722 Vol. 9, 722–728, February 2003 Clinical Cancer Research Immunohistochemical Expression of CD23 and CD40 May Identify Prognostically Favorable Subgroups of Diffuse Large B-cell Lymphoma: A Nordic Lymphoma Group Study1 Johan Linderoth,2 Mats Jerkeman, to a germinal center origin or attributable to increased Eva Cavallin-Ståhl, Stein Kvaløy, and apoptosis via induction of bax and/or enhanced T-cell inter- Emina Torlakovic action, resulting in improved autologous tumor response. Confirmatory studies are necessary. Department of Oncology, Lund University Hospital, 221 85 Lund, Sweden [J. L., M. J., E. C-S.]; Department of Oncology, the Norwegian Radium Hospital, Oslo, Norway [S. K.]; and Department INTRODUCTION of Pathology, The Norwegian Radium Hospital, Oslo, Norway [E. T.] DLBCL3 is the most frequent lymphoma subtype and en- compasses the majority, ϳ60–70%, of the aggressive lympho- mas. Biologically and clinically, it shows considerable hetero- ABSTRACT geneity. Eventually, 30–40% of the patients with advanced Purpose: In search for subgroups of diffuse large B-cell stage DLBCL who are treated with current therapies will be lymphoma (DLBCL) with different histogenetic origin and long-time survivors, whereas the rest will succumb to the dis- prognosis, as has been described by gene expression profil- ease. There is an obvious need for prognostic information to ing, we examined tumor specimens from 125 patients with design a more risk-adapted primary therapy. The IPI is one DLBCL, uniformly treated by either cyclophosphamide- available, validated tool, based on clinical features (age, stage, Adriamycin-vincristine-prednisone or methotrexate, doxo- level of lactate dehydrogenase, performance status, and number rubicin, cyclophosphamide, vincristine, prednisone, and of extranodal sites; Ref. 1). However, the IPI is not an ultimate bleomycin in a multicenter trial set by the Nordic Lym- instrument because a considerable proportion of patients classi- phoma Group 1989–1994. fied as “low” or “low-intermediate risk” eventually shows ther- Experimental Design: Bcl-6, CD10, and CD40 were cho- apeutic failure. Consequently, attempts have been made to iden- sen as markers for a germinal center phenotype, CD23 as a tify other biological prognostic factors to be used separately or marker of pre/early germinal center origin, and CD138 as a combined with the IPI. According to the recent WHO classifi- marker for postgerminal center origin. In addition, expres- cation, there are several morphological variants of DLBCL: (a) sion of the apoptotic regulators bcl-2 and bax was analyzed. centroblastic; (b) immunoblastic; (c) T-cell/histiocyte-rich; (d) Immunohistochemical analysis was performed using the En- anaplastic; (e) plasmablastic; (f) lymphomatoid granulomatosis Vision method. type; and (g) a variant expressing full-length anaplastic large Results: CD10 was positive in 51%, bcl-6 in 97%, and cell lymphoma (2). The prognostic importance of morphological CD138 only in 2% of the cases. No prognostic conclusions subclassification remains a disputable issue. could be drawn from analysis of these factors. CD40 was By DNA microarray analysis, Alizadeh et al. (3, 4) iden- positive in 76% of the cases. This group was associated with tified two main groups of DLBCL with different prognosis: (a) ؍ superior time to treatment failure (P 0.027) and overall “germinal center DLBCL,” expressing genes characteristic of ؍ survival (OS; P 0.0068). By Cox regression analysis, pos- germinal center B cells (with ongoing somatic mutations); and itivity for CD40 was shown to be a prognostic factor for OS, (b) “activated B-like type DLBCL,” expressing genes normally independent of International Prognostic Index. CD23 was induced during in vitro activation of peripheral blood B cells. positive in 16% of the cases (all CD5 negative and all CD40 The result of this study brought about a new challenge: a positive). This group showed a strong tendency for better OS search for a protein expression pattern which would best distin- ؍ (P 0.033). CD40 expression correlated with bax but not guish between the two groups. Although immunohistochemical with bcl-2 expression. characteristics of the non-neoplastic germinal center are known, Conclusions: CD23 and CD40 expression seems to be a normal counterpart of “activated B cell” is not easily defined. prognostically favorable in DLBCL. This may be secondary Trying to identify subgroups of DLBCL with different histogenetic origin and prognosis, the following immunohisto- chemical markers were used: (a) bcl-6; and (b) CD10, as well as established markers of the germinal center (5–8) together with Received 6/20/02; revised 10/5/02; accepted 10/7/02. The costs of publication of this article were defrayed in part by the CD40, essential in the germinal center reaction. CD23 was payment of page charges. This article must therefore be hereby marked chosen as a possible pre/early germinal center marker. CD138/ advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported with grants from the Swedish Cancer Society. 