Limited Reliability of Ig/TCR Based MRD Monitoring in BCR/ABL-Positive Childhood ALL: Comparison to Quantitative Fusion Transcript Detection.
Authors: Krejcikova Katerina , Muzikova Katerina , Fronkova Eva , Kalinova Marketa , Reznickova Leona , Zuna Jan , Stary Jan , Trka Jan 
Published: October 2019

Abstract
Leukemias with the t(9;22) translocation resulting in BCR/ABL fusion protein expression comprise 3–5% of childhood ALL. Despite modern therapeutic regimens, their prognosis is inferior. Minimal residual disease (MRD) based on leukemia-specific immunoglobulin (Ig) and T-cell receptor (TCR) gene rearrangements has become a tool influencing clinical decisions in many therapeutic trials for childhood ALL. The presence of BCR/ABL fusion gene offers a possibility of the fusion transcript detection - a faster and cheaper alternative to Ig/TCR-based MRD monitoring. Up to now, no direct comparison based on a sufficient number of samples has been done. We analyzed 350 follow-up samples from 16 children (aged 4–17 years) with BCR/ABL-positive ALL by Ig/TCR-based real-time quantitative PCR (RQ-PCR) and by reverse-transcriptase (RT) RQ-PCR for BCR/ABL transcripts. Beta-2 microglobulin housekeeping gene was used for cDNA quality normalization. WBC, age, immunophenotype and blast proportion in the bone marrow (BM) and peripheral blood (PB) showed no relation to the initial BCR/ABL level. All children expressed m-BCR/ABL transcript at the time of diagnosis; 3 of 16 children expressed both m-BCR/ABL and M-BCR/ABL transcripts representing the p190 and p210 variant of BCR/ABL protein, respectively. The expression levels of m-BCR/ABL in diagnostic samples differed up to 3 logs, being the lowest in patients expressing both variants of the fusion gene. In 38 samples from those patients, M-BCR/ABL expression was generally higher than m-BCR/ABL expression, being negative by m-BCR/ABL and positive by M-BCR/ABL in 13 samples. For further analysis we used the higher value of m- and M-BCR/ABL as the BCR/ABL MRD level. For the comparison with Ig/TCR-based method, MRD levels in follow-up samples were related to the expression levels in diagnostic samples, which were set to 1. In total, 133 (38%) and 127 (36%) samples were negative and positive by both methods, respectively. The quantitative levels differed by more than 1 log in 46 (36%) double-positive samples, being underestimated by Ig/TCR method in 25 cases and by m-BCR/ABL quantification in 21 cases. With the same sensitivity of both methods we found significantly more false-negative samples by Ig/TCR approach (70 samples) compared to BCR/ABL quantification (20 samples). Altogether, we tested 219 bone marrow (BM), 130 peripheral blood (PB) and 1 cerebrospinal fluid samples. The PB samples showed significantly worse correlation between the two methods compared to BM (p=0.02). Interestingly, some patients had higher MRD levels in PB compared to BM as shown by corresponding BM and PB samples. Our data suggest that BCR/ABL-positive childhood ALL is a biologically heterogeneous group. We show that all diagnostic samples should be screened for the simultaneous m- and M- BCR/ABL expression to avoid false-negativity when using m-BCR/ABL quantification only. In our hands, the quantification of BCR/ABL transcripts appears to be a more reliable method than the generally accepted Ig/TCR-based MRD monitoring as the number of false-negative samples by BCR/ABL quantification is significantly lower. This contention is further supported by our pilot data on transplanted patients where BCR/ABL positivity preceding transplantation seems to be a better predictor of subsequent relapse than Ig/TCR approach. Support: MSM0021620813, MZ00064203 and 62/2004 GAUK CR. KK and KM contributed equally to this work.