Diffuse large B-cell lymphoma (DLBCL) is one of the

Practical Applications in Immunohistochemistry Evaluation of Diffuse Large B-Cell Lymphoma and Related Large B-Cell Lymphomas Dennis P. O Malley, MD; Aaron Auerbach, MD; Lawrence M. Weiss, MD Context. Diffuse large B-cell lymphoma is the most commonly diagnosed subtype of lymphoma worldwide. The current World Health Organization (WHO) classification includes several subtypes, based on a combination of clinical, immunohistochemical, and genetic differences. Immunohistochemical staining is essential in evaluating diffuse large B-cell lymphoma and many related large B- cell lymphomas and aggressive B-cell lymphomas. Objective. To address different immunohistochemical features used for identification, subclassification, prognosis and in some cases, therapy, of diffuse large B-cell lymphoma and related lymphomas. Data Sources. The information outlined in this review article is based on our experiences with routine cases, on the current WHO classification of hematopoietic and lymphoid tumors, and on a review of English-language articles published throughout 2014. Conclusions. Features and diagnostic criteria of diffuse large B-cell lymphoma, aggressive variants of B-cell lymphomas, including Burkitt lymphoma and doublehit lymphomas, are discussed. Identification of cell of origin (germinal center type versus activated B-cell type) is discussed at length. Finally, practical approaches for diagnosis are discussed. (Arch Pathol Lab Med. 2015;139:1094 1107; doi: 10.5858/arpa.2014-0451-CP) Diffuse large B-cell lymphoma (DLBCL) is one of the most common diagnoses in hematopathology, typically including approximately 30% to 40% of non-hodgkin lymphoma cases. In spite of being a common diagnosis, it represents a heterogeneous group of disorders that are related by the identification of a diffuse proliferation of B cells, which are typically large. However, from this simple starting point, distinguishing DLBCL from related disorders and proper determination of prognostic markers can add a degree of complexity to this diagnosis, which can be daunting to the practicing pathologist. In all cases, to confirm a diagnosis of DLBCL, one must confirm the B-cell lineage of the lymphoma cells. Therefore, a de facto standard for B-cell identification is expression of CD20. In untreated lymphomas, CD20 is expressed in the most mature B-cell neoplasms, with some notable exceptions Accepted for publication November 14, 2014. Published as an Early Online Release January 2, 2015. From Clarient Diagnostic Services, Aliso Viejo, California (Drs O Malley and Weiss); and Joint Pathology Center, Silver Spring, Maryland (Dr Auerbach). The authors have no relevant financial interest in the products or companies described in this article. This article is provided for educational purposes only and is not intended to suggest either a practice standard or the only acceptable pathway for diagnostic evaluation. The views presented reflect the authors opinions. The application of these opinions to a particular medical situation must be guided by the informed medical judgment of the responsible pathologist(s), based on the individual circumstances presented by the patient. The College of American Pathologists has no responsibility for the content or application of the views expressed herein. Reprints: Dennis P. O Malley, MD, Clarient Diagnostic Services, 31 Columbia, Aliso Viejo, CA 92656 (e-mail: domalley@clarientinc. com). discussed below. Since CD20 is a target for a successful and commonly used monoclonal antibody therapy (rituximab) for most B-cell lymphomas, the use of this antibody should be a first consideration. Rituximab-treated B-cell lymphomas may no longer express CD20 and they require additional immunostains, such as PAX5, CD79a, CD19, or CD22, to identify B cells. Confirming the presence of CD20 þ large B cells with an appropriate histologic pattern could be considered a minimal standard for the diagnosis of DLBCL. It is however incumbent on the practicing pathologist to recognize that several variants and subtypes of DLBCL, and related disorders, rely on a more extensive evaluation of clinical, histopathologic, immunohistochemical, and genetic features for their accurate identification (Table 1). The current classification of DLBCL and related disorders from the 2008 World Health Organization (WHO) classification is highlighted in Table 2. Further, some groups have established guidelines for lymphoma diagnosis and/or reporting, including the College of American Pathologists (CAP) (Table 3). 1 3 Currently, nationally recognized clinical guidelines for the treatment of patients with lymphoma emphasize the use of the 2008 WHO Classification of Tumors of Hematopoietic and Lymphoid Tissues. 1,4 Based on this guidance, it is appropriate to provide subclassification and as specific a diagnosis as possible assuming that one has arrived at a diagnosis of diffuse large cell lymphoma. While this review emphasizes immunohistochemical studies, where relevant, other ancillary tests to identify subtypes of DLBCL and variants will also be discussed. DIAGNOSIS, PROGNOSIS, AND THERAPY As pathologists, our role in the care of patients is expanding rapidly. While in the past our focus was mainly 1094 Arch Pathol Lab Med Vol 139, September 2015 IHC in DLBCL O Malley et al

