Monitoring multiple myeloma by next-generation sequencing of V(D)J rearrangements from circulating myeloma cells and cell-free myeloma DNA

Abstract

Recent studies suggest that circulating tumor cells and cell-free DNA may represent powerful non-invasive tools for monitoring disease in patients with solid and hematologic malignancies. Here, we conducted a pilot study in 27 myeloma patients to explore the clonotypic V(D)J rearrangement for monitoring circulating myeloma cells and cell-free myeloma DNA. Next-generation sequencing was used to define the myeloma V(D)J rearrangement and for subsequent peripheral blood tracking after treatment initiation. Positivity for circulating myeloma cells/cell-free myeloma was associated with conventional remission status (P<0.001) and 91% of non-responders/progressors versus 41% of responders had evidence of persistent circulating myeloma cells/cell-free myeloma DNA (P<0.001). About half of the partial responders showed complete clearance of circulating myeloma cells/cell-free myeloma DNA despite persistent M-protein, suggesting that these markers are less inert than the M-protein, rely more on cell turnover and, therefore, decline more rapidly after initiation of effective treatment. Positivity for circulating myeloma cells and for cell-free myeloma DNA were associated with each other (P=0.042), but discordant in 30% of cases. This indicates that cell-free myeloma DNA may not be generated entirely by circulating myeloma cells and may reflect overall tumor burden. Prospective studies need to define the predictive potential of high-sensitivity determination of circulating myeloma cells and DNA in the monitoring of multiple myeloma.

Figure 1.

Scheme of V(D)J DNA amplification and next-generation sequencing from peripheral blood cellular and cell-free DNA. Illumina adapters are shown in green and blue, barcode sequences are shown in red. cfDNA: cell-free DNA; IGH: immunoglobulin heavy chain; IGK/L: immunoglobulin kappa/lambda chain; FW: forward; RV: reverse; R1/R2: read 1/2; NGS: next-generation sequencing.

Figure 2.

Schematic illustration of study workflow. BMMC: bone marrow mononuclear cells; IGH: immunoglobulin heavy chain; IGK/L: immunoglobulin kappa/lambda chain; gDNA: genomic DNA; cfDNA: cell-free DNA.

Figure 3.

Representative V(D)J bone marrow and peripheral blood repertoires of patient MM123 at diagnosis. Every dot represents a clonotypic V(D)J rearrangement within the immunoglobulin repertoire. The size of each dot represents the size of the clone. The malignant plasma cell clone is highlighted in the bone marrow as well as in the cellular and cell-free peripheral blood compartments. The plot was generated using R statistical software tools. BM: bone marrow; PB: peripheral blood.

Figure 4.

Monitoring of circulating myeloma cells [(cmc-V(D)J)] and cell-free myeloma DNA [(cfm-V(D)J)] after myeloma treatment by next-generation sequencing. (A) Positivity of patients’ samples for cmc-V(D)J and cfm-V(D)J at diagnosis/relapse and after treatment, respectively. Remission status is indicated according to the IMWG criteria. (B) Positivity of patients’ samples for V(D)J at diagnosis/relapse and after treatment. Time points were considered V(D)J-positive if the malignant clone was detectable in at least one compartment (cellular or cell-free). (C) Quantification of cmc-/cfm-V(D)J per global number of V(D)J rearrangements per compartment. Patients with PD and SD were summarized as non-responders/progressors and patients with PR, vgPR and CR were summarized as responders. PD: progressive disease; SD: stable disease; PR: partial response; vgPR: very good partial response; CR: complete response.

Figure 5.

Overlap of V(D)J repertoires from cellular and cell-free peripheral blood compartments. Shared V(D)J clonotypes from different compartments were calculated using the tcR tool and overlap repertoires were plotted using the R statistical software tool. The V(D)J rearrangement of the malignant plasma cell clone [(cmc- and/or cfm-V(D)J)] is shown in red.