Abd Elaziz, Amany M.a; Mounir, Bahaa I.b; El-Naggar, Samia I.a; Darweesh, Mohamed F.b
Colonic adenocarcinoma is the most frequent neoplasia of the intestine. This pathology is the third highest cause of death from cancer, with 430 000 deaths globally per year (Huo et al., 2009).
Colonic adenocarcinoma is one of the most common malignancies in industrialized countries, and 875 000 or more people are diagnosed with colonic adenocarcinoma annually (Castiglione et al., 2008).
The large majority of colorectal malignancies develop from an adenomatous polyp (adenoma). These can be defined as well-demarcated masses of epithelial dysplasia, with uncontrolled crypt cell proliferation. When neoplastic cells pass through the muscularis mucosa and infiltrate the submucosa, they are considered as malignant (Di Leo et al., 2008).
Adenocarcinomas usually originate from pre-existing adenomas, but this does not imply that all polyps undergo malignant changes and does not exclude de-novo oncogenesis (Tanaka, 2009).
The prognosis for patients with colonic adenocarcinoma is heavily dependent on the stage at diagnosis, and almost all patients require surgical resection. The 5-year survival rate is more than 90% for Dukes’ stage A, but only 5% for Dukes’ stage D. Therefore, early diagnosis and prevention against colonic adenocarcinoma is extremely important (Mostafaie et al., 2009).
Estrogen receptor (ER)-β is the predominant ER in the colonic epithelium, suggesting that effects of estrogen in the colon are mediated by ER-β (Deroo and Korach, 2006). ER-β plays a physiological role in colonic tissue. Crypt shape and number of cells per crypt-villus unit. ER-β is also important for the maintenance of quiescence of hematopoietic stem cells (Barone et al., 2008).
ER-β might reduce cell proliferation by inhibiting the cyclin D1 gene, a key factor controlling the G1 to S transition of the cell cycle, and thus cell proliferation.
ER-β-induced inhibition of proliferation could be explained by the inhibition of the bcl-2 gene (Pietras and Márquez-Garbán, 2007).
Immunohistochemical staining showed prominent expression of ER-β in colonic superficial epithelium, and this led us to consider that ER-β could play a significant role in differentiation (Zhao et al., 2008).
ER-β is the predominant ER form in malignant human colon tissue and several human colon cancer-derived cell lines. ER-β protein levels are reportedly lower in colon tumors compared with normal colon tissue, and loss of ER-β is associated with advanced stages of colon cancer and tumor cell dedifferentiation, suggesting a protective role for ER-β in colon tumorigenesis (Nüssler et al., 2008).
Men are more susceptible than women to colon cancer. Furthermore, hormone replacement therapy (HRT) provides an additional protective effect for postmenopausal women, and when these women do develop cancer, they typically have less aggressive disease (Kennelly et al., 2008).
Data from the Women’s Health Initiative indicate a significantly reduced incidence of colon cancer in postmenopausal women receiving combined HRT (estrogen plus progestin), consistent with a protective role of ER-β (Deroo and Korach, 2006).
In vitro, raloxifene inhibits the proliferation and viability of colon cancer cell lines, which may indicate the value of selective ER modulators (tamoxifen and raloxifene), in the prevention and treatment of colon cancer (Janakiram et al., 2009).
Materials and methods
A total of 60 archival retrospective randomly selected formalin-fixed, paraffin-embedded blocks were obtained from 40 patients who underwent colectomy for colorectal cancer and 20 biopsies diagnosed as adenomatous polyps. Specimens were obtained from the Department of Pathology, Cairo University, during the period from September 2009 to August 2010. For all specimens, clinical data were available on computer files including age, sex, and size and site of the mass. Consent is unavailable as the materials of this study were archival paraffin blocks taken by code number (patients’ identities were unavailable).
All specimens were formalin-fixed, routinely processed, and embedded in paraffin. Two 5-μm-thick sections were prepared from each tissue block, one of them stained with haematoxylin and eosin for re-evaluation, including the tumor stage, which was classified according to the Dukes’ staging system (1932). Patterns of tumor growth were numbered in order of increasing malignancy as stages A, B, and C.
The other section was mounted on poly-L-lysine-coated slides (Superfrost slides) and subjected to ER-β immunohistochemical staining.
After routine deparaffinization in xylene, the sections were hydrated through a series of graded alcohols, distilled water and PBS at pH 7.2–7.4. Antigen retrieval was performed using Tris-EDTA (pH 9) in a pressure cooker. Slides were placed in Dako autostainer S3400 (Dako, New York, USA), which performed the rest of the steps as follows:
Incubation in 3% H2O2 for 5 min to inhibit the endogenous peroxidase activity.
Washing the slides with PBS at pH 7.2–7.4.
Incubation of slides with monoclonal rabbit antihuman ER-β diluted at 1 : 50 in Dako antibody diluent S3022 for 30 min at room temperature.
