Expression of bcl-2 in Classical Hodgkin's Lymphoma: An Independent Predictor of Poor Outcome
http://www.100md.com
《临床肿瘤学》
the Department of Clinical Pathology, and Department of Hematology and Medical Oncology, Cleveland Clinic Foundation, Cleveland, OH
ABSTRACT
PURPOSE: Although most classical Hodgkin's lymphoma (CHL) patients are cured, a significant minority fails primary therapy and may die as a result of their disease. Age, stage, and other basic clinical and laboratory parameters, which comprise the International Prognostic Score (IPS), are used at diagnosis to predict outcome. To date, there is no consensus on biologic markers that add value to these parameters.
PATIENTS AND METHODS: We evaluated 107 CHL patients for bcl-2, p53, and p21 expression by immunohistochemistry using tissue microarrays and correlated the results with outcome. The median follow-up of the 79 surviving patients was 6.8 years.
RESULTS: Univariate analysis showed that age 45 years, stage III or IV, and IPS 3 were associated with a poor failure-free survival (FFS) and overall survival (OS). bcl-2 was expressed in 26% of patients and was associated with poor FFS and a trend for OS. p53 expression in combination with lack of p21 expression was not associated with outcome. Multivariate analysis showed that three factors were independently associated with both FFS and OS: age 45 years, stage III or IV, and bcl-2 expression. Using these three parameters, a scoring system was devised that stratified patients into three risk groups (with zero, one, or two to three of these risk factors) and a progressively worse FFS and OS (P < .001).
CONCLUSION: Expression of bcl-2 in CHL is a useful, independent prognostic marker and can be used in association with clinical parameters to identify newly diagnosed patients with a good, intermediate, or poor prognosis.
INTRODUCTION
The majority of patients with classical Hodgkin's lymphoma (CHL) are cured. Nevertheless, a significant minority experiences treatment failure after primary therapy, and many of these patients ultimately succumb to their disease or complications of its treatment. The ability to identify more accurately patients at risk for primary treatment failure might allow the application of novel therapies in high-risk patients and at the same time minimize the exposure to potentially toxic therapies in low-risk patients. Many clinical parameters, such as older age, advanced stage, and bulky disease, have been associated with a worse prognosis. Several prognostic models have been used to stratify patients into good- and poor-risk groups. The International Prognostic Score (IPS), which incorporates seven clinical and laboratory parameters, was recently developed for patients with advanced-stage Hodgkin's lymphoma (HL) but has also been applied to patients with early-stage disease.1,2 In addition, some investigators have studied biologic markers that might provide additional prognostic information to these clinical models. Several candidate molecules, including bcl-2 and p53, have been proposed. The independent predictive power of these markers is controversial and none has gained wide acceptance.3-11 To further evaluate the prognostic significance of these biologic markers in CHL, we assessed bcl-2 and p53 expression in Hodgkin's and Reed-Sternberg (HRS) cells of archived specimens using tissue microarrays. We analyzed these results in conjunction with clinical and laboratory parameters and correlated them with patient outcomes.
PATIENTS AND METHODS
The archives of the Division of Pathology and Laboratory Medicine and medical records of the Department of Hematology and Medical Oncology were searched for patients with CHL diagnosed at the Cleveland Clinic Foundation from 1986 to 2000. Patients were included in this study if the initial diagnostic biopsy specimen was available for analysis, a diagnosis of CHL was confirmed, and adequate clinical and laboratory data from the time of diagnosis as well as follow-up information were available. Of the original 137 patients identified for review, 30 were excluded because the diagnosis was changed, the biopsy sample was from the time of disease recurrence, or the clinical information was inadequate. This study was performed after approval by the Cleveland Clinic Foundation Institutional Review Board.
Patients
Patients were staged at initial diagnosis with radiographic studies and bone marrow biopsies according to the Ann Arbor staging system.12 Medical records were reviewed and the following clinical and laboratory data from the time of diagnosis were collected: age, sex, stage, bulky tumors (defined as a mediastinal widening one third of the thoracic diameter or a mass 10 cm in maximum dimension), hemoglobin, WBC, lymphocyte count, and albumin. The IPS was calculated based on the seven defined high-risk features (ie, serum albumin < 4 g/dL, hemoglobin < 10.5 g/dL, male sex, stage IV disease, age 45 years, WBC 15,000/μL, and lymphocyte count < 600/μL), and patients were divided into a low-risk group (zero to two factors) and a high-risk group (three to seven factors), as previously described.2
Initial treatment for each patient was reviewed. All patients were treated with curative intent. During the study period, standards of care changed and, consequently, patients received a variety of treatments. In general, newly diagnosed patients with early-stage disease received radiation therapy alone or combination chemotherapy with or without radiation therapy, and patients with advanced-stage disease received combination chemotherapy with or without radiation therapy. Patients who experienced treatment failure after primary radiation therapy received combination chemotherapy, and patients who experienced treatment failure after initial chemotherapy received salvage chemotherapy and/or high-dose therapy with autologous stem-cell transplantation. Follow-up information was obtained from the medical records or the Cleveland Clinic Taussig Cancer Center Tumor Registry. The dates of diagnosis, disease progression, and/or death were recorded.
