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ORIGINAL ARTICLE  
Year : 2011  |  Volume : 54  |  Issue : 3  |  Page : 476-481
Hepsin immunohistochemical expression in prostate cancer in relation to Gleason's grade and serum prostate specific antigen


1 Department of Pathology, CSM Medical University, Lucknow, UP, India
2 Department of Urology, CSM Medical University, Lucknow, UP, India

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Date of Web Publication20-Sep-2011
 

   Abstract 

Background: Hepsin (HPN), a type II trans-membrane serine protease, has been reported to be one of the most up regulated genes in prostate cancer. The aim of the present study was to find out immuno-histochemistry based phenotypic expression of HPN in formalin fixed paraffin embedded sections of prostate cancer compared with that in benign prostatic hyperplasia, in a prospective clinical setting, to know the differential status of HPN expression in benign and malignant prostatic disease. Materials and Methods: Tissue biopsies of histologically proven cases of prostatic cancers (48), benign prostatic hyperplasia (42), benign prostatic hyperplasia with prostatic intraepithelial neoplasia (7) and 4 cases of benign prostatic hyperplasia with prostatitis, were subjected to immunohistochemical staining with HPN antibody by strepavidin biotin method. Results: Hepsin expression was 100% in prostate carcinoma, 11.9% (5/42) in benign prostatic hyperplasia, 57.14% (4/7) in benign prostatic hyperplasia with prostatic intraepithelial neoplasia (PIN), and none in benign prostatic hyperplasia with prostatitis. Hepsin staining showe higher expression in high grade tumor in comparison to low grade tumor. Conclusions: Positive immunohistochemical expression of hepsin in cent percent cases of prostate cancer cases is intriguing, underscoring the significance of hepsin gene expression in prostate cancer.

Keywords: Hepsin, immunohistochemistry, prostate cancer

How to cite this article:
Goel MM, Agrawal D, Natu S M, Goel A. Hepsin immunohistochemical expression in prostate cancer in relation to Gleason's grade and serum prostate specific antigen. Indian J Pathol Microbiol 2011;54:476-81

How to cite this URL:
Goel MM, Agrawal D, Natu S M, Goel A. Hepsin immunohistochemical expression in prostate cancer in relation to Gleason's grade and serum prostate specific antigen. Indian J Pathol Microbiol [serial online] 2011 [cited 2014 Apr 24];54:476-81. Available from: http://www.ijpmonline.org/text.asp?2011/54/3/476/85078



   Introduction Top


Prostate needle biopsy remains the current gold standard for detecting prostate carcinoma in patients with elevated serum prostate specific antigen (PSA) or positive digital rectal examination (DRE). Clinical and pathologic stage, histological Gleason grade and serum PSA levels are firmly established parameters used for prognostication and guidance of prostate cancer management. [1],[2] The sensitivity and specificity of serum PSA remain low because the antigen is secreted by both diseased and normal prostate tissue, leading to unnecessary biopsies in men having benign hyperplasia of prostate (BPH) but have high circulating PSA and also missed diagnosis of prostate cancer in men with smaller prostate glands showing low circulating PSA. [3],[4],[5] The subjectivity of histological parameters in prediction of prognosis and the perceived need of objective markers have led to the intense biomarker discovery drive. [1]

Review of several published studies shows that gene hepsin (HPN), a type II transmembrane serine protease is consistently up-regulated in prostate cancer. [6],[7],[8],[9],[10],[11],[12],[13],[14] In a gene expression profiling study using cDNA microarray of more than 6500 genes, 210 genes were observed to be significantly associated in prostate cancer. HPN was found to have one of the highest differences in expression ratio between normal/BPH and prostate cancer. [9],[14]

The present study was undertaken to find out the differences in immunohistochemistry based phenotypic expression of HPN in formalin fixed paraffin embedded (FFPE) tissue biopsies of prostate cancer and benign hyperplasia of prostate (BPH) received for routine histological diagnostic workup, in relation to conventional Gleason's grading and serum PSA levels.


