Clinical Cancer Research Vol. 11, 5084-5089, July 15, 2005
© 2005 American Association for Cancer Research
Estrogen Receptor 
and ß are Prognostic Factors in Non–Small Cell Lung Cancer
Hideki Kawai1,
Akira Ishii2,
Kiyotada Washiya2,
Toshiko Konno2,
Hiroto Kon2,
Chiharu Yamaya2,
Iwao Ono2,
Yoshihiro Minamiya1 and
Junichi Ogawa1
Authors' Affiliations: 1 Division of Thoracic Surgery, Department of Surgery, Akita University School of Medicine, and 2 Department of Pathology, Nakadori General Hospital, Akita, Japan
Requests for reprints: Hideki Kawai, Division of Thoracic Surgery, Department of Surgery, Akita University School of Medicine, 1-1-1 Hondo Akita 010-8543, Japan. Phone: 81-18-884-6132; Fax: 81-18-836-2615; E-mail: hkawai{at}doc.med.akita-u.ac.jp.
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Abstract
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Purpose: Estrogen receptor-
(ER-) and -ß (ER-ß) play important roles in the carcinogenesis of breast tumors. Similarly, there have been several reports of ER expression in lung cancers, but the results have not been consistent, and the receptors' prognostic value remains unclear. Our goal was to investigate ER expression in non–small cell lung cancer (NSCLC) and to assess whether their expression correlates with prognosis.
Experimental Design: ER expression was examined using immunohistochemical methods with sections from 132 resected NSCLC specimens. Kaplan-Meier survival curves were analyzed to determine the significance of ER expression in the prognosis of NSCLC patients.
Results: ER- was detected in the cytoplasm of 73% of the specimens analyzed, whereas ER-ß was detected in the nucleus of 51%. ER- expression correlated with poorer overall survival (P<0.001), as did the absence of ER-ß expression (P = 0.048). Likewise, at histopathologic stage I, ER- expression (P = 0.028) or the absence of ER-ß (P = 0.037) correlated with a poorer prognosis, and ER-(+)ER-ß(–) patients had a significantly worse prognosis than ER-(–)ER-ß(+) patients (P = 0.00007). Multivariate Cox regression analysis revealed the absence of ER-ß to be an independent factor predictive of poor disease outcome (hazard ratio, 1.9; 95% confidence interval, 1.1-3.4; P = 0.0264).
Conclusions: ER- expression and the absence of ER-ß expression are associated with a poorer prognosis among NSCLC patients. In particular, the absence of ER-ß could serve as a marker identifying patients at high risk even at an early clinical stage.
Lung cancer is now the leading cause of cancer death throughout the world. Despite recent improvements in its treatment, the prognosis for lung cancer patients remains poor. The pathologic staging of non–small cell lung cancer (NSCLC) is a key determinant of the patient's prognosis and the treatment options. For instance, in Japan, the 5-year survival rate after surgery at stage IA is 
80%, whereas the 5-year survival rate following surgery at stage IIIA is only 30%. Nevertheless, we have often encountered patients in whom the course of their disease differed substantially from what would be predicted based on their clinical staging, which highlights the need to consider additional predictive factors.
A focus on estrogen receptors (ER) as targets of hormonal therapy has played a significant role in the treatment of breast cancer; in general, the prognosis of patients expressing ERs is better than that of patients not expressing them (1, 2). There have been several studies examining ER expression in lung cancers, but their results remain inconclusive, and the relationship between ER expression and prognosis remains unclear (3–12). Some clinical analyses have shown the incidence of ER expression to be as high as 90% among lung cancer patients (3, 4), whereas others detected little or no ER expression (5–12). At the cellular level, Stabile et al. observed that lung cancer cells express both ER- and ER-ß; the former is localized in the cytoplasm, the latter in the nucleus (13). The purpose of the present study was to determine whether ER expression influences the prognosis of NSCLC patients and to evaluate the role of the interaction between ER- and ER-ß in these patients.
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Patients and Methods
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Patients. This study was approved by the Human Ethics Committee of Nakadori General Hospital, Akita, Japan. The study participants included 76 males and 56 females, ranging in age from 38 to 81 years (median, 66 years), that were diagnosed with NSCLC in Nakadori General Hospital between 1995 and 1997. The clinical features of these patients are summarized in Table 1.