Tissue Microarray
Paraffin tissue blocks from the initial biopsies were retrieved and a tissue microarray was constructed (Beecher Instruments, Sun Prairie, WI). The biopsies were sampled using 1.5-mm-diameter tissue cores selected from areas of the whole tissue biopsy with the highest concentrations of HRS cells. The cores were arrayed in triplicate in the vast majority of samples, with a few samples having two or four cores.
Immunohistochemistry
Immunohistochemistry was performed using automated immunohistochemical stainers (Ventana, Tucson, AZ) and antibodies to bcl-2 (clone 100/D5; Ventana), p53 (clone DO7; DAKO, Carpinteria, CA), and p21 (clone SX118; BD Pharmingen, San Diego, CA). p21 expression was assessed because the expression of p53 in the absence of p21 (p53 positive/p21 negative) has been suggested as a better surrogate for mutated p53 than p53 expression alone.13 Samples were reviewed for expression of these proteins in HRS cells by S.S. and E.H., who were blinded as to outcome. Samples were considered positive only if 10% or more of the malignant cells strongly (at least as intense as small lymphocytes) expressed the protein. Expression of these proteins in positive samples varied between 10% and 75% of HRS cells. Samples in which only weak expression was present were scored as negative. Appropriate cellular localization for immunostaining was cytoplasmic for bcl-2 and nuclear for p53 and p21 (Fig 1).
Statistical Analyses
Overall survival (OS) was measured from the date of initial diagnosis to the date of death or the date of last follow-up. Failure-free survival (FFS) was measured from the date of diagnosis to the date of disease progression or death as a result of any cause, whichever came first. Patients alive and free of disease at analysis were censored as of their last follow-up. The method of Kaplan and Meier was used to summarize FFS and OS data and to estimate 5-year survival rates. The generalized Wilcoxon rank sum test was used to compare FFS and OS for individual factors, and the Cox proportional hazards model was used to assess multiple factors simultaneously. All analyses were performed using Statistical Analysis Software (SAS, version 8.0; SAS Institute Inc, Cary, NC).
RESULTS
A total of 107 patients formed the basis of this study. The patient and disease characteristics are summarized in Table 1. Eighty-three patients (78%) had nodular sclerosis, 17 patients (16%) had mixed cellularity, and seven patients (6%) had either lymphocyte-rich CHL or CHL not further specified (due to specimen limitations). Twenty-eight patients (26%) were bcl-2 positive. Eleven patients (10%) were p53 positive and nine patients (8%) were both p53 positive and p21 negative (p53 positive/p21 negative). Sixteen patients received radiation therapy only, 45 patients received chemotherapy only, and 40 patients received combined-modality therapy. Exact details of therapy were not available for six patients. Twenty-eight patients experienced disease progression during or after initial therapy and 20 patients died. Median follow-up for the 79 patients still alive and without evidence of disease is 6.8 years (range, 0.9 to 16.9 years). The estimated 5-year FFS was 75% ± 4% and the estimated 5-year OS was 85% ± 4% (Fig 2).
Table 1 lists the univariate survival analysis for sex, age, stage, bulky disease, IPS, bcl-2 positive status, and p53 positive/p21 negative status. As expected, age 45 years, stage III or IV, and IPS 3 were associated with significantly worse FFS and OS. bcl-2 expression was also associated with a significantly worse FFS (P = .02) and some indication of an association with worse OS (P = .14). Survival curves of the univariate analyses are shown in Figure 3.
Stepwise multivariate analysis was then performed using all of the factors in Table 1 to identify independent predictors of FFS and OS. On the basis of this analysis, three independent prognostic factors for both FFS and OS were identified: bcl-2 status, age, and stage. These data are summarized in Table 2.
these results, a simple scoring system using these two standard clinical parameters and a biologic marker was devised. On the basis of the number of adverse prognostic factors (ie, bcl-2 positivity, age 45 years, and stage III or IV), patients were stratified into three distinct groups each having a different prognosis. Patients with no poor prognostic factors were considered good risk; patients with one poor prognostic factor were considered intermediate risk; and patients with two or three poor prognostic factors were considered high risk. Using these definitions, 31% of patients in this series were considered good risk, 50% were considered intermediate risk, and 19% were considered high risk. Only one of the good-risk patients has relapsed and none have died (median follow-up for 32 patients alive and disease-free, 93 months). In contrast, the estimated 5-year FFS and OS are 69% ± 7% and 85% ± 5%, respectively, for the intermediate-risk group, and 54% ± 11% and 58% ± 11%, respectively, for the poor-risk group (P < .001, trend test). FFS and OS curves for the three groups are shown in Figure 4.
