Produção Nacional
Revisado por pares
CD137 Is Expressed in Follicular Dendritic Cell Tumors and in Classical Hodgkin and T-Cell Lymphomas
2012; Elsevier BV; Volume: 181; Issue: 3 Linguagem: Inglês
10.1016/j.ajpath.2012.05.015
ISSN1525-2191
AutoresMatthew W. Anderson, Shuchun Zhao, Aharon G. Freud, Debra K. Czerwinski, Holbrook E. Kohrt, Ash A. Alizadeh, Roch Houot, Denize Azambuja, Irène Biasoli, José Carlos Morais, Nelson Spector, Hernan Molina‐Kirsch, Roger A. Warnke, Ronald Levy, Yasodha Natkunam,
Tópico(s)T-cell and B-cell Immunology
ResumoCD137 (also known as 4-1BB and TNFRSF9) is a member of the tumor necrosis factor receptor superfamily. Originally identified as a costimulatory molecule expressed by activated T cells and NK cells, CD137 is also expressed by follicular dendritic cells, monocytes, mast cells, granulocytes, and endothelial cells. Anti-CD137 immunotherapy has recently shown promise as a treatment for solid tumors and lymphoid malignancies in preclinical models. We defined the expression of CD137 protein in both normal and neoplastic hematolymphoid tissue. CD137 protein is expressed by follicular dendritic cells in the germinal center and scattered paracortical T cells, but not by normal germinal-center B cells, bone marrow progenitor cells, or maturing thymocytes. CD137 protein is expressed by a select group of hematolymphoid tumors, including classical Hodgkin lymphoma, T-cell and NK/T-cell lymphomas, and follicular dendritic cells neoplasms. CD137 is a novel diagnostic marker of these tumors and suggests a possible target for tumor-directed antibody therapy. CD137 (also known as 4-1BB and TNFRSF9) is a member of the tumor necrosis factor receptor superfamily. Originally identified as a costimulatory molecule expressed by activated T cells and NK cells, CD137 is also expressed by follicular dendritic cells, monocytes, mast cells, granulocytes, and endothelial cells. Anti-CD137 immunotherapy has recently shown promise as a treatment for solid tumors and lymphoid malignancies in preclinical models. We defined the expression of CD137 protein in both normal and neoplastic hematolymphoid tissue. CD137 protein is expressed by follicular dendritic cells in the germinal center and scattered paracortical T cells, but not by normal germinal-center B cells, bone marrow progenitor cells, or maturing thymocytes. CD137 protein is expressed by a select group of hematolymphoid tumors, including classical Hodgkin lymphoma, T-cell and NK/T-cell lymphomas, and follicular dendritic cells neoplasms. CD137 is a novel diagnostic marker of these tumors and suggests a possible target for tumor-directed antibody therapy. CD137 (also known as 4-1BB and TNFRSF9) is a transmembrane glycoprotein of the tumor necrosis factor receptor superfamily. CD137 is broadly expressed by cells of the human immune system, including activated CD8+ and CD4+ T cells, activated natural killer (NK) cells, follicular dendritic cells (FDCs), monocytes, and a minor subpopulation of activated B cells within the germinal center (GC).1Garni-Wagner B.A. Lee Z.H. Kim Y.J. Wilde C. Kang C.Y. Kwon B.S. 4-1BB is expressed on CD45RAhiROhi transitional T cell in humans.Cell Immunol. 1996; 169: 91-98Crossref PubMed Scopus (38) Google Scholar, 2Zhang X. Voskens C.J. Sallin M. Maniar A. Montes C.L. Zhang Y. Lin W. Li G. Burch E. Tan M. Hertzano R. Chapoval A.I. Tamada K. Gastman B.R. Schulze D.H. Strome S.E. CD137 promotes proliferation and survival of human B cells.J Immunol. 2010; 184: 787-795Crossref PubMed Scopus (53) Google Scholar, 3Lin W. Voskens C.J. Zhang X. Schindler D.G. Wood A. Burch E. Wei Y. Chen L. Tian G. Tamada K. Wang L.X. Schulze D.H. Mann D. Strome S.E. Fc-dependent expression of CD137 on human NK cells: insights into "agonistic" effects of anti-CD137 monoclonal antibodies.Blood. 