Immunohistologic staining. Specimens of resected lung cancer tissue obtained from surgical treatment were fixed in formalin, embedded in paraffin and cut into 5-µm sections. The sections were then deparaffinized in xylene and ethanol, placed in 0.1 mol/L citrate buffer (pH 6.0) and irradiated with microwaves (750 W) for 15 minutes. The primary antibodies used were rabbit polyclonal anti-ER- (HC-20, Santa Cruz Biotechnology, Santa Cruz, CA; 1:50 dilution in PBS) or anti-ER-ß (H-150, Santa Cruz Biotechnology; 1:10 dilution in PBS) antibody. The immunostaining was carried out using the EnVision method (DAKO, Kyoto, Japan) according to the manufacturer's instructions. Brown staining was considered positive. The positive control for both ER- and ER-ß was breast cancer tissue; the negative control consisted of using PBS instead of primary antibody. All controls gave satisfactory results. Staining was categorized into eight grades according to previously described immunohistochemical scores (14–16). Briefly, each slide was examined in its entirety under a light microscope, and initially a proportion score was assigned, which represented the estimated proportion of positive tumor cells (0, none; 1, <1/100; 2, 1/100 to 1/10; 3, 1/10 to 1/3; 4, 1/3 to 2/3; and 5, >2/3). Next, an intensity score was assigned, which represented the average intensity of the positive tumor cells (0, none; 1, weak; 2, intermediate; and 3, strong). The proportion and intensity scores were then added to obtain a total score, which ranged from 0 to 8. Slides were scored by pathologists with no knowledge of the ligand-binding results or patient outcomes.
Statistical analysis. Two groups were compared using the 
2 test. The probabilities of overall survival were calculated using the Kaplan-Meier method and compared using the log-rank test. For determination of factors related to overall survival, a Cox proportional hazard model was used. These analyses yielded hazard ratios, their 95% confidence intervals and P values. Values of P < 0.05 were considered significant.
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Results
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Expression of ER- and ER-ß. Figure 1 shows representative immunohistochemical staining of ER- in the cytoplasm of NSCLC cells (Fig. 1A and B) and of ER-ß in the nucleus (Fig. 1C and D). Ninety-six (73%) of the tumors tested were ER--positive, whereas 67 (51%) were ER-ß-positive. ER- expression occurred significantly more frequently among moderate or poorly differentiated carcinomas than among well differentiated ones (P = 0.017). On the other hand, the incidence of ER-ß expression was significantly greater among squamous cell carcinomas than adenocarcinomas (P = 0.033). There was no correlation between the expression of ER- or ER-ß and any of the clinical variables listed in Table 1.
Clinical outcome. The overall Kaplan-Meier survival curves for ER- and ER-ß expression are shown in Fig. 2. Univariate analysis revealed expression of ER- to be linked to poor overall survival (P < 0.001; Fig. 2A), whereas expression of ER-ß was associated with better overall survival (P = 0.048; Fig. 2B). The immunohistochemical scores and the correlation between the expression of ER- and ER-ß in NSCLC tumors are shown in Tables 2 and 3, respectively. Univariate analysis showed that ER-(+)ER-ß(–) patients had a significantly poorer prognosis than ER-(–)ER-ß(+) patients (P = 0.00007; Fig. 3). Moreover, at histopathologic stage I, patients whose cancer received an ER- immunohistochemical score of 5 to 8 (Ia, n = 23; Ib, n = 10) had a significantly poorer prognosis than those whose cancers received a score of 0 to 4 (Ia, n = 23; Ib, n = 10; P = 0.028; Fig. 2C). Conversely, patients whose cancer received an ER-ß immunohistochemical score of 5 to 8 (Ia, n = 21; Ib, n = 7) had a significantly better prognosis than those whose cancer received a score of 0 to 4 (Ia, n = 26; Ib, n = 13; P = 0.037; Fig. 2D). There was no significant difference between patients with ER- or ER-ß immunohistochemical scores of 0 to 4 and 5 to 8 at histopathologic stages II or III (data not shown).
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Fig. 3. Kaplan-Meier analysis showing overall survival among NLCLC patient subtypes defined by ER immunohistochemistry. P values were calculated using the log-rank test.
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Multivariate analysis of overall survival. We then carried out a multivariate analysis of overall survival using the Cox proportional hazards model to determine whether the prognostic value of ER- or ER-ß expression persisted even when other prognostic factors were considered. Of the factors listed in Table 4, histologic grade, stage, and ER-ß were found to be significant independent variables that correlated with overall survival (P = 0.0107, P < 0.001, and P = 0.0264, respectively). The ER-ß-negative value for overall survival yielded a hazard ratio of 1.9, with a 95% confidence interval ranging from 1.1 to 3.4.
