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May 1998, Volume 48, Issue 5

Original Article

Immuno-Histochemical Analysis of Estrogen Receptors on the Malignant Gallbladder Tissue

Imtiaz A. Malik  ( Department of Medical Oncology, National Cancer Institute, Aga Khan University Hospital, Karachi. )
Zaigharn Abbas  ( Departments of Medicine, Aga Khan University Hospital, Karachi. )
Zia Shamsi  ( Departments of Medicine and Surgery, Ziauddin Medical University, Karachi. )
Irfan Daudi  ( Departments of Medicine and Surgery, Ziauddin Medical University, Karachi. )
Hasnain All Shah  ( Departments of Medicine, Aga Khan University Hospital, Karachi. )
Imran Moid  ( Departments of Medicine, Aga Khan University Hospital, Karachi. )
Zubair K. Khan  ( Department of Medical Oncology, National Cancer Institute, Aga Khan University Hospital, Karachi. )
Hizbullah Shaikh  ( Departments of Pathology, Aga Khan University Hospital, Karachi. )

Abstract

Sex steroids play an important part in the functioning of normal gallbladder, formation of gallstones and possibly in the pathogenesis of gailbiaddercancer. Steroids receptors have been previously demonstrated on normal and malignant gall bladder tissues. To study this phenomenon further, we correlated clinico­pathological features.and survival with estrogen receptor (ER) status of the tumour in 30 patients with histologically- proven adenocarcinoma of the gallbladder. Estrogen receptor assay was performed im­muno-histochemically utilizing Universal Immunoperoxidase Staining Kit. Tumor tissue was obtained either surgically orwith fine needle aspiration of the gallbladder mass. There were 27 females and 3 males. Eighteen patients had estrogen receptors expressed on the malignant tissue, 12 were negative. Comparison of cinicopathological characteristics and survival between the two groups demonstrated no significant difference in gender, mean age, marital status and parity. Similarly, presence of gallstones, histologic grade or survival did not correlate with the estrogen receptor status. There is, however, a trend in favour of poorly differentiated tumors being more often receptor negative. Further studies are necessary to elucidate the biologic significance of these receptors (JPMA 48:123, 1998).

Introduction

Several studies have suggested the importance of female sex hormones in the pathogenesis of gallbladder disease. Sex steroids have been demonstrated to influence the functioning of normal gallbladder1-6. Gallbladder emptying is impaired during lutcal phase of the menstrual cycle and pregnancy. Development of cholelithiasis has been associated with the use of oral contraceptive drugs. Furthermore. gallstones are more frequently observed in women and are particularly common in multiparous females7. This suggests a significant role played by the sex steroids in the functioning of normal gallbladder and the development of gallstones.
Gallbladder cancer is an uncommon malignancy. It accounts for less than 1% of all cancers in the United States8. Incidence of gallbladder cancer, however, exhibits marked geographic variability9,10. This cancer is mere frequently observed in Israel, Central and Eastern Europe, Japan and Latin America. Particularly high incidence has been reported in Hispanics and Native Indians11-15. In Pakistani females, gallbladder cancer is the second commonest malignancy of gastrointestinal tract origin16. This makes gallbladder cancer, a formidable health problem, atleast in certain population. Gallbladder cancer is more frequently observed in women. Risk increases with the number of pregnancies. Additionally, gallstones am present in majority of the patients with gallbladder cancer17. This raises the possibility that sex steroids may influence the pathogenesis of gallbladder cancer.
Sex steroids have been demonstrated to influence the growth of tumors arising from the target tissues such as breast and prostate. Biologic effect of sex steroids is mediated by the receptors present on the target cells1. Presence of these receptors on the malignant mammary tissue influences the biologic behaviour of breast cancer. Therapeutic responses to anti- estrogens are more frequently observed in tumors with higher experssion of the steroid receptors18,19.
Gallbladder cancer carries a very poor prognosis. It is usually diagnosed at an advanced stage, response to therapy is poor and survival is short17. Due to the high incidence of gallbladder cancer in Pakistan, lack of any effective therapy, poor prognosis and a likelihood that sex steroids may influence the pathogenesis and management of gallbladder cancer, we investigated 30 patients with galibladdercancerfor estrogen receptor (ER) expression on the malignant tissue and correlated it with the clinicopathological characteristics and survival.

Patients and Methods

This study was carried out on 30 consecutive patients who were histologically confirmed to have adenocarcinoma of the gallbladder. Twenty patients had tissue obtained at the time of laparotomy. Other ten had diagnosis based upon fine needle aspiration of the gallbladder mass. In all cases, samples were obtained from the primary site and enough tissue was available for histopathologic examination and ER analysis. Tissue was immediately fixed and embedded. Receptors were assayed subsequently, usually within two weeks of obtaining the tissue.
Histopathologic analysis was canied out on tissue stained with hematoxylin and eosin. Degree of differentiation of the tumor was noted. ER assay was performed immnuno-histochemically utilizing Univeisal Immunoperoxidase Staining Kit (Signet Laboratories, Dedham, MA)20. For this purpose, tissue sections were deparaffinized, rehydrated and separately incubated with two blocking agents (hydrogen peroxide and normal saline) to reduce non-specific background staining. Monoclonal antibody against estradiol was used to demonstrate cytoplasmic binding to the receptors. Peroxidase-antiperoxidase (PAP) method was used. Two negative and positive control slides were run in each case. Antibody reactivity was observed in the cytoplasm and scored as negative, mild, moderate or strongly positive depending upon the percentage of positive cells and intensity of staining. These values were expressed numerically.
Statistical analysis was performed using Fisher’s exact test. Unless otherwise specified all values are expressed as the mean±one. standard deviation. A probability value of 0.05 or less was considered significant.

