EZH2 expression is associated with inferior overall survival in mantle cell lymphoma.

Enhancer of zeste homolog 2 (EZH2) is a catalytic component of the polycomb repressive complex 2 (PRC2) which reduces gene expression via trimethylation of a lysine residue of histone 3 (H3K27me3). Expression of EZH2 has not been assessed systematically in mantle cell lymphoma (MCL). Expression of EZH2 was assessed by immunohistochemistry in 166 patients with MCL. We also assessed other PRC2 components and H3K27me3. Fifty-seven (38%) of MCL patients were positive for EZH2 using 40% cutoff. EZH2 expression was associated with aggressive histologic variants (65% vs. 29%, p < 0.001), high Ki-67 proliferation rate (median, 72% vs. 19%, p < 0.001), and p53 overexpression (43% vs. 2%, p < 0.001). EZH2 expression did not correlate with expression of other PRC2 components (EED and SUZ12), H3K27me3, MHC-I, and MHC-II. Patients with EZH2 expression (EZH2+) had a poorer overall survival (OS) compared with patients without EZH2 expression (EZH2-) (median OS: 3.9 years versus 9.4 years, respectively, p < 0.001). EZH2 expression also predicted a poorer prognosis in MCL patients with classic histology (median OS, 4.6 years for EZH2+ and 9.6 years for EZH2-negative, respectively, p < 0.001) as well as aggressive histology (median OS, 3.7 years for EZH2+ and 7.9 years for EZH2-negative, respectively, p = 0.046). However, EZH2 expression did not independently correlate with overall survival in a multivariate analysis. Gene expression analysis and pathway enrichment analysis demonstrated a significant enrichment in cell cycle and mitotic transition pathways in MCL with EZH2 expression. EZH2 expression detected by immunohistochemistry is present in 38% of MCL cases and it is associated with high proliferation rate, p53 overexpression, aggressive histologic variants, and poorer OS. Based on gene expression profiling data, EZH2 expression could potentiate cell cycle machinery in MCL. These data suggest that assessment of EZH2 expression could be useful to stratify MCL patients into low- and high-risk groups.

Lymphoid enhancer binding factor 1 (LEF1) expression is significantly higher in Hodgkin lymphoma associated with Richter syndrome relative to de novo classic Hodgkin lymphoma.

Lymphoid enhancer binding factor 1 (LEF1) is consistently upregulated in chronic lymphocytic leukemia (CLL) and in a subset of large B cell lymphoma. Knowledge of LEF1 expression in Hodgkin lymphoma is limited. In this study, we used immunohistochemistry to survey LEF1 expression in various subsets of Hodgkin lymphoma, de novo classic Hodgkin lymphoma (CHL) (n = 43), Hodgkin lymphoma associated with Richter syndrome (HL-RS) (n = 20), and nodular lymphocyte predominant Hodgkin lymphoma (NLPHL) (n = 9). LEF1 expression was significantly higher in HL-RS compared with de novo CHL (12/20, 60% vs. 12/43, 28%; p = 0.0248). Only a single case (1/9; 11%) of NLPHL showed LEF1 expression. Epstein-Barr virus encoded RNA (EBER) was detected in 17 (40%) cases of de novo CHL and 14 (70%) HL-RS. Notably, we identified a correlation between LEF1 expression and EBER positivity (p = 0.0488). We concluded that LEF1 is commonly positive in CHL but not in NLPHL, and such a distinction may be helpful in this differential diagnosis. The higher frequency of LEF1 upregulation in HL-RS relative to de novo CHL suggests that these neoplasms might have different underlying pathogenic mechanisms and warrants further investigation.

The life and death of the germinal center.

