Leukemic presentation and progressive genomic alterations of MCD/C5 diffuse large B-cell lymphoma (DLBCL).

Diffuse large B-cell lymphoma (DLBCL) is a heterogenous group of lymphoid malignancies. Based on gene expression profiling, it has been subdivided into germinal center (GC)-derived and activated B-cell (ABC) types. Advances in molecular methodologies have further refined the subclassification of DLBCL, based on recurrent genetic abnormalities. Here, we describe a distinct case of DLBCL that presented in leukemic form. DNA sequencing targeting 275 genes revealed pathogenically relevant mutations of CD79B, MyD88, TP53, TBL1XR1, and PIM1 genes, indicating that this lymphoma would be best classified as MCD/C5 DLBCL, an ABC subtype. Despite an initial good clinical response to BTK inhibitor ibrutinib, anti-CD20 antibody rituxan, alkylating agent bendamustine, and hematopoietic stem-cell transplant, the lymphoma relapsed, accompanied by morphologic and molecular evidence of disease progression. Specifically, the recurrent tumor developed loss of TP53 heterozygosity (LOH) and additional chromosomal changes central to ABC DLBCL pathogenesis, such as PRDM1 loss. Acquired resistance to ibrutinib and rituxan was indicated by the emergence of BTK and FOXO1 mutations, respectively, as well as apparent activation of alternative cell-activation pathways, through copy-number alterations (CNAs), detected by high-resolution chromosomal microarrays. In vitro, studies of relapsed lymphoma cells confirmed resistance to standard BTK inhibitors but sensitivity to vecabrutinib, a noncovalent inhibitor active against both wild-type as well as mutated BTK. In summary, we provide in-depth molecular characterization of a de novo leukemic DLBCL and discuss mechanisms that may have contributed to the lymphoma establishment, progression, and development of drug resistance.

Progression of follicular lymphoma and related entities: Report from the 2021 SH/EAHP Workshop.

OBJECTIVES: The 2021 Society for Hematopathology and European Association for Haematopathology Workshop addressed the molecular and cytogenetic underpinnings of transformation and transdifferentiation in lymphoid neoplasms. METHODS: Session 4, "Transformations of Follicular Lymphoma," and session 5, "Transformations of Other B-Cell Lymphomas," included 45 cases. Gene alteration analysis and expression profiling were performed on cases with submitted formalin-fixed, paraffin embedded tissue. RESULTS: The findings from session 4 suggest that "diffuse large B-cell lymphoma/high-grade B-cell lymphoma with rearrangements of MYC and BCL2" is a distinct category arising from the constraints of a preexisting BCL2 translocation. TdT expression in aggressive B-cell lymphomas is associated with MYC rearrangements, immunophenotypic immaturity, and a dismal prognosis but must be differentiated from lymphoblastic -lymphoma. Cases in session 5 illustrated unusual morphologic and immunophenotypic patterns of transformation. Additionally, the findings support the role of cytogenetic abnormalities-specifically, MYC and NOTCH1 rearrangements-as well as single gene alterations, including TP53, in transformation. CONCLUSIONS: Together, these unique cases and their accompanying molecular and cytogenetic data suggest potential mechanisms for and unusual patterns of transformation in B-cell lymphomas and indicate numerous opportunities for further study.

Progression and transformation of chronic lymphocytic leukemia/small lymphocytic lymphoma and B-cell prolymphocytic leukemia: Report from the 2021 SH/EAHP Workshop.

