On-Going Evolution Of IGH In B-Cell Precursor Acute Lymphoblastic Leukemia Does Not Substantially Affect Day 29, Post-Treatment MRD Quantification By High-Throughput Sequencing

High-throughput sequencing (HTS) of immunoglobulin heavy chain genes (IGH) may be useful for detecting minimal residual disease (MRD) in B-cell precursor acute lymphoblastic leukemia (BPC-ALL), particularly in the context of massive clonal evolution at the IGH locus, as previously identified by others (Gawad et al., Blood 120(22):4407-17, 2012; and Faham et al., Blood 120(26):5173-80, 2012). This on-going rearrangement of IGH may limit detection of MRD in post-treatment samples by traditional molecular-based methods, typically real-time PCR using patient-specific primers or probes. Here, we examine the extent to which evolution of IGH in unselected pre-treatment samples from patients with BPC-ALL affects detection of MRD in day 29 post-treatment samples by high-throughput sequencing of IGH.

Of 99 samples from an unselected series from the Children’s Oncology Group trial AALL0932, we find that 92 of 98 samples have a clonal IGH gene rearrangement in pre-treatment samples. One sample failed at the outset during the DNA extraction step. Of the remaining 92 cases with pre-treatment VDJ or D-J rearrangements, 82 had evidence of on-going recombination in which V_H replacement was identified in clones, each having conserved D-J rearrangements. The average number of clones was 192, but ranged from 1 to over 2000 unique sequences. In cases with V_H replacement, an average of 4.12% of IGH sequences was made up of V_H-replaced sequences. In post-treatment samples that were MRD positive, the predominant clone in pre-treatment samples was typically the most frequent clone. Clones consistent with V_H replacement were found in 19 patients; in one patient, the only MRD detected was a single clone consistent with V_H replacement at a level of ∼1 in 1,000,000. In the other 18 post-treatment MRD positive cases, the dominant clone identified pre-treatment was also dominant post-treatment: on average, 3.2% of total IGH rearrangements matched the dominant clone post-treatment, while only 0.027% of IGH rearrangements were consistent with V_H replacement of the major clone. Among pre-treatment samples in which V_H replaced clones were detected, all V_H replaced clones together were 12% as large as the dominant clone on average. Among post-treatment samples, V_H replaced clones were on average 14% as large as the dominant clone, indicating little change in the relative proportions of the dominant clone and V_H replaced sub-clones.

These findings together suggest that on-going rearrangement of the IGH locus is not likely to be important for clonal tumor evolution within the time frame of initial chemotherapy, as no substantial change in clonal diversity as assessed by IGH sequencing is evident. In other words, on-going rearrangement of IGH appears to be neutral with respect to therapy-induced selection of tumor clones that may represent early (day 29) relapse.