oesophageal adenocarcinoma ( oac ) is the focus of intense research into the cause of the disease and treatment . in the context of oac screening , a biomarker capable of reliably predicting progression to dysplasia or cancer by increased expression is highly valuable . oesophageal cancer develops symptoms late in the course of this disease and carries a poor prognosis . in the uk , oesophageal cancer is the eleventh most commonly diagnosed cancer and , of greater concern , the sixth most common cause of death from cancer , which represented 1  :  20 of all cancer deaths in the uk ; 8,161 new cases were diagnosed in 2009 and the mortality rate was 7,610 in 2010 . the role of inflammation in the development of oac is not well understood , unlike its role in colon and breast cancers . however , this role in the progression of oac is now beginning to gain greater attention . proinflammatory cytokines and their pathways are associated with inflammation in barrett 's oesophagus and tumourigenesis . elucidating the association between inflammation and oac may contribute to the eventual prevention of oac . further assessment of these cytokines has recently been recommended , as these may be important targets for early diagnosis and chemoprevention drugs [ 6 , 7 ] . inflammatory factors are commonly associated with contributing to the development of tumours ; chronic inflammation is often associated with malignancy . abdel - latif et al .   concluded that chronic inflammation plays a crucial role in the pathogenesis of the disease where inflammatory cytokines are linked with advancement of tumourigenesis . il-17 induces a wide variety of factors , which include chemokines , cytokines , acute phase proteins , tissue remodelling proteins , antimicrobial proteins , and nitric oxide . cytokines that induce il-17 include il-6 and il-8 , and expression increases in both as the disease progresses . -defensin , indirectly induced by il-17 , recruits dendritic cells and t lymphocytes to the inflamed site in the oesophagus through interaction with chemokine receptor ( ccr ) 6 . chemokine ( c - c motif ) ligand ( ccl ) 20 is up - regulated by il-17 , a chemokine that also attracts dendritic cells and t lymphocytes to the inflamed site . -defensins and ccl20 serve to recruit and extend the th17 population at the inflamed site . il-17 is implicated in several chronic inflammatory diseases , including the autoimmune diseases , rheumatoid arthritis ( ra ) , and psoriasis [ 10 , 11 ] . recent evidence suggests that the presence of infiltrating il-17 + cells , which include lymphocytes , mast cells , and neutrophils , in patients with oac , may have a pathogenic role in this disease . several studies have demonstrated a high frequency of il-17 + cells in inflammation - related cancers including oac , nonsmall cell lung carcinoma , where il-17 has been implicated in metastasis of lung cancer by promoting lymphangiogenesis   and hepatocellular carcinoma where il-17 is also believed to promote angiogenesis . the aim of this pilot study was to evaluate the expression of the proinflammatory cytokine il-17 along the barrett 's metaplasia - dysplasia - carcinoma sequence by establishing the expression levels of il-17 in columnar epithelium , intestinal metaplastic cells , and dysplastic / glandular neoplastic cells . ethics statementthis study , which included the use of archived human tissue samples , was approved in writing by the proportionate review sub - committee of the national research ethics service committee east midlands - nottingham 2 research ethics committee in the united kingdom , reference 11/em/0384 , on november 23 2011 . written permission for this study was also received from the research and development directorate at aintree university hospital national health service foundation trust , reference 451/11 , on november 28 2011.forty distal oesophageal biopsy samples embedded in formalin - fixed paraffin blocks , originally collected between 2010 and 2011 , were retrieved from the cellular pathology department archive at aintree university hospitals national health service foundation trust , uk ; ten from each of the following endoscopic examination result categories : normal oesophagus , reflux oesophagitis , barrett 's oesophagus , and oesophageal adenocarcinoma . the mean age of the patients involved with this study was 71 years ( range : 4988).the il-17 expression in columnar epithelium , columnar metaplastic cells , and dysplastic / glandular neoplastic cells was compared . a negative control was established using an internal control on each slide ( squamous mucosa ) , and a positive control was established , which was taken from normal kidney tissue . the rabbit polyclonal antibodies against human origin il-17 sc7927 from santa cruz biotechnology ( scbt ) have been used extensively in recent studies , including human immunohistochemical studies ; therefore , this il-17 antibody was selected for use by this study . the dilutions 1  :  50 , 1  :  100 , and 1  :  200 of the il-17 antibody were prepared as recommended by scbt , and the stained tissue sections were examined via microscopy to find the initial optimum dilution , which was 1  :  50 . further dilutions were prepared , which included 1  :  30 , 1  :  40 , 1  :  50 , and 1  :  60 , and of these , the 1  :  40 dilution was the optimum to visualise il-17 expression in the patient tissue samples . the same batch of this primary antibody was used throughout . all positive controls were taken from a human kidney biopsy .prior to staining , the antigens were unmasked at 97c with a target retrieval solution at ph 9.4 . 