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The role of TMPRSS2–ERG gene fusion in prostate cancer prognostication remains controversial. We evaluated the prognostic role of TMPRSS2–ERG fusion using fluorescence in situ hybridization analysis in a case–control study nested in The Johns Hopkins retropubic radical prostatectomy cohort. In all, 10 tissue microarrays containing paired tumors and normal tissues obtained from 172 cases (recurrence) and 172 controls (non-recurrence) matched on pathological grade, stage, race/ethnicity, and age at the time of surgery were analyzed. All radical prostatectomies were performed at our institution between 1993 and 2004. Recurrence was defined as biochemical recurrence, development of clinical evidence of metastasis, or death from prostate carcinoma. Each tissue microarray spot was scored for the presence of TMPRSS2–ERG gene fusion and for ERG gene copy number gains. The odds ratio of recurrence and 95% confidence intervals were estimated from conditional logistic regression. Although the percentage of cases with fusion was slightly lower in cases than in controls (50 vs 57%), the difference was not statistically significant (P=0.20). The presence of fusion due to either deletion or split event was not associated with recurrence. Similarly, the presence of duplicated ERG deletion, duplicated ERG split, or ERG gene copy number gain with a single ERG fusion was not associated with recurrence. ERG gene polysomy without fusion was significantly associated with recurrence (odds ratio 2.0, 95% confidence interval 1.17–3.42). In summary, TMPRSS2–ERG fusion was not prognostic for recurrence after retropubic radical prostatectomy for clinically localized prostate cancer, although men with ERG gene copy number gain without fusion were twice more likely to recur.

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

Objective: To test the hypothesis that total liquid ventilation enables a more effective and better tolerated lavage than a bronchoalveolar lavage performed with diluted surfactant in a newborn ovine model of severe acute meconium aspiration syndrome. Design: Prospective, randomized, interventional study. Setting: Animal research laboratory at the Faculte de medecine et des sciences de la sante de I'universite de Sherbrooke, Sherbrooke, Canada. Subjects: Twenty-three newborn lambs

Purpose: Cysteine-rich angiogenic inducer 61 (Cyr61) is an extracellular matrix protein involved in the transduction of growth factor and hormone signaling. Previous studies have suggested that Cyr61 may be a marker for a more aggressive phenotype. In this study, we evaluated the association between Cyr61 staining intensity and subsequent recurrence after surgical treatment of clinically localized prostate cancer. Experimental Design: A study of 229 men with recurrence and 229 controls matched on age, race, pathologic stage, and Gleason sum nested in a cohort of men who underwent radical prostatectomy for clinically localized prostate cancer, utilizing immunohistochemistry analysis of tissue microarray (TMA) sections, was conducted. Odds ratios (OR) of recurrence and 95% confidence intervals (CIs) were estimated using conditional logistic regression. Results: Recurrence was identified in 12.2% of cases, and in 24.0% of controls that had at least 1 TMA spot containing cancer with a staining intensity of 3 (P = 0.001). Taking into account age, pathologic stage and grade, presurgery prostate-specific antigen concentration, and calendar of surgery as a measure of tissue block storage time, men with a Cyr61 staining intensity of 3 were 56% less likely to recur than men with a lower staining intensity (OR = 0.44, 95% CI = 0.22–0.90). Conclusions: High Cyr61 staining intensity in adenocarcinoma was associated with a lower risk of recurrence after surgical treatment of prostate cancer independent of pathologic tumor characteristics. If validated in other sample sets, Cyr61 may serve as a tissue biomarker for stratifying men for risk of recurrence and thus could inform treatment decision making. Clin Cancer Res; 16(23); 5908–13. ©2010 AACR.