   Materials and Methods Top


Cases

The study included a total of 101 consecutive cases reporting to Urology outpatient and inpatient Departments. These were histological proven 53 cases of BPH, 4 cases of BPH with prostatitis, 7 cases of BPH with prostatic intraepithelial neoplasia (PIN) and 48 cases of prostatic cancers. Histopathological examinations of prostate biopsies were done Haematoxylin and Eosin stained FFPE tissues sections. Prostate carcinoma were first assigned the Gleason's score and then subcategorized into 3 groups: score less than 7, score of 7 and score more than 7. Serum PSA and creatinine levels were done in all cases at the time of first diagnosis and follow up after surgery.

Immunohistochemistry

Immunohistochemical detection of Hepsin expression on BPH and cancer tissue sections were done manually by Streptavidin Biotin method, using HPN Polyclonal Antibody-Affinity purified (aa 241-260), Cayman Chemicals, Ann Arbor, MI, USA and Ultratech HRP (DAB) Streptavidin Biotin detection System, Immunotech, Bechman Coulter, Inc, France, with appropriate positive, negative and reagent controls. Sections were deparaffinised and microwave antigen retrieval was done followed by incubation with 3% hydrogen peroxide, washed, and subsequently treated with blocking antibody. Primary antihepsin antibody was applied and incubated overnight at 4 0 C. Biotinylated link antibody was applied to the sections on day 2, followed by the enzyme conjugate streptavidin horse radish peroxidise and with diaminobenzidine, followed by counterstaining with hematoxylin. Sections were dehydrated through graded ethanol and xylene, and mounted. Scoring of immunostaining was done independently by two pathologists and composite scores of HPN staining were generated. Brown coloured cytoplasmic staining was recorded as positive staining. Intensity of staining was recorded as weak, moderate and strong. Overall percentage positivity of staining was recorded by counting the number of positive cells per 500 cells in at least 10 fields and a range was noted follows as: negative (0), range of percentage positive area (≥25, 26-50, 51-75, >75).

Statistical Analysis

Statistical analysis for difference among HPN expression in BPH and prostate cancer was done by STATA 9.2 (Stata Corp LP College Station Texas, USA) statistical software. Data was described in term of mean ± standard deviation and proportions. Associations between two categorical variables were done by Chi-square test and the means compared by using two sample 't' test/Mann Whitney/Analysis of variance/Kruskal-Wallis test as per the nature of data. P value less than 0.05 was considered as significant.


   Results Top


Significant difference was observed (χ2 = 70.6097, P = 0.000) in immunohistochemical expression of HPN positivity in benign and malignant prostatic conditions [Table 1] and [Table 2]. Benign conditions showed HPN positivity only in 9 of 53 cases (16.98%), of which 4 cases had BPH with PIN. HPN expression was positive in all prostatic carcinomas.
Table 1: Hepsin expression in diff erent prostatic conditions

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Table 2: Comparison of Hepsin immunostaining in benign prostatic hyperplasia and prostate cancer

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The percentage area positivity and intensity of HPN staining in benign and malignant lesions [Table 2] also revealed a significant difference (χ2 = 71.9996, P = 0.000; χ2 = 6.8976, P = 0.032 respectively).

Comparison of intensity of HPN staining in different Gleason's scores of prostate cancer did not reveal any correlation between pathological grade and intensity of HPN positivity whereas comparison of percentage area positivity of HPN with different Gleason's scores revealed significant difference (χ2 = 13.575, P = 0.035) with increasing Gleason's grade [Table 3].
Table 3: Comparison of Hepsin immunostaining in diff erent Gleason's score of prostatic cancers

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Positive HPN expression was mainly cytoplasmic. However 11 cases of prostate cancer showed both cytoplasmic and nuclear HPN expression. Two cases of BPH and two cases of BPH with PIN also showed nuclear positivity in addition to cytoplasmic expression. Examples of HPN positive and negative immunostainng are shown in [Figure 1] (a-f).
Figure 1: Hepsin immunostaining in FFPE sections. (a) Negative staining in BPH. (HPN immunostaining, x100). (b) BPH with PIN showing nuclear and cytoplasmic positivity. (HPN immunostaining, x400. (c) Cancer Gleason score > 7 with trabecular and solid pattern. (Hematoxylin and Eosin x100). (d) Cancer (Gleason score > 7) showing HPN positivity in trabecular and solid pattern. (HPN immunostaining x100). (e) Cancer (Gleason score 7) showing malignant and fused glands (Hematoxylin and Eosin, x100). (f) Cancer (Gleason score 7) showing HPN positivity in malignant glands. (HPN immunostaining, x100).

