-Author name in bold denotes the presenting author
-Asterisk * with author name denotes a Non-ASH member
Clinically Relevant Abstract denotes an abstract that is clinically relevant.

PhD Trainee denotes that this is a recommended PHD Trainee Session.

Ticketed Session denotes that this is a ticketed session.

872 Single-Cell RNA Sequencing of Circulating Tumor Cells in Precursor Myeloma Patients Reveals Mechanisms of Disease Dissemination

Program: Oral and Poster Abstracts
Type: Oral
Session: 651. Multiple Myeloma and Plasma Cell Dyscrasias: Basic and Translational: Multiple Myleoma Circulating Tumor Cells, Novel Mechanisms, and Immune Interactions
Hematology Disease Topics & Pathways:
Research, Translational Research, Plasma Cell Disorders, genomics, Diseases, Lymphoid Malignancies, Biological Processes, Technology and Procedures, profiling, Study Population, Human
Monday, December 11, 2023: 3:00 PM

Elizabeth D. Lightbody, PhD1,2,3, Romanos Sklavenitis-Pistofidis, MD1,2,3, Danielle T. Firer, BS2*, Junko Tsuji, PhD2*, Mike P. Agius, PhD1,2,3*, Ankit K. Dutta, PhD1,2,3*, Ting Wu, MS2*, Hadley Barr, BSc1*, Sungjae Kim, PhD2*, Tim Coorens, PhD2*, Jean-Baptiste Alberge, PhD1,2,3, Michelle P. Aranha, PhD1,2,3*, Nicholas J Haradhvala, PhD2*, Nang Kham Su, MSc1*, Cody J. Boehner, BSc1*, Kelly Walsh, PA-C4*, Mahshid Rahmat, PhD1,2,3*, Yoshinobu Konishi, MD, PhD1,2,3, Laura Hevenor, BS1*, Katherine Towle, BS1*, Erica M. Horowitz, BA1*, Jacqueline Perry, MPH1*, Grace Fleming, BSc1*, Habib El-Khoury, MD1*, Michael Vinyard, BA2*, Annie Cowan, BA1*, Daniel Auclair, PhD5, John E. Ready, MD3,4*, Catherine R. Marinac, PhD1, Gad Getz, PhD2,3,6* and Irene M. Ghobrial, MD1,2,3

1Dana-Farber Cancer Institute, Boston, MA
2Broad Institute of MIT and Harvard, Cambridge, MA
3Harvard Medical School, Boston, MA
4Department of Orthopaedic Surgery, Brigham and Women's Hospital, Boston, MA
5AstraZeneca, Waltham, MA
6Cancer Center, Massachusetts General Hospital, Boston, MA

Multiple Myeloma (MM) is preceded by the precursors Monoclonal Gammopathy of Undetermined Significance (MGUS) and Smoldering Multiple Myeloma (SMM), where some patients with precursor disease progress to active MM faster than others. BM biopsies are useful to monitor disease progression, however, they cannot be repeated often for continuous monitoring of tumor burden especially in the precursor disease management setting. Numerous studies have identified circulating tumor cell (CTC) levels in peripheral blood (PB) are a powerful biomarker of disease aggressiveness; CTC levels have associated with disease stage, PFS and OS in various trials and even outperformed BM to assess tumor burden in the SMM setting. Genomic profiling also demonstrated CTCs harbor the same copy number abnormalities, translocations and mutations as BM tumor cells, and CTCs can be serially monitored to detect the emergence of high-risk subclones in the blood. Further investigations of the molecular profile of both normal plasma cells (NPCs) and CTCs are needed to improve our understanding of myeloma pathogenesis and mechanisms of CTC dissemination across the MM disease continuum.

A cohort of 309 samples including CD138-enriched paired PB and BM aspirates and matched CD138- BM aspirates were collected from 103 individuals, including patients with MGUS (n=11), SMM (n=46), NDMM (n=17) and healthy donors (n=29). Malignant PCs from BM and PB underwent 5' single-cell RNA sequencing (scRNA-seq) and single-cell B-cell receptor sequencing (scBCR-seq) (10x Genomics), and CD138- BM immune cells underwent 5’ scRNA-seq and single-cell T-cell receptor sequencing (scTCR-seq) to study immune alterations related the tumors’ ability to circulate. 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.

We successfully captured and profiled 774,647 BM tumor cells and 93,878 CTCs from the PB. The percent malignant plasma cells within the PB increased with disease stage, where NDMM patients had significantly more CTCs compared to MGUS patients (p=0.017) and low-risk SMM patients (p=0.0052). A median of 13.51%, 15.53%, and 18.34% CTCs were present from low (n=20), intermediate (n=10), and high-risk (n=16) 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. A cytogenetic classifier model was developed to determine the accuracy of translocation calling using minimal numbers of CTCs, where the classifier’s performance was able to consistently identify translocations downsampled to 50 CTCs. 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.

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.

Disclosures: Rahmat: AstraZeneca: Current Employment. Auclair: AstraZeneca: Current Employment, Other: may own stock or stock options. Marinac: Exact Sciences: Membership on an entity's Board of Directors or advisory committees. Getz: Scorpion Therapeutics: Consultancy, Current equity holder in private company; IBM: Research Funding; Pharmacyclics: Research Funding; Ultima Genomics: Research Funding. Ghobrial: Bristol-Myers Squibb: Consultancy, Honoraria; Disc Medicine: Other: Spouse is Chief Medical Officer and holds equity in the company; Takeda: Consultancy, Honoraria; Amgen: Consultancy; Janssen: Consultancy, Honoraria; Vor Biopharma: Ended employment in the past 24 months, Honoraria, Speakers Bureau; Novartis: Consultancy, Honoraria, Research Funding; Sanofi: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; Menarini Silicon Biosystems: Consultancy, Honoraria; The Binding Site: Consultancy; GlaxoSmithKline: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; 10x Genomics: Honoraria; Regeneron: Consultancy, Honoraria; Adaptive: Honoraria; Aptitude Health: Consultancy; Huron Consulting: Consultancy; Oncopeptides: Consultancy; Window Therapeutics: Consultancy.

*signifies non-member of ASH