prognostic significance of epithelial stromal caveolin 1 expression in prostatic hyperplasia high grade prostatic intraepithelial hyperplasia and prostatic carcinoma and its correlation with microvessel density

Journal of the Egyptian National Cancer Institute xxx (2017) xxx–xxx Contents lists available at ScienceDirect Journal of the Egyptian National Cancer Institute journal homepage: www.sciencedirect.com Full length article Prognostic significance of epithelial/stromal caveolin-1 expression in prostatic hyperplasia, high grade prostatic intraepithelial hyperplasia and prostatic carcinoma and its correlation with microvessel density Dareen A Mohammed, Duaa S Helal ⇑ Department of Pathology, Faculty of Medicine, Tanta University, Egypt a r t i c l e i n f o Article history: Received 18 October 2016 Received in revised form January 2017 Accepted 10 January 2017 Available online xxxx Keywords: Caveolin-1 Prostatic carcinoma BPH HGPIN Microvessel density CD34 a b s t r a c t Caveolin-1 may play a role in cancer development and progression The aim was to record the expression and localization of caveolin-1 in benign prostatic hyperplasia (BPH), high grade prostatic intraepithelial neoplasia (HGPIN) and prostatic carcinoma (PCa) Microvessel density was evaluated with CD34 immunostain Correlations with known prognostic factors of PCa were recorded Immunohistochemical expression of caveolin-1 and the MVD was evaluated in 65 cases; BPH (25), HGPIN (20) and PCa (20) Stromal caveolin-1expression was significantly higher in BPH than HGPIN and PCca There was significant inverse relation between stromal caveolin-1 expression and extension to lymph node and seminal vesicle in carcinoma cases Epithelial caveolin-1 was significantly higher in carcinomas than in BPH and HGPIN Epithelial expression in carcinoma was significantly associated with preoperative PSA, Gleason score and lymph node extension MVD was significantly higher in PCa than in BPH and HGPIN There were significant relations between MVD and preoperative PSA, Gleason score, lymph node and seminal vesicle extension Stromal caveolin-1 was associated with low MVD while epithelial caveolin-1 with high MVD Conclusions: Caveolin-1 plays an important role in prostatic carcinogenesis and metastasis Stromal expression of caveolin-1 in PCa is lowered in relation to BPH and HGPIN In PCa; stromal caveolin-1 was associated with good prognostic parameters Epithelial caveolin-1 is significantly increased in PCa than BPH and HGPIN It is associated with clinically aggressive disease Caveolin-1 may play a role in angiogenesis Ó 2017 National Cancer Institute, Cairo University Production and hosting by Elsevier B.V This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/) Introduction Prostatic carcinoma (PCa) is the second diagnosed malignancy and the sixth cause of cancer mortality in men Incidence rates vary more than 25-fold worldwide, with the highest rates in the developed countries this may be due to the widespread use of prostatespecific antigen screening and prostatic tissue biopsy [1] Overall, the complex morphology, histologic heterogeneity, and the early metastatic ability of localized PCa show the requirement for extra clinical and pathologic tests for the evaluation of PCa and its early diagnosis [2] Distant spread of primary malignant cells to local and distant sites was explained by several models Cumulative research shows a pivotal role of tumor microenvironment in the process of malig- Peer review under responsibility of The National Cancer Institute, Cairo University ⇑ Corresponding author at: Faculty of Medicine, Tanta University, El Geish Street, Tanta, Egypt E-mail address: Duaahelal@yahoo.com (D.S Helal) nant cell dissemination This role occurs through angiogenesis and epithelial-stromal interactions which induce an epithelialmesenchymal transition which result in increased migration of epithelial cells [3] Caveolin protein is an important component of caveolae structure that are found in smooth muscle cells, adipocytes and endothelium Caveolin-1 was overexpressed in mouse and human metastatic malignant prostate cells Moreover, decreased caveolin1 expression with stably transfected antisense caveolin-1 cDNA converted androgen-insensitive metastatic mouse prostate cancer cells to an androgen-sensitive phenotype [4] Accordingly, Caveolin-1 was considered a metastasis-related gene as well as a representative gene for hormone-resistant human prostate cancer Recent studies