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Short communication| Volume 179, ISSUE 4, P1630-1637, October 2011

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p52 Activation in Monomorphic B-Cell Posttransplant Lymphoproliferative Disorder/Diffuse Large B-Cell Lymphoma without BAFF-R Expression

Open ArchivePublished:August 25, 2011DOI:https://doi.org/10.1016/j.ajpath.2011.07.003
      Posttransplantation lymphoproliferative disorders (PTLD) are associated with Epstein-Barr virus (EBV) and activate the NF-κB pathway. B-cell activating factor (BAFF) modulates cell growth and survival in non-Hodgkin's lymphomas. However, there are few studies of EBV, BAFF/BAFF-R signaling, and NF-κB1 and NF-κB2 pathway activation in PTLD. Diffuse large B-cell lymphomas (DLBCL) in two different clinical contexts, immunocompetent patients (DLBCL/IC; n = 30) or posttransplantation solid-organ recipients (DLBCL/PTLD; n = 21), were characterized histogenically as germinal center (GC) or non–germinal center (NGC). Expression of BAFF, BAFF-R, and NF-κB proteins p50 and p52 and the presence or absence of EBV were compared in these clinical contexts. Regardless of the GC or NGC pattern of DLBCL, BAFF-R was expressed in 37% of DLBCL/IC but in only 4.8% of DLBCL/PTLD. p52 was expressed in DLBCL/PTLD/NGC (12 of 19 cases) as compared with DLBCL/IC/NGC (0 of 18 cases). This pattern might be related to the presence of EBV and latent membrane protein 1 because p52 expression was observed primarily in EBV-positive DLBCL/PTLD cases expressing latent membrane protein 1. Thus, the activation profile or NGC pattern of DLBCL/PTLD was not associated with BAFF/BAFF-R expression, whereas nuclear p52 related to NF-κB2 pathway activation might be linked to EBV.
      Posttransplantation lymphoproliferative disorders (PTLD) are among the worst prognostic complications after solid-organ transplantation.
      • Jacobson C.A.
      • LaCasce A.S.
      Lymphoma: risk and response after solid organ transplant [review].
      These lymphoid proliferations are characterized by their heterogeneity. Several categories of PTLD have been identified by the World Health Organization (World Health Organization classification). Among these, the B-cell monomorphic subcategory fulfills the criteria for diffuse large B-cell lymphomas (DLBCL) described in immunocompetent individuals.
      • Jaffe E.S.
      • Harris N.L.
      • Stein H.
      • Vardiman J.W.
      WHO Classification of Tumours.
      Studies of the histogenesis of PTLD also reflect this heterogeneity.
      • Capello D.
      • Rossi D.
      • Gaidano G.
      Post-transplant lymphoproliferative disorders: molecular basis of disease histogenesis and pathogenesis.
      Several studies of histogenesis origin using immunohistochemistry expression of CD10, BCL6, MUM1/IRF4, and CD138 demonstrated two patterns, germinal center (GC) and non–germinal center (NGC), including late GC/early post-GC and post-GC.
      • Carbone A.
      • Gloghini A.
      • Larocca L.M.
      • Capello D.
      • Pierconti F.
      • Canzonieri V.
      • Tirelli U.
      • Dalla-Favera R.
      • Gaidano G.
      Expression profile of MUM1/IRF4 BCL-6, and CD138/syndecan-1 defines novel histogenetic subsets of human immunodeficiency virus-related lymphomas.
      • Hans C.P.
      • Weisenburger D.D.
      • Greiner T.C.
      • Gascoyne R.D.
      • Delabie J.
      • Ott G.
      • Müller-Hermelink H.K.
      • Campo E.
      • Braziel R.M.
      • Jaffe E.S.
      • Pan Z.
      • Farinha P.
      • Smith L.M.
      • Falini B.
      • Banham A.H.
      • Rosenwald A.
      • Staudt L.M.
      • Connors J.M.
      • Armitage J.O.
      • Chan W.C.
      Confirmation of the molecular classification of diffuse large B-cell lymphoma by immunohistochemistry using a tissue microarray.
      • Johnson L.R.
      • Nalesnik M.A.
      • Swerdlow S.H.
      Impact of Epstein-Barr virus in monomorphic B-cell posttransplant lymphoproliferative disorders: a histogenetic study.
      • Chang C.C.
      • McClintock S.
      • Cleveland R.P.
      • Trzpuc T.
      • Vesole D.H.
      • Logan B.
      • Kajdacsy-Balla A.
      • Perkins S.L.
      Immunohistochemical expression patterns of germinal center and activation B-cell markers correlate with prognosis in diffuse large B-cell lymphoma.
      Epstein-Barr virus (EBV)–positive cases were primarily associated with the NGC phenotype.
      • Johnson L.R.
      • Nalesnik M.A.
      • Swerdlow S.H.
      Impact of Epstein-Barr virus in monomorphic B-cell posttransplant lymphoproliferative disorders: a histogenetic study.
      EBV-negative PTLD are primarily described as late complications and are more aggressive than EBV-positive PTLD.
      • Nelson B.P.
      • Nalesnik M.A.
      • Bahler D.W.
      • Locker J.
      • Fung J.J.
      • Swerdlow S.H.
      Epstein-Barr virus–negative post-transplant lymphoproliferative disorders: a distinct entity?.
      • Paessler M.
      • Kossev P.
      • Tsai D.
      • Raghunath P.
      • Majewski M.
      • Zhang Q.
      • Ramalingam P.
      • Schuster S.
      • Tomaszewski J.
      • Arber D.A.
      • Hsi E.
      • Wasik M.A.
      Expression of SHP-1 phosphatase indicates post–germinal center cell derivation of B-cell posttransplant lymphoproliferative disorders.
      EBV is a member of the herpesvirus family and is usually associated with mononucleosis and a wide variety of malignant lesions.
      • McClain K.L.
      Epstein-Barr virus and HIV-associated diseases.
      • Young L.S.
      • Rickinson A.B.
      Epstein-Barr virus: 40 years on.
      Latent infection leads to expression of nine proteins including latent membrane proteins LMP1, LMP2A, and LMP2B and nuclear proteins EBNA1, EBNA2, EBN3A, EBN3B, and EBN3C, as well as the small nonencoded RNAs EBER1 and EBER2.
      • Nelson B.P.
      • Nalesnik M.A.
      • Bahler D.W.
      • Locker J.
      • Fung J.J.
      • Swerdlow S.H.
      Epstein-Barr virus–negative post-transplant lymphoproliferative disorders: a distinct entity?.
      Therefore, the transcriptional activator protein EBNA2 has been described as essential for B-cell transformation and LMP1 expression. The cytoplasmic C-terminal–located CTAR1 and CTAR2 regions of LMP1 activate the NF-κB2 and NF-κB1 pathways, respectively (canonical and non-canonical pathways).
      • Wu L.
      • Nakano H.
      • Wu Z.
      The C-terminal activating region 2 of the Epstein-Barr virus–encoded latent membrane protein 1 activates NF-kappaB through TRAF6 and TAK1.