OBJECTIVES: Session 3 of the 2021 Workshop of the Society for Hematopathology/European Association for Haematopathology examined progression and transformation of chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) and B-cell prolymphocytic leukemia (B-PLL). METHODS: Thirty-one cases were reviewed by the panel. Additional studies such as immunohistochemistry and molecular genetic testing, including whole-exome sequencing and expression profiling, were performed in select cases. RESULTS: Session 3 included 27 CLL/SLL cases and miscellaneous associated proliferations, 3 cases of B-PLL, and 1 case of small B-cell lymphoma. The criteria for -accelerated CLL/SLL are established for lymph nodes, but extranodal disease can be diagnostically challenging. Richter transformation (RT) is a broad term and includes true transformation from original CLL/SLL clone(s) and clonally unrelated neoplasms. The morphologic, immunophenotypic, and genetic spectrum is diverse with classical and highly unusual examples. T-cell proliferations can also be encountered in CLL/SLL. B-cell prolymphocytic leukemia is a rare, diagnostically challenging disease due to its overlaps with other lymphoid neoplasms. CONCLUSIONS: The workshop highlighted complexity of progression and transformation in CLL/SLL and B-PLL, as well as diagnostic caveats accompanying heterogeneous presentations of RT and other manifestations of disease progression. Molecular genetic studies are pivotal for diagnosis and determination of clonal relationship, and to predict response to treatment and identify resistance to targeted therapy.

Progression of Hodgkin lymphoma and plasma cell neoplasms: Report from the 2021 SH/EAHP Workshop.

OBJECTIVES: To summarize cases submitted to the 2021 Society for Hematopathology/European Association for Haematopathology Workshop under the categories of progression of Hodgkin lymphoma, plasmablastic myeloma, and plasma cell myeloma. METHODS: The workshop panel reviewed 20 cases covered in this session. In addition, whole-exome sequencing (WES) and whole-genome RNA expression analysis were performed on 10 submitted cases, including 6 Hodgkin lymphoma and 4 plasma neoplasm cases. RESULTS: The cases of Hodgkin lymphoma included transformed cases to or from various types of B-cell lymphoma with 1 exception, which had T-cell differentiation. The cases of plasma cell neoplasms included cases with plasmablastic progression, progression to plasma cell leukemia, and secondary B-lymphoblastic leukemia. Gene variants identified by WES included some known to be recurrent in Hodgkin lymphoma and plasma cell neoplasm. All submitted Hodgkin lymphoma samples showed 1 or more of these mutations: SOCS1, FGFR2, KMT2D, RIT1, SPEN, STAT6, TET2, TNFAIP3, and ZNF217. CONCLUSIONS: Better molecular characterization of both of these neoplasms and mechanisms of progression will help us to better understand mechanisms of progression and perhaps develop better prognostic models, as well as identifying novel therapeutic targets.

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.

SETD2 Haploinsufficiency Enhances Germinal Center-Associated AICDA Somatic Hypermutation to Drive B-cell Lymphomagenesis.

SETD2 is the sole histone methyltransferase responsible for H3K36me3, with roles in splicing, transcription initiation, and DNA damage response. Homozygous disruption of SETD2 yields a tumor suppressor effect in various cancers. However, SETD2 mutation is typically heterozygous in diffuse large B-cell lymphomas. Here we show that heterozygous Setd2 deficiency results in germinal center (GC) hyperplasia and increased competitive fitness, with reduced DNA damage checkpoint activity and apoptosis, resulting in accelerated lymphomagenesis. Impaired DNA damage sensing in Setd2-haploinsufficient germinal center B (GCB) and lymphoma cells associated with increased AICDA-induced somatic hypermutation, complex structural variants, and increased translocations including those activating MYC. DNA damage was selectively increased on the nontemplate strand, and H3K36me3 loss was associated with greater RNAPII processivity and mutational burden, suggesting that SETD2-mediated H3K36me3 is required for proper sensing of cytosine deamination. Hence, Setd2 haploinsufficiency delineates a novel GCB context-specific oncogenic pathway involving defective epigenetic surveillance of AICDA-mediated effects on transcribed genes. SIGNIFICANCE: Our findings define a B cell-specific oncogenic effect of SETD2 heterozygous mutation, which unleashes AICDA mutagenesis of nontemplate strand DNA in the GC reaction, resulting in lymphomas with heavy mutational burden. GC-derived lymphomas did not tolerate SETD2 homozygous deletion, pointing to a novel context-specific therapeutic vulnerability. This article is highlighted in the In This Issue feature, p. 1599.

Oncogenic Integration of Nucleotide Metabolism via Fatty Acid Synthase in Non-Hodgkin Lymphoma.

