Molecular Analysis Of Circulating Tumor Cells Identifies Mutations That Are Distinct From Those Present In The Bone Marrow Of Patients With Multiple Myeloma

Clonal evolution involves simultaneous evolution of multiple co-existant subclones. Recent studies have suggested that clonal heterogeneity is critical during the progression of Multiple Myeloma (MM). Circulating tumor cells (CTCs) have been identified in many patients with solid tumors and hematological malignancies. Recent studies have suggested that CTCs can be identified in patients with Multiple Myeloma. The aims of this study were to identify the phenotypic characteristics of CTCs in patients with Mutliple Myeloma at different stages of the disease, to determine whether somatic mutations present in the bone marrow clones are also identified in CTCs or whether specific subclones are more prone to enter the systemic circulation. These subclones may have a higher likelihood of inducing dissemination into extramedullary sites and potential for drug resistance.

We analyzed the peripheral blood samples of 466 patients diagnosed with Multiple Myeloma at different stages of progression. Two plasma cell leukemia patients were included in the study. Freshly collected peripheral blood was processed to obtain white blood cell fractions. The cells were stained with eight antibodies including CD19, CD38, CD138, CD45, CD56, CD28, CD44, and CD183 and CTCs were purified by gating on CD19-/CD38+/CD138+ cells. Among them, ten of the CTC samples were selected to analyze somatic mutation using whole exome sequencing. Briefly, 1µg of genomic DNA was extracted form sorted cells followed by shearing, end repair, and ligation to barcoded adaptors. The DNA was size-selected, subjected to exonic hybrid capture and sequenced on Illumina HiSeq flow cells with an average depth of coverage of 100x.

Of the 466 samples analyzed, the number of CTCs identified ranged from 0.01% to 61% of total WBC count. CTCs were detected in 61.4% of all samples analyzed. CTCs were detected in 64.5% of relapsed MM, 63.4% of newly diagnosed MM, 24.0% of smoldering MM, and 25.0% of MGUS patients. Significant differences of the surface markers including CD45, CD28, CD56, and CD44 were not observed in the different stages of MM disease progression. For further characterization of CTCs, we performed whole exome sequencing of CTCs in 10 MM samples, of which 5 had sequencing of their matched tumor cells collected from BM as well as matched normal germline cells to examine whether circulating tumor cells possess any distinctive somatic mutations. The sequence analysis revealed that both CTCS and marrow restricted tumor cells have substantial numbers of protein-coding mutations. CTCs and bone marrow cells shared 5-38% similar mutations, while interestingly the rest of the mutations were exclusively present in either the CTCs or bone marrow samples. We identified a total of 347 somatic mutations, which included 199 CTC specific mutations. Several known driver mutations were observed, i.e. BRaf V600E mutation present in the CTC samples but not in the matching bone marrow samples in one patient. Twelve of these CTC mutations were shared at least in two patients including ZNF721, NBPH10, F5, and PRDM15.

These data suggest subclonal out growth of CTCs from one of the parent clones with acquisition of additional mutation over time outside of the bone marrow microenvironment. Further validation of the unique mutations in CTCs may provide mechanistic insight into myeloma cell dissemination, and so potentially inform treatment strategies.

Tai:Onyx: Consultancy. Anderson:celgene: Consultancy; onyx: Consultancy; gilead: Consultancy; sanofi aventis: Consultancy; oncopep: Equity Ownership; acetylon: Equity Ownership. Munshi:Celgene Corporation: Consultancy, Membership on an entity’s Board of Directors or advisory committees; Millennium: The Takeda Oncology Company: Consultancy, Membership on an entity’s Board of Directors or advisory committees; Novartis Pharmaceuticals Corporation: Consultancy, Membership on an entity’s Board of Directors or advisory committees; Onyx Pharmaceuticals Inc: Membership on an entity’s Board of Directors or advisory committees. Ghobrial:Noxxon: Research Funding; BMS: Advisory board, Advisory board Other, Research Funding; Onyx: Advisoryboard Other; Sanofi: Research Funding.