Risk of Second Tumors and T-Cell Lymphoma after CAR T-Cell Therapy

Abstract

Background: The risk of second tumors after chimeric antigen receptor (CAR) T-cell therapy, especially the risk of T-cell neoplasms related to viral vector integration, is an emerging concern.

Methods: We reviewed our clinical experience with adoptive cellular CAR T-cell therapy at our institution since 2016 and ascertained the occurrence of second tumors. In one case of secondary T-cell lymphoma, a broad array of molecular, genetic, and cellular techniques were used to interrogate the tumor, the CAR T cells, and the normal hematopoietic cells in the patient.

Results: A total of 724 patients who had received T-cell therapies at our center were included in the study. A lethal T-cell lymphoma was identified in a patient who had received axicabtagene ciloleucel therapy for diffuse large B-cell lymphoma, and both lymphomas were deeply profiled. Each lymphoma had molecularly distinct immunophenotypes and genomic profiles, but both were positive for Epstein-Barr virus and were associated with DNMT3A and TET2 mutant clonal hematopoiesis. No evidence of oncogenic retroviral integration was found with the use of multiple techniques.

Conclusions: Our results highlight the rarity of second tumors and provide a framework for defining clonal relationships and viral vector monitoring. (Funded by the National Cancer Institute and others.).

Figure 1:. Development of post-CAR19 T-cell lymphoma is rare.

A) Screening 724 patients treated with CAR T-cell therapy at Stanford reveals a single T-cell lymphoma developed after standard of care axi-cel infusion. Experimental products that were eventually approved for commercial distribution are included under the commercial name in this figure for the purpose of clarity. B) The index patient was a 59-year-old female with an EBV⁺ DLBCL refractory to RCHOP and RGemOx chemotherapy. She subsequently developed an EBV⁺ PTCL-NOS noted on day 54 leading to hemophagocytic lymphohistiocytosis and death. Axi-cel – axicabtagene autoleucel. Timepoints for major sequencing experiments used in this manuscript are listed in the timeline. DLBCL – Diffuse large B-cell lymphoma, TCL – T-cell lymphoma, RCHOP – Rituximab, cyclophosphamide, hydroxydaunorubicin (Adriamycin), oncovin/vincristine, and prednisone, R-RGemOx – rituximab, gemcitabine, oxaliplatin, CRS – cytokine release syndrome, ICANS – immune effector cell associated neurotoxicity syndrome, MTX – methotrexate, MMF – mycophenolate mofetil, HLH – hemophagocytic lymphohistiocytosis.

Figure 2:. Clinical and molecular characteristics of the index TCL suggest molecular distinction from the DLBCL clone.

A) Immunohistochemistry (IHC) of the original DLBCL tumor in the lymph node demonstrated an abnormal large cell population that was CD19⁺CD20⁺EBER⁺. B) IHC of the CD3⁺CD4⁺CD8⁻EBER⁺ index T-cell lymphoma C) BCR Clonotype analysis; loss of DLBCL BCR detection after axi-cel infusion measured by cellular profiling of the LN, blood and bone marrow (clonoSEQ). Longitudinal data points depicted in order from: 1) DLBCL lymph node at Day −88, 2) peripheral blood at Day −50, 3) peripheral blood at Day 25, 4) Bone marrow harboring TCL. TCRB tracing was performed by identifying the dominant clone in the index bone marrow specimen and determining clonal abundance in prior samples normalized as clones per million nucleated cells (see Methods). D) CAPP-Seq profiling of the plasma. There was low-level detection of the original DLBCL at the time of axi-cel infusion which subsequently became undetectable by. At D+28 a novel TCR clone associated with the index TCL was detectable in the plasma by DNA capture profiling. This clone dominated circulation at the time the TCL became clinically apparent. E) Axi-cel expansion and retraction measured by hybridization capture sequencing (CAPP-Seq, solid line) and flow cytometry (dotted line). F) Concurrent with development of the index TCL EBV titers rose dramatically in clinical profiling of the plasma. G) Comprehensive molecular profiling of longitudinal samples by CAPP-Seq. Panel 1 demonstrates increased malignant TCR clonal load and increased EBV expression in conjunction with gain of CD3 and CD4 expression measured by TSS entropy (EPIC-seq). Grey boxes with diagonal line indicate no data available. Panels 2 and 3 demonstrate the molecular mutational spectrum associated with the original DLBCL and the index TCL are distinct providing no evidence of trans-differentiation of the original B-cell malignancy. Grey boxes indicate the mutation is not detected. Panel 4 demonstrates high burden of pre-existing DNMT3A and TET2 clonal hematopoiesis which was present at the time of diagnosis and remained at high levels throughout the patient’s course. Panel 5 demonstrates the presence of copy number variation in the original DLBCL and the index TCL. DLBCL has a distinct chr3q gain whereas the TCL has a distinct chr1q and chr6q loss. BCR – B-cell receptor. CAPP-Seq - CAncer Personalized Profiling by deep Sequencing. TCR – T-cell receptor. EBV – Epstein-Barr virus. TSS – transcription start site. LN – lymph node. BM – bone marrow.