      NF-κB is a dimeric transcription factor that is regulated by the IκB protein family. Two pathways have been identified: the canonical pathway (NF-κB1), which generates a p50/p65 dimer through Iκbα degradation by IKKβ, and the non-canonical pathway (NF-κB2) leading to p52/RelB dimer release via involvement of IKKα. These two pathways lead to translocation into the nucleus of these various dimers to modulate several genes.
      • Vallabhapurapu S.
      • Karin M.
      Regulation and function of NF-kappaB transcription factors in the immune system.
      To date, several publications have reported constitutive expression of p65 in DLBCL subtypes.
      • Ramalingam P.
      • Chu W.S.
      • Tubbs R.
      • Rybicki L.
      • Pettay J.
      • Hsi E.D.
      Latent membrane protein 1, tumor necrosis factor receptor-associated factor (TRAF) 1, TRAF-2, TRAF-3, and nuclear factor kappa B expression in posttransplantation lymphoproliferative disorders.
      • Jost P.J.
      • Ruland J.
      Aberrant NF-kappaB signaling in lymphoma: mechanisms, consequences, and therapeutic implications.
      BAFF (B cell–activating factor), a member of the tumor necrosis factor family, increases the amount of peripheral B cells and is implicated in development of autoimmune diseases in a transgenic mice model overexpressing BAFF.
      • Schneider P.
      • MacKay F.
      • Steiner V.
      • Hofmann K.
      • Bodmer J.L.
      • Holler N.
      • Ambrose C.
      • Lawton P.
      • Bixler S.
      • Acha-Orbea H.
      • Valmori D.
      • Romero P.
      • Werner-Favre C.
      • Zubler R.H.
      • Browning J.L.
      • Tschopp J.
      BAFF, a novel ligand of the tumor necrosis factor family, stimulates B cell growth.
      • Mackay F.
      • Woodcock S.A.
      • Lawton P.
      • Ambrose C.
      • Baetscher M.
      • Schneider P.
      • Tschopp J.
      • Browning J.L.
      Mice transgenic for BAFF develop lymphocytic disorders along with autoimmune manifestations.
      Moreover, BAFF modulates cell growth and survival in multiple myeloma and B-cell chronic lymphocytic leukemia.
      • Novak A.J.
      • Darce J.R.
      • Arendt B.K.
      • Harder B.
      • Henderson K.
      • Kindsvogel W.
      • Gross J.A.
      • Greipp P.R.
      • Jelinek D.F.
      Expression of BCMA TACI, and BAFF-R in multiple myeloma: a mechanism for growth and survival.
      • Novak A.J.
      • Bram R.J.
      • Kay N.E.
      • Jelinek D.F.
      Aberrant expression of B-lymphocyte stimulator by B chronic lymphocytic leukemia cells: a mechanism for survival.
      BAFF is expressed in non–Hodgkin's lymphoma–like mantle and marginal zones, follicular lymphoma, and DLBCL.
      • Novak A.J.
      • Grote D.M.
      • Stenson M.
      • Ziesmer S.C.
      • Witzig T.E.
      • Habermann T.M.
      • Harder B.
      • Ristow K.M.
      • Bram R.J.
      • Jelinek D.F.
      • Gross J.A.
      • Ansell S.M.
      Expression of BLyS and its receptors in B-cell non-Hodgkin lymphoma: correlation with disease activity and patient outcome.
      BAFF-R, also known as BR3, TNFRSF13C, or CD268, is a type III transmembrane protein that is considered the principal receptor of BAFF leading to the survival and maturation of primary B cells.
      • Treml J.F.
      • Hao Y.
      • Stadanlick J.E.
      • Cancro M.P.
      The BLyS family: toward a molecular understanding of B cell homeostasis.
      BAFF-R is the only specific receptor of BAFF.
      • Thompson J.S.
      • Bixler S.A.
      • Qian F.
      • Vora K.
      • Scott M.L.
      • Cachero T.G.
      • Hession C.
      • Schneider P.
      • Sizing I.D.
      • Mullen C.
      • Strauch K.
      • Zafari M.
      • Benjamin C.D.
      • Tschopp J.
      • Browning J.L.
      • Ambrose C.
      BAFF-R, a newly identified TNF receptor that specifically interacts with BAFF.
      Expression of BAFF-R was first detected in tonsils, and demonstrated strong staining on B cells of the mantle zone and weak staining of GC B lymphocytes.
      • Ng L.G.
      • Sutherland A.P.
      • Newton R.
      • Qian F.
      • Cachero T.G.
      • Scott M.L.
      • Thompson J.S.
      • Wheway J.
      • Chtanova T.
      • Groom J.
      • Sutton I.J.
      • Xin C.
      • Tangye S.G.
      • Kalled S.L.
      • Mackay F.
      • Mackay C.R.
      B cell–activating factor belonging to the TNF family (BAFF)-R is the principal BAFF receptor facilitating BAFF costimulation of circulating T and B cells.
      Similar staining has been also described in other studies. Furthermore, several non–Hodgkin's lymphomas have been investigated through BAFF-R expression, which was recently exhibited in most lymphoproliferative disorders.
      • Nakamura N.
      • Hase H.
      • Sakurai D.
      • Yoshida S.
      • Abe M.
      • Tsukada N.
      • Takizawa J.
      • Aoki S.
      • Kojima M.
      • Nakamura S.
      • Kobata T.
      Expression of BAFF-R (BR 3) in normal and neoplastic lymphoid tissues characterized with a newly developed monoclonal antibody.
      • Zhang X.
      • Park C.S.
      • Yoon S.O.
      • Li L.
      • Hsu Y.M.
      • Ambrose C.
      • Choi Y.S.
      BAFF supports human B cell differentiation in the lymphoid follicles through distinct receptors.
      • Rodig S.J.
      • Shahsafaei A.
      • Li B.
      • Mackay C.R.
      • Dorfman D.M.
      BAFF-R, the major B cell–activating factor receptor, is expressed on most mature B cells and B-cell lymphoproliferative disorders.
      BAFF-R signaling activates NEMO-independent processing of the NF-κB2 pathway.
      • Claudio E.
      • Brown K.
      • Park S.
      • Wang H.
      • Siebenlist U.
      BAFF-induced NEMO-independent processing of NF-kappa B2 in maturing B cells.
      Moreover, LMP1 activates the BAFF gene promoter via NF-κB. The ability of BAFF to increase cell survival and of BAFF-R and LMP1 to activate the NF-κB pathway, and the strong association of EBV in PTLD constitute at least three reliable reasons to study the role of EBV, NF-κB2, and BAFF-R in PTLD.
      To analyze some differences in BAFF-R expression according to the activation level of B cells, we tested the expression of BAFF and BAFF-R in 51 DLBCL cases from the general population (30 patients) and immunodeficient patients (21 with PTLD). All cases were characterized according to morphologic features, immunophenotype, and GC or NGC histogenesis pattern. In EBV-positive cases, LMP1, EBNA2, and ZEBRA, an immediate replicative protein, were tested. The detection of nuclear or cytoplasmic expression of p50 and p52 proteins to analyze the activation of NF-κB pathways demonstrated a correlation between p52 expression and the presence of EBV, as well as latency II or III, ie, expression of LMP1 in PTLD.