Metabolic dysfunctions enabling increased nucleotide biosynthesis are necessary for supporting malignant proliferation. Our investigations indicate that upregulation of fatty acid synthase (FASN) and de novo lipogenesis, commonly observed in many cancers, are associated with nucleotide metabolic dysfunction in lymphoma. The results from our experiments showed that ribonucleotide and deoxyribonucleotide pool depletion, suppression of global RNA/DNA synthesis, and cell cycle inhibition occurred in the presence of FASN inhibition. Subsequently, we observed that FASN inhibition caused metabolic blockade in the rate-limiting step of the oxidative branch of the pentose phosphate pathway (oxPPP) catalyzed by phosphogluconate dehydrogenase (PGDH). Furthermore, we determined that FASN inhibitor treatment resulted in NADPH accumulation and inhibition of PGDH enzyme activity. NADPH is a cofactor utilized by FASN, also a known allosteric inhibitor of PGDH. Through cell-free enzyme assays consisting of FASN and PGDH, we delineated that the PGDH-catalyzed ribulose-5-phosphate synthesis is enhanced in the presence of FASN and is suppressed by increasing concentrations of NADPH. Additionally, we observed that FASN and PGDH were colocalized in the cytosol. The results from these experiments led us to conclude that NADP-NADPH turnover and the reciprocal stimulation of FASN and PGDH catalysis are involved in promoting oxPPP and nucleotide biosynthesis in lymphoma. Finally, a transcriptomic analysis of non-Hodgkin's lymphoma (n = 624) revealed the increased expression of genes associated with metabolic functions interlinked with oxPPP, while the expression of genes participating in oxPPP remained unaltered. Together we conclude that FASN-PGDH enzymatic interactions are involved in enabling oxPPP and nucleotide metabolic dysfunction in lymphoma tumors.

Monocarboxylate transporter antagonism reveals metabolic vulnerabilities of viral-driven lymphomas.

Epstein-Barr virus (EBV) is a ubiquitous herpesvirus that typically causes asymptomatic infection but can promote B lymphoid tumors in the immune suppressed. In vitro, EBV infection of primary B cells stimulates glycolysis during immortalization into lymphoblastoid cell lines (LCLs). Lactate export during glycolysis is crucial for continued proliferation of many cancer cells-part of a phenomenon known as the "Warburg effect"- and is mediated by monocarboxylate transporters (MCTs). However, the role of MCTs has yet to be studied in EBV-associated malignancies, which display Warburg-like metabolism in vitro. Here, we show that EBV infection of B lymphocytes directly promotes temporal induction of MCT1 and MCT4 through the viral proteins EBNA2 and LMP1, respectively. Functionally, MCT1 was required for early B cell proliferation, and MCT4 up-regulation promoted acquired resistance to MCT1 antagonism in LCLs. However, dual MCT1/4 inhibition led to LCL growth arrest and lactate buildup. Metabolic profiling in LCLs revealed significantly reduced oxygen consumption rates (OCRs) and NAD+/NADH ratios, contrary to previous observations of increased OCR and unaltered NAD+/NADH ratios in MCT1/4-inhibited cancer cells. Furthermore, U-13C6-glucose labeling of MCT1/4-inhibited LCLs revealed depleted glutathione pools that correlated with elevated reactive oxygen species. Finally, we found that dual MCT1/4 inhibition also sensitized LCLs to killing by the electron transport chain complex I inhibitors phenformin and metformin. These findings were extended to viral lymphomas associated with EBV and the related gammaherpesvirus KSHV, pointing at a therapeutic approach for targeting both viral lymphomas.

Non-Hodgkin Lymphomas: Malignancies Arising from Mature B Cells.

Non-Hodgkin lymphomas (NHLs) are a diverse group of entities, both clinically and molecularly. Here, we review the evolution of classification schemes in B-cell lymphoma, noting the now standard WHO classification system that is based on immune cell-of-origin and molecular phenotypes. We review how lymphomas arise throughout the B-cell development process as well as the molecular and clinical features of prominent B-cell lymphomas. We provide an overview of the major progress that has occurred over the past decade in terms of our molecular understanding of these diseases. We discuss treatment options available and focus on a number of the diverse research tools that have been employed to improve our understanding of these diseases. We discuss the problem of heterogeneity in lymphomas and anticipate that the near future will bring significant advances that provide a measurable impact on NHL outcomes.

