Digital Droplet PCR Based Monitoring of CAR-T Cell Expansion in Lymphoma Patients: Impact on Treatment Outcome

Introduction In vivo CAR-T cell proliferation is central to clinical response and toxicity in patients with hematologic malignancies. CAR-T expansion has been evaluated by calculating the AUC in clinical trials. However, AUC requires longitudinal monitoring by flowcytometry or qPCR, which is time expensive, costly and operator dependent. Methods We evaluated CAR-T levels in a cohort of NHL patients through digital droplet PCR (ddPCR) on day 28 ± 7 post-commercial CAR T infusion. In this internal assay, the PCR primers target the Mammalian expression vector present in the CAR constructs as well as the ATP2B4 reference gene. The CAR-T and reference gene probes segregate differently depending on the intensities of the droplets’ signal. The concentration of CAR construct per microliter (uL) is then extrapolated using a Poisson law-based formula. The assay sensitivity is ∼0.003% of CAR construct per reference gene. We collected outcome data retrospectively. Results Between October 2020 and May 2022, 77 patients (60% male) with DLBCL (75%), MCL (12%) and FL (13%) received axi-cel (83%), brexu-cel (12%), or tisa-cel (5%), with flu/cy (58%) or bendamustine (42%) lymphodepletion and had ddPCR testing within the timeframe. Median follow-up was 5.5 months (IQR 3.1-9 mo) with day 28 overall response rate 84%, complete remission 61%, and progressive disease 15%.47% of patients experienced grade 0-1 CRS, while 53% had a grade 2+. Median CAR copies/uL was significantly lower (p=0.002) in the former (1/uL, range 0 – 474) compared to the latter (4/uL, range 0 – 180). 13% of patients had a grade 2+ ICANS and no significant association was found between ICANS grade and day 28 CAR copies.Excluding the 7 patients who had progression at the same time the sample was drawn, Day 28 landmarked 3 and 6 month progression free survival (PFS) for patients with non-detectable (18%) vs detectable (82%) ddPCR was 31% (95% CI 12-76) vs 82% (95% CI 71-93) and 31% (95% CI 12-76) vs 73% (95% CI 59-90), respectively (p<0.001, Figure 1A). Median CAR copies at day 28 for detectable patients was 3.39/uL. PFS at 6 months was significantly higher in patients above the median, being 82% (95% CI 67-100), compared to 66% (95% CI 47-94) in detectable <3.39/ul and 31% (95% CI 12-76) in those with non-detectable CAR copies (p=0.003, figure 1B)Univariable analysis for day 28 detection >0 CAR copies/ul was performed including age, sex, elevated LDH, lines of therapy, bridging and specific histology: none of them reached statistical significance. Of note, patients receiving bendamustine lymphodepletion had a significant lower rate of CART cell detection at day 28 by Fisher Exact Test (p<0.001). Conclusion Assessment of PB CAR-T concentration performed by ddPCR at a single timepoint reliably predicted CAR-T cells efficacy with detectable CAR construct at day 28 predicting better 6-month PFS. Further research is needed to define the most accurate timepoint.