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ANTICANCER RESEARCH 24: 2919-2924 (2004)

Expression of -Beta Isoforms in Barrett’s Metaplasia, Dysplasia and Esophageal Adenocarcinoma

LIANG LIU, MINNI CHIRALA and MAMOUN YOUNES

Departments of Pathology, Baylor College of Medicine and The Methodist Hospital, Houston, TX 77030, U.S.A.

Abstract. We have previously shown that the majority of and beta (ER-B). ER-A is mainly expressed in female sex esophageal adenocarcinomas (EA), and its precursor Barrett’s organs, such as breast and (2), whereas ER-B is metaplasia (BM), express beta (ER-B). Several expressed in both sex organs and other tissues, such as isoforms of ER-B have been described and are presumed to have prostate, lung, thyroid, adrenal cortex and testis (3). Several different functions, but their distribution in BM and EA is not ER-B isoforms have been identified and characterized in known. The aim of this work was to determine which ER-B many non-gynecologic tumors including carcinomas of the isoforms are expressed in EA and BM. Sections of formalin-fixed lung (4), prostate (5), colon (6, 7) and stomach (8). and paraffin-embedded esophageal tissue from 33 esophageactomy , a specific ER-B antagonist, has been successfully specimens, of which 27 had invasive EA, were stained for the ER- used in the treatment of patients with breast carcinoma B isoforms ER-B1, ER-B2, ER-B3 and ER-B5 utilizing the (9,10) and it has been shown that ER-B status is a significant immunoperoxidase method. ER-B1 was detected in 23 out of 27 predictor of survival in women with breast carcinoma treated (85%) EA compared to 3 out of 14 (21%) Barrett’s metaplasia with mastectomy and adjuvant tamoxifen (11). negative for dysplasia (BMND) (p=0.0001); ER-B2 was expressed The incidence of esophageal adenocarcinoma has been rising in 22 out of 27 (81%) EA in contrast to 3 out of 14 (21%) BMND in the United States and Western Europe since the 1970s (12). (p=0.0004); ER-B3 was positive in 27 out of 27 (100%) EA in Despite advances in cancer therapy, mortality of this cancer contrast to only 1 out of 14 (7%) BMND (p<0001); ER-B5 was remains high with an overall 2-year survival of only 20% detected in 27 out of 27 (100%) EA compared to 9 out of 14 (12,13). We recently showed that ER-B is expressed in Barrett’s (62%) of BMND (p=0.0027). High- and low-grade dysplasia metaplasia and associated esophageal adenocarcinoma (14), showed a similar ER-beta isoform expression profile to that of EA. raising the possibility that EA may respond to treatment with Cancers invasive through the esophageal wall had a higher percent . The purpose of this study was to determine which of cells with cytoplasmic expression of ER-B1 than tumors limited ER-B isoforms are expressed in esophageal adenocarcinomas. to the wall (T3 vs. T1 and T2, p=0.051). We conclude that ER- B1, ER-B2, ER-B3 and ER-B5 are overexpressed in EA compared Materials and Methods to its precursor lesion BMND, suggesting a significant biological role for hormones in EA. This study was approved by the Institutional Review Board for Baylor College of Medicine and Affiliated Hospitals. Estrogen receptors (ER) are intracellular protein receptors Sections of formalin-fixed and paraffin-embedded tissue from activated by to initiate serial reactions associated 32 randomly selected esophagectomy cases were used in this study. Of these cases, 27 had esophageal adenocarcinoma (EA), 11 high- with cell differentiation and proliferation (1). ERs are grade dysplasia (HGD), 14 low-grade dysplasia (LGD) and 14 of classified into two subtypes, (ER-A) Barrett’s metaplasia negative for dysplasia (BMND). The expression of ER-B isoforms was determined by immunoperoxidase staining as we have recently described (15), utilizing a DAKO automated immunostainer (DAKO Corporation, Correspondence to: Mamoun Younes, M.D., Department of Carpinteria, CA, USA). The tissue sections were evaluated for ER- Pathology, Baylor College of Medicine, One Baylor Plaza, B isoform immunoreactivity, which was scored as follows: 0 (0%), Houston, TX 77030, U.S.A. Tel: (713) 394-6485, Fax: (713) 793- 1(1% to 10%), 2 (11% to 25%), 3 (26% to 50%), 4 (51% to 75%) 1603, e-mail: [email protected] and 5 (>75%). Nuclear and cytoplasmic immunoreactivity were determined separately. Statistical analysis (Fisher’s exact test and Key Words: , esophagus cancer, Barrett’s unpaired t-test) was performed utilizing InStat version 3.0a metaplasia. software (GraphPad Software, San Diego, CA, USA).

