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Purpose: Recurrent prostate cancer remains a major problem. Staging, grading and prostate specific antigen level at surgery are helpful but still imperfect predictors of recurrence. For this reason there is an imperative need for additional biomarkers that add to the prediction of currently used prognostic factors.Materials and Methods: We evaluated the extent of promoter methylation of genes previously reported as aberrantly methylated in prostate cancer (AIM1, APC, CCND2, GPX3, GSTP1, MCAM, RARβ2, SSBP2 and TIMP3) by quantitative fluorogenic methylation-specific polymerase chain reaction. We used cancer tissue from a nested case-control study of 452 patients surgically treated for prostate cancer. Recurrence cases and controls were compared and the association between methylation extent and recurrence risk was estimated by logistic regression adjusting for patient age at prostatectomy, prostatectomy year, stage, grade, surgical margins and preprostatectomy prostate specific antigen. All statistical test...

The need for effective targeted therapies for renal cell carcinomas (RCCs) has fueled the interest for understanding molecular pathways involved in the oncogenesis of kidney tumors. Aiming to analyze the expression status and prognostic significance of mTOR and hypoxia-induced pathway members in patients with clear cell RCC (ccRCC), tissue microarrays were constructed from 135 primary and 41 metastatic ccRCCs. Immunoexpression levels were compared and correlated with clinicopathologic parameters and outcome. PTEN levels were significantly lower in primary and metastatic ccRCCs compared with benign tissues (P

Background: Hemangioblastomas account for up to 2.5% of all intracranial tumors. They may occur sporadically or as a part of the multisystem genetic syndrome of Von Hippel-Lindau syndrome (VHL). Patients with VHL are also at an increased risk of developing clear cell renal cell carcinoma (ccRCC). Distinguishing hemangioblastomas from metastatic ccRCC to the central nervous system (CNS) can be challenging at times when based solely on hematoxylin and eosin-stained sections. We propose an immunohistochemistry (IHC) panel of combination of PAX2, PAX8, and inhibin A as a helpful approach in distinguishing the 2 lesions. Design: Archival tissues from 20 hemangioblastomas and 16 ccRCCs metastatic to the CNS were retrieved from our surgical pathology files (2001 to 2010). IHC for PAX2, PAX8, and inhibin A was performed on routine or tissue microarray sections using standard IHC protocol. The intensity of nuclear staining was evaluated for each marker and was assigned an incremental 0, 1+, 2+, and 3+ score. The extent of staining was categorized as focal ( 75%). Result: (1) Hemangioblastoma: The Von Hippel-Lindau syndrome was diagnosed in 4 of 16 (25%) patients, 2 of whom developed multiple hemangioblastomas. All 20 (100%) hemangioblastomas were positive for inhibin A (cytoplasmic). The staining intensity was moderate or strong (2+ or 3+) in 19 cases (95%), all of which were multifocal or diffuse in extent. Nuclear PAX2 staining was present in 1 of 19 evaluable lesions (5%), whereas PAX8 staining was not present in any of the 20 examined lesions. (2) Metastatic ccRCC to the CNS: Fourteen of 16 (88%) examined ccRCCs were positive for PAX2, whereas 15 of 16 (94%) lesions showed PAX8 staining. None of 16 (0%) examined ccRCCs were positive for inhibin A. Conclusions: We propose the use of the combination of PAX2, PAX8, and inhibin A as a helpful ancillary IHC panel to resolve the differential diagnosis of hemangioblastoma versus metastatic ccRCC. The immunoprofile of PAX2(+) or PAX8(+) and inhibin A(� ) supports the diagnosis of metastatic ccRCC with a sensitivity of 94%, specificity of 100%, and positive predictive value of 100%. The PAX2(� ), PAX8(� ), and inhibin A(+) profile supports the diagnosis of hemangioblastoma with a sensitivity of 95%, specificity of 100%, and positive predictive value of 100%.

