|Year : 2017 | Volume
| Issue : 4 | Page : 115-120
Evaluation of androgen receptor and HER2/neu receptor in tumor tissue of transitional cell carcinoma of urinary bladder and normal bladder mucosa
Avtar Pachauri, HS Pahwa, Awanish Kumar
Department of General Surgery, King George Medical University, Lucknow, Uttar Pradesh, India
|Date of Web Publication||29-Oct-2018|
Dr. Avtar Pachauri
Department of General Surgery, King George Medical University, Room No 103E, Gautam Buddha Hostel, KGMU, Lucknow - 226 003, Uttar Pradesh
Source of Support: None, Conflict of Interest: None
Background: Transitional cell carcinoma (TCC) is the most prevalent, singly accounting for 90% of all bladder cancer cases. Various markers involved in cell cycle but none of them proven its capacity as a prognostic indicator. The discovery of new biological markers may lead to early clinical prediction and may help in individualization of therapeutic approach in management of bladder cancer patients. The human epidermal growth factor receptors (EGFRs) – HER2/neu play a role in neoplastic cell growth and its role proven in breast and ovarian cancer while its role in bladder cancer still not explored its potential. Bladder cancer incidence is higher in male as compared to female. This gender related difference is still not clear in humans while it is proven in animal studies. In this study, we investigated the potential role and association of HER2/neu and androgen receptors (ARs) in etiology and prognosis of TCC.
Patients and Methods: The study included 20 histopathologically confirmed cases of TCC of urinary bladder attending the Department of General Surgery, KGMU, Lucknow and 20 healthy individuals were taken as control. Immunohistochemical analysis for AR and HER2/neu receptor was done using paraffin-embedded tissue blocks treated with monoclonal mouse anti-human antibodies against AR and HER2/neu receptor, respectively.
Results: HER2/neu over expression was observed in 14 (73%) of which 12 were males and 2 were females. Among these 16.7%, 33.3%, and 37.5% cases were in Grade I, II, and III, respectively. The AR overexpression was observed in 4 (20%) of which all were males. About 16.7% and 27.3% cases were in Grade II and III, respectively. Peak incidence was observed in the age group of 50–70 years (75%).
Conclusions: We conclude that HER2/neu and AR can be considered as bad prognostic factor in TCC. These results suggested that targeting AR and HER2/neu may provide novel chemopreventive and therapeutic approaches for the management of bladder cancer.
Keywords: Androgen receptor, bladder cancer, HER2/neu, transitional cell carcinoma
|How to cite this article:|
Pachauri A, Pahwa H S, Kumar A. Evaluation of androgen receptor and HER2/neu receptor in tumor tissue of transitional cell carcinoma of urinary bladder and normal bladder mucosa. Arch Int Surg 2017;7:115-20
|How to cite this URL:|
Pachauri A, Pahwa H S, Kumar A. Evaluation of androgen receptor and HER2/neu receptor in tumor tissue of transitional cell carcinoma of urinary bladder and normal bladder mucosa. Arch Int Surg [serial online] 2017 [cited 2019 Mar 19];7:115-20. Available from: http://www.archintsurg.org/text.asp?2017/7/4/115/244416
| Introduction|| |
Worldwide bladder cancer is a major and the fifth most common type of cancer. In India, according to the recent reports of the National Cancer Registry Program, the overall incidence rate of the urinary bladder cancer is 2.25% (per 100,000 annually): 3.67% among males and 0.83% for females. Among the various types of urinary bladder cancers, the transitional cell carcinoma (TCC) is the most prevalent which singly constitute 90% of all bladder cancer cases.,, Despite such a high prevalence, the molecular mechanism involved in the induction and progression of bladder carcinoma is still poorly understood.
Bladder cancer etiology involves several molecular alterations and complex biological pathways that regulate cellular processes, such as proliferation, differentiation, angiogenesis, metastasis and apoptosis. Till date, many studies have concentrated on the evaluation of certain markers involved in the regulation of the cell cycle. The discovery of new biological markers may lead to early clinical prediction and may help in individualization of therapeutic approach in management of bladder cancer patients with the aim of reducing the chances of progression. The human EGFRs are one among various biological markers which can represent as promising therapeutic targets.,
HER2/neu is a member of the EGFR family. The HER2 gene is localized to chromosome 17q and encodes a glycoprotein with intrinsic tyrosine kinase activity. The HER2/neu-encoded protein molecule occupies a critical position in the biochemical pathways responsible for the transduction of mitogenic signals from a variety of growth factor receptors. In addition to its role in regulating normal cellular proliferation, overexpression of the HER2/neu gene appears to play a role in neoplastic cell growth. HER2/neu gene amplification and/or the protein over expression have been considered as bad prognostic factors in several carcinomas, particularly in breast and ovarian carcinomas since long., Many studies have shown the role of HER2/neu in the pathogenesis of bladder carcinoma. However, its expression has been found to be variable in different studies, ranging between 9 to 81%.,,, Beside from its prognostic significance, accurate determination of HER2/neu status in patients with bladder cancer has therapeutic implications also.
