Funda Vakar-Lopez

Androgen Receptor Variants Occur Frequently in Castration Resistant Prostate Cancer Metastases

Background Although androgens are depleted in castration resistant prostate cancer (CRPC), metastases still express nuclear androgen receptor (AR) and androgen regulated genes. We recently reported that C-terminal truncated constitutively active AR splice variants contribute to CRPC development. Since specific antibodies detecting all C-terminal truncated AR variants are not available, our aim was to develop an approach to assess the prevalence and function of AR variants in prostate cancer (PCa). Methodology/Principal Findings Using 2 antibodies against different regions of AR protein (N- or C-terminus), we successfully showed the existence of AR variant in the LuCaP 86.2 xenograft. To evaluate the prevalence of AR variants in human PCa tissue, we used this method on tissue microarrays including 50 primary PCa and 162 metastatic CRPC tissues. RT-PCR was used to confirm AR variants. We observed a significant decrease in nuclear C-terminal AR staining in CRPC but no difference between N- and C-terminal AR nuclear staining in primary PCa. The expression of the AR regulated proteins PSA and PSMA were marginally affected by the decrease in C-terminal staining in CRPC samples. These data suggest that there is an increase in the prevalence of AR variants in CRPC based on our ability to differentiate nuclear AR expression using N- and C-terminal AR antibodies. These findings were validated using RT-PCR. Importantly, the loss of C-terminal immunoreactivity and the identification of AR variants were different depending on the site of metastasis in the same patient. Conclusions We successfully developed a novel immunohistochemical approach which was used to ascertain the prevalence of AR variants in a large number of primary PCa and metastatic CRPC. Our results showed a snapshot of overall high frequency of C-terminal truncated AR splice variants and site specific AR loss in CRPC, which could have utility in stratifying patients for AR targeted therapeutics.

Predictors of cancer in repeat extended multisite prostate biopsy in men with previous negative extended multisite biopsy

OBJECTIVES To evaluate the factors influencing the cancer detection rate in men whose initial and repeat biopsies were both performed using an extended multisite biopsy scheme. Sextant biopsy of the prostate is associated with a significant false-negative rate, as evident from the high cancer detection rate after repeat prostate biopsy. Extended multisite biopsy schemes have therefore been recommended to maximize cancer detection. METHODS Between June 1997 and August 2001, 939 men underwent prostate biopsy for early detection of prostate cancer using the extended multisite scheme (10 or 11 cores incorporating the anterior horn of the peripheral zone with or without midline peripheral zone and/or the transition zone). Of these 939 men, 89 (9.5%) underwent a repeat extended multisite prostate biopsy. The median prostate-specific antigen level was 6.9 ng/mL (range 0.7-36.1). Twenty-four men (27%) had an abnormal digital rectal examination at presentation. Most men (86%) in the group undergoing repeat biopsy had two or more risk factors for a positive biopsy. The median interval between biopsies was 4 months. RESULTS Of the 89 men, 15 (17%) had prostate cancer in the repeat biopsy specimen. Seven cancers (47%) were found only in the alternate biopsy sites, 5 (33%) cancers were found only in the sextant sites, and 3 in both sextant and alternate sites. Cancer was present in only one biopsy core in 11 (73%) of the 15 men, and the median Gleason score was 6 (range 6-8). On multivariate analysis, the presence of atypical glands suspicious for carcinoma (AGSC) was the only independent predictor of cancer in repeat biopsy (P <0.004). Of the 79 men without AGSC in the initial biopsy, 8 (10%) had a positive repeat biopsy. The total and percent free prostate-specific antigen level, digital rectal examination, ultrasound findings, and presence of high-grade prostatic intraepithelial neoplasia were not predictive of cancer detection. CONCLUSIONS The probability of a positive result for a repeat prostate biopsy is lower after an initial extended multisite biopsy compared with an initial sextant biopsy. The presence of AGSC was the only significant predictor of cancer in the repeat biopsy. Because nearly 50% of cancers detected in the repeat biopsy were in alternate sites only, using a sextant biopsy scheme for repeat biopsy would have missed these cancers.