Washing the slides with PBS at pH 7.2–7.4.
Applying the EnVision Dual Link Kit (K5007, New York, USA) optimized for Dako cytomation automated systems for 30 min.
Washing the slides with PBS at pH 7.2–7.4.
Applying DAB (3,3′-di-amino-benzidinetetrahydrochloride) as the chromogen for 5 min.
Rinsing the slides well in distilled water for 5 min.
The slides in the autostainer were removed, and haematoxylin counterstaining was performed. Slides were dehydrated in ascending grades of alcohol and were cleared in xylene for three changes, and cover slips were applied.
Evaluation of estrogen receptor-β expression
Nuclear staining within the cell, whether weak or strong, was considered positive. Given previous evidence that ER-β is a nuclear-localized steroid receptor, only positive nuclear immunostaining was scored. Tumors were scored as positive for ER-β if 5% or more of the nuclei were stained by the antibody (in 10 HPFs) Xie et al. (2004).
Data were statistically described in terms of frequencies (the number of cases) and relative frequencies (%). The χ2-test was performed to compare the different study variables between the study groups. The Yates correction and the Fisher exact tests were used only when the expected frequency was found to be less than 5. A P value of less than 0.05 was considered statistically significant.
All statistical calculations were performed using Microsoft Excel version 7 (Microsoft Corporation, New York, USA) and statistical package for the social science (SPSS; SPSS Inc., Chicago, Illinois, USA) statistical program.
In the present retrospective study, 60 cases were studied: 40 adenocarcinomas and 20 adenomatous polyps. Clinicopathological features of the studied cases are shown in Table 1. The highest frequency of colonic adenocarcinoma cases (37.5%) occurred between 50 and 60 years of age, and the highest frequency of adenomatous polyps cases (40%) occurred at age 40 years or less in the current study. Most of the colonic adenocarcinoma cases (62.5%) occurred in men, whereas there was no difference with respect to sex in the case of the adenomatous polyps studied. Seventy-five percent of both colonic adenocarcinoma and adenomatous polyps cases studied occurred in rectosigmoid. Most of the colonic adenocarcinoma cases studied (75%) had a size in the range of 5–10 cm, whereas all adenomatous polyps cases studied (100%) had a size less than 5 cm. With regard to the histopathological grade of colonic adenocarcinoma (75%), the studied cases were well differentiated. Half of the studied cases of colonic adenocarcinoma (50%) were of modified Dukes’ stage B1. Most of the colonic adenocarcinoma cases studied (75%) were not associated with lymph node metastasis.
On comparing the immunohistochemical expressions of ER-β in colorectal carcinoma and adenoma, the results revealed that loss of ER-β expression was present in a larger number of adenocarcinoma cases (25 cases, 75%) than in adenomatous polyps cases (five cases, 25%) (Table 2).
Correlations of ER-β immunohistochemical expression with different clinicopathological features in the studied cases showed significant correlation between the site and the size distribution in the studied cases of colonic adenocarcinoma and adenomatous polyps. However, age and sex distribution in the studied cases of colonic adenocarcinoma and adenomatous polyps showed no significant correlation (Tables 3 and 4).
The highest frequency of positive ER-β expression in the studied cases of colonic adenocarcinoma occurred in well-differentiated tumors (25%) and in modified Dukes’ stage B1 (20%; P<0.05, significant) (Table 4). There was insignificant correlation between the expression of ER-β in the studied cases of colonic adenocarcinoma that occurred in tumors associated with positive lymph nodes (P>0.05) (Figs 1–8).
Colon cancer is the second most common cancer and the second most common cause of cancer death in the USA. In women, it is responsible for ∼11% of all new cancer cases and 11% of cancer deaths; in men, it is 10% for both measures (Weidner et al., 2009).
Estrogens, through the interaction with their receptors, play important roles in the control of cellular growth and differentiation. Specifically, estrogens regulate the growth and development of the mammary gland in embryogenesis as well as in prepubertal and postpubertal periods, and of ovarian follicles during the reproductive cycle. Although it was initially thought that estrogens mediate their action through a single receptor, the ER-α, a second ER, has been identified from a rat prostate library and termed ER-β (Acconcia and Marino, 2011).
ER-β seems to be essential for the maintenance of cellular homeostasis and for driving cellular differentiation in the colon. Cells of the colon are continuously renewed through a process initiated by stem cell division. The daughter cells produced differentiate and migrate from the bottom to the top of the crypt, lose the capacity to divide, and are shed within several days. Immunohistochemical staining showed prominent expression of ER-β in colonic superficial epithelium, prompting us to suggest that ER-β plays a significant role in differentiation. In the hyperproliferative state, a progressive accumulation of somatic mutations occurs in the stem cells, which have a long residence period in the mucosa. This cumulative damage may contribute to the development of a malignant phenotype that possesses a growth advantage over other stem cells within the crypt (Mostafaie et al., 2009).
