High-Throughput Sequencing of Antibody Genes Successfully Identifies Clonal Ig Rearrangements in Multiple Myeloma Patients

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 application of Ig HTS to clinical multiple myeloma (MM) samples has not been demonstrated previously.

Approach: We conducted HTS of PCR-amplified IgH (VDJ and DJ) and κ/λ (VJ) rearrangements from bone marrow aspirates (BMA) of patients with MM (n=9), MGUS (n=1), and lymphoplasmacytic lymphoma (n=1), and peripheral blood (PB) of a patient with plasma cell leukemia (n=1). In 9/12 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. Disease burden in un-enriched samples was also evaluated by microscopy (BMA/PB smear) and ranged from 0 (hemodilute) to 37%.

Results: In 11/12 samples, a clearly dominant IgH and/or κ/λ rearrangement (>2.7% of total sequences, range 2.7-99.9%) was identified with clear separation from background frequency (at least 2.7-fold higher frequency than next most common clone). One sample exhibited an oligoclonal repertoire with no clearly dominant sequence. In 9/9 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. Results were largely consonant with clinical data, though in one IgG-λ MM sample, no dominant, productive λ rearrangement was detected, and in one IgG-κ MM sample, no dominant, productive heavy chain rearrangement was detected. This may be due to mutations at primer-binding sites in these rearrangements. In both cases, alternative clone-tracking sequences were available from the other loci (i.e., IgH in the first case and κ in the second). In 7/12 cases, >1 dominant sequence among the IgH (VDJ and DJ), κ, and λ rearrangements was identified that would be suitable for longitudinally tracking the malignant clone.

Conclusion: HTS of Ig heavy and light chain rearrangements can successfully identify the MM clone in clinical specimens, including those with low MM burden. Application of this technique to MRD evaluation in MM warrants further development.