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    MRD and Biomarkers

    Category: MRD and Biomarkers

    Profiling Minimal Residual Disease with Single-Cell Whole-Genome Sequencing of Circulating Tumor Cells in Patients Receiving BCMA- or GPRC5D-Targeted Agents

    (PA-195) Profiling Minimal Residual Disease with Single-Cell Whole-Genome Sequencing of Circulating Tumor Cells in Patients Receiving BCMA- or GPRC5D-Targeted Agents

    Wednesday, September 17, 2025
    Introduction: Genetic profiling can guide the use of targeted therapies in relapsed/refractory multiple myeloma (RRMM); however, it typically requires bone marrow (BM) biopsies, which are often not performed in advanced stages of the disease. Consequently, the use of immunotherapies like anti-BCMA or GPRC5D CAR-T cells and bispecific T-cell engagers (TCEs) is rarely informed by relevant somatic mutations. To address this critical unmet need, we explored whole-genome sequencing (WGS) on (single) circulating tumor cells (CTCs) isolated from peripheral blood (PB) of RRMM patients before and during therapy, aiming to generate clinically relevant genomic data without relying on invasive BM sampling.

    Methods: CTCs were enriched for CD138+ cells using autoMACS® columns and sorted by 9-color FACS based on abnormal immunophenotypes. DNA was extracted from single cells and mini-pools (up to 25 cells) using primary template-directed amplification (ResolveDNA®, Bioskryb Genomics), or directly from pools of >50 CTCs. Libraries were sequenced on NovaSeq X10B flowcells, and tumor-specific variants were identified via comparison to matched germline controls. Clinical data were assessed at baseline; response was assessed by IMWG Uniform Response Criteria.

    Results: We profiled CTCs from 16 RRMM patients (median 6 prior lines; range 3–12), treated with anti-BCMA CAR-T (n=8), anti-BCMA TCE (n=5), or anti-GPRC5D TCE (n=3). Tumor origin of CTCs was confirmed in all cases (median tumor purity 99% [22-100%]), and clonotypic BCRs were detected in 88% (14/17). Initiating events—translocations and hyperdiploidy—were observed in 81% (13/17). Somatic driver mutations were identified in 81% (13/17), including NRAS/KRAS (6/17), TP53 (3/17), DIS3 (3/17), SP140 (1/17). Of note, no mutations in GPRC5D or TNFRSF17 (BCMA) were found, even among those previously treated with anti-BCMA CAR-T (ide-cel n=4, cilta-cel n=1). The absence of mutation in immunotherapy targets aligned with treatment response (CR n=13; SD n=1; PD n=2; non-relapsed death n=1), suggesting that previous lines of treatment did not select for resistant clones. To investigate whether resistant clones were present before disease relapse, we longitudinally characterized CTCs in 5 patients and 7 timepoints undergoing treatment with immunotherapies. In one anti-BCMA TCE-treated patient (SD), a TP53 R342* clone with complex karyotype expanded (cancer cell fraction 16% to 45%) despite reduced CTC counts. In another post-CAR-T patient (MRD+ at 10⁻⁶, CR), WGS of 7 and 30 single CTCs at 10 and 18 months post-infusion revealed persistent tumor-derived cells lacking BCMA mutations and driving relapse after 20 months.

    Conclusions: Single-cell WGS of CTCs enables high-resolution, longitudinal genomic monitoring in RRMM. This approach enables the detection of mutant clones, tracking of increasing subclones, and supports treatment adaptation, all without requiring a bone marrow biopsy.