DISCUSSION
The pathogenesis of CHL remains poorly understood. Single-cell techniques have proven that HRS cells are derived from germinal center B cells, which harbor clonally rearranged and somatically mutated immunoglobulin genes but lack immunoglobulin expression.14 Although normal germinal center B cells that lack functional high-affinity antibody undergo apoptosis within the germinal center, HRS cells may escape this programmed cell death.15-17 Indeed, expression of bcl-2, other bcl-2 family members (eg, bcl-XL, BAX), and apoptosis regulators are altered in HL.5,6,8-11,15,16,18-20 The altered expression of these proteins in HRS cells may prevent apoptosis caused by the absence of the functional B-cell receptor and explain resistance to treatment-induced apoptosis and treatment failure.
Many biologic markers associated with apoptosis (including bcl-2, BAX, bcl-XL, and p53) have been studied in an attempt to identify clinically relevant predictors of outcome in HL.3,5-10,15,18,20,21 Several (but not all) investigators have reported that high expression of bcl-2 and p53 in HRS cells of HL patients confers a worse prognosis, but their independent predictive value has varied.5,7,9,10,20 In our study, bcl-2 (but not p53) expression in HRS cells was an independent and statistically significant predictor of poor outcome in patients with CHL. Furthermore, this biologic marker in conjunction with age and stage identified three distinct groups of patients with a good, intermediate, and poor prognosis. Because of the relatively small number of treatment failures, these results require additional follow-up and, ideally, confirmation in an independent set of patients.
Several other investigators have also studied one or more of these biologic markers along with clinical parameters in an attempt to identify different prognostic groups.5,9,10,15,20,21 Rassidakis et al5 evaluated bcl-2 expression along with clinical and laboratory parameters in 412 patients with CHL who were treated with doxorubicin, bleomycin, vinblastine, and dacarbazine or equivalent regimens. As in our study, bcl-2 expression, older age, and advanced stage (as well as low albumin and high lactate dehydrogenase) were independent prognostic factors. A higher number of these five adverse factors correlated with an increasingly worse FFS.
Brink et al9 quantified the percentage of HRS cells that expressed bcl-2 and p53 in 78 patients with nodular sclerosis or mixed cellularity HL. They reported that patients in whom p53 was expressed in a high percentage of HRS cells had a 100% 5-year survival (which was independent of bcl-2 expression), and that patients in whom p53 and bcl-2 were both expressed in a low percentage of HRS cells had a 90% 5-year survival. Conversely, patients in whom p53 was expressed in a low percentage and bcl-2 was expressed in a high percentage of HRS cells had only a 57% 5-year survival. They suggested that the patients with high p53 expression might contain wild-type (and not mutated) p53, thus rendering these cells more susceptible to chemotherapy- or radiotherapy-induced apoptosis. This combined p53 low/bcl-2 high profile was associated with poor outcome on univariate analysis but failed to maintain significance in multivariate analysis.
Smolewski et al6,10 studied p53, bcl-2, and proliferating cell nuclear antigen along with clinical and laboratory parameters. They also found that advanced stage, older age, high bcl-2 expression as well as high p53 expression and poor performance status were independent predictors of poor disease-free survival, whereas advanced stage, older age, poor performance status, and high p53 expression, but not high bcl-2 expression, were independent predictors of poor OS. This study also included patients with nodular lymphocyte–predominant HL (NLPHL). Because the expression of bcl-2 and p53 in NLPHL is rare, the inclusion of 41 NLPHL patients (12.5%) in their series may have affected the results of their analysis.6,10,22
Recent series from Spain show variable results. Morente et al21 examined 140 patients with HL for p53, bcl-2, Rb, Ki-67, CD15, and Epstein-Barr virus. p53 and bcl-2 expression were not found to correlate with outcome. However, absence of Epstein-Barr virus and high proliferative fraction were independent adverse prognostic factors. Garcia et al15 initially reported that the expression of bcl-2 and p53 as well as several other markers was significantly related to a shorter OS. In addition, they noted a trend (although it was not statistically significant) for low p21. In a recent update, Montalban et al20 analyzed 40 biologic markers involved in cell differentiation and activation, cell cycle control, apoptosis regulation, and host response along with the IPS in 259 CHL patients and correlated them all with outcome. By univariate analysis, expression of p53 and p21 (as well as multiple other genes) and IPS was associated with an adverse outcome. Expression of bcl-2 was associated with a tendency toward a shorter disease-specific survival, which was not statistically significant. By multivariate analysis, expression of p53, bcl-XL, and apoptosis (as measured by terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate-biotin nick-end labeling), but not bcl-2 or p21, was associated with an adverse outcome.