Purpose: Given discrepancies between preclinical and clinical observations of mammalian target of rapamycin (mTOR) inhibition in prostate cancer, we sought to determine the pharmacodynamic effects of the mTOR/TORC1 inhibitor rapamycin in men with intermediate- to high-risk prostate cancer undergoing radical prostatectomy. Experimental Design: Rapamycin was given at 3 or 6 mg orally for 14 days before radical prostatectomy in men with multifocal Gleason sum ≥7 prostate cancer; 10 untreated control subjects were included. The primary outcome was inhibition of phosphorylation of ribosomal S6 in posttreatment radical prostatectomy versus pretreatment biopsy tumor tissue, evaluated using a Simon two-stage design for pharmacodynamic efficacy. Results: Thirty-two subjects were accrued: 20 at 3 mg, 2 at 6 mg, and 10 controls. No dose-limiting toxicities were observed at 3 mg; however, two of two men enrolled at 6 mg experienced dose-limiting toxicities including thrombocytopenia and fever with grade 3 stomatitis. Adverse events observed at 3 mg included stomatitis, rash, ileus, and neutropenia. Pharmacodynamic studies showed tumor S6 phosphorylation inhibition in 50% of 10 evaluable rapamycin-treated men with sufficient paired tissue [median 58% inhibition (P = 0.049) versus 2% inhibition in controls (P = 0.75)] with no significant effect on AKT activity. We observed no change in Ki-67 or caspase-3 cleavage but noted a reduction in cytoplasmic p27 staining with increased nuclear localization with rapamycin treatment. Prostate tissue rapamycin concentrations were 3- to 4-fold higher than blood. Conclusions: At 3 mg daily, rapamycin successfully and safely inhibited prostate cancer S6 phosphorylation and achieved relatively high prostate tissue concentrations. No effect on AKT phosphorylation, tumor proliferation, or apoptosis was observed. Clin Cancer Res; 16(11); 3057–66. ©2010 AACR.

This study sought to assess putative pathways involved in the anti-inflammatory effects of 17,18-epoxyeicosatetraenoic acid (17,18-EpETE), as measured by a decrease in the contractile reactivity and Ca(2+) sensitivity of TNF-α-pretreated human bronchi. Tension measurements performed in the presence of 12-(3-adamantan-1-yl-ureido)-dodecanoic acid (AUDA), a soluble epoxide hydrolase (sEH)-specific inhibitor, demonstrated that 17,18-EpETE reduced the reactivity of TNF-α-pretreated tissues. The overexpression of sEH detected in patients with asthma and TNF-α-treated bronchi contributed to the maintenance of hyperresponsiveness in our models, which involved intracellular proinflammatory cascades. The inhibition of peroxisome proliferator-activated receptor (PPAR)γ by GW9662 abolished 17,18-EpETE + AUDA-mediated anti-inflammatory effects by inducing IκBα degradation and cytokine synthesis, indicating that PPARγ is a molecular target of epoxy-eicosanoids. Western blot analysis revealed that 17,18-EpETE pretreatment reversed the phosphorylation of p38 mitogen-activated protein kinase (p38-MAPK) induced by TNF-α in human bronchi. The Ca(2+) sensitivity of human bronchial explants was also quantified on β-escin permeabilized preparations. The presence of SB203580, a p38-MAPK inhibitor, reversed the effect induced by epoxy-eicosanoid in the presence of AUDA on TNF-α-triggered Ca(2+) hypersensitivity by increasing the phosphorylation level of PKC Potentiated Inhibitor Protein-17 (CPI-17) regulatory protein. Moreover, PPARγ ligands, such as rosiglitazone and 17,18-EpETE, decreased the expression of CPI-17, both at the mRNA and protein levels, whereas this effect was countered by GW9662 treatment in TNF-α-treated bronchi. These results demonstrate that 17,18-EpETE is a potent regulator of human lung inflammation and concomitant hyperresponsiveness, and may represent a valuable asset against critical inflammatory bronchial disorder.

