Archival Prostate Cancer Research Articles

Prostate cancer immunohistochemical (IHC) stains and survival in stage D3 patients (pts)

22187 Background: We investigated expression of pathways in archival prostate cancer biopsies by IHC staining. Methods: In an IRB approved protocol, primary prostate cancer specimens were obtained from archival formalin-fixed, paraffin-embedded tissue collected between 1992 and 2006 at VA New Jersey Health Care System. The most representative tumor tissue block was chosen from each of the 42 cases evaluated. ICH stains to detect tumor expression of S6 (ribosomal), p70s6, pTEN, AKT-1, BCL-1 (Cyclin D1), VEGF, c-KIT (CD117), PDGFR-alpha (C-20), and PDGFR-beta (P20) were done by US LABS (Irvine, CA). All immunostains were evaluated by two pathologists. Immunoreactivity was scored using a semiquantitative system for intensity of staining and percentage of positive cells. Staining intensity was scored as 0 (no detectable stain), 1 (weak staining detected at intermediate to high power), 2 (moderate detected at low to intermediate power) to 3 (strong detected at low power). Percentage of positive cells was scored as 0 (0%), 1 (1–33% positive cells), 2 (34–66%), or 3 (67–100%). The total score was obtained by adding the scores of intensity and percent of positive cells. Medical records were reviewed for dates of diagnosis, and demographic and laboratory information. A Cox survival model for each stain was developed with known survival variables, Gleason score, Hemoglobin (Hgb), Alkaline Phosphatase (Alk Phos), Prostate Specific Antigen (PSA), Lactate Dehydrogenase (LDH) levels at the time of D3 stage, and the ICH grade. Results: Data from 39 patients were available. The median, age was 75 yrs (range 56–94), Gleason score was 8(6–10), LDH was 186(134–899), Hgb was 12.3(6.3–15.5), PSA was 66(0.4–2675), Alk Phos was 121(45–1119).The median survival was 204 days (18–2085). None of the stains was predictive of survival on a univariate or multivariate analysis. Conclusions: The over expression of S6, p70s6, pTEN, AKT-1, BCL-1, VEGF, c-KIT, PDGFR-alpha and PDGFR-beta by ICH on archival tissue was not predictive of survival. No significant financial relationships to disclose.

Correlation between prostate cancer immunohistochemical stains (IHC) and survival in stage D2 patients(pts)

16093 Background: The expression of 9 signaling transduction pathways, important for angiogenesis and apoptosis was identified by IHC in archival prostate cancer biopsies and correlated with survival in pts with metastatic disease. All pts had androgen deprivation and were followed at 3 month intervals. Methods: In an IRB approved protocol, 42 pts with paraffin-embedded tissue collected between 1992 and 2006 at our Institution were identified for chart review. The most representative tumor tissue block was chosen for each of the 42 cases evaluated. IHC stains to detect tumor expression of S6 (ribosomal), p70s6, pTEN, AKT-1, BCL-1 (Cyclin D1), VEGF, c-KIT (CD117), PDGFR-alpha (C-20), and PDGFR-beta (P20) were done by US LABS (Irvine, CA). All results were independently evaluated by two pathologists. Immunoreactivity was scored using a semiquantitative system combining intensity of staining(0–3+) and percentage of cells staining positive(0–3+). The total score was obtained by adding the scores for intensity and the percentage of positive cells, then averaging the results obtained by each reader. A Cox regression survival model for each stain was developed with variables known to predict survival: Gleason score, Hemoglobin (Hgb), Alkaline Phosphatase (Alk Phos), Prostate Specific Antigen (PSA), Lactate Dehydrogenase (LDH) levels. Results: The medians were: age 70 yrs(56–92), Gleason score 8(6–10), LDH 171 IU/L(97–350), Hgb 12.9 gm/dl(6.8–16.3), PSA 188 ng/ml(2–5677), Alk Phos 139 U/L(60–1756), survival 851 days(163–6102). In univariate analysis, VEGF staining was predictive of D2 survival (p<0.037) but not in multivariate analysis. None of the other stains were predictive of survival. Conclusions: In this sample the overexpression of S6,p70s6,pTEN,AKT-1,BCL-1,VEGF,c-KIT,PDGFR-alpha and PDGFR-beta by IHC staining in archival tissue is not independent predictors of survival. The VEGF staining, however, was predictive for survival in univariate analysis. No significant financial relationships to disclose.