30  ml of dako envision flex target retrieval solution ( high ph 50x ) was added to 1.5  l of distilled water and treated at 97c for 20 minutes using the dako pt link machine ( dako).the primary il-17 antibody ( 1  :  40 dilution in dako real antibody diluent , santa cruz biotechnology , usa ) was used in this study . staining was conducted using dako autostainer link 48 ( dako ) , which added 300  l of undiluted dako envision flex peroxidase - blocking reagent to each slide for 5 minutes . 300  l of diluted il-17 antibody was subsequently added to each slide for 30 minutes . the treatment continued by adding 300  l of undiluted dako envision flex+ rabbit ( linker ) to each slide for a further 15 minutes . after the linker treatment , 300  l of undiluted dako envision flex / horseradish peroxidase ( hrp ) was added to the slides for 20 minutes . this was followed by adding a 300  l of diluted chromogen reagent to each slide for 5 minutes , followed by a buffer wash , and then by a second application of 300  l of diluted chromogen for 5 minutes . the diluted chromogen was prepared by placing 20 drops of dako envision flex dab+ chromogen into 20  ml of dako envision flex substrate buffer . the final step of the automated autostainer process was to add 300  l of dako envision flex haematoxylin ( link ) to each slide for 5 minutes . between each treatment , the diluted wash buffer consisted of 0.5  l dako envision flex wash buffer ( 20x ) and 10  l of distilled water . this buffer was placed into a large container that was connected to the autostainer.before the cover slips were mounted , the sections were dehydrated by the leica st4040 system ( leica ) . the sections were dehydrated in ascending alcohols ( 50% , 70% , and 99% twice ) for 15 seconds each using alcohol methylated spirit 99% ( genta medical ) diluted with distilled water . after the section dehydration , the slides were placed into the automated cover slipper , the leica cv5000 ( leica ) . this study , which included the use of archived human tissue samples , was approved in writing by the proportionate review sub - committee of the national research ethics service committee east midlands - nottingham 2 research ethics committee in the united kingdom , reference 11/em/0384 , on november 23 2011 . written permission for this study was also received from the research and development directorate at aintree university hospital national health service foundation trust , reference 451/11 , on november 28 2011 . forty distal oesophageal biopsy samples embedded in formalin - fixed paraffin blocks , originally collected between 2010 and 2011 , were retrieved from the cellular pathology department archive at aintree university hospitals national health service foundation trust , uk ; ten from each of the following endoscopic examination result categories : normal oesophagus , reflux oesophagitis , barrett 's oesophagus , and oesophageal adenocarcinoma . the mean age of the patients involved with this study was 71 years ( range : 4988 ) . the il-17 expression in columnar epithelium , columnar metaplastic cells , and dysplastic / glandular neoplastic cells was compared . a negative control was established using an internal control on each slide ( squamous mucosa ) , and a positive control was established , which was taken from normal kidney tissue . the rabbit polyclonal antibodies against human origin il-17 sc7927 from santa cruz biotechnology ( scbt ) have been used extensively in recent studies , including human immunohistochemical studies ; therefore , this il-17 antibody was selected for use by this study . the dilutions 1  :  50 , 1  :  100 , and 1  :  200 of the il-17 antibody were prepared as recommended by scbt , and the stained tissue sections were examined via microscopy to find the initial optimum dilution , which was 1  :  50 . further dilutions were prepared , which included 1  :  30 , 1  :  40 , 1  :  50 , and 1  :  60 , and of these , the 1  :  40 dilution was the optimum to visualise il-17 expression in the patient tissue samples . the same batch of this primary antibody was used throughout . 30  ml of dako envision flex target retrieval solution ( high ph 50x ) was added to 1.5  l of distilled water and treated at 97c for 20 minutes using the dako pt link machine ( dako ) . the primary il-17 antibody ( 1  :  40 dilution in dako real antibody diluent , santa cruz biotechnology , usa ) was used in this study . staining was conducted using dako autostainer link 48 ( dako ) , which added 300  l of undiluted dako envision flex peroxidase - blocking reagent to each slide for 5 minutes . 