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Ann Thorac Surg 1999;68:343-347
© 1999 The Society of Thoracic Surgeons

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Original Articles

Significance of P53 and Rb protein expression in surgically treated non-small cell lung cancers

^a Division of Thoracic Surgery, Department of Surgery, College of Medicine, National Taiwan University, Taipei, Taiwan

Address reprint requests to Dr Lee, Department of Surgery, National Taiwan University Hospital, No. 7 Chung-Shan South Rd, Taipei 10016, Taiwan, ROC

	Abstract

Top Abstract Introduction Material and methods Results Comment References

 
Background. Alterations of the P53 or Rb gene are among the most frequently observed genetic changes in primary lung cancer. Nevertheless, there has been no final conclusion on the relationship between P53 or Rb protein expression and clinico-pathological parameters in primary lung cancer. A large-scale study was performed to examine the clinicopathological and prognostic significance of P53 and Rb expressions in 207 surgically resected non-small cell lung cancer (NSCLC) patients.

Methods. Tumor specimens were obtained from 207 primary NSCLC surgically resected from January 1990 through December 1994. The avidin-biotin-peroxidase method was used to determine the expression of P53 or Rb of tumor cells using anti-P53 or anti-Rb monoclonal antibodies. The relationships between P53 or Rb protein expression and the clinicopathological parameters were analyzed.

Results. Expression of P53 or Rb protein was detected in 115 (55.6%) and 136 (65.7%) of the 207 lung tumors, respectively. P53 had significantly higher positive results in patients with regional lymph node metastasis and advanced tumor stage. Rb expression was significantly lower in lung cancers with a macro- or microscopic picture of tumor necrosis. Additionally, an inverse correlation between the expression of Rb and P53 was found. By multiple variate analysis, P53 expression and pathological stage were independent, significant prognostic factors. Further analysis demonstrated P53 expression was an independent prognostic factor in stage 1, but not in other stages.

Conclusions. P53 expression is especially useful as a prognostic factor in stage 1 lung cancer.

	Introduction

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Lung cancer is one of the most common causes of cancer death in Taiwan [1]. Although the etiologies of lung cancer are not clear, studies of the genetic changes in these lung cancers are important for understanding the possible mechanisms of pathogenesis of lung cancer in Taiwan. Alterations of the p53 and Rb genes have been among the most commonly observed tumor genetic changes that have been closely related to the initiation and progression of a diverse group of human cancers, including lung cancer [2–4]. A p53 gene mutation can result in an increase in cellular p53 protein levels, because mutated p53 protein has a much longer half-life than normal p53 protein [5]. Accordingly, detectable levels of p53 protein production by immunohistochemistry suggest the existence of genetic alteration at this locus [6]. Indeed, overexpression of p53 protein as determined by immunohistochemistry has been demonstrated to be closely correlated with the presence of p53 gene mutation in several types of malignancies [7]. It has also been reported that p53 alterations have played a significant role in the carcinogenesis, proliferation, and invasion of lung cancer [8–11].

Alteration of Rb gene expression was observed originally in retinoblastoma [12]. Subsequent studies showed abnormality in the Rb gene, and altered expression of Rb gene protein was frequently observed in certain types of malignancies, including small cell lung cancer [13, 14]. It has been suggested that alteration of the Rb gene in small cell lung cancer may be correlated not only with initiation, but also with proliferation and invasion of the tumor [15]. In non-small cell lung cancer (NSCLC), alteration of Rb gene protein expression was less frequently seen. It has also been shown that the alteration of the Rb gene may be related to proliferation, differentiation, and development of the tumor [16, 17]. However, no final conclusion on the relationship between p53 or Rb protein expression and the clinical parameters in NSCLC has been reached.

In this study, we investigated the expression of p53 and Rb protein by immunohistochemistry in 207 surgically resected NSCLC. Various clinical parameters of the patients as well as the pathological characteristics of the tumors were collected for correlation with the extent of expression of p53 or Rb protein in these tumors. The relationship of p53 and Rb protein expressions to the patient’s survival was analyzed to define the prognostic significance.

	Material and methods

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Lung cancer patients and specimens
Two hundred and seven lung cancer specimens were obtained from patients who underwent surgical resection for NSCLC at the National Taiwan University Hospital during the period from January 1990 through December 1994. All of the specimens were formalin fixed and sectioned for microscopic examination after staining with hematoxylin-eosin. Histological diagnosis and pathological features were obtained including tumor cell type, degree of differentiation, vascular invasion or emboli, regional lymph node metastasis, macro- or microscopic picture of tumor necrosis, desmoplastic reaction, and direct invasion to surrounding structures. Pathological staging was done according to the International Union Against Cancer (UICC, 1987) based on tumor size and involvement, lymph node metastases, and distant metastases.

