Identifying a malignant B-cell Lymphoma clone in the peripheral blood using Immunoglobulin high-throughput sequencing and lineage tree analysis

Roughly 80% of aggressive B-cell lymphoma patients respond to frontline therapy but only 50%-60% are cured, necessitating longitudinal monitoring. Post-therapy surveillance PET-CT scans have not been shown to improve the outcome of patients. Identifying the malignant B-cell clone using Immunoglobulin (Ig) high-throughput sequencing (HTS) from plasma DNA was shown to be feasible as a method to detect minimal residual disease (MRD), but has not been standardized for MRD detection in lymphomas. We aimed to follow malignant B-cell clones using HTS of original lymph node (LN) biopsies, bone marrow (BM) samples collected during screening and serial blood samples from patients treated for relapsed/refractory aggressive B-cell lymphoma in a phase II clinical trial with ibrutinib, bendamustine and rituximab (NCT02747732). Genomic DNA was extracted and IgV libraries were produced and sequenced. Sequences were pre-processed using pRESTO and annotated using IMGT/HighV-QUEST. Clones were assigned using Change-O. Lineage trees were constructed using IgTree© and analyzed using PopTree© and custom R scripts. Analyzing 10 samples from one patient, 98 of the 24,328 clones included the same VJ as the malignant clone; two of them included sequences from both the original LN biopsy and other blood samples. Without lineage tree analysis, we would have not been able to track our sequences to their correct ancestor in the malignant lymph node. Thus, using HTS combined with lineage tree analysis it is possible to reconstruct and follow a malignant B cell clone with blood sampling. We therefore suggest to use these methods for MRD tracking in B-cell malignancies.