The cancer cell microenvironment includes complex interactions between the cell and the extracellular matrix. Expression of the CCN family of extracellular matrix associated proteins is often modified in disease states. Depending on cancer type these changes are linked with enhanced or inhibited tumor growth. We characterized Cyr61 in prostate cancer. Cyr61 is an integrin binding matricellular protein with altered expression in many cancer types.Cyr61 expression in prostate cancer, benign prostatic hyperplasia and normal tissues was evaluated by microarray analysis, quantitative real-time polymerase chain reaction and tissue microarray. Immunoblots were analyzed to assess endogenous protein expression in prostate cancer cell lines.On genomic analysis Cyr61 up-regulation was observed in prostate cancer tissue and in normal prostate tissue adjacent to tumor vs that in prostate donor tissue. In 174 matched tumors and normal prostate tissues adjacent to tumor tissue microarray revealed significantly up-regulated Cyr61 protein expression in cancer tissue vs normal prostate tissue adjacent to tumor. Also, increased Cyr61 expression correlated with Gleason sum 8 or greater cancer. Staining in high grade prostatic intraepithelial neoplasia was moderately up-regulated vs that in normal prostate tissue adjacent to tumor but generally less intense than in carcinoma tissue.In addition to the correlation with more advanced disease, the strong association between Cyr61 expression and prostate cancer supports the potential usefulness of Cyr61 as a novel biomarker for prostate cancer. This warrants further analysis to determine the mechanisms by which Cyr61 may contribute to prostate cancer development and progression.

Renal medullary carcinoma is an aggressive renal neoplasm without currently available effective therapy to our knowledge. Topoisomerase II alpha is a gyrase involved in cell proliferation, and DNA maintenance and repair. Topoisomerase II alpha is a target of inhibiting agents such as anthracyclines. Triggered by a recent response to topoisomerase II alpha inhibitors in a patient with renal medullary carcinoma, we evaluated topoisomerase II alpha expression in relation to the proliferation index and topoisomerase II alpha gene copy number status in a larger series of patients with renal medullary carcinoma.Archival tissues from 14 renal medullary carcinomas were retrieved from our 3 institutions. Immunohistochemistry was performed using monoclonal antibodies for topoisomerase II alpha and Ki67. The percent of cells with positive nuclear staining was assessed in the highest area of expression for each marker. A previously suggested greater than 5% cutoff was used for topoisomerase II alpha over expression. The topoisomerase II alpha gene copy number was evaluated using fluorescence in situ hybridization. Locus specific topoisomerase II alpha gene and chromosome 17 centromere probes were used. The total number of topoisomerase II alpha and chromosome 17 centromere signals was counted in 150 cells per tumor and a topoisomerase II alpha-to-chromosome 17 centromere signal ratio was calculated in each tumor. A topoisomerase II alpha-to-chromosome 17 centromere ratio of 2.0 or greater and less than 0.8 was used as a cutoff for amplification and deletion, respectively. The percent of tumor cells with polysomic, eusomic or monosomic chromosome 17 status was also determined.On immuno-expression analysis topoisomerase II alpha immunohistochemistry was technically inconclusive in 1 renal medullary carcinoma. Topoisomerase II alpha was over expressed in 11 of 13 renal medullary carcinomas (85%) (median 50%, range 1% to 80%). As expected, a high Ki67 proliferation index was noted in 13 of 14 tumors (median 87.5%, range 2% to 100%). Ki67 expression was greater than topoisomerase II alpha expression in all 13 informative tumors. A strong, statistically significant correlation was found for topoisomerase II alpha and Ki67 expression (pairwise CC 0.9, p = 0.0000). Topoisomerase II alpha over expression was associated with shorter survival (p = 0.000). On fluorescence in situ hybridization no topoisomerase II alpha amplification was detected in any of the 14 renal medullary carcinomas, including the 11 with topoisomerase II alpha over expression. Topoisomerase II alpha gene deletions were noted in 4 tumors. Two of 4 deletions were associated with chromosome 17 monosomy and 2 were in eusomic chromosome 17 tumors.Topoisomerase II alpha is over expressed in 85% of renal medullary carcinomas, potentially supporting the use of topoisomerase II alpha inhibitor agents to treat this aggressive renal tumor. Our findings suggest that topoisomerase II alpha over expression in our renal medullary carcinoma cohort was not due to gene amplification, but rather to transcriptional or post-transcriptional modifications. The significance of the incidentally found topoisomerase II alpha deletions in 28% of renal medullary carcinomas requires further evaluation.

