Single-Cell Sequencing Illuminates Thymic Development: An Updated Framework for Understanding Thymic Epithelial Tumors.

Thymic epithelial tumors (TETs) are rare tumors for which treatment options are limited. The ongoing need for improved systemic therapies reflects a limited understanding of tumor biology as well as the normal thymus. The essential role of the thymus in adaptive immunity is largely effected by its epithelial compartment, which directs thymocyte (T-cell) differentiation and immunologic self-tolerance. With aging, the thymus undergoes involution whereby epithelial tissue is replaced by adipose and other connective tissue, decreasing immature T-cell production. Against this natural drive toward involution, a fraction of thymuses will instead undergo oncologic transformation, leading to the formation of TETs, including thymoma and thymic carcinoma. The rarity of these tumors restricts investigation of the mechanisms of tumorigenesis and development of rational treatment options. To this end, the development of technologies which allow deep molecular profiling of individual tumor cells permits a new window through which to view normal thymic development and contrast the malignant changes that result in oncogenic transformation. In this review, we describe the findings of recent illuminating studies on the diversity of cell types within the epithelial compartment through thymic differentiation and aging. We contextualize these findings around important unanswered questions regarding the spectrum of known somatic tumor alterations, cell of origin, and tumor heterogeneity. The perspectives informed by single-cell molecular profiling offer new approaches to clinical and basic investigation of thymic epithelial tumors, with the potential to accelerate development of improved therapeutic strategies to address ongoing unmet needs in these rare tumors.

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- naive 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.

TP63 fusions drive multicomplex enhancer rewiring, lymphomagenesis, and EZH2 dependence.

Gene fusions involving tumor protein p63 gene (TP63) occur in multiple T and B cell lymphomas and portend a dismal prognosis for patients. The function and mechanisms of TP63 fusions remain unclear, and there is no target therapy for patients with lymphoma harboring TP63 fusions. Here, we show that TP63 fusions act as bona fide oncogenes and are essential for fusion-positive lymphomas. Transgenic mice expressing TBL1XR1::TP63, the most common TP63 fusion, develop diverse lymphomas that recapitulate multiple human T and B cell lymphomas. Here, we identify that TP63 fusions coordinate the recruitment of two epigenetic modifying complexes, the nuclear receptor corepressor (NCoR)-histone deacetylase 3 (HDAC3) by the N-terminal TP63 fusion partner and the lysine methyltransferase 2D (KMT2D) by the C-terminal TP63 component, which are both required for fusion-dependent survival. TBL1XR1::TP63 localization at enhancers drives a unique cell state that involves up-regulation of MYC and the polycomb repressor complex 2 (PRC2) components EED and EZH2. Inhibiting EZH2 with the therapeutic agent valemetostat is highly effective at treating transgenic lymphoma murine models, xenografts, and patient-derived xenografts harboring TP63 fusions. One patient with TP63-rearranged lymphoma showed a rapid response to valemetostat treatment. In summary, TP63 fusions link partner components that, together, coordinate multiple epigenetic complexes, resulting in therapeutic vulnerability to EZH2 inhibition.

Intralesional microbleeding in resected thymic cysts indeterminate on imaging.