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Table I. Expression of estrogen receptor beta isoforms 1, 2, 3 and 5 in Endothelial cells and nerves were always positive for ER- esophageal adenoarcinoma (EA), high-grade dysplasia (HGD), low-grade B2, as reported previously in breast tissues (15). dysplasia (LGD) and Barrett’s metaplasia negative for dysplasia (BMND). ER-B3 was detected in 100% of EA (27/27), 91% of HGD EA HGD LGD BMND p (EA vs. (10/11), 93% of LGD (13/14) but only 7% (1/14) in BMND, BMND) and it showed more nuclear (52%) than cytoplasmic (22%) distribution in cases of EA. Although many mitotic figures ER-B1 85% 73% 57% 21% 0.0001 were positive for ER-B3, not all mitoses were positive, and (23/27) (8/11) (8/14) (3/14) some nuclei that were not in mitosis were also positive. There ER-B2 81% 73% 50% 21% 0.0004 was no significant correlation between the percent of ER-B3- (22/27) (8/11) (7/14) (3/14) positive cancer cells and the number of mitotic figures per 10 high power microscopic fields (proliferative activity). ER-B3 100% 91% 93% 7% <0.0001 ER-B5 was expressed in 100% of EA (27/27), 100% of (27/27) (10/11) (13/14) (1/14) HGD (11/11), 93% of LGD (13/14) and in 64% of BMND ER-B5 100% 100% 93% 62% 0.0027 (9/14). Unlike the other isoforms, ER-B5 showed more (27/27) (11/11) (13/14) (9/14) nuclear pattern of expression (52%) and less cytoplasmic pattern (4%) in cases of EA, and was negative in mitosis. ER-B1, ER-B2, ER-B3 and ER-B5 were expressed in significantly more cases of EA than in its precursor lesion Results BMND (p=0.0001, p=0.0004, p<0001and p=0.0027, respectively), suggesting a significant role for steroid ER-B isoforms immunoreactivity in esophageal hormones in the development and maintenance of EA. adenocarcinoma (EA), high-grade dysplasia (HGD), low- In all cases of BMND, all isoforms showed only nuclear grade dysplasia (LGD) and BMND is detailed in Table I and expression, whereas cytoplasmic expression was seen in the intracellulr distribution of these isoforms in the positive dysplasia and adenocarcinoma (Table II). cases is shown in Table II and Figure 1. The percentage of There was no correlation between the expression of any of ER-B-positive cells in EA is detailed in Figure 2. the ER-B isoforms and tumor proliferative activity, ER-B1 is expressed in 23 out of 27 (85%) EA, 8 out of 11 determined as the number of mitotic figures in 10 consecutive (73%) HGD, 8 out of 14 (57%) LGD and 3 out of 14 (21%) high-power microscopic fields, or with lymph node metastasis. BMND. Slightly more cancers showed cytoplasmic ER-B1 Tumors invasive through the wall of the esophagus (pT3) had immunoreactivity (44%) than nuclear immunoreactivity a higher cytoplasmic ER-B1 score (mean 3.4, median score (30%). ER-B1 was not expressed in any mitotic figures. 4) than tumors limited to the wall (pT1 and pT2, mean 1.7, Occasional stromal cells and rare blood vessels () median 0) and the difference almost reached statistical were positive for ER-B1, but nerves were always negative. significance (p=0.051, unpaired t-test, two-tailed). ER-B2 was positive in 22 out of 27 (81%) EA, 8 out of 11 (73%) HGD, 7 out of 14 (50%) LGD and 3 out of 14 Discussion (21%) BMND. In EA, ER-B2 immunoreactivity was cytoplasmic in most cases (73%) and nuclear in only a few The incidence of esophageal adenocarcinoma (EA) in the (9%). ER-B2 was not detected in any mitotic figures. United States has been steadily increasing since the mid

