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Hematopathology / BCL-3 IN CLL
Expression of bcl-3 in Chronic Lymphocytic Leukemia Correlates With Trisomy 12 and Abnormalities of Chromosome 19
Ellen Schlette, MD, George Z. Rassidakis, MD, PhD, Ozlem Canoz, MD,* and L. Jeffrey Medeiros, MD
Key Words: bcl-3; Immunohistochemistry; Chronic lymphocytic leukemia; Trisomy 12
DOI: 10.1309/6Q27Q3NDGV8LW1BU

Abstract

The bcl-3 gene at chromosome 19q13 encodes a member of the IB family involved in regulating the nuclear factor B pathway. Originally identified by its involvement in the t(14:19)(q32;q13), bcl-3 expression recently has been reported in 12% of non-Hodgkin lymphomas and 41% of Hodgkin lymphomas. Because the t(14;19) is detected most commonly in chronic lymphocytic leukemia (CLL), we assessed for bcl-3 expression using immunohistochemical analysis in 72 CLL cases with immunophenotypic and cytogenetic data. Of 72 CLL cases, 12 (17%) were bcl-3+. Expression of bcl-3 correlated with an atypical immunophenotype, defined using the World Health Organization scoring system. Expression also correlated with trisomy 12 and chromosome 19 abnormalities but was not limited to cases with the t(14:19)(q32;q13). Although the mechanism of bcl-3 expression is unclear, these results raise the possibility that bcl-3 may be involved in the pathogenesis of this subset of tumors and could be a potential target for investigational therapies.
The bcl-3 gene, located at chromosome 19q13, originally was identified by its involvement in the t(14:19)(q32;q13). In this translocation, bcl-3 is juxtaposed with the immunoglobulin heavy chain gene locus at 14q32, and bcl-3 expression is up-regulated. The t(14;19) originally was described in 1983,1 and the bcl-3 gene first was cloned by McKeithan and colleagues2 in 1987. The bcl-3 gene encodes a member of the IB protein family, and the members of this family are known to regulate the transcription factor nuclear factor (NF) B.3 The mammalian NF-B family consists of 5 known members: p50/p105, p52/p100, RelA (p65), c-Rel, and RelB.3 These proteins normally are intracytoplasmic and are complexed with inhibitory IB molecules, but when IB proteins are degraded, free NF-B family proteins can translocate to the nucleus and activate expression of a variety of genes. The IB family is composed of 5 members: bcl-3, IB, IB, IB, and IB. In this family, bcl-3 is unique because it is located predominantly in the nucleus, it interacts with p50 and p52 homodimers of NF-B, and it binds to other nuclear proteins. The cellular functions of bcl-3 remain obscure. It has been suggested that bcl-3 is an adapter protein between members of the NF-B family, particularly p50 and p52, and other transcriptional regulators (eg, JAB1, Pirin). bcl-3 can form stable complexes with p50 or p52 on NF-B DNA binding sites to activate transcription or dissociate these molecules from DNA. In addition, the phosphorylation status or concentration of bcl-3 also can affect its interaction with p50 and p52.4 The t(14;19) or bcl-3 gene rearrangement has been reported rarely in hematologic neoplasms, most often in cases of chronic lymphocytic leukemia/small lymphocytic
Am J Clin Pathol 2005;123:465-471
American Society for Clinical Pathology

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Schlette et al / BCL-3 IN CLL
lymphoma (CLL/SLL), but in fewer than 5% of cases based on conventional cytogenetics and molecular genetic analysis.5-9 The t(14;19) or bcl-3 gene rearrangement is even more rare in other types of non-Hodgkin lymphoma and acute leukemias.6,9-14 Although the t(14;19) is rare, bcl-3 expression is more common and has been shown in 12% of nonHodgkin lymphomas, 6% of B-cell and 23% of T-cell lineage, and 41% of Hodgkin lymphomas.15 In previous studies of CLL/SLL, the t(14;19) has been associated with atypical morphologic features and trisomy 12. For the present study, we hypothesized that bcl-3 expression also might be associated with atypical morphologic features and trisomy 12. Thus, we studied bcl-3 expression in 72 cases of CLL for which immunophenotypic and cytogenetic data were available.

Morphologic Examination The morphologic features of all cases were examined using Wright-Giemsastained bone marrow aspirate smears and were classified as atypical or typical using adapted criteria for atypical morphology on peripheral blood smears in CLL, as described by Matutes et al.17 Specifically, cases with more than 15% lymphoplasmacytoid cells or clefted nuclear contours were considered atypical. Cases with more than 10% prolymphocytes were classified as CLL/prolymphocytic leukemia, not atypical CLL in agreement with the WHO classification.16 In each case, the percentage of each lymphocyte type was determined by a manual 100-cell differential count. Immunohistochemical Methods Tumors were analyzed using full tissue sections (n = 30) or 2 tissue microarrays (n = 42). The tissue microarrays included duplicate cores from each tumor and were constructed using a manual tissue arrayer (Beecher Instruments, Silver Spring, MD) as described previously.15 All cases were fixed in 10% buffered formalin, processed routinely, and embedded in paraffin. Tissue or microarray sections (3 or 4 m thick) were deparaffinized in xylene and rehydrated in a graded series of ethanols. Heat-induced epitope retrieval was performed by placing sections in plastic Coplin jars containing preheated target retrieval solution (DAKO, Carpinteria, CA) heated in a household vegetable steamer (model Sunbeam 4713/5710, 900 W; Sunbeam-Oster, Boca Raton, FL) for 35 minutes. Sections then were allowed to cool at room temperature for at least 15 minutes. We used a monoclonal antibody specific for bcl-3 (clone 1E8, Novocastra, Newcastle upon Tyne, England) at a dilution of 1:50. Subsequent steps of the immunostaining procedure were performed in a humidity chamber as previously described.15

Materials and Methods

Study Group Bone marrow aspirate smears and clot or biopsy specimens from 72 patients with the diagnosis of CLL and for which the results of flow cytometry immunophenotypic and conventional cytogenetic analysis were available were collected from the files of the Department of Hematopathology, The University of Texas M.D. Anderson Cancer Center, Houston, from September 1999 through December 2001. The diagnosis of CLL was based on morphologic and immunophenotypic criteria outlined in the World Health Organization (WHO) classification.16 There were 47 men and 25 women; the median age was 57 years (range, 24-79 years). Fifteen patients had been treated with chemotherapy before the bone marrow sample included in this study was obtained. The clinical and laboratory data for these patients are summarized in Table 1.

Table 1 Summary of Clinical and Laboratory Information*
CLL With Trisomy 12 (n = 22) Median age at diagnosis (y) Sex (M/F) Lymphadenopathy Splenomegaly Median WBC count, /L ( 109/L) Median absolute lymphocyte count, /L ( 109/L) 2-Microglobulin, g/mL (nmol/L) Median time from diagnosis to last follow-up, mo Follow-up
CLL Without Trisomy 12 (n = 50) 56 (range, 24-79) 33/17 36/47 (77) 19/45 (42) 34,600 (34.6); range, 3,800-299,500 (3.8-299.5) 29,500 (29.5) range, 1,300-287 ,500 (1.3-287.5) 3.6 (306); range, 1.6-10.7 (136-910) 58; range, 15-156 Died, 12; alive, 24

P.87.6.52.29.075.12.072

55 (range, 27-73) 13/9 19/22 (86) 6/22 (27) 68,300 (68.3); range, 4,000-226,000 (4.0-226.0) 65,400 (65.4) range, 2,400-225,500 (2.4-225.5) 2.6 (221); range, 1.2-5.8 (102-493) 47 range, 28-149.5; Died, 3; alive, 15
Data are given as number/total (percentage) unless otherwise indicated. Mann-Whitney test. Fisher exact test.

