Clinical Cancer Research 13, 4046, July 15, 2007. doi: 10.1158/1078-0432.CCR-07-0449
© 2007 American Association for Cancer Research
Significance of Immunohistochemical Expression of Estrogen Receptors 
and ß in Squamous Cell Carcinoma of the Esophagus
Tadahiro Nozoe,
Tsunehiro Oyama,
Mitsuhiro Takenoyama,
Takeshi Hanagiri,
Kenji Sugio and
Kosei Yasumoto
Authors' Affiliation: Second Department of Surgery, University of Occupational and Environmental Health, Kitakyushu, Japan
Requests for reprints: Tadahiro Nozoe, Second Department of Surgery, University of Occupational and Environmental Health, 1-1, Iseigaoka, Yahata-NishiWard, Kitakyushu 807-0855, Japan. Phone: 81-093-691-7442; Fax: 81-093-692-4004; E-mail: ntvb{at}med.uoeh-u.ac.jp.
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Abstract
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Purpose: Possible significance of sex hormone estrogen as an antitumor therapeutic arm toward esophageal squamous cell carcinoma (ESCC) cells has been suggested. The aim of the current study was to clarify the clinicopathologic significance of an immunohistochemical expression of estrogen receptors 
and ß (ER and ERß) in ESCC.
Experimental Design: Expression of ER and ERß were examined using an immunohistochemical methods in 73 paraffin-embedded sections collected from patients with ESCC who had been subjected to esophageal resection and digestive reconstruction without any preoperative induction therapy.
Results: Forty-seven (64.4%) ESCCs had a positive cytoplasmic expression of ER and 21 (28.8%) ESCCs had a positive nuclear expression of ERß. Univariate analysis showed that both positive ER expression (P = 0.0001) and negative ERß expression (P = 0.026) were unfavorable prognostic indicators in ESCC. Moreover, multivariate analysis showed that ER-positive/ERß-negative expression (P = 0.003) and progression of tumor stage (P = 0.014) were found to be independent unfavorable prognostic indicators in ESCCs.
Conclusions: Immunohistochemical expression of ER and ERß were found to be observed in ESCC. Positive expression of ER in addition to negative expression of ERß proved to be an unfavorable independent prognostic indicator in ESCC.
Previous reports have indicated that there is a possible correlation of estrogen with the biological potential of esophageal squamous carcinoma cells (1, 2). An epidemiologic investigation showed that the male to female rate of the incidence of esophageal squamous cell carcinoma (ESCC) reached to 3:1 or 4:1 (3).
The previous investigation showed that the prognosis of esophageal carcinoma was found to be significantly better in female than in male (4). Therefore, estrogen and estrogen receptors (ER) are considered to be possibly involved in tumorigenesis and/or progression of squamous cell carcinoma of the esophagus. Previous report showed that inhibitory effect against the proliferation of esophageal squamous carcinoma cells might be regulated by the function of ER (5). However, an immunohistochemical expression of ER on esophageal squamous carcinoma cells has not been studied fully.
Subtypes of ER, ER and ERß, were initially cloned in 1986 and 1996, respectively. ER and ERß are found to be located on human chromosome 6q25 (6) and chromosome 14q22-24 (7), respectively. These ERs function as the factor for ligand-activated transcription. Both ER and ERß contain some functional domains, including a central DNA-binding domain conversed between ER and ERß. Therefore, it is suggested that these sex hormone receptor might contain different biological function. Since the discovery of these ERs, the biological significance of these sex hormone receptors in human tumors, including breast carcinoma, has been investigated, and the relationship between the expression of ER subtypes and the malignant potential of the tumors have been reported (8–10). ERß is known to bind to ER and to have a suppressive function toward ER (11, 12). Therefore, these sex hormones have been considered to mutually contain inverse biological function.
The purpose of this study is to elucidate the clinicopathologic and/or prognostic significances of an immunohistochemical expression of ER and ERß in ESCC.
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Materials and Methods
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Patients and samples. Seventy-three specimens of ESCCs collected from the patients, who had been subjected to esophageal resection and reconstruction of the digestive tract in our department between 1993 and 2004, were enrolled. Neoadjuvant therapy was not done to any patients. These 66 men and 7 women had a median age of 64 years (range, 42-83 years).
The follow-up for patients was continued until their death and only patients who died of ESCC were included in the tumor-related deaths. Follow-up periods after surgical treatment ranged from 3 months to 11 years and 3 months with a mean of 2 years and 8 months.