Table II. Differential expression of estrogen receptor beta isoforms 1, 2, 3 and 5 in positive cases of adenoarcinoma (EA), high-grade dysplasia (HGD), low-grade dysplasia (LGD) and Barrett’s esophagus negative for dysplasia (BMND).

EA HGD LGD BMND

N1 C2 N&C3 N C N&C N C N&C N C N&C

ER-B1 30% 44% 26% 75% 12.5% 12.5% 75% 0 25% 100% 0 0 ER-B2 9% 73% 18% 62.5% 25% 12.5% 71% 29% 0 100% 0 0 ER-B3 52% 22% 26% 80% 10% 10% 77% 15% 8% 100% 0 0 ER-B5 52% 4% 44% 100% 0 0 100% 0 0 100% 0 0

1. N: nuclear only 2. C: cytoplasmic only 3. N&C: both nuclear and cytoplasmic

2920 Liu et al: Estrogen Receptor Beta Isoforms in Barrett’s and Esophageal Adenocarcinoma

Figure 1. Examples of immunohistochemical staining for ER-B isoforms in esophageal adenocarcinoma: (A) nuclear staining, (B) cytoplasmic staining, (C) nuclear and cytoplasmic staining and (D) staining in mitosis, seen only in some cases with ER-B3.

only 25% of patients surviving 3 years after surgical resection (12). The precursor lesion of EA is Barrett’s metaplasia (BM), which is a complication of gastroesophageal reflux disease (GERD) (20). In order to detect EA at an early stage when surgical resection is associated with good prognosis (21-23), patients with BM are enrolled in endoscopic surveillance programs performed at intervals determined mostly by the grade of epithelial dysplasia in endoscopic biopsies (24-25). The risk of EA in BM increases with the grade of dysplasia. Patients with BM progress to EA through the successive appearance of LGD, followed by HGD and finally EA (21, 26, 27). Tamoxifen is a nonsteroidal drug with potent anti- estrogen properties that is believed to be due to its competition with estrogen for binding sites present in target Figure 2. Extent of ER-B isoforms expression in esophageal adenocarcinoma. tissues. Tamoxifen has been successfully used for over two decades in the treatment of estrogen receptor (ER)-positive , and more recently in chemoprevention of 1970 (12, 16-19), not only in White males but also in White breast cancer (28). Most of our current knowledge of the females and Hispanic males and females (12). Despite tissue distribution of estrogen receptors (ER) came from recent advances in the detection and treatment of human IHC studies utilizing monoclonal anti-ER antibodies. Based cancers, the mortality of EA remains significantly high with on these studies, it was believed that, with rare exception,

