T-cell Neoplasms Research Articles

Preclinical evaluation of anti-CD38 therapy in mature T-cell neoplasms.

Preclinical evaluation of anti-CD38 therapy in mature T-cell neoplasms.

What's new in hematopathology 2023: updates on mature T-cell neoplasms in the 5th edition of the WHO classification.

The overview of the upcoming Blue Book of the 5th edition of the World Health Organization Classification of Hematolymphoid Tumors was published in Leukemia in June 2022. The updates on mature T-/NK-cell lymphomas and leukemias are organized in nine groups based on cell of origin, morphology, clinical scenario, and localization, and are highlighted in this newsletter.

Analysis and therapeutic targeting of the IL-1R pathway in anaplastic large cell lymphoma

AbstractAnaplastic large cell lymphoma (ALCL), a subgroup of mature T-cell neoplasms with an aggressive clinical course, is characterized by elevated expression of CD30 and anaplastic cytology. To achieve a comprehensive understanding of the molecular characteristics of ALCL pathology and to identify therapeutic vulnerabilities, we applied genome-wide CRISPR library screenings to both anaplastic lymphoma kinase positive (ALK+) and primary cutaneous (pC) ALK− ALCLs and identified an unexpected role of the interleukin-1R (IL-1R) inflammatory pathway in supporting the viability of pC ALK− ALCL. Importantly, this pathway is activated by IL-1α in an autocrine manner, which is essential for the induction and maintenance of protumorigenic inflammatory responses in pC-ALCL cell lines and primary cases. Hyperactivation of the IL-1R pathway is promoted by the A20 loss-of-function mutation in the pC-ALCL lines we analyze and is regulated by the nonproteolytic protein ubiquitination network. Furthermore, the IL-1R pathway promotes JAK-STAT3 signaling activation in ALCLs lacking STAT3 gain-of-function mutation or ALK translocation and enhances the sensitivity of JAK inhibitors in these tumors in vitro and in vivo. Finally, the JAK2/IRAK1 dual inhibitor, pacritinib, exhibited strong activities against pC ALK− ALCL, where the IL-1R pathway is hyperactivated in the cell line and xenograft mouse model. Thus, our studies revealed critical insights into the essential roles of the IL-1R pathway in pC-ALCL and provided opportunities for developing novel therapeutic strategies.

SAM-Competitive EZH2-Inhibitors Induce Platinum Resistance by EZH2-Independent Induction of ABC-Transporters.

T-cell lymphomas are heterogeneous and rare lymphatic malignancies with unfavorable prognosis. Consequently, new therapeutic strategies are needed. The enhancer of zeste homologue 2 (EZH2) is the catalytic subunit of the polycomb repressive complex 2 and responsible for lysine 27 trimethylation of histone 3. EZH2 is overexpressed in several tumor entities including T-cell neoplasms leading to epigenetic and consecutive oncogenic dysregulation. Thus, pharmacological EZH2 inhibition is a promising target and its clinical evaluation in T-cell lymphomas shows favorable results. We have investigated EZH2 expression in two cohorts of T-cell lymphomas by mRNA-profiling and immunohistochemistry, both revealing overexpression to have a negative impact on patients' prognosis. Furthermore, we have evaluated EZH2 inhibition in a panel of leukemia and lymphoma cell lines with a focus on T-cell lymphomas characterized for canonical EZH2 signaling components. The cell lines were treated with the inhibitors GSK126 or EPZ6438 that inhibit EZH2 specifically by competitive binding at the S-adenosylmethionine (SAM) binding site in combination with the common second-line chemotherapeutic oxaliplatin. The change in cytotoxic effects under pharmacological EZH2 inhibition was evaluated revealing a drastic increase in oxaliplatin resistance after 72 h and longer periods of combinational incubation. This outcome was independent of cell type but associated to reduced intracellular platinum. Pharmacological EZH2 inhibition revealed increased expression in SRE binding proteins, SREBP1/2 and ATP binding cassette subfamily G transporters ABCG1/2. The latter are associated with chemotherapy resistance due to increased platinum efflux. Knockdown experiments revealed that this was independent of the EZH2 functional state. The EZH2 inhibition effect on oxaliplatin resistance and efflux was reduced by additional inhibition of the regulated target proteins. In conclusion, pharmacological EZH2 inhibition is not suitable in combination with the common chemotherapeutic oxaliplatin in T-cell lymphomas revealing an EZH2-independent off-target effect.

Primary Cutaneous Marginal Zone Lymphoproliferative Disorder of Donor Origin after Allogeneic Hematopoietic Stem Cell Transplantation.

