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Ann Thorac Surg 2000;70:913-917
© 2000 The Society of Thoracic Surgeons

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Original articles: general thoracic

Combined analysis of p53 and retinoblastoma protein expressions in esophageal cancer

^a Department of Surgery I, Faculty of Medicine, Tottori University, Yonago, Japan

Address reprint requests to Dr Ikeguchi, Department of Surgery I, Faculty of Medicine, Tottori University, 36–1 Nishicho, Yonago 683–8504, Japan

	Abstract

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Background. p53 gene mutation and abnormal p53 protein expression, also loss of the retinoblastoma gene and protein expression are frequently associated with esophageal squamous cell carcinoma (ESCC). Recently, the prognostic significance of the combined analysis of p53 protein and retinoblastoma protein (pRB) has been reported in non–small cell lung cancer. However, in ESCC, the prognostic significance of the combined analysis of these proteins remains unclear. In this study, we immunohistochemically analyzed the p53 protein and pRB expressions in surgically resected ESCC, and we evaluated the prognostic significance of the combination of these proteins.

Methods. We analyzed p53 protein and pRB expressions immunohistochemically in 191 surgically resected ESCC cases. Overexpression of p53 and loss of pRB were considered abnormal.

Results. Overexpression of p53 protein was detected in 79 patients (41%) and decreased pRB nuclear staining occurred in 82 (43%). The Kaplan-Meier survival curve showed that absence of pRB expression was significantly associated with shortened survival (p = 0.001), whereas expression of p53 was not significantly associated with survival. Moreover, p53 and pRB status individually were not independent prognostic factors in multivariate survival analysis. With respect to pRB and p53, the tumors could be grouped into four categories: p53-/pRB+ (31%); p53-/pRB- (27%); p53+/pRB+ (26%); and p53+/pRB- (16%). Favorable prognosis was observed in patients with p53-/pRB+ tumors. Multivariate analysis showed p53-/pRB+ status to be an independent prognostic factor.

Conclusions. The combination of p53 protein loss and pRB expression was associated with good prognosis in patients with ESCC.

	Introduction

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The prognosis of patients with esophageal cancer is poor, with a 5-year survival rate of patients with surgically treated esophageal cancer of only about 30% [1]. Many patients die from cancer recurrence by way of lymph node or hematogenic metastasis soon after operation. The biologic characteristics associated with the high malignant potential of esophageal squamous cell carcinomas are widely researched, but useful biologic prognostic indicators of this carcinoma have not yet been reported [2, 3].

Esophageal cancer is now thought to result from the accumulation of inactivating mutations in tumor suppressor genes, such as the p53 and retinoblastoma (RB) genes. The products of these genes play important roles in control of cell cycle. When the DNA of cells is damaged, wild-type p53 protein induces the expression of p21 (Waf1/Cip1/Sdi1), which is an inhibitor of the phosphorylation of the cyclin-cdk complex. Under this condition, retinoblastoma protein (pRB) strongly binds to E2F and regulates the cell cycle by inducing G₁ arrest [4]. During this G₁ arrest, cells undergo apoptosis or repair of their DNA. Mutation of the p53 gene and overexpression of p53 protein have been reported in esophageal squamous cell carcinoma (ESCC) [5, 6]. Also, loss of the RB gene and loss of retinoblastoma protein (pRB) expression were frequently observed in ESCC [7]. So, the abnormality in this p53-pRB pathway might correlate with tumorigenesis and tumor progression. In non-small cell carcinoma of the lung, the prognostic significance of altered expression of p53 protein and pRB has been analyzed extensively. Reports using multivariate analysis showed that positive or negative p53 protein or pRB expressions, as individual factors, were not statistically correlated with survival or prognosis of patients [8, 9]. However, they reported that the combined immunohistochemical analysis of p53 protein and pRB expression might have potential synergistic effects for determining survival and prognosis of patients.

The combined analysis of both p53 protein and pRB has not been reported and the clinical significance of this analysis in ESCC is not known. In this study, we investigated whether this combined analysis was a useful prognostic indicator of ESCC.

	Material and methods

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Patients
From the beginning of 1981 to the end of 1998, 191 patients with primary ESCC had esophagectomies at our hospital. All 191 tumors were investigated. There were 171 men and 20 women, and they ranged in age from 39 to 84 years (mean, 63 years; median, 65 years). None of them had chemotherapy or radiation therapy preoperatively. All patients had a total or subtotal esophagectomy, performed by right thoracotomy and laparotomy, with mediastinal and abdominal lymphadenectomy. Reconstruction was done by esophagogastrostomy using a gastric tube through the retrosternal route.