suggested a possible role for caveolin-1 in the resistance of various malignances to multiple antineoplastic drugs [5] Previous reports have shown different roles of caveolin protein in tumor stroma and epithelium In breast cancer, absence of stromal caveolin-1 is associated with ductal carcinoma in situ (DCIS) progression and metastatic breast carcinoma [6] While in PCa, http://dx.doi.org/10.1016/j.jnci.2017.01.002 1110-0362/Ó 2017 National Cancer Institute, Cairo University Production and hosting by Elsevier B.V This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/) Please cite this article in press as: Mohammed DA, Helal DS Prognostic significance of epithelial/stromal caveolin-1 expression in prostatic hyperplasia, high grade prostatic intraepithelial hyperplasia and prostatic carcinoma and its correlation with microvessel density J Egyptian Nat Cancer Inst (2017), http://dx.doi.org/10.1016/j.jnci.2017.01.002 D.A Mohammed, D.S Helal / Journal of the Egyptian National Cancer Institute xxx (2017) xxx–xxx mouse models studies found overexpressed epithelial caveolin-1 gene [7] Angiogenesis is common denominator between benign prostatic hyperplasia (BPH) and PCa It is the process of new blood vessel formation Angiogenesis is important for tumor growth and metastasis in various malignancies Non-vascular cells in tumoral microenvironments such as macrophages, mast cells and fibroblasts can regulate angiogenesis by secreting angiogenic factors Microvessel density (MVD) is an indicator of the neoangiogenetic process, which helps local growth and distant metastases of malignant cells Microvessel density (MVD) denotes the number of small blood vessels in BPH, high grade prostate intraepithelial neoplasia (HGPIN) and PCa and is evaluated by endothelial marker CD34 immunostaining [8] Aim of the study was to record the expression and localization of caveolin-1 in BPH, HGPIN and PCa Microvessel density was evaluated with CD34 expression Correlations with known prognostic factors of PCa were recorded Material and methods The present retrospective study included 25 cases of BPH, 20 cases of HGPIN and 20 cases of PCa surgically removed by radical prostatectomy Formalin fixed paraffin embedded blocks were obtained from archives of pathology department, Tanta university in the period from 2012 till 2015 Data about preoperative PSA, Gleason score, seminal vesicle invasion and lymph node metastasis were obtained from clinical and pathologic files of the patients All cases were subjected to routine hematoxylin and eosin staining and examined by two pathologists to confirm the diagnosis Immunohistochemical procedures Heat-mediated antigen retrieval was performed using a citrate buffer after initial deparaffinization and rehydration Afterwards to inactivate endogenous peroxidase, sections were treated for 10 with 2% hydrogen peroxide and blocked with 3% normal goat serum in 0.2 M phosphate-buffered saline, then incubated for h with rabbit polyclonal antibody to caveolin-1 (antiCaveolin-1 antibody (ab2910) Abcam UK, dilution of 1:10000) and CD-34 (Rabbit monoclonal antibody Biogenex, San Ramon, CA, USA, diluted 1:150) Standard avidin–biotin immunostaining was performed Epithelial caveolin-1 evaluation Sections were evaluated at a high power Positive epithelial caveolin-1 staining was considered if 50% of the tumor cells in any microscopic field showed caveolin-1 positive granular brown stain in their cytoplasm Stromal caveolin-1 evaluation Caveolin-1 stromal expression was scored as negative (0; no staining), weak (1; either diffuse weak staining or strong staining in less than 30% of stromal cells per core), or strong (2; defined as strong staining of 30% or more of the stromal cells) MVD evaluation by CD34 expression Tumor sections stained for CD34 were examined using computer-aided image analysis [ImageJ software (version 4.10.03, Nikon, Tokyo, Japan)] to count CD34 +ve vessels per high-power field 4–5 areas that had the greatest density of positively stained vessels (hot spots) were labeled The median value of microvessel count (MVC) was 90 microvessels For statistical analysis, MVD was divided into two groups: low (median or less) and high (greater than the median) Statistical analysis of the collected data Results were collected, tabulated and statistically analyzed with SPSS statistical package version 20 (SPSS Inc Released 2011 IBM SPSS statistics for windows, version 20.0, Armnok, NY: IBM Corp.) Two types of statistical analysis