four tips The last process of branching isknown as orthogonal bifurcation In this process the initial planar bifurcation is followed by a rotation around the planar axis which createstwo new tips through bifurcation A critical gene in this processSprouty has been found to attenuate Erk12 signaling thereby alteringthe orientation of cell division and future tube elongation [] Othercritical genes and regulatory networks associated with FGF signalingalso contribute to controlling the periodicity of the branched network[] Although elements such as domain speciï¬cation bifurcation rotation and branch generation remain largely undetermined [] newtechnologies involving highresolution live imaging tension sensingand forcemapping are opening paths to further explore and explainthe branching morphogenesis phenomenon []The early structure of the lung gives rise to a striking architecturalseparation of future SOX2 proximal lineages and SOX9 distal lineages at least in mice [] The diameter of tube generated duringbranching morphogenesis in the pseudoglandular and canalicularstages has a small degree of variance within each stage as measuredfrom electron micrograph sources of fetal human tissue [] This suggests that the branching program is rigorous in its control of lung structure and that tubes themselves may have instructive potential on thedeveloping epithelia Once the basic an structure has formed thelung continues to be exposed to mechanical cues as it continues to mature In several cases these cues have been shown to be essential forcorrect an function In utero the fetal lung is a secretory an thatonly converts to an absorptive one to prepare for breathing afterbirth through a change in the activity of chloride and sodium channelslate in development Fetal lung secretions result in a static ï¬uid pressureof around cmH2O in the developing terminal sacs of the fetus whichpropels branching morphogenesis outwards into the developing thoracic cavity [] Lack of amniotic ï¬uid in the developing lung alters the expression of distal epithelial markers and consequentlyresults in the creation of smaller than normal lungs pulmonary hypoplasia [] highlighting the importance of this mechanical pressureduring lung development In addition cyclic strain is generated fromfetal breathing movements FBM in utero that prime the airway foruse after birth FBM are detectable from the tenth week of pregnancyand begin as infrequent and erratic activity with long quiescent periodsAs development continues these quiescent periods decrease andsustained periods of fetal breathing occur These breathing movementsvary with the fetal sleep cycle and can be chemically tuned [] andalter the volume of terminal sacs by around [] againhighlighting the importance of mechanical signals uencing lung development Finally a novel FGF10FGFR2dependenttensionalmechanism has been shown by which distal epithelial cells in the lungaccumulate motor proteins at the apex of the cell thereby becoming resistant to compression from increasing ï¬uid pressure within the tubelumen Cells under this tension are more likely to become AEC II cellswhile those under compression become AEC I cells [] Interestinglywhile the above examples highlight the importance of speciï¬c mechanical signals in the growth development and differentiation of the lungPSC directed differentiation protocols of the lung are primarily based onmimicking the sequential chemical changes that occur during lungdevelopment Directed differentiation of lung epithelia inspired by embryologyEarly attempts to create lung epithelia from PSCs began in mouseand did not attempt to mimic the stepwise changes in chemical signaling that occur during development Rather groups focused on applyinglunglike physical cues such as airliquid interface [] These protocols while successful in generating NKX21 positive populationsalso produced contaminating cells expressing pluripotency markersOCT4 NANOG SSEA4 TRA160 TRA181 These early attempts solidiï¬ed that further optimization particularly related to the chemical cuesapplied was needed to reliably create lung progenitors from pluripotentsources without remnant pluripotent contaminating cells More successful directed differentiation protocols were rationalized from the detailed understanding of the chemical changes during lung embryologyIn this section we describe in detail the different differentiation protocols currently available that evolved from this approach Mouse embryonic stem cell derived lung epitheliaAlthough mouse models do not fully recapitulate human lung development they have served as guides for earlier iterations of PSC directeddifferentiation protocols and have identiï¬ed critical chemical cues forlung anogenesis Broadly speaking these protocols begin by drivingstem cells towards a deï¬nitive endoderm fate SOX17 and FOXA2mimicking the preembryonic period of human lung developmentweeks through high doses of the nodal activating molecule ActivinA [] Foregut endoderm is then induced via transforminggrowth factor beta TGFβ inhibition either alone [] or with BMP inhibition [] for a short period a process called anteriorization as duringthe embryonic period of human lung development weeks Thisforegut endoderm FOXA2SOX2 is subsequently induced to generate NKX21 cells putative lung progenitors by stimulating theretinoic acid RA BMP WNT and FGF signaling pathways []These lung progenitors are further matured as demonstrated by increased NKX21 expression through application of corticosteroids[] In brief each protocol begins with PSCs guided through deï¬nitiveendoderm followed by anteriorization to foregut endoderm and subsequent ventralization to generate NKX21 cells [] These protocolsformed the basis and backbone for the creation of human lung epitheliafrom PSCsGiven the structural and cellular complexity of the lung it is reasonable that the earliest protocols focused on mouse However there aresubtle differences that highlight how human models are different interms of structure patterning and differentiation For example thePlease cite this as R Varma JP Soleas TK Waddell Current strategies and opportunities to manufacture cells for modelinghuman lungs Adv Drug Deliv Rev 101016jaddr202008005 0cR Varma Advanced Drug Delivery Reviews xxx xxxentire human conducting airway is comprised of a pseudostratiï¬ed epithelium even at diameters less than 05mm [] In contrastconducting airways in mice only exhibit pseudostratiï¬ed epitheliumwith accompanying submucosal glands and cartilage in the most proximal portion of the airway and transition directly into alveolar sacs []This difference in histology affects the residing cell populations as evidenced by the lack of basal cells in the lower portion of the proximal airways of mice [] Similarly mouse models suggest thatSOX2SOX9 progenitors are quite rare and their cell fate is ambiguous [] However evidence from directed differentiation of humanlung epithelia [] which has been conï¬rmed in vivo []reveals that SOX2SOX9 progenitors are common in the developinglung buds and that branch tips of the pseudoglandular staged lunggive rise to both proximal and distal epithelia [] Moreover speciï¬cprotein markers have been found to differ in both timing and locationof expression between human and mouse models proSPC in mouseis expressed early and throughout the developing mouse epithelium[] while in human proSPC is rarely detected early in development and is only robustly found later in distal epithelia [] These examples highlight that while there are similarities development andpatterning of mouse and human lungs is different and these differencesrequire human models to be fully appreciated Human pluripotent stem cellderived lung epitheliaHuman PSC protocols have generally followed the same differentiation chronology as that of mouse directed differentiation wherein deï¬nitive endoderm anterior foregut endoderm and NKX21 lungprogenitors are produced sequentiallyDifferent groups have adhered to their own methods of generatingdeï¬nitive endoderm which primarily involves exposing PSCs to highconcentrations of Activin A Slight variations such as introducing WNTagonism through WNT3a or CHIR99021 prior to [] or alongside[] Activin A or additional exposure to BMP4 and FGF2[] during this stage exist across protocols for differentially inducing primitive streak and its anteriorization towards producing deï¬nitive endoderm In addition the use of embryoid bodies which arelimited by user experience and technique has resulted in a widerange of production efï¬ciencies for achieving this stage from CKITCXCR4EPCAM cells [] to CKIT CXCR4 cells []Recent advances in commercial products have led to development ofstandardized 2D culturebased media STEMdiff Deï¬nitive EndodermKit STEMCELL Technologies which allow reliable derivation of of deï¬nitive endoderm []Similarly generation of both anterior foregut endoderm andtoventralized lung progenitor populations has been subjectmuch investigation and modiï¬cation Earlier work suggested thatSOX2FOXA2 ± in their case anterior foregut endoderm canonly be induced by subjecting deï¬nitive endoderm to TGFβ and BMP inhibition [] Subsequent studies however attempted to anteriorize deï¬nitive endoderm to foregut endoderm through TGFβ inhibition alone SOX2 a combination of endogenous WNT TGFβ and BMP inhibition not quantiï¬ed [] and via Sonic Hedgehog SHH and FGF2signaling FOXA2 EPCAM [] A comparison of the lattertwo strategies demonstrated that SHH and FGF2 are insufï¬cient inproducing reliable NKX21 lung progenitors [] possibly becauseFGF2 is involved in promoting thyroid lineages [] In general TGFβand BMP inhibition [] is the basis for currently applied endodermanteriorization strategies []Factors involved in early versions of ventralization in directed differentiation protocols included WNT3a FGF7 FGF10 BMP4 epidermalgrowth factor EGF and RA have now been reduced based on elimination studies [] As such CHIR99021 CHIR WNT agonist BMP4and RA are necessary and sufï¬cient for producing lung progenitorsfrom anterior foregut endoderm derived from both mouse and humanPSCs [] Despite ï¬nding that FGF7 and FGF10 are nonessential for inducing NKX21 expression they continue to be used forventralization in some protocols [] Although each protocol differsin terms of the duration of each phase NKX21 lung progenitors aregenerally achieved by days with the exception of a study by deCarvalho in which they maintained their cultures for an additional days in FGF7 FGF10 and CHIR99021 to attain NKX21FOXA2 lung progenitors In all cases these lung progenitors are theneither sorted or directly guided towards proximal or distal progeny in2D or 3D culture systems Ideally products of directed differentiationprotocols should mimic the cell proportions present in human airwaysand lungs Table however current protocols have not progressedthat far While these protocols continue to be reï¬ned the percentageof select cell populations generated from these protocols have beensummarized in Table Creation of human proximal lung epitheliaProtocols to create proximal lung epithelia have focused on the production of the four major cells types present ciliated goblet club andbasal cells see Table for a summary of markers for each cell type Motivation for creating proximal epithelia in the ï¬eld has primarily been todevelop patientspeciï¬c cystic ï¬brosis CF models [] andor toproduce epithelia with multiciliated cell populations for protocol validation [] A shift towards human PSCderived CF models hasbeen critical as mouse models do not accurately represent CF diseaseprogression and phenotypes seen in humans [] As such theï¬rst evidence of human PSC proximalization using CF patientderivedPSCs was shown by Mou who exposed anterior foregut endodermto BMP4 GSK3iXV WNT agonist FGF2 and RAsupplemented B27 togenerate NKX21 cells by Day Although contaminatingneuroectodermal and distal lung NKX21SOX9 cells were presentday populations included proximal NKX21SOX2 progenitorsSubcutaneous implantation of this population in immunodeï¬cientmice for days resulted in emergence of NKX21P63 cells howeverno mature epithelial markers for ciliated goblet and club cells werefoundWong employed a longer 2D differentiation approach to produce mature proximal airway epithelia in vitro Through a processthey called proximal speciï¬cation they generated day lung progenitors via low levels of BMP4 mimicking signaling gradients in theairway FGF7 and FGF10 which began expressing proximal genes Further culture with FGF7 FGF10 and FGF18 resulted in upregulated geneexpression of KRT5 P63 FOXJ1 SOX17 cystic ï¬brosis transmembraneconductance regulator CFTR and SCGB1A1 to a lesser extent alongwith low levels of distal SOX9 and SPC by day Protein expressionamounted to NKX21 panKRT P63 FOXJ1 cells These cells were subsequently matured in airliquid interface ALI culture for weeks week of submerged culture withFGF18 followed by weeks of ALI culture to generate CFTRpanKRT FOXJ1 with coexpressing CFTR and CFTRLHS28 cells The resulting epithelium ranged from being squamous to cuboidal with sparse pseudostratiï¬ed regions implying thatthis protocol lacked speciï¬c maturation cues Contaminating thyroidthyroglobulin and PAX9 liver HNF4 and AFP and pancreaticPDX1 lineages were detected through quantitative PCR while percentages of goblet club and basal cell populations barring gene expression analysis were not evaluatedA similar 2D culture approach was employed by Firth to generate proximal lung progenitors which were subsequently matured intomulticiliated epithelia They optimized lower concentrations of BMP4required during the ventralization phase day | 2 |
" oral administration is the most common way to deliver drugs to the systemic circulation or targetans orally administered drugs are absorbed in the intestine and metabolized in the intestine and liver in theearly stages of drug development it is important to predict firstpass metabolism accurately to select candidatedrugs with high bioavailability the caco2 cell line derived from colorectal cancer is widely used as an intestinalmodel to assess drug membrane permeability however because the expression of major drugmetabolizingenzymes such as cytochrome p450 cyp is extremely low in caco2 cells it is difficult to predict intestinalmetabolism which is a significant factor in predicting oral drug bioavailability previously we constructed a mouseartificial chromosome vector carrying the cyp cyp2c9 cyp2c19 cyp2d6 and cyp3a4 and p450 oxidoreductasepor 4cypsmac genes and increased cyp expression and metabolic activity in hepg2 cells via transfer of thisvectorresults in the current study to improve the caco2 cell assay model by taking metabolism into account weattempted to increase cyp expression by transferring the 4cypsmac into caco2 cells the caco2 cells carryingthe 4cypsmac showed higher cyp mrna expression and activity in addition high metabolic activity availabilityfor permeation test and the potential to assess drugdrug interactions were confirmeds the established caco2 cells with the 4cypsmac are expected to enable more accurate prediction ofthe absorption and metabolism in the human intestine than parental caco2 cells the mammalian artificialchromosome vector system would provide useful models for drug developmentkeywords mammalian artificial chromosome chromosome transfer cytochrome p450 intestinal metabolismcaco2 cell correspondence kazukitottoriuacjp1division of genome and cellular functions department of molecular andcellular biology school of life science faculty of medicine tottoriuniversity nishicho yonago tottori japan2chromosome engineering research center cerc tottori university nishicho yonago tottori japanfull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cohta bmc biotechnology page of bioavailability is an important area of concern in drugdevelopment poor oral bioavailability has led to drugwithdrawal oral drug bioavailability is often limited bymetabolizing enzymes and efflux transporters in the gut the caco2 cell line derived from human colon carcinoma is a commonly used model for estimating the intestinal absorption of new drug candidates althoughcaco2 cells express a variety of efflux and uptake transporters they have an absence or low levels of cytochrome p450 cyp isoforms such as cyp3a4 andcyp2c that are typically expressed in the human intestinal epithelium therefore caco2 cells are of limited use in evaluating the role of metabolism inintestinal absorption after oral administration to predict the intestinal absorption of drugs more accurately itis necessary to modify caco2 cells to increase their expression of cyp isoformssome studies reported that cyp3amediated metabolism in caco2 cells was enhanced by transfection withboth cyp3a4 and cyp oxidoreductase por [ ] treatment with 1α25dihydroxyvitamin d3 [ ] or the combination of transfection with cyp3a4 and treatment withboth sodium butyrate and 12otetradecanoylphorbol13acetate in contrast few studies aimed at enhancingmultiple cyp isoforms in caco2 cells have been performed honkakoski and his collaborators created caco2cell lines expressing nuclear receptors pregnane x receptor and constitutive androstane receptor [] thesenuclear receptors upregulated the expression of somecyp isoforms in caco2 cells but cyp activities remainedvery low in the absence of 1α25dihydroxyvitamin d3therefore a new approach is needed to introduce multiple cyp isoforms in caco2 cellsmammalian artificial chromosome ac vectors derived from native chromosomes have several advantagesover conventional vectors acs segregate freelyfrom host chromosomes through a set of cell divisionsand are adapted to carry multiple target genes with a desired copy number and mbsized genomic regions withendogenous regulatory elements furthermore acs carrying genes of interest can be transferred into varioustarget celllines via microcellmediated chromosometransfer mmct considering these advantages acshave been used to generate several model cells for pharmacokinetic and toxicokinetic studies previously a lack of cyp3a4 expression in the caco2cell line was addressed through the introduction of exogenous cyp3a4 and por which is a coenzyme ofcyps via a human artificial chromosome hac vectorderived from human chromosome [ ] the haccarrying cyp3a4 and por genes conferred sufficientcyp3a activity to parental caco2 cells to be useful forpredicting the intestinal extraction ratio in humansrecently a mouse artificial chromosome mac vectorconstructed from native mouse chromosome wasused to increase the activity of multiple cyps in hepg2cells which are a liver cancer cell line typically exhibiting low cyp activity in this study four cyp genescyp3a4 cyp2c9 cyp2c19 cyp2d6 and a pene were loaded on the mac 4cypsmac and transferred to hepg2 cells tchepg2 to make the cellsmore suitable as a model to evaluate drugdrug interactions ddis and hepatotoxicity in the initial screeningof candidate drugs the expression and activity ofcyps in tchepg2 were comparable to those in humanhepatocytes and this expression was sustained after along culture period because of the stability of the macin human cells regarding the assessment of ddisthe activity of cyps in tchepg2 was reduced in a concentration and timedependent manner by specific inhibitors which reflects the conditions in primary humanhepatocytes furthermore metabolic toxicity of aflatoxinb1 which is converted to its active metabolite viacyp3a4 and exerts hepatotoxicity through dna damage was clearly recapitulated in tchepg2 cells rather than parental hepg2 cells this study suggestedthat tchepg2 can provide a useful model to assess notonly hepatic metabolism but also cypmediated hepatotoxicity during the early stages of drug development andthe system using the mac can improve the existingcellbased modelin the current study we aimed to utilize previouslyconstructed 4cypsmac to generate a novel caco2 cellline with increased activity of multiple major cyps the4cypsmac was transferred to caco2 cells via mmctto establish caco2 cells carrying the 4cypsmac andthe caco2 4cypsmac cells were examined to determine whether they exhibited sufficient cyp activity foruse in initial drug screeningresultsmmct and analyses of acquired clonescho cells carrying a mac vector with cyp2c9cyp2c19 cyp2d6 cyp3a4 por and gfp genes wereprepared 4cypsmac fig 1a using cho cells asdonor cells and caco2 cells as recipient cells weattempted to generate caco2 cells carrying the 4cypsmac via mmct fig 1a after selection four drugresistant gfppositive clones were obtained caco24cypsmac fig 1b to examine whether the cypand por genes were introduced into the obtainedclones genomic pcr analyses were performed chocells with the 4cypsmac and caco2 cells were usedas positive and negative controls respectively consequently a band of the desired size was observed for eachprimer set in the candidate clones fig 1c next achromosome specimen was prepared from the acquired 0cohta bmc biotechnology page of fig introduction of the 4cypsmac into caco2 cells a transfer of the 4cypsmac into caco2 cells the structure of the mac carrying fourcyps and por is shown at the top a cag promoter was placed upstream of each gene a schematic view of the transfer of the 4cypsmac tocaco2 cells is shown at the bottom the 4cypsmac was transferred from cho cells to caco2 cells through the mmct method b an image ofgfp fluorescence in parental caco2 cells and caco2 4cypsmac cells the gfp fluorescence indicates the presence of the 4cypsmac in thecaco2 cells the white bars indicate a distance of μm c results of genomic pcr analyses to detect four cyp and por transgenes on the macin caco2 cells donor cho cells and caco2 cells are positive and negative controls respectively d images of fish analyses of caco2 cellscarrying the 4cypsmac red and green signals indicate the mac and transgenes respectively the arrow shows the 4cypsmac and the insetshows an enlarged image of the 4cypsmacclones and fish analysis was performed to check thekaryotype fish analysis revealed that a single copy of the4cypsmac existed in the candidate clones fig 1drtqpcr analysis was performed to examine themrna expression level of the introduced cyps and porin the obtained clones compared with that in parentalcaco2 cells the gene expression level was particularlyhigh in the caco2 4cypsmac and caco2 4cypsmac clones fig among the introduced genespor expression was only slightly enhanced in theseclones basal expression of por in parental caco2 cells ishigh as observed in a previous study caco2 4cypsmac showed high expression of the majority of genescompared with caco2 4cypsmac the caco2 cellline consists of a heterogeneous population of cells therefore difference of the gene expression levels between obtained clones may partly depend on the population into which the 4cypsmac has been introducedalthough the other two clones obtained by mmct stillshowed higher expression levels than the parental caco2cells the level of increase was moderate therefore we selected caco2 4cypsmac and caco2 4cypsmac clones for further analyses to evaluate the availability asan improved model system these results suggest that wesuccessfully transferred the 4cypsmac to caco2 cellsand the genes on the mac were highly expressedmonolayer formation of caco2 4cypsmac cellswe seeded caco2 4cypsmac and caco2 4cypsmac cells at a concentration of à cellswell 0cohta bmc biotechnology page of fig gene expression analyses of caco2 cells with the 4cypsmac the expression levels of the four cyps and por in caco2 cells with the4cypsmac the relative expression levels of the four cyps and por genes of the parental caco2 cells and acquired clones were analyzedthrough rtqpcr gapdh was used for normalization mean ± se n in a millicell 24well cell culture insert plate the caco 4cypsmac cells spread across the entire membrane and formed a cell layer while the caco2 4cypsmac cells did not spread and there were gaps in thecell layer fig 3a caco2 4cypsmac appeared toaggregate and form multiple layers rather than spreadand form a single layer it was reported that multilayeredareas appeared in the cell population for late passagecells the teer value was measured using amillicellers fig 3b the teer value is an index oftight junction formation and when the value is almostconstant it is considered that a cell layer has formedwith the exception of the caco2 4cypsmac clonethe caco2 cells and caco2 4cypsmac cellsshowed an increase in teer value untilit plateauedafter d the caco2 cells and caco2 4cypsmac showed almost equivalent teer values because monolayer formation is essential for the permeation test thesubsequenttests were performed using the caco24cypmac cellsculture timedependent change in gene expressiontotal rna was extracted from caco2 cells andcaco2 4cypsmac cells on the 4th 11th and22nd days after seeding we compared the expressionlevel of each gene on each day and confirmed thatthe expression levels of the four cyps and por increased in both the caco2 cells and caco2 4cypsmac cells fig 3c the expression levels of allgenes analyzed were significantly higher in the caco24cypsmac cells on the 22nd day than those inparental caco2 cells the gene expression levelincreased depending on the culture time and the geneexpression levels of the caco2 4cypsmac cellsestablished in the current study were higher thanthose of parental caco2 cells with the exceptions ofcyp3a4 and cyp2d6 the expression levels in parental caco2 cells were higher in all cases until day the parental caco2 cell line appears to have higherpotential to enhance the expression of cyp2c9 andcyp2c19 during differentiation 0cohta bmc biotechnology page of fig monolayer formation assay a images of bright and gfp fluorescence from cells seeded on the membrane of a millicell24 plate in caco24cypsmac cells did not spread throughout the membrane and did not form a cell layer but in caco2 4cypsmac there were no gapsbetween cells and they formed a cell layer the white bars indicate a distance of μm b transepithelial electrical resistance teer values ofcaco2 cells caco2 4cypsmac and caco2 4cypsmac cells c culture timedependent change in gene expression the relative expressionlevel was evaluated in caco2 and caco2 4cypsmac mean ± se n the expression levels in caco2 and caco2 4cypsmac at day were compared with those in humanadult intestine additional file figure s1 in caco24cypsmac the expression levels of cyp2c9 andcyp2c19 were comparable and that of cyp2d6 washigher than in human adult intestine although cyp3a4expression was significantly enhanced in caco2 4cypsmac compared with that in parental caco2 cellsthe expression was stilllower than in human adultintestinecyp metabolic activity measurementa p450glo assay with each specific substrate wasemployed to measure the metabolic activity of cyps inthe caco2 4cypsmac cells which had high geneexpression levels as confirmed through rtqpcr analysis the activities of all four cyps were higher in thecaco2 4cypsmac clone than in caco2 cellsfig 4a the resultsthe introducedindicate that4cypsmac expressed functional cyps and increasedthe total activity of each cyp in the caco2 cells theenhancements in the rates of metabolic activity ofcyp2c9 cyp2c19 and cyp2d6 were generally correlated with those of mrna expression however therewas a gap between the enhancement of the rate ofcyp3a4 mrna expression and that of metabolic activity this may have been because the parental caco2 originally had extremely low expression of cyp3a4mdz permeability testa permeation test was conducted using midazolammdz a cyp3a substrate to examine whether the cellsreflected the behavior of small intestinal epithelial cellsin terms of mdz permeation the penetration test wasperformed on day after cell seeding when the teervalue plateaued we measured the amount of ²ohmdz in each of the donor side apical recipient sidebasal and intracellularly the amounts of ²oh mdz 0cohta bmc biotechnology page of fig activity of each cyp in the caco2 4cypsmac cells a the metabolic activity of each cyp in caco2 4cypsmac cells the relative activityfor each cyp was measured by comparing the parental caco2 cells and the caco2 4cypsmac mean ± se n b permeability test usingmdz the permeability test was performed d after seeding caco2 cells and caco2 4cypsmac whereby μm mdz was added to theapical side and after min the apical intracellular and basal supernatants were collected the ²oh mdz in the supernatant was measuredthrough lcmsms c cyp3a4 inhibition test ketoconazole an inhibitor of cyp3a4 was added to the caco2 4cypsmac and incubated for h a luminescent substrate was measured to detect cyp3a4 activity with different concentrations of ketoconazolein alllayers of the caco2 4cypsmac cells werehigher than in those of caco2 cells fig 4b moreoverer was calculated using eq and the results were and for caco2 and caco2 4cypsmac respectively er indicates the rate of metabolism during cellpermeation cyp3a4 was scarcely expressed in parentalcaco2 cells so the er value was extremely low howevercaco2 4cypsmac cells showed an er of which was higher than in the caco2 cells and mdz wasmetabolized by cyp3a4 when passing through the cellsinhibition testto determine the availability of the established clone forthe assessment of ddis we added ketoconazole an inhibitor of cyp3a4 to the caco2 4cypsmac cells andexamined whether the metabolic activity was reduced ketoconazole at and μm was addedand cells were incubated at °c for h followed by themeasurement of metabolic activity the metabolic activityof cyp3a4 decreased as the inhibitor concentration increased fig 4c the activity of cyp3a4 in caco2 4cypsmac appeared to be sufficient for the inhibition test compared with that in parental caco2 cells in addition to thepermeation test for cyp3a4 inhibition of cyp3a4s function by ketoconazole in caco2 4cypsmac cells was alsoconfirmed this suggests that the established cells could beused for ddi testing of drugs that are substrates ofcyp3a4 therefore the caco2 4cypsmac cells moreaccurately reflect the behavior of cyp3a4 substrates in human epithelial cells than parental caco2 cellsdiscussionin the current study we introduced four cyps and porinto caco2 cells to increase their drug metabolic 0cohta bmc biotechnology page of abilities which are typically low this study was intendedto establish a better human cell model to more preciselyevaluate the behavior of drugs in the small intestine the4cypsmac was successfully introduced into the caco2cells and successfully increased cyp activityin contrastin this studythe gene expression and activity of cyps in tchepg2 carrying the 4cypsmac are either comparableto or higher than those in primary human hepatocytes to the other cypscyp3a4 mrna expression was still low in caco2 carrying the 4cypsmac compared with the level in human adult intestine despite the significant enhancementof mrna expression regarding the genes on the4cypsmac each is present as a single copy becausethe nature of gene regulation is supposed to differ between hepg2 and caco2 changing copy number of thecyp3a4 gene may further optimize the expression profile of caco2 cells to that of human intestinethe established caco2 4cypsmac cells with particularly high gene expression showed high activity in all cypsin the future we will conduct metabolic tests inhibitiontests and permeation tests using drugs that are substratesfor other cyps and investigate whether the caco2 4cypsmac cells reflect the behavior of drugs in small intestinal epithelial cells the cyp expression level in the humansmall intestine is reported to be approximately forcyp3a4 approximately for cyp2c9 approximately for cyp2c19 and approximately for cyp2d6 it will be necessary to evaluate whether the proportion ofcyp expression in the caco2 4cypsmac is close tothat of the human small intestineif cyp metabolic capacity is guaranteed in the established clones the established cell line can be used asnew human small intestine model cells in recent yearssmall intestine model cells prepared from induced pluripotent stem cells have been reported but such cells areconsidered difficult to use for screening large quantitiesof drug candidate compounds however caco2cells are easy to handle therefore it is possible to usecypmodified caco2 cells to test large numbers of candidate compounds as a first screeningwako osaka japan supplemented with fetal bovine serum fbs and μgml g418 parental caco2cells atcc® htb37¢ atcc manassas va usawere maintained in dulbeccos modified eagles mediumdmem wako supplemented with fbs memnonessential amino acids gibco thermo fisher scientific waltham ma usa m hepes gibco mmsodium pyruvate gibco mm glutamax gibcoand penicillinstreptomycin wako caco2 cells withthe 4cypsmac were maintained in the above mediumsupplemented with μgml g418 these cells werecultured at °c in co2microcellmediated chromosome transfertransfer of 4cypsmac from cho cells to caco2 cellswas performed using a standard procedure brieflydonor cho cells were cultured in f12 medium supplemented with fbs and μgml colcemid after h microcells were isolated through centrifugation withdmem containing cytochalasin b and filtration thenmicrocells suspended in phytohemagglutinin p phapdmem were poured onto caco2 cells in a 6cm dishand incubated for min the cells were treated withpolyethylene glycol peg solution g of peg1000 ml of dmem ml of dimethyl sulfoxide for minfollowed by washing with dmem after h of recoveryculture cells were seeded in a 24well collagencoatedplate corning ny usa and maintained with selectionmedium h after seeding thereafter the medium waschanged twice a week to obtain drugresistant clonesbecause the mac carries a gfp gene gfppositiveclones were selected from the drugresistant clonesgenomic pcr analyseswe extracted genomic dna from cell lines using a genomic dna extraction kit with dnasefree rnase gentra systems minneapolis mn usa the primers forthe genomic pcr are listed in additional file tables1 they amplified each gene region on the 4cypsmac we used kod fx takara otsu japan in accordance with the manufacturers instructionsthe mammalian artificial chromosome vector systemwould provide useful models for drug development theestablished caco2 cells with the 4cypsmac are expected to more accurately predict absorption and metabolism in the human intestine than parental caco2 cellsmethodscell culturechinese hamster ovary cho cells jcrb0218 jcrbcell bank nibiohn osaka japan carrying the 4cypsmac were maintained in hams f12 nutrient mixturefish analysestrypsinized cells were incubated for min in m kcland fixed with methanol and acetic acid and thenslides were prepared using standard methods fish analyses were performed using the fixed metaphase of each cellhybrid using digoxigeninlabeled roche germany dna[mouse cot1 dna invitrogen carlsbad ca usa] andbiotinlabeled dna [pac 4cypspor] essentially as described previously chromosomal dna was counterstained using dapi sigmaaldrich st louis mo usaimages were captured using an axioimagerz2 fluorescencemicroscope carl zeiss germany 0cohta bmc biotechnology page of rtqpcrwe extracted mrna using the rneasy mini kit qiagen germany and synthesized firststrand cdna usingthe high capacity cdna reverse transcription kit applied biosystems foster city ca usa the primersare listed in additional file table s1 for rtqpcranalysis tb green premix ex taq takara was usedand relative mrna expression was evaluated throughthe δδct method gapdh was used for normalizationculture timedependent expression level change of fourcypscaco2 cells and caco2 4cypsmac were seededin a 6cm dish at a concentration of à cellswell cells were lysed using trizol invitrogen causa on days and after seeding and rnawas extracted and purified using an rneasy mini kitqiagen thereafter cdna synthesis was performedusing the highcapacity cdna reverse transcriptionkit applied biosystemsactivity test of the four cypswe tested the metabolic activity ofthe four cypsusing the p450glo¢ assay promega madison wiusa the luminogenic substrates used for the testwere luciferinipa cyp3a4 luciferinme egecyp2d6 luciferinh cyp2c9 and luciferinhege cyp2c19 cells wereseeded in 48wellcollagencoated plates corning at a density of à cellswell after h the medium was changedand h later we added transport medium tm containing substrate tm was prepared using hanks balanced salt solution hbss with mm nahco3 mm glucose and mm hepes which was adjusted to ph after incubation we added detectionreagent and measured the luminescence using an infiniteandcyp2c19 required h of incubation while cyp2d6and cyp3a4 required h after the measurementthe cells were washed with pbs dissolved in lysisbuffer and diluted fivefold the same amount of celltiter glo promega was added to μl of the lysateand luminescence measurement was performed tonormalize data to the number of viable cellswako cyp2c9f500 platereadermidazolam mdz permeability testthe obtained clones were assessed in an mdz permeation test each cell was seeded on a 24well cell cultureinsert plate millipore billerica ma usa at a concentration of à cellswell the medium was changedonce a week after seeding and every d after the secondweek transepithelial electrical resistance teer wasmeasured using millicellers millipore before mediumexchange the test was performed d after seedingfor the test tm ph prepared by adding mmnahco3 mm glucose and mm hepes tohbss at ph was used the donor side solutionwas prepared by dissolving μm mdz in tm ph the acceptor side solution was prepared by adding fbs to tm ph on the test day themedium was removed from the culture insert seededwith the cells and the cells were rinsed twice withtm ph tm ph and tm ph wereadded to the apical and basal chambers respectivelyand cells were incubated at °c for min the testwas started by adding the donor side solution to thedonor side chamber and the acceptor side solution tothe acceptor side chamber thirty minutes after thestart of the test the solution in the donor side andacceptor side chambers was collected moreover tomeasure the amount of mdz and ²hydroxy mdz²oh mdz in the cells after the test the cultureinsert was quickly rinsed three times with icecoldtm ph the membrane was cut using a cutterand μl of icecold tm ph was added cellswere detached from the membrane through sonicationand a cell suspension was used as a sample thesesamples were deproteinized and stored at °cuntil measurementlcmsms was used for the measurement of mdzand ²oh mdz in the samples qtrap5500 sciexframingham ma usa and a prominence uflc system shimadzu kyoto japan were combined for measurement the hplc conditions and msms conditionsare shown in additional file table s2 quantitativeanalysis was performed in multiple reaction monitoringmode mass transitions mz were formdz for ²oh mdz and for ²oh mdz d4 data were analyzed usinganalyst software sciexequation formula to calculate extraction ratio erer ¼metabolite donorþreceiverþintracellularpþparent receiverþintracellularððþ þ pmetabolite donorþreceiverþintracellularðþtokyo chemicalinhibition testketoconazoleindustry tokyojapan was used as an inhibitor against cyp3a4 andchanges in metabolic activity were measured using ap450glo assay with luciferinipa cells were seededin a 48well collagencoated plate at à cellswell and the medium was changed after d thenext daythe medium was collected cells werewashed twice with pbs and then μl of tm ph containing ketoconazole tokyo chemical industry at and μm was added toeach set of three wells tm was adjusted to ph byadding mm nahco3 mm glucose and mm 0cohta bmc biotechnology page of received april accepted august referencesbenet l wu c hebert m wacher v intestinal drug metabolism andantitransport processes a potential paradigm shift in oral drug delivery jcontrol release xie f ding x zhang qy an update on the role of intestinal cytochromep450 enzymes in drug disposition acta pharm sin b takenaka t kazuki k harada n kuze j chiba m iwao t matsunaga t abes oshimura m kazuki y development of caco2 cells coexpressingcyp3a4 and nadphcytochrome p450 reductase using a human artificialchromosome for the prediction of intestinal extraction ratio of cyp3a4substrates drug metab pharmacokinet hu m li y davitt cm huang sm thummel k penman bw crespi cltransport and metabolic characterization of caco2 cells expressing cyp3a4and cyp3a4 plus oxidoreductase pharm res schmiedlinren p thummel ke fisher jm paine mf lown ks watkins pbexpression of enzymatically active cyp3a4 by caco2 cells grown onextracellular matrixcoated permeable supports in the presence of1alpha25dihydroxyvitamin d3 mol pharmacol fan j liu s du y morrison j shipman r pang ks upregulation oftransporters and enzymes by the vitamin d receptor ligands 1alpha25dihydroxyvitamin d3 and vitamin d analogs in the caco2 cell monolayer jpharmacol exp ther cummins cl mangravite lm benet lz characterizing the expression ofcyp3a4 and efflux transporters pgp mrp1 and mrp2 in cyp3a4transfected caco2 cells after induction with sodium butyrate and thephorbol ester 12otetradecanoylphorbol13acetate pharm res korjamo t honkakoski p toppinen mr niva s reinisalo m palmgren jjmonkkonen j absorption properties and pglycoprotein activity of modifiedcaco2 cell lines eur j pharm sci korjamo t monkkonen j uusitalo j turpeinen m pelkonen o honkakoskip metabolic and efflux properties of caco2 cells stably transfected withnuclear receptors pharm res kublbeck j hakkarainen jj petsalo a vellonen ks tolonen a reponen pforsberg mm honkakoski p genetically modified caco2 cells withimproved cytochrome p450 metabolic capacity j pharm sci mes to hbss the cells were preincubated for h at °c and 1000fold diluted cyp3a4 substrate wasadded one hour later μl of the supernatant wascollected from the well mixed with μl of detectionreagent and the luminescence value was measuredusing an infinite f500 plate reader wakosupplementary informationsupplementary information accompanies this paper at httpsdoi101186s12896020006378additional file figure s1 comparison of gene expression betweenhuman small intestine and day culture of caco2 and caco2 4cypsmac table s1 primer sequences for genomic pcr and rtqpcrtable s2 lcmsms analysis conditions mdz ²oh mdzabbreviationscyp cytochrome p450 ac artificial chromosome mmct microcellmediated chromosome transfer por p450 oxidoreductase hac humanartificial chromosome mac mouse artificial chromosome ddi drugdruginteraction cho chinese hamster ovary mdz midazolamteer transepithelial electrical resistance er extraction ratioacknowledgmentswe thank satoru iwado at tottori university for technical assistance with theexperiments we also thank dr hiroyuki kugoh dr masaharu hiratsuka drhiroyuki satofuka and dr takahito ohira at tottori university for criticaldiscussions this research was partly performed at the tottori bio frontiermanaged by tottori prefecture we thank edanz group wwwedanzeditingcomac for editing a draft of this manuscriptauthors contributionsall authors conceived and designed the experiments yo and kka performedthe experiments yo sa kko and yk wrote the paper mo and yksupervised the study all authors read and approved the final manuscriptfundingthis work was supported in part by the basis for supporting innovative drugdiscovery and life science research binds from the japan agency formedical research and development amed under grant numberjp18am0301009 ykavailability of data and materialsthe data and materials used andor analyzed during the current study areavailable from the corresponding author on reasonable requestethics approval and consent to participatenot applicableconsent for publicationnot applicablecompeting interestsdr mitsuo oshimura is ceo and a shareholder of trans chromosomics incdr satoshi abe is a member of trans chromosomics inc and the otherauthors declare no conflict of interestauthor details1division of genome and cellular functions department of molecular andcellular biology school of life science faculty of medicine tottoriuniversity nishicho yonago tottori japan 2chromosomeengineering research center cerc tottori university nishicho yonagotottori japan 3trans chromosomics inc nishicho yonagotottori japan 4laboratory of biopharmaceutics meijipharmaceutical university noshio kiyose tokyo japan oshimura m uno n kazuki y katoh m inoue t a pathway fromchromosome transfer to engineering resulting in human and mouseartificial chromosomes for a variety of applications to biomedicalchallenges chromosom res satoh d abe s kobayashi k nakajima y oshimura m kazuki y human andmouse artificial chromosome technologies for studies of pharmacokineticsand toxicokinetics drug metab pharmacokinet kazuki y hoshiya h takiguchi m abe s iida y osaki m katoh m hiratsukam shirayoshi y hiramatsu k ueno e kajitani n yoshino t kazuki k ishiharac takehara s tsuji s ejima f toyoda a saka | 0 |
American Joint Committee on Cancer AJCC Cancer Staging Manual 8th edition we explored the characteristics of central lymph node metastasis CLNM of papillary thyroid microcarcinoma PTMC in elderly patients ¥ years of age Our goal was to provide references for establishing a lymph node dissection scheme in such patientsMethods We retrospectively analyzed the clinical data of thyroid cancer patients admitted to the Head and Neck Surgery Center of Sichuan Cancer Hospital Chengdu China from January to September Then we screened and analyzed eligible PTMC cases in strict accordance with our inclusion and exclusion criteriaResults The study included patients including men and women Median age was ± years The maximum diameter range of the cancer foci was mm and the median was ± mm Unilateral lobectomy had been performed in cases total thyroidectomy in cases and lateral cervical lymph node dissection in cases There were cases of CLNM and cases of lateral cervical lymph node metastasis The sensitivity of preoperative ultrasound in predicting CLNM was but its accuracy was only Multivariate logistic regression analysis showed that multiple cancer foci area under the curve [AUC] extrathyroidal expansion of cancer focus AUC and irregular nodules AUC were independent risk factors for CLNM of PTMC in elderly patients P Overall predictability for PTMCCLNM was Conclusion Preoperative color Doppler ultrasound is not recommended as the basis for cervical lymph node dissection in PTMC patients For multiple cancer foci irregular nodules and elderly patients with PTMC extrathyroidal expansion we recommend a prophylactic central lymph node dissecting Nonsurgical observation of PTMC in elderly patients with low risk should be carefully selectedKeywords elderly patients thyroid cancer papillary carcinoma microcarcinoma central lymph node metastasisIntroductionIn the World Health anization defined thyroid cancer TC with a maximum tumor diameter of mm as microcarcinoma Up to now doctors in different countries and regions of the world still differ significantly on how to treat such diseases including on whether to perform preventive lymph node dissection in the central region of the cancer As the AJCC raised the age factor in the clinical staging of TC from to in it can be seen that the medical community Cancer Management and Research Fu This work is published and licensed by Dove Medical Press Limited The full terms of this license are available at wwwdovepresscomtermsphp and incorporate the Creative Commons Attribution Non Commercial unported v30 License httpcreativecommonslicensesbync30 By accessing the work you hereby accept the Terms Noncommercial uses of the work are permitted without any further permission from Dove Medical Press Limited provided the work is properly attributed For permission for commercial use of this work please see paragraphs and of our Terms wwwdovepresscomtermsphp 0cFu Dovepresstends to be more conservative in general on surgical treatment of TC Based on clinical staging of TC in the AJCC Cancer Staging Manual 8th edition this study aimed to explore the characteristics of CLNM of PTMC in the elderly population ¥ years so as to provide some references for developing clinical treatment plans for such patients mm underwent concurrent total thyroidectomy and bilateral central lymph node dissection Centralregion lymph nodes were dissected in the following areas upper boundarylower hyoid margin lower boundarysuperior sternal fossa upper margin of the unknown artery and externallateral margin of the carotid sheath and lower boundaryanterior vertebral fasciaMethodsPatientsWe retrospectively analyzed the data of patients with PTMC admitted to the Head and Neck Surgery Center of Sichuan Cancer Hospital Chengdu China from January to September Eligible patients were screened in strict accordance with our inclusion and exclusion criteria for relevant data statistics and analysis Inclusion criteria were as follows Patient age was ¥ years All of the patients had been newly diagnosed and newly treated with no previous history of thyroid surgery Postoperative pathological diagnosis based on paraffinembedded sections was papillary carcinoma Benign nodules with or without pathology and mm in maximum diameter suggested by preoperative color ultrasound Patients complete medical records were available All of the surgeons had years experience in thyroid surgery Patients had undergone surgical resection of glandular lobes and isthmus on the affected side as well as lymph node dissection in the central region with or without lateral cervical lymph node dissection Exclusion criteria were as follows Postoperative pathology indicated mixed carcinoma with papillary and other types of carcinoma Patient had refused lymph node dissection in the central area on the affected side There were multiple cancer lesions with the sum of the maximum diameter mmSurgical TechniqueAll of the patients had been operated on according to the at least principle namely lymph node dissection at least in the central area on the cancerous side Total thyroidectomy bilateral centralarea lymph node dissection with or without lateral cervical lymph node dissection were performed on patients with preoperative cytological confirmation of bilateral multilobed carcinoma or lateral cervical lymph node metastasis Those with papillary microcarcinoma in one glandular lobe and benign nodules in the other glandular lobe ie multiple nodules with maximum diameter of Statistical AnalysisWe used SPSS software version SSPS Inc Chicago Illinois US to statistically analyze all of the data In our analysis of risk factors for lymph node metastasis in the central region we performed singlefactor analysis using Ï2 test We ran multivariate logisticregression analysis on statistically significant positive univariate influencing factors as well as univariate and multivariate receiver operating curve ROC analysis on the previously analyzed risk factors to predict lymph node metastasis in the central regionResultsWe screened a total of PTMC cases Of these met the inclusion criteria including men and women with a maletofemale ratio of Patients age range was years old with a median of ± years The maximum diameter range of the cancer lesion was mm median ± mm There were cases with singleleaf singlefocus with singleleaf multifocus and with multileaf multifocus Unilateral lobectomy was performed in cases and total thyroidectomy in cases and lateral cervical lymph node dissection in cases In terms of staging of cases were in stage T1 in stage T3 and in stage T4 Postoperative pathology showed CLNM in cases and lateral cervical lymph node metastasis in cases Evaluation of Central Lymph Nodes by Color Doppler UltrasoundPreoperative ultrasound examination indicated stage cN1a cases and stage cN0 cases Postoperative pathology confirmed stage pN1a cases and stage pN0 cases which were significantly different Predictive sensitivity specificity positive predictive value PPV and negative predictive value NPV were and respectively Table submit your manuscript wwwdovepresscom DovePress Cancer Management and Research 0cDovepress Fu et alTable Comparison of Preoperative Ultrasound Predictions of CentralRegion Lymph Node MetastasisNTotalUltrasonic StagingcN1acN0Pathological stagepN1apN0TotalSensitivity Specificity PPV NPV Matching rate Abbreviations PPV positive predictive value NPV negative predictive valueUnivariate AnalysisIn this study singlefactor analysis of patients with PTMC age ¥ years showed that distribution nodule morphology calcification and extrathyroidal expansion of cancer focus significantly influenced centralregion lymph node metastasis P However patients gender thyroid stimulating hormone TSH levels thyroglobulin Tg levels nodular goiter Hashimotos thyroiditis maximum diameter of cancer focus nodular boundary and nodular blood flow had no statistical significance on such metastasis Table Multivariate LogisticRegression AnalysisFactors that had been statistically significant in univariate analysis results were further included in multivariate logisticregression analysis variables that might be clinically relevant but had been negative in univariate analysis were also included We found that distribution morphology and extrathyroidal expansion of cancer focus were independent risk factors for CLNM P while gender TSH Tg nodular goiters Hashimotos thyroiditis nodular boundary blood flow calcification and maximum diameter had no predictive significance Table ROC Curve AnalysisWe performed ROC curve analysis according to the independent risk factors obtained in our multivariate logistic regression analysis of CLNM as discussed above and we calculated areas under the curve AUCs Figures and DiscussionDisease Status of TCThe incidence of TC has been on the rise globally over the past years which has been confirmed by most current to International Agency studies1 According for Research on Cancer IARC data for a total of new cases malefemale and deaths malefemale were reported in countries around the world respectively accounting for and of all new cancer cases and deaths4 On the one hand due to the great influence of medical ultrasound and cytologicalpuncture diagnosis the proportion of PTMC in new cases of TC has increased significantly According to the data the overall incidence of PTMC in the United States has increased by over the past years with average annual new cases accounting for about On the other hand PTMC incidence in the elderly is significantly higher than that in the general adult population Some studies have shown that the average annual growth rate of PTMC in patients years old is times that in adults years old8 These results indicate that we should pay sufficient attention to elderly PTMC patients As the 8th edition of the AJCC Cancer Staging Manual raise PTC staging age from to years old it further confirms this viewTherapeutic ControversiesAs we all know diagnosis and treatment of PTMC have always been controversial especially in elderly patients In Japans Kuma hospital Ito defined the maximum diameter of thyroid cancer foci ¤10cm no cervical and distant lymph node metastasis and cytological biopsy of thyroid cancer foci as nonhighly malignant and no invasion of the trachea and recurrent laryngeal nerve as the judgment criteria for lowrisk PTMC After analyzing the data of patients they concluded that immediate surgical treatment of all PTMC patients was more harmful than beneficial so they suggested that lowrisk PTMC patients should choose active observation Among them elderly PTMC patients over years old were considered to be the most suitable group for observation1112 Conversely Megwalu UC of the National Cancer Center Plainview New York US reviewed cases in which senile PTMC patients age ¥ received nonoperative therapy His data analysis shows that the overall 5year survival rate was and the surgery was P which suggests that surgery for such patients has a survival advantage although more highquality investigative studies are necessary1 American Thyroid Association Management Guidelines for Adult Patients with Thyroid Nodules and Differentiated Thyroid Cancer suggested that suspicious malignant thyroid nodules with a maximum diameter of cm be followed up to cm for cytological Cancer Management and Research submit your manuscript wwwdovepresscom DovePress 0cFu DovepressTable SingleFactor Analysis of CentralRegion Lymph Node MetastasisFactorsGenderMaleFemaleTSH levelsNormalAbnormalTg levelsNormalAbnormalNodular goiterYesNoHashimotos thyroiditisYesNoDistribution of carcinomaUnilateral glandular lobes single fociUnilateral glandular lobes multiple fociBilateral glandular lobes multiple fociMaximum diameter¤ mm mm x ¤ mmBoundaryClearUnclearEchoLow or noStrong or mixedExtrathyroidal expansionYesNoCalcificationYesNoBlood flowRichNot richNumber n107Central Lymph Node Metastasis CLNMÏ2PYes n No n Abbreviations TSH thyroidstimulating hormone Tg thyroglobulinpuncture and other treatments but immediate surgical treatment is still recommended for highrisk patients In this study we performed surgical treatment on all PTMC patients with clear diagnoses including stage T1 T3 and T4 Although the study sample size needs to be further expanded we still believe that microcarcinoma is not equal to early cancer The percentage of differentiated tumor cells in elderly PTC patients is relatively higher than in youth and children leading to shorter life expectancy Choosing followup for middleaged and elderly submit your manuscript wwwdovepresscom DovePress Cancer Management and Research 0cDovepress Fu et alTable Multivariate LogisticRegression Analysis of Lymph Node Metastasis in the Central RegionFactorsGenderDistribution of carcinomaMaximum diameterTumor formation patternBoundaryExtrathyroidal expansionCalcificationBlood flowβSEWaldPOR CI ORAbbreviations SE Standard error OR odds ratio CI confidence intervalPTMC patients may be feasible but for those with longer life expectancy early surgery can significantly reduce future progress of tumors may not only but also reduced the forward of surgery as a result of basic diseases such as cardiovascular increase intolerance Therefore for PTMC patients age ¥ with good survival expectations we believe surgical intervention is still necessary which is also consistent with Shindo et al13Risk FactorsIn the past there have been many studies analyzing PTMCCLNM but few such reports address elderly patients with PTMC Due to air interference in the tracheal cavity it is relatively difficult to diagnose CLNM of the neck using ultrasound which has a high falsenegative rate In this study the accuracy of ultrasound in predicting CLNM was Therefore it is questionable whether dissection of such lymph nodes can be performed only by preoperative ultrasound Chung et al14 found that in young PTMC patients multiple cancer foci enlarged nodules extrathyroidal expansion of cancer focus and vascular invasion are independent risk factors for PTMCCLNM and lateral cervical lymph node metastasis but they did not clearly identify calcified nodules as an independent risk factor By analyzing the data of PTMC patients Oh et al15 showed that the rate of lymph node metastasis in patients with calcified nodules was higher than in patients whose nodules were not calcified they concluded that calcified nodules were an important risk factor for PTMC cervical lymph node metastasis Haugen et al16 have a similar view In this study the metastasis rates of the central cervical region and lateral cervical lymph nodes were and respectively The Ï2 test showed that distribution nodular morphology calcification and extrathyroidal expansion of cancer focus were risk factors for centralregion lymph nodes P Figure Areas under the curve AUC Distribution of carcinoma AUC extrathyroidal expansion AUC tumor formation pattern AUC Cancer Management and Research submit your manuscript wwwdovepresscom DovePress 0cFu DovepressAcknowledgmentsAll authors made substantial contributions to conception and design acquisition of data or analysis and interpretation of data took part in drafting the article or revising it critically for important intellectual content gave final approval of the version to be published and agree to be accountable for all aspects of the work We thank LetPub for its linguistic assistance during the preparation of this manuscriptDisclosureThe authors report no conflicts of interest for this workFigure Multiple independent risk factors predicted lymph node metastasis in the central region Areas under the curve AUC which was consistent with Liu et al17 However our multivariate logisticregression analysis found that only distribution of cancer lesions Ï2 P nodule morphology Ï2 P and extra thyroidal expansion of cancer focus Ï2 P were independent risk factors for such metastasis The AUCs of these factors were and respectively and overall predictability was In summary we believe that active followup and observation should be carefully selected for elderly patients with PTMC especially for those with multiple cancer foci extrathyroidal expansion of cancer focus and irregular morphology preventive centralarea lymph node dissection is also appropriate Although we did not find nodular calcification maximum tumor diameter Hashimotos thyroiditis or other variables to be independent risk factors we believe this result may have a certain relationship with the small sample size which we will further expand in the future for related studies and supplementsEthical ApprovalThis study was approved by the Institutional Review Board of Sichuan Cancer Hospital and Institutional Ethics Committee and performed according to the ICH GCP principleInformed ConsentWe obtained written informed consent from all of the individual participants included in the studyReferences Megwalu UC Observation versus thyroidectomy for papillary thyroid microcarcinoma in the elderly J Laryngol Otol doi101017S0022215116009762 Davies L Welch HG Current thyroid cancer trends in the United States JAMA Otolaryngol Head Neck Surg doi101001jamaoto20141 Kilfoy BA Zheng T Holford TR et al International patterns and trends in thyroid cancer incidence Cancer Causes Control doi101007s1055200892604 Bray F Ferlay J Soerjomataram I et al Global cancer statistics GLOBOCAN estimates of incidence and mortality worldwide for cancers in countries CA Cancer J Clin doi103322caac21492 Hughes DT Haymart MR Miller BS et al The most commonly occurring papillary thyroid cancer in the United States is now a microcarcinoma in a patient older than years Thyroid doi101089thy20100137 Davies L Welch HG Increasing incidence of thyroid cancer in the JAMA United States doi101001jama295182164 Kuo EJ Goffredo P Sosa JA Aggressive variants of papillary thyroid microcarcinoma are associated with extrathyroidal spread and lymphnode metastases a populationlevel analysis Thyroid doi101089thy20120563 Hay ID Hutchinson ME GonzalezLosada T Papillary thyroid microcarcinoma a study of cases observed in a 60year period Surgery discussion doi101016j surg200808035 Simard EP Ward EM Siegel R et al Cancers with increasing incidence trends in the United States through CA Cancer J Clin doi103322caac20141 Cramer JD Fu P Harth KC Analysis of the rising incidence of thyroid cancer using the surveillance epidemiology and end results national cancer data registry Surgery doi101016jsurg201010016 Ito Y Miyauchi A Kudo T et al Trends in the implementation of active surveillance for lowrisk papillary thyroid microcarcinomas at Kuma Hospital gradual increase and heterogeneity in the acceptance of this new management option Thyroid doi101089thy20170448 Ito Y Miyauchi A Kihara M Patient age is significantly related to the progression of papillary microcarcinoma of the thyroid under observation Thyroid doi101089thy20130367 Shindo M Wu JC Park EE The importance of central compartment elective lymph node excision in the staging and treatment of thyroid cancer Arch Otolaryngol Head Neck Surg papillary doi101001archotol1326650submit your manuscript wwwdovepresscom DovePress Cancer Management and Research 0cDovepress Fu Liu LS Liang J Li JH et al The incidence and risk factors for central lymph node metastasis in cN0 papillary thyroid microcarcinoma a metaanalysis Eur Arch Otorhinolaryngol doi101007s0040501643020 Chung YS Kim JY Bae JS Lateral lymph node metastasis in papillary thyroid carcinoma results of therapeutic lymph node dissection Thyroid doi101089thy20080244 Oh EM Chung YS Song WJ The pattern and significance of the calcifications of papillary thyroid microcarcinoma presented in preoperative neck ultrasonography Ann Surg Treat Res doi104174astr2014863115 Haugen BR Alexander EK Bible KC American Thyroid Association Management Guidelines for adult patients with thyroid nodules and differentiated thyroid cancer the American Thyroid Association Guidelines task force on thyroid nodules and differenthyroid cancer Thyroid doi101089 tiated thy20150020Cancer Management and Research Publish your work in this journal Cancer Management and Research is an international peerreviewed access journal focusing on cancer research and the optimal use of preventative and integrated treatment interventions to achieve improved outcomes enhanced survival and quality of life for the cancer patient The manuscript management system is completely online and includes a very quick and fair peerreview system which is all easy to use Visit httpwwwdovepresscomtestimonialsphp to read real quotes from published authors Dovepress Submit your manuscript here wwwdovepresscomcancermanagementandresearchjournalCancer Management and Research submit your manuscript wwwdovepresscom DovePress 0c' | 2 |
