Background Chimeric antigen receptor (CAR) T-cells directed against CD19 (CART19) are effective in B-cell malignancies, but little is known about the molecular factors predicting clinical outcome of CART19 therapy. The increasingly recognized relevance of epigenetic changes in cancer immunology prompted us to determine the impact of the DNA methylation profiles of CART19 cells on the clinical course. Methods We recruited 114 patients with B-cell malignancies, comprising 77 acute lymphoblastic leukemia (ALL) and 37 non-Hodgkin lymphoma (NHL) patients, who were treated with CART19 cells. Using a comprehensive DNA methylation microarray, we determined the epigenomic changes that occur in the patient T-cells upon transduction of the CAR vector. The effects of the identified DNA methylation sites on clinical response, cytokine release syndrome (CRS), immune effector cell-associated neurotoxicity syndrome (ICANS), event-free survival (EFS) and overall survival (OS) were assessed. All statistical tests were 2-sided. Results We identified 984 genomic sites with differential DNA methylation between CAR-untransduced and CAR-transduced T-cells before infusion into the patient. Eighteen of these distinct epigenetic loci were associated with complete response (CR) adjusting by multiple testing. Using the sites linked to CR, the EPICART signature was established in the initial discovery cohort (n = 79), which was associated with CR (Fisher’s exact test, P<.001) and enhanced EFS (HR = 0.36, 95% CI = 0.19 to 0.70, P=.002; log-rank P=.003) and OS (HR = 0.45, 95% CI = 0.20 to 0.99, P=.047; log-rank P=.04;). Most important the EPICART profile maintained its clinical course predictive value in the validation cohort (n = 35) where it was associated with CR (Fisher’s exact test, P<.001) and enhanced OS (HR = 0.31, 95% CI = 0.11 to 0.84, P=.02; log-rank P=.02). Conclusions We show that the DNA methylation landscape of patient CART19 cells influences the efficacy of the cellular immunotherapy treatment in patients with B-cell malignancy.

No abstract

DNA methylation, one of the best characterized epigenetic marks in the human genome, plays a pivotal role in gene transcription regulation and other biological processes in humans. On top of that, the DNA methylome undergoes profound changes in cancer and other disorders. However, large-scale and population-based studies are limited by high costs and the need for considerable expertise in data analysis for whole-genome bisulphite-sequencing methodologies. Following the success of the EPIC DNA methylation microarray, the newly developed Infinium HumanMethylationEPIC version 2.0 (900K EPIC v2) is now available. This new array contains more than 900,000 CpG probes covering the human genome and excluding masked probes from the previous version. The 900K EPIC v2 microarray adds more than 200,000 probes covering extra DNA cis-regulatory regions such as enhancers, super-enhancers and CTCF binding regions. Herein, we have technically and biologically validated the new methylation array to show its high reproducibility and consistency among technical replicates and with DNA extracted from FFPE tissue. In addition, we have hybridized primary normal and tumoural tissues and cancer cell lines from different sources and tested the robustness of the 900K EPIC v2 microarray when analysing the different DNA methylation profiles. The validation highlights the improvements offered by the new array and demonstrates the versatility of this updated tool for characterizing the DNA methylome in human health and disease.