Treatment Outcomes of Small Cell Carcinoma of the Prostate A Single-Center Study

The current study was conducted to determine the clinical characteristics and prognostic features associated with prostatic small cell carcinoma (SCC).

The Effects of Aging on the Molecular and Cellular Composition of the Prostate Microenvironment

Background Advancing age is associated with substantial increases in the incidence rates of common diseases affecting the prostate gland including benign prostatic hyperplasia (BPH) and prostate carcinoma. The prostate is comprised of a functional secretory epithelium, a basal epithelium, and a supporting stroma comprised of structural elements, and a spectrum of cell types that includes smooth muscle cells, fibroblasts, and inflammatory cells. As reciprocal interactions between epithelium and stromal constituents are essential for normal organogenesis and serve to maintain normal functions, discordance within the stroma could permit or promote disease processes. In this study we sought to identify aging-associated alterations in the mouse prostate microenvironment that could influence pathology. Methodology/Principal Findings We quantitated transcript levels in microdissected glandular-adjacent stroma from young (age 4 months) and old (age 20–24 months) C57BL/6 mice, and identified a significant change in the expression of 1259 genes (p<0.05). These included increases in transcripts encoding proteins associated with inflammation (e.g., Ccl8, Ccl12), genotoxic/oxidative stress (e.g., Apod, Serpinb5) and other paracrine-acting effects (e.g., Cyr61). The expression of several collagen genes (e.g., Col1a1 and Col3a1) exhibited age-associated declines. By histology, immunofluorescence, and electron microscopy we determined that the collagen matrix is abundant and disorganized, smooth muscle cell orientation is disordered, and inflammatory infiltrates are significantly increased, and are comprised of macrophages, T cells and, to a lesser extent, B cells. Conclusion/Significance These findings demonstrate that during normal aging the prostate stroma exhibits phenotypic and molecular characteristics plausibly contributing to the striking age associated pathologies affecting the prostate.

Bcl-2 IS SIGNIFICANTLY OVEREXPRESSED IN LOCALIZED RADIO- RECURRENT PROSTATE CARCINOMA, COMPARED WITH LOCALIZED RADIO-NAIVE PROSTATE CARCINOMA

PURPOSE We set out to determine whether patients who underwent prostatectomy for recurrence after external-beam radiotherapy for prostate cancer had a higher incidence of alterations in the apoptotic pathway than did patients who underwent surgery as initial treatment. MATERIALS AND METHODS Twenty patients who underwent unsuccessful full-dose external-beam radiotherapy for prostate cancer and subsequently underwent salvage radical surgery (radio-recurrent group), and 20 patients matched for various clinical parameters who underwent only radical prostatectomy for localized or locally advanced prostate cancer (radio-naive group), were studied. Tissue samples were examined for immunoreactivity for p53, p21, Bcl-2, and Ki-67 proteins. Statistically, the two groups were compared using exact logistic regression. RESULTS Fifty-five percent of the tumors from patients initially treated with radiotherapy were noted to overexpress Bcl-2; whereas, in the radio-naive group, no patient had Bcl-2 overexpression (p = 0.0004). More patients who underwent salvage radical surgery were found to have a higher mean proliferative index (Ki-67 staining) (39.6%), compared with patients undergoing prostatectomy alone (22.1%), p = 0.0800. No significant difference was noted in immunohistochemical expression of p53 and p21 between the two groups. CONCLUSIONS Patients undergoing radical prostatectomy after radiotherapy had a significantly higher rate of Bcl-2 overexpression than did patients who underwent surgery as the initial treatment. Alterations in the apoptotic pathway may be important in the development of local recurrence after radiation therapy.

The potential impact of reproducibility of Gleason grading in men with early stage prostate cancer managed by active surveillance: a multi-institutional study.

