Comprehensive genetic profiling reveals frequent alterations of driver genes on the X chromosome in extranodal NK/T-cell lymphoma.

Extranodal NK/T-cell lymphoma (ENKTCL) is an Epstein-Barr virus (EBV)-related neoplasm with male dominance and a poor prognosis. A better understanding of the genetic alterations and their functional roles in ENKTCL could help improve patient stratification and treatments. Here, we performed comprehensive genetic analysis of 177 ENKTCL cases to delineate the landscape of mutations, copy number alterations (CNAs), and structural variations, identifying 34 driver genes including six previously unappreciated ones, namely HLA-B, HLA-C, ROBO1, CD58, POT1, and MAP2K1. Among them, CD274 (24%) was the most frequently altered, followed by TP53 (20%), CDKN2A (19%), ARID1A (15%), HLA-A (15%), BCOR (14%), and MSN (14%). Chromosome X (chrX) losses were the most common arm-level CNAs in females (~40%), and alterations of four X-linked driver genes (MSN, BCOR, DDX3X, and KDM6A) were more frequent in males and females harboring chrX losses. Among X-linked drivers, MSN was the most recurrently altered, and its expression was lost in approximately one-third of cases using immunohistochemical analysis. Functional studies of human cell lines demonstrated that MSN disruption promoted cell proliferation and NF-κB activation. Moreover, MSN inactivation increased sensitivity to NF-κB inhibition in vitro and in vivo. In addition, recurrent deletions were observed at the origin of replication in the EBV genome (6%). Finally, by integrating the 34 drivers and 19 significant arm-level CNAs, non-negative matrix factorization and consensus clustering identified two molecular groups with different genetic features and prognosis irrespective of clinical prognostic factors. Together, these findings could help improve diagnostic and therapeutic strategies in ENKTCL.

PD1 inhibits PKCθ-dependent phosphorylation of cytoskeleton-related proteins and immune synapse formation.

The inhibitory cell surface receptor programmed death 1 (PD1) is a major target for antibody-based cancer immunotherapies. Nevertheless, a substantial number of patients fail to respond to the treatment or experience adverse effects. An improved understanding of intracellular pathways targeted by PD1 is thus needed to develop better predictive and prognostic biomarkers. Here, via unbiased phosphoproteome analysis of primary human T cells, we demonstrate that PD1 triggering inhibited the phosphorylation and physical association with PKC theta (PKCof a variety of cytoskeleton-related proteins. PD1 blocked activation and recruitment of PKCθ to the forming immune synapse (IS) in a SHP1/SHP2 tyrosine phosphatase-dependent manner. Consequently, PD1 engagement led to impaired synaptic phosphorylation of cytoskeleton-related proteins and formation of smaller IS. TCR-induced phosphorylation of the PKCθ substrate and binding partner Vimentin was long-lasting and it could be durably inhibited by PD1 triggering. Vimentin phosphorylation in intratumoral T cells also inversely correlated with PDL1 levels in human lung carcinoma. Thus, PKCθ and its substrate Vimentin represent important targets of PD1-mediated T-cell inhibition, and low levels of Vimentin phosphorylation may serve as a biomarker for the activation of the PD1 pathway.

Romidepsin Plus Cyclophosphamide, Doxorubicin, Vincristine, and Prednisone Versus Cyclophosphamide, Doxorubicin, Vincristine, and Prednisone in Patients With Previously Untreated Peripheral T-Cell Lymphoma: Final Analysis of the Ro-CHOP Trial.

