nucleophosmin mutations ( npm1 m ) occur in about one - third of acute myeloid leukaemias ( amls ) , and the current classification of myeloid neoplasms defined a recent entity of npm1-mutated aml with distinct biological , clinical , and prognostic features . moreover , the detection and quantification of npm1 m represents a major specific marker for the molecular monitoring of minimal residual disease ( mrd ) in aml , since it appears as an early initiating event in leukaemogenesis [ 3 , 4 ] . the expression of this marker is very stable during disease evolution , and the detection of increasing npm1 m expression levels seems strongly predictive for impending haematological relapse [ 5 , 6 ] . finally , patients ' stratification in international clinical protocols and the development of new targeted therapies rely on the npm1 status in aml . thus , the identification of npm1 m is of critical importance for the aml patients ' admission process . most of the npm1 m identified to date , as the type a mutation ( 7580% of cases ) , are exon 12 frameshift mutations [ 1 , 5 , 8 ] leading to an aberrant accumulation of the protein in the cytoplasm . several protocols and methods have been developed for the detection of npm1 m including dna sequencing of different mutation - specific rt - pcr assays [ 1013 ] , denaturing high - performance liquid chromatography , capillary electrophoresis , locked nucleic acid - mediated polymerase chain reaction clamping , and high - resolution melting analysis . although these methods possess a high specificity to assess npm1 mutational status at diagnosis , they always require direct sequencing for npm1 m characterization , a more expensive and time - consuming method . we therefore investigated a new strategy where ( i ) mutational status , ( ii ) distinction between npm1 mutation types , and ( iii ) quantitative value of the identified mutation at diagnosis would be rapidly obtained . a series of 337 aml patients ' samples were referred to our laboratory for the initial diagnosis of aml from march 2007 to july 2011 . mononuclear cells from bone marrow or blood samples were separated by ficoll - hypaque density gradient centrifugation ( histopaque ficoll-1077 , sigma - aldrich , saint louis , mi , usa ) and stored as cellular suspensions containing 10 cells . we extracted genomic dna from aliquots of 10 mononuclear cells using the qiaamp dna mini kit and the qiacube instrument ( qiagen , hilden , germany ) according to the manufacturer 's instructions , and aliquots containing dna at 5  ng/l were prepared . rna was extracted using the nucleospin rna ii kit ( macherey - nagel , dren , germany ) , aliquots containing 1  g rna were prepared , and reverse transcription was performed as previously described . figure 1 presents an overview of the 2-step strategy we suggest to detect ( first step ) and identify ( second step ) the presence of npm1 m . first , detection of npm1 m was carried out on genomic dna by pcr and high - resolution melting ( hrm ) analysis . pcr reactions were performed in a 20  l final volume containing 5  l of genomic dna and 300  nm of each primer ( eurogentec , seraing , belgium ) ( table 1 )   with 10  l of lightcycler 480 probe master 2x ( roche diagnostics , mannheim , germany ) , containing 3.2  mm mgcl2 and with 1  l of resolight dye 20x ( roche diagnostics ) as a nucleotide binding dye . amplification ( defining a 232  bp amplicon ) was achieved by 45 cycles of 95c for 10  secs , 56c for 15  secs , and 72c for 15  secs followed by a gene scanning analysis according to the manufacturers instructions by using the lightcycler 480 real - time pcr system instrument ( roche diagnostics ) . when hrm analysis revealed the presence of npm1 m , we proceeded to the second step ( figure 1 ) . identification and quantification of the different mutation types by allele - specific oligonucleotide real - time quantitative polymerase chain reaction ( aso - rq - pcr ) were performed by using 5 distinct rq - pcr tubes containing a common forward primer , one of five different reverse primers ( eurogentec ) designed to specifically target types a , b , c , d , and p mutations [ 11 , 12 ] , and a common probe ( life technologies corporation applied biosystems , carlsbad , ca , usa ) ( table 1 ) . each rq - pcr mixture reaction contained 5  l cdna , 300  nm of each primer , 200  nm probe , and 10  l of lightcycler 480 probe master 2x in a total volume of 20  l . preheating of the mixture at 95c for 5 minutes was followed by 45 cycles of a 3-step cycle procedure ( 10 seconds at 95c , 40 seconds at 62c , and 1 second at 72c ) . rq - pcr of the endogenous reference gene abl was accomplished as previously described [ 19 , 20 ] . all quantitative pcrs were performed using ipsogen plasmids ( ipsogen cancer profiler , new haven , ct , usa ) , and the assays were found to be linear over at least 5 orders of magnitude ( slope : 3.350 , 3.480 , 3.349 , 3.373 , 3.305 ; intercept : 40.27 , 40.53 , 39.83 , 39.66 , 39.93 for mutations a , b , c , d , and p , resp . ) . analysis was performed by a comparative cycle threshold ( ct ) method of relative quantification giving the amount of target , normalized to the abl gene as follows : ct = ct(npm1 m )  ct(abl ) . the mutation type of each sample was identified using table 2 , which indicates the ct profile obtained with each rq - pcr primer depending on the mutation type . this table had been previously built using a few patients ( 13 depending on mutation types ) carrying known npm1 m . considering that the smallest ct value ( as the ct values ) corresponds to the most specific primer , each mutation must be defined by the set of the different primers ' ct values obtained : mutations a , b , c , and p are clearly identified because the lowest ct values are obtained with primer a , b , c , or p , respectively , as compared to those obtained with the other specific primers ; in case of mutation d , both primers a and d have low ct value . these two mutations can be discriminated since the ct values of primers b , c , and p are far higher in case of type a than type d. mutations a , b , c , and p are clearly identified because the lowest ct values are obtained with primer a , b , c , or p , respectively , as compared to those obtained with the other specific primers ;   in case of mutation d , both primers a and d have low ct value . these two mutations can be discriminated since the ct values of primers