ANTICANCER RESEARCH 24: 2919-2924 (2004) Expression of Estrogen Receptor-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 uterus (2), whereas ER-B is metaplasia (BM), express estrogen receptor 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 Tamoxifen, 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 antiestrogens. 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 steroid 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 estradiol 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, estrogen receptor alpha (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: Estrogen receptor beta, esophagus cancer, Barrett’s unpaired t-test) was performed utilizing InStat version 3.0a metaplasia. software (GraphPad Software, San Diego, CA, USA). 0250-7005/2004 $2.00+.40 2919 ANTICANCER RESEARCH 24: 2919-2924 (2004) 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 (endothelium) 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).