In this work, evaluation of ER-β expression in 20 cases of colonic adenoma and 40 adenocarcinoma cases and its correlation with the clinicopathologic features of such cases was performed to determine the relation of ER-β expression with tumorigenesis and prognostic factors in colonic adenocarcinoma.
With regard to the age, the highest frequency of adenomatous polyps cases in this study (40%) occurred at age 40 years or less. Di Leo et al. (2008) demonstrated that the frequency of these tumors increases markedly after 40 years of age and reaches a peak at an age of 70 years.
The highest frequency of colonic adenocarcinoma cases (37.5%) in this study occurred between 50 and 60 years of age. While the peak incidence occurred between the 60 and 79 years of age, fewer than 20% of cancers occurred in patients less than 50 years of age according to the American Cancer Society study (2011). This difference could be explained by the prevalence of screening programs and early detection of colonic lesions in western communities, whereas it is difficult in our country because of its high cost and the higher proportion of elderly people in their population than in our country.
In the present study, the highest frequency of colonic adenocarcinoma cases (62.5%) occurred in men, whereas no difference was observed with respect to sex in the adenomatous polyps cases studied. This is demonstrated by the studies of Weidner et al. (2009), who found that colonic carcinoma occurs more often in men (male-to-female ratio, 3 : 2). In contrast, Acconcia and Marino (2011) demonstrated that colon cancer occurred at an approximately equal frequency between the sexes.
The highest frequency of colonic adenocarcinoma cases (75%) and highest frequency of adenomatous polyps cases (75%) in this study occurred in rectosigmoid. These results agree with Weidner et al. (2009), who reported that cancers of the left colon (distal to the splenic flexure) outnumber those of the right colon.
Most of the colonic adenocarcinoma cases studied (75%) had a size of 5–10 cm, whereas all adenomatous polyps cases studied (100%) had a size less than 5 cm; this result differs from those found by Weidner et al. (2009), who stated that most colonic adenocarcinomas are less than 5 cm, but agreed with us regarding the size of adenomas. We can explain this by the screening programs for the early detection of carcinoma in western communities, whereas in our country, patients delay in seeking medical advice.
Regarding the histopathological grading in the studied cases of colonic adenocarcinoma, most of the studied cases (75%) are well differentiated, keeping with the results of a study conducted by Jankowski et al. (2008).
Also, we found that half of the colonic adenocarcinoma cases studied (50%) were of modified Dukes’ stage B1. In contrast, the American Cancer Society study (2011) demonstrated that the most common stage was modified Dukes’ stage A. This could be explained by the success of screening programs in early detection of the tumor.
In our study, we found that most of the colonic adenocarcinoma cases studied (75%) were not associated with lymph nodes metastasis. This is in keeping with the results of a study conducted by Turner and Vollmer (2006).
Twenty-five (62.5%) of the colonic adenocarcinoma cases studied showed negative expression of ER-β, whereas five (25%) of the adenomatous polyps cases studied showed negative expression of ER-β. Our results are in line with those of Xie et al. (2004), Wada-Hiraike et al. (2006), and Koo and Leong (2010).
Regarding adenoma, Di Leo et al. (2008) demonstrated a significant reduction of ER-β expression in the precancerous phase of colon carcinogenesis (adenoma). This result suggested a role of selective ER-β agonists in the prevention of colorectal cancer. These results are keeping with our results in this study.
The correlation between the expression of ER-β and sex distribution in the studied cases of colonic adenocarcinoma and adenomatous polyps were not significant (P>0.05).
The correlation between the expression of ER-β and site distribution in the studied cases of colonic adenocarcinoma and adenomatous polyps were significant as the highest frequency of positive expression of ER-β in studied cases of colonic adenocarcinoma (20%) occurred in rectosigmoid; these results agree with those of Xie et al. (2004) and Di Leo et al. (2008).
The correlation between expression of ER-β and size of the tumor in the studied cases of colonic adenocarcinoma was significant, as the highest frequency of positive expression of ER-β in the studied cases of colonic adenocarcinoma (30%) occurred in tumors of size 5–10 cm; these results differ from those found by Wada-Hiraike et al. (2006).
Regarding the correlation between the expression of ER-β and the distribution of histopathological grading, we found that the highest frequency of positive expression of ER-β in the studied cases of colonic adenocarcinoma (25%) occurred in well-differentiated tumors (P<0.05, significant). Our results are in agreement with the results of the study of Xie et al. (2004).