Clinical trials frequently include multiple end points that mature at different times. The initial report, typically based on the primary end point, may be published when key planned co-primary or secondary analyses are not yet available. Clinical Trial Updates provide an opportunity to disseminate additional results from studies, published in JCO or elsewhere, for which the primary end point has already been reported.The primary analysis of the Ro-CHOP phase III randomized controlled trial (ClinicalTrials.gov identifier: NCT01796002) established that romidepsin (Ro) plus cyclophosphamide, doxorubicin, vincristine, prednisone (CHOP) did not yield an increased efficacy compared with CHOP alone as first-line treatment of peripheral T-cell lymphoma. We report the planned final analysis 5 years after the last patient enrolled. With a median follow-up of 6 years, median progression-free survival (PFS) was 12.0 months compared with 10.2 months (hazard ratio [HR], 0.79 [95% CI, 0.62 to 1.005]; P = .054), while median overall survival was 62.2 months (35.7-86.6 months) and 43.8 months (30.1-70.2 months; HR, 0.88 [95% CI, 0.68 to 1.14]; P = .324) in the Ro-CHOP and CHOP arms, respectively. In an exploratory analysis, the median PFS in the centrally reviewed follicular helper T-cell lymphoma subgroup was significantly longer in the Ro-CHOP arm (19.5 v 10.6 months, HR, 0.703 [95% CI, 0.502 to 0.985]; P = .039). Second-line treatments were given to 251 patients with a median PFS2 and OS2 after relapse or progression of 3.3 months and 11.5 months, respectively. Within the limits of highly heterogeneous second-line treatments, no specific regimen seemed to provide superior disease control. However, a potential benefit was observed with brentuximab vedotin in association with chemotherapy even after excluding anaplastic large-cell lymphoma subtype or after adjusting for histology and international prognostic index in a multivariate model (HR for PFS, 0.431 [95% CI, 0.238 to 0.779]; P = .005).

Follicular Lymphoma Presenting With Symptomatic Bone Involvement: A Clinicopathologic and Molecular Analysis of 16 Cases.

Primary bone lymphoma (PBL) is rare and mostly represented by diffuse large B-cell lymphomas (DLBCL). Follicular lymphoma (FL), albeit commonly disseminating to the bone marrow, rarely presents primarily as bone lesions. Here, we studied 16 patients (12 men:4 women, median age 60 years) who presented with bone pain and/or skeletal radiologic abnormalities revealing bone FL. Lesions were multifocal in 11 patients (spine ± appendicular skeleton), and unifocal in 5 patients (femoral, tibial, or vertebral). An infiltrate of centrocytes and centroblasts (CD20+ CD5- CD10+ BCL2+ BCL6+) with abundant reactive T cells and an increased reticulin fibrosis massively replaced the marrow spaces between preserved bone trabeculae. The pattern was diffuse ± nodular, often with paratrabecular reinforcement and/or peripheral paratrabecular extension. Ki-67 was usually <15%. Two cases had necrosis. BCL2 rearrangement was demonstrated in 14 of 14 evaluable cases (with concomitant BCL6 rearrangement in one). High-throughput sequencing revealed BCL2, KMT2D, and TNFRSF14 to be the most frequently mutated genes. After staging, 5 qualified for PBL (3 limited stage) and 11 had stage IV systemic FL. All patients received rituximab ± polychemotherapy as firstline treatment, and 7 received local therapy (6 radiotherapy and 2 surgery). Three patients experienced transformation to DLBCL. At the last follow-up (15/16, median 48 months), 11 patients achieved complete remission, including all cases with PBL and most patients with limited extraosseous disease (3-year progression-free survival 71%). One patient died of unrelated cause (3-year overall survival 91%). FL may manifest as a localized or polyostotic bone disease. A minority represent PBL, whereas most reveal systemic disease.

Angioimmunoblastic T-cell lymphoma and Kaposi sarcoma: A fortuitous collision?