Background: The propensity of thymic cysts to mimic solid thymic epithelial tumors (TETs) on computed tomography (CT), on account of attenuation values greater than water and thickened or calcified walls, can lead to non-therapeutic thymectomy. These lesions can fluctuate in volume, CT attenuation, and magnetic resonance imaging (MRI) signal over time. We hypothesized that spontaneous hemorrhage and resorption may contribute to their variable appearance over time. Methods: Completely excised thymic cysts were identified retrospectively over a 20-year period by their pathologic diagnosis. Cysts were excluded if they did not have available presurgical imaging, were not prevascular, were located within or contained an enhancing mass by imaging, or were of non-thymic origin upon microscopic review. Histopathological analysis of all available resected thymic cyst material and radiologic analysis of the cysts on pre-operative imaging were performed. Results: Upon application of exclusion criteria, we identified 18 thymic cysts from the initial 85 mediastinal cystic specimens. Most cysts were unilocular (11/15, 73%), showed turbid-to-semisolid, hemorrhagic fluid (10/12, 83%) and showed histopathological findings suggestive of intralesional microbleeding (14/18, 78%), remodeling (8/18, 44%), pathological wound healing/scarring of the capsule (16/18, 89%), and fat necrosis in the surrounding thymic tissue (12/18, 67%). On CT, 6/17 (35%) cysts demonstrated wall calcification. Sixty-five percent (11/17) had attenuation values ≥20 Hounsfield units (HU). Two of the 4 cysts imaged by MRI were T1-isointense, one was mixed hyper- and isointense, and one T1-hypointense to muscle, with iso- and hyperintensity indicating hemorrhagic or proteinaceous content. Twenty-five percent (1/4) of cyst walls imaged by MRI were T1/T2-hypointense, indicating presence of calcification, hemosiderin, and/or fibrosis. Conclusions: Resected thymic cysts in this cohort often showed features suggestive of intralesional microbleeding, inflammation, and fibrosis, which may explain their appearance and behavior over time on CT and MRI.

Computer vision quantitation of erythrocyte shape abnormalities provides diagnostic, prognostic, and mechanistic insight.

Examination of red blood cell (RBC) morphology in peripheral blood smears can help diagnose hematologic diseases, even in resource-limited settings, but this analysis remains subjective and semiquantitative with low throughput. Prior attempts to develop automated tools have been hampered by their poor reproducibility and limited clinical validation. Here, we present a novel, open-source machine-learning approach (denoted as RBC-diff) to quantify abnormal RBCs in peripheral smear images and generate an RBC morphology differential. RBC-diff cell counts showed high accuracy for single-cell classification (mean AUC, 0.93) and quantitation across smears (mean R2, 0.76 compared with experts, interexperts R2, 0.75). RBC-diff counts were concordant with the clinical morphology grading for 300 000+ images and recovered the expected pathophysiologic signals in diverse clinical cohorts. Criteria using RBC-diff counts distinguished thrombotic thrombocytopenic purpura and hemolytic uremic syndrome from other thrombotic microangiopathies, providing greater specificity than clinical morphology grading (72% vs 41%; P < .001) while maintaining high sensitivity (94% to 100%). Elevated RBC-diff schistocyte counts were associated with increased 6-month all-cause mortality in a cohort of 58 950 inpatients (9.5% mortality for schist. >1%, vs 4.7% for schist; <0.5%; P < .001) after controlling for comorbidities, demographics, clinical morphology grading, and blood count indices. RBC-diff also enabled the estimation of single-cell volume-morphology distributions, providing insight into the influence of morphology on routine blood count measures. Our codebase and expert-annotated images are included here to spur further advancement. These results illustrate that computer vision can enable rapid and accurate quantitation of RBC morphology, which may provide value in both clinical and research contexts.

Navigating the Heterogeneity of Follicular Lymphoma and its Many Variants: An Updated Approach to Diagnosis and Classification.

Follicular lymphoma (FL) is a lymphoid neoplasm composed of follicle center (germinal center) B cells, with varying proportions of centrocytes and centroblasts, that usually has a predominantly follicular architectural pattern. Over the past decade, our understanding of FL has evolved significantly, with new recognition of several recently defined FL variants characterized by distinct clinical presentations, behaviors, genetic alterations, and biology. This manuscript aims to review the heterogeneity of FL and its variants, to provide an updated guide on their diagnosis and classification, and to describe how approaches to the histologic subclassification of classic FL have evolved in current classification schemes.

Disease-Defining Molecular Features of Primary Cutaneous B-Cell Lymphomas: Implications for Classification and Treatment.

Primary cutaneous B-cell lymphoma-primary cutaneous follicle center lymphoma; primary cutaneous marginal zone lymphoma; and primary cutaneous diffuse large B-cell, leg type-is a heterogeneous group with a variety of clinical and histological presentations. Until recently, the molecular bases of these disease subtypes have been unclear. We and others have identified the specific genetic characteristics that distinguish these subtypes from their respective systemic counterparts. These molecular features can improve diagnoses, determine the likelihood of concurrent or future systemic disease, and enable the rational design of novel clinical trials.