Cyclooxygenase‐2 (cox‐2) expression is an independent predictor of prostate cancer recurrence

Lack of reliable prognostic markers hinders accurate prediction of disease progression in prostate cancer. The inducible proinflammatory enzyme cyclooxygenase-2 (COX-2) is implicated in prostate carcinogenesis, but its role in cancer progression is less clear. We examined whether COX-2 expression evaluated by immunohistochemistry (IHC) in radical prostatectomy (RP) specimens can predict biochemical recurrence. Archival prostate cancer specimens (n = 60) were obtained from patients who underwent RP, but had not received neoadjuvant hormonal therapy. Twenty-three patients had biochemical or clinical recurrence (mean time of recurrence: 38.2 months), and 37 patients were recurrence free (mean follow-up: 95 months). COX-2 expression was determined by IHC, using an anti-COX-2 antibody. Three individuals scored the staining independently, as high- or low-expression. COX-2 was expressed in prostate cancer cells, in adjacent normal glands and in specimens from patients who later progressed. At 62-months follow-up, COX-2 staining predicted progression with 82.4% sensitivity and 81.3% specificity. Sensitivity (86.4%) and specificity (86.7%) improved at > or = 100-months follow-up. In univariate analysis, Gleason score, preoperative PSA, extraprostatic extension, margin, seminal vesicle invasion, and high COX-2 expression were significant predictors of biochemical recurrence (p < 0.05). In multivariate analysis, preoperative PSA (hazard ratio/unit PSA change 1.080; p = 0.0036) and COX-2 expression (hazard ratio 16.442; p < 0.0001) were independent prognostic indicators. Patients with PSA > 7 ng/ml and high COX-2 expression had the highest probability of recurrence (Kaplan-Meier analysis). COX-2 expression is an independent predictor of prostate cancer progression following RP and underscores the significance of inflammatory factors in this process.

Somatic Mosaicism and Cancer: A Micro-Genetic Examination into the Role of the Androgen Receptor Gene in Prostate Cancer

Recent evidence has shown that the androgen receptor (AR) plays a major role in all prostate cancer stages, including both androgen-dependent and -independent tumors. A large number of studies have examined the possible effects of a functional polymorphism in the AR gene, a variable-length CAG repeat, on the development of prostate cancer, but the results to date have been inconclusive. We have considered the fact that the tissue heterogeneity present in almost all prostate cancer tumors has rarely been regarded as an indicator of AR genetic heterogeneity. To determine if genetic heterogeneity exists and is a significant event in prostate cancer development, we have examined prostate cancer tumors for somatic shortening of the AR gene CAG repeat. All 72 laser capture microdissected samples from archival prostate cancer tissues, as well as samples from freshly prepared prostate cancer tissues, showed some genetic heterogeneity (somatic mosaicism) for AR CAG repeat length. Cancerous tissues showed a much greater degree of genetic heterogeneity than adjacent benign tissues, as well as a very significant shortening of their CAG repeat lengths. However, CAG repeat length heterogeneity was not observed in normal prostate tissues. It is hypothesized that somatic mosaicism of the AR CAG repeat in prostate cancer tumors may be found to be an important genetic event in precancerous tissue, which may subsequently lead to the development of prostate cancer.

FGF8 isoform b expression in human prostate cancer.

