Expression of Estrogen Receptor-ß (ER-ß) in Bengin and Malignant Prostatic Epithelial Cells and its Correlation with the Clinico-Pathological Features

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1 Journal of the Egyptian Nat. Cancer Inst., Vol. 9, No., December: 9-8, Expression of Estrogen Receptor-ß () in Bengin and Malignant Prostatic Epithelial Cells and its Correlation with the Clinico-Pathological Features SAMIA M. GABAL, M.D.; FAHIMA M. HABIB, M.D.; DINA O. HELMY, M.D.; MOHAMMAD F. IBRAHIM, M.Sc. The Department of Pathology, Faculty of Medicine, Cairo University. ABSTRACT The role of estrogen and its receptors in the etiology and progression of prostate cancer (PC) is poorly understood. In normal and malignant human prostate, estrogen receptor-α is expressed only in the stroma, whereas estrogen receptor-ß () is present in both normal stroma and epithelium. Because loss of expression is associated with prostate hyperplasia in null mice, this study determined patterns of ERß expression in hyperplastic, PIN (prostatic intraepithelial neoplasia) and malignant human prostate and associations with tumor progression. Formalin-fixed, paraffin-embedded blocks were obtained from thirty-five patients who underwent radical prostatectomy and pelvic lymphadenectomy for prostate cancer, core biopsies diagnosed as PIN and TURP (transurethral prostatic resection) diagnosed as BPH (benign prostatic hyperplasia) were assessed for expression using immunohistochemistry. positivity was defined as % of cells demonstrating nuclear immunoreactivity. Nine (9%) of the studied BPH showed positive expression where they showed % positive cells, while eight (%) of the studied PIN were negative for expression. Twenty-nine (8.8%) of the studied adenocarcinoma were negative for ERß expression, p<.. The loss of expression is associated with progression from hyperplastic prostate epithelium to PC. The expression is inversely proportional to PCs stage and grade. From these data we can conclude that estrogen can affect prostatic cancerogenesis and neoplastic progression through an mediated process in human prostate tissue. Our data also identify the need for additional studies to address the potential role of ERß in the regulation of prostate epithelial cell proliferation at different stages in the development of PC. Key Words: ER ß Prostatic hyperplasia PIN Prostatic carcinoma. Correspondence: Dr Samia M. Gabal, Department of Pathology, Faculty of Medicine, Cairo University, ns_gabal@yahoo.com INTRODUCTION Prostatic carcinoma (PC) is the most frequent malignant tumor among men over years of age. Its incidence varies according to countries and ethnic groups []. It accounts for.% of all cancers in Egypt [] and the reported at the National Cancer Institute, Cairo University, during the years - were (9.8% of the male cancers during this period) []. The prostate is absolutely dependent on androgens to maintain its size and secretory function. Although the androgen-dependency of the prostate gland has long been known, the role of estrogens in the prostate and in human prostate carcinogenesis has been only recently recognized [,]. Epidemiological studies of circulating hormone levels in men from different ethnic groups have provided limited evidence for a link between PC risk and high circulating levels of estrogens, but nested case-control studies have failed to confirm this finding []. The diethylstilbestrol, a synthetic estrogen, is directly cytotoxic to both hormone-sensitive and hormone-insensitive PC cell lines, implying that ERs are important throughout PC progression. Estrogen signaling may represent an alternative mitogenic stimulus in androgenindependent PC and as such may represent a possible therapeutic target at a stage when the disease becomes largely refractory to therapy []. Estrogens are believed to play a significant role in the pathogenesis of prostate cancer (PCa) 9