Collecting duct carcinoma (CDC) is a relatively rare but aggressive type of renal malignancy with variable morphologic features. One of the World Health Organization diagnostic criteria for CDC is the exclusion of urothelial carcinoma of renal pelvis from the differential diagnosis. PAX8 is a novel lineage restricted transcription factor expressed in renal tubules. We investigated the expression pattern of PAX8 in CDC and its utility, in combination with p63, in resolving the differential diagnosis of CDC versus upper tract urothelial carcinoma (UUC).Archival tissues from 21 CDC and 34 UUC were retrieved from our institutional files. Immunohistochemistry for PAX8 and p63 were performed on routine and tissue microarray sections using standard immunohistochemistry protocol. Intensity of nuclear staining was evaluated for each marker and assigned an incremental 0, 1+, 2+, and 3+ score. Extent of staining was categorized as focal (25%), nonfocal (25% to 75%), or diffuse (75%).CDC: All 21 (100%) CDC were positive for PAX8. Intensity of expression was moderate to strong (2+/3+) in 19 cases (90%). Extent of staining was diffuse in 13 of 21 tumors. The p63 was positive in 3 of 21 (14%) CDC cases (PAX8+/p63+). UUC: The 34 UUC included 5 pT1, 4 pT2, and 25 pT3/pT4 tumors. Thirty-one of 34 (91.2%) UUC were negative for PAX8, whereas 33 of 34 (97%) were p63 positive. Staining intensity was moderate in 15 cases (44%), of which 12 were nonfocal or diffuse. The unique p63-negative UUC was a pT1 tumor that was also negative for PAX8 (PAX8-/p63-).We propose the use of the combination of PAX8 and p63 in the diagnosis of poorly differentiated renal sinus epithelial neoplasms where the differential diagnosis includes CDC versus UUC. The immunoprofile of PAX8+/p63- supports the diagnosis of CDC with a sensitivity of 85.7% and a specificity of 100%. In contrast, a (PAX8-/p63+) profile supports the diagnosis of UUC with a sensitivity of 88.2% and a specificity of 100%. The inverse PAX8/p63 expression seen in CDC and UUC supports a renal tubular rather than an urothelial differentiation in CDC given the nephric lineage restriction of PAX8.

Histone modifications have been linked to DNA replication, transcription and repair. The phosphorylation of histone H2AX at serine 139 (gamma-H2AX) is associated with DNA breaks. gamma-H2AX has been shown to be expressed in bladder urothelial carcinoma. To our knowledge studies of the relationship of gamma-H2AX expression and the clinical outcome of urothelial carcinoma are lacking. Hence, we evaluated the rate of H2AX phosphorylation in low grade bladder urothelial carcinoma and assessed its potential role for predicting recurrence and/or progression.Immunohistochemical expression of gamma-H2AX using a polyclonal antibody was retrospectively assessed in 2 groups of patients from The Johns Hopkins Hospital with low grade bladder urothelial carcinoma. Group 1 consisted of transurethral resection biopsies from 18 patients from 2004 to 2006 that were retrieved from our surgical pathology files. Group 2 consisted of 42 archival transurethral biopsies obtained between 1971 and 1995 with longer followup that were used to construct a tissue microarray.On univariate analysis recurrence in the entire cohort was more likely to develop in gamma-H2AX negative than in gamma-H2AX positive cases (24 of 32 or 81% vs 13 of 28 or 46%). The difference in recurrence was statistically significant (p = 0.02). The same was true in group 2 (16 of 21 cases or 76% vs 9 of 21 or 43%, p = 0.02). Female gender and intravesical therapy were also associated with a higher recurrence rate in our cohort. A higher progression rate was noted in group 2 patients and in the entire cohort in association with negative gamma-H2AX staining. However, the difference in progression between gamma-H2AX negative and positive tumors was not statistically significant. On multivariate analysis only patient gender and prior intravesical treatment remained predictive of recurrence (p0.03).Our data suggest that epigenetic alterations may have an important role in the mechanism of bladder tumor recurrence. Analysis in a larger cohort is needed to further assess our current preliminary findings of the role of gamma-H2AX expression for predicting outcome in low grade urothelial carcinoma cases.