The 5th edition of the World Health Organization Classification of Haematolymphoid Tumours: Lymphoid Neoplasms.

We herein present an overview of the upcoming 5th edition of the World Health Organization Classification of Haematolymphoid Tumours focussing on lymphoid neoplasms. Myeloid and histiocytic neoplasms will be presented in a separate accompanying article. Besides listing the entities of the classification, we highlight and explain changes from the revised 4th edition. These include reorganization of entities by a hierarchical system as is adopted throughout the 5th edition of the WHO classification of tumours of all organ systems, modification of nomenclature for some entities, revision of diagnostic criteria or subtypes, deletion of certain entities, and introduction of new entities, as well as inclusion of tumour-like lesions, mesenchymal lesions specific to lymph node and spleen, and germline predisposition syndromes associated with the lymphoid neoplasms.

Sex-Biased ZRSR2 Mutations in Myeloid Malignancies Impair Plasmacytoid Dendritic Cell Activation and Apoptosis.

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is an aggressive leukemia of plasmacytoid dendritic cells (pDC). BPDCN occurs at least three times more frequently in men than in women, but the reasons for this sex bias are unknown. Here, studying genomics of primary BPDCN and modeling disease-associated mutations, we link acquired alterations in RNA splicing to abnormal pDC development and inflammatory response through Toll-like receptors. Loss-of-function mutations in ZRSR2, an X chromosome gene encoding a splicing factor, are enriched in BPDCN, and nearly all mutations occur in males. ZRSR2 mutation impairs pDC activation and apoptosis after inflammatory stimuli, associated with intron retention and inability to upregulate the transcription factor IRF7. In vivo, BPDCN-associated mutations promote pDC expansion and signatures of decreased activation. These data support a model in which male-biased mutations in hematopoietic progenitors alter pDC function and confer protection from apoptosis, which may impair immunity and predispose to leukemic transformation. SIGNIFICANCE: Sex bias in cancer is well recognized, but the underlying mechanisms are incompletely defined. We connect X chromosome mutations in ZRSR2 to an extremely male-predominant leukemia. Aberrant RNA splicing induced by ZRSR2 mutation impairs dendritic cell inflammatory signaling, interferon production, and apoptosis, revealing a sex- and lineage-related tumor suppressor pathway.This article is highlighted in the In This Issue feature, p. 275.

The molecular ontogeny of follicular lymphoma: gene mutations succeeding the BCL2 translocation define common precursor cells.

Relapsed follicular lymphoma (FL) can arise from common progenitor cells (CPCs). Conceptually, CPC-defining mutations are somatic alterations shared by the initial and relapsed tumours, mostly B-cell leukaemia/lymphoma 2 (BCL2)/immunoglobulin heavy locus (IGH) translocations and other recurrent gene mutations. Through complementary approaches for highly sensitive mutation detection, we do not find CPC-defining mutations in highly purified BCL2/IGH-negative haematopoietic progenitor cells in clinical remission samples from three patients with relapsed FL. Instead, we find cells harbouring the same BCL2/IGH translocation but lacking CREB binding protein (CREBBP), lysine methyltransferase 2D (KMT2D) and other recurrent gene mutations. Thus, (i) the BCL2/IGH translocation can precede CPC-defining mutations in human FL, and (ii) BCL2/IGH-translocated cells can persist in clinical remission.

Integrated genomic analyses of cutaneous T-cell lymphomas reveal the molecular bases for disease heterogeneity.