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Hematopathology / ORIGINAL ARTICLE
Scoring of Positive Cells Only nuclear staining was considered positive for bcl-3, irrespective of intensity, because the protein has been shown to be located predominantly within the nucleus.4 Positivity was arbitrarily defined as 20% or more positive cells. However, almost all negative tumors were completely negative or tumors had fewer than 5% positive cells and positive tumors showed a range of positive cells, always more than 20% and, in several cases, more than 50%. Flow Cytometry Immunophenotypic Analysis The samples were assessed using 3- or 4-color flow cytometric analysis and a FACScan (or FACScalibur) instrument (Becton Dickinson, San Jose, CA). Lymphocytes were gated for analysis using CD45 expression and right-angle light scatter. Fluorescein isothiocyanate and phycoerythrin-conjugated IgG1 and IgG2 antibodies were used for negative control experiments, and cursors were set to include more than 95% of events as negative. The panel of antibodies, conjugated to fluorescein isothiocyanate, phycoerythrin, or allophycocyanin, included reagents specific for CD3, CD5, CD11c, CD19, CD20, CD22, CD23, CD38, CD79b, FMC-7, and immunoglobulin and light chains (Becton Dickinson). The intensity of expression of CD20 and immunoglobulin light chains was also assessed. As described in the WHO classification,16 the typical CLL immunophenotype is defined as a monotypic B-cell neoplasm positive for CD5 and CD23 with low intensity (dim) positivity for CD20 and surface immunoglobulin and negative for CD22, CD79b, and FMC-7.

Conventional Cytogenetics Conventional G-band karyotype analysis was performed on bone marrow aspirate specimens from all patients. Cells were placed in 10 mL of Ham F10 medium with 20% fetal calf serum at a concentration of 2 to nucleated cells per milliliter. The culture was incubated overnight at 37C (approximately 24 hours). Standard harvesting procedures were used. Colcemid (0.1 mL/10 mL) was added to the culture for 30 minutes. For the hypotonic treatment, a 0.075mol/L concentration of potassium chloride was used for 30 minutes at room temperature. The fixation procedure consisted of 3 changes of methanol:glacial acetic acid (3:1) with a 10-minute waiting period between each change. The Thermaton drying chamber (Thermaton Industries, Holland, MI) was used for slide preparation. Slides were placed in a 60C oven overnight in preparation for Giemsa trypsin Gbanding. A maximum of 30 metaphases was analyzed. The karyotypes were written using the International System for Human Cytogenetic Nomenclature.

Results

Expression of bcl-3 in CLL Of 72 cases of CLL, 12 (17%) were bcl-3+ Image 1. The majority of positive cases had 50% or more neoplastic cells that were positive, and staining intensity was moderate to strong. No difference in pattern or expression was noted between cases assessed by tissue microarrays and those assessed by full tissue sections. No significant association was
Image 1 A, The PAX-5 stain highlights the interstitial pattern of involvement in a case of trisomy 12 chronic lymphocytic leukemia (CLL) without abnormalities of chromosome 19 (400). B, Nuclear bcl-3 expression is illustrated in this case (400).
American Society for Clinical Pathology 467

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found between bcl-3 expression and age, sex, laboratory findings, or history of chemotherapy. Correlation of bcl-3 Expression With Morphology and Immunophenotype In bone marrow aspirate smears, atypical morphologic features were detected in 10 cases (14%). One of 10 atypical CLL cases compared with 9 of 62 typical CLL cases were bcl3+ (P = 1; Fisher exact test). All cases tested were positive for monotypic immunoglobulin light chain, CD5, CD19, and CD20 and were negative for CD3. Of 70 CLL cases, 66 (94%) were positive for CD23 and 11 (16%) were positive for FMC-7. Most cases assessed expressed CD11c (54/61 [89%]). CD38 was positive in 25 (38%) of 66 cases assessed. For 58 patients, sufficient immunophenotypic data were available to generate a score using the antigens and system described in the WHO classification.16 Of 11 bcl-3+ cases, 6 (55%) had a score of 3 or less (ie, atypical for CLL) compared with 8 (17%) of 47 bcl-3 cases (P =.017; Fisher exact test). Expression of bcl-3 also correlated with CD79b positivity (P =.008) and the combination of CD11c and CD22 (P =.026; Fisher exact test) Table 2. CD11c, CD22, CD23, CD38, and FMC-7 positivity were not associated significantly with bcl-3 expression. Correlation Between bcl-3 Expression and Cytogenetic Findings Based on the karyotype, the 72 CLL cases in this study can be divided into 3 groups: diploid cases (n = 24), cases with trisomy 12 with or without additional abnormalities (n = 22), and cases with cytogenetic abnormalities other than trisomy

Table 2 Expression of bcl-3 Correlated With Immunophenotypic and Cytogenetic Findings*
bcl-3+ Cases (n = 12) CD79b Positive 7/10 (70) Negative 3/10 (30) CD11c/CD22 Positive 6/11 (55) Negative 5/11 (45) Immunophenotypic score 3 6/11 (55) 4 or 5 5/11 (45) Trisomy 12 Present 9/12 (75) Absent 3/12 (25) Chromosome 19 abnormalities Present 3/12 (25) Absent 9/12 (75)
12 (n = 26). The karyotypic findings in the bcl-3+ CLL cases are summarized in Table 3. When bcl-3 expression in these cytogenetic groups was compared, a significant association between bcl-3 expression and trisomy 12 was identified (P =.003; Fisher exact test). Nine (75%) of 12 bcl-3+ CLL cases had trisomy 12, while 13 (28%) of 47 bcl-3 CLL cases had trisomy 12. In addition, 3 of 5 CLL cases with abnormalities of chromosome 19, including 1 with the t(14;19) and trisomy 12, were positive for bcl-3, also showing a positive correlation (P =.03; Fisher exact test). One tumor with a diploid karyotype and 1 with cytogenetic abnormalities other than trisomy 12 or chromosome 19 were positive for bcl-3 (Tables 2 and 3).

Discussion

The t(14;19)(q32;q13) is a rare recurrent translocation that juxtaposes the bcl-3 gene, normally at chromosome 19q13, with the immunoglobulin heavy chain gene on chromosome 14q32. The bcl-3 gene encodes a member of the IB family. Although most of the IB proteins are located in the cytoplasm and are known to be inhibitors of transcription by sequestering NF-B, it has been shown that bcl-3 is predominantly nuclear and interacts with general transcription factors, transcriptional coactivators, and other DNA binding factors, all of which support a general role for bcl-3 in transcriptional activation.3,4 bcl-3 is unique in having an ankyrin repeat domain, like inhibitory IB proteins, while also possessing domains capable of transcriptional activation. Expression of bcl-3 is induced by many cell growth or survival-promoting factors in lymphocyte cell lines, suggesting that bcl-3 is involved
Table 3 Karyotypes of bcl-3+ Chronic Lymphocytic Leukemia Cases
Case No. Karyotype 47 ,XX,+12,t(14;19)(q32;q13)[7] idem,add(13)(q34)[6] idem,5,18 [cp5] 48,XY,+12,+19[19] 48,XY,+12,t(13;19)(q21;q13),+19[1] 47 ,XX,+12[2] idem,del(1)(p34),add(6)(p25),del(10)(p12),add(13)(p12)[1] 45,XY,der(1)t(1;7)(p31;q33),der(3)t(3;7)(q21;p22),del(6)(q21), der(7)t(3;7)t(1;7),del(10)(q24),der(13;17)(q10;q10)[7] 47 ,XY,del(6)(q21),+12[2] 47 ,XY,+12,del(14)(q22q32)[18] 47 ,XX,+12[3] 47 ,XY,+12[3] 47 ,XY,+12[3] 47 ,XX,+12[9] 43-47 ,XX,add(3)(q29),del(3)(p24),del(6)(q21),del(11) (q14q23),17 ,+18,add(19)(p13.3),+mar[cp17] 46-47 ,XY,add(14)(q32)[cp2]45,X-Y[27] 46,XY[20]

bcl-3 Cases (n = 60)