The highest frequency of positive expression of ER-β in the studied cases of colonic adenocarcinoma (20%) occurred in modified Dukes’ stage B1 (P<0.05, significant), which is compatible with the study of Barone et al. (2010), who stated that ER-β expression is related to the severity of the disease, supporting the role of ER-β as a relevant biomarker of tumor progression. However, it differs from the results of the study of Xie et al. (2004), who also detected that the highest frequency of positive expression of ER-β in cases of colonic adenocarcinoma (66.7%) occurred in modified Dukes’ stage A, whereas the lowest frequency (57.5%) occurred in modified Dukes’ stage C.
The correlation between the expression of ER-β and lymph node involvement in the studied colonic adenocarcinoma cases was insignificant (P>0.05).
Conclusion and recommendations
ER-β may protect normal colonic epithelial cells from undergoing unscheduled cell proliferation and neoplastic transformation. ER-β expression is related to tumorigenesis and prognostic factors in colonic adenocarcinoma. ER-β expression decreases in adenomas and colonic adenocarcinoma. Further studies are recommended to prove the protective effect of estrogen HRT against colonic adenocarcinoma in high-risk patients.
Conflicts of interest
There are no conflicts of interest.
Acconcia F, Marino M.The effects of 17β-estradiol in cancer are mediated by estrogen receptor signaling at the plasma membrane.Front Physiol2011;2:30.
Barone M, Tanzi S, Lofano K, Scavo MP, Guido R, Demarinis L, et al..Estrogens, phytoestrogens and colorectal neoproliferative lesions.Genes Nutr2008;3:7–13.
Barone M, Tanzi S, Lofano K, et al..Dietary-induced ERβ upregulation counteracts intestinal neoplasia development in intact male ApcMin/+mice.Carcinogenesis2010;31:269–274.
Castiglione F, Taddei A, Degl’Innocenti DR, Buccoliero AM, Bechi P, Garbini F, et al..Expression of estrogen receptor β in colon cancer progression.Diagn Mol Pathol2008;17:231–236.
Deroo BJ, Korach KS.Estrogen receptors and human disease.J Clin Invest2006;116:561–570.
Di Leo A, Barone M, Maiorano E, Tanzi S, Piscitelli D, Marangi S, et al..ER-β expression in large bowel adenomas: implications in colon carcinogenesis.Dig Liver Dis2008;40:260–266.
Huo Q, Kinugasa T, Wang L, Huang J, Zhao J, Shibaguchi H, et al..Claudin-1 protein is a major factor involved in the tumorigenesis of colorectal cancer.Anticancer Res2009;29:851–858.
Janakiram NB, Steele VE, Rao CV.Estrogen receptor-β as a potential target for colon cancer prevention: chemoprevention of azoxymethane-induced colon carcinogenesis by raloxifene in F344 rats.Cancer Prev Res (Phila)2009;2:52–59.
Jankowski J, Sampliner R, Kerr D, Fong Y.Gastrointestinal oncology: a critical multidisciplinary team approach2008.Oxford, UK:Blackwell Publishing.
Kennelly R, Kavanagh DO, Hogan AM, Winter DC.Oestrogen and the colon: potential mechanisms for cancer prevention.Lancet Oncol2008;9:385–391.
Koo JH, Leong RWL.Sex differences in epidemiological, clinical and pathological characteristics of colorectal cancer.J Gastroenterol Hepatol2010;25:33–42.
Mostafaie N, Kállay E, Sauerzapf E, Bonner E, Kriwanek S, Cross HS, et al..Correlated downregulation of estrogen receptor beta and the circadian clock gene Per1 in human colorectal cancer.Mol Carcinog2009;48:642–647.
Nüssler NC, Reinbacher K, Shanny N, Schirmeier A, Glanemann M, Neuhaus P, et al..Sex-specific differences in the expression levels of estrogen receptor subtypes in colorectal cancer.Gend Med2008;5:209–217.
Pietras RJ, Márquez-Garbán DC.Membrane-associated estrogen receptor signaling pathways in human cancers.Clin Cancer Res2007;13:4672–4676.
Tanaka T.Colorectal carcinogenesis: Review of human and experimental animal studies.J Carcinog2009;8:5.
Turner J, Vollmer RT.Lymph nodes in colorectal carcinoma: the Poisson probability paradigm.Am J Clin Pathol2006;125:866–872.
Wada-Hiraike O, Imamov O, Hiraike H, Hultenby K, Schwend T, Omoto Y, et al..Role of estrogen receptor β in colonic epithelium.Proc Natl Acad Sci USA2006;103:2959–2964.
Weidner N, Cote RJ, Suster S, Weiss LM.Modern surgical pathology2009:2nd ed..Philadelphia, PA:Saunders.
Xie L-Q, Yu J-P, Luo H-S.Expression of estrogen receptor β in human colorectal cancer.World J Gastroenterol2004;10:214–217.
Zhao C, Dahlman-Wright K, Gustafsson JA.Estrogen receptor beta: an overview and update.Nucl Recept Signal2008;6:e003.