Activating mutations in genes related to TCR signaling in angioimmunoblastic and other follicular helper T-cell-derived lymphomas.

Angioimmunoblastic T-cell lymphoma (AITL) and other lymphomas derived from follicular T-helper cells (TFH) represent a large proportion of peripheral T-cell lymphomas (PTCLs) with poorly understood pathogenesis and unfavorable treatment results. We investigated a series of 85 patients with AITL (n = 72) or other TFH-derived PTCL (n = 13) by targeted deep sequencing of a gene panel enriched in T-cell receptor (TCR) signaling elements. RHOA mutations were identified in 51 of 85 cases (60%) consisting of the highly recurrent dominant negative G17V variant in most cases and a novel K18N in 3 cases, the latter showing activating properties in in vitro assays. Moreover, half of the patients carried virtually mutually exclusive mutations in other TCR-related genes, most frequently in PLCG1 (14.1%), CD28 (9.4%, exclusively in AITL), PI3K elements (7%), CTNNB1 (6%), and GTF2I (6%). Using in vitro assays in transfected cells, we demonstrated that 9 of 10 PLCG1 and 3 of 3 CARD11 variants induced MALT1 protease activity and increased transcription from NFAT or NF-κB response element reporters, respectively. Collectively, the vast majority of variants in TCR-related genes could be classified as gain-of-function. Accordingly, the samples with mutations in TCR-related genes other than RHOA had transcriptomic profiles enriched in signatures reflecting higher T-cell activation. Although no correlation with presenting clinical features nor significant impact on survival was observed, the presence of TCR-related mutations correlated with early disease progression. Thus, targeting of TCR-related events may hold promise for the treatment of TFH-derived lymphomas

Super-enhancer-based identification of a BATF3/IL-2R-module reveals vulnerabilities in anaplastic large cell lymphoma.

Anaplastic large cell lymphoma (ALCL), an aggressive CD30-positive T-cell lymphoma, comprises systemic anaplastic lymphoma kinase (ALK)-positive, and ALK-negative, primary cutaneous and breast implant-associated ALCL. Prognosis of some ALCL subgroups is still unsatisfactory, and already in second line effective treatment options are lacking. To identify genes defining ALCL cell state and dependencies, we here characterize super-enhancer regions by genome-wide H3K27ac ChIP-seq. In addition to known ALCL key regulators, the AP-1-member BATF3 and IL-2 receptor (IL2R)-components are among the top hits. Specific and high-level IL2R expression in ALCL correlates with BATF3 expression. Confirming a regulatory link, IL-2R-expression decreases following BATF3 knockout, and BATF3 is recruited to IL2R regulatory regions. Functionally, IL-2, IL-15 and Neo-2/15, a hyper-stable IL-2/IL-15 mimic, accelerate ALCL growth and activate STAT1, STAT5 and ERK1/2. In line, strong IL-2Rα-expression in ALCL patients is linked to more aggressive clinical presentation. Finally, an IL-2Rα-targeting antibody-drug conjugate efficiently kills ALCL cells in vitro and in vivo. Our results highlight the importance of the BATF3/IL-2R-module for ALCL biology and identify IL-2Rα-targeting as a promising treatment strategy for ALCL

The Genetic Basis of Hepatosplenic T-cell Lymphoma

Hepatosplenic T cell lymphoma (HSTL) is a rare and lethal lymphoma; the genetic drivers of this disease are unknown. Through whole exome sequencing of 68 HSTLs, we define recurrently mutated driver genes and copy number alterations in the disease. Chromatin modifying genes including SETD2, INO80 and ARID1B were commonly mutated in HSTL, affecting 62% of cases. HSTLs manifest frequent mutations in STAT5B (31%), STAT3 (9%), and PIK3CD (9%) for which there currently exist potential targeted therapies. In addition, we noted less frequent events in EZH2, KRAS and TP53. SETD2 was the most frequently silenced gene in HSTL. We experimentally demonstrated that SETD2 acts as a tumor suppressor gene. In addition, we found that mutations in STAT5B and PIK3CD activate critical signaling pathways important to cell survival in HSTL. Our work thus defines the genetic landscape of HSTL and implicates novel gene mutations linked to HSTL pathogenesis and potential treatment targets

Gene Expression Profiling Using a Reverse Transcriptase-Multiplex Ligation Dependant Probe Amplification Assay Allows for an Accurate Classification of Peripheral T-Cell Lymphoma and Highlights Novel Subgroups within the PTCL-NOS Category