This study included 142 male and 65 female patients, and the mean age was 58.3 years (range 32 to 81 years). The clinical data of these patients, including sex, age, smoking status, location of the tumor, and multiplicity of the tumor also, were recorded and analyzed with the result of p53 or Rb protein expression in each tumor.

Immunohistochemistry
For immunohistochemical demonstration of the p53 protein or Rb protein expression in the tumor tissue, 4-µm-thick sections from each formalin-fixed, paraffin-embedded tissue block were treated with 0.3% hydrogen peroxide in methanol, and heated in a microwave oven for 20 minutes to block endogenous peroxidase. The tissue sections were then incubated with normal nonimmune goat serum. After excessive goat serum had been blotted, the slides were incubated with a specific mouse anti-p53 protein antibody "p53 (Ab-6), pantropic" (diluted 1:50), (Oncogene Science, Cambridge, MA) or a specific mouse anti-Rb protein antibody "Rb (Ab-5)," (diluted 1:50), (Calbiochem, Cambridge, MA) for 1 hour at room temperature. After washing with phosphate-buffered solution (PBS) three times, the sections were incubated with bionylated goat anti-mouse antibody for 20 minutes at room temperature. The sections were again washed three times with PBS, then incubated with peroxidase-conjugated steptavidin for 15 minutes at room temperature. After a third triple washing with PBS, the sections were stained with 0.05% (3', 3) diamino benzidine tetrachloride freshly prepared in 0.05M Tris-HCl (pH = 7.6) containing 0.01% hydrogen peroxide. Finally, the sections were counterstained with methylene green and then mounted.

The expression of p53 or Rb protein was considered as positive when a strong coloration was evident in the tumor cell, mostly in the nucleus (Fig 1). For each tumor tissue, 500 tumor cells per 10 high-power fields were counted. The percentage of positive staining cells in each tumor was also estimated. A tumor was considered p53 or Rb positive if more than 1% of all visible tumor cells were stained.

View larger version (125K): [in this window] [in a new window]   Fig 1. Immunohistochemical study of a pulmonary adenocarcinoma in a 35-year-old female patient, who died 23 months postoperatively. (A) A moderately differentiated adenocarcinoma shows nuclear staining for P53 in the lung tumor (66x). (B) Negative staining for Rb in the lung tumor (66x).

	Results

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In the current study, P53 nuclear staining was detected in 115 of the 207 lung tumors (56.7%). The relationship of clinical parameters of the patients with the presence of P53 expression is shown in Table 1. A borderline significance of higher positive rate of P53 expression was noted in lung cancer patients with age under 45 years. There was no significant difference of P53 expression in relation to gender, smoking status, tumor location, or multiplicity of the tumor. Compared with various pathological characteristics of the tumor, P53 had significantly higher positive results in patients with regional lymph node metastasis (p = 0.002), and advanced tumor stage (p = 0.008), whereas no correlation was found with histological type, grade of differentiation, desmoplastic reaction, tumor necrosis, vascular invasion, or direct invasion (Table 2).

The influences of various clinico-pathological parameters, including the expression of P53 or Rb protein, on patient survival was analyzed. By single variate analysis of survival function, both P53 (Fig 2) (p < 0.0001) and Rb expression (Fig 3), (p < 0.05), in addition to tumor stage (p < 0.0001), histological type (p < 0.001), and tumor location, (p < 0.05), were significant prognostic factors affecting survival. By multiple variate analysis, only advanced stage (stage 3) and P53 protein expression remained as independent, significant prognostic factors (p < 0.0005, p < 0.005). Further analysis investigated the influence of P53 expression on patient’s survival in different pathological stages of lung cancer. The results showed P53 expression was an independent, significant prognostic factor only in stage 1 patients (Fig 4), but not in stage 2 or stage 3 patients (Figs 5 and 6).

View larger version (14K): [in this window] [in a new window]   Fig 2. Survival curves of P53-positive and P53-negative NSCLC patients after operation (p < 0.0001).

View larger version (14K): [in this window] [in a new window]   Fig 3. Survival curves of Rb-positive and Rb-negative NSCLC patients after operation (p < 0.05).

View larger version (14K): [in this window] [in a new window]   Fig 4. Survival curves of P53-positive and P53-negative stage 1 NSCLC patients after operation (p < 0.0001).

View larger version (13K): [in this window] [in a new window]   Fig 5. Survival curves of P53-positive and P53-negative stage 2 NSCLC patients after operation (p = 0.13).

