The predictive strength of next-generation sequencing MRD detection for relapse compared with current methods in childhood ALL.

The predictive strength of next-generation sequencing MRD detection for relapse compared with current methods in childhood ALL Minimal residual disease (MRD) monitoring via antigen receptor quantitative polymerase chain reaction (qPCR) is an important pre-dictor of outcome in childhood acute lymphoblastic leukemia (ALL), is rigorously standardized within the EuroMRD consortium and has a greater sensitivity than flow cytometry (FC), which has been used in other trials. 1 However, qPCR is laborious, expensive, and time consuming because of the development of patient-specific assays. MRD detection based on next-generation sequencing (NGS) of an-tigen receptor gene rearrangements is a promising tool that permits sequencing of large numbers of rearranged V-(D)-J segments and thus provides the picture of not only residual leukemia but also the normal immune repertoire of respective cells. To date, no correlation study on larger number of samples has been performed to determine how the discrepancies between NGS and qPCR data would affect clinical data. Moreover, there are no data investigating the impact of normal B-cell compartment reconstitu-tion after induction treatment in ALL on prognosis. We developed a simple, cost effective, and easily adoptable approach using 2-round PCR amplifying virtually all immunoglobulin heavy chain (IgH) rearrangements 6 and NGS on Ion Torrent/Proton sequencers (supplemental Methods; available on the Blood Web site). We compared the NGS-MRD results with current techniques 7 in patients treated by Berlin-Frankfurt-Munster (BFM)-based protocol and investigated the changes in the treatment stratification using the new method. Altogether, we sequenced 210 samples from 76 patients (623 day 15, 733 day 33, 753 day 78) with a median coverage of 729 842 reads per sample. The overall correlation of NGS-and qPCR-MRD in all samples was satisfactory (R 2 5 0.72; Figure 1A). A total of 32 samples (15%) were positive by NGS/negative by qPCR or vice versa, causing a shift in BFM risk stratification in 25 patients (33%), mainly between standard-risk (SR) and intermediate-risk (IR) groups in the low-positive patients. Five patients would be reassigned from the IR to the slow early responder (SER) group (4 of them relapsed), and 2 patients from SER to IR (1 of them relapsed). NGS-MRD positivity at day 33 provided a more accurate prediction of relapse than qPCR-MRD positivity (Figure 1B-C) (5-year Figure 1. The comparison of MRD as measured by qPCR and NGS, the prognostic significance of NGS, qPCR and FC at various treatment time points and the impact of IgH repertoire diversity on …