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Table 4. Univariate and multivariate Cox proportional hazard analysis for overall survival of 132 patients with NSCLC
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Discussion
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Although there have been a number of studies investigating the expression of ERs in lung cancer, the results remain inconclusive, and the relationship between ER expression and prognosis remains unclear (3–13). Using monoclonal and polyclonal anti-ER antibodies, Stabile et al. confirmed ER expression in lung cancer cells, identifying ER- and ER-ß in the cytoplasm and nucleus, respectively (13). Our findings obtained using polyclonal antibodies against the COOH terminus of the ERs are consistent with those earlier findings, but we observed no positive staining when we used a monoclonal antibody against the NH2 terminus of ER- (1D5, DAKO; data not shown). This suggests that the ER- isoform localized in the cytoplasm of lung cancer cells is a variant lacking part of the NH2 terminus (17–19), which is noteworthy, as the expression of such an ER- variant in hepatocellular carcinomas is indicative of extremely aggressive tumors that are unresponsive to the estrogen antagonist tamoxifen (20).
The other main question addressed in this study was whether expression of ER- or ER-ß is a prognostic factor in lung cancer, and whether that prognosis is affected by an interaction between the two receptors. In breast cancer, expression of ER- is generally indicative of a better prognosis (1, 2), which we anticipated would be the case in lung cancer as well. Our univariate analysis showed the exact opposite, however, which may mean that the ER- variant expressed in the cytoplasm of lung cancer cells contributes to the aggressiveness of the tumor, as it does in hepatocellular carcinomas. Because the number of patients negative for ER- was relatively small in the present study, ER- could not be shown to be an independent prognostic factor of overall survival in lung cancer in our multivariate analysis. ER-ß, by contrast, was found to be an independent prognostic factor of overall survival. Similarly, our results suggest that the expression of ERs is an important prognostic factor for NSCLC at histopathologic stage I.
Evidence suggests that in breast cancer cells, ER-ß may bind to ER-, thereby inhibiting its activity (21–24). Although our data reflect the opposing actions of ER- and ER-ß, their respective localizations within cells differ, suggesting that if ER-ß acts to inhibit ER- function, it works in some way other than through direct binding.
Recently, Fasco et al. reported that the expression of ERs in lung cancer is gender-dependent, and that ER- expression occurs more often in the lungs of women than men (25). We found no such distinction, although the number of women smokers in our sample was small.
At present, there are few therapeutic options available for lung cancer patients. Our findings suggest that hormone therapy may be a useful new strategy for the treatment of lung cancer, although further study is needed to determine the sensitivity of the cytoplasmic ER- variant to such therapy, and to determine the mechanism by which ER-ß inhibits the ER- variant in these cells.
In summary, expression of ER- and the absence of ER-ß expression were each associated with poor prognosis. In particular, the absence of ER-ß was found to be an important indicator that could serve as a marker identifying patients at high risk even at an early clinical stage.
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Footnotes
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Grant support: Nakadori General Hospital.
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 1/27/05;
revised 4/ 5/05;
accepted 4/22/05.