Pathologic features were shown based on the guidelines for clinical and pathologic studies on carcinoma of the esophagus established by the Japanese Society for Esophageal Diseases (13, 14).
Immunohistochemical expression of ER and ERß. Four-micrometer-thick sections sliced from paraffin-embedded specimen were prepared on the slide glasses precoated with silane. After removing the paraffin with xylene and washing in a graded series of ethanol, the sections were placed in TBS for 10 min. Endogenous peroxidase activity was blocked for 10 min in methanol containing 0.3% H2O2, and then the slides were placed in TBS. The sections were incubated with TBS including 1% concentration of bovine serum albumin for 10 min to block nonspecific binding of the immunoreagents. After washing in TBS, the sections were incubated with 1:50 diluted rabbit anti-human polyclonal ER antibody (HC-20; Santa Cruz Biotechnology) or 1:20 diluted rabbit anti-human polyclonal ERß antibody (H-150; Santa Cruz Biotechnology). All incubations proceeded overnight at 4°C. After washing in TBS, an immunoperoxidase staining was done by an EnVision antibody complex method (15) using Envision kit (DAKO Ltd.). Finally, the localization of ERs was visualized with diaminobenzidine tetrahydrochloride.
In accordance with the criteria in the report of Wu et al. (16) about ER expression in lung carcinoma, ESCCs with at least 50% of the tumor cells having cytoplasmic expression of ER and nuclear expression of ERß were regarded as ER and ERß positive, respectively (Fig. 1
).
Statistical analysis. The 
2 test and Student's t test were used to compare the clinicopathologic data. The cumulative survival rates were calculated by the Kaplan-Meier method and the survival curves were tested by the Mantel-Cox method. The multivariate survival analysis was calculated according to Cox's proportional hazards model in a forward stepwise manner. A P value of <0.05 was considered significant.
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Results
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Forty-seven (64.4%) ESCCs had a positive cytoplasmic expression of ER and the other 26 (35.6%) did not (Fig. 1A and B). In the normal mucosa concomitant with carcinoma tissue, cytoplasmic expression of ER was observed in only some cells (Fig. 1C). Relationship between ER expression and clinicopathologic features was shown in Table 1
. Proportion of male among patients with ESCCs expressing ER was significantly higher than that among patients without ER expression (P = 0.004). Lymph node metastasis and venous invasion of ESCCs with ER expression were significantly more frequent than those of tumors without ER expression (P = 0.023 and 0.017, respectively). Stage of tumors was significantly more advanced in ESCCs with ER expression (P = 0.027). Five-year survival rate of patients with ESCCs with ER expression (23.2%) was significantly worse than that of patients with ESCCs without ER expression (68.6%; P = 0.0001; Fig. 2
).
Twenty-one (28.8%) ESCCs had a positive nuclear expression of ERß and the other 52 (71.2%) did not (Fig. 1D and E). In the normal mucosa concomitant with carcinoma tissue, nuclear expression of ERß was observed in only some cells (Fig. 1F). There was no significant difference about clinicopathologic features between ERß-positive and ERß-negative patients (Table 2
). Five-year survival rate of 57.5% in patients with ESCCs with ERß expression was significantly better than that of 32.6% in patients with ESCCs without ERß expression (P = 0.026; Fig. 3
).
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Fig. 3. Survival curves of patients with ESCCs with and without ERß expression. Survival rates of patients with ESCCs with ERß expression (bold line) was significantly better than that of patients with ESCCs without ERß expression (thin line; P = 0.026).
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Proportion of positive and negative expression of ER among ESCCs with positive ERß expression was 42.9% (9 of 21) and 57.1% (12 of 21), respectively. On the other hand, proportion of positive and negative expression of ER among ESCCs with negative ERß expression was 73.1% (38 of 52) and 26.9% (14 of 52), respectively. There was a significantly inverse correlation between ER and ERß expression (P = 0.15; Table 3
).
Comparison of clinicopathologic features between ER-positive/ERß-negative ESCCs and tumors showing other expression patterns (both positive, both negative, and ER negative/ERß positive) was shown in Table 4
. Proportion of male among patients with ER-positive/ERß-negative ESCCs was significantly higher than that among patients with ESCCs of other expressing patterns (P = 0.028). Lymph node metastasis and venous invasion of ER-positive/ERß-negative ESCCs were significantly more frequent than those with other expressing patterns (P = 0.028 and 0.045, respectively). Five-year survival rate of 22.1% in patients with ER-positive/ERß-negative ESCCs was significantly worse than that of patients with ESCCs with other expressing patterns (58.1%; P < 0.0001; Fig. 4
).