      Materials and Methods

      Patients

      Fifty-one DLBCL cases were collected. Patients enrolled in the present study were followed up in two solid-organ transplantation centers, the Bicêtre and Paul Brousse University hospitals. All lymphomas in this series were monomorphic B-cell lymphomas according to the criteria of the World Health Organization classification. Written informed consent was obtained from all included patients. The DLBCL cases comprised two subgroups: 30 immunocompetent patients with DLBCL (DLBCL/IC) and 21 solid-organ recipients with DLBCL (DLBCL/PTLD). Patient clinical data are given in Table 1. Most DLBCL cases, either DLBCL/IC or DLBCL/PTLD, were localized in lymph nodes (34 of 51). Mean age of patients with DLBCL/PTLD at diagnosis of lymphoma was 39 years (range, 1 to 74 years), and of patients with DLBCL/IC was 61.5 years (range, 18 to 92 years).
      Table 1Clinical Data in Patients with DLBCL
      PatientsAge at diagnosis (year)SexGraft typeDelay of lymphoma occurrence (mo)LocalizationHistologic type
      135MNRNRLymph nodesDLBCL/IC
      255FNRNRCavumDLBCL/IC
      332MNRNRTonsilsDLBCL/IC
      463FNRNRLymph nodesDLBCL/IC
      581FNRNRLymph nodesDLBCL/IC
      681FNRNRLymph nodesDLBCL/IC
      765MNRNRLymph nodesDLBCL/IC
      860MNRNRLymph nodesDLBCL/IC
      975MNRNRLymph nodesDLBCL/IC
      1092FNRNRLymph nodesDLBCL/IC
      1118MNRNRLymph nodesDLBCL/IC
      1273MNRNRLymph nodesDLBCL/IC
      1352MNRNRLymph nodesDLBCL/IC
      1451FNRNRLymph nodesDLBCL/IC
      1585MNRNRLymph nodesDLBCL/IC
      1675MNRNRLymph nodesDLBCL/IC
      1782FNRNRORLDLBCL/IC
      1877FNRNRLymph nodesDLBCL/IC
      1979FNRNRLymph nodesDLBCL/IC
      2058FNRNRORLDLBCL/IC
      2180FNRNRLymph nodesDLBCL/IC
      2234FNRNRKidneyDLBCL/IC
      2346FNRNRCavumDLBCL/IC
      2458FNRNRLymph nodesDLBCL/IC
      2571MNRNRLymph nodesDLBCL/IC
      2653MNRNRLymph nodesDLBCL/IC
      2758MNRNRGutDLBCL/IC
      2856MNRNRGallbladderDLBCL/IC
      2960MNRNRLymph nodesDLBCL/IC
      3089MNRNRLymph nodesDLBCL/IC
      3151MLiver109LiverDLBCL/PTLD
      3216MLiver5CavumDLBCL/PTLD
      3322MLiver1Lymph nodesDLBCL/PTLD
      3451MLung6LungDLBCL/PTLD
      3564FKidney114Lymph nodesDLBCL/PTLD
      363FLiver27ORLDLBCL/PTLD
      372FLiver6LiverDLBCL/PTLD
      385FLiver32TonsilDLBCL/PTLD
      3956MLiver109Lymph nodesDLBCL/PTLD
      403FLiver2CavumDLBCL/PTLD
      4166FKidney259Small intestineDLBCL/PTLD
      4239MKidney84Lymph nodesDLBCL/PTLD
      4374MLiver49Lymph nodesDLBCL/PTLD
      442FLiver7TonsilsDLBCL/PTLD
      4554MKidney + pancreas5BrainDLBCL/PTLD
      463MLiver5Lymph nodesDLBCL/PTLD
      4749MKidney + pancreas162Lymph nodesDLBCL/PTLD
      481MLiver10TonsilDLBCL/PTLD
      4936FKidney152PeritoneumDLBCL/PTLD
      5053FKidney192Lymph nodesDLBCL/PTLD
      5159MKidney81BrainDLBCL/PTLD
      DLBCL, diffuse large B-cell lymphoma; DLBCL/IC, DLBCL in immunocompetent patients; F, female; M, male; NR, not relevant; ORL, otorhinolaryngology; PTLD, post-transplantation lymphoproliferative disorder.