PURPOSE We evaluated the reproducibility of Gleason grading as relevant to the clinical treatment of men on active surveillance. MATERIALS AND METHODS Three sets of digital images of prostatic adenocarcinoma in biopsies were reviewed and assigned Gleason scores by a total of 11 pathologists from 7 institutions. Interobserver and intra-observer reproducibility were assessed for assignment of the highest Gleason pattern (3 vs 4 or higher). We also identified 97 consecutive patients on active surveillance. Prostate biopsy glass slides from 82 of the patients were available for re-review and the frequency of carcinoma requiring the distinction of tangentially sectioned Gleason pattern 3 from 4 was determined. RESULTS Interobserver reproducibility for classic Gleason patterns was substantial (Lights κ 0.76). Interobserver reproducibility for the histological distinction of tangentially sectioned Gleason pattern 3 from Gleason pattern 4 was only fair (Lights κ 0.27). Intra-observer reproducibility ranged from 65% to 100% (mean 81.5%). Of the 82 patients on active surveillance 61 had carcinoma and 15 (24.5%) had a set of biopsies with at least 1 focus in which the distinction between tangentially sectioned Gleason pattern 3 and poorly formed pattern 4 glands had to be considered. CONCLUSIONS The reproducibility of grading classic Gleason patterns is high. However, variability in grading occurred when distinguishing between tangentially sectioned pattern 3 glands and the poorly formed gland subset of pattern 4. Developing universally accepted histological and/or molecular criteria to distinguish these patterns and subsequently characterizing their natural history would be useful when treating patients on active surveillance.

Selenium and Vitamin E: Cell Type– and Intervention-Specific Tissue Effects in Prostate Cancer

BACKGROUND Secondary analyses of two randomized, controlled phase III trials demonstrated that selenium and vitamin E could reduce prostate cancer incidence. To characterize pharmacodynamic and gene expression effects associated with use of selenium and vitamin E, we undertook a randomized, placebo-controlled phase IIA study of prostate cancer patients before prostatectomy and created a preoperative model for prostatectomy tissue interrogation. METHODS Thirty-nine men with prostate cancer were randomly assigned to treatment with 200 microg of selenium, 400 IU of vitamin E, both, or placebo. Laser capture microdissection of prostatectomy biopsy specimens was used to isolate normal, stromal, and tumor cells. Gene expression in each cell type was studied with microarray analysis and validated with a real-time polymerase chain reaction (PCR) and immunohistochemistry. An analysis of variance model was fit to identify genes differentially expressed between treatments and cell types. A beta-uniform mixture model was used to analyze differential expression of genes and to assess the false discovery rate. All statistical tests were two-sided. RESULTS The highest numbers of differentially expressed genes by treatment were 1329 (63%) of 2109 genes in normal epithelial cells after selenium treatment, 1354 (66%) of 2051 genes in stromal cells after vitamin E treatment, and 329 (56%) of 587 genes in tumor cells after combination treatment (false discovery rate = 2%). Validation of 21 representative genes across all treatments and all cell types yielded Spearman correlation coefficients between the microarray analysis and the PCR validation ranging from 0.64 (95% confidence interval [CI] = 0.31 to 0.79) for the vitamin E group to 0.87 (95% CI = 0.53 to 0.99) for the selenium group. The increase in the mean percentage of p53-positive tumor cells in the selenium-treated group (26.3%), compared with that in the placebo-treated group (5%), showed borderline statistical significance (difference = 21.3%; 95% CI = 0.7 to 41.8; P = .051). CONCLUSIONS We have demonstrated the feasibility and efficiency of the preoperative model and its power as a hypothesis-generating engine. We have also identified cell type- and zone-specific tissue effects of interventions with selenium and vitamin E that may have clinical implications.