We used tissue microarray and considered a sample positive for bcl-2, p53, or p21 if 10% or more of the RS cells strongly expressed the gene by immunohistochemistry. We used this criterion because the expression of these proteins in even a small percentage of HRS cells might ultimately result in primary treatment failure. The techniques and criteria used in the other studies varied considerably. Consequently, the percentage of samples considered positive for protein expression in HRS cells also varied and may in part explain the different results reported in the five other studies detailed in Table 3.
Overall, the evidence suggests that bcl-2 expression in HRS cells is an independent biologic marker associated with unfavorable outcome. However, p53 protein expression may not be a strong predictor. The great variability in p53-positive samples (10% to more than 90%) among various studies suggests problems in evaluating expression.7,9,10,15,20,21 Overexpression of p53 protein in HRS cells may be common, technical considerations not withstanding, but it is probably associated with wild-type p53 gene.15,23 p53 mutations in HRS cells are detected only sporadically and may not correlate with p53 overexpression.7,23-25 Thus, p53 expression by immunohistochemical staining is an imperfect surrogate for mutational status. In non-Hodgkin's lymphomas, expression of p53 without p21 (p53 positive/p21 negative) is indicative of mutated p53, whereas expression of both p53 and p21 (p53 positive/p21 positive) is associated with the wild-type p53.13 We also applied these criteria as surrogates for distinguishing mutated from wild-type p53. Nevertheless, we found no correlation with patient outcome.
In summary, bcl-2 is overexpressed in a significant proportion of CHL patients and is an independent adverse prognostic factor. Our findings support the use of this biologic marker in conjunction with clinical parameters such as age and stage to identify patients at diagnosis who are at a low, intermediate, or high risk for primary treatment failure. These risk groups might then be used to guide standard therapy and develop risk-stratified protocols. In addition, these data provide a rationale for novel therapies targeting bcl-2 expression in CHL.
Authors' Disclosures of Potential Conflicts of Interest
The authors indicated no potential conflicts of interest.
NOTES
Authors' disclosures of potential conflicts of interest are found at the end of this article.
REFERENCES
Franklin J, Paulus U, Lieberz D, et al: Is the International Prognostic Score for advanced stage Hodgkin's disease applicable to early stage patients German Hodgkin Lymphoma Study Group. Ann Oncol 11:617-623, 2000
Hasenclever D, Diehl V: A prognostic score for advanced Hodgkin's disease: International Prognostic Factors Project on Advanced Hodgkin's Disease. N Engl J Med 339:1506-1514, 1998
Naresh KN, O'Conor GT, Soman CS, et al: A study of p53 protein, proliferating cell nuclear antigen, and p21 in Hodgkin's disease at presentation and relapse. Hum Pathol 28:549-555, 1997
Wang J, Taylor CR: Apoptosis and cell cycle-related genes and proteins in classical Hodgkin lymphoma: Application of tissue microarray technique. Appl Immunohistochem Mol Morphol 11:206-213, 2003
Rassidakis GZ, Medeiros LJ, Vassilakopoulos TP, et al: BCL-2 expression in Hodgkin and Reed-Sternberg cells of classical Hodgkin disease predicts a poorer prognosis in patients treated with ABVD or equivalent regimens. Blood 100:3935-3941, 2002
Smolewski P, Niewiadomska H, Blonski JZ, et al: Expression of proliferating cell nuclear antigen (PCNA) and p53, bcl-2 or C-erb B-2 proteins on Reed-Sternberg cells: Prognostic significance in Hodgkin's disease. Neoplasma 45:140-147, 1998
Xerri L, Bouabdallah R, Camerlo J, et al: Expression of the p53 gene in Hodgkin's disease: Dissociation between immunohistochemistry and clinicopathological data. Hum Pathol 25:449-454, 1994
Dukers DF, Meijer CJ, ten Berge RL, et al: High numbers of active caspase 3-positive Reed-Sternberg cells in pretreatment biopsy specimens of patients with Hodgkin disease predict favorable clinical outcome. Blood 100:36-42, 2002
Brink AA, Oudejans JJ, van den Brule AJ, et al: Low p53 and high bcl-2 expression in Reed-Sternberg cells predicts poor clinical outcome for Hodgkin's disease: Involvement of apoptosis resistance Mod Pathol 11:376-383, 1998
