Quantification of cell-free DNAfor the analysis of CD19-CAR-T cells during lymphoma treatment

Thomas Mika; Julia Thomson; Verena Nilius-Eliliwi; Deepak Vangala; Alexander Baraniskin; Gerald Wulf; Susanne Klein-Scory; Roland Schroers

doi:10.1016/j.omtm.2021.10.009

Quantification of cell-free DNAfor the analysis of CD19-CAR-T cells during lymphoma treatment

Mol Ther Methods Clin Dev. 2021 Oct 28:23:539-550. doi: 10.1016/j.omtm.2021.10.009. eCollection 2021 Dec 10.

Authors

Thomas Mika¹, Julia Thomson², Verena Nilius-Eliliwi¹, Deepak Vangala¹, Alexander Baraniskin^{1

3}, Gerald Wulf², Susanne Klein-Scory^{1

4}, Roland Schroers¹

Affiliations

¹ Department of Medicine, Hematology and Oncology, Ruhr University Bochum, 44892 Bochum, Germany.
² Clinic for Hematology and Medical Oncology, Georg-August University, 37075 Göttingen, Germany.
³ Department of Hematology, Oncology and Palliative Care, Evangelisches Krankenhaus Hamm, 59063 Hamm, Germany.
⁴ IMBL, Universitätsklinikum Knappschaftskrankenhaus Bochum, 44892 Bochum, Germany.

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.

Keywords: CAR-T cells; CD19-directed chimeric antigen receptor T cells; axi-cel; cancer immunotherapy; cell-free DNA; digital-droplet PCR (ddPCR); liquid biopsy; lymphoma.