11/47 (23) 36/47 (77).026 10/51 (20) 41/51 (80).017 8/47 (17) 39/47 (83).003 13/60 (22) 47/60 (78).03 2/60 (3) 58/60 (97)
Data are given as number/total (percentage). Fisher exact test. Includes 1 case with t(14;19) that was bcl-3+.

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closely in cell proliferation and survival.18 In 1 study, transgenic mice expressing bcl-3 were found to have an expansion of B cells in vivo,19 further suggesting a role for bcl-3 in cell proliferation or survival. In addition, bcl-3 can bind to the promoter of cyclin D1, enhancing progression of the cell cycle from G1 to S phase.3,20 Furthermore, although bcl-3deficient mice appear developmentally normal, they show defects in B- and T-cell responses to antigens, suggesting a role of bcl-3 in maintaining B cells in wild-type mice.21,22 Thus, bcl-3 expression might confer a survival advantage for CLL cells. Previous studies have reported that the t(14;19) in CLL is associated with atypical morphologic features and immunophenotype and trisomy 12. In a review of 23 cases of lymphoproliferative disorders with the t(14;19) by Michaux and colleagues,B-cell CLL cases were described. Young patient age and progressive disease were common, and atypical lymphocyte morphologic features were reported in 41% of cases.14 McKeithan and colleagues13 also reported an association between young age or progressive disease and the t(14;19) or bcl-3 gene rearrangements. On the basis of these data, we hypothesized that bcl-3 expression also might correlate with trisomy 12 and atypical morphologic and immunophenotypic features, and this seems to be true, in part. Atypical morphologic features in CLL have been associated with various factors, including atypical immunophenotype, abnormal cytogenetic findings, and, in particular, trisomy 12, and have been reported by others as an indicator of poor prognosis.17,23-33 In the present study, we did not find an association between atypical morphology and bcl-3 expression, as only 1 (8%) of the 12 bcl-3+ CLL cases had atypical morphology. In the present study, 12 (17%) of 72 CLL cases expressed bcl-3, and expression correlated with trisomy 12, present in 9 (75%) of 12 bcl-3+ cases. Trisomy 12 is a common cytogenetic abnormality in CLL, reported in 15% to 30% of cases in various studies, and has previously been associated with atypical morphology, atypical immunophenotype, other karyotypic abnormalities, and poorer prognosis.23-25,34-38 Expression of bcl-3 also correlated with atypical immunophenotype (score 3) using the immunophenotypic scoring system for CLL described in the WHO classification16 and based on earlier studies by Matutes et al39 and Moreau and colleagues.40 Fifty-eight cases had the prerequisite antigens needed to generate this score, which is based on expression of CD5 (1 point), CD23 (1 point), low (dim) levels of surface immunoglobulin (1 point), absent or weak expression of CD79b/CD22 (1 point), and FMC-7 (1 point). Cases with scores of 4 or 5 are considered typical for CLL, whereas scores of 3 or less are atypical and more often occur in other types of B-cell leukemia.16 In the present study, 6 (55%) of 11

bcl-3+ CLL cases had a score of 3 or less compared with 8 (17%) of 47 bcl-3 CLL cases (P =.017, not significant). Specific antigens associated with bcl-3 expression were CD79b and the combination of CD11c and CD22. CD79b expression previously has been associated with trisomy 12 in CLL.25 When CD11c and CD22 expression were analyzed separately, no significant correlation with bcl-3 expression was identified for either antigen. Expression of bcl-3 in CD22+ CLL might have clinical importance because antiCD22 monoclonal antibody therapy may be considered as a treatment for patients with CD22+ CLL. However, no clinical data using this antibody are available in CLL.41 Although only a small number of cases had karyotypic abnormalities involving chromosome 19 and these abnormalities were associated with trisomy 12, chromosome 19 abnormalities correlated with bcl-3 expression. A single CLL/SLL case with t(14;19) was included in the study. This case showed strong expression of bcl-3. In CLL/SLL, the prevalence of t(14;19) is low, less than 1% by conventional cytogenetic analysis and up to 2% by molecular genetic analysis.5-9 Thus, the frequency of the t(14;19) in the present study is consistent with earlier studies. Because bcl-3 expression is not limited to cases with the t(14;19), other mechanisms might up-regulate bcl-3 in positive cases or the translocation may be present in a subset of the neoplastic cells that was not detected by conventional cytogenetic analysis. In a study by Lishner and colleagues42 using fluorescence in situ hybridization (FISH), 4 (20%) of 20 consecutive CLL patient samples had evidence of bcl-3/IgH fusion sequences in 6% to 93% (median, 16%) of nuclei counted. This study suggests that the t(14;19) is identified more commonly by FISH than by conventional cytogenetics and can be present in only a subset of cells. Because the present study used cytogenetic data obtained retrospectively, FISH for t(14;19) was not attempted. In summary, we performed an immunohistochemical study for bcl-3 in 72 cases of CLL. Our results indicated that a subset of CLL cases, most of which have trisomy 12 or chromosome 19 abnormalities, express bcl-3. The relatively high frequency of bcl-3 expression in these cases raises the possibility that bcl-3 has an oncogenetic role in development of these neoplasms and might be a potential target for investigational therapies. While the t(14;19) has been reported to be associated with atypical morphologic features and progressive disease requiring early treatment, in this study bcl-3 expression did not correlate with atypical morphologic features or overall survival. Expression of bcl-3 did, however, correlate with atypical immunophenotypic findings, as has been suggested by others.

From the Department of Hematopathology, The University of Texas M.D. Anderson Cancer Center, Houston.

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Address correspondence to Dr Schlette: Dept of Hematopathology, Box 72, University of Texas M.D. Anderson Cancer Center, 1515 Holcombe Blvd, Houston, TX 77030. * Dr Canoz participated in this work as a visiting pathologist at M.D. Anderson Cancer Center and currently is with the Department of Pathology, Erciyes University Medical Faculty, Kayseri, Turkey. Acknowledgment: We thank Andrea Sedo for help with manuscript preparation.