      Conventional Morphologic Analysis and Immunostaining

      Formalin-fixed, paraffin-embedded tissue sections from DLBCL biopsy specimens were stained using H&E for conventional morphologic analysis. The slides were reviewed by two hematopathologists (M.R. and T.L.) to reclassify cases according to the World Health Organization criteria. Immunostaining was performed on paraffin sections using an immunoperoxidase technique (ChemMate EnVision Detection Kit, DakoCytomation A/S, Glostrup, Denmark, or the Vectastain Elite ABC kit, Vector Laboratories, Inc., Burlingame, CA) according to the manufacturer's recommendations. To identify GC or NGC pattern, the following B-cell markers were tested: CD20 (clone L26; dilution 1:1950; DakoCytomation A/S), CD10 (clone 56C6; dilution 1:40; Novocastra, Newcastle-upon-Tyne, England), BCL6 (clone PG-B6p; dilution 1:40; DakoCytomation A/S), IRF4/MUM1 (clone M-17; dilution 1:400; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), and CD138 (syndecan-1; clone B-A38; dilution 1:100; Diaclone, Stamford, CT).
      Using the same technique, expression of EBV latency proteins LMP1 (clone CS.1–4; dilution 1:100), EBNA2 (clone PE2; dilution 1:100), and the replicative protein ZEBRA (clone BZ.1; dilution 1:25) (all three from DakoCytomation A/S) were studied, as was expression of BAFF (clone buffy-2; dilution 1:100) and BAFF-R (clone 11C1; dilution 1:600) (both from Alexis Biochemicals Corp., Lausen, Switzerland). For BAFF and BAFF-R, cases exhibiting more than 30% of immunostained tumor cells were considered positive.
      • Hans C.P.
      • Weisenburger D.D.
      • Greiner T.C.
      • Gascoyne R.D.
      • Delabie J.
      • Ott G.
      • Müller-Hermelink H.K.
      • Campo E.
      • Braziel R.M.
      • Jaffe E.S.
      • Pan Z.
      • Farinha P.
      • Smith L.M.
      • Falini B.
      • Banham A.H.
      • Rosenwald A.
      • Staudt L.M.
      • Connors J.M.
      • Armitage J.O.
      • Chan W.C.
      Confirmation of the molecular classification of diffuse large B-cell lymphoma by immunohistochemistry using a tissue microarray.
      Protein expression of p50 (dilution 1:40; Santa Cruz Biotechnology, Inc.) and p52 (dilution 1:400; Millipore Corp., Billerica, MA) was also assessed using immunoperoxidase to explore NF-κB pathways. Localization of staining was specified as nuclear (activated NF-κB pathway) or cytoplasmic.

      Detection and Characterization of EBV

      EBV status of all DLBCL was performed via in situ hybridization using EBER1 and EBER2 probes with the PNA In Situ Hybridization Detection Kit (DakoCytomation A/S) according to the manufacturer's recommendations. In EBV, EBER-positive cases, latency was defined as latency I (EBER-positive, LMP1-negative, or EBNA2-negative), latency II (EBER-positive, LMP1-positive, or EBNA2-negative), and latency III (EBER-positive, LMP1-positive, or EBNA2-positive).

      Controls

      External controls tested included reactive lymph nodes or tonsils and lymphomatous tissues known to express BAFF and BAFF-R: follicular lymphoma, 6 cases; mantle cell lymphoma, 5 cases; marginal zone B-cell lymphoma, 4 cases; MALT (mucosa-associated lymphoid tissue) lymphoma, 6 cases; and Burkitt's lymphoma, 6 cases.