2 [8]. The steroid hormone receptor, estrogen receptor (ER)-α, was thought to mediate all estrogen actions. However in 99, a second ER,, was identified and found to differ significantly from ER-α [9]. Unlike ER-α, which is the predominant ER in female reproductive organs, the ß-isotype is highly expressed in the male reproductive tract, including the prostate. Although the precise biological function of ERß is incompletely defined, it has been suggested that the receptor, acting through estrogens, may protect the normal prostate epithelium from undergoing unscheduled cell proliferation, neoplastic transformation and from oxidative injuries [,]. In mice, the absence of leads to failure of the prostatic epithelium to differentiate fully, whereas the epithelial cells continue to proliferate. If the is included as a factor in the endocrine control of prostate growth, we have androgen receptors causing proliferation and secretion and suppressing proliferation and promoting differentiation. Since proliferation and differentiation oppose each other, cellular homeostasis must be the result of a dynamic balance. Until recently, a key component of this balance the natural ligand for in the prostate was missing []. However, the comparative expression of the protein in human prostate carcinoma and benign prostatic hyperplasia (BPH) tissues has not been reported. Furthermore, the mechanisms of inactivation of the gene in prostate carcinoma are not known []. Hence, the aim of this study was to define the pattern of protein expression in hyperplastic, PIN and malignant human prostate tissue and to determine whether changes in expression were associated with disease progression. MATERIAL AND METHODS Formalin-fixed, paraffin-embedded, blocks were obtained from thirty five patients who underwent radical prostatectomy and pelvic lymphadenectomy for prostate cancer, core biopsies diagnosed as PIN and TURP diagnosed as BPH. Specimens were obtained from the Department of Pathology-Cairo University during the time period from September to August. Expression of Estrogen Receptor-ß () in Bengin For all, the clinical data were available on the computer files including age, PSA level and extent of tumor including capsule penetration, surgical margin, seminal vesicles invasion and lymph node involvement. Histological evaluation: Two micron-thick sections were prepared from each tissue block. One section was stained by Haematoxylin and Eosin (H & E) for reevaluation, including the tumor grade classified according to Gleason's grading system. Patterns of tumor growth were numbered in order of increasing malignancy, grades to. In each case, a predominant and a secondary pattern grade were also recorded. The sum of the two grades yielded a Gleason's score that ranged from to. The of prostatic adenocarcinoma were classified according to Rubin et al. [] into well (Gleason's up to ), moderately (Gleason's or ) and poorly (Gleason's 8, 9 and ). The tumor staging was done according to the AJCC th edition []. The PIN were classified into low grade (PIN ) and high grade (PIN ) according to Bostwick []. The other section was mounted on poly-llysine-coated slides (Superfrost slides) and subjected to estrogen receptor ß immunohistochemical staining. Immunohistochemical staining: After routine deparaffinization in xylene, the sections were hydrated through a series of graded alcohols, distilled water and (PBS) at ph..-.. Antigen retrieval was performed using Tris-EDTA (ph 9) in a pressure cooker. The slides were put in DAKO autostainer S which performed the rest of the steps which are: Incubation in % H O for minutes to inhibit the endogenous peroxidase activity. Washing the slides with PBS at ph..-.. Incubation with monoclonal rabbit anti-human estrogen receptor ß diluted : in DAKO antibody diluent S for minutes at room temperature. Washing the slides with phosphate-buffered saline (PBS) at ph..-.. Applying the EnVision TM dual link kit (K) optimized for DAKO cytomation automated systems for minutes.

