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Clinical Cancer Research Vol. 12, 3740-3745, June 15, 2006
© 2006 American Association for Cancer Research

Imaging, Diagnosis, Prognosis

Expression of p53R2 Is Related to Prognosis in Patients with Esophageal Squamous Cell Carcinoma

Hiroshi Okumura, Shoji Natsugoe, Naoya Yokomakura, Yoshiaki Kita, Masataka Matsumoto, Yasuto Uchikado, Tetsuro Setoyama, Tetsuhiro Owaki, Sumiya Ishigami and Takashi Aikou

Authors' Affiliation: Department of Surgical Oncology and Digestive Surgery, Graduate School of Medicine, Kagoshima University, Sakuragaoka, Kagoshima, Japan

Requests for reprints: Hiroshi Okumura, Department of Surgical Oncology and Digestive Surgery, Graduate School of Medicine, Kagoshima University, Sakuragaoka 8-35-1, Kagoshima 890-8520, Japan. Phone: 81-992-75-5361; Fax: 81-992-65-7426. E-mail: hokumura{at}m.kufm.kagoshima-u.ac.jp.


   Abstract
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 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Purpose: The p53 gene and its family are important factors for carcinogenesis, prognosis, and chemoresistance in esophageal squamous cell carcinoma. A recently identified ribonucleotide reductase, p53R2, is regulated by p53 for supplying nucleotides to repair damaged DNA. In the present study, we analyzed the expression and clinicopathologic significance of p53 and p53R2 in esophageal squamous cell carcinoma.

Experimental Design: We immunohistochemically investigated the relationship between p53 and p53R2 expressions in surgical specimens of primary tumors in 222 patients with esophageal squamous cell carcinoma.

Results: The positive expression rate of p53 was 47.1% and that of p53R2 was 61.7%. Positive p53R2 expression was significantly correlated with depth of invasion, lymph node metastasis, stage, and poor prognosis. In the p53-negative group, the 5-year survival rate was better in patients with negative p53R2 expression than in those with positive p53R2 expression. Multivariate analysis indicated that the negative expression of both p53 and p53R2 was an independent prognostic factor along with tumor depth nodal metastasis and stage.

Conclusions: We showed that positive p53R2 expression was related to tumor development and that alteration of p53R2 expression in p53-negative tumors was closely related to prognosis. Evaluation of the expressions of p53 and p53R2 proteins should be useful for determining the tumor properties, including prognosis, in patients with esophageal squamous cell carcinoma.

Esophagus squamous cell carcinoma is one of the most aggressive carcinomas of the gastrointestinal tract. Many studies have identified various biological factors in the malignant potential of esophagus squamous cell carcinoma. The tumor suppressor gene p53 and its target genes play a key role in a wide range of human cancers including esophagus squamous cell carcinoma, regulating cell cycle and triggering apoptosis following DNA damage (1–4). A recently identified ribonucleotide reductase, p53R2, is regulated by p53 for supplying nucleotides to repair damaged DNA (5). Ribonucleotide reductase is an enzyme in DNA synthesis that catalyzes the conversion of ribonucleotide diphosphates into their corresponding deoxyribonucleotides and is important in DNA replication and repair (6, 7). For cell survival, p53 activated by DNA damage then induces p53R2 expression to repair the damaged DNA. Synthesis of DNA in cells arrested in G1 or G2 after DNA damage is mediated by p53R2 (5, 8).

The aims of this retrospective study were to examine the expressions of p53 and p53R2 in surgical specimens of esophagus squamous cell carcinoma and to evaluate whether such expressions are useful for predicting the outcome.