Genome-defined African ancestry is associated with distinct mutations and worse survival in patients with diffuse large B-cell lymphoma.

BACKGROUND: Significant racial differences have been observed in the incidence and clinical outcomes of diffuse large B-cell lymphoma (DLBCL) in the United States, but to the authors' knowledge it remains unclear whether genomic differences contribute to these disparities. METHODS: To understand the influences of genetic ancestry on tumor genomic alterations, the authors estimated the genetic ancestry of 1001 previously described patients with DLBCL using unsupervised model-based Admixture global ancestry analysis applied to exome sequencing data and examined the mutational profile of 150 DLBCL driver genes in tumors obtained from this cohort. RESULTS: Global ancestry prediction identified 619 patients with >90% European ancestry, 81 patients with >90% African ancestry, and 50 patients with >90% Asian ancestry. Compared with patients with DLBCL with European ancestry, patients with African ancestry were aged >10 years younger at the time of diagnosis and were more likely to present with B symptoms, elevated serum lactate dehydrogenase, extranodal disease, and advanced stage disease. Patients with African ancestry demonstrated worse overall survival compared with patients with European ancestry (median, 4.9 years vs 8.8 years; P = .04). Recurrent mutations of MLL2 (KMT2D), HIST1H1E, MYD88, BCL2, and PIM1 were found across all ancestry groups, suggesting shared mechanisms underlying tumor biology. The authors also identified 6 DLBCL driver genes that were more commonly mutated in patients with African ancestry compared with patients with European ancestry: ATM (21.0% vs 7.75%; P < .001), MGA (19.7% vs 5.33%; P < .001), SETD2 (17.3% vs 5.17%; P < .001), TET2 (12.3% vs 5.82%; P = .029), MLL3 (KMT2C) (11.1% vs 4.36%; P = .013), and DNMT3A (11.1% vs 4.52%; P = .016). CONCLUSIONS: Distinct prevalence and patterns of mutation highlight an important difference in the mutational landscapes of DLBCL arising in different ancestry groups. To the authors' knowledge, the results of the current study provide the first-ever characterization of genetic alterations among patients with African descent who are diagnosed with DLBCL.

Identifying transcriptional profiles and evaluating prognostic biomarkers of HIV-associated diffuse large B-cell lymphoma from Malawi.

Lymphoma incidence in sub-Saharan Africa (SSA) is increasing due to HIV and population aging. Diffuse Large B-cell lymphoma (DLBCL), the most common lymphoma in SSA and worldwide, is highly associated with HIV, but molecular studies of HIV-associated DLBCL are scarce globally. We describe profiling of DLBCL from Malawi, aiming to elucidate tumor biology and identify clinically meaningful biomarkers specifically for SSA. Between June 1, 2013 and June 1, 2016, 59 cases of DLBCL (32 HIV+/27 HIV-) enrolled in the Kamuzu Central Hospital Lymphoma Study were characterized, of which 54 (92%) were negative for Epstein-Barr virus. Gene expression profiling (GEP) by whole transcriptome sequencing was performed on the first 36 cases (22 HIV+/14 HIV-). Immunohistochemistry (IHC) and GEP results were compared with published data and correlated to clinical outcome and pathologic features. Unsupervised clustering strongly segregated DLBCL by HIV status (p = 0.0003, Chi-squared test), indicating a marked contribution of HIV to expression phenotype. Pathway analysis identified that HIV-associated tumors were enriched in hypoxia, oxidative stress, and metabolism related gene expression patterns. Cell-of-origin subtype, determined by sequencing and IHC, did not associate with differences in overall survival (OS), while Ki-67 proliferation index ≥80% was associated with inferior OS in HIV+ DLBCL only (p = 0.03) and cMYC/BCL2 co-expression by IHC was negatively prognostic across the entire cohort (p = 0.01). This study provides among the first molecular characterizations of DLBCL from SSA, demonstrates marked gene expression differences by HIV status, and identifies genomic and immunophenotypic characteristics that can inform future basic and clinical investigations.