AIMS: Follicular helper T-cell (TFH) lymphoma of the angioimmunoblastic-type (AITL), one of the most prevalent T-cell lymphomas, typically encompasses proliferation of high endothelial venules and Epstein-Barr virus-positive immunoblasts, but neither infection with HHV8 nor association with Kaposi's sarcoma (KS) have been described. The aims of this study are to characterise the association between AITL and HHV8 infection or KS. METHODS AND RESULTS: Three male patients aged 49-76 years, HIV-negative, with concurrent nodal involvement by AITL and KS, were identified from our files and carefully studied. Two patients originated from countries where endemic KS occurs, including one with cutaneous KS. The lymphomas featured abundant vessels, expanded follicular dendritic cells and neoplastic TFH cells [PD1+ (three of three), ICOS+ (three of three), CXCL13+ (three of three), CD10+ (two of three), BCL6 (two of three)] but lacked EBV+ immunoblasts. The foci of KS consisted of subcapsular proliferations of ERG+, CD31+ and/or CD34+ , HHV8+ spindle cells. High-throughput sequencing showed AITL-associated mutations in TET2 (three of three), RHOA (G17V) (three of three) and IDH2 (R172) (two of three), which were absent in the microdissected KS component in two cases. Relapses in two patients consisted of AITL, without evidence of KS. No evidence of HHV8 infection was found in a control group of 23 AITL cases. CONCLUSION: Concurrent nodal involvement by AITL and KS is rare and identification of both neoplastic components may pose diagnostic challenges. The question of whether the association between AITL and KS may be fortuitous or could reflect the underlying immune dysfunction in AITL remains open.

What is new in the classification of peripheral T cell lymphomas?

In this review focus article, we highlight the main modifications introduced in the latest 2022 International Consensus Classification and World Health Organization classification (ICC and WHO-HAEM5) of mature T (and NK) cell neoplasms (PTCLs) and consequent implications for diagnostic practice. The changes result from recent advances in the genomic and molecular characterization of PTCLs and enhanced understanding of their pathobiology. Specifically, consideration is given to the following groups of diseases: Epstein-Barr virus (EBV)-associated neoplasms; follicular helper T cell lymphoma; anaplastic large cell lymphomas; primary intestinal T and NK cell lymphomas and lymphoproliferative disorders; and PTCL, not otherwise specified.

Pathobiology of nodal peripheral T-cell lymphomas: current understanding and future directions.

Predominantly nodal is the most common clinical presentation of peripheral T- (and NK-) cell lymphomas (PTCL), which comprise three main groups of diseases: (i) systemic anaplastic large cell lymphomas (ALCL), whether positive or negative for anaplastic lymphoma kinase (ALK); (ii) follicular helper T-cell lymphomas (TFHL); and (iii) PTCL, not otherwise specified (NOS). Recent advances in the genomic and molecular characterization of PTCL, with enhanced understanding of pathobiology, have translated into significant updates in the latest 2022 classifications of lymphomas. ALK-negative ALCL is now recognized to be genetically heterogeneous, with identification of DUSP22 rearrangements in approximately 20-30% of cases, correlated with distinctive pathological and biological features. The notion of cell-of-origin as an important determinant of the classification of nodal PTCL is best exemplified by TFHL, considered as one disease or a group of related entities, sharing oncogenic pathways with frequent recurrent epigenetic mutations as well as a relationship to clonal hematopoiesis. Data are emerging to support that a similar cell-of-origin concept might be relevant to characterize meaningful subgroups within PTCL, NOS, based on cytotoxic and/or Th1 versus Th2 signatures. The small group of primary nodal Epstein-Barr virus-positive lymphomas of T- or NK-cell derivation, formerly considered PTCL, NOS, is now classified separately, due to distinctive features, and notably an aggressive course. This review summarizes current knowledge of the pathology and biology of nodal-based PTCL entities, with an emphasis on recent findings and underlying oncogenic mechanisms.

Nitrogen isotopic composition as a gauge of tumor cell anabolism-to-catabolism ratio.