   Materials and Methods
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 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Study groups. The present study involved 222 consecutive patients with esophageal squamous cell carcinoma who underwent curative surgery at the Kagoshima University Hospital between January 1990 and December 1998. All of these patients underwent an esophagectomy with lymph node dissection. The patients ranged in age from 36 to 85 years (mean, 64.2 years). None of these patients underwent endoscopic mucosal resection, palliative resection, preoperative chemotherapy, or radiotherapy, and none of them had synchronous or metachronous multiple cancers in other organs. Ninety-seven patients had relapsed disease in their follow-up period. Among them, 31 patients received chemotherapy and 44 patients received radiation therapy after surgery. Specimens of cancer and adjacent noncancerous tissues were collected from the patients according to the institutional guidelines of our hospital after informed consent had been obtained. Classifications of the specimens were determined according to the International Union against Cancer tumor-node-metastasis classification system (9). All of the M1 tumors had distant lymph node metastases. All patients were followed up after discharge with a chest X-ray every 1 to 3 months, computed tomography every 3 to 6 months, and ultrasonography every 6 months. Follow-up data after surgery were available for all patients with a median follow-up period of 30 months (range, 1-181 months).

Immunohistochemistry. Tumor samples were fixed with 10% formaldehyde in PBS, embedded in paraffin, and sectioned into 4-µm-thick slices. After deparaffinization of the sections, the endogenous peroxidase was blocked by immersing the slides in a 0.3% hydrogen peroxidase-methanol solution for 30 minutes at room temperature. As preparation for staining with anti-p53 and anti-p53R2 antibodies, sections were treated with citrate buffer for 10 minutes at 100°C in a microwave oven. The sections were washed thrice with PBS, each for 5 minutes, and then blocked by treatment with PBS containing 3% skim milk for 30 minutes at room temperature. The blocked sections were incubated with primary antibody p53 (DO7, Novocastra Laboratories, Newcastle upon Tyne, United Kingdom; 1:50) or p53R2 (sc-10840, Santa Cruz Biotechnology, Inc., Santa Cruz, CA; 1:100), diluted in PBS at 4°C overnight, followed by staining with a streptavidin-biotin peroxidase kit (Nichirei, Tokyo, Japan). The sections were washed in PBS for 5 minutes thrice and the immune complex was visualized by incubating the sections with diaminobenzidine tetrahydrochloride. The sections were rinsed briefly in water, counterstained with hematoxylin, and mounted. The breast cancer cell line (MCF-7) and esophageal cancer cell line (TE-8) expressing p53R2 were used as positive controls. Negative controls were done by replacing the primary antibodies with PBS. Evaluation of immunohistochemistry was independently carried out by two investigators (H.O. and S.N.). To evaluate the expressions of p53 and p53R2, 10 fields (within the tumor and at the invasive front) were selected, and the expression in 1,000 tumor cells (100 per field) was evaluated using high-power (x200) microscopy. For p53, a distinct nuclear immunoreaction in >10% of the cancer cells was judged positive, and for p53R2, a distinct immunoreaction in the perinuclear and other cytoplasmic regions of >10% of the cancer cells was judged positive as previously described (10).

Statistical analysis. Statistical analysis of group differences was done using the {chi}2 test and t test. The Kaplan-Meier method was used for survival analysis and differences in survival were estimated using the log-rank test. Prognostic factors were examined by univariate and multivariate analyses (Cox proportional hazards regression model). P < 0.05 was considered to be statistically significant. The P values in this study were two sided. All statistical analyses were done using the software package StatView version 5.0 (Abacus Concepts, Berkeley, CA).


   Results
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 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Expressions of p53 and p53R2 in esophageal squamous cell carcinoma
The p53 expressions were detected as nuclear staining; p53 had 46.8% positive expression. The p53R2 expression was distinct detectable in cytoplasmic regions; p53R2 had 61.7% positive expression (Fig. 1 ).


Figure 1
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  		Fig. 1. Expressions of p53R2 proteins in esophageal squamous cell carcinoma. A, positive expression of p53R2 was detectable in cytoplasmic regions (x200). B, negative expression of p53R2 (x200).

