Comparison of NGS and RT-PCR approaches for MRD monitoring in acute lymphoblastic leukemia

Background:

Being the major cause of relapse in leukemia, minimal residual disease (MRD) is considered to be the strongest prognostic factor, allowing to evaluate the efficiency of a treatment, and to make a decision on the subsequent therapy. Detection of MRD by Next generation sequencing (NGS) is proven to be one of the most efficient and sensitive techniques. Detection of clonal rearrangements of immunoglobulin (Ig) and T-cell receptor (TCR) genes is widely used for clonality assessment and for MRD monitoring along with RT-PCR of fusion transcripts, and unlike the latter one, is applicable in most cases of lymphoblastic malignancies.

Aims:

The purpose of this study is to compare a new approach for monitoring MRD in acute lymphoblastic leukemia (ALL) by targeting massive sequencing of clonal rearrangements of immunoglobulin (IG) and T-cell receptor (TR) genes with MRD monitoring via RT-PCR of fusion transcripts.

Methods:

A cohort of uniformely treated patients consisted of children aged 4 to 13 years diagnosed with ALL. DNA was extracted from patients' bone marrow (BM) samples on the 1^st and 36^th day of treatment. Initial detection of patient specific clonal rearrangements was carried out by 8 multiplex PCRs of Ig and TCR loci followed by NGS. MRD detection included targeted NGS of previously detected rearrangements in post-treatment follow-up samples in serial dilutions. The rearrangements chosen for future MRD diagnostics were chosen using 5% cut-off. Quantitative analysis was based on “Digital PCR”-like statistical approach.

Results:

We identified over 500 leukemic rearrangements in 97 patients using NGS technique. All cases had detectable Ig and/or TCR rearrangements with a median number of 3 per sample. 65% of samples contained rearranged TCRG, and in 85% of samples IGH was rearranged. Complete VDJ rearrangements dominated over incomplete DJ rearrangements in both IGH and TRB. We compared the MRD levels of 21 follow-up samples with data obtained by RT-PCR of fusion transcripts including t(12;21)(p13;q22) ETV6/RUNX1, t(5;14)(q35;q32) BCL11B/TLX3, and t(4;11)(q21;q23) KMT2A/AFF1. Three KMT2A/AFF1 positive samples from a 9-year-old boy revealed a strong correlation between MRD level measured by RT-PCR and NGS. Among 17 RT-PCR negative results, 14 were confirmed negative by NGS, however 3 of those follow-up samples showed significant MRD level, verified by flow cytometry.

Conclusions:

RT-PCR of fusion genes is a reliable and widely used MRD detection technique, however, it is only applicable for patients with known fusion transcripts. Moreover, our data showed that in 17% of cases MRD negativity by RT-PCR was not confirmed by NGS, clearly showing the presence of tumor clones in analysed samples, undetectable by RT-PCR. Therefore, NGS-based detection of rearrangements of Ig and TCR loci, being a sensitive, specific, and widely applicable method, allowing for clonality evaluation and MRD quantification, is likely to become a routine procedure for MRD diagnostics in the nearest future.

Russian Foundation for Basic Research (grant № 17 – 29 – 06052); Russian Science Foundation (grant № 17 – 75 – 10113); The Russian President's Fellowship SP-671.2018.4.