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References
|
|---|
- Osborne CK, Yochmowitz MG, Knight WA III, McGuire WL. The value of estrogen and progesterone receptors in the treatment of breast cancer. Cancer 1980;46:2884–8.[CrossRef][Medline]
- Thorpe SM, Rose C, Rasmussen BB, Mouridsen HT, Bayer T, Keiding N. Prognostic value of steroid hormone receptors: multivariate analysis of systemically untreated patients with node negative primary breast cancer. Cancer Res 1987;47:6126–33.[Abstract/Free Full Text]
- Canver CC, Memoli VA, Vanderveer PL, et al. Sex hormone receptors in non-small-cell lung cancer in human beings. J Thorac Cardiovasc Surg 1994;108:153–7.[Abstract/Free Full Text]
- Mollerup S, Jorgensen K, Berge G, Haugen A. Expression of estrogen receptors and ß in human lung tissue and cell lines. Lung Cancer 2002;37:153–9.[CrossRef][Medline]
- Nunno LD, Larsson LG, Rinehart JJ, Beissner RS. Estrogen and progesterone receptor in non-small cell lung cancer in 248 consecutive patients who underwent surgical resection. Arch Pathol Lab Med 2000;124:1467–70.[Medline]
- Su JM, Hsu HK, Chang H, et al. Expression of estrogen and progesterone receptors in non-small-cell lung cancer: Immunohistochemical study. Anticancer Res 1996;16:3803–6.[Medline]
- Beattie CW, Hansen NW, Thomas PA. Steroid receptors in human lung cancer. Cancer Res 1985;45:4206–14.[Abstract/Free Full Text]
- Cagle PT, Mody DR, Schwartz MR. Estrogen and progesterone receptors in bronchogenic carcinoma. Cancer Res 1990;50:6632–5.[Abstract/Free Full Text]
- Ollayos CW, Riordan GP, Rushin JM. Estrogen receptor detection in paraffin sections of adenocarcinoma of the colon, pancreas, and lung. Arch Pathol Lab Med 1994;118:630–2.[Medline]
- Chaudhuri PK, Thomas PA, Walker MJ, et al. Steroid receptors in human lung-cancer cytosols. Cancer Lett 1982;16:327–32.[CrossRef][Medline]
- Kobayashi S, Mizuno T, Tobioka N, et al. Sex-steroid receptors in diverse human tumors. Gann 1982;73:439–45.[Medline]
- Kaiser U, Hofmann J, Schilli M, et al. Steroid-hormone receptors in cell lines and tumor biopsies of human lung cancer. Int J Cancer 1996;67:357–64.[CrossRef][Medline]
- Stabile LP, Davis ALG, Gubish CT, et al. Human non-small cell lung tumors and cells derived from normal lung express both estrogen receptor and ß and show biological responses to estrogen. Cancer Res 2002;62:2141–50.[Abstract/Free Full Text]
- Harvey JM, Clark GM, Osborne CK, Allred DC. Estrogen receptor status by immunohistochemistry is superior to the ligand-binding assay for predicting response to adjuvant endocrine therapy in breast cancer. J Clin Oncol 1999;17:1474–81.[Abstract/Free Full Text]
- Allred DC, Harvey JM, Berardo MD, et al. Prognostic and predictive factors in breast cancer by immunohistochemical analysis. Mod Pathol 1998;11:155–68.[Medline]
- Allred DC, Clark GM, Elledge R, et al. Association of p53 protein expression with tumor cell proliferation rate and clinical outcome in node-negative breast cancer. J Natl Cancer Inst 1993;85:200–6.[Abstract/Free Full Text]
- Jazaeri O, Shupnik MA, Jazaeri AA, Rice LW. Expression of estrogen receptor mRNA and protein variants in human endometrial carcinoma. Gynecol Oncol 1999;74:38–47.[CrossRef][Medline]
- Huang A, Leygue E, Dotzlaw H, Murphy LC, Watson PH. Influence of estrogen receptor variants on the determination of ER status in human breast cancer. Breast Cancer Res Treat 1999;58:219–25.[Medline]
- Murphy LC, Cherlet T, Lewis A, Banu Y, Watson PH. New insights into estrogen receptor function in human breast cancer. Ann Med 2003;35:614–31.[CrossRef][Medline]
- Villa E, Colantoni A, Grottola A, et al. Variant estrogen receptors and their role in liver disease. Mol Cell Endocrinol 2002;193:65–9.[CrossRef][Medline]
- Liu MM, Albanese C, Anderson CM, et al. Opposing action of estrogen receptors and ß on cyclin D1 gene expression. J Biol Chem 2002;277:24353–60.[Abstract/Free Full Text]
- Schultz JR, Loven MA, Melvin VMS, et al. Differential modulation of DNA conformation by estrogen receptors and ß. J Biol Chem 2002;277:8702–7.[Abstract/Free Full Text]
- Hayashi S, Eguchi H, Tanimoto K, et al. The expression and function of estrogen receptor and ß in human breast cancer and its clinical application. Endocr Relat Cancer 2003;10:193–202.[Abstract]
- Omoto Y, Eguchi H, Yamamoto Y, Hayashi S. Estrogen receptor (ER) ß1 and ERßcx/ß2 inhibit ER function differently in breast cancer cell line MCF-7. Oncogene 2003;22:5011–20.[CrossRef][Medline]
- Fasco MJ, Hurteau GJ, Spivack SD. Gender-dependent expression of and ß estrogen receptors in human nontumor and tumor lung tissue. Mol Cell Endocrinol 2002;188:125–40.[CrossRef][Medline]
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Abstract
Patients and Methods
