Japanese Journal of Clinical Oncology 32:393-397 (2002)
© 2002 Foundation for Promotion of Cancer Research
Prognostic Evaluation of the Expression of p53 and bcl-2 Oncoproteins in Patients with Surgically Resected Non-small Cell Lung Cancer
Departments of 1 Medicine, 3 Pathology and 4 Surgery, Kaohsiung Veterans General Hospital, Kaohsiung and 2 School of Medicine, National Yan-Ming University, Taipei, Taiwan
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONAcknowledgmentsREFERENCES |
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Background: Tumor staging remains the most important prognostic predictor in patients with non-small cell lung cancer (NSCLC). However, the prognostic significance of expression of oncoproteins involved in regulation of cellular uncontrolled proliferation remains controversial.
Methods: In this retrospective study, we investigated the expression of bcl-2 and p53 oncoproteins in 114 surgically resected NSCLC patients (46 stage I, 39 stage II and 29 stage IIIa) using immunohistochemical analysis and correlated the molecular markers with survival.
Results: Positive bcl-2 immunoreactivity was detected in 26 of 114 (22.8%) NSCLC, including 15 of 43 (34.9%) squamous cell carcinoma and 11 of 71 (15.5%) adenocarcinoma cases. Nuclear staining for p53 was observed in 59 of 114 (51.8%) NSCLC, including 26 of 43 (60.5%) squamous cell carcinoma and 33 of 71 (46.5%) adenocarcinoma patients. There was no correlation between pathological staging and expression of bcl-2 and p53. However, the expression frequency of bcl-2 was significantly higher in squamous cell carcinoma than in adenocarcinoma (P < 0.02). The presence of bcl-2 expression did not provide a favorable prognosis (P = 0.23) and the overexpression of p53 oncoprotein was also not significantly associated with adverse prognosis (P = 0.09). No inverse relationship was found between bcl-2 and p53 expression (P = 0.83).
Conclusion: Expressions of bcl-2 and p53 using immunohistochemical staining are not independent prognostic predictors in patients undergoing surgery for NSCLC.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONAcknowledgmentsREFERENCES |
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Lung cancer is the major cause of cancer mortality worldwide. For non-small cell lung cancer (NSCLC), surgery is the mainstay in the treatment of patients without demonstrable metastatic disease. Staging is the most accurate means to estimate prognosis. Although surgery achieves long-term survival in early-stage patients, a significant proportion may suffer from regional or distant recurrence (1). Obviously, considerable discrepancies arise in survival between patients within the same stage of lung cancer. This illustrates the need to identify more molecular markers, which play a role in tumor behavior and may help clinicians to assess the survival probability better.
Recent evidence suggests that the genetic regulation of apoptosis may be of critical importance during tumorigenesis. The bcl-2 gene is a negative regulator of programmed cell death (apoptosis) (2). The avoidance of apoptosis via bcl-2 may result in a survival advantage. The p53 gene may block the progression of cell growth cycle and trigger apoptosis in response to DNA damage. The mutation of p53 gene causes a loss of tumor-suppressor function, promotes cellular proliferation and inhibits apoptosis. Besides follicular lymphoma (3), the bcl-2 oncoprotein has been detected in some non-lymphomatous malignancy, such as breast (4), colorectal (5) and lung cancer (6–13). p53 mutation has also been detected in many types of human malignancy. Expressions of both oncoproteins bcl-2 and p53 have been reported as a prognostic predictor in NSCLC with conflicting results in a number of studies.
Based on the involvement of apoptosis regulation and the controversial prognostic value of bcl-2 and p53, we investigated the expression frequency of both genes and correlated oncoprotein expression with clinicopathological parameters and survival in patients with radically resected NSCLC.
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PATIENTS AND METHODS |
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TOPABSTRACTINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONAcknowledgmentsREFERENCES |
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Patients
The study included 114 patients with NSCLC (79 male, 35 female, mean age 68 ± 3 years), who were diagnosed and treated at Kaohsiung Veterans General Hospital, Taiwan. All specimens were obtained from exploratory thoracotomy with lung resection. The pathological classification of the specimens and staging of the disease were based on the World Health Organization (14) and TNM (15) systems. The distribution of pathological staging and the types of surgical intervention are listed in Table 1. After having excluded four cases who died during the post-operative period, the median follow-up period was 34 months.