Smolewski P, Robak T, Krykowski E, et al: Prognostic factors in Hodgkin's disease: Multivariate analysis of 327 patients from a single institution. Clin Cancer Res 6:1150-1160, 2000
Vassallo J, Metze K, Traina F, et al: The prognostic relevance of apoptosis-related proteins in classical Hodgkin's lymphomas. Leuk Lymphoma 44:483-488, 2003
Carbone PP, Kaplan HS, Musshoff K, et al: Report of the Committee on Hodgkin's Disease Staging Classification. Cancer Res 31:1860-1861, 1971
Chilosi M, Doglioni C, Magalini A, et al: p21/WAF1 cyclin-kinase inhibitor expression in non-Hodgkin's lymphomas: A potential marker of p53 tumor-suppressor gene function. Blood 88:4012-4020, 1996
Marafioti T, Hummel M, Foss HD, et al: Hodgkin and Reed-Sternberg cells represent an expansion of a single clone originating from a germinal center B-cell with functional immunoglobulin gene rearrangements but defective immunoglobulin transcription. Blood 95:1443-1450, 2000
Garcia JF, Camacho FI, Morente M, et al: Hodgkin and Reed-Sternberg cells harbor alterations in the major tumor suppressor pathways and cell-cycle checkpoints: Analyses using tissue microarrays. Blood 101:681-689, 2003
Kuppers R: Molecular biology of Hodgkin's lymphoma. Adv Cancer Res 84:277-312, 2002
Rajewsky K: Clonal selection and learning in the antibody system. Nature 381:751-758, 1996
Rassidakis GZ, Medeiros LJ, McDonnell TJ, et al: BAX expression in Hodgkin and Reed-Sternberg cells of Hodgkin's disease: Correlation with clinical outcome. Clin Cancer Res 8:488-493, 2002
Hinz M, Loser P, Mathas S, et al: Constitutive NF-kappaB maintains high expression of a characteristic gene network, including CD40, CD86, and a set of antiapoptotic genes in Hodgkin/Reed-Sternberg cells. Blood 97:2798-2807, 2001
Montalban C, Garcia JF, Abraira V, et al: Influence of biologic markers on the outcome of Hodgkin's lymphoma: A study by the Spanish Hodgkin's Lymphoma Study Group. J Clin Oncol 22:1664-1673, 2004
Morente MM, Piris MA, Abraira V, et al: Adverse clinical outcome in Hodgkin's disease is associated with loss of retinoblastoma protein expression, high Ki67 proliferation index, and absence of Epstein-Barr virus-latent membrane protein 1 expression. Blood 90:2429-2436, 1997
Lauritzen AF, Hou-Jensen K, Ralfkiaer E: P53 protein expression in Hodgkin's disease. APMIS 101:689-694, 1993
Elenitoba-Johnson KS, Medeiros LJ, Khorsand J, et al: P53 expression in Reed-Sternberg cells does not correlate with gene mutations in Hodgkin's disease. Am J Clin Pathol 106:728-738, 1996
Chen WG, Chen YY, Kamel OW, et al: p53 mutations in Hodgkin's disease. Lab Invest 75:519-527, 1996
Trumper LH, Brady G, Bagg A, et al: Single-cell analysis of Hodgkin and Reed-Sternberg cells: Molecular heterogeneity of gene expression and p53 mutations. Blood 81:3097-3115, 1993(Stephen J. Sup, Carlos A.)
ABSTRACT
PURPOSE: Although most classical Hodgkin's lymphoma (CHL) patients are cured, a significant minority fails primary therapy and may die as a result of their disease. Age, stage, and other basic clinical and laboratory parameters, which comprise the International Prognostic Score (IPS), are used at diagnosis to predict outcome. To date, there is no consensus on biologic markers that add value to these parameters.
PATIENTS AND METHODS: We evaluated 107 CHL patients for bcl-2, p53, and p21 expression by immunohistochemistry using tissue microarrays and correlated the results with outcome. The median follow-up of the 79 surviving patients was 6.8 years.
RESULTS: Univariate analysis showed that age 45 years, stage III or IV, and IPS 3 were associated with a poor failure-free survival (FFS) and overall survival (OS). bcl-2 was expressed in 26% of patients and was associated with poor FFS and a trend for OS. p53 expression in combination with lack of p21 expression was not associated with outcome. Multivariate analysis showed that three factors were independently associated with both FFS and OS: age 45 years, stage III or IV, and bcl-2 expression. Using these three parameters, a scoring system was devised that stratified patients into three risk groups (with zero, one, or two to three of these risk factors) and a progressively worse FFS and OS (P < .001).
CONCLUSION: Expression of bcl-2 in CHL is a useful, independent prognostic marker and can be used in association with clinical parameters to identify newly diagnosed patients with a good, intermediate, or poor prognosis.