 
Relationship between the expressions of p53 and p53R2 and clinicopathologic findings
The expression of p53 was associated with depth of tumor invasion and lymph node metastasis. The tumors with positive p53 expression invaded deeper and had more lymph node metastasis than the tumors with negative p53 expression although the significances were not strong (P = 0.048 and P = 0.022, respectively). The expression of p53R2 was significantly associated with depth of tumor invasion, lymph node metastasis, and stage. The tumors that were positive for p53R2 expression invaded deeper, had more lymph node metastasis, and had more advanced stages than those that were negative for p53R2 expression (P < 0.0001, P = 0.029, and P < 0.0001, respectively). The expression of p53R2 was significantly correlated with the expression of p53. Of the 104 tumors with positive p53 expression, 74 (71.2%) also had positive p53R2 expression (P = 0.0066). However, p53R2 expression did not correlated with age, sex, tumor location, histology, and smoking history (Table 1 ).


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  		Table 1. Relationship between p53R2 expression and clinicopathologic findings

 
Relationship between p53R2 expression and clinicopathologic findings according to p53 expression
The tumors were divided into the p53-positive group and the p53-negative group. Although the expression of p53R2 had no correlation with the clinicopathologic variables in the p53-positive group, it was related to depth of tumor invasion (P < 0.0001) and stage (P = 0.0002) in the p53-negative group (Table 2 ).


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  		Table 2. Relationship between p53R2 expression and clinicopathologic findings according to p53 expression

 
Relationship between clinical outcome and expressions of p53 and p53R2
Relapse and survival data correlated with expressions of p53 and p53R2. The expression of p53 was associated with relapse, disease-free survival, and overall survival. The patients with positive p53 expression tumors had more relapse, shorter median disease-free survival, and shorter overall survival than those with negative p53 expression tumors (P = 0.006, P = 0.007, and P = 0.023, respectively; Table 3 ). The expression of p53R2 was significantly associated with relapse, disease-free survival, and overall survival and had weak association with death. Tumors that were positive for p53R2 expression had more relapse, more death, shorter median disease-free survival, and shorter overall survival than tumors with negative p53R2 expression (P = 0.0002, P = 0.063, P = 0.007, and P = 0.013, respectively; Table 3).


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  		Table 3. Relapse and survival correlate with p53 and p53R2 expressions

 
Relationship between prognosis and expressions of p53 and p53R2. There was a significant difference in overall survival rates between negative and positive expressions of p53 (P = 0.023; Fig. 2A ) and of p53R2 (P = 0.013; Fig. 2B).


Figure 2
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  		Fig. 2. The postoperative overall survival curves according to the expression of p53 (A) or p53R2 (B) proteins. There was a significant difference in survival between the patients with positive (+) and negative (–) expressions of p53 (P = 0.023). There was also a significant difference in survival between the patients with p53R2 (–) and p53R2 (+) expressions (P = 0.013). The postoperative overall survival curves between the patients with p53R2 (+) or p53R2 (–) expression according to p53 expression. C, in the p53 (–) group, the patients with p53R2 (–) expression had a better outcome than those with p53R2 (+) expression (P = 0.035). D, in the p53 (+) group, there was no difference between the patients with p53R2 (+) or p53R2 (–) tumors.

 
In the p53-negative group, the overall survival rate was significantly higher in patients with negative p53R2 expression than in those with positive p53R2 expression (P = 0.035; Fig. 2C). However, in the p53-positive group, no significant difference was found between patients with positive and negative p53R2 expressions (Fig. 2D).

Univariate and multivariate analyses of survival
Univariate analysis showed that the following factors were significantly related to postoperative survival: sex, tumor depth, lymph node metastasis, stage, p53 expression, p53R2 expression, and the combination of p53 and p53R2 expression (P < 0.05). Multivariate regression analysis indicated that depth of invasion, lymph node metastasis, stage, and combination of p53 and p53R2 expression were independent prognostic factors (Table 4 ).


