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CD20 Expression in Hodgkin and Reed-Sternberg Cells of Classical Hodgkin’s Disease: Associations With Presenting Features and Clinical Outcome

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.

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{at}mail.mdanderson.org

	ABSTRACT

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
PURPOSE: CD20 can be expressed in Hodgkin and Reed-Sternberg (HRS) cells of classical Hodgkin’s disease (HD), but its clinical significance 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 immunodeficiency virus-1 negative, had biopsy-proven classical HD, and if pretreatment paraffin-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 identified 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.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
ALTHOUGH HODGKIN’S 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, leukocyte and lymphocyte counts, and serum levels of albumin, lactate dehydrogenase (LDH), and ß₂-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-cell–specific activator protein (BSAP), a transcription factor specific 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 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-Hodgkin’s 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 significantly 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 significance of CD20 expression in classical HD is controversial. Recently, the Memorial Sloan-Kettering 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

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
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 confirmed by review of available slides at the time CD20 expression was evaluated, according to criteria defined by the Revised European-American Lymphoma and World Health Organization classifications.^43,44 In all cases, the neoplastic cells were positive for CD30 and/or CD15. Patients with antibodies to human immunodeficiency virus-1 by standard enzyme-linked immunoassays were excluded from the analysis.

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 defined as low if less than 3.5 g/dL.⁴ Anemia was defined as hemoglobin less than 12 g/dL for female patients and less than 14 g/dL for male patients.⁴ Serum ß₂-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 defined as absence of disease for at least 1 month as determined by physical examination and appropriate laboratory and imaging studies. Partial response (PR) was defined as more than 50% reduction of tumor mass measurable in two dimensions. Progressive disease (PD) was defined as enlargement (> 25%) of a preexisting site of disease or development of disease in a previously uninvolved site. Primary treatment failure was defined as failure to achieve CR or PR during initial therapy. Relapse was defined as progression occurring at least 1 month after CR or PR.

Immunohistochemistry
Method. Tissue sections were deparaffinized 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-induced epitope retrieval used a modification 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 protein-blocking solution (Dako, Carpinteria, CA) for 15 minutes to block nonspecific protein binding sites. Subsequently, the slides were incubated with the L26 primary monoclonal antibody specific for CD20 (Dako) in a dilution of 1:100 (final concentration of the monoclonal immunoglobulin, 0.3 µg/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 confirmation of diagnosis of HD according to criteria defined by the Revised European-American Lymphoma and World Health Organization classifications.^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 fields were present and were counted to determine the percentage of CD20-positive 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 identified: 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.

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 significance 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 Fisher’s 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

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Study Group
We identified 1,568 untreated patients with classical HD who presented to the participating institutions between 1984 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 ß₂-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.

View larger version (188K): [in this window] [in a new window]   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.

View larger version (20K): [in this window] [in a new window]   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.

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 ß₂-microglobulin and anemia, but these associations did not reach statistical significance (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 significance (P = .05 and .05, respectively).

View larger version (21K): [in this window] [in a new window]   Fig 3. FFS and CD20 expression in classical Hodgkin’s 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.

View larger version (21K): [in this window] [in a new window]   Fig 4. FFS and CD20 expression for patients with classical Hodgkin’s 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.

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, five different cutoffs for the percentage of CD20-positive HRS cells were examined, 5%, 10%, 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 significant 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

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

Our results on frequency of CD20 expression are generally in agreement with most previous studies.³⁶ We also report for first 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 findings 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 reflect 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 significant. CD20 expression was not associated with significantly different FFS in homogeneously treated patient cohorts in our patient population.

The prognostic significance 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 reclassified after pathology review; however, 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 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 significantly, 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. However, 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.

	ACKNOWLEDGMENTS

 
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.

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Submitted April 6, 2001; accepted November 9, 2001.

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