Primary cutaneous posttransplant lymphoproliferative disorders (PTLDs) after allogeneic hematopoietic stem cell transplant (allo-HSCT) are exceedingly rare, with only 6 published cases, all of them consisting in T-cell neoplasms. In this report, we present for the first time a donor-derived B-cell PTLD consisting in a primary, cutaneous, B-cell, marginal zone, lymphoproliferative disorder (PCMZLPD). The patient, a 37-year-old woman with a history of Hodgkin lymphoma received an allo-HSCT from her healthy, matched, related father, achieving complete host chimerism in the bone marrow and peripheral blood. However, 8 years after the allo-HSCT, she presented asymptomatic skin lesions consisting in oval, well-defined, slightly raised erythematous plaques, located on the arms, trunk, and legs. Skin biopsies of 2 lesions demonstrated a class-switched IgG+, EBV-, PCMZLPD, showing kappa light chain restriction and monoclonal rearrangement of the IgH gene. Microsatellite genotyping and 2-color fluorescence in situ hybridization (X and Y chromosomes) confirmed that the origin of the neoplastic cells was the donor graft. The lesions showed an indolent behavior, good response to topical corticosteroids, and no need for systemic treatment. Our case broadens the spectrum of PTLD, a diverse group of lymphoid and/or plasmacytic proliferations with variable clinical presentations and histopathological features.

CHROMATIN ACCESSIBILITY PROFILING TO INCREASE DIAGNOSTIC ACCURACY AND REFINE CELL‐OF‐ORIGIN CLASSIFICATION OF MATURE T‐CELL LYMPHOMAS

Mature T-cell neoplasms (MTCN) are heterogeneous diseases with dismal prognosis. Differentiating between the many entities requires specialized pathology expertise, and studies show up to 30% of minor or major diagnostic reclassifications following expert review of cases. Assay for transposase-accessible chromatin sequencing (ATAC-seq) is a simple technique to profile open chromatin regions, which has been shown to be highly discriminative for clustering solid tumors and acute myeloid leukemias. We applied ATAC-seq to MTCN to explore the epigenetic landscape of these different entities, and built a predictive model to aid in diagnosis. FACS-sorted tumor cells from primary MTCN samples and 50µm sections of frozen tumor tissue from the French TENOMIC T-cell lymphoma consortium were processed according to previously published FAST- and OMNI-ATAC protocols, respectively. In parallel, we applied FAST-ATAC to several normal T and NK cell subsets sorted from PBMC or lymph node suspensions of healthy donors. Sequencing data were processed by an adapted version of the ENCODE ATAC-seq pipeline using a custom Hg38 genome version including HTLV1 sequence. A total of 678 normal and tumor samples were sequenced to provide a comprehensive landscape of chromatin accessibility in MTCN. Unsupervised clustering of normal NK and T cell subtypes (N = 49) and sorted tumoral lymphoma cells (N = 104) confirmed that AITL are derived from TFH cells, HSTL and LGL are closely segregated with NK and gd-T cells. We also found that T-PLL likely derive from the transformation of naïve T cells. Epigenetic profiling by ATAC-seq of FACS-sorted tumor samples resulted in a complete segregation of the known MTCN entities (TFH, ALK+ and ALK- ALCL, HSTL, CTCL, ATLL, LGL and T-PLL). Most PTCL-NOS (13/17) clustered with a predefined MTCN subtype (mainly AITL/TFH-phenotype PTCL, CTCL and lymphomas exhibiting cytotoxic features), showing that this waste basket subgroup is merely virtual, at least from an epigenetic point of view. Using unsupervised deconvolution approaches, we were able to discriminate different MTCN subtypes from 223 processed frozen bulk samples. All known MTCN subtypes were identified by ATAC-seq but a novel subset of PTCL-NOS representing ∼5% of cases was pinpointed, showing high GATA3 transcription factor activity. Subsequent exome sequencing revealed numerous copy number alterations and TP53 (8/12) and NCOR1 mutations (7/12). A support vector machine model was trained to predict diagnosis and showed accurate classification performance by cross-validation and on external validation cohort collected from 5 tertiary care centers. ATAC-seq is a rapid and cost-effective technique with high classification accuracy for PTCL subtypes. Among GATA3-expressing PTCL that spread across multiple epigenetic subgroups, we identified a specific entity with recurrent NCOR1 mutations. Keywords: aggressive T-cell non-Hodgkin lymphoma, genomics, epigenomics, and other -omics, pathology and classification of lymphomas No conflicts of interests pertinent to the abstract.

