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Ann Thorac Surg 2002;74:222-226
© 2002 The Society of Thoracic Surgeons

Original article: general thoracic

Expression of heat shock protein 70 in grossly resected esophageal squamous cell carcinoma

Tsuyoshi Noguchi, MD, PhDa, b, c, Shinsuke Takeno, MD, PhD*a, Tomotaka Shibata, MDa, b, c, Yuzo Uchida, MD, PhDa, b, c, Shigeo Yokoyama, MD, PhDa, b, c, Wolfram Müller, MDa, b, c

a Department of Surgery II, Oita Medical University, Oita, Japan
b Department of Pathology I, Oita Medical University, Oita, Japan
c Institute of Pathology, Heinrich-Heine University, Düsseldorf, Germany

Accepted for publication March 25, 2002.

* Address reprint requests to Dr Takeno, Department of Surgery II, Oita Medical University, Idaigaoka 1-1, Hasama-machi, Oita 879-5593, Japan
e-mail: surg2{at}oita-med.ac.jp


   Abstract
Top
 Abstract
Introduction
 Material and methods
 Results
 Comment
 Acknowledgments
 References
 
Background. The aim of the present study was to use immunohistochemical methods to clarify the clinical implication of heat shock protein (HSP) 70 expression in esophageal squamous cell carcinoma and to investigate the function of HSP70 as a chaperone for p53.

Methods. Seventy-one patients with esophageal squamous cell carcinoma were admitted in the present study. Expression of HSP70 was analyzed by immunohistochemistry and correlated with TNM classification, vessel invasion, p53 expression, and clinical outcome after operation.

Results. Overexpression of HSP70 was related to sex (p < 0.05), tumor configuration (p < 0.05), lymph node metastasis (p < 0.01), and lymphatic vessel invasion (p < 0.05). Expression of p53 and HSP70 were not correlated with each other (p = 0.824). Esophageal squamous cell carcinoma with HSP70 expression exhibited a significantly better prognosis compared with HSP70-negative esophageal squamous cell carcinoma in univariate analysis (p < 0.05), but no significance was found in multivariate analysis.

Conclusions. We suggest that HSP70 expression might be of use to assess the progression, lymph node metastasis, and lymphatic vessel invasion of esophageal squamous cell carcinoma. Inasmuch as both lymph node metastasis and HSP70 expression are prognostic variables in esophageal squamous cell carcinoma, examination of HSP70 expression may be of use to assess clinical outcome after operation.


   Introduction
Top
 Abstract
 Introduction
Material and methods
 Results
 Comment
 Acknowledgments
 References
 
Heat shock proteins (HSPs) are highly conserved proteins. Heat shock proteins consists of at least five groups and are induced by various stresses, including heat, drugs, infection, or ischemia. Heat shock proteins play an important role in the response made by cells in a variety of biologic circumstances. When the cells are damaged, HSPs prevent cell death by binding to and refolding and renaturing abnormal proteins [1, 2].

We are particularly interested in the role of HSPs in tumor cell biology, especially in tumors of the gastrointestinal tract where they have been documented to be involved in cytoprotection, resistance to chemotherapy, and radiotherapy as well as cancer immunology [3–6].

Of the various proteins within the HSP family, HSP70 was identified as a critical protein that is part of the initial cellular response to heat-induced stress as well as to various other stresses [7].

It is of interest that the prognostic implication of elevated HSP70 expression by tumors varies according to the organ of the primary lesion. For example, tumor positivity for HSP70 is a poor prognostic factor in patients with carcinoma of the rectum [8] but is of no prognostic importance in patients with gastric cancer [9]. Concerning esophageal squamous cell carcinoma (ESCC), which exhibits the poorest outcome of all cancers arising from the digestive tract, Kawanishi and associates [10] and Shiozaki and coworkers [11] reported that increased expression of HSP70 correlated with a better prognosis. In a previous report, we suggested that HSP70 might play a critical role in the presentation of tumor-specific antigen and reduce lymph node metastasis secondary to the accumulation of macrophages and CD8+ T cells in ESCC [6]. Thus, HSP70 expression levels may play an important role in determining the clinical outcome in patients with ESCC.

Furthermore, the role of HSP70 as a molecular chaperone involved in p53 regulation has been reported, and it is suggested that mutant p53, which exhibits a prolonged half-life, forms a complex with HSP70 [12–14]. However, the exact nature of the correlation between immunohistochemical expression of HSP70 and p53 is still controversial as disparate results have been reported in colorectal, gastric, and head and neck cancers [8, 9, 15–17]. However, to date, there is no report regarding the expression of both HSP70 and p53 in ESCC.

The present study was designed to clarify the clinical implications of HSP70 expression levels in patients with ESCC as well as to address the question of whether HSP70 does play a chaperoning role for p53 in ESCC.