Marker Minimal Evaluation Table 1. Evaluation of Diffuse Large B-Cell Lymphoma by Immunohistochemistry a,b NCCN Clinical Practice Guidelines in Oncology (NCCN Guidelines) Adequate NCCN Guidelines Useful Basic Basic With Hans Basic With GC/NGC Thorough CD20 þ þ þ þ þ þ þ CD3 þ þ þ þ þ þ þ CD5... þ þ þ þ þ þ CD10... þ þ þ þ þ CD45... þ þ BCL2... þ þ þ þ þ þ BCL6... þ þ þ þ þ MUM1 c þ þ þ þ þ Ki-67... þ þ þ þ þ þ MYC... þ þ þ þ þ þ Cyclin D1...... þ......... þ j/k c... þ............ CD138 c... þ c c c c EBER-ISH...... þ......... þ ALK...... þ............ HHV8...... þ............ GCET1...... (þ) e...... þ þ FOXP1...... (þ) e...... þ þ LMO2...... (þ) e...... þ þ CD30...... (þ) e...... þ PAX5 d...... d d d d CD79a d...... d d d d Abbreviations: ALK, anaplastic lymphoma kinase; EBER, Epstein-Barr virus encoded small RNAs; GC, germinal center; HHV8, human herpesvirus 8; ISH, in situ hybridization; NCCN, National Comprehensive Cancer Network; NGC, nongerminal center; þ, positive;, negative. a Minimal evaluation would be performed if there were limited sample or if resources were limited, and a minimal evaluation would suffice for therapeutic evaluation, such as in the case of a recurrence of a known lymphoma. The NCCN Guidelines specify adequate immunophenotype to establish diagnosis and GC versus NGC origin. Other markers are useful under certain circumstances to establish lymphoma subtype. Additional evaluations are based on the practice patterns of the authors and include a basic evaluation panel (without GC/non-GC), the basic panel with addition of markers for applying the Hans classifier, and the basic panel with markers for both Hans and Tally classifiers. The thorough panel addresses Epstein-Barr virus expression as well as cyclin D1 expression. b Adapted from Zelenetz et al 1 with permission from the NCCN Clinical Practice Guidelines in Oncology (NCCN Guidelines) for Non-Hodgkin s Lymphoma V.4.2014. Copyright 2014 National Comprehensive Cancer Network, Inc. All rights reserved. The NCCN Guidelines and illustrations herein may not be reproduced in any form for any purpose without the express written permission of the NCCN. To view the most recent and complete version of the NCCN Guidelines, go online to NCCN.org. NATIONAL COMPREHENSIVE CANCER NETWORK, NCCN, NCCN GUIDELINES, and all other NCCN Content are trademarks owned by the National Comprehensive Cancer Network, Inc. c CD138 and/or CD38 should be used in all cases with evidence of plasmacytic or plasmablastic differentiation. Light-chain staining may also be of benefit in these cases. d If there is an indication of previous anti-cd20 therapy (such as rituximab) or a lack of staining for CD20 in what would otherwise be a typical diffuse large B-cell lymphoma, then alternative B-cell markers, such as PAX5 or CD79a, should be used to confirm B-cell differentiation. e When available, may be useful per NCCN guidelines. on diagnosis, currently we are seeing an expansion of our role in establishing prognosis for patients, which may play an important role in directing therapy. Many prognostic factors may directly drive selection of the type of therapy (more or less aggressive) that is recommended (Table 4). The 2008 WHO classification includes at least 26 types of large B-cell lymphomas, guiding classification of clinicopathologic entities based on histologic features, immunohistochemical features, and genetics. 4 Evaluation of this large group of diagnoses often requires careful correlation of several clinical and pathologic findings. Immunohistochemistry is a keystone in identifying many of the different types of large B-cell lymphomas. In almost all cases, primary identification is made by a histologic pattern along with the presence of pan B-cell antigens. In the appropriate context, the combination of large cells in a diffuse pattern, with clear expression of CD20 may be adequate for identification of DLBCL, not otherwise specified (NOS). While this could be considered as a minimal standard of care, evaluation should take other morphologic, immunophenotypic, genetic, and clinical findings into account to completely classify DLBCL and related lymphomas. It is important to note that there are several specific subtypes of DLBCL that do not have distinctive histologic findings and as such can only be distinguished from DLBCL, NOS, by considering clinical, genetic, or immunophenotypic findings. Several markers may be expressed by specific subtypes of DLBCL but are not necessarily associated with specific morphologic features, including Epstein-Barr virus (EBV), CD5, anaplastic lymphoma kinase (ALK), CD30, and CD138. Prognosis in DLBCL has been a fertile area of research for several decades. A wide range of individual markers may be associated with prognosis in DLBCL and related lymphomas, supporting the idea that this is a complex and heterogeneous group of neoplasms, and each year additional markers are put forward for evaluation 5 18 (Table 5). While a plethora of unique aspects of DLBCL is prognostic, only those markers that have been vetted by extensive clinical study and can be routinely applied are the primary focus of this report. DIFFUSE LARGE B-CELL LYMPHOMA, NOS Most cases of DLBCL, NOS, express pan B-cell markers CD20, PAX5, and CD79a, although the plasmablastic types may lack CD20 and PAX5 in many cases. 19 Most cases also strongly express the B-cell transcription factors BOB1 and OCT2. Several markers are associated with general evaluation of large cell lymphomas and may be of prognostic value, Arch Pathol Lab Med Vol 139, September 2015 IHC in DLBCL O Malley et al 1095

Table 2. 2008 World Health Organization Classification: Diffuse Large B-Cell Lymphoma (DLBCL) Variants, Subgroups, and Subtypes a DLBCL, not otherwise specified Common morphologic variants Centroblastic Immunoblastic Anaplastic Rare morphologic variants Molecular subgroups Germinal center B-cell like Activated B-cell like Immunohistochemical subgroups CD5 þ DLBCL Germinal center B-cell like Nongerminal center B-cell like Diffuse large B-cell subtypes T-cell/histiocyte rich large B-cell lymphoma Primary DLBCL of the CNS Primary cutaneous DLBCL, leg type EBV þ DLBCL of the elderly Other lymphomas of large B cells Primary mediastinal (thymic) large B-cell lymphoma Intravascular large B-cell lymphoma DLBCL associated with chronic inflammation Lymphomatoid granulomatosis ALK þ DLBCL Plasmablastic lymphoma Large B-cell lymphoma arising in HHV8-associated multicentric Castleman disease Primary effusion lymphoma Borderline cases B-cell lymphoma, unclassifiable, with features intermediate between DLBCL and Burkitt lymphoma B-cell lymphoma, unclassifiable, with features intermediate between DLBCL and classical Hodgkin lymphoma Abbreviations: ALK, anaplastic lymphoma kinase; CNS, central nervous system; EBV, Epstein-Barr virus; HHV8, human herpesvirus 8. a Reproduced with permission from the World Health Organization, from Table 10.14 from Swerdlow et al. 4 therapeutic relevance, or are used in combination with other markers or studies for prognosis or subclassification. Ki-67 Some older studies 20 have shown that proliferation rate, as measured by Ki-67, is associated with prognosis in DLBCL. However, more recent studies have not shown these same associations. Nonetheless, Ki-67 is a valuable stain in the diagnostic approach to lymphomas and can provide considerable additional information besides prognosis. 