3 Samia M. Gabal, et al. Washing the slides with phosphate-buffered saline (PBS) at ph..-.. Applying DAB (, -di-amino-benzidin etetrahydrochloride) as chromogen for minutes. Rinsing well in distilled water for minutes. 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 changes and cover slips were applied. Evaluation of estrogen receptor ß expression: Nuclear staining within the cell, whether weak or strong, was considered positive. Given previous evidence that is a nuclearlocalized steroid receptor, only positive nuclear immunostaining was scored. Counting of epithelial cells (basal and secretory) in each case was undertaken to determine the percentage of immunostained nuclei across all the cancer areas present. The primary analysis was based on a cut-off of % and classified as positive if % of cell nuclei were immunoreactive. ERß scores were expressed as the percentage of cells demonstrating nuclear immunoreactivity in scores as, -, -, -, > []. Statistical analysis: Data were statistically described in terms of frequencies (number of ) and relative frequencies (percentages). The Chi square (X ) test was performed to compare the different study variables between the study groups. Yates correction and Fisher exact tests were used only when the expected frequency was found to be less than five. A probability value (p value) less than. was considered statistically significant. All statistical calculations were done using Microsoft Excel version (Microsoft Corporation, NY, USA) and SPSS (Statistical Package for the Social Science; SPSS Inc., Chicago, IL, USA) statistical program. RESULTS Sixty specimens, including thirty-five specimens of prostatic adenocarcinoma, ten specimens of benign prostatic hyperplasia, Fig. (), and fifteen specimens of PIN have been studied in this work. Tables (,) show the age distribution and PSA levels of the studied as reported in the clinical sheets of the patients. Nine (%) of the studied PIN were of high grade, (Fig. ), while (%) were of low grade atypia. Most of the adenocarcinoma of this study, 8 (%), were poorly according to Gleason scoring, (Figs. -), while seven (%) were well, (Fig. ) and (9%) were moderately (Fig. ; Table ). The presence or absence of capsular, vascular, seminal vesicle, surgical margins and lymph node involvement in the studied prostatic carcinoma are shown in Table (), and the TNM staging is shown in (Table ). Results of expression in the studied : The expression of in the studied is shown in Table (). Nine (9%) of the studied BPH showed positive expression where they showed % positive cells, (Fig. ). Eight (%) of the studied PIN were negative for expression. Twenty-nine (8.8%) of the studied adenocarcinoma were negative for expression, while only (.%) showed positive expression, (Fig. 8) with highly significant difference, p<.. The distribution of scores of this expression is presented in (Table ). The relation between the expression and the prognostic factors in the studied : There was high expression in of BPH ng/ml PSA level, negative expression in PIN and in adenocarcinoma with > ng/ml PSA level (Tables 8-). Most of the PIN with positive expression were of low grade PIN and PIN with negative or low expression were of high grade PIN, (tables, ). The relation between the, the Gleason score, and tumour stage in the studied adenocarcinoma showed that most of the moderately and poorly were negative for. Also, there was negative expression in stage IV and low expression scores in stages II & III. Tables (-) charts (&). All the with lymph node metastasis, presence of vascular and seminal vesicle and surgical margin invasion and most of the with capsular invasion tables showed negative ERß expression (tables,8).

4 Expression of Estrogen Receptor-ß () in Bengin Table (): Age distribution in studied. Age group -9 years - years > years Table (): PSA level in studied BPH, PIN and prostatic adenocarcinoma. psa level ng/ml > ng/ml of BPH (%) (%) (%) of PIN (%) (8%) (%) of prostatic adenocarcinoma (%) (88%) (%) Most BPH (%) presented with PSA level ( ng/ml) and more than half of adenocarcinoma in this study (%) presented with PSA level > ng/ml. Table (): No. of prostatic adenocarcinoma according to Gleason's score. Gleason score Well (score up to ) Moderately (score & ) Poorly (score 8,9 & ) of BPH (%) 8 (8%) of PIN (8%) (%) of prostatic adenocarcinoma (%) (9%) 8 (%) (%) More than half (%) of the prostatic adenocarcinoma in this study showed high Gleason's grade. 8 (8%) (%) No (%) (.%) 8 (8%) (.%) (%) The highest frequency of BPH, PIN and adenocarcinoma (8%) occurred between and years. Table (): Extent of tumour in studied prostatic adenocarcinoma. State of invasion Negative Positive Capsule penetration (%) (%) (%) Extent of the tumor Seminal vesicles invasion 8 (8%) (%) (%) Lymph node involvement (%) (%) (%) Vascular invasion 8 (8%) (%) (%) Positive surgical margin (9%) (%) (%) Table (): Pathological TNM staging of the studied prostatic adenocarcinoma. Stage Stage I: Ta N M Gleason - Stage II: Ta N M Gleason - or (Tb, Tc or T) N M any Gleason score Stage III: T N M any Gleason score Stage IV: T or N or M No & % (%) 8 (%) (%) (%) (%) Table (): The expression in the studied. Studied BPH PIN Adenocarcinoma expression, No. (%) 9 (9%) (%) (.%) (%) 8 (%) 9 (8.8%) (%) (%) (%) The loss of expression in adenocarcinoma was significantly present in a larger number of than in PIN and BPH., p value <.. Table (): Distribution of expression in the studied. scores > BPH No. (%) (%) (%) (%) (%) (%) PIN No. (%) (%) (%) (%) (%) (%) Adenocarcinoma No. (%) (.%) (.%) (.%) (%) (%) 9% of studied BPH were positive for ER. Four (%) of PIN were - and no were >. Most of the prostatic adenocarcinoma in this study showed negative expression ( score). Table (8): Relation of scores to PSA level in studied BPH. ng/ml 8 (%) 8 (%) PSA >ng/ml (%) (%) (%) (%) 9 (9%) (%) BPH showed higher expression in have ng/ml PSA level.