      Results

      Characterization and Histogenesis of DLBCL/IC and DLBCL/PTLD

      The definition of GC origin means a GC B-cell pattern having the expression of CD10 and/or BCL6 or activated GC B-cell pattern according to expression of at least one GC B-cell marker, either BCL6 or CD10, and at least one activation marker, either IRF4/MUM1 or CD138. The activated NGC B-cell pattern means expression of activation markers IRF4/MUM1 and/or CD138 but not GC B-cell markers.
      • Chang C.C.
      • McClintock S.
      • Cleveland R.P.
      • Trzpuc T.
      • Vesole D.H.
      • Logan B.
      • Kajdacsy-Balla A.
      • Perkins S.L.
      Immunohistochemical expression patterns of germinal center and activation B-cell markers correlate with prognosis in diffuse large B-cell lymphoma.
      DLBCL/PTLD specimens generally demonstrated an activated pattern (Table 2, Table 3): of 21 cases, 19 expressed the NGC phenotype, and 2 could not be characterized because of nondetection of the four markers.
      • Vakiani E.
      • Basso K.
      • Klein U.
      • Mansukhani M.M.
      • Narayan G.
      • Smith P.M.
      • Murty V.V.
      • Dalla-Favera R.
      • Pasqualucci L.
      • Bhagat G.
      Genetic and phenotypic analysis of B-cell post-transplant lymphoproliferative disorders provides insights into disease biology.
      Of 30 DLBCL/IC cases, 12 expressed the GC pattern, and 18 expressed the NGC pattern (Table 3).
      Table 2Immunohistochemical Staining of DLBCL Samples
      DLBCLCD10BCL6IRF4CD138BAFFBAFF-REBERLMP1EBNA2ZEBRAP52P50Origin
      1+++++++NRNRNRGC
      2+++++NRNRNRNuNuGC
      3++++++NRNRNRGC
      4++++NRNRGC
      5++++NRNRNuGC
      6++-+++NRNRNRGC
      7++++NRNRNRGC
      8+++++NRNRNRNuNuGC
      9++++NRNRNRGC
      10+++++NRNRNRNuGC
      11++-++++++NRNRNRNuGC
      12+++++NRNRNRNuGC
      13+++++++NRNRNRNGC
      14++++++NRNRNRNGC
      15++++++NRNRNRNGC
      16+++NRNRNRNGC
      17++++NRNRNRNGC
      18++++NRNRNRNGC
      19++++NRNRNRNGC
      20++++NRNRNRNuNGC
      21++++NRNRNRNGC
      22++++++++NuNGC
      23+++++NRNRNRNGC
      24+++++NuNGC
      25+++NRNRNRNGC
      26++++++NRNRNRNGC
      27+++++++NRNRNRNGC
      28+++NRNRNRNGC
      29++++NRNRNRNGC
      30+++NRNRNRNGC
      31+++++NRNRNRNGC
      32+++++NRNRNRNuNuNGC
      33++++ND+NuNDNGC
      34+++++NRNRNRNuNGC
      35++NRNRNRNGC
      36++++++++NuNGC
      37++++++++NuNuNGC
      38++++++++NuNuNGC
      39++NRNRNRNDNGC
      40+++++++NuNuNGC
      41+++NGC
      42+NRNRNRNGC
      43++NRNRNRNuNGC
      44+++++++NuNGC
      45+++++++NuNuNGC
      46++++++++NuNGC
      47+++NRNRNRNuNGC
      48++++++++NuNGC
      49+++++NRNRNRNuNGC
      50-++++NuNuND
      51+++++++NuND
      Nos. 1 to 30, DLBCL/IC; 31 to 51, DLBCL/PTLD.
      BAFF/BAFF-R staining, semiquantitative evaluation: +++, >60%; ++, 30% to 60%; +, <30%; −, negative. For other markers: +, >30%; −, negative.
      GC, germinal center; ND, not determined; NGC, non–germinal center; NR, not relevant; Nu, nuclear staining.
      Table 3Expression of BAFF/BAFF-R, NF-κB Proteins, and EBV in Patients with DLBCL
      Lymphoma typeOriginNo. of patientsBAFFBAFF-Rp52p50EBVLMP1
      ICGC1211 of 126 of 125 of 123 of 120ND
      ICNGC1817 of 185 of 180 of 183 of 182/182/2
      PTLDNGC1917 of 191 of 19
      Statistically significant: P < 2.10−4 for p52, P < 0.008 for BAFF-R.
      12 of 19
      Statistically significant: P < 2.10−4 for p52, P < 0.008 for BAFF-R.
      7 of 1710 of 199 of 10
      PTLDND22 of 20 of 22 of 21 of 22 of 21 of 2
      GC, germinal center; IC, immunocompetent; EBV, Epstein-Barr virus; LMP1, latent membrane protein 1; ND, not determined; NGC, non–germinal center; p50 and p52 positive staining, nuclear localization (>10%); PTLD, post-transplantation lymphoproliferative disorders.
      low asterisk Statistically significant: P < 2.10−4 for p52, P < 0.008 for BAFF-R.

      Detection and Characterization of EBV

      EBV was detected in 12 of 21 DLBCL/PTLD cases (57%) versus only 2 of 30 DLBCL/IC cases that were EBV-positive. The EBV latency status was characterized in 11 of 12 DLBCL/PTLD EBV-positive cases exhibiting different types of latency: latency I, 2 cases; latency II, 3 cases; and latency III, 6 cases (Table 2).
      Nine cases expressed ZEBRA: one DLBCL/IC case with latency II, and eight DLBCL/PTLD cases, of which five cases demonstrated latency III, two exhibited latency II, and latency in one case was undetermined.

