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chronic lymphocytic leukemia ( cll ) is the most frequent leukemia in adults in the western society and is characterized by accumulation of clonal mature b cells [ 13 ] . in most patients ,
cll is preceded by a monoclonal b cell lymphocytosis ( mbl ) , characterized by less than 5000 monoclonal b  cells/l in the peripheral blood .
although most cll patients have an indolent clinical course , some cases present with a more aggressive evolution , leading to high morbidity and mortality .
although several frequent mutations have been found in cll , there is no common translocation or mutation known .
recently , a reciprocal fusion between ypel5 ( yippee - like 5 ) and ppp1cb ( serine / threonine protein phosphatase-1 beta catalytic subunit ) was described to be exclusively expressed in more than 90% of primary cll samples   and not in control samples or samples from patients with other hematological malignancies .
the two chimeric rna transcripts appeared be the result of trans - splicing as no sign of genomic reorganization could be detected .
the discovery of a common , cll - specific molecular defect is interesting as it provides a unique target for drug discovery and molecular monitoring . here
, we show that the ypel5ppp1cb chimeric transcript is not specific for cll as it is also detected in primary patient samples of the cll - precursor mbl , acute myeloid leukemia ( aml ) , acute lymphocytic leukemia ( all ) , chronic myeloid leukemia ( cml ) and normal samples . as such
, it should not be used as a diagnostic marker and the relevance for this fusion transcript in cll as a drug target and molecular marker is questionable .
diagnostic or follow - up blood and bone marrow samples from patients with cll , mbl , aml , cml and all were collected from february 2012 to september 2013 in the course of normal treatment .
permission to use left - over samples was obtained from the ethical committee of ghent university hospital and all samples were treated according to the declaration of helsinki .
rna was isolated using the rneasy kit ( qiagen ) according to the manufacturer 's instructions .
cdna was made using superscript ii or superscript iii ( life technologies ) according to the manufacturer 's instructions .
briefly , 1  g rna was reverse transcribed in a total volume of 40 ( superscript ii ) or 50  l ( superscript iii ) . for the nupcr ( illumina ;
product discontinued ) , primers and mnazyme were designed with the designstudio . ypel5-ppp1cb and ppp1cb
 ypel5 pcrs ( both with fam - labeled probe ) were performed in a two multiplex pcrs , each in combination with an abl1 pcr ( hex - labeled probe ) .
the primers used are : 5-taaacctggagactcgggtgg-3 and 5-ctttcctggacgacatcctcgta-3 for ypel5-ppp1cb , 5-agtctgtgccgacaagatggc-3 and 5-tccatctgtcaagacgacgtagaataaaa-3 for ppp1cb - ypel5 and 5-cagaggtccatctcgctgagata-3 and 5-ctcggaggagacgtagagctt-3 for abl1 .
briefly , 5  l cdna was used in a 20  l total reaction volume .
cycling was done on a cfx96 cycler ( bio - rad ) following this protocol : 95  c 2  min , 40 cycles of 95  c for 15  s , 50  c for 30  s and 72  c for 30  s. for the evagreen qpcr , we used the published sybr green assay primers   for ypel5ppp1cb and 5-tgacaggggacacctacaca-3 and 5-atactccaaatgcccagacg-3 for abl1 in combination with the ssofast evagreen supermix ( bio - rad ) . for the pcr ,
5  l cdna was used in a 20  l total reaction volume using 300  nm primers .
cycling was done on a cfx96 cycler ( bio - rad ) with the following pcr protocol : 95  c for 30  s and 50 cycles of 95  c for 5  s and 60  c for 20  s. following the pcr , a melting curve from 65  c to 95  c was done ( 0.5  c/5  s ) . for both pcr strategies ,
samples were analyzed for tumor - specific markers ( e.g. bcr  abl1 expression in cml samples ) to confirm their identity as non - cll samples .
diagnostic or follow - up blood and bone marrow samples from patients with cll , mbl , aml , cml and all were collected from february 2012 to september 2013 in the course of normal treatment .
permission to use left - over samples was obtained from the ethical committee of ghent university hospital and all samples were treated according to the declaration of helsinki .
rna was isolated using the rneasy kit ( qiagen ) according to the manufacturer 's instructions .
cdna was made using superscript ii or superscript iii ( life technologies ) according to the manufacturer 's instructions .
briefly , 1  g rna was reverse transcribed in a total volume of 40 ( superscript ii ) or 50  l ( superscript iii ) .
for the nupcr ( illumina ; product discontinued ) , primers and mnazyme were designed with the designstudio . ypel5-ppp1cb and ppp1cb
 ypel5 pcrs ( both with fam - labeled probe ) were performed in a two multiplex pcrs , each in combination with an abl1 pcr ( hex - labeled probe ) .