Studies have suggested that cancerous tissue has a lower 15N/14N ratio than benign tissue. However, human data have been inconclusive, possibly due to constraints on experimental design. Here, we used high-sensitivity nitrogen isotope methods to assess the 15N/14N ratio of human breast, lung, and kidney cancer tissue at unprecedented spatial resolution. In lung, breast, and urothelial carcinoma, 15N/14N was negatively correlated with tumor cell density. The magnitude of 15N depletion for a given tumor cell density was consistent across different types of lung cancer, ductal in situ and invasive breast carcinoma, and urothelial carcinoma, suggesting similar elevations in the anabolism-to-catabolism ratio. However, tumor 15N depletion was higher in a more aggressive metaplastic breast carcinoma. These findings may indicate the ability of certain cancers to more effectively channel N towards growth. Our results support 15N/14N analysis as a potential tool for screening biopsies and assessing N metabolism in tumor cells.

The many faces of nodal and splenic marginal zone lymphomas. A report of the 2022 EA4HP/SH lymphoma workshop.

Session 3 of the lymphoma workshop of the XXI joint meeting of the European Association for Haematopathology and the Society for Hematopathology took place in Florence, Italy, on September 22, 2022. The topics of this session were splenic and nodal marginal zone lymphomas, transformation in marginal zone lymphomas, and pediatric nodal marginal zone lymphomas and their differential diagnosis as well as related entities. Forty-two cases in these categories were submitted to the workshop, including splenic lymphomas (marginal zone and diffuse red pulp lymphomas), transformed marginal zone lymphomas (splenic and nodal), nodal marginal zone lymphomas with increased TFH-cells, and pediatric nodal marginal zone lymphomas. The case review highlighted some of the principal problems in the diagnosis of marginal zone lymphomas, including the difficulties in the distinction between splenic marginal zone lymphoma, splenic diffuse red pulp lymphoma, and hairy cell leukemia variant/splenic B-cell lymphoma with prominent nucleoli which requires integration of clinical features, immunophenotype, and morphology in blood, bone marrow, and spleen; cases of marginal zone lymphoma with markedly increased TFH-cells, simulating a T-cell lymphoma, where molecular studies (clonality and mutation detection) can help to establish the final diagnosis; the criteria for transformation of marginal zone lymphomas, which are still unclear and might require the integration of morphological and molecular data; the concept of an overlapping spectrum between pediatric nodal marginal zone lymphoma and pediatric-type follicular lymphoma; and the distinction between pediatric nodal marginal zone lymphoma and "atypical" marginal zone hyperplasia, where molecular studies are mandatory to correctly classify cases.

A CD64/FcγRI-mediated mechanism hijacks PD-1 from PD-L1/2 interaction and enhances anti-PD-1 functional recovery of exhausted T cells.

Therapeutic monoclonal antibodies (mAb) targeting the immune checkpoint inhibitor programmed cell death protein 1 (PD-1) have achieved considerable clinical success in anti-cancer therapy through relieving T cell exhaustion. Blockade of PD-1 interaction with its ligands PD-L1 and PD-L2 is an important determinant in promoting the functional recovery of exhausted T cells. Here, we show that anti-PD-1 mAbs act through an alternative mechanism leading to the downregulation of PD-1 surface expression on memory CD4+ and CD8+ T cells. PD-1 receptor downregulation is a distinct process from receptor endocytosis and occurs in a CD14+ monocyte dependent manner with the CD64/Fcγ receptor I acting as the primary factor for this T cell extrinsic process. Importantly, downregulation of surface PD-1 strongly enhances antigen-specific functional recovery of exhausted PD-1+CD8+ T cells. Our study demonstrates a novel mechanism for reducing cell surface levels of PD-1 and limiting the inhibitory targeting by PD-L1/2 and thereby enhancing the efficacy of anti-PD-1 Ab in restoring T cell functionality.

Cytotoxic peripheral T-cell lymphomas and EBV-positive T/NK-cell lymphoproliferative diseases: emerging concepts, recent advances, and the putative role of clonal hematopoiesis. A report of the 2022 EA4HP/SH lymphoma workshop.