Results

Thirty patients were included in this study. Clinical characteristics are provided in Table I.

There were 27 females. Most of them were married, multiparous and post-menopausal. Majority (80%) had gallstones. Most of the patients had moderately differentiated tumors and two-thirds presented with stage V disease (Table I). Nevin’s staging system was used21.. Eighteen tumors had cytoplasmic expression of estrogen receptors, 12 were negative (Table II).

Table II provides a comparison of clinico-pathological characteristics and survival between the two groups. There was no significant difference in gender, mean age at the time of presentation, marital status and parity. Most of the patients in both groups were post-menopausal. Menopause, however, did not influence ER status. Vast majority of the patients in both the groups had cholelithiasis. ER positive tumors were more frequently well-to moderately-well differentiated (89% vs 58%). This difference, however, did not achieve statistical significant. Most of the ER negative patients had advanced disease. Overall survival was poor and similar between the two groups.

Discussion

It has previously been demonstrated that sex steroids influence the formation and growth of tumors arising from their target organs. This effect is mediated through some specific receptors on the malignant cells. In breast cancer, ER status is an important determinant of response to anti-estrogenic therapy and survival18,19. Estrogen receptors have also been demonstrated on other hormone-dependent tumors such as ovarian and endomet.rial cancer22,23. Interestingly, a variety of tumors arising from tissues not generally considered hormone-dependent also express these receptors. This includes tumors of pancreas, stonmch colon, liver, thyrnus, thyroid, brain, kidney and skin24-30. The patho-physiologic significance and clinical relevance of these receptors, however, remain uncertain31-34.
Steroid receptors have been demonstrated on benign and malignant gallbladder tissue. Singletaiy etalobserved the presence of both estrogen and progesterone receptors in the cytosolic and nuclear fractions of benign gallbladder tissue obtained from patients undergoing cholecystectomy during liver transplantation or for cholelithiasis. These were high-affinity receptors and specific for their respective ligands35. Daignault et al reported the presence of progesterone receptors in 60% of patients who had cholelithiasis36. More recently, similar results were reported by others37-38. These studies suggest that estrogen and progesterone receptors are present on the benign gallbladder tissue obtained from patients with cholelithiasis. Since estrogenic effect is required for the expression of progesterone receptors, these studies also suggest that these receptors are functional39. Veiy few studies have been performed on the malignant gallbladder tissue. Nakarnura et a! studied 21 patients with gallbladder cancer40. Immuno-histochemical analyses revealed that 58.6% of the tumors had cytoplasmic and 52.4% had nuclear ER expression. In this study, moderately-well and poorly-differentiated tumors were more often ER positive. This difference, however, was not statistically significant. Similarly, gender did not influence the receptor status. Yamamoto et al demonstrated that 23% of the patients with gallbladder cancer had ER positive tumors41. Well differentiated tumors were more often ER positive. Receptors status, however, did not influence survival. Our study demonstrates the presence of cytoplasmic estrogen receptors in 60% of patients with gallbladder cancer. These figures are similar to Nakamura et al but more frequent than the nuclear receptor positivity reported by Yamomoto et al. Variability of ER expression has been previously documented35-38. This may reflect heterogeneity in the patient population, methodology employed to determine ER status and interpretation of the histologic findings. Source of the malignant tissue for histologic examination i.e. primary site versus metastases may also play an important role. In breast carcinoma, it has been demonstrated that metastatic sites are less frequently ER positive as compared to the primary site19. All patients in our study had tissues obtained from the primary site. We utilized anti-estradiol antibiotics to demonstrate cytoplasmic receptor binidng. Although less specific than anti ER antibodies, it was the only method available at the Aga Khan University Hospital at that time. Additionally, other authors have used similar methods and demonstrated cytosolic binding36,37,40. Even the more widely used dextran-coated charcoal method demonstrates cytosolic binding35,36,38. However, using more specific monoclonal antibody against nuclear estrogenic receptors may be more advantageous. Such a study is in progress.
In this study, we observed no correlation between ER status and age, parity, menopausal status, presence of cholelithiasis, degree of differentiation of turnoror stage of the disease. Overall survival is also not influenced by the receptor status. Although these observations are similar to the previous two reports. number of patients is small. Larger studies are required to substantiate these preliminary findings.
Biologic significance of these receptors remains unknown. Only a few studies have utilized anti-estrogenic therapy in patients with tumors arising from the “non-target” organs30. In general, such trials have been negative. This may be due to several reasons. Level of ER expression in “non-target” tissues is generally low. Degree of positivity is an important detenuinant of response to anti-estrogenic therapy. It is also possible that, atleast in some cases,these receptors are not functional. This has been demonstrated in patients with melanoma42,43. Furthennore, improper selection of patients may have contributed to the poor results. Restricting the use of anti-estrogens to individuals with ER positive tumors may be more appropriate to evaluate the worth of such an approach. Anti-estrogen therapy has not been previously evaluated in patIents with gallbladder cancer. A well controlled prospective clinical trial of hormonal therapy in patients with ER positive gallbladdercancer may be the best way to evaluate the pathophysiological significance of these receptors. Such a trial is in progress at our institution.

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