significant genebiomarkers of primary colorectal cancerJing Han Xue Zhang Yan Liu Li Jing Yibing Liu andDepartment of Medical Oncology The Fourth Hospital of Hebei Medical University Jiankang Road Shijiazhuang Hebei PR ChinaLi FengCorrespondence Li Feng lifeng191gqsinacomBackground Primary colorectal cancer PCRC is a common digestive tract cancer in theelderly However the treatment effect of PCRC is still limited and the longterm survival rateis low Therefore further exploring the pathogenesis of PCRC and searching for specificmolecular targets for diagnosis are the development trends of precise medical treatmentwhich have important clinical significanceMethods The public data were downloaded from Gene Expression Omnibus GEOdatabase Verification for repeatability of intragroup data was performed by Pearsons correlation test and principal component analysis Differentially expressed genes DEGs between normal and PCRC were identified and the proteinprotein interaction PPI networkwas constructed Significant module and hub genes were found in the PPI network A total of PCRC patients were recruited between and from the Fourth Hospital of Hebei Medical University RTPCR was used to measure the relative expression ofCLCA4 and MS4A12 Furthermore the study explored the effect of expression of CLCA4and MS4A12 for overall survivalResults A total of DEGs were identified between PCRC and normal colorectal tissuesTen hub genes concerned to PCRC were screened namely CLCA4 GUCA2A GCG SSTMS4A12 PLP1 CHGA PYY VIP and GUCA2B The PCRC patients with low expressionof CLCA4 and MS4A12 has a worse overall survival than high expression of CLCA4 andMS4A12 P005Conclusion The research of DEGs in PCRC DEGs hub genes especially CLCA4and MS4A12 and related signaling pathways is conducive to the differential analysis of themolecular mechanism of PCRCIntroductionPrimary colorectal cancer PCRC is a common digestive tract cancer in the elderly The primary lesioncan be seen in the left colon the right colon the upper or lower rectum [] PCRC is the second mostcommonly diagnosed cancer in women and the third most commonly diagnosed cancer in men and theprevalence of male is higher than that of female in most areas [] With the social environment lifestyleand dietary structure changes the incidence of PCRC is on the rise and there is a trend of rejuvenationThis is a social issue worthy of attention [] At present there is controversy about the pathogenesis ofPCRC It is generally believed that smoking drinking greasy diet obesity lack of exercise colorectalinflammation and genetic factors are all involved in the onset of cancer But these factors are also the causeof many other tumors Therefore the specific etiological mechanism of PCRC has not yet been elucidated[] Some scholars believe that some genes or molecules are involved in the development of PCRC Thesefindings promote the research and treatment of PCRC [] At present the treatment of PCRC includestraditional surgery chemotherapy radiotherapy emerging immunotherapy molecular targeted therapyetc Clinically the single or combination therapy that best suits the condition is usually selected accordingto the actual situation of the patient [] However the treatment effect of PCRC is still limited andReceived March Revised August Accepted August Accepted Manuscript online August Version of Record published August The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20200963101042BSR20200963the longterm survival rate is low The early prognosis of patients with early diagnosis is often better [] Thereforefurther exploring the pathogenesis of PCRC searching for specific molecular targets for diagnosis and treatment realizing early diagnosis targeted treatment and individualized treatment are the development trends of precise medicaltreatment which have important clinical significancePersonalized medicine refers to the treatment of existing diseases based on the information of each persons disease genome [] It is now widely believed that majority of individual differences in drug response are due to geneticfactors Personalized medicine is a discipline that emphasizes studying the effect of genetic factors on a drug [] Recently due to the smooth implementation of the human genome project and the rapid development of bioinformaticspersonalized medicine has been strongly promoted and the concept has been gradually developed []Bioinformatics is a method to process and analyze biological data by combining biological knowledge with information processing technology It is commonly used in highthroughput data analysis such as gene and proteomicsAs a frontier interdisciplinary subject bioinformatics analysis technology can realize the biological analysis of thestructure and function of histological data find the genes or proteins most relevant to diseases and further analysis may find the molecules most relevant to diseases and can be used as disease markers [] At present a largenumber of scholars have applied this technology to tumor research that is processing gene sequence or omics databy bioinformatics analysis technology to find genes or molecular markers most relevant to tumors []Therefore the present study aimed to use the bioinformatics to identify the hub genes of PCRC and to verify theirrole on the overall survival of patients with PCRC based on the clinical data And the research would provide novelinsights for the personalized medicine on the treatment of patients with PCRCMaterial and methodsLease start with dates and time location of study and the recruitmentsof patientsThe present study recruited a total of PCRC patients between and from the Fourth Hospital of HebeiMedical University Shijiazhuang of Hebei province Clinical and histopathological characteristics and followup andsurvival information were available for all patients and were collected retrospectively from medical records Patientsaged years old histologically confirmed as PCRC not received tumor treatment and no history of gastrointestinal surgery will be screened for inclusion criteria Exclusion criteria included age years old or yearsold combined with other malignant tumors operation time more than one month after the last examination andsevers heart diseaseEthical clearance and informed contentThe research conformed to the Declaration of Helsinki and was authorized by the Human Ethics and Research EthicsCommittees of the Fourth Hospital of Hebei Medical University The written informed consents were obtained fromall participatesDownload public dataThe Gene Expression Omnibus GEO database [] httpwwwncbinlmnihgovgeo is the largest most comprehensive and publicly available source of gene expression data It contains information about the expression levels ofmultiple genes in different groups of clinical samples such as the differences in gene expression between tumor tissues and normal tissues GSE41258 GPL96 [HGU133A] Affymetrix Human Genome U133A Array and GSE81558GPL15207 [PrimeView] Affymetrix Human Gene Expression Array were obtained from the GEO database A totalof samples including tumor colorectum tissues from PCRC patients and normal colorectum tissues wereselected from GSE41258 A total of samples including tumor colorectum tissues from PCRC patients and normal colorectum tissues were selected from GSE81558Veriï¬cation for repeatability of intragroup dataFirst repeatability of intragroup data were verified by the Pearsons correlation test The heatmap was drew via the Rlanguage environment and presented the correlation among intragroup data Second principal component analysisPCA was the general method for sample clustering and is commonly performed for diversity analysis resequencinggene expression and other sample clustering based on various variable information The verification for repeatabilityof intragroup data was executed by PCA The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20200963101042BSR20200963Differentially expressed genes DEGs between normal and PCRCGEO2R [] httpwwwncbinlmnihgovgeogeo2r could import data of the GEO database into the R languageand perform differential analysis essentially through the following two R packages including limma packages andGEOquery Therefore through the GEO2R tool DEGs were identified between normal and PCRC group The adjusted Pvalues adj P and the fold change FC ¥ or ¤ were defined as significance SangerBoxshengxinren one tool was used to draw volcano maps [] Venn diagrams were delineated using anonline Venn tool httpbioinformaticspsbugentbewebtoolsVenn which would visualize common DEGs sharedbetween GSE41258 and GSE81558Proteinprotein interaction PPI networkThe common DEGs shared between GSE41258 and GSE81558 were converted into differently expressed proteinsThe STRING Search Tool for the Retrieval of Interacting Genes online database tringdb could construct PPI network which was visualized by Cytoscape version []GO and KEGG analysis via DAVID toolOne online tool DAVID davidncifcrfgovhomejsp version Maryland America was applied to carriedout the functional annotation for DEGs Gene Ontology GO [] generally perform enrichment analysis of genomesAnd there are mainly cellular components CC biological processes BP and molecular functions MF in theGO analysis Kyoto Encyclopedia of Genes and Genomes KEGG wwwkeggjp [] is a comprehensivedatabase of genomic chemical and systemic functional information Therefore DAVID was used to make analysisof GO and KEGGSigniï¬cant module and hub genesMolecular Complex Detection tool MCODE version [] an plugin of Cytoscape was performed toidentify tested most significant module from the PPI network and the criteria was that the maximum depth MCODE scores cutoff kscore and node score cutoff Then cytoHubba [] a free plugin of Cytoscape was applied to authorize the hub genes when the degree ¥ChiaHao Chins [] research introduce a novel Cytoscape plugin cytoHubba for ranking nodes in a network by theirnetwork features CytoHubba provide a userfriendly interface to explore important nodes in biological networksWhen the degree¥ in the cytoHubba the hub genes would be obtained And in the former publications []numerous researchers chose hub genes out of the DEGs Therefore the present study chose hub genes out of DEGsInteraction between the hub genesPearsons correlation analysis was also performed to present the interaction between the hub genes The cBioPortalhttpwwwcbioportal [] one online software constructed the coexpression network of these hub genesSimultaneously the coexpression network of hub genes in the field of PCRC was also analyzed via Coexpedia a freeand online toolhttpwwwcoexpedia []Expression analysis of hub genesUCSC Xena xenaucsceduwelcometoucscxena could integrate the public genomic data sets to analyzeand visualize the expression level of hub genes Then the clustering analysis of expression level of hub genes wasperformed using heatmaps based on the GSE41258 and GSE81558 Also the expression profiles of hub genes in thehuman different ans were displayed with Gene Expression Profiling Interactive Analysis GEPIA httpgepiacancerpkucn [] In order to compare the expression of hub genes in the various tumors GEPIA was used Andthe expression profiles of hub genes in the PCRC and normal groups were analyzed using GEPIAEffect of hub gene expression for pathological stage and overall survivalEffect of hub gene expression for pathological stage and overall survival was analyzed by the GEPIA Finally the correlation and linear regression analysis between PCRC and hub gene expression were performed And the receiveroperator characteristic ROC curve analysis was performed to test the sensitivity and specificity of hub gene expression for diagnose PCRC The SPSS software version IBM New York America was used to conduct all thestatistical analysis A Pvalue was defined as statistical significance The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20200963101042BSR20200963Table Summaries for the function of hub genesNoGene symbolFull nameFunctionCLCA4GUCA2AChloride channel accessory Guanylate cyclase activator 2AGCGSSTGlucagonSomatostatinMay be involved in mediating calciumactivated chloride conductanceEndogenous activator of intestinal guanylate cyclase It stimulates this enzyme through thesame receptor binding region as the heatstable enterotoxinsRegulates blood glucose by increasing gluconeogenesis and decreasing glycolysisGLP1 is a potent stimulator of glucosedependent insulin release GLP2 stimulatesintestinal growth concomitant with increased crypt cell proliferationSomatostatin inhibits the release of somatotropin This hormone is an important regulatorof the endocrine system through its interactions with pituitary growth hormone thyroidstimulating hormone and most hormones of the gastrointestinal tractMS4A12Membrane spanning 4domainsMay be involved in signal transduction as a component of a multimeric receptor complexA12Silencing of this gene in colon cancer cells inhibits the proliferation cell motility andchemotactic invasion of cellsPLP1CHGAPYYVIPProteolipid protein This is the major myelin protein from the central nervous system It plays an important rolein the formation or maintenance of the multilamellar structure of myelinChromogranin AThis gene product is a precursor to three biologically active peptides vasostatinpancreastatin and parastatinPeptide YYThis gut peptide inhibits exocrine pancreatic secretion has a vasoconstrictory action andinhibitis jejunal and colonic mobilityVasoactive intestinal peptideVIP causes vasodilation lowers arterial blood pressure stimulates myocardial contractilityincreases glycogenolysis and relaxes the smooth muscle of trachea stomach andgallbladderGUCA2BGuanylate cyclase activator 2B May be a potent physiological regulator of intestinal ï¬uid and electrolyte transport May bean autocrineparacrine regulator of intestinal salt and water transportRTqPCR assayTotal RNA was extracted from tumor colorectum tissues from PCRC patients and adjacent normal colorectum tissuesby the RNAiso Plus Trizol kit Thermofisher Massachusetts America and reverse transcribed to cDNA RTqPCRwas performed using a Light Cycler® System with specific primers for the ten hub genes Table presents theprimer sequences used in the experiments The RQ values 01 01Ct where Ct is the threshold cycle of each samplewere calculated and are presented as fold change in gene expression relative to the control group GAPDH was usedas an endogenous control The expression level of CLCA4 and MS4A12 in PCRC patients was measured by RTqPCROverall survival analysis of the PCRCThe KaplanMeier method was performed to analyze the overall survival All statistical analyses were conductedusing SPSS software version and P005 was considered statistically significantResultsHigh repeatability of dataThere exist strong correlations among samples in the PCRC group and also strong correlations among samples in thecontrol group in the GSE41258 via the Pearsons correlation test Supplementary Figure S1A And there also existstrong correlations among samples in the PCRC group and also strong correlations among samples in the controlgroup in the GSE81558 via the Pearsons correlation test Supplementary Figure S1B Furthermore PCA was performed to verify the repeatability of data Through the PCA the repeatability of the data in GSE41258 was fine Thedistances between per samples in the PCRC group were close and the distances between per samples in the controlgroup were also close in the dimension of PC1 Supplementary Figure S1C Through the PCA the repeatability ofthe data in GSE81558 was fine The distances between per samples in the PCRC group were close and the distancesbetween per samples in the control group were also close in the dimension of PC1 Supplementary Figure S1DDEGs between control and PCRCThere are plenty of DEGs on the all chromosomes between PCRC and control samples Supplementary Figure S1EOne volcano plot presents the DEGs in the GSE41258 Figure 1A and another volcano plot presents the DEGs in theGSE81558 Figure 1B In the volcano plots the green nodes indicate the downregulated DEGs and the red nodesindicate the upregulated DEGs The Venn diagram manifested that a total of DEGs were exist in the two datasetsGSE41258 and GSE81558 simultaneously Figure 1C After construction of PPI network for the common DEGsthere are nodes and edges in the PPI network Figure 1D The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20200963101042BSR20200963Figure The differently expressed genes and PPI networkA One volcano plot presents the DEGs in the GSE41258 B Another volcano plot presents the DEGs in the GSE81558 In thevolcano plots the green nodes indicate the downregulated DEGs and the red nodes indicate the upregulated DEGs C TheVenn diagram manifested that a total of DEGs were exist in the two datasets GSE41258 and GSE81558 simultaneously DThe PPI network of the common DEGs The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20200963101042BSR20200963The functional enrichment analysis of DEGs via GO and KEGGGO analysis manifested that variations in DEGs related with biological processes BP were significantly enrichedin bicarbonate transport onecarbon metabolic process cell surface receptor signaling pathway collagen catabolicprocess transport xenobiotic transport body fluid secretion axon development positive regulation of guanylate cyclase activity drug transmembrane transport response to steroid hormone response to tumor necrosis factor positiveregulation of peptidylthreonine phosphorylation cell proliferation and regulation of intracellular pH Figure 2AThe variations in DEGs related with cellular components CC were significantly enriched in extracellular space extracellular region anchored component of membrane proteinaceous extracellular matrix plasma membrane apicalplasma membrane integral component of plasma membrane apical part of cell extracellular exosome and basolateralplasma membrane Figure 2B The variations in DEGs related with molecular functions MF were significantly enriched in hormone activity carbonate dehydratase activity xenobiotictransporting ATPase activity arylesterase activity metalloendopeptidase activity neuropeptide hormone activity and hydrolase activity hydrolyzing Oglycosylcompounds Figure 2C KEGG pathway enrichment analysis showed that the top pathways related with DEGs werenitrogen metabolism bile secretion proximal tubule bicarbonate reclamation and pancreatic secretion Figure 2DSigniï¬cant module network and identiï¬cation of hub genesA significant module was screened from the PPI network and the module network consisted of nodes and edgesFigure 2E And ten hub genes were identified including CLCA4 GUCA2A GCG SST MS4A12 PLP1 CHGAPYY VIP and GUCA2B Figure 2F The function of hub genes were summarized in the Table Strong interaction among the hub genesThrough the Pearsons correlation test heat maps manifested that there were strong correlations among hub genes inthe GSE41258 Supplementary Figure S2A and GSE81558 Supplementary Figure S2B datasets PYY SST GCG andVIP existed simultaneously in the coexpression network via cBioPortal Supplementary Figure S2C And throughthe analysis of Coexpedia there were strong interactions among PYY SST GCG CHGA CLCA4 GUCA2B andMS4A12 Supplementary Figure S2DDifference of expression of hub genes between PCRC and controlsamplesHeat map showed that the expressions of all the hub genes were lower in the PCRC samples than the control samplesSupplementary Figure S2E Hierarchical clustering allowed for simple differentiation of PCRC tissues from normalcolorectal tissues via the expression levels of hub genes in the GSE41258 Supplementary Figure S3A and GSE81558Supplementary Figure S3B datasets The expressions of all the hub genes were lower in the PCRC group than thecontrol groupThe analysis of expression level of hub genesThe hub genes in the human different ans were expressed in the Supplementary Figure S3C The pink presents thetumor individuals and the green presents the normal individuals The expression of hub genes in the colorectum washigher in the normal individuals compared with the tumor samples Supplementary Figure S3C When we comparedthe expression of hub genes in the various tumors the all hub genes were downregulated in the PCRC samples alsonamed colon adenocarcinoma COAD Supplementary Figure S3D Through GEPIA analysis the expressions ofhub genes in the PCRC patients were lower than the normal individuals Supplementary Figure S4AAssociation between hub gene expression pathological stage andoverall survivalThe results of GEPIA manifested that the expression of VIP was significantly positively related with pathological stageP0027 while the expression of CLCA4 GUCA2A GCG SST MS4A12 PLP1 CHGA PYY and GUCA2B wasnot as Supplementary Figure S4B KaplanMeier analysis showed that PCRC patients with low expression levelsof CLCA4 and MS4A12 had poorer overall survival times than those with high expression levels P005 Figure3AE PCRC patients with high expression levels of GCG SST PLP1 and CHGA had poorer overall survival timesthan those with low expression levels P005 Figure 3CDF Supplementary Figure S5A However there was nostatistically significant effect on OS associated with the expression of GUCA2A PYY VIP and GUCA2B P005Figure 3B Supplementary Figure S5BD The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20200963101042BSR20200963Figure The enrichment analysis for DEGs and the identiï¬cation of hub genesA Detailed information relating to changes in the biological processes BP of DEGs in PCRC and control colorectal samplesB Detailed information relating to changes in the cellular components CC of DEGs in PCRC and control colorectal samples CDetailed information relating to changes in the molecular functions MF of DEGs in PCRC and control colorectal samples D KEGGpathway analysis for DEGs E The significant module of the PPI network F The hub genes identified from the PPI network The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20200963101042BSR20200963Figure The overall survival KaplanMeier of six hub genesA CLCA4 B GUCA2A C GCG D SST E MS4A12 F PLP1 The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20200963101042BSR20200963Table The correlation and linear regression analysis between PCRC and relevant gene expressionPCRCMultiple linear regressionVIFODTGene symbolPearsons correlation coefï¬cientPvalueÏaCLCA4GUCA2AGCGSSTMS4A12PLP1CHGAPYYVIPGUCA2BPvalueaPearsons correlation coefï¬cient between PCRC and relevant characteristics Ï Pearsons correlation coefï¬cientbMultiple linear regression analysis PCRC primary colorectal cancer P005 P001 P0001Table Receiver operator characteristic curve analysis of hub gene expression for PCRCGene symbolPCRCCLCA4GUCA2AGCGSSTMS4A12PLP1CHGAPYYVIPGUCA2BAUC0987maxPvalue95CIAUC area under curve max the maximum of AUC Signiï¬cant variables ODT Optimal diagnostic thresholdPCRC primary colorectal cancer P0001Correlation linear regression and ROC analysisThe Pearsons correlation coefficient was used in the correlation analysis and CLCA4 Ï P0001GUCA2A Ï P0001 GCG Ï P0001 SST Ï P0001 MS4A12 Ï P0001 PLP1 Ï P0001 CHGA Ï P0001 PYY Ï P0001 VIP Ï P0001 and GUCA2B Ï P0001 were significantly correlated with PCRC Table In themultivariate linear regression model holding all other variables at a fixed value the natural logarithmic DN remainedassociated with CLCA4 GUCA2A SST MS4A12 PLP1 CHGA PYY and GUCA2B P005 Table To identify accurate thresholds for hub genes to predict PCRC we constructed ROC The expression of all hubgenes was associated with a diagnosis of PCRC AUC Pvalue0001 Table and Figure The ROCcurve of CLCA4 was shown in Figure 4A and the area under curve of CLCA4 was maximal The ROC curve ofGUCA2A was shown in Figure 4B The ROC curve of GCG was shown in Figure 4C The ROC curve of SST wasshown in Figure 4D The ROC curve of MS4A12 was shown in Figure 4E The ROC curve of PLP1 was shown inFigure 4F The ROC curve of CHGA was shown in Figure 4G The ROC curve of PYY was shown in Figure 4H TheROC curve of VIP was shown in Figure 4I The ROC curve of GUCA2B was shown in Figure 4J The ROC curves ofper hub genes are shown in Figure 4K The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20200963101042BSR20200963Figure ROC curves of hub genes for PCRCA CLCA4 B GUCA2A C GCG D SST E MS4A12 F PLP1 G CHGA H PYY I VIP J GUCA2B K ROC curves of allhub genes The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20200963101042BSR20200963Table Clinicopathological variables and the expression status of CLCA4 and MS4A12CLCA4PMS4A12PLow High Low High SexAgeMaleFemale years¥ yearsOverall survival months¥ monthsPearsons chisquared test was usedP005Figure The veriï¬cation of expression and overall survival analysis for CLCA4 and MS4A12A The relative expression of CLCA4 based on PCR B The relative expression of MS4A12 based on PCR C The overall survivalof PCRC based the expression of CLCA4 D The overall survival of PCRC based the expression of MS4A12Basic information of PCRC patientsPatients basic information were presented in Table The mean patient age was years old range yearsand the median OS was months range monthsRTqPCR analysis validation of hub genesAs presented in the result CLCA4 P005 Figure 5A and MS4A12 P005 Figure 5B were markedly The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20200963101042BSR20200963downregulated in PCRC samples when compared with the adjacent normal colorectum tissues It should be notedthat the expression situation of CLCA4 and MS4A12 were consistent in above results of bioinformaticsLow expression of CLCA4 and MS4A12 in PCRC patients wereindependent prognostic factors for the poor overall survivalThe KaplanMeier OS curves were analyzed Low expression of CLCA4 was predictive of a shorter OS in the PCRC patients P005 Figure 5C Low expression of MS4A12 was predictive of a shorter OS in the PCRC patients P005Figure 5DDiscussionPCRC is a common digestive tract cancer which seriously affects the life expectancy and quality of life of patients Inrecent years the survey results show that the morbidity and mortality rate are on the rise [] The clinical manifestations of patients with PCRC are related to the location and pathological type of the tumor The most common typeof pathology is adenocarcinoma The primary lesion located in the colon often causes diarrhea obstruction bleedingin the rectum anemia and cachexia in the later stage of cancer patients [] The current treatment is mainly surgerycombined with chemotherapy or radiotherapy while advocated exercise to enhance the bodys immunity and preventinfection [] Gavrilas et al found that combination of dietary preparations such as curcumin and resveratrol withchemotherapeutic drugs contributed to the prognosis of PCRC [] Clinical application benefit and safety of epidermal growth factor EGFRrelated targeted therapy and PD1PDL1 immunotherapy are still to be further studied[] The investigation found that the cost of PCRC treatment is high and it takes up a lot of medical resources andthe prognosis of patients is not necessarily proportional to the input The early treatment of early treatment patientshas a relatively low total cost of treatment and a good prognosis [] Therefore to further explore the pathogenesisof PCRC to find possible therapeutic targets to achieve early diagnosis targeted therapy individualized treatmenthas important clinical value and market prospectsBioinformatics has been widely used as a new means of exploring disease mechanism and searching fordiseaserelated genetic molecules Zhang et al found genes related to hepatocellular carcinoma by bioinformaticsanalysis Further analysis confirmed the correlation between these differential genes and diseases suggesting thatthese molecules may be used as molecular targets for early diagnosis and treatment [] Zhang et al found the mostrelevant molecules of gastric cancer miR19b3p and miR165p by analyzing the genomewide miRNA microarraydata of gastric cancer patients which provided a new idea for the diagnosis and treatment of gastric cancer [] Sunfound molecules related to the pathogenesis of colorectal cancer by screening from public databases Further analysisshowed that differentially expressed genes such as PPBP CCL28 and CXCL12 are likely to be involved in the development of colorectal cancer and may be potential diagnostic and therapeutic targets [] We found genes thatwere differentially expressed in patients with PCRC by bioinformatics analysis Low expression of PLP1 VIP SSTGCG PYY MS4A12 CLCA4 GUCA2A CHGA and GUCA2B in tumor patients compared with normal subjectsAt the same time we performed survival analysis on patients with PCRC The results showed that CLCA4 GUCA2AGCG SST MS4A12 and PLP1 genes were significantly associated with the survival of patients with PCRCCLCA4 is the chloride channel accessory CLCA4 is a member of the calciumsensitive chloridetransportingprotein family involved in intracellular ion channel activity chloride ion transmembrane transport and proteolysis Members of the calcium activated chloride channel CLCA gene family are thought to have multiple functionsincluding cell adhesion and tumor suppression Ye et al found that CLCA4 is low expressed in patients with oraltongue squamous cell carcinoma through genomewide transcriptional mapping which provides ideas for diagnosisand targeted therapy [] Bundela also found multiple differentially expressed genes in oral cancer patients in Indiaand suggested that CLCA4 may be a potential therapeutic target [] Yu et al found that CLCA4 is low expressedin breast cancer patients Further analysis revealed that CLCA4 is a marker of breast epithelial differentiation andmay be involved in tumor proliferation and metastasis Clinical data analysis showed that patients with breast cancerwith low expression of CLCA4 had lower recurrencefree survival rate suggesting that it may serve as a diagnosticand therapeutic target [] Hu found that CLCA4 was low expressed in bladder cancer tissues Further analysis revealed that CLCA4 may be involved in the proliferation and invasion of bladder cancer through PI3KAKT signaltransduction suggesting that CLCA4 may be a target for diagnosis and treatment [] Liu found that CLCA4 mayinhibit epithelialmesenchymal transition EMT by affecting PI3KATK phosphorylation thereby inhibiting cellmigration and invasion of hepatoma cells [] Yang found that patients with colorectal cancer CRC had low expression of CLCA1 and CLCA4 and further experiments confirmed that CLCA1 is involv | 2 |
emergence of promising targeted therapies for the treatment of hepatocellular carcinoma HCCSorafenib has been the mainstay of treatment for a decade and newer modalities were ineffective and did not confer any increasedtherapeutic beneï¬t until the introduction of lenvatinib which was approved based on its noninferiority to sorafenib Thesubsequent success of regorafenib in HCC patients who progress on sorafenib treatment heralded a new era of secondlinetreatment and was quickly followed by ramucirumab cabozantinib and the most uential immune checkpoint inhibitors ICIsOver the same period combination therapies including antiangiogenesis agents with ICIs dual ICIs and targeted agents inconjunction with surgery or other locoregional therapies have been extensively investigated and have shown promise andprovided the basis for exciting clinical trials Work continues to develop additional novel therapeutic agents which could potentiallyaugment the presently available options and understand the underlying mechanisms responsible for drug resistance with the goalof improving the survival of patients with HCCSignal Transduction and Targeted Therapy 101038s4139202000264xINTRODUCTIONPrimary liver cancer remains a major problem for all health caresystems worldwide and is associated with a significant clinicaleconomic and psychological burden Hepatocellular carcinomaHCC accounts for of cases of nonmetastatic tumors of theliver1 During the past decades research has shed light on theepidemiology risk factors and molecular and genetic proï¬les ofHCC contributing to the evolution of strategies for preventionsurveillance early diagnosis and treatment23 Liver resectionablation and liver transplantation are potentially curative butrequire diagnosis at a sufï¬ciently early stage Unfortunately asignificant proportion of HCC patients present with intermediateand advanced stage disease often despite diligent surveillanceand curative treatments are frequently not possible4 In thesepatients systemic therapy remains essential and its pivotal roleand potential have stimulated considerable research over the pastdecade In this review we examine recent advances in targetedtherapy and discuss the impact this has had on the managementof HCC We also provide an overview of the most important areasof HCC research including novel clinical trials and technicalplatforms which promise to facilitate substantial progress withinthe next decadeAPPROVED FIRSTLINE AGENTS FOR HCCSorafenibThe success of SHARP and AsiaPaciï¬c trial promoted the approvalof sorafenib as ï¬rstline targeted therapy for advanced HCC5ushering in the era of systemic treatment Subsequently virtuallyall trials were centered around sorafenib and it was used as acontrol with which novel ï¬rstline agents were compared andevaluated in an attempt to improve the prognosis of patients withHCC Unfortunately despite a number of trials which comparedthese novel agents including sunitinib10 brivanib11 cediranib12linifanib13 dovotinib14 and immunecheckpoint inhibitors ICIs tosorafenib none achieved the predeï¬ned primary end points Fig In addition during the decade when these agents were investigated the median overall survival OS of sorafenib monotherapy asï¬rstline treatment for advanced HCC increased from monthsSHARP to months CheckMate459 further consolidating itsposition Meanwhile the antitumor activity and safety of sorafenibhave been validated in realworld setting Subanalyses of the SHARPand AsiaPaciï¬c trials found sorafenib was effective and safeirrespective of disease etiology disease burden ECOG EasternCooperative Oncology Group performance status status etc15The safety of sorafenib was consistent across ChildPugh A and Bpatients in clinical practice18 and the occurrence of side effects suchas handfoot syndrome and diarrhea were associated with animproved OS19 Baseline hepatic function clinicopathologicalfactors and etiology also affect the prognosis in HCC patientstreated with sorafenib20 In addition sorafenib exerts antitumoreffects with recurrenttransplantationconferring a survival advantage when compared with bestsupportive care BSC21 Noticeably the application of sorafenibin clinical practice displays significantregional variations andincompliance with guidelines besides its usage as ï¬rstline therapyIt is common that initially unresectable HCCs got downstaged aftersorafenib treatment and underwent curativeintent surgery24 andlocoregional therapies before sorafenib were commonly encountered in realworld settings2930tumors following liver1Department of Liver Surgery and Transplantation Liver Cancer Institute Zhongshan Hospital Fudan University Shanghai China 2Key Laboratory of Carcinogenesis and CancerInvasion Fudan University Ministry of Education Shanghai China 3Shanghai Key Laboratory of an Transplantation Zhongshan Hospital Fudan University Shanghai China4Department of Hepatobiliary and Pancreatic Surgery University Hospitals of Leicester NHS Trust Leicester UK 5Institute of Biomedical Sciences Fudan University ShanghaiChina and 6State Key Laboratory of Genetic Engineering Fudan University Shanghai ChinaCorrespondence Jian Zhou zhoujianzshospitalshcnThese authors contributed equally Ao Huang XinRong YangReceived April Revised July Accepted July The Authors 0cTargeted therapy for hepatocellular carcinomaHuang et alFirstlineSorafenibSharpAsiaPacificCediranibNCT00427973SunitinibNCT0069937BrivanibBRISKFLLinifanibNCT01009593NintedanibNCT01004003DovitinibNCT01232296ATEZOBEVGO30140IMbrave150LenvatinibREFLECTPEMLENKEYNOTE524NivolumabCheckMate459DonofenibChina SecondlineBrivanibBRISKPSEverolimusEVOLVE1AxitinibNCT01210495RamucirumabREACHRegorafenibRESORCENivolumabCheckMate040PEMKEYNOTE224TivantinibMETIVHCCS1SCUBEPEMKEYNOTE240CabozantinibCELESTIALRamucirumabREACH2NIVIPICheckMate040CAMChinaApatinibChinaFig Overview of the targeted agents approved for HCC ATEZO atezolizumab BEV bevacizumab CAM camrelizumab LEN lenvatinib PEMpembrolizumab NIV nivolumab IPI ipilimumabThe clinical beneï¬t of sorafenib however remains modest andthe complex molecular pathogenesis of HCC stimulated theinvestigation of combinations of sorafenib with other moleculartargeting drugs Sorafenib has been combined with antiangiogenic agents MEKERK pathway inhibitors mTOR pathwayinhibitors histone deacetylase inhibitors EGFEGFR pathwayinhibitors and HGFcMet pathway inhibitors31 Other agentssuch as interferon32 selumetinib33 capecitabine34 tegafururacil35 gemcitabine and oxaliplatin GEMOX3637 and gemcitabinealone38 have also been evaluated but to date no treatmentsinvolving combinations containing sorafenib have succeeded inphase III trialsSince sorafenib and TACE are both recommended therapies foradvanced HCC it is reasonable to expect that their combined usewould confer beneï¬ts when compared with monotherapy Resultshowever highlighted regional differences and the heterogeneityof the trial protocolsIn TACE the multicenter randomizedplacebocontrolled phase European trial when compared withTACE alone the addition of concurrent sorafenib unlike the SPACEtrial39 did not improve progression free survival PFS40 It was alsoshown that the addition of sorafenib did not confer any survivalbeneï¬t in patients with unresectable HCCs who had alreadyresponded to TACE41 Contrasting with these ï¬ndings retrospective studies from China have shown that combination therapywith sorafenib and TACE increased OS by more than compared with TACE alone42 which was supported by theï¬ndings from a number of other groups5455 RecentlytheTACTICS trial a randomized multicenter prospective trial fromJapan reported an improved PFS for TACE plus sorafenibcompared with TACE alone vs months p although this trial used a redeï¬ned PFS not conventional PFS buttime until unTACEable progression The TACTICS trial also usedtime to any cause of death plus OS as primary endpoints resultsnot reported and compared with sorafenib monotherapy TACEplus sorafenib was only superior in controlling tumor progressionand did not prolong OS5758The acceptance of sorafenib as the standard to which othernewer agents and nonsurgical interventions are compared hasresulted in studies comparing its use as monotherapy with TACEplus external beam radiotherapy59 and TACE plus intensitymodulated radiotherapy combined with sorafenib60 In the SARAHstudy selective internal radiotherapy with yttrium90 resin microspheres did not produce any survival beneï¬t compared withsorafenib in locally advanced and inoperable HCC median OS vs p and did not meet the primary endpoint of OS61Similarly the addition of selective internal radiation therapy tosorafenib did not result in a significant improvement in OScompared with sorafenib alone62 Bettinger et al63 however diddemonstrate that stereotactic body external beam radiotherapyemployed as monotherapy SBRT was able to improve OScompared with sorafenib and SBRT with TACE also providedimproved OS and PFS when compared with sorafenib and TACE incombination64 In a recentinfusionchemotherapy HAIC NCT02774187 He et al65 reported thatsorafenib plus HAIC with FOLFOX improved OS compared withsorafenib alone in advanced HCC when portal vein invasion waspresent which was supported by other studies6667 Although theSCOOP2 trial found sequential HAIC with cisplatin and sorafenibdid not improve the survival beneï¬t compared with sorafenibalone this is likely to have resulted from the study beingunderpowered for the primary and secondary endpoints68trial of hepatic arterialDue to the high recurrence rates following hepatectomy fortherapy has been extensivelyHCC approaches to adjuvantinvestigated although previous attemptsincluding the use ofantiviral agents have been largely unsuccessful Based on thepalliative use and success of sorafenib its potential in the adjuvantsetting was investigated and improved survivals following surgeryanticipated Unfortunately this has not been demonstrated and itfailed to reduce postoperative tumor recurrence in the STORMtrial69 and other western studies70 Explanations for the negativeoutcome in the STORM trial include highdose modiï¬cation ratesshort treatment durations and the enrollment of patients whowere not at high risk of tumor recurrence with no evidenceof tumor satellites with one lesion and with nomicroscopic vascular invasion71 Consistent with this viewpointWang et al72 reported no case of recurrence during the sorafenibdosing period whereas patients suffered recurrence of theirtumor within months of discontinuation of sorafenib72 andpersistent sorafenib intake following postoperative recurrenceimproved OS73 Considering that patients who respond tosorafenib may belong to limited clinical or biological subsetsthe effectiveness of sorafenib in an unselected population cohortsupports its use in the adjuvant setting A number of studies fromthe Far East including China Japan and Korea include patientswith HCCs who are treated with hepatectomy despite their tumorsbeing outside Barcelona Clinic Liver Cancer Classiï¬cation BCLCguidelines and although the results are difï¬cult to compare dueto heterogeneity ofthe protocols the results are positiveSorafenib significantly reduces tumor recurrence in BCLC stage Cpatients7475 and increases diseasefree survivalDFS76 andSignal Transduction and Targeted Therapy 0cZhuang et al77 demonstrated that adjuvant therapy increaseddisease free survival DFS and OS Sorafenib treatment followinghepatectomy significantly prolonged the OS of advanced HCCthan intermediate HCC78 In addition to BCLC stageratherpatients who underwent hepatectomy and were pathologicallydiagnosed with microvascular invasion MVI also beneï¬ted fromadjuvant sorafenib treatment79 In line with these results a largerecent study with propensity score matching analysis alsodemonstrated that sorafenib significantly improved overall andrecurrencefree survival following resection80 The results fromthese studies which include all eligible patients suggest that moreprecise stratiï¬cation would enable the identiï¬cation of thosepatients who will beneï¬t most from the use of adjuvant sorafeniband those in where additional treatment is not appropriateOngoing trials are attempting to evaluate the role of sorafenib inpatients with MVI following radical resection NCT02867280 andNCT02537158LenvatinibFollowing the approval of sorafenib for use in the treatment ofHCC it takes more than a decade before the second ï¬rstlinetargeted agent for HCC emerged Lenvatinib was approved for theï¬rstline therapy in advanced HCC following the results of theREFLECT trial a randomized phase III noninferiority trial publishedby Kudo et al81 Although not approved for long further multicenter data from realworld conditions conï¬rmed the efï¬cacy oflenvatinib regardless of previous tyrosine kinase inhibitor TKItherapies8283 and lenvatinib monotherapy demonstrated antitumor activity for more than years in unresectable HCC whenportal vein invasion was present84 In intermediatestage HCCpatients with tumors exceeding the uptoseven criteria for whomTACE is not helpful lenvatinib could provide significant longer OS vs months and PFS vs months85 Lenvatinibpharmacokinetics in HCC is affected by body weight8687 and asufï¬cient dose relative dose intensity RDI is required to achieve agood therapeutic effect and consequently improved outcomesand prognosis are associated with the preservation ofliverfunction which reduces the number of patients who need todiscontinue their treatment88 With lenvatinib unlike other TKIsthere are issues with thyroid toxicity and surveillance for thyroidabnormalities during treatment is important92 Hypothyroidism isnot unusual and there are also fewer common reports ofthyrotoxicosis and destructive thyroiditis93 From a healtheconomics standpoint however lenvatinib is more cost effectivethan sorafenib9495Secondline targeted agents for HCCStill sorafenib displays limited antitumor activity and someinitially sorafenibsensitive would eventually succumb to thedisease indicating the acquired resistance to sorafenib reducesits beneï¬cial effects and an urgent need for secondline therapyRegorafenibInitial attempts to discover effective secondline agents wereunsuccessful and mirrored attempts to develop ï¬rstline agentswhich were superior to sorafenib96 The RESORCE trial was arandomized double blind placebocontrolled and phase III trialdemonstrating the effectiveness of regorafenib in patients whohad progressed on sorafenib treatment Thisstudy ï¬nallyconï¬rmed the potential of secondline agents and ushered inthe era of secondline and sequential therapy97 Regorafenibprovided survival beneï¬tthe rate of diseaseprogression during prior sorafenib treatment or since the lastsorafenib dose98 This was conï¬rmed by Yoo et al99 in aretrospective study of safety and efï¬cacy in Korean patientswhere data were consistent with those from the RESORCE trialRegorafenib was even shown to be effective in patients with HCCrecurrence following liver transplantation with a median OS ofregardless ofSignal Transduction and Targeted Therapy Targeted therapy for hepatocellular carcinomaHuang et al months following regorafenib initiation and monthsfollowing sorafenib initiation CI for the sorafenibfollowed by regorafenib sequential therapy100However not all patients who progress on sorafenib arecandidates for secondline therapy101 In clinical practice only of patients are eligible forsecondline regorafenibtreatment102 Good liver functional reserve and ECOG performance status during sorafenib treatment contributed to theefï¬cacy and better outcomes of subsequent treatment103104including lenvatinib105 This may in part be due to the RDIrequired to achieve a clinically significantinprognosis106 This is supported by the demonstration that thenew liver reserve function biomarker albuminbilirubin gradeALBI107 successfully identiï¬ed regorafenib candidates and thatin the selected cohort a median OS of months was achievedcompared with months for noncandidates108 Even in patientsnot eligible for regorafenib the ones with an ECOGPS score of the absence of MVI and TTP time to progression ¥ monthscould still have acceptable postprogression survival109 Longtermtreatment with regorafenib has also been shown to reduceangiogenesis and improve portal hypertension PHT and acuteadministration ameliorates portal haemodynamics suggestingthat it may be especially suitable for patients with PHT andpreserved liver function110improvementCabozantinibCabozantinib is another small molecule inhibitor of the tyrosinekinases which are implicated in the progression of HCC and theacquired resistance to sorafenib Cabozantinib blocks the receptors involved in oncogenesis and angiogenesis including VEGFR hepatocyte growth factor receptor MET AXL and theangiopoietin receptors TIE2 RET cKit and FLT3 in vitro andin vivoIn CELESTIAL trial cabozantinib achieved the primaryendpoint with median OS of months compared with months for the placebo group111 and was consequentlyapproved in the EU and USA There remains a paucity of datahowever from realworld clinical practice examining the sequentialtreatment utilizing cabozantinib as the secondline agent it is acostly option associated with frequent highgrade adverse eventsConsequently several studies have addressed the costeffectivenessof cabozantinib using the cost and utility data extracted from theCELESTIAL trial The conclusion from these studies is consistent andconï¬rms that at its current cost point the gain of qualityadjustedlifeyears for cabozantinib QALYs and the incrementalcosteffectiveness ratio ICER mean that it isnot a costeffective treatment option for patients with sorafenibrefractory HCC112 compared with regorafenib QALY and ICER RamucirumabRamucirumab is a fully human recombinant IgG1 monoclonalantibody targeting the VEGF2 receptor Although ramucirumabfailed to meet its primary endpoint as secondline treatment in theREACH trial117 subgroup analysis found survival beneï¬t in patientswith AFP of ngml or higher118 This was later conï¬rmed inthe REACH2 trial122 which led to the approval of ramucirumab assecondline treatment for advanced HCC REACH2 is the ï¬rstpositive phase trialin patients with HCC performed in abiomarkerselected patient cohort and more recent ï¬ndingsdemonstrated that AFPenriched HCCs displayed significantactivation of VEGF which suggests the underlying mechanism ofaction and conï¬rms the potential value of biomarkerdrivenclinical trials123Immune checkpoint therapy and TKI inhibitorsICIs stand as the mainstream of immunotherapy The CheckMate and KEYNOTE224125 studies evaluated the safety andefï¬cacy of nivolumab and pembrolizumab in patients with 0cTargeted therapy for hepatocellular carcinomaHuang et alphaseIIrandomizedparallelgrouptislelizumab sintilimabrealworld cohort studyadvanced HCC refractory to previous sorafenib treatment whichestablished the basis for accelerated approval by the FDA assecondline treatment Subanalysis of CheckMate040 data validated the safety and efï¬cacy of nivolumab in Asian cohort126 Inan internationalICIs have showedpromising efï¬cacy and safety in advanced HCCs as systemicï¬rstsecondthirdfourthline treatment with median OS andPFS of and months respectively127 and an excellentresponse to antiPD1 therapy has also been described in casereport128 Although the subsequent phase III KEYNOTE240 trialdid not meet its prespeciï¬ed statistical signiï¬cance in respect ofimproved PFS and OS the results were consistent with previousKEYNOTE224129 The KEYNOTE394 presently underway in Asianpatients may clarify the role of pembrolizumab in cases ofadvanced HCC with a viral background NCT03062358 RecentlyCheckMate459 the multicenter phase III randomized sorafenibcontrolled trial evaluating nivolumab as ï¬rstline treatment foradvanced HCC failed to achieve its endpoints ESMO butnivolumab did prolong OS vs months and achievelongtime disease control less adverse events AEs and survivalbeneï¬t regardless of the level of PDL1 expression Furthermorenivolumab improved the survival of HCC patients whose etiologywas HBVHCV and did not reactivate hepatitis CamrelizumabSHR1210 Hengrui Pharmaceutical is an antiPD1 inhibitor fromChina investigated for the treatment of Hodgkin lymphoma andHCC It has been shown to have antitumor activity in previouslytreated Chinese patients with advanced HCC in a multicenteropenlabeltrialNCT02989922130 providing evidence for the effectiveness ofPD1 therapy for HBV related HCC in Chinese patients The resultsICIs including durvalumabfrom other trials investigating novelavelumabtremelimumabipilimumabspartalizumab and toripalimab will hopefully yield positive resultsand provide further options for the treatment of patients withHCC particularly those who have relapsed on ï¬rstline treatmentsFurther efforts to enhance the treatment effect of ICIs includedual ICIs treatment and combination therapy of ICIs with otherkinds of targeted agents For dual ICIs treatment the initial resultsfrom CheckMate 9DW were astonishing the objective responserate was higher than monotherapy of any ICIs alone FDA hasapproved nivolumab in combination with ipilimumab for patientswith HCC previously treated with sorafenib Treatment modalitiessuch as radiotherapy and antiangiogenesis agents which affectantigen release or modulate the tumor microenvironments havethe potential to increase the efï¬cacy of immunotherapy and thecombination oftargeted agents with ICIs are attracting theattention of a number of research groups and in vitro studies andearlyphase clinical trials assessing combination treatments haveshown promising antitumor effects in patients with advancedIn vitro evidence by Qui et al131 demonstrated thatHCClenvatinib and regorafenib could affect the expression of PDL1and realtime PCR results suggested that the mRNA expression ofPDL1 in the lenvatinib group was significantly higher than that inthe control group while its expression in the regorafenib groupwas significantly lower When combined with antiPD1 lenvatinibcan modulate cancer immunity in the tumor microenvironmentand enhance antitumor activity132133 In July the FDAannounced its approval of the ï¬rst combination therapy employing the TKI lenvatinib with the ICI pembrolizumab based on theresults from the KEYNOTE524Study NCT03006926 for thetreatment of HCC Recently results from Study Phase IbNCT03418922 showed marginally better results for lenvatinibwith nivolumab than lenvatinib with pembrolizumab METmediated phosphorylation leads to a decreased expression ofPDL1 using the combination of MET inhibitors tivantinib andcapmatinib antiPD1 and antiPDL1 produced an additive effectwhich slows the growth of HCCs in mice134 Clinically based onthe results from the experimental arm A of the GO studyNCT02715531 the FDA approved atezolizumab plus bevacizumab as breakthrough therapy for untreated advanced ormetastatic HCC135 Individual case studies also reported promisingresults for the use of combined TKI and antiPD1PDL1 agents foradvanced HCC136 Such results were conï¬rmed in the phase IIItrialIMbrave study NCT03434379 which reported thatatezolizumab combined with bevacizumab resulted in better OSand PFS than sorafenib in patients with unresectable HCC139Other combination therapies include Galunisertib with nivolumabNCT02423343 spartalizumab with and without capmatinibNCT02795429 FGF401 with spartalizumab NCT02325739regorafenib with pembrolizumab NCT03347292 cabozantinibwithaxitinibNCT03289533 ramucirumab with durvalumab NCT02572687and XL888 with pembrolizumab NCT03095781 Table avelumab withNCT03299946nivolumabImmunerelated adverse events IRAEs occur frequently duringtreatment with ICIs and the clinical consequences can besignificant140 Activation of the immune system leads to damageof normal healthy tissues and IRAEs can have myriad effects andinvolve a number of different ans and have been reported toproduce colitis hepatitis pneumonitis dermatitis myocarditisendocrine glands ammation and rheumatic and musculoskeletal phenotypesincluding ammatory arthritis arthralgiamyositis and sicca syndrome141 Although the precise pathophysiology underlying the IRAEs side effects during treatment withICIs remains unknown discontinuing administration and the useof steroids is generally effectiveIn severe cases howeveradditional immunosuppressants may be required but based oncurrent available evidence immunosuppression for IRAEs does notappearresponse to the ICItreatment142143to compromise the antitumorPromising agents and treatment regimensDespite abovementioned targeted drugs novel agents have beencontinuously under development Table Of note apatinib anovel inhibitor of VEGFR2 tyrosine kinase has attracted considerable attention and there is now a significant body of workdescribing clinical experience with its use Although less effectivethan sorafenib as a ï¬rstline treatment in a retrospective study144apatinib still displayed promising antitumor effects in sorafenibresistant HCC145 where portal vein invasion was present148when metastases have occured149150 and for unresectable andrelapsed HCCs151152 Combination therapy in studies utilisingapatinib with TACE have achieved better clinical effectivenessthan TACE alone with tolerable AEs153 Recentlythecombination of apatinib with the antiPD1 monoclonal antibodycamrelizumab achieved partial response rates of Theresults of other ongoing trials including the phase IIItrialcomparing TACE and apatinib with sorafenib as ï¬rstline treatmentfor locally advanced or metastatic and unresectable HCC NCT and the adjuvant apatinib after hepatectomy for theprevention of tumor recurrence NCT03722875 and NCT03261791will hopefully prove effective and add to the presently availabletherapeutic optionsThese promising results have stimulated the investigation ofother new agents the combinations of agents and regimenswhich have been thoroughly discussed in a recent review fromZhu and Sun154 The combination of bevacizumab and erlotinibhas been extensively evaluated as ï¬rst155 or secondline inadvanced HCCs156 but unfortunately the heterogeneousnature of the results precludes ï¬rm conclusions and recommendations Recently a singlearm metaanalysis of prospectivestudies found that combination therapy with bevacizumab anderlotinib used as secondline treatment was associated with afavorable PFS weeks P and OS months P suggesting that future welldesigned and sufï¬ciently poweredlargescale RCTsshould be able to identify the potentialcontribution of these agents163Signal Transduction and Targeted Therapy 0cTable Trials investigating the combination therapy of ICIs and TKIs in HCCTrial nameidentiï¬erPatient No Study type LineInterventionsPrimaryendpointStudy statusTargeted therapy for hepatocellular carcinomaHuang et alLEAP002NCT03713593Phase IIIFirstLenvatinib pembrolizumab vs lenvatinib PFSOSPhase IIIFirstPhase IIIPhase IIIPhase IIIFirstFirstFirstPhase IIIFirstFirstPhase IIFirstPhase IIPhase Ib II FirstFirstPhase IIPhase IbFirstNivolumab ipilimumab vs lenvatinib orsorafenibCabozantinib atezolizumab vs sorafenib PFSOSSintilimab IBI305 vs sorafenibOS ORRCamrelizumab apatinib vs sorafenibOSPFSOSCamrelizumab apatinib vs FOLFOX orsorafenibNivolumab lenvatinibNivolumab sorafenibSorafenib pembrolizumabAnlotinib sintilimabAvelumab axitinibOSORR AEMTD ORRORRORR AEAEActive notrecruitingOngoingOngoingOngoingOngoingOngoingOngoingOngoingOngoingOngoingCompletedCheckMate 9DWNCT04039607COSMIC312NCT03755791ORIENT32NCT03794440SHR1210III310NCT03764293SHR1210III305NCT03605706IMMUNIBNCT03841201NCT03439891NCT03211416KEEPG 04NCT04052152VEGF Liver NCT03289533KN743NCT03347292GOINGNCT04170556REGOMUNENCT03475953NCT02423343aaRegorafenib pembrolizumabFirstPhase ISecond Regorafenib nivolumabPhase IISecond Regorafenib avelumabPhase IIIPhase Ib II Second Galunisertib nivolumabAEAECR PRPhase Ib MTDOngoingOngoingOngoingOngoingPFS progressionfree survival OS overall survival ORR objective response rate AE adverse events MTD maximum tolerated dose CR complete response PRpartial responseaTrials enroll not only HCC patientsTable Trials investigating targeted therapy in advanced HCCTrial nameidentiï¬erPatient noStudy typeLineInterventionsPrimary endpointStudy statusIMbrave150 NCT03434379ZGDH3NCT02645981HIMALYYA NCT03298451RATIONALE301 NCT03412773PHOCUSNCT04344158ALTER0802 NCT02809534AHELPNCT02329860KEYNOTE394 NCT03062358NCT04080154Phase IIIPhase IIIIIPhase IIIPhase IIIPhase IIIPhase IIIPhase IIPhase IIIPhase IIIPhase IIFirstFirstFirstFirstFirstFirstFirstSecond Apatinib vs placeboSecond Pembrolizumab BSC vs placebo BSCSecond AnlotinibAtezolizumab bevacizumab vs Sorafenib OS PFSDonafenib vs sorafenibOSDurvalumab tremelimumab vs sorafenib OSOSTislelizumab vs sorafenibPexaVec sorafenib vs sorafenibOSAK105 anlotinib vs sorafenibOSPFS 12WAnlotinibOSOSPFSOS overall survival PFS progressionfree survival BSC best supportive careCompletedCompletedOngoingOngoingOngoingOngoingOngoingCompletedOngoingOngoingundergoingevaluationandPreclinical evidence for cyclindependent kinase CDK targetingtherapies in HCC has showed promise and supports theirinvestigation164 especially with the potential ability toabrogate the emergence of sorafenib resistance167 and sensitizeHCC to regorafenib treatment168 A number of CKD inhibitors arepalbociclibpresentlyNCT01356628 milciclibribociclibNCT02524119 The antiMET monoclonal antibody emibetuzumab exhibited the greatest antitumor activity in HCC whencombined with ramucirumab and had an excellentsafetyproï¬le169 and for HCC with high MET expression there was analmost 3fold increase in PFS vs months relative to thosewith low MET expression suggesting the potential for furtherbiomarkerdriven clinical trials Rigosertib is a synthetic benzylstyryl sulfone small molecule inhibitor which has been used in theNCT03109886includingtreatment of monomyelocytic leukemia and due to its activity as aRAS and PLK1signaling inhibitorit was investigated in HCCpatients who demonstrate upregulation of PLK1 during tumordevelopment and HRAS expression in advanced HCC Highexpression levels of PLK1 are also significantly correlated withpoor patient survival and the multiple effects of rigosertib couldbe beneï¬cially employed to produce a therapeutic dualhitapproach in selected patients170 Donafenib is a novel multikinaseinhibitor which is similar to sorafenib displaying comparable orbetter safety and efï¬cacy when treating advanced HCC in phase1b trial and phase studies using sorafenib as the controlNCT02645981171 There are ongoing trials evaluating novelagents such as anlotinib another multikinase inhibitor which isorally administered and targets VEGFR ï¬broblast growth factorreceptor FGFR plateletderived growth factor receptors PDGFRSignal Transduction and Targeted Therapy 0cTargeted therapy for hepatocellular carcinomaHuang et alTable Molecular classiï¬cation of HCCResearcherBoyault et alHoshida et alSchulze et alSia et alKurebayashi et alShinata et alJiang et alYearClassiï¬cationG1G6S1S3Msig iC1iC3 HCCs with adaptive or exhausted immune responsesImmunehigh mid and lowMS1 SI SII and SIIITypeTranscriptomeTranscriptomeExome sequencingMultiomocisImmune cell proï¬lingImmunomicroenvironmentTranscriptome and gonomeProteomicsCase no and ckit NCT02809534 Tivozanib is another oralinhibitor ofVEGFR123 with promising activity against HCC in vivoNCT01835223 and TRC105 which despite demonstrating clinicaltolerated in HCC patients followingactivity and being wellsorafenib has notto date met prespeciï¬ed criteria and itsdevelopment in HCC continues as combination therapy withsorafenib NCT02560779Biomarkerdriven targeted therapyDespite extensive research investigating potential biomarkers to aidthe development of protocols for the treatment of HCC none haveso far been identiï¬ed to be able to predict the effect of or responseto treatment with sorafenib172 Although the molecularclassiï¬cation of HCC has been widely reported Table to date itremains unclear whether this basic genomic and proteomic datawill prove valuable in guiding targeted therapies183The continued belief that the future lies with personalizedtreatment which will be made possible through the rapiddevelopments in next generation sequencing and the precisionmedicine that it underpins have encouraged the development ofnovel trial designs191 These novel trials designs offer new hopethat biomarkerdriven targeted therapies can be modul | 2 |
"radiotherapy in combination with anti cytotoxic T lymphocyte associated antigen monoclonal antibody ipilimumab in patients with metastatic melanoma Nathalie Chaput Gras2 Emilie Lanoy3 Alicia Larive3 Celine Boutros Christine Mateus4 Emilie Routier4 Roger Sun5 Yun Gan Tao5 Christophe Massard6 Rastilav Bahleda6 Dominique Schwob3 Nathalie Ibrahim7 Rita Maria Khoury Abboud7 Caroline Caramella8 Andrea Lancia Lydie Cassard2 Severine Roy4 J C Soria10 Caroline Robert11 Eric Deutsch12 To cite Boutros a0C Chaput Gras a0N Lanoy a0E et a0al Dose escalation phase study of radiotherapy in combination with anti cytotoxic T lymphocyte associated antigen monoclonal antibody ipilimumab in patients with metastatic melanoma Journal for ImmunoTherapy of Cancer 20208e000627 101136jitc2020000627 º Additional material is published online only To view please visit the journal online http dx jitc CR and ED contributed equallyAccepted June Authors or their employers Re use permitted under CC BY NC No commercial re use See rights and permissions Published by BMJFor numbered affiliations see end of Correspondence toDr Eric Deutsch eric deutsch gustaveroussy frBackground A synergy between radiotherapy and anti cytotoxic T lymphocyte associated antigen anti CTLA4 monoclonal antibody has been demonstrated preclinically The Mel Ipi Rx phase study aimed to determine the maximum tolerated dose MTD and safety profile of radiotherapy combined with ipilimumab in patients with metastatic melanomaPatients and methods A dose escalation design was used with and Gy dose of radiotherapy at week combined with mgkg ipilimumab every weeks for four doses Patients with evidence of clinical benefit at week were eligible for maintenance with ipilimumab mgkg every weeks starting at week until severe toxicity or disease progression The database lock occurred on April Tumor growth rate of irradiated lesions and non irradiated lesions were analyzed to assess the systemic immunologic antitumor response Blood immune monitoring was performed before and during treatment to determine if radiotherapy could modify ipilimumab pharmacodynamicsResults patients received ipilimumab between August and July Nine patients received the four doses of ipilimumab All patients received the combined radiotherapy Grade adverse events occurred in nine patients the most common being colitis and hepatitis No drug related death occurred Dose limiting toxicity occurred in two of six patients in the cohort receiving Gy The MTD was Gy Two patients had complete response three had partial response response and seven had stable disease giving an objective response rate of and a clinical benefit rate of at week The median duration of follow up was years Q145 Q368 The median overall survival CI was estimated at years The median progression free survival PFS CI was Radiotherapy combined with ipilimumab was associated with increased CD4 and CD8ICOS T cells Increased CD8 was significantly associated with PFSConclusion When combined with ipilimumab at mgkg the MTD of radiotherapy was Gy This combination of ipilimumab and radiotherapy appears to be associated with antitumor activity Increased CD8 was significantly associated with PFS Thus immune biomarkers may be useful for early response evaluationTrial registration number NCT01557114INTRODUCTIONImmunotherapy using immune checkpoint inhibition has revolutionized the management of patients with advanced stage melanoma and is an emerging approach for many other cancers1 The first immune checkpoint inhibitor that was developed was ipilimumab2 Ipilimumab targets the cytotoxic T lymphocyte associated antigen CTLA4 and significantly improves the overall survival in patients with metastatic melanoma2 However the objective response rate ORR and the disease control rates proportion of patients with a partial response PR or complete response CR or stable disease SD are relatively low and respectively3 As such increased interest has been drawn to enhance the induction of systemic immune responses of ipilimumab by combining it with radiotherapy4 One of the rationale put forward to combine these therapies is that ipilimumab is able to deplete T regulatory cells Treg cells through antibody dependent cell cytotoxicity and consequently increases the CD8 T cell to Treg ratio whereas radiotherapy promotes the diversity of the T cell receptor TCR Boutros a0C et a0al J Immunother Cancer 20208e000627 101136jitc2020000627 0c access repertoire of intratumoral T cells7 When combined ipilimumab promotes the expansion of T cells while radiation enhances the TCR repertoire of the expanded peripheral clones4 Ipilimumab at a dose of mgkg is approved in several countries for the treatment of unresectable or metastatic melanoma In a recent prospective phase study patients were treated with ipilimumab mgkg combined with concurrent or sequential stereotactic ablative radiotherapy11 Dose limiting toxicities DLTs and grade toxicities were reported in and of patients respectively PR and clinical benefit were reported in and of patients respectively However few clinical studies were conducted to assess the efficacy of ipilimumab mgkg combined with radiotherapy11 although overall survival was significantly improved with the ipilimumab mgkg monotherapy compared with the mgkg dose2 Here we present the phase Mel Ipi Rx study investigating the safety and efficacy of ipilimumab mgkg combined with radiotherapy in patients with metastatic melanoma Although the treatment landscape of patients with advanced melanoma has changed since this study was initiated the increased survival benefit of ipilimumab mgkg compared with mgkg suggests that the clinical utility of ipilimumab in refractory patients with high unmet medical need could warrant further assessment The primary objective of this study was to determine the maximum tolerated dose MTD DLT and the recommended phase dose RPTD of radiotherapy administered in combination with ipilimumab at a dose of mgkg in patients with metastatic melanoma The secondary objectives were to determine the adverse event AE profiles to describe the preliminary antitumor activity following escalating doses of radiation combined to ipilimumab using the immune related response criteria irRC and to evaluate the overall survival in patients treated with this combination The exploratory objectives were to evaluate the systemic immunologic antitumor response and factors influencing this responseMETHODSPatientsEligible patients had unresectable locally advanced or metastatic melanoma with at least one measurable metastasis accessible to radiotherapy Subcutaneous nodules and lymph nodes were considered as targets for irradiation Tumor lesions located within vital ans or gastrointestinal tract were not considered as target for radiotherapy All patients had measurable and non measurable disease evaluable according to irRC Patients were ¥ years of age were able to give written informed consent had Eastern Cooperative Oncology Group ECOG performance status of to and had normal renal and liver functions on blood tests Patients were excluded if they had one of the following criteria suspected or known central nervous system tumors including brain metastasis any other malignancy with a disease free for less than years an autoimmune disease a history of prior treatment with ipilimumab prior radiotherapy within the same body area or radiotherapy in fields containing flat bones volume If chemotherapy or immunotherapy were previously used a wash out period of weeks at least was required before the first administration of ipilimumabStudy designThis phase I dose escalation study evaluated the MTD of radiotherapy in combination with ipilimumab in patients with unresectable locally advanced or metastatic malignant melanoma Eligible patients received ipilimumab at mgkg at weeks and for a total of four doses at week intervals online supplementary figure S1 Patients without progressive disease PD who tolerated the treatment continued ipilimumab dosing in week intervals until progression or withdrawal of consent Radiotherapy was delivered on week on Monday Tuesday and Friday at the time of the second cycle of ipilimumab on measurable superficial lesions including subcutaneous nodules and lymph nodes Radiotherapy was delivered using MV photons or electrons with standard field encompassing Maximal field had to be at least à cm but not be more than à cm maximal dimensions on a lesion Millimetric margins were used to take into account microscopic spreading and patient movements clinical and planning target volumes of mm Dose escalation of ionizing radiation was planned in cohorts of three to six patients depending on the occurrence of DLTs during the first combination treatment cycle A hypofractionated radiation regimen higher doses per fraction was used Dose escalation was used with total doses of and Grays Gy administered in three fractions every Monday Wednesday and Friday and planned in cohorts of three to six patients depending on the occurrence of DLTs from week to week A minimum deadline of hours between two radiotherapy sessions had to be respected If necessary the radiotherapy could be put back to Tuesday Thursday Saturday Given the small size of the radiation field and the fact that irradiated lesions were superficial vital ans and gastrointestinal tract were not involved in the irradiated fieldToxicities were evaluated according to the Common Terminology Criteria for Adverse Events CTCAE version DLT observation period ranged from week to Any toxicity before the start of radiotherapy was not considered as a DLT but as a toxicity from ipilimumab alone The DLT was defined by the appearance of at least one of the following study combination related event within weeks grade vomiting diarrhea or gastrointestinal bleeding grade or non hematologic toxicity excluding grade nausea vomiting diarrhea and transient fever or grade or radiation dermatitis except if return to grade ¤ within weeks If none of the first three subjects in a given dosing cohort had experienced DLT dose escalation was realized If Boutros a0C et a0al J Immunother Cancer 20208e000627 101136jitc2020000627 0cone of the first three subjects in a given dosing cohort had experienced DLT an additional three subjects were enrolled to that dose level before further escalation was considered A dose escalation was realized if none of these additional three subjects had experienced DLT If two of the first three subjects in a given dosing cohort had experienced DLT dose escalation was stopped Dose escalation was continued until one third or more of the subjects at a particular dose level experienced DLT or up to the fourth cohort This was considered the maximum administered dose MAD The MTD was defined as the highest dose at which less than one third of the subjects experienced DLT The RPTD was defined as the MTD or the dose that was considered to give the optimal clinical andor immunological results Tumor assessments consisting of CT scans and assessment of skin lesions were performed at weeks and every weeks thereafter until the patient withdrew from the study or entered the follow up phase MRI or CT scan of the brain had to be realized when clinically indicated Evidence of PD was confirmed by a second assessment performed weeks later Definitions of lesions were based on irRC Measurable lesions were defined as lesions that could be accurately measured in two perpendicular diameters with at least one diameter ¥ mm and the other dimension ¥ mm It was possible to consider skin lesions if they were measurable All measurable lesions up to a maximum of lesions per an and lesions in total were identified as index lesions measured and recorded at screening and follow up The index lesions were representative of all involved ans Non index lesions corresponded to measurable lesions that were recorded and evaluated at the same assessment time points as the index lesions and that were irradiated according to the trial irradiation designBlood immune monitoringBlood samples were collected at baseline T0 at week W4 before second injection of ipilimumab and at week W6 after radiotherapy and before third injection of ipilimumab online supplementary figure S2 Phenotyping was performed on fresh whole blood and peripheral blood mononuclear cells PBMCs isolated by Ficoll density gradient were frozen for later analyses Data analysis from standard blood tests was realized to estimate the derived neutrophil to lymphocyte ratio dNLR at baseline W4 and W6 to see whether or not granulocytes could impact the prognosis of patients Whole blood or PBMCs were incubated with fluorochrome conjugated antibodies for min at room temperature or at °C respectively followed by min of lysis Versalyse Beckman Coulter Mervue Galway Ireland The following fluorochrome conjugated antibodies were used fluorescein isothiocyanate anti ICOS CD278 clone DX29 phycoerythrin PE conjugated anti CD25 clone B1499 allophycocyanin cyanine APC Cy7 conjugated CD45RA clone HI100 phycoerythrin cyanine PE Cy7 conjugated anti CD45RA clone access2H4 allophycocyanin Alexa Fluor AA700 conjugated anti CD3 clone UCHT1 Pacific Blue PB conjugated anti CD4 clone 13B82 and Krome orange conjugated anti CD8 clone B911 were obtained from Beckman Coulter Stained cells were acquired using a FACS Canto II cytometer BD Bioscience or a Gallios Cytometer Beckman Coulter and analyzed using Kaluza software Beckman Coulter Conventional T cells CD3CD4 and CD3CD8 were respectively defined by CCR7CD45RA for naïve T cells CCR7CD45RA for central memory T cells TCM CCR7CD45RA for effector T cells TEM and CCR7CD45RA for terminally differentiated T cells TEMRA Treg cells were defined as CD3CD127lowCD25Tumor growth rate analysisTo assess the systemic immunologic antitumor response tumor growth rate TGR of irradiated lesions and non irradiated lesions were analyzed Briefly TGR estimates the variation in tumor volume over time using each patient as his own control and has shown to be interesting to identify the specific therapeutic effect of a treatment regardless of the disease course of each patient16 TGR was expressed as a percentage increase in tumor volume during month in accordance with Hiniker et al11 using the following formula TGR100 [exp[3LogDtDt0t] ] with D0tumor size defined as the sum of the longest diameters of the lesions at baseline Dttumor size defined as the sum of the longest diameters of the lesions at the time t of evolution evaluation in monthsTGR100 [exp3LogDtDt0t]The TGR was computed for irradiated lesions TGRirr and non irradiated target lesions defined by the radiologist for the irRC evaluation TGRnon irr for two treatment periods when data were available the Reference TGR REF TGR assessed before the onset of the treatment using the baseline CT and a CT realized before the baseline prebaseline CT the Experimental TGR EXP TGR assessed between the onset of the treatment and the first evaluation The TGR was computed using the same target lesions at each evaluation time A positive value of the EXP TGR reflected a bigger lesion at the first evaluation than at baseline Therefore new lesions related to PD were not included in the TGR computation Difference between EXP TGR and REF TGR was used to assess the effect of the treatment ÎTGREXP TGRREF TGR a negative value reflecting a slowdown of the natural course of the disease ie a slower tumor growth or a tumor response between the two periodsStatistical analysisDemographic and baseline characteristics were summarized for all registered subjects using descriptive statistics Toxicity grades per subject were tabulated for AEs and on study laboratory measurements by using the NCI CTCAE version Analyses of efficacy endpoints were based on all subjects evaluable for efficacyBoutros a0C et a0al J Immunother Cancer 20208e000627 101136jitc2020000627 0c access The Kaplan Meier method was used to estimate overall survival and immune related progression free survival PFS Overall survival was defined as the time from treatment initiation to the date of death or date of last follow up in persons alive at last follow up Immune related progression was defined according to irRC An event was defined as progression or death whichever comes first PFS was defined as the time from treatment initiation to the date of occurrence of the event Follow up of patients who did not experience was censored at the date of last evaluation Duration of follow up was estimated using the Schemper and Smith method18 All statistical analyses were done using SAS software V94Evolution of innate immune cells was analyzed using GraphPAD PRISM software values at W4 and at W6 were described and compared with baseline by using Friedman test followed by Dunn's multiple comparisons testStudy oversightThe study was conducted in accordance with the protocol Good Clinical Practice guidelines and the provisions of the Declaration of Helsinki All patients provided written informed consentRESULTSPatient populationNineteen patients with advanced melanoma were treated at Gustave Roussy in the Mel Ipi Rx phase trial between August and July online supplementary table S1 The database lock occurred on April Nine patients received the four doses of ipilimumab and two patients received maintenance ipilimumab one and two cycles respectively All patients received the combined radiotherapy at week in three fractions Thirteen patients were enrolled at dose level Gy and six patients at dose level Gy Demographic data of patients are summarized in table Overall patients presented visceral metastases M1c had elevated level of lactate dehydrogenase LDH tumors were BRAF mutated and none had a history of brain metastases All patients had received systemic therapy previously Prior therapy included chemotherapy in patients BRAF inhibitors in patients radiotherapy in patient and surgery in patients respectively and all of them were immune checkpoint inhibitor naïve Irradiation was delivered on subcutaneous lesions in patients and patients in the cohorts treated with and Gy respectively and on lymph nodes in patients and patients in the cohorts treated with and Gy respectively online supplementary table S2 The average irradiated tumor volumes were relatively homogeneous with a median of mm IQR SafetyAll patients presented at least one AE of any grade These AEs were felt to be probably related to ipilimumab The role of concurrent radiotherapy on these AEs was difficult Table Patient baseline characteristicsCharacteristics SexMaleFemaleAge yearsMedianRangeMutation BRAF statusNon mutantMutantUnknownLactate dehydrogenaseNormalElevatedM staging extent of metastasesM0M1aM1bM1cBrain metastasesNoYesSite of irradiated lesionsLymph nodesSubcutaneous nodulesVisceral ansPrevious treatments No prior systemic treatmentIpilimumabChemotherapyAnti BRAFRadiotherapySurgeryOverall populationN19 No The normal range for lactate dehydrogenase is UIL All patients were naïve to immune checkpoint inhibitorsto assess However no radiation induced necrosis or local symptoms were observed inside the radiation field AEs of all grades are summarized in table regardless of their attributionAsthenia was the most commonly reported AE of any grade It occurred in patients Among them nine patients were treated in the Gy cohort and five patients in the Gy cohort The median time to onset of asthenia was Q1 Q34 weeks and the median duration was Q1 Q34 weeks The other most common AEs of Boutros a0C et a0al J Immunother Cancer 20208e000627 101136jitc2020000627 0cTable Adverse events of all grade safety populationNo of patients with at least one adverse eventsFatigueDiarrheaDisease related painFeverPainNauseavomitingAnorexiaConstipationColitisPruritusWeight lossAnemiaEdema of limbsDyspneaSkin eruptionVitiligoHypereosinophiliaAlanineaspartate aminotransferase increasedCoughLymphedemaLevel dose N13 Level dose N6 TotalN19 any grade included diarrhea disease related pain fever nausea and vomitingThirteen patients discontinued the study owing to treatment related AEs nine patients in the cohort receiving Gy and four patients in the cohort receiving Gy Nine of these patients had AEs of grade Multiple AEs of grade occurred in some patients AEs of grade included colitis n2 hepatitis n2 asthenia n2 thyroid disorders n1 DRESS drug rash with eosinophilia and systemic symptoms syndrome n1 and nauseavomiting n1 Online supplementary table S3 presents the grade and grade events for each dose level for all events There were no treatment related deathsFour of the patients who discontinued the study did not have grade or AEs Indeed three patients had grade colitis associated with either contraindication to corticosteroids because of uncontrolled diabetes mellitus or complete remission after four cycles of ipilimumab leading to regular follow up without maintenance ipilimumab One patient had a grade asthenia after four cycles of ipilimumab leading to regular follow up without maintenance ipilimumab accessDose escalation and DLTsEighteen patients were evaluable for DLTs in this study patients in the cohort treated with Gy and patients in the cohort treated with Gy Among them four patients experienced DLTs All the DLTs occurred outside the radiation fieldIn the cohort treated with Gy of evaluable patients experienced DLTs after two cycles of ipilimumab combined with radiotherapy Indeed one of these patients presented grade hepatitis and the other one presented grade colitis with grade hypokalemia grade anorexia and grade thyroid disorders In both patients the three fractions of radiotherapy on Monday Wednesday and Friday were delivered on week DLTs led to permanent ipilimumab discontinuation in both patientsIn the cohort treated with Gy two of the six evaluable patients experienced a DLT after two and three cycles of ipilimumab respectively combined with radiotherapy One of these patients presented grade hepatitis and the other one presented grade colitis with unusually normal macroscopic colonoscopy but a total villous atrophy mimicking celiac disease on biopsies In both patients the radiotherapy fractions on Monday Wednesday and Friday were delivered on week DLTs led to permanent ipilimumab discontinuation in both patientsThe MTD of radiotherapy was thus Gy when combined with ipilimumab mgkg in the present design Consequently the RPTD of radiotherapy administered in combination with ipilimumab at mgkg in patients with metastatic melanoma was GyClinical outcomesAt the time of analysis the median duration of follow up was years Q145 Q368 The median overall survival CI was estimated at years The median PFS CI was Figure shows Kaplan Meier median overall survival and PFS curves with CI According to irRC the best response within the trial was CR for two patients both in the cohort receiving Gy PR for three patients two patients in the cohort receiving Gy and one patient in the cohort receiving Gy and SD for seven patients six patients in the cohort receiving Gy and one patient in the cohort receiving Gy giving an ORR of and a clinical benefit rate of at week online supplementary table S2 The initial melanoma staging of the patients who had CR was M1a for one patient multiple subcutaneous nodules and M1c multiple subcutaneous nodules and lymph nodes associated with an elevated LDH for the other patient The initial melanoma staging of the patients who had PR was M1a for one patient multiple subcutaneous nodules and M1c for two patients one of these patients had a subcutaneous nodule a lymph node and elevated LDH The other patient had multiple lymph nodes and bone metastasisBoutros a0C et a0al J Immunother Cancer 20208e000627 101136jitc2020000627 0c access Figure A Waterfall plot of patients according to the variation of tumor growth rate ÎTGR between reference before treatment and experimental period on treatment For each patient specific ÎTGR of irradiated and non irradiated lesions are represented ÎTGR on treatment lesions at the first evaluation are bigger than at baseline ÎTGR of non irradiated lesions was superior to the irradiated lesion B Changes of the sum of diameters of the target lesions irradiated and non irradiated respectively at months in compared with the baselineVariation of tumor growth rate across lesionsTGR variation for irradiated and non irradiated lesions before and after the treatment was evaluable for patients figure A total of non irradiated lesions with up to lesions for one patient were evaluated for the REF and EXP TGRnon irr median IQR non irradiated lesions by patient and irradiated lesions for the REF and EXP TGRirr one patient had two irradiated lesions Median reference and experimental period were IQR and IQR months respectively The sum of diameters of lesions at the three evaluation times prebaseline baseline first evaluation and corresponding TGR are presented in table The EXP TGR on treatment was not correlated with the REF TGR before treatment Spearmans rho011 p073 Decrease of the TGR was of the tumor sizemonths IQR to for irradiated lesions and month IQR to for non irradiated lesions although the difference was not significantly different p082 Response Figure Kaplan Meier median overall survival A and progression free survival B curves with CINo pseudoprogression was observed among the patients treated in this study nor radiation induced necrosis or edema Of note three patients were not evaluable for response because they progressed and died before the radiologic assessment scheduled in this studyTreatments administered after the Mel Ipi Rx study in patients with disease progression are summarized in online supplementary figure S3Boutros a0C et a0al J Immunother Cancer 20208e000627 101136jitc2020000627 0cTable Characteristics of lesions evaluated for tumor growth rate TGRPatients nLesions n sumLesions n median IQRSum of diameters prebaseline median IQRSum of diameters baseline median IQRSum of diameters at first evaluation median IQRREFperiod median IQREXPperiod median IQRREF TGR median IQREXP TGR median IQRTGRdiff median IQR accessWilcoxon p valueOverallIrradiated lesionsNon irradiated lesions to to to to to to to to to to to to to to to to to to to to to REF TGR corresponds to TGR before the start of the treatment EXP TGR corresponds to TGR between the start of the treatment and the first evaluation at monthsof non irradiated lesions seemed more representative of patient outcomes vs irradiated lesion EXP TGRnon irr was significantly higher in patients with PD Although the number of patients was too low for statistical test p values are shown for information in table figure and online supplementary figure S4Immune analysesWe analyzed immune parameters in the blood that have been previously described to be changed during ipilimumab treatment to determine if radiotherapy could modify ipilimumab pharmacodynamics19 For CD4 T cell counts and as expected ipilimumab alone W4 could favor accumulation of TEM Treg and ICOSCD4 T cells Interestingly at W6 after ipilimumabradiotherapy only TEM and ICOSCD4 T cells remained significantly increased suggesting that combination favored accumulation of activated memory CD4 T cells rather than Treg cells For CD8 T cell counts no accumulation could be observed at W4 while augmentation of TCM and TEMRA could be depicted between W4 and W6 suggesting that adjunction of radiotherapy to ipilimumab was more prone to boost these CD8 T populations online supplementary figure S2 High fold change in CD8 from baseline to week was significantly correlated with PFS p00223 but not significantly correlated with overall survival p02355 online supplementary figure S5Innate immune cells and NLR absolute neutrophils count ANC divided by the number of lymphocytes or dNLR ANC divided by the number of white blood cellsANC have been shown to have a prognostic role in patients treated with immunotherapy and even might represent a predictive biomarker of response We took advantage of standard blood tests to determine if Table Univariate analysis of tumor growth rate TGRAll lesions n37Progressive disease WilcoxonNop valueYesIrradiated lesion n13Progressive diseaseNoYesWilcoxon p valueNon irradiated lesions n24Progressive diseaseNoWilcoxon p valueYesSum of diameters prebaseline meanSum of diameters baseline meanSum of diameters at first evaluation meanREF TGR meanExp TGR meanÎTGR meanTGR is evaluated in percentage per months ÎTGR EXP TGR REF TGR A negative value corresponds to a slowdown of the tumor growthBoutros a0C et a0al J Immunother Cancer 20208e000627 101136jitc2020000627 0c access neutrophils monocytes NLR or dNLR at baseline W4 and W6 could correlate with the prognosis in our study ANC or monocytes did not correlate with the prognosis of patients while absolute count of lymphocytes significantly increased at W6 compared with baseline in only patients with a clinical benefit CRPRSD Both NLR and dNLR were significantly lower at W6 only in patients with clinical benefit online supplementary figure S6 Note that we did not found an association with the dose of radiotherapy data not shownDISCUSSIONIn this dose escalation phase study four patients experienced DLTs All the DLTs occurred outside the radiation field Therefore it was difficult to assess the role of concurrent radiotherapy on the DLTs The MTD of radiotherapy combined with ipilimumab at mgkg was Gy The RPTD of radiotherapy administered in combination with ipilimumab at mgkg in patients with metastatic melanoma was Gy A hypofractionated radiation regimen higher doses per fraction was used in this study It is usually preferred for melanoma which displays low alphabeta ratio Our three fractions hypofractionated regimen is in line with the radiation standard in the metastatic setting Moreover it has been shown recently that radiation doses per session inferior to Gy combine more favorably with immunotherapy through interferon type induction21 Of note our study is the only one that combines radiotherapy and high dose of ipilimumab mgkgThe incidence of treatment related AEs was high with this combination in our study but numerically similar to the incidence reported previously with ipilimumab monotherapy at mgkg Grade or AEs occurred in of patients in our study whereas they occurred in of patients treated with ipilimumab monotherapy at mgkg2 The overall AE spectrum of the combination in this study was consistent with previous findings with the drugs immune based mechanism of action The high rate of AEs might be partially attributed to the high dose of ipilimumab The dose of mgkg was chosen based on data from the randomized phase trial that compared various | 2 |