5 Samia M. Gabal, et al. Table (9): Relation of scores to PSA level in studied PIN. ng/ml (8.%) (.%) (%) PSA >ng/ml (.%) (.%) (%) 8 (.%) (.%) (%) Most of the PIN showed negative expression with PSA value in ng/ml. Table (): Relation of scores to PSA level in studied prostatic adenocarcinoma. ng/ml 9 (%) (%) (%) PSA >ng/ml (%) (%) 9 (8.9%) (.%) (%) Prostatic adenocarcinoma showed negative expression with PSA levels > ng/ml. Table (): Relation of scores to histopathological grading in studied of PIN. Low grade (.%) (.%) (%) Pin grade High grade (.%) (.%) 9 (%) Most of the positive pin were high grade. Table (): scores in grades of PIN. scores Low grade (%) Histopathological grading High grade 8 (.%) (.%) (%) Most of the of high grade PIN showed no or low expression of. 9 (%) (%) Table (): Relation of scores to gleason score in studied prostatic adenocarcinoma. Well (.%) (.9%) (%) Gleason score Moderately (%) (%) (%) Poorly (8.%) (.%) 8 (%) 9 (8.9%) (.%) (%) Most of the poorly were negative for. Table (): scores in different gleason scores in the studied prostatic adenocarcinoma. scores Well (%) Gleason score Moderately All the adenocarcinoma with positive expression showed Gleason low scores. (8.%) Poorly 8 (.%) (%) Table (): Relation of scores to primary tumor stage in the studied prostatic adenocarcinoma. Stage Stage II Stage III Stage IV (8.%) (8.%) (%) scores (.%) (.%) 8 (%) (%) (%) Most of the stage IV showed negative expression. Table (): scores in different tumor stages of the studied prostatic adenocarcinoma. Stage Stage II Stage III Stage IV (%) (8.%) (8%) scores - (.%) (%) - - The prostatic adenocarcinoma showed negative expression in stage IV and low expression scores in stage II & III. (.%) (.%) 8 (%) (%) (%)

6 Expression of Estrogen Receptor-ß () in Bengin Table (): The relation between expression and extent of the tumor in the studied prostatic adenocarcinoma. Capsule penetration 9 9% %.%.% Seminal vesicles invasion % Extent of the tumor 8.%.% Lymph node involvement % % % Vascular invasion % 8.%.% Surgical margin % 8.8% 8.% % % % 8 % % % % 8 % % % The was negative in all the with lymph node metastasis, presence of vascular and seminal vesicle and surgical margin invasion and most of the with capsular invasion. Table (8): scores in different modes of tumor extention in the studied prostatic adenocarcinoma. Extent of the tumor > Capsule penetration 8 9% % %.% %.% Seminal vesicles invasion % 8.%.%.9%.% Lymph node involvement % % % % % Vascular invasion % 8.%.%.9%.% Surgical margin % 9.%.%.% % (%) (%) (%) 8 (%) (%) (%) (%) 8 (%) (%) (%) 8 8 Low grade Moderate grade High grade Stage I Stage II Stage III Stage IV > > Chart (): Relation of scores to gleason grade in the studied prostatic adenocarcinoma. Chart (): Relation of scores to primary tumor (T) stage in the studied prostatic adenocarcinoma.