International audienceIntroductionVarious Peripheral T-cell lymphoma (PTCL) entities are recognized in the World Health Organization (WHO) classification based on clinical, histopathological, phenotypic and molecular criteria. Their diagnosis is however often challenging for pathologists, and up to 30% of cases, currently not classifiable, are recognized as not otherwise specified (NOS). Recent gene expression profiling (GEP) studies have significantly improved the molecular and ontogenic characterization of these tumors, but such high-throughput technologies are hardly feasible in the routine clinical setting. There is therefore an important need for alternative diagnostic strategies to allow for the development of specific therapies. Here, we sought to create a parsimonious and robust GEP assay to differentiate the different PTCL entities and to better characterize the heterogeneity of PTCL-NOS.MethodsA Reverse Transcriptase-Multiplex Ligation dependant Probe Amplification (RT-MLPA) assay addressing 20 markers was applied to a cohort of 227 PTCLs biopsies enriched in PTCL-NOS (n=126). This assay determines the expression of seventeen genes routinely tested as immunohistochemical (IHC) markers or selected from high throughput GEP studies, together with the EBV infection status (EBER1 expression) and the presence of RHOAG17V and IDH2R172K/T mutations.ResultsUnsupervised hierarchical clustering analysis of 101 control cases representing the main PTCL entities other than PTCL-NOS by RT-MLPA accurately classified 28/29 Angioimmunoblastic T-cell lymphomas (AITL), 21/21 Anaplastic large T-cell lymphomas (ALCL) ALK+, 16/16 NK/T-cell lymphomas (NKTCL), 6/6 Hepatosplenic T-cell lymphomas (HSTL) and 12/12 Adult T-cell Leukemia/Lymphomas (ATLL)(Figure). AITL were classified according to the expression of Tfh markers (CXCL13, CXCR5, ICOS, BCL6) and RHOA mutations (n=18); NKTCLs according to EBER1, GZMB and Th1 markers (TBX21, IFNγ); HSTLs to CD56, GATA3, TBX21 and BCL6; ALCL ALK+ according to CD30, ALK and cytotoxic markers (PRF, GZMB); ATLLs to ICOS and Th2 markers (GATA3, CCR4). Interestingly, ALCL ALK- cases (n=17) were divided into 2 subgroups: one, associated with high expression of CD30 and cytotoxic markers (PRF, GZMB), clustered with ALCL ALK+ cases (n=11), the other showed absence of PRF and GZMB, but expression of CD30 and Th2 markers (n=6).Applied to 126 nodal PTCL-NOS, the RT-MLPA classifier identified 33 AITL-Like cases expressing Tfh markers and often presenting with RHOA mutations (15 cases). It also identified 5 NKTCL-like cases (EBV-cytoxic) and 1 ALCL-like case (cytotoxic-CD30).The CD30-Th2 signature was found in 15 cases, reinforcing the hypothesis that it may delineate a novel PTCL entity, at the frontier between ALCL ALK- and other PTCLs. In agreement with previous GEP studies, 23 cases expressed Th2 markers but no CD30 (often in association with a significant Tfh signature, probably reflecting a contribution from the microenvironment). Twenty-five other cases showed a hybrid cytotoxic-Th1 signature. The remaining 14 cases did not reveal any recognizable gene expression profile.Finally, we observed a strong correlation between RT-MLPA and IHC for most markers evaluated by both methods (p<10-3 for CD8, CD30, GZMB, PRF, ALK, CD56, CXCL13, ICOS and GATA3), indicating that our assay is reliable and may constitute a valuable alternative to IHC.ConclusionThis study demonstrates the applicability of a parsimonious RT-MLPA classifier for the classification of PTCLs. Its simplicity of use and its applicability on FFPE samples makes it an attractive alternative to high throughput GEP approaches and IHC. Its implementation in clinical trials, in combination with conventional pathological evaluation, may thus participate to improve the classification of PTCLs and therefore the management of PTCL patients

ALK-negative anaplastic large cell lymphoma with DUSP22 rearrangement has distinctive disease characteristics with better progression-free survival: a LYSA study.

peer reviewedALK-negative anaplastic large cell lymphoma (ALCL) comprises subgroups harboring rearrangements of DUSP22 (DUSP22-R) or TP63 (TP63-R). Two studies respectively reported 90% and 40% 5-year overall survival (OS) in 21 and 12 DUSP22-R/TP63-not rearranged (NR) patients, making the prognostic impact of DUSP22-R unclear. Here, 104 newly diagnosed ALK-negative ALCL patients (including 37 from first-line clinical trials) from the LYSA TENOMIC database were analyzed by break-apart FISH assays for DUSP22-R and TP63-R. There were 47/104 (45%) DUSP22-R and 2/93 (2%) TP63-R cases, including one DUSP22-R/TP63-R. DUSP22-R tumors showed more frequent CD3 expression (62% versus 35%, P=0.01), and less commonly a cytotoxic phenotype (27% versus 82%; P