The formation, development and dissolution of germinal centers is a major part of immune system function. It is important to differentiate neoplastic processes from follicular hyperplasia and regressive follicular changes. Better understanding of germinal center development and dissolution also provides diagnostic clues to the underlying pathologic process. It is also important in identifying the immune basis of different pathologic entities as well as in immunotherapy decision making and follow up. In this study, we characterize the immunoarchitecture of lymphoid follicles with a focus on germinal center in one representative case, each of commonly encountered benign and malignant lymph node disorders, with morphologic and immunohistochemical alterations of germinal centers. The cases include reactive follicular hyperplasia (FH), florid follicular hyperplasia (FFH), follicular lymphoma (FL), angioimmunoblastic T-cell lymphoma (AITL), hyaline-vascular Castleman disease (HVCD), progressive transformation of germinal centers, nodular lymphocyte predominant Hodgkin lymphoma (NLPHL), lymphocyte-rich classic Hodgkin lymphoma (LR-CHL), human immunodeficiency virus (HIV)-associated follicular dissolution and chronic lymphocytic leukemia (CLL) with proliferation centers (PC). A panel of antibodies were used namely CD3, CD20, CD10, BCL2, BCL6, CD21, CD23, CD35, FOXP1, GCET1, HGAL/GCET2, LMO2, MUM1, IgD, Ki67, PD1 and PD-L1. We found that these entities show distinct immunoarchitectural patterns of germinal center formation, development and regression, particularly, the distribution of mantle zone B-cells, follicular helper T cells (Tfh) and FDC meshworks, confirming the influence of antigenic stimulation and status of immune system in these changes. This also confirms the interrelationship of underlying immunologic mechanisms in these disease processes.

EBV-Positive Primary Large B-Cell Lymphoma: The Role of Immunohistochemistry and XPO1 in the Diagnosis of Mediastinal Lymphomas.

Primary mediastinal (thymic) large B-cell lymphoma (PMBL) is described as almost always negative for Epstein-Barr virus (EBV). In the context of a mediastinal lymphoma, the distinction between PMBL, classical Hodgkin lymphoma, diffuse large B-cell lymphoma, and mediastinal gray-zone lymphoma can be very difficult; hence, EBV positivity often argues against PMBL. We present a 19-year-old man with mediastinal mass morphologically consistent with PMBL. The tumor expressed classic immunophenotype, including positivity for CD20, CD19, MAL, OCT2, BOB1, BCL6, CD79a, and subset positivity for CD30. However, the tumor was EBV-positive by in situ hybridization. Next-generation sequencing detected somatic mutations in XPO1 (E571K), SMARCB1 (L356fs), and MYCC (T73A). Although the immunophenotype and XPO1 mutation are characteristic of PMBL, EBV expression is uncommon. Since EBV positivity can occur in rare PMBLs, it should not be the deciding factor in the diagnosis. This is the first EBV-positive PMBL in which mutational profiling has been reported. Aside from providing diagnostic support, the finding of the XPO1 E571K mutation may suggest a targeted therapeutic option.

Clinicopathologic characteristics and novel biomarkers of aggressive B-cell lymphomas in the nasopharynx.

BACKGROUND: The most common nasopharyngeal lymphoma in the United States are B-cell non-Hodgkin lymphomas (B-NHL). Relatively little is known about the clinicopathologic features of these cases. In this study, we characterize a bi-institutional cohort of aggressive B-NHL primary to the nasopharyngeal area. We compare and contrast EBV-positive versus EBV-negative cases and evaluate expression of SSTR2, CD30, and PD-L1, potential markers for targeted therapeutics. METHODS AND RESULTS: We retrieved 53 cases of aggressive B-NHL from the two institutions. Staining was performed for in situ EBV (EBER), CD30, SSTR2 and PD-L1. The response to initial therapy, disease-free interval, and survival at two- and five-year following initial diagnosis were used as primary clinical outcome. Overall, 13 out of 53 cases (23%) were EBV positive. CD30 expression was more frequent in EBV-positive than in EBV-negative cases (4/6 vs 1/17). Seven of 14 (50%) cases tested demonstrated expression of PD-L1 within tumor cells; the two EBV-positive DLBCL tested showed substantial PD-L1 reactivity. Six of 15 (40%) cases tested were positive for SSTR2. The three EBV-positive patients with available outcome data died within one year of diagnosis; in contrast, the EBV-negative cases showed survival rate of 100% (8/8) and 83% (5/6) at two- and five-year follow-up, respectively. DISCUSSION: The aggressive B-NHLs of the nasopharynx show differences between EBV-positive versus EBV-negative cases. The association of EBV-positive cases with expression of CD30 and PD-L1 may be particularly informative for targeted therapies. A significant number of cases expresses SSTR2, which could render them susceptible to somatostatin analogue and peptide receptor radionuclide therapies. Finally, our limited case series suggest that EBV negativity may be associated with a better prognosis.

Practical Applications in Immunohistochemistry: An Immunophenotypic Approach to the Spleen.