References

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16. Muller-Hermelink HK, Montserrat E, Catovsky D, et al. Chronic lymphocytic leukemia/small lymphocytic leukemia. In: Jaffe ES, Harris NL, Stein H, et al, eds. World Health Organization Classification of Tumours of Haematopoietic and Lymphoid Tissue. Lyon, France: IARC Press; 2001:127-130. 17. Matutes E, Oscier D, Garcia-Marco J, et al. Trisomy 12 defines a group of CLL with atypical morphology: correlation between cytogenetic, clinical and laboratory features in 544 patients. Br J Haematol. 1996;92:382-388. 18. Rebollo A, Dumoutier L, Renauld JC, et al. bcl-3 expression promotes cell survival following interleukin-4 deprivation and is controlled by AP1 and AP1-like transcription factors. Mol Cell Biol. 2000;20:3407-3416. 19. Ong ST, Hackbarth ML, Degenstein LC, et al. Lymphadenopathy, splenomegaly, and altered immunoglobulin production in bcl3 transgenic mice. Oncogene. 1998;16:23332343. 20. Westerheide SD, Mayo MW, Anest V, et al. The putative oncoprotein bcl-3 induces cyclin D1 to stimulate G1 transition. Mol Cell Biol. 2001;21:8428-8436. 21. Franzoso G, Carlson L, Scharton-Kersten T, et al. Critical roles for the bcl-3 oncoprotein in T cellmediated immunity, splenic microarchitecture, and germinal center reactions. Immunity. 1997;6:479-490. 22. Schwarz EM, Krimpenfort P, Berns A, et al. Immunological defects in mice with a targeted disruption in bcl-3. Genes Dev. 1997;11:187-197. 23. Criel A, Verhoef G, Vlietinck R, et al. Further characterization of morphologically defined typical and atypical CLL: a clinical, immunophenotypic, cytogenetic and prognostic study on 390 cases. Br J Haematol. 1997;97:383391. 24. Oscier DG, Matutes E, Copplestone A, et al. Atypical lymphocyte morphology: an adverse prognostic factor for disease progression in stage A CLL independent of trisomy 12. Br J Haematol. 1997;98:934-939. 25. Schlette E, Medeiros LJ, Keating M, et al. CD79b expression in chronic lymphocytic leukemia: association with trisomy 12 and atypical immunophenotype. Arch Pathol Lab Med. 2003;127:561-566. 26. Que TH, Marco JG, Ellis J, et al. Trisomy 12 in chronic lymphocytic leukemia detected by fluorescence in situ hybridization: analysis by stage, immunophenotype, and morphology. Blood. 1993;82:571-575. 27. Finn WG, Thangavelu M, Yelavarthi KK, et al. Karyotype correlates with peripheral blood morphology and immunophenotype in chronic lymphocytic leukemia. Am J Clin Pathol. 1996;105:458-467. 28. OConnor SJ, Suut L, Morgan GJ, et al. The relationship between typical and atypical B-cell chronic lymphocytic leukemia: a comparative genomic hybridizationbased study. Am J Clin Pathol. 2000;114:448-458. 29. DArena G, DellOlio M, Musto P, et al. Morphologically typical and atypical B-cell chronic lymphocytic leukemias display a different pattern of surface antigenic density. Leuk Lymphoma. 2001;42:649-654. 30. Frater JL, McCarron KF, Hammel JP, et al. Typical and atypical chronic lymphocytic leukemia differ clinically and immunophenotypically. Am J Clin Pathol. 2001;116:655-664. 31. Domingo-Domenech E, Domingo-Claros A, Gonzalez-Barca E, et al. CD38 expression in B-chronic lymphocytic leukemia: association with clinical presentation and outcome in 155 patients. Haematologica. 2002;87:1021-1027.

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32. Mauro FR, Gentile M, Mancini F, et al. Prognostic significance of lymphocyte morphology in patients with advanced chronic lymphocytic leukemia treated with first line therapy of fludarabine + prednisone. Haematologica. 2002;87:602-608. 33. Cuneo A, Rigolin GM, Bigoni R, et al. Chronic lymphocytic leukemia with 6q shows distinct hematological features and intermediate prognosis. Leukemia. 2004;18:476-483. 34. Geisler CH, Philip P, Christensen BE, et al. In B-cell chronic lymphocytic leukaemia chromosome 17 abnormalities and not trisomy 12 are the single most important cytogenetic abnormalities for the prognosis: a cytogenetic and immunophenotypic study of 480 unselected newly diagnosed patients. Leuk Res. 1997;21:1011-1023. 35. Byrd JC, Smith L, Hackbarth ML, et al. Interphase cytogenetic abnormalities in chronic lymphocytic leukemia may predict response to rituximab. Cancer Res. 2003;63:36-38. 36. Karhu R, Tobin G, Thunberg U, et al. More extensive genetic alterations in unmutated than in hypermutated cases of chronic lymphocytic leukemia. Genes Chromosomes Cancer. 2003;37:417-420.
37. Wiestner A, Rosenwald A, Barry TS, et al. ZAP-70 expression identifies a chronic lymphocytic leukemia subtype with unmutated immunoglobulin genes, inferior clinical outcome, and distinct gene expression profile. Blood. 2003;101:4944-4951. 38. Thornton PD, Fernandez C, Giustolisi GM, et al. CD38 expression as a prognostic indicator in chronic lymphocytic leukaemia. Hematol J. 2004;5:145-151. 39. Matutes E, Owusu-Ankomah K, Morilla R, et al. The immunological profile of B-cell disorders and proposal of a scoring system for the diagnosis of CLL. Leukemia. 1994;8:1640-1645. 40. Moreau EJ, Matutes E, AHern RP, et al. Improvement of the chronic lymphocytic leukemia scoring system with the monoclonal antibody SN8 (CD79b). Am J Clin Pathol. 1997;108:378-382. 41. Mavromatis BH, Cheson BD. Novel therapies for chronic lymphocytic leukemia. Blood Rev. 2004;18:137-148. 42. Lishner M, Lalkin A, Klein A, et al. The bcl-1, bcl-2, and bcl-3 oncogenes are involved in chronic lymphocytic leukemia: detection by fluorescence in situ hybridization. Cancer Genet Cytogenet. 1995;85:118-123.
American Society for Clinical Pathology 471