Cytotoxic peripheral T-cell lymphomas and EBV-positive T/NK-cell lymphoproliferative diseases were discussed at the 2022 European Association for Haematopathology/Society for Hematopathology lymphoma workshop held in Florence, Italy. This session focused on (i) primary nodal EBV-positive T and NK-cell lymphomas (primary nodal-EBV-TNKL), (ii) extranodal EBV-positive T/NK lymphoproliferative diseases (LPD) in children and adults, (iii) cytotoxic peripheral T-cell lymphomas, NOS (cPTCL-NOS), EBV-negative, and (iv) miscellaneous cases. Primary nodal-EBV-TNKL is a newly recognized entity which is rare, aggressive, and associated with underlying immune deficiency/immune dysregulation. All cases presented with lymphadenopathy but some demonstrated involvement of tonsil/Waldeyer's ring and extranodal sites. The majority of tumors are of T-cell lineage, and the most frequent mutations involve the epigenetic modifier genes, such as TET2 and DNMT3A, and JAK-STAT genes. A spectrum of EBV-positive T/NK LPD involving extranodal sites were discussed and highlight the diagnostic challenge with primary nodal-EBV-TNKL when these extranodal EBV-positive T/NK LPD cases demonstrate predominant nodal disease either at presentation or during disease progression from chronic active EBV disease. The majority of cPTCL-NOS demonstrated the TBX21 phenotype. Some cases had a background of immunosuppression or immune dysregulation. Interestingly, an unexpected association of cPTCL-NOS, EBV-positive and negative, with TFH lymphomas/LPDs was observed in the workshop cases. Similar to a published literature, the genetic landscape of cPTCL-NOS from the workshop showed frequent mutations in epigenetic modifiers, including TET2 and DNMT3A, suggesting a role of clonal hematopoiesis in the disease pathogenesis.

Emerging entities: high-grade/large B-cell lymphoma with 11q aberration, large B-cell lymphoma with IRF4 rearrangement, and new molecular subgroups in large B-cell lymphomas. A report of the 2022 EA4HP/SH lymphoma workshop.

Emerging entities and molecular subgroups in large B-cell lymphomas (LBCLs) were discussed during the 2022 European Association for Haematopathology/Society for Hematopathology workshop in Florence, Italy. This session focused on newly recognized diseases and their diagnostic challenges. High-grade/large B-cell lymphoma with 11q aberration (HG/LBCL-11q) is defined by chromosome 11q-gains and telomeric loss. FISH analysis is recommended for the diagnosis. HG/LBCL-11q can occur in the setting of immunodeficiency, including ataxia-telangiectasia, and predominates in children. The morphological spectrum of these cases is broader than previously thought with often Burkitt-like morphology and coarse apoptotic bodies. It has a Burkitt-like immunophenotype (CD10+, BCL6+, BCL2-) but MYC expression is weak or negative, lacks MYC rearrangement, and is in contrast to Burkitt lymphoma 50% of the cases express LMO2. LBCL with IRF4 rearrangement (LBCL-IRF4) occurs mainly in the pediatric population but also in adults. LBCL-IRF4 has an excellent prognosis, with distinguishing molecular findings. IRF4 rearrangements, although characteristic of this entity, are not specific and can be found in association with other chromosomal translocations in other large B-cell lymphomas. Other molecular subgroups discussed included primary bone diffuse large B-cell lymphoma (PB-DLBCL), which has distinctive clinical presentation and molecular findings, and B-acute lymphoblastic leukemia (B-ALL) with IGH::MYC translocation recently segregated from Burkitt lymphoma with TdT expression. This latter disorder has molecular features of precursor B-cells, often tetrasomy 1q and recurrent NRAS and KRAS mutations. In this report, novel findings, recommendations for diagnosis, open questions, and diagnostic challenges raised by the cases submitted to the workshop will be discussed.

Cavity-based lymphomas: challenges and novel concepts. A report of the 2022 EA4HP/SH lymphoma workshop.