CONTEXT.­: Even though immunohistochemistry is routinely used by pathologists, evaluation of immunohistochemistry in splenic lesions remains difficult for many. Classification of benign and splenic lesions often requires a combination of hematoxylin-eosin evaluation, immunophenotyping, and sometimes molecular testing. Immunohistochemical staining is essential in evaluating many splenic lesions, and requires an understanding of the normal compartments of the spleen. OBJECTIVE.­: To address different immunohistochemical features used for identification and subclassification of different lesions of the spleen, as well as in the normal compartments of the spleen. DATA SOURCES.­: The information outlined in this review article is based on our experiences with a variety of spleen cases, on the current World Health Organization classification of hematopoietic and lymphoid tumors, and on a review of English-language articles published during 2018. CONCLUSIONS.­: Features for phenotyping normal spleen as well as a variety of splenic lesions, including littoral cell angioma and splenic marginal zone lymphoma, are discussed. Suggested immunopanels are provided to assist in the diagnosis of different lesions of the spleen.

American Registry of Pathology Expert Opinions: Immunohistochemical evaluation of classic Hodgkin lymphoma.

The diagnosis of classic Hodgkin lymphoma requires immunohistochemical confirmation in most cases and one can argue for these studies as standard-of-care in the diagnostic workup. The authors propose a panel of studies for primary identification of CHL to include: CD3, CD20, CD15, CD30 and PAX5. When pattern discordances are identified, additional assessment is recommended. In the case of overexpression of B lineage markers by Hodgkin/Reed-Sternberg cells, or a differential diagnosis that includes large B-cell lymphoma or variants, additional markers recommended are: CD45, OCT2, BOB1, CD79a and MUM1/IRF4. If primary mediastinal large B cell lymphoma is considered in the differential diagnosis, suggested additional markers include: P63, CD23, CD45 and CD79a. When considering a differential diagnosis that includes anaplastic large cell lymphoma we suggest: ALK, CD45, pan T cell antigens (such as CD2, CD5, CD7, and CD43), and cytotoxic markers (granzyme, perforin, and TIA1). If peripheral T cell lymphoma or T cell lymphomas of follicular helper origin are considered in the differential diagnosis, the following panel is recommended: pan T cell antigens, CD4, CD8, one or more follicular dendritic cell markers, and assessment for Epstein-Barr virus (EBV) infection, preferably EBV encoded RNA (EBER) as assessed by in situ hybridization When the differential diagnosis includes nodular lymphocyte predominant Hodgkin lymphoma, recommended additional studies include OCT2, CD21 and/or CD23, PD1, and assessment for EBV infection. The authors recognize that these panels may not be adequate to completely characterize other lymphomas, but these panels will usually be sufficient to distinguish classic Hodgkin lymphoma from other lymphoma types.

Immunohistochemical detection of PD-L1 among diverse human neoplasms in a reference laboratory: observations based upon 62,896 cases.

Targeting of the PD1/PD-L1 immune checkpoint pathway has rapidly gained acceptance as a therapeutic strategy for a growing number of malignancies. Testing for expression of PD-L1 in tumor cells and immune cells has been used as a companion or complementary test for drugs targeting the PD1/PD-L1 pathway. We evaluated the results of PD-L1 testing in a large reference lab cohort. Using Food and Drug Administration-approved methods and interpretive instructions for each individual test, 62,896 cases were evaluated for PD-L1 using antibody clone 22C3, 28-8, SP142, or SP263. Case data analyzed included test results and information on tumor location and clinical history. No clinical outcome information was available and no attempt was made to correlate PD-L1 results with any other tests performed. The following numbers of cases were evaluated: 22C3 with tumor proportion score [n = 52585], 22C3 with combined positive score [n = 2631], 28-8 [n = 4191], SP142 [n = 850], and SP263 [n = 70]. In 22C3/tumor proportion score cases, the general results were as follows: negative 33.1% (n = 17,405), (low) expression 33.9% (n = 17,822), and high expression 29.5% (n = 15,486). In cases identified as metastatic, the results were as follows: negative 35.9% (n = 1411), (low) expression 30.8% (n = 1211), and high expression 30.7% (n = 1208). We found broad ranges of expression in tumor types with increasing positivity, as adenocarcinomas were reported as poorly differentiated, whereas squamous cell carcinomas showed more positivity as tumors were described as well-differentiated. The results of many individual tumor types were evaluated and showed, in general, high levels of positive expression. Practical challenges and observations of PD-L1 stain results and interpretation are also discussed.