2008; 112: 699-707Crossref PubMed Scopus (87) Google Scholar, 4Maniar A. Zhang X. Lin W. Gastman B.R. Pauza C.D. Strome S.E. Chapoval A.I. Human gammadelta T lymphocytes induce robust NK cell-mediated antitumor cytotoxicity through CD137 engagement.Blood. 2010; 116: 1726-1733Crossref PubMed Scopus (150) Google Scholar, 5Lindstedt M. Johansson-Lindbom B. Borrebaeck C.A. Expression of CD137 (4-1BB) on human follicular dendritic cells.Scand J Immunol. 2003; 57: 305-310Crossref PubMed Scopus (28) Google Scholar, 6Kienzle G. von Kempis J. CD137 (ILA/4-1BB), expressed by primary human monocytes, induces monocyte activation and apoptosis of B lymphocytes.Int Immunol. 2000; 12: 73-82Crossref PubMed Scopus (73) Google Scholar, 7Pauly S. Broll K. Wittmann M. Giegerich G. Schwarz H. CD137 is expressed by follicular dendritic cells and costimulates B lymphocyte activation in germinal centers.J Leukoc Biol. 2002; 72: 35-42PubMed Google Scholar CD137 can also be expressed by mesenchymal cells, including endothelial cells, chondrocytes, and cells of the central nervous system.8Drenkard D. Becke F.M. Langstein J. Spruss T. Kunz-Schughart L.A. Tan T.E. Lim Y.C. Schwarz H. CD137 is expressed on blood vessel walls at sites of inflammation and enhances monocyte migratory activity.FASEB J. 2007; 21: 456-463Crossref PubMed Scopus (86) Google Scholar, 9von Kempis J. Schwarz H. Lotz M. Differentiation-dependent and stimulus-specific expression of ILA, the human 4-1BB-homologue, in cells of mesenchymal origin.Osteoarthritis Cartilage. 1997; 5: 394-406Abstract Full Text PDF PubMed Google Scholar, 10Reali C. Curto M. Sogos V. Scintu F. Pauly S. Schwarz H. Gremo F. Expression of CD137 and its ligand in human neurons, astrocytes, and microglia: modulation by FGF-2.J Neurosci Res. 2003; 74: 67-73Crossref PubMed Scopus (32) Google Scholar Although CD137 has diverse roles in the immune response, one key function is to promote the survival of both T cells and dendritic cells by binding the cognate ligand CD137L (4-1BBL).11Wang C. Lin G.H. McPherson A.J. Watts T.H. Immune regulation by 4-1BB and 4-1BBL: complexities and challenges.Immunol Rev. 2009; 229: 192-215Crossref PubMed Scopus (230) Google Scholar In T cells and FDCs, engagement of CD137 by CD137L leads to activation of the NF-κB pathway and up-regulation of antiapoptotic members of the Bcl-2 family including Bfl-1 and Bcl-XL.12Lee H.W. Park S.J. Choi B.K. Kim H.H. Nam K.O. Kwon B.S. 4-1BB promotes the survival of CD8+ T lymphocytes by increasing expression of Bcl-xL and Bfl-1.J Immunol. 2002; 169: 4882-4888PubMed Google Scholar, 13Choi B.K. Kim Y.H. Kwon P.M. Lee S.C. Kang S.W. Kim M.S. Lee M.J. Kwon B.S. 4-1BB functions as a survival factor in dendritic cells.J Immunol. 2009; 182: 4107-4115Crossref PubMed Scopus (52) Google Scholar In addition to prosurvival signals, CD137 expression by FDCs has been shown to promote T-cell-dependent humoral immune responses by stimulating the proliferation of GC T and B cells.7Pauly S. Broll K. Wittmann M. Giegerich G. Schwarz H. CD137 is expressed by follicular dendritic cells and costimulates B lymphocyte activation in germinal centers.J Leukoc Biol. 2002; 72: 35-42PubMed Google Scholar, 14Wilcox R.A. Chapoval A.I. Gorski K.S. Otsuji M. Shin T. Flies D.B. Tamada K. Mittler R.S. Tsuchiya H. Pardoll D.M. Chen L. Cutting edge: expression of functional CD137 receptor by dendritic cells.J Immunol. 