The whole-genome landscape of Burkitt lymphoma subtypes.

Burkitt lymphoma (BL) is an aggressive, MYC-driven lymphoma comprising 3 distinct clinical subtypes: sporadic BLs that occur worldwide, endemic BLs that occur predominantly in sub-Saharan Africa, and immunodeficiency-associated BLs that occur primarily in the setting of HIV. In this study, we comprehensively delineated the genomic basis of BL through whole-genome sequencing (WGS) of 101 tumors representing all 3 subtypes of BL to identify 72 driver genes. These data were additionally informed by CRISPR screens in BL cell lines to functionally annotate the role of oncogenic drivers. Nearly every driver gene was found to have both coding and non-coding mutations, highlighting the importance of WGS for identifying driver events. Our data implicate coding and non-coding mutations in IGLL5, BACH2, SIN3A, and DNMT1. Epstein-Barr virus (EBV) infection was associated with higher mutation load, with type 1 EBV showing a higher mutational burden than type 2 EBV. Although sporadic and immunodeficiency-associated BLs had similar genetic profiles, endemic BLs manifested more frequent mutations in BCL7A and BCL6 and fewer genetic alterations in DNMT1, SNTB2, and CTCF. Silencing mutations in ID3 were a common feature of all 3 subtypes of BL. In vitro, mass spectrometry-based proteomics demonstrated that the ID3 protein binds primarily to TCF3 and TCF4. In vivo knockout of ID3 potentiated the effects of MYC, leading to rapid tumorigenesis and tumor phenotypes consistent with those observed in the human disease.

TET2 Deficiency Sets the Stage for B-cell Lymphoma.

TET2 is a well-established tumor suppressor in the context of myeloid malignancies, but its role in lymphoma development has been less clear. In this issue of Cancer Discovery, Dominguez and colleagues report that TET2 function is critical for germinal center exit and plasma cell differentiation, and its deficiency can lead to B-cell lymphoma phenotypes.See related article by Dominguez et al., p. 1632.

Gene essentiality landscape and druggable oncogenic dependencies in herpesviral primary effusion lymphoma.

Primary effusion lymphoma (PEL) is caused by Kaposi's sarcoma-associated herpesvirus. Our understanding of PEL is poor and therefore treatment strategies are lacking. To address this need, we conducted genome-wide CRISPR/Cas9 knockout screens in eight PEL cell lines. Integration with data from unrelated cancers identifies 210 genes as PEL-specific oncogenic dependencies. Genetic requirements of PEL cell lines are largely independent of Epstein-Barr virus co-infection. Genes of the NF-κB pathway are individually non-essential. Instead, we demonstrate requirements for IRF4 and MDM2. PEL cell lines depend on cellular cyclin D2 and c-FLIP despite expression of viral homologs. Moreover, PEL cell lines are addicted to high levels of MCL1 expression, which are also evident in PEL tumors. Strong dependencies on cyclin D2 and MCL1 render PEL cell lines highly sensitive to palbociclib and S63845. In summary, this work comprehensively identifies genetic dependencies in PEL cell lines and identifies novel strategies for therapeutic intervention.

Plasmodium parasite exploits host aquaporin-3 during liver stage malaria infection.

Within the liver a single Plasmodium parasite transforms into thousands of blood-infective forms to cause malaria. Here, we use RNA-sequencing to identify host genes that are upregulated upon Plasmodium berghei infection of hepatocytes with the hypothesis that host pathways are hijacked to benefit parasite development. We found that expression of aquaporin-3 (AQP3), a water and glycerol channel, is significantly induced in Plasmodium-infected hepatocytes compared to uninfected cells. This aquaglyceroporin localizes to the parasitophorous vacuole membrane, the compartmental interface between the host and pathogen, with a temporal pattern that correlates with the parasite's expansion in the liver. Depletion or elimination of host AQP3 expression significantly reduces P. berghei parasite burden during the liver stage and chemical disruption by a known AQP3 inhibitor, auphen, reduces P. falciparum asexual blood stage and P. berghei liver stage parasite load. Further use of this inhibitor as a chemical probe suggests that AQP3-mediated nutrient transport is an important function for parasite development. This study reveals a previously unknown potential route for host-dependent nutrient acquisition by Plasmodium which was discovered by mapping the transcriptional changes that occur in hepatocytes throughout P. berghei infection. The dataset reported may be leveraged to identify additional host factors that are essential for Plasmodium liver stage infection and highlights Plasmodium's dependence on host factors within hepatocytes.

