Absence of Evidence for Pervasive CAR19 Driven T-Cell Lymphomagenesis Revealed By Comprehensive Genomic Profiling of an Index Tumor.

Despite the success of chimeric antigen receptor (CAR) T-cell therapies, concerns over toxicity remain. Recent reports indicate development of post-infusion T-cell lymphoma (TCL) after CAR therapy. There is minimal data regarding TCL development after commercially available CAR19 products. We analyzed 234 cases of lymphoma (189 LBCL, 20 FL, 25 MCL) treated with commercial CAR19 (100% axi-cel and brexu-cel). One patient, a 59-year-old female with CD19+/CD20+/EBV+ DLBCL, developed a post-infusion CD3+/CD4+/EBV+ T-cell lymphoma (Fig 1) diagnosed in the bone marrow (BM) on day 55 (D55). We comprehensively characterized this patient using longitudinal cfDNA samples (CAPP-seq) and single cell RNA sequencing (scRNA). Peripheral blood viral profiling by qPCR and cell free DNA (cfDNA) sequencing demonstrated post-infusion EBV viral expansion (Fig 2A). Despite this EBV expansion, Clonoseq MRD analysis for the original tumor immunoglobulin clonotype promptly became negative in the blood and bone marrow after CAR19 (Fig 2B). cfDNA analysis suggested retraction followed by subsequent re-expansion of circulating tumor DNA (ctDNA) with good expansion of the axi-cel vector (Fig 2C). A TCR clone first detectable at D28 then dominated the cfDNA at D55 consistent with a new TCL (Fig 2D). Copy number profiling of ctDNA demonstrated novel amplification of chr1q and deletion of chr6q in the D55 sample that was not present in the pre-infusion sample possibly indicating development of a novel driver mutation post-infusion (D0, Fig 2E). To better define the biology of the post-CAR19 TCL we performed scRNA-Seq profiling of the bone marrow in the index patient and 4 healthy controls (Fig 3A). The post-CAR TCL demonstrated the presence of a highly clonal T-cell population (Fig 3B) with TCR sequence consistent with that found in the peripheral blood (Fig 3C). This tumor was universally negative for CAR19 RNA (Fig 3D). Inferred copy number variation analysis revealed novel amplification of chromosome 1q and deletion of chromosome 6q associated with the malignant clone (Fig 3E). This study highlights what is to our knowledge the first comprehensive genomic profiling of a post-CAR TCL after commercial CAR19. We find a novel TCL population that does not demonstrate evidence of CAR19 mRNA expression. Instead, the likely pre-existent TCL clone arises on D28 with novel amplification of chr1q, deletion of ch6q, and expansion of EBV virus in the peripheral blood. Additional profiling of the B- and T-cell tumors is ongoing and will be presented to discern a shared lineage implicating infidelity or trans-differentiation versus an unrelated secondary malignancy. These additional studies in progress include direct DLBCL vs TCL comprehensive genotyping, scDNA sequencing of marrow lymphocytes and progenitors, EBV genotyping and terminal repeat analysis, and retroviral insertion site mapping.