Figure 3.. RNA and DNA profiling of the index TCL at single cell resolution shows no evidence of axi-cel vector integration.

A) scRNA analysis of the index patient marrow versus five healthy control marrows demonstrates distinct clustering of the pathologic marrow consistent with the presence of TCL and HLH. B) Single cell TCR sequencing reveals the previously described TCL clone found in the plasma matches the T-cell cluster present in the index lesion. C) There is complete absence of axi-cel mRNA expression at single cell resolution in the index TCL. D) Cell surface antibody profiling combined with single cell DNA profiling again reveals the characteristic CD3⁺CD4⁺CD8− malignant clone clustering separately from myeloid cell types. E) scDNA profiling of the pre-existing DNMT3A R882C, TET2 deletion, and TET2 L1212Vfs clone. The myeloid populations also have the same clonal hematopoiesis mutations detected. The TET2 L1212Vfs clone is hemizygous following an initiating TET2 single copy deletion that preceding the DNMT3A and TET2 mutations. Annotations as follows: TET2 del/wt – TET2 deletion only, TET2 del/wt + DNMT3A mut/wt – TET2 deletion + DNMT3A R882C, TET2 del/mut + DNMT3A mut/wt – TET2 deletion + DNMT3A R882C + TET2 L1212Vfs. F) scDNA sequencing of axi-cel using an 11-amplicon panel tiled across the axi-cel vector again fails to demonstrate any evidence of axi-cel integration into the index TCL. scRNA – single cell RNA. scDNA – single cell DNA. UMAP – Uniform manifold approximation and projection. Red circles indicate T-cell lymphoma cluster location in each UMAP plot. G) Schematic of parallel development of LBCL and TCL from a common hematopoietic progenitor cell population. Single cell DNA sequencing data indicates a linear accumulation of clonal hematopoiesis (TET2 deletion → DNMT3A R882C mutation → TET2 L1212fs mutation). The presence of this mutation in all cell populations in scDNA sequencing data indicates the founder population was in hematopoietic stem cells which propagated into the common lymphoid progenitor and common myeloid progenitor (CLP and CMP). Both the DLBCL and TCL tumors contain recombined BCR and TCR respectively consistent with mature lymphoid neoplasms and indicating the DLBCL was derived after germinal center maturation and the TCL was post-thymic. EBV-activation due to immune suppression were likely common events in both tumors but led to unique subsequent mutation and structural variation that resulted in the mature neoplasm. The malignant clones can be traced both by their respective BCR and TCR sequences as well as their unique mutational and structural changes. TCR – T-cell receptor, BCR – B-cell Receptor, BM – bone marrow, CH – clonal hematopoiesis.