7 Samia M. Gabal, et al. Fig. (): Benign prostatic hyperplasia. (H&Ex). Fig. (): High grade prostatic intra-epithelial neoplasia (PIN) (H &Ex). Fig. (): Prostatic adenocarcinoma Gleason grade (grade III) showing tumour cells arranged mostly in strands and solitary cells (H&E x). Fig. (): Prostatic adenocarcinoma Gleason score 9 (grade III) showing focal comedo pattern [central necrosis] (H&E x). Fig. (): Prostatic adenocarcinoma Gleason score (grade I) (H&E x). Fig. (): A- positive expression score (>) in benign prostatic hyperplasia. B- positive expression, score (-) in benign prostatic hyperplasia (Immunoperoxidase, DAB chromogen x). Fig. (): Prostatic adenocarcinoma Gleason score 8 (grade III) with focal colloid areas (H&Ex). Fig. (8): (A & B) positive expression, score (-) in two of prostatic adenocarcinoma. (Immunoperoxidase, DAB chromogen x and x).

8 DISCUSSION Estrogens are believed to play a significant role in the pathogenesis of prostate cancer (PCa) [8]. Although the precise biological function of is incompletely defined, it has been suggested that the receptor, acting through estrogens, may protect the normal prostate epithelium from undergoing unscheduled cell proliferation, neoplastic transformation, and from oxidative injuries [,]. In this work, evaluation of the expression in BPH, PIN and prostatic adenocarcinoma and its correlation with the clinicopathological features of such was done to determine the relation of the expression to tumorigenesis and prognostic factors in PCs. The highest frequency of BPH in this study (8%) occurs between - years. This is demonstrated by the study of Oesterling [] who found clinical evidence of BPH in almost 9% of autopsied men at the age of 8-9. Also, the highest frequency of PIN and adenocarcinoma in this study (8%) occurs between - years. A urology practice study of ultrasound guided biopsies of hypoechoic lesions identified of PIN (.%) and cancers (.%). Interestingly, those with PIN had a mean age of years, whereas those with cancer were older with a mean age of year [8]. Most of the PIN in this study (%) were high grade and almost half of prostatic adenocarcinoma showed high Gleason's scores, which constitute 8% of all studied. Humphrey et al. [9] mentioned that Gleason's score 8- is the most common pattern of growth of prostatic adenocarcinoma. In this work, most of the BPH (%) presented with PSA levels < ng/ml while more than half of the adenocarcinoma in this study (%) presented with PSA levels > ng/ml. Lorente et al. [], made the observation that men with prostatic cancers and metastatic prostate cancers had high serum levels of acid phosphatase. Their work was the foundation of the clinical utility of acid phosphatase and prostate-specific antigen (PSA) for the presence of cancer prostate []. Expression of Estrogen Receptor-ß () in Bengin Nine (9%) of BPH, (%) of PIN in this study showed positive expression, while (.%) of the prostatic adenocarcinoma were positive for expression with highly significant difference. Nearly similar results were given by Horvath et al. [] who identified that was highly expressed in normal human prostate and that there was progressive loss of expression in prostatic hyperplasia and, to a greater extent, invasive cancer. was found in both the basal and secretory compartments of the epithelium, whereas >% of PCs did not express. Latil et al. [] mentioned similar results where they demonstrated a decrease in mrna expression in the majority of prostate tumors relative to normal tissue. Interestingly, a few in each group showed no change or increased expression in the tumor compared with normal tissue. Gallardo et al. [] also found that labelling was seen in 8% of epithelial cells of normal glands and in lower levels of such cells in glands showing hyperplastic and neoplastic changes. Pasquali et al. [] also demonstrated that the loss of, in conjunction with other unknown molecular events, may promote cell proliferation and possibly carcinogenesis. Such a hypothesis is compatible with the loss of ERß expression in prostatic hyperplasia and carcinoma observed in their study. The possible mechanism is that down-regulation of androgen receptor levels by results in limited epithelial cell proliferation. Bonkhoff et al. [] were unable to detect expression in 8 primary PCs by IHC. The low rate of.% positivity in a larger cohort may explain why Bonkhoff et al. [] were unable to demonstrate expression in their much smaller cohort. An alternative explanation may relate to different sensitivities of the antibodies used in the two studies []. Studies of breast and colon cancers demonstrate similar patterns of loss of expression with development of carcinoma. mrna and protein have been detected in normal breast epithelium, but there was a trend toward loss of mrna expression in adjacent cancers []. Similarly, selective loss of protein in colon cancers compared with matched normal tissue has been demonstrated []. As regard the scores of expression in this work, half of the of BPH showed scores >, (%) of PIN were -