Bladder carcinoma is three times more common in men than women.,, The etiology for this difference in incidence by sex is unknown. This gender difference was already proven in animal experiment in which male are more prone to develop bladder cancer and prevent this tumor by treating with androgen deprivation therapy. In humans, excessive exposure to cigarette smoke and industrial chemicals in males has been suggested as an explanation, although a previous study demonstrated that the gender-related risk of bladder cancer persisted even after controlling for these factors.
To investigate the potential role of sex steroids in human bladder cancer, the expression of hormone receptors in human bladder cancer specimens has been evaluated. Androgen and its receptor have been found a crucial role in cancer development and its progression.,, Therefore, androgen receptor (AR) may represent a potential therapeutic target.
| Patients and Methods|| |
Total number of 20 histopathologically confirmed cases of TCC of urinary bladder attending the Department of General Surgery, Urology and Surgical Oncology, KGMU, Lucknow and institutional ethical clearance was taken. Twenty healthy individuals were taken as control. Cases of TCC were classified according to the WHO grading system in three grades: Grade I, Grade II and Grade III and Stage T1, T2, T3 and T4. Cases were further divided according to age into two age groups: <50 years and >50 years.
For androgen receptor: Formalin fixed paraffin-embedded tissue blocks were cut in 5 microns thick serial sections. The sections were de-paraffinized, rehydrated and rinsed in phosphate-buffered saline (PBS). An immunohistochemical assay for AR was performed on consecutive paraffin sections using biotin method. Monoclonal mouse anti human antibodies were used as primary antibodies for AR. After antigen retrieval, slides were incubated with primary antibody followed by secondary antibody. Sections were washed in PBS and then incubated with peroxidase. Finally chromogendiaminobenzidine (DAB) was used and sections were counter stained with hematoxylin.
For HER2/neu receptor: Tumor specimen fixed in buffered formalin embedded in paraffin and sectioned in 3–5 microns thick slices for the immunohistochemistry (IHC). Antigen retrieval for HER2/neu IHC was performed by heating the slides submerged in citrate buffer at pH 6 at 95–99°C for 40 min in microwave oven. Antigen retrieval was followed by incubation with primary antibodies for 30 min. Incubation was followed by diaminobenzidine visualization of the final reaction product in brown color.
For androgen receptor: Tumors were classified as AR negative (i.e. low expression) if ≤10% of cells displayed positivity and AR positive (i.e. high expression) if >10% of cells displayed positivity.
For HER2/neu receptor: The scoring percentage was assessed by counting the percentage of positive cell in 100 malignant cells at 40 × total magnification for at least five fields.
- Score 0: completely negative
- Score 1: faint staining of membrane (<10 malignant cell)
- Score 2: moderate staining membrane (>10 malignant cell)
- Score 3: strong circumferential staining of entire membrane creating fishnet pattern.
Chi-square (χ2) test was performed to find out the possible correlation between hormonal receptors and other clinical parameters. Statistical significance was defined as P < 0.05.
| Results|| |
In our study included IHC proven transitional cell carcinoma of urinary bladder 20 cases and 20 controls including male and female. Cases of TCC classified according to WHO grading system Grade I, Grade II and Grade III. Cases were further divided according to age groups: <50 years and >50 years.
The age of the cases (male – 56.33 ± 8.96; female – 54.50 ± 0.70) and controls (male – 51.71 ± 10.85; female – 55.33 ± 8.73) was similar among both male and female patients. The tumor Grade I, II and III was in 16.7%, 33.3% and 37.5% among male patients. All females were in Grade III [Table 1].