Aggressive B cell lymphomas in the 2017 revised WHO classification of tumors of hematopoietic and lymphoid tissues.

The recent 2017 update of the World Health Organization classification of lymphomas has significant changes from the previous edition. Subtypes of large B cell lymphoma and related aggressive B cell lymphomas are addressed. Clinicopathological features of entities as related to morphology, immunophenotype, cell of origin, and molecular/genetic findings are reviewed with emphasis on changes or updates in findings. Specific subtypes addressed include: T cell/histiocyte-rich large B cell lymphoma, primary diffuse large B cell lymphoma (DLBCL) of the CNS, primary cutaneous DLBCL leg-type, EBV-positive DLBCL, NOS, DLBCL associated with chronic inflammation, primary mediastinal large B cell lymphoma, intravascular large B cell lymphoma, ALK-positive large B cell lymphoma, plasmablastic lymphoma, primary effusion lymphoma, HHV8-positive diffuse large B-cell lymphoma, NOS, Burkitt lymphoma, Burkitt-like lymphoma with 11q aberration, high-grade B cell lymphoma with MYC and BCL2 and/or BCL6 rearrangements, high grade B cell lymphoma, NOS, B cell lymphoma, unclassifiable, with features intermediate between DLBCL and classic Hodgkin lymphoma and large B cell lymphoma with IRF4 translocation. In addition, EBV positive mucocutaneous ulcer is addressed.

Diagnostically relevant updates to the 2017 WHO classification of lymphoid neoplasms.

The recent 2017 WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues contains a number of updates under the category of lymphoid neoplasms. These changes include introduction of new entities, amended classification or terminology, and addition of newly discovered diagnostic and molecular features. In this review, we perform a focused, concise summary of selected lymphoid neoplasms and discuss changes in their classification. Rather than a comprehensive overview, we place specific emphasis on important and diagnostically relevant aspects of each entity that are novel or different from the previous WHO iteration and bring the practicing pathologist quickly up to speed with the updated classification.

Autoimmune and medication-induced lymphadenopathies.

This article will provide a discussion of some common autoimmune disorders that could affect the lymph nodes and potentially mimic B and T-cell lymphomas. Some of these disorders are more characteristic of individuals in the pediatric age group (autoimmune lymphoproliferative syndrome, Kawasaki disease), while others present in older individuals (rheumatoid arthritis, lupus erythematosus, sarcoidosis). A common finding that groups all of these disorders together is the overall relative preservation of the architecture, a feature that can be particularly helpful to distinguish them from many B and T-cell lymphomas. Another area of interest, that will be discussed in this review, is the pathologic manifestations that can be present in lymph nodes secondary to medications. Such alterations range from 'reactive' forms of follicular, interfollicular or paracortical hyperplasia, to specific B and T-cell lymphoproliferative disorders (particularly documented in association with methotrexate and TNF-inhibitors).

Lymphadenopathy associated with IgG4-related disease: Diagnosis & differential diagnosis.

IgG4-related sclerosing disease, which now encompasses diverse organ-related disorders with various prior eponymic designations, may also present with solitary or multifocal lymph node enlargement. This review considers the histopathologic features of IgG4 lymphadenopathy (IgG4LAD), which has been subdivided by Cheuk & Chan into 5 microscopic subtypes. Those include variants that are typified by multicentric Castleman disease (MCD)-like changes, follicular hyperplasia, interfollicular lymphoplasmacytic proliferation, progressive transformation of germinal centers, and formation of inflammatory pseudotumor (IPT)-like lesions. All of them demonstrate an excess of IgG4-immunoreactive plasma cells in the inflammatory cell population. Differential diagnostic considerations for IgG4LAD include true MCD, true IPT, luetic lymphadenitis, Rosai-Dorfman disease, and inflammatory myofibroblastic tumor, among others. An interpretative distinction between malignant lymphoma and IgG4LAD is also crucial.

Genetic evaluation of juvenile xanthogranuloma: genomic abnormalities are uncommon in solitary lesions, advanced cases may show more complexity.