Case Report: A rare case of CD20-positive T-cell large granular lymphocyte leukemia with renal cell carcinoma: a challenging diagnosis

Large granular lymphocytic leukemia (LGLL) is a clonal lymphoproliferative disease with a slow course and considerable difficulties in correct diagnosis and therapy. T-LGLL is the most prevalent subtype of LGLL, accounting for approximately 85%. T-LGLL co-existence with solid malignancies is relatively rare. CD20-positive T-cell neoplasm is a rare disease in clinics. In this study, we report a case of CD20-positive T-LGLL with renal cell carcinoma (RCC) that was eventually diagnosed by splenectomy and nephrectomy. The accumulation of cases will contribute to diagnosing and treating CD20-positive T-LGLL complicated with solid tumors.

Transdifferentiation, phenotypic infidelity, progression, and transformation in T/NK-cell neoplasms: Report from the 2021 SH/EAHP Workshop.

Sessions 8 and 9 of the 2021 Society for Hematopathology and the European Association for Haematopathology Workshop aimed to collect examples of transdifferentiation, lineage infidelity, progression, and transformation in precursor and mature T/natural killer (NK)-cell neoplasms. Twenty-eight cases were submitted and analyzed, with whole-exome sequencing and genome-wide RNA expression analysis performed in a subset of the cases. In session 8, 7 T-lymphoblastic lymphoma/leukemia cases were received that showed transdifferentiation to clonally related mature myeloid hematopoietic neoplasms, including 6 histiocytic/dendritic cell lineage neoplasms and a mast cell sarcoma. Session 9 included 21 mature T-cell neoplasms that were grouped into 3 themes. The first one addressed phenotypic infidelity in mature T-cell lymphomas (TCLs) and included 8 TCLs expressing aberrant antigens, mimicking classic Hodgkin and non-Hodgkin B-cell lymphomas. The second theme addressed disease progression in TCL and included 5 cutaneous T-cell lymphoproliferative disorders and 2 T-cell large granular lymphocyte proliferations with subsequent progression to systemic TCL. The third theme included 6 patients with TCL with T-follicular helper phenotype, mainly angioimmunoblastic T-cell lymphoma, with concurrent/subsequent clonal hematopoiesis or myeloid neoplasms and/or subsequent/concomitant diffuse large B-cell lymphoma. This cohort of cases allowed us to illustrate, discuss, and review current concepts of transdifferentiation, aberrant antigen expression, and progression in various T/NK-cell neoplasms.

Immunotherapy for Peritoneal Carcinomatosis: Challenges and Prospective Outcomes

Peritoneal metastasis, also known as peritoneal carcinomatosis (PC), is a refractory cancer that is typically resistant to conventional therapies. The typical treatment for PC is a combination of cytoreductive surgery (CRS) and hyperthermic intraperitoneal chemotherapy (HIPEC). Recently, research in this area has seen significant advances, particularly in immunotherapy as an alternative therapy for PC, which is very encouraging. Catumaxomab is a trifunctional antibody intraperitoneal (IP) immunotherapy authorized in Europe that can be used to diminish malignant ascites by targeting EpCAM. Intraperitoneal (IP) immunotherapy breaks immunological tolerance to treat peritoneal illness. Increasing T-cell responses and vaccination against tumor-associated antigens are two methods of treatment. CAR-T cells, vaccine-based therapeutics, dendritic cells (DCs) in combination with pro-inflammatory cytokines and NKs, adoptive cell transfer, and immune checkpoint inhibitors are promising treatments for PC. Carcinoembryonic antigen-expressing tumors are suppressed by IP administration of CAR-T cells. This reaction was strengthened by anti-PD-L1 or anti-Gr1. When paired with CD137 co-stimulatory signaling, CAR-T cells for folate receptor cancers made it easier for T-cell tumors to find their way to and stay alive in the body.

Delineation of clinical course, outcomes, and prognostic factors in patients with T-cell prolymphocytic leukemia.