CD20 Expression in Hodgkin and Reed-Sternberg Cells of Classical Hodgkins Disease: Associations With Presenting Features and Clinical Outcome
By George Z. Rassidakis, L. Jeffrey Medeiros, Simonetta Viviani, Valeria Bonfante, Gian-Paolo Nadali, Theodoros P. Vassilakopoulos, Ofelia Mesina, Marco Herling, Maria K. Angelopoulou, Roberto Giardini, Marco Chilosi, Christos Kittas, Peter McLaughlin, M. Alma Rodriguez, Jorge Romaguera, Gianni Bonadonna, Alessandro M. Gianni, Giovanni Pizzolo, Gerassimos A. Pangalis, Fernando Cabanillas, and Andreas H. Sarris
Purpose: CD20 can be expressed in Hodgkin and Reed-Sternberg (HRS) cells of classical Hodgkins disease (HD), but its clinical signicance remains controversial. Therefore, we correlated CD20 expression with presenting features and clinical outcome of untreated patients with classical HD. Patients and Methods: Patients were eligible if they were previously untreated and human immunodeciency virus-1 negative, had biopsy-proven classical HD, and if pretreatment parafn-embedded tumor tissue was available. CD20 expression was determined by immunohistochemistry without knowledge of clinical outcome. A tumor was considered positive if any HRS cells expressed CD20, but other cutoffs for number of CD20-positive HRS were also investigated. Results: We identied 598 patients whose median age was 30 years and of whom 55% were male. HRS cells expressed CD20 in 132 (22%) of 598 patients with classical HD. When any percentage of CD20 expression in HRS cells was used as a cutoff, the 5-year failure-free survival (FFS) for positive versus negative tumors was 86% versus 84%, respectively, for 302 patients treated with doxorubicin, bleomycin, vinblastine, and dacarbazine or equivalent regimens (P 7. by log-rank test), 74% versus 77%, respectively, for 181 patients treated with mitoxantrone, vincristine, vinblastine, and prednisone and radiotherapy (P 7. by log-rank test), 74% versus 84%, respectively, for 54 patients treated with MOPP (P 4. by log-rank test), and 77% versus 88% for 53 patients treated only with radiotherapy (P 5. by log-rank test). The 5-year FFS was not statistically different when cutoffs of 5% up to 50% for CD20-positive HRS cells were used. Conclusion: CD20 is expressed by HRS cells in 22% of patients with classical HD but is not associated with different FFS after treatment with equivalent regimens. J Clin Oncol 20:1278-1287. 2002 by American Society of Clinical Oncology.
LTHOUGH HODGKINS disease (HD) is a curable lymphoma, up to 30% of patients relapse and eventually die of disease or treatment complications.1-3 Various clinical and laboratory features have been used to predict failure-free survival (FFS) and overall survival (OS) to identify patients destined to relapse. These include age, sex, peripheral or mediastinal bulk, stage IV disease, involvement of bone marrow or inguinal lymph nodes, anemia,
From the Departments of Lymphoma-Myeloma and Hematopathology, The University of Texas M.D. Anderson Cancer Center, Houston, TX; Departments of Medical Oncology and Pathology, Istituto Tumori, Milan, and Departments of Hematology and Pathology, University of Verona, Verona, Italy; and First Department of Internal Medicine and Laboratory of Histology and Embryology, National and Kapodistrian University of Athens, Athens, Greece. Submitted April 6, 2001; accepted November 9, 2001. Supported in part by Cancer Center support grant no. CA-16672 to A.H.S. G.Z.R. is a recipient of an Alexander S. Onassis Foundation scholarship. Address reprint requests to Andreas H. Sarris, MD, PhD, Department of Lymphoma-Myeloma, Box 429, The University of Texas M.D. Anderson Cancer Center, 1515 Holcombe Blvd, Houston, TX 77030; email: asarris@mail.mdanderson.org. 2002 by American Society of Clinical Oncology. 0732-183X/02/2005-1278/$20.00

leukocyte and lymphocyte counts, and serum levels of albumin, lactate dehydrogenase (LDH), and 2-microglobulin.4-11 Recently, we and other investigators have shown that elevated serum interleukin-10 levels are also associated with inferior FFS for HD.12-15 However, additional prognostic factors related to the biology of HD need to be evaluated to improve prediction of clinical outcome and provide a rational basis for experimental therapy. Immunologic and molecular studies have shown that most Hodgkin and Reed-Sternberg (HRS) cells of classical HD are derived from germinal center B cells with rearranged immunoglobulin genes bearing crippling mutations.16-19 Previous immunohistologic studies have detected B-cell markers in HRS cells, including CD20 and CD79a.20-28 Recently, the PAX-5 gene product, also known as B-cellspecic activator protein (BSAP), a transcription factor specic for B cells, has been detected in HRS cells of classical HD.29,30 CD20 is a transmembrane protein involved in the regulation of human B-cell growth and differentiation.31,32 It has been suggested that CD20 may function as a calcium channel, thus initiating intracellular signals important for differentiation and cell-cycle progression of B lymphocytes.33 CD20 is detectable on the surface of most mature
Journal of Clinical Oncology, Vol 20, No 5 (March 1), 2002: pp 1278-1287 Downloaded from jco.ascopubs.org on June 8, 2011. For personal use only. No other uses without permission. Copyright 2002 American Society of Clinical Oncology. All rights reserved.
CD20 IN CLASSICAL HODGKINS DISEASE
were positive for CD30 and/or CD15. Patients with antibodies to human immunodeciency virus-1 by standard enzyme-linked immunoassays were excluded from the analysis.
normal and neoplastic B lymphocytes. In addition, CD20 is detected in the malignant lymphocytic and histiocytic (L&H) cells of almost all HD of nodular lymphocyte predominance (LPHD) type.20,34,35 The neoplastic HRS cells of classical HD also express CD20 with a reported frequency ranging from less than 5% to more than 50% of tumors.22-25,27,36 The expression of CD20 by B-cell non-Hodgkins lymphomas has been targeted by the monoclonal antibody Rituximab, with good response rates in indolent and aggressive lymphomas.37-39 The combination of Rituximab and chemotherapy in CD20-positive diffuse large B-cell lymphomas has signicantly improved clinical outcome.40 Preliminary reports have indicated that Rituximab is active in relapsed LPHD, which expresses CD20.41 However, its activity in classical HD is unknown, as is the activity of chemotherapy combined with Rituximab. The prognostic signicance of CD20 expression in classical HD is controversial. Recently, the Memorial SloanKettering group reported that CD20 expression in HRS cells is associated with inferior clinical outcome in previously untreated adults with HD.42 The German Hodgkin Study Group reported inferior FFS and OS for patients with classical HD expressing only CD20 but not CD30 and CD15 in HRS cells.27 These tumors represented 1.6% of the whole patient population, and the authors raised the possibility that they might not be HD. By contrast, in the same study, CD20 expression was not associated with different clinical outcome in HD patients with HRS cells that expressed CD30.27 Because this analysis included patients treated with different regimens, it is not clear what the outcome would be in uniformly treated patients. We therefore decided to investigate CD20 expression in previously untreated patients with classical HD and determine its association with presenting clinical and laboratory features and clinical outcome. To minimize the effect of heterogeneous therapy, we determined the FFS of patients treated with equivalent regimens.

PATIENTS AND METHODS

Staging
All patients underwent physical examination, chest radiography, bone marrow biopsy, and computed tomography of the chest, abdomen, and pelvis. When clinically indicated, lymphangiograms, gallium scans, and computed tomography scans of the head and neck were also obtained according to the individual practices of the participating institutions. The ratio of mediastinal mass to thoracic diameter was measured at the T4 to T5 interspace, as previously described,4 and was considered high if it was 0.45 or greater. The Ann Arbor stage45 and treatment according to either standard or investigational protocols were determined by the attending physician at each institution. Serum LDH was considered high if the levels were 150% or higher than the upper normal limit, as previously described,12 and serum albumin was dened as low if less than 3.5 g/dL.4 Anemia was dened as hemoglobin less than 12 g/dL for female patients and less than 14 g/dL for male patients.4 Serum 2-microglobulin measured by radioimmunoassay (Pharmacia Diagnostics, Uppsala, Sweden) was considered high when greater than the upper limit of normal at each institution.12

Therapy

Treatment was either standard or according to investigational protocols active during the time the patient was diagnosed at the participating institutions. Informed consents (signed or oral) were obtained before all procedures and before the administration of all investigational therapy according to local practice guidelines. Regimens included doxorubicin, bleomycin, vinblastine, and dacarbazine (ABVD) or epirubicin, bleomycin, vinblastine, and dacarbazine (EBVD)46,47; mitoxantrone, vincristine, vinblastine, and prednisone (NOVP) followed by radiotherapy48; nitrogen mustard, vincristine, prednisone, and procarbazine (MOPP)1; MOPP alternating with ABVD3; cyclophosphamide, vinblastine, prednisone, and procarbazine (CVPP) alternating with doxorubicin, bleomycin, dacarbazine, prednisone, and carmustine (ABDIC)48; and vinblastine, etoposide, epirubicin, bleomycin, cyclophosphamide, and prednisone (VEBEP).49 Radiotherapy was administered alone or after completion of chemotherapy with various ports and dosages according to local standard or investigative protocols. For the purposes of this analysis, ABVD, EBVD, CVPP/ABDIC, MOPP/ABVD, and VEBEP were considered equivalent regimens.3,12 Complete remission (CR) was dened as absence of disease for at least 1 month as determined by physical examination and appropriate laboratory and imaging studies. Partial response (PR) was dened as more than 50% reduction of tumor mass measurable in two dimensions. Progressive disease (PD) was dened as enlargement ( 25%) of a preexisting site of disease or development of disease in a previously uninvolved site. Primary treatment failure was dened as failure to achieve CR or PR during initial therapy. Relapse was dened as progression occurring at least 1 month after CR or PR.