   Material and methods
Top
 Abstract
 Introduction
 Material and methods
Results
 Comment
 Acknowledgments
 References
 
Patients
Seventy-one patients with ESCC (63 men and 8 women, mean age, 63.8 years; age range, 43 to 84 years) who underwent surgical treatment at the Department of Surgery II of the Oita Medical University between January 1990 and December 1997 were included in the study. Surgical treatment comprised resection of the esophagus with lymph node dissection without preoperative adjuvant therapy. Patients who had no macroscopic residual cancer except distant metastasis were used in the study because several authors reported that residual cancer at the time of operation correlated with unfavorable outcome after operation [18–20].

Resected specimens were classified according to the TNM classification system by UICC [21] as well as to the presence or absence of vessel invasion.

Immunohistochemical staining
Immunohistochemical staining was performed as described previously [5, 6]. Briefly, formalin-fixed, paraffin-embedded specimens were cut, and tissue sections were placed on silane-coated slides. After deparaffinization and antigen retrieval, the sections were incubated with normal goat or rabbit serum and then incubated with either a polyclonal anti-HSP70 antibody (1:150, ScyTek, Logan, UT) or a monoclonal anti-p53 antibody (DO-7, 1:50, Dako, Carpinteria, CA). Immunohistochemical staining was performed using the SAB-PO (R) or (M) kit (Nichirei, Tokyo, Japan).

Positive control tissue included ESCC (for HSP70 staining) and gastric cancer (for p53 staining), whereas negative control tissue included omission of the primary antibodies.

Samples that reacted positively for HSP70 in more than 50% of the area of ESCC were evaluated as positive for HSP70. By contrast, when more than 10% of cancer cells were stained for p53, these samples were evaluated as positive for p53 [22, 23]. Immunohistochemical staining was evaluated by two independent observers without access to clinical information.

Clinicopathologic factors
Factors used in the present study included patient age, sex, tumor location (upper, middle, or lower esophagus), configuration (early [flat spread], protuberant, or ulcerating lesions), diameter, histologic grade (differentiation) of the tumor, TNM classification, and the degree of lymphatic or blood vessel invasion. Patients were divided into two groups according to the mean patient age (63.8 years) and the mean tumor diameter (45.7 mm).

Statistical methods
Correlation between HSP70 expression levels and clinicopathologic factors was analyzed using the Mann-Whitney U test or {chi}2 test. The correlation between HSP70 and p53 expression was analyzed using the {chi}2 test.

The Kaplan-Meier method was used to assess clinical outcome after operation. The differences were examined using the log-rank-test. Multivariate analysis was assessed using the Cox proportional hazard model. A probability value less than 0.05 was considered to be statistically significant.


   Results
Top
 Abstract
 Introduction
 Material and methods
 Results
Comment
 Acknowledgments
 References
 
Expression of heat shock protein 70 and correlation with clinicopathologic factors
Various patterns in HSP70 expression were evident in ESCC. Cell nuclei were positively stained in the basal, spinous, and granular layers whereas the superficial layer of normal squamous epithelium was HSP70 negative (Fig 1a). In contrast, dysplastic epithelium exhibited a positive cytoplasmic and nuclear expression of HSP70 (Fig 1b). Tumor cells within ESCC also exhibited a predominantly cytoplasmic staining pattern, although some cancer cells showed additional positive nuclei staining (Fig 1c). Forty-eight of 71 tissue samples examined revealed positive reaction for HSP70.



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  		Fig 1. Immunohistochemical staining for heat shock protein 70 in normal squamous epithelium (A), dysplasia (B), and squamous cell carcinoma (C). Heat shock protein 70 localized in the nuclei of normal squamous epithelium. In contrast, cytoplasmic and nuclear expression of heat shock protein 70 was observed in squamous dysplasia. Esophageal squamous cell carcinoma also exhibited a predominantly cytoplasmic staining pattern.

 
Heat shock protein 70 expression exhibited a significant correlation with sex (p < 0.05), tumor configuration (p < 0.05), tumor diameter (p < 0.05), pN (p < 0.01), and lymphatic vessel invasion (p < 0.05; Table 1). Male patients, early tumors, ulcerating esophageal tumors, and large tumors (>45.7 cm) also tended to exhibit HSP70 positivity. Heat shock protein 70 overexpression in ESCC was associated with lymphatic metastatic spread, pN (p < 0.01), and lymphatic vessel invasion (p < 0.05).


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  		Table 1. Immunohistochemical Status of Heat Shock Protein 70 in Esophageal Cancer

 
Correlation between heat shock protein 70 and p53 expression
Thirty tissue samples revealed positive immunostaining for both p53 and HSP70, but no significant correlation between HSP70 and p53 expression was detected (p = 0.824; Table 2).


