A Clonally Expanded Nodal T Cell Population Diagnosed As T Cell Lymphoma after CAR-T Therapy

Reports of secondary malignancies after chimeric antigen receptor (CAR)-T and possible CAR-T derived malignant transformation necessitate caution. In November 2023, the FDA issued a warning regarding risk of T cell malignancies deriving from the CAR-T manufacture process and several case reports have identified T cell malignancies occurring after BCMA- or CD19-directed CAR-T cell therapy, some identifying the CAR transgene.

We systematically assessed our clinical database of patients with non-Hodgkin lymphoma (NHL), MM, and B-cell acute lymphoblastic leukemia (ALL) receiving one of the six FDA-approved CAR-T products to quantify incidence of secondary malignancy at our center from 2017-2023 with a focus on possible T-cell lymphomas (TCLs). We identified 43 cases of secondary malignancy (6.9%, n=12 myelodysplastic syndrome [MDS]/acute myeloid leukemia [AML], n=28 solid tumors, n=3 possible TCL) from 626 total patients; six cases were in MM patients and 37 in NHL.

One possible TCL case was a patient treated with third-line axicabtagene ciloleucel (Axi-cel) for transformed follicular lymphoma (FL). The patient achieved complete metabolic response on the first restaging PET/CT thirty days after CAR-T, which was subsequently maintained. Two years after CAR T, the patient developed a protracted COVID-19 infection complicated by recurrent superimposed bacterial pneumonias. In this context, the patient presented with weight loss and fatigue, prompting a PET/CT, which revealed highly FDG-avid right cervical lymphadenopathy, concerning for disease relapse or new malignancy. H&E stain of a core needle biopsy demonstrated architecture effaced by an infiltrate of medium to large cells with irregular/folded nuclei, condensed chromatin, variably prominent nucleoli, and scant eosinophilic cytoplasm with small scattered lymphocytes in the background. By immunohistochemical staining, the abnormal appearing cells expressed mature T cell markers (CD3, CD2, CD5, CD7), with many positive for both CD4 and CD8 and positive for TCR-BF1. Scattered cells were also positive MUM1 and BCL-6, Stathmin, PD-1, and BCL-2 suggestive of a T follicular helper (TFH) neoplasm. T cell receptor (TCR) gene rearrangement studies demonstrated a clonal process. This was diagnosed as TFH TCL. A repeat PET/CT demonstrated spontaneous resolution of the node.

To determine the cellular etiology and molecular characteristics of this aberrant T cell population, whole genome sequencing (WGS) was performed on remaining lymph node (LN) sections and genomic DNA from blood. The CAR construct was not identified by WGS. Several mutations in TET2 were identified in both LN and blood. To interrogate the clonality, phenotype, and spatial microenvironment of this hyperproliferative T cell population, we performed single-nuclei spatial transcriptomics on a fresh-frozen core needle biopsy using a novel approach with 5’ Slide-tags to recover TCR sequences. This revealed a LN dominated by transcriptionally abnormal CD4+CD8+ double positive T cells (51% of profiled nuclei). The rest of the LN was composed of 20% macrophages, 10% regulatory T cells, 5% fibroblasts, 5% CD4+ T cells, and smaller proportions of CD8+ T cells, NK cells, endothelial cells, and dendritic cells. No B cells were detected. The CD4+CD8+ T cells were distributed across the full area of the profiled LN in a spatially disorganized manner. The TFH diagnostic markers probed histologically (Ki67, PD1, BCL6, CD10) were not specific to the CD4+CD8+ T cell population. Next, we examined the clonality of T cells in the LN. The majority of CD4 or CD8 single positive T cells belonged to single member, small, or medium sized clonotypes. The CD4+CD8+ T cells belonged to a highly expanded clonotype, consisting of two alpha chains (CASPGGLTGGGNKLTF, CALSHPFRNSGNTPLVF) and two beta chains (CASSLVVWGRGLNEQFF, CASSQQDSRNTIYF).

In the current era of treatment success with CAR-T therapy, the field is now grappling with unprecedented clinical presentations of immunologic sequelae of CAR-T, including infectious, inflammatory, and malignant complications. Our findings underscore the need for careful clinicopathologic correlation in evaluating patients with unusual presentations after CAR-T therapy.