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Introduction Primary mediastinal large B cell lymphoma (PMBCL) is a rare non-Hodgkin lymphoma (NHL) with a female predominance; often presenting with a large anterior mediastinal mass. Though PMBCL has clinical and molecular features overlapping with Hodgkin lymphoma, it is a distinct entity defined by the World Health Organization classification. PMBCL is heterogeneously treated, and most patients receive front line therapy with either rituximab, cyclophosphamide, doxorubicin, vincristine, prednisone (R-CHOP) with radiotherapy (RT), or the more intensive etoposide, prednisone, vincristine, cyclophosphamide, doxorubicin with rituximab (EPOCH-R) regimen. Diagnosis of PMBCL is made using clinicopathologic criteria and radiographic imaging, however gene expression profiling (GEP) studies reveal a characteristic genotypic signature distinct from diffuse large B cell lymphoma (DLBCL). Molecular classification of PMBCL using the Lymph3Cx assay from formalin-fixed paraffin-embedded tissue (FFPE) is feasible, reproducible, and highly concordant in a training and validation cohort (Mottok et al. Blood 2018). Using a multicenter cohort of patients, we sought to estimate the rate of mis-match among patients with a clinical diagnosis of PMBCL using Lymph3Cx, and describe treatment selections and outcomes for each group. Methods Patients were identified from a cohort of patients with newly diagnosed NHL from the University of Iowa and Mayo Clinic Molecular Epidemiology Resource, and the Lymphoma Epidemiology of Outcomes cohort. Patients were enrolled between 2002-2019, and included if they had clinically defined PMBCL. FFPE was retrieved from hematopathology archives of participating academic centers. All diagnoses of PMBCL were based on expert hematopathology review at the time of therapy, and all cases underwent classification by GEP using the Lymph3Cx assay. Lymph3Cx was performed in the clinical lab at the Mayo Clinic in Arizona: Contiguous unstained sections were deparaffinized and macrodissected to enrich for tumor content before RNA isolation;100-200 ng of total RNA was used in an nCounter Elements XT, hybridized, and processed the following day using the nCounter FLEX system. Raw counts were processed through the Lymph3Cx algorithm and results reported as probability of PMBCL (≥0.90 as PMBCL, ≤0.10 as DLBCL all other results "Unclear PMBCL/DLBCL") (A. Mottok et al, Blood, 2018). For cases classified as DLBCL, the Lymph2Cx cell-of-origin classifier results was reported (Scott et al, JCO, 2016). Time to event endpoints were described with Kaplan-Meier plots by groups defined by mismatch status and compared with a logrank test. Binary outcomes will be presented with 90% exact confidence intervals. Results Fifty patients were identified. Median age was 35 years (range 19-70). Sixty four percent were women. Median follow up was 47 months. Treatments included R-CHOP (44%), EPOCH-R (44%), and MACOP-B [methotrexate with leucovorin rescue, doxorubicin, cyclophosphamide, vincristine, prednisone, and bleomycin] (6%), other (4%). Ten patients (20%) had events (defined as progression or death). Three patients in the entire cohort (6%) died. The Kaplan-Meier estimated survival at 47 months (median follow-up) is 92%. The Lymph3Cx assay yielded gene expression data of sufficient quality in 47/50 cases (94%, 90% CI=85.2, 98.3%). Of 47 cases clinically identified as PMBCL, 5 unclear were DLBCL/PMBCL and 1 was Germinal Center B cell subtype of DLBCL. Among these 6 patients, 4 received R-EPOCH (66%), 1 received R-CHOP (16.6%). One patient had missing treatment data. One patient had an event requiring subsequent therapy; all patients remain alive. Conclusions Using 47 patients with PMBCL defined by histology, clinical and radiographic findings, and molecular features, we demonstrate high concordance between clinical phenotype and molecular genotype of PMBCL by Lymph3Cx. Among the 6 patients not classified as PMBCL, most received R-EPOCH. Differences in outcome by mis-match status await longer follow-up and further accrual of subjects to our data base. Our data suggest molecular genotyping may have a role in mediastinal presentations of large cell lymphoma to optimize treatment decision making. Disclosures Maurer: Nanostring: Research Funding; Pfizer: Membership on an entity's Board of Directors or advisory committees; Celgene / BMS: Research Funding; Morphosys: Membership on an entity's Board of Directors or advisory committees; Kite: Membership on an entity's Board of Directors or advisory committees. Cerhan:BMS/Celgene: Research Funding; NanoString: Research Funding. Flowers:AbbVie: Consultancy, Research Funding; Kite: Research Funding; Burroughs Wellcome Fund: Research Funding; Genentech, Inc./F. Hoffmann-La Roche Ltd: Consultancy, Research Funding; Denovo Biopharma: Consultancy; Celgene: Consultancy, Research Funding; Cancer Prevention and Research Institute of Texas: Research Funding; TG Therapeutics: Research Funding; Eastern Cooperative Oncology Group: Research Funding; V Foundation: Research Funding; Bayer: Consultancy; National Cancer Institute: Research Funding; Millennium/Takeda: Consultancy, Research Funding; Gilead: Consultancy, Research Funding; Acerta: Research Funding; Spectrum: Consultancy; Pharmacyclics/Janssen: Consultancy; Karyopharm: Consultancy; OptumRx: Consultancy; Leukemia and Lymphoma Society: Membership on an entity's Board of Directors or advisory committees; BeiGene: Consultancy. Friedberg:Acerta Pharma - A member of the AstraZeneca Group, Bayer HealthCare Pharmaceuticals.: Other; Astellas: Consultancy; Bayer: Consultancy; Kite Pharmaceuticals: Research Funding; Portola Pharmaceuticals: Consultancy; Roche: Other: Travel expenses; Seattle Genetics: Research Funding.