Histopathologic examination
The resected specimens were fixed in 10% buffered formalin for 24 hours. All specimens were cut into 5-mm slices after fixation. The slices were embedded in paraffin and cut into 4-µm-thick sections for hematoxylin-eosin and immunostaining. The histopathologic diagnoses were defined according to the guidelines for TNM classification [10]. All of 191 tumors were diagnosed as squamous cell carcinoma (SCC), 14 tumors as well differentiated SCC (G1), 91 tumors as moderately differentiated SCC (G2), and 86 tumors as poorly differentiated SCC (G3). The depth of tumor invasion of 47 tumors was diagnosed as pT1, that of 32 tumors as pT2, that of 65 tumors as pT3, and that of 47 tumors as pT4. Lymph node metastasis was detected in 102 cases. Of the 191 ESCCs there were 38 in stage I, 61 in stage II, 78 in stage III, and 14 in stage IV.

Immunohistochemical detection of p53 and RB protein
The paraffin-embedded sections of the tumors were stained by a monoclonal antibody raised against p53 (BP53 to 12; diluted 1:100; Novocastra Laboratories Ltd, Newcastle, UK) and by a monoclonal antibody raised against human pRB (clone Rb1, diluted 1:40; DACO, Glostrup, Denmark). Briefly, 4-µm-thick sections were dewaxed using xylene and transferred to alcohol. The slides were then placed in citric acid buffer (10 mmol/L) and heated in a microwave oven (700 W) for 12 minutes to expose antigens. Endogenous peroxidase activity was blocked by incubating sections with 0.3% hydrogen peroxide in methanol for 30 minutes. Slides were then washed three times in phosphate-buffered saline (PBS) and incubated in 10% normal goat serum for 20 minutes to reduce nonspecific antibody binding. After washing with PBS, slides were incubated with the primary antibodies overnight at 4°C. Nonspecific mouse immunoglobulin G1 was used as a negative control. After washing the slides with PBS, biotinylated antibodies against mouse immunoglobulin were applied as second antibodies (Histofine ABC Kit; Nichirei, Tokyo, Japan) for 30 minutes. The reaction products were visualized with diaminobenzidine as the chromogen, and the slides were counterstained with methyl green.

p53 and pRB expression was determined by two independent observers (SO and YG) who had no knowledge of the patients. The extent of p53 immunoreactivity was classified into the following three categories based on the percentage of tumor cells showing nuclear reactivity: less than 10% (n = 64), 10% to 50% (n = 42), and more than 50% (n = 85). The extent of p53 immunointensity also was classified into three categories: tumor cells showing no immunostaining (n = 59), weak immunostaining (n = 52), and strong immunostaining (n = 80). We decided that tumors with more than 50% of tumor cells showing strong nuclear reactivity of p53 were p53 positive. In pRB immunostaining, adequate nuclear staining was obtained in the normal esophageal epithelia in all cases, which represented a positive control. When immunostaining results were assessed, we took into account the pRB-positive cancer cells in the tumors: if more than 50% of tumor cells stained for pRB, tumors were considered pRB positive.

Statistical analysis
The differences of distributions of patients between p53 negative and positive and between pRB negative and positive in individual variables were evaluated by Mann-Whitney U test. Also, in individual variables, the differences of distributions of patients among the four groups classified according to the p53 status and the pRB status of tumors were evaluated by Kruskal-Wallis test. The survival distributions were estimated using the method of Kaplan and Meier [11]. Corrected survival rates were used; that is, only deaths caused by esophageal cancer were taken as outcome events and all other deaths were considered censored events. Differences between survival distributions were tested for statistical significance by log rank analysis [12]. The influence of each variable on survival was assessed by the Cox proportional hazard model. All statistical analyses were performed using the StatView 5.0 software package for Macintosh (Abacus Concepts Inc, Berkeley, CA). A p value of less than 0.05 was considered statistically significant.

	Results

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Seventy-nine of 191 tumors (41%) were positive for p53, and 109 (57%) were positive for pRB. p53 and pRB status was analyzed in relation to various clinicopathologic characteristics (Table 1). The percentage of tumors with invasion to the adventitia and that of tumors with lymph node metastasis were significantly higher in the 82 pRB-negative tumors than in the 109 pRB-positive tumors. However, there were no significant differences in clinicopathologic characteristics between p53 positive and negative tumors (Table 1). All 191 patients were divided into four subgroups according to p53 and pRB status of tumors (p53-/pRB+, n = 60; p53-/pRB-, n = 52; p53+/pRB+, n = 49; and p53+/pRB-, n = 30). Clinicopathologic characteristics were compared among the four groups (Table 2). The incidence of lymph node metastasis was significantly lower in patients with p53-/pRB+ tumors (40%) compared with those of other groups (p53-/pRB-, 69%; p53+/pRB+, 53%; and p53+/pRB-, 73%).