b , c , and p are far higher in case of type a than type d. to validate our method , we performed direct sequencing on a proportion of positive and negative cases with primer npm1-as . pcr - amplified fragments from 20 hrm - negative samples and 38 hrm - positive samples ( cases with mutation detected by hrm analysis and identified by aso - rq - pcr using their ct profile ) were sequenced to confirm the results obtained with our strategy . among the 337 samples of aml diagnosis , the hrm screening revealed the presence of npm1 mutation in 88 of them ( 26.1% ) . typical results of hrm analysis are shown in figure 2(a ) , allowing distinction between mutated and nonmutated samples . to confirm the absence of mutations and make sure that the new assay does not give false negative results , we investigated by direct sequencing 20 cases that were considered as npm1 m negative with the hrm analysis . all the cases proved to be wild - type sequences , which allowed us to consider our strategy as highly specific .   among the 88 hrm - positive samples , the aso - rq - pcr and the use of table 2 allowed us to identify the mutation type in 86 samples : 69 carried type a , 10 type b , 1 type c , 5 type d , and 1 type p. the different mutation types obtained using the ct method were confirmed by direct sequencing in 36 samples ( 30 with mutation type a , 2 type b , 1 type c , 2 type d , and 1 type p ) , and none of them revealed any other mutation other than the one we identified with our ct method . two samples identified by the hrm screening step showed ct values which did not correspond to any of these mutation profiles showing the following values:ct = 0 with primer a and ct = 16 with primers b , c , d , and p for the first sample;ct = 15 with primer d and ct = 16 with primers a , b , d , and p for the second one . ct = 0 with primer a and ct = 16 with primers b , c , d , and p for the first sample ; ct = 15 with primer d and ct = 16 with primers a , b , d , and p for the second one . we then performed direct sequencing that revealed rare type q ( first sample ) and m ( second sample ) npm1 mutations . the determination of the npm1 mutation status in patients with aml is a new urgent requirement for patients enrolled in clinical trials , in order to stratify patients . although the presence of mutation is currently associated with better outcome , irrespective of the type , its characterization at diagnosis is absolutely necessary for the monitoring of residual disease ( mrd ) during followup to assess the effectiveness of treatment and may help to identify patients likely to relapse prior to any haematological relapse . for each npm1 m patient , the mrd was performed from rna with a high - sensitivity rq - pcr method using npm1 m specific primers as described above ( 79 follow - up samples ranging from 0.0014% to 2800% ; figure 2(b ) provides an example ) and demonstrates that the assays are also suitable for the mrd . although sanger sequencing represents so far the reference method to identify the mutation types for the first time at diagnosis , this expensive and labor - intensive technique does not represent the ideal way to routinely screen large numbers of patients . using our strategy , mutation types can be identified since the global ct profiles are unique and surely define exclusively one type of mutation , without requiring sequencing . besides , this method can be reproduced by each laboratory since it is based on the comparison of ct and not only on raw ct values which could fluctuate between laboratories . recently , barakat et al .   described a unique q - pcr strategy to detect 6 of the most common npm1 m . this method presents the advantage to perform only one pcr reaction in a single tube . nevertheless , it does not allow the identification of the mutation type which must be determined with an additional sequencing step to ensure the mrd . in addition , their assay was designed to screen only the most common npm1 mutations and can fail to detect other rare types . furthermore , among our 88 samples , we detected two rare mutation types ( 2.3% ) that could have been missed or incorrectly identified with a one - tube q - pcr strategy . although other mutations are rare , they must not be missed given the importance of npm1 m for the molecular followup and therapeutic approaches in clinical trials . we could also avoid the hrm screen step , since samples without npm1 m were not amplified by the use of the different specific primers ( 101 negative remission samples tested as negative control ) but , even if the m and q mutations were amplified by our aso - rq - pcr approach , the first screening hrm step avoids missing truly rare but real npm1 mutations . these results allow us to consider that our strategy is highly specific , and demonstrate in a large group of patients a reliable alternative to npm1 sequencing in order to identify the most common npm1 m . this method provides a useful and inexpensive tool , easy to use in routine practice , and thus could be included in the genetic diagnosis workup of aml disease .
somatic mutations in the npm1 gene , which encodes for nucleophosmin , have been reported to be the most frequent genetic abnormalities found in acute myeloid leukaemia ( aml ) . 
 their identification and quantification remain crucial for the patients ' residual disease monitoring . 
 we investigated a new method that could represent a novel reliable alternative to sequencing for its identification . 
 this method was based on high - resolution melting analysis in order to detect mutated patients and on an allele - specific oligonucleotide real - time quantitative polymerase chain reaction ( aso - rq - pcr ) for the identification and quantification of the transcripts carrying npm1 mutations ( npm1 m ) . 
 few patients carrying known npm1 m enabled us to set up a table with the different primers ' ct values , identifying a profile for each mutation type . 
 we then analysed a series of 337 aml patients ' samples for npm1 mutational status characterization and confirmed the aso - rq - pcr results by direct sequencing . 
 we identified some mutations in 86 samples , and the results were fully correlated in 100% of the 36 sequenced samples . 
 we also detected other rare npm1 m in two samples , that we confirmed by direct sequencing . 
 this highly specific method provides a novel quick , useful , and costless tool , easy to use in routine practice .