"ammatory bowel disease ibd is a long life disease with remission and relapse periods ibd arises as a result of inappropriateimmune response to intestinal commensal anisms in individualswith genetic predisposition and consequently causes ammation andintestinal ulcers in addition ibd has a complex pathogenesis andmany factors such as dysbiosis oxidative stress and epigenetics thatmay also be involved in disease pathogenesis [] ulcerative colitisuc and crohn's diseases cd are known as two main forms of ibdaccordingly these diseases cause intestinal ulcers and some annoyingsymptoms such as diarrhea abdominal pain and rectal bleeding occasionally the severity of these symptoms is very high which can leadpatients to be hospitalized in this regard therapeutic approaches totreat these diseases mainly focus on prolonging remission and are almost similar however diï¬erential diagnosis can also help to treat thedisease in a more eï¬ective way for example 5asa which is acommon drug in the treatment of ibd is less eï¬ective on maintainingremission in cd patients on the other hand antibiotic therapy is notrecommended for the treatment uc but it can be eï¬ective on cd patients diï¬erential diagnosis is a serious challenge because cdand uc have significant similarities in terms of their clinical endoscopic and histological features however there are some diï¬erencesbetween uc and cd which are summarized in table1 in addition tointestinal complications uc and cd also have significant extraintestinal manifestations for example it was shown that uc is significantly associated with primary sclerosing cholangitis and cd is alsoassociated with cholelithiasis especially in cases that the ileum is involved furthermore cd can cause ï¬stulas to the urinary systemwhich leads intestinal bacteria to enter the urethra and recurrent urinary tract infections both cd and uc can cause several disorderssuch as arthritis erythema nodosum pyoderma gangrenosum andanemia which are known as the most important extraintestinal manifestations of ibd the latest statistics showed that the global corresponding author at department of clinical biochemistry and laboratory medicine faculty of medicine tabriz university of medical sciences daneshgahstreet po box tabriz iranemail address vagharimtbzmedacir m vagharitabari101016jcca202008025received july received in revised form august accepted august available online august elsevier bv all rights reserved 0cf khakikhatibi table1clinical endoscopic and histological features of cd and ucclinical featuresfeaturesrectal bleedingabdominal painfevermucus defectionintestinal obstructionperineal diseasepostoperative recurrenceasca positiveanca positiveendoscopic featurescdoccasionallyfrequentlyfrequentlyoccasionallyyesyesyesfrequentlynot commonucfrequentlyoccasionallynot commonfrequentlynonononot commonfrequentlyfeaturescduclocationmucosal involvementdepth of ulcerationï¬stulacobblestone appearanceaphthous ulcerationmucosal friabilityhistological featuresfeaturesgranulomascrypt abscessespatchinessany part of gi tractdiscontinuousdeepyesyesfrequentlynot commoncdfrequentlynot commonfrequentlycolon and rectumcontinuoussuperï¬cialnonooccasionallyfrequentlyucrarefrequentlynot commonprevalence of ibd currently is on the rise and it is not an exaggerationif we consider it as a global serious health problem according to areport published in ibd has the highest prevalence rate ineurope and its prevalence in the newly industrialized countries of asiaafrica and south america also appears to be increased over the pastthree decades unfortunately the peak of the disease is at the young age of years old therefore in addition to the suï¬ering from icts on the patients it also has many negative eï¬ects on societymoreover many ï¬nancial burdens are annually imposed on countriesfor controlling and treating this chronic disease the invasive diagnosticand therapeutic measures are currently undertaken to diagnose andmanage ibd which are unpleasant for patients as well as having thehigh associated costs now the gold standard method for diagnosingibd and monitoring patient status is performing colonoscopy examination and histopathologic evaluation which are invasive measures therefore in recent years many studies have been conducted toï¬nd a suitable laboratory marker with suï¬cient sensitivity and speciï¬city for the purpose of diagnosing and noninvasive management ofibd a high proportion of these studies have investigated the eï¬cacy offecal calprotectin in diagnosing and monitoring patients althoughsome of these studies reported auspicious results there are still somedoubts on the eï¬ectiveness of fecal calprotectin on diagnosing andmonitoring ibd patients so in this review we addressed the advantages and limitations of fecal calprotectin for the diagnosis andmanagement of ibd the role of fecal calprotectin in diagnosis and management ofibdthe eï¬cacy of fecal calprotectin as an laboratory marker in various areas of ibd diagnosis and management have been studied including ibd diï¬erentiation from irritable bowel syndrome ibs evaluation of endoscopic activity of the disease evaluation of histologicalactivity of the disease and prediction of disease recurrence andclinica chimica acta response to treatment in following after a brief introduction andmentioning the important points regarding laboratory measurement offecal calprotectin we reviewed the most interesting ï¬ndings in all ofthe abovementioned areas calprotectin a clinically valuable proteincalprotectin is an antimicrobial protein mainly secreted by neutrophils this protein competes with bacteria over zinc thus kills thebacteria however this is not the only contribution that it has to antimicrobial activity moreover this protein has many potential clinicalapplications such as the elevated serum levels that have been observedunder various immunological and immunopathological conditionsserum calprotectin levels rapidly increase in response to bacterial infections in the kidney and heart or during transplant rejection at theearly stages of ammation of the lung serum calprotectin can also beconsidered as a reliable marker besides plasma levels of calprotectinappear to be useful in reï¬ecting disease activity in ammation of thejoints in addition it has been demonstrated that serum calprotectin levels are increased in patients with bacterial sepsis so it can beconsidered as a reliable biomarker in neonatal sepsis the serumlevel of calprotectin increases as well as a sensitivity of and aspeciï¬city of that have been reported for serum calprotectin indiagnosis of neonatal sepsis it has been recently shown thatserum calprotectin levels increase in patients with aneurysmal subarachnoid hemorrhage and higher levels in the ï¬rst of onset areassociated with a poor prognosis at the ï¬rst three months serumcalprotectin levels also increase in patients with rheumatoid arthritisand even in patients with a moderate to high disease activity who havenormal or low crp levels so they appear to be more eï¬cient at reï¬ecting disease activity some studies have also investigated the eï¬cacy of serum calprotectin in the diagnosis of cancers correspondingly in one of thesestudies it was shown that serum calprotectin levels significantly increased in patients with laryngeal carcinoma compared with healthyindividuals and those with benign laryngeal pathologies moreover inthis study a direct relationship was also observed between serum levelsof calprotectin and stage of cancer another study showed that theserum level of calprotectin increased in patients with papillary thyroidcarcinoma but it significantly decreased after operation alsoregarding the eï¬cacy of serum and saliva calprotectin for the diagnosisof ibd impressive results have been reported a study onpatients with ibd both uc and cd have shown that serum calprotectinlevels were directly correlated with fecal calprotectin levels and weremore potent in ibd diagnosis compared to crp and albumin this studyalso indicated that the combination of serum calprotectin with crp oralbumin can be helpfulin the prediction of treatment escalationespecially in patients with cd however no significant correlationwas observed between serum calprotectin and fecal calprotectin levelsin patients with cd and uc as well as a slight correlation betweenserum calprotectin level and crp that was observed only in patientswith uc another study showed that the serum level of calprotectin was significantly higher in patients with cd compared to healthyindividuals in addition although a significant correlation was observedwith the clinical activity of the disease no significant correlation wasfound between the level serum calprotectin and endoscopic activity ofthe disease the eï¬cacy of salivary calprotectin in the diagnosisof ibd has also been studied which showed that salivary calprotectinsignificantly increased in patients with ibd compared to healthy individuals in this study auc values for unstimulated saliva and stimulated saliva to distinguish ibd patients from healthy individualswere reported to be and respectively however thepopularity of calprotectin is mainly due to the use of fecal calprotectinin the diagnosis and management of ibd that is discussed in the following 0cf khakikhatibi clinica chimica acta laboratory measurement and reference intervalfecal calprotectin is a stable protein that remains stable for daysat room temperature this property is an excellent advantage for alaboratory marker also it seems that keeping the specimen at refrigerated temperature °c can increase the stability of fecal calprotectin however evidence has been obtained regarding thatthe stability of this protein decreases after staying for three days atroom temperature on the other hand it is not also recommended tokeep samples in the refrigerator for more than days it seemsthat fecal calprotectin remains stable up to one year at °c measurement of fecal calprotectin can be done both qualitatively andquantitatively accordingly in the qualitative measurement monoclonal antibodies are used to detect fecal calprotectin and the positiveresults are characterized by the appearance of colored lines on the testcassette however in the qualitative one only positive or negative results are reported and despite of sensitivity test speciï¬city in theevaluation of disease activity was reported to be only it seems thatthe main application of this test is to diï¬erentiate healthy individualsfrom ibd patients rapidly however some studies have shown that it isnot accurate enough in this case as well nevertheless asignificant concordance has been reported between home test resultsibdoc and fecal calprotectin laboratory measurement results whenquantum blue calprotectin elisa kit was used notably the agreements between results were and depending on the selectedcutoï¬s several commercial kits are also available for fecal calprotectin qualitative test known as rapid calprotectin these tests reportpositive results ranged from to µgg there are also severalcommercial kits that can be used for the quantitative measurement offecal calprotectin these kits are usually designed in terms of the elisamethod and some have a measurement range between and µgg moreover the chemiluminescence immunoassays cliamethod can also detect values between and µgg fluoro enzyme immunoassays feia and particle enhanced turbidimetric immunoassays petia can also be used for the measurement of fecalcalprotectin in this regard one of the most serious challenges to thelaboratory evaluation of fecal calprotectin is the determination of theupper limit in healthy individuals among healthy adults there is asignificant agreement on µgg as an upper limit one study suggested values up to µgg in people over years old and up to µgg in children aged between and years old as referenceranges of fecal calprotectin in healthy individuals fecal calprotectin levels appear to be higher in healthy infants andchildren under four years old than in adults and further studies areneeded in this regard to determine the acceptable upper limit for diagnosis of pediatric ibd table lists the median levels of fecalcalprotectin in healthy individuals with diï¬erent ages reported in somestudies according to these reports age can aï¬ect fecal calprotectinlevels fecal calprotectin and ibd diagnosisonly a small percentage of patients complaining of abdominal painand diarrhea have ibd in many cases ibs as a functional gastrointestinal disorder is known as the cause of such clinical symptomspatients with ibs have normal colonoscopy results while ibd patientsindicate abnormal colonoscopy results and have intestinal ulcersunfortunately the significant prevalence of ibs and the overlap between clinical symptoms and ibd can increase the colonoscopy ratetherefore a noninvasive diagnostic marker can be very helpful in thisregard notably the ï¬rst evidence of the eï¬cacy of fecal calprotectin inthe diagnosis of ibd was obtained in the 1990s røseth in proposed a method for measuring calprotectin in stool specimens one of the ï¬rst and most interesting studies regarding fecal calprotectinutility in ibd diagnosis was the study by røseth published in in this study patients with ulcerative colitis were studied and according to their results fecal calprotectin levels are higher in patientswith ulcerative colitis compared to healthy controls this study havealso shown that even patients with low disease activities had higherlevels of fecal calprotectin compared to healthy individuals subsequent studies somehow conï¬rmed and complemented the ï¬ndings of this study in another study published in auc values of ci were reported for fecal calprotectin in thediagnosis of colorectal ammation moreover in a study onchildren with ibd it was shown that the level of fecal calprotectin washigher in these patients compared to healthy children so it can beconcluded that it is also directly correlated with esr levels in astudy published in kolho reported auc values of ci for fecal calprotectin in the diagnosis of pediatric ibd in a study on patients with crohn disease a sensitivity of and a speciï¬city of at cutoï¬ of μgg have been reportedfor fecal calprotectin in diagnosis of the disease the results of ourrecent study along with other studies showed that fecal calprotectin ispreferred over traditional ammatory biomarkers such as crp andesr in the diagnosis of ibd diamanti reported a sensitivity of and a speciï¬city of for fecal calprotectin at a cutoï¬ of μgg in ibd diagnosis in our recent study a sensitivityof and a speciï¬city of at a cutoï¬ of μgg were observed for fecal calprotectin in the diagnosis of ibd however oursample size was and the majority of patients were in the active phaseof the disease in another study conducted on patients with ulcerative colitis asensitivity of and a speciï¬city of at cutoï¬ of μgg havebeen reported in this regard in one study it was shown that fecalcalprotectin in cutoï¬ of μgg is able to distinguish patients withibd from patients without ibd patients with diseases other than ibdpatients with ibs and healthy persons with sensitivity and speciï¬city caviglia in their study reported a sensitivity of and a speciï¬city of at a cutoï¬ of μgg for fecalcalprotectin in diï¬erentiating between ibs and ibd howeversome studies have reported significantly lower values accordingly in astudy on patients with ulcerative colitis kalantari reported asensitivity of and a speciï¬city of at a cutoï¬ of μgg besides there is a considerable agreement between fecal calprotectinand capsule endoscopy ï¬ndings in patients with crohn's disease asensitivity of and a speciï¬city of have also been reported at acutoï¬ of mgkg for fecal calprotectin in predicting ce ï¬ndings anddiagnosis of crohn's disease in another study lower sensitivityand speciï¬city rates sensitivity speciï¬city were reportedfor fecal calprotectin in this regard furthermore in one studythat examined the eï¬cacy of fecal calprotectin in predicting wirelesscapsule endoscopy ï¬ndings a sensitivity of and a speciï¬city oftable reported median levels of fecal calprotectin in healthy individuals of diï¬erent agesagesmedian levels of fecal calprotectin range µggnumber of subjectsused kitup to monthchildren yearschildren yearsadultsover years bühlmann elisabühlmann elisacalpro® calprotectin elisa test alpphicalphicalreference 0cf khakikhatibi were reported for this biomarker at μgg in the diagnosis ofsmall bowel ammation in crohn's disease given these ï¬ndings it seems that fecal calprotectin has no ideal sensitivity and speciï¬city for the diagnosis of ibd where the small intestine is involvedbesides there are some preanalytical limitations which are explainedin the next sections therefore optimistically speaking fecal calprotectin measurement can eliminate the need for colonoscopy howeverin a metaanalysis performed to evaluate the eï¬cacy of fecal calprotectin and some other ammatory markers to diï¬erentiate betweenibd and ibs the probability of ibd was less than at fecal calprotectin values lower than µgg or crp values lower than mgdl therefore it seems that fecal calprotectin can be helpful at leastin ruling out the possibility of ibd in patients with ibslike symptoms aswell as reducing the rate of colonoscopy moreover it should be notedthat although a systematic review has reported pooled sensitivity andspeciï¬city above for fecal calprotectin to diï¬erentiate between ibdand ibs it emphasized more on the possibility of falsepositive resultsin low cutoï¬ points hence performing extensive studies indiï¬erent countries on the healthy population and the ibd patient is beneeded to determine a suitable cutoï¬ with maximum sensitivity andspeciï¬city and minimum falsepositive resultstable summarizes the results of various clinical investigationsregarding fecal calprotectin utility in the diï¬erential diagnosis of ibdfrom ibs and table4 summarizes some metaanalysis results in thisregard as shown in table the most important limitation of the majority of clinical studies conducted to date is the small sample size alarge global study may be helpful in providing a more precise evaluation of fecal calprotectin clinical value in discrimination between ibdand nonibd diseases fecal calprotectin and endoscopic and histologic activity evaluationundoubtedly one of the most serious challenges in the managementof ibd is evaluating the endoscopic and histologic activities of thedisease nowadays colonoscopy and histopathologic examinations arethe routine tools for the assessment of mucosal healing in patients withibd as noted earlier several scoring systems have been devised toscore disease activity based on the ï¬ndings of colonoscopy and histopathologic examinations in recent years many promising results havebeen reported regarding the correlation between these scores and fecalcalprotectin levels in addition many studies have been performed inthe last decade all of which cannot be reviewed in this article the ï¬rstevidence of a link between fecal calprotectin and disease endoscopicactivity was obtained in the late 1990s in one of the ï¬rst studiesroseth found a significant correlation between fecal calprotectinlevels and endoscopic and histologic activities in patients with ulcerative colitis furthermore in another study they observed that ibdpatients who were in remission clinically and had normal fecal calprotectin levels less than mgl had normal colonoscopy results these interesting ï¬ndings indicate that fecal calprotectin can beconsidered as a biomarker in the evaluation of endoscopic activity andclinica chimica acta table4summarized results of some metaanalysis regarding the utility of fecal calprotectin in discrimination between patients with ibd and without ibdsample sizepooled sensitivitypooled speciï¬cityreferences mucosal healing in ibd patients also these studies were the startingpoint of extensive studies that have been conducted up to now in astudy conducted on patients with crohn's disease sipponen investigated the sensitivity and speciï¬city of fecal calprotectin in predicting endoscopic activity of crohn's disease correspondinglythe researchers used the crohn's disease endoscopic index of severitycdeis scoring system in their study to evaluate the endoscopic activity of crohn's disease as a result they found that there was a significant correlation between the endoscopic activity of the disease andthe level of fecal calprotectin besides the ï¬ndings of this study demonstrated that fecal calprotectin at µgg cutoï¬ can predict theendoscopic activity of crohn's disease with sensitivity and speciï¬city in another study cdeis and mayo disease activity indexmdai were used to evaluate the endoscopic activity of crohn's diseaseand ulcerative colitis respectively according to the results of thatstudy on ibd patients there was a significant correlation between fecalcalprotectin levels and disease endoscopic activity another studyshowed that fecal calprotectin is more strongly correlated with theendoscopic activity of the disease in ulcerative colitis compared to therachmilewitz clinical activity index in addition in this study theoverall accuracy of fecal calprotectin for endoscopically active diseaseidentiï¬cation was obtained as some studies have also shown the superiority of fecal calprotectinover traditional ammatory markers like crp besides one studyfound that fecal calprotectin was more strongly correlated with thesimple endoscopic score for crohn's disease sescd compared to thecrp and even crohn's disease activity index cdai the modiï¬edbaron index was also used in another study to evaluate the endoscopicactivity of ulcerative colitis as a result it was shown that calprotectinis more strongly correlated with the endoscopic activity of ulcerativecolitis compared to crp and clinical activity of the disease in thisregard similar results were also observed in our recent study in whichthe ulcerative colitis endoscopic index of severity uceis and sescdwere used therefore fecal calprotectin appears to be superior totraditional ammatory markers in the prediction of ibd endoscopicactivity the high values of sensitivity and speciï¬city that were mentioned earlier have raised the hope that using fecal calprotectin canreduce colonoscopy rate for patients monitoring however severalrecent studies have reported some significantly lower values accordingly in a recent study in which mayo endoscopic score [mes] wasused to evaluate the endoscopic activity of ulcerative colitis atable summary of the results of some studies regarding the utility of fecal calprotectin in discrimination between patients with ibd and without ibdnumber of ibd patientsage grouplocationcut oï¬sensitivityspeciï¬city cd and uc cd and uc cd and uc and unclassiï¬ed68cd and uc cd and uc and unclassiï¬ed cd and uc cd and uc uc cd ucadultsadultsadultsboth adult and pediatricpediatricadultspediatricadultsadultsboth adult and pediatrictaiwanchinaitalyspainfinlandiranitalyirandenmarkindia48µgg µgg150µgg150µgg595µgg784µgg160µgg164µgg150µgg188µggaucreferences spsreï¬dbib60 0cf khakikhatibi clinica chimica acta table summary of the results of some studies regarding the correlation of fecal calprotectin with endoscopic activity in ibd patientsage groupstudylocationusedendoscopicactivity indexcorrelationcoeï¬cientrreferencenumberof ibdpatients cd uc uc cd ucadultsadultsadultsadultsadultsfinlandiranswitzerlandswitzerlandswitzerland modiï¬edcdeisuceisrachmilewitzsescd uc cdadultsadults uc cd uc cd cd uc ucadultsadultsadultsadultsadultsadultsadultsbaron scorerachmilewitzsescdgermanyusa andcanadajapanitalyitalybrazilfrancefrancesouth korea uceismattssescdmayo scoresescdcdeismayo score sensitivity of and a speciï¬city of were reported for fecalcalprotectin at µgg to diï¬erentiate active endoscopic from inactivemes or from mes or in another study the sensitivityand speciï¬city of fecal calprotectin at a cutoï¬ of µgg for diï¬erentiating mes ¤ in patients with ulcerative colitis were and respectively overall as presented in table several studiesperformed in diï¬erent countries reported the correlation between fecalcalprotectin and ibd endoscopic activity although some of these studies reported a strong correlation some others reported a relativelyweak correlation as noted earlier there are significant diï¬erencesbetween the reports on the sensitivity and speciï¬city of fecal calprotectin to predict the endoscopic activity of ibd undoubtedly a widerange of factors from sample size and the inclusionexclusion criteriato preanalysis variables and indexes used to evaluate the endoscopicactivity may also contribute to these diï¬erences however fecal calprotectin does not appear to be a very reliable marker for the predictionof ibd endoscopic activity so currently it seems a bit optimistic toconsider fecal calprotectin as a reliable alternative for colonoscopy inthis regard further studies are still needed however under some certain circumstances such as pregnancy or pandemics the use of fecalcalprotectin to evaluate ibd endoscopic activity can be helpfulpregnant patients with ibd have serious limitations for colonoscopyexamination and it has been recommended that colonoscopy should beonly performed in the second trimester of pregnancy and where there isa strong indication therefore noninvasive markers such as fecalcalprotectin can be helpful during pregnancy in one study physicianglobal assessment [pga] which is a clinical symptombased criterionwas used to evaluate ibd activity and subsequently the associationbetween fecal calprotectin and this criterion was investigated in pregnant women with ibd the results of this study showed a significantcorrelation between fecal calprotectin and pga levels at prepregnancyduring pregnancy and postpartum stages in another study asignificant association was reported between fecal calprotectin levelsand clinical activity of ibd in pregnant women moreover it was shownthat stool calprotectin at a cutoï¬ of mgkg had a sensitivity between and as well as a speciï¬city between and in the assessment of ibd clinical activity at diï¬erent stages ofpregnancy a recently published systematic review has also conï¬rmed the conclusions obtained from these studies according tothese results it seems that fecal calprotectin is not aï¬ected by physiological changes during pregnancy however it is significantly correlatedwith ibd clinical activity during pregnancy therefore from the viewpoint of relatively acceptable sensitivity and speciï¬city in predictingthe endoscopic activity of ibd fecal calprotectin may be considered as anoninvasive biomarker for the evaluation of ibd endoscopic activity inpregnant women in addition under pandemic conditions fecal calprotectin can be very helpful following the covid19 pandemicwhich began in late and is still ongoing healthcare systems indiï¬erent countries were forced to impose significant limitations oncolonoscopy therefore noninvasive ibd management and fecal calprotectin as a noninvasive laboratory marker have become moreimportant than before the combination between disease clinical activity and fecal calprotectin has been recommended as a noninvasiveapproach that can help in making decisions on treatment duringcovid19 pandemic therefore it seems that fecal calprotectincan be considered as an alternative for colonoscopy used for ibd endoscopic activity evaluation during pandemic fecal calprotectin appears to be associated with ibd histologic activity as well given thediï¬culty in the evaluation of the histologic activity of crohn's disease some studies have been focused on the ulcerative colitis andmany scoring systems have been devised so far correspondingly thesesystems score the disease's histologic activity based on histologic observationstherefore for this purpose a biopsy of the intestinal tissue is required which can be prepared by colonoscopy and then sent to thelaboratory in this regard one of these histologic scoring systems isroberts score that was used in one of our recent studies where weobserved a significant correlation between the level of fecal calprotectinand the histologic activity of ulcerative colitis which was calculatedbased on the roberts scoring system theede also used themodiï¬ed harpaz index and performed some interesting studies in thisregard in one of their studies fecal calprotectin was found to be significantly associated with the histologic activity of the ulcerative colitisand it was shown that it could predict histological mucosal healingauc ci95 sensitivity speciï¬city andcutoï¬ mgkg in another study on patients with endoscopically inactive ulcerative colitis mayo endoscopic score the researchers showed that patients with ulcerative colitis who were inendoscopic remission but had histologically active disease had higherlevels of fecal calprotectin compared to patients with no histologicallyactive disease versus mgkg p also despite thehigh speciï¬city the sensitivity of fecal calprotectin in theprediction of score of histological activity was achieved as at mgkg in a recent study the geboes index has been used toevaluate histologic activity in patients with clinically quiescent ulcerative colitis as a result this study reported relatively low sensitivityand speciï¬city for fecal calprotectin in the prediction of geboesscore sensitivity speciï¬city and cut oï¬ µgg in another recent study the nancy index has been used toevaluate the histologic activity of ulcerative colitis and a high sensitivity and a low speciï¬city were ï¬nally reported for fecalcalprotectin at a cutoï¬ of µgg in the prediction of histologic activity however some studies have reported both high sensitivityand speciï¬city for fecal calprotectin in the prediction of histologicalremission for example one study reported a sensitivity of aswell as a speciï¬city of for fecal calprotectin at a cutoï¬ of µggin the prediction of gs it seems that the cause of theseconï¬icts should be explored in the endoscopic and clinical activity ofthe disease the inclusion and exclusion criteria of these studies andpossibly in the diï¬erent indexes used for the evaluation of the histologicactivity of the disease another notable issue is that all of these studieshave been conducted on a relatively low number of patients with ulcerative colitis so the need for a study with a large sample size is stillstrongly felt moreover a large global study may be helpful in this regard prediction of relapse and response to treatmentas mentioned previously ibd has recurrence and relief periods sopredicting response to treatment and relapse is one of the significant 0cf khakikhatibi clinica chimica acta challenges in ibd management the ï¬rst evidence of the eï¬cacy offecal calprotectin to predict recurrence dates back to the early 2000saccordingly a study published in by tibble is one of the ï¬rststudies in this regard in this study ibd patients in clinical remissionwere followed up for one year for the assessment of recurrence afterpreparing a stool sample to measure calprotectin this study has alsoshown that fecal calprotectin levels were higher in ibd patients withrecurrent disease and it was found that fecal calprotectin had a sensitivity of and a speciï¬city of at a cutoï¬ of mgl to predictibd recurrence in a study published in costa showedthat the sensitivity and speciï¬city of fecal calprotectin to predict ulcerative colitis recurrence are more than that of crohn's disease asensitivity of and a speciï¬city of versus a sensitivity of and a speciï¬city at a cutoï¬ of μgg another studyconducted on patients with ulcerative colitis has also reported appropriate sensitivity and speciï¬city rates for fecal calprotectin in the prediction of relapse a sensitivity of and a speciï¬city at a cutoï¬of μgg however another study was conducted on patientswith ulcerative colitis who have been followedup for year and ï¬nally a lower sensitivity was reported this study have shown that fecalcalprotectin at cutoï¬ of μgg could predict diseas | 0 |
"The main complexity of modeling and interpreting such phenomena lies in the additional temporal dimension needed to express the association as the risk depends on both intensity and timing of past exposures. This type of dependency is defined here as exposurelagresponse association. In this contribution I illustrate a general statistical framework for such associations established through the extension of distributed lag non-linear models originally developed in time series analysis. This modeling class is based on the definition of a cross-basis obtained by the combination of two functions to flexibly model linear or nonlinear exposure-responses and the lag structure of the relationship respectively. The methodology is illustrated with an example application to cohort data and validated through a simulation study. This modeling framework generalizes to various study designs and regression models and can be applied to study the health effects of protracted exposures to environmental factors drugs or carcinogenic agents among others. 2013 The Authors. Statistics in Medicine published by John Wiley & Sons Ltd. latency distributed lag models exposurelagresponse delayed effects splines 1. Introduction In biomedical research it is commonly appreciated that an exposure event produces effects lasting well beyond the exposure period with an increase in risk occurring from few hours to many years later depending on the physiological processes linking the exposure and the health outcome. The problem is made even more complicated in the presence of protracted time-varying exposures when the health effect measured at a given time can be described as the result of multiple exposure events of different intensities sustained in the past. This phenomenon common to various research fields has been associated for example with peak 1 or chronic exposures 2 to environmental stressors drug intake 34 or occupational exposures to carcinogenic substances 5. The main complexity of modeling and interpreting such dependencies lies in the additional temporal dimension needed to express the association beyond the usual exposureresponse relationship as the risk depends on both intensity and timing of past exposures. Nonetheless the appropriate representation of the temporal pattern of risks may provide further insights on the association of interest in particular regarding the underlying pathophysiological mechanisms and prevent biases in estimates and predictions. Revising previous terminology 6 I define these dependencies as exposurelagresponse associations. In particular this issue has been debated in cancer epidemiology 79. Analytical approaches extend simple indices such as cumulative exposure in order to accommodate the temporal variation in risk because of protracted exposures. In particular the pioneering work by Thomas 106 helped develop sophisticated statistical methods on the basis of weighting past exposures through specific functions whose parameters are estimated by the data. Vacek 11 Langholz and colleagues 12 and Richardson 13 provided interesting applications in case-control studies with weights represented through simple parametric functions. The methodology was improved by Hauptmann and colleagues in a series of papers 1416 by using flexible and smooth spline functions. Sylvestre and Abrahamowicz 17 and Abrahamowicz and colleagues 18 extended the spline methods to the analysis of time-to-event data with a cohort design and presented their applications in pharmaco-epidemiology. The main limitation of the statistical techniques described in these papers is the assumption of a linear exposureresponse relationship. Models for nonlinear dependencies introduce further nontrivial complexities from both statistical and interpretational perspectives as the problem becomes inherently bidimensional. Abrahamowicz and Mackenzie 19 proposed a model for analyzing the nonlinear time-dependent effects of fixed exposures while Vacek 11 and Berhane and colleagues 20 extended this scheme to the case of protracted time-varying exposures. However the modeling techniques illustrated in these other papers still face some limitations as they are based on complex estimation routines with convergence issues and problems in producing uncertainty measures such as standard errors and confidence intervals. Interestingly equivalent approaches were previously established in time series analysis on the basis of distributed lag models (DLMs) a methodology originally formulated in econometrics 21 then applied in epidemiological research 22. These models involve the definition of a distributed lag function analogous to the weighting function described before. In particular Armstrong 23 generalized the method to distributed lag non-linear models (DLNMs) a class of models with different options for the functions applied to model nonlinearity and distributed lag effects. The theory of DLMs and DLNMs have been recently re-evaluated 24 offering a well-grounded statistical tool and a comprehensive scheme for interpretation. In this paper I aim to establish a general conceptual and statistical framework for modeling exposurelagresponse associations built upon the paradigm of DLMs and DLNMs. This modeling class extended beyond time series analysis provides a unified methodology applicable in different study designs data structures and regression models including most of the previous methods as specific cases. Also the statistical framework is defined by completely parametric functions and fitted through standard regression methods with measures of uncertainty and fit statistics easily available. The R package dlnm originally developed for time series data 25 is extended in parallel offering a easy-to-use implementation of the modeling approach. The manuscript is structured as follows. The development and algebraic definition of the modeling framework is described in Section 2. As an illustrative example in I apply the method for investigating the relationship between occupational exposure to radon and lung cancer mortality by using the data from the Colorado Plateau miners cohort. The modeling framework is then validated in a simulation study n. A final discussion is provided in. Information on data and software implementation is included in. The R code and data are included in the supporting information together with additional details making the results of the illustrative example and of the simulation study entirely reproducible. 2. Modeling framework The modeling skeleton is derived by extending the class of DLNMs beyond the time series context. This extension provides a neat algebraic representation and a comprehensive statistical definition. The focus is on a function here defined s(xt) which describes the dependency in terms of the exposure history to x evaluated at time t. The function s(xt) is commonly included in regression models in order to estimate the association while controlling for potential confounders. Although the regression model varies depending on the study design and the type of data the definition of s(xt) provided later and the related modeling framework generally apply. 2.1. Models for linear exposureresponse relationships Previous studies on the topic have defined the function s(xt) by using slightly different algebraic formulae 1026111417. Assuming a linear exposureresponse relationship a general notation can be given by (1a) (1b) (1c) In (1a) the increase in risk at time t is defined as the integral of the instantaneous exposure intensity xu over the period ?t = [t0t1] with t0 and t1 representing the times of the first and last relevant exposures. Here w(t ? u) is the weighting function previously described in which assigns weights to past exposures experienced at time t ? u on the basis of their contribution to the risk at time t. The model can be reparameterized as in (1b) where the risk is now expressed along the lag with ? ? [?0L]. Here L ? ?0 = t1 ? t0 is interpreted as the lag period over which an exposure to x is assumed to affect the risk at time t usually with ?0 = 0. This parameterization offers the advantage that the function w is now directly defined in the new dimension of lag ? and it is independent of the time axis chosen for t which may represent different time scales depending on the study design. The function w(?) termed from here on as the lagresponse function models the lagresponse curve associated with exposure x. Finally for computational purposes the integral is approximated in (1c) by a sum of terms derived by partitioning the lag interval in equally spaced discrete units and assuming the protracted exposure as a sequence of exposure events xt ? ? at lags ? = ?0 ¦ L. A statistical model for (1) can be defined by expressing the lagresponse function w(?) as a linear combination of terms obtained through basis transformation with related parameters. By using matrix notation let the vector qxt of exposure history be defined by (2) Such exposure history changes along time depending on the time t at which the vector qxt is computed. Given (2) the cumulative function s(xt) in (1) can be written using a compact and general matrix notation as (3) The (L ? ?0 + 1) à v? matrix C is obtained from the transformation of the lag vector ? = [?0 ¦ ? ¦ L]T by choosing a specific basis with dimension v? for w(?) which defines the related basis functions. In this parameterization the function s(xt) representing the integral of x · w(?) over the interval [?0L] is defined as a lagbasis function with parameters ?. Interestingly the equation in (3) is almost identical to that defining DLMs 24 Eq. (4). The different indexing in the original version reflects the specific application in time series where the data are perfectly ordered in time and the matrix Q has a structure such that qt? ? qt + 1? + 1. However this is a specific case of the general representation in (2)(3). The theory and software already developed for DLMs can be therefore extended in parallel. Alternative lagbasis functions for representing s(xt) are derived through different lagresponse functions w(?) in (1). In particular the traditional index of unweighted cumulative exposure is a specific case of (3) where reduces to with w(?) equal to a constant c. This is obtained by specifying C as an (L ? ?0 + 1)-dimensional vector of 1's with v? = 1. More sophisticated models with splines or other functions such as those illustrated in publications cited in only require the application of different bases for deriving C but are nevertheless represented by 3). 2.2. Extension to nonlinear exposureresponse relationships The extension to the nonlinear case presents further complexities as anticipated earlier. The model in (1) can be extended by defining an additional exposureresponse function f(x) to express the potentially nonlinear exposureresponse curve along the dimension of the predictor. An intuitive generalization of (1) is: (4) with f(x) as the standard exposureresponse function. However the function f(x) · w(?) in 4) previously proposed 1119 is not easily represented as a linear combination of basis variables and generates models that are not linear in their parameters and thus require ad hoc optimization routines. More importantly this representation is based on the strong assumption of independency between f(x) and w(?) namely that the exposureresponse shape is the same at each lag ? and vice versa that the lag structure is the same at each value of x. This assumption can be relaxed by expressing s(xt) as a truly bivariate function with the more flexible representation: (5) Here the bidimensional function f · w(x?) is defined as the exposurelagresponse function and models simultaneously the exposureresponse curve along x and lagresponse curve along ? namely an exposurelagresponse surface. Differently from 4) the exposurelagresponse function in 5) can be expressed as a linear combination of basis variables and related parameters through a special tensor product. As anticipated earlier Armstrong 23 proposed the same approach for time series data within the DLNM framework generalizing this tensor product parameterization through the concept of cross-basis. Specifically two sets of basis functions are independently chosen to represent f(x) and w(?) respectively. The cross-basis is the bidimensional space of functions obtained by the combination of the two sets integrated over the lag dimension and represents the core of DLNMs. The algebraic representation has been previously presented 24 and a revised version is proposed here. Briefly the simpler lag-basis for DLMs in 3) can be extended by choosing an additional basis with dimension vx for representing f(x). The application of the related basis functions to the vector of exposure history qxt obtained by 2) generates a (L ? ?0 + 1) à vx matrix Rxt. Let Axt be: (6) with 1v as a v-dimensional vector of 1's and C defined in 3). The cross-basis function s(xt;?) can be defined as (7) In this case the dimension of the cross-basis is determined by the product of the dimensions of the bases for the two spaces and the association is expressed through vx · v? values W and related parameters ?. The cross-basis function s(xt) represents the integral of f · w(x?) over the interval [?0L] cumulating the contributions of events representing the exposure history. In spite of the relatively complex algebraic form the definition of cross-basis and the specification of DLNMs only amount to the choice of the bases for the functions f(x) and w(?). These can be independently selected between several options such as splines linear threshold or piecewise constant (step) functions. The DLNM modeling class comprises the simpler DLMs from Section 2.1. For example the bidimensional exposurelagresponse function f · w(x?) in 5) reduces to a non-linear function for un-weighted cumulative exposure f(x) · c when w(?) is a constant function c and to the lagresponse function x · w(?) in (1) when f(x) is simply an linear function of the untransformed x. The model proposed by Berhane and colleagues 20 can be written in the form of 6)7) when both f(x) and w(?) are cubic B-splines. 2.3. Estimation and prediction Although the lag-basis and cross-basis functions in (1)(3) and (5)(7) involve a nonstandard parameterization in terms of exposure histories DLMs and DLNMs do not require specialized estimation procedures. The association is entirely expressed by the vx à v? parameters ? of the cross-basis values W. The computation of the exposure history in (2) can be extended to all N observations with x measured at time t producing an N à (L ? ?0 + 1) matrix of exposure histories Q. The matrix of transformed variables W in (3) and (7) is consequently derived. This matrix can be included in the design matrix of standard regression models to estimate the parameters ?. In the completely parametric development proposed here the number of coefficients vx à v? represents the degrees of freedom (df) used to model the association. Inference on the parameters ? and interpretation of the estimated association is aided by the prediction of specific risk measures. For simpler DLMs that assume a linear exposureresponse relationship this step reduces to the computation of a series of estimated risk contributions at lag ?p with ?0 ? ?p ? L and the associated (co)variance matrix . The series of risk contributions is provided by (8) with Cp obtained from the vector of lag ?p used for prediction by applying the same basis functions for w(?) used for estimation. These estimated risk contributions compose the lagresponse curve and can be interpreted using either a forward or backward perspective. Namely represents the risk contribution at time t + ?p in the future from a unit increase in exposure x at time t or the contribution from a unit increase in exposure x occurring at time t ? ?p in the past to a given risk measured at time t. The estimated risk contributions associated with different exposure increases are easily derived. The equations in (8) only apply to DLMs with lag-bases as defined in (3). For DLNMs the association is allowed to vary nonlinearly in the space of x. Moreover the specification in (5)(7) allows the lag-response curve to change depending on the level of the exposure. The prediction of risk contributions corresponding to a specific exposure intensity xp at lag ?p involves a more complex procedure. First let be the (L ? ?0 + 1)-dimensional vector of exposure history with constant exposure xp. The related matrices and are derived from (6) substituting qxt and C with and Cp by applying the same two sets of basis functions for f · w(x?) chosen for estimation. The exposure-specific risk contributions and associated (co)variance matrix are provided by (9) The estimated risk contributions may be interpreted as a lag-response curve similar to in (8) but this time associated with a specific exposure level xp instead of a unit increase. These measures may be used to define a grid of predicted risk contributions defined within the ranges of the exposure x and the lag ? thus obtaining a bi-dimensional representation of the association. From this grid besides above it is also possible to derive the vector of lag-specific risk contributions expressing the exposure-response curve for lag ?p. As noted in Section 2.2 the truly bivariate definition of (7) allows both the lag-response curve and exposure-response curve defined by and respectively to change depending on the specific exposure and lag values xp and ?p. The grid is interpreted as a risk surface along x and ? representing the exposurelagresponse. In addition predictions in (8)(9) may be extended to a generic exposure history qh. Substituting it into in (9) provides the vector of lag-specific risk contributions for each exposure that occurred within the lag period. The overall cumulative effect of such exposure history with associated (co)variance matrix may be computed with: (10) The Equation (10) can be used to estimate the predicted cumulative risk for a given pattern of exposure qh. This method can also be applied to investigate how the risk progressively evolves along an exposure profile computing the cumulative risk at each time associated with the time-varying exposure history qh. 2.4. Identifiability and constraints The tensor product structure of the cross-basis defined in (5)(7) poses some identifiability issues. In particular each of the vx basis variables in R is multiplied by each of the v? basis variables in C. If an intercept is included in f(x) the related matrix of cross-basis variables W is not of full rank and the parameters of the regression model are not identifiable even when a common intercept is not included. Therefore the cross-basis in (7) should always be defined without an intercept in the basis functions for x. Also these basis functions can be centered on a specific exposure value x0 which will represent the reference for the risk summaries computed by (8)(10). The bidimensional shape of the exposurelagresponse can be constrained to follow a prespecified pattern. In particular a priori assumptions on the lag structure can be imposed through functional constraints on the basis for the space of ?. Left and right constraints on the extremes of the supporting interval ?0L are particularly meaningful for smooth functions. A left constraint can be imposed by excluding the intercept from the basis. This step will force the lagresponse curve to predict a null risk at the beginning of the lag period. A right constraint on a B-splines basis can be produced by excluding specific basis variables as previously described for linear exposureresponse relationships 17. The constraint produces a smooth dependency which approaches a null risk at the end of the lag period. Such constraints are particularly useful in the presence of sparse data in order to limit the flexibility of the model under specific assumptions about the lagresponse curve. However biases can be introduced if these assumptions are not met. Additional information is provided in Section D1 of the supporting information. The functional constraints discussed in this section can be specified without introducing customized optimization methods for estimating the parameters ? in (3)(7). More sophisticated methods are required for example to constrain the lagresponse curve to be non-negative in the whole lag period L. These approaches have been previously proposed for linear dependencies 141718 and introduce further complexities in the bidimensional context of DLNMs. This development is not pursued here. 2.5. Model selection and inferential procedures The framework described in Sections 2.12.2 includes a fairly large number of models defined by different functions for each of the two dimensions and by different choices regarding each function such as number and location of knots in splines. This raises the issue of selecting the optimal model for describing the exposurelagresponse association. Previous studies on temporal dependencies have proposed selection procedures on the basis of profile likelihood 15 AIC 141620 or BIC 17. Simulation studies seems to indicate a better performance of AIC when compared with BIC in this context 18 a result consistent with unpublished simulations performed on time series data for DLNMs. Inference on the models illustrated in the previous sections primarily focuses on the specification of confidence intervals for the risk measures in Section 2.3 and on the definition of tests for a set of null hypotheses. Confidence intervals for lagresponse curves exposureresponse curves and cumulative risks obtained through and can be easily derived from the diagonal of the related (co)variance matrices in (8)(10) assuming a multivariate normal distribution of the estimators. Regarding hypothesis testing two null hypotheses are particularly relevant in this framework. The first one postulates a linear exposureresponse relationship namely H0 : f(x) = x. The second one assumes a constant risk namely H0 : w(?) = c. Tests on constrained models can be also defined. The assumption of independency is not easily tested as the form in (4) cannot be expressed as a model linear in its parameters. However defining general inferential procedures in this setting is not straightforward. First the null hypotheses H0 : f(x) = x and H0 : w(?) = c are not independent and an incorrect assumption about the association in one dimension may bias the test estimator for the hypothesis related to the other space as previously reported 19. In addition estimates are usually conditional on a posteriori selection of a best-fitting model based on the selection methods discussed before. Under these conditions the estimators for the (co)variance matrices in (8)(10) are likely to underestimate the true sampling (co)variance and the distribution of the test statistics may be different from that assumed unconditional on the selection procedure. This may generate undercoverage of confidence intervals and inflated type I error for tests 1727. Given these complexities a general framework for hypothesis testing embedded in the model selection procedure is not provided here. An assessment through simulations of the performance of estimators generated by AIC and BIC-selected models will be presented in. Specifically simulations will provide an empirical evaluation of the ability of the information criteria to identify the correct model between those defining the null or alternative hypotheses about linearity and constant effects and measures of performance such as bias coverage and root mean square error. 3. An application The conceptual and statistical framework of DLNMs described in extended beyond time series data is general and applicable in different study designs. As an illustrative example I propose here an application in survival analysis of time-to-event data. This represents one of the most complex settings as the temporal pattern of risk is produced by exposure histories that vary during the follow-up of each subject. Specifically the methodology is used to investigate the association between occupational exposure to radon and mortality for lung cancer. The analysis is based on data from the Colorado Plateau uranium miners cohort already used in previous methodological contributions 121520. Section A of the supporting information provides a list of the main steps to replicate the analysis in other real-life examples. 3.1. Data The cohort data used in this example were collected by the National Institute for Occupational Safety and Health. Detailed information on the cohort is given elsewhere 12. Briefly subjects were eligible to enter the cohort if they worked in mines within the Colorado Plateau area between 1950 and 1960 and provided demographic personal and occupational information during their working period. Vital status and cause of death were ascertained by linkage with different sources. The data used in this example refer to the follow-up of the cohort on December 311982 including 3347 subjects and 258 lung cancer deaths. Exposure data available in the data set include cumulative measures of radon and smoking in 5-year age intervals. The radon exposure history for each subject expressed in working-level months (WLM) was reconstructed by linking employment information with measured or predicted levels in each mine in each year. The smoking history expressed in the number of cigarettes packs à 100 was reported by each subject during his working period and assumed constant after the last reporting age. A summary of the data is provided in Table I. Table I Descriptive statistics of the Colorado Plateau uranium miners cohort. The data included here refer to the follow-up on December 31 1982. Exposure to radon is measured in working level months (WLM) while smoking is reported as packs of cigarettes/100 Full cohort Lung cancer cases N % N % Subjects 3347 100.0 258 7.7 Deaths (%) 1258 37.6 258 100.0 Ever smokers (%) 2656 79.4 238 92.2 Median Min 25th 75th Max Median Min 25th 75th Max Age at entry 34.0 15.8 25.8 44.0 80.0 41.6 18.6 34.3 48.0 63.9 Follow-up time (years) 23.9 0.1 19.6 25.5 32.5 18.3 0.3 12.9 22.0 30.8 Exposure to radon Exposure period (years) 6.7 0.1 2.7 11.8 53.0 12.8 0.1 7.8 17.6 39.5 Total cumulative exposure (WLM/year) 429.0 0.0 153.5 1016.8 10000.0 1231.9 8.0 553.7 2528.6 10000.0 Yearly exposure (WLM/year) All 60.2 0.1 26.7 122.2 3245.3 81.6 1.0 42.3 165.4 1295.7 Lag 09 52.4 0.1 23.8 102.5 2994.0 61.4 3.9 31.3 144.7 1110.8 Lag 1019 53.8 0.1 24.3 112.5 3245.3 78.3 1.0 42.9 164.0 1295.7 Lag 2029 74.0 0.1 33.0 141.7 3245.3 104.7 4.1 52.2 180.0 1295.7 Lag 3040 95.7 0.2 48.0 151.6 2994.0 104.7 5.5 60.0 175.3 860.2 Smoking Exposure period (years) 38.0 5.0 31.0 46.0 75.0 40.0 14.0 33.0 48.0 72.0 Total cumulative exposure (packs à 100) 131.6 0.4 94.5 174.5 676.3 147.4 21.8 109.5 188.1 567.2 Yearly exposure (packs à 100) 3.6 0.0 2.5 3.6 24.4 3.6 0.0 3.5 4.2 13.4 3.2. Modeling strategy For this illustrative example the analysis is performed through a Cox proportional-hazard model with time-varying covariates by using age as the time axis. Effect measures are reported as a hazard ratio (HR). The model is represented by the following: (11) where the log-hazard log [h(t)] is expressed as a sum of baseline log-hazard log [h0(t)] and contributions of additional covariates. These comprise cross-basis functions sx(xt) and sz(zt) for radon and smoking respectively as defined in (1)(7) and a linear term for calendar time u in order to control for secular trends in lung cancer risk not accounted for by the delayed effects of the two exposures. Radon is the exposure of interest and is modeled with different combinations of bases for f(x) and w(?) in the cross-basis sx(xt). Given the limited information on smoking histories in this analysis the cross-basis sz(zt) is a priori defined with a natural cubic B-spline with one knot at the median of 2.5 yearly packs à 100 for the exposureresponse and a step function with a single cut-off at lag 20 for the lag structure with lag period 240 years. However different cross-basis functions can be applied. The model spends 5 df controlling for confounders and a different amount for modeling the effect of radon depending on the chosen cross-basis sx(xt). Modeling exposurelagresponse associations in time-to-event data assumes the definition of an extended version of continuous time-varying predictors namely the varying exposure history for each subject at the ages he contributes to different risk sets 28. The lag scale is chosen as years with lag 0 identifying the exposure during the last year. The lag period is fixed at 240 assuming no effect of exposure after 40 years and in the last 2 years consistently with previous analyses. Multiple exposure histories are computed for each subject at the ages he contributed to each risk set given his exposure profile reconstructed from the 5-year periods. This step produced matrices of exposure histories Qx and Qz for radon and smoking respectively as defined in (2). These matrices are used to specify the lag-bases or cross-bases matrices Wx and Wz from (3)(7) included in the design matrix of the Cox model." | 1 |
