Chronic active Epstein-Barr virus (EBV) infection (CAEBV) is an orphan disease characterized by the proliferation and infiltration of EBV-infected T/natural killer (NK) cells into multiple organs. Although CAEBV is a heterogeneous disease with diverse clinical courses, its pathogenesis remains poorly understood. In this study, we explored the molecular mechanisms underlying CAEBV by performing a comprehensive multiomics analysis, including genome, transcriptome, epigenome, and single-cell transcriptome and surface proteome analyses, of 65 patients with CAEBV. Methylation analysis identified 2 distinct subtypes of NK cell–type CAEBV based on the CpG island methylator phenotype (CIMP). In CIMP-positive CAEBV, regions associated with enhancer of zeste homolog 2 binding sites and histone H3 lysine 27 trimethylation exhibited increased DNA hypermethylation, resulting in downregulation of tumor suppressor and antiherpesvirus genes. CIMP-positive CAEBV had a particularly poor prognosis and displayed a “neoplastic” phenotype with a DNA methylation pattern similar to that of extranodal NK/T-cell lymphoma, a higher tumor mutation burden, and frequent copy number alterations. In addition, both in vitro and in vivo functional assays demonstrated that 5-azacytidine, a hypomethylating agent, was a potentially effective agent for high-risk CIMP-positive CAEBV. Finally, we established a method to effectively detect EBV-infected cells in single-cell analysis, suggesting that EBV-infected NK cells have tissue-resident properties and that innate and adaptive immunity to EBV is compromised in patients with CAEBV. The present findings provide insight into the complex molecular features of CAEBV and suggest potential molecular therapies.

KMT2A-rearranged infant acute lymphoblastic leukemia (ALL) represents the most refractory type of childhood leukemia. To uncover the molecular heterogeneity of this disease, we perform RNA sequencing, methylation array analysis, whole exome and targeted deep sequencing on 84 infants with KMT2A-rearranged leukemia. Our multi-omics clustering followed by single-sample and single-cell inference of hematopoietic differentiation establishes five robust integrative clusters (ICs) with different master transcription factors, fusion partners and corresponding stages of B-lymphopoietic and early hemato-endothelial development: IRX-type differentiated (IC1), IRX-type undifferentiated (IC2), HOXA-type MLLT1 (IC3), HOXA-type MLLT3 (IC4), and HOXA-type AFF1 (IC5). Importantly, our deep mutational analysis reveals that the number of RAS pathway mutations predicts prognosis and that the most refractory subgroup of IC2 possesses 100% frequency and the heaviest burden of RAS pathway mutations. Our findings highlight the previously under-appreciated intra- and inter-patient heterogeneity of KMT2A-rearranged infant ALL and provide a rationale for the future development of genomics-guided risk stratification and individualized therapy.

As chimeric antigen receptor (CAR) T cell therapy targeting CD19 has shown favorable outcomes in patients with relapsed or refractory (r/r) mature B cell lymphomas and B cell acute lymphoblastic leukemia (B-ALL), an increasing number of patients are waiting to receive these treatments. Optimized protocols for T cell collection by lymphapheresis for chimeric antigen receptor (CAR) T cell therapy are urgently needed to provide CAR T cell therapy for patients with refractory and progressive disease and/or a low number of lymphocytes owing to prior chemotherapy. The predicted efficiency of CD3⁺ cell collection in apheresis can guide protocols for apheresis, but a clinically applicable model to produce reliable estimates has not yet been established. In this study, we prospectively analyzed 108 lymphapheresis procedures for tisagenlecleucel therapy at 2 centers. The apheresis procedures included 20 procedures in patients with B cell acute lymphoblastic leukemia and 88 procedures in patients with diffuse large B cell lymphoma, with a median age at apheresis of 58 years (range, 1 to 71 years). After lymphapheresis with a median processing blood volume of 10 L (range, 3 to 16 L), a median of 3.2 × 10⁹ CD3⁺ cells (range, .1 to 15.0 × 10⁹ cells) were harvested. Collection efficiency 2 (CE2) for CD3⁺ cells was highly variable (median, 59.3%; range, 11.0% to 199.8%). Multivariate analyses revealed that lower hemoglobin levels, higher circulating CD3⁺ cell counts, and higher platelet counts before apheresis significantly decreased apheresis CE2. Based on multivariate analyses, we developed a novel formula that estimates CE2 from precollection parameters with high accuracy (r = .56; P < .01), which also suggests the necessary processing blood volume. Our strategy for lymphapheresis should help reduce collection failure, as well as achieve efficient utilization of medical resources in clinical practice, thereby allowing delivery of CAR T cell therapy to more patients in a timely manner.

Spi-1 Proto-Oncogene (SPI1) fusion genes are recurrently found in T-cell acute lymphoblastic leukemia (T-ALL) cases but are insufficient to drive leukemogenesis. Here we show that SPI1 fusions in combination with activating NRAS mutations drive an immature T-ALL in vivo using a conditional bone marrow transplant mouse model. Addition of the oncogenic fusion to the NRAS mutation also results in a higher leukemic stem cell frequency. Mechanistically, genetic deletion of the β-catenin binding domain within Transcription factor 7 (TCF7)-SPI1 or use of a TCF/β-catenin interaction antagonist abolishes the oncogenic activity of the fusion. Targeting the TCF7-SPI1 fusion in vivo with a doxycycline-inducible knockdown results in increased differentiation. Moreover, both pharmacological and genetic inhibition lead to down-regulation of SPI1 targets. Together, our results reveal an example where TCF7-SPI1 leukemia is vulnerable to pharmacological targeting of the TCF/β-catenin interaction.