Juvenile xanthogranuloma is a rare histiocytic proliferation primarily affecting infants and young children, characterized by aberrant infiltration of histiocyte-derived cells in the skin, soft tissues and more rarely, visceral organs. Juvenile xanthogranuloma is generally considered to be a benign disorder; most lesions are solitary cutaneous nodules that resolve spontaneously without treatment. However, cases with extracutaneous involvement, multiple lesions, and/or systemic disease often require aggressive therapy. Though molecular studies have provided evidence of clonality in juvenile xanthogranuloma, in support of a neoplastic process, little is known about the genetic profile of juvenile xanthogranuloma. We used molecular inversion probe array technology to evaluate the genomic characteristics (copy number alterations or copy neutral-loss of heterozygosity) of 21 archived cases of juvenile xanthogranuloma (19 solitary, 1 diffuse cutaneous, 1 systemic). Four cases (19%) showed acquired, clonal alterations. Two lesions from a case of diffuse cutaneous juvenile xanthogranuloma showed distinct profiles: JXG-1a contained trisomy 5 and 17 and JXG-1b contained loss of heterozygosity in 5q. The systemic juvenile xanthogranuloma (JXG-2) showed multiple genomic alterations. Only two of 19 solitary juvenile xanthogranulomas showed abnormal genomic profiles: JXG-3 showed gains on 1q and 11q and JXG-4 showed a 7.2 Mb loss in 3p. No recurrent abnormalities were observed among these cases. The presence of non-recurrent copy number alterations in a subset of samples implies that copy number changes are unlikely driving pathogenesis in juvenile xanthogranuloma, but may be acquired during disease progression. The presence of genomic abnormalities in more advanced cases (ie, systemic and diffuse cutaneous juvenile xanthogranuloma) supports this notion, particularly as the advanced cases of juvenile xanthogranuloma presented more genomic complexity.

Predominance of CD4+ T Cells in T-Cell/Histiocyte-Rich Large B-Cell Lymphoma and Identification of a Subset of Patients With Peripheral B-Cell Lymphopenia.

OBJECTIVES: T-cell/histiocyte-rich large B-cell lymphoma (THRLBCL) is a morphologic variant of large B-cell lymphoma whose flow cytometry findings are not well characterized. METHODS: Nineteen cases with flow cytometric immunophenotyping were identified from the case records of four institutions between 2001 and 2016. RESULTS: In most cases, neoplastic B cells were not detected by flow cytometry. Overall, cases showed a predominance of CD4+ T cells, which in some cases was marked. Significant coexpression of CD57 was seen on CD4+ T cells where this marker was analyzed, which correlated with PD-1 expression. Two cases also showed a profound systemic B-cell lymphopenia, which was associated in one case with hypogammaglobulinemia. CONCLUSIONS: Overall, our work challenges previous findings that cases of THRLBCL are rich in CD8+ T cells and highlights parallels between THRLBCL and nodular lymphocyte-predominant Hodgkin lymphoma (NLPHL). Also, an association of THRLBCL with systemic B-cell lymphopenia has not been previously reported but may represent an underrecognized manifestation.

Genomic analysis of follicular dendritic cell sarcoma by molecular inversion probe array reveals tumor suppressor-driven biology.

Follicular dendritic cell sarcoma is a rare malignant neoplasm of dendritic cell origin that is currently poorly characterized by genetic studies. To investigate whether recurrent genomic alterations may underlie the biology of follicular dendritic cell sarcoma and to identify potential contributory regions and genes, molecular inversion probe array analysis was performed on 14 independent formalin-fixed, paraffin-embedded samples. Abnormal genomic profiles were observed in 11 out of 14 (79%) cases. The majority showed extensive genomic complexity that was predominantly represented by hemizygous losses affecting multiple chromosomes. Alterations of chromosomal regions 1p (55%), 2p (55%), 3p (82%), 3q (45%), 6q (55%), 7q (73%), 8p (45%), 9p (64%), 11q (64%), 13q (91%), 14q (82%), 15q (64%), 17p (55%), 18q (64%), and 22q (55%) were recurrent across the 11 samples showing abnormal genomic profiles. Many recurrent genomic alterations in follicular dendritic cell sarcoma overlap deletions that are frequently observed across human cancers, suggesting selection, or an active role for these alterations in follicular dendritic cell sarcoma pathogenesis. In support of a tumor suppressor-driven biology, homozygous deletions involving tumor suppressor genes CDKN2A, RB1, BIRC3, and CYLD were also observed. Neither recurrent gains nor amplifications were observed. This genomic characterization provides new information regarding follicular dendritic cell sarcoma biology that may improve understanding about the underlying pathophysiology, provide better prognostication, and identify potential therapeutic markers for this rare disease.