9 Samia M. Gabal, et al. and no were >, (%) of the studied carcinoma showed score and (.%) showed score - with statistically significant difference. These results were close to those found by Horvath et al. [] where all the studied normal prostatic tissues were >, most of the PCs (%) were score and % were -. Although Bergan et al. [8] mentioned that antiestrogens had little success in the past as a therapy for PC, these results may identify a group of positive PC patients in which these agents may have therapeutic benefit. In this work, there was high expression in of BPH with ng/ml PSA level, negative expression in PIN and in adenocarcinoma with > ng/ml PSA level. Most of the PIN with positive expression were of low grade PIN while negative or low expression was in high grade PIN. Similar results were given by Chang and Prins [9] whose also mentioned that the underlying mechanism(s) for these findings in dysplasias awaits further investigation, based on the proposed antiproliferative function of the receptor. The presence of in secretory cells of low/ moderategrade lesions may represent a transient abortive attempt to counter growth these cells. In contrast, the attrition of receptor-positive basal cells in the high-grade dysplasias may signify a continuing loss of the growth inhibitory function mediated by in these precursor lesions. In this work, the correlation between the, the Gleason score, and tumor stage in the studied adenocarcinoma showed that most of the moderately and poorly were negative for. Also, there was negative expression in stage IV and low expression scores in stages II & III. All the with lymph node metastasis, presence of vascular, seminal vesicle and surgical margin invasion and most of the with capsular invasion showed negative expression. Li, et al. [] also correlated expression scores to Gleason grade in their studied prostatic adenocarcinoma which showed low expression in the high grade group. staining was present in the majority of grade carcinomas of the peripheral zone but was greatly diminished or absent in most grade / carcinomas and, interestingly, nuclear membrane staining was evident in many cells in high-grade dysplasias and grade / carcinomas. The precise meaning of this finding is currently undefined but seems to represent the localization of that occurs with neoplastic progression and could reflect alteration in receptor function. Zhu et al. [] demonstrated that expression declined as PCa developed and reached higher grades in the primary site and they postulate that the receptor plays distinct and different roles at various stages in the evolution and progression of PCa. Thus, a better understanding of how expression is regulated throughout the natural history of the disease may yield new strategies for the diagnosis, prevention, and treatment of PCa. Also Dotzlaw et al. [] and Speirs et al. [] stated that the expression of mrna in breast tumors was associated with clinicopathological markers of poor prognosis. mrna expression in breast cancer biopsies correlated inversely with progesterone-receptor expression. In addition, higher rates of mrna expression was demonstrated in node-positive breast cancers compared with node-negative and in tamoxifen-insensitive breast cancers compared with tamoxifen-sensitive tumors. These relationships suggest that breast cancers that express are likely to have a poorer prognosis, but this has yet to be proven. From these data we can conclude that estrogen can affect prostatic cancerogenesis and neoplastic progression through an -mediated process in human prostate tissue and identify the need to additional studies to address the potential role of in the regulation of prostate epithelial cell proliferation at different stages in the development of PC. A better understanding of the function of in the evolution of PC could potentially impact on the therapeutic options for patients who have ERß expressing tumors. REFERENCES - Cancel-Tassin G, Cussenot O. Genetic susceptibility to prostate cancer. BJU Int., 9 (9): Inas Elattar I. Cancer in the Arab World: Magnitude of the Problem. ICC%Mar.. - Moktar N, Gouda I, Adel I. Cancer pathology registry, - and time trend analysis book. Pathology department, National Cancer Institute., Chapter, p Horvath LG, Henshall SM, Lee CS, Head DR, Quinn DI, Makela S, et al. Frequent Loss of Estrogen Receptor-ß Expression in Prostate Cancer. Cancer Re- Search., (): -.

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