Single-Cell RNA Sequencing of Rare Circulating Tumor Cells in Precursor Myeloma Patients Reveals Molecular Underpinnings of Tumor Cell Circulation

Introduction

Multiple Myeloma (MM) is a hematological malignancy characterized by abnormal proliferation of terminally differentiated plasma cells (PCs) in the bone marrow (BM). MM is almost always 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 patients for disease monitoring during precursor stages. Profiling circulating tumor cells (CTCs) from peripheral blood (PB) may provide information for the non-invasive surveillance of precursor myeloma and nominate novel PB-based biomarkers to identify high-risk patients that may benefit from early therapeutic intervention.

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. Matched CD138- BM immune cells underwent 5’ scRNA-seq to study immune alterations related to the tumors’ capacity to circulate. 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 patients with MGUS (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 commonly 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 their BM and CTCs, indicating scRNA-seq can detect missed prognostically relevant cytogenetic abnormalities, which we further confirmed by whole genome sequencing. 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. Of note, TNF-a and NF-κB signaling, commonly induced by extrinsic factors in the BM milieu, was significantly upregulated or downregulated in CTCs vs. BM tumor cells, suggesting variability in how CTCs differ from BM tumor cells. Remarkably, within each SMM risk group, we observed patients with a higher fraction of CTCs (CTC-high), suggesting CTC enumeration captures interpatient variability not explained by BM tumor burden alone. To determine whether CTC-high status was associated with changes in the BM immune cell composition, we compared immune cells between CTC-high and CTC-low patients and observed a significant increase in the abundance of Granzyme K- and Granzyme B-expressing CD8+ T-cells in the BM of CTC-high patients, suggesting that the CTC fraction may reflect both tumor-intrinsic and tumor-extrinsic alterations.

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, provide novel insights into PC circulatory potential, and uncover novel associations between CTC burden and BM immune cell composition. 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.