Cutaneous T-cell lymphomas (CTCLs) are a clinically heterogeneous collection of lymphomas of the skin-homing T cell. To identify molecular drivers of disease phenotypes, we assembled representative samples of CTCLs from patients with diverse disease subtypes and stages. Via DNA/RNA-sequencing, immunophenotyping, and ex vivo functional assays, we identified the landscape of putative driver genes, elucidated genetic relationships between CTCLs across disease stages, and inferred molecular subtypes in patients with stage-matched leukemic disease. Collectively, our analysis identified 86 putative driver genes, including 19 genes not previously implicated in this disease. Two mutations have never been described in any cancer. Functionally, multiple mutations augment T-cell receptor-dependent proliferation, highlighting the importance of this pathway in lymphomagenesis. To identify putative genetic causes of disease heterogeneity, we examined the distribution of driver genes across clinical cohorts. There are broad similarities across disease stages. Many driver genes are shared by mycosis fungoides (MF) and Sezary syndrome (SS). However, there are significantly more structural variants in leukemic disease, leading to highly recurrent deletions of putative tumor suppressors that are uncommon in early-stage skin-centered MF. For example, TP53 is deleted in 7% and 87% of MF and SS, respectively. In both human and mouse samples, PD1 mutations drive aggressive behavior. PD1 wild-type lymphomas show features of T-cell exhaustion. PD1 deletions are sufficient to reverse the exhaustion phenotype, promote a FOXM1-driven transcriptional signature, and predict significantly worse survival. Collectively, our findings clarify CTCL genetics and provide novel insights into pathways that drive diverse disease phenotypes.

Histiocytic and Dendritic Cell Sarcomas of Hematopoietic Origin Share Targetable Genomic Alterations Distinct from Follicular Dendritic Cell Sarcoma.

BACKGROUND: Histiocytic and dendritic cell neoplasms are a diverse group of tumors arising from monocytic or dendritic cell lineage. Whereas the genomic features for Langerhans cell histiocytosis and Erdheim-Chester disease have been well described, other less common and often aggressive tumors in this broad category remain poorly characterized, and comparison studies across the World Health Organization diagnostic categories are lacking. METHODS: Tumor samples from a total of 102 patient cases within four major subtypes of malignant histiocytic and dendritic cell neoplasms, including 44 follicular dendritic cell sarcomas (FDCSs), 41 histiocytic sarcomas (HSs), 7 interdigitating dendritic cell sarcomas (IDCSs), and 10 Langerhans cell sarcomas (LCSs), underwent hybridization capture with analysis of up to 406 cancer-related genes. RESULTS: Among the entire cohort of 102 patients, CDKN2A mutations were most frequent across subtypes and made up 32% of cases, followed by TP53 mutations (22%). Mitogen-activated protein kinase (MAPK) pathway mutations were present and enriched among the malignant histiocytosis (M) group (HS, IDCS, and LCS) but absent in FDCS (72% vs. 0%; p < .0001). In contrast, NF-κB pathway mutations were frequent in FDCSs but rare in M group histiocytoses (61% vs. 12%; p < .0001). Tumor mutational burden was significantly higher in M group histiocytoses as compared with FDCSs (median 4.0/Mb vs. 2.4/Mb; p = .012). We also describe a pediatric patient with recurrent secondary histiocytic sarcoma treated with targeted therapy and interrogated by molecular analysis to identify mechanisms of therapeutic resistance. CONCLUSION: A total of 42 patient tumors (41%) harbored pathogenic mutations that were potentially targetable by approved and/or investigative therapies. Our findings highlight the potential value of molecular testing to enable precise tumor classification, identify candidate oncogenic drivers, and define personalized therapeutic options for patients with these aggressive tumors. IMPLICATIONS FOR PRACTICE: This study presents comprehensive genomic profiling results on 102 patient cases within four major subtypes of malignant histiocytic and dendritic cell neoplasms, including 44 follicular dendritic cell sarcomas (FDCSs), 41 histiocytic sarcomas (HSs), 7 interdigitating dendritic cell sarcomas (IDCSs), and 10 Langerhans cell sarcomas (LCSs). MAPK pathway mutations were present and enriched among the malignant histiocytosis (M) group (HS, IDCS, and LCS) but absent in FDCSs. In contrast, NF-κB pathway mutations were frequent in FDCSs but rare in M group histiocytosis. A total of 42 patient tumors (41%) harbored pathogenic mutations that were potentially targetable by approved and/or investigative therapies.

Clinicopathologic Features of Peripheral T-Cell Lymphoma in Sub-Saharan Africa.