2002; 168: 4262-4267PubMed Google Scholar Given the important role of CD137 in providing costimulatory and prosurvival signals, the use of anti-CD137 antibodies as a therapeutic strategy for both solid-organ and hematolymphoid malignancies has been the subject of intense investigation.15Lynch D.H. The promise of 4-1BB (CD137)-mediated immunomodulation and the immunotherapy of cancer.Immunol Rev. 2008; 222: 277-286Crossref PubMed Scopus (100) Google Scholar Preclinical data using agonistic blocking anti-CD137 antibodies has shown promising antitumor efficacy in mouse models of sarcoma, mastocytoma, and plasma cell myeloma.16Melero I. Shuford W.W. Newby S.A. Aruffo A. Ledbetter J.A. Hellstrom K.E. Mittler R.S. Chen L. Monoclonal antibodies against the 4-1BB T-cell activation molecule eradicate established tumors.Nat Med. 1997; 3: 682-685Crossref PubMed Scopus (759) Google Scholar, 17Murillo O. Arina A. Hervas-Stubbs S. Gupta A. McCluskey B. Dubrot J. Palazón A. Azpilikueta A. Ochoa M.C. Alfaro C. Solano S. Pérez-Gracia J.L. Oyajobi B.O. Melero I. Therapeutic antitumor efficacy of anti-CD137 agonistic monoclonal antibody in mouse models of myeloma.Clin Cancer Res. 2008; 14: 6895-6906Crossref PubMed Scopus (71) Google Scholar More recently, members of our research group demonstrated that agonistic antibodies to CD137 could also eradicate established B-cell lymphomas in a murine model.18Houot R. Goldstein M.J. Kohrt H.E. Myklebust J.H. Alizadeh A.A. Lin J.T. Irish J.M. Torchia J.A. Kolstad A. Chen L. Levy R. Therapeutic effect of CD137 immunomodulation in lymphoma and its enhancement by Treg depletion.Blood. 2009; 114: 3431-3438Crossref PubMed Scopus (104) Google Scholar The antitumor effect of the agonistic anti-CD137 antibody was shown to be mediated by NK cells and CD8+ T cells, because the lymphoma cells did not express CD137. Also, in tumor samples that included both tumor cells and infiltrating immune cells, the expression of the mRNA encoding CD137 (TNFRSF9) and a marker of germinal-center B cells (LMO2) predicted outcome in patients with diffuse large B-cell lymphoma (DLBCL).19Alizadeh A.A. Gentles A.J. Alencar A.J. Liu C.L. Kohrt H.E. Houot R. Goldstein M.J. Zhao S. Natkunam Y. Advani R.H. Gascoyne R.D. Briones J. Tibshirani R.J. Myklebust J.H. Plevritis S.K. Lossos I.S. Levy R. Prediction of survival in diffuse large B-cell lymphoma based on the expression of two genes reflecting tumor and microenvironment.Blood. 2011; 118: 1350-1358Crossref PubMed Scopus (158) Google Scholar The prognostic effect of CD137 was contributed by the tumor microenvironment and likely correlates with the frequency of an activated T-cell subset, because no correlation was seen between CD137 expression and total T-cell infiltration within tumors. Although the precise mechanism of the antitumor effect of CD137 immunomodulation remains unclear, there is evidence to suggest that CD137 expression by dendritic cells and intratumoral blood vessels may also play a role in the antitumor effect of CD137.20Murillo O. Dubrot J. Palazón A. Arina A. Azpilikueta A. Alfaro C. Solano S. Ochoa M.C. Berasain C. Gabari I. Pérez-Gracia J.L. Berraondo P. Hervás-Stubbs S. Melero I. In vivo depletion of DC impairs the anti-tumor effect of agonistic anti-CD137 mAb.Eur J Immunol. 2009; 39: 2424-2436Crossref PubMed Scopus (46) Google Scholar, 21Zhang L. Wang Q. Wang X. Ding P. Song J. Ma C. Sun W. Anti-CD137 monoclonal antibody promotes the direct anti-tumor effect mediated by peripheral blood-derived human dendritic cells in vitro.Cell Mol Immunol. 2004; 1: 71-76PubMed Google Scholar, 22Palazón A. Teijeira A. Martínez-Forero I. Hervás-Stubbs S. Roncal C. Peñuelas I. Dubrot J. Morales-Kastresana A. Pérez-Gracia J.L. Ochoa M.C. Ochoa-Callejero L. Martínez A. Luque A. Dinchuk J. Rouzaut A. Jure-Kunkel M. Melero I. Agonist anti-CD137 mAb act on tumor endothelial cells to enhance recruitment of activated T lymphocytes.Cancer Res. 2011; 71: 801-811Crossref PubMed Scopus (105) Google Scholar, 23Broll K. Richter G. Pauly S. Hofstaedter F. Schwarz H. CD137 expression in tumor vessel walls High correlation with malignant tumors.Am J Clin Pathol. 