21 Importantly, in cases with a near 100% proliferation rate, it may raise the diagnostic consideration of Burkitt lymphoma (BL) or other aggressive large B-cell lymphomas, but it is not useful alone in identifying double-hit lymphomas (DHLs). 22 BCL2 BCL2 expression in DLBCL is an adverse prognostic marker, particularly in cases of activated B-cell (ABC) type or with coexpression of MYC protein. 23 25 Its expression is not necessarily related to the presence or absence of the t(14;18) IgH/BCL2 genetic abnormality seen in follicular lymphomas and a subset of DLBCLs. The role of BCL2 in germinal center (GC) and ABC types differs and this may have an impact on therapeutic choices for these patients. MYC Protein Expression in Large B-Cell Lymphomas Because of its association with high proliferation, the MYC gene has been of considerable interest in DLBCL. In most cases, the presence of a MYC translocation, identified by classical cytogenetics, fluorescence in situ hybridization (FISH), or other molecular studies, has been of benefit in evaluating lymphomas. 26 The presence of MYC translocations is most often associated with a diagnosis of BL. However, DLBCLs may also have MYC translocations (8% 16%), as do a subset of DLBCLs with features intermediate between BL and DLBCL, DHLs (lymphomas with the combination of IGH/MYC translocations and either IGH/ BCL2 and/or BCL6 translocation) (Table 6), and rare B lymphoblastic lymphomas. 27 MYC amplifications have also been associated with poor prognosis. 28 MYC immunohistochemistry is associated with prognosis in DLBCL, especially in association with BCL2 protein expression (Figure 1, a through c). 29,30 High nuclear expression of MYC protein by immunohistochemistry (.40% 70%) in aggressive B-cell lymphomas may provide an appropriate triage step to indicate the need for additional genetic studies to evaluate for DHL or MYC translocations. 31 33 DLBCL and Cyclin D1 Expression Cyclin D1 expression can be seen in approximately 2% (30 of 1435) of DLBCLs. 34 However, in contrast to the staining seen in mantle cell lymphoma, it is only weakly positive and often seen in only a subset of lymphoma cells. It is not associated with differences in prognosis or pathologic features. Its primary use in the evaluation of DLBCL is to exclude the possibility of the blastoid or pleomorphic variants of mantle cell lymphoma, which may at times appear similar to DLBCL. 35 DLBCL and CD30 Expression CD30 expression can be seen in 10% to 21% (35 of 167) of cases of typical DLBCL, and the most recent studies suggest CD30 þ DLBCL may be associated with an overall better prognosis, with a unique gene expression profile. 36,37 It is seen with a high frequency in specific lymphoma types such as lymphomatoid granulomatosis, primary mediastinal large B-cell lymphoma (PMLBCL), plasmablastic lymphoma (PBL), and gray zone lymphomas. At present, its evaluation is especially relevant because brentuximab vedotin, an anti- CD30 monoclonal antibody, is currently in clinical trials and may be useful in the treatment of some cases of DLBCL. 36,38 COMMON AND RARE MORPHOLOGIC VARIANTS The common and rare morphologic variants in DLBCL do not require a specific evaluation distinct from DLBCL, NOS. Studies 39 have attempted to correlate immunoblastic morphology with prognosis in DLBCL. However, reproducibility of morphologic subtyping can be difficult in routine practice. CELL-OF-ORIGIN SUBGROUPS: MOLECULAR Gene expression microarray studies have led to the recognition that most DLBCLs may be separated into GC and ABC/nongerminal center (NGC) types, by RNA expression patterns. 40 43 Gene expression arrays are not currently applied in routine diagnosis, and as such, other immunohistochemical systems have been proposed to act as surrogate markers for gene expression arrays (see below). 44 1096 Arch Pathol Lab Med Vol 139, September 2015 IHC in DLBCL O Malley et al

Table 3. Summaries of Published Guidelines/Reporting Templates for Evaluation of Diffuse Large B-Cell Lymphoma a,b,c A. NCCN Clinical Practice Guidelines in Oncology (NCCN Guidelines) for Non-Hodgkin Lymphoma 1. Adequate immunophenotyping to establish diagnosis and GC versus non-gc origin 2. IHC panel: CD20, CD3, CD5, CD10, CD45, BCL2, BCL6, Ki-67, MUM1, MYC or Cell surface marker analysis by flow cytometry: j/k, CD45, CD3, CD5, CD19, CD10, CD20 3. Useful under certain circumstances Additional immunohistochemical studies to establish lymphoma subtype IHC panel: cyclin D1, j/k, CD30, CD138, EBER-ISH, ALK, HHV8 Cytogenetics or FISH: t(14;18), t(3;v), t(8;14), t(8;v) B. Alberta Provincial Hematology Tumour Team Lymphoma 1. CD20 2. May include CD45/CD3/CD20/CD30/CD15/PAX5/MUM1 3. Routine use of immunohistochemical stains for the express purpose of subtyping ABC versus GC is not recommended 4. CD5 staining should be performed for all patients with DLBCL 5. EBER analysis should be performed for patients with immune suppression related lymphomas, or those who possibly have EBVrelated DLBCL of the elderly 6. Ki-67 (high proliferation rate by Ki-67. 80%) 7. MYC rearrangement testing by FISH is required 8. If MYC is rearranged, the case should be followed up with BCL2 and BCL6 rearrangement testing by FISH d C. College of American Pathologists Diffuse Large B-Cell Lymphoma, Not Otherwise Specified, Biomarker Reporting Template 1. Immunohistochemical staining BCL2, CD5, CD20, CD30, Ki-67, MYC 2. GC versus NGC e Hans (CD10, BCL-6, MUM1) Choi (GCET1, CD10, MUM1, BCL6, FOXP1) Tally (CD10, GCET1, MUM1, FOXP1, LMO2) Gene expression profiling 3. FISH MYC, BCL2, BCL6 Abbreviations: ABC, activated B cell; ALK, anaplastic lymphoma kinase; DLBCL, diffuse large B-cell lymphoma; EBER, Epstein-Barr virus encoded small RNAs; EBV, Epstein-Barr virus; FISH, fluorescence in situ hybridization; GC, germinal center; HHV8, human herpesvirus 8; IHC, immunohistochemistry; ISH, in situ hybridization; NCCN, National Comprehensive Cancer Network, Inc; NGC, nongerminal center. a Section A: Adapted with permission from Zelenetz et al, 1 the NCCN Clinical Practice Guidelines in Oncology (NCCN Guidelines) for Non- Hodgkin s Lymphoma V.4.2014. Copyright 2014 National Comprehensive Cancer Network, Inc. All rights reserved. The NCCN Guidelines and illustrations herein may not be reproduced in any form for any purpose without the express written permission of the NCCN. To view the most recent and complete version of the NCCN Guidelines, go online to NCCN.org. NATIONAL COMPREHENSIVE CANCER NETWORK, NCCN, NCCN GUIDELINES, and all other NCCN Content are trademarks owned by the National Comprehensive Cancer Network, Inc. b Section B: Copyright 2014 Alberta Health Service. 