The 2022 European Association for Haematopathology/Society for Hematopathology lymphoma workshop session on cavity-based lymphomas included sixty-eight cases in seven sections. The disease entities discussed include primary effusion lymphomas (PEL), extracavitary primary effusion lymphomas and confounding entities (ECPEL), HHV8-negative B-lineage lymphomas-effusion based (EBV-negative, EBV-positive, and plasmablastic types), diffuse large B-cell lymphoma associated with chronic inflammation, fibrin-associated diffuse large B-cell lymphoma (FA-DLBCL), breast implant-associated anaplastic large cell lymphoma (BIA-ALCL), and other lymphomas presenting as an effusion. All entities above are discussed; however, three are delved into greater detail given the challenges with classification: ECPEL, HHV8-negative effusion-based lymphomas, and FA-DLBCL. Cases exemplifying the diagnostic difficulty in differentiating ECPEL from HHV8-positive diffuse large B-cell lymphoma and germinotropic lymphoproliferative disorder were discussed. The more recently recognized effusion-based HHV8-negative large B-cell lymphoma is explored, with several cases submitted raising the question if this subset should be carved out as a specific entity, and if so, what should be the refining diagnostic criteria. Case submissions to the FA-DLBCL section yielded one of the largest case series to date, including classic cases, cases furthering the discussion on disease sites and prognosis, as well as novel concepts to be considered in this entity. The 2022 EA4HP/SH workshop cases allowed for further confirmation of the characteristics of some of the more historically accepted cavity-based lymphomas, as well as further inquiry and debate on relatively new or evolving entities.

Multilevel human secondary lymphoid immune system compartmentalization revealed by complementary imaging approaches.

Secondary human lymphoid tissue immune reactions take place in a highly coordinated environment with compartmentalization representing a fundamental feature of this organization. In situ profiling methodologies are indispensable for the understanding of this compartmentalization. Here, we propose a complementary experimental approach aiming to reveal different aspects of this process. The analysis of human tonsils, using a combination of single cell phenotypic analysis based on flow cytometry and multiplex imaging and mass spectrometry-based methodologies, revealed a compartmentalized organization at the cellular and molecular levels. More specifically, the skewed distribution of highly specialized immune cell subsets and relevant soluble mediators was accompanied by a compartmentalized localization of several lipids across different anatomical areas of the tonsillar tissue. The performance of such combinatorial experimental approaches could lead to the identification of novel in situ interactions and molecular targets for the in vivo manipulation of lymphoid organ, particularly the germinal center, immune reactions.

Follicular helper T-cell lymphomas: disease spectrum, relationship with clonal hematopoiesis, and mimics. A report of the 2022 EA4HP/SH lymphoma workshop.

Follicular helper T-cell lymphomas (TFH lymphomas) were discussed in session V of the lymphoma workshop of the European Association for Haematopathology (EA4HP)/Society for Hematopathology (SH) 2022 meeting in Florence, Italy. The session focused on the morphologic spectrum of TFH lymphoma, including its three subtypes: angioimmunoblastic-type (AITL), follicular-type, and not otherwise specified (NOS). The submitted cases encompassed classic examples of TFH lymphoma and unusual cases such as those with early or indolent presentations, associated B-cell proliferations, or Hodgkin/Reed-Sternberg-like cells. The relationship between TFH lymphoma and clonal hematopoiesis was highlighted by several cases documenting divergent evolution of myeloid neoplasm and AITL from shared clonal mutations. The distinction between TFH lymphoma and peripheral T-cell lymphoma, not otherwise specified (PTCL, NOS), was stressed, and many challenging examples were presented. Various cases highlighted the difficulties of differentiating TFH lymphoma from other established types of lymphoma and reactive conditions. Cutaneous T-cell lymphoma expressing TFH markers, particularly when resulting in lymph node involvement, should be distinguished from TFH lymphomas. Additional immunophenotyping and next-generation sequencing studies were performed on various cases in this session, highlighting the importance of these technologies to our current understanding and classification of TFH lymphomas.