Monitoring Plasma Cells and Clonal Emergence through Genome Sequencing of Circulating Tumor Cells for Minimally Invasive Molecular Characterization of Multiple Myeloma

Introduction Multiple Myeloma (MM) develops from well-defined precursor stages, however, bone marrow (BM) biopsy limits screening and monitoring strategies for patients. Precursor conditions are incidentally diagnosed and require invasive BM biopsies for complete characterization, highlighting the need for improved early detection methods.

Minimally invasive blood biopsies to measure circulating tumor cells (CTCs) as markers of MM disease development are a promising solution to this need. CTCs are amenable to repeatable enrichment, enumeration, and molecular characterization for early detection, real-time monitoring, and assessment of treatment response and clonal evolution.

We present our novel method, MinimuMM-seq, for enumeration and whole genome sequencing (WGS) of CTCs that enables genomic profiling and WGS-based cytogenetic analyses from enriched liquid biopsy samples. CTCs were enumerated from 241 patients, with 50 patients undergoing WGS that revealed that CTCs were of tumor origin and could faithfully match BM sequencing results, detecting 100% of clinically reported events. Longitudinal sampling of CTCs tracked clonal dynamics over time and detected the emergence of high-risk genetic subclones.

Methods Peripheral blood from 241 precursor patients (82 MGUS, 159 SMM) from the PCROWD observational study (IRB #14-174) was collected in CellRescuepreservation tubes and processed on the CellSearch® system (Menarini Silicon Biosystems) using the Circulating Multiple Myeloma Cell (CMMC) assay kit with 4mL of blood. Enrichment and enumeration of CTCs was based on CD138⁺38⁺CD45^-19^- immunophenotype. Nucleated cells were identified using DAPI staining. In 50 patients, CTCs were sorted for library construction, quantification, and WGS on Illumina NovaSeq6000. Mutation analyses were performed with the cancer genome analysis pipelines of the Broad Institute. Tumor phylogeny was reconstructed with the PhylogicNDT algorithm in patients with matched CTC and BM sequencing (n=27), as well as serial CTCs derived from PB only (n=10).

Results CTCs were detected in 75% of MGUS and 86% of SMM patients enrolled in the study, with a median count of 2.5 (range 0 to 1328) and 13 CTCs (range 0 to 43836), respectively. An increased number of CTCs correlated with progressive disease staging from MGUS, to low, intermediate and high-risk SMM defined by IMWG 2/20/20 risk stratification.

MinimuMM-seq yielded a median tumor purity of 98% (range 45 to 100%) in CTCs. Our method detected 100% of clinically reported BM biopsy events by fluorescence in situ hybridization (FISH), including translocations and copy number abnormalities. It could replace molecular cytogenetics for diagnostic yield and risk classification in patients with paired BM and CTC samples (n=27). Additional diagnostic yield compared to clinical BM FISH was found in 88% of patients. Our method detected novel events and unknown/cryptic translocations, such as t(8;14) and t(14;20), and MM driver mutations, including KRAS, NRAS, and TP53.

As peripheral blood continuously circulates, liquid biopsy sampling may be affected by the phenomena of spatial and temporal heterogeneity in CTC burden. In 10 patients longitudinal CTC sampling was carried out to monitor clonal evolution of disease. We validated stability of the major clone and described shifting dynamics of subclones over time with the emergence of potentially high-risk CTC subclones. Expansion of subclones harboring MM events (such as 13q deletion) and driver mutations (such as KRAS and DIS3) was observed, indicating their potential to confer selective advantage for clonal fitness and independence for circulation. Anecdotally, one SMM patient received early interventional treatment, and we identified the emergence of the new fittest clone (bearing KRAS p.G12S) before any potential resistance was clinically observed.

Conclusion We have established an approach for WGS of CTCs that can replace standard molecular cytogenetics. CTCs harbored the same pathognomonic MM abnormalities as BM plasma cells. We tracked clonal dynamics over time and detected the emergence of high-risk CTC subclones. Our findings show that capture and genomic profiling of CTCs could be a robust surrogate for BM biopsy, allowing minimally invasive detection and monitoring of disease and unlocking the clinical potential of liquid biopsies for MM diagnostics.

Nadeem:Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Research Funding; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Karyopharm: Membership on an entity's Board of Directors or advisory committees; GSK: Membership on an entity's Board of Directors or advisory committees; Adaptive Biotechnologies: Membership on an entity's Board of Directors or advisory committees; GPCR Therapeutics: Membership on an entity's Board of Directors or advisory committees. Auclair:AstraZeneca: Current Employment, Other: No conflicts to declare during the conduct of this study. Now an employee of AstraZeneca. Getz:IBM: Research Funding; MSMutSig: Patents & Royalties; MSMuTect: Patents & Royalties; Scorpion Therapeutics: Consultancy, Current equity holder in publicly-traded company, Other: Founder; SignatureAnalyzer-GPU: Patents & Royalties; Pharmacyclics: Research Funding; MSIDetect: Patents & Royalties; POLYSOLVER: Patents & Royalties. Ghobrial:Celgene: Research Funding; Window Therapeutics: Other: Advisory board participation; Veeva Systems: Honoraria; Vor Biopharma: Honoraria; Huron Consulting: Honoraria; Pfizer: Honoraria; Oncopeptides: Honoraria; Sanofi: Honoraria; Janssen: Honoraria; Menarini Silicon Biosystems: Honoraria; Sognef: Honoraria; GSK: Honoraria; Takeda: Honoraria; Bristol Myers Squibb: Honoraria; Aptitude Health: Honoraria; Amgen: Honoraria; Novartis: Research Funding; AbbVie: Honoraria; The Binding Site: Honoraria; Adaptive: Honoraria.