AbstractObjectivesTo report the findings of the 2019 Society for Hematopathology/European Association for Haematopathology Workshop within the categories of reactive eosinophilia, hypereosinophilic syndrome (HES), germline disorders with eosinophilia (GDE), and myeloid and lymphoid neoplasms associated with eosinophilia (excluding entities covered by other studies in this series).MethodsThe workshop panel reviewed 109 cases, assigned consensus diagnosis, and created diagnosis-specific sessions.ResultsThe most frequent diagnosis was reactive eosinophilia (35), followed by acute leukemia (24). Myeloproliferative neoplasms (MPNs) received 17 submissions, including chronic eosinophilic leukemia, not otherwise specified (CEL, NOS). Myelodysplastic syndrome (MDS), MDS/MPN, and therapy-related myeloid neoplasms received 11, while GDE and HES received 12 and 11 submissions, respectively.ConclusionsHypereosinophilia and HES are defined by specific clinical and laboratory criteria. Eosinophilia is commonly reactive. An acute leukemic onset with eosinophilia may suggest core-binding factor acute myeloid leukemia, blast phase of chronic myeloid leukemia, BCR-ABL1–positive leukemia, or t(5;14) B-lymphoblastic leukemia. Eosinophilia is rare in MDS but common in MDS/MPN. CEL, NOS is a clinically aggressive MPN with eosinophilia as the dominant feature. Bone marrow morphology and cytogenetic and/or molecular clonality may distinguish CEL from HES. Molecular testing helps to better subclassify myeloid neoplasms with eosinophilia and to identify patients for targeted treatments.

Abstract Diffuse large B-cell lymphoma (DLBCL) is a molecularly heterogeneous group of malignancies with frequent genetic abnormalities. G-quadruplex (G4) DNA structures may facilitate this genomic instability through association with activation-induced cytidine deaminase (AID), an antibody diversification enzyme implicated in mutation of oncogenes in B-cell lymphomas. Chromatin immunoprecipitation sequencing analyses in this study revealed that AID hotspots in both activated B cells and lymphoma cells in vitro were highly enriched for G4 elements. A representative set of these targeted sequences was validated for characteristic, stable G4 structure formation including previously unknown G4s in lymphoma-associated genes, CBFA2T3, SPIB, BCL6, HLA-DRB5 and MEF2C, along with the established BCL2 and MYC structures. Frequent genome-wide G4 formation was also detected for the first time in DLBCL patient-derived tissues using BG4, a structure-specific G4 antibody. Tumors with greater staining were more likely to have concurrent BCL2 and MYC oncogene amplification and BCL2 mutations. Ninety-seven percent of the BCL2 mutations occurred within G4 sites that overlapped with AID binding. G4 localization at sites of mutation, and within aggressive DLBCL tumors harboring amplified BCL2 and MYC, supports a role for G4 structures in events that lead to a loss of genomic integrity, a critical step in B-cell lymphomagenesis.