300  l of diluted il-17 antibody was subsequently added to each slide for 30 minutes . the treatment continued by adding 300  l of undiluted dako envision flex+ rabbit ( linker ) to each slide for a further 15 minutes . after the linker treatment , 300  l of undiluted dako envision flex / horseradish peroxidase ( hrp ) was added to the slides for 20 minutes . this was followed by adding a 300  l of diluted chromogen reagent to each slide for 5 minutes , followed by a buffer wash , and then by a second application of 300  l of diluted chromogen for 5 minutes . the diluted chromogen was prepared by placing 20 drops of dako envision flex dab+ chromogen into 20  ml of dako envision flex substrate buffer . the final step of the automated autostainer process was to add 300  l of dako envision flex haematoxylin ( link ) to each slide for 5 minutes . between each treatment , the diluted wash buffer consisted of 0.5  l dako envision flex wash buffer ( 20x ) and 10  l of distilled water . this buffer was placed into a large container that was connected to the autostainer . before the cover slips were mounted , the sections were dehydrated by the leica st4040 system ( leica ) . the sections were dehydrated in ascending alcohols ( 50% , 70% , and 99% twice ) for 15 seconds each using alcohol methylated spirit 99% ( genta medical ) diluted with distilled water . after the section dehydration , the slides were placed into the automated cover slipper , the leica cv5000 ( leica ) . two experienced pathology laboratory staff independently assessed tissue sections for the staining intensity of il-17 , with scoring from 0 to 3 ( 0no staining , 1week intensity , 2medium intensity , and 3high intensity ) .   the one - way nonparametric anova ( kruskal - wallis test ) , and the mann - whitney u test were used to determine if there was a difference in il-17 expression . pairwise comparisons were made between the columnar epithelium , intestinal metaplastic cells , and dysplastic cells / glandular neoplastic cells . the association between disease progression and the level of expression of il-17 was evaluated using spearman 's rank correlation method to determine whether there was an association between disease progression and the expression of il-17 . the original haematoxylin and eosin ( h&e ) slides were used to compare the morphological features of the samples with the il-17 staining . expression levels of il-17 in columnar epithelium , intestinal metaplasia , and dysplasia / glandular neoplasia were compared . there was significant difference between the expression of il-17 between the three different cell types ( columnar epithelium , intestinal metaplastic cells , and dysplastic / glandular neoplastic cells ) , ( x = 21.852 , df = 2 , p < 0.001 , n = 29 , figure 1 ) . squamous epithelium included in the paraffin - embedded blocks demonstrated absent cytoplasmic staining and acted as an internal negative control ( figure 2 ) . kidney - tissue positive controls demonstrated strong il-17 expression with the collecting ducts ( figure 2 ) . at the optimal concentration , columnar epithelium demonstrated absent il-17 staining in 3 of 7 ( 43% ) or absent / weak staining ( figure 2 ) in 4 of 7 ( 57% ) . interestingly , the staining was distributed throughout the cytoplasm , and the stains were uniform for all the cell types examined ( n = 29 ) . for the 11 intestinal metaplasia samples , 5 of 11 ( 46% ) demonstrated moderate staining ( figure 2 ) , 4 of 11 ( 36% ) demonstrated weak staining , and 2 of 11 ( 18% ) demonstrated absent / weak staining . these staining scores were elevated when compared with columnar epithelium , as demonstrated by the staining intensity scores , and the difference was highly significant difference ( mwu , z = 3.242 , p = 0.001 , n = 18 ) . for the 11 dysplasia and glandular neoplasia regions , 6 of 11 ( 55% ) demonstrated strong staining ( figure 2 ) , 4 of 11 ( 36% ) demonstrated moderate staining , and 1 of 11 ( 9% ) demonstrated moderate / strong staining . these staining scores were elevated when compared with columnar epithelium , and also with intestinal metaplasia ; these were ( mwu , z = 3.598 , p < 0.001 , n = 18 ) and ( mwu , z = 3.483 , p < 0.001 , n = 22 ) , respectively . neutrophils ( n = 6 ) and mast cells ( n = 7 ) found in normal , barrett 's oesophagus , and oac cases were all strongly positive . surprisingly , only one case of plasma cells ( n = 1 ) was found in an oesophagitis case , and it was strongly positive . the spearman rank correlation coefficient ( non - parametric correlation analysis method ) revealed that there was a highly significant correlation ( rs = 0.883 , p < 0.001 , n = 29 ) between il-17 expression and the progression of the disease , where the disease progresses stepwise from columnar epithelium to intestinal metaplasia , and onwards towards dysplasia and glandular neoplasia . there was a strong positive correlation between disease progression and il-17 expression , and this suggests that il-17 expression increases as the disease progresses . ( k = 1.00 , n = 29 ) between the intensity scores provided by dr . their long working relationship together within the trust may have led to the high scoring concordance . the findings , that there is a highly significant increase in il-17 expression between columnar epithelium and intestinal metaplastic cells , and between intestinal metaplastic cells and dysplastic / glandular neoplastic cells in the distal oesophagus , are novel ( figure 1 ) . amongst the native squamous oesophagus and oesophageal columnar epithelium , there appears to be a stage in the transition between columnar epithelium to intestinal columnar epithelium where expression of il-17 becomes apparent . during the investigation some il-17 staining of the nuclei , but not cytoplasm , was observed in the columnar epithelium and squamous mucosa ; this may be an indication of the transition phase where il-17 becomes apparent . th17 cells are associated with mucosal immunity , particularly within the gastrointestinal tract , and autoimmune diseases . a study examining gastric cancer conducted by zhang et al .   