2001; 115: 543-549Crossref PubMed Scopus (75) Google Scholar In contrast to the well-characterized expression of CD137 by normal immune-cell subsets, the expression of CD137 by neoplastic hematolymphoid cells is poorly understood. CD137 expression is inducible in human T-cell leukemia cell lines in vitro,1Garni-Wagner B.A. Lee Z.H. Kim Y.J. Wilde C. Kang C.Y. Kwon B.S. 4-1BB is expressed on CD45RAhiROhi transitional T cell in humans.Cell Immunol. 1996; 169: 91-98Crossref PubMed Scopus (38) Google Scholar, 24Zhou Z. Kim S. Hurtado J. Lee Z.H. Kim K.K. Pollok K.E. Kwon B.S. Characterization of human homologue of 4-1BB and its ligand.Immunol Lett. 1995; 45: 67-73Crossref PubMed Scopus (42) Google Scholar but few data have been available on the expression pattern of CD137 in primary human hematolymphoid tumors. In the present study, we characterized CD137 expression in normal and neoplastic hematolymphoid tissue through the use of immunohistochemistry and immunofluorescence on tissue sections and cell suspensions. Formalin-fixed, paraffin-embedded tissue samples were obtained from the archives of the Department of Pathology, Stanford University Medical Center (SUMC). Tissue microarrays (TMAs) incorporating SUMC pathological material were constructed as described previously.25Natkunam Y. Warnke R.A. Montgomery K. Falini B. van De Rijn M. Analysis of MUM1/IRF4 protein expression using tissue microarrays and immunohistochemistry.Mod Pathol. 2001; 14: 686-694Crossref PubMed Scopus (164) Google Scholar TMAs encompassing 265 cases of classical Hodgkin lymphoma from the Federal University of Rio de Janeiro were also used.26Azambuja D. Lossos I.S. Biasoli I. Morais J.C. Britto L. Scheliga A. Pulcheri W. Natkunam Y. Spector N. Human germinal center-associated lymphoma protein expression is associated with improved failure-free survival in Brazilian patients with classical Hodgkin lymphoma.Leuk Lymphoma. 2009; 50: 1830-1836Crossref PubMed Scopus (14) Google Scholar All tissues were obtained before treatment, and Institutional Review Board approval was obtained for the study. For expression in normal hematopoietic tissue, whole-tissue sections of normal human spleen, thymus, bone marrow, and tonsil were used. Hematolymphoid neoplasia was classified according to the current, 2008 World Health Organization (WHO) classification.27Swerdlow S.H. Campo E. Harris N.L. Jaffe E.S. Pileri S.A. Stein H. Thiele J. Vardiman J.W. WHO Classification of Tumors of Haematopoietic and Lymphoid Tissues. IARC Press, Lyon2008Google Scholar TMAs, whole sections of human lymphoma and leukemia samples, and normal human hematopoietic tissue samples were sectioned at 0.4-μm thickness, deparaffinized in xylene, and hydrated in graduated alcohols. Slides were pretreated with 10 mmol/L citrate buffer (pH 6.0). Slides were then stained with anti-CD137 antibody (clone BBK-2; Thermo Scientific, Fremont, CA) at 1:30 dilution. Multiple different commercially available antibodies were tested, but the most consistent staining results were obtained with clone BBK-2. Slides were developed using the Dako Envision method (Dako, Carpinteria, CA) and were coverslipped with aqueous-based mounting medium. Immunohistologic staining for CD20 (L26, dilution 1:1000; Dako), CD3 (rabbit polyclonal, dilution 1:50; Cell Marque, Rocklin, CA), CD21 (IF8, dilution 1:20; Dako), CD23 (1B12, dilution 1:50; Novocastra Laboratories, Newcastle upon Tyne, UK), CD4 (IF6, dilution 1:20; Novocastra Laboratories), CD8 (C8/144B, dilution 1:200; Dako), CD30 (BerH2, dilution 1:40; Dako), CD15 (Carb3, dilution 1:250; Dako), and ALK1 (clone ALK1, dilution 1:75; Dako) were performed on a BenchMark XT (Ventana Medical Systems, Tucson, AZ), or a Leica Bond-Max (Leica Microsystems, Buffalo Grove, IL; Wetzlar, Germany) automated