2 This material is protected by Canadian copyright law. Except as otherwise provided for under Canadian copyright law, this material may not be copied, published, or distributed without the prior written permission of the copyright owner. This material was originally published by Alberta Health Services and has been reprinted with permission. These materials are intended for general information only and are provided on an as is, where is basis. Although reasonable efforts were made to confirm the accuracy of the information, Alberta Health Services does not make any representation or warranty, express or implied, oral or written, statutory or otherwise, as to the accuracy, reliability, completeness, applicability or fitness for the particular purpose of such information, including without limitation implied warranties or warranties of noninfringement or merchantability. These materials are not a substitute for the advice of a qualified health professional. Alberta Health Services expressly disclaims all liability for the use of these materials, and for any claims, actions, demands or suits arising from such use. c Section C: Data derived from Duncavage et al. 3 d In patients younger than 70 years and in cases with high proliferation rate by Ki-67, with atypical (eg, Burkitt-like) morphology or with aggressive clinical behavior. e Germinal center type versus nongerminal center type. Specific classifier is not endorsed and Hans, Tally, and Choi classifiers are listed as possible options. CELL-OF-ORIGIN SUBGROUPS: IMMUNOHISTOCHEMISTRY Germinal Center Type Versus Nongerminal Center Type When considering DLBCL, NOS, determination of GC versus ABC type is of prognostic relevance. In general, GCtype DLBCL is associated with a better prognosis compared to ABC-type DLBCL. In the pre-rituximab era, the average 5-year survival associated with GC type was 60% versus 35% for ABC type; with the addition of rituximab to standard therapy, 5-year survival is 87% to 92% for GC type and 44% for ABC type. 42,45 Moreover, responses to newer therapeutic options may be significantly different for GC versus NGC types, making this classification even more important. 46 However, in the special types of DLBCL, those with a poor prognosis are typically of ABC type, and determination of GC versus ABC may not be relevant. Testing of GC versus ABC type of DLBCL is best accomplished by gene expression profiling. However, since this technique is not available for routine practice, other surrogate systems have been and continue to be tested. 44,45,47 51 While these immunohistochemical approaches are not without controversy, in general there is relatively good concordance with gene expression profiling results. 44,51 54 There are several schemes that allow for immunohistochemical classification of DLBCL, NOS (Figure 2, a through f). The first and most well known is the Hans classifier. 47 It is based on the application of 3 antibodies (CD10, BCL6, and MUM1). The overall concordance with gene expression array signatures is 80% (122 of 152). Other systems use combinations of other markers ([Choi et al 45 : GCET1, MUM1, CD10, BCL6, FOXP1], [Muris et al 48 : BCL2, CD10, MUM1], [Natkunam et al 49 : LMO2], [Nyman et al 50 : MUM1, Arch Pathol Lab Med Vol 139, September 2015 IHC in DLBCL O Malley et al 1097

Table 4. Large B-Cell Lymphoma Types and Association With Therapy and Prognosis Based on Suggested Therapies a Lymphoma Type Specialized Therapy b Difference in Prognosis b Centroblastic No No Immunoblastic No No Anaplastic No No GC type (GEP) Likely Yes ABC-like (GEP) Likely Yes CD5 þ DLBCL No Yes GC type (IHC) Likely Yes ABC type (IHC) Likely Yes TCHRLBCL No Yes Primary CNS DLBCL Yes Yes Primary cutaneous DLBCL Yes Yes EBV þ DLBCL of elderly No Yes Primary mediastinal large B-cell lymphoma Yes Yes IVLBCL No Yes DLBCL arising from chronic inflammation No Yes Lymphomatoid granulomatosis Yes Yes ALK þ DLBCL c No Yes Plasmablastic lymphoma Yes Yes LBCL associated with MCD Yes Yes Primary effusion lymphoma Yes Yes BCL, intermediate DLBCL/BL Likely Yes BCL, intermediate DLBCL/CHL Likely Yes Abbreviations: ABC, activated B cell; ALK, anaplastic lymphoma kinase; BCL, B-cell lymphoma; BL, Burkitt lymphoma; CHL, classical Hodgkin lymphoma; CNS, central nervous system; DLBCL, diffuse large B-cell lymphoma; EBV, Epstein-Barr virus; GC, germinal center; GEP, gene expression profiling; IHC, immunohistochemistry; IVLBCL, intravascular large B-cell lymphoma; LBCL, large B-cell lymphoma; MCD, multicentric Castleman disease; TCHRLBCL, T-cell/histiocyte rich large B-cell lymphoma. a Data derived from Zelenetz et al. 1 b Compared to typical de novo DLBCL. c Although not specific currently, anti-alk therapies (ie, crizotinib) may be used for patients with ALK þ DLBCL. FOXP1], and [Meyer et al 51 : CD10, GCET1, MUM1, FOXP1, LMO2]) to achieve a more accurate prediction of the gene expression profile results (Figure 3, a through n). There are differences suggested in the prognostic significance of BCL6 protein expression versus BCL6 translocation. If GC versus ABC type is relevant to prognosis, then expression of BCL6 generally supports GC type. However, genetic abnormalities of the BCL6 gene may be seen in ABC-type DLBCL. CD5 þ Diffuse Large B-Cell Lymphoma CD5 þ DLBCL has been described in the 2008 WHO classification and represents 5% to 10% of DLBCLs. 