A Novel Evolutionary Pattern Revealed Using Deep Sequencing of Immunoglobulin Loci at Diagnosis and over the Course of Treatment in Multiple Myeloma Patients

Introduction: Immunoglobulin (Ig) gene rearrangement is a hallmark of early B-cell development. As multiple myeloma is considered a clonal disease originating from the transformation of a single plasma cell, myeloma cells are traditionally thought to have one clonal Ig gene sequence that remains stable throughout the course of the disease. We previously observed that multiple Ig sequences related by somatic hypermutation (SHM) may be present in some MM patients at diagnosis. Here we provide an expanded observation in a very large cohort of the patients, and perform mutational analysis of the oligoclonal myeloma clonotypes observed at diagnosis and post-treatment, revealing changes in the relative frequency of the MM clonotypes and emergence of new Ig clones.

Methods: 620 MM patientsenrolled in IFM/DFCI and Hospital 12 de Octubre trials were included in this analysis. The next-generation sequencing (NGS)-based immunosequencing platform was used to detect evidence of oligoclonality at the Ig heavy chain loci. Using universal primer sets, we amplified IGH variable, diversity, and joining gene segments from DNA and/or RNA isolated from purified CD138+ MM cells collected at the time of diagnosis. MM-specific clonotypes were identified for each patient based on their high frequency (5%) within the B-cell repertoire in the diagnostic (dx) sample. The highest frequency MM clonotype in a dx sample is termed the "index clonotype." DNA and/or RNA isolated from dx AND post-treatment bone marrow samples were assessed for evidence of evolved MM clonotypes.

Results: We identified Ig clones in 367 RNA samples and 430 DNA samples from the cohort. We first looked for cases with evidence that myeloma cells have two unrelated origins. We found 11/620 (1.8%) cases at diagnosis, which had evidence of unrelated clones as evident by having three IgH or two functional sequences. In 8 of the 11 cases (72.6%), we had multiple samples to analyze, including two samples at diagnosis or diagnosis/post-treatment pairs. In 4 of the 8 samples, we saw dramatically different relative frequencies of unrelated clones in these samples suggesting that these unrelated clones are likely to be present in two distinct cells. We then considered cases where we found two IgH sequences that are related to each other by SHM at diagnosis. Overall 79 (12.7%) of 620 samples had more than one evolved clone; of these 63/367 (17.2%) of RNA dx samples showed evidence of evolved clones via SHM, while 22/430 patients (5.1%) showed evidence of evolved clones related to the index clone via SHM in DNA samples from diagnosis. Mutant clonotypes had an average of 3.9 to 4.5 mutations in the CDR3 region. We also noted mixed isotypes in 13 clones from 13 patients at diagnosis. The majority of related clones observed in the RNA samples are present at very low frequencies (<10^-4), as the greater sequencing depth in RNA allows for identification of low frequency clones. 304 post-treatment samples from 206 patients were MRD positive and were assessed for the presence of clonal evolution. In 27/304 follow-up samples (8.8%) and 7/206 patients (3.4%), an evolved clone related to the index clone was observed even though the period between diagnosis and post-treatment samples was only 6 months. In 6 patients, a substantial change in the relative index and unrelated clone frequencies was observed from the dx to post-treatment time points suggesting a differential sensitivity to treatment.

Conclusions: We confirm presence of multiple evolved clonotypes in a substantial percentage of diagnostic MM samples in a large cohort of patients. The evolution of multiple clones related by SHM indicates that SHM remains active after myeloma development and may also impact other non-Ig sites. These findings shed light on the biology and pathogenesis of MM and may provide prognostic information. The very high depth of our sequencing also indicates that the emergence of new IgH clones may be newly acquired mutations in the Ig gene, driven by some ongoing genomic mutation process. Thus, these evolved myeloma clonotypes may be useful as surrogate markers for other oncogenic mutations providing resistance to therapy.