immunostainer, using standard retrieval conditions. In situ hybridization with the EBER probe was performed using an INFORM EBER kit on a BenchMark XT immunostainer (Ventana Medical Systems). Stains for CD21 and CD23 were performed on sections of normal tonsil and histiocytic/dendritic cell neoplasms, whereas stains for CD30, CD15, and EBER were performed on a TMA composed of classical Hodgkin lymphoma cases. The remaining immunohistochemical stains (CD3, CD4, CD8, CD20, and ALK1) were performed to further characterize select T-cell lymphoma cases. TMAs were scored as 0 = no staining, 1 = uninterpretable (loss of sample tissue or high background), 2 = weak staining (5% to 20% of cells positive), and 3 = strong staining (>20% of cells positive). For the immunohistochemistry data on tumor cell expression of CD137 (Table 1), cases were scored positive if >5% of the lesional cells stained positive for CD137. Each sample was independently scored by three pathologists (Y.N., M.W.A., and A.G.F.), with a concordance rate of 98%. Discrepant cases were jointly reviewed using a multiheaded microscope and a final score was assigned.Table 1Tumor Cell Expression of CD137 Protein Determined by ImmunohistochemistryTumor subtypeTotal positive (n/N)% PositiveHistiocytic and dendritic cell neoplasms (n = 23)6/2326 Follicular dendritic cell sarcoma6/786 Interdigitating dendritic cell tumor0/10 Langerhans cell histiocytosis0/100 Rosai-Dorfman disease0/30 Histiocytic sarcoma0/20Hodgkin lymphoma (n = 225)179/22580 Classical Hodgkin lymphoma (CHL)179/20886 Nodular lymphocyte predominant Hodgkin lymphoma (NLPHL)0/170T-cell lymphoma (n = 80)46/8058 T-lymphoblastic lymphoma/leukemia0/70 Peripheral T-cell lymphoma, not otherwise specified (PTCL NOS)9/1656 Angioimmunoblastic T-cell lymphoma (AITL)18/3256 Mycosis fungoides6/786 Large T-cell lymphoma, transformed from mycosis fungoides7/978 Subcutaneous panniculitis-like T-cell lymphoma1/1100 Anaplastic large cell lymphoma (ALK+ and ALK−)5/863Extranodal NK/T-cell lymphoma, nasal type34/9337B-cell lymphoma (n = 359)12/3593 Follicular lymphoma0/1340 Grade 1 and 20/840 Grade 3A and 3B0/500 Diffuse large B-cell lymphoma (DLBCL)0/950 Primary mediastinal large B-cell lymphoma (PMBL)7/4615 B-cell lymphoma, unclassifiable (features intermediate between DLBCL and CHL)⁎Referred to in the text as BCLU/DLBCL/CHL.5/1050 Burkitt's lymphoma0/30 Extranodal marginal zone lymphoma0/100 Splenic marginal zone lymphoma0/50 Nodal marginal zone lymphoma0/30 Mantle cell lymphoma0/120 Chronic lymphocytic leukemia/small cell lymphocytic leukemia (CLL/SLL)0/300 Lymphoplasmacytic lymphoma0/40 B-lymphoblastic lymphoma/leukemia0/50 Post-transplant lymphoproliferative disorder0/20Plasma cell neoplasms (n = 158)0/1580 Monoclonal gammopathy of undetermined significance (MGUS)0/80 Multiple myeloma0/1310 Plasma cell leukemia0/110 Plasmacytoma0/80Other (n = 17) Systemic mast cell disease0/20 Acute myeloid leukemia (various WHO subtypes)0/110 Blastic plasmacytoid dendritic cell neoplasm1/425 Referred to in the text as BCLU/DLBCL/CHL. Open table in a new tab Paraffin-embedded whole-tissue sections of normal human tonsil (n = 12), classical Hodgkin lymphoma (n = 10), peripheral T-cell lymphoma not otherwise specified (PTCL NOS) (n = 3), and angioimmunoblastic T-cell lymphoma (AITL) (n = 1) were sectioned at 0.4-μm thickness and deparaffinized; microwave-induced antigen retrieval was performed in 0.1 mol/L sodium citrate (pH 6.0). Slides were then stained with mixtures of two of three primary antibodies [CD137 (mouse anti-human) (1:30 dilution; Thermo Scientific, Fremont, CA), CD3 (rabbit anti-human) (1:100 dilution; Cell Marque), and Oct-2 (1:100 dilution; Santa Cruz Biotechnology, Santa Cruz, CA)]. Slides were then washed in PBS (pH 7.5) and incubated in the