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Immunohistochemistry
Serial sections from the formalin-fixed, paraffin-embedded blocks were used for the detection of bcl-2 and p53 oncoprotein by immunohistochemical methods. Monoclonal antibodies against p53 (clone DO-7, diluted 1:100, DAKO) and bcl-2 (clone 100, diluted 1:40, Oncogen) were applied. The p53 antibody may react with wild and mutant types of p53 protein.
Immunohistochemical staining for bcl-2 and p53 was performed using a microwave-induced antigen retrieval method. Sections of 4 mm thickness were deparaffined with xylene and graded ethanol. Dewaxed sections, immersed in a citric acid buffer containing NP-40 (0.01 M, pH 6.0; Sigma) were heated in a microwave oven (600 W) for 10 min and allowed to stand for a further 15 min. Endogenous peroxidase was blocked with 2% hydrogen peroxide in 70% methanol for 10 min at room temperature. Using a standard avidin–biotin complex method, sections were incubated with monoclonal antibodies against bcl-2 and p53 oncoprotein for 2 h and with biotinylated secondary antibodies for 20 min at room temperature and subsequently processed with 3,3-diaminobenzidine tetrahydrochloride (DAB) (7 min) as the chromogen. Sections were lightly counterstained with hematoxylin and viewed under a light microscope. Positive control consisted of follicular lymphoma for bcl-2 and known p53-positive cases of oral squamous cell carcinoma. Negative control included sections incubated without primary antibodies. The number of positively immunostained cells was recorded semiquantitatively for each tumor. bcl-2 immunoreactivity (cytoplasmic staining) was initially graded on a scale of 0–3 (scale 0, all tumor cells negative; scale 1, <10% weakly positive tumor cells; scale 2, multifocal aggregates of uniformly staining tumor cells, 10–75%; scale 3, diffuse positive staining throughout the tumor, >75%). Reactive lymphocytes served as internal positive controls. p53 immunostaining was evaluated using a similar scoring system. Only those tumors with scales of 2 and 3 were considered to be bcl-2 or p53 positive.
Statistics
Survival for each patient was calculated from the day of surgery until death or the last date seen alive by the patient’s doctor. Patients who died within 30 days of surgery were excluded from survival correlation.
All statistical analyses were performed using the SPSS software system (v10.0, SPSS, Chicago, IL). The correlation between the presence of oncoprotein expression and clinicopathological parameters (gender, tumor status, node status, stage and histology) was examined by the chi-squared test. The relationship between p53 and bcl-2 expression was evaluated by the chi-squared test and by a linear regression coefficient test. Survival analysis was calculated by the Kaplan–Meier method and the statistical significance of differences was determined by a log-rank test.
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RESULTS |
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TOPABSTRACTINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONAcknowledgmentsREFERENCES |
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Immunohistochemical Staining for bcl-2 and p53
The expression frequencies of bcl-2 and p53 oncoprotein in squamous cell carcinoma (SQC) and adenocarcinoma (ADC) are shown in Table 2. Staining of bcl-2 was confined to nuclear membrane and cytoplasm (Fig. 1a). Twenty six of 114 (22.8%) NSCLC were bcl-2 positive; including 15 of 43 (34.9%) SQC and 11 of 71 (15.5%) ADC. Nuclear staining for p53 (Fig. 1b) was observed in 26 of 43 (60.5%) SQC and 33 of 71 (46.5%) ADC. In all, 59 of 114 (51.8%) of NSCLC were p53 positive.
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Clinicopathological Correlation
No statistical difference was found between bcl-2 expression and pathological staging, including T (tumor) and N (nodal) status (Table 3). However, the frequency of bcl-2 expression was significantly higher in squamous cell carcinoma than that in adenocarcinoma (P < 0.02). Similarly, no correlation was observed between p53 expression and clinicopathological parameters. The data did not reveal an inverse correlation between the presence of p53 and bcl-2 expression (P = 0.8). Regardless of cell types, the overall survival was significantly related to the pathological staging in NSCLC (P < 0.001); however, the presence of immunohistochemical staining for bcl-2 and p53 gene product was not critical for prediction of outcome. There was no significant difference in life span between the patients with oncoprotein expression and those without (Kaplan–Meier method with log-rank test; P = 0.23 in bcl-2 and P = 0.09 in p53) (Fig. 2). Even in the patients with early-stage NSCLC (45 stage I), no difference was observed between molecular markers (bcl-2 and p53) expression and life span (P = 0.26 in bcl-2 and P = 0.16 in p53). Having compared patients with p53 (+)/bcl-2 (–) and p53 (–)/bcl-2 (+), no significantly elevated risk for p53 (+)/bcl-2 (–) can be demonstrated (P = 0.13).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONAcknowledgmentsREFERENCES |
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In the present study of NSCLC, bcl-2 and p53 were detected using immunohistochemistry in about 23 and 52% of the cases, respectively. The expression of both oncoproteins revealed no correlation with pathological staging. The presence of bcl-2 expression did not provide a favorable prognosis and p53 overexpression was also not significantly associated with adverse prognosis in NSCLC.