Abstract Purpose: Mantle cell lymphoma (MCL) is characterized by the t(11;14)(q13;q32) translocation leading to cyclin D1 overexpression. Cyclin D1 is a major cell-cycle regulator and also regulates transcription, but the impact of cyclin D1–mediated transcriptional dysregulation on MCL pathogenesis remains poorly understood. The aim of this study was to define a cyclin D1–dependent gene expression program and analyze its prognostic value. Experimental Design: We integrated genome-wide expression analysis of cyclin D1–silenced and overexpressing cells with cyclin D1 chromatin-binding profiles to identify a cyclin D1–dependent transcriptional program in MCL cells. We analyzed this gene program in two MCL series of peripheral blood samples (n = 53) and lymphoid tissues (n = 106) to determine its biological and clinical relevance. We then obtained a simplified signature of this program and evaluated a third series of peripheral blood MCL samples (n = 81) by NanoString gene expression profiling to validate our findings. Results: We identified a cyclin D1–dependent transcriptional program composed of 295 genes that were mainly involved in cell-cycle control. The cyclin D1–dependent gene program was overexpressed in MCL tumors directly proportional to cyclin D1 levels. High expression of this program conferred an adverse prognosis with significant shorter overall survival of the patients. These observations were validated in an independent cohort of patients using a simplified 37-gene cyclin D1 signature. The cyclin D1–dependent transcriptional program was also present in multiple myeloma and breast tumors with cyclin D1 overexpression. Conclusions: We identified a cyclin D1–dependent transcriptional program that is overexpressed in MCL and predicts clinical outcome.

Abstract Objectives To summarize cases submitted to the 2019 Society for Hematopathology/European Association for Haematopathology Workshop under the category of myeloid/lymphoid neoplasms with eosinophilia and PDGFRA, PDGFRB, or FGFR1 or with PCM1-JAK2 rearrangements, focusing on recent updates and relevant practice findings. Methods The cases were summarized according to their respective gene rearrangement to illustrate the spectrum of clinical, laboratory, and histopathology manifestations and to explore the appropriate molecular genetic tests. Results Disease presentations were heterogeneous, including myeloproliferative neoplasms (MPNs), myelodysplastic syndromes (MDSs), MDS/MPN, acute myeloid leukemia, acute B- or T-lymphoblastic lymphoma/acute lymphoblastic lymphoma (ALL/LBL), or mixed-lineage neoplasms. Frequent extramedullary involvement occurred. Eosinophilia was common but not invariably present. With the advancement of RNA sequencing, cryptic rearrangements were recognized in genes other than PDGFRA. Additional somatic mutations were more frequent in the FGFR1-rearranged cases. Cases with B-ALL presentations differed from Philadelphia-like B-ALL by the presence of an underlying MPN. Cases with FLT3 and ABL1 rearrangements could be potential candidates for future inclusion in this category. Conclusions Accurate diagnosis and classification of this category of myeloid/lymphoid neoplasms has important therapeutic implications. With the large number of submitted cases, we expand our understanding of these rare neoplasms and improve our ability to diagnose these genetically defined disorders.

Abstract Objectives The 2019 Society for Hematopathology and European Association for Haematopathology Workshop reviewed the spectrum of neoplastic, nonneoplastic, and borderline entities associated with reactive eosinophilia in tissue. Methods The workshop panel reviewed 46 cases covered in 2 workshop sessions. Results The 46 cases were presented with their consensus diagnoses during the workshop. Reactive eosinophilia in lymph nodes and other tissues may be accompanied by or be distinct from peripheral blood eosinophilia. Reactive etiologies included inflammatory disorders such as Kimura disease and IgG4-related disease, which may show overlapping pathologic features and reactions to infectious agents and hypersensitivity (covered in a separate review). Hodgkin, T-cell, and B-cell lymphomas and histiocytic neoplasms can result in reactive eosinophilia. The spectrum of these diseases is discussed and illustrated through representative cases. Conclusions Reactive eosinophilia in lymph nodes and tissues may be related to both nonneoplastic and neoplastic lymphoid proliferations and histiocytic and nonhematolymphoid processes. Understanding the differential diagnosis of reactive eosinophilia and the potential for overlapping clinical and pathologic findings is critical in reaching the correct diagnosis so that patients can be treated appropriately.

