Pediatric large B-cell lymphomas (LBCLs) share morphological and phenotypic features with adult types but have better prognosis. The higher frequency of some subtypes such as LBCL with IRF4 rearrangement (LBCL-IRF4) in children suggests that some age-related biological differences may exist. To characterize the genetic and molecular heterogeneity of these tumors, we studied 31 diffuse LBCLs (DLBCLs), not otherwise specified (NOS); 20 LBCL-IRF4 cases; and 12 cases of high-grade B-cell lymphoma (HGBCL), NOS in patients ≤25 years using an integrated approach, including targeted gene sequencing, copy-number arrays, and gene expression profiling. Each subgroup displayed different molecular profiles. LBCL-IRF4 had frequent mutations in IRF4 and NF-κB pathway genes (CARD11, CD79B, and MYD88), losses of 17p13 and gains of chromosome 7, 11q12.3-q25, whereas DLBCL, NOS was predominantly of germinal center B-cell (GCB) subtype and carried gene mutations similar to the adult counterpart (eg, SOCS1 and KMT2D), gains of 2p16/REL, and losses of 19p13/CD70. A subset of HGBCL, NOS displayed recurrent alterations of Burkitt lymphoma–related genes such as MYC, ID3, and DDX3X and homozygous deletions of 9p21/CDKN2A, whereas other cases were genetically closer to GCB DLBCL. Factors related to unfavorable outcome were age >18 years; activated B-cell (ABC) DLBCL profile, HGBCL, NOS, high genetic complexity, 1q21-q44 gains, 2p16/REL gains/amplifications, 19p13/CD70 homozygous deletions, and TP53 and MYC mutations. In conclusion, these findings further unravel the molecular heterogeneity of pediatric and young adult LBCL, improve the classification of this group of tumors, and provide new parameters for risk stratification.

Diffuse large B-cell lymphoma (DLBCL) with aberrant coexpression of CD10+BCL6+MUM1+ (DLBCL-AE), classified as germinal center B cell (GCB) type by the Hans algorithm (HA), was genetically characterized. To capture the complexity of DLBCL-AE, we used an integrated approach that included gene expression profiling (GEP), fluorescence in situ hybridization, targeted gene sequencing, and copy number (CN) arrays. According to GEP, 32/54 (59%) cases were classified as GCB-DLBCL, 16/54 (30%) as activated B-cell (ABC) DLBCL, and 6/54 (11%) as unclassifiable. The discrepancy between HA and GEP was 41%. Three genetic subgroups were identified. Group 1 included 13/50 (26%) cases without translocations and mainly showing and ABC/MCD molecular profile. Group 2 comprised 11/50 (22%) cases with IRF4 alterations (DLBCL-IRF4), frequent mutations in IRF4 (82%) and NF-κB pathway genes (MYD88, CARD11, and CD79B), and losses of 17p13.2. Five cases each were classified as GCB- or ABC-type. Group 3 included 26/50 (52%) cases with 1 or several translocations in BCL2/BCL6/MYC/IGH, and GCB/EZB molecular profile predominated. Two cases in this latter group showed complex BCL2/BCL6/IRF4 translocations. DLBCL-IRF4 in adults showed a similar copy number profile and shared recurrent CARD11 and CD79B mutations when compared with LBCL-IRF4 in the pediatric population. However, adult cases showed higher genetic complexity, higher mutational load with frequent MYD88 and KMT2D mutations, and more ABC GEP. IRF4 mutations were identified only in IRF4-rearranged cases, indicating its potential use in the diagnostic setting. In conclusion, DLBCL-AE is genetically heterogeneous and enriched in cases with IRF4 alterations. DLBCL-IRF4 in adults has many similarities to the pediatric counterpart.

Abstract Primary mediastinal large B-cell lymphoma (PMBCL) is recognized as a distinct entity in the World Health Organization classification. Currently, the diagnosis relies on consensus of histopathology, clinical variables, and presentation, giving rise to diagnostic inaccuracy in routine practice. Previous studies have demonstrated that PMBCL can be distinguished from subtypes of diffuse large B-cell lymphoma (DLBCL) based on gene expression signatures. However, requirement of fresh-frozen biopsy material has precluded the transfer of gene expression–based assays to the clinic. Here, we developed a robust and accurate molecular classification assay (Lymph3Cx) for the distinction of PMBCL from DLBCL subtypes based on gene expression measurements in formalin-fixed, paraffin-embedded tissue. A probabilistic model accounting for classification error, comprising 58 gene features, was trained on 68 cases of PMBCL and DLBCL. Performance of the model was subsequently evaluated in an independent validation cohort of 158 cases and showed high agreement of the Lymph3Cx molecular classification with the clinicopathological diagnosis of an expert panel (frank misclassification rate, 3.8%). Furthermore, we demonstrate reproducibility of the assay with 100% concordance of subtype assignments at 2 independent laboratories. Future studies will determine Lymph3Cx’s utility for routine diagnostic purposes and therapeutic decision making.

Diffuse large B cell lymphomas (DLBCL) represent a clinically heterogeneous group of lymphomas that are classified together based on similarities in morphology and immunophenotype. Gene expression profiling further classifies DLBCL into distinct molecular subgroups based on cell-of-origin (COO), including germinal center B cell type, activated B cell type, and unclassified type. COO assignment of DLBCL has important biological and prognostic significance, as well as emerging therapeutic implications. Herein, we describe the first clinical validation of a digital gene expression-profiling assay (Lymph2Cx) to perform COO assignment in the routine work-up of DLBCL using formalin-fixed paraffin-embedded (FFPE) tissue sections and describe the results of 90 consecutive DLBCL cases analyzed prospectively by a College of American Pathologists/Clinical Laboratory Improvement Amendments (CAP/CLIA)-certified clinical molecular diagnostics laboratory.

Diffuse large B‐cell lymphoma (DLBCL) is up to 17‐fold more likely to occur, follows a more aggressive clinical course and frequently presents at advanced stages in HIV infected (+) individuals compared to HIV negative (−) individuals. However, the molecular pathology underpinning the clinical features of DLBCL in HIV(+) patients relative to the general population is poorly understood. We performed a retrospective study examining the transcriptional, genomic and protein expression differences between HIV(+) and HIV(−) germinal center B‐cell (GCB) DLBCL cases using digital gene expression analysis, array comparative genomic hybridization (CGH) and immunohistochemistry (IHC). Genes associated with cell cycle progression (CCNA2, CCNB1, CDC25A, E2F1), DNA replication (MCM2, MCM4, MCM7) and DNA damage repair, including eight Fanconi anemia genes (FANCA, FANCD1/BRCA2, FANCE, FANCG, FANCR/RAD51, FANCS/BRCA1, FANCT/UBE2T, FANCV/MAD2L2), were significantly increased in HIV(+) GCB‐DLBCL tumors compared to HIV(−) tumors. In contrast, genes associated with cell cycle inhibition (CDKN1A, CDKN1B) as well as apoptosis regulating BCL2 family members (BCL2, BAX, BIM, BMF, PUMA) were significantly decreased in the HIV(+) cohort. BCL2 IHC confirmed this expression. Array CGH data revealed that HIV(+) GCB‐DLBCL tumors have fewer copy number variations than their HIV(−) counterparts, indicating enhanced genomic stability. Together, the results show that HIV(+) GCB‐DLBCL is a distinct molecular malignancy from HIV(−) GCB‐DLBCL; with an increased proliferative capacity, confirmed by Ki67 IHC staining, and enhanced genomic stability, the latter of which is likely related to the enhanced expression of DNA repair genes.