Besides normal expression in tissues that have proliferating ability (16), bcl-2 may also be detectable in a variety of malignancies. In lung cancer, the prevalence of bcl-2 oncoprotein expression has been reported to be higher in small cell lung cancer (17) than in NSCLC (8). Among different histological types of NSCLC, squamous cell carcinoma was predominant (7) (SQC 34.9% vs ADC 15.5% in the present study). There has been controversy about the association between bcl-2 expression and its prognostic importance in NSCLC. Some studies revealed longer survival in patients with bcl-2 positive tumors than in those with bcl-2 negative tumors (6–9). The explanation for the better prognosis for bcl-2 positive tumors has not yet been determined. However, it has been reported that bcl-2 may play a role in controlling the development of tumor angiogenesis in NSCLC with mediation by vascular endothelial growth factor (VEGF) (18). Our results, like those of other studies (10–13), do not confirm that bcl-2 immunoactivity is an independent prognostic factor in NSCLC. The fact that bcl-2 protein was expressed in about 20–60% of NSCLC indicates that bcl-2 may not be a sensitive marker in practical use.
Abnormal p53 gene products may be detected by immunohistochemistry in cancer cells as a consequence of p53 alterations. The frequency of p53 mutation may be up to 50% in NSCLC and 80% in SCLC (19). Some studies revealed a favorable prognosis in patients with negative immunohistochemical staining for p53 oncoprotein (20–24). However, other research (7,25,26), including the present results, did not demonstrate that positive p53 immunostaining is a predictor of poor prognosis in patients with NSCLC. To evaluate p53 as an independent prognostic predictor, it seems reasonable to apply immunohistochemical analysis in stage I NSCLC. However, we did not find better prognosis in early-stage NSCLC with p53 (–)/bcl-2 (+). Besides, if p53 (+) indicated poor prognosis, it would be expressed with higher frequency in advanced NSCLC. However, the present and previous studies (27) did not support this fact. As revealed in studies by Hashimoto et al. (28), tumors with p53 (–) staining by null mutation gave a poor prognosis. Therefore, if an examined group of tumor specimens with negative p53 staining contained many null mutations, the group would not indicate a favorable prognosis. In other words, immunostaining of p53 products alone could not characterize the tumors precisely. Since multiple oncogenes or tumor suppression genes may be involved in the tumorgenesis, the results for the prognostic value of p53 or bcl-2 oncoproteins may be complex and conflicting (29). The different immunohistochemical results between studies, including bcl-2 and p53, may be due to the procedures used, monoclonal antibodies, interpretation of expression levels, etc.
Both bcl-2 and p53 have been implicated in the regulation of apoptosis. An inverse relation has been observed between the expression of p53 and bcl-2 oncoproteins in some studies of breast cancer (30), colon cancer (31) and NSCLC (9). However, some studies (18,32), and the present results, did not support such a relationship. bcl-2 and p53 are only two of many genes that regulate complex apoptotic process.
In conclusion, the expression of p53 and bcl-2 oncoproteins is not correlated with clinicopathological parameters in NSCLC. Our results also do not indicate that bcl-2 and p53 expression would be a relevant prognostic predictor in NSCLC. More research is necessary to understand the complex interaction between multiple genes and the identification of new biological markers that can more accurately predict the risk of tumor progression.
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Acknowledgments |
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TOPABSTRACTINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONAcknowledgmentsREFERENCES |
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This study was supported by grant VGHKS 89-40 from Kaohsiung Veterans General Hospital, Taiwan. The authors are grateful to Miss Lin Yi-Ping for statistical assistance.
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FOOTNOTES |
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+ For reprints and all correspondence: Ruay-Sheng Lai, Division of Chest Medicine, Department of Medicine, Kaohsiung Veterans General Hospital, 386 Ta-Chung 1st Road, Kaohsiung, Taiwan. E-mail: lairs@seed.net.tw
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Received January 7, 2002; accepted June 19, 2002
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