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HPN expression was maintained in 3 cases of prostate cancer with bone metastasis [Table 4]. [Table 5] shows PSA levels before and after surgery in all cases. The correlation of significance between Hepsin and PSA in BPH and prostate cancer is shown in [Table 6].
Table 4: Details of 3 cases of prostate carcinoma which had metastasized to bone

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Table 5: Comparison of total PSA in diff erent prostatic conditions and Gleason's score at initial presentation and 3 follow-ups

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Table 6: Comparison of statistically significant variables in different prostatic conditions

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   Discussion Top


With the development of molecular global gene expression profiles of prostate cancer and accumulated evidence in documented literature, HPN has been found to be a most upregulated gene. The present study was undertaken to study the immunohistochemical expression of HPN in formalin fixed paraffin embedded tissue (FFPE) sections of prostate cancer compared with BPH. Significant difference was observed in HPN expression in BPH and prostate cancer. All the 48 cases of prostate cancer in this study showed HPN over expression. Percentage area positivity of HPN revealed significant difference with increasing Gleason's grade. Intensity of HPN immnostaining in different Gleason's scores of prostate cancer did not reveal any correlation between pathological grade. Majority of BPH (37 of 42; 88%) did not show HPN expression whereas 4 out of 7 cases of BPH with PIN showed HPN positivity.

The published studies on HPN in prostate are microarray based studies done on cell lines or frozen tissues from patients of BPH and prostate cancer. IHC has been done on tissue microarray as a part of phenotypic expression of HPN with other expressed genes after validation with real time polymerase Chain Reaction (RT-PCR). The summarized details of these studies are shown in [Table 7].
Table 7: Summarized review of literature showing Hepsin expression studies

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Dhanasekaran et al. [6] reported IHC studies in tissue microarray demonstrating strongest protein expression in high grade PIN and lower HPN expression in patients with post-operative PSA failure, and higher Gleason scores, consistent with an inverse correlation of HPN expression with poor prognosis. In our study three cases of prostate cancer had bone metastasis (diagnosed by bone scan) in which 2 cases had Gleason Score of >7 and in 1 case it was <7. Although we observed positive HPN expression in these three cases, the number is too small to draw any inference. An association of decreased HPN expression with elevated PSA levels has been observed in hormone refractory metastatic prostate cancer patients. Results of established prostate cancer cell lines on HPN functions in prostate cancer suggest that HPN over expression might represent some type of protective feed back response. HPN may have anti tumorigenic effects on cancer prostate cells and it is possible that HPN might degrade the collagen of the tumour extracellular matrix thereby inhibiting cell migration. [12]

The results of cent percent positivity in prostate cancer cases in the present study, in a situation where IHC may vary with formalin fixation and tissue processing, are intriguing, underscoring the significance of HPN gene expression in prostate cancer. A possible explanation for consistently high expression of HPN could be because majority of prostate cancer cases in the present study were advanced cancer with high serum PSA levels. This is in contrast to the patient population in the west where reported serum PSA levels are comparatively low and cancers are detected at an earlier stage as compared to Indian patients. A long term prospective study in a large series of cases is warranted comparing HPN expression in early cancer, BPH with PIN and advanced prostate cancer with high Gleason score, androgen refractory, and Prostate specific antigen failure cases. This might stratify the candidate prostate cancer patients suitable for monitoring and future targeted therapy. [15],[16] HPN may emerge as a prognostic marker of prostate cancer and also a molecule for targeted therapy. Anatomic pathologist will play a significant role in monitoring of these cases.