T-cell prolymphocytic leukemia (T-PLL) is a rare, post-thymic T-cell neoplasm with a diverse clinical course. T-PLL is typically associated with a poor prognosis; however, a subset of patients have inactive disease on initial presentation. There is a lack of accurate delineation of the disease based on initial clinical presentation and pathological assessment, hindering clinical decision-making. To characterize and delineate disease subtypes based on initial clinical presentation and pathologic assessment, we retrospectively reviewed 81 patients with T-PLL treated at our institution. We compared patients with T-PLL who initially presented with a relatively indolent or stable disease course to those with an aggressive disease course. Clinicopathologic characteristics, overall survival (OS), and prognostic factors were analyzed. Patients with inactive disease had a significantly longer OS than patients with active disease. At diagnosis, presence of B symptoms, low hemoglobin, low platelet count, lymphocyte doubling time of fewer than 3 months, and abnormal cytogenetics were associated with shorter OS. Cell morphology, immunophenotype, absolute lymphocyte count, lactate dehydrogenase levels, involvement of liver, spleen, skin or central nervous system, presence of TCL1 rearrangement or inv (14)/t(14;14), presence of chromosome 8 abnormalities, and presence of deletion of 11q were not associated with significant OS difference among the patients. Receiving alemtuzumab as first-line treatment and consolidation with allogeneic hematopoietic stem cell transplant were associated with better outcomes. T-PLL inactive and active disease subtypes can exhibit overlapping yet different clinical and pathological features. We describe several prognostic factors at diagnosis that can be used for risk stratification and aid in guiding treatment decisions.

A Comprehensive Study of the Immunophenotype and its Clinicopathologic Significance in Adult T-Cell Leukemia/Lymphoma

Adult T-cell leukemia/lymphoma (ATLL) is a mature T-cell tumor caused by human T-lymphotropic virus type 1 (HTLV-1). The typical ATLL immunophenotypes are described in the 2017 World Health Organization Classification of Tumours of Haematopoietic and Lymphoid Tissues (positive: CD2, CD3, CD5, CD4, and CD25; negative: CD7, CD8, and cytotoxic markers; and partially positive: CD30, CCR4, and FOXP3). However, limited studies are available on the expression of these markers, and their mutual relationship remains unknown. Furthermore, the expression status of novel markers associated with T-cell lymphomas, including Th1 markers (T-bet and CXCR3), Th2 markers (GATA3 and CCR4), T follicular helper markers (BCL6, PD1, and ICOS), and T-cell receptor (TCR) markers, and their clinicopathologic significance is unclear. In this study, we performed >20 immunohistochemical stains in 117 ATLL cases to determine the comprehensive immunophenotypic profile of ATLL, which were compared on the basis of clinicopathologic factors, including morphologic variants (pleomorphic vs anaplastic), biopsy locations, treatments, Shimoyama classification-based clinical subtype, and overall survival. CD3+/CD4+/CD25+/CCR4+ was considered a typical immunophenotype of ATLL, but approximately 20% of cases did not conform to this pattern. Simultaneously, the following new findings were obtained: (1) most cases were negative for TCR-β and TCR-δ (104 cases, 88.9%), indicating the usefulness of negative conversion of TCR expression to provide differentiation from other T-cell tumors; (2) the positivity of CD30 and CD15 and the negativity of FOXP3 and CD3 were significantly associated with anaplastic morphology; and (3) atypical cases, such as T follicular helper marker-positive (12 cases, 10.3%) and cytotoxic molecule-positive cases (3 cases, 2.6%), were identified. No single markers could predict the overall survival among patients with acute/lymphoma subtypes of ATLL. The results of this study illustrate the diversity of ATLL phenotypes. In T-cell tumors occurring in HTLV-1 carriers, the possibility of ATLL should not be eliminated even when the tumor exhibits an atypical phenotype, and the confirmation of HTLV-1 in the tissue is recommended.

Clinicopathologic characteristics, genetic features, and treatment options for acute lymphoblastic leukemia with JAK2 rearrangement—A 10-case study and literature review

JAK2 rearrangement (JAK2-R) in acute lymphoblastic leukemia (ALL) is rare and often categorized as B-ALL with BCR::ABL1-like features based on the World Health Organization classification. We report 10 patients with JAK2-R ALL, 9 males and 1 female, with a median age 40.5 years. Eight patients presented with marked leukocytosis (median WBC, 63 × 10 9/L) and hypercellular (>95%) bone marrow with increased lymphoblasts (72%-95%). There was no evidence of bone marrow fibrosis or hypereosinophilia. Immunophenotypic analysis showed 9 B-cell and 1 T-cell neoplasms. Using fluorescence in situ hybridization (FISH) and RNA sequencing analysis, JAK2 partners were identified for 7 cases and included PCM1 (n=4), ETV6 (n=2) and BCR (n=1). All patients received upfront polychemotherapy. Additionally, 2 patients received ruxolitinib, 2 received allogeneic stem cell transplant, and 1 received CAR-T therapy. The 1- and 3-year overall survival rates were 55.6% and 22.2%, respectively. A literature review identified 24 B-ALL and 4 T-ALL cases with JAK2-R reported, including 16 males, 6 females and 6 gender not stated. Many JAK2 partner-genes were reported with the most common being PAX5 (n=7), ETV6 (n=4), BCR (n=3) and PCM1 (n=2). Survival data or 13 reported cases showed 1- and 3-year overall survival rates of 41.7% and 41.7%, respectively. In summary, JAK2-R ALL occurs more often in adult males, are mostly of B-cell lineage, and associated with an aggressive clinical course. Absence of eosinophilia and bone marrow fibrosis and no evidence of preexisting/concurrent JAK2-R myeloid neoplasms distinguish JAK2-R ALL from other myeloid/lymphoid neoplasms with eosinophilia and JAK2-R.