Activation by NaturalPhytochemicals An OverviewConcetta Iside Marika Scafuro Angela Nebbioso and Lucia Altucci Department of Precision Medicine University of Campania Luigi Vanvitelli Naples ItalySirtuins are class III histone deacetylases whose enzymatic activity is dependent on NADas a cofactor Sirtuins are reported to modulate numerous activities by controlling geneexpression DNA repair metabolism oxidative stress response mitochondrial functionand biogenesis Deregulation of their expression andor action may lead to tissuespeciï¬cdegenerative events involved in the development of several human pathologies includingcancer neurodegeneration and cardiovascular disease The most studied member of thisclass of enzymes is sirtuin SIRT1 whose expression is associated with increasinginsulin sensitivity SIRT1 has been implicated in both tumorigenic and anticancerprocesses and is reported to regulate essential metabolic pathways suggesting thatits activation might be beneï¬cial against disorders of the metabolism Via regulation of p53deacetylation and modulation of autophagy SIRT1 is implicated in cellular response tocaloric restriction and lifespan extension In recent years scientiï¬c interest focusing on theidentiï¬cation of SIRT1 modulators has led to the discovery of novel small moleculestargeting SIRT1 activity This review will examine compounds of natural origin recentlyfound to upregulate SIRT1 activity such as polyphenolic products in fruits vegetablesand plants including resveratrol ï¬setin quercetin and curcumin We will also discuss thepotential therapeutic effects of these natural compounds in the prevention and treatmentof human disorders with particular emphasis on their metabolic impactKeywords sirtuin natural compounds oxidative stress human disorders polyphenolsEdited byCecilia BattistelliSapienza University of Rome ItalyReviewed byNarasimham L ParinandiThe Ohio State UniversityUnited StatesCarmen JeronimoPortuguese Oncology InstitutePortugalCorrespondenceAngela NebbiosoangelanebbiosounicampaniaitLucia Altucciluciaaltucciunicampaniait These authors share last authorshipINTRODUCTIONSpecialty sectionThis was submitted toTranslational Pharmacologya section of the journalFrontiers in PharmacologyReceived April Accepted July Published August CitationIside C Scafuro M Nebbioso A andAltucci L SIRT1 Activation byNatural Phytochemicals An OverviewFront Pharmacol 103389fphar202001225Epigenetic modiï¬cations are associated with genome stability gene transcription and metabolicregulation Acetylation is one of the most characterized histone modiï¬cations Histoneacetyltransferase HAT and histone deacetylase HDAC enzymes control the levels of histoneacetylation modulating gene expression Cavalli and Heard Sirtuins SIRT are enzymes classiï¬ed as class III HDACs They exhibit different subcellularlocalizations SIRT1 SIRT6 and SIRT7 are nuclear although SIRT1 isoforms were also identiï¬ed inAbbreviations SIRT1 Sirtuin HATs Histone acetyl transferases HDACs Histone deacetylases ROS Reactive oxygenspecies PPAR Receptor peroxisome proliferatoractivated receptor NRF Nuclear respiratory factor TFAM Mitochondrialtranscription factor A SOD Superoxide dismutase TNFa Tumor necrosis factor a IAP Apoptosis protein inhibitor Bcl2Bcell lymphoma2 family MnSOD Manganese superoxide dismutase RSV Resveratrol Que Quercetin oxLDL OxidizedLDL BBR Berberine Cur Curcumin COX Cytochrome c oxidase T2D Type II diabetes NAFLD Nonalcoholic fatty liverdisease CRM Caloric restriction mimeticFrontiers in Pharmacology wwwfrontiersinAugust Volume 0cIside et alNatural SIRT1 Activatorsthe cytoplasm SIRT2 is mainly cytosolic SIRT3 SIRT4 andSIRT5 are mitochondrial and can shuttle to the nucleus Changand Guarente The enzymatic activity of SIRTs is dependent on NAD as acofactor and plays an important role in controlling geneexpression DNA repair metabolism oxidative stress responsemitochondrial function and biogenesis Deregulation of theiractivity may lead to tissuespeciï¬c degenerative events thatunderlie several human pathologiesincluding cancerdiabetes and cardiovascular diseases Haigis and Sinclair OCallaghan and Vassilopoulos Waldman The most studied member of this enzymatic class isSIRT1 SIRT1 regulates metabolic pathways cell survivalcellular senescence and ammation and acts in thepathogenesis of chronic conditions such as diabetes as well aspulmonary neurodegenerative and cardiovascular diseasesIndeed SIRT1 has been reported to play a key role intumorigenesis as an oncogene or tumor suppressor dependingon the context speciï¬city BiasonLauber It is able tocontrol these processes via deacetylation of lysine groups ofhistone and nonhistone proteins including known transcriptionfactors FOXO MyoD p53 PGC1a Kupis Chronic ammation caused by oxidative damage increasesthe risk of many chronic disordersincluding heartcardiovascular and neurodegenerative diseases obesity insulinresistance and type diabetes T2D Geto Oxidative stress plays a key role in the pathogenesis of theseconditions The overproduction of reactive oxygen speciesROS including free radicals and reactive nitrogen speciesRNS can lead to damage of cellular components such aslipids proteins and DNA The imbalance between oxidantsand antioxidants can result in cellular dysfunction apoptosisand necrosis Liguori SIRT1 guards against oxidative stress by activating genetranscription of PGC1a via deacetylation and by regulatingtranscription of factors such as the nuclear receptor peroxisomeproliferatoractivated receptor PPAR nuclear respiratory factorNRF and mitochondrial transcription factor A TFAMinvolved in modulation of biogenesis and mitochondrialfunction Ren and metabolism of glucose and lipidsRodgers SIRT1 is also able to regulate the expressionof superoxide dismutase SOD and glutathione peroxidase Sun In addition since mitochondrial dysfunction leads tothe activation of apoptosis SIRT1 can directly regulate theapoptotic process by modulating acetylation of PGC1a Zhang SIRT1 also regulates ammatory responseKauppinen By modulating the acetylation level ofNFkB p65 SIRT1 is able to control transcription of genes such asIL interleukin1 tumor necrosis factor a TNFa IL8 IL6and other ammatory factors Rodgers Ren Yeung Through NFkB SIRT1 also regulatesthe expression of genes such as inhibitor of apoptosis proteinIAP and Bcell lymphoma2 Bcl2 and tumor necrosis factorreceptor TNFR Ren SIRT1 protects against oxidative stress via regulation ofFOXO protein acetylation which is involved in antioxidantprocesses apoptosis and cell proliferation Wong andWoodcock By activating FOXOMsSOD pathwaySIRT1 increases the expression of manganese superoxidedismutase MnSOD and catalase counteracting oxidativestress and promoting damage repair Gu SIRT1also increases the expression of MnSOD by deacetylating p53thus enhancing cellular antioxidant capacity Brunet Zhang Ren Over the past few years the evergrowing awareness that goodhealth goes hand in hand with a healthy and balanced diet hasencouraged people to eat more fruit and vegetables and to takesupplements to make up for any deï¬ciency DAngelo Bioactive compounds in the diet can act as antioxidantand antiammatory agents thereby reducing the negativeeffects of oxidative stress and the incidence of chronicdiseases such as obesity diabetes and cardiovascular disordersWang Several moleculesincluding naturalphytochemical compounds can modulate SIRT1 activityMiceli Numerous studies have provided evidenceof the protective effects of natural polyphenolic substances suchas resveratrol quercetin curcumin and ï¬setin and ofnatural nonpolyphenolic substances such as berberineMcCubrey Natural polyphenols are the largestgroup of phytonutrients and are considered potential agents forthe prevention and treatment of stressrelated oxidative diseasesThey are found in many plants and foods such as fruitsvegetables tea cereals and wine and longterm intake isassociated with health beneï¬ts Mediterranean diets are in factlinked to a reduced risk of chronic diseases due to theconsumption of olive oil and red wine which contain highamounts of polyphenols Romagnolo and Selmin Most of the evidence supporting the beneï¬cial effects ofphytochemical compounds comes from in vitro or animalstudies while human studies evaluating the longterm impact ofphytomolecules are particularly few or inconsistent Interventionalstudies are in fact limited by issues of bioavailability andmetabolism However in vitro studies aimed at identifyingcellular targets linked to the beneï¬cial actions of phytonutrientrich foods at concentrations ranging from nM to µM challenge thetranslatability of data After ingestion these compounds are in factdetected as phase II metabolites and their blood level does notexceed concentrations in the nM range Substantial amounts of thecompounds and their metabolites are degraded in the colon byintestinal microbiota giving rise to small phenolic acids andaromatic catabolises which are absorbed by the circulatorysystem Del Rio Interesting studies showed thatthese natural polyphenol and nonpolyphenol substances couldaffect SIRT1 expressionactivity Table de Boer Themain mechanisms of action common to polyphenol and nonpolyphenol molecules that lead to antioxidant and antiammatory effects via SIRT1 activation are reported in Figure Here we focus on the natural molecules resveratrolquercetin ï¬setin curcumin and berberine and elucidate theireffect on SIRT1 activation and their potential to treat andorprevent several human pathologies mainly associated withmetabolic disorders Figure Frontiers in Pharmacology wwwfrontiersinAugust Volume 0cIside et alNatural SIRT1 ActivatorsTABLE Classiï¬cation of nutraceuticals based on their action and food sourceNaturalSIRT1activatorsEffectSourceReferencesResveratrol Positive effect on bloodlipid proï¬le antioxidantDark grapesraisins peanutsQuercetinBerberineCurcuminFisetinAnticancer positiveeffect on blood lipidproï¬le antioxidant antiammatoryAntioxidant antiammatoryAnticancer antioxidantantiammatoryAnticancercardiovascularpreventive antiammatoryantioxidantFruits vegetablesnutsNatural componentof traditionalChinese herbCoptidis rhizomaActive componentin Curcuma longaApplespersimmonsgrapes onionskiwi kalestrawberriesDAngelo Zordoky Hung Nabavi Nabavi Wu Hung Zendedel Kim Chen NATURAL COMPOUNDS ENHANCINGSIRT1 EXPRESSION AND ACTIVITYResveratrolResveratrol RSV a nonï¬avonoid polyphenol found in grapesand grape products such as red wine exerts an antioxidant actionwith reported cancer preventive properties KrisEtherton RSV also has antiammatory anticancer andantineurodegenerative effects Piotrowska The roleof RSV as an immune response modulator was demonstrated inboth in vitro and in vivo studies where it reversed immunesenescence in older rats reduced ammatory responses inrodents and improved immunological activity against cancercells Malaguarnera RSV was shown to be involved in theactivation of macrophages T cells and natural killer cells as wellas in the suppression processes of CD4 CD25 regulatory T cellsYang Svajger and Jeras All these effects aredue to its ability to remove ROSinhibit cyclooxygenaseCOX and trigger antiammatory pathways via SIRT1activation Miceli Malaguarnera ActivatedSIRT1 interrupts TLR4NFkBSTAT axis reduces cytokineproduction by inactivated immune cells and inhibits proammatory factors derived from macrophagesmast cellssuch as plateletactivating factor and TNFa Capiralla RSVSIRT1 interaction modiï¬es SIRT1 structure andpromotes binding activity with its substrates including p65RelA Yeung a component of the NFkB complexwhich regulates activation of leukocytes and ammatorycytokines SIRT1 activated by RSV inhibits acetylation of RelAby reducing the expression of ammatory factors such as TNFa IL1b IL6 metalloprotease MMP1 MMP3 and NFkBmediated Cox2 Malaguarnera AMP activatedprotein kinase AMPK is also a target of RSV as it controlsSIRT1 activity via regulation of cellular levels of NAD thusacting as an energy sensor Price Cyclicadenosine monophosphate cAMP levels activate proteinkinase A resulting in phosphorylation and activation of SIRT1FIGURE Basic mechanisms and effects of SIRT1 activation by polyphenol and nonpolyphenol moleculesFrontiers in Pharmacology wwwfrontiersinAugust Volume 0cIside et alNatural SIRT1 ActivatorsFIGURE Nutraceutical action on SIRT1 expression Natural substances have beneï¬cial effects on human health by regulating SIRT1 action in different cellularprocesses wwwpubchemncbinlmnihgovWan Activated SIRT1 catalyzes the deacetylationand activation of PGC1a thereby promoting beneï¬cial effectsin the metabolism Ren In different anisms S cerevisiae C elegans and Dmelanogaster expressing SIRT1 or its homologous genesRSV treatment is able to extend life span In mammaliansRSV administration can improve SIRT1dependent cellularprocesses such as axonal protection Araki fatmobilization Chaplin and inhibition of NFkBdependent transcription Yeung these effects areabolished in SIRT1 knockdown models Numerous studiesinvestigated the beneï¬cial effects of RSV in cardiovasculardiseases including hypertension Theodotou cardiac ischemia Fourny and atherosclerosisChassot RSV has an effect on blood vesselsreduces ammation and prevents thrombus formation andplatelet oxidation Zordoky It can also reducecardiac dysfunction oxidative stress ï¬brosis and apoptosis inthe heart Gupta Yamagata In addition RSVwas found to improve heart and kidney damage in rats Li et al2020a The protective effect of RSV is associated with anincrease in SIRT1 activity which deacetylates FOXO1 andactivates MnSOD downstream RSVinduced MnSOD alsoreduces oxidative stress Li 2020a A recent in vitrostudy showed that RSV reduces hypoxiainduced apoptosis inH9C2 cells through activation of SIRT1miR30d5pNFkB axisHan RSV treatment decreased cortical andhippocampal malondialdehyde levels while increasing SODactivity and SIRT1 expression in a diabetic rat model Ma RSV was shown to activate SIRT1 and improve endothelialfunction in obese mice via upregulation of PPARd expressionactivity in PPARd mutant mice Cheang It hadpreviously been observed that Akt activation together withPPARd is involved in vascularization of dbdb mice Tian RSV was subsequently reported to increasephosphorylation of Akt and transcriptional activity of PPARdin the aorta of wildtype mice thus supporting the hypothesis ofSIRT1PPARd interaction and to strongly decrease LPCinduced mitochondrial ROS in the aortic endothelium ofC57BL6 mice Cheang Taken together theseï¬ndings highlight the beneï¬cial effects of RSV against oxidativestress which is involved in major pathologies such as heart andmetabolic disorders Although RSV is beneï¬cialin manycontexts its pleiotropic actions need to be better studied inorder to understand which of its described activities are directlydue to SIRT1 modulation and whether this effect is always directBecause of the pleiotropic actions of RSV clinical trials arecurrently testing its therapeutic potential in a wide range ofhuman diseases However of all the mechanisms described in invitro and in vivo studies only a few have been conï¬rmed inhumans such as gene and protein regulation in blood or musclecells and Akt signaling pathways Ghanim Brasnyo Many clinical studies conducted in healthy patientsand volunteers using both high and low doses of RSV highlightits potential cardioprotective beneï¬t through improvement ofendothelial functionammatory markers and glucosemetabolism Nevertheless the mechanisms of action are notyet well deï¬ned Despite clinical evidence of its effects thepoor bioavailability and rapid metabolism of RSV severelylimit the potential use of this molecule in the clinic Futurescientiï¬c research should focus on identifying actual metabolitesor mediators of these observed effectsTo date clinical trials have tested the efï¬cacy safety andpharmacokinetics of RSV in the prevention and treatment of different pathological conditions wwwclinicaltrialsgovFrontiers in Pharmacology wwwfrontiersinAugust Volume 0cIside et alNatural SIRT1 ActivatorsRestricting the search to interventional phase studiescompleted and terminated clinical trials addressed theability of RSV to improve the pathological conditions ofpatients affected by several diseases Most of these studiestested RSVmediated effects in central nervous systemdisorders Friedreich ataxia Alzheimers disease Parkinsonsdisease metabolic disorders [T2Dinsulin resistancedyslipidemia hypercholesterolemia metabolic syndrome Xnonalcoholic fatty liver disease NAFLD] A phase clinicaltrial NCT01640197 tested the effects of chronic resveratrolsupplementation mg daily for days in healthy humansand found considerable improvements in cognitive performancecerebral blood ï¬ow subjective sleep mood health and bloodpressure A list of completed and terminated clinical trials inwhich RSV was tested for metabolic disorders is reported inTable Focusing on completed clinical trials with availableresults NCT02114892 evaluated the effect of RSV on metabolicsyndrome demonstrating that when administered three timesper day mgdie before meals RSV was able to treat andprotect from obesity and diabetes with beneï¬cial effects onglucose and lipid metabolism blood pressure and bodyweight Another phase study NCT02095873 evaluated theeffects of a formulation composed of RSV and hesperetin inobese subjects and found that these molecules are dietaryinducers of glyxalase improving metabolic and vascularhealth of obese subjects Xue QuercetinThe ï¬avonoid polyphenol quercetin Que ²²pentahydroxyï¬avone is a natural safe dietary supplementfound in a glycoside form in fruits vegetables and nuts whichhas antioxidant and antiammatory properties Nabavi Wu In recent years the scientiï¬c community has focused on thepotential antiproliferative chemopreventive and anticarcinogenicactivities of Que as well as on its role as a modulator of geneexpression However Que was also found to have potentially toxiceffects including mutagenicity prooxidant activity mitochondrialtoxicity and inhibition of enzymes involved in hormonalmetabolism Li Due to its poor solubility short halflife and low bioavailability its medical use is limited Konrad andNieman In humans Que bioavailability is very low and absorption varies from to in subjects receiving mgdie Costa Que may reduce infection Li hepatic lipemicoxidative damage Cui Zhang et al2016b and antioxidant risk Xu In addition Que isknown to exert a modulating action on immunity Galleggiante As regards its mechanism of action in some cell linesQue was able to inhibit the production of TNF in macrophagesTang IL8 in A549 lung cells induced bylipopolysaccharide LPS Geraets and TNFa andIL1a mRNA levels in glial cells causing a decrease in neuronal celldeath induced by microglial activation Li MainlyTABLE Resveratrol in clinical trials for metabolic disordersStatusStudy TitleConditionsInterventionPhaseNCT NumberCompleted Effects of Resveratrol in Patients With TypeType Diabetes DiabetesTerminated Effect of Administration of Resveratrol onType Diabetes Mellitus mg to a maximum dose of g daily mg times dailyPhase NCT01677611Phase NCT02549924Glycemic Variability in Individuals With Type Diabetes MellitusCompleted Effect of Resveratrol on Agerelated InsulinResistance and Inï¬ammation in HumansCompleted Regulation of Intestinal and HepaticLipoprotein Secretion by ResveratrolType Diabetes Mellitus InsulinResistanceDyslipidaemia Insulin ResistanceCompleted Effects of Dietary Antioxidants to PreventHypercholesterolemia HealthyCardiovascular DiseaseHealthy Aging Through Functional FoodCompletedwith resultsCompleted Effects of Resveratrol on Inï¬ammation inType Diabetic PatientsCompletedwith resultsEffect of Resveratrol Administration onMetabolic Syndrome Insulin Sensitivity andInsulin SecretionCompleted Resveratrol for the Treatment of NonAlcoholic Fatty Liver Disease and InsulinResistance in Overweight AdolescentsGlucose Intolerance Aortic Stiffness VasodilationType Diabetes Mellitus Inï¬ammation Insulin Resistance Other Disorders ofBone Density and StructureMetabolic Syndrome XNAFLD Type Diabetes MetabolicSyndromeCompleted A Study of Resveratrol as Treatment forFriedreich AtaxiaFriedreich AtaxiaCompleted Effect of Banaba Lagerstroemia Speciosaon Metabolic Syndrome Insulin Secretionand Insulin SensitivityMetabolic Syndrome X mg twice daily for daysPhase NCT01354977 mg for week followed by g for weekDietary Supplement red wine for monthDietary Supplementresveratrol for monthTransresveratrol mg hesperetin mg combination months mg daily then months mg daily mg times daily beforemeals with a total dose of mg daily mg twice daily for a total dailydose of mg for days g daily mg twice daily for weeks then g daily gtwice daily for weeksBanaba capsules mg times daily before meals for daysPhase NCT01451918Phase NCT02409537Phase NCT02095873Phase NCT02244879Phase NCT02114892Phase NCT02216552Phase NCT01339884Phase NCT02767869Frontiers in Pharmacology wwwfrontiersinAugust Volume 0cIside et alNatural SIRT1 Activatorsin immunity and ammation Que acts on leukocytes and targetsmany intracellular signaling kinases and phosphatases as well asenzymes and membrane proteins Li Theimmunostimulating effect of Que is due to induction of theexpression of interferong IFNg derived from Th1 andinhibition of IL4 derived from Th2 in normal peripheral bloodmononuclear cells Nair In addition Que reduces T cellproliferation by blocking IL12induced tyrosine phosphorylationof JAK2 TYK2 STAT3 and STAT4 Muthian and Bright Nabavi In ammation Que inhibits the enzymesCOX and lipoxygenase Lee and Min Savikin Inthe RAW cell line Que was also shown to counteract LPSinduced ammation by phosphorylation of tyrosinephosphatidylinositol3kinase PI3Kp85 and complexformation of tolllike receptor TLR4MyD88PI3K Endale Oxidative stress occurs following an imbalance of the bodysantioxidant defence mechanisms and excessive generation of freeradicals and is involved in various pathologies such as diabetesatherosclerosis hypertension neurodegenerative diseasesammation and cancer Oboh Que is apowerful ROS scavenger and its antioxidant action is due tothe presence of two pharmacophores within the molecularstructure which confer a favorable conï¬guration for freeradical elimination Costa Generally Que reducesthe effects of free radicals by transferring the hydrogen atom andstabilizing the radicals a feature that has a structurefunctionrelationship Oboh Que can also act as both an antioxidant and prooxidant agentAt low concentrations µM Que displayed a protective effectagainst oxidative DNA damage in vitro in human lymphocytes Li At concentrations between µM and µM Que wasable to directly eliminate ROS in vitro Costa Howeverits effect in vivo is very likely not direct but due to its ability tomodulate the cells antioxidant defense mechanisms moderateoxidative stress can in fact increase the cells antioxidant defensesresulting in general cytoprotection Halliwell Recentresearch showed that oxidized LDL oxLDL induces oxidativestress LaraGuzman Oxidative injuries promote ROSgeneration in human endothelial cells and SIRT1 regulatesendothelial function Therefore enhancement of SIRT1 activityand SIRT1AMPK axis upregulation inhibits oxidative injuryinducing endothelial dysfunction Chen Shentu Que may reduce oxLDLinduced oxidative damageby upregulating SIRT1 and AMPK Hung thereforepotentially preventing oxLDLimpaired SIRT1 inhibition linked toendothelial dysfunction These ï¬ndings indicate that SIRT1 canfunction as a regulator to improve AMPK activity under oxLDLstimulation Hung It was very recently shown that Que mgkg can reduceinsulin resistance and improve glucose metabolism by reducingsensitivity to T2Dinsulin resistance in obob mice via SIRT1activation Hu In this context another study showedthat in streptozotocininduced diabetic rats Que mgkginhibits oxidative damage by increasing SIRT1 expression anddecreasing levels of NFkB a SIRT1 substrate Iskender In recent years the scientiï¬c community has focused on therole of apoptosis in cardiovascular disease showing thatoxidative stress myocardial ischemia hypoxia and ischemiareperfusion injury may induce myocardial apoptosis Donniacuo Tang and colleagues evaluated the effects of Que inimproving myocardialischemiareperfusion injury MIRinduced cell apoptosis both in vitro and in vivo SIRT1 andPGC1a expression levels were decreased in rat MIR groups butwere signiï¬cantly increased after treatment with Que Tang Furthermore activation of SIRT1PGC1a pathwayupregulated Bcl2 expression and downregulated Bax exertingantiapoptotic effects The authors hypothesized that Que mightimprove MIRinduced myocardial damage via regulation ofSIRT1PGC1a and Bcl2Bax pathways Tang Que is also reported to regulate ROS generation and mitigatemitochondrial dysfunction by promoting their biogenesisSpeciï¬cally in a study to develop a therapeutic strategy forosteoarthritis Que was shown to increase expression levels ofSIRT1 PGC1a NRF1 and NFR2 TFAM and phosphoAMPKa in osteoarthritis rats conï¬rming the hypothesis that Quemight act via the AMPKSIRT1 signaling pathway Qiu Overall these ï¬ndings suggest that Que may counteractcardiovascular disease and oxidative damageThe growing body of evidence supporting the beneï¬cial effects ofQue has led to its clinical use as demonstrated by the number ofclinical trials studies on ClinicalTrialsgov A list of completedstudies using Que in different metabolic and ammatoryconditions is reported in Table Speciï¬cally a phase clinicaltrial NCT01839344 measured the effect of Que on glucosetolerance and postprandial endothelial function in subjects withT2D compared to the effect of an alphaglusidase inhibitor acarboseThe administration of g of Que led to a decrease in postprandialblood glucose NCT01839344 Given its antioxidative and antiammatory capacities this ï¬avonoid was considered a goodcandidate for antioxidant therapy in mucositis NCT01732393hepatitis C NCT01438320 idiopathic pulmonary ï¬brosisNCT02874989 osteoporosis NCT00330096 uric acidmetabolism NCT01881919 cytokine release NCT01106170and chronic obstructive pulmonary disease NCT01708278 Inthe latter study Que supplementation was safely tolerated bypatients with mildtosevere chronic obstructive pulmonarydisease opening the way towards the potential use of Que as atherapeutic agent for this conditionHowever as for RSV and nutraceuticals in general the resultsof molecular studies on Que obtained from in vitro investigationsand animal models are often inconsistent with data from clinicaltrials Concentration factor dose and timing of administrationand bioavailability are the two main issues that require furtherclariï¬cation Additional studies are needed to identify theoptimal concentration of Que for it to exert a beneï¬cial effectfor example on insulin sensitivityBerberineBerberine BBR is an isoquinoline alkaloid reported to haveanalgesic anticancer antiammatory and myocardialprotective properties Cicero and Baggioni It was foundFrontiers in Pharmacology wwwfrontiersinAugust Volume 0cIside et alNatural SIRT1 ActivatorsTABLE Quercetin in clinical trials for metabolic and ammatory disordersStatusStudy TitleConditionsInterventionPhaseNCT NumberCompleted Effect of Quercetin in Prevention andTreatment of Oral MucositisBeneï¬cial Effects of Quercetin in ChronicObstructive Pulmonary Disease COPDCompletedwith resultsCompleted QTrial in Patients With Hepatitis CCompleted Effects of Quercetin on Blood Sugar andChemotherapy Induced OralMucositisChronic ObstructivePulmonary DiseaseChronic Hepatitis CDiabetes Mellitus Type mg daily for weeksPhase NCT01732393 to mg daily for weekPhase NCT01708278 days mg oral single dose of mgPhase Phase NCT01438320NCT01839344Blood Vessel Function in Type DiabetesCompleted Effect of Quercetin Supplements onCompletedHealthy Males a 4Week RandomizedCrossOver TrialTargeting ProInï¬ammatory Cells inIdiopathic Pulmonary Fibrosis a HumanTrialCompleted Efï¬cacy of Provex CV Supplement toReduce Inï¬ammation Cytokines andBlood PressureHyperuricemia Gout KidneyCalculi DiabetesCardiovascular DiseaseIdiopathic Pulmonary FibrosisIPFBlood Pressure mg tablet for days with meal breakfastpreferredEarly PhaseNCT01881919 doses administered over consecutive days in consecutive weeks oral administration ofquercetin mg daily mg of Provex CV supplement by mouth perday for weeksPhase NCT02874989Phase NCT01106170Completed Effects of Hesperidin on Bone MineralOsteoporosis OsteopeniaPhase NCT00330096Density and Bone Metabolism ofPostmenopausal Womento exert protective antioxidative effects in different physiologicand pathologic conditions Huang Li 2020bHowever the mechanisms underlying these effects remainunclear BBR was described as a potential antitumor agent thatinduces cell cycle arrest in G0G1 phase increases Cipp21 andKipp27 protein expression decreases expression of cyclin D1D2 and DE and the cyclindependent kinases Cdk2 Cdk4 andCdk6 promoting apoptosis in HL60 human leukemia cellsLi BBR can also deregulate telomerase activityand promote mitochondriadependent apoptosis in HepG2human hepatocarcinoma cells through caspase and caspase activation PARP cleavage induction increased expression ofthe proapoptotic protein Bax through activation of FOXOtranscription factors and inhibition of Bcl2 and Bclx antiapoptotic protein expression Hwang BBR wasobserved to exert an apoptotic effect by inducing ROSproduction and increasing MAPK and JNK activity of p38 inSW620 human colon carcinoma cells and by increasing Ca andcytochrome C release in HSC3 squamous cells Song In addition BBR inhibits the proliferation of cancer cellsthrough an antiammatory pathway In oral carcinoma celllines and in SCC4 cells BBR inhibits expression of COX2 andAP1 bond decreases prostaglandin E2 PGE2 production andsuppresses NFkB IKK ERK and JNK activities FurthermoreBBR can inhibit colon cancer cell growth by activating retinoid Xreceptor a RXRa which binds RXRa and promoting bcatenin degradation Ruan However some studieshighlighted the potential ability of BBR to prevent oxidativestressinduced senescence by activating AMPK and restoringNAD levels Song Initial research revealed a signiï¬cant role of SIRT1 signalingin mediating the antioxidant effect of BBR in diabetes Pang and in lipid metabolism Hasanein Thelipidlowering activity mediated by cotreatment with BBR andRSV was investigated in mice exposed to a high fat diet Zhu In vivo data showed that BBR combined with RSVlowered total cholesterol triglyceride and LDL cholesterol levelsin mice These ï¬ndings were also conï¬rmed in vitro with 3T3L1adipocytes treated with BBR or RSV alone Speciï¬cally BBR andRSV cotreatment was able to reduce lipid accumulation morerobustly than single treatments BBR in combination with RSVdisplayed hypolipidemic effects likely mediated by SIRT1expression regulation Moreover BBR pretreatment seemed tocounteract SIRT1 downregulation Zhu The antioxidant and antiammatory effects of BBR werealso investigated in heart Yu BBRmediated SIRT1activation reduced MIR injury by affecting oxidative damageand ammation signaling Speciï¬cally BBR exerted anantioxidant effect by decreasing the generation of cardiacsuperoxide and gp91phox expression and by increasing SODlevels Yu A previous study had also shown thatSIRT1 activation promotes antioxidant molecule production anddecreases proapoptotic proteins through FOXO1 activationthus protecting against MIR lesions Hsu As well as activating SIRT1 BBR is also able to decreaseFOXO1 acetylation triggering antiapoptotic signaling pathwaysvia Bcl2 expression and Bax and caspase3 downregulation Yu A very recent report described the protective effect of BBRagainst doxorubicininduced cardiovascular damage Wu This effect is | 2 |
we report the case of a 24yearold man who presented with chief complaintsof shortness of breath and haemoptysis chest radiography revealed completecollapse of the left lung bronchoscopy revealed an endobronchial tumourwith complete obstruction of the left main bronchus cryosurgical excisionwas performed tissue pathology conï¬rmed the diagnosis of metastatic embryonal carcinoma the patient underwent a right orchiectomy followed by ableomycin etoposide cisplatin bep chemotherapy regimenkeywordscryosurgery embryonal carcinoma endobronchialmetastases endobronchial tumourcorrespondencewenchien cheng division of pulmonary and critical care medicine department of internal medicinechina medical university hospital no yuderoad north dis taichung city taiwanemail wcchengdrgmailcomreceived july revised july accepted july associate editor james horespirology case reports e00644 101002rcr2644introductionlung metastases from extrapulmonary malignancies areobserved frequently in clinical practice by contrastendobronchial metastases ebms from extrapulmonarymalignancies are rare and may have distinct histopathological etiologies [] primary lung cancer is the most common cause of endobronchialtumours extrapulmonarymalignancies that are frequently associated with metastasesto the central airways include tumours of breast colorectalthyroid origin [] although mediastinalrenal orlymphadenopathy is frequently observed in associationwith testicular seminoma ebms from embryonal carcinomas are extremely rare in this report we present acase of ebm from a primary embryonal carcinomacase reporta 24yearold man with no relevant past medical historypresented to our hospital with a chief complaint of shortness of breath lasting for several days upon questioningthecoughexperiencedrevealedpatientthatheanendobronchialrevealedleft main bronchushaemoptysis shortness of breath and occasional chest painfor the past several days he reported no fever chills coldsweats weight loss or decreased appetite a chest radiograph at admission revealed complete collapse of the leftlung fig 1a computed tomography ct was notable forleft pleural masses an endobronchial tumour obstructingthe left main bronchus and complete collapse of the leftlung and a soft tissue mass lesion in the right scrotumbronchoscopytumourobstructing thefig 1b theendobronchialtumour was excised with bronchoscopiccryosurgery a followup chest radiograph revealed someimprovement in the status of the left lung immunohistochemical staining of the tumour tissue revealed that the cellswere thyroid transcription factor1 ttf1negative sallike protein sall4positive and cluster of differentiation cd30positive these ï¬ndings were consistentwith a ï¬nal diagnosis of metastatic embryonal carcinomafig we checked the levels of tumour markers in thepatient including those of alphafetoprotein afp betahuman chorionic gonadotropin bhcg and lactate dehydrogenase ldh each tumour marker was found to be the authors respirology case reports published by john wiley sons australia ltdon behalf of the asian paciï¬c society of respirologythis is an open access under the terms of the creative commons attributionnoncommercialnoderivs license which permits use and distribution in any mediumprovided the original work is properly cited the use is noncommercial and no modiï¬cations or adaptations are made vol iss e00644page 0cebm from embryonal carcinomack teng figure chestradiograph and bronchoscopic view of the endobronchial metastasesebm a complete collapse of the left lungon chest radiograph b bronchoscopic viewof the endobronchial tumour within the leftmain bronchusfigure tumour pathology of metastatic embryonal carcinoma a embryonal carcinoma with a complex glandular growth pattern the characteristic large cohesive and highly pleomorphic tumour cells with moderate amounts of amphophilic cytoplasm overlapping nuclei vesicular chromatinand frequent mitoses are shown as indicated by the arrows original magniï¬cation b immunohistochemical staining with antithyroid transcription factor1 ttf1 highlighting cells in the alveolar space original magniï¬cation c immunohistochemical staining with antisallikeprotein sall4 revealed diffuse nuclear staining original magniï¬cation d immunohistochemical staining with anticluster of differentiation cd30 highlighting diffuse membranous staining original magniï¬cation presentin high levels afp ngml bhcg miuml and ldh iul the patientunderwent a right orchiectomy followed by a bepbleomycin etoposide cisplatin chemotherapy regimendiscussionwe report here the case of a young man with an ebmfrom a primary embryonal carcinoma who presentedshortness of breath and haemoptysis chest radiography atpresentation revealed complete collapse of the left lungtumourslikewise manykindslung metastases from extrapulmonary malignancies arereported relatively frequently in clinical practice howeverebms from extrapulmonary malignancies are rare [] primary lung cancer is the most common cause ofendobronchialofextrapulmonary primary tumours have been associatedwith ebm primarily breast colon and renal carcinomas[] ebms from testicular seminomas are also extremelyrare the majority of testicular tumours arise from testicular germ cells and are frequently composed of multiple celltypesaretumours most ofie mixedtypethese the authors respirology case reports published by john wiley sons australia ltdon behalf of the asian paciï¬c society of respirology 0cck teng ebm from embryonal carcinomatable reports of previous cases of ebmslocationdiagnostic methodpathology¶zt¼rk moreirameyer case case the oriï¬ce of right upper loberight main bronchusleft main bronchus main carina andright main bronchus fibreoptic bronchoscopy mixed gctfibreoptic bronchoscopy mixed gctvideobronchoscopyembryonal carcinoma¶zsu the oriï¬ce of the right upper lobefibreoptic bronchoscopytesticular seminomaturan varkey our caseand right intermediary loberight intermediate bronchusleft main bronchusleft main bronchusebm endobronchial metastases gct germ cell tumourembryonic carcinomas or seminomas there are only a few published reports of primary testicularembryonic carcinomas resulting in ebms []the mostofcommon symptomsendobronchialtumours are haemoptysis and coughing with shortness ofbreath and wheezing reported less frequently howeversome patients are asymptomatic in our patient symptoms on presentation included haemoptysis and shortnessof breathresults from chest radiography in patients with ebmcan be quite variable and may include mediastinal lymphadenopathy hilar masses atelectasis and multiple pulmonary nodules chest radiographs may also be normal onpresentation our patient presented with complete collapse of the left lung that was revealed initially by chestradiographyhowever the full diagnosis cannot be made based onlyon symptoms and chest radiographs it can be difï¬cult todistinguish between primary lung cancer and tumours ofextrapulmonary origin based on these ï¬ndings alone toconï¬rm the diagnosis we obtained a bronchoscopic biopsyspecimen to examine the tumour tissue the ï¬exible bronchoscopy ï¬breoptic bronchoscopy can be performedunder sedation without general anaesthesia as comparedwith rigid bronchoscopy the former is a simple techniquewhich is well tolerated and most commonly performed asan outpatient day procedure the patient underwentbronchoscopic cryosurgery under sedation in our bronchoscopy room to excise the tumour the ï¬nal pathology reportconï¬rmed the diagnosis of metastatic embryonal carcinomawe had evaluated the presence of afp bhcg andldh tumour markers elevated afp levels can be secretedby germ cell tumours gcts including embryonal carcinoma yolk sac tumour or teratoma in gcts detectablerigid bronchoscopybronchoscopyfibreoptic bronchoscopyand cryosurgerysomatictype gctembryonal carcinomaembryonal carcinomabhcg elevation is observed in both seminomas and nonseminomas the serum level of ldh was directly correlated with tumour burden in nonseminomatous gctswhich is also useful for the surveillance of patients withadvanced seminoma the tumour markers in ourpatient showed elevated levels of afp bhcg and ldhthis was compatible with the diagnosis of embryonal carcinoma moreirameyer also evaluated the patienttumour markers and found elevated levels of afp ngml and bhcg miuml the elevatedlevels of both tumour markers contribute to the diagnosisof metastatic embryonal carcinoma ¶zsu onlyevaluated the patients bhcg level which was found to beelevated miuml and the ï¬nal diagnosis wasmetastatic testicular seminoma on comparison with previous case reports table ours was the ï¬rst case in which the tissue was obtainedusing cryosurgery other reports obtained tissue samplesusing forceps biopsy alone or forceps biopsy combinedwith argon plasma coagulation apc to control bleedingcryobiopsy provided us with enough sample to performmore extensive immunohistochemical staining cryobiopsyhas more successful diagnostic results than forceps biopsiesdue to larger and highquality artefactfree sampleshaemorrhage was observed to be similar during both procedures further study on this topic will be needed toevaluate which of the diagnostic methods result in superioroutcomesin conclusion ebms from primary gcts notably thoseassociated with total or partial collapse are extremely rarewe have presented this case to emphasize the importanceof distinguishing ebm from primary lung carcinoma andto report the ï¬rst case in which metastatic embryonalcarcinoma was diagnosed by bronchoscopic cryosurgery the authors respirology case reports published by john wiley sons australia ltdon behalf of the asian paciï¬c society of respirology 0cebm from embryonal carcinomadisclosure statementappropriate written informed consent was obtained forpublication ofand accompanyingimagesreportcasethisreferences ¶zt¼rk a aktas¸ z and yılmaz a endobronchialmetastasis of mixed germ cell tumors two cases tuberktoraks lee sh jung jy kim dh endobronchialmetastases from extrathoracic malignancy yonsei med j ikemura k lin dm martyn cp endobronchialmetastasis from extrapulmonary neoplasms analysis ofclinicopathologic features and cytological evaluation bybronchial brushing lung moreirameyer a bautistaherrera d hern¡ndezembryonal endobronchialgonz¡lez mck teng carcinomaj bronchologyinterv pulmonol ¶zsu s erol mm oztuna f endobronchial metastasis from testicular seminoma med princ pract tumoraltesticular germ cell turan d akif ¶zg¼l m kirkil gendobronchial metastasis ofeurasian j pulmonol et varkey b and heckman mg diagnosis of a case ofembryonal carcinoma by bronchial biopsy chest paradis tj dixon j and tieu bh the role of bronchoscopy in the diagnosis of airway disease j thorac dis aktas z gunay e hoca nt endobronchialcryobiopsy or forceps biopsy for lung cancer diagnosisann thorac med barlow lj badalato gm and mckiernan jm serumtumor markers in the evaluation of male germ cell tumorsnat rev urol the authors respirology case reports published by john wiley sons australia ltdon behalf of the asian paciï¬c society of respirology 0c' | 0 |
"analyse the quality of information included in websites aimed at the public on COVID19Methods Yahoo Google and Bing search engines were browsed using selected keywords on COVID19ï¬rst websites from each search engine for each keyword were evaluated Validated tools wereThe used to assess readability [Flesch Reading Ease Score FRES] usability and reliability LIDA tool andquality DISCERN instrument Nonparametric tests were used for statistical analysesResults Eightyfour eligible sites were analysed The median FRES score was range 00 Themedian LIDA usability and reliability scores were range 00 and 37range14 00 respectively Alow overall LIDA score was recorded for n of the websites The median DISCERN score 14 was found in websites The DISCERNwas range The DISCERN score of score was significantly associated with LIDA usability and reliability scores p and the FRES scorep Conclusion The majority of websites on COVID19 for the public had moderate to low scores with regardsto readability usability reliability and qualityPractice Implications Prompt strategies should be implemented to standardize online health informationon COVID19 during this pandemic to ensure the general public has access to good quality reliableinformationï¾ Elsevier BV All rights reserved Introductionfor The coronavirus COVID19 pandemic has become the greatestglobal health crisis of the st century [] During this pandemicthe demand information on COVID19 has skyrocketedInformation such as latest news updates on the pandemic itssymptoms prevention and mechanism of transmission are highlysought by the public [] On the other hand free access toinformation especially through social media which is accessed bythe majority [] has led to an increase in misinformation and panicassociated with COVID19 [] Although high quality healthinformation is known to be related to lower stress levels andbetter psychological health [] previous studies have shown thatonline information on many medical disorders to be of substandard quality []A previous study done on websites related to COVID19 hasreported substandard quality information that could potentiallymislead the public [] However this study has used a limitedsearch strategy and had not assessed some important areasincluding usability and reliability of the information Thereforethis topic remains a knowledge gap in COVID19 research []Therefore we conducted this study to analyse the current COVID websites targeting the general public in terms of qualityusability readability and reliability using a wide search strategyand validated instruments MethodsAbbreviations USA United States of America FRES Flesch reading ease scoreHONcode Health on the net Code of Conduct SPSS Statistical package for socialsciences NICE National Institute for Health and Care Excellence WHO WorldHealth anization Corresponding author at Department of Surgery Faculty of MedicineUniversity of Colombo PO Box Kynsey Road Colombo Western ProvinceSri LankaEmail addresses ravindrijayasinghegmailcom R Jayasingheranasigmcgmailcom S Ranasinghe umeshejayagmailcom U Jayarajahsanjeewasrgcmbaclk S SeneviratneYahoo Google and Bing were searched using the keywordssevere acute respiratory synnovel coronavirusSARSCoV2 and coronavirus The searchdrome coronavirus2 COVID19was performed during the ï¬rst week of May The details ofthe search strategy and the piloting process are provided in thesupplementary material File S1 []Two independent investigators with previous experience ofconducting similar studies assessed the selected websites []Prior to the assessment a pilot run was conducted to ensure101016jpec20200800107383991ï¾ Elsevier BV All rights reservedPlease cite this in press as R Jayasinghe et al Quality of online information for the general public on COVID19 Patient Educ Couns 101016jpec202008001 0cModelGPEC No of Pages R Jayasinghe et al Patient Education and Counseling xxx xxxxxxuniformity and accuracy The information on symptoms investigations public health measures and available treatmentmodalities were collected The accuracy of the content wasassessed using the national institute for health and care excellenceNICE guidelines on COVID19 [] A rating was given as all ornone based on congruence with the guidelinesValidated instruments were used to assess the quality ofwebsites Readability was assessed using the Flesch Reading EaseScore FRES [] The LIDA Instrument 2007Version12 wasused to analyse the content and the design of the websites usingthe usability and reliability domains [] The quality of thecontent was assessed using the DISCERN questionnaire which has questions in two separate groups [] The detailed assessmentcriteria and the scoring system is included in the supplementarymaterial File S1A website was classiï¬ed as governmental if it was maintainedby the countrys public health authority If managed by privateinstitutions nongovernmental anizations or voluntary institutions independent from the government they were consideredas nongovernmental The online healthrelated websites arestandardized in terms of their credibility and reliability by onlinecertiï¬cation sites We chose the Health on the Net code of conductHONcode which is the oldest and widely used out of the qualityevaluation tools available []Data analysis was performed using SPSS Version20 softwareand the associations were determined with non parametric testsA pvalue of was considered statistically significant ResultsOf the retrieved websites were excluded and thein the analysis Theremaining websites were characteristics of the websites are mentioned in Table included Half were governmental websites and only n were HON accredited websites The median FRES was range 00 10th12th grade readability level which is classiï¬ed asfairly difï¬cult to read Only three websites had a readabilityscore of above equivalent to 7th grade which is therecommended standardThe overall median LIDA score was range whilethe median LIDA usability and reliability scores were range 00 and range 00 respectively The median DISCERNscore was range which classiï¬es websites as being offair quality Excellent Good Fair Poor Very poor However the top websitesTable A were of excellent qualityTable Website characteristicsWebsite Characteristics Frequency PercentageUsability Governmental websites Notforproï¬t and private websites HONcode accredited Readability score of above equivalent to 7th grade Readability Date of publication stated References mentioned Disclosure statement by authors Infographics Moderate Low Moderate 00 Low score Moderate 00 Low Used Textonly Reliability Table Correlation between DISCERN scores and other factorsDISCERN SCORELow Mean High 00 Mean Range 00 00 00 00 Range 00 00 00 00 N P valueP0001P0001P0001P P P LIDA Usability LIDA Reliability LIDA Overall FRES Score Government HON Certiï¬cation No N No Yes Yes Signiï¬cant correlations were observed between the DISCERNscore and the overall LIDA score as well as LIDA usability andreliability scores Table p HONcode certiï¬ed websitessites obtained significantly higher DISCERN scores p Pertaining to the currency of information only publishers stated the date of the publication Most websites n did not declare the sources of evidence This was furtherestablished by the median reliability score of Nevertheless the authors have included a disclosure statement in mostn websiteslowFigures A1 and A2 summarize the rating of websites onindividual criteria assessed by the DISCERN tool The speciï¬cinformation provided regarding COVID19 is shown in Fig More than half of the websites failed to discuss the treatmentoptions available n beneï¬ts or risks n and effects of no treatment n Furthermore potentialcomplications and prognosis were stated only in and websites respectively Discussion and conclusion DiscussionThis study has shown that still most of the websites on onlinehealth information on COVID19 are of suboptimal quality exceptfor a few credible sources of goodquality health informationNevertheless the websites ranked among the top according tothe DISCERN score Table A2 had high scores indicating thepotential for publishing credible highquality information onlinewhich would beneï¬t the publicin turn causes panic which ranges Misinformation is a major concern during this pandemic aspeople fail to spend adequate time to critically analyse the onlineinformation This fromhoarding medical supplies to panic shopping and using drugswithout prescription with negative social and medical consequences [] Therefore measures implemented to ensure the qualityand accuracy of online information by the responsible authoritiesmay help negate these adverse consequencesinformation Stating the methods of content production with names of thecontributing authors may help increase the credibility of onlinehealth information while displaying the date of the publicationprovides an idea of the currency of the information Absence ofin over half of the websites was a majorsuch drawback especially for COVID19 where new information isgenerated almost daily Health authorities should therefore ensurethat the patient information websites provide the above information and certify websites based on such details so that the publiccan get information from trusted sources []Most users of the worldwide web only have an average level ofeducation and reading skills [] Guidance from the NationalPlease cite this in press as R Jayasinghe et al Quality of online information for the general public on COVID19 Patient Educ Couns 101016jpec202008001 0cModelGPEC No of Pages R Jayasinghe et al Patient Education and Counseling xxx xxxxxx Fig Website characteristics evaluated outside the DISCERN toolInstitute of Health NIH had shown that the readability should bebelow the level of seventh grade for the lay public to adequatelyunderstand the content [] However the median readabilitylevel was found to be equivalent to 10th12th grade readabilitySuch complexities with the readability of information mayincrease the risk of misunderstandings or misinterpretation Usingshort sentences in writing using the active voice using 12point orlarger font size using illustrations and nontextual media asappropriate and accompanying explanations with examples wouldbe helpful to overcome this problem []So far only a limited number of studies have been done to assessthe quality of health information websites related to COVID19 Thestudy by CuanBaltazar et al prior to February reported poorquality information with approximately of included websiteswith low DISCERN scores [] Our study done three months latershows similar results with only a minimal improvement in thequality of information Furthermore the Cuan Baltazar study hadseveral limitations which includes the limited search strategy andnoninclusion of key quality parameters including readabilityFurthermore out of the sites they had includedwere online news sites that are not considered as patientinformation websites In that study the HONcode seal waspresent only in n websites whereas in our study ofthe sites were HONcode certiï¬edThere were several limitations in this study Although mostpopular search engines were used in this study under defaultsettings they may produce variable results depending on manyfactors including geographical location and popularity of websitesat a given point of time The algorithms unique to those searchengines are subjected to constant change and therefore the exactresults of our study may not be reproducible However we believethe general patterns observed in our study are validproviding health information to the general public are to be ofsubstandard quality Practice implicationsTo improve the credibility of the content the websites shouldstate methods of content production and display the date of thepublication to give an idea about the currency of the informationTo improve the readability of the content the websites shouldincorporate more nontextual media write in short sentencesusing the activevoice and use larger font sizes The patientinformation websites should display scores of reliability qualityand readability as a guidance for its users Furthermore it is vitalfor medical regulatory authorities and the government to imposeregulations to ensure quality and to prevent the spread ofmisinformationAvailability of data and materialsOn reasonable request from the corresponding author the dataused in the above study can be made availableEthics approval and consent to participateUnnecessary in this type of studyInformed consent and patient detailsNot applicable in this type of studyFundingNone ConclusionCRediT authorship contribution statementThis study has shown the quality readability usability andreliability of the information on COVID19 on majority of websitesRavindri Jayasinghe Conceptualization Methodology Datacuration Writing original draft Visualization InvestigationPlease cite this in press as R Jayasinghe et al Quality of online information for the general public on COVID19 Patient Educ Couns 101016jpec202008001 0cModelGPEC No of Pages R Jayasinghe et al Patient Education and Counseling xxx xxxxxxValidation Formal analysis Resources Sonali RanasingheConceptualization Methodology Data curation Writing originaldraft Visualization Investigation Validation Formal analysisResources Umesh Jayarajah Conceptualization MethodologyData curation Writing original draft Visualization InvestigationValidation Formal analysis Resources Sanjeewa SeneviratneConceptualization Methodology Writing review editingSupervision Project administrationDeclaration of Competing InterestThe authors report no declarations of interestAcknowledgementsNone declaredAppendix A Supplementary dataSupplementary material related to this can be found in101016jversion at online the pec202008001References[] IJ Bes do Nascimento N Cacic HM Abdulazeem Novel Coronavirusinfection COVID19 in humans a scoping review and metaanalysis JClinical Med [] HT Le DN Nguyen AS Beydoun XTT Le TT Nguyen QT Pham NTK Ta QT Nguyen AN Nguyen MT Hoang Demand for health information onCOVID19 among Vietnamese Int J Environ Res Public Health [] C Wang R Pan X Wan Y Tan L Xu RS McIntyre FN Choo B Tran R Ho VK Sharma A longitudinal study on the mental health of general populationduring the COVID19 epidemic in China Brain Behav Immun [] CS Ho CY Chee RC Ho Mental health strategies to combat the psychologicalimpact of COVID19 beyond paranoia and panic Ann Acad Med Singapore [] C Wang R Pan X Wan Y Tan L Xu CS Ho RC Ho Immediate psychologicalresponses and associated factors during the initial stage of the coronavirus disease COVID19 epidemic among the general population inChina Int J Environ Res Public Health [] RH Waidyasekera U Jayarajah DN Samarasekera Quality and scientiï¬caccuracy of patientoriented information on the internet on minimallyinvasive surgery for colorectal cancer Health Policy Technol [] R Jayasinghe S Ranasinghe U Jayarajah S Seneviratne Quality of patientoriented webbased information on oesophageal cancer J Cancer Educ In press[] JY CuanBaltazar MJ MuñozPerez C RobledoVega MF PÃrezZepeda ESotoVega Misinformation of COVID19 on the internet Infodemiology studyJMIR Public Health Surveill 2020e18444[] BX Tran GH Ha LH Nguyen GT Vu MT Hoang HT Le CA Latkin CS HoR Ho Studies of novel coronavirus disease COVID19 pandemic a globalanalysis of literature Int J Environ Res Public Health [] G Eysenbach C Köhler How do consumers search for and appraise healthinformation on the world wide web Qualitative study using focus groupsusability tests and indepth interviews Brit Med J [] AS Prasanth U Jayarajah R Mohanappirian SA Seneviratne Assessment ofthe quality of patientoriented information over internet on testicular cancerBMC Cancer [] V Udayanga U Jayarajah SD Colonne SA Seneviratne Quality of thepatientoriented information on thyroid cancer in the internet Health PolicyTechnol [] National Institute for Health and Care Excellence Coronavirus COVID19 Accessed April wwwniceukcovid19[] Readable The Flesch Reading Ease and FleschKincaid Grade Level Accessed February readablecomblogtheï¬eschreadingeaseandï¬eschkincaidgradelevel[] Minervation The Minervation Validation Instrument for Healthcare WebsitesLIDA Tool Accessed February httpwwwminervationcomwpcontentuploads201104MinervationLIDAinstrumentv12pdf[] Minervation Is the Lida Website Assessment Tool Valid Accessed February httpwwwminervationcomdoeslidawork[] Discern Online The DISCERN Instrument Accessed February httpwwwdiscernukdiscern_instrumentphp[] E Fahy R Hardikar A Fox S Mackay Quality of patient health information onthe Internet reviewing a complex and evolving landscape Australas Med J [] J Kluger As Disinfectant Use Soars to Fight Coronavirus So Do AccidentalPoisonings Accessed April timecom5824316coronavirusdisinfectantpoisoning[] BX Tran AK Dang PK Thai HT Le XTT Le TTT Do TH Nguyen HQPham HT Phan GT Vu Coverage of health information by different sourcesin communities implication for COVID19 epidemic response Int J EnvironRes Public Health [] National Institutes of Health How to Write EasyToRead Health Materials Accessed April wwwscribdcomdocument261199628HowtoWriteEasyToReadHealthMaterialsMedlinePlus[] DM DAlessandro P Kingsley J JohnsonWest The readability of pediatricpatient education materials on the world wide web Arch Pediatr AdolescMed Please cite this in press as R Jayasinghe et al Quality of online information for the general public on COVID19 Patient Educ Couns 101016jpec202008001 0c" | 2 |
Purpose of Review Recognize which are the elements that predict why a person is aging faster or slower and which interventionwe can arrange to slow down the process which permits to prevent or delay the progression of multimorbidity and disabilityRecent Findings Aging is a complex process that leads to changes in all the systems of the body and all the functions of theperson however aging develops at different rates in different people and chronological age is not always consistent withbiological ageSummary Gerontologists are focused not only on finding the best theory able to explain aging but also on identifying one or moremarkers which are able to describe aging processes These biomarkers are necessary to better define the agingrelated pathologies manage multimorbidity and improve the quality of life The aim of this paper is to review the most recent evidence onaging biomarkers and the clusters related to them for personalization of treatmentsKeywords Biomarker of aging Frailty syndrome Aging phenotype Quality of life Multimorbidity Life expectancy SocialneedsIntroductionMost people dont grow up Most people age They findparking spaces honour their credit cards get married havechildren and call that maturity What that is is agingMaya Angelou One of the biggest megatrends impactingthe world today is population aging Aging is a topic thathas captivated both scientists and philosophers throughouthistory but aging as a population scenario emerged on aThis is part of the Topical Collection on Geriatric Oncology Beatrice Di Capuabeatricedicapuagmailcom UOC di Radioterapia Oncologica Dipartimento Diagnostica perImmagini Radioterapia Oncologica e Ematologia FondazionePoliclinico Universitario A Gemelli IRCCS Rome Italy Dipartimento di Scienze dellinvecchiamento neurologicheortopediche e della testacollo Fondazione Policlinico UniversitarioA Gemelli IRCCS Rome Italy Moffitt Cancer Center Tampa FL USAworldwide scale for the first time in the last century Thus itis hard to really identify a definition of aging It is a decrease infitness with chronological age it is a developmental phasebeyond the normal life trajectory and it is a time of the increased risk of physical and psychological disabilities testingthe limits of resilienceAging occurs at a different rate in varying geographic regions of the worldEurope is currently the oldest region with of thetotal population aged and older However the Asia andLatin America older population is growing fast with Asiasolder population almost tripling in size from million in to million in []All these data do not consider aging as an epiphenomenonbut an individual data of the global population just a chronological number Aging is intrinsically a complex scenariocharacterized by changes that take place at different levels ofbiological systems Biological age is of course influenced bychronological age but chronological age is by itself not representative of biological age biological age is determined byphysiological reserve and functional status Assessing biological age is essential to predict life expectancy and resilience to 0c Page of Curr Oncol Rep stressors [] If any definition of aging may appear incompleteand insufficient much more difficult and complex is to findthe marker or biomarker that can identify itMany theories currently trying to explain aging processesand many biomarkers are identified to measure aging and itsevolutionary stages Theories and biomarkers are not studiedto extend life span but to guide therapeutic choices and optimize patient management and personalization of careThe purpose of this paper is not purely to list which biomarkers are able to identify the various stages of aging ratherexplain how an epiphenomenon natural and physiological isso complex [] how many factors are protagonists in its development and how many actors and characters play in maximizing its individual features taking into account social andmorbidity biomarker These factors such as frailty loss ofautonomy essential needs and comorbidities influence theaging process and are able to justify why the biological ageof a person living in a country does not correspond to the ageof another person living in a country with better sociosanitaryconditionsClinical and Biological Aging PhenotypesThe aging phenotype can be described as a complex mosaicresulting from the interaction of a variety of environmentalstochastic and geneticepigenetic eventsstimuli impinginglifelong on our body [ ]There is no clear evidence which molecular cellular orphysiological changes are the most important drivers of theaging process andor how they influence one another [] In itsbroadest sense aging merely refers to the changes that occurduring an anisms life span though the rate at which thesetake place varies widely [] Despite its enormous complexityinvolving combinations of these variables a small number ofbasic molecular mechanisms underpin the aging process including a set of evolutionary highly conserved basic biological mechanisms responsible for body maintenance and repairOne of the key mechanisms is inflammation a typical featureof the aging process is the development of a chronic lowgrade inflammatory status named inflammaging [8cid129] whichemerged as critical in the pathogenesis of major agerelatedchronic diseases such as atherosclerosis type diabetes and neuro degeneration Inflammaging plays a pivotal role in themost important geriatric conditions such as sarc ia [9cid129cid129]osteoporosis [] frailty and disability thus contributing tomortality [] Interestingly a variety of tissues adipose tissue muscle ans brain liver systems immune systemand ecosystems gut microbiota of the body indicated assubsystems can contribute to the onset and progressionof such a systemic inflammatory state [] by increasing theproduction of several proinflammatory mediators or loweringthat of the antiinflammatory ones [8cid129]To differentiate the innocuous changes from those leadingto increased risk of disease disability or death biogerontologists tend to use a more precise termsenescencewhen describing aging [] Senescence is thereforethe progressive deterioration of bodily functions over time andnormal human aging has been associated with a loss of complexity in a wide range of physiological processes and anatomic structures [] including blood pressure [] strideintervals [] respiratory cycles [] and vision [] amongothers such as postural dynamics [] ultimately leading todecreased fertility and increased risk or mortality []Systemic consequences of aging are widespread but theycan be clustered into four domains Fig Changing in body compositionThe balance between energy availability and energydemandSignaling networks that maintain homeostasis NeurodegenerationThese changes develop in parallel and affect each otherthrough many feedforward and feedback loopThe phenotype that results from the aging process is characterized by increased susceptibility to disease high risk ofmultiple coexisting diseases impaired response to stress theemergence of geriatric syndromes altered response to treatment high risk of disability and loss of personal autonomywith all its psychological and social consequences On theother hand all these factors influence aging itself in a dynamic and parallel way so that they can be considered as not onlya consequence of aging but also an integral part of the agingprocessTheories of AgingHuman aging is currently defined as a dynamic process involving the continual adaptation of the body to lifelong exposure tointernal and external damaging as conceptualized in the remodelling theory of aging [21cid129cid129] Theories of aging are generallyclassified as either program or damage theories Programmedaging theories suggest that there is a deliberate deterioration withage because a limited life span results in evolutionary benefits[] This plan could be a result of aging genes The firstdescribed mutation to yield a significant extension in the life spanof Caenorhabditis elegans was in the ageI gene which wasshown to result in a increase in mean life span and a increase in maximum life span of this anism []Evolutionary biologists may argue that aging occurs due to theabsence of natural selection at the postreproductive stage of life[] Although such aging theories are subjectively appealing asthey convey a cure for aging the accumulation of damage is aspontaneous entropydriven process [] Among the damage 0cCurr Oncol Rep Fig Systemic consequences ofagingPage of theories a prevailing idea is that of oxidative damage Reactiveoxygen species ROS are generated during metabolism throughseveral interrelated reactions The supposition that aging may becaused by ROS has been further substantiated by studies involving transgenic animals for genes encoding antioxidants The lifespan of Drosophila melanogaster has been extended by overexpression of both superoxide dismutase SOD and catalase bothantioxidant enzymes [] Since mitochondria are the major producer of ROS in mammalian cells mitochondrial DNAmtDNA is therefore particularly susceptible to oxidative damage [] Mitochondrial maintenance is therefore essential topreserve cellular homeostasis and impaired mitochondrial maintenance has been described as a shared hallmark of numeroushuman pathologies and aging [] Mitochondrial DNA varieswith age and it is commonly considered that DNA hypomethylation is a typical aspect of the aging process [] ROS are activeintermediates of DNA methylation as well as histone modification These reactive oxygen species may play a role in epigeneticprocesses physiological phenotypic variations caused by external or environmental factors that switch genes onoff throughreactions of nucleophilic substitution at the DNA levelConsequently it has been suggested that better preservation ofDNA methylation levels slower cell metabolism and improvedcontrol in signal transmission through epigenetic mechanismscould be key processes involved in human longevity Oxidativedamage to proteins is irreversible and irreparable [] and mustbe degraded by the proteasome The proteasome is the mostimportant proteolytic machinery in eukaryotic cells largely responsible for the removal of oxidized proteins and the preventionof its aggregation [] However it has been shown that theactivity of proteasome is impaired during aging leading to theaccumulation of oxidizing proteins aggresome and lipofuscinsocalled the age pigment Similarly to oxidative damage nitrosamine damagethat caused by reactive nitrogen speciesRNS such as nitric oxidehas been suggested to also contribute to agerelated diseases namely hepatic steatosis and apoptosis [] as well as functional and structural changes in the cardiovascular system [ ] sleep homeostasis [] psychological disorders [] and dementia []Most supporters of the genomic instability theory of agingrefer to telomere shortening [] and mutation in DNA mitochondrial Telomeres are the repeated DNA sequences at theends of linear chromosomes which are unable to be fullyreplicated by DNA polymerasesMutations in mtDNA cause a wide range of human mitochondrial diseases and have been implicated in agerelateddiseases and agingBiomarker FeaturesFinding the biomarker of aging is one of the most importantgoals of medicine The National Institutes of HealthBiomarkers Definitions Working Group defined a biomarkeras a characteristic that is objectively measured and evaluatedas an indicator of normal biological processes pathogenicprocesses or pharmacologic responses to a therapeutic intervention []The American Federation for Aging Research AFAR recommends the following criteria for biomarkers of aging [39cid129]It must predict a persons physiological cognitive andphysical function in an agerelated way independentlyof chronological age 0c Page of Curr Oncol Rep It must be testable and not harmful to test subjects forexample a blood test or an imaging technique it mustalso be technically simple to perform and it must be accurate and reproducibly without the need for specializedequipment or techniquesIt should work in laboratory animals as well as humanssince preliminary testing is always done in nonhumansubjectsFerrucci et al reviewed the biomarkers proposed as elements of a theory based on the balance between resiliencemechanisms and accumulated damages where biomarkersact in reducing resilience mechanisms or increasing damages[40cid129] Tables and The pathways eligible to become biomarkers are thefollowingGenomic Instability Endogenous and exogenous agents continuously challenge the integrity of DNA when DNA repairmechanisms cannot manage the repeated damage the result isan accumulation of DNA somatic mutations This phenomenon causes dysregulation of gene expression and the production of altered proteins that lead to cellular damage Somaticmutation accumulation has been observed in skeletal musclecells neurons and lymphocytes B related to aging []nevertheless quantification of DNA repair capacity in humanshas yet to be finalized []Telomere Attrition Telomeres are the DNA sequences that areplaced at the end of the DNA chain and protect theTable Biological changesunderlying agingGenomic instabilityTelomere attritionEpigenetic alterationscid129 DNA methylationcid129 Histone modificationcid129 Noncoding RNALoss of proteostasisMitochondrial dysfunctionCellular senescenceDeregulated nutrientsensingSteam cell exhaustionAltered intercellular communicationchromosome ends from damage During each replicationtelomeres are reproduced but not completely so with agingthey become shorter and contribute to cellular senescence[] To date different techniques are available to detecttelomere length in circulating cells however no techniqueshave been validated for evaluating aging because of the heterogeneity between different cells between individuals andhigh measurement errors that make these techniques not yetvalid in clinical practice []Epigenetic Alterations Epigenetics refers to those mechanismsexternal to DNA that modulate gene expression in cells theregulation of gene expression determines the phenotypic characteristics of the different cells and tissues The main mechanismsare DNA methylation histone modification and noncodingRNA While DNA methylation is easily measured in circulatingcells and seems to be correlated to aging [ ] measuringhistone modification or noncoding RNA is difficult and expensive Recent evidence correlates DNA methylation with agingand agerelated chronic diseases in humans [ ]Individuals with higher levels of DNA methylation have a higherrisk of developing several agerelated diseases and prematuremortality for all causes and cardiovascular diseases [] as wellas physical and cognitive functions [ ]Loss of Proteostasis The repair of damaged structures or theirelimination is fundamental to maintain cell integrity and function [] Studies suggest that proteostasis becomes defectivewith aging and contributes to immunosenescence [] and thatautophagy appears to be more functional in longlived peopleAccumulation of DNA somatic mutationsDysregulation of gene expressionAltered proteins productionTelomere shortening contribute tocellular senescenceAltered gene expressionRelated to agerelated chronic diseasesAccumulation of damaged structuresAltered energy productionIncreased ROS productionApoptosisprogrammed cell deathActivation of pathways leading to apoptosisProduction of SASPIncrease of life span in dietary restrictionDecline of regenerative potentialInflammagingDysfunction of endocrine neuronaland immune systems 0cCurr Oncol Rep Page of Table Measurable biomarkers classified by respective hallmarksPathways measuredMeasurable biomarkersHallmarkGenomic instabilityTelomere shorteningCellular senescencecid129 DNA repair mechanismscid129 DNA modificationscid129 Telomere lengthcid129 Markers of DNA damage responsecid129 Telomerase activitycid129 Senescent markers in blood and tissueEpigenetic changes or epigenetic clockcid129 DNA methylationcid129 Histone acetylationcid129 Noncoding RNAMitochondrialDecreased autophagy proteostasiscid129 Mitochondrial volumenumbershapecid129 Mito respirationcid129 Markers of biogenesiscid129 mtDNA copy number and haplotypescid129 Autophagy markerscid129 Chaperon proteinsStem cell exhaustionDeregulated nutrientsensingAltered intercellular communicationcid129 Proliferative capacity in vitrocid129 Resistance to stresscid129 Growth hormone GH axiscid129 Metabolism alterationscid129 Measures of inflammationcid129 yH2AX immunohistochemistrycid129Leukocyte telomere lengthcid129MIR31HGcid129 p16INK4acid129 Senescenceassociated secretoryphenotype SASP proteinscid129 Measures of DNA methylationcid129 SIRT1 SIRT2 SIRT3 SIRT6 SIRT7cid129 Dosage of circulating microRNAs miR34aMiR21 miR1263p miR151a3pmiR181a5p miR1248cid129 p31 MRI spectroscopycid129 Growth differentiating factor GDF15cid129 NADcid129 Target of rapamycin TORcid129 Protein carbamylationcid129 Advanced glycation end productscid129 Insulinlike growth factor IGF1cid129 HGBA1ccid129 IL6cid129 TNFαcid129 CRP Creactive proteincid129 TNFRII tumor necrosis factorα RII[] Measuring the loss of proteostasis mechanism could be agood biomarker but to date there are no valid techniques forthis purposeMitochondrial Dysfunction The main role of mitochondria isto guarantee energy for the cell through the production ofATP They are also involved in signaling by the productionof ROS and in apoptosisprogrammed cell deathMitochondrial dysfunction is a good biomarker of aging andis associated with disability in older persons through the reduction of muscle strength [65cid129]Many techniques are measuring oxidative phosphorylationand ROS generation that have been associated with chronicdisease [ ] nevertheless the relation with aging is notcompletely validatedCellular Senescence Genomic instability telomere shorteningand other endogenous and exogenous mechanisms can inducethe cell to activate specific pathways that lead to apoptosis[] This process is called cellular senescence and is characterized by structural and functional changes in the cell []Senescent cells produce proinflammatory cytokines andchemokines growth factors and matrix proteases called senescenceassociated secretory phenotype SASP [ ]which may induce some agerelated diseases [] Thedetection of SASP has been proposed as a biomarker of aging[]Deregulated NutrientSensing Genetic mutations in growthhormone and the insulinlike growth factor have been linkedto longevity [] Moreover dietary restriction showed to increase life span in primates [ ] For these reasons thispathway has been proposed as biomarkers of agingSteam Cell Exhaustion The decline in the regenerative potential is one of the elements at the base of aging [] Despitepharmacological interventions being explored to counteractthis phenomenon [] evidences are still poor 0c Page of Curr Oncol Rep Altered Intercellular Communication With aging we also observe changes in intercellular communication as inflammatory reaction increases the other communication ways becomedysfunctional endocrine neuronal immune system []As we discussed earlier inflammation can be inappropriately increased in aging and this has been related to agerelated disease [ ]Indeed the pathways described as potential biomarkers ofaging are strongly related to inflammation for this reasonmeasuring circulating levels of cytokines is considered anew field of research [ 84cid129 ]Aging and Life ExpectancyAging and life expectancy are closely related In a broadsense determining an individuals life expectancy is also away of schematizing his or her aging process Life expectancyis a statistical measure of the average time an anism isexpected to live based on the year of its birth LEB itscurrent age and demographic factors including gender []In the last decades life expectancy has increased in high income country the rise in human life expectancy has involveddeclines in intrinsic and extrinsic mortality processes associated respectively with senescence and environmental challenges []In association to this increased longevity there are diseasescalled agerelated that increase quadratically with age andcause a progressive loss of physical mental and cognitiveintegrities leading to impaired function and increased vulnerability to morbidity mortality [] and disability in additionto increasing care needs and agerelated burden measuredthrough the sum of disabilityadjusted life years DALYs ofthese diseases among these adults Fig Ninetytwo of the of the Global Burden of Disease causes were identified asagerelated diseases In particular cardiovascular diseaseneoplasm and chronic respiratory disorders are those withhigher agerelated disease burden []Determinants of Frailty Syndrome as AgingBiomarkerFrailty can be defined as a state of increased vulnerabilityto stressors or a loss of capacity to resolve homeostasisperturbation Frailty condition is closely related to aging[88cid129cid129] and the frailty indexes can consequently be considered biomarkers of aging themselves In frail individuals it is possible to find both changing in body composition and balance between energy availability and energydemand Moreover in the definition of frailty it is welldescribed how signaling networks maintain homeostasisand association with neurodegeneration These fouraspects all refer to the hallmarks of aging Frailty is associated with adverse clinical outcomes including falls institutionalization and death [88cid129cid129]Two principal models emerged in the last decades that areable to conceptualize and consequently measure frailty in everyday clinical practice and research the frailty phenotypemodel and the cumulative deficits modelThe frailty phenotype was first described by Fried and colleagues in analyzing data from the CardiovascularHealth Study CHS involving men and women aged years and older In this study it was investigated whichcharacteristics of the population were predictive of falls disability hospitalization and death Their operational definitionof frailty included a cluster of at least three of the followingvariables unintentional weight loss selfreported exhaustionlow energy expenditure slow gait speed and weak gripstrength This model does not take into consideration cognitive impairment as a cause of increased vulnerability as thiscould contribute to functional decline and adverse events inolder people [ ]The cumulative deficits model was developed byRockwood and colleagues as part of the prospectiveCanadian Study of Health and Aging CSHA involvinga cohort of older adults [] The authors identified parameters including diseases disabilities signssymptoms and laboratory values which were defined asdeficits The sum of the deficits in a single individualallowed for the calculation of a frailty index ie thenumber of deficits divided by Frailty in this modelis not considered as a cluster of symptoms but is conceptualized as a gradable syndrome with a higher number ofdeficits implying an increased vulnerability state The twomodels of frailty show significant overlap although theycapture slightly different sides of the same problem It isimportant to notice that physical frailty is frequently associated with multimorbidity [ 93cid129 ]It has been observed that the frailty phenotype construct is intrinsically related to mobility issues Indeedin older adults physical performance measures are a robust and consistent predictor for disability hospitalization institutionalization and death both in the researchand in the clinical setting Lower physical performance isfrequently associated with loss of skeletal muscle massand quality causing reduced strength and functional impairment [95cid129cid129] This process has been called sarc iaEven though sarc ia has been long associated withaging it has to be acknowledged that it can develop muchearlier in life [] Different definitions exist for this condition for the operational definition of sarc ia both inthe clinic and for research purposes that prioritize theassessment of muscle strength over muscle mass to identity sarc ic patients Strength is more closely related tosurvival and functional decline compared with muscle 0cCurr Oncol Rep Page of CARDIOVASCULAR DISEASESAtrial ï¬brillaÆon and ï¬uÆ©er endocardiÆs hypertensive heart disease intracerebralhaemorrhage ischaemic heart disease ischaemic stroke myocardiÆs nonrheumaÆc valve disease other cardiomyopathy other cardiovascular and circulatory diseases peripheralartery diseaseNEOPLASMSLeukaemia lymphoma mulÆple myeloma myelodysplasÆc syndroms and other hematopoieÆc neoplasms brain and nervous system cancer breastcancer prostate cancer larynx cancer lip and oral cavity cancer oesophagealcancer stomach cancer colon and rectum cancer liver cancer gallbladder and biliary tract cancer pancreaÆc cancer kidney cancer bladder cancer melanoma and nonmelanoma skin cancer ovarian cancer uterine cancer thyroid cancer tracheal bronchus and lung cancer mesothelioma othermalignant neoplasms other benign and insitu neoplasmsGASTROINTESTINAL ENDOCRINE AND KIDNEY DISEASESChronic kidney disease type diabetes mellitus cirrhosis due to nonalcoholicsteatohepaÆÆs pancreaÆÆs paralyÆc ileus and intesÆnal obstrucÆon pepÆculcer disease vascular intesÆnal disorders diarrhoeal diseasesSKIN AND SUBCUTANEOUS DISEASESCelluliÆs decubitus ulcer fungal skin diseases pyoderma other skin and subcutaneousdiseasesFig Agerelated diseases adapted from Chang et al []mass [95cid129cid129] According to EWGSOP criteria sarc iais defined by the presence of low muscle strength criterion and either or low muscle quantity or quality criterion or low physical performance criterion [95cid129cid129]The physical performance parameters used in the identification of frailty syndrome both integrated eg SPPB andalone walking speed handgrip strength can be used as agingperformance biomarkersDetermination of Medical and Social NeedsWhy consider medical and social needs aging biomarkersIn Robert J Havighurst said In considering theneeds of older people it is well first to remember that olderpeople have the needs that are common to all people andsecond that they have special needs due to the fact that theyare old people This sentence describes everything there is toknow about the need for the elderly and answers the questionbeforeIn every society and age there is what is meant by normality An elderly person in this scenario needs what is needed tomaintain this level of normalcy Activity of daily living andinstrumental activity of daily living ADL and IADL aloneremodelled according to the context and gender can identifythe minimum necessary Conducting needs assessment various areas must be considered including physical health mental health emotional care social cultural economic nutritional service security legal and educationalCHRONIC RESPIRATORY DISEASESAsbestosis chronic obstrucÆve pulmonary disease coal worker pneumoconiosis intersÆÆallung disease and pulmonary sarcoidosis other pneumoconiosis silicosis lower respiratoryinfecÆonsNEUROLOGICAL DISORDERSAlzheimers disease and other demenÆas motor neuron disease Parkinsonsdisease encephaliÆs pneumococcal meningiÆsAGE RELATED DISEASESENSE AN DISEASESHearing loss vision loss ex agerelated macular degeneraÆon cataract glaucoma other sense an diseases refracÆon disorders trachomaINJURIESDrowning environmental heat and cold exposure falls foreign body in other body part other transport injuries other unintenÆonal injuriesOTHER DISEASESCongenital musculoskeletal and limb anomalies digesÆve congenital anomalies endocrine metabolic blood and immune disorders other haemoglobinopathies and haemolyÆcanaemiasMany tools are used to evaluate peoples needs The majority of these tools are focused on physical performance ableto maintain autonomy few studies focus on social needs andthe costs of care In the West World of patients accountfor of total health care expenditures This is represented by older people individuals with multiple chronic conditions many medications frequent hospitalizations and limitations on their ability to perform basic daily functions due tophysical mental or psychosocial challenge []Since the health care and social needs of older adults differfrom that of other adults it is necessary to identify the needs ofthe elderly to make proper plans that will promote their healthCurrently most of the conducted studies had mainly focused on the elderly physical health needs and had neglectedto take into account other needs such as social and health careneeds Furthermore in addition to quantitative studies discovering the older adults perceptions of their own health needsis also necessaryConclusionThere is a large interest of researchers in biomarkers of agingand despite some of them seem to be very promising biological biomarkers are still far from a clinical application to datethere is no technique that meets the mentioned criteria of theideal biomarker [40cid129] Moreover we know that the biologicalpathways are the final agents of aging but on one side theycan be influenced by social economic and environmental factors and on the other side they express in various disease and 0c Page of Curr Oncol Rep disabilities of the person physical and cognitive impairmentsagerelated disease systems functions sensory functions etcFig To date more than a single biomarker to assess agingwe should consider a cluster of biomarkers that comprisethe various elements that we analyzed social and educational aspects economic factors country of origin presence of agerelated disease presence of dependence indaily activities physical capability cognitive functionlung and cardiovascular function and presence of sensorydysfunctions In Table we propose several clinical andlaboratory biomarkers that can be used in clinical practiceand researchThe geriatric assessment GA can currently be considered a system capable of monitoring multiple biomarkersclinical and laboratory of aging and at the same timeable to relate them to each other Through the GA it ispossible to make a prediction of the risk of toxicity of atreatment of life expectancy of social needs and of compliance with the treatments GA is composed indeed byseveral evaluations made through standardized toolswhich examine various aspects of the person a multidimensional assessmentAlthough it seems difficult to imagine a geriatric assessment as a biomarker currently for its characteristicsand for the high predictivity it has it can be consideredthe gold standard in the management of the older individual and instrumenttoward which other biomarkersshould be evaluatedThe purpose of this paper was to evaluate the multipleaspects that distinguish the aging process Aging must noFig Mechanisms connectingdifferent clusters of biomarkerslonger be described as a simple demographic event butas a complex mosaic in which several tesserae relate toeach other some in a very evident way others often in amore subdued but all fundamental way Each aging theory has attempted to justify this process effectively however there is no single biomarker to date that has beenfound able to identify the stage of this process At thesame time clinical clusters have been added to purelybiological markers and social ones should certainly beconsidered It therefore becomes important not to consider biomarkers only as life span but to try to overcomethis link and focus on the set of factors that influencingeach other are able to guide aging in good health andgood quality of life towards a lived aging as a slowdecline At the time we are writing this paper COVID infection is reaping victims especially in Italy Thehighest mortality is observed among the older adultsbut surprisingly it seems to maintain similar values between the youngest and oldest old over yearsCurrently no plausible justification is provided for thesedata In frailty the number of comorbidities the reducedfunctional reserve was the most used reasons Indirectlythis infection is highlighting the need to use parametersthat can more easily identify the aging process regardlessof chronological ageThe studies analyzed in the literature show that if on theone hand there are physiological biomarkers able ofhighlighting some features of aging other functionalmarkers performance social and economic status somepathologies and the presence of addiction are able ofspeed it up or slow it | 2 |
