MRD and Biomarkers

Category: MRD and Biomarkers

Identification of a CAR–Derived Clone by NGS-based MRD After Fully Human BCMA CAR T-Cell Therapy in Multiple Myeloma

(PA-198) Identification of a CAR–Derived Clone by NGS-based MRD After Fully Human BCMA CAR T-Cell Therapy in Multiple Myeloma

Wednesday, September 17, 2025

Wenqiang Yan, MD, PhD

Resident physician
State Key Laboratory of Experimental Hematology, National Clinical Research Center for Blood Diseases, Haihe Laboratory of Cell Ecosystem, Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College

Introduction: B cell maturation antigen (BCMA)-directed chimeric antigen receptor T-cell (CAR-T) therapy has significantly improved survival outcomes in relapsed/refractory multiple myeloma (RRMM), frequently inducing deep responses with high rates of minimal residual disease (MRD) negativity. However, during routine next-generation sequencing (NGS)-based MRD assessments in our real-world cohort, we identified a persistent novel clone signal in a subset of patients, despite sustained remission and clearance of the original myeloma clone. This raises concerns about MRD interpretation in the setting of genetically engineered T cell therapies.

Methods: We retrospectively analyzed 55 myeloma patients who received BCMA CAR-T cell therapy at our center and had at least one NGS-MRD assessment post-infusion, as part of real-world observational studies or investigator-initiated trials (IIT). NGS-MRD analysis was conducted using the Neo-MRD® assay (Neoimmune, Shenzhen, China), targeting V(D)J rearrangements in IGH, IGK, and IGL genes. Lentiviral vector copy number (VCN) was quantified using digital droplet PCR (ddPCR) in 27 bone marrow (BM) DNA samples collected at MRD timepoints. Single-cell RNA, BCR, and TCR sequencing were performed on bone marrow mononuclear cells from P22 at T4 (D28 post-infusion), to explore the clonal origin of the novel clone.

Results: Among 55 patients analyzed, 25 (45.5%) exhibited this novel clone following infusion of equecabtagene autoleucel (eque-cel), a fully human-derived BCMA CAR-T product. The sequence was identical across patients and aligned with the single-chain variable fragment (scFv) of the eque-cel construct. It was absent in recipients of non-human-derived CAR-T products. The novel clone abundance strongly correlated with CAR vector copy number quantified by digital droplet PCR (R=0.88, P< 0.001). Single-cell multiomic sequencing confirmed its origin from CAR+ T cells, excluding endogenous derivation. This previously unrecognized NGS-MRD artifact reflects CAR transgene persistence rather than residual disease.

Conclusions:

Collectively, our findings demonstrate that persistent novel clone observed post-eque-cel therapy represents an analytical artifact derived from the CAR transgene, not residual disease or secondary lymphoproliferative processes. Awareness of this signal is essential to prevent misinterpretation of disease status and to ensure accurate clinical decision-making in the post-CAR-T setting.