OBJECTIVES: Peripheral T-cell lymphomas (PTCLs) are heterogeneous, clinically aggressive, and rare. Subtype distribution varies by geographic location; however, data from sub-Saharan Africa (SSA) are lacking. We sought to elucidate clinicopathologic features of PTCL in SSA. METHODS: We reviewed PTCL consultation cases from three SSA countries. PTCL subtype was determined per 2017 World Health Organization classification. Cases with sufficient material were evaluated by polymerase chain reaction for human T-cell leukemia virus type 1 (HTLV-1) and T-cell receptor γ (TCRG) rearrangement. RESULTS: Among 32 cases, median age was 45 years and male-to-female ratio was 1.7. Thirty (94%) of 32 cases required additional workup for subclassification. PTCL, not otherwise specified (PTCL-NOS) was the most common subtype (13/32, 41%), followed by PTCL with T-follicular helper phenotype (6/32, 19%) and systemic anaplastic large cell lymphoma (6/32, 19%). Four (16%) of 25 cases were Epstein-Barr virus positive (EBV+) (2/2 extranodal natural killer/T-cell lymphoma, 1/13 PTCL-NOS, and 1/4 angioimmunoblastic T-cell lymphoma with EBV+ immunoblasts). Two (15%) of 13 patients with PTCL-NOS were human immunodeficiency virus positive. No cases with evaluable DNA (0/15) were HTLV-1 positive, and 9 of 10 showed clonal TCRG rearrangements. CONCLUSIONS: In comparison to Western studies, PTCLs from SSA show similar subtype distribution and male predominance but a younger age at diagnosis. Appropriate diagnosis of PTCL requires extensive ancillary testing not readily available in low-income countries, including much of SSA.

Genomic landscape of cutaneous follicular lymphomas reveals 2 subgroups with clinically predictive molecular features.

Primary cutaneous follicle center lymphomas (PCFCLs) are indolent B-cell lymphomas that predominantly remain skin restricted and manageable with skin-directed therapy. Conversely, secondary cutaneous involvement by usual systemic follicular lymphoma (secondary cutaneous follicular lymphoma [SCFL]) has a worse prognosis and often necessitates systemic therapy. Unfortunately, no histopathologic or genetic features reliably differentiate PCFCL from SCFL at diagnosis. Imaging may miss low-burden internal disease in some cases of SCFLs, leading to misclassification as PCFCL. Whereas usual systemic FL is well characterized genetically, the genomic landscapes of PCFCL and SCFL are unknown. Herein, we analyzed clinicopathologic and immunophenotypic data from 30 cases of PCFCL and 10 of SCFL and performed whole-exome sequencing on 18 specimens of PCFCL and 6 of SCFL. During a median follow-up of 7 years, 26 (87%) of the PCFCLs remained skin restricted. In the remaining 4 cases, systemic disease developed within 3 years of diagnosis. Although the SCFLs universally expressed BCL2 and had BCL2 rearrangements, 73% of the PCFCLs lacked BCL2 expression, and only 8% of skin-restricted PCFCLs had BCL2 rearrangements. SCFLs showed low proliferation fractions, whereas 75% of PCFCLs had proliferation fractions >30%. Of the SCFLs, 67% had characteristic loss-of-function CREBBP or KMT2D mutations vs none in skin-restricted PCFCL. Both SCFL and skin-restricted PCFCL showed frequent TNFRSF14 loss-of-function mutations and copy number loss at chromosome 1p36. These data together establish PCFCL as a unique entity with biological features distinct from usual systemic FL and SCFL. We propose 3 criteria based on BCL2 rearrangement, chromatin-modifying gene mutations (CREBBP, KMT2D, EZH2, and EP300), and proliferation index to classify cutaneous FL specimens based on the likelihood of concurrent or future systemic spread.

Simultaneous Identification of Cell of Origin, Translocations, and Hotspot Mutations in Diffuse Large B-Cell Lymphoma Using a Single RNA-Sequencing Assay.