Patients

Patients were eligible if they had presented from 1984 to 1996 without any prior treatment to the University of Texas M.D. Anderson Cancer Center (Houston, TX), Istituto Nationale Tumori (Milan, Italy), University of Verona (Verona, Italy), or National and Kapodistrian University of Athens (Athens, Greece). It was required that pathologic diagnosis be made on the basis of tissue biopsy, that tumor tissue be available for immunohistochemical determination of CD20 expression, and that the histologic diagnosis be conrmed by review of available slides at the time CD20 expression was evaluated, according to criteria dened by the Revised European-American Lymphoma and World Health Organization classications.43,44 In all cases, the neoplastic cells

Immunohistochemistry

Method. Tissue sections were deparafnized in xylene and rehydrated in a graded series of ethanols. Endogenous peroxidase was blocked by 3% hydrogen peroxide in 140 mmol/L NaCl, 2 mmol/L KCl, 12 mmol/L NaPO4, and 1.7 mmol/L KH2PO4 (pH 7.4) (phosphate-buffered saline) for 10 minutes at room temperature. Heat-
Downloaded from jco.ascopubs.org on June 8, 2011. For personal use only. No other uses without permission. Copyright 2002 American Society of Clinical Oncology. All rights reserved.
induced epitope retrieval used a modication of a previously published method.50 Tissue sections were heated in10 mmol/L sodium citrate buffer (pH 6.4) in a household vegetable steamer (Sunbeam 4713/5710, 900 W; Sunbeam-Oster, Boca Raton, FL) for 30 minutes and allowed to cool to room temperature for at least 15 minutes. After equilibration with phosphate buffered saline, the slides were incubated with proteinblocking solution (Dako, Carpinteria, CA) for 15 minutes to block nonspecic protein binding sites. Subsequently, the slides were incubated with the L26 primary monoclonal antibody specic for CD20 (Dako) in a dilution of 1:100 (nal concentration of the monoclonal immunoglobulin, 0.3 g/mL) in 0.1% bovine serum albumin in 50 mmol/L TrisHCl buffer (pH 7.6) for 1 hour. The LSAB system (Dako), based on streptavidin-biotin-peroxidase complex, was applied for the detection of immunoreaction. Color was developed with 3,3'-diaminobenzidine and hydrogen peroxide (Dako), and all slides were subsequently counterstained with hematoxylin. Each reaction set included external controls, a CD20-positive follicular lymphoma, and a CD20-negative cell block of K562 cells. In addition, coexisting small reactive lymphocytes served as internal positive and negative controls in each slide. Evaluation. Evaluation of all immunostained slides was performed by two pathologists (G.Z.R and L.J.M) without knowledge of the clinical outcome. All slides were reviewed at the time of immunohistochemical analysis for conrmation of diagnosis of HD according to criteria dened by the Revised European-American Lymphoma and World Health Organization classications.43,44 HRS cells in this study included mononuclear variants (in mixed cellularity), lacunar cells (in nodular sclerosis), and typical Reed-Sternberg cells. Slides were considered assessable if all concurrent internal and external controls stained appropriately. Any membranous or cytoplasmic CD20 staining of the malignant HRS cells was considered positive. In most cases, at least 100 HRS cells in representative elds were present and were counted to determine the percentage of CD20positive HRS cells. In a small subset of cases, less than 100 HRS cells were present. In these tumors, we counted all HRS cells and calculated the percentage. The following three categories of staining intensity were identied: absent, when no HRS cells expressed CD20; weak to moderate, when HRS cells stained for CD20 less intensely than the small reactive B lymphocytes; and strong, when HRS cells stained for CD20 with the same or greater intensity than small reactive B lymphocytes.

RASSIDAKIS ET AL

and 1996. Archival pretreatment biopsy material was available for 598 patients who constitute the study group. Most presenting clinical and laboratory features of patients with known versus unknown CD20 expression are similar, as shown in Table 1. Patients with known CD20 expression status were more likely to have high serum 2-microglobulin, low serum albumin, and anemia than those without available tissue. However, the extent of differences was small, even though the large number of patients suggested that the differences were unlikely to be random. Therefore, we consider the analyzed group to be representative of the whole patient population. The histologic subtypes of HD with known CD20 expression were nodular sclerosis in 472, mixed cellularity in 123, and lymphocyte depletion in three patients (Table 1). Treatment was ABVD or equivalent regimen in 302, NOVP and radiotherapy in 169, MOPP in 53, and radiotherapy alone in 53 patients. CD20 Expression CD20 was detected in HRS cells of 132 (22%) of 598 classical HD tumors (Table 2), where it was present in the membrane and cytoplasm of HRS cells, but with variable staining intensity (Fig 1). Reactive small B lymphocytes, present in variable numbers in all cases, were strongly positive for CD20 and served as internal positive controls (Fig 1). At rst statistical analysis, a tumor was considered positive for CD20 if any HRS cells were immunoreactive, regardless of the percentage or the staining intensity of positive HRS cells. However, the expression of CD20 in HRS cells of classical HD varied considerably between tumors both in terms of percentage of positive HRS cells (Fig 2a) and their staining intensity (Fig 2b). The median percentage of HRS cells expressing CD20 was 55% in the CD20-positive tumors (Fig 2a). Strong staining for CD20, as dened in the Methods section, in HRS cells was found in 33% of the cases of classical HD (Fig 2b). The percentage of CD20-positive HRS cells was strongly associated with the intensity of staining. High percentage of CD20-positive HRS cells was statistically associated with strong staining intensity (P .0001 by Mann-Whitney test). The correlation between CD20 expression and various presenting clinical and laboratory characteristics is shown in Table 3. CD20 immunoreactivity was slightly more frequent in patients with stage I disease and less frequent in patients with high levels of serum LDH and 2-microglobulin and anemia, but these associations did not reach statistical signicance (Table 3). Patients with involvement of inguinal or iliac lymph nodes or involvement of the bone marrow had a lower frequency of CD20 expression in HRS cells. However, the P values were of marginal signicance (P .05 and.05, respectively).

Statistical Analysis

FFS was measured according to the method of Kaplan and Meier,51 from the beginning of treatment to primary treatment failure, relapse, or last follow-up. Patients who died during treatment without evidence of progressive disease or after the end of therapy without prior evidence of relapse were censored. The statistical signicance of differences in FFS between groups of patients was estimated by the log-rank test.52 The comparisons between CD20 expression and clinical or laboratory parameters were based on 2 and Fishers exact tests. Nonparametric Mann-Whitney test was used to evaluate the correlation between patient age and CD20 expression. All statistical calculations were performed using StatView (Abacus Concepts, Berkeley, CA).