View larger version (15K): [in this window] [in a new window]   Fig 6. Survival curves of P53-positive and P53-negative stage 3 NSCLC patients after operation (p = 0.44).

	Comment

Top Abstract Introduction Material and methods Results Comment References

 
It is well documented that specific genetic alteration and aberrant gene expression in tumor cells may influence the biological behaviors of a tumor [2, 6]. P53 is a tumor suppressor gene, involved in cell cycle control and preservation of genomic integrity [2, 5]. Alteration of the p53 gene may be involved in the initiation, development, progression, and invasion of lung cancer [6, 8, 10, 11]. The correlation of p53 protein expression with clinical and biological characteristics of lung cancer has been extensively studied. However, the results of these studies are quite diverse [18–23]. Our results indicated that p53 expression was closely related with invasiveness of the tumor. And p53 was especially useful as a prognostic factor in the stage 1 lung cancer patients.

The Rb gene encodes a group of nuclear phosphoproteins associated with DNA-binding activity, acting in a cell cycle-specific way to control cellular proliferation and differentiation [15]. Abnormalities at the Rb gene locus, such as mutations or deletions and inactivation of expression, may have an uncontrolling effect on the cell cycle. Alteration of the Rb gene or its protein may play a significant role in the proliferation, differentiation, and development of the tumor [12, 15]. In the present study, Rb gene expression was found to be inversely correlated with tumor necrosis, which could be explained by loss of the effect of Rb gene on the inhibition of rapid tumor proliferation, in Rb-negative tumor, which potentially induced tumor ischemic necrosis. An inverse correlation of the expression of p53 and Rb protein expressions in the same tumors might reflect that genetic changes in the tumor initiation and progression were multifactorial and complicated. Alterations of p53 and Rb gene may be closely related in certain patients with primary lung cancer.

The definition of cut-off values in the analysis of immunohistochemical staining is an obvious problem. In the current study, a low cut-off level of 1% positive cells was used to define p53 and Rb positivity. Similar results can be obtained in this study using three grades of categorization: negative (0% to 1% positive), weakly positive (1% to 60% positive), and strongly positive ( 60% positive) (data not shown). The observed prevalence rate of P53 positivity (55.6%) is slightly higher than that of previous studies using 1% positive cells as a cut-off value [18, 19, 22]. Absence of Rb protein expression occurred in 34.3% of our lung cancer subjects, a finding similar to previous reports [13, 14, 17].

In this study, a higher rate of P53 protein expression was noted in lung cancer patients with age under 45 years. This phenomenon was not related to the difference of the tumor stage, because the distribution of tumor stages in these two groups (age > 45 years and age 45 years) was quite similar. It is suggested that alteration of P53 suppressor gene might play a more significant role in the pathogenesis of primary lung cancer in the younger age group. Although a relationship between smoking and P53 over-expression in lung cancer has been reported [24–26], the difference in P53 positivity in lung cancer patients with smoking habits was only slightly higher than nonsmokers (57.9% vs 52.9%) in the current study. Regarding the relationship of P53 or Rb with pathological characteristics of the tumor, the results reported in the literature have been controversial [18–23]. In this study, we found that lung cancer with lymph node metastasis has a significantly higher rate of P53 protein expression (p < 0.005), and has a slightly higher rate with negative Rb protein expression. There was also a significant correlation between P53 protein expression and advanced tumor stage (p < 0.01), which was similar to several previous reports in the literature [10, 11].

The relationship of P53 alterations with the prognosis of lung cancer has been extensively studied, but the results of research have been conflicting. The reasons for such a controversial difference are not yet clear, but several possible causes might be posed, including varying methods of detection, antibodies used for staining, antigen-retrieval techniques, and cut-off values for positivity. In this study, using multivariate analysis of patient survival, we found P53 protein expression to be an independent prognostic factor after surgical resection, especially in the stage 1 lung cancer patients. These results are similar to the findings of the study recently reported by Dalquen and associates [19].

In conclusion, the results of the present study show an inverse relationship between P53 and Rb protein expression in primary lung cancer. A higher percentage of lung tumors with regional lymph node metastasis, advanced tumor stage, or younger age group have positive P53 protein expression than other lung tumors. Rb expression is significantly lower in lung cancers with a histological picture of tumor necrosis. P53 expression is also an independent prognostic parameter in stage 1 lung cancer. A longer period of follow-up is required to reach a final conclusion.

	Acknowledgments

 
This study was supported by research grant NSC-86-2314-B002-045 from the National Science Council, ROC.

	References

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