 
   References Top

1.Emerging biomarkers for the detection and management of prostate carcinoma. chapter 19, Biopsy Interpretation Series. Biopsy Interpretation of the Prostate. In: Epstein JI and Netto GJ, editors, 4 th ed., USA: Lippincott Williams and Wilkins; 2008.p.330-9.  Back to cited text no. 1
    
2.Epstein JI, Potter SR. The pathologic interpretation and significance of prostate needle biopsy findings: Implications and current controversies. J Urol 2001;166:402-10.  Back to cited text no. 2
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3.Stamey TA, Caldwell M, McNeal JE, Nolley R, Hemenez M, Downs J. The prostate specific antigen era in the United States is over for prostate cancer: What happened in the last 20 years? J Urol 2004;172:1297-301.  Back to cited text no. 3
    
4.Polascik TJ, Oesterling JE, Partin AW. Prostate specific antigen: A decade of discovery-what we have learned and where we are going. J Urol 1999;162:293-306.  Back to cited text no. 4
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5.Sardana G, Dowell B, Diamandis EP. Emerging biomarkers for the diagnosis and prognosis of prostate cancer. Clin Chem 2008;54:1951- 60.  Back to cited text no. 5
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6.Dhanasekaran SM, Barrette TR, Ghosh D, Shah R, Varambally S, Kurachi K, et al. Delineation of prognostic biomarkers in prostate cancer. Nature 2001;412:822-6.  Back to cited text no. 6
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7.Magee JA, Araki T, Patil S, Ehrig T, True L, Humphrey PA, et al. Expression profiling reveals hepsin overexpression in prostate cancer. Cancer Res 2001;61:5692-6.  Back to cited text no. 7
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8.Kelly KA, Setlur SR, Ross R, Anbazhagan R, Waterman P, Rubin MA, et al. Detection of early prostate cancer using a hepsin-targetted imaging agent. Cancer Res 2008;68:2286-91.  Back to cited text no. 8
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9.Luo J, Duggan DJ, Chen Y, Sauvageot J, Ewing CM, Bittner ML, et al. Human prostate cancer and benign prostatic hyperplasia molecular dissection by gene expression profiling. Cancer Res 2001;61:4683-8.  Back to cited text no. 9
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10.Klezovitch O, Chevillet J, Mirosevich J, Roberts RL, Matusik RJ, Vasioukhin V. Hepsin promotes prostate cancer progression and metastasis. Cancer Cell 2004;6:185-95.  Back to cited text no. 10
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11.Pal P, Xi H, Kaushal R, Sun G, Jin CH, Jin L, et al. Variants in the Hepsin genes are associated with prostate cancer in men of the European origin. Hum Genet 2006;120:187-92.  Back to cited text no. 11
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12.Srikantan V, Valladares M, Rhim JS, Moul JW, Srivastava S. Hepsin inhibits cell growth/invasion in prostate cancer cells. Cancer Res 2002;62:6812- 6.  Back to cited text no. 12
    
13.Xuan JA, Schneider D, Toy P, Lin R, Newton A, Zhu Y, et al. Antibodies neutralizing hepsin protease activity do not impact cell growth but inhibit invasion of prostate and ovarian tumor cells in culture. Cancer Res 2006;66:3611-9.  Back to cited text no. 13
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14.Wu Q, Parry G. Hepsin and prostate cancer. Front Biosci 2007;12:5052-9.  Back to cited text no. 14
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15.Chevillet JR, Park GJ, Bedalov A, Simon JA, Vasioukhin VI. Identification and characterization of small-molecule inhibitors of hepsin. Mol Cancer Ther 2008;7:3343-51.  Back to cited text no. 15
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16.Huang HC, Zheng S, VanBuren V, Zhao Z. Discovering disease-specific biomarker genes for cancer diagnosis and prognosis. Technol Cancer Res Treat 2010;9:219-30.  Back to cited text no. 16
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Correspondence Address:
Madhu Mati Goel
Department of Pathology, CSM Medical University, Lucknow, UP 226 003
India
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DOI: 10.4103/0377-4929.85078

PMID: 21934206

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    Figures

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    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6], [Table 7]

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