Tax-targeted dendritic cell vaccine therapy for long-term remission of adult T-cell leukemia-lymphoma

Adult T-cell leukemia-lymphoma (ATL) is a highly aggressive peripheral T-cell neoplasm caused by human T-cell leukemia virus type 1 (HTLV-1) infection occurring in approximately 5% of patients after prolonged latent period. ATL relapses within a short period despite its transient response to multiagent chemotherapy and the prognosis is extremely poor due to anticancer drug resistance and immunodeficiency. Although novel agents with different mechanisms, such as molecular targeted agents, have improved the prognosis, the number of cured patients remains limited. Hematopoietic stem cell transplantation resulted in long-term remission, whereas its indication is limited due to treatment-related mortality. As most ATL patients are of advanced age, development of a lesser toxic treatment is necessary. Therefore, we developed a novel therapeutic dendritic cell vaccine targeting the HTLV-1 Tax antigen. The safety profile has been confirmed in a pilot and phase I clinical studies, and a promising long-term clinical efficacy has also been obtained. This novel vaccine is a noninvasive, long-lasting therapy for ATL and can potentially be extended to different applications for low-grade ATL and high-risk HTLV-1 carriers.

ANK Therapeutic Prospects and Usefulness of PD-L1 and NK Activity as Biomarkers for Predicting Treatment Efficacy Revealed from the Treatment Course of Patients with HTLV-1-Associated Bronchioloalveolar Disease

Adult T-cell leukemia/lymphoma (ATL) is a peripheral T-cell neoplasm with poor prognosis that can present as HTLV-1associated bronchioloalveolar disease (HABA). Chemotherapy is recommended for ATL; however, it is not very effective against all types of ATL. Furthermore, there are no effective treatments for smoldering HABA-associated ATL. We present a case in which amplified natural killer cell (ANK) therapy was effective in a woman in her early 80s who was previously diagnosed with ATL-related smoldering HABA and presented with dyspnea and productive cough on exertion. The symptoms were suppressed for approximately 10 months after the first treatment, but then gradually worsened. About a year later, a second treatment was followed by mild side effects. Suppression of ATL cell proliferation by repeated doses of ANK therapy appears to be effective in this patient. The therapeutic effect was high even with long treatment intervals, and the efficacy and safety of repeated treatments have been demonstrated. ANK therapy is expected to be the mainstay of treatment ATL and HABA. ANK therapy has been reported to kill PD-L1 positive tumor cells and some solid tumors with excellent responses have many PDL1-positive tumor cells. ANK therapy is thought to be effective for ATL because there are many PD-L1-positive tumor cells. Furthermore, administration of activated NK cells may increase tumor-killing activity in those patients with reduced NK activity. While future studies are needed, PD-L1 positive rate and NK activity may be biomarkers for the effectiveness of ANK therapy.

ANK Therapeutic Prospects and Usefulness of PD-L1 and NK Activity as Biomarkers for Predicting Treatment Efficacy Revealed from the Treatment Course of Patients with HTLV-1-Associated Bronchioloalveolar Disease

Adult T-cell leukemia/lymphoma (ATL) is a peripheral T-cell neoplasm with poor prognosis that can present as HTLV-1associated bronchioloalveolar disease (HABA). Chemotherapy is recommended for ATL; however, it is not very effective against all types of ATL. Furthermore, there are no effective treatments for smoldering HABA-associated ATL. We present a case in which amplified natural killer cell (ANK) therapy was effective in a woman in her early 80s who was previously diagnosed with ATL-related smoldering HABA and presented with dyspnea and productive cough on exertion. The symptoms were suppressed for approximately 10 months after the first treatment, but then gradually worsened. About a year later, a second treatment was followed by mild side effects. Suppression of ATL cell proliferation by repeated doses of ANK therapy appears to be effective in this patient. The therapeutic effect was high even with long treatment intervals, and the efficacy and safety of repeated treatments have been demonstrated. ANK therapy is expected to be the mainstay of treatment ATL and HABA. ANK therapy has been reported to kill PD-L1 positive tumor cells and some solid tumors with excellent responses have many PDL1-positive tumor cells. ANK therapy is thought to be effective for ATL because there are many PD-L1-positive tumor cells. Furthermore, administration of activated NK cells may increase tumor-killing activity in those patients with reduced NK activity. While future studies are needed, PD-L1 positive rate and NK activity may be biomarkers for the effectiveness of ANK therapy.