      Controls of BAFF and BAFF-R Immunostaining

      BAFF and BAFF-R expression obtained in different lymphomatous tissues demonstrated the same positivity as has been previously described in the literature (Figure 1). In brief, all lymphomas expressed BAFF, whereas BAFF-R was expressed in follicular, mantle, and marginal zone lymphomas but was negative in Burkitt's lymphoma.
      • Nakamura N.
      • Hase H.
      • Sakurai D.
      • Yoshida S.
      • Abe M.
      • Tsukada N.
      • Takizawa J.
      • Aoki S.
      • Kojima M.
      • Nakamura S.
      • Kobata T.
      Expression of BAFF-R (BR 3) in normal and neoplastic lymphoid tissues characterized with a newly developed monoclonal antibody.
      • Rodig S.J.
      • Shahsafaei A.
      • Li B.
      • Mackay C.R.
      • Dorfman D.M.
      BAFF-R, the major B cell–activating factor receptor, is expressed on most mature B cells and B-cell lymphoproliferative disorders.
      Figure thumbnail gr1
      Figure 1BAFF, BAFF-R, and p52 staining. A–C: BAFF staining. D–F: BAFF-R staining. G–I: p52 staining. A: Tonsil control. High expression in GC cells, weak expression in mantle cells, and heterogeneous staining in the interfollicular zone. B: Positive staining. C: Weak staining. D: Tonsil control. High expression in mantle cells and heterogeneous staining in GC cells. E: Positive staining. F: Negative staining. G: Tonsil control. Nuclear (arrow) and cytoplasmic staining in GC cells. H: Positive staining, nuclear localization (arrow). I: Negative staining, cytoplasmic localization (arrow).

      Expression of BAFF and BAFF-R

      BAFF expression was predominantly detected in almost all cases in the entire series of DLBCL [47 of 51 cases (92%)]. Only two DLBCL/PTLD cases exhibiting the NGC pattern and two DLBCL/IC cases exhibiting the NGC and GC patterns, respectively, did not express BAFF. Of note, regardless of the histogenesis pattern, BAFF-R was weakly expressed in only 1 of 21 (4.88%) DLBCL/PTLD cases, in contrast with DLBCL/IC cases, of which 11 of 30 (37%) were positive for BAFF-R. This distinct BAFF-R expression was statistically significant (P < 0.008).

      Expression of NF-κB Pathways

      In the entire series of DLBCL (DLBCL/IC or DLBCL/PTLD) demonstrating the NGC pattern (37 cases), LMP1 expression observed in 11 cases was associated with NF-κB activation, regardless of the pathway (p50 and/or p52). Among these 11 LMP1-positive cases, 8 (73%) expressed nuclear p52 protein. Nine of these 11 cases were from the subgroup DLBCL/PTLD, associated with p50 in 4 cases. In contrast, in the LMP1-negative subgroup (EBV-positive or EBV-negative), p52 nuclear expression was observed in only 4 of 26 (15%) NGC DLBCL cases (Figure 1). This difference was statistically significant (P = 0.0014). No correlation was observed between p52 and BAFF-R protein expression. p52 expression with the NGC pattern was positive in 12 of 19 cases (63%). Furthermore, none of 18 DLBCL/IC cases with the NGC pattern expressed p52 (Table 3). This distinct p52 expression was highly statistically significant (P < 2.10−4). p52 expression between DLBCL/PTLD and DLBCL/IC with the GC profile could not be compared because of the absence of GC pattern in our series of DLBCL/PTLD cases. Moreover, p52 expression seems to be associated with ZEBRA expression in DLBCL/PTLD because almost all cases expressing ZEBRA [7 of 8 cases (lsqb]87%)] demonstrated an NF-κB2–activated profile at nuclear p52 staining.