the primers used are : 5-taaacctggagactcgggtgg-3 and 5-ctttcctggacgacatcctcgta-3 for ypel5-ppp1cb , 5-agtctgtgccgacaagatggc-3 and 5-tccatctgtcaagacgacgtagaataaaa-3 for ppp1cb - ypel5 and 5-cagaggtccatctcgctgagata-3 and 5-ctcggaggagacgtagagctt-3 for abl1 .
briefly , 5  l cdna was used in a 20  l total reaction volume .
cycling was done on a cfx96 cycler ( bio - rad ) following this protocol : 95  c 2  min , 40 cycles of 95  c for 15  s , 50  c for 30  s and 72  c for 30  s. for the evagreen qpcr , we used the published sybr green assay primers   for ypel5ppp1cb and 5-tgacaggggacacctacaca-3 and 5-atactccaaatgcccagacg-3 for abl1 in combination with the ssofast evagreen supermix ( bio - rad ) . for the pcr ,
5  l cdna was used in a 20  l total reaction volume using 300  nm primers .
cycling was done on a cfx96 cycler ( bio - rad ) with the following pcr protocol : 95  c for 30  s and 50 cycles of 95  c for 5  s and 60  c for 20  s. following the pcr , a melting curve from 65  c to 95  c was done ( 0.5  c/5  s ) . for both pcr strategies ,
samples were analyzed for tumor - specific markers ( e.g. bcr  abl1 expression in cml samples ) to confirm their identity as non - cll samples .
the apparent frequent and exclusive occurrence of the trans - splicing of ypel5 and ppp1cb in cll   could be a good target for directed therapy and molecular monitoring of this disease . to maximize the sensitivity and specificity in the detection of this fusion transcript
the combination of two specific primers and a specific mnazyme ensures a high specificity of the pcr in combination with a sensitive detection method ( fig .
1 ) . initially , we tested four samples of cll patients for the expression of both ypel5ppp1cb and ppp1cb 
the panel of primary diagnostic cll samples was expanded and in total , we detected variable expression of the ypel5ppp1cb transcript in 17/18 cll samples ( 94% ) ( fig .
2a  black bars ) , confirming the recurrent expression of the ypel5ppp1cb transcript in cll . to test the specificity of the expression of ypel5ppp1cb ,
we tested diagnostic samples of patients with aml ( n=3 ) , all ( n=4 ) , mbl ( n=7 ) , cml ( n=3 ) , different hematological cell lines ( n=10 ; data not shown ) and samples from healthy individuals ( n=3 ) with the nupcr for ypel5ppp1cb .
all patient samples and three cell lines ( me-1 , kasumi-1 , and monomac-6 ) tested positive , albeit at different levels ( fig .
black bars ) , showing that the presence of ypel5ppp1cb is not specific for cll . the expression levels in the different sample types were highly variable , but correspond to the levels observed in primary cll samples . to investigate the discrepancy between the nupcr and the results obtained by velusamy et al .
, we repeated the analysis on the same samples with the primers from the published sybr green assay   ( fig . 1 ; further referred to as evagreen method ) .
after the pcr , one melting peak was obtained and sequence analysis confirmed the identity of the ypel5ppp1cb fusion ( supplementary fig .
the patient samples showed similar expression with the evagreen method as obtained by the nupcr ( figs .
this confirmed the wide expression in non - cll samples , disproving the statement that the ypel5ppp1cb is specific for cll .
globally , the expression level was comparable regardless of the pcr technique used ( figs .
a report on high - throughput mrna sequencing in cll patients   also described a low - level expression of the ypel5ppp1cb transcript , both in normal and cancer samples .
although the expression level was below the threshold set by the authors , this finding is concordant with our results .
is potentially explained by the recent finding that incubation - induced gene dysregulation affects the expression level and splicing of numerous genes in leukemic samples . as the expression level of ypel5ppp1cb is very low ,
even small differences in splicing efficiency , as a result of different sample processing times , could make the difference between being able to detect the transcript or not . to conclude
, we find that the ypel5ppp1cb transcript is not exclusively present in cll samples and its usability as a molecular marker and the relevance for cll therapeutics should be scrutinized thoroughly before taking steps in the development of target - specific drugs .
kv : designed the study , performed experiments , drafted the manuscript , approved the final version .
jp , bd : designed the study , revised the manuscript , approved the final version .
chronic lymphocytic leukemia ( cll ) is the most frequent leukemia in western adults . 
 it was suggested that transcripts from a reciprocal trans - splicing event between ypel5 and ppp1cb were present exclusively in cll patients ( more than 90% ) . 
 here we show that the ypel5ppp1cb fusion is not specific for cll but is also detected in other hematological malignancies such as chronic myeloid leukemia , monoclonal b cell lymphocytosis or acute leukemia and also in normal samples . 
 as such , it is unlikely that the ypel5ppp1cb fusion is a good drug target in cll or a suitable target to monitor disease .