found il-17 mrna expression in the majority of the tumour samples , and expression was not detected in the patient 's normal gastric tissue . this concurs with the finding of this study in that il-17 expression is elevated in tissue affected by oac at a level significantly greater level than that found in normal tissues . studied serum il-17 levels in patients with colorectal carcinoma and found significantly higher il-17 serum levels in patients with colorectal carcinoma than found in healthy subjects . their study indicated that il-17 , combined with other serum markers , is a valuable tumour marker in patients with colorectal carcinoma , which may provide additional information about the characteristics of the tumour . examining similar serum markers against a possible case of barrett 's oesophagus or oesophageal adenocarcinoma may also provide valuable diagnostic and prognostic information , which offers the prospect of an alternative cost - effective and noninvasive approach to the clinician . recent research suggests that there is a diverse range of il-17 producing cells amongst the immune system community , and innate immunity cells may secrete il-17 . mast cells and neutrophils were found in normal , barrett 's oesophagus , and oac cases . mast cells and neutrophils were associated with strong il-17 expression in this study , suggesting that these innate immune cells have an active role in maintaining high levels of il-17 amongst the disease - affected tissues . lin et al .   studied the pathogenesis of psoriasis and demonstrated that mast cells and neutrophils , rather than t lymphocytes , are the prominent immune cell types that express il-17 in the human skin . release of il-17 from mast cells and neutrophils may be important to the pathogenesis of oac and may have a similar pattern of chronic inflammation as seen in psoriasis . clear evidence of any lymphocyte involvement in the normal , barrett 's oesophagus , or oac cases was not found . however , the stroma was affected by strong background staining , and it was difficult to differentiate between stroma and inflammatory cells via microscopy . difficulties were also experienced in attempting to make this distinction in the corresponding archived haematoxylin and eosin stained slides ; there was significant background staining in most of these cases , and because of these problems , the stroma regions found in the cases examined were not included in the results . reducing the concentration of the il-17 antibody may have aided interpretation within the stroma , but at the expense of being able to interpret regions outside the stroma ; these regions ( outside the stroma ) would deviate from their optimum concentration . this study demonstrated that il-17 expression increases as the disease progresses along the metaplasia - dysplasia - carcinoma sequence , which potentially has a significant diagnostic , prognostic , and therapeutic value . understanding the differential expression of il-17 between benign and malignant tissue may allow diagnostic , prognostic , and therapeutic strategies to be developed . the evidence presented by this study suggests that il-17 has a role in the development of oac , which merits further investigation .
introduction . 
 this pilot study evaluated the expression of the proinflammatory cytokine il-17 along the barrett 's metaplasia - dysplasia - adenocarcinoma sequence by establishing the expression levels of il-17 in columnar epithelium , intestinal metaplastic cells , and dysplastic / glandular neoplastic cells . 
 immunohistochemical techniques were used to examine the accumulation of the proinflammatory cytokine il-17 in forty ( n = 40 ) formalin - fixed , paraffin - embedded oesophageal archived specimens across a range of endoscopic diagnostic categories , and a highly significant difference was found , where p  0.001 , in il-17 expression ( kruskall wallis and mann - whitney u ) between all the cell types examined . 
 there was also a strong positive correlation ( spearman 's rank correlation ) between disease progression and il-17 expression ( rs = 0.883 , p < 0.001 , n = 29 ) , il-17 expression was absent or absent / weak in columnar epithelium , weak to moderate in columnar metaplastic cells , and moderate to strong in dysplastic / neoplastic cells , which demonstrated that the elevation of il-17 expression occurs in the progression of the disease . 
 understanding the differential expression of il-17 between benign and malignant tissue potentially has a significant diagnostic , prognostic , and therapeutic value . 
 ultimately , this selective biomarker may be employed in routine clinical practice for the screening of oesophageal adenocarcinoma .