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Table 4. Comparison of clinicopathologic features between patients with ER-positive/ERß-negative tumors and the others
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Fig. 4. Survival curves of patients with ER-positive/ERß-negative ESCCs and other patients. Survival rates of the former (bold line) was significantly worse than that of the latter (thin line; P < 0.0001).
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Multivariate analysis showed that stage of the tumor (P = 0.014) and ER-positive/ERß-negative expression (P = 0.003) proved to be independent prognostic indicators of patients with ESCCs (Table 5
).
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Table 5. Multivariate analysis to determine factors independently associated with unfavorable prognosis of patients
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Discussion
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In spite of the recent remarkable progress in diagnosis and therapeutic options, including surgical treatment in ESCC, the prognosis of the disease still remains dismal (17).
Previous experimental investigations showed that sex hormone estrogen might have a biological function to suppress the progression and proliferation of ESCC (1, 2). Utsumi et al. (5) reported that growth of transplanted tumors derived from ESCC cells expressing ER in female nude mice were significantly enhanced when they were oophorectomized, which suggested the inhibitory effect of estrogen to the proliferation of ESCC. These results also suggested the clinical application of sex hormone estrogen as a possible antitumor agent in ESCC. Experimental investigations to date have shown the antitumor effect of tamoxifen for squamous cell carcinoma, mainly of the head and neck. However, the effect could not be ascribed to the estrogen antagonism but to some other biological genetic alteration (18, 19).
Whereas estrogen has been known to accelerate the malignant potential of breast cancer, the ER has been chiefly focused as the target of the hormonal therapy in breast cancer. Moreover, the prognosis of patients with breast cancer–expressing ER has been reported to be more favorable than that of patients with tumors without ER expression, and the expression of ER has been acknowledged as a marker of candidates for hormonal therapy in patients with breast carcinoma (20). According to the panel of the investigations, showing a difference between functions of sex hormone to squamous cell carcinoma of some organs and breast carcinoma, the biological significance of ERs in ESCC might differ from that in breast carcinoma.
Recently, expression of ER subtypes, ER and ERß, in human malignant tumors has come to be focused. Clinical and experimental investigations to date have generally shown that expression of ER and/or loss of ERß expression could be correlated with malignant potential of human tumors, including non–small lung cancer, colorectal cancer, and prostate cancer (8–10, 16, 21). However, to the best of our knowledge, investigation for an immunohistochemical expression of these ER subtypes in ESCC has not been reported and this is the first report about this point.
Both ER and ERß are ligands to estradiol, but they differ in effect on transcription at activator protein-1 sites (22). Although ER activates the transcription at activator protein-1 site, ERß inversely inhibits the transcription. ERß is known to bind to ER and to have a suppressive function toward ER (11, 12).
In the current study, expression of ER was found to be also inversely correlated with that of ERß in ESCC. Similar results have been shown about lung cancer (10). Expression of ER has been reported to be crucially correlated with progression of colorectal carcinoma (8), and the significant correlation between loss of ERß expression and advanced Dukes' stage was shown, which might suggest the significance of ERß expression as an indicator of favorable prognosis and a protective role of ERß against colon cancer progression (9).
Expression of ER and ERß were observed in the cytoplasm and the nucleus of the ESCC cells, respectively, which was consistent with the previous reports (16, 23). However, the reason for an intracellular localization of ER and ERß remains unclear.
In lung cancers, the proportion of ER expression in female patients has been reported to be higher than in men, showing that the ER expression could be gender dependent (24). Although the number of female patients enrolled in the current study is small, the proportion of male among patients with ESCCs expressing ER-positive and ERß-negative expression was significantly higher than in female.
In conclusion, positive expression of ER in addition to negative expression of ERß was found to be an unfavorable independent prognostic indicator in ESCC. Sex hormone (estrogen) therapy can be considered as one of candidates to improve prognosis of ESCC. Although results in the current study might provide the hint for possibility of sex hormone therapy for selected patients with ESCC, relationship between expression of ER and ERß and possible antitumor effect derived by sex hormone in ESCC should be established using experimental and clinical investigations in the future proposition.
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Footnotes
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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 2/21/07;
revised 4/16/07;
accepted 5/ 7/07.
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Abstract
Materials and Methods
