P-097: Single-Cell RNA Sequencing of Circulating Tumor Cells in Precursor Myeloma Patients Reveals Early Mechanisms of Disease Dissemination

Introduction: Multiple Myeloma (MM) is preceded by the precursors Monoclonal Gammopathy of Undetermined Significance (MGUS) and Smoldering Multiple Myeloma (SMM). BM biopsies are useful to monitor disease progression, but they are not routinely collected from precursor patients for disease monitoring. Profiling circulating tumor cells (CTCs) from peripheral blood (PB) may provide information for the non-invasive surveillance of precursors and nominate novel PB-based biomarkers to identify high-risk patients.

Methods: Paired PB and BM aspirates were collected from 73 individuals, including patients with MGUS (n=9), SMM (n=40), NDMM (n=12) and healthy individuals (n=12). Malignant PCs underwent 5' single-cell RNA sequencing (scRNA-seq) and single-cell B-cell receptor sequencing (scBCR-seq) (10x Genomics). To differentiate malignant from normal PCs, we used clonal V(D)J rearrangements. Differential expression (DE) and composition analyses were conducted using Wilcoxon's rank-sum tests.

Results: We profiled 351,225 BM tumor cells and 55,110 CTCs. High-risk SMM patients had significantly more CTCs compared to MGUS patients (p=0.024) and low-risk SMM (p=0.042). A median of 5, 26, and 47 CTCs were present per mL of blood from low (n=15), intermediate (n=10), and high-risk (n=15) SMM patients as defined by the International Myeloma Working Group's 2/20/20 criteria, suggesting sequencing-based CTC enumeration captures prognostically relevant differences in tumor burden. High expression of driver genes upregulated in patients with translocations, including CCND1, NSD2, and MAF, were detected in both BM tumor cells and CTCs in patients with t(11;14), t(4;14), and t(14;16) as identified by fluorescence in situ hybridization (FISH). In 5 patients with normal or inconclusive FISH results, we observed high levels of CCND2 and MAF in both BM and CTCs, indicating scRNA-seq can detect missed prognostically relevant cytogenetic abnormalities. DE analysis of CTCs vs. BM tumor cells highlighted transcriptional similarity between BM and CTCs, validating their utility as a surrogate for analyzing BM tumor cells. DE analysis also revealed 8 genes significantly upregulated and 3 genes significantly downregulated in CTCs compared to BM tumor cells, providing novel insights into genes involved in PC circulatory potential. Pathway enrichment analysis revealed genes upregulated in CTCs were associated with epithelial mesenchymal transition, interferon response, and inflammation, consistent with CTC studies on MM patients, suggesting these pathways are dysregulated earlier in the disease continuum.

Conclusions: In the largest scRNA-seq study on CTCs to date, we demonstrate the utility of CTC-based molecular profiling for prognostication of patients with early-stage disease and provide novel insights into PC circulatory potential. Additional analyses are ongoing to gain further insight into intra-patient CTC heterogeneity and define high-risk disease CTC signatures that emerge throughout the MM disease continuum.