INTRODUCTION
The majority of patients with classical Hodgkin's lymphoma (CHL) are cured. Nevertheless, a significant minority experiences treatment failure after primary therapy, and many of these patients ultimately succumb to their disease or complications of its treatment. The ability to identify more accurately patients at risk for primary treatment failure might allow the application of novel therapies in high-risk patients and at the same time minimize the exposure to potentially toxic therapies in low-risk patients. Many clinical parameters, such as older age, advanced stage, and bulky disease, have been associated with a worse prognosis. Several prognostic models have been used to stratify patients into good- and poor-risk groups. The International Prognostic Score (IPS), which incorporates seven clinical and laboratory parameters, was recently developed for patients with advanced-stage Hodgkin's lymphoma (HL) but has also been applied to patients with early-stage disease.1,2 In addition, some investigators have studied biologic markers that might provide additional prognostic information to these clinical models. Several candidate molecules, including bcl-2 and p53, have been proposed. The independent predictive power of these markers is controversial and none has gained wide acceptance.3-11 To further evaluate the prognostic significance of these biologic markers in CHL, we assessed bcl-2 and p53 expression in Hodgkin's and Reed-Sternberg (HRS) cells of archived specimens using tissue microarrays. We analyzed these results in conjunction with clinical and laboratory parameters and correlated them with patient outcomes.
PATIENTS AND METHODS
The archives of the Division of Pathology and Laboratory Medicine and medical records of the Department of Hematology and Medical Oncology were searched for patients with CHL diagnosed at the Cleveland Clinic Foundation from 1986 to 2000. Patients were included in this study if the initial diagnostic biopsy specimen was available for analysis, a diagnosis of CHL was confirmed, and adequate clinical and laboratory data from the time of diagnosis as well as follow-up information were available. Of the original 137 patients identified for review, 30 were excluded because the diagnosis was changed, the biopsy sample was from the time of disease recurrence, or the clinical information was inadequate. This study was performed after approval by the Cleveland Clinic Foundation Institutional Review Board.
Patients
Patients were staged at initial diagnosis with radiographic studies and bone marrow biopsies according to the Ann Arbor staging system.12 Medical records were reviewed and the following clinical and laboratory data from the time of diagnosis were collected: age, sex, stage, bulky tumors (defined as a mediastinal widening one third of the thoracic diameter or a mass 10 cm in maximum dimension), hemoglobin, WBC, lymphocyte count, and albumin. The IPS was calculated based on the seven defined high-risk features (ie, serum albumin < 4 g/dL, hemoglobin < 10.5 g/dL, male sex, stage IV disease, age 45 years, WBC 15,000/μL, and lymphocyte count < 600/μL), and patients were divided into a low-risk group (zero to two factors) and a high-risk group (three to seven factors), as previously described.2
Initial treatment for each patient was reviewed. All patients were treated with curative intent. During the study period, standards of care changed and, consequently, patients received a variety of treatments. In general, newly diagnosed patients with early-stage disease received radiation therapy alone or combination chemotherapy with or without radiation therapy, and patients with advanced-stage disease received combination chemotherapy with or without radiation therapy. Patients who experienced treatment failure after primary radiation therapy received combination chemotherapy, and patients who experienced treatment failure after initial chemotherapy received salvage chemotherapy and/or high-dose therapy with autologous stem-cell transplantation. Follow-up information was obtained from the medical records or the Cleveland Clinic Taussig Cancer Center Tumor Registry. The dates of diagnosis, disease progression, and/or death were recorded.
Tissue Microarray
Paraffin tissue blocks from the initial biopsies were retrieved and a tissue microarray was constructed (Beecher Instruments, Sun Prairie, WI). The biopsies were sampled using 1.5-mm-diameter tissue cores selected from areas of the whole tissue biopsy with the highest concentrations of HRS cells. The cores were arrayed in triplicate in the vast majority of samples, with a few samples having two or four cores.
Immunohistochemistry
Immunohistochemistry was performed using automated immunohistochemical stainers (Ventana, Tucson, AZ) and antibodies to bcl-2 (clone 100/D5; Ventana), p53 (clone DO7; DAKO, Carpinteria, CA), and p21 (clone SX118; BD Pharmingen, San Diego, CA). p21 expression was assessed because the expression of p53 in the absence of p21 (p53 positive/p21 negative) has been suggested as a better surrogate for mutated p53 than p53 expression alone.13 Samples were reviewed for expression of these proteins in HRS cells by S.S. and E.H., who were blinded as to outcome. Samples were considered positive only if 10% or more of the malignant cells strongly (at least as intense as small lymphocytes) expressed the protein. Expression of these proteins in positive samples varied between 10% and 75% of HRS cells. Samples in which only weak expression was present were scored as negative. Appropriate cellular localization for immunostaining was cytoplasmic for bcl-2 and nuclear for p53 and p21 (Fig 1).
Statistical Analyses
Overall survival (OS) was measured from the date of initial diagnosis to the date of death or the date of last follow-up. Failure-free survival (FFS) was measured from the date of diagnosis to the date of disease progression or death as a result of any cause, whichever came first. Patients alive and free of disease at analysis were censored as of their last follow-up. The method of Kaplan and Meier was used to summarize FFS and OS data and to estimate 5-year survival rates. The generalized Wilcoxon rank sum test was used to compare FFS and OS for individual factors, and the Cox proportional hazards model was used to assess multiple factors simultaneously. All analyses were performed using Statistical Analysis Software (SAS, version 8.0; SAS Institute Inc, Cary, NC).