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ER expression is limited to female sex-related organs (such especially the case with ER-B2. We have previously observed as breast and uterus) and cancers arising in these organs. In that in the normal colonic ER-B expression was gastrointestinal cancers, especially cancers of the stomach limited to the nucleus, whereas cytoplasmic ER-B was and colon, there were no good explanation for why estrogen expressed only in colon cancer, although not in all cases, receptors can be demonstrated in these cancers utilizing the where it had a tendency to be present mostly in the invasive biochemical assay while IHC studies generally failed to part of the cancer (6). We speculated then that perhaps there demonstrate ER positivity (29-37). Interestingly, estrogen is a different level of expression of ER-B isoforms between "binding sites" have been previously detected in human normal colonic epithelium and colon cancer where certain colon cancer cell lines and tumors (38), and estradiol was isoforms with predominantly cytoplasmic immunoreactivity found to activate the growth of colon cancer cell lines in are associated with the malignant phenotype (6). ER-B5 vitro (39,40), suggesting that an as yet unrecognized type of shows mostly nuclear localization, and is the most widely estrogen receptors is expressed in gastrointestinal cancers. expressed isoform in BMND, in contrast to ER-B2, which Prior to 2002, there were no published papers on ER shows mostly cytoplasmic immunoreactivity, and is mostly expression in human esophagus tissue, BM, or EA. present in the cancers and rarely in BMND. ER-B1 A new type of estrogen receptor has been discovered and cytoplasmic immunoreactivity appears to be most prominent called estrogen receptor beta (ER-B) (41-44). This receptor in the most invasive EAs. has several isoforms, and appears to have a wide tissue Finally, and similar to what we have previously reported distribution at the mRNA level (45). The traditional ER, in breast cancer tissue (15), we found ER-B2 to be the detected by IHC in all previous studies, is now called only isoform detected in nerve fibers and the most estrogen receptor alpha (ER-A). ER-B has been identified prominently and consistently expressed in endothelial in several human cancer types that were traditionally known cells. This is may have implications for future drug to be ER-negative based on previous assays using ER-A- development whereby ER-B1- or ER-B5-specific agonists specific antibodies, including carcinomas of the colon, or antagonists would not be expected to have significant esophagus, stomach, lung and prostate (4-8, 14, 46-48). neurological effects, or may have no significant vascular While tamoxifen acts as partial agonist on ER-A, it has effects. An ER-B2-specific agonist or antagonist, on the been suggested to have a pure antagonist effect on ER-B other hand, is likely to have a significant neurological and (49). We have previously shown that, in women with breast vascular action. cancer treated with mastectomy and adjuvant hormonal In conclusion, our results show that ER-B isoforms are therapy with tamoxifen, ER-B expression determined by overexpressed in EA, compared to BMND, suggesting a IHC was a significant predictor of survival (9,50). This possible role for antiestrogens in the treatment of EA which indicates that ER-B expression in human cancer tissue, deserves further investigation. The finding of ER-B isoform detected by IHC, predicts response to treatment with expression in LGD and HGD at a frequency similar to that tamoxifen. Furthermore, tamoxifen was found to inhibit observed in EA suggests a possible role for antiestrogens in growth and induce apoptosis in cultured human colon the chemoprevention of EA, which is currently being cancer cell lines in a dose-dependent manner through its explored by our group. action on ER-B (51-55). Utilizing the IHC method, and the same anti-ER-B Acknowledgements antibody used in our previous studies on human breast cancer tissue (9,50), we found ER-B to be expressed in EA, This study was supported by National Institutes of Health grant BMND, LGD and HGD (14), suggesting a potential role for R01 CA81570-01A2. antiestrogens, such as tamoxifen, in the treatment and/or Presented in part at Digestive Diseases Week and the 104th chemoprevention of EA. Annual Meting of the American Gastroenterological Association, Utilizing affinity-purified antibodies to ER-B isoforms 1, May 17-22,2003, Orlando, FL, USA. 2, 3 and 5, two major differences appeared between EA and its precursor lesion BMND. The first difference is that all References isoforms are expressed in significantly more cases, and there is a higher percentage of positive cells per case, in EA as 1 Greene GL, Sobel NB, King WJ and Jensen EV: compared to BMND, indicating a significant role for ER-B Immunochemical studies of estrogen receptors. J Steroid isoforms in the maintenance, and possibly the evolution, of Biochem 20: 51-56, 1984. 2 Enmark E and Gustafsson JA: Oestrogen receptors - an EA. The second difference is in the localization of the ER-B overview. J Intern Med 246: 133-138, 1999. immunoreactivity. In BMND, ER-B isoforms are detected only 3 Taylor AH and Al-Azzawi F: Immunolocalisation of oestrogen in the nuclei of BM cells. By contrast, a significant number of receptor beta in human tissues. J Mol Endocrinol 24: 145-155, carcinomas show cytoplasmic ER-B immunoreactivity; this is 2000.

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