" rectus sheath block rsb is known to attenuate postoperative pain and reduce perioperative opioidconsumption thus a retrospective study was performed to examine the effects of bilateral rectus sheath blockbrsb in cytoreductive surgery crs combined with hyperthermic intraperitoneal chemotherapy hipecmethods a total of patients undergoing crshipec at our hospital were included patient information andanaesthesiarelated indicators were collected from the electronic medical record emr system all subjects weredivided into the following two groups the g group general anaesthesia and the gr group rsb combined withgeneral anaesthesia patients in the gr group received ropivacaine for brsb before surgery the primaryoutcomes included the total amount of remifentanil and rocuronium the total consumption of dezocine aftersurgery the visual analogue scale vas score and the patientcontrolled intravenous analgesia pcia input dose at h t6 h t7 h t8 h t9 and h t10 after surgery other outcomes were also recorded such aspatient demographic data the intraoperative heart rate hr and mean arterial pressure map and postoperativecomplicationsresults compared with the g group the gr group showed a shorter time to tracheal extubation p adecreased total amount of remifentanil and rocuronium p and a reduced vas score pcia input dose andnumber of pcia boluses at h h and h after surgery p however at h and h after surgery therewere no differences in the vas score of pain at rest or during motion between the two groups p moreoverthe incidence of hypertension emergence agitation delayed recovery hypercapnia and nausea and vomiting waslower in the gr group than in the g group p there were no differences in the changes in map and hrduring the surgery between the two groups p no complications associated with nerve block occurred brsb could provide shortterm postoperative analgesia reduce perioperative opioid consumption andreduce the incidence of postoperative complications it is an effective and safe procedure in crshipeckeywords cytoreductive surgery hyperthermic intraperitoneal chemotherapy rectus sheath block generalanaesthesia analgesia correspondence trmzltz126comdepartment of anesthesiology beijing shijitan hospital capital medicaluniversity beijing china the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cwang bmc anesthesiology page of radical cytoreductive surgery crs combined withhyperthermic intraperitoneal chemotherapy hipec isconsidered a standard for the treatment of peritonealcancer such as rectal cancer ovarian cancer peritonealpseudomyxoma and peritoneal mesothelioma thistechnique could prolong the longterm survival of patients with a decreased recurrence rate although thepositive results of this treatment have been proven inprevious studies [] because of the large peritonealsurface area involved in this kind of surgery crshipecis time consuming and complex which presents agreat challenge for the anaesthesiologist in terms of perioperative managementdue to the stable respiratory and circulatory supportgeneral anaesthesia is the preferred choice in this surgery however long periods of general anaesthesia leadto drug accumulation in the body followed by increasedanaesthesiarelated complications including delayed recovery respiratory inhibition and cognitive dysfunction consequently exploring better anaesthesia methodsfor this surgery is still a major concerna new approach called ultrasoundguided bilateral rectus sheath block brsb has been proven to amelioratepostoperative pain and reduce the consumption of morphine [] nonetheless there have been no reportson the application of general anaesthesia combined withbrsb in patients undergoing crshipec based on theinformation presented above this retrospective observational study was conducted to examine the efficacy andsafety of brsb in patients treated with crs and hipecmethodssubjectsall patients who underwent crs and hipec at beijingshijitan hospital between august and december were retrieved from the institutional database inthis study the exclusion criteria were as follows laparoscopic surgery with crshipec intraoperativeblood loss volume greater than ml mechanicventilation required after surgery and use of analgesictechniques apart from brsb and general anaesthesiaaccording to this standard a total of patients wereincluded and divided into the following groups generalanaesthesia g group n and general anaesthesiacombined with posterior rsb gr group n anaesthesia methodgeneral anaesthesia was consistently induced in all patients with intravenous propofol mgkg sufentanil μgkg and rocuronium mgkg invasive arterialpressure and central venous pressure were monitored byradial artery puncture flotracvigileo® edwards lifesciences irvine ca usa and internaljugular veinsitetargeteffectpuncture respectively after anaesthesia induction anaesthesia was maintained with sevoflurane and remifenconcentration ngmltanilkeeping the bispectral index bis between and rocuronium mgkg wasintermittently used tomaintain muscle relaxation in the gr group before anaesthesia induction patients received brsb under ultrasound guidance the puncture site was placed at theouter edge of the bilateral rectus abdominis at the levelof the umbilicus fig a a total of ropivacaine ml was injected into each side the spindleshapedspread of ropivacaine was observed between the posterior sheath of the rectus abdominis and the rectus abdominis itself implying success of the procedure fig b c patientcontrolled intravenous analgesia pciawas applied in both groups after the surgery sufentanil μgkg palonosetron hydrochloride mg was diluted to ml the dose was mlh and asingle dose was mlh with a 15min lockout intervalafter the surgery all patients were sent to the surgicalintensive care unit sicu if the visual analogue scalevas score at rest after surgery was ¥ dezocine mgwas used as a rescue analgesicdata collectionall the indicators we needed were obtained from theemr system the records included patient demographicdata patient medical history american society of anesthesiologists asa grade and new york heart association nyha grade the hr and map were recordedat the time before brsb t1 the time of anaesthesiat2 the time of skin incision t3 the time of peritoneal thermochemotherapy t4 and the end of surgeryt5 in addition the duration of the surgery time totracheal extubation the time after skin closure totalamount of remifentanil and muscle relaxants total fluidvolume urine volume and the total volume of allogeneicerythrocytes and plasma infused during the surgery wereall recorded moreover after surgery the occurrence ofhypertension the systolic blood pressure dropped bymore than of baseline blood pressure beforeanesthesia or the sbp mmhg during surgery nausea and vomiting hypoxemia spo2 or pao2 mmhg hypercapnia paco2 mmhg and emergence agitation during the recovery period were recorded the recovery period was considered as the timefrom switching off inhalation anaesthetics remifentaniland muscle relaxant to recovery of the patients abilitiesto command movement orientation as well as conscious state when the recovery period of patients is beyond min it was considered as delayed recovery thevas score for pain at rest and during motion the pciainput dose and the number of boluses at h t6 ht7 h t8 h t9 and h t10 after surgery 0cwang bmc anesthesiology page of the incomplete datachemotherapy crshipec during the year from to in our hospital one hundred and six patientsreceived brsb seventeen patients were excluded because ofincluding patientsundergoing intraoperative haemorrhage blood ml and other patients received mechanic ventilationbecause of acute respiratory distress syndrome ardsallergic shock and cardiac insufficiency thus patients with brsb were eventually obtained finally patients without brsb were randomly selected to analysis in this study fig statistical analysisspss software was used for statistical analysisnormal distribution data were recorded as the mean ±standard deviation sd and analysed by independentsamples t test for comparison between the two groupsnonnormally distributed data are presented as themedian range and were analysed by kruskalwallistest chisquared test or fishers exact test was used forcategorical data a p value of was considered statistically significantresultscharacteristics of study populationin total patients were included in the study thebaseline demographic and surgical variables of patientsare presented in table there were no significant differences in age sex body mass index bmi basic diseases asa grade nyha grade total surgery time totalfluid volume urine volume total volume of allogeneicerythrocyte infusion or total volume of plasma p however the time to tracheal extubation was shorter inthe gr group than in the g group p the totalamount of both remifentanil and rocuronium used wasless in the gr group than in the g group p thus posterior rsb could reduce the use of remifentaniland rocuronium during surgerychanges in haemodynamic parametersthe changes in hr and map are presented in table there were no significant differences in hr or map atany point in time t1 to t5 between the two groupsp the results suggest that brsb did not affectthe haemodynamics ofthe patient undergoing crshipecpainrelated indicatorstable shows the postoperative vas score the pca input dose and the number of pca boluses at h t6 ht7 h t8 h t9 and h t10 after surgeryas well as the dose of dezocine used as a rescue analgesic from t6 to t8 compared with the g group thegr group showed significantly decreased vas scores offig ultrasoundguided brsbas well as the dose of dezocine used as a rescue analgesic were also recordedin addition brsbrelatedcomplications such as peritoneal punctureinternalan injury and systemic toxicity were all recordeda total of patients underwent cytoreductive surintraperitonealcombined withgeryhyperthermic 0cwang bmc anesthesiology page of fig flow chart showing patient consecutive enrolment and analysis abbreviations crshipec cytoreductive surgery and hyperthermicintraperitoneal chemotherapy ga general anesthesia brsb bilateral rectus sheath block ards acute respiratory distress syndrome vas visualanalogue scalepain at rest and during motion p however at h and h after surgery there were no significant differences in the vas scores of pain at rest and duringmotion between the two groups p from t6 tot10 the pcia input dose and the number of pca boluses were also obviously reduced in the gr group compared with the g group p in addition as arescue analgesic the dose of dezocine after surgery inthe gr group was significantly lower than that in the ggroup p postoperative adverse eventsadverse events that occurred in the sicu are presentedin table after surgery there were cases withhypertension cases of emergence agitation cases ofdelayed recovery cases of hypercapnia and cases ofnausea and vomiting in the g group fewer cases of allof these events occurred in the gr group p there were no differences in the incidence of hypoxemiabetween the two groups p there were no complications associated with nerve block in either group 0cwang bmc anesthesiology page of table demographic and surgical variables mean ± sdage ysex malefemalebmi kgm2medical historydiabetes mellitus n yesnohypertension n yesnocoronary heart disease n yesnoasa grade iiiiiinyha grade iiitotal surgery time mintime to tracheal extubation minremifentanil mgrocuronium mgtotal fluid volume mlurine volume mltotal volume of allogeneic erythrocyte infusion mltotal volume of plasma mlg group n ± ± ± ± ± ± ± ± ± ± gr group n ± ± ± ± ± ± ± ± ± ± p value asa american society of anesthesiologists bmi body mass index calculated as weight in kilograms divided by height in metres squared nyha new york heartassociation g general anaesthesia gr bilateral rectus sheath block combined with general anaesthesia before bilateral rectus sheath block t1 the time ofanaesthesia t2 the time of skin incision t3 the time of peritoneal thermochemotherapy t4 and the end of surgery t5discussionin this retrospective study we examined the efficacy andsafety of brsb combined with general anaesthesia in patients undergoing crshipec regarding efficacy theresults show that ultrasoundguided brsb significantlyreduced the total dose of remifentanil used during thesurgery and shortened the time to tracheal catheter extraction which is consistent with the findings of previous studies of other surgeries [ ] in addition rsbreduced the total dose of rocuronium in this studytable haemodynamic parameters in both groups mean ± sdindextimepointgn ± grn ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± pvaluemmhghrbpmt1t2t3t4t5t1t2t3t4t5map mean arterial pressure hr heart rate g general anaesthesia gr bilateralrectus sheath block combined with general anaesthesiawhich may be associated with the high concentration ofropivacaine used in the studyrsb also effectively relieved postoperative pain in thisstudy we found that the vas scores of pain at rest andduring motion were all lower in the gr group than inthe g group at h after surgery however at h and h after surgery there were no differences in the vasscores of pain at rest and during motion between thetwo groups suggesting that the analgesic effects of asingle brsb remained within h after surgery thisresult may be different from the findings of others cho reported that at h after surgerythere were no differences in the vas scores of painat rest and during motion between the rsb and nonrsb groups the discrepant results may be related todifferences in the concentration of ropivacaine andthe physical constitution of patients a high concentration can prolong the duration of action of a localanaesthetic in this study we selected not ropivacaine additionallythese patientsundergoing crshipec may have been adaptive topain furthermore compared with the control groupthe rsb group showed a reduced totalinfused doseof sufentanil as pcia number of pca boluses within h after surgery and total dose of dezocine used asa rescue analgesic after surgery these results furtherprove the role of rsb in providing shortterm postoperative analgesia 0cwang bmc anesthesiology page of table painrelated indicators in both groups median [range]vas score of pain at rest [median range]t6t7t8t9t10vas score of pain during motion [median range]t6t7t8t9t10total infused dose of pcia [ml median range]t6t7t8t9t10cumulative number of pcia boluses [median range]t6t7t8t9t10total dose of dezocine as a rescue analgesic mggn [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] ± grn [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] ± p value vas visual analogue scale pcia patientcontrolled intravenous analgesia g general anaesthesia gr bilateral rectus sheath block combined with generalanaesthesia the time at h after surgery t6 h after surgery t7 h after surgery t8 h after surgery t9 and h after surgery t10table postoperative adverse events in both groupsadverse eventsn gn grn hypertensionemergence agitation delayed recoveryhypoxemiahypercapnia nausea and vomiting peritoneal punctureinternal an injurysystemic toxicity g general anaesthesia gr bilateral rectus sheath block combined withgeneral anaesthesiawe also examined the safety of rsb during the surgery ultrasoundguided brsb had no significant effectson the haemodynamics of patients during surgery compared with general anaesthesia alone in terms of postoperative adverse events the results show that comparedwith the control group the rsb group showed a reducedincidence of hypertension emergence agitation delayedrecovery hypercapnia and nausea and vomiting whichmight be correlated with the decreased analgesic andmuscle relaxant doses no rsbrelated complications occurred in any patient these data indicate that rsbcould reduce the risk of complications associated withgeneral anaesthesia and is safe for patientsrsb an established technique has regained popularityin clinical applications [] previous studies havedemonstrated that this technique could achieve relaxation of the anterior abdominal wall [ ] bashandyreported that anterior branches of the t7t12 thoracicnerve and the l1 lumbar nerve travelled through thepvalue 0cwang bmc anesthesiology page of plane of the transverse abdominis muscle entered the rectus abdominis sheath and distributed on the surface of theskin the main process of rsb is to inject local anaesthetics between the rectus abdominis and the posteriorsheath of the rectus abdominis therefore rsb exerteda good effect in terms of perioperative analgesia for medianabdominal incisions a midline incision is required inthis kind of surgery thus based on these results rsbcould meet the need for analgesia in these patientsin addition for a long time epidural analgesia eawas thought to be an effective method for abdominalsurgery [] studies have proved that epidural analgesia could maintain a good analgesic effect and reduceperioperative opioid consumption including in this typeof surgery [ ] however the safety of ea in crshipec remains controversial especially regarding effectson coagulation and circulatory function coagulationdysfunction and profound fluid loss are the main characteristics of patients with peritoneal cancer [ ]which might limit the administration of eaalthough epidural catheter is standard of care insolankis guideline in our hospital epidural catheter in not the standard of care we performed generalanesthesia combined with epidural anesthesia in somepatients to reduce the consumption of intravenous drugsand provide perfect analgesia but coagulation dysfunction and profound fluid loss are the main characteristicsof patients with peritoneal cancer in our previous studywe found that the mean arterial pressure of patientsundergoing epidural anesthesia was difficult to be maintained in the surgery besides there were many patientswith coagulation dysfunction before surgery who werenot suitable for the epidural anesthesia these results wefound in clinical were similar with others researcheskajdi and colleagues reported a case of epidural haematoma in their study godden found that the incidence of hypotension in the ea group was obviouslyhigher than that in the rsb group consequentlyrsb could be a better choice than ea in crshipecadditionally there are some limitations to this studyfirst all the data of this study were collected from theemr system as this was a retrospective study the findings are not as persuasive as those of a randomized controlled study we plan to conduct prospective studies toexplore the comprehensive influence of rsb in this surgery second we only examined the application of brsbestablished with a single injection which provides only ashortterm analgesic effect the efficacy of continuousanalgesia with brs catheters in crshipec remains unclear and needs further exploration third in our the results are initially presented according to the different aspects the primary outcome of this study is thetotal consumption of remifentanil during the surgeryother indicators were belonged to second outcomessin brsb could provide good postoperativeanalgesia reduce perioperative opioid consumption andreduce the incidence of postoperative complicationsthis is an easily applicable and safe procedure in crshipecabbreviationsrsb rectus sheath block brsb bilateral rectus sheath blockcrs cytoreductive surgery hipec hyperthermic intraperitonealchemotherapy emr electronic medical record vas visual analogue scalepcia patientcontrolled intravenous analgesia hr heart rate map meanarterial pressure bis bispectral index sicu surgical intensive care unitasa american society of anesthesiologists nyha new york heartassociation spo2 pulse oximetry paco2 partial pressure of carbon dioxidepao2 oxygen partial pressure bmi body mass indexacknowledgementsi would like to express my heartfelt thanks to the staff of the informationdata center of beijing shijitan hospital affiliated to capital medical universityauthors contributionswsh study design data collection writing paper lpf gt data collectionand data analysis gl coordinated the study and manuscript revision ltzstudy design and manuscript revision all authors read and approved thefinal manuscript all authors ensure the accuracy of the manuscript andagree to take personal responsibility for their contributionsfundingno fundingavailability of data and materialsthe datasets used andor analyzed during the current study are availablefrom the corresponding author on reasonable requestethics approval and consent to participatethis study was approved by the ethics committee of beijing shijitan hospitalaffiliated to capital medical university approval code research ethicsno69 this study is retrospective only anonymous data sources were usedand informed consent was not requiredconsent for publicationnot applicablecompeting intereststhe authors declare that they have no competing interestsreceived january accepted july referencesklaver ce musters gd bemelman wa punt cj verwaal vj dijkgraaf mgaalbers ag van der bilt jd boerma d bremers aj adjuvanthyperthermic intraperitoneal chemotherapy hipec in patients with coloncancer at high risk of peritoneal carcinomatosis the colopec randomizedmulticentre trial bmc cancer 201515undefined428van oudheusden tr braam hj nienhuijs sw wiezer mj van ramshorst bluyer md lemmens ve de hingh ih cytoreduction and hyperthermicintraperitoneal chemotherapy a feasible and effective option for colorectalcancer patients after emergency surgery in the presence of peritonealcarcinomatosis ann surg oncol passot g vaudoyer d villeneuve l kepenekian v beaujard ac bakrin ncotte e gilly fn glehen o what made hyperthermic intraperitonealchemotherapy an effective curative treatment for peritoneal surfacemalignancy a 25year experience with procedures j surg oncolarjonasánchez a barrios p boldoroda e camps b carrascocampos jmartÃn vc garcÃafadrique a gutiérrezcalvo a morales r ortegapérez g hipect4 multicentre randomized clinical trial to evaluate safety andefficacy of hyperthermic intraperitoneal chemotherapy hipec with 0cwang bmc anesthesiology page of huepenbecker sp cusworth se kuroki lm lu p samen cd woolfolk cdeterding r wan l helsten dl bottros m continuous epiduralinfusion in gynecologic oncology patients undergoing exploratorylaparotomy the new standard for decreased postoperative pain and opioiduse gynecol oncol teoh da hutton mj else s walker a lee a mack la epidural analgesia aprospective analysis of perioperative coagulation in cytoreductive surgeryand hyperthermic intraperitoneal chemotherapy am j surg schmidt c creutzenberg m piso p hobbhahn j bucher m perioperativeanaesthetic management of cytoreductive surgery with hyperthermicintraperitoneal chemotherapy anaesthesia kajdi me beckschimmer b held u kofmehl r lehmann k ganter mtanaesthesia in patients undergoing cytoreductive surgery withhyperthermic intraperitoneal chemotherapy retrospective analysis of asingle centre threeyear experience world j surg oncol solanki sl mukherjee s agarwal v thota rs balakrishnan k shah sb desain garg r ambulkar rp bhorkar nm society of oncoanaesthesia andperioperative care consensus guidelines for perioperative management ofpatients for cytoreductive surgery and hyperthermic intraperitonealchemotherapy crshipec indian j anaesth godden ar marshall mj grice as daniels ir ultrasonography guidedrectus sheath catheters versus epidural analgesia for open colorectal cancersurgery in a single centre ann r coll surg engl publishers notespringer nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliationsmitomycin c used during surgery for treatment of locally advancedcolorectal carcinoma bmc cancer baratti d kusamura s iusco d gimondi s pietrantonio f milione mguaglio m bonomi s grassi a virzì s hyperthermic intraperitonealchemotherapy hipec at the time of primary curative surgery in patientswith colorectal cancer at high risk for metachronous peritoneal metastasesann surg oncol chua tc robertson g liauw w farrell r yan td morris dl intraoperativehyperthermic intraperitoneal chemotherapy after cytoreductive surgery inovarian cancer peritoneal carcinomatosis systematic review of currentresults j cancer res clin oncol li y zhou yf liang h wang hq hao jh zhu zg wan ds qin lx cui szji jf chinese expert consensus on cytoreductive surgery andhyperthermic intraperitoneal chemotherapy for peritoneal malignanciesworld j gastroenterol willschke h bosenberg a marhofer p johnston s kettner sc wanzel o kaprals ultrasonographyguided rectus sheath block in paediatric anaesthesiaanew approach to an old technique br j anaesth azemati s khosravi mb an assessment of the value of rectus sheath blockfor postlaparoscopic pain in gynecologic surgery j minim invasive gynecol dingeman rs barus lm chung hk clendenin dj lee cs tracy s johnsonvm dennett kv zurakowski d chen c ultrasonographyguided bilateralrectus sheath block vs local anesthetic infiltration after pediatric umbilicalhernia repair a prospective randomized clinical trial jama surg relland lm tobias jd martin d veneziano g beltran rj mckee c bhalla tultrasoundguided rectus sheath block caudal analgesia or surgical siteinfiltration for pediatric umbilical herniorrhaphy a prospective doubleblinded randomized comparison of three regional anesthetic techniques jpain res 201710undefined2629 xu l hu z shen j pm mq efficacy of usguided transversus abdominisplane block and rectus sheath block with ropivacaine anddexmedetomidine in elderly highrisk patients minerva anestesiol cho s kim yj jeong k moon hs ultrasoundguided bilateral rectus sheathblock reduces early postoperative pain after laparoscopic gynecologicsurgery a randomized study j anesth li t ye q wu d li j yu j doseresponse studies of ropivacaine in bloodflow of upper extremity after supraclavicular block a doubleblindrandomized controlled study bmc anesthesiol bell jc rylah bg chambers rw peet h mohamed f moran bjperioperative management of patients undergoing cytoreductive surgerycombined with heated intraperitoneal chemotherapy for peritoneal surfacemalignancy a multiinstitutional experience ann surg oncol landmann a visoiu m malek mm development of a novel technique forbilateral rectus sheath nerve blocks under laparoscopicguidance j pediatrsurg kumar a wilson ga engelhardt te ultrasound guided rectus sheathblockade compared to perioperative local anesthetic infiltration in infantsundergoing supraumbilical pyloromyotomy saudi journal of anaesthesia bashandy gm elkholy ah reducing postoperative opioid consumption byadding an ultrasoundguided rectus sheath block to multimodal analgesiafor abdominal cancer surgery with midline incision anesthesiol pain med201443e18263 dowidar aerm ezz haa shama aae eloraby ma postoperative analgesiaof ultrasound guided rectus sheath catheters versus continuous woundcatheters for colorectal surgery a randomized clinical trial egypt journalof anaesth karaarslan e topal a avci o tuncer uzun s research on the efficacy of therectus sheath block method agri piccioni f casiraghi c fumagalli l kusamura s baratti d deraco m arientif langer m epidural analgesia for cytoreductive surgery withperitonectomy and heated intraperitoneal chemotherapy int j surg 16pt a99 vesterandersen m lundstrøm lh møller mh the association betweenepidural analgesia and mortality in emergency abdominal surgery apopulationbased cohort study acta anaesthesiol scand httpsdoi101111aas13461 0c" | 0 |
EpidemiologyEPIDEMIOLOGICAL SCIENCERisk factors for hospital admissions related to COVID19 in patients with autoimmune inflammatory rheumatic diseasesDalifer D Freites Nu±ez1 Leticia Leon Arkaitz Mucientes1 Luis Rodriguez Rodriguez Judit Font Urgelles3 Alfredo Madrid Garca1 Jose I Colomer1 Juan A Jover34 Benjamn Fernandez Gutierrez3 Lydia Abasolo1Handling editor Josef S Smolen1Rheumatology Department and IDISSC La Fundacion para la Investigacion Biomedica del Hospital Clinico San Carlos Madrid Spain2Department of Health and Education Universidad Camilo Jose Cela Villafranca del Castillo Madrid Spain3Rheumatology Department Hospital Clinico San Carlos Madrid Spain4Medicine Department Universidad Complutense de Madrid Madrid Comunidad de Madrid SpainCorrespondence toDr Leticia Leon IdISSC and Rheumatology La Fundacion para la Investigacion Biomedica del Hospital Clinico San Carlos Madrid Spain lleon hcsc salud madrid Received May Revised July Accepted July Objectives To describe patients with autoimmune inflammatory rheumatic diseases AIRD who had COVID19 disease to compare patients who required hospital admission with those who did not and assess risk factors for hospital admission related to COVID19Methods An observational longitudinal study was conducted during the pandemic peak of severe acute respiratory syndrome coronavirus March to April All patients attended at the rheumatology outpatient clinic of a tertiary hospital in Madrid Spain with a medical diagnosis of AIRD and with symptomatic COVID19 were included The main outcome was hospital admission related to COVID19 The covariates were sociodemographic clinical and treatments We ran a multivariable logistic regression model to assess risk factors for the hospital admissionResults The study population included patients with AIRD and COVID19 Of these patients required hospital admission related to COVID19 The mean age on admission was years and the median time from onset of symptoms to hospital admission was days The median length of stay was days A total of patients died during admission Compared with outpatients the factors independently associated with hospital admission were older age OR p000 and autoimmune systemic condition vs chronic inflammatory arthritis OR p001 No statistically significant findings for exposure to disease modifying antirheumatic drugs were found in the final modelConclusion Our results suggest that age and having a systemic autoimmune condition increased the risk of hospital admission whereas disease modifying antirheumatic drugs were not associated with hospital admission Authors or their employers No commercial re use See rights and permissions Published by BMJTo cite Freites Nu±ez a0DD Leon a0L Mucientes a0A et a0al Ann Rheum Dis Epub ahead of print [please include Day Month Year] 101136annrheumdis2020217984INTRODUCTIONSevere acute respiratory syndrome coronavirus SARS CoV2 causes a myriad of clinical signs and symptoms together with typical laboratory abnormalities that manifest as the disease COVID191Since the confirmation of the first patient infected with SARS CoV2 in Spain in January the current COVID19 outbreak has had a considerable impact especially in the Madrid region where the highest incidence of COVID19 cases has been Key messagesWhat is already known about this subject º The epidemiological scenario is changing daily There is little evidence for risk factors of poor outcome with COVID19 specific to autoimmune inflammatory rheumatic diseasesWhat does this study add º Patients with an autoimmune systemic condition have a higher risk of hospital admission related to COVID19 compared with those with chronic inflammatory arthritis º Disease modifying agents were not associated with a higher risk of hospital admission related to COVID19How might this impact on clinical practice or future developments º Our data show that in a real world setting a high percentage of patients with autoimmune inflammatory rheumatic diseases and COVID19 required hospital admission The patients were mainly elderly with comorbidities and a systemic autoimmune conditionrecorded with more than patients admitted to the hospital until the first week of May2The incidence and severity of COVID19 disease seem to be higher in patients with risk factors such as advanced age and associated comorbidities mainly hypertension diabetes heart disease and previous respiratory diseases3 It is not clear whether patients with rheumatic diseases are more susceptible to SARS CoV2 infection or when they are infected whether they have more severe disease or a poorer outcome Previous outbreaks caused by coronaviruses did not yield overwhelming evidence that patients with rheumatic diseases are at an increased risk4 although some patients are candidates for a higher number of infections owing to their rheumatic disease predominantly systemic or the treatment they are receiving for rheumatic diseases5 Preliminary experiences in patients with COVID19 show that those with chronic arthritis treated with synthetic conventional or targeted syntheticbiologic disease modifying antirheumatic Freites Nu±ez DD et a0al Ann Rheum Dis 101136annrheumdis2020217984 0cEpidemiologydrugs DMARDs do not seem to be at a greater risk of respiratory or life threatening complications from SARS CoV2 than the general population6 The epidemiological scenario is changing and evidence on the risk factors of poor outcome with COVID19 specific to inflammatory rheumatic disease is scarce In addition there are little data on how the hospital admissions of these patients with severe COVID19 infection have evolved8The aim of our study was to describe patients with autoimmune inflammatory rheumatic diseases AIRD who had COVID19 during the pandemic peak We also explored possible risk factors associated with hospital admission related to COVID19 disease in patients with AIRD from a tertiary hospital in Madrid SpainMETHODSSetting study design and patientsThe study was performed in a public tertiary hospital Hospital Clnico San Carlos HCSC in Madrid Spain The catchment area is home to almost peopleWe performed a prospective observational study from March when our health area had the first hospital admission related to COVID19 to April We preselected all patients attended at the rheumatology outpatient clinic of our centre during the study period whose data were recorded in the electronic clinical history of our department HCR Penelope The inclusion criteria were age years a medical diagnosis according to International Classification of Diseases ICD10 of inflammatory rheumatic disease and symptomatic COVID19 disease assessed by medical diagnosis or confirmed with a positive SARS CoV2 PCR diagnostic testPatient data were obtained during routine clinical practice The study was conducted in accordance with the Declaration of Helsinki and the principles of Good Clinical Practice and was approved by the HCSC Ethics Committee approval number E BSVariablesThe primary outcome was admission to hospital with a medical diagnosis of COVID19 andor a positive PCR result between March and April compared with outpatients with symptomatic COVID19 diseaseThe covariables recorded were as follows sociodemographic baseline characteristics including sex age and rheumatic disease duration Type of AIRD including systemic autoimmune conditions polymyalgia rheumatica mixed connective tissue disease systemic sclerosis Sjogrens syndrome vasculitis Raynaud phenomenon polymyositis polychondritis sarcoidosis antiphospholipid syndrome autoinflammatory syndromes and systemic lupus erythematosus and chronic inflammatory arthritis rheumatoid arthritis inflammatory polyarthritis juvenile idiopathic arthritis psoriatic arthritis axial spondyloarthritis uveitis and inflammatory bowel disease Baseline comorbid conditions including hypertension dyslipidaemia depression diabetes mellitus smoking habit kidney disease chronic liver disease respiratory diseases chronic obstructive pulmonary disease and interstitial lung disease thyroid disease heart disease valve disease arrythmias cardiomyopathy heart failure and pericarditis ischaemic vascular disease stroke cardiovascular and peripheral vascular disease venous thrombosislung embolism and cancer Treatment for inflammatory rheumatic disease a glucocorticoids b non steroidal anti inflammatory drugs NSAIDs c conventional synthetic disease modifying antirheumatic drugs csDMARDs antimalarials hydroxychloroquine and chloroquine azathioprine cyclophosphamide cyclosporine colchicine leflunomide methotrexate mycophenolate mofetilmycophenolic acid and sulfasalazine d targeted syntheticbiologic DMARDs tsbDMARDs including antitumour necrosis factor TNF alpha drugs infliximab adalimumab etanercept certolizumab and golimumab other biologics anti interleukin IL6 tocilizumab and sarilumab rituximab abatacept belimumab anti IL1723 anti IL17 ustekinumab ixekizumab and secukinumab Janus kinase JAK inhibitors tofacitinib and baricitinibTreatment had to start at least month before the beginning of the study and continue during the study period until the end of the study or hospital admission for antimalarial therapy glucocorticoids sulfasalazine NSAIDs or colchicine Regarding csDMARDs and tsbDMARDs treatment had to start at least month before the beginning of the study and continue until at least 21st March the end of the study or hospital admission In the case of rituximab the last infusion had to be at least in JanuaryData sourcesPatient sociodemographic clinical laboratory and data on treatment of rheumatic disease were obtained through HCR PenelopePatients with COVID19 were detected by warning calls to our rheumatologists or nurses or via routine telephone consultation Other infected patients were detected through their sick leave forms for COVID19 The results of SARS CoV2 PCR diagnostic assays were obtained from the microbiologyinfectious service of HCSC In addition our Hospital Central Services registered all medical admissions to HCSC This information was provided from March to AprilThe researchers carried out an exhaustive review of the clinical histories of admitted patients to identify COVID19 cases and rule out patients admitted for other reasons Once the COVID19 cases were identified we collected clinical laboratory and treatment data during admission until the end of admission either discharge or death in order to describe the progress of the disease The review was performed until 24th April in order to include follow up data from patients admitted to the hospital with COVID19Statistical analysisPatient characteristics are expressed as mean and SD or median and IQR for continuous variables categorical variables are expressed as percentages Statistical tests were performed to compare characteristics between patients admitted with COVID19 and those without hospital admissions Continuous variables were analysed using the Mann Whitney test or t test and discrete variables were analysed using the Ï2 or Fisher exact test Univariable logistic regression analyses were performed to assess differences between hospital admissions related to COVID19 risk and covariates Multivariable logistic regression models adjusted for age sex and comorbidity were run in a stepwise manner to examine the possible effect of sociodemographic clinical and therapeutic factors on hospital admissions related to COVID19 The model also included csDMARDs and all other variables with a p02 from the simple regression analysis The results were expressed as the OR with its respective CIAll analyses were performed in Stata V13 statistical software Stata Corp A two tailed p value was considered to indicate statistical significanceFreites Nu±ez DD et a0al Ann Rheum Dis 101136annrheumdis2020217984 0cRESULTSA total of patients with AIRD with symptomatic COVID19 disease were included in the study table The tests were performed as an exploratory measure of the association between a variable and the outcomeMost of the patients were women with a mean age of years and a mean time since diagnosis of years The main diagnosis was rheumatoid arthritis followed by axial spondyloarthritis Many patients had at least one baseline comorbid condition the most prevalent being hypertension dyslipidaemia and lung disease Most patients were taking csDMARDs Half of the patients were taking glucocorticoids a quarter were taking NSAIDs and were taking tsbDMARDs of which adalimumab was the most frequently prescribed followed by rituximab Only one patient was taking a JAK inhibitor Interestingly of the patients taking tsbDMARDs were taking the drug in combination with a csDMARDA total of patients had to be admitted to the hospital because of COVID19 Of these were evaluated in the HCSC Emergency Department were admitted to HCSC and were transferred to the Institucion Ferial de Madrid IFEMA support hospital owing to the lack of capacity in our hospital at that time The remaining three patients were evaluated and admitted to other hospitals in the Autonomous Community of Madrid Table presents data for the patients admitted to HCSCOf the patients admitted to our hospital were women with a mean age at admission of years and median lag time from the onset of symptoms to the admission of days The median length of stay was days table At admission the median haemoglobin was gdL and the median total lymphocyte count was ngmL The median D dimer value was ngmL In of patients median interleukin IL6 levels were pgmL Patients received various antibiotics mainly azithromycin levofloxacin and third generation cephalosporinsMost patients were treated with hydroxychloroquine during admission About half received glucocorticoids Eighteen were treated with lopinavirritonavir and received the anti IL 6R antibody tocilizumab table 2FEDERA total of patients developed relevant complications during admission the most frequent being myocarditis thrombosis and kidney failure Only two patients were admitted to the intensive care unit during admission The first was a patient in 50s with mixed connective tissue disease and associated comorbidities who developed acute respiratory insufficiency and bilateral pneumonia The patient was treated with antibiotic therapy lopinavirritonavir hydroxychloroquine and βinterferon Finally the patient was extubated days later and is recovering The other was a young adult patient with systemic lupus erythematosus treated with methotrexate rituximab hydroxychloroquine and glucocorticoids who days after being diagnosed with COVID19 PCR developed an erythematous rash and generalised urticaria requiring hospitalisation in the intensive care unit owing to general clinical and laboratory worsening elevated D dimer values The patient was treated with methylprednisolone heparin and a cephalosporin A few days later the patients condition improved and he recovered completely at dischargeOf the patients admitted to HCSC were sent to another care centre converted hotel hospitalIFEMA support hospital when their condition improved A further patients Epidemiologywere discharged home to continue self isolation after improvement At the end of the study five patients remained in hospital A total of patients died during admission men and women with a median age of years Of the patients who died had relevant comorbidity diabetes mellitus pulmonary disease ischaemic vascular disease hypertension venous thrombosislung embolism lung disease and or liver disease The main diagnoses were rheumatoid arthritis followed by spondyloarthritis polymyalgia rheumatica vasculitis and Sjogrens syndrome The results of the univariable analysis are shown in table Older age systemic autoimmune conditions vs chronic inflammatory arthritis OR CI p0014 hypertension diabetes mellitus lung disease heart disease and glucocorticoids were associated with statistically significant greater risk of admission to the hospital Female sex NSAIDs and anti TNF drugs vs non use were associated with a statistically significant lower risk The differences reported for the remaining variables did not reach statistical significanceThe multivariable analysis was adjusted for gender age and comorbidities related to COVID19 These comorbidities were diabetes mellitus pulmonary disease ischaemic vascular disease hypertension venous thrombosislung embolism lung disease andor liver disease table Age and systemic autoimmune conditions had more probability of hospital admissions regardless of other factors Differences in exposure to glucocorticoids were not statistically significant The type of exposed DMARDs did not reach statistical significance in the multivariate model In fact long term treatment with antimalarials OR CI p066 other csDMARDs including methotrexate leflunomide and azathioprine OR CI p09 and NSAIDs OR CI p05 dropped from the final model The variable tsbDMARDs was also eliminated from the final model anti TNF vs none OR CI p016 and non anti TNF vs none OR CI p03DISCUSSIONOur study aims to shed light on rheumatologists concerns regarding their patients We found that in a real world setting of patients with AIRD and COVID19 required hospital admission These were mainly elderly patients with more comorbidities and systemic autoimmune conditions Our data show that patients exposed to disease modifying agents do not seem to be at higher risk of hospital admission related to COVID19Of the patients included in the study with COVID19 required hospital admission Comparison of the characteristics of patients admitted to hospital because of COVID19 and those who did not require hospital admission were as follows admitted patients had a median age close to years that is more than years older than patients who were not admitted Moreover those who were admitted more frequently had baseline comorbidities and systemic autoimmune conditions As for therapy admitted patients were less frequently exposed to antimalarial and anti TNF alpha agentsThe median lag time from onset of symptoms to admission was days and almost of patients had pneumonia at admission The baseline laboratory results for admitted patients in our study are consistent with those published elsewhere9 and are characterised by lymphopenia and elevated acute phase reactants In fact of the patients had elevated D dimers normal and elevated IL6 normal pgmL Treatment during admission varied widely as the disease proved Freites Nu±ez DD et a0al Ann Rheum Dis 101136annrheumdis2020217984 0cEpidemiologyTable Baseline demographic and clinical characteristics of patients with AIRD and with COVID19 admitted vs no admitted at the hospitalAIRDCOVID19 patientsAIRDCOVID non admitted patientsAIRDCOVID admitted patientsVariableN123N69N54P value Positive Negative Not performed Women n Age years mean SDTime since diagnosis years mean SDPCR test n Smoking habit active vs noneDiagnosis AIRD n Rheumatoid arthritis Axial spondyloarthritis Polymyalgia rheumatica Psoriatic arthritis Systemic lupus erythematosus Mixed connective tissue disease Sjogrens syndrome Vasculitis Uveitis Systemic sclerosis Inflammatory polyarthritis Polychondritis Polymyositis Raynaud phenomenon OtherComorbidities n NSAIDs n Glucocorticoids n csDMARDs n TsbDMARDs n JAKi n Others inflammatory bowel disease antiphospholipid syndrome juvenile idiopathic arthritis autoinflammatory syndromes and sarcoidosis Heart disease arrhythmiasvalve disease cardiomyopathy and heart failure Ischaemic vascular disease stroke cardiovascular and peripheral vascular diseaseAIRD autoimmune inflammatory rheumatic disease Anti TNF tumour necrosis factor alpha COPD chronic obstructive pulmonary disease csDMARD conventional synthetic disease modifying antirheumatic drug ILD interstitial lung disease JAKi JAK inhibitor tsbDMARDs target syntheticbiologic disease modifying antirheumatic drug Hypertension Dyslipidaemia Depression Diabetes mellitus Heart disease Vascular disease Liver disease Kidney disease Lung disease ILDCOPD Cancer Venous thrombosislung embolism Thyroid disease Anti TNF alpha agent Other biologics Abatacept Tocilizumab Belimumab Rituximab Methotrexateleflunomideazathioprine Sulfasalazine AntimalarialsFreites Nu±ez DD et a0al Ann Rheum Dis 101136annrheumdis2020217984 0cTable Hospital admissions related to COVID19 among patients with AIRDVariableValueTable OR of hospital admission related to COVID19 in patients with AIRD univariable analysisVariable CIORPEpidemiology Haemoglobin gdL D dimer ngmL Neutrophil count 109L Lymphocyte count 109L CRP mgdL LDH UL Platelet count 109L Creatinine mgdL Ferritine ngmLAdmissions nLag time from onset of symptoms to admission days median IQRPneumonia at admission n Systemic autoimmune conditions n Laboratory data at admission median IQR COVID19 related treatments during admission n Admitted by intensive care unit during hospital admission Length of stay days median IQRDischarge reason n Azithromycin Other antibiotics Glucocorticoids Lopinavirritonavir Remdesivir Darunavircobicistat Tocilizumab Interferon HCQ Immunoglobulin Improvement home isolation Other care centre medicalised hotelIFEMA hospital Death End of study no discharge No Yes Data for patients patients were treated in other support centres after referral or admission in other centresCRP C reactive protein HCQ hydroxychloroquine LDH lactate dehydrogenase challenging for specialists who prescribed various combinations of drugs based on little published evidence In this sense the anti IL 6R antibody tocilizumab has proven to be beneficial in patients with COVID1912 Treatment may also be successful in the early stages of cytokine release syndrome if it can effectively block the signal transduction pathway of IL6 therefore tocilizumab and sarilumab are likely to emerge as effective drugs for patients with moderate to severe COVID1913 In our study almost of the patients were treated with tocilizumabThe patients who eventually died had a median age of years This finding is in line with data for the general population where over of deaths occurred in persons years and more than of all deaths were in people aged ¥ years7The multivariable regression model showed that only age increasing by per year and systemic autoimmune conditions continued to be risk factors for hospital admission related to COVID19 Rheumatoid arthritis Inflammatory polyarthritis Systemic lupus erythematosus Psoriatic arthritis Spondyloarthritis MTCD Sjogren syndrome Hypertension Dyslipidaemia Depression Diabetes mellitus Heart disease Vascular disease Liver disease Kidney disease Lung disease ILDCOPD Cancer Venous thrombosislung embolism Thyroid diseaseGender womenAge yearsDiagnosis AIRD one category vs the rest Disease durationSmoking habit active vs noneComorbidities yes NSAIDsGlucocorticoidscsDMARDSs TsbDMARDs JAKisOther biologics anti IL6 tocilizumab sarilumab rituximab Rtx anti IL1723 anti IL17Othercategories could not be represented polymalgia rheumatica systemicsclerosis vasculitis Raynaud phenomenon polychondritis Beh§et diseasepolymyositis uveitis inflammatory boweldisease antiphospholipid syndrome juvenile idiopathic arthritis autoinflammatorysyndromes and sarcoidosisAIRD autoimmune inflammatory rheumatic disease anti TNF tumour necrosis factor csDMARD Conventional synthetic disease modifying antirheumatic drug IL6 interleukin6 JAKi JAK inhibitors tsbDMARDs target syntheticbiologic disease modifying antirheumatic drug Methotrexateleflunomideazathioprine Sulfasalazine Antimalarial agents None Anti TNF agents Other biologicsAs for the association between sex and risk of hospital admission we did not find a higher risk of admission in women despite the fact that rheumatic diseases are more prevalent in this group The type of diagnosis seems to play an important role in the probability of hospital admission and patients with systemic autoimmune conditions seem to have the highest risk compared with chronic inflammatory arthritisAs it has been reported elsewhere comorbidities play an important role in the risk of hospital admission15 Clinical outcomes are poorer in patients with COVID19 with a comorbid condition than in those without and a greater number of comorbidities correlates with poorer clinical outcomes16 Diabetes is a major comorbidity in COVID19 and patients history of diabetes is an independent risk factor for morbidity and mortality in this condition17 Diabetes has been associated with admissions to Freites Nu±ez DD et a0al Ann Rheum Dis 101136annrheumdis2020217984 0cEpidemiologyTable Multivariable analysis risk factors for hospital admission related to COVID19 in patients with AIRDVariableOR CIP valueGender womenAge yearsAIRD systemic autoimmune conditions vs chronic inflammatory arthritisCOVID comorbidities yesGlucocorticoidsSystemic autoimmune conditions polymyalgia rheumatica mixed connective tissue disease systemic sclerosis Sjogrens syndrome vasculitis Raynaud polymyositis polychondritis sarcoidosis antiphospholipid syndrome autoinflammatory syndromes and systemic lupus erythematosus vs chronic inflammatory arthritis rheumatoid arthritis inflammatory polyarthritis juvenile idiopathic arthritis psoriatic arthritis axial spondyloarthritis uvetis and inflammatory bowel disease Comorbidities including the presence of at least one of the follows hypertension heart disease vascular disease diabetes mellitus venous thrombosislung embolism chronic kidney disease liver disease and lung disease ILDCOPDAIRD autoimmune inflammatory rheumatic disease COPD chronic obstructive pulmonary disease ILD interstitial lung diseasethe intensive care unit due to COVID19 in recent series19 and has been shown to increase mortality6 Therefore we adjusted for comorbidity in the multivariable analysisTreatment with glucocorticoids lost its statistical significance in the multiple regression model However the dose was not reported in our data and in the case of these agents the risk could be dose dependent In a recent publication from a European registry the authors found that exposure to mgday was associated with a greater probability of hospitalisation21The exposure to DMARDs regardless of whether they were biological or synthetic does not seem to be associated with a higher hospital admission related to COVID19 Although we have to consider the limited number of patients in our study our results are in concordance with data reported elsewhere8 Our results should be interpreted taking into account other limitations First patients were included from a single centre Second of all the patients with COVID19 who did not require admission one third contacted the rheumatology service to report the disease and the remainder were detected through the COVID19 discharge reports sent to their primary care physician Elderly persons and homemakers who did not contact us can be considered missing Consequently there may be some selection bias between those admitted and those not admitted although this problem was addressed by adjusting for confounders in the multivariable analysis Third while it is acknowledged that clinical suspicion must be confirmed by PCR assay almost of patients admitted did not undergo PCR owing to the lack of tests or the extreme healthcare overload Nevertheless all cases included were clinically compatible and managed as COVID19The key strength of our study is that it was performed in real life conditions during then pandemic peak with access to complete sociodemographic and clinical data from our rheumatology electronic clinical history including thorough hospital admission data such as laboratory abnormalities and COVID19 treatment data from the hospital computer services Consequently this has allowed us to analyse the risk of hospital admission related to COVID19 adjusted for confounders thus avoiding possible biasAlthough we are unable to modify the factors reported here knowing them can help rheumatologists to treat and advise their patients during this new and challenging period Results provided by our study are preliminary and should be corroborated with other real life studies but we consider our findings helpful to increase the knowledge in the management of patients with AIRD and COVID19Twitter Benjamn Fernandez Gutierrez Fergutbe2001Acknowledgements The authors would like to thank Ana M Perez for her help with data collection They would like to say a special thanks to all the rheumatologists and nurses who contributed to the care of the patients in such an innovative and conscientious wayContributors BF G LL JAJ LR R and LA conceived and designed the study DDFN JFU AMG JIC and LL collected data LA and LL performed the data analysis and interpreted the data All of the authors were involved in the drafting andor revising of the manuscriptFunding This work was supported by the Instituto de Salud Carlos III ISCIII Ministry of Health Spain CP1600916 PI1801188 and RD1600120014 and cofunded by el Fondo Europeo de Desarrollo Regional FEDER The funders had no role in study design data collection analysis manuscript preparation or decision to publishCompeting interests None declaredPatient and public involvement Patients andor the public were not involved in the design or conduct or reporting or dissemination plans of this researchPatient consent for publication Not requiredEthics approval The study was approved by the Hospital Clnico San Carlos institutional ethics committee approval number E BS This study was conducted according to the principles of the Declaration of HelsinkiProvenance and peer review Not commissioned externally peer reviewedData availability statement Data are available upon reasonable requestThis article is made freely available for use in accordance with BMJs website terms and conditions for the duration of the covid19 pandemic or until otherwise determined by BMJ You may use download and print the article for any lawful non commercial purpose including text and data mining provided that all copyright notices and trade marks are retainedORCID iDsLeticia a0Leon http orcid Luis a0Rodriguez Rodriguez http orcid REFERENCES Fernandez Gutierrez B COVID19 with pulmonary involvement An autoimmune disease of known cause Reumatol Clin COVID19 Situaci³n actual en La Comunidad de Madrid Informe de situaci³n del de Mayo Available httpswww comunidad madrid sites default files doc sanidad 200508_ cam_ covid19 pdf [Accessed May ] Chen N Zhou M Dong X et a0al Epidemiological and clinical characteristics of cases of novel coronavirus pneumonia in Wuhan China a descriptive study Lancet Figueroa Parra G Aguirre Garcia GM Gamboa Alonso CM et a0al Are my patients with rheumatic diseases at higher risk of COVID19 Ann Rheum Dis Favalli EG Ingegnoli F De Lucia O et a0al COVID19 infection and rheumatoid arthritis Faraway so close Autoimmun Rev Zhou F Yu T Du R et a0al Clinical course and risk factors for mortality of adult inpatients with COVID19 in Wuhan China a retrospective cohort study Lancet Monti S Balduzzi S Delvino P et a0al Clinical course of COVID19 in a series of patients with chronic art | 2 |