Overexpression of fibroblast growth factor 8 (FGF8) mRNA has been previously described in prostate cancer. Of its four isoforms, FGF8b is thought to be the most important in carcinogenesis. We hypothesised that immunodetection of FGF8b in archival prostate cancer specimens is of potential prognostic value. Using a selected cohort of prostate tumours from transurethral (n=30) and radical prostatectomies (n=59), an optimised protocol for FGF8b immunoreactivity was used to corroborate expression with clinical parameters. No expression was observed in benign prostates (n=10). In prostate cancer, immunoreactivity was localised to the malignant epithelium with weak signals in the adjacent stroma. Expression of FGF8b in stage T1 and T2 cancers were 40 and 67%, respectively. In contrast, FGF8b expression was present in 94% of T3 and 100% of T4 cancers. By histological grade, FGF8b was found in 41% of low-grade cancers (Gleason score 4–6), 60% of intermediate-grade cancers (Gleason score 7 and 92% of high-grade cancers (Gleason score 8–10). The intensity of expression was significantly associated with stage (P=0.0004) and grade (P<0.0001) of disease. We further hypothesised that FGF8b overexpression resulted from enhanced transcription and translation rather than from abnormalities involving the FGF8 gene locus. This was tested by means of fluorescent in situ hybridisation in 20 cancer specimens to map the FGF8 gene locus. FGF8 gene copy number in benign and malignant nuclei was found to be similar (2.33±0.57 and 2.0±0.81, respectively P=0.51). Based on these findings, we propose a multicentre study on cohorts of patients to further evaluate FGF8b as a potential prognostic marker in prostate cancer.

Application of a yeast assay to detect functional p53 mutations in archival prostate cancer tissue.

Detection and functional evaluation of mutant p53 alleles using a yeast assay could yield significant information for predicting the prognosis of patients with prostate cancer (CaP). Since the current version of this yeast assay is not applicable to archival tissues, we developed a modified assay for use on formalin-fixed, paraffin-embedded tissue and have applied it to the study of patient samples. Using this modified assay, we examined archival CaP samples from 10 patients for mutations in exons 5-8 of p53 gene. Mutations were detected in four samples: three resulted in the formation of red yeast colonies indicating complete loss of function, while one gave pink yeast colonies, indicating that this mutant retained partial function. In parallel, we analyzed these samples for p53 abnormalities using a single-strand conformational polymorphism (SSCP) approach. Only three of the four yeast-positive samples gave abnormal SSCP bands. In each case where abnormal p53 was found by both methods, DNA sequencing revealed the identical base change. These results suggest that the modified yeast assay may be more sensitive than SSCP for detection of p53 mutations, and demonstrate that the modified method can be used to detect and evaluate the function of p53 mutants present in archival tissue.

HER-2/neu oncogene amplification in prostate cancer

Oncogene amplification is a manifestation of genetic instability, which has been implicated in the pathogenesis of many cancers. Work performed in this laboratory has focused on HER-2/neu amplification in breast cancer, ovarian cancer, cervical cancer and rhabdomyosarcoma, all of which revealed HER-2/neu amplification in at least a proportion of tumors. In the present project, we studied HER-2/neu oncogene amplification in prostate carcinoma, 244,000 new cases of which are diagnosed yearly, and which affects a significant number of men. Fluorescent in situ hybridization (FISH) using the Vysis HER-2/neu probe with a chromosome 17 centromere control probe was performed on formalin-fixed paraffin-embedded archival prostate cancer tissues. Out of a total of 85 cases studied, 7 cases (8.2%) were found to be amplified. Of those 7 amplified cases, 2 were also trisomic for chromosome 17. Amplification is defined as a signal ratio of HER-2/neu to chromosome 17 of >1.5, after Mark et al. (1998). Clinicopathologic characteristics of the amplified cases versus the nonamplified cases will be compared. Although the results of the present study still need to be confirmed and extended by additional cases in other centers, the data thus far do indicate that a small subset of prostate cancer is characterized by HER-2/neu gene amplification, as in other cancers. (This study was partially funded by Vysis, Inc., Downers Grove, IL).

Immunohistochemical determination of tumor angiogenesis measured by the maximal microvessel density in human prostate cancer.