post-DRE first-catch urine specimens were collected prior to prostate biopsy. Progensa PCA3 and T2:ERG (quantitative nucleic acid amplification assay to detect T2:ERG mRNA) were assessed and correlated (individually and as a marker panel) to prostate cancer diagnosis, Gleason score, and clinical T stage. This was compared to serum PSA. In a subgroup (n 61) prostatectomy outcome was evaluated. The independent sample T-test and the non-parametric Mann Whitney test were used. RESULTS: Of the 497 men that were included, urine samples of 443 men contained sufficient mRNA for marker analysis. Table 1 shows the diagnostic and prognostic values of the markers. Prostate cancer was diagnosed in 196/443 men. Serum PSA, PCA3 and T2: ERG were all significantly correlated with diagnosis prostate cancer. PCA3 using cutoff 25 had the strongest correlation (p 6.3 10-13), exceeding serum PSA. By combining PCA3 and T2:ERG the correlation improved significantly (p 5.4 10-17). The AUC (area under the curve) increased from 0.720 (only PCA3) to 0.760 for the marker panel PCA3 T2:ERG. The sensitivity increased from 68% to 76%. T2:ERG was significantly correlated with biopsy Gleason score, clinical T stage, and extracapsular extension. PCA3 had no correlation with these prognostic parameters. CONCLUSIONS: PCA3 is a strong diagnostic marker for prostate cancer. By combining PCA3 and T2:ERG the diagnostic value and the AUC increases evidently. In this prospective multicenter study T2:ERG had significant prognostic value, there was no correlation of PCA3 with Gleason score and T stage.

BACKGROUND—TMPRSS2-ERG fusions have been identified in about one-half of all prostatic adenocarcinomas (PCa). Fluorescence in situ hybridization (FISH) and reverse transcription polymerase chain reaction (RT-PCR) have been the most commonly used techniques in this setting. The aim of the present study is to evaluate the utility of ERG immunoexpression as a surrogate for TMPRSS2-ERG fusion in a large series of PCa cases. MATERIAL & METHODS—427 radical retropubic prostatectomy tissue samples were used to construct 10 tissue microarrays (TMA). FISH analysis was previously performed using dual-color interphase break-apart probes for the 5′ and 3′ regions of the ERG gene. ERG expression was evaluated using a commercial rabbit anti-ERG monoclonal antibody (clone EPR3864; Epitomics, Burlingame CA). Each TMA spot was independently assessed and any nuclear staining positivity was considered as indicative of ERG expression. RESULTS—TMPRSS2-ERG fusions were detected by FISH in 195 (45.7%) of the PCa cases. ERG immunoexpression was found in 192 (45.0%) of the PCa cases and in none of the nontumoral tissue samples. Mean ERG H-scores were significantly higher in tumors harboring FISH-detected TMPRSS2-ERG fusions (P

1339 EXPRESSION OF THE EXTRACELLULAR MATRIX ASSOCIATED PROTEIN CYR61 IS ASSOCIATED WITH THE DEVELOPMENT OF PROSTATE CANCER Katherine D’Antonio*, Antoun Toubaji, Roula Albadine, George J Netto, Robert H Getzenberg. Baltimore, MD. INTRODUCTION AND OBJECTIVE: Prostate cancer is a disease that comprises more than the cancer cells themselves. The microenvironment in which the cancer cells exist is central to the disease process. Genomic studies of cancer have revealed that the genes encoding the CCN family of extracellular matrix associated proteins consistently exhibit altered expression. One of the changes most frequently associated with cancer development is an integrin binding extracellular matrix protein, cystein rich angiogenic inducer 61 (Cyr61), also known as CCN1. The objective of these studies is to examine the expression of Cyr61 in prostate cancer, normal prostate, and the associated tissues as well as to study its role in prostate cancer development. METHODS: Cyr61 gene expression in prostate cancer and normal tissues was evaluated by microarray analysis. Immunoblot analysis was used to assess endogenous Cyr61 protein expression in prostate cell lines and further substantiated by tissue microarray analysis of prostatectomy samples. 200 radical prostatectomy samples were collected and Cyr61 expression was evaluated by tissue microarray analysis (TMA). In vitro functional studies were also performed on prostate cancer cell lines to gain insight into the role of Cyr61 in prostate cancer, particularly regarding growth and adhesion. RESULTS: Cyr61 expression is up-regulated at both the mRNA