RESULTS
A total of 107 patients formed the basis of this study. The patient and disease characteristics are summarized in Table 1. Eighty-three patients (78%) had nodular sclerosis, 17 patients (16%) had mixed cellularity, and seven patients (6%) had either lymphocyte-rich CHL or CHL not further specified (due to specimen limitations). Twenty-eight patients (26%) were bcl-2 positive. Eleven patients (10%) were p53 positive and nine patients (8%) were both p53 positive and p21 negative (p53 positive/p21 negative). Sixteen patients received radiation therapy only, 45 patients received chemotherapy only, and 40 patients received combined-modality therapy. Exact details of therapy were not available for six patients. Twenty-eight patients experienced disease progression during or after initial therapy and 20 patients died. Median follow-up for the 79 patients still alive and without evidence of disease is 6.8 years (range, 0.9 to 16.9 years). The estimated 5-year FFS was 75% ± 4% and the estimated 5-year OS was 85% ± 4% (Fig 2).
Table 1 lists the univariate survival analysis for sex, age, stage, bulky disease, IPS, bcl-2 positive status, and p53 positive/p21 negative status. As expected, age 45 years, stage III or IV, and IPS 3 were associated with significantly worse FFS and OS. bcl-2 expression was also associated with a significantly worse FFS (P = .02) and some indication of an association with worse OS (P = .14). Survival curves of the univariate analyses are shown in Figure 3.
Stepwise multivariate analysis was then performed using all of the factors in Table 1 to identify independent predictors of FFS and OS. On the basis of this analysis, three independent prognostic factors for both FFS and OS were identified: bcl-2 status, age, and stage. These data are summarized in Table 2.
these results, a simple scoring system using these two standard clinical parameters and a biologic marker was devised. On the basis of the number of adverse prognostic factors (ie, bcl-2 positivity, age 45 years, and stage III or IV), patients were stratified into three distinct groups each having a different prognosis. Patients with no poor prognostic factors were considered good risk; patients with one poor prognostic factor were considered intermediate risk; and patients with two or three poor prognostic factors were considered high risk. Using these definitions, 31% of patients in this series were considered good risk, 50% were considered intermediate risk, and 19% were considered high risk. Only one of the good-risk patients has relapsed and none have died (median follow-up for 32 patients alive and disease-free, 93 months). In contrast, the estimated 5-year FFS and OS are 69% ± 7% and 85% ± 5%, respectively, for the intermediate-risk group, and 54% ± 11% and 58% ± 11%, respectively, for the poor-risk group (P < .001, trend test). FFS and OS curves for the three groups are shown in Figure 4.
DISCUSSION
The pathogenesis of CHL remains poorly understood. Single-cell techniques have proven that HRS cells are derived from germinal center B cells, which harbor clonally rearranged and somatically mutated immunoglobulin genes but lack immunoglobulin expression.14 Although normal germinal center B cells that lack functional high-affinity antibody undergo apoptosis within the germinal center, HRS cells may escape this programmed cell death.15-17 Indeed, expression of bcl-2, other bcl-2 family members (eg, bcl-XL, BAX), and apoptosis regulators are altered in HL.5,6,8-11,15,16,18-20 The altered expression of these proteins in HRS cells may prevent apoptosis caused by the absence of the functional B-cell receptor and explain resistance to treatment-induced apoptosis and treatment failure.
Many biologic markers associated with apoptosis (including bcl-2, BAX, bcl-XL, and p53) have been studied in an attempt to identify clinically relevant predictors of outcome in HL.3,5-10,15,18,20,21 Several (but not all) investigators have reported that high expression of bcl-2 and p53 in HRS cells of HL patients confers a worse prognosis, but their independent predictive value has varied.5,7,9,10,20 In our study, bcl-2 (but not p53) expression in HRS cells was an independent and statistically significant predictor of poor outcome in patients with CHL. Furthermore, this biologic marker in conjunction with age and stage identified three distinct groups of patients with a good, intermediate, and poor prognosis. Because of the relatively small number of treatment failures, these results require additional follow-up and, ideally, confirmation in an independent set of patients.
Several other investigators have also studied one or more of these biologic markers along with clinical parameters in an attempt to identify different prognostic groups.5,9,10,15,20,21 Rassidakis et al5 evaluated bcl-2 expression along with clinical and laboratory parameters in 412 patients with CHL who were treated with doxorubicin, bleomycin, vinblastine, and dacarbazine or equivalent regimens. As in our study, bcl-2 expression, older age, and advanced stage (as well as low albumin and high lactate dehydrogenase) were independent prognostic factors. A higher number of these five adverse factors correlated with an increasingly worse FFS.