The AR positivity was 16.7% in tumor Grade II patients and 27.3% in Grade III patients. However, the association between tumor grade and AR positivity was statistically not significant (P > 0.05). The AR positivity was 22.2% in male patients and it was nil in females. The AR positivity was significantly (P = 0.02) lower among the patients of age >50 years (7.1%) than ≥50 years (50%) [Table 2].
| Discussion|| |
In carcinoma of the urinary bladder HER2/neu expression has been reported 10 years ago and although the staining pattern is well known, the prognostic significance of HER2/neu expression in bladder cancer is highly controversial, with several conflicting results in the published literature. The success of trastuzumab therapy in patients with breast carcinoma has stimulated interest in exploring the potential of this therapy for patients with urothelial carcinoma.
In our study HER2/neu overexpression correlated with higher tumor grades i.e. invasive tumor (Grade II/III or Stage T3/T4) which was in accordance with many studies.,, However, in one literature it was also found that HER2/neu expression decreases with increase in tumor grade.
There are various methods for the assessment of HER2/neu expression. (i.e. gene amplification, FISH, IHC, serum analysis, PCR) out of which IHC is most commonly used because it is cheaper, easy to use, availability and its most commonly used in breast cancer. In our study HER2/neu expression was assessed by IHC. Majority of studies on HER2/neu expression assessment are done by IHC technique.,, In our study, we found 73% HER2/neu expression. Various reports of HER2/neu expression in bladder cancer ranged from 2% to 81%. So this rate was in range of following study [Table 3].
In this series we found that HER2/neu overexpression was significantly associated with tumor differentiation grade as well as the depth of tumor invasion with the overexpression more prevalent in the high grade and deeply invasive specimens reflecting the aggressiveness of the tumor cells. Majority of studies in favor of increase of HER2/neu expression with increase in tumor grade. Various studies showed HER2/neu has fundamental role in progression and proliferation of malignant cell.,
In our study we found the AR expression is 20%. Earlier study analyzing >20 samples each detected AR in 12–78% of bladder cancer.,, However more recent studies analyzing larger samples showed AR expression in almost half of bladder tumors.
In our study when we analyzed AR expression with respect to sex, it was found that in TCC of females there is no AR expression while among males it was 22.2%. While in some studies AR expression was equal in both the genders and found no correlation between them.,, A possible explanation of this gender differences is differing hormone levels. The lower incidence in females may be attributable to some interaction between tumor's sex hormone, genetic and non-genetic factors. Many animal experiments have shown that bladder cancer is induced by chemical carcinogens more easily in males than females.,
In our study the peak incidence of bladder cancer was observed in the age group of 50–70 years (75%). This result is in accordance with previous study in which similar result was found. The reason for this remains unclear, but it might be due to the cumulative effects of long time exposures to carcinogens, the failing of DNA repair mechanisms and aging.
In our study we did not find AR expression in normal urinary bladder or indicating no or undetectable level of expression which is in deep contrast to high expression of AR in the TCC of urinary bladder. This finding is also in confirmation with other study.
In our study the expression pattern was not only high (20%), but also showed that expression pattern of AR was rising from Grade I (0) to Grade II (16.7%) and further to Grade III (27.3%). Our finding indicates that AR expression increases with tumor grade but we do not get any significant association between AR receptor expression and tumor grade. This correlation was also seen in other studies. While many studies also shows that decrease in AR expression with increase in tumor grade/stage. So the prognostic significance of AR expression in patient with bladder cancer is controversial [Table 4]. The reason for such discrepancies between studies may be result of different staining condition, criteria for overexpression and patient population.
|Table 4: Prognostic significance of AR expression in patient with bladder cancer in various studies|
Click here to view
The AR expression in males of 50 years and above age group was (75%) which was significantly higher in males >50 years (7.1%). The reason might be due to cumulative effects of long time exposures to carcinogens, failing of DNA repair mechanisms with aging.
| Conclusion|| |
Our study including IHC proven TCC of urinary bladder cases for the assessment of HER2/neu and AR concludes that HER2/neu overexpression can be considered as a factor associated with higher tumor grades of TCC. Therefore, our results suggest that HER2/neu overexpression could offer supplementary information for prognosis in patients with TCC. And also the patient with high grade tumors owing to HER2/neu overexpression may benefit from anti HER2/neu targeted therapies.
AR expression is associated with bad prognosis. These result suggested that targeting AR and androgen may provide novel chemopreventive and therapeutic approaches for the management of bladder cancer.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Consolidated report of population-based cancer registries 2001-2004. National Cancer Registry Programme (Indian Council of Medical Research), Bangalore, 2006.
Dinney CP, McConkey DJ, Millikan RE, Wu X, Bar-Eli M, Adam L, et al
. Focus on bladder cancer. Cancer Cell 2004;6:111-6.
