Characterization of Leukemic Resistance to CD19-Targeted CAR T-cell Therapy through Deep Genomic Sequencing

Abstract Chimeric antigen receptor (CAR) T-cell therapy targeting CD19 has been a clinical breakthrough for pediatric B-cell acute lymphoblastic leukemia (B-ALL), and loss of the CD19 target antigen on leukemic cells represents a major mechanism of relapse. Previous studies have observed CD19 mutations specific to CD19− relapses, and we sought to clarify and strengthen this relationship using deep whole-exome sequencing in leukemic cells expanded in a patient-derived xenograft. By assessing pre-treatment and relapse cells from 13 patients treated with CAR T-cell therapy, 8 of whom developed CD19− relapse and 5 of whom developed CD19+ relapse, we demonstrate that relapse-specific single-nucleotide variants and small indels with high allele frequency combined with deletions in the CD19 gene in a manner specific to those patients with CD19− relapse. Before CAR T-cell infusion, one patient was found to harbor a pre-existing CD19 deletion in the context of genomic instability, which likely represented the first hit leading to the patient's subsequent CD19− relapse. Across patients, preexisting mutations and genomic instability were not significant predictors of subsequent CD19− relapse across patients, with sample size as a potential limiting factor. Together, our results clarify and strengthen the relationship between genomic events and CD19− relapse, demonstrating this intriguing mechanism of resistance to a targeted cancer immunotherapy.