OBJECTIVES: Diffuse large B-cell lymphoma (DLBCL) is an aggressive non-Hodgkin lymphoma with a heterogenous genetic landscape that can require multiple assays to characterize. We reviewed a 1-step RNA-based assay to determine cell of origin (COO), detect translocations, and identify mutations and to assess the role of the assay in diagnosis. METHODS: Using a single custom Archer FusionPlex Lymphoma panel, we performed anchored multiplex polymerase chain reaction-based RNA sequencing on 41 cases of de novo DLBCL. Each case was subclassified by COO, and gene fusions and hotspot mutations were identified. The findings were then compared with COO classification by the Hans immunohistochemical algorithm and NanoString technology, cytogenetics, and fluorescence in situ hybridization results. RESULTS: Concordant COO classification by the FusionPlex panel and NanoString was observed in 35 of 41 cases (85.3%), with NanoString and Hans concordant in 33 of 41 cases (80.5%) and FusionPlex and Hans concordant in 33 of 41 cases (80.5%). The FusionPlex assay also detected 6 of 11 BCL6 translocations (4 cryptic), 2 of 3 BCL2 translocations, and 2 of 4 MYC translocations. Mutations were detected in lymphoma-related genes in 24 of 41 cases. CONCLUSION: This FusionPlex assay offers a single method for COO classification, mutation detection, and identification of important translocations in DLBCL. Although not replacing traditional testing, it could offer useful data when limited tissue is available.

Reduced Mitochondrial Apoptotic Priming Drives Resistance to BH3 Mimetics in Acute Myeloid Leukemia.

Acquired resistance to BH3 mimetic antagonists of BCL-2 and MCL-1 is an important clinical problem. Using acute myelogenous leukemia (AML) patient-derived xenograft (PDX) models of acquired resistance to BCL-2 (venetoclax) and MCL-1 (S63845) antagonists, we identify common principles of resistance and persistent vulnerabilities to overcome resistance. BH3 mimetic resistance is characterized by decreased mitochondrial apoptotic priming as measured by BH3 profiling, both in PDX models and human clinical samples, due to alterations in BCL-2 family proteins that vary among cases, but not to acquired mutations in leukemia genes. BCL-2 inhibition drives sequestered pro-apoptotic proteins to MCL-1 and vice versa, explaining why in vivo combinations of BCL-2 and MCL-1 antagonists are more effective when concurrent rather than sequential. Finally, drug-induced mitochondrial priming measured by dynamic BH3 profiling (DBP) identifies drugs that are persistently active in BH3 mimetic-resistant myeloblasts, including FLT-3 inhibitors and SMAC mimetics.

Pan-Cancer Landscape Analysis Reveals Recurrent KMT2A-MAML2 Gene Fusion in Aggressive Histologic Subtypes of Thymoma.

PURPOSE: Thymomas are epithelial neoplasms that represent the most common thymic tumors in adults. These tumors have been shown to harbor a relatively low mutational burden. As a result, there is a lack of genetic alterations that may be used prognostically or targeted therapeutically for this disease. Here, we describe a recurrent gene rearrangement in type B2 + B3 thymomas. PATIENTS AND METHODS: A single index case of thymoma was evaluated by an RNA-based solid fusion assay. Separately, tissues from 255,008 unique advanced cancers, including 242 thymomas, were sequenced by hybrid capture-based next-generation DNA sequencing/comprehensive genomic profiling of 186 to 406 genes, including lysine methyltransferase 2A (KMT2A) rearrangements, and a portion were evaluated for RNA of 265 genes. We characterized molecular and clinicopathologic features of the pertinent fusion-positive patient cases. RESULTS: We identified 11 patients with thymomas harboring a gene fusion of KMT2A and mastermind-like transcriptional coactivator 2 (MAML2). Fusion breakpoints were identified between exon 8, 9, 10, or 11 of KMT2A and exon 2 of MAML2. Fifty-five percent were men, with a median age of 48 years at surgery (range, 29-69 years). Concurrent genomic alterations were infrequent. The 11 thymomas were of B2 or B3 type histology, with 1 case showing foci of thymic carcinoma. The frequency of KMT2A-MAML2 fusion was 4% of all thymomas (10 of 242) and 6% of thymomas of B2 or B3 histology (10 of 169). CONCLUSION: KMT2A-MAML2 represents the first recurrent fusion described in type B thymoma. The fusion seems to be specific to type B2 and B3 thymomas, the most aggressive histologic subtypes. The identification of this fusion offers insights into the biology of thymoma and may have clinical relevance for patients with disease refractory to conventional therapeutic modalities.