Deep Sequencing Reveals Oligoclonality At The Immunoglobulin Locus In Multiple Myeloma Patients

Immunoglobulin (Ig) gene rearrangement is a hallmark of early B-cell development. Multiple myeloma (MM) is a malignancy of the plasma cells, which are at the terminal stage of B cell development. MM is a clonal disease originating from the transformation process of a single plasma cell and, thus, myeloma cells are traditionally thought to have one clonal Ig gene sequence that remains stable throughout the course of the disease. Based on preliminary evidence of oligoclonality, we utilized the LymphoSIGHT™ platform, a high-throughput sequencing method, to detect evidence of oligoclonality at the Ig heavy and kappa chain (IGH and IGK) loci. The sequencing approach can be used to examine two general models for oligoclonality. In the first model, two unrelated clonal Ig sequences are observed indicating the presence of two independent myelomas. Alternatively, in the second model, two related myeloma clonal Ig sequences are observed indicating that both myeloma clones are derived from a common ancestor that arose after the pro B cell stage when VDJ recombination is completed. The common ancestor can be a cell with premalignant lesion or after the MM has developed. Using the sequencing platform, we looked for evidence of these two models of oligoclonality in 193 MM patients.

Two cohorts of newly diagnosed MM patients were included in this analysis (N=125, N=68). Using universal primer sets, we amplified IGH and IGK variable, diversity, and joining gene segments from genomic DNA or RNA from bone marrow collected at initial diagnosis. Amplified products were sequenced and analyzed using standardized algorithms for clonotype determination (Faham et al, Blood 2012). In the first cohort (N=125), we assessed gene rearrangement at the IGH-VDJ and IGK loci in 120 patients using RNA only and in 5 patients, we used both DNA and RNA to assess the IGH-VDJ, IGH-DJ and IGK loci. In the second cohort (N=68), we analyzed gene rearrangement at the IGH-VDJ, IGH-DJ and IGK loci using genomic DNA. Myeloma-specific clonotypes were identified for each patient based on their high frequency (5%) within the B-cell repertoire in the diagnostic sample. To identify clonotypes that are present in more than one cell we looked for patterns that are not consistent with having a maximum of one functional and one non-functional clonotype in a cell.

We observed oligoclonality in 23 of 193 (12%) MM patients. Unrelated Ig sequences, which are consistent with the first model of oligoclonality, were present in 8 of the 193 (4%) patients. Fifteen of 193 (8%) patients exhibited related Ig clones, which is consistent with the second model of oligoclonality. In 4 of the 15 patients clones were related to each other via a somatic hypermutation process and differed by only a few bases (Figure 1), while in other 11 patients, the same VDJ sequence was associated with two distinct isotypes (IgA and IgG). Interestingly, in cases with both RNA and DNA sequencing and oligoclonality, we observed differential expression levels compared to clonal content at the DNA level, suggesting that a low frequency clone could be contributing as a predominant secretory clone.

Figure 1

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The germline V, D, J sequences are shown on top with the boxed sequences present in the clonotypes and the unboxed bases representing the sequences that were deleted during the VDJ recombination to form the clonotypes. The middle and lower sequences represent two detected clones present at 49% and 40% frequency. The untemplated base additions (N bases) in the clones are those that do not correspond to the germline bases in the first line. The red bases are those that have undergone somatic hypermutation. Three mutations are present in clone 2 that are not present in clone 1.

Figure 1

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The germline V, D, J sequences are shown on top with the boxed sequences present in the clonotypes and the unboxed bases representing the sequences that were deleted during the VDJ recombination to form the clonotypes. The middle and lower sequences represent two detected clones present at 49% and 40% frequency. The untemplated base additions (N bases) in the clones are those that do not correspond to the germline bases in the first line. The red bases are those that have undergone somatic hypermutation. Three mutations are present in clone 2 that are not present in clone 1.

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This study demonstrates frequent oligoclonality in MM patients and suggests that this phenomenon does occur due to two distinct processes, either as unrelated sequences consistent with independent clones or as related sequences consistent with evolution after the MM malignant lesions occur. These findings shed light on the biology and pathogenesis of MM and may provide prognostic information. Currently, this analysis was limited to high frequency clones, using a threshold of 5% for identification of the myeloma-specific clones. Additional analysis is being performed to assess the presence of lower frequency clones, and data will be presented.

Carlton:Sequenta, Inc. : Employment, Equity Ownership. Moorhead:Sequenta, Inc.: Employment, Equity Ownership. Faham:Sequenta, Inc. : Employment, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees.