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

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.).

Indolent CD4+ CAR T-Cell Lymphoma after Cilta-cel CAR T-Cell Therapy.

Indolent CD4+ cytotoxic chimeric antigen receptor (CAR) T-cell lymphoma involving the small intestine was diagnosed in a patient who had previously received ciltacabtagene autoleucel (cilta-cel) CAR T-cell therapy for treatment of myeloma. Targeted messenger RNA sequencing revealed the presence of CAR gene product in tumor cells. Whole-genome sequencing of samples of tumor and peripheral blood identified a single lentiviral insertion site within the second intron of the SSU72 gene. In addition, numerous genetic alterations that may have contributed to malignant transformation were identified in the tumor sample. (Funded by MedStar Georgetown University Hospital.).

[Interpretation of T-cell and NK-cell lymphoid proliferations and lymphomas in the 5th edition of the WHO classification of haematolymphoid tumours].

The 5th edition of the World Health Organization (WHO) classification of haematolymphoid tumours used the hierarchical system to classify T-cell and NK-cell lymphoid proliferations and lymphomas (T/NK-LPD/LYM) based on research advances and clinicopathological characteristics of the diseases. In this edition of classification, tumour-like lesions were included, some tumors were added/deleted, the names or terms of certain diseases were refined, and the diagnostic criteria or subtypes of some diseases were revised. This group of diseases was reintegrated from non-clonal hyperplasia to highly aggressive lymphoma, which would further reflect the nature of T/NK-LPD/LYM and benefit to clinical application.

The Fate(s) of CAR T-Cell Therapy: Navigating the Risks of CAR+ T-Cell Malignancy.

The introduction of chimeric antigen receptor (CAR) T-cell therapy represents a landmark advancement in treating resistant forms of cancer such as leukemia, lymphoma, and myeloma. However, concerns about long-term safety have emerged following an FDA investigation into reports of second primary malignancies (SPM) after CAR-T cell treatment. This review offers a thorough examination of how genetically modified T cells might transform into CAR+ SPM. It explores genetic and molecular pathways leading to T-cell lymphomagenesis, the balance between CAR T-cell persistence, stemness, and oncogenic risk, and the trade-off of T-cell exhaustion, which may limit therapy efficacy but potentially reduce lymphomagenesis risk. Significance: An FDA probe into 22 cases of second primary T-cell malignancies following CAR T-cell therapy stresses the need to investigate their origins. Few may arise from preexisting genetic and epigenetic alterations and those introduced during therapeutic engineering. Technological advances, regulatory oversight, and patient monitoring are essential to mitigate potential risks.

Epigenetic Priming by Hypomethylation Enhances the Immunogenic Potential of Tolinapant in T-cell Lymphoma.

Programmed cell death mechanisms are important for the regulation of tumor development and progression. Evasion of and resistance to apoptosis are significant factors in tumorigenesis and drug resistance. Bypassing apoptotic pathways and eliciting another form of regulated cell death, namely necroptosis, an immunogenic cell death (ICD), may override apoptotic resistance. Here, we present the mechanistic rationale for combining tolinapant, an antagonist of the inhibitor of apoptosis proteins (IAP), with decitabine, a hypomethylating agent (HMA), in T-cell lymphoma (TCL). Tolinapant treatment alone of TCL cells in vitro and in syngeneic in vivo models demonstrated that ICD markers can be upregulated, and we have shown that epigenetic priming with decitabine further enhances this effect. The clinical relevance of ICD markers was confirmed by the direct measurement of plasma proteins from patients with peripheral TCL treated with tolinapant. We showed increased levels of necroptosis in TCL lines, along with the expression of cancer-specific antigens (such as cancer testis antigens) and increases in genes involved in IFN signaling induced by HMA treatment, together deliver a strong adaptive immune response to the tumor. These results highlight the potential of a decitabine and tolinapant combination for TCL and could lead to clinical evaluation. SIGNIFICANCE: The IAP antagonist tolinapant can induce necroptosis, a key immune-activating event, in TCL. Combination with DNA hypomethylation enhances tolinapant sensitivity and primes resistant cells by re-expressing necrosome proteins. In addition, this combination leads to increases in genes involved in IFN signaling and neoantigen expression, providing further molecular rationale for this novel therapeutic option.

Single-cell T-cell receptor repertoire profiling in dogs.

Spontaneous cancers in companion dogs are robust models of human disease. Tracking tumor-specific immune responses in these models requires reagents to perform species-specific single cell T cell receptor sequencing (scTCRseq). scTCRseq and integration with scRNA data have not been demonstrated on companion dogs with cancer. Here, five healthy dogs, two dogs with T cell lymphoma and four dogs with melanoma are selected to demonstrate applicability of scTCRseq in a cancer immunotherapy setting. Single-cell suspensions of PBMCs or lymph node aspirates are profiled using scRNA and dog-specific scTCRseq primers. In total, 77,809 V(D)J-expressing cells are detected, with an average of 3498 (348 - 5,971) unique clonotypes identified per sample. In total, 29/34, 40/40, 22/22 and 9/9 known functional TRAV, TRAJ, TRBV and TRBJ gene segments are observed respectively. Pseudogene or otherwise defective gene segments are also detected supporting re-annotation of several as functional. Healthy dogs exhibit highly diverse repertoires, T cell lymphomas exhibit clonal repertoires, and vaccine-treated melanoma dogs are dominated by a small number of highly abundant clonotypes. scRNA libraries define large clusters of V(D)J-expressing CD8+ and CD4 + T cells. Dominant clonotypes observed in melanoma PBMCs are predominantly CD8 + T cells, with activated phenotypes, suggesting possible anti-tumor T cell populations.

TRBC1-targeting antibody-drug conjugates for the treatment of T cell cancers.

Antibody and chimeric antigen receptor (CAR) T cell-mediated targeted therapies have improved survival in patients with solid and haematologic malignancies1-9. Adults with T cell leukaemias and lymphomas, collectively called T cell cancers, have short survival10,11 and lack such targeted therapies. Thus, T cell cancers particularly warrant the development of CAR T cells and antibodies to improve patient outcomes. Preclinical studies showed that targeting T cell receptor ß-chain constant region 1 (TRBC1) can kill cancerous T cells while preserving sufficient healthy T cells to maintain immunity12, making TRBC1 an attractive target to treat T cell cancers. However, the first-in-human clinical trial of anti-TRBC1 CAR T cells reported a low response rate and unexplained loss of anti-TRBC1 CAR T cells13,14. Here we demonstrate that CAR T cells are lost due to killing by the patient's normal T cells, reducing their efficacy. To circumvent this issue, we developed an antibody-drug conjugate that could kill TRBC1+ cancer cells in vitro and cure human T cell cancers in mouse models. The anti-TRBC1 antibody-drug conjugate may provide an optimal format for TRBC1 targeting and produce superior responses in patients with T cell cancers.

KLRG1 Cell Depletion as a Novel Therapeutic Strategy in Patients with Mature T-Cell Lymphoma Subtypes.

PURPOSE: Develop a novel therapeutic strategy for patients with subtypes of mature T-cell and NK-cell neoplasms. EXPERIMENTAL DESIGN: Primary specimens, cell lines, patient-derived xenograft models, commercially available, and proprietary anti-KLRG1 antibodies were used for screening, target, and functional validation. RESULTS: Here we demonstrate that surface KLRG1 is highly expressed on tumor cells in subsets of patients with extranodal NK/T-cell lymphoma (ENKTCL), T-prolymphocytic leukemia (T-PLL), and gamma/delta T-cell lymphoma (G/D TCL). The majority of the CD8+/CD57+ or CD3-/CD56+ leukemic cells derived from patients with T- and NK-large granular lymphocytic leukemia (T-LGLL and NK-LGLL), respectively, expressed surface KLRG1. The humanized afucosylated anti-KLRG1 monoclonal antibody (mAb208) optimized for mouse in vivo use depleted KLRG1+ TCL cells by mechanisms of ADCC, ADCP, and CDC rather than apoptosis. mAb208 induced ADCC and ADCP of T-LGLL patient-derived CD8+/CD57+ cells ex vivo. mAb208 effected ADCC of subsets of healthy donor-derived KLRG1+ NK, CD4+, CD8+ Tem, and TemRA cells while sparing KLRG1- naïve and CD8+ Tcm cells. Treatment of cell line and TCL patient-derived xenografts with mAb208 or anti-CD47 mAb alone and in combination with the PI3K-δ/γ inhibitor duvelisib extended survival. The depletion of macrophages in vivo antagonized mAb208 efficacy. CONCLUSIONS: Our findings suggest the potential benefit of a broader treatment strategy combining therapeutic antibodies with PI3Ki for the treatment of patients with mature T-cell and NK-cell neoplasms. See related commentary by Varma and Diefenbach, p. 2300.

High CD8+tumor-infiltrating lymphocytes indicate severe exhaustion and poor prognosis in angioimmunoblastic T-cell lymphoma.

Background: Exhaustion of CD8+ tumor-infiltrating lymphocytes (TILs), characterized by the overexpression of immune checkpoints (IC), is a major impediment to anti-tumor immunity. However, the exhaustion status of CD8+TILs in angioimmunoblastic T cell lymphoma (AITL) remains unclear. Therefore, we aimed to elucidate the exhaustion status of CD8+TILs in AITL and its influence on prognosis. Methods: The correlation between CD8+TILs and IC expression in AITL was analyzed using single-cell RNA sequencing (n = 2), flow cytometry (n = 20), and RNA sequencing (n = 20). Biological changes related to CD8+TILs exhaustion at different cytotoxic T lymphocyte (CTL) levels (mean expression levels of CD8A, CD8B, GZMA, GZMB, and PRF1) in AITL were evaluated using RNA sequencing (n = 20) and further validated using the GEO dataset (n = 51). The impact of CD8 protein expression and CTL levels on patient prognosis was analyzed using flow cytometry and RNA sequencing, respectively. Results: Our findings demonstrated that the higher the infiltration of CD8+TILs, the higher was the proportion of exhausted CD8+TILs characterized by the overexpression of multiple IC. This was accompanied by extensive exhaustion-related biological changes, which suggested severe exhaustion in CD8+TILs and may be one of the main reasons for the poor prognosis of patients with high CD8+TILs and CTL. Conclusion: Our study comprehensively reveals the exhaustion status of CD8+TILs and their potential negative impact on AITL prognosis, which facilitates further mechanistic studies and is valuable for guiding immunotherapy strategies.

High-dimensional and single-cell transcriptome analysis of the tumor microenvironment in angioimmunoblastic T cell lymphoma (AITL).

Angioimmunoblastic T-cell lymphoma (AITL) is an aggressive lymphoid malignancy associated with a poor clinical prognosis. The AITL tumor microenvironment (TME) is unique, featuring a minority population of malignant CD4+ T follicular helper (TFH) cells inter-mixed with a diverse infiltrate of multi-lineage immune cells. While much of the understanding of AITL biology to date has focused on characteristics of the malignant clone, less is known about the many non-malignant populations that comprise the TME. Recently, mutational consistencies have been identified between malignant cells and non-malignant B cells within the AITL TME. As a result, a significant role for non-malignant populations in AITL biology has been increasingly hypothesized. In this study, we have utilized mass cytometry and single-cell transcriptome analysis to identify several expanded populations within the AITL TME. Notably, we find that B cells within the AITL TME feature decreased expression of key markers including CD73 and CXCR5. Furthermore, we describe the expansion of distinct CD8+ T cell populations that feature an exhausted phenotype and an underlying expression profile indicative of dysfunction, impaired cytotoxicity, and upregulation of the chemokines XCL2 and XCL1.

Clinicopathological and molecular genomic features of monomorphic epitheliotropic intestinal T-cell lymphoma in the Chinese population: a study of 20 cases.

BACKGROUND: Monomorphic epitheliotropic T-cell lymphoma (MEITL) is an aggressive non-Hodgkin lymphoma with a high fatality rate. This study was aimed to explore the clinicopathological and molecular genetic features of MEITL in the Chinese population. METHODS: A retrospective analysis was performed based on the clinical manifestations and pathological features of 20 Chinese MEITL. 9 cases with paired diseased-normal tissues were also analyzed for molecular information by whole-exome sequencing. RESULTS: There were 14 men and 6 women with a median age of 58.5 (28-81) years. 17(17/20) lesions were located in the jejunum or ileum; 13(13/20) cases had ulcers or perforations. Microscopically, except for 1(1/20) case of pleomorphic cells, the monomorphic, middle-sized tumor cells infiltrating into the intestinal epithelial and peripheral intestinal mucosa recess could be seen in the other 19 cases. Immunohistochemistry showed that most of the tumor cells in MEITL were positive for CD3(20/20), CD8(17/20), CD43(19/20), and CD56(15/20), but negative for CD5(20/20). The most frequently mutated genes of these Chinese cases were STAT5B (4/9) and TP53 (4/9), not SETD2(2/9). JAK3 mutations (3/9) were also detected with a high mutated frequency. We demonstrated that mutations of JAK-STAT pathway-related genes and the amplification of Chromosome 9q appeared at the same time in most cases(5/9). CONCLUSIONS: The clinicopathological features were consistent with that in previous western studies, but a special case with pleomorphic cells was found in this study. The co-occurrence of JAK-STAT pathway-related gene mutations and the amplification of Chr9q is a molecular feature of MEITL.

Capecitabine Can Induce T Cell Apoptosis: A Potential Immunosuppressive Agent With Anti-Cancer Effect.

Capecitabine (CAP) is now widely used in the comprehensive treatment of digestive system tumors. Some clinical observations have shown that CAP may have immunosuppressive effects, but there is still a lack of clear experimental verification. In this study, different doses of CAP were administered to normal mice by gavage. Our results confirmed that CAP did not cause myelosuppression in bone marrow tissue; CAP selectively reduced the proportion of T cells and the concentration of related pro-inflammatory cytokines, while it increased the concentration of anti-inflammatory cytokines. Thymidylate phosphorylase (TP) is the key enzyme for the transformation of CAP in vivo; this study confirmed that T cells express TP, but the bone marrow tissue lacks TP expression, which explains the selectivity in pharmacodynamic effects of CAP. In addition, it was confirmed that CAP can induce T cell apoptosis in vivo and in vitro. In vitro experiments showed that CAP-induced T cell apoptosis was related to TP expression, endoplasmic reticulum stress (ERS) induction, reactive oxygen species (ROS) production, and mitochondria-mediated apoptosis activation. Therefore, this study confirmed that the differential expression of TP in cells and tissues explains why CAP avoids the toxic effects of myelosuppression while inducing T cell apoptosis to exert the immunosuppressive effect. Therefore, CAP may become an immunosuppressive agent with a simultaneous anti-cancer effect, which is worthy of further studies.

Inter-cellular CRISPR screens reveal regulators of cancer cell phagocytosis.

Monoclonal antibody therapies targeting tumour antigens drive cancer cell elimination in large part by triggering macrophage phagocytosis of cancer cells1-7. However, cancer cells evade phagocytosis using mechanisms that are incompletely understood. Here we develop a platform for unbiased identification of factors that impede antibody-dependent cellular phagocytosis (ADCP) using complementary genome-wide CRISPR knockout and overexpression screens in both cancer cells and macrophages. In cancer cells, beyond known factors such as CD47, we identify many regulators of susceptibility to ADCP, including the poorly characterized enzyme adipocyte plasma membrane-associated protein (APMAP). We find that loss of APMAP synergizes with tumour antigen-targeting monoclonal antibodies and/or CD47-blocking monoclonal antibodies to drive markedly increased phagocytosis across a wide range of cancer cell types, including those that are otherwise resistant to ADCP. Additionally, we show that APMAP loss synergizes with several different tumour-targeting monoclonal antibodies to inhibit tumour growth in mice. Using genome-wide counterscreens in macrophages, we find that the G-protein-coupled receptor GPR84 mediates enhanced phagocytosis of APMAP-deficient cancer cells. This work reveals a cancer-intrinsic regulator of susceptibility to antibody-driven phagocytosis and, more broadly, expands our knowledge of the mechanisms governing cancer resistance to macrophage phagocytosis.

Angioimmunoblastic T-cell lymphoma diagnosed from pleural fluid by integration of morphologic, immunophenotypic, and molecular findings.