RESULTS

Study Group We identied 1,568 untreated patients with classical HD who presented to the participating institutions between 1984
Table 1. Presenting Clinical and Laboratory Characteristics of Patients With Classical HD
CD20 Expression Known No. % CD20 Expression Unknown No. %
All patients Age Median Range Male sex B symptoms Histology Nodular sclerosis Mixed cellularity Lymphocyte depletion Ann Arbor stage I II III IV High serum LDH High serum 2-microglobulin Inguinal or iliac node involvement Mediastinal mass ratio 0.45 Low serum albumin Anemia
6-216/49/556 92/422 104/598 63/484 71/568 250/595

100 NA NA 42

4-345/60/640 67/415 159/947 24/262 138/753 460/949

100 NA NA 2.48.4*.7.9

.6.04.8.4.03.01
Abbreviation: NA, not applicable. *Evaluated by Mann-Whitney test. Fisher exact test was used for all other comparisons.
FFS Analysis After a median follow-up of 65 months for the survivors, 46 of the 302 patients treated with ABVD or equivalent regimen had refractory disease or relapsed. The 5-year FFS for patients with CD20-positive HRS cells was 86% versus 84% for patients with CD20-negative HRS cells (P .7 by log-rank test) (Fig 3a). When the analysis was restricted to the 155 patients with stage I or II disease, the 5-year FFS was almost identical for tumors with versus without CD20 expression (90% v 92%, P .9 by log-rank test; Fig 3b). Similarly, for patients with stage III or IV disease, CD20 expression was not associated with signicantly different 5-year FFS (82% v 76%, P .5 by log-rank test; Fig 3c). Forty-four of 169 patients with stage I through III disease who were treated with NOVP and radiotherapy had refractory disease or relapsed. The 5-year FFS was 74% versus
77% for those with CD20-positive versus CD20-negative tumors, respectively (P .7 by log-rank test; Fig 4a). Nine of the 53 patients treated with MOPP had refractory disease or relapsed. The 5-year FFS for MOPP-treated patients with any stage of disease did not signicantly differ between patients with CD20-positive and CD20-negative tumors (74% v 84%, P .4 by log-rank test; Fig 4b) Nine of 53 patients treated only with radiotherapy had refractory disease or relapsed. The 5-year FFS was 77% for patients with CD20-positive tumors versus 88% for patients with CD20-negative tumors (P .5 by log-rank test; Fig 4c). Cutoffs for CD20 Expression Because the frequency of CD20 expression seemed to be a continuous variable, ve different cutoffs for the percentage of CD20-positive HRS cells were examined, 5%, 10%,

Table 2.

Patients (n)
CD20 Expression in HRS Cells According to Histologic Subtype
CD20-Positive Patients No. % % of HRS Cells Expressing CD20* Mean Range

Histology

All patients Nodular sclerosis Mixed cellularity Lymphocyte depletion

2-100 2-100 8-99 0

*For the 132 tumors with any CD20 expression in HRS cells. P .2 by Mann-Whitney test.
Fig 1. CD20 expression in classical HD. (a, b) Mixed cellularity HD with CD20-negative HRS cells. (c, d) Nodular sclerosis HD with weak CD20 expression in HRS cells. (e, f), Nodular sclerosis HD with a large number of HRS cells strongly expressing CD20. Small reactive lymphocytes stained positively for CD20 and served as internal positive controls in all panels.
20%, 30%, and 50%. The selection of these cutoffs was based on the distribution of CD20-positive HRS cells (histogram) in the cases of classical HD of the present series (Fig 2a). Table 4 summarizes the survival analysis results according to the percentage of CD20-positive HRS cells. No signicant difference in FFS was found between the patient groups with CD20-positive versus CD20-negative tumors using different percentages as cutoffs when uniform treatment was considered (Table 4). Similarly, CD20 staining intensity (strong v weak or moderate) was not statistically associated with FFS in the entire study group or in any of
the patient groups treated with equivalent regimens (data not shown).

DISCUSSION

In this study, we report that CD20 is expressed by HRS cells in 22% of patients with classical HD, if we consider the presence for any CD20-positive HRS cells as a criterion for positivity. However, with this or other cutoffs, there was no correlation between CD20 expression in HRS cells and clinical outcome. This conclusion is based on analysis of 598 patients derived from an international database of 1,568
Fig 2. (a) Frequency of HRS cells expressing CD20 among classical HD patients. Only positive tumors are shown. (b) Staining intensity of CD20 in tumors with classical HD.
untreated patients with biopsy-proven classical HD. Thus, we eliminated most selection biases toward presenting features and clinical outcome, which might arise by entry of patients presenting at relapse. Statistical analysis showed that the presenting clinical and laboratory characteristics of these 598 patients and of the 970 without available tissue for immunohistochemical analysis were similar. Therefore, we considered the population with available tissue to be representative of the entire patient population. Our results on frequency of CD20 expression are generally in agreement with most previous studies.36 We also

Table 3.

Correlation of CD20 Expression With Presenting Clinical and Laboratory Findings in Classical HD
Parameter % of Tumors Expressing CD20 P*
Age, 45 v 45 years Sex, male v female Stage, I v II v III v IV Stage, I v II through IV Stage, I and II v III and IV Serum albumin, low v normal B symptoms, present v absent Anemia, present v absent Serum 2-microglobulin, high v normal Serum LDH, high v normal Bone marrow involvement, present v absent Mediastinal mass ratio, .45 v .45 Inguinal or iliac nodes, involved v uninvolved

30 v 15

v v v v v v v v v v v v v

21 v 24

.2.6.3.1.2.18.3.1.1.06.05.5.05
*All P values were calculated with Fishers exact test, except for the P value between CD20 expression and stage I versus II versus III versus IV, which was calculated with the 2 test. As dened in Patients and Methods.
report for rst time the distribution of the percentage of CD20-positive HRS cells and the staining intensity, providing novel and detailed information for the expression of CD20 by HRS cells in classical HD. Our ndings support the published molecular single-cell and immunohistochemical studies, suggesting that the HRS cells arise from germinal center B cells with rearranged immunoglobulin genes.16,19,29 However, it is unknown why only a proportion of HRS cells express CD20 in a given tumor, because they are clonal. Theoretically, this might reect the position of HRS cells in B-cell differentiation.29,53 It is also not known whether the clonogenic HRS cells responsible for tumor propagation express CD20 in tumors where the frequency of CD20 expression is not high and its staining intensity is weak. Expression of CD20 was not associated with major differences in presenting clinical or laboratory features. Patients without involvement of iliac or inguinal lymph nodes or without bone marrow involvement had statistically higher frequency of CD20 expression. However, the P values were marginally signicant. CD20 expression was not associated with signicantly different FFS in homogeneously treated patient cohorts in our patient population. The prognostic signicance of CD20 expression by HRS cells of classical HD is controversial. In a previous study, the German Hodgkin Study Group used a cutoff of 20% to distinguish CD20-positive versus CD20-negative tumors.27 There were 21 tumors among 1,286 that expressed only CD20 but not CD30 or CD15. These patients had inferior FFS and overall survival.27 The authors indicated that these tumors might be reclassied after pathology review; how-

Fig 3. FFS and CD20 expression in classical Hodgkins disease for patients treated with ABVD or equivalent regimens for all patients (a), patients with stage I or II disease (b), and patients with stage III or IV disease (c). Circles indicate CD20 positive; diamonds, CD20 negative.
Fig 4. FFS and CD20 expression for patients with classical Hodgkins disease. (a) Patients with stage I through III disease treated with NOVP and radiotherapy; (b) patients with all stages of disease treated with MOPP; (c) patients with stage I through III disease treated only with radiotherapy. Circles indicate CD20 positive; diamonds, CD20 negative.
ever, to our knowledge, the results of this retrospective pathology re-evaluation have not yet been reported. By contrast, CD20 expression was not related to prognosis among the patients with HRS cells expressing CD15 or
CD30.27 Recently, the group from Memorial Sloan-Kettering reported an association of CD20 expression in HRS cells with worse clinical outcome in classical HD.42 However, CD20 was detected in only 9% of tumors with

Table 4.