Expression of LEF1 in mantle cell lymphoma.

Small lymphocytic lymphoma/chronic lymphocytic leukemia (CLL/SLL) and mantle cell lymphoma (MCL) usually are distinctly different in regard to clinical presentation, morphology, immunophenotype and molecular/genetic findings. In spite of this, select cases may show overlapping characteristics and represent a diagnostic challenge. Recently LEF1 staining was identified as a fairly characteristic finding in CLL/SLL, with positivity identified in up to 95% of cases. LEF1 staining has not been reported as being present in cases of MCL, making this stain a useful tool in distinguishing these diagnoses. We identified an index case of MCL with cyclin D1 expression and the presence of the typical t(11;14) IGH-CCND1, which expressed LEF1. Subsequently, we assessed LEF1 immunohistochemical staining in a series of 23 cases of MCL, as confirmed by staining for cyclin D1 and/or SOX11. We found expression present in one additional case, and evaluated some published literature suggesting a frequency of 4-9% expression of LEF1 by MCL. LEF1 expression by immunohistochemistry in MCL is unusual but can be seen rarely, and could represent a potential diagnostic pitfall.

EBV-associated Peripheral T-Cell Lymphoma of Gastrointestinal Tract Presented With Widespread Chronic Inflammation: A Case Report.

We report a case of Epstein-Barr virus (EBV)-associated T-cell lymphoma of gastrointestinal (GI) tract from a 70-year-old white woman who initially presented with a widespread GI inflammation and gastric obstruction. Initial biopsies of the GI tract showed severe chronic inflammation in the esophagus, stomach, and the small intestine. Celiac disease and inflammatory bowel disease were ruled out. The patient was treated with partial gastrectomy. Histology showed gastric wall thickening with EBV-positive, mixed lymphocytic and plasma cell infiltration in the mucosa, and thickening and fibrosis of the submucosa. She developed EBV-associated T-cell lymphoma of the GI tract one and a half years later and expired due to multiorgan failure. The T-cell lymphoma diffusely infiltrated the GI wall without forming a mass lesion. The lymphoma expressed EBV and cytotoxic molecules but lacked common features of extranodal natural killer/T-cell lymphoma nasal type, such as angioinvasion/angiodestruction, necrosis, or CD56 expression. Immunoglobulin heavy chain (IGH) gene and T-cell receptor-γ gene rearrangements and EBV-positive cells were detected at the early stage of the disease. While IGH clones were transient, T-cell clones and EBV-positive cells progressively increased over the disease course. We conclude that this case is best classified as EBV-associated peripheral T-cell lymphoma of GI tract. Age-related immune senescence may have contributed to the uncontrolled GI inflammation and subsequent transformation to T-cell lymphoma.

Aggressive B-cell lymphomas: frequency, immunophenotype, and genetics in a reference laboratory population.

Diffuse large B-cell lymphoma (DLBCL) is the most common type of lymphoma worldwide. The current World Health Organization classification includes several subtypes based on a combination of clinical, immunohistochemical, and genetic differences. Other aggressive variants of B-cell lymphomas, including Burkitt lymphoma and double-hit lymphomas are part of the differential diagnosis and often have overlapping features with DLBCL. In this study, we evaluated 760 of cases of DLBCL and other aggressive B-cell lymphomas using a relatively uniform immunohistochemical panel and genetic methods. We assessed the frequency of different subtypes and locations and documented distinctive immunophenotypic and genetic findings of these cases. Most cases in the study group were DLBCL (89%), including 38 CD5+ DLBCL, 28 T-cell/histiocyte-rich large B-cell lymphomas, and 33 Epstein-Barr virus-positive DLBCL (including 6 cases in elderly patients). The study also included 39 Burkitt lymphoma and 39 cases of double-hit lymphoma. In general, our results support the World Health Organization classification approach as well as other studies of DLBCL. In this study, we focus on specific issues of interest including cell-of-origin classification testing, comparing the Hans classifier with the tally classifier, correlation of MYC immunohistochemistry with MYC fluorescence in situ hybridization, and Epstein-Barr virus positivity in aggressive B-cell lymphomas.