19,55,56 CD5 þ DLBCL is noted to have some differences from other typical DLBCL. In general, these cases are more often associated with a more aggressive clinical course, poor outcome, and are seen at a higher frequency in patients with human immunodeficiency virus (HIV)/AIDS. They express BCL2, with most expressing MUM1 and one-half expressing BCL6. 55 There are 4 recognized morphologic subtypes: common variant (centroblastic monomorphic) (76%; 91 of 120); giant cell rich variant, with intravascular involvement (11%; 13 of 120); polymorphic variant (12%; 14 of 120); and immunoblastic variant (1%; 2 of 120). 57 Approximately 25% (3 of 13) of the giant cell variant cases express CD30. 57 The expression of CD5 raises the differential diagnosis of mantle Table 5. Selected Markers Associated With Prognosis in Diffuse Large B-Cell Lymphoma Marker Source, y Thymidine phosphorylase Nie et al, 6 2013 TNF-a Nakayama et al, 8 2014 CIP2A Lilja et al, 7 2013 Cyclin E Frei et al, 5 2013 MET Koh et al, 9 2013 RON Koh et al, 9 2013 PD1-L1 Chen et al, 15 2013 DcR3 Bedewy et al, 10 2013 IL9R Lv et al, 14 2013 IRF8 Tinguely et al, 13 2014 MYD88 Choi et al, 11 2013 MCL1 Wenzel et al, 12 2013 MLL2 Morin et al, 16 2011 PI3K Cui et al, 17 2014 AKT Cui et al, 17 2014 p105/p50 Montes-Moreno et al, 18 2012 p100/p52 Montes-Moreno et al, 18 2012 cell lymphoma and chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL). Conventional mantle cell lymphoma can be distinguished by morphology and in cases of blastoid mantle cell lymphoma, the uniform expression of cyclin D1 would distinguish this entity from DLBCL. The possibility of CD5 expression in a large cell transformation of CLL/SLL (ie, Richter syndrome) is dependent on an antecedent history of the low-grade disorder with subsequent transformation. DLBCL SUBTYPES T-Cell/Histiocyte Rich Large B-Cell Lymphoma T-cell/histiocyte rich large B-cell lymphoma (TCHRLBCL) has distinctive morphologic and clinicopathologic findings. 58 60 It is a rare large B-cell lymphoma. It is most commonly seen in middle-aged men, and is associated with an aggressive clinical course. By morphology, scattered large abnormal B cells (10% or less of the overall cellularity) are seen in a background rich in small lymphocytes (T cells) or histiocytes. Because of this distinctive appearance, the differential diagnosis of classical Hodgkin lymphoma (CHL) is frequently a consideration. The large B cells are positive for pan B-cell antigens including CD19, CD20, CD79a, PAX5, OCT2, and BOB1 with expression of CD45. 60 CD30 expression is seen in a small subset of cases, but the large cells do not express CD15. They do not express CD5, CD43, or CD138. They are usually positive for BCL6 and BCL2, without expression of CD10. Epithelial membrane antigen (EMA) may show variable positivity in some cases. In contrast to many cases of CHL, staining for EBV is exceedingly rare in TCHRLBCL. If EBV is present, other diagnoses should be considered including CHL or EBV-positive DLBCL of the elderly. The background small lymphocytes are usually T cells that are positive for pan T-cell antigens, typically with more CD8 þ T cells compared to CD4 þ, in contrast to nodular lymphocyte predominant Hodgkin lymphoma (NLPHL), which is typically rich in CD4 þ T cells. The histiocytes are positive for CD68 and CD163. Staining with follicular dendritic cell markers (eg, CD21, CD23) may be of benefit; follicular dendritic cell networks are absent in TCHRLBCL but are present in cases of NLPHL. Immunoglobulin (Ig) D is expressed in many of the small lymphocytes associated 1098 Arch Pathol Lab Med Vol 139, September 2015 IHC in DLBCL O Malley et al

Table 6. with NLPHL. Recently, gene expression arrays comparing the microdissected large cells of TCHRLBCL and NLPHL were performed and no consistent differences were identified between these two entities. While not specific, it does lend some credence to the suggestions that at least some cases of TCHRLBCL may arise from previous NLPHL. 61 Primary DLBCL of the Central Nervous System and Primary Cutaneous DLBCL, Leg Type Primary central nervous system lymphoma and diffuse large B-cell lymphoma, leg type are lymphomas of activated B cells (ABC type), and are negative for CD10. The leg type diffuse large B-cell lymphoma characteristically has very strong BCL2 expression, often stronger than the admixed T cells. In these cases, because of independent poor prognosis of the special type, complete evaluation of cell of origin is not necessary, provided CD10 expression is excluded in leg type. EBV-Associated DLBCL of the Elderly: DLBCL and Epstein-Barr Virus In the 2008 WHO classification the new entity, EBVassociated DLBCL of the elderly, was added. These lymphomas are always associated with EBV infection and by definition, are seen in patients 50 years or older. 62 66 These lymphomas are more common in Asian patients (8% 10%) and less common in Western populations (5% or less). The morphologic findings include monomorphous large transformed B cells with most having areas of geographic necrosis, although this is not a universal feature. 66 These lymphomas are associated with a poor prognosis compared to typical DLBCLs. They are identified most reliably by in situ staining for EBV (Epstein-Barr virus encoded small RNAs; EBER). 64 In the diagnosis of EBV-positive DLBCL, exclusion of impairment of the immune system secondary to immunosuppression, transplantation, autoimmune disease, medications, or previous lymphoma is necessary. The immunophenotype of EBV-associated DLBCL of the elderly is positive for the pan B-cell antigens CD79a, CD20, CD19, and PAX5. It is negative for CD15 and CD10. Epstein-Barr virus should show positivity in greater than 50% of cases and is usually present in virtually all of the lymphoma cells. 