Tumor growth beyond a certain size requires angiogenesis. Experimental evidence shows that once tumors leave the pre-angiogenic phenotype to become angiogenic, metastases often start to evolve. The aim of this study was to develop a reproducible immunohistochemical technique and method to characterize the neovascularization in archival prostate cancer tissue by quantifying the microvessel density (MVD). Archival tumor specimens from 64 consecutively diagnosed prostate cancer patients were immunostained for von Willebrand Factor (vWF), endothelial antigen and for CD31 combined with the use of different digestive enzymes (trypsin and pronase) and heating in a microwave oven. Both the mean and the maximal MVD, and the reproducibility of the method were estimated. Finally, the mean MVD, the maximal MVD, and clinical characteristics were correlated with the crude survival of the patient population. The immunohistochemical staining for vWF to measure the maximal MVD was found to be a reproducible method of characterizing the individual tumor. Both a univariate and a multivariate analysis demonstrated that the maximal MVD, in contrast to the mean MVD, was significantly associated with survival in prostate cancer patients. We conclude that evaluation of angiogenesis by immunostaining the endothelial cells for vWF measured by the MVD in the most vascularized areas of the tumor is a reproducible method of characterizing the individual prostate tumor. Maximal MVD proved to be an independent prognostic parameter useful in conjunction with other known prognostic markers in human prostate cancer.

HER-2/ neu gene amplification status in prostate cancer by fluorescence in situ hybridization

HER-2/ neu expression has been established as a prognostic factor in breast and other cancers. In prostate cancer (PC), a similar predictive role has been hindered by variable immunohistochemical (IHC) results. The authors studied DNA amplification of the HER-2/ neu gene on 4-μm sections obtained from 62 formalin-fixed, paraffin-embedded PCs by fluorescence in situ hybridization (FISH). The results were compared with HER-2/ neu protein expression as determined by IHC and correlated by logistic regression analysis with Gleason tumor grade, DNA ploidy, serum prostate specific antigen (PSA), and pathological stage. The HER-2/ neu gene was localized using the Oncor (Gaithersburg, MD) digoxigenin-labeled unique sequence probe. Amplified PCs had at least 20 malignant cells, with 5 or more copies of the sequence. Amplification of HER-2/ neu correlated with Gleason score ( P = .0001). The mean Gleason score of unamplified tumors was 5.7 and that of amplified tumors was 7.5. Nondiploid tumors had a significantly greater rate of HER-2/ neu amplification compared with diploid tumors ( P = .0003). Of the 62 cases evaluated by IHC and FISH, 18 cases (29%) were overexpressed by IHC, and 27 cases (44%) were amplified by FISH. A trend for similar HER-2/ neu status in each PC by the two methods did not reach statistical significance ( P = .23). HER-2/ neu amplification by FISH was associated with advanced pathological stage; however, this relationship reached only near-statistical significance ( P = .06). There was no correlation of HER-2/ neu amplification by FISH with patient age or preoperative serum PSA levels. The authors conclude that HER-2/ neu gene amplification status can be determined by FISH on archival prostate cancer specimens, significantly correlates with high tumor grade and nondiploid DNA content, and is more frequently encountered in tumors with advanced pathological stage. Also, FISH is more sensitive than IHC for detection of abnormalities in the HER-2/ neu gene, and further studies should be undertaken to determine whether a FISH-based HER-2/ neu detection method may prove of importance in the prediction of prognosis and planning of therapy in prostate cancer patients.

Vascular Endothelial Growth Factor (VEGF) Expression in Human Prostate Cancer: In Situ and in Vitro Expression of VEGF by Human Prostate Cancer Cells