dark for 30 minutes with a mixture of two secondary antibodies: goat anti-mouse IgG labeled with Alexa Fluor 568 and goat anti-rabbit IgG labeled with Alexa Fluor 488 (Invitrogen, Carlsbad, CA). Slides were then washed in PBS (pH 7.5) and counterstained by incubation with Vectashield DAPI (Vector Laboratories, Burlingame, CA). The slides were then coverslipped with an aqueous-based mounting medium. Expression of CD137 on the surface of lymphoid cells from normal tonsil (n = 2) and from T-cell lymphoma samples (n = 4) was determined using antibodies against CD20 FITC, CD3 Pacific Blue, CD4 PerCP, CD8 APC-H7, CD45RO PE-Cy7, and CD137 PE (Pharmingen; BD Biosciences, San Jose, CA), according to standard protocols for surface staining. Cells were interrogated on an LSR II flow cytometer using FACSDiva software (BD Biosciences), and data were analyzed via Cytobank web-based software (http://www.cytobank.org). Images of normal human hematolymphoid tissue and TMA immunohistochemical staining results were acquired using a Nikon Eclipse E1000 microscope (Nikon, Tokyo, Japan) equipped with 4×, 10×, 20×, 40×, and 60× objective lenses with numerical apertures ranging from 0.05 to 0.90. Images were captured with a SPOT Flex mosaic digital camera (imaging area, 15.2 × 15.2 mm) and SPOT Basic software (SPOT Imaging Solutions; Diagnostic Instruments, Sterling Heights, MI). Immunofluorescence images were acquired using a Nikon Eclipse E800 microscope and a DXM1200C Nikon digital camera. Digitized images were processed using Adobe Illustrator software (Adobe Systems, San Jose, CA). Confocal images were obtained using a Nikon D-Eclipse C1 confocal system and processed with Nikon EZ-C1 3.90 software and Adobe Photoshop software. The stained lymphoma TMA slides were scanned and stored as high-resolution images using an automated slide scanner (Bacus Laboratories; Olympus, Center Valley, PA). Images of primary TMA staining data are available online (http://tma.stanford.edu/tma_portal/CD137). To define the expression pattern of CD137 protein in normal human lymphoid tissue, we performed immunofluorescence microscopy on paraffin-embedded tissue sections of normal tonsil. Antibodies to CD3 and the B-cell transcription factor OCT-2 highlighted a normal tonsillar immunoarchitecture of T-cell-rich interfollicular zones and B cells localized predominantly to the GC and mantle zone (Figure 1, A and B). Antibody to CD137 highlighted spindled-appearing cells localized exclusively to the GC, morphologically consistent with FDCs (Figure 1, A and B, insets). Although rare T cells in the interfollicular zones coexpressed CD137, we did not observe expression of CD137 by either GC T cells (Figure 1A, inset) or by OCT-2-expressing GC B cells (Figure 1B). Consistent with prior reports,5Lindstedt M. Johansson-Lindbom B. Borrebaeck C.A. Expression of CD137 (4-1BB) on human follicular dendritic cells.Scand J Immunol. 2003; 57: 305-310Crossref PubMed Scopus (28) Google Scholar, 7Pauly S. Broll K. Wittmann M. Giegerich G. Schwarz H. CD137 is expressed by follicular dendritic cells and costimulates B lymphocyte activation in germinal centers.J Leukoc Biol. 2002; 72: 35-42PubMed Google Scholar these results suggested that CD137 expression is restricted to FDCs in normal human tonsil. To confirm the expression of CD137 by FDCs, sections of normal tonsil were also stained with antibodies against CD21, a well-characterized marker of FDCs.28Liu Y.J. Xu J. de Bouteiller O. Parham C.L. Grouard G. Djossou O. de Saint-Vis B. Lebecque S. Banchereau J. Moore K.W. Follicular dendritic cells specifically express the long CR2/CD21 isoform.J Exp Med. 1997; 185: 165-170Crossref PubMed Scopus (117) Google Scholar Indeed, tonsillar GCs showed identical patterns of immunohistochemical reactivity using