64 These lymphomas are usually of NGC B- cell type, lacking BCL6 and CD10, but expressing MUM1 and usually also CD30. The EBER marker consistently shows positivity, and EBV-latent membrane protein (EBV-LMP) also shows positivity in almost all cases. Several other large B-cell lymphomas are occasionally or always positive for EBV, including PBL, primary effusion lymphoma, DLBCL associated with chronic inflammation, Possible Genetic Abnormalities Seen in Double-Hit Lymphomas MYC-Associated Translocation Additional Translocation Additional Translocation Note IGH/MYC IGH/BCL2... Most common combination MYC/X IGH/BCL2...... MYC/X BCL6/X...... IGH/MYC IGH/BCL2 BCL6/X Triple hit a MYC/X IGH/BCL2 BCL6/X Triple hit a MYC/X BCL3/X...... IGH/BCL2 BCL6... Controversial b Abbreviation: X, unidentified translocation partner (a subset of cases are j or k light-chain genes, others are nonimmunoglobulin genes). a Triple-hit lymphomas are considered to be equivalent to double-hit lymphomas and may be referred to as double-hit lymphomas. b It is unclear if combinations of translocations without involvement of MYC have comparable prognostic implications to those with MYC translocation, although technically these are still referred to as double-hit lymphomas. and lymphomatoid granulomatosis. Subsets of cases of BL are also positive for EBV. 67 Most cases of posttransplant lymphoproliferative disorders, including those of large cell, monomorphic type (eg, DLBCL) also show positivity. In all cases, consideration of these specific subtypes, clinical history, and overall pathologic findings are necessary to render an accurate diagnosis. Some types of large cell lymphomas that are associated with chronic EBV activity are positive for immunohistochemical staining for EBV-LMP; however, many types of EBV-positive lymphoproliferative disorders do not express EBV-LMP consistently. When possible, in situ stains for EBV (EBER) are recommended for evaluation of EBV expression in lymphoproliferative disorders. OTHER LYMPHOMAS OF LARGE B CELLS Primary Mediastinal Large B-Cell Lymphoma Primary mediastinal large B-cell lymphoma is associated with a fairly unique clinicopathologic presentation. 68 It is seen most frequently in young adults, with a slight female predominance and a rapidly growing mediastinal mass. The lymphoma cells are large, with round to slightly irregular nuclei, occasional pleomorphic multilobated nuclei, with moderate amounts of pale cytoplasm, imparting a clear cell appearance. This finding is not seen in all cases, and is only focal in some cases. In general, PMLBCL has an equivalent or slightly better prognosis than typical DLBCL. In most cases, the differential diagnosis is between PMLBCL, CHL, and other types of diffuse large B-cell lymphomas. Because of its distinct prognosis and a proposed origin from thymic B cells, it often has a distinctive immunophenotype. However, gene expression arrays in PMLBCL show considerable overlap with CHL, and overlapping diagnostic features can be seen. 69,70 In rare cases, collision tumors of PMLBCL and CHL, and synchronous and metachronous presentations of both, have been noted. The immunophenotype shares features with other typical large B-cell lymphomas. The lymphoma cells are positive for pan B-cell antigens including CD20, CD19, CD22, and PAX5. Distinctive features include expression of CD23 and MUM1 in most cases. Most cases are also positive for p63. 71 CD30 expression is present in most cases, but is usually weak and heterogeneous, as compared to uniform and strong expression seen in CHL. CD15 is not typically identified and if present should raise the possibility of an overlap diagnosis (see below). Expression of BCL2 and BCL6 is variable and CD10 is typically negative. In comparing CHL and PMLBCL, Hoeller et al 72 suggest that Arch Pathol Lab Med Vol 139, September 2015 IHC in DLBCL O Malley et al 1099

expression of BOB1 favors PMLBCL and expression of cyclin E favors CHL. Although these immunohistochemical stains are not routinely evaluated, most cases of PMLBCL express MAL, CD54, CD95, and TRAF1. 72 Intravascular Large B-Cell Lymphoma Intravascular large B-cell lymphoma (IVLBCL) is an extremely rare lymphoma. 73,74 It occurs slightly more frequently in men (male to female ratio: 1.1:1) with a median age of presentation of 67 years. This lymphoma has an unusual range of clinical presentations dependent on tissue types involved, and the diagnosis is often unexpected. It can be seen in several tissue types, including skin, bone marrow, spleen, lung, brain, prostate, and many other sites. Hemophagocytosis is seen in a subset of cases and may be associated with significant cytopenias. As implied in the name, clusters and aggregates of large atypical lymphoid cells are seen in an intravascular distribution. Although classically associated with a poor prognosis, the advent of rituximab therapy has resulted in improved outcomes for many patients. 73 IVLBCLs express pan B-cell antigens, including CD19 (85%), CD20 (96%), CD79a (100%), and PAX5. They express CD5 in 38% and CD10 in 12% of cases. 73 Most cases are positive for MUM1 (95%), except those that are CD10 þ. BCL2 expression is seen in 91% of cases. IVLBCL likely has unique markers that account for its intravascular distribution (such as CXCR3), but surface proteins that are critical for endothelial transmigration or adhesion are absent (CD29, CD54); however, use of these markers are not required for lymphoma diagnosis. DLBCL Associated With Chronic Inflammation In patients with long-standing chronic inflammation, DLBCL may develop. The prototype of this type of lymphoma is pyothorax-associated lymphoma, although other clinical scenarios including cysts and pseudocysts, and association with joint replacement, have also been described. 75 77 These cases usually express pan B-cell antigens CD20, CD79a, and PAX5, although cases with plasmacytic or plasmablastic differentiation may lose expression of mature B-cell markers. In these cases expression of plasma cell associated markers, including MUM1 and CD138, may be seen. These cases will express EBV, and while EBER in situ staining may be more accurate, most cases will express EBV- LMP. They express BCL2 and lack CD10 and BCL6. 76 CD30 is expressed in a subset of cases. 77 Lymphomatoid Granulomatosis Lymphomatoid granulomatosis is a rare EBV-driven lymphoproliferative disorder involving extranodal sites in patients with inherited or acquired immunodeficiencies. 78,79 Pulmonary involvement is most common, followed by brain, kidney, liver, and skin. The morphologic features include a polymorphous lymphohistiocytic infiltrate associated with angioinvasive and angiodestructive lesions. Within this background are variable numbers of atypical Figure 1. High-grade B cell lymphoma (a) with expression of BCL2 (b) and a high percentage of MYC expression (c). Cases with BCL2 and high MYC expression are associated with an aggressive clinical course (hematoxylin-eosin, original magnification 3400 [a]; original magnification 3400 [b and c]). 1100 Arch Pathol Lab Med Vol 139, September 2015 IHC in DLBCL O Malley et al

