Detection of minimal residual disease in B lymphoblastic leukemia by high-throughput sequencing of IGH

Abstract

Purpose: High-throughput sequencing (HTS) of immunoglobulin heavy-chain genes (IGH) in unselected clinical samples for minimal residual disease (MRD) in B lymphoblastic leukemia (B-ALL) has not been tested. As current MRD-detecting methods such as flow cytometry or patient-specific qPCR are complex or difficult to standardize in the clinical laboratory, sequencing may enhance clinical prognostication.

Experimental design: We sequenced IGH in paired pretreatment and day 29 post-treatment samples using residual material from consecutive, unselected samples from the Children's Oncology Group AALL0932 trial to measure MRD as compared with flow cytometry. We assessed the impact of ongoing recombination at IGH on MRD detection in post-treatment samples. Finally, we evaluated a subset of cases with discordant MRD results between flow cytometry and sequencing.

Results: We found clonal IGH rearrangements in 92 of 98 pretreatment patient samples. Furthermore, while ongoing recombination of IGH was evident, index clones typically prevailed in MRD-positive post-treatment samples, suggesting that clonal evolution at IGH does not contribute substantively to tumor fitness. MRD was detected by sequencing in all flow cytometry-positive cases with no false-negative results. In addition, in a subset of patients, MRD was detected by sequencing, but not by flow cytometry, including a fraction with MRD levels within the sensitivity of flow cytometry. We provide data that suggest that this discordance in some patients may be due to the phenotypic maturation of the transformed cell.

Conclusion: Our results provide strong support for HTS of IGH to enhance clinical prognostication in B-ALL.

Figure 1. Pre- and day 29 post-treatment B lymphoblast frequencies by high-throughput sequencing (HTS) versus multi-parametric flow cytometry (mpFC)

Pre-treatment and Day 29, post-treatment, clonal B lymphoblasts were identified by HTS (red) or by mpFC (blue) (N=91). HTS data are reported as the frequency of the clonal sequence of total rearranged IGH sequences; mpFC data are reported as the percentage of total nucleated cells by mpFC. Cases without a complete IGH gene rearrangement in pre-treatment samples (n=7) are not shown. (A) High-throughput sequencing identifies pre-treatment, clonal IGH sequences in 91 of 98 patients. (B) High-throughput sequencing of IGH identifies post-treatment MRD detected by flow cytometry and in an additional subset of patients for whom flow cytometry analysis was negative. Day 29, post-treatment samples (n=91) includes only those cases with complete V-J rearrangement and excludes 1 case with only a D-J rearrangement and 5 cases for which no clonal IGH rearrangement was seen. Three subsets of cases are identified in post-treatment samples (left to right): 1) MRD not detected by either HTS or mpFC, 2) MRD detected by HTS, but not by mpFC, and 3) MRD detected by both HTS and mpFC.

Figure 2. Comparison of clonal frequency of MRD in cases with presumed bi-allelic rearrangement of IGH versus mpFC

In samples with bi-allelic rearrangement, tracking of both alleles provides enhanced specificity for quantification of MRD. 26 diagnostic samples generated clear evidence for bi-allelic rearrangements (exactly 2 dominant IGH sequences): in these samples, the frequency of each allele was tracked in post-treatment samples. In 10 samples, neither allele was observed post-treatment. In 4 samples, only one allele was observed post-treatment (at an average frequency of 1.2 × 10^-5). In the remaining 12 samples, both alleles were observed post-treatment. The frequencies of the dominant and secondary alleles in post-treatment samples are charted. Post-treatment allele frequencies in bi-allelic samples are highly correlated (r² = 0.97).