Brink et al9 quantified the percentage of HRS cells that expressed bcl-2 and p53 in 78 patients with nodular sclerosis or mixed cellularity HL. They reported that patients in whom p53 was expressed in a high percentage of HRS cells had a 100% 5-year survival (which was independent of bcl-2 expression), and that patients in whom p53 and bcl-2 were both expressed in a low percentage of HRS cells had a 90% 5-year survival. Conversely, patients in whom p53 was expressed in a low percentage and bcl-2 was expressed in a high percentage of HRS cells had only a 57% 5-year survival. They suggested that the patients with high p53 expression might contain wild-type (and not mutated) p53, thus rendering these cells more susceptible to chemotherapy- or radiotherapy-induced apoptosis. This combined p53 low/bcl-2 high profile was associated with poor outcome on univariate analysis but failed to maintain significance in multivariate analysis.
Smolewski et al6,10 studied p53, bcl-2, and proliferating cell nuclear antigen along with clinical and laboratory parameters. They also found that advanced stage, older age, high bcl-2 expression as well as high p53 expression and poor performance status were independent predictors of poor disease-free survival, whereas advanced stage, older age, poor performance status, and high p53 expression, but not high bcl-2 expression, were independent predictors of poor OS. This study also included patients with nodular lymphocyte–predominant HL (NLPHL). Because the expression of bcl-2 and p53 in NLPHL is rare, the inclusion of 41 NLPHL patients (12.5%) in their series may have affected the results of their analysis.6,10,22
Recent series from Spain show variable results. Morente et al21 examined 140 patients with HL for p53, bcl-2, Rb, Ki-67, CD15, and Epstein-Barr virus. p53 and bcl-2 expression were not found to correlate with outcome. However, absence of Epstein-Barr virus and high proliferative fraction were independent adverse prognostic factors. Garcia et al15 initially reported that the expression of bcl-2 and p53 as well as several other markers was significantly related to a shorter OS. In addition, they noted a trend (although it was not statistically significant) for low p21. In a recent update, Montalban et al20 analyzed 40 biologic markers involved in cell differentiation and activation, cell cycle control, apoptosis regulation, and host response along with the IPS in 259 CHL patients and correlated them all with outcome. By univariate analysis, expression of p53 and p21 (as well as multiple other genes) and IPS was associated with an adverse outcome. Expression of bcl-2 was associated with a tendency toward a shorter disease-specific survival, which was not statistically significant. By multivariate analysis, expression of p53, bcl-XL, and apoptosis (as measured by terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate-biotin nick-end labeling), but not bcl-2 or p21, was associated with an adverse outcome.
We used tissue microarray and considered a sample positive for bcl-2, p53, or p21 if 10% or more of the RS cells strongly expressed the gene by immunohistochemistry. We used this criterion because the expression of these proteins in even a small percentage of HRS cells might ultimately result in primary treatment failure. The techniques and criteria used in the other studies varied considerably. Consequently, the percentage of samples considered positive for protein expression in HRS cells also varied and may in part explain the different results reported in the five other studies detailed in Table 3.
Overall, the evidence suggests that bcl-2 expression in HRS cells is an independent biologic marker associated with unfavorable outcome. However, p53 protein expression may not be a strong predictor. The great variability in p53-positive samples (10% to more than 90%) among various studies suggests problems in evaluating expression.7,9,10,15,20,21 Overexpression of p53 protein in HRS cells may be common, technical considerations not withstanding, but it is probably associated with wild-type p53 gene.15,23 p53 mutations in HRS cells are detected only sporadically and may not correlate with p53 overexpression.7,23-25 Thus, p53 expression by immunohistochemical staining is an imperfect surrogate for mutational status. In non-Hodgkin's lymphomas, expression of p53 without p21 (p53 positive/p21 negative) is indicative of mutated p53, whereas expression of both p53 and p21 (p53 positive/p21 positive) is associated with the wild-type p53.13 We also applied these criteria as surrogates for distinguishing mutated from wild-type p53. Nevertheless, we found no correlation with patient outcome.
In summary, bcl-2 is overexpressed in a significant proportion of CHL patients and is an independent adverse prognostic factor. Our findings support the use of this biologic marker in conjunction with clinical parameters such as age and stage to identify patients at diagnosis who are at a low, intermediate, or high risk for primary treatment failure. These risk groups might then be used to guide standard therapy and develop risk-stratified protocols. In addition, these data provide a rationale for novel therapies targeting bcl-2 expression in CHL.
Authors' Disclosures of Potential Conflicts of Interest
The authors indicated no potential conflicts of interest.
NOTES
Authors' disclosures of potential conflicts of interest are found at the end of this article.
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