antibodies to either CD137 or CD21, suggesting that CD137 is a specific marker of FDCs (Figure 1, C and D). Next, we investigated CD137 protein expression in normal human thymus, spleen, and bone marrow using immunohistochemistry. In normal human thymus, only rare cells within the thymic medulla expressed CD137 protein (Figure 1E). No expression of CD137 was detected in the thymic cortex, suggesting that neither immature T cells nor thymic epithelial cells express CD137. In normal human spleen, CD137 expression was restricted to spindled cells with morphological features of FDCs within GCs of the white pulp (Figure 1F); CD137 expression was not detected within the mantle zones or red pulp. Sections of normal human bone marrow showed an absence of CD137 staining in hematopoietic precursors and in bone marrow stromal elements (data not shown). To evaluate the expression of CD137 in nonhematolymphoid tissue, a TMA containing samples from 61 cases of both normal and neoplastic tissues derived from human skin, soft tissue, placenta, skeletal muscle, heart, adrenal gland, salivary gland, breast, ovary, uterus, prostate, bladder, colon, stomach, pancreas, lung, kidney, liver, parathyroid gland, thyroid gland, testis, and brain was stained for CD137. No significant CD137 expression was observed in any of these nonhematopoietic tissues (see Supplemental Table S1 at http://ajp.amjpathol.org). Although previous reports identified CD137 expression in intratumoral blood vessels,23Broll K. Richter G. Pauly S. Hofstaedter F. Schwarz H. CD137 expression in tumor vessel walls High correlation with malignant tumors.Am J Clin Pathol. 2001; 115: 543-549Crossref PubMed Scopus (75) Google Scholar we did not detect significant CD137 expression by endothelial cells in either neoplastic or normal tissue. Dendritic and histiocytic cell tumors are a heterogeneous group of neoplasms that are often difficult to diagnose solely by morphological criteria. Because CD137 was strongly expressed by normal FDCs, we investigated the expression of CD137 across a diverse group of histiocytic and dendritic cell neoplasms by immunohistochemistry. We found strong and consistent cytoplasmic and cell membrane expression of CD137 in FDC neoplasms, confirming that CD137 expression is maintained during neoplastic transformation (Figure 2A and Table 1). In our series, an equivalent number of cases were positive for CD21 (6/7, or 86%) (Table 2). In one case, CD137 was positive but CD21 and CD23 were focal or negative, suggesting that CD137 may be a useful adjunct marker to diagnose FDC tumors in the absence of CD21 and CD23 staining. In contrast, CD137 expression was not observed in other histiocytic neoplasms, including Langerhans cell histiocytosis (Figure 2B) and interdigitating dendritic cell tumor (Table 1). Other spindle cell neoplasms, such as dermatofibrosarcoma protuberans and leiomyosarcoma, failed to stain with CD137 (see Supplemental Table S1 at http://ajp.amjpathol.org). One case of blastic plasmacytoid dendritic cell tumor showed weak positive staining for CD137 (Table 1). CD137 was not expressed in any subtype of acute myeloid leukemia that was evaluated (Table 1). Reactive histiocytic proliferations such as Rosai-Dorfman disease also failed to express CD137 (Table 1).Table 2Immunohistochemical Characterization of Follicular Dendritic Cell TumorsCaseCD137CD21CD23CD351+++ (weak)n/d2++ (focal)−+3+−++ (weak)4++n/d+5+ (weak)+n/dn/d6−+−−7++++n/d, not done. Open table in a new tab n/d, not done. TMAs and whole-tissue sections were used to investigate the expression of CD137 in both classical Hodgkin lymphoma (CHL, 208 cases) and nodular lymphocyte predominant Hodgkin lymphoma (NLPHL, 17 cases). We found expression of CD137 protein in the Hodgkin and Reed-Sternberg cells of 179/208 (86%) CHL cases (Figure 2C and Table 1). By immunohistochemistry, CD137 staining was localized to the membrane and cytoplasm, often with punctate paranuclear staining similar to the typical pattern of reactivity of CD15 (Figure 2C). CD137 was also expressed in a considerable proportion of cases that lacked CD15 staining (63/80, or 79%), suggesting that CD137 is a useful additional marker for CHL diagnosis with lacking CD15 expression (see Supplemental Table S2 at http://ajp.amjpathol.org). CD137 was frequently coexpressed with CD30 (176/197, 89%), but CD137 expression did not correlate with the presence of Epstein-Barr virus (EBV) as identified by in situ hybridization for EBER RNA (P = 0.98). The expression of CD137 did not correlate with overall survival (P = 0.504) or progression-free survival (P = 0.235) in our series of cases (see Supplemental Figures S1 and S2 at http://ajp.amjpathol.org). In cases of NLPHL, CD137 highlighted the expanded and disrupted FDC networks that are often prominent in this diagnosis (Figure 2D), but the atypical lymphocyte predominant cells (Figure 2D, inset) failed to stain for CD137. Immunofluorescence staining of CD137 showed crisp membranous localization with faint cytoplasmic and strong paranuclear staining when visualized using either standard (Figure 2, E and F) or confocal microscopy (Figure 2G). Very little CD137 staining was observed in the background cellular infiltrate, including infiltrating T lymphocytes (Figure 2, F and G). We next asked whether CD137 was expressed by other tumors of the lymphoid lineage, in a broad survey of 359 non-Hodgkin B-cell lymphomas and 158 plasma cell neoplasms (Table 1). In contrast to the frequent expression of CD137 in CHL, no significant CD137 expression was detected in immature B-cell tumors or the majority of mature B-cell lymphomas, including follicular lymphoma (Figure 3A), DLBCL (Figure 3B), and chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL; Figure 3C). CD137 was also not expressed by the tumor cells of marginal zone lymphoma, mantle cell lymphoma, or plasma cell neoplasms (Table 1). In contrast, we observed CD137 expression in a subset of cases of primary mediastinal large B-cell lymphoma (PMBL) (7/46, or 15%; Table 1). Of note, CD137 staining in the positive cases of PMBL highlighted large atypical Hodgkin-appearing cells but not all lymphoma cells within the biopsy specimen. Because PMBL and CHL share overlapping morphological, immunophenotypic, and molecular features, we stained an additional 10 cases of B-cell lymphoma, unclassifiable, with features intermediate between diffuse large B-cell lymphoma and classical Hodgkin lymphoma (BCLU/DLBCL/CHL), a new WHO 2008 diagnostic category that was created to formally recognize and better define cases exhibiting overlap between those two categories (the so-called gray-zone lymphoma).29Traverse-Glehen A. Pittaluga S. Gaulard P. Sorbara L. Alonso M.A. Raffeld M. Jaffe E.S. Mediastinal gray zone lymphoma: the missing link between classic Hodgkin's lymphoma and mediastinal large B-cell lymphoma.Am J Surg Pathol. 2005; 29: 1411-1421Crossref PubMed Scopus (260) Google Scholar, 30Quintanilla-Martinez L. de Jong D. de Mascarel A. Hsi E.D. Kluin P. Natkunam Y. Parrens M. Pileri S. Ott G. Gray zones around diffuse large B cell lymphoma Conclusions based on the workshop of the XIV meeting of the European Association for Hematopathology and the Society of Hematopathology in Bordeaux, France.J Hematop. 2009; 2: 211-236Crossref PubMed Scopus (62) Google Scholar Notably, we observed CD137 expression in 5 of 10 (50%) of these cases (Table 1; see also Supplemental Figure S3 at http://ajp.amjpathol.org). Although CD137 was not found in the majority of B-cell ly
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