Purpose A growing body of literature supports the role of angiogenesis in the development and spread of a variety of human cancers including prostate cancer (Pca). Angiogenesis is controlled by chemical signals known as angiogenic factors (AF) however, little is known about angiogenesis factors in prostate cancer. We evaluated the in situ and in vitro expression of vascular endothelial growth factor (VEGF), a potent angiogenic factor, in archival prostate cancer specimens and prostate cancer cell cultures during unstimulated and cytokine stimulated conditions. Methods Ex-vivo studies involved immunohistochemical analysis for VEGF expression and distribution in 25 archival specimens including, prostate cancer, benign prostatic hyperplasia (BPH) and normal prostate tissue. In-vitro studies utilized prostate cancer cells (DU-145) grown in culture and stimulated with cytokines thought to induce VEGF (i.e. IL-1 alpha, IL-1 beta, TNF-alpha and TNF-beta). Cell culture supernatants were analyzed by ELISA for VEGF levels. Results Immunohistochemical studies demonstrated that in 20 of 25 specimens prostate cancers cells stained positively for VEGF. BPH and normal prostate cells displayed little staining for VEGF. DU-145 prostate cancer cells produced low levels of VEGF in unstimulated conditions. Induction of DU-145 cells with cytokines resulted in differential stimulation whereby TNF was a potent inducer of VEGF, and IL-1 produced lesser but statistically significant increases in VEGF expression. Conclusions Our immunohistochemical results indicate that significant levels of VEGF are present in prostate cancer, but not in BPH or normal prostate cells in-vivo. In-vitro studies suggests that differential regulation of angiogenesis factor expression by IL-1 and TNF occurs in prostate cancer. Identifying the angiogenesis factors involved in prostate cancer growth and understanding their regulation will lead to the development of anti-angiogenic strategies useful for diagnostic studies and therapeutic interventions.

Presence and enzymatic activity of prostate-specific antigen in archival prostate cancer samples

Patients with advanced prostate cancer frequently have a poor prognosis as a result of metastasis. The serum prostate-specific antigen (PSA) test is widely used for the diagnosis of prostate cancer. The enzymatic activity of PSA may be involved in the invasion of prostate cancer. We set out to determine the prevalent form of PSA in human prostate adenocarcinoma samples by ELISA and Western blot analysis and its enzymatic activity using a synthetic substrate S-2586 and fibronectin. Our results show that in serum from prostate cancer patients and in tumour homogenates, the prevalent form was PSA bound to alpha1-antichymotrypsin. All homogenates showed enzymatic activity towards a synthetic PSA substrate, whereas only five samples showed activity at 28 kDa towards fibronectin as determined by enzymography, which is most likely due to active PSA. Human prostate cancer LNCaP cells produced largely inactive PSA. In comparison, 22Rv1 cells produced 29-fold less PSA, but with high specific activity. Similarly, our results from the human prostate cancer tissue samples also show that free PSA appears to exist in diverse forms of very different specific activity. Since PSA, as a serine protease, may be involved in the invasion of prostate cancer, our results suggest that prostate cancers have potentially diverse invasive capacity due to differences in specific enzymatic activity of PSA.

Human papillomavirus types 16 and 18 are not involved in human prostate carcinogenesis: analysis of archival human prostate cancer specimens by differential polymerase chain reaction.

Human papilloma viruses (HPV) have been implicated in the pathogenesis of a variety of malignancies, especially in carcinomas of the female genital tract. Recently, based on observations using the polymerase chain reaction amplification assay, HPV types 16 and 18 specific DNA sequences have been detected in prostate cancer specimens obtained by transurethral resection. Since HPV types 16 and 18 have been shown to possess oncogenic potential, an association between HPV infection and prostatic carcinoma has been suggested. In order to exclude potentially HPV-colonized urethral mucosa from analysis and restrict our study to predominantly malignant tissue, cancerous areas from a series of 30 paraffin-embedded prostate adenocarcinomas were microdissected and analyzed for the presence of HPV 16 or HPV 18 specific sequences by a modification of PCR (D-PCR) and Southern blot analysis. Despite the high sensitivity of our analytical technique, we found no evidence of HPV-DNA of either type in any of the 30 primary prostate cancers. In contrast, both HPV 16 (2/8 specimens) and HPV 18 (2/8 specimens) DNA was detected in randomly chosen cervical carcinomas using the D-PCR methodology. Our data would indicate that the oncogenic HPV-types 16 and 18 are unlikely effectors of prostate carcinogenesis.