Abstract Objectives The 2019 Workshop of the Society for Hematopathology/European Association for Haematopathology received and reviewed cases covering the spectrum of mastocytosis and related diseases, including morphologic mimics, focusing on recent updates and relevant findings for pathologists. Methods The workshop panel reviewed 99 cases of cutaneous and systemic mastocytosis (SM) and SM and associated hematologic neoplasms (SM-AHN). Results Despite a common theme of KIT mutation (particularly D816V), mastocytosis is a heterogeneous neoplasm with a wide variety of presentations. This spectrum, including rare subtypes and extramedullary organ involvement, is discussed and illustrated by representative cases. Conclusions In the age of targeted treatment aimed at KIT, the accurate diagnosis and classification of mastocytosis has major implications for therapy and further interventions. Understanding the clinical, pathologic, and genetic findings of mastocytosis is crucial for selecting the proper tests to perform and subsequent arrival at a correct diagnosis in this rare disease.

Objective: The increased risk for persons living with HIV to develop diffuse large B-cell lymphoma (DLBCL) even in the post-antiretroviral therapy eras suggests a role beyond immunosuppression in lymphoma development. However, the mechanisms leading to lymphoma in the HIV setting are not fully understood. HIV is known to induce activation-induced cytidine deaminase (AID) levels in nonneoplastic B cells in vitro and chronic AID expression may play an important role in lymphomagenesis. Although AID expression is observed in B-cell lymphoma, studies in HIV-associated DLBCL are limited. Design: In this study, we conducted a retrospective review of DLBCL tissues from patients with and without HIV infection to compare expression of AID and B-cell receptors potentially involved in HIV and B-cell interaction. Methods: We evaluated DLBCL formalin-fixed paraffin-embedded tissues from 72 HIV-seropositive and 58 HIV-seronegative patients for AID, DC-SIGN, and CD40 protein expression. BCL2 and MYC, two well established prognostically significant oncoproteins in DLBCL, were also assessed at the protein and mRNA levels. Subset analysis was performed according to DLBCL subtype and EBV status. Results: Of note, AID expression was more frequent in HIV-associated DLBCL compared with non-HIV-associated DLBCL regardless of cell-of-origin subtype, and also displayed significantly less BCL2 expression. Despite no direct correlation with AID expression, the HIV-DLBCL tissues also exhibited high levels of the DC-SIGN receptor. Conclusion: Collectively, these findings support a potential role for AID in the pathogenesis of HIV-associated lymphomas and suggest the need of further investigations into the involvement of the DC-SIGN receptor-signaling pathway.

PURPOSEDiffuse large B-cell lymphoma (DLBCL) presents as a limited-stage disease in 25% to 30% of patients, with better overall survival (OS) than that for advanced-stage disease but with continuous relapse regardless of treatment approach. The preferred treatment is abbreviated rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) and radiation therapy. On the basis of promising results of positron emission tomography (PET)–directed treatment approaches, we designed a National Clinical Trials Network (NCTN) study to improve outcomes and decrease toxicity.METHODSPatients with nonbulky (< 10 cm) stage I/II untreated DLBCL received 3 cycles of standard R-CHOP therapy and underwent a centrally reviewed interim PET/computed tomography scan (iPET). Those with a negative iPET proceeded with 1 additional cycle of R-CHOP, whereas those with a positive iPET received involved field radiation therapy followed by ibritumomab tiuxetan radioimmunotherapy.RESULTSOf 158 patients enrolled, 132 were eligible and 128 underwent iPET, which was positive in 14 (11%) of the patients. With a median follow-up of 4.92 years (range, 1.1-7.7 years), only 6 patients progressed and 3 died as a result of lymphoma. Eleven patients died as a result of nonlymphoma causes at a median age of 80 years. The 5-year progression-free survival estimate was 87% (95% CI, 79% to 92%) and the OS estimate was 89% (95% CI, 82% to 94%), with iPET-positive and iPET-negative patients having similar outcomes.CONCLUSIONTo our knowledge, S1001 is the largest prospective study in the United States of limited-stage DLBCL in the rituximab era, with the best NCTN results in this disease subset. With PET-directed therapy, 89% of the patients with a negative iPET received R-CHOP × 4, and only 11% had a positive iPET and required radiation, with both groups having excellent outcomes. The trial establishes R-CHOP × 4 alone as the new standard approach to limited-stage disease for the absolute majority of patients.