Vol. 9, 1381–1386, April 2003 Clinical Cancer Research 1381 Prognostic Significance of CD20 Expression in Classical Hodgkin Lymphoma: A Clinicopathological Study of 119 Cases1 Alexandar Tzankov,2 Jens Krugmann, INTRODUCTION Falko Fend, Martina Fischhofer, Richard Greil, HRS3 cells represent the neoplastic population of cHL. and Stephan Dirnhofer Recent immunological and molecular studies have shown that Institute of Pathology [A. T., J. K.] and Department of Hematology HRS cells originate from mature germinal center B cells that and Oncology and the Tyrolean Cancer Research Institute [M. F., have undergone clonal immunoglobulin gene rearrangement, R. G.], University of Innsbruck, A-6020 Innsbruck, Austria; Institute but have crippling mutations or other molecular alterations, of Pathology, Klinikum Rechts der Isar, Technical University of preventing immunoglobulin transcription (1–7). Although cHL Munich, Munich, Germany [F. F.]; and Institute of Pathology, University of Basel, Basel, Switzerland [S. D.] is genotypically considered a B-cell lymphoma, the classical B-cell marker CD20 is expressed only in ϳ20–30% of such cases. Moreover, CD20 is the only marker of B-cell differenti- ABSTRACT ation that can be repeatedly and reproducibly detected on HRS Purpose: Recent evidence has demonstrated that classi- cells in paraffin sections (7–9). CD20 is a membrane-embedded, cal Hodgkin lymphoma (cHL) originates from mature ger- nonglycosylated, phosphorylated protein (10, 11) that appears minal center B cells. However, only ϳ25% of cHLs express on the B-cell surface after immunoglobulin light chain rear- the classical B-cell marker CD20. There is very little, and rangement and before expression of intact surface immunoglob- controversial, information on the prognostic significance of ulin (12). CD20 resembles a Ca2ϩ ion channel (11, 13) and is CD20 expression in cHL with regard to failure-free (FFS) involved in signal transduction for B-cell differentiation and and overall survival (OS). proliferation (14), as well as for G0-G1 cell cycle transition (15). Experimental Design: CD20 expression was investigated Clinically established prognostic factors in cHL are stage, in a series of 119 cases of cHL treated at a single institution age, presence of B-symptoms, bulky disease, hemoglobin con- where complete clinical follow-up was available. The results centration, erythrocyte sedimentation rate, and serum levels of were correlated to FFS and OS by the Kaplan-Maier ␤ albumin, lactate dehydrogenase, 2-microglobulin, and interleu- method and uni- and multivariate analyses. kin-10 as well as association with HIV (16–20). The prognostic Results: Hodgkin and Reed-Sternberg cells expressed significance of CD20 expression in cHL is controversial and a CD20 in 20% (24 of 119) of the cases based on a cutoff of matter of ongoing debate (19–23). The German Hodgkin Study 10% positivity. Within a mean follow-up period of 12 years, Group reported that the CD30ϩ/CD20ϩ immunophenotype of univariate analysis revealed a significantly higher frequency HRS cells has a positive but statistically insignificant impact on of disease relapses in the CD20-negative group (30 of 95; FFS in cHL (20). In the same study, however, CD30Ϫ/CD15Ϫ/ 32%) compared with CD20-positive tumors (2 of 24; 8%; CD20ϩ cHL had an inferior clinical outcome. In a recent -Compared by the log-rank test, the mean FFS in investigation, Rassidakis et al. (19) found no association be .(0.022 ؍ P CD20-negative cases (202 months) was considerably shorter tween CD20 status and FFS or OS in patients with stages I–IV ؍ than in the CD20-positive cases (286 months; P 0.0195). In cHL treated by at least seven efficacy-equivalent chemotherapy a multivariate analysis, CD20 expression was an independ- regimens. In line with these findings, Molot et al. (22) reported ent positive prognostic factor for FFS in cHL patients no prognostic role for CD20 expression in Hodgkin lymphoma, ؍ treated from 1974 to 1980 (P 0.035). This effect disap- whereas the group from Memorial Sloan-Kettering Cancer Cen- ؍ peared in the period from 1981 to 1999 (P 0.266). ter (23) found an inferior clinical outcome in CD20-positive Conclusion: CD20-positive cHL shows a trend for bet- cHL patients. ter FFS and OS. However, improved treatment modalities To further assess the prognostic significance of CD20 seem to abolish these differences. expression in cHL, we performed a retrospective single institu- tional study of 119 cases with a mean clinical follow-up of 12 years. Received 8/19/2002; revised 11/15/2002; accepted 11/15/2002. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to 3 The abbreviations used are: HRS, Hodgkin and Reed-Sternberg; indicate this fact. cHL, classical Hodgkin lymphoma; FFS, failure-free survival; 1 This study was supported by the Austrian National Bank (Project OS, overall survival; EBV, Epstein-Barr virus; LMP-1, latent 5621) and by the Krebshilfe Tirol. membrane protein-1 of Epstein-Barr virus; ABVD, adriamycin- 2 To whom requests for reprints should be addressed, at Institute of bleomycin-vincristine-dacarbatine; COPP, cyclophosphamide-vin- Pathology, University of Innsbruck, Mu¨llerstrasse 44, A-6020 Inns- cristine-procarbazine-prednisone; BEACOPP, bleomycin-etoposide- bruck, Austria. Phone: 43 512 507 36 92; Fax: 43 512 58 20 88; E-mail: adriamycin-cyclophosphamide-vincristine-procarbatine-prednisone; [email protected]. LPHL, lymphocyte-predominant Hodgkin lymphoma. Downloaded from clincancerres.aacrjournals.org on September 26, 2021. © 2003 American Association for Cancer Research. 1382 CD20 in Classical Hodgkin Lymphoma Table 1 Antibodies and antigen retrieval techniques used Antibody Antigen retrieval Dilution Incubation Source CD15 Pressure cooker; 5 min; 121°C 1:400 30 min; 20°C DAKO CD20 Microwave oven; 10 min; 800 W 1:700 30 min; 20°C DAKO CD30 Pressure cooker; 5 min; 121°C 1:50 30 min; 20°C DAKO LMP-1 0.1% pronase; 4 min 1:1000 30 min; 20°C DAKO Fig. 1 Expression of CD20 in Hodgkin cells of cHL. Cells are stained with immunoperoxidase stain. Magnification, ϫ400. PATIENTS AND METHODS used as chromogen. For positive control staining, tonsils with Patients. We analyzed 119 formalin-fixed, paraffin-em- follicular hyperplasia and with paracortical hyperplasia from bedded samples from cHL cases from the files of the Depart- patients with florid infectious mononucleosis were used. For ment of Pathology at the University of Innsbruck, diagnosed negative controls, the primary antibodies were omitted. A case between 1974 and 1999 (24). All cases were reclassified ac- was considered CD20-positive if there was specific membra- cording to the updated WHO classification (8). Clinical data nous staining in Ͼ10% of the HRS cells (Fig. 1). The staining were obtained by reviewing the charts and contacting the treat- intensity of small B lymphocytes within the cHL infiltrates ing physicians. Treatment was either standard or consistent with served as internal control. Taking into account previous studies the investigational protocols active during the time the patients investigating the expression pattern of CD20 in cHL and its were diagnosed. In brief, patients were staged surgically in association with clinical outcome (19–21), we considered a stages I and II when radiotherapy was administered as the sole cutoff value of 10% the most appropriate. treatment modality. Otherwise, patients were staged clinically Statistical Analysis. Statistical analysis was performed and mostly treated with risk-adapted treatment strategies ac- using the Statistical Package of Social Sciences (SPSS). FFS cording to the protocols of the German Hodgkin Study Group. was analyzed with the Kaplan-Maier method and compared by 2 In advanced stages, radiotherapy was used only for treatment of the log-rank test. The Pearson ␹ test was applied to demonstrate residual disease or primary bulky disease (24). Disease remis- correlations between expression of CD20 and relapse rate as sion was defined as absence of disease for at least 1 month as well as different clinical and laboratory parameters. To compare determined by laboratory and imaging studies as well as phys- differences for patients treated before and after 1980, we used ical examination. Disease relapse was defined as disease pro- the independent sample t test. Multivariate analysis for the effect gression occurring at least 1 month after achieving disease of expression of CD15 and CD20 as well as for age, sex, Ann remission. Treatment failure was defined as disease relapse or Arbor stage, EBV association, B-symptoms, and histological primary treatment resistance. subtype was performed using a general linear model. P Ͻ 0.05 Immunohistochemistry. The primary antibodies used in was considered significant. this study and types of antigen retrieval performed are listed in Table 1. Standard immunoperoxidase techniques were used with RESULTS an automated immunostainer (Nexes; Ventana, Tucson, AZ) Clinical Data, Histopathology, and OS Analysis. Our exactly as described previously (25). Diaminobenzidine was study group consisted of 65 (55%) male and 54 (45%) female Downloaded from clincancerres.aacrjournals.org on September 26, 2021. © 2003 American Association for Cancer Research. Clinical Cancer Research 1383 Table 2 Clinicopathological characteristics of cHL patients with respect to CD20 expression All patients CD20-positive (n) cases, n (%) Histology Nodular sclerosis 70 11 (16) Mixed cellularity 39 9 (23) Lymphocyte depleted 3 1 (33) Lymphocyte rich 7 3 (43) Sex M 65 14 (22) F 54 10 (19) Ann Arbor stage I–II 68 17 (25) III–IV 42 5 (12) Age (years) Ͻ45 86 13 (15) Ͼ45 33 11 (33) Therapy Radiotherapy 24 6 (25) Chemotherapy/Radiochemotherapy 84 16 (19) ABVD 27 6 (22) COPP 15 1 (7) COPP ϩ ABVD or BEACOPP 42 8 (19) B-Symptoms 51 11 (22) Fig. 2 FFS of cHL patients correlated with the expression of CD20. Total 119 24 (20) CD20ϩ versus CD20Ϫ was compared by the log-rank test.