were done: a) Descriptive statistics e.g was expressed in: Number (No), percentage (%) mean ( x) and standard deviation (SD) b) Analytic statistics e.g - Chi-square test (v2) was used to study association between qualitative variables Whenever any of the expected cells were less than five, Fischer’s Exact test was used - P-value of 0.05) Stromal caveolin-1 positive tumors had no extension to lymph nodes or seminal vesicles There were significant inverse relations between stromal caveolin-1 expression and lymph node metastasis and seminal vesicle extension Analysis of epithelial caveolin-1 immunostaining In studied cases of BPH, 20% were positive for epithelial caveolin-1 Whereas; 60% of HGPIN cases and 90% of PCa cases were positive Epithelial caveolin-1 expression was significantly higher in PCa than HGPIN and BPH (p value < 0.001) (Table 1) (Fig 2) The association between epithelial caveolin-1 expression and the clinicopathologic data are shown in Table Cases with high preoperative PSA exhibited higher epithelial caveolin-1 expression then low and moderate groups This relation was statistically significant (p value < 0.05) Epithelial caveolin-1 positivity was Please cite this article in press as: Mohammed DA, Helal DS Prognostic significance of epithelial/stromal caveolin-1 expression in prostatic hyperplasia, high grade prostatic intraepithelial hyperplasia and prostatic carcinoma and its correlation with microvessel density J Egyptian Nat Cancer Inst (2017), http://dx.doi.org/10.1016/j.jnci.2017.01.002 D.A Mohammed, D.S Helal / Journal of the Egyptian National Cancer Institute xxx (2017) xxx–xxx Table Immunostaining results of caveolin-1 and CD34 MVD Stromal caveolin-1 BPH (25 cases) PIN (20 cases) PC (20 cases) v2 P value * Epithelial caveolin-1 MVD +ve Àve +ve Àve High Low 22 (88%) (25%) (15%) 29.03 10 were positive for epithelial caveolin-1 Our results showed a significant direct correlation of epithelial caveolin-1 expression with the preoperative PSA level These results coincided with others [16] yet contradicted with others’ [2] In our study, epithelial caveolin-1 expression in PCa was correlated with tumor grade Epithelial caveolin-1 was overexpressed in PCa with higher Gleason score compared to those with moderate and low Gleason score Our results were concordant with previous reports [2,16,17] confirming that epithelial caveolin-1 plays an important role in prostatic carcinogenesis Moreover, previous experimental studies of PCa in mouse models identified overexpressed epithelial caveolin-1 gene and demonstrated that decreased levels of caveolin initiated apoptosis [7] This study showed that epithelial caveolin-1 expression was significantly associated with lymph node metastasis which demonstrates the possible role of epithelial caveolin-1 in promoting tumor growth and metastasis as reported by others [17–19] Our results showed an inverse relation between stromal and epithelial caveolin-1 Stromal expression of caveolin-1 was associated in our study with good prognostic parameters On the other hand, epithelial expression of caveolin-1 was correlated with clinically aggressive disease with higher preoperative PSA & Gleason score and extension to lymph node Our result was in line with previous publication [19,20] This suggests that caveolin plays different roles in prostatic carcinogenesis according to its location which N % 10 P = 0.93 10.0 70.0 Low (4) 5.0 15.0 P = 0.87 75.0 5.0 15.0 5.0 P = 0.85 lead to failed treatment and may be an important therapeutic target Angiogenesis is the formation of new blood vessels from preexisting ones Endothelial marker (CD34) has been used to assess angiogenesis in different cancer stages [21] CD34 immunostain was used to evaluate the Microvessel density (MVD) in the studied cases of BPH, HGPIN and PCa In agreement with others [22–24], MVD was significantly higher in carcinoma (80% with high MVD) compared to HGPIN (70%) and BPH (28%) Within the carcinoma cases; All cases with high preoperative PSA showed high MVD while only one case (25%) with low preoperative PSA showed high MVD A significant direct correlation was found between high MVD and the preoperative PSA levels This finding coincided with others [25] In contrast, a study has shown no relationship [26] Regarding tumor differentiation; In agreement with previous publications [13,22,25,26] MVD was higher in carcinoma cases with high Gleason score (92.3%) while all cases with low Gleason score (100%) showed low MVD This