A Dual Role for FADD in Human Precursor T-Cell Neoplasms

A reduction in FADD levels has been reported in precursor T-cell neoplasms and other tumor types. Such reduction would impact on the ability of tumor cells to undergo apoptosis and has been associated with poor clinical outcomes. However, FADD is also known to participate in non-apoptotic functions, but these mechanisms are not well-understood. Linking FADD expression to the severity of precursor T-cell neoplasms could indicate its use as a prognostic marker and may open new avenues for targeted therapeutic strategies. Using transcriptomic and clinical data from patients with precursor T-cell neoplasms, complemented by in vitro analysis of cellular functions and by high-throughput interactomics, our results allow us to propose a dual role for FADD in precursor T-cell neoplasms, whereby resisting cell death and chemotherapy would be a canonical consequence of FADD deficiency in these tumors, whereas deregulation of the cellular metabolism would be a relevant non-canonical function in patients expressing FADD. These results reveal that evaluation of FADD expression in precursor T-cell neoplasms may aid in the understanding of the biological processes that are affected in the tumor cells. The altered biological processes can be of different natures depending on the availability of FADD influencing its ability to exert its canonical or non-canonical functions. Accordingly, specific therapeutic interventions would be needed in each case.

Copy Number Loss at Chromosome 14q11.2 Correlates With the Proportion of T Cells in Biopsies and Helps Identify T-Cell Neoplasms.

Evidence of T-cell clonality is often critical in supporting the diagnosis of a T-cell lymphoma. To retrospectively explore the significance of copy number losses at the 14q11.2 T-cell receptor α locus in relation to the presence of a T-cell neoplasm and proportion of T cells by targeted next-generation sequencing. Targeted next-generation sequencing data from 139 tissue biopsies, including T-cell lymphomas, B-cell lymphomas, classic Hodgkin lymphomas, nonhematopoietic malignancies, and normal samples, were reviewed for copy number losses involving the T-cell receptor α gene segments at chr14q11.2. We found that biallelic or homozygous deletion of 14q11.2 was found in most (28 of 33, 84.8%) T-cell lymphomas. The magnitude of 14q11.2 loss showed a statistically significant correlation with the proportion of T cells in lymphoma tissue samples. Copy number losses could also be detected in other lymphomas with high numbers of T cells (8 of 32, 25% of B-cell lymphomas, 4 of 4 classical Hodgkin lymphomas), though biallelic/homozygous deletion of 14q11.2 was not significantly observed outside of T-cell lymphomas. Most nonhematopoietic neoplasms and normal tissues (59 of 64, 92.2%) showed no significant copy number losses involving the T-cell receptor α locus at chr14q11.2. Analysis of copy number losses at the T-cell receptor α locus chr14q11.2 with targeted next-generation sequencing can potentially be used to estimate the proportion of T cells and detect T-cell neoplasms.

High-Throughput Screening to Elucidate In Vivo Oncogenicity of Loss-of-Function Alterations in Lymphoid Malignancies

