Digital Polymerase Chain Reaction (dPCR) Versus Flow Cytometry (FC), a Validation Study for CAR-T Cell Product Optimal Detection

Abstract

CONCLUSIONS Adoptive immunotherapy with Chimeric Antigen Receptor - T cells (CAR-T), has revolutionized hematological treatment mainly in relapsed/refractory (r/r) B-cell malignancies. CAR-T cells constitute challenging living drugs with heterogeneous in vivo cellular kinetics and adverse side effects that emphasizes the importance of its accurate detection and monitoring. Diagnostic assays are required for precise CAR-T quantification. Digital PCR (dPCR) is characterized by a high specificity, sensitivity and reproducibility, therefore we developed a study to implement this technique for CAR-T detection and compare its efficacy against Flow Cytometry (FC). Duplex dPCR assays were performed to concomitantly amplify CAR and RPP30 (reference gene) sequences using ddPCR Expert Design Assay: CD-19 CAR-T Assay (Bio-Rad, California, USA) with QIAcuity Digital PCR System and data analysis with QIAcuitySoftware Suite (QIAGEN, Germany). To assess sensitivity, a Axicabtagene Ciloleucel (Axi-Cel ®) original product obtained from leftovers's infusion bag patient was employed to perform dPCRs on serial dilutions using genomic DNA(gDNA) from a wild type (WT) patient. Specificity was ensured using WT and no template controls (NTC). Two independent dPCR assays with replicates for each sample were conducted to assess reproducibility. For FC comparative study, CAR-T cells percentage (%CAR-T) was determined by both techniques at variable post-infusion intervals (n=210) in different sample types, peripheral blood (n=205) and cerebrospinal fluid (n=5). Anti-FMC63 scFv Antibody, Mouse IgG1 (Acro biosystems) was employed, using FACSCanto II®, Becton-Dickinson Biosciences with FACSDiva software ® (V8.0; BD) and InfinicytTM software (Cytognos SL, Salamanca, Spain), for data analysis. Correlation coefficients were determined using two-tailed Pearson statistics with a confidence interval of 95%. Statistical analyses were made with SPSS Software (IBM, Germany). The dPCR data collected of 2 independent dPCR assays revealed a good split between the negative and positive partitions for the targets evaluated, showing a progressive decrease of the %CAR-T detected (63,88% - 4,12% - 0,77% - 0,08% - 0,01% - 0,00%). This reduction is correlated with the dilution factor applied to the series with an initial concentration of 50ng/µL both for CAR-T original product and WT gDNA. WT and NTC samples were negative with a SD=0; %CV=0, intra and inter-assays, evidencing thus the specificity of the technique. Positivity of the last (1 pg/µL) samples revealed a maximum sensitivity of 0.009 %CAR-T, threshold below which false positive results would be considered. WT gDNA did not influence the assay's sensitivity. 3 replicates of each sample and 2 independent dPCR assay were performed to study potential variations, exhibiting an excellent intra and inter-assay reproducibility with a minimal SD across dilutions ( Table.1.) Comparative study with FC revealed a light correlation between techniques with a Pearsons's correlation coefficient (R=0,553) ( p<0,001). Differences among both were enhanced for Minimal Residual Disease (MRD) evaluation from 60 days post-infusion monitoring (R=0,58) ( p=0,066) in which CAR-T cells can be detected applying dPCR up to 2 years and 9 months post-infusion, whereas being undetectable by FC. Complex CAR-T in vivo kinetics enhances the need for optimal diagnostic and monitoring techniques. dPCR is an ideal tool for precise CAR-T therapy evaluation with a higher grade of specificity, sensitivity and reproducibility than FC, mainly for MRD assessment. dPCR implementation and the establishment of standardized protocols across centers is required to improve CAR-T biology knowledge with a direct benefit on patient care.