Abstract
Chimeric antigen receptor (CAR)-T cells are increasingly used for the treatment of hematologic malignancies. Treatment success relies highly upon sufficient expansion of CAR-T effector cells. Accordingly, longitudinal quantification of CAR-T cells during therapy is clinically important. Techniques to quantify CAR-T cells in patient blood samples are based on flow cytometry and PCR. However, cellular kinetics of CAR-T cells are very complex and under current investigation. In this study, feasibility of CAR-T cell quantification by cell-free DNA (cfDNA) was analyzed. cfDNA isolated from 74 blood samples of 12 patients during lymphoma treatment with the anti-CD19 CAR-T cell product axicabtagene ciloleucel (axi-cel) were analyzed. Concentrations of cfDNA specific for the CAR-T gene construct (cfCAR-DNA) and a reference gene were quantified by a newly designed digital-droplet PCR (ddPCR) assay. Detection and quantification of cfCAR-DNA was feasible and reliable for all patients included. Relative quantification of cfCAR-DNA compared to a reference gene, suitable for genomic DNA analysis, was heterogeneous in treatment responders and non-responders. In contrast, parallel analyses of cfCAR-DNA and reference cfDNA in a patient-specific approach gave insight into active lymphoma killing and treatment responses. In summary, plasma cfDNA determination in lymphoma patients is a promising tool for future clinical decision making.
Highlights
Chimeric antigen receptor (CAR)-T cells are increasingly used for the treatment of hematologic malignancies
The modified digital-droplet PCR (ddPCR) assay was evaluated by analyzing cell-free DNA (cfDNA) obtained from patients treated with axi-cel and by analyzing conditioned medium (CM) in cell culture experiments. cfDNA was reliably detected with the primerprobe pairs used, which distinctly discriminated between FAM-labeled (CD19-CAR) and Hexlabeled (TERT as reference gene) events in patients’ cfDNA samples (Figure 1A)
The assay had a limit of detection of at least 3 CAR-T cells in a background of 10,000 cells when used for cellular DNA analysis (0.03%; Figure S5A)
Summary
Chimeric antigen receptor (CAR)-T cells are increasingly used for the treatment of hematologic malignancies. CAR-T cells are approved in B cell neoplasia including diffuse large B cell lymphoma (DLBCL), mantle-cell lymphoma, B-lineage acute lymphatic leukemia (B-ALL), and multiple myeloma.[1,2,3,4] In most studies, sufficient expansion of the CAR-T cells is associated with overall response rates and side effects, such as cytokinerelease syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS).[5,6,7,8] quantification of CAR-T cells in patients during treatment is important in research and routine clinical settings. Techniques including flow cytometry and qPCR are commonly used to detect CAR-T cells in patients’ blood samples.[1,2,6,9] In these studies, CAR-T cell expansion followed a distinct pattern, reaching a maximum peak within the first 7 to 14 days, followed by subsequent reduction and loss of CAR-T cell signal in a proportion of patients.[1,6] Recently, our group and others have developed digital-droplet PCR (ddPCR) assays to detect CAR-T cells in peripheral blood.[10,11] The advantages of ddPCR compared to quantitative real-time PCR (qPCR) are high interlaboratory reproducibility, omission of calibration curves, and feasibility of the assay.[12,13] ddPCR assays have low limits of detection and are already applied in multiple settings, including analyses of chimerism and minimal residual disease.[12,14] The handling of qPCR and ddPCR data is currently discussed, because multiple factors influence the amount and composition of cellular (genomic) DNA in patients’ blood samples during CAR-T therapy.[15,16]
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