Detection of the Malignant B Cell Clone in Multiple Myeloma Via High Throughput Sequencing Is Robust to Significant Levels of Somatic Hypermutation

Background: High-throughput sequencing (HTS) of antibody gene rearrangements is an emerging tool for minimal residual disease (MRD) monitoring in B cell malignancies in which the malignant clone harbors a monoclonal Ig heavy chain (IgH) and/or light chain (κ or λ) rearrangement. This approach has shown promise in B-ALL and CLL, but experience with this technique applied to samples from multiple myeloma patients is limited.

Approach: We conducted HTS of PCR-amplified IgH (VDJ and DJ) rearrangements from bone marrow aspirates of 21 patients with various plasma cell dyscrasias (MM, MGUS, LPL) and peripheral blood of a patient with plasma cell leukemia. In 17/21 samples, an aliquot was enriched for CD138+ cells by immunomagnetic separation and analyzed separately. Dominant clones from enriched and un-enriched aliquots were compared to verify the malignant clonotype sequence(s). Disease burden in un-enriched samples was also evaluated by microscopy of the bone marrow aspirate smear and ranged from 0 (hemodilute) to 37% plasma cells.

Results: In 19 out of 21 samples, a clearly dominant IgH gene rearrangement (>2.5% of total sequences, range 2.9-99.9%) was identified with clear separation from background frequency (at least 2.7-fold higher frequency than next most common clone). In 17/17 cases with paired CD138-enriched samples, the dominant sequences in the enriched and un-enriched samples were identical, indicating successful identification of the malignant clonal Ig rearrangements in the un-enriched sample. More than one IgH rearrangement suitable for longitudinally tracking the malignant clone was identified in 8 of 21 cases. The two cases without an expected, productive IgH rearrangement were IgG-κ and IgG-λ. This suggested that somatic hypermutation (SHM) in the primer binding sites might interfere with some clonal amplifications, so we investigated the degree of SHM in the VH segment of the 19 cases with at least one detected dominant Ig rearrangement. A total of 18 productive VDJ rearrangements were identified, and had SHM frequencies ranging from 2% to 19% in the sequenced portions of the rearranged VH gene. 8 myeloma clones harbored an identifiable DJ rearrangement, none of which showed evidence of SHM. Finally, 3 myeloma clones harbored nonproductive VDJ rearrangements, two with no SHM, and one with 2 SHM in 84 bp of the sequenced VH gene.

Conclusion: HTS of Ig heavy and light chain rearrangements can successfully identify the malignant plasma cell clone in clinical specimens, including those with low disease burden and significant SHM. Application of this technique to MRD evaluation in multiple myeloma warrants further development.