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  		Table 4. Univariate and multivariate analyses of prognostic factors

 

   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
In the present study, we examined the expression of p53 and p53R2 proteins and its clinical significance in esophageal squamous cell carcinoma. In this series, the positive expression of p53 was observed in 46.8%. This result was consistent with previous studies on esophageal squamous cell carcinoma using an immunohistochemical method in which the p53-positive rate was detected as 48.1% to 67.2% (11–14). Some authors reported a close relationship between p53 overexpression and poor prognosis in esophageal squamous cell carcinoma (11, 12) whereas other authors found no relationship (13, 14). In our study, correlations were found between p53 expression and tumor depth, nodal metastasis, and prognosis.

In this study, the positive expression of p53R2 was observed in 61.7%. The expression of p53R2 is induced by wild-type p53 in response to various genotoxic stresses (5, 8). The synthesis of DNA in cells arrested in G1 or G2 after DNA damage is mediated through the ribonucleotide reductase activity of p53R2. For cell survival, p53 activated by DNA damage then induces p53R2 expression to repair the damaged DNA. For cell death, either severe DNA damage or inactivation of the p53R2-dependent DNA synthesis pathway induces the apoptosis signaling pathway to eliminate the unrepaired cells (5). In the present study, p53R2 expression was strongly associated with depth of tumor invasion, lymph node metastasis, stage, and p53 expression. In the p53-negative group, p53R2 expression was also strongly associated with depth of tumor invasion and stage. These results suggest that the expression rate of p53R2 in esophageal squamous cell carcinoma increases in accordance with the degree of tumor invasion and stage, resulting in the acquired efficiency of the DNA repair function in esophageal squamous cell carcinoma closely related to the ability of invasion. In patients with oral squamous cell carcinoma, p53R2 mRNA expression was related to tumor size and lymph node metastasis (15); these data were in agreement with ours.

In a recent article, Smeds et al. screened nine exons of p53R2 gene for mutations and polymorphisms in 14 esophageal squamous cell carcinoma cases. They found no mutation in any of the nine exons although in the 5' untranslated region of the gene, they detected a novel polymorphism in four cases. Among those four cases, one sample with no p53 mutation showed a specific loss of the wild-type allele in the tumor compared with the corresponding benign tissue that contained both alleles. However, the rest of the polymorphic cases had both intact alleles (16). Those data suggested that the polymorphic esophageal squamous cell carcinoma tumors with loss of the wild-type allele of p53R2 gene might have overexpression of p53R2 protein as a possible mechanism.

In the survival analysis, sex, depth of tumor invasion, lymph node metastasis, stage, and negative expressions of p53 and p53R2 were prognostic factors in all patients of this study. Multivariate analysis revealed that the negative expression of both p53 and p53R2, as well as tumor depth, nodal metastasis, and stage, was an independent prognostic factor. Fan et al. (17) reported that altered ribonucleotide reductase levels have been shown to be involved in tumorigenicity. Abid et al. (18) reported that active ribonucleotide reductase might increase deoxyribonucleotide triphosphate production and stimulate cell division. In the overexpression of p53R2 as ribonucleotide reductase in wild-type p53 tumors, activation of its function stimulates the tumorigenicity or cell division under the p53 signaling pathway. Thus, in our study, p53R2 expression might correlate with tumor depth and poor prognosis in p53-negative patients. Further studies should be carried out to clarify the mechanisms underlying those results.

In conclusion, we showed that the prognostic value of p53R2 expression was important, particularly in patients with p53-negative esophageal squamous cell carcinoma. An examination of p53 and p53R2 expressions is useful for determining malignant properties, including clinical outcome in patients with esophageal squamous cell carcinoma.


   Footnotes
 
Grant support: Ministry of Education, Science, Sports and Culture, Japan, grant 17390373.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 11/ 8/05; revised 4/13/06; accepted 4/20/06.


   References
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 Materials and Methods
 Results
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