CD20 Cutoff (%) % of Patients CD20 Positive
FFS at 5 Years According to the Frequency of CD20 Expression in HRS Cells of Classical HD

5-Year FFS*

ABVD or Equivalent (n 302)
NOVP Radiotherapy (n 169)

MOPP (n 53)

Radiotherapy Alone (n 53)
v P .7 v P .8 v P .9 v P .10 v P .10 v P .3
v P .8 v P .8 v P .11 v P .13 v P .15 v P .7
v P .12 v P .13 v P .15 v P .15 v P .15 v P .4
v P .10 v P .11 v P .14 v P .13 v P .13 v P .9
*The rst value in each column is the FFS of patients with CD20-positive HRS cells greater or equal to the cutoff, and the second value is the FFS of patients with CD20-positive HRS cells less than the cutoff. Log-rank (Mantel-Cox) P values were used for all statistical comparisons.
classical HD histology. Furthermore, it is not clear which cutoff was used for CD20 positivity.42 Because CD20 has no known function that could confer resistance to chemotherapy, it is not immediately apparent why its expression should be associated with a different FFS. Even when various cutoffs are used by us to distinguish positive from negative classical HD tumors, the FFS does not change signicantly, whereas the percentage of the CD20-positive tumors declines (Table 4). Our results may have clinical implications for the treatment of HD with Rituximab. Preliminary results suggest that Rituximab is very active against LPHD,41 where CD20 is expressed by most L&H cells in more than 90% of tumors.35 Anecdotal experience also suggests that Rituximab is active against classical HD-expressing CD20. How-
ever, the relationship between response rate and the frequency and intensity of CD20 expression by HRS cells remains unknown. Present studies in our laboratory are exploring the possibility of experimental modulation of CD20 expression in HRS cells to increase the expression of CD20 as a possible target of Rituximab. We conclude that CD20 is expressed by HRS cells in 22% of patients with classical HD, but with a variable staining pattern, both in the percentage of CD20-positive HRS cells and the intensity of CD20 expression. However, expression of CD20 was not associated with different presenting clinical and laboratory features or prognosis among uniformly treated patients. The expression of CD20 in HRS cells may serve as a guide for the treatment of HD patients with regimens including anti-CD20 antibody and chemotherapy.

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RASSIDAKIS ET AL 29. Foss HD, Reusch R, Demel G, et al: Frequent expression of the B-cell-specic activator protein in Reed-Sternberg cells of classical Hodgkins disease provides further evidence for its B-cell origin. Blood 94:3108-3113, 1999 30. Krenacs L, Himmelmann AW, Quintanilla-Martinez L, et al: Transcription factor B-cell-specic activator protein (BSAP) is differentially expressed in B cells and in subsets of B-cell lymphomas. Blood 92:1308-1316, 1998 31. Einfeld DA, Brown JP, Valentine MA, et al: Molecular cloning of the human B cell CD20 receptor predicts a hydrophobic protein with multiple transmembrane domains. EMBO J 7:711-717, 1988 32. Tedder TF, Klejman G, Schlossman SF, et al: Structure of the gene encoding the human B lymphocyte differentiation antigen CD20 (B1). J Immunol 142:2560-2568, 1989 33. Tedder TF, Engel P: CD20: A regulator of cell-cycle progression of B lymphocytes. Immunol Today 15:450-454, 1994 34. von Wasielewski R, Werner M, Fischer R, et al: Lymphocytepredominant Hodgkins disease: An immunohistochemical analysis of 208 reviewed Hodgkins disease cases from the German Hodgkin Study Group. Am J Pathol 150:793-803, 1997 35. Anagnostopoulos I, Hansmann ML, Franssila K, et al: European Task Force on Lymphoma project on lymphocyte predominance Hodgkin disease: Histologic and immunohistologic analysis of submitted cases reveals 2 types of Hodgkin disease with a nodular growth pattern and abundant lymphocytes. Blood 96:1889-1899, 2000 36. Weiss LM, Chan JKC, MacLennan K, et al: Pathology of classical Hodgkins disease, in Mauch PM, Armitage JO, Diehl V, et al (eds): Hodgkins Disease. Philadelphia, PA, Lippincott Williams & Wilkins, 1999, pp 101-120 37. Maloney DG, Grillo-Lopez AJ, White CA, et al: IDEC-C2B8 (rituximab) anti-CD20 monoclonal antibody therapy in patients with relapsed low-grade non-Hodgkins lymphoma. Blood 90:2188-2195, 1997 38. McLaughlin P, Grillo-Lopez AJ, Link BK, et al: Rituximab chimeric anti-CD20 monoclonal antibody therapy for relapsed indolent lymphoma: Half of patients respond to a four-dose treatment program. J Clin Oncol 16:2825-2833, 1998 39. Colombat P, Salles G, Brousse N, et al: Rituximab (anti-CD20 monoclonal antibody) as single rst-line therapy for patients with follicular lymphoma with a low tumor burden: Clinical and molecular evaluation. Blood 97:101-106, 2001 40. Coifer B, Lepage E, Herbrecht R, et al: Mabthera (rituximab) plus CHOP is superior to CHOP alone in elderly patients with diffuse large B-cell lymphoma (DLCL): Interim results of a randomized GELA trial. Blood 96:223a, 2000 (abstr) 41. Lukas JB, Hoppe RT, Horwitz SM, et al: Rituximab is active in lymphocyte predominance Hodgkins disease. Blood 96:508a, 2000 (abstr) 42. Donnelly GB, Filippa D, Moskowitz CH, et al: Increased treatment failure in patients with CD20 positive classic Hodgkins disease (HD). Blood 94:2662, 1999 (part 1, suppl 1, abstr) 43. Harris NL, Jaffe ES, Stein H, et al: A revised EuropeanAmerican classication of lymphoid neoplasms: A proposal fromthe International Lymphoma Study Group. Blood 84:1361-1392, 1994 44. Stein H, Mann R, Delsol G, et al: Classical Hodgkin lymphoma, in Jaffe ES, Harris NL, Stein H, et al (eds): Pathology & Genetics of Tumours of Haematopoietic and Lymphoid TissuesWorld Health Organization Classication of Tumours. Lyon, France, IARC Press, 2001, pp 244-253

CD20 IN CLASSICAL HODGKINS DISEASE 45. Carbone PP, Kaplan HS, Musshoff K, et al: Report of the committee on Hodgkins disease staging classication. Cancer Res 31:1860-1861, 1971 46. Bonadonna G, Valagussa P, Santoro A: Alternating non-cross-resistant combination chemotherapy or MOPP in stage IV Hodgkins disease: A report of 8-year results. Ann Intern Med 104:739-746, 1986 47. Angelopoulou MK, Vassilakopoulos TP, Siakantaris MP, et al: EBVD combination chemotherapy plus low dose involved eld radiation is a highly effective treatment modality for early stage Hodgkins disease. Leuk Lymphoma 37:131-143, 2000 48. Hagemeister FB, Fuller L, McLaughlin P, et al: NOVP and radiotherapy for early-staged Hodgkins disease: An interim analysis. Ann Oncol 4:87-90, 1992 49. Viviani S, Bonfante V, Santoro A, et al: Long-term results of an intensive regimen: VEBEP plus involved-eld radiotherapy
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