Figure 2. Diagrams of classification systems (in general, markers are considered to show positivity if.30% of cells express the marker). a, Hans classifier. b, Visco-Young classifier. c, Choi classifier. d, Muris classifier (Note: group 1 is comparable to germinal center type and group 2 is comparable to nongerminal center/activated B-cell type). e, Nyman classifier. f, Tally classifier. Light gray shading: germinal center type or equivalent; medium gray shading: nongerminal center or equivalent. *Other type in Nyman classifier is poorly defined. Abbreviations: GC, germinal center; NGC, nongerminal center; þ, positive,, negative. EBV-positive B cells. These may appear Hodgkin-like, or as pleomorphic large lymphocytes. The large abnormal cells are positive for EBV (EBER, EBV- LMP) and CD20. They express CD30 in most cases but are negative for CD15, in contrast to CHL. ALK-Positive Large B-Cell Lymphoma Anaplastic lymphoma kinase positive large B-cell lymphoma (ALK-LBCL) is exceedingly rare, is more common in men (male to female ratio: 5:1) and presents in a wide age range (14 85 years). 80,81 It is composed of large cells with large round nuclei, with prominent nucleoli and often with large amounts of pale-staining cytoplasm. These cells express the ALK protein but lack T-cell antigen expression and T-cell receptor gene rearrangements seen in anaplastic large (T) cell lymphoma. 82 Staining with ALK is typically granular and cytoplasmic, although rare cases with cytoplasmic and nuclear staining can be seen, depending on the specific translocation present. These lymphomas will often express plasma cell associated markers, including CD138, CD38, IgA (88%; 29 of 33), and cytoplasmic restricted light chains. 81 They will often lack expression of pan B-cell antigens (CD19, CD20, and PAX5). 81,83 CD45 expression is seen in most cases (82%; 19 of 23) and EMA expression is seen in almost all cases. CD30 is rarely expressed (6%; 2 of 36) and in contrast to plasmablastic lymphoma, there is no association with human herpesvirus 8 (HHV8) or EBV (EBER). ALK-LBCL may be susceptible to the relatively recently developed anti-alk therapy, crizotinib. 38,84 Arch Pathol Lab Med Vol 139, September 2015 IHC in DLBCL O Malley et al 1101

Plasmablastic Lymphoma Plasmablastic lymphoma is an aggressive B-cell neoplasm, which has immunophenotypic features of plasma cells with large cell morphology, and varying degrees of associated, more typical mature plasma cells. 85,86 Most cases are associated with immunodeficiency, particularly HIV/ AIDS, although a subset of cases is seen in immunocompetent patients. Plasmablasts are large and round, with round nuclei, prominent nucleoli, and small or minimal amounts of cytoplasm. The immunophenotypic features of PBL include expression of CD138, CD38, and CD79a (50% 85%), but PBLs usually lack expression of CD45, CD20, and PAX5. They will also express monoclonal cytoplasmic light chain in approximately more than one-half of cases. The proliferation rate by Ki-67 is greater than 90%. They lack expression of CD56, which is seen more frequently in high-grade transformation of plasma cell myeloma. The EMA and CD30 markers often show positivity. Importantly, most cases are positive for EBV by in situ staining (EBER). Human herpesvirus 8 is not seen, in contrast to some plasma cell neoplasms associated with HIV/AIDS. 85 Expression of CD56, cyclin D1, or bone/bone marrow involvement would suggest plasmablastic plasma cell myeloma rather than PBL, while the presence of EBV positivity or a MYC translocation would favor PBL. 18 Large B-Cell Lymphoma Arising in HHV8-Associated Multicentric Castleman Disease An exceedingly rare lymphoma, this is a large B-cell lymphoma that arises in HHV8-associated multicentric Castleman disease. 87 89 Most cases occur in HIV/AIDS patients or in areas with endemic HHV8 infection, including Africa and the Mediterranean area. These lymphomas have a plasmablastic appearance and start as clusters and cell aggregates in a background of multicentric Castleman disease (so-called microlymphomas). The abnormal cells will have variable expression of CD20 and typically lack expression of CD79a and CD138. They show expression of HHV8-associated antigens (LANA-1) and viral interleukin 6. They also express cytoplasmic IgM and monotypic k light chain. In contrast to some other plasmablastic lymphomas seen in HIV/AIDS, there is no evidence of EBV infection. Primary Effusion Lymphoma Primary effusion lymphoma (PEL) is another exceedingly rare B-lineage lymphoma, most often seen in immunocompromised patients, such as those with HIV infection. 90 92 As its name implies, most cases are seen in fluid effusions of the pleural or peritoneal cavities and exhibit overlapping morphologic features of immunoblastic, anaplastic, or plasmablastic large cell lymphomas. Some rare cases of Figure 3. Immunohistochemical staining in diffuse large B-cell lymphomas: contrasting differences in immunophenotype and subsequent classification by Hans and Tally classifiers. Case 1: Left column (a, hematoxylin-eosin [H&E]; c, CD10; e, BCL6; g, MUM1; i, FOXP1; k, GCET1; m, LMO2); by Hans classifier, this case would be considered of germinal center (GC) type (CD10 þ, BCL6 þ, MUM1 ); by Tally classifier, this case would be of GC type (CD10 þ, GCET1 þ, MUM1, FOXP1 þ ;GC. NGC). Case 2: Right column (b, H&E; d, CD10; f, BCL6; h, MUM1; j, FOXP1; l, GCET1; n, LMO2); by Hans classifier, this case would be of non-gc (NGC) type (CD10, BCL6 þ, MUM1 þ ); by Tally classifier, this case would also be of NGC type (CD10, GCET1, MUM1 þ, FOXP1 þ ) (original magnification 3400). 1102 Arch Pathol Lab Med Vol 139, September 2015 IHC in DLBCL O Malley et al