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  		Table 2. Correlation Between Heat Shock Protein 70 and p53 Overexpression

 
Univariate and multivariate analysis of prognostic variables
Expression of HSP70 was significantly correlated with a favorable prognosis in the univariate analysis (p < 0.05; Fig 2). In contrast, p53 positivity of tumors had no implication on the outcome after operation in the present series (data not shown).



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  		Fig 2. Heat shock protein 70 expression correlated with a favorable prognosis in univariate analysis (p < 0.05).

 
Conventional prognostic factors including pT, pN, pM, UICC stage, lymphatic and blood vessel invasion, and expression of HSP70 and p53 were analyzed using multivariate analysis. Only pM exhibited an independent prognostic implication (p = 0.0081), whereas HSP70 expression failed to reach independent significance (Table 3).


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  		Table 3. Multivariate Analysis of Prognostic Outcome

 

   Comment
Top
 Abstract
 Introduction
 Material and methods
 Results
 Comment
Acknowledgments
 References
 
Esophageal cancer is a common cancer and exhibits a very poor prognosis [24]. It is therefore apparent that studies clarifying the biologic behavior of ESCC may well be of significant clinical benefit. The prognostic implications of the molecular marker HSP70 are still unclear.

Concerning carcinogenesis, Kaur and associates [25] report HSP70 expression owing to biologic stress in premalignant lesions of the oral cavity. The status of HSP70 expression varied in dysplasia compared with normal squamous mucosa of the esophagus in the present study. The change of localization from nucleus to cytoplasm may reflect some roles of HSP70 in esophageal carcinogenesis are the same as in oral cavity.

Kawanishi and colleagues [10] and Shiozaki and coworkers [11] suggested that HSP70 expression was related to increased survival in patients with ESCC. However, in contrast to ESCC, HSP70 expression was reported to indicate a poor prognosis in patients with HSP70-positive rectal cancer and to be of no prognostic significance in patients with gastric cancer [8, 9].

Our study revealed that HSP70 expression does implicate a prolonged survival, which is in agreement with the findings of Kawanishi and colleagues [10] and Shiozaki and associates [11], although it was not an independent prognostic factor in multivariate analysis. In our previous report, HSP70 expression was related to tumor-specific antigen presentation and accumulation of CD8+ T cells and macrophages [6]. As a result of the ensuing immunologic response, lymph node metastasis was inhibited. In the present study, HSP70 expression was correlated with a reduced frequency of lymph node metastasis and lymphatic vessel invasion as well as a reduced tumor size. Because lymph node metastasis is a critically important prognostic factor and is correlated with lymphatic vessel invasion in ESCC, the expression of HSP70 may be a critical molecular marker of prognosis [26]. Thus, the different clinical influence of HSP70 expression in ESCC compared with rectal or gastric cancer may be secondary to fundamental histologic differences between squamous cell carcinoma and adenocarcinoma.

Heat shock protein 70 plays an important role as a molecular chaperone and is reported to bind to mutant p53 and form a complex [12–14]. Mutant p53 is believed to exhibit a prolonged half-life, which may result in increased immunohistochemical expression of p53 [27, 28]. The present study attempted to clarify the interaction between HSP70 and p53 expression but showed no significant correlation between them. It is of interest that significant correlation was found in breast cancer and colorectal cancer but not in squamous cell carcinoma of the head and neck or cancer of the tongue or stomach [8, 9, 15–17]. Heat shock protein 70 expression requires the constitutive expression of heat shock cognate coupled with the expression of HSP70, which is induced by heat or other biologic stresses [29]. A p53 mutant resulting from a missense mutation is reported to form a complex with heat shock cognate and accumulate predominantly in the nucleus. Our results are derived from immunohistochemistry using a polyclonal anti-HSP70 antibody that reacts with both heat shock cognate and HSP70. In addition, anti-p53 monoclonal antibody reacts not only to mutant type but also to a part of wild type. Furthermore, HSP70 plays a role as a chaperone not only for mutant p53 but also for other proteins, including peptide antigen as described in our previous report [6]. These may be reasons why the significant correlation between HSP70 and p53 expression was not determined in the present study.

In the present study, HSP70 expression was of use in predicting longer survival and reduced frequency of lymph node metastasis in patients with ESCC. In addition, it may be possible to use the function of HSP70 for gene or immunotherapy because HSP70 expression plays an important role in the immunologic response to ESCC [6, 30, 31]. Further analysis of HSP70 in ESCC will be of future benefit to patients with ESCC.


   Acknowledgments
Top
 Abstract
 Introduction
 Material and methods
 Results
 Comment
 Acknowledgments
References
 
The authors thank Naomi Kawano, Yoko Iwata, and Kaori Soe in the Department of Surgery II, Oita Medical University, for technical assistance in immunohistochemical staining.


   References
Top
 Abstract
 Introduction
 Material and methods
 Results
 Comment
 Acknowledgments
 References
 

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