Large-scale genetic studies utilizing next-generation sequencing have identified many recurrently altered genes and revealed their overall picture in lymphoid malignancies. Although limited genes mutated at high frequency have been extensively investigated using genetically engineered mouse models, less attention has been directed to hundreds of rarely mutated genes (so-called long tail driver genes). Here we performed in vivo CRISPR loss-of-function screening to examine oncogenicity of somatic alterations, cooperability between them, and association between genotype and lineage in lymphoid malignancies. As many driver candidates mutated with various frequencies have been reported in diffuse large B-cell lymphomas (DLBCL), we constructed a custom lentiviral CRISPR library targeting 86 genes (12 sgRNAs each) in which loss-of-function alterations (mutations and deletions) are recurrently (> 5%) observed in DLBCL. These sgRNA-targeted genes also covered the majority of driver candidates of other precursor and mature lymphoid neoplasms, such as 71% and 87% of those observed in B-acute lymphoblastic leukemia (B-ALL/LBL) and peripheral T-cell lymphoma, not otherwise specified (PTCL, NOS). Transplantation of the CRISPR library-transduced murine hematopoietic stem/progenitor (Lin-c-Kit+Sca-1+) cells caused a variety of hematologic malignancies in 180 (90%) out of 199 recipient mice, resulting in earlier death (with a median of 228 [range, 37‒476] days) of transplanted mice than control mice. Hematologic, immunophenotypic, and pathologic analyses of 132 tumors revealed that these malignancies consisted of B- and T-cell lymphomas (17% and 20%), B- and T-ALL/LBL (14% and 15%) and myeloid malignancy (14%) according to Bethesda criteria, although the diagnosis of 22 (17%) tumors could not be determined. sgRNA sequencing of 109 tumors showed that at least 1 sgRNA-targeted genes were detected in almost all (>99%) tumors, with a median of 3 (range, 1‒10) genes per tumor. Enrichment analysis of sgRNA-targeted genes demonstrated significant overrepresentation of 13 genes, such as Trp53 and Irf2bp2, pointing to stronger oncogenic capacity of these driver genes. These sgRNA-targeted genes showed lineage and tissue specificity, such as Pax5, Kmt2d, Atm, and S1pr2 for B-cell lineage and Zc3h12a and Socs1 for T-cell lineage, while only weak enrichment was observed in myeloid neoplasms. These tumors showed characteristic disease phenotype according to sgRNA-targeted genes: Pax5-targeted tumors were usually B-ALL/LBL, whereas Kmt2d-targeted tumors developed as B-cell lymphomas with earlier onset than those harboring other targeted genes. All of Zc3h12a-targeted tumors were T-cell lymphoma with CD4+CD8- immunophenotype and low clonality, while Socs1-targeted tumors primarily developed as T-ALL/LBL with CD4+CD8+ immunophenotype and frequent thymus involvement. Whole-exome sequencing (n = 108) not only confirmed targeted disruption by sgRNAs but also identified additional driver alterations in 76% and 40% of B-cell and T-cell neoplasms, respectively, suggesting the necessity of second-hit alterations in these tumors. These additional driver alterations were associated with a specific lineage. Frequent C-terminal truncating mutations of Notch1 and loss-of-function mutations of Ikzf1 were found in T-cell malignancies, while Jak1 and Jak3 hotspot missense mutations and Flt3 internal tandem duplications were common in B-cell malignancies, suggesting cooperative relationship between specific alterations and their association with lineage. In addition, hierarchical clustering of RNA-sequencing data (n = 105) identified several clusters associated with disease phenotypes and sgRNA-targeted genes. These clusters showed distinct expression signatures, including germinal center B-cell-like signatures in Kmt2d-targeted tumors, MYC target signatures in Pax5-targeted tumors, and inflammation-related signature in Zc3h12a-targeted tumors. In summary, our in vivo CRISPR loss-of-function screening enables high-throughput evaluation of the tumorigenic potential of many genetic abnormalities, their cooperability and association with lineage in lymphoid malignancies. Such approach accelerates understanding of genotype-phenotype association and discovery of potential therapeutic targets. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Characteristics and Outcomes of Adult Patients with T Prolymphocytic Leukemia: A Real World Study of the French Innovative Leukemia Group (FILO)