solid tissue based PEL have been reported. Virtually all cases have evidence of HHV8 infection, and cases not associated with HIV/AIDS are typically seen in areas with endemic HHV8 infection. Coinfection with EBV (as identified by EBER staining) is seen in several cases (60%). Exceedingly rare cases of HHV8-negative PEL have been identified. 93 The immunophenotype includes expression of CD45 (88%; 38 of 43) but lack of expression of most pan B-cell markers (CD20: 67% [51 of 76]; CD19: 92% [45 of 49]; CD79a: 88% [21 of 24]; and CD22: 97% [32 of 33]). 90,91 Plasma cell associated markers including CD138 (77%; 17 of 22), CD38 (100%; 48 of 48), MUM1 (92%; 47 of 51), VS38c, and EMA show positivity. 90,91 CD30 often shows positivity (69% 81%; 62 of 76). Primary effusion lymphoma cells lack T-cell associated antigen expression, although exceedingly rare cases of PEL of T-cell lineage have been reported. 90,91 Almost all cases show evidence of clonal B-cell gene rearrangements by polymerase chain reaction. BURKITT LYMPHOMA Burkitt lymphoma is a distinctive, but relatively rare, aggressive B-cell lymphoma. 67 It can present in both extranodal and nodal sites. Patients are generally younger than 30 years, but BL may be seen in older patients as well. It has a distinctive morphology of intermediate-sized lymphocytes with round nuclei, mature chromatin, and small to moderate amounts of deeply staining cytoplasm. Mitotic figures and apoptotic bodies are frequently seen. Tingible body macrophages will often impart the classic starry-sky pattern at low magnification. The characteristic immunophenotype of BL shows pan Bcell antigen expression (CD20, PAX5, CD19, CD22, CD79a), with strong expression of CD10, and a very high proliferation rate by Ki-67 (approximately 100%). Burkitt lymphoma lacks expression of terminal deoxynucleotidyl transferase (to exclude B lymphoblastic leukemia/lymphoma). BCL2 usually shows negativity, but may show weak positivity in approximately 25% of cases. Admixed CD3 þ T cells are infrequent but CD68 þ /CD163 þ macrophages are consistently seen. The BL cells consistently express germinal center associated markers including BCL6, GCET1, and HGAL. Some cases will show expression of MUM1, and CD43 is expressed in about one-half of cases. Burkitt lymphoma lacks expression of cyclin D1, CD5, and CD23. Flow cytometry will often show strong expression of CD38, a reflection of the high proliferative cell fraction in BL. 67,94 A classic immunophenotype can accurately diagnose BL in approximately 94% of cases (CD20 þ, CD10 þ, Ki-67 expression of almost 100%, CD38 þ ; BCL2 ). 94 In situ staining for EBV (EBER) will be positive in some cases, more often in those associated with immunosuppression. In the current classification, the presence of a MYCassociated translocation (t(8;14) MYC/IGH) or variants is necessary to confirm all but the most classic cases. In some cases, BCL2 expression may be seen, and when present, particularly when strongly positive, other diagnoses, including MYC translocation positive DLBCL, B cell lymphoma, unclassifiable, with features intermediate between DLBCL and BL, and DHL should be excluded. In MYC translocation positive lymphomas, a simple karyotype (IGH/MYC, with no or 1 other genetic abnormality) is supportive of a diagnosis of BL, while multiple genetic abnormalities support a diagnosis of either DHL or DLBCL. 26 BORDERLINE CASES B-Cell Lymphoma, Unclassifiable, With Features Intermediate Between Diffuse Large B-Cell Lymphoma and Burkitt Lymphoma (BCLUWFIBDLBCLABL) and Double-Hit Lymphoma Within the category of aggressive B-cell lymphomas, a subset of cases with the morphologic appearance of DLBCL, or cases that fall in the BCLUWFIBDLBCLABL provisional category, will have evidence of a combination of genetic findings, leading to a diagnosis of DHL. This is estimated to occur in 11% of cases of DLBCL and B-cell lymphoma, unclassifiable. 27,95 99 Double-hit lymphoma is characterized by the presence of a translocation of MYC and another classic B-cell lymphoma driver translocation. The most commonly identified combination is the presence of an IGH/ MYC translocation and IGH/BCL2. Other possible combinations are shown in Table 6. The immunophenotype of DHL is not characteristic. CD10 is generally expressed (76% 100%), as well as BCL2 (63% 96%), which usually shows strong positivity. 95 BCL6 is expressed in 63% to 96% of cases. While Ki-67 expression is usually 90% or higher, several studies 22,95 have shown wide variation (15% 100%) caused by biologic factors, suggesting that Ki-67 alone is not adequate for triaging cases for DHL workups. B-Cell Lymphoma, Unclassifiable, With Features Intermediate Between Diffuse Large B-Cell Lymphoma and Classical Hodgkin Lymphoma Occasional lymphomas have features that show considerable overlap in clinical, histologic, and/or immunophenotypic features between diagnoses of CHL and DLBCL. 100 In general, these occur in the mediastinum. These have been included in a provisional diagnosis in the 2008 WHO classification, B cell lymphoma, unclassifiable, with features intermediate between DLBCL and classical Hodgkin lymphoma (BCLUWFIBDLBCLACHL). As mentioned, there are considerable molecular similarities between PMLBCL and CHL, suggesting that lymphomas with overlapping features might exist. These are often referred to as gray zone lymphomas when distinction between DLBCL and CHL cannot be resolved with immunohistochemical staining. Table 7 highlights immunohistochemical findings which would help to resolve the differential diagnosis. In cases with equivocal results, a diagnosis of BCLUWFIBDLBCLACHL would be appropriate. When studied as a group, gray zone lymphomas are more aggressive than either PMLBCL or CHL. 68 PRACTICAL GUIDELINES FOR APPLICATION TO DIAGNOSIS With this myriad of entities and possibilities, some guidance to the practical application of this information is appropriate. Clinical scenarios are discussed below. Small/Limited Sample Small-gauge needle core biopsies of lymphoma are common practice and may present inherent diagnostic problems. However, in cases of a limited sample, with the minimal acceptable morphologic evidence of a large cell lymphoma (eg, diffuse sheets of large lymphocytes), a minimal evaluation for diagnostic confirmation would Arch Pathol Lab Med Vol 139, September 2015 IHC in DLBCL O Malley et al 1103