Identification of Sp2 as a Transcriptional Repressor of Carcinoembryonic Antigen-Related Cell Adhesion Molecule 1 in Tumorigenesis

Down-regulation of carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) tumor suppressor gene expression is common in several malignancies including prostate, colon, and breast cancer. The mechanism that mediates this down-regulation is not known. Here, we report that down-regulation of CEACAM1 expression in prostate cancer cells occurs primarily at the transcriptional level and is mediated by Sp2, a member of the Sp family of transcription factors. Sp2 binds to the CEACAM1 promoter in vitro and in vivo, and transient overexpression of Sp2 down-regulates endogenous CEACAM1 expression in normal prostate epithelial cells. Sp2 appears to repress CEACAM1 gene expression by recruiting histone deacetylase activity to the CEACAM1 promoter. In human prostate cancer specimens, Sp2 expression is high in prostate cancer cells but low in normal prostate epithelial cells and is inversely correlated with CEACAM1 expression. Our studies show that transcriptional repression by Sp2 represents one mechanism by which CEACAM1 tumor suppressor gene is down-regulated in prostate cancer.

Therapy Tolerance in Selected Patients With Androgen-Independent Prostate Cancer Following Strontium-89 Combined With Chemotherapy

PURPOSE Clinicians may have reservations about using strontium-89 for the treatment of bone metastases because of concerns that it may limit future use of chemotherapy. We assessed the rate of bone marrow failure in patients with prostate cancer who had received a dose of strontium-89. PATIENTS AND METHODS This subgroup analysis involved 34 patients with androgen-independent prostate cancer who had been given a dose of strontium-89 and six weekly doses of doxorubicin after response to induction chemotherapy. We assessed subsequent hematotoxicity in terms of bone marrow failure and the ability to tolerate additional treatments during a median of 25 months (range, 7 to 76 months) after the strontium-89 was administered. RESULTS No patients developed bone marrow failure within 6 months of receiving strontium-89. Five (15%) of 34 patients developed bone marrow failure at a median 23 months (range, 6 to 53 months) after the strontium-89 treatment. Bone marrow biopsy performed in two of these five patients showed complete replacement of the marrow by tumor. Thirty-one patients (91%) received subsequent cytotoxic treatments at a median 11 months (range, 1 to 33 months) after the strontium-89 treatment. CONCLUSION This analysis demonstrated that a single dose of strontium-89 combined with chemotherapy did not affect the delivery of subsequent courses of chemotherapy in a select group of patients. However, a majority of these therapies were given off protocol and were administered at a dose schedule that might be considered inappropriate or inadequate. The clinical role and safety profile of radiopharmaceuticals combined with chemotherapy in prostate cancer therapy deserve further exploration.

Effect of artemisinin derivatives on apoptosis and cell cycle in prostate cancer cells

Artemisinin is a plant-derived anti-malarial drug that has relatively low toxicity in humans and is activated by heme and/or intracellular iron leading to intracellular free radical formation. Interestingly, artemisinin has displayed anti-cancer activity, with artemisinin dimers being more potent than monomeric artemisinin. Intracellular iron uptake is regulated by the transferrin receptor (TfR), and the activity of artemisinin depends on the availability of iron. We examined the level of TfR in prostate cancer (PCa) tumor cells, synthesized two new artemisinin dimers, and evaluated the effect of dihydroartemisinin and artemisinin dimers, ON-2Py and 2Py, on proliferation and apoptosis in PCa cells. TfR was expressed in the majority of PCa bone and soft tissue metastases, all 24 LuCaP PCa xenografts, and PCa cell lines. After treatment with dihydroartemisinin, ON-2Py, or 2Py all PCa cell lines displayed dose-dependent decrease in cell number. 2Py was most effective in decreasing cell number. An increase in apoptotic events and growth arrest was observed in the C4-2 and LNCaP cell lines. Growth arrest was observed in PC-3 cells, but no significant change was observed in DU 145 cells. Treatment with 2Py resulted in a loss of the anti-apoptotic protein survivin in all four cell lines. 2Py treatment also decreased androgen receptor and prostate-specific antigen expression in C4-2 and LNCaP cells, with a concomitant loss of cell cycle regulatory proteins cyclin D1 and c-Myc. This study shows the potential use of artemisinin derivatives as therapeutic candidates for PCa and warrants the initiation of preclinical studies.