Construction of a novel prognosticpredicting modelcorrelated to ovarian cancerWeichun Tang12 Jie Li3 Xinxia Chang12 Lizhou Jia1 Qi Tang12 Ying Wang4 Yanli Zheng4 Lizhou Sun5 andZhenqing Feng121National Health Commission Key Laboratory of Antibody Technique Nanjing Medical University Nanjing Peoples Republic of China 2Department of Pathology Nanjing MedicalUniversity Nanjing Peoples Republic of China 3Department of Nursing The Second Afï¬liated Hospital of Nantong University Nantong Peoples Republic of China 4Department ofGynaecology and Obstetrics The Second Afï¬liated Hospital of Nantong University Nantong Peoples Republic of China 5Department of Obstetrics and Gynecology First Afï¬liatedHospital of Nanjing Medical University Nanjing Peoples Republic of ChinaCorrespondence Zhenqing Feng fengzhenqingnjmueducn or Lizhou Sun lizhou sun163comBackground Ovarian cancer OC is one of the most lethal gynecological cancers worldwide The pathogenesis of the disease and outcomes prediction of OC patients remainlargely unclear The present study aimed to explore the key genes and biological pathwaysin ovarian carcinoma development as well as construct a prognostic model to predict patients overall survival OSResults We identified upregulated and downregulated differentially expressedgenes DEGs associated with OC Gene Ontology GO term enrichment showed DEGsmainly correlated with spindle microtubes For Kyoto Encyclopedia of Genes and GenomesKEGG pathways cell cycle was mostly enriched for the DEGs The proteinprotein interaction PPI network yielded nodes and edges Top three modules and ten hub geneswere further filtered and analyzed Three candidiate drugs targeting for therapy were alsoselected Thirteen OSrelated genes were selected and an eightmRNA model was presentto stratify patients into high and lowrisk groups with significantly different survivalConclusions The identified DEGs and biological pathways may provide new perspective onthe pathogenesis and treatments of OC The identified eightmRNA signature has significantclinical implication for outcome prediction and tailored therapy guidance for OC patientsBackgroundOvarian cancer OC is the most lethal malignant disease in the female reproductive system with over new cases and deaths each year worldwide [] Epithelial OC accounts for ofovarian malignancies listed as the most common histological type Since the ovaries locate in the deeppelvis with mere symptoms emerging at the beginning of ovarian morbid change the early detectionfor the malignancy is truly difficult Hence when OC is detected the patient is usually at an advancedstage with invasion and metastasis accompanied [] For patients in the early stage the 5year survivalrate can reach whereas for advancedstage patients the number is mere ¼ [] Thereforeit is imperative to explore the molecular mechanisms of malignant biological behavior of OC cells andto develop more reliable molecular markers for predicting recurrence and evaluating prognosis furtherguiding clinicians for therapyAt present various highthroughput microarrays and nextgeneration sequence genomic datasetswhich were deposited in the Gene Expression Omnibus GEO [] and The Cancer Genome Atlas TCGAdatabases have been widely analyzed for identifying differentially expressed genes DEGs which couldserve as candidate diagnostic or prognostic markers further effectively improving our understanding ofthe disease from genetic perspective Whereas since the existence of tissue or sample heterogeneity inThese authors contributedequally to this workReceived April Revised July Accepted July Accepted Manuscript online July Version of Record published August The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20201261101042BSR20201261each independent experiment as well as the discrepancy of different data processing methods and technology platforms the DEGs identified from a singlecohort study may generate false positives Herein the Robust Rank Aggregation RRA method which analyzes the significant probability of all elements by a probabilistic model is developedto identify statistically significant genes from multiple datasets and provide more accurate and valuable informationfor clinical use far beyond one gene list [] To date a bunch of novel prognostic markers have been discovered topotentially improve the efficacy of diagnosis and prognosis of OC [] However these identified markers were onlyeffective for partial stages or grades and were difficult to apply widely Hence a prognostic model which is basedon signature gene expression level with high discriminating power to effectively assist prognosis prediction for eachpatient is required in clinical practiceIn the present study we downloaded six primary microarray datasets from the GEO database which containeda total of samples with OC samples and normal samples The geneset and relative clinical information on ovary tissues of OC patients and healthy females from TCGA and GTEx portal were also downloadedIntegrated DEGs between cancerous and normal ovarian samples were screened using the limma R package andRRA method Gene Ontology GO and Kyoto Encyclopedia of Genes and Genomes KEGG pathways enrichmentof DEGs were performed for nextstep functional analysis The Search Tool for the Retrieval of Interacting GenesDatabase STRING and the Connectivity Map CMap online database were then used to analyze the association ofDEGs and explore the molecular mechanisms as well as drugs involved in tumorigenesis Through survival analysisprognostic mRNAs were also selected By performing Cox regression analysis we identified an eightmRNA signature model with the ability to predict the prognosis of OC patients and independent from clinical factors Our studyprovides reliable molecular markers and prognostic models for early detection and outcome prediction as well aseffective drug targets for treating OCMethodsData collectionThrough searching on the GEO Repository with ovarian cancer we downloaded the gene expression profiles ofGSE54388 GSE40595 GSE38666 GSE27651 GSE18520 and GSE14407 and the corresponding annotation files fromthe GPL570 [HGU133 Plus ] Affymetrix Human GenomeU133 Plus Array platform GSE54388 contains ovarian tissue samples with normal ovarian surface epithelium and tumor epithelial components from highgradeserous OC patients GSE40595 includes OCassociated stroma and epithelium samples which consist of cancerassociated stroma samples and epithelial tissues from highgrade serous OC patients along with stromal component and ovarian epitheliums from the normal ovary GSE38666 comprises stroma and matchedovarian epitheliums from healthy females as well as cancer stroma and matched cancer epitheliums from OCpatients GSE27651 incorporates normal ovarian surfaces epithelial and serous borderline ovarian tumors lowgrade serous ovarian carcinomas and highgrade serous ovarian carcinomas GSE18520 covers advancedstage highgrade primary OC specimens and normal ovarian surface epithelium tissues GSE14407 involves healthy ovarian surface epithelial samples and paired serous OC epithelium Note that all samples from these GEOdatasets are classified into the cancerous or normal part to be clear the normal stromal and surface epithelium is defined as normal ovarian tissues whereas the borderline tumors as well as cancerous stromal and epithelial tissues areconsidered as malignancies Besides we also downloaded the FPKM format gene expression data and relative clinicalinformation of OC patients samples and normal ovarian tissues from TCGA and GTEx portal respectivelyScreening for DEGs and integration of microarray dataWe used the limma R package [] to integrate the expression profiles from TCGA and GTEx portal standardize the data from the integrated TCGA and GTEx expression matrix as well as six GEO datasets and furtherscreen the DEGs between ovarian cancerous and normal samples The list of DEGs obtained from six GEO microarray datasets by limma analysis was further integrated by the RRA method to get prioritized commonly upor downregulated gene list The final overlapped DEGs for subsequent biological function analysis were the combination of prioritized jointly dysregulated genes from six GEO microarrays and the results from TCGA and GTExdatabases The cutoff criteria were set as FDR and log2fold change FC GO term and KEGG pathway enrichment analysisGO classified the known genes into three main biological progress Molecular Function MF Cellular ComponentCC and Biological Process BP [] KEGG provides researchers highlayer functions of the biological system frommolecular level information [] The Enrichr online tool amppharmmssmeduEnrichr allows for GO term The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20201261101042BSR20201261Figure The expression heatmap of the top signiï¬cantly dysregulated genes in six GEO datasetsHierarchical clustering that shows the expression profiles of mRNAs from A GSE14407 B GSE18520 C GSE27651 DGSE38666 E GSE40595 F GSE54388annotation and KEGG pathway for a cluster of genes [] We explored the biological functions of overlappedDEGs and hub modules from our proteinprotein interaction PPI network using Enrichr website Pvalue was considered as significant enrichment Likewise the functional biological pathways of the top ten hub genes fromPPI network were also analyzed by the FunRich tool version [] and the top five enriched pathways of up anddownregulated genes were displayed as bar charts respectively We set the Pvalue as statistically significant The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20201261101042BSR20201261PPI network construction and analysisPPI networks display the relationships of various proteins according to their physical or biochemical propertiesSTRING is a database that encompasses the interaction information between known proteins and potentially interacted proteins [] In order to explore the correlations between the DEGs we used the STRING database to constructa PPI network and visualize our results by Cytoscape software [] Confidence score was set as significantMolecular Complex Detection MCODE was utilized to select hub modules of PPI networks in Cytoscape [] Weset the degree cutoff node score cutoff kscore and max Depth was set as the criterion Thenthe significant modules were performed by GO and KEGG analyses Top ten genes were defined according to thehigh degree of connectivity in STRING network [] The coexpression analysis of ten hub genes was performed bySTRING eitherValidation of the hub genesWe downloaded the raw geneset of OC patients from TCGA to explore the expression differences of hub genes inlow and highgrade tumor tissues of OC and draw the survival plot using KaplanMeier plotter webtool [] Thegene and protein expression level of graderelated hub genes were then confirmed by Oncomine and The HumanProtein Atlas HPA database [] Meanwhile we explored the genetic alteration information of the selected tenhub genes in OC patients by plugin cBioPortal cBio Cancer Genomics Portal tool which deposits the genomicsprofiles of various cancer types and provides analysis and visualization of the genomics datasets []Identiï¬cation of candidate small molecule drugsThe CMap database was able to predict potential drugs which might reverse or induce the biological state encoded incertain gene expression signatures in OC [] The DEGs from our study were used to query the CMap databaseThe enrichment scores which represent the similarity were calculated ranging from to The positive connectivity score means an inducing influence on the input signature whereas drugs with negative connectivity score presentreversion impact on the characteristic in human cell lines and are considered as candidate therapeutic molecules After sorting all instances the connectivity score of various instances was filtered by Pvalue Next we investigatethe structures of these candidate molecular drugs from the Pubchem database pubchemncbinlmnihgovEstablishment and evaluation of the prognostic modelThe OC patients from the TCGA project were randomly classified into the training cohort n188 and the testingcohort n186 OSrelated genes were determined by performing univariate Cox regression analysis in the trainingcohort with the Survival R package and further selected for the nextstep screening Least Absolute Shrinkage andSelection Operator LASSO is a parameter selection algorithm which shrinks all highdimensional regression coefficients and generates the penalty regularization parameter λ via the crossvalidation routine by glmnet R packageTo select the optimal prognostic mRNAs we adopted LASSO regression among the selected candidate genes and further perform multivariate Cox proportional hazards regression to evaluate their independent prognostic values Theriskscore model for predicting outcomes of OC patients was the sum of each optimal prognostic mRNA expressionlevel multiplying relative regression coefficient weight calculated from the multivariate Cox regression modelRisk score patient cid2Coefficient mRNAi à Expression mRNAiiAll training cohort patients were classified into high and lowrisk groups according to the median risk score TheKaplanMeier curves of two diverse groups were plotted using survfit function and the receiver operating characteristic ROC curve was unfolded for OS prediction to estimate the sensitivity and specificity of the prognostic modelCox multivariate analysis was also performed to examine whether the risk score was independent of the clinical characters such as age tumor stage and grade Next we used the testing group to check the efficacy of the prognostic riskmodel Each individual in the testing cohort was also categorized as high or lowrisk case by comparing the patientsrisk score with the cutoff value calculated from the training cohort KaplanMeier curve analysis timedependentROC analysis and Cox multivariate analysis were performed eitherSearching tumorinï¬ltrating immune cells associated with patientsprognostic signaturesThe TIMER webtool allows for systematical evaluations of the relationship between the six immune cell types inthe tumor microenvironment which are B cell CD4 T cell CD8 T cell neutrophil macrophage as well as dendritic The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20201261101042BSR20201261Figure The volcano plot of all gene expression distribution in six GEO datasetsVolcano plot of differentially expressed mRNAs of A GSE14407 B GSE18520 C GSE27651 D GSE38666 E GSE40595 FGSE54388cell and clinical impact in various cancer types via a novel statistical method [] To further explore the prognosticsignature we used the TIMER online tool to search the most significant tumorinfiltrating immune cells according tothe TCGA OC gene data To be clear the relative gene expression levels of six types of immune cells for each patientin high and lowrisk groups from training and testing cohort were measuredResultsThe DEGs among six GEO microarray datasetsThe top significantly up and downregulated genes from each microarray dataset were displayed in the heatmapsFigure 1AF and the distribution of all gene expression was presented in volcano plots Figure 2AF ThroughRRA analysis of expression microarrays we identified DEGs which consisted of upregulated and downregulated genes and displayed the top dysregulated genes by pheatmap R package in Figure Next weanalyzed the expression profiles of TCGA and GTEx getting dysregulated genes Intriguingly when these DEGs were combined with the DEGs from GEO datasets we found that genes were commonly dysregulatedin these two databases with upregulated Figure 4A and downregulated genes Figure 4BGO term and KEGG pathway enrichment analysis of DEGsTo study the potential biological function of the DEGs we performed biological pathway analysis and identifiedsignificantly enriched pathways via Enrichr web tools In GO term Figure 5A for the BP group the DEGs weremostly enriched in regulation of attachment of spindle microtubes to kinetochore cellular response to laminar fluidshear stress and microtubule cytoskeleton anization involved in mitosis In MF group the dysregulated geneswere highly correlated to microtubulebinding microtubule motor activity and tubulinbinding As for CC group The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20201261101042BSR20201261Figure Heatmap showing the top upregulated genes and top downregulated genes according to PvalueEach row represents one gene and each column indicates one dataset Red indicates upregulation and blue represents downregulation The numbers in the heatmap indicate log FC in each dataset calculated by the limma R package Abbreviation log FClogarithmic fold changethe DEGs were closely related to condensed nuclear chromosome kinetochore and mitotic spindle KEGG pathwayanalysis showed DEGs highly enriched in cell cycle and Alanine aspartate and glutamate metabolism Figure5B The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20201261101042BSR20201261Figure The intersection of up and downregulated DEGs of GEO and TCGA datasetsA upregulated intersected DEGs in both datasets B downregulated intersected DEGs in both GEO and TCGA dataset Theintersected DEGs were defined as the significant DEGsPPI network construction and modules analysisUsing the STRING database and Cytoscape software a total of DEGs were mapped into the PPI network whichincluded nodes and edges Figure 6A The PPI enrichment Pvalue was 10e16 The top three key modules Figure 6CE within PPI network were then selected Module MCODE score Module MCODEscore Module MCODE score and the biological function of Module which consisted of nodesand edges was further analyzed GO analysis indicated that Module1 was mainly associated with regulation ofattachment of spindle microtubules to kinetochore condensed nuclear chromosome kinetochore and microtubulemotor activity KEGG analysis showed that cell cycle and oocyte meiosis were the most highly enriched pathwaysSupplementary Figure S1The screening of Hub genes and their characteristicsThe top ten hub genes with the highest degree of connectivity were CDC45 CDK1 TOP2A CDC20 CCNB1CEP55 UBE2C HMMR FOXM1 and TPX2 Figure 6B The coexpression analysis results of the hub genes demonstrated that these genes actively interacted with each other Supplementary Figure S2 Besides we established theinteraction network of ten hub genes with their related genes and explored the biological role Supplementary Figure S2ACF of the network by FunRich The gene alteration type and frequency as well as the most frequentlyaltered neighbor genes were also exhibited Figure Gene alteration frequency of ten hub genes among TCGAOC samples was beyond with most genes showed amplified and multiple altered Figure 7AB The top threemost frequently altered genes were FOXM1 CDC20 and CCNB1 with FOXM1 and CDC20 largely amplified whileCCNB1 deep deleted Through analysis of OC patients geneset from TCGA we found that CCNB1 UBE2C CDK1CEP55 as well as FOXM1 expressed significantly higher in highgrade tumors and predicted worse outcomes sincepatients overexpressed above genes owned lower overall survival OS and diseasefree survival DFS rates Figure The Oncomine database showed results from various studies were consistent to our finding Supplementary Figure S3 The HPA website also demonstrate that proteins translated by such five hub genes were overexpressed in OCtissues Supplementary Figure S4 HMMR and TPX2 were also negatively correlated to patients prognosis while noexpression difference was observed in diverse tumor grades and CDC20 was positively associated with tumor gradebut not correlated to patients outcomesRelated small molecule drugs screeningIn total DEGs were analyzed in CMap to screen small molecule drugs and the candidate molecules with top tenconnectivity score are listed in Table Five of these molecules showed a negative correlation and suggested potentialin clinical applications Among them Trichostatin A pyrvinium and 8azaguanine showed a significantly negativecorrelation and the stuctures of such candidate molecule drugs was found in the PubChem database and shown inSupplementary Figure S5 The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20201261101042BSR20201261Figure GO and KEGG functional annotation for the signiï¬cant DEGsA The top ten enriched BP of the DEGs B The top ten enriched CC of the DEGs C The top ten enriched MF of the DEGs DThe top ten enriched KEGG pathways of the DEGs The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20201261101042BSR20201261Figure The PPI network and top hub genes as well as top three modules were constructedA The PPI network of the DEGs B The hub genes of the DEGs CE Top three hub modules were identified by Cytoscapeplugin MCODE C module1 D module2 E module3Table The top ten OCrelated small molecules with highly signiï¬cant correlations in results of CMap analysisRankCMap nametrichostatin A8azaguaninepyrviniumisoï¬upredonequinpirolevorinostatgenisteinantimycin AheptaminolmidodrineMeanNEnrichmentP value The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20201261101042BSR20201261Figure The gene mutation overview of ten hub genes in TCGA OC patientsA Ten hub genes are altered in of queried patients B The summary of mutation type of ten hub genes in OC patientsC The network of hub genes and the most frequently altered neighbor genesTable Univariate cox regression identiï¬ed DEGs correlated to patients OSGene IDCCND1SYNE4CCDC80TMC4MCCFOXQ1KRTCAP3CXCR4IL4I1DEFB1CSGALNACT1KLHL14MCUR1HRAbbreviation HR hazard ratioHR95LHR95HPvalueConstruction of prognostic model and evaluation of its predictive abilityUnivariate Cox regression analysis revealed that of DEGs were significantly correlated to patients OS in thetraining cohort Table The OSrelated genes were listed as follows CCND1 SYNE4 CCDC80 TMC4 MCC The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20201261101042BSR20201261Figure The clinical characteristics of CCNB1 CEP55 CDK1 FOXM1 and UBE2C in OC patientsA Five genes were overexpressed in high grade G1 and G2 compared with low grade G3 and G4 in OC BC The OS time Band DFS time C of five genes in OC patients The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20201261101042BSR20201261Figure Identiï¬cation of prognosisrelated mRNAs using LASSO regression modelA LASSO coefficient profiles of the mRNAs associated with the OS of OC B Plots of the crossvalidation error rates Each dotrepresents a λ value along with error bars to give a confidence interval for the crossvalidated error rateTable Multivariate Cox regression selected eight DEGs correlated to patients OSGene IDTMC4KLHL14CXCR4CCDC80KRTCAP3DEFB1SYNE4FOXQ1HRHR95LHR95HPvalue845E08Abbreviation HR hazard ratioFOXQ1 KRTCAP3 CXCR4 IL4I1 DEFB1 CSGALNACT1 KLHL14 and MCUR1 Through LASSO Cox regression we narrowed the number of prognosisassociated genes to according to the minimum criteria Figure Next based on the multivariate Cox model of candidate mRNAs retained their prognostic significance and werefinally selected as independent remarkable prognostic factors which were TMC4 KLHL14 CXCR4 CCDC80 KRTCAP3 DEFB1 SYNE4 and FOXQ1 Table To predict patients outcomes we developed an individuals risk scoremodel as follows risk score à expression value of TMC4 à expression value of KLHL14 à expression value of CXCR4 à expression value of CCDC80 à expression value of KRTCAP3 à expression value of DEFB1 à expression value of SYNE4 à expression value of FOXQ1 On the basis of the median risk score patients were divided into high orlowrisk groups KaplanMeier curve analysis showed that the OS time of the lowrisk group was significantly longerthan the highrisk group P1147e07 Figure 10E ROC curve analysis revealed the area under the ROC curveAUC of the prognostic model was Figure 10D Meanwhile the risk scores Figure 10A of OC patients inthe training group were ranked for displaying their distribution and the survival status Figure 10B was marked onthe dot plot The expression pattern of eight prognostic mRNAs between high and lowrisk groups was also shown inthe heatmap Figure 10C Univariate and multivariate Cox regression analyses concerning the risk score and clinicalfactors showed that the prognostic model was able to serve as an independent prognostic indicator Figure 11ABROC curve analysis also showed that the AUC value of the model was much significantly higher than the tumor stage AUC grade AUC and patients age AUC Figure 11C Interestingly when The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20201261101042BSR20201261Figure Prognostic analysis of the TCGA training modelA The risk score B survival status C expression heatmap D timedependent ROC curves and E KaplanMeier survival ofthe prognostic model for the TCGA OC training cohort The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20201261101042BSR20201261Figure The clinical independency of the risk model in training cohortUnivariate A and multivariate B regression analyses as well as timedependent ROC curve analysis CD of the prognostic valuebetween the training model and OC patients OS status when compared with or combined with clinical factorscombined the risk score with clinical factors the ROC curve of combination model was much higher than each aloneFigure 11DAs for the testing cohort we divided the group into highrisk and lowrisk individuals based on the trainingcohort cutoff risk score The outcomes of low and highrisk groups patients of the testing cohort were also measuredand the OS time of the highrisk group was significantly shorter than the lowerrisk group P1721e02 Figure12E The AUC of the prognostic model was Figure 12D The risk scores distribution Figure 12A and survivalstatus Figure 12B of OC patients as well as the eightprognostic gene expression heatmap Figure 12C in the testinggroup were also present Meanwhile the independency of the prognostic model was confirmed in testing cohort sinceunivariate and multivariate Cox regression analyses showed the model correctly predicted high or lowrisk factroups patients outcomes without relying on any clinical factors Figure 13AB ROC curve analysis showed that theprognostic model exhibited better sensitivity and specificity when compared with tumor stage grade and patientsage for the AUC value of the model was much higher than later Figure 13C In accordance with results from trainingcohort the combination of risk score and clinical factors showed better OS prediction capability Figure 13DThe prognostic signature correlating to immune cells inï¬ltrationThrough TIMER webtool we analyzed the relative gene expression levels of six types of immune cells for each patientand found that genes concerning macrophage fraction were expressing significantly higher in the highrisk groupP005 compared with the lowrisk group in training cohort Figure Interestingly same result was also observed in the testing cohort Figure The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20201261101042BSR20201261Figure Prognostic analysis of the TCGA testing modelA The risk score B survival status C expression heatmap D timedependent ROC curves and E KaplanMeier survival ofthe prognostic model for the TCGA OC testing cohort The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20201261101042BSR20201261Figure The clinical independency of the prognostic risk signature in testing cohortUnivariate A and multivariate B regression analyses as well as timedependent ROC curve analysis CD of the prognostic valuebetween the testing model and OC patients OS status when compared with or combined to clinical factorsDiscussionIn the present study we used the RRA methods to jointly analyze six GEO OC microarrays which contained OC and normal samples identifying DEGs and overlapped them with dysregulated genes of OC cohort fromTCGA and GTEx portal finally getting upregulated and downregulated genes Functional analysis showedthat DEGs were significantly enriched in the cell division cycle to be clear in the process of the mitotic spindleSpindle microtubules have been proved to play crucial role in physiological and pathological processes As for celldivision only when all chromosomes linked to spindle microtubules through kinetochores and the spindle assemblycheckpoint is satisfied this process could step to anaphase [] Suraokar et al found that the mitotic spindle assemblycheckpoint and microtubule network were significantly altered in malignant pleural mesothelioma MPM whileusing epothiloneB a nontaxane small molecule inhibitor targeting the microtubules could greatly decrease theviability of MPM cell lines [] Rogalska et al compared the antiproliferative capacity of epothilone B with paclitaxelon OC cell line SKOV3 found that this effect of Epo B was greater than latter [] The researches above wereconsistent with our study that the mitotic spindle process was dysregulated in OC progression playing importantroles in OC cell proliferation and tumor developmentPPI network construction of DEGs included nodes and edges among which we identified three keymodules Interestingly the top1 module was also highly associated with spindle microtubules and chromosome kinetochore confirming the role of cell cycle in OC pathogenesis The top ten hub genes from the PPI network were alsorecognized which were CDC45 CDK1 TOP2A CDC20 CCNB1 CEP55 UBE2C HMMR FOXM1 and TPX2 The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20201261101042BSR20201261Figure The expression level of immune cells related genes in high and lowrisk groups of the training cohortA Bcell fraction B dendritic fraction C CD4 Tcell fraction D CD8 Tcell fract | 2 |
"what clinicians know about the diagnosis treatment and prevention of disease originates from studies mostly done on male cells male mice and men1 historically for multiple reasons including the purported safety of women and their offspring women of childbearing age were excluded from clinical trials as a result medical research and care have been centred on male physiology the assumption was that male and female cells and animals were biologically identical and evidencebased medicine was defined by clinical trials done predominantly in men1 in the us national institutes of health nih mandated the inclusion of women in nihfunded clinical trials but many investigators did not follow this mandate and many of those who did include women did not analyse the results by sex23 minimising the effectiveness of this policy preclinical research and drug development studies have also predominantly used male animal models and cells4 it is not surprising that a us government accountability office report found that eight of the ten prescription drugs withdrawn from the market between and posed greater health risks for women than for men most funding agencies from europe and north america have implemented policies to support and mandate researchers to consider sex and gender at all levels of medical research8 still the field of sexbased biology and medicine is often viewed as a specialised area of interest rather than a central consideration in medical research essential for the success of clinical care and translational science is awareness by clinicians and researchers that the diseases they are treating and studying are characterised by differences between women and men in epidemiology pathophysiology clinical manifestations psychological effects disease progression and response to treatmentthis review explores the role of sex biological constructs and gender social constructs as modifiers of the most common causes of death and morbidity and articulates the genetic biological and environmental determinants that underlie these differences we aim to guide clinicians and researchers to better understand and harness the importance of sex and gender as genetic wwwthelancetcom vol august biological and environmental modifiers of chronic disease ultimately it is a necessary and fundamental step towards precision medicine that will benefit women and mensex as a genetic modifier of biology and diseasesex differences in disease prevalence manifestation and response to treatment are rooted in the genetic differences between men and women genetic sex differences start at conception when the ovum fuses with a sperm cell carrying an x or a y chromosome resulting in an embryo carrying either xx or xy chromosomes this fundamental difference in chromosome complement eg genes outside the testisdetermining sry gene generates ubiquitous sex differences in the molecular makeup of all male and female cells9 first the y chromosome carries genes that exhibit subtle functional differences from their xlinked homologues eg zfy vs zfx and uty vs utx and also carries genes with no homologue at all eg sry in addition in men the x chromosome carries only maternal imprints ie epigenetic modifications made by the parent in generating the sex cellswhich alter the expression of genes in the offspring as women have x chromosomes from both parents they carry maternal and paternal imprints which target a different set of genes random inactivation of one of the x chromosomes in female cells which prevents sex differences in x chromosome gene dosage causes another degree of sex difference in gene expression as some of these xlinked genes escape inactivation in women those genes are often expressed at higher levels in women than in men9 sexspecific gene expression due to genomic search strategy and selection criteriawe searched pubmed for papers published in english between jan and june using sex or gender and the name of the disease of interest as search terms although we tried to cite seminal studies when necessary because of space limitation representative reviews were often selectedlancet diabetes discovery sexbased medicine laboratory section of endocrinology john w deming department of medicine tulane university school of medicine and southeast louisiana veterans health care system medical center new orleans la usa prof f mauvaisjarvis md barbra streisand womens heart center cedarssinai smidt heart institute los angeles ca usa prof n bairey merz md national heart lung institute imperial college london london uk prof p j barnes md department of pharmacology and department of neurology college of medicine center for innovation in brain science university of arizona tucson az usa prof r d brinton phd department of medical epidemiology and biostatistics and center for gender medicine karolinska institutet stockholm sweden prof jj carrero phd channing division of network medicine and the division of pulmonary and critical care medicine department of medicine brigham and womens hospital harvard medical school boston ma usa d l demeo md neuroscience institute and department of biology geia state university atlanta ga usa prof g j devries phd department of psychiatry university of colorado school of medicine anschutz medical campus aurora co usa prof c n epperson md division of oncology department of medicine washington university school of medicine st louis mo usa prof r govindan md w harry feinstone department of molecular microbiology and immunology the johns hopkins bloomberg school of public health baltimore md usa prof s l klein phd department of biomedical metabolic and neural sciences university of modena andreview 0creggio emilia azienda ospedalierouniversitaria di modena ospedale civile di baggiovara modena italy prof a lonardo md department of psychiatry department of psychology and department of obstetrics gynecology university of illinois at chicago chicago il usa prof p m maki phd department of neurology mcgovern medical school university of texas health science center houston tx usa prof l d mccullough md berlin institute of gender medicine charituniversittsmedizin berlin berlin germany prof v regitzzagrosek md department of cardiology university hospital z¼rich university of z¼rich switzerland prof v regitzzagrosek center for womens health research divisions of general internal medicine and cardiology university of colorado school of medicine aurora co usaimprinting extends to autosomes as well and these imprinted genes exhibit sexspecific and tissuespecific expression in humans10 thus fundamental sex differences deriving directly from genetic heterogeneity between the x and y chromosome complements and parentoforigin inher itance exist at the molecular level in all human cells these sex differences persist throughout life and are independent of sex hormones figure arguably the greatest source of differences between men and women comes from the y chromosomal sry gene which directs the development of a testis in men the ensuing developmental surge of testicular testosterone permanently masculinises the reproductive tract and the anisation of brain circuits affecting male behaviour at puberty1112 in humans the first surge occurs at the end of the first trimester of pregnancy because it alters cellular gene expression and tissue structure in multiple ans of men via epigenetic mechanisms this testosterone surge is also paramount in programming sex differences in physiology and susceptibility to diseases that will manifest in adulthood after this initial testicular testosterone surge gonadal hormone concentrations remain low until puberty which triggers lasting sex differences in circulating oestrogens and testosterone concentrations after puberty cells with androgen or afemale sexbrandom x chromosomeinactivation and escapexxxxxxxxxxxxxxxxxxxy chromosome complementmale sexsryctesticular testosterone surgefetal testistestosteroneohogenetic diï¬erences of male and female cellsepigenetic programming of male cellsfigure genetic causes of sex differencesa genetic sex differences start with cells carrying either xx or xy chromosome complement eg genes outside the testisdetermining sry gene which generates ubiquitous sex differences in the molecular makeup of all male and female cells b random inactivation of one x chromosome in female cells causes another level of sex differences in gene expression some xlinked genes escape inactivation in female individuals and have a higher expression in female than male individuals c the y chromosomal sry gene directs the development of a testis in male individuals which produces a surge of testicular testosterone at the end of pregnancy the testosterone surge programmes cellular gene expression and tissue structure in multiple ans of male individuals via epigenetic remodelling the combination of these genetic and developmental events programmes sex differences in physiology and susceptibility to diseases that will manifest in adulthoodoestrogen receptors will be affected differ ently in men and women the combination of all genetic and hormonal causes of sex differences aforementioned culminates in two different biological systems in men and women that translate into differences in disease predisposition manifestation and response to treatment therefore sex is an important modifier of physiology and disease via genetic epigenetic and hormonal regulations figure gender as a determinant of patients and doctors behaviour and as a modifier of health disease and medicinegender according to the global health definition refers to the socially constructed norms that impose and determine roles relationships and positional power for all people across their lifetime13 gender interacts with sex the biological and physical characteristics that define women men and those with intersex identities13 gender is not a binary term it includes the understanding that in many people traits of masculinity or femininity coexist and are expressed to different degrees gender attributes are fluid more than two thirds of women and men report genderrelated characteristics traditionally attributed to the opposite sex1415 in transgender people gender identity differs with the sex they were assigned at birth so far transgender people have generally been underrepresented in clinical studies to date although this underrepresentation is changing gender is an equally important variable as biological sex in human health and influences the behaviour of communities clinicians and patients1415 gender roles represent the behavioural norms applied to men and women in society which influence individuals everyday actions expectations and experiences including diet perceived stress smoking and physical activity and affect health and disease susceptibility gender identity describes the fluidity of how a person perceives oneself as a woman or a man which affects feelings and behaviours gender relations refer to how we interact with or are treated by people on the basis of our ascribed gender institutionalised gender reflects the distribution of power between men and women in the political educational and social institutions in society and shapes social norms that define perpetuate and often justify different expectations and opportunities for women and men1617 as such the distribution of genderrelated charac teristics within populations of men and women can influence health differently than biological sex together these gender constructs determine access to health care helpseeking behaviours and individual use of the healthcare system being perceived as a man or a woman triggers different responses from clinicians who might diagnose and suggest interventions differently according to gender as such gender largely determines the use of preventive measures and referral for or acceptance of invasive therapeutic strategies genderrelated behaviours contribute to risk exposure and preventive behaviour in several wwwthelancetcom vol august review 0cdiseases this postulation is well exemplified in the cardiovascular field in which women often underestimate their risk compared with men and seek consultation later than men in the clinic for treatment of myocardial infarction1819 in the genesispraxy prospective study mortality year after an acute coronary event was more strongly associated with gender than with biological sex1415 similarly control of cardio vascular risk factors hypertension diabetes depres sive symptoms was better predicted by gender than by biological sex1415 therefore including a gender dimension in clinical studies and practice will contribute to the understanding of different clinical manifestations and outcomes of diseases in women and men1617 although beyond the scope of this review it is also important to consider that regarding health and disease gender intersect with race or ethnicity and age20 sex and gender are fundamentally and frequently reciprocally interrelated in biology and disease24 sex influences behaviours eg towards more aggressive or caring phenotypes on the other hand genderrelated behaviours eg smoking lifestyle perceived stress and pain and nutritional habits might produce epigenetic modifications that modulate gene expression and biological phenotypes figure summarises how sex and gender are interrelated in biology and diseasesex and gender differences in major chronic diseaseshaving established the importance of sex and gender in disease we will summarise their influences on the most common causes of death and debilitating diseases in the usa as an example figure note that these sex and gender disparities are relevant to other highincome countries as well as lowincome and middleincome countries where the burden of these diseases becomes increasingly like those in highincome countries26 in most diseases efforts to separate the effects of sex and gender are still incomplete so that we just refer to the differences among women and men because current knowledge about pathophysiology diagnosis and treatment of disease is primarily based on men as representative of the human species this review focuses on how women differ from men we discuss some key aspects regarding the dimensions of men in a dedicated sectionheart diseaseepidemiology pathogenesis manifestations and diagnosisheart disease is the leading cause of death in the usa in heart disease accounted for · of all deaths for men and for · of all deaths for women figure ischaemic heart disease and heart failure are major contributors to heart disease mortality and have important sex and gender differences for example heart failure disproportionately contributes to coronary heart disease mortality in women27 potentially due to undiagnosed ischaemic heart disease in women the strength of the association with cardiovascular risk factors differ by sex biological sexsex chromosomesepigeneticeï¬ectssex hormonesohohohobehaviour of patients and doctorssocietygender constructslifestylenutritional habitsexerciseperceived stresssmokingdiseasepathophysiologymanifestationresponse to treatmentdisease perceptionhelpseeking behaviouruse of health caredecision makingtherapeutic responsesex and gender diï¬erences in health disease and medicinefigure interrelation between sex and gender in health diseases and medicinebiological sex causes sex differences through genetic and hormonal influences in disease pathophysiology clinical manifestations and response to treatment sex also influences behaviours towards more aggressive or caring phenotypes on the other hand genderrelated behaviours eg smoking lifestyle perceived stress and nutritional habits produce epigenetic modifications that modulate the expression of biological sex gender constructs determine patients perception of disease helpseeking behaviour and individual use of health care gender constructs also influence decision making and trigger different therapeutic responses from providers biased by gendersystolic blood pressure and hypertension smoking and diabetes are associated with higher hazard ratios for myocardial infarction in women than in men28ischaemic heart disease is the most recognised example for integrating the concept of gender and sex which shape divergent or distinct disease outcomes compared with men women suffering from ischaemic heart disease are older this difference is historically believed to be due to the protection of endogenous oestrogens29 although contemporary study refutes this simplistic explanation30 and associations cannot be inferred to be causation still women suffering from ischaemic heart disease are underdiagnosed3132 and less likely to have a prehospital diagnosis of myocardial infarction33 the reasons for this disparity reflect the intersection between sex and gender first biological sex differences exist in the pathogenesis of ischaemic heart disease whereas men are more likely to be affected by obstructive coronary artery disease of large vessels than women coronary microvascular dysfunction36 leading to chronic myocardial ischaemia without obstructive coronary artery disease has a higher prevalence in women than men37 a metaanalysis reported that following acute myocardial infarction both sexes most often presented with chest pain but compared with men women were more likely to present with pain between the shoulder blades nausea or vomiting and shortness of breathprof j g regensteiner phd department of medicine department of paediatrics and department of neuroscience washington university school of medicine st louis mo usa prof j b rubin md center for the study of sex differences in health aging and disease geetown university washington dc usa prof k sandberg phd division of gastroenterology duke university medical center durham nc usa a suzuki md and durham va medical center durham nc usa a suzukicorrespondence to prof franck mauvaisjarvis diabetes discovery sexbased medicine laboratory section of endocrinology john w deming department of medicine tulane university school of medicine new orleans la usa fmauvaistulaneeduwwwthelancetcom vol august review 0cmale individualsother·heart disease·protection might disappear after menopause45 by contrast testosterone induces adverse cardiac remod elling in the male heart44chronic liver disease ·inï¬uenza and pneumonia ·suicide ·alzheimers disease ·type diabetes ·stroke ·cpd ·injuries ·female individualsother·septicaemia ·chronic kidney disease ·inï¬uenza and pneumonia ·type diabetes ·injuries ·alzheimers disease ·stroke ·cpd ·cancer·heart disease·cancer·figure percent distribution of the ten leading causes of death by sex usa adapted from heron25 cpdchronic pulmonary diseasesecond a gender bias appears to be responsible for the absence of recognition of ischaemic heart disease presentation in women38 men and women with ischaemic heart disease who score high on feminine roles and personality traits on questionnaires designed to ascertain aspects of gender are at an increased risk of recurrent ischaemic heart disease independent of female sex39heart failure affects of adults aged years and older and more women than men in absolute numbers4041 heart failure occurs at an older age and with less ischaemic causes in women than in men however hypertension and diabetes predispose older women to heart failure to a greater extent than men heart failure with preserved ejection fraction a form of heart failure with normal systolic function is twice as prevalent in women as in men by contrast heart failure with reduced ejection fraction affects more men than women women who have heart failure with preserved ejection fraction have smaller and stiffer hearts than men inflammation and the resulting fibrosis play a sexspecific role in the pathogenesis of heart failure with preserved ejection fraction under stress premenopausal womens hearts develop less inflammation resulting in less fibrosis than mens hearts4243 this difference is partially driven by sex hormones as oestrogens produce antiinflammatory actions on endothelial and immune cells and promote cardioprotective effects in premenopausal women44 this response to treatmentcompared with men women suffering from ischaemic heart disease are less likely to receive evidencebased treatment3132 and when suffering from acute myocardial infarction they are less likely to receive reperfusion33 an stelevation myocardial infarction registry revealed that compared with men women exhibit delayed reperfusion leading to higher mortality46 women suffering from acute myocardial infarction treated by male emergency physicians have a higher mortality rate than those treated by female physicians38 additionally male physicians are more effective at treating female patients with acute myocardial infarction when they work with female colleagues and when they have experience in treating female patients38 this treatment disparity between women and men can be corrected by improving emergency recognition of stelevation myocardial infarction in women and acceleration of percutaneous coronary intervention which equilibrates gender mortality47guidelines for the treatment of heart failure are similar for women and men24 however evidence suggests that optimal survival in women occurs with lower doses of β blockers angiotensin receptor blockers and angiotensin converting enzyme inhibitors than in men48 finally fewer women undergo heart transplantation than men although women are more frequently donors suggesting a referral bias could exist41cancersepidemiology pathogenesis manifestations and diagnosiscancers are the second leading cause of death dominated by lung cancer accounting for · of deaths in men and · of deaths in women figure more men develop cancer than women49 with few exceptions eg meningioma thyroid cancer lung cancer in nonsmokers nonreproductive cancers exhibit a male predominance though for some cancers oropharynx larynx oesophagus and bladder the male versus female incidence ratios can be higher than a male predominance in cancers that affect both sexes is evident around the world in all races and at all ages50 survival is also shorter for men than women across multiple cancer types the higher cancer risk in men is partially explained by gender constructs like dietary habits or risk behaviours such as smoking and alcohol consump tion51 it is unlikely to be the only cause after appropriate adjustment for these risk factors adult men still have a higher cancer risk than women52 moreover a similar male bias in incidence and survival is seen in paediatric cancers before puberty and the adoption of highrisk cancerpromoting behaviours eg smoking53the universal male predominance in cancer incidence and differential outcomes argues for a fundamental role wwwthelancetcom vol august review 0cin inutero tumour suppressors of sex in addition to gender in cancer biology sexspecific biology includes genetic differences xx vs xy chromosomes the incomplete xinactivation in female individuals which results in biallelic expression of xencoded female cells54 y chromosomeencoded oncogenes such as the rnabinding motif on y chromosome in male cells55 and the chromatin remodelling effects of testicular testosterone in male cells56 these mechanisms have an influence on several of the hallmarks of cancer57 including metabolism growth regulation58 angiogenesis and immunity which all contribute to cancer predisposition59 a crucial example is the male predisposition to glioblastoma which is the most common form of brain cancer in glioblastoma there is a cellintrinsic predisposition of male astrocytes a subtype of glial cell to malignant transformation60 after puberty sex hormones produce additional epigenetic and acute effects on cells that further influence sex disparities in cancer for example the increased frequency and aggressive phenotype of hepatocellular carcinoma in male individuals has been linked to the stimulatory effects of androgens in male individuals and the protective effects of oestrogens in female individuals61 importantly the biology of cancer is not the same across histological and genetic diagnostic groups or even within single histol ogical subtypes thus the interaction between sex gender and cancer mechanisms cannot be expected to be constant take colon cancer the second leading cause of cancerrelated death for example although women have a lower overall incidence of colon cancer than men they have a higher incidence of rightsided colon cancers which have the worst outcomes62 tumours from women with rightsided colon cancers exhibit a distinct molecular signature of energy metabolism compared with those of women with leftsided colon cancers63 this molecular signature is not observed when comparing tumours from men with rightsided colon cancers to men with leftsided colon cancers thus overall the male predisposition to cancer is probably the consequence of genetic program ming of male cells and the effect of sex hormones after puberty interacting with genderspecific behaviours to establish cancer risksresponse to treatmentin the future cancer prevention and treatment will be improved by sexspecific and genderspecific approaches for example immune checkpoint inhibitors can improve survival for men with advanced melanomas and nonsmallcell lung cancers more than for women64 the molecular subtyping of glioblastomas based on sexspecific transcriptomes has the potential to enhance chemotherapy in a sexspecific manner65 in colon cancer sex differences in xenobiotic metabolism regulatory networks might underlie greater treatment response in women than men and require modification of approaches for men with colon cancer66 other elements of cancer metabolism are also ripe for novel sexspecific targeting in treatment cellular nutrient partitioning is sexually dimorphic so approaches such as ketogenic diets or glutaminase inhibition might be associated with substantial sexspecific responses67 furthermore data from models of development ageing and cancer all indicate that molecular pathways such as the enzyme phosphatidylinositol 3kinase and tumour suppressors such as p53 and retinoblastoma protein are sexually dimorphic and require sexspecific targeting545968chronic pulmonary diseaseepidemiology pathogenesis manifestations and diagnosischronic pulmonary disease is the third leading cause of death for women · of deaths and the fourth for men · figure it is mostly accounted for by chronic obstructive pulmonary disease and to a lesser extent by asthma chronic obstructive pulmonary disease is characterised by irreversible airflow limitation and is associated with previous exposure to smoking or air pollutants women are overrepresented among individuals with chronic obstructive pulmonary disease especially among those with earlyonset disease or those who have never smoked69 women are also twice as likely to have chronic obstructive pulmonary disease with chronic bronchitis and women with severe chronic obstructive pulmonary disease have as much emphysema as men countering the misconception of emphysema as a male form of chronic obstructive pulmonary disease70 the female lung is more susceptible to chronic obstructive pulmonary disease than the male lung and women develop symptoms of the disease at a younger age with less tobacco exposure than men71 the genetic epidemiology of chronic obstructive pulmonary disease copdgene study72 suggests that earlyonset chronic obstructive pulmonary disease might originate in utero in susceptible women from alterations in lung development potentiated by maternal asthma and smoking genetic factors or hormonal influences future studies should focus on the contribution of maternally inherited factors such as mitochondrial and x chromosome genes to understand disease pathogenesis it is important to consider gender constructs as well smoking advertising campaigns targeting women rose in the 1960s and the resulting higher smoking rates influenced womens risk for developing chronic obstructive pulmonary disease73 from a disease severity vantage point chronic obstructive pulmonary disease exacerbation rates are also higher in women than men especially at a younger age74 additionally despite the burden of symptomatology and increased rates of hospitalisations and deaths women with chronic obstructive pulmonary disease are often misdiagnosed and disproportionally suffer from comorbid conditions including anxiety and depression therefore physicians should consider chronic obstructive pulmonary disease in the differential diagnosis of women with pulmonary symptoms regardless of tobacco or pollutant exposure historieswwwthelancetcom vol august review 0casthma characterised by variable airflow obstruction and chronic airway inflammation also affects men and women differently asthma is more prevalent in prepubertal boys than girls regarding asthma both male biological sex lung development and atopy and male gender constructs related to outdoor play and indoor pet exposure are factors contributing to the devel opment of asthma and sex versus gender contributions could be difficult to separate75 from puberty onwards more female than male individuals have asthma with an increased severity in middleaged women and a higher mortality rate76 this phe nomenon could be secondary to gender differences eg symptoms perception or healthseeking behaviours however biological sex plays a crucial role in asthma and sex hormones have a major impact on female asthma symptoms and severity after puberty75 worsening of asthma occurs in women before menstruation and is known as premenstrual asthma premenstrual asthma is more common in women with severe rather than mild asthma obesity rather than normal weight and a long rather than a short duration of asthma77 premenstrual asthma is hypoth esised to be due to a fall in progesterone and patients with a severe disease respond to progestogens78 during pregnancy approximately a third of asthmatic women exhibit a worsening of asthma a third show an improvement and the remainder are unaffected79response to treatmentthe menopausal transition represents a pivotal time of accelerated decline in lung function in women with chronic obstructive pulmonary disease and thus represents a sexspecific window for treatment intensification these observations also suggest that oestrogens protect from chronic obstructive pulmonary disease women exhibit greater expression of m2 over m3 muscarinic receptors and accordingly show greater improvements in lung function than men in response to the muscarinic anticholinergic bronchodilator ipatroprium80 pooled analyses of drug studies also suggest that women experience a greater improvement in quality of life than men after treatment of chronic obstructive pulmonary disease with a β2adrenoreceptor agonist combined with an anti cholinergic drug eg indacaterol and glycopyrronium81unlike chronic obstructiv | 0 |
Oral squamous cell carcinoma OSCC is a common kind of squamous cell carcinoma of the head and neck which is a threat to public health Long noncoding RNAs lncRNAs are associated with the development of various diseases including cancers LncRNA titin antisense RNA TTNAS1 is known as a crucial regulatory factor in several cancers Nevertheless the specific functions of TTNAS1 in OSCC remains obscureMethods The expression of TTNAS1 in OSCC samples or cells was analyzed through qRTPCR Colony formation assay EdU assay flow cytometry assay TUNEL assay and wound healing assay were conducted to estimate the functions of TTNAS1 in OSCC cells RIP and luciferase reporter assays were utilized to detect the interaction between TTNAS1 and miR3p as well as between miR3p and NFAT5Results TTNAS1 expression was stronger in OSCC cells Knockdown of TTNAS1 effectively restrained cell proliferation and migration but had inductive role in apoptosis Moreover TTNAS1 could function as the miR3p sponge in OSCC and miR3p exerted the inhibitory functions on OSCC cell growth In addition NFAT5 was proven as the target of miR3p Rescue assay indicated that overexpressing NFAT5 could reverse the inhibitory function of TTNAS1 depletion on cell growthConclusion lncRNA TTNAS1 contributed to the progression of OSCC via miR3pNFAT5 axisKeywords TTNAS1 miR3p NFAT5 Oral squamous cell carcinomaBackgroundOral squamous cell carcinoma OSCC is one of the commonest squamous cell carcinomas occurs in the head and neck It ranks sixth in occurrence and had a high mortality rate [ ] According to many years of investigation and research the pathogenesis of OSCC is related to the internal factors such as drinking and smoking but its specific pathogenesis is still unclear [ ] Although the surgery for OSCC is effective the situation for the overall survival of OSCC patients is still unfavorable [ ] Thus Correspondence fusuwei2009163comDepartment of Stomatology Henan Provincial Peoples Hospital Peoples Hospital of Zhengzhou University No7 Weiwu Road Zhengzhou Henan Chinaindepth study of the potential molecular mechanisms of OSCC is of great significance for developing new therapeutic strategiesLong noncoding RNAs lncRNAs are classified as the subgroup member of noncoding RNAs family with over nucleotides in length which are not able to encode proteins [ ] Recently lncRNAs are confirmed to involve in different cell progression such as cell proliferation and cell apoptosis Moreover the crucial functions of lncRNAs in the occurrence and development of assorted cancers have also been reported through a flow of researches [ ] Different kind of lncRNAs exerted different functions in cancers For example PVT1 accelerated esophageal carcinoma cell migration and invasion via sponging miR145 and regulating FSCN1 [] The Authors This is licensed under a Creative Commons Attribution International License which permits use sharing adaptation distribution and reproduction in any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate if changes were made The images or other third party material in this are included in the s Creative Commons licence unless indicated otherwise in a credit line to the material If material is not included in the s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtain permission directly from the copyright holder To view a copy of this licence visit httpcreat iveco mmons licen sesby40 The Creative Commons Public Domain Dedication waiver httpcreat iveco mmons publi cdoma inzero10 applies to the data made available in this unless otherwise stated in a credit line to the data 0cFu a0et a0al Cancer Cell Int Page of SARCC alters he androgen receptormiRNA1433p signals thereby suppresses the progression of renal cell carcinoma [] And GAPLINC facilitated gastric cancer cell growth through serving as a sponge of miR378 to regulate MAPK1 [] Titin antisense RNA TTNAS1 is a novel lncRNA that takes part in the regulation of cancer development in accordance with existing researches For illustration TTNAS1 with high expression in lung adenocarcinoma cells can expedite cellular functions of lung adenocarcinoma through serving as a sponge of miR1425p to regulate CDK5 [] Nevertheless its specific function of TTNAS1 in OSCC remains unclearHere we selected TTNAS1 as the object of our research and investigated the regulatory mechanisms and functions in OSCCMethodsTissues samplesPaired tissues adjacent normal and tumor were collected from patients with OSCC who were diagnosed at Henan Provincial Peoples Hospital Patients participated in this study didnt receive any kind of therapy before surgery All patients enrolled in this study had signed informed consent This study received the approval of the Ethics Committee of Henan Provincial Peoples Hospital Samples were stored at a0 °C until useCell linesHuman normal squamous epithelial cell line NOK obtained from Shanghai Honsun Biological Technology CoLtd Shanghai China human tongue squamous carcinoma cell lines including SCC4 SCC9 CAL27 procured from ATCC Manassas VA USA and BICR cell obtained from European Collection of Authenticated Cell Cultures ECACC UK were used in current study NOK cell was cultured in DMEM Gibco Rockville MD USA with antibiotics and FBS Gibco CAL27 cell was cultured in DMEM containing FBS SCC4 cell was cultured in DMEM F12 Medium containing a0ngml hydrocortisone and FBS SCC9 cell was cultured in DMEM F12 Medium containing a0mM a0lglutamine a0gL sodium bicarbonate a0mM HEPES a0 mM sodium pyruvate supplemented with a0 ngml hydrocortisone and FBS BICR16 cell was cultured in DMEM with 500ugml G418 and FBS Cell culture was conducted under a condition with CO2 and a0°CTotal RNA extraction and a0qRTPCRTRIzol Reagent Invitrogen Carlsbad CA was responsible for total RNA extraction from samples or cells Afterwards RNA samples were converted into cDNA Japan PowerUp¢ SYBR® Green Master Mix Life Techby employing Reverse Transcriptase Kit Takara Shiga nologies Grand Island NY USA was utilized for PCR analysis [] After amplification ÎÎCt method was applied to quantify PCR products U6 snRNA or GAPDH was used as the internal control for lncRNA mRNA or miRNA All primers used in this experiments were provided in Additional file a0 Table a0 S1 Each samples were assayed for more than triplicateTransfectionsThe shRNAs designed for TTNAS1 or NFAT5 and nonspecific shRNAs as well as pcDNA31NFAT5 and empty vector theses transfection plasmids were procured from GenePharma Shanghai China In addition the miR4113p mimicsinhibitor and NC mimicsinhibitor were procured from Genechem Shanghai China SCC4 and SCC9 cells were collected for a0h of plasmid transfections by use of Lipofectamine Invitrogen Sequence for all plasmids used in current study were listed in Additional file a0 Table a0 S1 Each samples were assayed for more than triplicateCCK assayAs previously described [] CCK8 Kit Beyotime Shanghai China was applied to detect cell viability under manufacturers protocols Cells cellswell were planted in 96well plates After and a0h the CCK8 reagents were added into each well Cell viability was detected using a microplate reader to measure the absorbance at the wave length of a0nm Each samples were assayed for more than triplicateColony formation assayAfter indicated transfections SCC4 and SCC9 cells were planted into 6well plates with cells in each well Following 14day of cell culture the resulting colonies were fixed using PFA for a0min stained using crystal violet solution for a0min and finally counted manually [] Each samples were assayed for more than triplicateEdU assayfor cell proliferation detection by use of BeyoClick¢ EdU assay was undertaken in cells of SCC4 and SCC9 EdU Cell Proliferation Kit Beyotime Shanghai China with Alexa Fluor [] The DAPI staining solution was acquired from Beyotime for detecting cell nucleus After washing in PBS cells were studied using inverted microscope Olympus Tokyo Japan Each samples were assayed for more than triplicate 0cFu a0et a0al Cancer Cell Int Page of Flow cytometryCell apoptosis of transfected SCC4 and SCC9 cells was assayed employing the flow cytometer BD Biosciences Franklin Lakes NJ in the presence of Annexin VPI double staining kit Invitrogen Cell samples were collected from 6well plates via centrifugation then stained in Binding Buffer and assayed with flow cytometry [] Each samples were assayed for more than triplicateTUNEL assayThe transfected cell samples of SCC4 and SCC9 were washed employing PBS and fixed using PFS for TUNEL assay [] in the presence of TUNEL assay reagent Merck KGaA Darmstadt Germany Following addition of DAPI staining solution cell samples were analyzed using optical microscopy Olympus Each samples were assayed for more than triplicateWound healingThe transfected cell samples of SCC4 and SCC9 were seeded in 6well plates and cultivated until confluence [] Then the artificial wounds were created with a0μL of pipette tip At and a0h after incubation in serumfree medium the distance of wound healing were imaged under microscope Olympus Each samples were assayed for more than triplicateSubcellular fractionationThe TTNAS1 content in cytoplasmic and nuclear fracPARIS¢ Kit Invitrogen as requested by provider Cell tions of SCC4 and SCC9 cells was studied by use of samples were lysed with cell fractionation buffer and cell disruption buffer then centrifuged for separating cell cytoplasm and cell nucleus [] For quantification GAPDH and U6 served as the cytoplasmic indicator and nuclear indicator respectively Each samples were assayed for more than triplicateFISHThe subcellular location of TTNAS1 in SCC4 and SCC9 cells was also studied with FISH assay using the deigned specifically TTNAS1probe Ribobio Guangzhou China After fixation the digested cells were airdried and cultured with probes in the hybridization buffer then treated in DAPI staining buffer [] Olympus fluorescence microscope was used for imaging Each samples were assayed for more than triplicateApplying the Magna RIP¢ RNABinding Protein Immunoprecipitation Kit [] RIP assay was conducted RNA immunoprecipitation RIPfor RNA interaction in SCC4 and SCC9 cells as guided by provider Millipore Bedford MA RIP lysis buffer Thermo Fisher Scientific Waltham MA USA was applied to obtain the lysates Lysis was incubated with the magnetic beads Invitrogen Carlsbad CA USA conjugated with antiAgo2 antibody or antiIgG antibody at a0 °C overnight Complex was washed and purified according to the protocol of RIP kit used in this experiment The enrichment of RNAs were examined via RTqPCR Each samples were assayed for more than triplicateLuciferase reporter assayTTNAS1 fragment covering wildtype or mutant miR4113p binding sites were employed to construct TTNAS1WT or TTNAS1Mut vectors by use of the pmirGLO dualluciferase vectors Promega Madison WI SCC4 and SCC9 cells were cotransfected with miR4113p mimics or NC mimics and TTNAS1WT or TTNAS1Mut vectors for a0h followed by analysis of dualluciferase reporter assay system Promega [] Renilla luciferase activity was used as the internal control Each samples were assayed for more than triplicateWestern blotCells were lysed via RIPA buffer BCA Protein Assay kit Pierce Biotechnology Rockford IL was used to assess the concentration of protein Separation of equal amount of proteins was conducted via SDSPAGE BioRad Laboratories Hercules CA followed by the transformation to PVDF membranes Millipore Bedford MA The membranes were blocked with skim milk and incubated with primary and secondary antibodies All antibodies were obtained from Abcam Cambridge MA USA Protein bands were detected using a ECL detection kit Pierce Biotechnology Rockford IL Each samples were assayed for more than triplicateAnimal studySix 4weekold BALBc nude mice Shanghai Laboratory Animal Center was subjected to animal study in line with the ethical standards and guidelines of Henan Provincial Peoples Hospital SCC6 cells à stably transfected with shNC or shTTNAS11 were injected into the right dorsal flanks of six mice Tumor sizes and volume were monitored by a caliper every a0days Four weeks later the mice were killed followed with the resection of tumors for measuring tumor weightStatistical analysesData of three or more independent assays were exhibited as the mean ± SD In addition Students ttest or onewaytwoway ANOVA followed by Tukey post hoc test 0cFu a0et a0al Cancer Cell Int Page of use of GraphPad Prism ® GraphPad Software Inc La was employed for comparing the group difference by Jolla CA USA Experimental data were collected when p ResultsKnockdown of a0TTNAS1 restrains the a0proliferation and a0migration of a0OSCC cellsAt first the relative higher level of TTNAS1 was observed in OSCC samples rather than adjacent normal ones Additional file a0 Fig S1A Next we detected the expression of TTNAS1 in OSCC cells through qRTPCR analysis We discovered that TTNAS1 expression was extremely high in OSCC cells in comparison of normal human squamous epithelial cell NOK cell Fig a01a At the same time we also found that TTNAS1 expression in SCC4 and SCC9 cells was highest Thus we knocked down TTNAS1 expression in SCC4 and SCC9 cells and identified that the TTNAS1 expression was exactly declined Fig a0 1b Following functional experiments were implemented to test the influence of inhibiting TTNAS1 on cells proliferation apoptosis and migration CCK8 assay unveiled that TTNAS1 depletion had significantly suppressive effect on cell viability Additional file a0 Fig S1B The number of colonies and EdU positive cells were reduced after silencing TTNAS1 indicating that cell proliferation could be restrained by TTNAS1 depletion Fig a01c d Then it was found by flow cytometry and TUNEL experiments that apoptosis was accelerated when decreased the level of TTNAS1 Fig a01e f Finally wound healing assay revealed that the migrated capability of SCC4 and SCC9 cells was hampered by silencing TTNAS1 Fig a0 1g In a word knockdown of TTNAS1 restrained cell proliferation and migration of OSCCTTNAS1 acts as a0miR3p sponge in a0OSCCThen we tested the distribution of TTNAS1 in SCC4 and SCC9 cells The results indicated that TTNAS1 tended to be located in the cytoplasm of SCC4 and SCC9 cells Fig a0 2a b indicating the potential posttranscriptional regulatory role of TTNAS1 in OSCC A flow of evidence suggested that lncRNA could serve as a ceRNA to regulate mRNAs through sponging miRNAs at posttranscriptional level [ ] Then we utilized starBase website to predict the possible miRNA which could have the binding site of TTNAS1 and one potential miRNA miR4113p was found out Fig a02c Then qRTPCR analysis was implemented to test the expression of miR4113p in OSCC samples and cells And the results indicated that miR4113p expression was lower in OSCC tissues and cells Additional file a0 Fig S1C and Fig a0 2d The lowest level of miR4113p was detected in SCC4 and SCC9 cells After that we discovered the binding site of miR4113p and TTNAS1 from starBase website Fig a02e and conducted Ago2RIP assay to evaluate the binding possibility of them We discovered that miR4113p and TTNAS1 were markedly enriched in antiAgo2 group Fig a02f and Additional file a0 Fig S1D which indicated that they were coexisted in RISC Following we overexpressed miR4113p and conducted the luciferase reporter assay We discovered that miR4113p overexpression caused a notable reduction on the luciferase activity of TTNAS1WT while the luciferase activity of TTNAS1Mut displayed no visible change Fig a02g h indicating that TTNAS1 could bind to miR4113p Overall TTNAS1 sponges miR4113p in OSCCUpregulation of a0miR3p represses OSCC cell growth and a0migrationIn order to search the role of miR4113p in OSCC functional experiments were implemented Firstly colony formation and EdU assays indicated that overexpressing miR4113p suppressed the proliferation of SCC4 and SCC9 cells Fig a0 3a b Moreover apoptosis of SCC4 and SCC9 cells was accelerated by miR4113p mimics through flow cytometry analysis and TUNEL assays Fig a03c d As illustrated in Fig a03e overexpression of miR4113p visibly reduced cell migration Taken together overexpression of miR4113p suppressed growth and migration in OSCCNFAT5 is a0the a0downstream target of a0miR3p in a0OSCCFor the sake of further verifying ceRNA hypothesis we searched the targets of miR4113p Combining the searching results from miRmap microT and PicTar databases candidate target genes were found under the condition Program number programs Fig a0 4a Then qRTPCR assay was applied to detect the influence of miR4113p overexpression and TTNAS1 inhibition on the levels of these mRNAs The results displayed a significant downregulation of mRNAs TLL2 MGAT4A RAB21 and NFAT5 when miR4113p was overexpressed and TTNAS1 was knocked down while other mRNAs were almost unchanged Fig a0 4b Then we tested the expressions of TLL2 MGAT4A RAB21 and NFAT5 in OSCC cells through qRTPCR for further detection We discovered that only NFAT5 displayed a high expression in OSCC cells Fig a04c High level of NFAT5 was further determined in OSCC tissues compared to adjacent normal ones Additional file a0 Fig S2A Thus we selected NFAT5 to conduct the further experiments Following we discovered the binding site of NFAT5 and miR4113p from starBase Fig a04d And RIP assays were implemented to evaluate the relationship of TTNAS1 NFAT5 and miR4113p The results 0cFu a0et a0al Cancer Cell Int Page of Fig Knockdown of TTNAS1 restrains the proliferation and migration of OSCC cells a The expression of TTNAS1 was tested through qRTPCR in OSCC cells b The interference efficiency of TTNAS1 was detected by qRTPCR in SCC and SCC cells c d Cell proliferation ability was measured by colony formation and EdU experiments when TTNAS1 was inhibited e f Cell apoptosis was evaluated through flow cytometry and TUNEL experiments after silencing TTNAS1 g Wound healing assays were utilized to estimate cell migration when TTNAS1 was subjected to knockdown P P 0cFu a0et a0al Cancer Cell Int Page of Fig TTNAS1 acts as the miR3p sponge in OSCC a b The cellular location of TTNAS1 was identified in SCC and SCC through Subcellular fractionation and FISH c StarBsae website was utilized to predict the possible miRNAs that could bind with TTNAS1 d MiR3p expression was detected through qRTPCR in OSCC cells e The binding site of TTNAS1 in miR3p f RIP assay was utilized to evaluate the relationship between miR3p and TTNAS1 g The efficiency of miR3p overexpression was tested through qRTPCR h Luciferase reporter assays were conducted to verify the correlation of miR3p and TTNAS1 P P showed that TTNAS1 NFAT5 and miR4113p were enriched in Ago2 indicating that TTNAS1miR4113pNFAT5 axis combined with RISC Fig a04e and Additional file a0 Fig S2B Then miR4113p was silenced and the interference efficiency was detected We could observe that miR4113p expression exactly declined after inhibition Fig a04f Following we detected the expression of NFAT5 when TTNAS1 and miR4113p were inhibited through qRTPCR Results indicated that NFAT5 expression could be hampered by TTNAS1 depletion but then recovered by miR4113p inhibition Fig a0 4g and Additional file a0 Fig S2C It demonstrated that NFAT5 and TTNAS1 were positively associated while NFAT5 and miR4113p were negatively correlated Then we investigated the function of NFAT5 in OSCC cells Firstly we knocked down the expression of NFAT5 in SCC4 and SCC9 cells and tested the knockdown efficiency Fig a04h and Additional file a0 Fig S2D NFAT5 expression could be hampered effectively after knockdown Then colony formation and EdU assays were carried out and the 0cFu a0et a0al Cancer Cell Int Page of Fig Upregulation of miR3p represses cell proliferation and migration in OSCC a b Cell proliferation was estimated through colony formation and EdU experiments when miR3p was overexpressed c d Flow cytometry and TUNEL experiments were implemented to measure cell apoptosis after overexpressing miR3p e Wound healing assays were adopted to test cell migration ability when miR3p was subjected to upregulation P See figure on next pageFig NFAT5 is a target gene of miR3p in OSCC a mRNAs which had the binding site with miR3p were predicted by starBase b The qRTPCR analysis was utilized to screen out the mRNAs which could be inhibited by NFAT5 depletion and miR3p overexpression c The expressions of TLL2 MGAT4A RAB21 and NFAT5 in SCC and SCC cells through qRTPCR d The binding site of NFAT5 and miR3p e RIP assay was adopted to test the relationship between TTNAS1 miR3p and NFAT5 f The interference efficiency of miR3p was tested by qRTPCR analysis g The expression of NFAT5 was detected when NFAT5 and miR3p was silenced h The interference efficiency of NFAT5 was tested by qRTPCR analysis i j Cell proliferation was evaluated through colony formation and EdU experiments when NFAT5 was knocked down k l Cell apoptosis was measured through flow cytometry and TUNEL experiments after inhibiting NFAT5 m Wound healing assays were carried out for estimating cell migration after NFAT5 was subjected to inhibition P 0cFu a0et a0al Cancer Cell Int Page of result indicated that silencing NFAT5 repressed the proliferation of SCC4 and SCC9 cells Fig a0 4i j Moreover cell apoptosis capability was expedited by NFAT5 depletion in flow cytometry and TUNEL assays Fig a04k l Finally wound healing assays indicated that silencing NFAT5 could hamper cell migration capability Fig a04m 0cFu a0et a0al Cancer Cell Int Page of Collectively NFAT5 was a target gene of miR4113p in OSCC and it accelerated the progression of OSCCTTNAS1 promotes OSCC progression via a0miR3pNFAT5 axisFor the sake of proving whether TTNAS1 could accelerate OSCC progression via miR4113pNFAT5 axis rescue assays were implemented Ahead of rescue assays qRTPCR was adopted to test the overexpression efficiency of NFAT5 in SCC4 and SCC9 cells The results displayed that NFAT5 expression was visibly increased after transfecting with pcDNA31NFAT5 Fig a05a Next we detected the mRNA and protein levels of NFAT5 in SCC4 and SCC9 cells after transfection It was uncovered that NFAT5 levels decreased by TTNAS1 depletion were rescued by the inhibition of miR4113p or the upregulation of NFAT5 Additional file a0 Fig S2E Then colony formation and EdU rescue assays were conducted we discovered that cell proliferation was hampered by TTNAS1 depletion but then it was recovered by NFAT5 overexpression or miR4113p inhibition Fig a0 5b c Through flow cytometry and TUNEL assays we found that knockdown of miR4113p or upregulation NFAT5 could reverse the cell apoptosis ability which was accelerated by TTNAS1 depletion Fig a05d e In the end it was indicated through wound healing assay that the inhibited cell migration caused by knockdown of TTNAS1 was restored by NFAT5 overexpression or miR4113p inhibition Fig a05f Thus we confirmed that TTNAS1 promoted OSCC cell growth and migration by miR4113pNFAT5 axisTTNAS1 promoted OSCC cell growth in a0vivoIn vivo study was conducted to support above in a0 vitro findings We observed that tumor size volume and weight in shNC group were all smaller than those in shTTNAS11 group Fig a06ac Importantly IHC staining indicated that silencing of TTNAS1 caused a reduction in the positivity of Ki67 and PCNA Fig a06d All these experiments unveiled that TTNAS1 promotes OSCC progression via miR4113pNFAT5 axisDiscussionOral squamous cell carcinoma OSCC is a common squamous cell carcinoma of the head and neck It has a relatively high incidence worldwide As the regulatory functions of lncRNA in assorted cancers are constantly being explored lots of lncRNAs have also been confirmed to play a crucial role in promoting the development of OSCC For example PLAC2 could promote cell growth through activating wntβcatenin pathway in OSCC [] CEBPAAS1 was considered to correlate with the bad prognosis and it also could facilitate tumorigenesis through CEBPABcl2 in OSCC [] Moreover P4713 was reported to contribute to the malignant phenotypes of OSCC through activating the JAKSTAT3 pathway [] In our research we investigated the functions of TTNAS1 in OSCC TTNAS1 was a novel lncRNA and it served as the oncogene in lung adenocarcinoma [] In this study TTNAS1 was discovered to be highly expressed in OSCC cells And TTNAS1 depletion impaired cell proliferation and migration but it accelerated cell apoptosis in OSCC Overall TTNAS1 exerted the carcinogenic effect in OSCCMiRNAs are small RNAs with nucleotides in length without ability of coding protein [] In recent years an increasing number of evidences discovered that lncRNA could function as a crucial element of competing endogenous RNA ceRNA network by sponging miRNA to regulate mRNA so as to take part in the regulation of cancer progression [ ] For example lncRNA ATB functioned as a ceRNA to expedite YAP1 through sponging miR5905p in malignant melanoma [] PAGBC acted as a sponge of miR133b and miR and accelerated gallbladder tumorigenesis [] AFAP1AS1 could act as a ceRNA of miR4235p to expedite nasopharyngeal carcinoma progression [] In our research we utilized bioinformatics tools to find the possible miRNA which could bind to TTNAS1 After screening miR4113p was selected With the conduction of RIP and luciferase experiments we proved that TTNAS1 could act as ceRNA to sponge miR4113p in OSCC MiR4113p was verified as the tumor suppressor gene in ovarian cancer and it could restrain cell proliferation migration and invasion of ovarian cancer [] Thus we investigated the functions of miR4113p in OSCC As we expected miR4113p could repress cell proliferation and migration but accelerate cell apoptosis in OSCC In short our research confirmed that TTNAS1 sponged miR4113p and overexpressing miR4113p could repress the progression of OSCCNFAT5 is a mRNA and it has been reported to be associated with several cancers For example NFAT5 was proved to conduce to the glycolytic phenotype rewiring and pancreatic cancer progression through transcription of PGK1 [] Moreover NFAT5 cpuld also promote glioblastoma celldriven angiogenesis through EGFL7 which was mediated via SBF2AS1 and miR3383p [] In our research we discovered that NFAT5 was highly expressed in OSCC cells And based on the mechanism experiments we also proved that NFAT5 was the target of miR4113p and overexpressing it could accelerate the progression of OSCC Rescue experiment indicated that upregulation of NFAT5 could offset TTNAS1 knockdownmediated functions on the progression of OSCC 0cFu a0et a0al Cancer Cell Int Page of Fig TTNAS1 promotes OSCC progression via miR3pNFAT5 axis a The qRTPCR analysis was utilized to examine the overexpression efficiency of NFAT5 in SCC and SCC cells b c Cell proliferation capability in SCC and SCC cells was measured by colony formation and EdU assay in different groups d e Cell apoptosis was tested through flow cytometry and TUNEL assays in different groups f Wound healing assays were implemented to detect the cell migration ability in different groups P 0cFu a0et a0al Cancer Cell Int Page of Fig TTNAS1 promoted OSCC cell growth in vivo a Tumors removed from the mice injected with shNCtransfected cells or shTTNAS11transfected cells b c Volume and weight in different groups were measured d IHC staining of tumor tissues collected from different groups with antiKi and antiPCNA P proving the functions of TTNAS1miR4113pNFAT5 axis in OSCCtransfected with shTTNAS11 was examined by qRTPCR and western blot analyses after cotransfection with miR3p inhibitor or pcDNA31NFAT5 P ConclusionTaken together TTNAS1 could contribute to the progression of OSCC via miR4113pNFAT5 axis which may provide the new idea for the exploration of OSCC treatmentsSupplementary informationSupplementary information accompanies this paper at https doi101186s1293 Additional file a0 Sequence for all plasmids used in current studyAdditional file a0 Figure S1 A TTNAS1 expression in adjacent normal and tumor tissues was examined by qRTPCR analysis B CCK assay was applied to analyze the viability of SCC and SCC cells transfected with shNC shTTNAS11 or shTTNAS12 C The level of miR3p was assessed in pairs of OSCC tissues and adjacent normal tissues D Agarose gel electrophoresis for the Ago2RIP assay in Fig 2F P Additional file a0 Figure S2 A NFAT5 expression in paired tissues obtained from OSCC patients B Agarose gel electrophoresis for the Ago2RIP assay in Fig 4E C Protein level of NFAT5 in cells transfected with shNC shTTNAS11 or cotransfected with shTTNAS11 and miR3p inhibitor D Protein level of NFAT5 in cells transfected with shNC shNFAT51 and shNFAT52 E mRNA and protein level of NFAT5 in cells AbbreviationsOSCC Oral squamous cell carcinoma TTNAS1 Titin antisense RNA lncRNAs Long noncoding RNAs ceRNAs Competing endogenous RNAs miRNAs microRNAs mRNA Messenger RNA ATCC American type culture collection DMEM Dulbeccos modified Eagles medium FBS Fetal bovine serum RIPA Radioimmunoprecipitation assay SDSPAGE Sulphatepolyacrylamide gel electrophoresis PVDF Polyvinylidene fluoride RTqPCR RNA extraction and quantitative realtime polymerase chain reaction HRP Horseradish peroxidase FISH Fluorescence in situ hybridization WT Wildtype Mut Mutant SD Standard deviation ANOVA Analysis of varianceAcknowledgementsWe appreciate all the people involved in this studyAuthors contributionSF project administration study design and review experiments YZ SL and ZS methods investigation data JZ and QH preparation draft manuscript All authors read and approved the final manuscriptFundingNoneAvailability of data and materialsNot applicable 0cFu a0et a0al Cancer Cell Int Page of Ethics approval and consent to participateAll patients enrolled in this study had signed informed consent This study received the approval of the Ethics Committee of Henan Provincial Peoples HospitalConsent for publicationAuthors confirmed that this work can be published The content of this manuscript is original and it has not yet been accepted or published elsewhereCompeting interestsNo competing interest existReceived February Accepted June References Krishna Rao SV Mejia G RobertsThomson K Logan R Epidemiology of oral cancer in Asia in the past decadean update Asian Pac J Cancer Prev APJCP Siegel RL Miller KD Jemal A Cancer statistics CA Cancer J Clin Warnakulasuriya S Global epidemiology of oral and oropharyngeal cancer Oral Oncol Sacco AG Cohen EE Current treatment options for recurrent or metastatic head and neck squa | 2 |