      Discussion

      In the present study, 51 DLBCL cases, divided into two subcategories, DLBCL/PTLD and DLBCL/IC, characterized by the level of activation of lymphomatous B cells and their histogenesis profile, ie, GC or NGC pattern, were investigated for BAFF-R and p52 expression.
      • Chang C.C.
      • McClintock S.
      • Cleveland R.P.
      • Trzpuc T.
      • Vesole D.H.
      • Logan B.
      • Kajdacsy-Balla A.
      • Perkins S.L.
      Immunohistochemical expression patterns of germinal center and activation B-cell markers correlate with prognosis in diffuse large B-cell lymphoma.
      In cases demonstrating the NGC pattern, a statistically significant difference of p52 expression was observed between DLBCL/PTLD [12 of 19 cases (63%)] and DLBCL/IC (0 of 18 cases). Despite BAFF-R expression, no association with p52 expression was observed. However, regardless of the histogenesis profile (GC versus NGC), BAFF-R expression, which was rarely observed in DLBCL/PTLD [1 of 21 cases (4.8%)], was statistically different from DLBCL/IC, in which 11 of 30 cases (37%) were positive.
      Among the 21 DLBCL/PTLD cases, the NGC phenotype was expressed in 19, whereas 18 of 30 DLBCL/IC cases demonstrated the NGC profile. EBV was present in 14 cases, 2 DLBCL/IC and 12 DLBCL/PTLD. All but two EBV-associated cases demonstrated the NGC histogenesis pattern. The percentage of EBV-associated DLBCL/PTLD was 57%. In the present series, this low percentage of EBV-positive PTLD can be related to the high number of PTLD cases occurring later than 24 months after transplantation, as has previously been described.
      • Leblond V.
      • Dhedin N.
      • Mamzer Bruneel M.F.
      • Choquet S.
      • Hermine O.
      • Porcher R.
      • Nguyen Quoc S.
      • Davi F.
      • Charlotte F.
      • Dorent R.
      • Barrou B.
      • Vernant J.P.
      • Raphael M.
      • Levy V.
      Identification of prognostic factors in 61 patients with posttransplantation lymphoproliferative disorders.
      BAFF expression was detected in 92% of the 51 DLBCL cases. This result could reflect ligand-receptor engagement as well as environmental- or cellular-secreted BAFF. Indeed, monoclonal antibodies against human BAFF recognized membrane-bound and soluble BAFF.
      • Schneider P.
      • MacKay F.
      • Steiner V.
      • Hofmann K.
      • Bodmer J.L.
      • Holler N.
      • Ambrose C.
      • Lawton P.
      • Bixler S.
      • Acha-Orbea H.
      • Valmori D.
      • Romero P.
      • Werner-Favre C.
      • Zubler R.H.
      • Browning J.L.
      • Tschopp J.
      BAFF, a novel ligand of the tumor necrosis factor family, stimulates B cell growth.
      The autocrine pathway could explain this expression, as described in other models such as chronic lymphocytic lymphoma and multiple myeloma.
      • Novak A.J.
      • Darce J.R.
      • Arendt B.K.
      • Harder B.
      • Henderson K.
      • Kindsvogel W.
      • Gross J.A.
      • Greipp P.R.
      • Jelinek D.F.
      Expression of BCMA TACI, and BAFF-R in multiple myeloma: a mechanism for growth and survival.
      • Kern C.
      • Cornuel J.F.
      • Billard C.
      • Tang R.
      • Rouillard D.
      • Stenou V.
      • Defrance T.
      • Ajchenbaum-Cymbalista F.
      • Simonin P.Y.
      • Feldblum S.
      • Kolb J.P.
      Involvement of BAFF and APRIL in the resistance to apoptosis of B-CLL through an autocrine pathway.
      Study of BAFF-R expression was performed to respond to this hypothesis of the autocrine signaling pathway.
      Little is known about BAFF-R expression relative to the level of DLBCL activation. Only one study has explored this correlation in association with Lck protein.
      • Paterson J.C.
      • Tedoldi S.
      • Craxton A.
      • Jones M.
      • Hansmann M.L.
      • Collins G.
      • Roberton H.
      • Natkunam Y.
      • Pileri S.
      • Campo E.
      • Clark E.A.
      • Mason D.Y.
      • Marafioti T.
      The differential expression of LCK and BAFF-receptor and their role in apoptosis in human lymphomas.
      Our results were similar to those of Paterson et al.
      • Paterson J.C.
      • Tedoldi S.
      • Craxton A.
      • Jones M.
      • Hansmann M.L.
      • Collins G.
      • Roberton H.
      • Natkunam Y.
      • Pileri S.
      • Campo E.
      • Clark E.A.
      • Mason D.Y.
      • Marafioti T.
      The differential expression of LCK and BAFF-receptor and their role in apoptosis in human lymphomas.
      ; indeed, no differences were detected between the histogenesis profile and BAFF-R expression in the DLBCL/IC subgroup. We observed that BAFF-R was expressed in 11 of 30 DLBCL/IC cases (37%), which corresponds to the mean percentage of previously published results.
      • Nakamura N.
      • Hase H.
      • Sakurai D.
      • Yoshida S.
      • Abe M.
      • Tsukada N.
      • Takizawa J.
      • Aoki S.
      • Kojima M.
      • Nakamura S.
      • Kobata T.
      Expression of BAFF-R (BR 3) in normal and neoplastic lymphoid tissues characterized with a newly developed monoclonal antibody.
      • Rodig S.J.
      • Shahsafaei A.
      • Li B.
      • Mackay C.R.
      • Dorfman D.M.
      BAFF-R, the major B cell–activating factor receptor, is expressed on most mature B cells and B-cell lymphoproliferative disorders.
      • Paterson J.C.
      • Tedoldi S.
      • Craxton A.
      • Jones M.
      • Hansmann M.L.
      • Collins G.
      • Roberton H.
      • Natkunam Y.
      • Pileri S.
      • Campo E.
      • Clark E.A.
      • Mason D.Y.
      • Marafioti T.
      The differential expression of LCK and BAFF-receptor and their role in apoptosis in human lymphomas.
