Single-Cell Whole-Genome Sequencing of Circulating Tumor Cells in Relapsed/Refractory Multiple Myeloma Patients Receiving BCMA- or GPRC5D-Targeted Immunotherapies
Authors: Toenges Rosa , Alberge Jean-Baptiste , Corrado Francesco , Bidikian Nayda , Cordas Dos Santos David M. , Dutta Ankit K. , Lightbody Elizabeth D. , Sklavenitis Pistofidis Romanos , Sperling Adam S. , Anderson Kenneth C. , Midha Shonali , Liu Yuxin , Laubach Jacob P. , Smith Eric L. , Munshi Nikhil C. , Nadeem Omar , Ghobrial Irene , Getz Gad 
Published: December 2024

Introduction: Detecting genetic abnormalities in relapsed/refractory multiple myeloma (RRMM) is crucial for guiding the selection of targeted therapies. This requires a bone marrow biopsy, a procedure often not performed in advanced disease settings. Consequently, the selection of subsequent therapies, such as anti-BCMA or GPRC5D chimeric antigen receptor T cells (CAR T) or bispecific T cell engagers (TCE), is typically not informed by the presence of somatic mutations known to impact treatment efficacy. In addition, disease relapse in RRMM is driven by the expansion of subclones under the selective pressure of treatment. To address these issues, we performed whole-genome sequencing (WGS) of (single) circulating tumor cells (CTCs) using peripheral blood (PB) samples from RRMM patients before and during treatment. Our goal is to inform clinical decisions more effectively without relying on bone marrow biopsies.
Methods: CTCs were first enriched for the CD138+ surface marker using autoMACS® columns and subsequently sorted based on their abnormal immunophenotype by FACS. DNA was then extracted from single cells and mini pools (max. of 25 cells) using a primary template-directed amplification method (ResolveDNA®, Bioskryb Genomics) or without amplification from pools of more than 50 CTCs. WGS libraries of CTCs were then sequenced on NovaSeq X10B flowcells and compared to their matched germline. Clinical characteristics of each patient were assessed at baseline, and responses to therapy were determined according to the IMWG Uniform Response Criteria for MM.
Results: We performed WGS of CTCs in 16 patients with RRMM after a median of 6 lines of therapy (range: 3 to 12) and before receiving an anti- BCMA CAR-T (n=8), an anti-BCMA TCE (n=5), or an anti-GPRC5D TCE (n=3). We confirmed that CTC samples originated from the tumor in all 16 cases (median tumor purity: 99%, range 22% to 100%) and B-cell receptor in most cases(n=14/16, 88%). Initiating events such as translocation and hyperdiploidy were detected in the majority of all cases (n=13/16, 81%). Twelve patients had a FISH test performed earlier during their disease course, we could confirm all of their cytogenetic findings and discover additional high-risk abnormalities at the RRMM timepoint (del(17p), n=1; gain(1q), n=1). Because WGS is unbiased, we also detected point mutations at expected rates in known MM drivers in 13/16 tumors (81%) showing the reliability of our assay (NRAS/KRAS [6/16; 40%], TP53 [3/16; 19%]; DIS3 [3/16; 19%]; SP140[1/16; 6%], etc.). Of note, none of those patients had a detectable mutation in GPRC5D or TNFRSF17 (codes for BCMA), even in those who had already received an anti-BCMA treatment (ide-cel CAR-T, n=4). Lack of target mutation was consistent with the depth of response achieved (CR, n=10; VGPR, n=1; PR, n=2; SD, n=2 [anti- GPRC5D TCE]; non relapsed death, n=1), including in one patient switching from an anti-BCMA CAR-T to an anti-BCMA TCE (VGPR). The absence of mutation(s) affecting BCMA after CAR-T suggests that the previous lines of treatment did not select for a mutated clone that expanded during therapy.
We then investigated whether resistant clones were present prior to disease relapse, which could inform decisions regarding early therapy changes. To that aim, we longitudinally characterized CTCs in 3 patients undergoing treatment with immunotherapies. The first patient receiving anti-BCMA TCE only achieved SD. Despite a 65% decrease in CTC counts, we already detected the selection of a clone bearing a TP53 nonsense mutation R342* and a complex karyotype (cancer cell fraction [CCF] increased from 16% to 45%) before progression. In another patient receiving anti-BCMA CAR-T who achieved a sCR (minimal residual disease [MRD] positive at 10-6), we could perform WGS on 7 single-cells. Of those, 5 were confirmed to originate from the tumor clone, but showed no mutation in TNFRSF17. Similar, the last patient receiving anti-BCMA TCE, we sequenced MRD cells after 6 cycles of therapy (CR) and did not find mutations in TNFRSF17, suggesting another mechanism of treatment escape at this stage.
Conclusion: Genomic characterization of the tumor cells in RRMM, even at the single-cell level at MRD post-therapy, can be used to predict response to immunotherapy, monitor the emergence of high-risk subclones, and inform subsequent treatment decisions for patients with disease relapse without the need for a bone marrow biopsy.