"Tumor microenvironment TME plays an important role in malignant tumors Our study aimed toinvestigate the effect of the TME and related genes in osteosarcoma patientsMethods Gene expression profiles and clinical data of osteosarcoma patients were downloaded from the TARGETdataset ESTIMATE algorithm was used to quantify the immune score Then the association between immune scoreand prognosis was studied Afterward a differential analysis was performed based on the high and lowimmunescores to determine TMErelated genes Additionally Cox analyses were performed to construct two prognosticsignatures for overall survival OS and diseasefree survival DFS respectively Two datasets obtained from the GEOdatabase were used to validate signaturesResults Eightyfive patients were included in our research The survival analysis indicated that patients with higherimmune score have a favorable OS and DFS Moreover genes were determined as TMErelated genes Theunsupervised clustering analysis revealed two clusters were significantly related to immune score and T cells CD4memory fraction In addition two signatures were generated based on three and two TMErelated genesrespectively Both two signatures can significantly divide patients into low and highrisk groups and were validatedin two GEO datasets Afterward the risk score and metastatic status were identified as independent prognosticfactors for both OS and DFS and two nomograms were generated The Cindexes of OS nomogram and DFSnomogram were and respectivelyConclusion TME was associated with the prognosis of osteosarcoma patients Prognostic models based on TMErelated genes can effectively predict OS and DFS of osteosarcoma patientsKeywords Tumor microenvironment Osteosarcoma Prognosis Immune features NomogramBackgroundOsteosarcoma is the most common bone tumor especiallyin children and adolescents [] It was reported that approximately of patients are between and yearsold and osteosarcoma is considered as the second leadingcause of death in this age group [] Currently surgery and Correspondence 407404159qqcom4Wenzhou Medical University Wenzhou ChinaFull list of author information is available at the end of the chemotherapy are still major treatments for osteosarcomapatients and these therapies are constantly improving inrecent years However due to the susceptibility of localaggressiveness and lung metastasis in osteosarcoma patients the prognosis of osteosarcoma remains unfavorable[] Previous studies indicated that the 5years survivalrates were and in metastatic and nonmetastaticpatients respectively [] Thereforeit is necessary to The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cHu BMC Cancer Page of investigate the mechanism of pathogenesis and progressionof osteosarcoma and accurately classify the risk of patientsRecently an increasing number of diagnostic and prognostic biomarkers of osteosarcoma patients have beenidentified For example Chen [] reported that tumorsuppressor p27 is a novel biomarker for the metastasis andsurvival status in osteosarcoma patients Moreover Huang [] discovered that dysregulated circRNAs serve asprognostic and diagnostic biomarkers in osteosarcomapatients and the relative potential mechanism mainly attributes to the regulation of downstream signaling pathwaysby sponging microRNA In addition lncRNA [] microRNA [] and many clinical data [] were also identified asprognostic biomarkers for osteosarcoma patients However osteosarcoma is one of the malignant cancers entitiescharacterized by the high level of heterogeneity in humansTherefore it is necessary to find accurate biomarkers forosteosarcomaIn recent years researchers have paid more and moreattention to the role of the tumor microenvironmentTME in malignant tumors The function of TME inthe tumorigenesis progression and therapy of tumorshave been initially understood [ ] More importantly Estimation of STromal and Immune cells in MAlignant Tumor tissues using Expression data ESTIMATE an algorithm to quantify the score of immune cellsand stromal cells by analyzing the gene expression datawas developed in [] Based on the algorithm theprognostic value of immune and stromal cells in bladdercancer acute myeloid leukemia gastric cancer cervicalsquamous cell carcinoma adrenocortical carcinomaclear cell renal cell carcinoma hepatocellular carcinomathyroid cancer and cutaneous melanoma have beenreported [] Generally the above research indicatedthat TME can serve as the prognostic biomarker in tumorsand many TMErelated genes were determined as the prognostic genes However the role of TME and TMErelatedgenes in osteosarcoma patients remains unclearIn the present study gene expression data and corresponding clinicopathologic data were obtained from TheTherapeutically Applicable Research to Generate EffectiveTreatments TARGET dataset Then the ESTIMATEalgorithm was performed to quantify the immune score ofosteosarcoma and the TMErelated genes were identifiedby the differential expression analysis Subsequently theprognostic value of TME and TMErelated genes weredetermined by a series of bioinformatics methodsMethodsGene expression datasetsLevel data of gene expression profiles and correspondingclinical data of osteosarcoma patients were downloadedfrom TARGET dataset ocgcancergovprogramstarget accessed on Oct The correspondingclinicopathologic data included in the present study wereage gender race ethnicity tumor site and metastaticstatus After data were extracted from the public domainthe ESTIMATE an algorithm inferring tumor puritystromal score and immune cell admixture from expression data was performed to evaluate the immune score byusing the estimate package in R software version [] Meanwhile the messenger RNAmRNA expressionprofiles and clinical data ofincludingGSE21257 [] and GSE39055 [] were obtained fromthe Gene Expression Omnibus as external validationcohortstwo cohortsSurvival analysis and correlation analysisAfter scores were obtained patients were divided intohighscore group and lowscore group according to themedian of the immune score The KaplanMeier survivalanalysis with logrank test was performed to estimatethe differences of overall survival OS and diseasefreesurvival DFS between high and lowscore cohorts Inaddition the association between clinicopathologic dataand TME score was also studied MannWhitney signedrank test was performed to compare the differences ofimmune score between each clinical group All statisticalanalyses in the present study were performed using Rsoftware Except for the special instructions p value twoside was identified as statistically significantin the present studyDEGexpressed geneDifferentially expressed gene analysisDifferentiallyanalysis wasperformed by comparing the proteincoding genesexpression between the lowimmune score group andthe highimmune score group The limma package in Rsoftware was used to perform the differential analysisand genes with log FC and adjusted pvalue qvalue were identified as DEGs []To further understand the function of DEGs identifiedin the present study Gene Ontology GOincludingbiological processes BP molecular functions MF andcellular componentsCC and Kyoto Encyclopedia ofGenes and Genomes KEGG analysis were performedby clusterProfiler package in R software []Evaluation of association with immune cellsTo further investigate the association between DEGs andimmune cells the CIBERSORT package was used toestimate the relative proportions of types of immunecells [] Meanwhile the ConsensusClusterPlus package was used to cluster in an unbiased and unsupervisedmanner based on the overlapping DEGs [] Cumulative distribution function CDF and relative change inarea under the CDF curve were used to determine theoptimal number of clusters k Then MannWhitney 0cHu BMC Cancer Page of signedrank test was performed to study the differenceof immune cells proportion between the clusters and theviolin plot was established to show the differences ofimmune cells among clusters []Survival analysis of DEGsBased on the DEGs the univariate COX analysis was performed to determine the prognostic value of immunerelated genes Then the OSrelated genes were validatedin the GSE21257 dataset while the DFSrelated geneswere validated in the GSE39055 dataset Only genes successfully validated were selected for further analysis Afterward based on the validated genes the multivariate COXanalysis was performed to establish the prognostic signature for predicting the prognosis of osteosarcoma patientsThe risk score for each patient was calculated based onthe coefficient from the multivariate COX analysis and thecorresponding gene expression Meanwhile all patientswere divided into the high and lowrisk groups accordingto the median of the risk score The survival receiver operating characteristic ROC curve was used to show the discrimination of signatures and the KaplanMeier survivalcurve with the logrank test was generated to show thedifferences of OS and DFS between high and lowriskgroups In addition the risk score of patients in the validation cohort was also calculated according to the aforementioned risk signature The KaplanMeier survivalcurve and survival ROC curve were generated to show thepredictive ability of the signature in the validation cohortDevelopment of a nomogram for osteosarcoma patientsNomogram is a tool to visualize the predictive model andconvenient for clinical practice Therefore we attemptedto develop a nomogram based on the TMErelated genessignature and clinicopathologic data to predict the prognosis of osteosarcoma patients Firstlythe univariateCOX analysis was performed to filter prognostic variableswhich will be further included in the multivariate COXanalysis Secondly based on independent prognostic variables two nomograms were established for predicting theOS and DFS respectively The Cindex was used to assessthe discriminatory performance of the nomogram whichrange from to [] A Cindex of means agreement by chance and a Cindex of represents perfectdiscriminatory performance The higher value of the Cindex the better performance of the nomogram is Furthermore the calibration curves of and 3year weredeveloped to evaluate the effectiveness of nomogramsResultsImmune significantly associated with the prognosis ofosteosarcoma patients osteosarcoma patients were included in the presentstudy including males and females The immunescore of the cohort range from to Tostudy the relationship between the immune score and theprognosis of osteosarcoma patients patients wereincorporated into the lowimmune score group while theremaining patients were incorporated into the highimmune score group The survival analysis indicated thatpatients with higher immune score had a favorable OSand DFS Fig 1a and b After adjusted age tumor siteand metastatic status the immune score still was a prognostic variable for both OS and DFSFig 1a and b Inaddition the relationship between immune score and clinical features was also investigated However there was nosignificant relationship between immune score and clinicalvariables Supplementary Figure 1A1CDifferential expression analysisAccording to the median of the immune score patients were divided into highscore n and lowFig Association between immune score and prognosis in osteosarcoma patients a KaplanMeier survival analysis of overall survival for patientswith high vs low immune score b KaplanMeier survival analysis of diseasefree survival for patients with high vs low immune score 0cHu BMC Cancer Page of score group n There were differentiallyexpressed genes between two groups which include upregulated genes and downregulated genesFig 2a b and Supplementary Table To furtherunderstand the function of DEGs GO analysisand KEGG analysis were performed The top significant results of GO analysis among three types wereillustrated in Fig 2c Interestingly we can find that theresults of GO analysis are mostly associated with immunity which further verify that the immunerelated DEGsare associated with immune features In addition the results of KEGG also confirmed it Such as PhagosomeAutoimmune thyroid disease Antigen processing andpresentation B cell receptor signaling pathway Intestinal immune network for IgA production Inflammatorybowel disease Primary immunodeficiency Th1 andTh2 cell differentiation Th17 cell differentiation Natural killer cell mediated cytotoxicity and NFkappa Bsignaling pathway Fig 2dconsensusunsupervisedEvaluation of DEGs and immune cellsTo further understand the molecular heterogeneity ofosteosarcomaanalysis wasperformed to divide patients into subgroups to explorewhether immunerelated genes presented discernable patterns Based on the consensus matrix heat map patientswere clearly divided into two clustersFig 3a In additionby comprehensively analyzing the relative change in areaunder the cumulative distribution function two clusterswere determined Fig 3bc The immune score betweentwo clusters was significantly different Fig 3d In additionthe proportion of types of immune cells in osteosarcomapatients was illustrated in a barplot Fig 3e Interestinglywe can see that the T cells CD4 memory activated ofcluster is significantly higher than cluster Fig 5fPrognostic value of TMErelated genesPrevious studies indicated that TMErelated genes canserve as the prognostic biomarker for tumor patientsFig Differentially expressed genes with the immune score in osteosarcoma patients a Heatmap of significantly differentially expressed genesbased on immune score b The volcano figure to show the upregulated and downregulated genes c GO analysis of differentially expressedgenes d KEGG of differentially expressed genes GO Gene Ontology KEGG Kyoto Encyclopedia of Genes and Genomes 0cHu BMC Cancer Page of Fig The immune landscape of the tumor microenvironment ac Unsupervised clustering of all samples based on the overlapping DEGs dComparison of immune score between two clusters e The distribution of types of immune cells in osteosarcoma patients f The comparisonof types of immune cells between clusters DEG Differentially expressed geneHence we performed the univariate COX analysis toidentify prognostic DEGs The results showed that and genes were identified as OS and DFSrelatedDEGs respectively Supplementary Table and Afterward five OSrelated genes were successfully validated inthe GSE21257 data set and five DFSrelated genes were successfully validated in the GSE39055 cohort Furthermoremultivariate COX analysis was performed and two prognostic signatures were generated for predicting the OS andDFS respectively The risk score for predicting the OS wasasrisk score FCGR2B0766 GFAP0702 MPP70387 In addition the risk score for predicting theDFS was as follows risk score CYP2S10574 ICAM3 The AUC values of OSrelated signature were follows 0cHu BMC Cancer Page of and in and 3year respectively Fig 4aand the AUC values of DFSrelated signature were and in and 3year respectively Fig 5aMoreover survival curves showed that patients in the highrisk group had worse OS and DFS compared with the lowrisk patients Figs 4b and 5b Heat maps risk score plotsand survival status were generated to show the distinctionbetween highrisk patients and lowrisk patients Figs 4ceand 5ce Then both signatures were validated in independent cohorts For OS signature the AUC values ofvalidation cohort were and at and3year Fig 4f For DFS signature the AUC values ofvalidation cohort were and at and3year Fig 5f Additionallyin both validation cohortssurvival curves showed that lowrisk patients were favorableprognosis than highrisk patients Figs 4g and 5gHeat maps risk score plots and survival status of validation cohorts were also generated to show the distinction between highrisk patients and lowrisk patientsFigs 4hj and f 5hjDevelopment of a nomogram for osteosarcoma patientsTo generate a nomogram for clinical use the COX analysiswas performed to select the clinical prognostic variables InFig Establishment and validation of the prognostic model for overall survival based on significant DEGs a Receiver operating characteristiccurves of prognostic signature in the training cohort b The survival curve showed the different overall survival status between high and lowriskpatients c The heat map showed the expression of prognostic genes in the training cohort d The risk curve of each sample reordered by riskscore e The scatter plot showed the overall survival status of osteosarcoma patients in the training cohort f Receiver operating characteristiccurves of prognostic signature in validation cohort g The survival curve showed the different overall survival status between high and lowriskpatients h The heat map showed the expression of prognostic genes in the validation cohort i The risk curve of each sample reordered by riskscore j The scatter plot showed the overall survival status of osteosarcoma patients in the validation cohort 0cHu BMC Cancer Page of Fig Establishment and validation of the prognostic model for diseasefree survival based on significant DEGs a Receiver operatingcharacteristic curves of prognostic signature in the training cohort b The survival curve showed the different diseasefree status between highand lowrisk patients c The heat map showed the expression of prognostic genes in the training cohort d The risk curve of each samplereordered by risk score e The scatter plot showed the diseasefree status of osteosarcoma patients in the training cohort f Receiver operatingcharacteristic curves of prognostic signature in validation cohort g The survival curve showed the different diseasefree status between high andlowrisk patients h The heat map showed the expression of prognostic genes in the validation cohort i The risk curve of each sample reorderedby risk score j The scatter plot showed the diseasefree status of osteosarcoma patients in the validation cohortthe univariate COX analysis risk score and metastatic status were identified as both OS and DFSrelated variablesFig 6a and e Afterward risk score and metastatic statuswere determined as both independent OS and DFSrelated variables in the multivariate COX analysis Fig 6band f Based on independent variables two nomogramswere established for predicting the OS and DFS in osteosarcoma patients respectively Fig 6c and g The Cindexvalues were and in OS nomogram and DFSnomogram respectively The results of Cindex mean thatboth two nomograms have good discrimination Meanwhile to evaluate the calibration of nomograms six calibration curves were generated and the results showed thatthe predictive curves were close to the ideal curve Fig 6dand h which indicated a good calibrationDiscussionThe relationship between TME and tumor have beenwidely studied in recent years In the present study ESTIMATE algorithm was utilized to quantify the immunescore based on gene expression profiles in osteosarcomapatients from TARGET database We confirmed that theTME is significantly associated with the prognosis ofosteosarcoma patientsInadditionfunctional enrichment analyses of TMErelated genes indicated that immunerelated processesincluding OS and DFS 0cHu BMC Cancer Page of Fig Nomograms based on the tumor microenvironment related genes for osteosarcoma patients a Univariate COX analysis of overall survivalrelated variables b Multivariate COX analysis of overall survivalrelated variables c Nomogram for predicting the overall survival in osteosarcomapatients d1 and 3year calibration curveS of overall survival nomogram e Univariate COX analysis of diseasefree survivalrelated variables fMultivariate COX analysis of diseasefree survivalrelated variables g Nomogram for predicting the diseasefree survival in osteosarcoma patientsh1 and 3year calibration curveS of diseasefree survival nomogramknown to contribute to tumor progression More importantly DEGs based on the TME were identified asimportant prognostic biomarkers for osteosarcoma patients and two nomograms were developed for predicting the OS and DFS of osteosarcoma patientsrespectivelyIn recent years an increasing number of studiesfocused on the carcinogenesis and progression of tumorsbased on the TME and the ESTIMATE algorithm is oneof the most important quantitative tools for this researchfield Based on the ESTIMATE algorithm the association between the prognosis and TME has been initially 0cHu BMC Cancer Page of elucidated in some tumors such as cervical squamouscell carcinoma gastric cancer cutaneous melanomaacute myeloid leukemia bladder cancer and clear cellrenal carcinoma [ ] However previousstudies indicated that TME scores serve as a differentrole in different tumors For example for hepatocellularcarcinoma gastric cancer acute myeloid leukemiabladder cancer and clear cell renal carcinoma patientswith high immune score have a worse prognosis [ ] However for cervical squamous cell carcinoma adrenocortical carcinoma and cutaneous melanoma patients with high immune score have a favorableprognosis [ ] Therefore we can find great heterogeneity among different tumors from the perspectiveof TME For osteosarcoma patients the present studyindicated that patients with higher immune score had abetter OS and DFS Hence the present study indicatedthat immune cells infiltrating tumor tissue may play animportant role in suppressing tumor progressionIn our research TMErelated genes were identified by comparing the highscore and lowscore osteosarcoma patients The functional enrichment includingGO and KEGG analyses showed that TMErelated geneswere mainly involved in the immune features such asregulation of leukocyte activation MHC protein complex MHC protein and complex binding More importantly the unsupervised cluster analysis based on DEGswas performed and all patients were divided into twoclusters Immune score and T cell CD4 memory activated fraction were significant difference between twoclusters which further elucidated the relationship between DEGs and immune featuresDue to the poor prognosis of osteosarcoma patientsidentifying robust prognostic biomarker is very importantThe tumor immune microenvironment is closely relatedto the prognosis of bone tumor patients Emilie etal []performed the first genomewide study to describe therole of immune cells in osteosarcoma and found thattumorassociated macrophages are associated with reduced metastasis and improved survivalin highgradeosteosarcoma Recently the prognostic signature based onTMErelated genes have been established for many tumors [ ] but only one study focused on osteosarcoma patients [] Compared with the study performedby Zhang [] we think that our research have someadvantages Firstly our signatures were established basedon several validated genes and both two signatures weresuccessfully validated in independent cohorts Secondlythe outcome of DFS was not reported in the previousstudy As reported in published studies tumor recurrenceis a terrible medical problem for osteosarcoma patientsand the 5year survival rate for osteosarcoma patients withmetastasis or relapse remains disappointing [ ]Hence the DFS nomogram can improve the managementof osteosarcoma patients Finally two nomograms incorporated TMErelated signature and clinical variables wereestablished in our research which further facilitated theclinical application of our findingsIn our research five genes were incorporated into thefinal prognostic signatures FCGR2B GFAP and MPP7were identified and validated as OSrelated biomarkerswhile CYP2S1 and ICAM3 were DFSrelated biomarkersThe role of these genes in tumor prognosis had beenwidely reported in previous studies [] FCGR2Bhas been confirmed as an immunerelated gene previously [] Although the relationship between FCGR2Band prognosis in sarcoma patients had not been reported the prognostic value of FCGR2B had been widelyconfirmed in other cancerssuch as hepatocellularcarcinoma and glioblastoma [ ] In addition NewM etal [] demonstrated that MPP7 is novel regulatorsof autophagy which was thought to be responsible forthe prognosis of pancreatic ductal adenocarcinomaCYP2S1 described as Cytochrome P450 Family Subfamily S Member was reported significantly associatedwith colorectal cancer In primary colorectal cancerCYP2S1 was present at a significantly higher level ofintensity compared with normal colon [] More importantly the presence of strong CYP2S1 immunoreactivity was associated with poor prognosis [] The roleof ICAM3 in cancer was also widely reported in published studies and the Akt pathway plays an importantrole in the impact of ICAM3 on tumors YG Kim etal[] reported that ICAM3 can induce the proliferationof cancer cells through the PI3KAkt pathway Additionally JK Park etal showed that the ICAM3 can enhancethe migratory and invasive potential of human nonsmall celllung cancer cells by inducing MMP2 andMMP9 via Akt pathway [] showed that the ICAM3can enhance the migratory and invasive potential ofhuman nonsmall celllung cancer cells by inducingMMP2 and MMP9 via Akt pathwayAlthough the role of TME and TMErelated genes inosteosarcoma patients have been initially studied by bioinformatic and statistical analyses in our research somelimitations should be elucidated Firstly the treatmentinformation cannot be obtained from the TARGET database which may influence the prognosis of osteosarcomapatients Secondly two nomograms were generated andshowed good performance in our study However externalvalidation by a large cohort is needed Thirdly many independent prognostic genes for osteosarcoma patients wereidentified in the present study but the potential mechanism to influence osteosarcoma remains unclear Finally inthe training cohort and DEGs were identified asOS and DFSrelated DEGs respectively However onlyfive OS and five DFSrelated genes were identified in thevalidation cohort The different age structures smaller 0cHu BMC Cancer Page of sample sizes and the platform covering only part of thegenes may contribute to this resultReceived February Accepted July ConclusionIn conclusion TME plays an important role in osteosarcoma patients and related with the progression of thetumor Moreover TMErelated genes can serve as prognostic biomarkers in osteosarcoma patients Howeverfurther researches are needed to study the potentialmechanism and validate the nomogram that developedin our present studySupplementary informationSupplementary information accompanies this paper at doi101186s12885020072162Additional file Additional file Additional file Additional file AbbreviationsTME Tumor microenvironment DEG Differentially expressed genesOS Overall survival DFS Diseasesfree survival ROC Receiver characteristiccurve ESTIMATE Estimation of STromal and Immune cells in MAlignantTumor tissues using Expression data TARGET Therapeutically ApplicableResearch to Generate Effective Treatments GO Gene Ontology BP Biologicalprocesses MF Molecular functions CC Cellular components KEGG KyotoEncyclopedia of Genes and Genomes CDF Cumulative distribution functionAcknowledgementsNoneAuthors contributionsC H L Y Sq T C L and Yh W conceived of and designed the study C H R Sand C L performed literature search R S L Y and B C generated the figuresand tables L Y Hl R X Y and Jy L analyzed the data C H wrote themanuscript and Sq T and L Y critically reviewed the manuscript L Ysupervised the research All authors have read and approved the manuscriptFundingWe received no external funding for this studyAvailability of data and materialsThe data of this study are from TARGET and GEO databaseEthics approval and consent to participateThe research didnt involve animal experiments and human specimens noethics related issuesConsent for publicationNot applicableCompeting interestsThe authors declare that they have no competing interestsAuthor details1Department of Joint Surgery the Affiliated Hospital of Qingdao UniversityQingdao China 2Department of Medical Oncology the First Hospital ofChina Medical University Shenyang China 3Department of Nursing Sir RunRun Shaw Hospital Affiliated to Zhejiang University Hangzhou China4Wenzhou Medical University Wenzhou ChinaReferencesJaffe N Bruland OS Bielack S Pediatric and adolescent osteosarcoma vol New York Springer Science Business Media Vander RG Osteosarcoma and its variants Orthopedic Clin North Am Biermann JS Adkins D Benjamin R Brigman B Chow W Conrad EU 3rdFrassica D Frassica FJ Gee S Healey JH Bone cancer J Natl ComprCancer Netw Simpson S Dunning MD de Brot S GrauRoma L Mongan NP Rutland CSComparative review of human and canine osteosarcoma morphologyepidemiology prognosis treatment and genetics Acta Vet Scand Chen X Cates JM Du YC Jain A Jung SY Li XN Hicks JM Man TKMislocalized cytoplasmic p27 activates PAK1mediated metastasis and is aprognostic factor in osteosarcoma Mol Oncol Huang X Yang W Zhang Z Shao Z Dysregulated circRNAs serve as prognosticand diagnostic markers in osteosarcoma by sponging microRNA to regulatethe downstream signaling pathway J Cell Biochem Liu M Yang P Mao G Deng J Peng G Ning X Yang H Sun H Long noncoding RNA MALAT1 as a valuable biomarker for prognosis in osteosarcoma asystematic review and metaanalysis Int J Surg Xu K Xiong W Zhao S Wang B MicroRNA106b serves as a prognosticbiomarker and is associated with cell proliferation migration and invasionin osteosarcoma Oncol Lett Zheng W Huang Y Chen H Wang N Xiao W Liang Y Jiang X Su W WenS Nomogram application to predict overall and cancerspecific survival inosteosarcoma Cancer Manag Res Kahlert C Kalluri R Exosomes in tumor microenvironment influence cancerprogression and metastasis J Mol Med Binnewies M Roberts EW Kersten K Chan V Fearon DF Merad M CoussensLM Gabrilovich DI OstrandRosenberg S Hedrick CC Understanding thetumor immune microenvironment TIME for effective therapy Nat Med Yoshihara K Shahmoradgoli M MartÃnez E Vegesna R Kim H TorresGarcia WTreviño V Shen H Laird PW Levine DA Inferring tumour purity and stromaland immune cell admixture from expression data Nat Commun Yang S Liu T Nan H Wang Y Chen H Zhang X Zhang Y Shen B Qian PXu S Comprehensive analysis of prognostic immunerelated genes inthe tumor microenvironment of cutaneous melanoma J Cell Physiol Deng Z Wang J Xu B Jin Z Wu G Zeng J Peng M Guo Y Wen Z MiningTCGA database for tumor microenvironmentrelated genes of prognosticvalue in hepatocellular carcinoma Biomed Res Int Zhao K Yang H Kang H Wu A Identification of key genes in thyroid Cancermicroenvironment Med Sci Monit Xu WH Xu Y Wang J Wan FN Wang HK Cao DL Shi GH Qu YYZhang HL Ye DW Prognostic value and immune infiltration of novelsignatures in clear cell renal cell carcinoma microenvironment AgingAlbany NY Chen B Chen W Jin J Wang X Cao Y He Y Data Mining of PrognosticMicroenvironmentRelated Genes in clear cell renal cell carcinoma a studywith TCGA database Dis Markers Li X Gao Y Xu Z Zhang Z Zheng Y Qi F Identification of prognostic genesin adrenocortical carcinoma microenvironment based on bioinformaticmethods Cancer Med Pan XB Lu Y Huang JL Long Y Yao DS Prognostic genes in the tumormicroenvironment in cervical squamous cell carcinoma Aging Albany NY Wang H Wu X Chen Y Stromalimmune scorebased gene signature aprognosis stratification tool in gastric Cancer F | 2 |
"adipogenesis is the process through which mesenchymalstem cells mscs commit to the adipose lineage and diï¬erentiate into adipocytes during this process preadipocytescease to proliferate begin to accumulate lipid droplets anddevelop morphologic and biochemical characteristics ofmature adipocytes such as hormoneresponsive lipogenesisand lipolytic programs currently there are mainly twomodels of benign adipocyte diï¬erentiation in vitro one isï¬broid pluripotent stem cells which can diï¬erentiate intonot only adipocytes but also muscle cartilage and othercells there are two kinds of ï¬broid pluripotent stem cellsbone marrow and adipose mesenchymal stem cells anothergroup is ï¬broblastic preadipocytes which have a single direction of diï¬erentiation namely lipid diï¬erentiation including3t3l1 and 3t3f422a cells cancer cells with tumorinitiation ability designated as cancer stem cells cscshave the characteristics of tumorigenesis and the expressionof speciï¬c stem cell markers as well as the longterm selfrenewal proliferation capacity and adipose diï¬erentiationpotential in addition to cscs cancer cells undergoing epithelialmesenchymaltransformation emt havebeen reported to be induced to diï¬erentiate into adipocytes[] lung cancer ncih446 cells can be induced to differentiate into neurons adipocytes and bone cells in vitro the adipogenesis diï¬erentiation treatment is promisingin the p53 gene deletion type of ï¬broblastderived cancer cancer cells with homologous recombination defectssuch as ovarian and breast cancer cells with breast cancersusceptibility genes brca mutations can be inducedto diï¬erentiate by poly adpribose polymerase parp 0cstem cells internationalinhibitors the nuclear receptor peroxisome proliferatoractivated receptor Î pparÎ agonist antidiabetic thiazolidinedione drug can induce growth arrest and adipogenicdiï¬erentiation in human mouse and dog osteosarcoma cells thyroid cancer cells expressing the pparÎ fusion proteinppfp can be induced to diï¬erentiate into adipocytes bypioglitazone adipogenesis can be induced in welldiï¬erentiated liposarcoma wdlps and dediï¬erentiatedliposarcoma ddlps cells by dexamethasone indomethacininsulin and 3isobutyl1methyl xanthine ibmx in this review we highlight some of the crucial transcription factors that induce adipogenesis both in mscs and inincluding the wellstudied pparÎ and ccaatcscsenhancerbinding proteins cebps as well as othercell factors that have been recently shown to have an important role in adipocyte diï¬erentiation we focus on understanding the complex regulatory mechanism of adipocytediï¬erentiation that can contribute to the clinical treatmentof human diseases including those caused by obesity andadipocytes dysfunction especially for the malignant tumorwhich can be transdiï¬erentiated into mature adipocytes adipocyte differentiationcell proliferation and diï¬erentiation are two opposingprocesses and there is a transition between these two processes in the early stages of adipocyte diï¬erentiation theinteraction of cell cycle regulators and diï¬erentiation factors produces a cascade of events which ultimately resultsin the expression of adipocyte phenotype adipogenesishas diï¬erent stages each stage has a speciï¬c gene expression pattern in general adipocyte diï¬erentiation ofpluripotent stem cells is divided into two phases the ï¬rstphase known as determination involves the commitment ofpluripotent stem cells to preadipocytes the preadipocytescannot be distinguished morphologically from their precursor cells but also have lost the potential to diï¬erentiate intoother cell types in the second phase which is known asterminal diï¬erentiation the preadipocytes gradually acquirethe characteristics of mature adipocytes and acquire physiological functions including lipid transport and synthesisinsulin sensitivity and the secretion of adipocytespeciï¬cproteins the diï¬erentiation of precursor adipocytes is also dividedinto four stages proliferation mitotic cloning early diï¬erentiation and terminal diï¬erentiation after the precursors are inoculated into the cell culture plates the cellsgrow exponentially until they converge after reaching contact inhibition the growth rate slows and gradually stagnatesand the proliferation of precursor adipocytes stops which isvery necessary for initiating the diï¬erentiation of precursoradipocytes adipocyte precursors exhibit transient mitosiscalled clonal expansion a process that relies on the actionof induced diï¬erentiation factors some preadipocyte cellsmouse cell lines 3t3l1 3t3f442a undergo one or tworounds of cell division prior to diï¬erentiation whereasother cell lines mouse c3h10t12 diï¬erentiating into adipocyte do not undergo mitosis clonal expansion whether mitotic clonal expansion is required for adiposediï¬erentiation remains controversial however it is certainthat some of the checkpoint proteins for mitosis regulateaspects of adipogenesis [ ] when cells enter the terminaldiï¬erentiation stage the de novo synthesis of fatty acidsincreases significantly the transcription factors and adipocyterelated genes work cooperatively to maintain precursor adipocyte diï¬erentiation into mature adipocytes containing largelipid droplets regulatory pathways inpreadipocytes commitmentadipocyte diï¬erentiation is a complex process in which geneexpression is ï¬nely regulated the most basic regulatory network of adipose diï¬erentiation has not been updated inrecent years but some factors and signaling pathways thatdo aï¬ect adipose diï¬erentiation have been continuouslyreported adipocyte diï¬erentiation is the result of the geneexpression that determines the phenotype of adipocyteswhich is a complex and delicate regulatory process figure wnt signal pathway in adipogenesis wnt signaling isimportant for adipocytes proliferation and diï¬erentiationboth in vitro and in vivo the wnt family of secretedglycoproteins functions through paracrine and autocrinemechanisms to uence cell fate and development wntprotein binding to frizzled receptors initiates signalingthrough catenindependent and independent pathways wnt signaling inhibits adipocyte diï¬erentiation in vitroby blocking the expression of pparÎ and cebpα constitutive wnt10b expression inhibits adipogenesis wnt10b isexpressed in preadipocytes and stromal vascular cells butnot in adipocytes in vivo transgenic expression of wnt10bin adipocytes results in a reduction in white adipose tissuemass and absent brown adipose tissue development wnt10a and wnt6 have also been identiï¬ed as determinantsof brown adipocyte development [ ] wnt5b is transiently induced during adipogenesis and promotes diï¬erentiation indicating that preadipocytes integrate inputs fromseveral competing wnt signals the hedgehog hh signaling pathway mechanismthree vertebrate hh ligands including sonic hedgehogshhindian hedgehog ihh and desert hedgehogdhh have been identiï¬ed and initiated a signaling cascademediated by patched ptch1 and ptch2 receptors [ ]hh signaling had an inhibitory eï¬ect on adipogenesis inmurine cells such as c3h10t12 ks483 calvaria mscslines and mouse adiposederived stromal cells thesecells were visualized by decreased cytoplasmic fat accumulation and the expression of adipocyte marker genes after hhsignaling was inhibited although it is generally agreedthat hh expression has an inhibitory eï¬ect on preadipocytediï¬erentiation the mechanisms linking hh signaling andadipogenesis remain poorly deï¬ned erkmapkppar signal pathway extracellularregulated protein kinase erk is required in the proliferativephase of diï¬erentiation erk activity blockade in 3t3l1 0cstem cells internationaldex insulin demxwnt 10band othersshhpbc smotgfð½p smad3 smad3testosteroneð½catentinarirspi3kaktcrebpkapcrebfoxo1a2tcflef gata23cebpð½mapkg3k3ð½p2cebpð½cebpαppará½»bmpssmad1srebpadipocytegenesfigure regulation pathways in preadipocytes commitment bmp and wnt families are mediators of mscs commitment to producepreadipocytes exposure of growtharrested preadipocytes to diï¬erentiation inducers igf1 glucocorticoid and camp triggers dnareplication leading to adipocyte gene expression due to a transcription factor cascade the dotted line indicates an uncertain molecularregulatory mechanismcells and embryonic stem cells can inhibit adipogenesis inthe terminal diï¬erentiation phase erk1 activity leads topparÎ phosphorylation which inhibits adipocyte diï¬erentiation this implies that erk1 activity must be reduced afteradipocyte proliferation so that diï¬erentiation can proceedthis reduction is mediated in part by mitogenactivatedprotein kinase mapk phosphatase1 mkp1 [ ]these extracellular and intracellular regulation factors causeadipocytespeciï¬c gene expression and eventually lead toadipocyte formation adipocyte differentiationregulatory proteins pparÎ and adipocyte diï¬erentiation pparÎ is a member of the nuclearreceptor superfamily and is both necessaryand suï¬cient for adipogenesis forced expression ofpparÎ is suï¬cient to induce adipocyte diï¬erentiation broblasts indeedthe proadipogenic cebps andkrüppellike factors klfs have all been shown to induceat least one of the two pparÎ promoters in contrast antiadipogenic transcription factor gata functioned in part byrepressing pparÎ expression pparÎ itself has twoisomers the relative roles of pparÎ1 and pparÎ2 in adipogenesis remain an open question pparÎ2 is mainlyexpressed in adipose tissue while pparÎ1 is expressed inmany other tissues although both can promote adipocytediï¬erentiation pparÎ2 could do so eï¬ectively at very lowligand concentration compared with pparÎ1 the twoprotein isoforms are generated by alternative splicing andpromoter usage and both are induced during adipogenesispparÎ1 can also be expressed in cell types other than adipocytes ren et al used engineered zincï¬nger proteins tothe expression ofthe endogenous pparÎ1 andinhibitpparÎ2 promoters in 3t3l1 cells ectopic expression ofpparÎ2 promotes adipogenesis whereas that of pparÎ1does not zhang et al reported that pparÎ2 deï¬ciencyimpairs the development of adipose tissue and insulin sensitivity there are transcriptional cascades between adipocytesgenes including pparÎ and cebpα which are the coreadipocyte diï¬erentiation regulators in the early stage of adipocyte diï¬erentiation the expression of cebp and cebpδincrease which upregulates cebpα expressionfurtheractivate pparÎ pparÎ activating cebpα in turn resultsin a positive feedback pparÎ binding with retinoic acid xreceptor rxr forms diï¬erent heterodimers the variousdimmers can combine with the pparÎ response elementppre and initiate the transcription of downstream genesfor diï¬erentiation into adipocytes cebps participate in adipogenesis and several cebpfamily members are expressed in adipocytesincludingcebpα cebp cebpÎ cebpδ and cebphomologous protein chop the temporal expression of thesefactors during adipocyte diï¬erentiation triggers a cascadewhereby early induction of cebp and cebpδ leads tocebpα expression this notion is further supported by thesequential binding of these transcription factors to severaladipocyte promoters duringadipocyte diï¬erentiationcebp is crucial for adipogenesis in immortalized preadipocyte lines cebp and cebpδ promote adipogenesis atleast in part by inducing cebpα and pparÎ cebpαinduces many adipocyte genes directly and plays an important role in adipose tissue development once cebpα isexpressed its expression is maintained through autoactivation despite the importance of cebps in adipogenesis 0cstem cells internationalthese transcription factors clearly cannot function eï¬cientlyin the absence of pparÎ cebp cannot induce cebpαexpression in the absence of pparÎ which is required torelease histone deacetylase1 hdac1 from the cebpαpromoter furthermore ectopic cebpα expressioncannot induce adipogenesis in pparÎï¬broblasts however cebpα also plays an important role in diï¬erentiated adipocytes overexpression of exogenous pparÎ incebpαdeï¬cient cells showed that although cebpα isnot required for lipid accumulation and the expression ofmany adipocyte genes it is necessary for the acquisition ofinsulin sensitivity [ ] figure human ï¬broblasts withthe ability to diï¬erentiate into adipocytes also do not undergomitotic cloning ampliï¬cation however pparÎ exogenousligands need to be added to promote adipocyte diï¬erentiation therefore it can be inferred that mitotic cloning expansion can produce endogenous ligands of pparÎ bmp and transforming growth factor tgf inadipocyte diï¬erentiation a variety of extracellular factorsaï¬ect the preadipocyte commitment of stem cells includingbone morphogenetic protein bmp transforminggrowth factor tgf insulininsulinlike growthfactor igf1 tumor necrosis factor α and interleukin matrix metalloproteinase ï¬broblast growthfactor fgf and fgf2 bmp and tgf have variedeï¬ects on the diï¬erentiation fate of mesenchymal cells the tgf superfamily members bmps and myostatinregulate the diï¬erentiation of many cell types includingadipocytes tgf inhibitor can promote adipose diï¬erentiation of cancer cells with a mesenchymal phenotypein vitro and transgenic overexpression of tgf impairsadipocyte development inhibition of adipogenesis couldbe obtained through blocking of endogenous tgf with adominantnegative tgf receptor or drosophila mothersagainst decapentaplegic protein smad inhibitionsmad3 binds to cebps and inhibits their transcriptionalactivity including their ability to transactivate the pparÎ2promoter [ ] exposure of multipotent mesenchymalcells to bmp4 commits these cells to the adipocyte lineageallowing them to undergo adipose conversion theeï¬ects of bmp2 are more complex and depend on the presence of other signaling molecules bmp2 alone has little eï¬ecton adipogenesis and it interacts with other factors such astgf and insulin to stimulate adipogenesis of embryonicstem cells bmp2 stimulates adipogenesis of multipotentc3h10t12 cells at low concentrations and can contribute tochondrocyte and osteoblast development at higher concentrations klfs in adipocyte diï¬erentiation during adipocyte differentiation some klf family members are overexpressedsuch as klf4 klf5 klf9 and klf15 while klf16 expression is reduced [ ] klf15 is the ï¬rst klf family members which were identiï¬ed to be involved in adipocytediï¬erentiation its expression increased significantly on thesixth day of 3t3l1 adipocyte diï¬erentiation and peakedon the second day of adipocyte induction in mscs andmouse embryonic ï¬broblasts inhibition of klf15 by sirnaor mutation led to a decrease in pparÎ cebpα fatty acidbinding protein fabp4 and glucose transporter glut4 however overexpression of klf15 in nih3t3cells was found to be associated with lipid accumulation aswell as increases in pparÎ and fabp4 mice with complete absence of klf5 showed embryonal lethality and micewith singlechromosome klf5 knockout showed a significant reduction in white fat in adulthood suggesting thatklf5 plays an important role in adipocyte diï¬erentiationklf5 can be activated by cebp or cebpδ which isinvolved in early adipocyte diï¬erentiation klf5 can beactivated by cebp or cebpδ which is involved in earlyadipocyte diï¬erentiation direct binding of klf5 to thepparÎ2 promoter in combination with cebps inducespparÎ2 expression transfection of klf5 dominantnegative mutants in 3t3l1 cells reduced lipid droplet accumulation and inhibited pparÎ and cebpα expressionwhereas overexpression of wild klf5 significantly promotedadipocyte diï¬erentiation even without exogenous hormonestimulation similar to klf5 klf9 knockdown can inhibitthe expression of a series of adipocyte diï¬erentiation genessuch as pparÎ cebpα and fabp4 hence inhibitingadipocyte diï¬erentiation however klf9 overexpressiondid not upregulate the expression of pparÎ and cebpα in addition klf4 can transactivate cebp by bindingto the region of kb upstream of the cebp promoter and promote lipid diï¬erentiation klf6 can forma complex with histone deacetylase3 hdac3 inhibitingpreadipocyte factor1 pref1 expression and promotinglipid diï¬erentiation klf2 is highly expressed in adiposeprogenitors and its expression decreases during the processof lipid diï¬erentiation overexpressed klf2 can bind to thecaccc region of pparÎ2 proximal promoter and inhibitlipid diï¬erentiation as well as the expression of pparÎcebpα and sterolregulated elementbinding proteinssrebp by inhibiting the promoter activity rnasequence analysisshowed that klfl6 expression wasdecreased on the ï¬rst day of adipocyte diï¬erentiation of3t3l1 cells adipocyte diï¬erentiation was promoted byklf16 knockdown but was inhibited by klf16 overexpression via inhibition of pparÎ promoter activity in addition klf3 and klf7 were also found to play a negativeregulatory role in adipocyte diï¬erentiation [ ] signal transducers and activators of transcriptionstats and adipocyte diï¬erentiation the activated statprotein enters the nucleus as a dimer and binds to the targetgene to regulate gene transcription in the adipocyte diï¬erentiation of mouse 3t3l1 cells the expression of stat1 andstat5 was significantly increased while that of stat3and stat6 was not significantly changed in the adipocyte diï¬erentiation of human subcutaneous adipose precursor cells stat1 expression was significantly decreased while the expression of stat3 and stat5 wasincreased and stat6 expression was unchanged therole of stat1 in adipocyte diï¬erentiation is not clearbecause its expression trend in humans and mice diï¬ersduring the adipocyte diï¬erentiation process early adipocytediï¬erentiation of 3t3l1 cells was inhibited by stat1 0cstem cells internationalklf5srebp1cklf15klf2chopcebpá½»krox20ligandcebpð½cebpð¿gata23ppará½»cebpð¼proadipogenicantiadipogenicgenes of terminaladipocytedifferentiationfigure a cascade of transcription factors that regulate adipogenesis pparÎ is one of the key transcription factors in adipogenesis and thecore of the transcriptional cascade that regulates adipogenesis pparÎ expression is regulated by several proadipogenic blue andantiadipogenic red factors cebpα is regulated through a series of inhibitory proteinprotein interactions some transcription factorfamilies include several members that participate in adipogenesis such as the klfs black lines indicate eï¬ects on gene expression violetlines represent eï¬ects on protein activityagonist interferon Î loss of stat1 in 3t3l1 cells can rescue the inhibition of adipocyte diï¬erentiation caused byprostaglandin factor 2α other studies have found thatstat1 is required for adipose diï¬erentiation and stat1overexpression in c3h10t12 cells can prevent the inhibition of lipid diï¬erentiation caused by bcell lymphoma6knockdown there was no abnormal adipose tissuein stat1 knockout mice stat3 not only aï¬ectsthe proliferation of 3t3l1 cells but also coregulates theiradipocyte diï¬erentiation with high mobility group protein the fabp4 promoter was used to speciï¬callyknock out stat3 in the adipose tissue of mice and theresults showed that mice weight significantly increasedand the adipocyte quantity increased compared with thewildtype mice stat5a and stat5b have diï¬erenteï¬ects on adipocyte diï¬erentiation abnormal adipose tissuewas found in the mice with stat5a or stat5b knockout ordouble knockout and the amount of adipose tissue was onlyoneï¬fth of the original adipose tissue in mice withoutknockdown histone modiï¬cation in adipocyte diï¬erentiation histone deacetylase sirtuin sirt plays an important rolein biological processes such as stress tolerance energymetabolism and cell diï¬erentiation during the adipocyte diï¬erentiation of c3h1012 cells sirt1 expressiondecreased overexpression of sirt1 activated thewnt signal which caused the deacetylation of cateninthe accumulation of catenin in the nucleus could inhibitadipocyte diï¬erentiation sirt1 knockdown resulted inincreased acetylation of the histones h3k9 and h4k16 inthe secreted frizzledrelated protein sfrp and sfrp2 promoters thereby promoting transcription of these genes andpromoting lipid diï¬erentiation forkhead box proteino foxo is a member of the transcription factor foxofamily it can recruit cyclic amp response elementbindingprotein cbphistone acetyltransferase p300 to initiate anacetylation the acetylated foxo1 can be phosphorylatedby phosphorylated protein kinase b pkbakt the phosphorylation of foxo1 by akt inhibits the transcriptionalactivation of foxo1 the acetylation of foxo1 lost the ability of dnabinding aï¬nity and promoted its shuttling fromnuclei to cytoplasm sirt1 and sirt2 can deacetylateand active foxo1 activated foxo1 nonphosphorylatednuclear foxo1 in the nucleus binds to the promoters of target genes encoding p21 p27 and pparÎ and initiates subsequent transcriptions sirt2 inhibits the acetylation andphosphorylation of foxo1 thereby induces the accumulation of activated foxo1 in the nucleus activated foxo1could inhibit adipogenesis via pparÎ [] lysinespeciï¬c histone demethylase lsd1 expression increasedduring the adipocyte diï¬erentiation of 3t3l1 cells lsd1could reduce the dimethylation levels of histone h3k9 andh3k4 in the cebpα promoter region thereby promotingadipocyte diï¬erentiation set domaincontaining setd8 catalyzed the monomethylation of h4k20 andpromoted pparÎ expression the activation of pparÎ transcriptional activity leads to the induction of monomethylatedh4k20 and modiï¬cation of pparÎ and its targets therebypromoting adipogenesis enhancer of zeste homolog ezh2 is a methyltransferase and can bind methyl groupsto histone h3k27 which is also necessary for lipid diï¬erentiation the absence of ezh2 in brown fat precursors results inreduced levels of the wnt promoter histone h3k27me3which is also saved by the ectopic ezh2 expression or theuse of a wntcatenin signal inhibitor in addition histone demethylases such as lysinespeciï¬c histone demethylase lsdkdm kdm6 and histone lysine demethylasephf2 are also involved in adipose diï¬erentiation andkdm2b inhibits transcription factor activator protein 2αpromoter via h3k4me3 and h3k36me2 role of microrna and long noncodingrna in adipogenesismicrorna mir can bind and cut target genes or inhibittarget gene translation endogenous sirna can be producedby the action of dicer enzyme and bind to a speciï¬c proteinto change its cellular location many kinds of mirsare involved in regulating adipocyte diï¬erentiation the 0cstem cells internationalexpression of mir143 increased during the diï¬erentiationof adipose progenitor cells overexpression of mir143promoted gene expression involved in adipose diï¬erentiationand triglyceride accumulation inhibition of mir143 prevented the adipose diï¬erentiation of human fat progenitorcells [ ] additionally mir8 promotes adipocyte diï¬erentiation by inhibiting wnt signaling moreover mir mir103 mir21 mir519d mir210 mir30mir204211 and mir375 also play a certain role in promoting adipocyte diï¬erentiation while mir130 mir448and let7y inhibit lipid diï¬erentiation [ ] in additionto mirs long noncoding rna lncrna is a type of noncoding rna and is important during epigenetic regulationand can form a doublestranded rna complex with mrnacauses protein transcription lncu90926 inhibits adipocytediï¬erentiation by inhibiting the transactivation of pparÎ2 as a novel lncrna hoxaas3 expression increasedduring the adipose diï¬erentiation of mscs and hoxaas3 silencing reduced the marker gene of adipose diï¬erentiation and inhibited the adipose diï¬erentiation zhu et al reported that hoxaas3 interacted with ezh2 toregulate lineage commitment of mscs hoxa as3 canregulate the trimethylation level of h3k27 in the runx2promoter region by binding to ezh2 therefore hoxaas3 is considered to be an epigenetic switch regulating mscslineage speciï¬city adipocyte diï¬erentiationassociatedlncrna can act as a competitive endogenous rna of mir in the process of lipid diï¬erentiation thereby promotingthe expression of sirt1 the target gene of mir204 and thusinhibiting lipid diï¬erentiation the lncrna neat1can also regulate adipocyte diï¬erentiation under the uence of mirna140 other lncrna including lncrnablnc1 and plnc are also involved in regulating adipocytediï¬erentiation [ ] other biochemical response involved inadipocyte differentiation unfolded protein responses in adipocyte diï¬erentiationin the endoplasmic reticulum of eukaryotes unfolded protein response involves three proteinsinositolrequiringenzyme 1α doublestranded rnadependent proteinkinaselike er kinase and activating transcription factoratf 6α knockdown of atf6α aï¬ects the expressionof adipocytes genes and inhibits c3h10t12 adipocyte differentiation the inhibitory eï¬ect of berberine on adipocyte diï¬erentiation of 3t3l1 cells is also due to inducedchop and decorin expressions and this inhibitory eï¬ectis ameliorated by chop knockout in the adipocytediï¬erentiation process of 3t3l1 cells increases in pparÎand cebpα as markers of adipocyte diï¬erentiation wereaccompanied by an increase in the corresponding proteinexpressions of phosphorylated eukaryotic translation initiaeif 2α phosphorylated endoribonucleasetion factorire1α atf4 chop and other unfolded protein responsesendoplasmic reticulum stress inducer or hypoxic endoplasmic reticulum stress can inhibit adipocyte diï¬erentiationadditionally eif2α mutation results in continuous activation or overexpression of chop which also inhibits adipocyte diï¬erentiation after the initiation of adiposediï¬erentiation numerous diï¬erentiationassociated proteinsare synthesized exogenous endoplasmic reticulum stressinducers can lead to excessive endoplasmic reticulumresponse which in turn aï¬ects the synthesis of proteinsrelated to diï¬erentiation and inhibits adipocyte formationfigure role of oxidative stress in adipogenesis during thedirectional diï¬erentiation of mscs mitochondrial complexi and iii and nadph oxidase nox4 are the main sourcesof oxygen species ros production currently it is believedthat ros aï¬ects not only the cell cycle and apoptosis but alsodiï¬erentiation through uencing the signaling pathwaysincluding the wnt hh and foxo signaling cascade duringmscs diï¬erentiation the diï¬erentiation ability ofstem cells is determined by the arrangement of perinuclearmitochondria which speciï¬cally manifests as low atpcellcontents and a high rate of oxygen consumption the lackof these characteristics indicates stem cell diï¬erentiation adipocyte diï¬erentiation is a highly dependent rosactivation factor related to mitosis and cell maturation schroder et al found that exogenous h2o2 could stimulate adipocyte diï¬erentiation of mouse 3t3l1 cells andhuman adipocyte progenitor cells in the absence of insulinh2o2 regulates adipocyte diï¬erentiation of 3t3l1 cells ina dosedependent manner high doses of h2o2 and μm promote adipocyte diï¬erentiation [ ] tormos et al found that ros synthesis increased in humanmscs at the early stage of adipose diï¬erentiation and targeted antioxidants could inhibit lipid diï¬erentiation byknocking down rieske ironsulfur protein and ubiquinonebinding protein ros produced by mitochondrial complexiii was found to be necessary in initiating adipose diï¬erentiation however other studies have shown that theexpression levels of adiponectin and pparÎ were decreasedby using h2o2 mm in 3t3l1 cells free radical nitric oxide no also promotes lipid diï¬erentiationbecause treatment with no inducer hydroxylamine or nosynthase nos substrate arginine can significantly induceadipose diï¬erentiation of rat adipose progenitor cells nosinduced adipose diï¬erentiation mainly via enos rather thaninos ros can induce adipose diï¬erentiation primarily by inhibiting wnt foxo and hh signaling pathwaysthat inhibit lipid diï¬erentiation autophagy in adipocyte diï¬erentiation the increase inautophagosomes during lipid diï¬erentiation indicates thatautophagy may play an important role in lipid diï¬erentiation baerga et al conï¬rmed that the adipocyte diï¬erentiation eï¬ciency was significantly inhibited in mouse embryonic ï¬broblasts lacking autophagyrelated gene atg agene encoding an essential protein required for autophagy knockdown of atg5 in 3t3l1 cells promotesproteasomedependent degradation of pparÎ2therebyinhibiting adipocyte diï¬erentiation zhang reportedthat autophagyrelated gene 7atg7 is also crucial for adipose development atg7deï¬cient mice were slim and onlyhad of white fat compared to wildtype mice and the 0cstem cells internationalcebpð½ geneebf1 geneklf4egr2cebpð½cytosolcebpð¿ genecebpð¿klf5geneppará½» geneklf5nr2f2nfkb11433relasrebf1a2rxrappará½»ppará½»rxra heterodimerppará½»rxracorepressor complexfabp4ligands of ppará½»fam120bthrap3ep300ncoa2ncoa3helz2ncoa1crebbpebf1adipoq geneaidrfcebpð¼ geneznf638znf467cebpð¼ncor1hdac3ncor2 slc2a4 geneglut4 genelep genefabp4 genecdk4ccnd3plin1 genepck1 genefabp4cd36 geneppararxracoactivator complexppará½»fatty acidrxramediatorcoactivator complexangptlgeneppargc1amediator complex consensuslpl genenucleoplasmproteins bind to gene promoterstranscription of genes into proteinsacting on proteins compoundingtgfð½1wnt1wnt10btnf77233adipoqglut4slc2a4 tetramerlepfabp4lipid dropletplin1pck1papa pa4xpalmccd36paangptl4lplfigure regulation of adipocyte diï¬erentiation a regulatory loop exists between pparÎ and cebp activation transcription factor coeebf activates cebpα cebpα activates ebf1 and ebf1 activates pparÎ cebp and cebpδ act directly on the pparÎ gene bybinding its promoter and activating transcription cebpα cebp and cebpδ can activate the ebf1 gene and klf5 the ebf1 and klf5proteins in turn bind the promoter of pparÎ which becomes activated other hormones such as insulin can aï¬ect the expression ofpparÎ and other transcription factors such as srebp1c pparÎ can form a heterodimer with the rxrα in the absence of activatingligands the pparÎrxrα complex recruits transcription repressors such as nuclear receptor corepressor ncor ncor1 andhdac3 upon binding with activating ligands pparÎ causes a rearrangement of adjacent factors corepressors such as ncor2 are lostand coactivators such as transcription intermediary factor tif2 cbp and p300 are recruited which can result in the expression of cyclicampresponsive elementbinding protein creb followed by pparÎ pparÎ expression initiates the expression of downstream genesincluding angiopoietinrelated protein pgar perilipin fabp4 cebpα fatty acid transportrelated proteins carbohydrate metabolismrelated proteins and energy homeostasisrelated proteinslipid metabolism and hormoneinduced lipolysis in the adipocytes were altered autophagy related gene atg4b isactivated by cebp in the process of lipid diï¬erentiationand autophagy activation is necessary for the degradationof klf2 and klf3 two negative regulators of lipid diï¬erentiation these results showed that adipose diï¬erentiation andautophagy are mutually complementary in 3t3l1cells autophagy was inhibited by aspartate ammonia or 0cstem cells internationalmethyladenine at diï¬erent lipid induction periods and days and only autophagy inhibition at days hindered the formation of lipid droplets and the expression of lipid marker genes indicating that autophagy wasvery important in the early stage of lipid diï¬erentiation recent studies showed that lc3 is overexpressed in3t3l1 cells further demonstrating the important role ofautophagy in lipid diï¬erentiation role of alternative splicing in adipogenesis selectivesplicing is uenced by splicing regulators which regulateadipocyte diï¬erentiation by regulating the selective splicingof genes speciï¬c to this process lipin1 is an important regulator in the process of adipocyte diï¬erentiation and includestwo isomers lipin1α and lipin1 which have diï¬erenteï¬ects high expression of lipin1α promotes adipocyte differentiation while that of lipin1 promotes lipid droplet formation in sam68deï¬cient mice the ï¬fth intron ofserinethreonineprotein kinase mtor was retained resulting in unstable and rapid mtor degradation and inhibitionof adipocyte diï¬erentiation furthermore there arefour isomers of pref1 pref1a and pr | 0 |