Introduction T-prolymphocytic leukemia (T-PLL) is a rare T-cell neoplasm of aggressive clinical course and poor outcome. Treatment with the monoclonal antibody to CD52-alemtuzumab (alem) has considerably improved outcomes, yet with transient responses. Bendamustine (benda) showed encouraging results, particularly in alem-refractory patients (pts). Allogeneic hematopoietic stem cells transplantation (Allo-HSCT) has been reported to be effective in T-PLL, yielding durable remissions and improved overall survival (OS). New approaches using well-tolerated therapies and drugs that target such growth and survival signals as that the JAK/STAT pathways or BCL2 have recently been tested in small series of pts ineligible to intensive therapy. Methods The present multicenter study aimed at evaluating clinical and biological characteristics, as well as outcome of a cohort of 157 T-PLL pts over 18 years old (yo) treated in any of 24 centers of the French Innovative Leukemia Organization (FILO) between 03/1997 and 05/2022. OS, relapse, progression and progression-fee survival (PFS) were evaluated. Molecular analyzes searched for JAK1, JAK3, STAT5B, IL2RG, NOCH1, NOTCH2, EZH2, TET2 and TP53 mutations/deletions in 39 pts. Results Pts characteristics are shown in Table 1. The median age at first therapy was 72 yo (range 30-94). Neuro-meningeal and extra-nodal involvement were present at diagnosis and first relapse in respectively 4.6% / 10.5% and 5.9% / 14.3% of pts. At diagnosis, 12% of pts had hemoglobin <100 g/L, 32.6% thrombocytopenia <100x109/L and 43% lymphocytosis >40x109/L. Flow cytometric immunophenotyping showed pan-T antigens expression (CD2,CD3, CD5, CD7) in nearly all pts (table). Alpha-beta TCR was present for 64/73 pts (87.7%) and CD25 for 17/42 (40.2%). The TPLL was CD4+ for 73/98 pts (74.5%), CD8+ for 7/93 (7.5%), double positive (CD4+/CD8+) for 15/93 (16.1%) and double negative (CD4-/CD8-) for 3/92 (3.2%). Cytogenetics/FISH showed recurrent changes involving chromosome 14 in 69/95 (72.6%) pts [inv(14) and t(14;14) in 47/69 (68.1%) and 7/69 (10.3%)] and complex karyotype in 67/95 (70.5%). C-MYC gain, ATM and TP53 deletions/mutations were identified in respectively 22.6%, 27.5% and 16.4% of the pts analyzed at diagnosis, while JAK1, JAK3, and STAT5B were mutated in 15.4 %, 64.3% and 21.4% of tested cases. Additional results will be presented at the meeting. Pts received 1 to 5 lines of treatment (median 1). In first line, 45/127 (35.4%) evaluable pts received alem 30 mg IVx3 per week, after ramp-up (median:6 cycles; 1-10), with for 7 of them a previous sequence of benda (90 mg/m2 on day 1 [D1]). Thirty pts (23.6%) received first line benda 90 mg/m2 on D1 then 2x6 cycles every 28 days (median 3; 1-6), 15 (11.8%) received pentostatin, 11 (8.6%) had a CHOP-like regimen. Best supportive care was provided to 21 pts (16.5%). No treatment was proposed to 3 indolent pts (2.3%). Various schedules were applied to the remaining pts. Post-CR Allo-HSCT was performed for 20 pts (15.7%). Median follow-ups are 9.5 months (0.03-217) for the whole cohort (Figure1) and 11.8 (0.03-221) for Allo-HSCT recipients. Median OS are respectively 12.5 (8.4-17.5) and 23.2 months (5.82-not reached [NR]). The median PFS of the whole cohort is 2.6 months (0-217). Median OS for patients treated with alem or benda in first line are respectively 14.1 (8.4-25.4) and 7.8 (4.3-21) months, while OS for pts who had a benda bridge is 8 months (3.22-NR). The overall response rate (ORR) after first line is 42.7 % (CR 25.6%, partial response [PR] 17.1%). ORRs after first line alem or benda are respectively 53.5% (CR 41.9%, PR 11.6%) and 35.7% (CR: 14.3%, PR 21.4%). In univariate analysis, platelets<100x109/L (HR: 1.95 [1.28-2.95]; p=0.002%) and complex karyotype (HR: 2.08 [1.19-3.63]; p=0.010) statistically significantly appear to impact survival. Conclusion This large study demonstrates that alem therapy significantly improves the survival of T-PLL pts. Consolidation by Allo-HSCT should however be performed for all eligible patients achieving CR. Other therapeutic agents should be reserved for relapse. Prospective trials are needed to define the most effective therapeutic strategy and improve clinical responses to frontline therapy. New approaches using well-tolerated targeted therapies involving growth and survival signals are needed for the majority of patients unable to receive intensive chemotherapy. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

GATA-3 is a proto-oncogene in T-cell lymphoproliferative neoplasms

Neoplasms originating from thymic T-cell progenitors and post-thymic mature T-cell subsets account for a minority of lymphoproliferative neoplasms. These T-cell derived neoplasms, while molecularly and genetically heterogeneous, exploit transcription factors and signaling pathways that are critically important in normal T-cell biology, including those implicated in antigen-, costimulatory-, and cytokine-receptor signaling. The transcription factor GATA-3 regulates the growth and proliferation of both immature and mature T cells and has recently been implicated in T-cell neoplasms, including the most common mature T-cell lymphoma observed in much of the Western world. Here we show that GATA-3 is a proto-oncogene across the spectrum of T-cell neoplasms, including those derived from T-cell progenitors and their mature progeny, and further define the transcriptional programs that are GATA-3 dependent, which include therapeutically targetable gene products. The discovery that p300-dependent acetylation regulates GATA-3 mediated transcription by attenuating DNA binding has novel therapeutic implications. As most patients afflicted with GATA-3 driven T-cell neoplasms will succumb to their disease within a few years of diagnosis, these findings suggest opportunities to improve outcomes for these patients.