Alon ZW, Shahrokh FS, John LH, Susan E, Seth PL. Metastatic transitional cell carcinoma of the urinary bladder to the shoulder girdle. Rev Urol 2002;4:97-9.
Reuter VE. The pathology of bladder cancer. Urology 2006;67:11-7.
Carlsson J, Forsssell AE, Hietala SO, Stigbrand T, Tennvall J. Tumor therapy with radionuclides. Assessment of progress and problems. Radiother Oncol 2003;66:107-17.
Chow NH, Chan SH, Tzai TS, Ho CL, Liu HS. Expression profiles of ErbB family receptors and prognosis in primary transitional cell carcinoma of the urinary bladder. Clin Cancer Res 2001;7:1957-62.
Grivas PD, Day M, Hussain M. Urothelial carcinomas: A focus on human epidermal receptors signaling. Am J Transl Res 2011;3:362-73.
Marin AP, Arranz EE, Sanchez AR, Aunon PZ, Baron MG. Role of anti-Her-2 therapy in bladder carcinoma. J Cancer Res Clin Oncol 2010;136:1915-20.
Slamon DJ, Godolphin W, Jones LA, Holt JA, Wong SG, Keith DE, et al
. Studies of the HER-2/neu proto-oncogene in human breast and ovarian cancer. Science 1989;244:707-12.
Pegram MD, Slamon DJ. Combination therapy with trastuzumab (Herceptin) and cisplatin for chemoresistant metastatic breast cancer: Evidence for receptor-enhanced chemosensitivity. Semin Oncol 1999;26:89-95.
Sato K, Moriyama M, Mori S, Saito M, Watanuki T, Terada K, et al
. An immunohistologic evaluation of C-erbB-2 gene product in patients with urinary bladder carcinoma. Cancer 1992;70:2493-8.
Gandour-Edwards R, Lara PN, Jr., Folkins AK, LaSalle JM, Beckett L, Li Y, et al
. Does HER2/neu expression provide prognostic information in patients with advanced urothelial carcinoma? Cancer 2002;95:1009-15.
Wester K, Sjostrom A, de la Torre M, Carlsson J, Malmstrom PU. HER-2--a possible target for therapy of metastatic urinary bladder carcinoma. Acta Oncol 2002;41:282-8.
Jimenez RE, Hussain M, Bianco FJ, Jr., Vaishampayan U, Tabazcka P, Sakr WA, et al
. Her-2/neu overexpression in muscle-invasive urothelial carcinoma of the bladder: Prognostic significance and comparative analysis in primary and metastatic tumors. Clin Cancer Res 2001;7:2440-7.
Madeb R, Messing EM. Gender, racial and age differences in bladder cancer incidence and mortality. Urol Oncol 2004;22:86-92.
Shishodia S, Aggarwal BB. Cyclooxygenase (COX)-2 inhibitor celecoxib abrogates activation of cigarette smoke-induced nuclear factor (NF)-kappaB by suppressing activation of IkappaBalpha kinase in human non-small cell lung carcinoma: Correlation with suppression of cyclin D1, COX-2, and matrix metalloproteinase-9. Cancer Res 2004;64:5004-12.
Jemal A, Siegel R, Ward E, Murray T, Xu J, Thun MJ. Cancer statistics, 2007. CA Cancer J Clin 2007;57:43-66.
Hartge P, Harvey EB, Linehan WM, Silverman DT, Sullivan JW, Hoover RN, et al.
Unexplained excess risk of bladder cancer in men. J Natl Cancer Inst 1990;82:1636-40.
Miyamoto H, Yang Z, Chen YT, Ishiguro H, Uemura H, Kubota Y, et al
. Promotion of bladder cancer development and progression by androgen receptor signals. J Natl Cancer Inst 2007;99:558-68.
Mir C, Shariat SF, van der Kwast TH, Ashfaq R, Lotan Y, Evans A, et al
. Loss of androgen receptor expression is not associated with pathological stage, grade, gender or outcome in bladder cancer: A large multi-institutional study. BJU Int 2010;108:24-30.
Kauffman EC, Robinson BD, Downes MJ, Powell LG, Lee MM, Scherr DS, et al
. Role of androgen receptor and associated lysine-demethylase coregulators, LSD1 and JMJD2A, in localized and advance human bladder cancer. Mol Carcino 2011;50;931-44.