Genetic convergence of rare lymphomas.

PURPOSE OF REVIEW: We review the genetic foundations of different rare lymphomas to examine their shared origins. These data indicate the potential application of genomics to improve the diagnosis and treatment of these rare diseases. RECENT FINDINGS: Next generation sequencing technologies have provided an important window into the genetic underpinnings of lymphomas. A growing body of evidence indicates that although some genetic alterations are specific to certain diseases, others are shared across different lymphomas. Many such genetic events have already demonstrated clinical utility, such as BRAF V600E that confers sensitivity to vemurafenib in patients with hairy cell leukemia. SUMMARY: The rareness of many lymphoma subtypes makes the conduct of clinical trials and recruitment of significant numbers of patients impractical. However, a knowledge of the shared genetic origins of these rare lymphomas has the potential to inform 'basket' clinical trials in which multiple lymphoma subtypes are included. These trials would include patients based on the presence of alterations in targetable driver genes. Such approaches would be greatly strengthened by a systematic assessment of significant patient numbers from each subtype using next generation sequencing.

Epstein-Barr Virus Induces Adhesion Receptor CD226 (DNAM-1) Expression during Primary B-Cell Transformation into Lymphoblastoid Cell Lines.

Epstein-Barr virus (EBV), an oncogenic herpesvirus, infects and transforms primary B cells into immortal lymphoblastoid cell lines (LCLs), providing a model for EBV-mediated tumorigenesis. EBV transformation stimulates robust homotypic aggregation, indicating that EBV induces molecules that mediate cell-cell adhesion. We report that EBV potently induced expression of the adhesion molecule CD226, which is not normally expressed on B cells. We found that early after infection of primary B cells, EBV promoted an increase in CD226 mRNA and protein expression. CD226 levels increased further from early proliferating EBV-positive B cells to LCLs. We found that CD226 expression on B cells was independent of B-cell activation as CpG DNA failed to induce CD226 to the extent of EBV infection. CD226 expression was high in EBV-infected B cells expressing the latency III growth program, but low in EBV-negative and EBV latency I-infected B-lymphoma cell lines. We validated this correlation by demonstrating that the latency III characteristic EBV NF-κB activator, latent membrane protein 1 (LMP1), was sufficient for CD226 upregulation and that CD226 was more highly expressed in lymphomas with increased NF-κB activity. Finally, we found that CD226 was not important for LCL steady-state growth, survival in response to apoptotic stress, homotypic aggregation, or adhesion to activated endothelial cells. These findings collectively suggest that EBV induces expression of a cell adhesion molecule on primary B cells that may play a role in the tumor microenvironment of EBV-associated B-cell malignancies or facilitate adhesion in the establishment of latency in vivo. IMPORTANCE Epstein-Barr virus (EBV) is a common human herpesvirus that establishes latency in B cells. While EBV infection is asymptomatic for most individuals, immune-suppressed individuals are at significantly higher risk of a form of EBV latent infection in which infected B cells are reactivated, grow unchecked, and generate lymphomas. This form of latency is modeled in the laboratory by infecting B cells from the blood of normal human donors in vitro. In this model, we identified a protein called CD226 that is induced by EBV but is not normally expressed on B cells. Rather, it is known to play a role in aggregation and survival signaling of non-B cells in the immune system. Cultures of EBV-infected cells adhere to one another in "clumps," and while the proteins that are responsible for this cellular aggregation are not fully understood, we hypothesized that this form of cellular aggregation may provide a survival advantage. In this article, we characterize the mechanism by which EBV induces this protein and its expression on lymphoma tissue and cell lines and characterize EBV-infected cell lines in which CD226 has been knocked out.