P1427: monitoring car-t cell in b-cell lymphoma patients through single chain variable fragment by digital pcr

Background: CD19-targeted chimeric antigen receptor (CAR)-T cell is a treatment for B-cell lymphoma patients. CAR-T cell monitoring is important to ensure a correct follow-up, being multiparametric flow cytometry (MFC) the actual gold standard technique. However, other molecular techniques such as digital PCR (dPCR) could add complementary information that could be valuable to predict CAR-T response. Both available commercial antiCD19 CAR-T cell therapies, Tisagenlecleucel (tisa-cel) (Kymriah®) and Axicabtagen Ciloleucel (axi-cel) (Yescarta®) share the single variable fragment domain (FMC63), which makes possible the measurement by dPCR. Aims: To evaluate the usefulness of digital PCR for monitoring CAR-T cell levels in comparison to multiparametric flow cytometer. Methods: Forty-five patients diagnosed with diffuse large B-cell lymphoma (36), transformed-follicular lymphoma (7) and mediastinal primary large B-cell lymphoma (2), treated consecutively with anti-CD19 CAR-T cell therapy between June 2019 and May 2021 were included in this study. One hundred and forty-two peripheral blood (PB) samples were collected at days +7, +14, +30, +90 (40, 39, 40 and 23 samples, respectively) after infusion. DNA was purified using the Maxwell® RSC Whole Blood DNA Kit (Promega, USA). dPCR assays were performed on the QIAcuity One platform (QIAgen, Germany). MFC analysis was performed on a DxFLEX cytometer (Beckman Coulter), using CD19 (20-291) protein-FITC (ACRO Biosystems). To assess the sensitivity of molecular methodologies, serial dilutions from 100% to 0.001% of CAR-T were analyzed by dPCR. Correlation of CAR-T cell detection between MFC and dPCR was calculated using Pearson’s test. Results: A high correlation between dPCR and MFC was found (r = 0.90), demonstrating the usefulness of dPCR to quantify absolute number of CAR copies (Figure 1A and 1B). dPCR improved the detection capacity of MFC, detecting CAR-T in 15 samples that were negative by MFC because of low CAR-T cell presence in PB (Figure 1C), as MFC measures CAR-T events in total PB the low content of CAR-T could reduce MFC sensitivity. Median number of CAR-T cells detected by dPCR was greater than that quantified by MFC in each days of the follow-up (Figure 1D). Different CAR-T cell products (Yescarta and Kymriah) differ in its expansion rates, therefore tisa-cel expand before axi-cel (near day 7th and day 14th, respectively) (Figure 1C). Image:Summary/Conclusion:FMC63 quantification through dPCR was effective for CAR-T measurement, even improving MFC sensitivity, especially when CAR-T counting in PB is low in late monitoring days. dPCR monitoring could complement data given by MFC and being a very sensible gold standard technique in late monitoring as its sensitivity is very superior to MFC in those follow-up days.