result denotes a significant direct correlation of MVD with the degree of tumor differentiation Among carcinoma cases included; this study found a significant elevation of CD34 +ve MVD in cases with lymph node and seminal vesicle extension These results were in agreement with Steiner et al whom reported that the density of CD34-marked microvessels showed significant associations with pT stage (P = 0.017) [13] Also, Nassif and Filho whom reported that the higher the MVD, the higher the stage (p < 0.0001) and the chance of recurrence (p = 0.002) in PCa [25] However, this relation was not reported by others [26,27] Caveolin-1 plays a role in regulation of endothelial cells proliferation and differentiation Increased caveolin-1 expression and MVD was found to be associated with metastasis and poor prognosis in clear cell renal cell carcinoma, denoting a proangiogenic role for caveolin-1 Previous reports provided evidence for a proangiogenic role of caveolin-1 in PCa [28] In the present study, 75% of epithelial caveolin-1 positive tumors showed high MVD by CD34 immunostain, while only 15% of epithelial caveolin-1 positive tumors showed low MVD This finding was in agreement with Yang et al who used dual immunofluorescent staining with CD34 and caveolin-1 on 56 cases of PCa obtained by radical prostatectomy They found that positive tumors for epithelial caveolin-1 had a significantly higher MVD than epithelial caveolin-1-negative tumor [28] These positive correlations provide new evidence for the involvement of caveolin-1 in mediating angiogenesis during prostate cancer progression [10] This result supports our previous finding that epithelial expression of caveolin-1 is correlated with clinically aggressive PCa Stromal caveolin-1 was inversely correlated with MVD in PCa cases Most of stromal caveolin-1 negative cases (70%) exhibited high MVD This further proves that loss of stromal Please cite this article in press as: Mohammed DA, Helal DS Prognostic significance of epithelial/stromal caveolin-1 expression in prostatic hyperplasia, high grade prostatic intraepithelial hyperplasia and prostatic carcinoma and its correlation with microvessel density J Egyptian Nat Cancer Inst (2017), http://dx.doi.org/10.1016/j.jnci.2017.01.002 D.A Mohammed, D.S Helal / Journal of the Egyptian National Cancer Institute xxx (2017) xxx–xxx expression of caveolin-1 promotes prostatic carcinogenesis and progression Limitation of the study The main limitation of our study was the small sample size and the lack of survival data (biochemical free survival, overall and disease specific survival) for PCa patients Conclusions Caveolin-1 plays an important role in prostatic carcinogenesis and metastasis Its role is complex and depends on its localization Stromal expression of caveolin-1 in carcinoma is reduced in relation to BPH and PIN In carcinoma; stromal caveolin-1 was associated with good prognostic parameters Epithelial caveolin-1 is significantly increased in carcinoma than BPH and PIN It is associated with clinically aggressive disease Stromal caveolin-1 was associated with low MVD while epithelial caveolin-1 with high MVD This shows a possible role for caveolin-1 in angiogenesis References [1] Jemal A, Bray F, Center MM, Ferlay J, Ward E, Forman D Global cancer statistics CA Cancer J Clin 2011;61(2):69–90 [2] Satoh T, Yang G, Egawa S, Addai J, Frolov A, Kuwao S, et al Caveolin-1 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clinically localized prostate adenocarcinoma after radical prostatectomy Rev Col Bras Cir 2010;37 (5):338–44 [26] Erbersdobler A, Isbarn H, Dix K, Steiner I, Schlomm T, Mirlacher M, et al Prognostic value of microvessel density in prostate cancer: a tissue microarray study World J Urol 2010;28(6):687–92 [27] Ho C, Huang P, Huang H, Chen Y, Yang P, Hsu S Up-regulated caveolin-1 accentuates the metastasis capability of lung adenocarcinoma by inducing filopodia formation Am J Pathol 2002;161(5):1647–56 [28] Yang G, Addai J, Wheeler TM, Frolov A, Miles BJ, Kadmon D, et al Correlative evidence that prostate cancer cell-derived caveolin-1 mediated angiogenesis Hum Pathol 2007;38:1688–95 Please cite this article in press as: Mohammed DA, Helal DS Prognostic significance of epithelial/stromal caveolin-1 expression in prostatic hyperplasia, high grade prostatic intraepithelial hyperplasia and prostatic carcinoma and its correlation with microvessel density J Egyptian Nat Cancer Inst (2017), http://dx.doi.org/10.1016/j.jnci.2017.01.002