2 To whom requests for reprints should be addressed, at Department of Oncology, Lund University Hospital, Getingeva¨gen 4, 221 85 Lund, 3 The abbreviations used are: DLBCL, diffuse large B-cell lymphoma; Sweden. Phone: 46 46 17 75 20; Fax: 46 46 17 60 80; E-mail: johan. IPI, International Prognostic Index; RR, relative risk; CI, confidence [email protected]. interval; TTF, time to treatment failure; OS, overall survival. Downloaded from clincancerres.aacrjournals.org on September 27, 2021. © 2003 American Association for Cancer Research. Clinical Cancer Research 723 Syndecan-1 is a plasma cell-associated antigen also present on Table 1 List of antibodies immunoblasts. When studied in HIV lymphomas, positivity for Antibody (clone) Dilution Source this antigen, in combination with negative bcl-6 expression, is CD5 (4C7) 1:40 NovoCastra Laboratories thought to represent a postgerminal center origin (9). Analysis (Newcastle upon Tyne, UK) of CD5 expression was included because it has been associated CD10 (56C6) 1:5 NovoCastra Laboratories with poor prognosis in several studies (10–12). CD23 (1B12) 1:50 NovoCastra Laboratories Analysis of bcl-2 protein expression was already per- CD40 (11E-9) 1:40 NovoCastra Laboratories CD138 (B-B4) 1:20 Serotec Ltd. (Oxford, UK) formed on this patient material confirming that bcl-2 expression Bcl-6 (PG-Bbp) 1:5 DAKO is associated with increased risk of therapeutic failure (13). In Bax (B-9) 1:120 Santa Cruz Biotechnology the present study, analysis of the proapoptotic bcl-2 analogue, (Santa Cruz, CA) bax, was included. PATIENTS AND METHODS Patients and Follow-Up. Tumor tissue was selected Immunohistochemistry. The paraffin blocks were cut at ␮ from a group of 405 patients with aggressive lymphomas, orig- 4–6 m, dried overnight at 60°C, and deparaffinised in xylene. Subsequently, sections were rehydrated through graded alcohol inally included in a trial set by the Nordic Lymphoma Group. in water. Heat-induced epitope retrieval was achieved by boiling Patients were included in this trial between 1989 and 1994 and sections in EDTA buffer at pH 8.9 in a microwave oven at 1000 randomized to treatment either with cyclophosphamide, W for 20 min (4 ϫ 5 min). After boiling, sections were allowed doxorubicin, vincristine, and prednisone or with methotrexate, to cool at room temperature for 20 min, rinsed thoroughly with doxorubicin, cyclophosphamide, vincristine, prednisone, and water, and placed in a Tris-buffered saline for 5 min. Endoge- bleomycin. No superiority of methotrexate, doxorubicin, cyclo- nous peroxidase was blocked with peroxidase block solution phosphamide, vincristine, prednisone, and bleomycin compared provided in the EnVision kit for 5 min, and slides were rinsed/ with cyclophosphamide, doxorubicin, vincristine, and predni- washed with Tris-buffered saline. The primary antibodies were sone could be demonstrated in this study (14). incubated for 30 min in room temperature. The list of the Inclusion criteria for the Nordic Lymphoma Group trial antibodies and their sources and dilutions are shown in Table 1. were aged between 18 and 67 years, high-grade lymphoma The immunostaining was performed using the EnVision method according to the updated Kiel classification, and stage II-IV (DAKO, Glostrup, Denmark) according to the manufacturer’s disease. Patients with central nervous system involvement, instructions. Scoring of intensity of the immunostaining was transformed low-grade, discordant, lymphoblastic, or Burkitt done semiquantitatively (0 ϭ no staining, 1ϩϭweak staining, lymphoma were excluded. OS was defined as time from ran- 2ϩϭmoderate staining, and 3ϩϭstrong staining of the tumor domization to death. TTF was defined as time from randomiza- cells). The cases were interpreted as negative if Ͻ10% of tumor tion to progression or relapse (15). The last clinical observations cells were positive. were made during spring 1998 when the original study was Multitissue blocks were prepared using 4-mm diameter analyzed. After this period, follow-up data concerning treatment modified, punch-biopsy needle. Generally, two different repre- failure are incomplete. Because median follow-up already was sentative areas were cut out from the paraffin blocks. In total, 67.5 months, this was not prolonged in the current study. Of this provided 25.5 mm2 for evaluation. originally 267 patients with diagnosis of DLBCL, paraffin Statistical Analysis. Survival analyses