      However, regardless of the histogenesis profile, BAFF-R expression was higher in DLBCL/IC (37%) than in DLBCL/PTLD (4.8%), with a statistically significant correlation. Thus, the hypothesis of the autocrine pathway through BAFF-R could be excluded. Nevertheless, receptors TACI and BCMA could also be implicated in this signaling pathway; however, their expression was not tested in the present series. The high level of BAFF expression in NHL, based on transcription enhancement through BAFF promoter, could be related to the possible effect of the polymorphism −871C→T (or other genetic variation) or to the nuclear translocation of the CD40-cRel dimer.
      • Paterson J.C.
      • Tedoldi S.
      • Craxton A.
      • Jones M.
      • Hansmann M.L.
      • Collins G.
      • Roberton H.
      • Natkunam Y.
      • Pileri S.
      • Campo E.
      • Clark E.A.
      • Mason D.Y.
      • Marafioti T.
      The differential expression of LCK and BAFF-receptor and their role in apoptosis in human lymphomas.
      • Novak A.J.
      • Grote D.M.
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      • Kline M.P.
      • Manske M.K.
      • Slager S.
      • Witzig T.E.
      • Shanafelt T.
      • Call T.G.
      • Kay N.E.
      • Jelinek D.F.
      • Cerhan J.R.
      • Gross J.A.
      • Harder B.
      • Dillon S.R.
      • Ansell S.M.
      Elevated serum B-lymphocyte stimulator levels in patients with familial lymphoproliferative disorders.
      • Novak A.J.
      • Slager S.L.
      • Fredericksen Z.S.
      • Wang A.H.
      • Manske M.M.
      • Ziesmer S.
      • Liebow M.
      • Macon W.R.
      • Dillon S.R.
      • Witzig T.E.
      • Cerhan J.R.
      • Ansell S.M.
      Genetic variation in B-cell–activating factor is associated with an increased risk of developing B-cell non-Hodgkin lymphoma.
      • Zhou H.J.
      • Pham L.V.
      • Tamayo A.T.
      • Lin-Lee Y.C.
      • Fu L.
      • Yoshimura L.C.
      • Ford R.J.
      Nuclear CD40 interacts with c-Rel and enhances proliferation in aggressive B-cell lymphoma.
      In addition, BAFF-R weak expression could be explained by possible down-regulation of BAFF-R arising from BAFF binding.
      • Sellam J.
      • Miceli-Richard C.
      • Gottenberg J.E.
      • Ittah M.
      • Lavie F.
      • Lacabaratz C.
      • Gestermann N.
      • Proust A.
      • Lambotte O.
      • Mariette X.
      Decreased B cell activating factor receptor expression on peripheral lymphocytes associated with increased disease activity in primary Sjogren's syndrome and systemic lupus erythematosus.
      • Nimmanapalli R.
      • Lyu M.A.
      • Du M.
      • Keating M.J.
      • Rosenblum M.G.
      • Gandhi V.
      The growth factor fusion construct containing B-lymphocyte stimulator (BLyS) and the toxin rGel induces apoptosis specifically in BAFF-R–positive CLL cells.
      • Bloom D.
      • Chang Z.
      • Pauly K.
      • Kwun J.
      • Fechner J.
      • Hayes C.
      • Samaniego M.
      • Knechtle S.
      BAFF is increased in renal transplant patients following treatment with alemtuzumab.
      EBV activates the NF-κB pathway through LMP1 protein, and the link between this activation pathway and lymphomas has also been described.
      • Wu L.
      • Nakano H.
      • Wu Z.
      The C-terminal activating region 2 of the Epstein-Barr virus–encoded latent membrane protein 1 activates NF-kappaB through TRAF6 and TAK1.
      • Jost P.J.
      • Ruland J.
      Aberrant NF-kappaB signaling in lymphoma: mechanisms, consequences, and therapeutic implications.
      As expected, in our series, all 11 NGC DLBCL cases positive for LMP1 were associated with NF-κB activation. Among these cases, p52 was generally expressed (8 of 11 cases); however, it was positive in only 4 of 26 cases in the LMP1-negative subgroup whether EBV was or was not present. BAFF-R expression could not explain these results because there was no correlation with p52 nuclear localization, which suggests that the BAFF/BAFF-R signaling pathway could not be directly implicated in DLBCL lymphomagenesis mediated by p52 signaling. The dissociation between BAFF/BAFF-R and p52 signaling pathways could be interpreted via repression of NF-κB activity mediated by BCL6 protein expression or the posttranslational modification of RelB.
      • Perez-Rosado A.
      • Artiga M.
      • Vargiu P.
      • Sanchez-Aguilera A.
      • Alvarez-Barrientos A.
      • Piris M.
      BCL6 represses NFkappaB activity in diffuse large B-cell lymphomas.
      • Neumann M.
      • Naumann M.
      Beyond IkappaBs: alternative regulation of NF-kappaB activity.
      The nuclear expression of p50 and/or p52 in six cases demonstrating the GC pattern in DLBCL/IC could be related to deregulation of the NF-κB pathway caused by multiple genetic events including negative and positive regulators of NF-κB, as previously reported by Compagno et al.
      • Compagno M.
      • Lim W.K.
      • Grunn A.
      • Nandula S.V.
      • Brahmachary M.
      • Shen Q.
      • Bertoni F.
      • Ponzoni M.
      • Scandurra M.
      • Califano A.
      • Bhagat G.
      • Chadburn A.
      • Dalla-Favera R.
      • Pasqualucci L.
      Mutations of multiple genes cause deregulation of NF-kappaB in diffuse large B-cell lymphoma.
      The higher expression of p52 related to the NF-κB2 activation pathway in DLBCL/PTLD, which is statistically different from the NGC pattern in DLBCL/IC, could be related not only to genetic events but also to activation of NF-κB2 via LMP1.
      Moreover, it must be emphasized that in seven of eight cases expressing ZEBRA in EBV-positive DLBCL/PTLD cases, p52 demonstrated nuclear staining, which suggests a relationship between the replicative cycle of EBV and p52 nuclear expression. However, this result must be confirmed in a larger series.
      Thus, our results demonstrating rare expression of BAFF-R and the frequency of NF-κB2 pathway activation in DLBCL/PTLD as compared with DLBCL/IC confirm the importance of EBV, especially LMP1, and suggest other mechanisms of NF-κB2 pathway deregulation in these tumors.

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