View larger version (20K): [in this window] [in a new window]   Fig 1. Five-year survival rates. a: p53-/pRB+ (n = 60), 50%; b: p53-/pRB- (n = 52), 15%; c: p53+/pRB+ (n = 49), 29%; and d: p53+/pRB- (n = 30), 23%. The survival curve of patients with p53-/pRB+ tumors was significantly better than that of the other groups (p = 0.003, by the log rank test).

	Comment

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The retinoblastoma (RB) gene product controls cell proliferation through regulation of the cell cycle at the G₁-S-phase transition. The pRB is bound to transcription factor E2F during the G₁ phase. However, when pRB is phosphorylated by the cyclin-dependent kinase complexes, E2F is released, and the cell can initiate DNA synthesis [13]. The p53 tumor suppressor gene, located on the short arm of chromosome 17, appears to have an important effect on cellular growth control. The p53 gene product is known to regulate cell growth and proliferation. The wild-type p53 protein suppresses cell growth by controlling the G₁ checkpoint. This effect is mediated by p21^WAF1 [14]. The wild-type p53 protein also has several additional physiologic functions, including control of the G₂ cell cycle checkpoint and mediation of apoptosis [14]. RB and p53 genes have been shown to alter frequently in ESCC. Wagata and colleagues [6] reported that p53 gene mutations were detected in 47% of ESCCs, and these tumors with p53 mutations showed overexpression of p53 protein. Also, Boynton and associates [15] reported that the loss of heterozygosity at the RB locus was detected in 54% of patients with ESCC. These reports indicate that considerable numbers of ESCC tumors might show abnormal p53 protein expression or reduced expression of pRB.

Mutations in the p53 gene usually result in p53 protein stabilization and accumulation; consequently, the gene product can be detected by immunohistochemistry. Overexpression of p53 has been found in a number of human malignancies and was associated with a poor prognosis. However, conflicting results, showing the absence of any correlation between p53 expression and prognosis, have been reported for ESCC [2, 16]. We found p53-specific immunoreactivity in 41% of tumors and overexpression of mutated p53 protein even in early stages of cancer. We also found that p53 status was not a prognostic factor for patients with ESCC. Further, the loss of pRB expression was detected in 43% of ESCCs, and the survival curve of patients with pRB-negative tumors was significantly lower than that of patients with pRB-positive tumors. These results indicate that reduced pRB expression in tumors strongly correlated with tumor progression in ESCC. However, pRB expression was not an independent prognostic factor for patients with ESCC. Similar results were found for non–small cell lung cancer [8]. We analyzed pRB abnormality by using immunohistochemistry, which is believed to be a sensitive and reliable method for detecting RB gene abnormality [17]. Xu and associates [8] reported that most known RB gene mutations result in total loss of RB protein expression, and even altered pRB is expressed rarely in tumors. Usually it does not enter the nucleus and so does not produce false-positive nuclear staining. However, Cote and colleagues [18] reported that, in bladder cancer patients with high levels of pRB, nuclear reactivity had essentially identical recurrence rates and the survival of such patients was as low as that of patients with no pRB-expressing tumors. pRB expression detected by immunohistochemical methods might show not only unphosphorylated RB protein (activated form) but also hyperphosphorylated RB protein (inactivated form). Thus, in our case, some pRB-positive tumors might have had hyperphosphorylated RB protein (inactivated form) and the prognosis of patients with such tumors should be as poor as that of patients with pRB-negative tumors. When a method that discriminates hyperphosphorylated RB protein from unphosphorylated RB protein is developed, the prognostic significance of pRB in ESCC should be more clearly explainable.

When neither p53 nor pRB was altered, patients had very low rates of recurrence and longer survival, regardless of the stage of the disease. This phenomenon has been recognized not only in ESCC, but also in lung cancer [8] and bladder cancer [18]. However, the survival and recurrence rates in patients with tumors showing alterations in both p53 and pRB were not different from those of patients with tumors showing alterations in either p53 or pRB. These results indicate that disorder in the RB pathway might have a more important effect on tumor progression than disorder in the p53 pathway in ESCC. However, even in tumors having no abnormal expression of pRB, the prognosis of patients with tumors showing abnormal p53 expression was poor. Thus, the combined analysis of p53 protein and pRB expression is a useful method to estimate prognosis of patients with ESCC.

	References

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