Freedman AN, Michalek AM, James RM, Asirwatham JE. The relationship between smoking exposure and p53 over-expression in colorectal cancer. Br J Cancer 1996;73:902-8.
Tryls G, Kennet W, Manuel De La T, Carlsson J, Malmström PU. Analysis of Her-2/neu expression in primary urinary bladder carcinoma and corresponding metastases. J Pathol 2005;95:982-6.
Atis G, Arisan S, Dalkilinc A. Determination of Her2 neu gene amplification and protein over expression in bladder transitional cell carcinoma. Adv Nol Biol 2007;1:89-94.
Naik DS, Sharma S, Ray A, Hedau S. Epidermal growth factor receptor expression in urinary bladder cancer. Indian J Urol 2011;27:208-14.
] [Full text]
Cancer V AJ, Rebecca S, Elizabeth W, Michael J. ThunClin 2008;58;71-96.
Hansel DE, Swain E, Dreicer R, Tubbs RR. HER2 over-expression and amplification in urothelial carcinoma of the bladder is associated with MYC coamplification in a subset of cases. Am J Clin Pathol 2008;130:274-81.
Voller RT, Humphrey PA, Swanson PE, Wick MR, Hudson ML. Invasion of the bladder by transitional cell carcinoma: Its relation to histologic grade and expression of p53, MIB-1, c-erb B-2, epidermal growth factor receptor, and bcl-2. Cancer 1998:82715-23.
Lipponen P, Eskelinen M, Syrjanen S, Tervahauta A, Syrjanen K. Use of immunohistochemically demonstrated c-erb B-2 oncoprotein expression as a prognostic factor in transitional cell carcinoma of the urinary bladder. Eur Urol 1991;20:238-42.
Mellon JK, Lunec J, Wright C, Home CH, Kelly P, Neal DE. C-erbB-2 in bladder cancer: Molecular biology, correlation with epidermal growth factor receptors and prognostic value. J Urol 1996;155:321-6.
Enache M, Cristiana Eugenia Simionescu, Stepan A. EGFR and Her2/neu immunoexpression in papillary urothelial bladder carcinomas. Rom J Morphol Embryol 2013;54:137-41.
Alexa A, Baderca F, Zăhoi DE, Lighezan R, Izvernariu D, Raica M. Clinical significance of Her2/neu overexpression in urothelial carcinomas. Rom J Morphol Embryol 2010;51:277-82.
Laor E, Schiffman ZJ, Braunstein JD, Reid RE, Tolia BM, Koss LG, et al
. Androgen receptors in bladder tumors. Urology 1985;25:161-3.
Noronha RF, Rao BR. Sex hormone receptors in localized and advanced transitional cell carcinoma of urinary tract in humans. Urology 1986;28:401-3.
Zhuang YH, Blauer M, Tammela T, Tuohimaa P. Immunodetection of androgen receptor in human urinary bladder cancer. Histopathology 1997;30:556-62.
Tuygun C, Kankaya D, Imamoglu A, Sertcelik A, Zengin K, Oktay M, et al
. Sex-specific hormone receptors in urothelial carcinomas of the human urinary bladder: A comparative analysis of clinicopathological features and survival outcomes according to receptor expression. Urol Oncol 2011;29:43.
Kirkali Z, Cowan S, Leake RE. Androgen receptors in transitional cell carcinoma. Int Urol Nephrol 1990;22:231-4.
Nam JK, Park SW, Lee SD, Chung MK. Prognostic value of sex-hormone receptor expression in non-muscle-invasive bladder cancer. Yonsei Med J 2014;55:1214-21.
Okajima E, Hiramatsu T, Iriya K, Ijuin M, Matsushima S. Effects of sex hormones on development of urinary bladder tumours in rats induced by N-butyl-N-(4-hydroxybutyl) nitrosamine. Urol Res 1975;3:73-9.
Imada S, Akaza H, Ami Y, Koiso K, Ideyama Y, Takenaka T. Promoting effects and mechanisms of action of androgen in bladder carcinogenesis in male rats. Eur Urol 1997;31:360-4.
Isola JJ. Immunohistochemical demonstration of androgen receptor in breast cancer and its relationship to other prognostic factors. J Pathol 1994;17:31-5.
Miyamoto H, Yang Z, Chen YT, Ishiguro H, Uemura H, Kubota Y, et al
. Promotion of bladder cancer development and progression by androgen receptor signals. J Natl Cancer Inst 2007;99:558-68.
[Table 1], [Table 2], [Table 3], [Table 4]