An 88-year-old man with end-stage renal disease on hemodialysis presented with shortness of breath and was found to have lower extremity edema and bilateral pleural effusions on a chest X-ray. A therapeutic and diagnostic thoracentesis was performed, and cytologic examination revealed atypical mononuclear cells. Based on this, flow cytometry was performed on the pleural fluid, along with immunostains on the cellblock and a next-generation sequencing (NGS) panel. A definitive diagnosis of angioimmunoblastic T-cell lymphoma (AITL) was made based on demonstrating an atypical T follicular helper cell population expressing CD10, BCL6, CXCL13, CD200, CD57, and PD1, and detection of pathogenic variants in RHOA, IDH2, and TET2. This case represents the first reported case where a primary diagnosis of AITL was made on a body fluid specimen and highlights how immunophenotyping and NGS can provide a definitive diagnosis of AITL on a cytologic specimen.

Effective Cytotoxicity of Dendritic Cells against Established T Cell Lymphomas in Mice.

T cell lymphomas arise in mice that constitutively express a single TCR in the absence of NK cells. Upon TCR engagement these lymphomas are able to corrupt tumor surveillance by decreasing NK cell numbers. In this study, we investigate the outcome of interactions between these T cell lymphomas and dendritic cells. Bone marrow-derived dendritic cells mediated effective killing of T cell lymphomas after activation with IFN-γ and TLR ligands in culture. This cytotoxicity was independent of MHC compatibility. Cell lysis was reduced by the presence of the peroxynitrite inhibitors FeTTPS and L-NMMA, whereas inhibitors of apoptosis, death receptors, and degranulation were without effect, suggesting NO metabolites as the main mediators. When injected together with GM-CSF and R848 into lymphoma-bearing mice, in vitro-expanded bone marrow-derived dendritic cells caused significant survival increases. These data show that dendritic cell adaptive immunotherapy can be used as treatment against T cell lymphomas in mice.

Clinicopathological findings of systemic Epstein-Barr virus-positive T-lymphoproliferative diseases in younger and older adults.

BACKGROUND: Systemic Epstein-Barr virus+ T-cell lymphoma (sEBV+ TCL) occurs in childhood and young adults, and is exceptionally rare in older adults. METHODS: We investigated clinicopathological features in 16 patients of various ages with systemic EBV+ CD8+ T-lymphoproliferative diseases. RESULTS: Eight younger patients and four of eight older adults had sEBV+ CD8+ TCL, with invasion by medium-sized to/or large atypical lymphocytes primarily in bone marrow and lymph nodes, hemophagocytic lymphohistiocytosis (HLH), and progressive clinicopathological course. A further two patients demonstrated EBV+ node-based CD8+ large TCL without HLH, while the remaining two had the systemic form of chronic active EBV infection (sCAEBV) with CD8+ small lymphocytes. Past history of sCAEBV-like lesions was observed in one sEBV+ TCL patient (8.3%). Immunohistologically, in 12 sEBV+ TCL patients, atypical lymphocytes were positive for phosphate signal transducer and activator of transcription 3 (66.7%), CMYC (83.3%), and p53 (75%). Strong reactions of programmed cell death-ligand (PD-L)1+ tumor or non-neoplastic cells were detected in nine sEBV+ TCL patients (75%). Clonal peaks of the T-cell receptor (TCR) γ gene were detected in eight sEBV+ TCL patients by polymerase chain reaction. Four younger patients in sEBV+ TCL (33.3%) are in remission with chemotherapies including etoposide, and three of the four underwent allogeneic stem cell transplantation (SCT). CONCLUSION: sEBV+ CD8+ TCL was observed in younger and older adults with less history of sCAEBV. HLH, tumor cell atypia, immunohistological findings, and progressive clinical course were characteristic of sEBV+ CD8+ TCL. Prompt chemotherapy and SCT induced tumor regression in sEBV+ CD8+ TCL patients.

Expression regulation and function of PD-1 and PD-L1 in T lymphoma cells.

T lymphoma cells may constitutively express PD-1 and PD-L1. The relative role of PD-1 and PD-L1 in T lymphoma is incompletely understood. We report here that PD-1+ PDL-1+ human T lymphoma cells exhibit constitutive hyperactivation of the TCR signaling and do not respond to PD-L1-mediated suppression in vitro. Knocking out PD-1 or PD-L1 has no effects on T lymphoma cell apoptosis and proliferation in vitro, but significantly increased tumor-bearing mouse survival. Our findings determine that the constitutively active TCR signaling pathway maintain T lymphoma cell growth in vitro and that both PD-1 and PD-L1 promote T lymphoma growth in vivo.