laparoscopic surgery for rectal cancer is commonly performed in china however compared with open surgerythe effectiveness of laparoscopic surgery especially the longterm survival has not been sufï¬ciently provedmethods data of eligible patients with nonmetastatic rectal cancer at nanfang hospital of southern medical university andguangdong provincial hospital of chinese medicine between and were retrospectively reviewed longterm survival outcomes and shortterm surgical safety were analysed with propensity score matching between groupsresults of cases collated from two institutes matched pairs were analysed after propensity score matching the estimated blood loss during laparoscopic surgery was significantly less than that during open surgery p¼ and the operativetime and hospital stay were shorter in the laparoscopic group both p the postoperative complications rate was inthe laparoscopic group and in the open group p¼ no significant difference was observed between the laparoscopicgroup and the open group in the 5year overall survival rate vs p¼ 5year relapsefree survival rate vs p¼ or 5year cancerspeciï¬c survival rate vs p¼ an elevated carcinoembryonic antigen harvested lymph nodes and perineural invasion were independent prognostic factors affecting overall survival and relapsefreesurvivals our ï¬ndings suggest that open surgery should still be the priority recommendation but laparoscopic surgery isalso an acceptable treatment for nonmetastatic rectal cancerkey words laparoscopic surgery open surgery propensity score matching rectal cancersubmitted february revised april accepted june vc the authors published by oxford university press and sixth afï¬liated hospital of sun yatsen universitythis is an open access distributed under the terms of the creative commons attribution noncommercial license httpcreativecommonslicensesbync40 which permits noncommercial reuse distribution and reproduction in any medium provided the original work is properly citedfor commercial reuse please contact spermissionsoupcom 0c kl tan introductioncolorectal cancer is the second most commonly diagnosedcancer and the fifth leading cause of cancerrelated death forboth sexes in china in a multidisciplinary approach thatcombines chemotherapy with radiotherapy for the treatmentof colorectal cancer surgery remains the major approach thefirst successful use of laparoscopy in colorectal surgery waspublished in by jacobs laparoscopic surgery hasbeen performed widely in colon cancer all over the world andseveral randomizedcontrolled trials have demonstrated thatlaparoscopic surgery for colon cancer is safe and feasible withbetter shortterm outcomes including a decrease in postoperative pain a shorter hospital stay and earlier recoveryand equivalent longterm results compared to open surgery[] however laparoscopic surgery for rectal cancer is morearduous than that for colon cancer so the early clinical trialsexcluded rectal cancer [] although a few clinical trialshave shown the advantages of laparoscopic rectalcancer resection compared with open surgery [] both the acosogz6051 and alacart trials did not support the use of laparoscopic surgery for rectal cancer [ ] it is still controversialwhether laparoscopic surgery is suitable for rectal cancer especially for low rectal cancer therefore we conducted thisretrospective cohort study to compare longterm survival outcomes and shortterm surgical safety between laparoscopicand open surgery for nonmetastatic rectal cancer in thechinese population propensity score matching psm was performed for the study designpatients and methodsstudy designall consecutive eligible patients with rectal cancer were confirmed from the department of general surgery of nanfanghospital of southern medical university and the department ofproctology of guangdong provincial hospital of chinesemedicine between january and december these twocenters were members of the southern chinese laparoscopiccolorectal surgery study group demographic clinical pathologic and imaging features together with the management andoutcomes were carefully reviewed written informed consentwas acquired from patients preceding the surgical proceduresthis study was approved by the ethical committee of nanfanghospital and guangdong provincial hospital of chinesemedicine no ze2019052study subjectsinclusion criteria were patients with clinical stage iiii rectalcancer who underwentrectal cancerexclusion criteria were patients with i combined operationsextending to the surrounding an ii multiple cancers iiiemergency operation iv conversion to open surgery or vpatients who received neoadjuvant therapyradical surgery forall included cases were classified into two groups based onthe surgical approach which was either laparoscopic or opensurgery the surgical approach was decided by the individualcolorectal surgeon based on a combined assessment of clinicalendoscopic and imaging featuresdata collectiondata were collected in a prospectively maintained databasefrom clinical report forms the demographic and clinicopathological data included age gender body mass index bmi preoperative carcinoembryonic antigen cealocationoperative time estimated blood loss surgical procedure protective ileostomy tumor grade tumor stage and hospital staypreoperative cea was defined as cea measured closest to theoperation time tumor location was divided into the followingthree sections upper rectum above cm from the anal vergemiddle rectum cm from the anal verge and lower rectumbelow cm from the anal verge surgical procedures consistedof three categories low anterior resection abdominoperinealtumorfigure flow diagram of patient disposition 0claparoscopic vs open surgery for rectal cancer table baseline characteristics of the study populationcharacteristictotal cohortmatched cohortlaparoscopic groupn¼ open groupn¼ pvaluelaparoscopic groupn¼ open groupn ¼ pvalueage years mean sdgender n malefemalebmi kgm2 mean sdpreoperative cea n 14 ngml ngmltumor location n upper rectummiddle rectumlower rectumtumor stage n iiiiii sd standard deviation bmi body mass index cea carcinoembryonic antigentable operative and pathological results in matched cohorts variablesurgical procedure n low anterior resectionabdominoperineal resectionhartmanns procedureprotective ileostomy n operative time min median iqrintraoperative blood loss ml median iqrhospital stay day median iqrtumor grade n wellmoderatepoorothersharvested lymph nodes n 15lymphovascular invasion n perineural invasion n tumor deposits n postoperative complications n wound infectionileusurinary dysfunctionanastomosis leakageintraabdominal bleedingpneumoniacardiac eventreoperation n mortality n iqr interquartile rangelaparoscopic group n¼ open group n ¼ pvalueresection and hartmanns procedure tumor grade was dividedinto three types well differentiated moderately differentiatedand poorly differentiated including signet or mucinous adenocarcinoma tumor stage was based on the final pathologic report and preoperative imaging examinationoutcome measurementsthe primary endpoint of this study was overall survival osrelapsefree survival rfs and cancerspecific survival cssos was defined as the time from operation to death from any 0c kl tan figure survival curve after laparoscopic surgery vs open surgery in matchedcohortscause or the last followup rfs was defined as the time fromoperation to identified recurrence or any cause of death csswas defined as the time from operation to death due to rectalcancer the last followup was january ileus urinary dysfunctionthe secondary endpoints were operative time estimated bloodloss hospital stay reoperation postoperative complications andmortality postoperative complications were defined as woundinfectionleakageintraabdominal bleeding pneumonia and cardiac eventsintraabdominal bleeding was defined in this study as bleeding requiring transfusion or reoperation all complications within daysafter surgery were recorded postoperative mortality was traditionally defined as any death occurring within days after surgeryanastomoticstatistical analysisdata are presented as mean standard deviation or medianwith interquartile range iqr for quantitative variables withparanormal distribution and numbers with percentages for categorical variables quantitative variables were compared usingthe students ttest or mannwhitney u test categorical variables were analysed using the chisquare test or fishers exacttest the estimates of the differences in age gender bmi preoperative cea level tumor location and tumor stage between thetwo groups were performed using psm [ ]survival rates were calculated by using the kaplanmeiermethod and comparisons between groups were performed withthe logrank test to identify the prognostic factors univariateand multivariate analyses were performed using the cox proportional hazards regression model and the results were presented as hazard ratios hrs with confidence intervalscis only factors with p in the univariate analysis wereevaluated in subsequent multivariate analysis using forwardstepwise selection for os and rfs a p was regarded asstatistically significant all statistical analyses were carried outwith ibmvr spssvr statistics version resultsbaseline characteristicsbetween january and december eligiblepatients were collected from hospitals in china of patients cases were excluded among the remaining cases underwent laparoscopic surgery and underwent open surgery after psm of pairs ofpatients were successfully matched figure baseline characteristics are outlined in table before psm there were differences in age and preoperative cea between the two groups afterpsm all variables were well balancedshortterm surgical outcomesthe perioperative and pathological results in matched cohortsare presented in table the estimated blood loss during laparoscopic surgery was significantly less than that during opensurgery p¼ in the laparoscopic group the operative timeand hospital stay were shorter than in the open groupp the incidence of postoperative complications was in the laparoscopic group and in the open groupp¼ in the open group the most common complicationswere wound infection and pneumonia followed byanastomosis leakage whereas in the laparoscopic groupthe most common complication was anastomosis leakage followed by pneumonia longterm survival outcomesin the matched cohorts the median followup period was months in the laparoscopic group iqr monthsand months in the open group iqr monthsduring the followup patients died among whom diedfrom rectal cancer and had locoregional recurrence or distantmetastasis no significant difference was observed between thelaparoscopic group and the open group in 5year os vs p¼ 5year rfs vs p¼ or 5yearcss vs p¼ figure subgroup analyses for os were conducted for gender agebmi tumor location and tumor stage compared with open surgery male patients or those with an intermediate bmi to who underwent laparoscopic surgery tended to show worseos figure 0claparoscopic vs open surgery for rectal cancer figure subgroup analysis of overall survival in matched cohortstable multivariate analysis for os and rfs in matched cohortsvariableosrfspreoperative cea vs 14 ngmlnumber of harvested lymph nodes 15 vs perineural invasion yes vs nohr cipvaluehr cipvalueos overall survival rfs relapsefree survival cea carcinoembryonic antigen hr hazard ratio ci conï¬dence intervalprognostic factors for longterm survivalprognostic factors affecting survival are presented in table univariate analyses revealed that an elevated cea ngml harvested lymph nodes perineural invasion and tumordeposits were associated with poor os and that an elevatedcea harvested lymph nodes perineural invasion and lymphovascular invasion were associated with poor rfs data notshown the surgical approach laparoscopic vs open was notassociated with os hr ci and rfs hr ci multivariate analyses testified that an elevated cea harvested lymph nodes and perineural invasionwere independent factors affecting os and rfs table discussionlaparoscopic surgery for rectal cancer is commonly performedin many countries nevertheless the evidence for laparoscopicsurgery for rectal cancer is insufficient this study focused onthe longterm survival outcomes and surgical safety of patientswho underwentlaparoscopic or open surgery for nonmetastatic rectal cancer in the chinese population in this twocenter study psm was performed to make selection balance between patients treated with laparoscopic and open surgery thesix factors of age gender bmi preoperative cea level tumor location and tumor stage were used as described in the protocolthe baseline characteristics were ideally balanced between thelaparoscopic and open groupssome studies have reported similar postoperative complications and mortality between laparoscopic surgery and opensurgery for rectal cancer [ ] and other studies have reportedfewer postoperative complications after laparoscopic surgerythan after open surgery [ ] in our study there were no significant differences in postoperative complications includingwound infectionileus urinarytract infection anastomosisleakageintraabdominal bleeding pneumonia and cardiacevent between the two groups the longer operative time is often considered a disadvantage of laparoscopic surgery according to some previous reports [ ] in contrast our studyshowed that the operative time of laparoscopic surgery wasshorter than that of open surgery the clasicc trial and colorii trial both showed that hospital stay was significantly shorterin the laparoscopic group [ ] similarly our study alsoshowed that the hospital stay for laparoscopic surgery wasshorter than for open surgerywith regard to longterm survival no largescale clinical trials have demonstrated a statistically significant difference between laparoscopic and open surgery for rectal cancer thecolor ii trial indicated no statistically significant differences indfs and os between laparoscopic and open surgeries inthe corean study dfs in laparoscopic surgery is noninferiorcompared to that in open surgery for mid or low rectal cancer consistently with previous studies os rfs and css didnot differ in both groups in our study interestingly subgroupanalyses for os showed that male and intermediate bmi to kgm2 subgroups were associated with unfavorable outcomes in the laparoscopicsurgery group vs the opensurgerygroup chinese male populations have a narrow pelvis whichmight affect the visualization of and access to the deep pelvic 0c kl tan anatomy during laparoscopic surgery kitano foundthat laparoscopic surgery might affect longterm outcomes inthe highbmi kgm2 subgroup in the current study thebmi subgroup unfavorable for laparoscopic surgery that weidentified was intermediate bmi not high bmi it might be dueto lower bmi in the chinese population compared to that in thewestern population and the small proportion of patientswith high bmi in our cohort further evaluation will be neededto determine which subgroups of patients require additional attention when undergoing laparoscopic surgerylymphovascularwe evaluated several possible prognostic factors that mayinfluence survival in patients with rectal cancer including tumor location tumor stage tumor grade surgical approach preoperative cea levelinvasion perineuralinvasion and tumor deposits [] as expected our studyshowed that perineural invasion was the significant prognosticfactor affecting os and rfs perineural invasion refers to the invasion of cancer cells into any of the layers of the nerve sheatha higher grade of perineural invasion was related to local recurrence and metastasis in distant ans such as the liver lungand peritoneum all patients in this study underwent radical surgery with lymphnode dissection a minimum of harvested lymph nodes is recommended to ensure adequatestaging and oncologic resection for colorectal cancer themore lymph nodes harvested the better the prognosis [ ]in this study the average number of harvested lymph nodeswas we found that patients with 15 harvested lymphnodes had better os and rfs than those with harvestedlymph nodes several studies have shown that elevated preoperative cea was a poor prognostic factor in colorectal cancer[] in our study we also found that patients with an elevated preoperative cea had poorer os and rfsour study has several limitations first a selection biasexisted due to its retrospective design to reduce this the twogroups were matched carefully using psm second the statistical power is insufficient because the number of patients enrolled may not be sufficient after matching third data aboutadjuvant therapy after surgery were not collected which mightbe different between both groups and thus have influenced survival outcomes fourth the exclusion of converted cases mayintroduce a bias in favor of laparoscopic surgery finally thebowelrecovery data could not be exactly assessed due to thelack of records in this retrospective study therefore further research with a large population is still awaitedanydifferencesinin our study revealed the benefit of laparoscopicsurgery on shortterm outcomes including less blood lossshorter operative time and shorter hospital stay we did notfindcomplicationslaparoscopic surgery was similar to open surgery in terms ofos rfs and css for patients however male patients and thosewith an intermediate bmi in the laparoscopic group tended toshow worse os than those in the open group findings fromthis study suggest that open surgery should still be the priorityrecommendation but laparoscopic surgery is also an acceptabletreatment for nonmetastatic rectal cancer in the chinesepopulationpostoperativeauthors contributionsklt hjd zqc and tym collected and analysed the dataklt hl and rsx performed statistical analysis klt andhjd drafted the manuscript gxl and xhf performed theprocedure conceived of and designed the study and criticallyrevised all the intellectual content of the manuscript allauthors read and approved the final manuscriptfundingthis work was supported by clinical research of guangdongprovincial hospital of chinese medicine [no yn10101902]and a scientific research project of guangdong provincialacademy of chinese medical sciences [no yn2018ml11]acknowledgementsthe authors thank the patients and their families for making this retrospective study possible we also thank all theinvestigators and staff who contributed to the patientfollowup and data collection in nanfang hospital ofsouthern medical university and guangdong provincialhospital of chinese medicineconflicts of interestthe authors declare that there is no conï¬ict of interests inthis studyreferences feng rm zong yn cao sm current cancer situation inchina good or bad news from the global cancerstatistics cancer commun jacobs m verdeja jc goldstein hs minimally invasive colonresection laparoscopic colectomy surg laparosc endosc hewett pj allardyce ra bagshaw pf shortterm outcomes of the australasian randomized clinical study comparing laparoscopic and conventional open surgical treatmentsfor colon cancer the alccas trial ann surg buunen m veldkamp r hop wc survival after laparoscopic surgery versus open surgery for colon cancer longterm outcome of a randomised clinical trial lancet oncol clinical outcomes of surgical therapy study group a comparison of laparoscopically assisted and open colectomy forcolon cancer n engl j med bonjer hj haglind e jeekel i laparoscopic surgery versusopen surgery for colon cancer shortterm outcomes of arandomised trial lancet oncol fleshman j sargent dj green e laparoscopic colectomyfor cancer is not inferior to open surgery based on 5year datafrom the cost study group trial ann surg van der pas mh haglind e cuesta ma laparoscopic versus open surgery for rectal cancer color ii shortterm outcomes of a randomised phase trial lancet oncol kang sb park jw jeong sy open versus laparoscopicsurgery for mid or low rectal cancer after neoadjuvant chemoradiotherapy corean trial shortterm outcomes of anopenlabel randomised controlled trial lancet oncol zhou zx zhao ly lin t longterm oncologic outcomesof laparoscopic vs open surgery for stages ii and iii rectal 0ccancer a retrospective cohort study world j gastroenteroliii colon cancer jcog0404 a phase randomised controlledtrial lancet gastroenterol hepatol laparoscopic vs open surgery for rectal cancer fleshman j branda m sargent dj effect of laparoscopicassisted resection vs open resection of stage ii or iii rectalcancer on pathologic outcomes the acosog z6051 randomized clinical trial jama stevenson ar solomon mj lumley jw effect oflaparoscopicassisted resection vs open resection on pathological outcomes in rectal cancer the alacart randomizedclinical trial jama austin pc an introduction to propensity score methods forreducing the effects of confounding in observational studiesmultivariate behav res wang h chen x liu h laparoscopyassisted colectomyas an oncologically safe alternative for patients with stage t4colon cancer a propensitymatched cohort study bmc cancer bonjer hj deijen cl abis ga a randomized trial of laparoscopic versus open surgery for rectal cancer n engl j med lujan j valero g biondo s laparoscopic versus opensurgery for rectal cancer results of a prospective multicentreanalysis of patients surg endosc landi f vallribera f rivera jp morbidity after laparoscopic and open rectal cancer surgery a comparative analysisof morbidity in octogenarians and younger patientscolorectal dis toda s kuroyanagi h laparoscopic surgery for rectal cancercurrent status and future perspective asian j endosc surg guillou pj quirke p thorpe h shortterm endpoints ofconventionalinpatients with colorectal cancer mrc clasicc trial multicentre randomised controlled trial lancet laparoscopicassisted surgeryversus jeong sy park jw nam bh open versus laparoscopicsurgery for midrectal or lowrectal cancer after neoadjuvantchemoradiotherapy corean trial survival outcomes of anopenlabel noninferiorityrandomised controlled triallancet oncol kitano s inomata m mizusawa j survival outcomes following laparoscopic versus open d3 dissection for stage ii or mehrkhani f nasiri s donboli k prognostic factors insurvival of colorectal cancer patients after surgery colorectaldis wiratkapun s kraemer m seowchoen f high preoperative serum carcinoembryonic antigen predicts metastatic recurrence in potentially curative colonic cancer results of aï¬veyear study dis colon rectum huang qs qin hb xiao j association of tumor differentiation and prognosis in patients with rectal cancer undergoingneoadjuvant chemoradiation therapy gastroenterol rep liebig c ayala g wilks ja perineural invasion is an independent predictor of outcome in colorectal cancer j clin oncol ueno h shirouzu k eishi y study group for perineuralinvasion projected by the japanese society for cancer of thecolon and rectum jsccr characterization of perineural invasion as a component of colorectal cancer staging am j surgpathol amin mb edge sb greene fl eds ajcc cancer stagingmanual 8th edn new york springer rosenberg r engel j bruns c the prognostic value oflymph node ratio in a populationbased collective of colorectal cancer patients ann surg sjo oh merok ma svindland a prognostic impact oflymph node harvest and lymph node ratio in patients withcolon cancer dis colon rectum tarantino i warschkow r worni m elevated preoperative cea is associated with worse survival in stage iiii rectalcancer patients br j cancer huang sh tsai ws you jf preoperative carcinoembryonic antigen as a poor prognostic factor in stage iiii colorectal cancer after curativeintent resection a propensity scorematching analysis ann surg oncol konishi t shimada y hsu m association of preoperativeand postoperative serum carcinoembryonic antigen and colon cancer outcome jama oncol 0c' | 0 |
" in thoracoscopic operations Ann Thorac Surg 1996 61 1070 1073 8607658 16 McConnell PI Feola GP Meyers RL Methylene blue-stained autologous blood for needle localization and thoracoscopic resection of deep pulmonary nodules J Pediatr Surg 2002 37 1729 1731 12483642 17 Hu J Zhang C Sun L Localization of small pulmonary nodules for videothoracoscopic surgery ANZ J Surg 2006 76 649 651 16813634 18 Wicky S Mayor B Cuttat JF Schnyder P CT-guided localizations of pulmonary nodules with methylene blue injections for thoracoscopic resections Chest 1994 106 1326 1328 7956378 19 Vandoni RE Cuttat JF Wicky S Suter M CT-guided methylene-blue labelling before thoracoscopic resection of pulmonary nodules Eur J Cardiothorac Surg 1998 14 265 270 9761435 20 Lenglinger FX Schwarz CD Artmann W Localization of pulmonary nodules before thoracoscopic surgery: value of percutaneous staining with methylene blue AJR Am J Roentgenol 1994 163 297 300 7518642 21 Ikeda K Nomori H Mori T Kobayashi H Iwatani K Yoshimoto K Kawanaka K Impalpable pulmonary nodules with ground-glass opacity: success for making pathologic sections with preoperative marking by lipiodol Chest 2007 131 502 506 17296654 22 Nomori H Horio H Naruke T Suemasu K Fluoroscopy-assisted thoracoscopic resection of lung nodules marked with lipiodol Ann Thorac Surg 2002 74 170 173 12118752 23 Watanabe K Nomori H Ohtsuka T Kaji M Naruke T Suemasu K Usefulness and complications of computed tomography-guided lipiodol marking for fluoroscopy-assisted thoracoscopic resection of small pulmonary nodules: experience with 174 nodules J Thorac Cardiovasc Surg 2006 132 320 324 16872957 24 Kim YD Jeong YJ I H Cho JS Lee JW Kim HJ Lee SH Kim DH Localization of pulmonary nodules with lipiodol prior to thoracoscopic surgery Acta Radiol 2011 52 64 69 21498328 25 Mayo JR Clifton JC Powell TI English JC Evans KG Yee J McWilliams AM Lam SC Finley RJ Lung nodules: CT-guided placement of microcoils to direct video-assisted thoracoscopic surgical resection Radiology 2009 250 576 585 19188326 26 Lee NK Park CM Kang CH Jeon YK Choo JY Lee HJ Goo JM CT-guided percutaneous transthoracic localization of pulmonary nodules prior to video-assisted thoracoscopic surgery using barium suspension Korean J Radiol 2012 13 694 701 23118567 27 Kamiyoshihara M Ishikawa S Morishita Y Pulmonary cryptococcosis diagnosed by video-assisted thoracoscopic surgery with CT-guided localization: report of a case Kyobu Geka 2000 53 795 797 10935411 28 Kwon WJ Kim HJ Jeong YJ Lee CH Kim KI Kim YD Lee JH Direct lipiodol injection used for a radio-opaque lung marker: stability and histopathologic effects Exp Lung Res 2011 37 310 317 21574876 29 Jang HS Effect of drugs for preoperative localization of thoracoscopy to histopathologic change in rabbit lung Seoul the Catholic University of Korea 2000 27 Dissertation 30 Okumura T Kondo H Suzuki K Asamura H Kobayashi T Kaneko M Tsuchiya R Fluoroscopy-assisted thoracoscopic surgery after computed tomography-guided bronchoscopic barium marking Ann Thorac Surg 2001 71 439 442 11235684 Fig. 1 Overview of the experimental design. Animals were randomly divided into two groups: Group A (n = 12) was sacrificed 6 hr after percutaneous injection and Group B (n = 12) was sacrificed 24 hr after a CT guided percutaneous injection of MLM and methylene blue. Fig. 2 Examples of evaluation of staining on the lung surface. Photographs show (A) the extensive staining (score 1) (B) localized dispersion of staining (score 2) and (C) minimal dispersion of staining (score 3). The white lines on the bottom of the figure are markings of the ruler. The distance between two lines is one centimeter. Fig. 3 Examples of assessment of radio-opacity on the fluoroscopic examinations. The fluoroscopic images show (A) a minimally increased opacity (arrow) (score 1) (B) a low density of increased opacity (arrow) (score 2) and (C) a compact nodular increased opacity (arrow) (score 3). Fig. 4 CT and corresponding photomicrograph of lung specimen. MLM in Group B (A-D); (A) discrete and compact nodular opacity (arrowheads) (B) focal neutrophil infiltration necrosis and hemorrhage (arrowheads) (H&E 12.5) (C) scattered small nodular opacities of lipiodol (long arrows) and faint nodular opacity (arrowheads) (D) focal hemorrhage and necrosis (arrowheads) with diffuse neutrophil infiltration (short arrows) (H&E 12.5). MLM in Group A (E F); (E) faint nodular lipiodol opacity (arrows) (F) focal hemorrhage (arrows) with diffuse neutrophil infiltration (arrowheads) (H&E 12.5). Methylene blue in Group A (G H); (G) faint nodular opacity (arrowheads) and (H) focal extent of neutrophil infiltration necrosis and hemorrhage (arrowheads) (H&E 12.5). Staining extent and localization ability of MLM versus methylene blue Data are mean±standard deviation. Numbers in parentheses are ranges. N/A indicates not available. *Non-parametric Mann-Whitney test was performed to compare the average score of MLM and methylene blue. MLM mixture of lipiodol and methylene blue. Localization ability score of staining and radio-opacity for MLM as well as methylene blue Data are numbers of subjects. Numbers in parentheses are percentages. MLM mixture of lipiodol and methylene blue. Comparison of localization ability between MLM and methylene blue in total subjects (n = 42) We considered a score of 2 or 3 as appropriate and 3 as excellent for localization respectively. Numbers in parentheses are percentages. *Fisher's exact test compared the proportion of appropriate or excellent staining between the mixture and methylene blue. MLM mixture of lipiodol and methylene blue. Localization ability of MLM: Evaluation of radio-opacity and staining score Data are given as numbers of subjects. Numbers in parentheses are percentages. MLM mixture of lipiodol and methylene blue. Histopathologic findings of lung specimens after percutaneous injections Data are numbers of subjects. Numbers in parentheses are percentages. N/A indicates not available. *Linear by linear association was performed between material and the extent of the histopathologic findings. Linear by linear association was performed between groups and the extent of the histopathologic findings. MLM mixture of lipiodol and methylene blue. PLoS One one 1932-6203 Public Library of Science San Francisco USA 24819391 4018408 PONE-D-13-46027 .0096911 Research Biology and Life Sciences Biochemistry Biomarkers Genetics Heredity Medicine and Health Sciences Diagnostic Medicine Epidemiology Biomarker Epidemiology Cancer Epidemiology Health Care Environmental Health Oncology Cancer Risk Factors Environmental Causes of Cancer Pathology and Laboratory Medicine Public and Occupational Health Pulmonology Environmental and Occupational Lung Diseases Single Nucleotide Polymorphism in ATM Gene Cooking Oil Fumes and Lung Adenocarcinoma Susceptibility in Chinese Female Non-Smokers: A Case-Control Study ATM Polymorphism and Risk of Lung Adenocarcinoma Shen Li 1 2 Yin Zhihua 1 2 Wu Wei 1 2 Ren Yangwu 1 2 Li Xuelian 1 2 Zhou Baosen 1 2 * 1 Department of Epidemiology School of Public Health China Medical University Heping District Shenyang Liaoning Province China 2 Key Laboratory of Cancer Etiology and Intervention University of Liaoning Province China Chang Jeffrey S. Editor National Health Research Institutes Taiwan * E-mail: bszhoumail.cmu.edu.cn Competing Interests: The authors have declared that no competing interests exist. Conceived and designed the experiments: LS. Performed the experiments: LS YR XL. Analyzed the data: LS WW ZY. Contributed reagents/materials/analysis tools: LS ZY XL BZ. Wrote the paper: LS. Obtained informed consent from subjects: Baosen Zhou. 2014 12 5 2014 9 5 e96911 3 11 2013 12 4 2014 2014 Shen et al This is an open-access distributed under the terms of the Creative Commons Attribution License which permits unrestricted use distribution and reproduction in any medium provided the original author and source are credited. Background The ataxia-telangiectasia mutated (ATM) gene plays an important role in the DNA double-strand breaks repair pathway. Single nucleotide polymorphisms (SNPs) of DNA repair genes are suspected to influence the risk of lung cancer. This study aimed to investigate the association between the ATM -111G>A (rs189037) polymorphism environmental risk factors and the risk of lung adenocarcinoma in Chinese female non-smokers. Methods A hospital-based case-control study of 487 lung cancer patients and 516 matched cancer-free controls was conducted. Information concerning demographic and environmental risk factors was obtained for each case and control by a trained interviewer. After informed consent was obtained 10 ml venous blood was collected from each subject for biomarker testing. Single nucleotide polymorphism was determined by using TaqMan method. Results This study showed that the individuals with ATM rs189037 AA genotype were at an increased risk for lung adenocarcinoma compared with those carrying the GA or GG genotype (adjusted odds ratios (OR) 1.44 95% confidence interval (CI) 1.022.02 P?=?0.039). The stratified analysis suggested that increased risk associated with ATM rs189037 AA genotype in individuals who never or seldom were exposed to cooking oil fumes (adjusted OR 1.89 95%CI 1.033.49 P?=?0.040). Conclusions ATM rs189037 might be associated with the risk of lung adenocarcinoma in Chinese non-smoking females. Furthermore ATM rs189037 AA genotype might be a risk factor of lung adenocarcinoma among female non-smokers without cooking oil fume exposure. This study was supported by grant no. 81272293 from National Natural Science Foundation of China grant no. 81102194 from National Natural Science Foundation of China and grant no. 00726 from China Medical Board. The funders had no role in study design data collection and analysis decision to publish or preparation of the manuscript. Introduction Lung cancer is the leading cause of cancer-related deaths both worldwide and in China. Although cigarette smoke is the major risk factor for lung cancer only a fraction of smokers develop this disease [1] suggesting that host genetic susceptibility may play an important role in the development of lung cancer. Recent genetic susceptibility studies of lung cancer have focused on single nucleotide polymorphisms (SNPs) in candidate genes among which DNA repair genes are increasingly studied because of their critical role in maintain genome integrity. Genetic variations in DNA repair genes are thought to affect DNA repair capacity and deficits in DNA repair capacity may lead to genetic instability and carcinogenesis [2] [3]. " | 1 |
"As a result the presence of ALK gene translocation was confirmed (Figure 2F). The patient underwent crizotinib treatment (250 mg/bid orally) for 26 days. After this period symptoms including neck constriction and cough were improved. Chest CT scan images demonstrated decrease in tumor size and metastases. According to the Response Evaluation Criteria in Solid Tumors (RECIST) guidelines (version 1.1) such tumor response to crizotinib was classified as partial response (PR) (Figure 1C). Follow-up chest CT scan performed 11 weeks after the beginning of the treatment revealed a dramatic reduction in tumor size and mediastinal lymph node with nearly no presence of metastases in both lungs (Figure 1D). IL-6 (3.34 vs 25.41 pg/mL) and CEA (1.84 vs 23.43 ng/mL) levels were also reduced 23 weeks after the beginning of the therapy which demonstrated continuous partial response. Discussion According to the National Comprehensive Cancer Network guidelines ALK testing is not routinely performed in patients with squamous cell lung cancer. Therefore this patient was not tested for ALK until crizotinib was approved for marketing by the CFDA (June 23 2013). Unfortunately neither chemotherapy nor EGFR-TKI treatment produced effective tumor response in this patient. ALK gene translocations have been previously detected in patients with lung adenocarcinoma and light or non smoking history [34]. To date two studies have previously reported cases of patients with mixed carcinoma and squamous cell component harboring ALK rearrangements [56] but these studies did not describe specific therapy or diagnostic procedures. Another report showed that a patient with squamous cell lung cancer harboring c-MET amplification had partially responded to crizotinib [7]. Herein we report the case of a non-smoking woman with squamous cell lung cancer and ALK gene rearrangement who experienced remarkable response to crizotinib treatment after failed chemotherapy. In concordance with other studies on patients with lung adenocarcinoma treated with crizotinib [89] this patient has remained in remission (PR). Most importantly such remarkable response was obtained after two courses of failed chemotherapy. Conclusion Despite the low reconstruction rate of ALK gene if applicable patients with squamous cell lung cancer should have the option of undergoing ALK testing and receiving crizotinib treatment. ALK testing and subsequent targeted therapy could be an effective option for patients with non-small cell lung cancer who present progression following chemotherapy radiotherapy or any other treatment. Consent The patient provided written consent for publication of this case report. Abbreviations EML4: Echinodermmicro tubule associated protein like 4; ALK: Anaplastic lymphoma kinase; NSCLC: Non-small-cell lung cancer; CT: Chest computed tomography; TKIs: Tyrosine kinase inhibitors; EGFR: Epidermal growth factor receptor; IL-6: Interleukin-6; CEA: Carcinoembryonic antigen; CFDA: China Food and Drug Administration; FISH: Fluorescent in situ hybridization; PR: Partial response; IHC: Immunohistochemistry; H & E: Hematoxylin and eosin; ARMS: Amplification refractory mutation system; RECIST: Response Evaluation Criteria in Solid Tumors. Competing interests The authors declare that they have no competing interests. Authors contributions QSW carried out the molecular identifications of EGFR and K-ras and drafted the manuscript. YH provided parts of the clinical treatment data and revised the manuscript. XY participated in the H & E IHC and FISH analyses. YBW provided parts of the clinical treatment data. HLX participated in the design and coordination of this study and helped to revise the manuscript. All authors read and approved the final manuscript. Pre-publication history The pre-publication history for this paper can be accessed here: http://www.biomedcentral.com/1471-2466/14/83/prepub Acknowledgements We would like to thank Li Lin for the assistance on IHC analysis. We would also like to thank Edanz Editing for language revision of the manuscript. Djalalov S Beca J Hoch JS Krahn M Tsao MS Cutz JC Leighl NB Cost effectiveness of EML4-ALK fusion testing and first-line crizotinib treatment for patients with advanced ALK-positive non-small-cell lung cancer J Clin Oncol 2014 32 10 1012 1019 10.1200/JCO.2013.53.1186 24567430 Rodig SJ Mino-Kenudson M Dacic S Yeap BY Shaw A Barletta JA Stubbs H Law K Lindeman N Mark E Janne PA Lynch T Johnson BE Iafrate AJ Chirieac LR Unique clinicopathologic features characterize ALK-rearranged lung adenocarcinoma in the western population Clin Cancer Res 2009 15 5216 5223 10.1158/1078-0432.CCR-09-0802 19671850 Shaw AT Yeap BY Solomon BJ Riely GJ Gainor J Engelman JA Shapiro GI Costa DB Ou SH Butaney M Salgia R Maki RG Varella-Garcia M Doebele RC Bang YJ Kulig K Selaru P Tang Y Wilner KD Kwak EL Clark JW Iafrate AJ Camidge DR Effect of crizotinib on overall survival in patients with advanced non-small-cell lung cancer harbouring ALK gene rearrangement: a retrospective analysis Lancet Oncol 2011 12 11 1004 1012 10.1016/S1470-2045(11)70232-7 21933749 Inamura K Takeuchi K Togashi Y Nomura K Ninomiya H Okui M Satoh Y Okumura S Nakagawa K Soda M Choi YL Niki T Mano H Ishikawa Y EML4-ALK fusion is linked to histological characteristics in a subset of lung cancers J Thorac Oncol 2008 3 1 13 17 10.1097/JTO.0b013e31815e8b60 18166835 Wong DW Leung EL So KK Tam IY Sihoe AD Cheng LC Ho KK Au JS Chung LP Pik Wong M The EML4-ALK fusion gene is involved in various histologic types of lung cancers from nonsmokers with wild-type EGFR and KRAS Cancer 2009 115 1723 1733 10.1002/cncr.24181 19170230 Shaw AT Yeap BY Mino-Kenudson M Digumarthy SR Costa DB Heist RS Solomon B Stubbs H Admane S McDermott U Settleman J Kobayashi S Mark EJ Rodig SJ Chirieac LR Kwak EL Lynch TJ Iafrate AJ Clinical features and outcome of patients with non-small-cell lung cancer who harbor EML4-ALK J Clin Oncol Off J Am Soc Clin Oncol 2009 27 4247 4253 10.1200/JCO.2009.22.6993 Schwab R Petaka I Kollar M Pinter F Varkondi E Kohank A Barti-Juhasz H Sch¶nleber J Brauswetter D Kopper L Urban L Major partial response to crizotinib a dual MET/ALK inhibitor in a squamous cell lung (SCC) carcinoma patient with de novo c-MET amplification in the absence of ALK rearrangement Lung Cancer 2014 83 1 109 111 10.1016/j.lungcan.2013.10.006 24192513 Toyokawa G Takenoyama M Watanabe S Toyozawa R Inamasu E Kojo M Shiraishi Y Morodomi Y Takenaka T Hirai F Yamaguchi M Taguchi K Seto T Ichinose Y Dramatic response to crizotinib in an ALK-positive adenocarcinoma patient with disseminated intravascular coagulation J Thorac Oncol 2013 8 11 e96 e98 10.1097/JTO.0b013e3182a008ed 24128725 Kim SJ Kim DW Kim TM Lee SH Heo DS Bang YJ Remarkable tumor response to crizotinib in a 14-year-old girl with ALK-positive nonsmall-cell lung cancer J Clin Oncol 2012 30 16 e147 e150 10.1200/JCO.2011.39.9766 22508824 15030100R 648 Ann Thorac Surg Ann. Thorac. Surg. The Annals of thoracic surgery 0003-4975 1552-6259 25106680 4185369 10.1016/j.athoracsur.2014.05.026 NIHMS619453 Intraoperative Near-Infrared Imaging Can Identify Pulmonary Nodules Okusanya Olugbenga T. MD 1 Holt David 2 Heitjan Daniel 3 Deshpande Charuhas 4 Venegas Ollin 1 Jiang Jack 1 Judy Ryan 1 DeJesus Elizabeth 1 Madajewski Brian 1 Oh Kenny Albelda Steven M. 5 Nie Shuming 6 Singhal Sunil MD 1 5 * 1Division of Thoracic Surgery Department of Surgery University of Pennsylvania School of Medicine Philadelphia Pennsylvania 2Department of Clinical Studies University of Pennsylvania School of Veterinary Medicine 3Department of Biostatistics & Epidemiology University of Pennsylvania 4Department of Pathology University of Pennsylvania School of Medicine 5Department of Medicine University of Pennsylvania School of Medicine 6Departments of Biomedical Engineering and Chemistry Emory University Atlanta Geia *Corresponding Author: Sunil Singhal Division of Thoracic Surgery University of Pennsylvania School of Medicine 6 White Building 3400 Spruce Street Philadelphia PA 19104 sunil.singhaluphs.upenn.edu 9 8 2014 5 8 2014 10 2014 01 10 2015 98 4 1223 1230 2014 The Society of Thoracic Surgeons. Published by Elsevier Inc. All rights reserved. 2014 Background Over 80000 people undergo pulmonary resection for a lung nodule in the United States each year. Small nodules are frequently missed or difficult to find despite preoperative imaging. We hypothesized that near-infrared (NIR) imaging technology could be used to identify and locate lung nodules during surgery. Methods We enrolled 18 patients who were diagnosed with a pulmonary nodule that required resection. All patients had a fine-cut 1mm computed tomography scan preoperatively. The patients were given systemic 5 mg/kg indocyanine green (ICG) and then underwent an open thoracotomy 24 hours later. NIR imaging was used to identify the primary nodule and search for additional nodules that were not found by visual inspection or manual palpation of the ipsilateral lung. Results Manual palpation and visual inspection identified all 18 primary pulmonary nodules and no additional lesions. Intraoperative NIR imaging detected 16 out of the 18 primary nodules. NIR imaging also identified 5 additional subcentimeter nodules: 3 metastatic adenocarcinomas and 2 metastatic sarcomas. This technology could identify nodules as small as 0.2 cm and as deep as 1.3 cm from the pleural surface. This approach discovered 3 nodules that were in different lobes than the primary tumor. Nodule fluorescence was independent of size metabolic activity histology tumor grade and vascularity. Conclusions This is the first-in-human demonstration of identifying pulmonary nodules during Thoracic surgery with NIR imaging without a priori knowledge of their location or existence. NIR imaging can detect pulmonary nodules during lung resections that are poorly visualized on computed tomography and difficult to discriminate on finger palpation. Mol Cancer Mol. Cancer Molecular Cancer 1476-4598 BioMed Central 24655544 3998010 1476-4598-13-68 10.1186/1476-4598-13-68 Research Downregulation of BRAF activated non-coding RNA is associated with poor prognosis for non-small cell lung cancer and promotes metastasis by affecting epithelial-mesenchymal transition Sun Ming 1 sunmingnjmu.edu.cn Liu Xiang-Hua 1 liuxianghuanjmu.edu.cn Wang Ke-Ming 2 422825636qq.com Nie Feng-qi 3 957714486qq.com Kong Rong 1 31815857qq.com Yang Jin-song 4 yangjinsongmedmail.com.cn Xia Rui 1 273459189qq.com Xu Tong-Peng 3 1034045558qq.com Jin Fei-Yan 1 759729211qq.com Liu Zhi-Jun 1 sm13776403108126.com Chen Jin-fei 4 1423594097qq.com Zhang Er-Bao 1 273459189qq.com De Wei 1 deweinjmu.edu.cn Wang Zhao-Xia 2 zhaoxiawang88hotmail.com 1Department of Biochemistry and Molecular Biology Nanjing Medical University Nanjing 210029 Peoples Republic of China 2Department of Oncology Second Affiliated Hospital Nanjing Medical University Nanjing Jiangsu 210011 Peoples Republic of China 3Department of Oncology First Affiliated Hospital Nanjing Medical University Nanjing Peoples Republic of China 4Department of Oncology Nanjing First Hospital Nanjing Medical University Nanjing P. R. China 2014 21 3 2014 13 68 68 16 9 2013 13 3 2014 Copyright 2014 sun et al.; licensee BioMed Central Ltd. 2014 sun et al.; licensee BioMed Central Ltd. This is an Open Access distributed under the terms of the Creative Commons Attribution License (http://creativecommons./licenses/by/2.0) which permits unrestricted use distribution and reproduction in any medium provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons./publicdomain/zero/1.0/) applies to the data made available in this unless otherwise stated. Background Recent evidence indicates that long noncoding RNAs (lncRNAs) play a critical role in the regulation of cellular processes such as differentiation proliferation and metastasis. These lncRNAs are found to be dysregulated in a variety of cancers. BRAF activated non-coding RNA (BANCR) is a 693-bp transcript on chromosome 9 with a potential functional role in melanoma cell migration. The clinical significance of BANCR and its molecular mechanisms controlling cancer cell migration and metastasis are unclear. Methods Expression of BANCR was analyzed in 113 non-small cell lung cancer (NSCLC) tissues and seven NSCLC cell lines using quantitative polymerase chain reaction (qPCR) assays. Gain and loss of function approaches were used to investigate the biological role of BANCR in NSCLC cells. The effects of BANCR on cell viability were evaluated by MTT and colony formation assays. Apoptosis was evaluated by Hoechst staining and flow cytometry. Nude mice were used to examine the effects of BANCR on tumor cell metastasis in vivo. Protein levels of BANCR targets were determined by western blotting and fluorescent immunohistochemistry. Results BANCR expression was significantly decreased in 113 NSCLC tumor tissues compared with normal tissues. Additionally reduced BANCR expression was associated with larger tumor size advanced pathological stage metastasis distance and shorter overall survival of NSCLC patients. Reduced BANCR expression was found to be an independent prognostic factor for NSCLC. Histone deacetylation was involved in the downregulation of BANCR in NSCLC cells. Ectopic expression of BANCR impaired cell viability and invasion leading to the inhibition of metastasis in vitro and in vivo. However knockdown of BANCR expression promoted cell migration and invasion in vitro. Overexpression of BANCR was found to play a key role in epithelial-mesenchymal transition (EMT) through the regulation of E-cadherin N-cadherin and Vimentin expression. Conclusion We determined that BANCR actively functions as a regulator of EMT during NSCLC metastasis suggesting that BANCR could be a biomarker for poor prognosis of NSCLC. Background Non-small cell lung cancers (NSCLCs) including adenocarcinomas and squamous cell carcinomas are the predominant forms of lung cancer and account for the majority of cancer deaths worldwide [1]. Despite recent advances in clinical and experimental oncology the prognosis of lung cancer remains poor with a 5-year overall survival rate of around 11% [2]. A continuing problem of NSCLC tumorigenesis is the metastasis of cancer cells which is the main cause of death in patients. Thus a detailed understanding of the mechanisms and molecular pathways activated in metastatic cells is crucial in identifying new treatment options for anticancer therapy that target metastasis. The invasion and metastasis of cancer cells are landmark events that involve many changes in cellular behavior and lead to different steps of the metastatic cascade [34]. One of the most crucial steps in the metastatic cascade is the acquisition of invasive capabilities including turnover of cell-cell junctions degradation of the cell matrix and activation of pathways that control cytoskeletal dynamics in cancer cells. This process is accompanied by multiple changes in gene expression such as the loss of epithelial markers and a gain in mesenchymal markers [56]. Over the past decade cell and tumor biologists have identified the key role of epithelial-mesenchymal transition (EMT) in cancer cell metastasis a biological process where epithelial cells lose their polarity and undergo transition into a mesenchymal phenotype [7]. EMT enhances tumor cell invasion in response to environmental triggers and augments invasive functions by promoting Rac-dependent mesenchymal migration and also contributes to cell growth and survival [89]. Important hallmarks of EMT include the loss of E-cadherin expression and increased expression of non-epithelial cadherins such as N-cadherin. The loss of E-cadherin expression is a fundamental event in EMT and a crucial step in the progression of papillomas to invasive carcinomas [10]. To date substantial effort has been devoted to understanding how EMT is regulated during cancer progression. It has been verified that EMT can be initiated by external signals such as hepatocyte growth factor (HGF) epidermal growth factor (EGF) transforming growth factor (TGF)-b and fibroblast growth factor (FGF) [11]. In addition to these signaling pathways triggered by membrane receptors recent studies have highlighted the importance of noncoding RNAs in the regulation of the epithelial phenotype by controlling EMT inducers. The miR-200 family has been found to control EMT by downregulating the expression of Zeb factors [12]. Furthermore the long noncoding RNA (lncRNA) MALAT-1 promoted EMT by regulating ZEB1 ZEB2 and Slug expression and activating Wnt signaling [13]. The lncRNAs are important new members of the ncRNA family that are greater than 200 nt and are unable to be translated into proteins. These lncRNAs are often expressed in a spatial- and temporal-specific pattern. Although very few lncRNAs have been characterized in detail they have been found to participate in a large range of biological processes including modulation of apoptosis and invasion reprogramming stem cell pluripotency and parental imprinting. These findings indicate that lncRNAs play a major role in the regulation of the eukaryotic genome [14-16]. Researchers have linked the dysregulation of lncRNAs with diverse human diseases in particular cancers [17-19]. Therefore identification of cancer-associated lncRNAs and investigation of their molecular and biological functions in controlling EMT are important in understanding the molecular biology of NSCLC metastasis and progression. BRAF-activated non-coding RNA (BANCR) an 693-bp lncRNA on chromosome 9 was firstly found by Ross J. Flockhart et.al via RNA-seq screen for transcripts affected by the expression of the oncogene BRAFV600E. BANCR is overexpressed in melanoma cells and crucial for melanoma cell migration [20]. In this study we investigated the effects of BANCR expression on NSCLC cell phenotypes in vitro and in vivo. Moreover we also showed that alteration of BANCR expression can influence E-cadherin N-cadherin and Vimentin protein levels which indicated that BANCR affected NSCLC cells invasion and metastasis partly via epithelial-mesenchymal transition. This study advances our understanding of the role of lncRNAs such as BANCR as a regulator of pathogenesis of NSCLC and facilitate the development of lncRNA-directed diagnostics and therapeutics. Results BANCR expression was downregulated and correlated with poor prognosis of NSCLC BANCR expression levels were investigated in 113 paired NSCLC samples and adjacent histologically normal tissues using quantitative polymerase chain reaction (qPCR) assays. BANCR expression was significantly downregulated (P?<?0.01) in 79% (89/113) of cancerous tissues compared with normal tissues (Figure 1A). BANCR expression levels in NSCLC were significantly correlated with tumor size (p?=?0.001) advanced pathological stage (p?<?0.001) and lymph node metastasis (p?=?0.001). However BANCR expression was not associated with other parameters such as gender (p?=?0.232) and age (p?=?0.616) in NSCLC (Table 1). The clinical data for all patients were summarized in Additional file 1: Table S1. Figure 1 Relative BANCR expression in NSCLC tissues and its clinical significance. (A) Relative expression of BANCR in NSCLC tissues (n?=?113) compared with corresponding non-tumor tissues (n?=?113). BANCR expression was examined by qPCR and normalized to GAPDH expression. Results were presented as the fold-change in tumor tissues relative to normal tissues. (B) BANCR expression was classified into two groups. (C D) KaplanMeier disease-free survival and overall survival curves according to BANCR expression levels. *P?<?0.05 **P?<?0.01. Table 1 Correlation between BANCR expression and clinicopathological characteristics of NSCLC patients (n?=?113) Characteristics BANCR P High no. cases (%) Low no. cases (%) Chi-squared test P-value Age(years) 0.616 ?65 29(54.7) 30(50.0) >65 24(45.3) 30(50.0) Gender 0.232 Male 35(66.0) 33(55.0) Female 18(34.0) 27(45.0) Histological subtype 0.466 Squamous cell carcinoma 30(56.6) 38(63.3) Adenocarcinoma 23(43.4) 22(36.7) TNM Stage " | 1 |
" IBDFecal calprotectinEndoscopic activityIBD noninvasive managementThe term IBD is usually used for referring to a group of ammatory gastrointestinal diseases mainly Crohn'sdisease and ulcerative colitis Accordingly IBD arises as a result of inappropriate immune response to intestinalcommensal anisms among genetically susceptible individuals Performing colonoscopy and histopathologicevaluation on an amed bowel biopsy specimen are currently considered as gold standards for diagnosis andmanagement of IBD Correspondingly these techniques are known to be invasive and costly In recent decadesfecal calprotectin as a biomarker has received much attention for the diagnosis and noninvasive managementof IBD Up to now many studies have investigated the eï¬cacy of fecal calprotectin in the areas of IBD diï¬erentiation from IBS prediction of endoscopic and histologic activities of IBD and prediction of disease recurrenceAlthough some of these studies have reported promising results some others have shown significant limitationsTherefore in this paper we reviewed the most interesting ones of these studies after a brief discussion of thelaboratory measurement of fecal calprotectin Moreover we attempted to provide an answer for the question ofwhether fecalcalprotectin could be considered as a potential surrogate marker for colonoscopy IntroductionInï¬ammatory bowel disease IBD is a long life disease with remission and relapse periods IBD arises as a result of inappropriateimmune response to intestinal commensal anisms in individualswith genetic predisposition and consequently causes ammation andintestinal ulcers [] In addition IBD has a complex pathogenesis andmany factors such as dysbiosis oxidative stress and epigenetics thatmay also be involved in disease pathogenesis [] Ulcerative colitisUC and Crohn's diseases CD are known as two main forms of IBDAccordingly these diseases cause intestinal ulcers and some annoyingsymptoms such as diarrhea abdominal pain and rectal bleeding Occasionally the severity of these symptoms is very high which can leadpatients to be hospitalized In this regard therapeutic approaches totreat these diseases mainly focus on prolonging remission and are almost similar however diï¬erential diagnosis can also help to treat thedisease in a more eï¬ective way For example 5ASA which is acommon drug in the treatment of IBD is less eï¬ective on maintainingremission in CD patients On the other hand antibiotic therapy is notrecommended for the treatment UC but it can be eï¬ective on CD patients [][] Diï¬erential diagnosis is a serious challenge because CDand UC have significant similarities in terms of their clinical endoscopic and histological features However there are some diï¬erencesbetween UC and CD which are summarized in Table1 In addition tointestinal complications UC and CD also have significant extraintestinal manifestations For example it was shown that UC is significantly associated with Primary sclerosing cholangitis and CD is alsoassociated with cholelithiasis especially in cases that the ileum is involved [] Furthermore CD can cause ï¬stulas to the urinary systemwhich leads intestinal bacteria to enter the urethra and recurrent urinary tract infections [] Both CD and UC can cause several disorderssuch as arthritis Erythema nodosum pyoderma gangrenosum andanemia which are known as the most important extraintestinal manifestations of IBD [][] The latest statistics showed that the global Corresponding author at Department of Clinical Biochemistry and Laboratory Medicine Faculty of Medicine Tabriz University of Medical Sciences DaneshgahStreet PO Box Tabriz IranEmail address vagharimtbzmedacir M VaghariTabari101016jcca202008025Received July Received in revised form August Accepted August Available online August Elsevier BV All rights reserved 0cF KhakiKhatibi et alTable1Clinical endoscopic and histological features of CD and UCClinical FeaturesFeaturesRectal bleedingAbdominal painFeverMucus defectionIntestinal obstructionPerineal diseasePostoperative recurrenceASCA positiveANCA positiveEndoscopic FeaturesCDOccasionallyFrequentlyFrequentlyOccasionallyYESYESYESFrequentlyNot commonUCFrequentlyOccasionallyNot commonFrequentlyNONONONot commonFrequentlyFeaturesCDUCLocationMucosal involvementDepth of ulcerationï¬stulaCobblestone appearanceAphthous ulcerationMucosal friabilityHistological featuresFeaturesGranulomasCrypt abscessesPatchinessAny part of GI tractDiscontinuousDeepYesYESFrequentlyNot commonCDFrequentlyNot commonFrequentlyColon and rectumContinuoussuperï¬cialNONOOccasionallyFrequentlyUCRareFrequentlyNot commonprevalence of IBD currently is on the rise and it is not an exaggerationif we consider it as a global serious health problem [] According to areport published in IBD has the highest prevalence rate inEurope and its prevalence in the newly industrialized countries of AsiaAfrica and South America also appears to be increased over the pastthree decades []Unfortunately the peak of the disease is at the young age of years old [] therefore in addition to the suï¬ering from icts on the patients it also has many negative eï¬ects on societyMoreover many ï¬nancial burdens are annually imposed on countriesfor controlling and treating this chronic disease The invasive diagnosticand therapeutic measures are currently undertaken to diagnose andmanage IBD which are unpleasant for patients as well as having thehigh associated costs Now the gold standard method for diagnosingIBD and monitoring patient status is performing colonoscopy examination and histopathologic evaluation which are invasive measures[] Therefore in recent years many studies have been conducted toï¬nd a suitable laboratory marker with suï¬cient sensitivity and speciï¬city for the purpose of diagnosing and noninvasive management ofIBD A high proportion of these studies have investigated the eï¬cacy offecal calprotectin in diagnosing and monitoring patients Althoughsome of these studies reported auspicious results there are still somedoubts on the eï¬ectiveness of fecal calprotectin on diagnosing andmonitoring IBD patients So in this review we addressed the advantages and limitations of fecal calprotectin for the diagnosis andmanagement of IBD The role of fecal calprotectin in diagnosis and management ofIBDThe eï¬cacy of fecal calprotectin as an laboratory marker in various areas of IBD diagnosis and management have been studied including IBD diï¬erentiation from irritable bowel syndrome IBS evaluation of endoscopic activity of the disease evaluation of histologicalactivity of the disease and prediction of disease recurrence andClinica Chimica Acta response to treatment In following after a brief introduction andmentioning the important points regarding laboratory measurement offecal calprotectin we reviewed the most interesting ï¬ndings in all ofthe abovementioned areas Calprotectin A clinically valuable proteinCalprotectin is an antimicrobial protein mainly secreted by neutrophils This protein competes with bacteria over zinc thus kills thebacteria However this is not the only contribution that it has to antimicrobial activity Moreover this protein has many potential clinicalapplications such as the elevated serum levels that have been observedunder various immunological and immunopathological conditionsSerum calprotectin levels rapidly increase in response to bacterial infections in the kidney and heart or during transplant rejection At theearly stages of ammation of the lung serum calprotectin can also beconsidered as a reliable marker besides plasma levels of calprotectinappear to be useful in reï¬ecting disease activity in ammation of thejoints [] In addition it has been demonstrated that serum calprotectin levels are increased in patients with bacterial sepsis so it can beconsidered as a reliable biomarker [] In Neonatal Sepsis the serumlevel of calprotectin increases as well as a sensitivity of and aspeciï¬city of that have been reported for serum calprotectin indiagnosis of Neonatal Sepsis [] It has been recently shown thatserum calprotectin levels increase in patients with aneurysmal subarachnoid hemorrhage and higher levels in the ï¬rst of onset areassociated with a poor prognosis at the ï¬rst three months [] Serumcalprotectin levels also increase in patients with rheumatoid arthritisand even in patients with a moderate to high disease activity who havenormal or low CRP levels so they appear to be more eï¬cient at reï¬ecting disease activity []Some studies have also investigated the eï¬cacy of serum calprotectin in the diagnosis of cancers Correspondingly in one of thesestudies it was shown that serum calprotectin levels significantly increased in patients with laryngeal carcinoma compared with healthyindividuals and those with benign laryngeal pathologies Moreover inthis study a direct relationship was also observed between serum levelsof calprotectin and stage of cancer [] Another study showed that theserum level of calprotectin increased in patients with papillary thyroidcarcinoma but it significantly decreased after operation [] Alsoregarding the eï¬cacy of serum and saliva calprotectin for the diagnosisof IBD impressive results have been reported [][] A study onpatients with IBD both UC and CD have shown that serum calprotectinlevels were directly correlated with fecal calprotectin levels and weremore potent in IBD diagnosis compared to CRP and albumin This studyalso indicated that the combination of serum calprotectin with CRP oralbumin can be helpfulin the prediction of treatment escalationespecially in patients with CD [] However no significant correlationwas observed between serum calprotectin and fecal calprotectin levelsin patients with CD and UC as well as a slight correlation betweenserum calprotectin level and CRP that was observed only in patientswith UC [] Another study showed that the serum level of calprotectin was significantly higher in patients with CD compared to healthyindividuals In addition although a significant correlation was observedwith the clinical activity of the disease no significant correlation wasfound between the level serum calprotectin and endoscopic activity ofthe disease [] The eï¬cacy of salivary calprotectin in the diagnosisof IBD has also been studied which showed that salivary calprotectinsignificantly increased in patients with IBD compared to healthy individuals In this study AUC values for unstimulated saliva and stimulated saliva to distinguish IBD patients from healthy individualswere reported to be and respectively [] However thepopularity of calprotectin is mainly due to the use of fecal calprotectinin the diagnosis and management of IBD that is discussed in the following 0cF KhakiKhatibi et alClinica Chimica Acta Laboratory measurement and reference intervalFecal calprotectin is a stable protein that remains stable for daysat room temperature [] This property is an excellent advantage for alaboratory marker Also it seems that keeping the specimen at refrigerated temperature °C can increase the stability of fecal calprotectin [] However evidence has been obtained regarding thatthe stability of this protein decreases after staying for three days atroom temperature On the other hand it is not also recommended tokeep samples in the refrigerator for more than days [] It seemsthat fecal calprotectin remains stable up to one year at °C []Measurement of fecal calprotectin can be done both qualitatively andquantitatively Accordingly in the qualitative measurement monoclonal antibodies are used to detect fecal calprotectin and the positiveresults are characterized by the appearance of colored lines on the testcassette However in the qualitative one only positive or negative results are reported and despite of sensitivity test speciï¬city in theevaluation of disease activity was reported to be only It seems thatthe main application of this test is to diï¬erentiate healthy individualsfrom IBD patients rapidly however some studies have shown that it isnot accurate enough in this case as well [][] Nevertheless asignificant concordance has been reported between home test resultsIBDoc and fecal calprotectin laboratory measurement results whenQuantum Blue calprotectin ELISA kit was used Notably the agreements between results were and depending on the selectedcutoï¬s [] Several commercial kits are also available for fecal calprotectin qualitative test known as rapid calprotectin These tests reportpositive results ranged from to µgg There are also severalcommercial kits that can be used for the quantitative measurement offecal calprotectin These kits are usually designed in terms of the ELISAmethod and some have a measurement range between and µgg Moreover the chemiluminescence immunoassays CLIAmethod can also detect values between and µgg Fluoro enzyme immunoassays FEIA and particle enhanced turbidimetric immunoassays PETIA can also be used for the measurement of fecalcalprotectin In this regard one of the most serious challenges to thelaboratory evaluation of fecal calprotectin is the determination of theupper limit in healthy individuals Among healthy adults there is asignificant agreement on µgg as an upper limit One study suggested values up to µgg in people over years old and up to µgg in children aged between and years old as referenceranges of fecal calprotectin in healthy individuals []Fecal calprotectin levels appear to be higher in healthy infants andchildren under four years old than in adults and further studies areneeded in this regard to determine the acceptable upper limit for diagnosis of pediatric IBD [] Table lists the median levels of fecalcalprotectin in healthy individuals with diï¬erent ages reported in somestudies According to these reports age can aï¬ect fecal calprotectinlevels Fecal calprotectin and IBD diagnosisOnly a small percentage of patients complaining of abdominal painand diarrhea have IBD In many cases IBS as a functional gastrointestinal disorder is known as the cause of such clinical symptomsPatients with IBS have normal colonoscopy results while IBD patientsindicate abnormal colonoscopy results and have intestinal ulcersUnfortunately the significant prevalence of IBS and the overlap between clinical symptoms and IBD can increase the colonoscopy rateTherefore a noninvasive diagnostic marker can be very helpful in thisregard Notably the ï¬rst evidence of the eï¬cacy of fecal calprotectin inthe diagnosis of IBD was obtained in the 1990s Røseth et al in proposed a method for measuring Calprotectin in stool specimens []One of the ï¬rst and most interesting studies regarding fecal calprotectinutility in IBD diagnosis was the study by Røseth et al published in In this study patients with ulcerative colitis were studied and according to their results fecal calprotectin levels are higher in patientswith ulcerative colitis compared to healthy controls This study havealso shown that even patients with low disease activities had higherlevels of fecal calprotectin compared to healthy individuals []Subsequent studies somehow conï¬rmed and complemented the ï¬ndings of this study In another study published in AUC values of CI were reported for fecal calprotectin in thediagnosis of colorectal ammation [] Moreover in a study onchildren with IBD it was shown that the level of fecal calprotectin washigher in these patients compared to healthy children so it can beconcluded that it is also directly correlated with ESR levels [] In astudy published in Kolho et al reported AUC values of CI for fecal calprotectin in the diagnosis of pediatric IBD [] In a study on patients with Crohn disease a sensitivity of and a speciï¬city of at cutoï¬ of μgg have been reportedfor fecal calprotectin in diagnosis of the disease [] The results of ourrecent study along with other studies showed that fecal calprotectin ispreferred over traditional ammatory biomarkers such as CRP andESR in the diagnosis of IBD [][] Diamanti et al reported a sensitivity of and a speciï¬city of for fecal calprotectin at a cutoï¬ of μgg in IBD diagnosis [] In our recent study a sensitivityof and a speciï¬city of at a cutoï¬ of μgg were observed for fecal calprotectin in the diagnosis of IBD however oursample size was and the majority of patients were in the active phaseof the disease []In another study conducted on patients with ulcerative colitis asensitivity of and a speciï¬city of at cutoï¬ of μgg havebeen reported in this regard [] In one study it was shown that fecalcalprotectin in cutoï¬ of μgg is able to distinguish patients withIBD from patients without IBD patients with diseases other than IBDpatients with IBS and healthy persons with sensitivity and speciï¬city [] Caviglia et al in their study reported a sensitivity of and a speciï¬city of at a cutoï¬ of μgg for fecalcalprotectin in diï¬erentiating between IBS and IBD [] Howeversome studies have reported significantly lower values Accordingly in astudy on patients with ulcerative colitis Kalantari et al reported asensitivity of and a speciï¬city of at a cutoï¬ of μgg []Besides there is a considerable agreement between fecal calprotectinand capsule endoscopy ï¬ndings in patients with Crohn's disease Asensitivity of and a speciï¬city of have also been reported at acutoï¬ of mgkg for fecal calprotectin in predicting CE ï¬ndings anddiagnosis of Crohn's disease [] In another study lower sensitivityand speciï¬city rates sensitivity speciï¬city were reportedfor fecal calprotectin in this regard [] Furthermore in one studythat examined the eï¬cacy of fecal calprotectin in predicting wirelesscapsule endoscopy ï¬ndings a sensitivity of and a speciï¬city ofTable Reported median levels of fecal calprotectin in healthy individuals of diï¬erent agesAgesMedian levels of fecal calprotectin range µggNumber of subjectsUsed kitUp to monthChildren yearsChildren yearsAdultsOver years Bühlmann ELISABühlmann ELISACALPRO® Calprotectin ELISA Test ALPPhiCalPhicalReference[][][][][] 0cF KhakiKhatibi et al were reported for this biomarker at μgg in the diagnosis ofsmall bowel ammation in Crohn's disease [] Given these ï¬ndings it seems that fecal calprotectin has no ideal sensitivity and speciï¬city for the diagnosis of IBD where the small intestine is involvedBesides there are some preanalytical limitations which are explainedin the next sections Therefore optimistically speaking fecal calprotectin measurement can eliminate the need for colonoscopy Howeverin a metaanalysis performed to evaluate the eï¬cacy of fecal calprotectin and some other ammatory markers to diï¬erentiate betweenIBD and IBS the probability of IBD was less than at fecal calprotectin values lower than µgg or CRP values lower than mgdL[] Therefore it seems that fecal calprotectin can be helpful at leastin ruling out the possibility of IBD in patients with IBSlike symptoms aswell as reducing the rate of colonoscopy Moreover it should be notedthat although a systematic review has reported pooled sensitivity andspeciï¬city above for fecal calprotectin to diï¬erentiate between IBDand IBS it emphasized more on the possibility of falsepositive resultsin low cutoï¬ points [] Hence performing extensive studies indiï¬erent countries on the healthy population and the IBD patient is beneeded to determine a suitable cutoï¬ with maximum sensitivity andspeciï¬city and minimum falsepositive resultsTable summarizes the results of various clinical investigationsregarding fecal calprotectin utility in the diï¬erential diagnosis of IBDfrom IBS and Table4 summarizes some metaanalysis results in thisregard As shown in Table the most important limitation of the majority of clinical studies conducted to date is the small sample size Alarge global study may be helpful in providing a more precise evaluation of fecal calprotectin clinical value in discrimination between IBDand nonIBD diseases Fecal calprotectin and endoscopic and histologic activity evaluationUndoubtedly one of the most serious challenges in the managementof IBD is evaluating the endoscopic and histologic activities of thedisease Nowadays colonoscopy and histopathologic examinations arethe routine tools for the assessment of mucosal healing in patients withIBD As noted earlier several scoring systems have been devised toscore disease activity based on the ï¬ndings of colonoscopy and histopathologic examinations In recent years many promising results havebeen reported regarding the correlation between these scores and fecalcalprotectin levels In addition many studies have been performed inthe last decade all of which cannot be reviewed in this article The ï¬rstevidence of a link between fecal calprotectin and disease endoscopicactivity was obtained in the late 1990s In one of the ï¬rst studiesRoseth et al found a significant correlation between fecal calprotectinlevels and endoscopic and histologic activities in patients with ulcerative colitis [] Furthermore in another study they observed that IBDpatients who were in remission clinically and had normal fecal calprotectin levels less than mgL had normal colonoscopy results[] These interesting ï¬ndings indicate that fecal calprotectin can beconsidered as a biomarker in the evaluation of endoscopic activity andClinica Chimica Acta Table4summarized results of some metaanalysis regarding the utility of fecal calprotectin in discrimination between patients with IBD and without IBDSample sizePooled SensitivityPooled Speciï¬cityReferences[][][][][]mucosal healing in IBD patients Also these studies were the startingpoint of extensive studies that have been conducted up to now In astudy conducted on patients with Crohn's disease Sipponen et alinvestigated the sensitivity and speciï¬city of fecal calprotectin in predicting endoscopic activity of Crohn's disease [] Correspondinglythe researchers used the Crohn's Disease Endoscopic Index of SeverityCDEIS scoring system in their study to evaluate the endoscopic activity of Crohn's disease As a result they found that there was a significant correlation between the endoscopic activity of the disease andthe level of fecal calprotectin Besides the ï¬ndings of this study demonstrated that fecal calprotectin at µgg cutoï¬ can predict theendoscopic activity of Crohn's disease with sensitivity and speciï¬city In another study CDEIS and Mayo Disease Activity IndexMDAI were used to evaluate the endoscopic activity of Crohn's diseaseand ulcerative colitis respectively According to the results of thatstudy on IBD patients there was a significant correlation between fecalcalprotectin levels and disease endoscopic activity [] Another studyshowed that fecal calprotectin is more strongly correlated with theendoscopic activity of the disease in ulcerative colitis compared to theRachmilewitz clinical activity index In addition in this study theoverall accuracy of fecal calprotectin for endoscopically active diseaseidentiï¬cation was obtained as []Some studies have also shown the superiority of fecal calprotectinover traditional ammatory markers like CRP Besides one studyfound that fecal calprotectin was more strongly correlated with theSimple Endoscopic Score for Crohn's disease SESCD compared to theCRP and even Crohn's disease activity index CDAI [] The modiï¬edBaron Index was also used in another study to evaluate the endoscopicactivity of ulcerative colitis As a result it was shown that calprotectinis more strongly correlated with the endoscopic activity of ulcerativecolitis compared to CRP and clinical activity of the disease [] In thisregard similar results were also observed in our recent study in whichthe Ulcerative Colitis Endoscopic Index of Severity UCEIS and SESCDwere used [] Therefore fecal calprotectin appears to be superior totraditional ammatory markers in the prediction of IBD endoscopicactivity The high values of sensitivity and speciï¬city that were mentioned earlier have raised the hope that using fecal calprotectin canreduce colonoscopy rate for patients monitoring However severalrecent studies have reported some significantly lower values Accordingly in a recent study in which Mayo Endoscopic Score [MES] wasused to evaluate the endoscopic activity of ulcerative colitis aTable Summary of the results of some studies regarding the utility of fecal calprotectin in discrimination between patients with IBD and without IBDNumber of IBD patientsAge groupLocationCut oï¬SensitivitySpeciï¬city CD and UC CD and UC CD and UC and unclassiï¬ed68CD and UC CD and UC and unclassiï¬ed CD and UC CD and UC UC CD UCAdultsAdultsAdultsBoth adult and pediatricpediatricAdultspediatricAdultsAdultsBoth adult and pediatricTaiwanChinaItalySpainFinlandIranItalyIranDenmarkIndia48µgg µgg150µgg150µgg595µgg784µgg160µgg164µgg150µgg188µggAUCReferences[][][]SPSreï¬dbib60[][][][][][][] 0cF KhakiKhatibi et alClinica Chimica Acta Table Summary of the results of some studies regarding the correlation of fecal calprotectin with endoscopic activity in IBD patientsAge groupStudylocationUsedendoscopicactivity indexCorrelationcoeï¬cientrReferenceNumberof IBDpatients CD UC UC CD UCAdultsAdultsAdultsAdultsAdultsFinlandIranSwitzerlandSwitzerlandSwitzerland Modiï¬edCDEISUCEISRachmilewitzSESCD UC CDAdultsAdults UC CD UC CD CD UC UCAdultsAdultsAdultsAdultsAdultsAdultsAdultsBaron ScoreRachmilewitzSESCDGermanyUSA andCanadaJapanItalyItalyBrazilFranceFranceSouth Korea UCEISMattsSESCDMayo scoreSESCDCDEISMayo score[][][][][][][][][][][][][][]sensitivity of and a speciï¬city of were reported for fecalcalprotectin at µgg to diï¬erentiate active endoscopic from inactiveMES or from MES or [] In another study the sensitivityand speciï¬city of fecal calprotectin at a cutoï¬ of µgg for diï¬erentiating MES ¤ in patients with ulcerative colitis were and respectively [] Overall as presented in Table several studiesperformed in diï¬erent countries reported the correlation between fecalcalprotectin and IBD endoscopic activity Although some of these studies reported a strong correlation some others reported a relativelyweak correlation As noted earlier there are significant diï¬erencesbetween the reports on the sensitivity and speciï¬city of fecal calprotectin to predict the endoscopic activity of IBD Undoubtedly a widerange of factors from sample size and the inclusionexclusion criteriato preanalysis variables and indexes used to evaluate the endoscopicactivity may also contribute to these diï¬erences However fecal calprotectin does not appear to be a very reliable marker for the predictionof IBD endoscopic activity so currently it seems a bit optimistic toconsider fecal calprotectin as a reliable alternative for colonoscopy Inthis regard further studies are still needed However under some certain circumstances such as pregnancy or pandemics the use of fecalcalprotectin to evaluate IBD endoscopic activity can be helpfulPregnant patients with IBD have serious limitations for colonoscopyexamination and it has been recommended that colonoscopy should beonly performed in the second trimester of pregnancy and where there isa strong indication [] Therefore noninvasive markers such as fecalcalprotectin can be helpful during pregnancy In one study physicianglobal assessment [PGA] which is a clinical symptombased criterionwas used to evaluate IBD activity and subsequently the associationbetween fecal calprotectin and this criterion was investigated in pregnant women with IBD The results of this study showed a significantcorrelation between fecal calprotectin and PGA levels at prepregnancyduring pregnancy and postpartum stages [] In another study asignificant association was reported between fecal calprotectin levelsand clinical activity of IBD in pregnant women Moreover it was shownthat stool calprotectin at a cutoï¬ of mgkg had a sensitivity between and as well as a speciï¬city between and in the assessment of IBD clinical activity at diï¬erent stages ofpregnancy [] A recently published systematic review has also conï¬rmed the conclusions obtained from these studies [] According tothese results it seems that fecal calprotectin is not aï¬ected by physiological changes during pregnancy however it is significantly correlatedwith IBD clinical activity during pregnancy Therefore from the viewpoint of relatively acceptable sensitivity and speciï¬city in predictingthe endoscopic activity of IBD fecal calprotectin may be considered as anoninvasive biomarker for the evaluation of IBD endoscopic activity inpregnant women In addition under pandemic conditions fecal calprotectin can be very helpful Following the COVID19 pandemicwhich began in late and is still ongoing healthcare systems indiï¬erent countries were forced to impose significant limitations oncolonoscopy Therefore noninvasive IBD management and fecal calprotectin as a noninvasive laboratory marker have become moreimportant than before The combination between disease clinical activity and fecal calprotectin has been recommended as a noninvasiveapproach that can help in making decisions on treatment duringCOVID19 pandemic [] Therefore it seems that fecal calprotectincan be considered as an alternative for colonoscopy used for IBD endoscopic activity evaluation during pandemic Fecal calprotectin appears to be associated with IBD histologic activity as well Given thediï¬culty in the evaluation of the histologic activity of Crohn's disease[] some studies have been focused on the ulcerative colitis andmany scoring systems have been devised so far Correspondingly thesesystems score the disease's histologic activity based on histologic observationsTherefore for this purpose a biopsy of the intestinal tissue is required which can be prepared by colonoscopy and then sent to thelaboratory In this regard one of these histologic scoring systems isRoberts score that was used in one of our recent studies where weobserved a significant correlation between the level of fecal calprotectinand the histologic activity of ulcerative colitis which was calculatedbased on the Roberts scoring system [] Theede et al also used themodiï¬ed Harpaz Index and performed some interesting studies in thisregard In one of their studies fecal calprotectin was found to be significantly associated with the histologic activity of the ulcerative colitisand it was shown that it could predict histological mucosal healingAUC CI95 Sensitivity Speciï¬city andCutoï¬ mgkg [] In another study on patients with endoscopically inactive ulcerative colitis Mayo endoscopic score the researchers showed that patients with ulcerative colitis who were inendoscopic remission but had histologically active disease had higherlevels of fecal calprotectin compared to patients with no histologicallyactive disease versus mgkg P Also despite thehigh speciï¬city the sensitivity of fecal calprotectin in theprediction of score of histological activity was achieved as at mgkg [] In a recent study the Geboes | 2 |
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