Alterations in methylation of CpG dinucleotides at the 5 position of deoxycytidine residues (5(m)C) are a hallmark of cancer cells, including testicular germ cell tumors. Virtually all testicular germ cell tumors are believed to be derived from intratubular germ cell neoplasia unclassified (IGCNU), which is thought to arise from primordial germ cells. Prior studies revealed that seminomas contain reduced levels of global DNA methylation as compared with nonseminomatous germ cell tumors. Smiraglia et al have proposed a model whereby seminomas arise from IGCNU cells derived from primordial germ cells that have undergone 5(m)C erasure, and nonseminomas arise from IGCNU cells derived from primordial germ cells that have already undergone de novo methylation after the original erasure of methylation and contain normal 5(m)C levels. Yet the methylation status of IGCNU has not been determined previously. We used immunohistochemical staining against 5(m)C to evaluate global methylation in IGCNU and associated invasive testicular germ cell tumors. Strikingly, staining for 5(m)C was undetectable (or markedly reduced) in the majority of IGCNU and seminomas, yet there was robust staining in nonseminomatous germ cell tumors. The lack of staining for 5(m)C in IGCNU and seminomas was also found in mixed germ cell tumors containing both seminomatous and nonseminomatous components. Lack of 5(m)C staining was not related to a lack of the maintenance methyltransferase (DNA methyltransferase 1) protein. We conclude that testicular germ cell tumors are derived in most cases from IGCNU cells that have undergone developmentally programmed 5(m)C erasure and that the degree of subsequent de novo methylation is most closely related to the differentiation state of the neoplastic cells. That is, IGCNU cells and seminoma cells remain unmethylated, whereas all other histological types appear to arise after de novo methylation.

Introduction: Lower Endoscopic Ultrasound-Guided Fine-Needle Aspiration (LEUS-FNA) of perirectal lesions is a safe, minimally invasive, and well tolerated procedure that provides valuable information which affects patient management. Herein, we presented our experience of LEUS -FNA to evaluate perirectal lesions. Materials and Methods: LEUS-FNAs were retrieved from the cytopathology archives of our University Hospital, from 2001February, 2014. The cytopathology reports, corresponding histology, immunohistochemistry when available, and clinical data were collected. The sensitivity and specificity of EUS-FNA were calculated in a subset of patients with available surgical pathology. Results: 114 specimens were retrieved. Masses measured 5e100 mm (mean: 27.5 mm) in diameter. Recurrent cancer was clinically suspected in 46% of cases (nZ53). For 37 cases histopatholgic material was available. The aspirated material showed malignant (n Z 48), benign (n Z 41), atypical/suspicious (nZ6) and nondiagnostic cytology (nZ 19). Malignant cases were adenocarcinoma (24), neuroendocrine tumor (3), squamous cell carcinoma (5), urothelial carcinoma (2), positive for malignant cells (12), gastrointestinal stromal tumor (1) and non Hodgkin lymphoma (1). The primary site of the tumors included colorectal, anal, urinary bladder, prostate, pancreas, ovary, and female lower genital tract. The benign cytology cases were negative for malignant cells (32), schwannoma (1), and 8 non neoplastic lesions including: abscess (3), endometriosis (2), hematoma (1), malacoplakia (1) and mucinous cyst (1). Histology confirmed 11/12 negative cytology; one false negative cytology of lymph node. Statistical analysis for LEUS-FNA showed 91% sensitivity, 100% specificity, diagnostic accuracy of 95%, and a positive predictive value of 100% and a negative predictive value of 88%. Discrepancies were likely due to cytology sampling errors. Conclusion: Lower EUS-FNA allows cytological examination and ancillary studies (immunohistochemistry, flow cytometry) of suspicious pelvic lesions in the gut wall and surrounding tissues. LEUS-FNA can detect local recurrences and also improve the staging accuracy of colorectal adenocarcinoma by proving nodal metastasis.

8096 Background: ALK gene rearrangement (ALK+) has been found in 3-5% of advanced non-small cell lung cancer patients. FISH is considered the “gold standard” for identification of ALK+ tumors, but its cost-effectiveness and adoption as a screening assay has been debated. Recent reports suggested that ALK IHC may serve as an alternative screening or possibly a diagnostic method. In this context, CALK was initiated to assess the feasibility of implementing ALK IHC and/or FISH assays across Canadian hospitals. Methods: FISH-confirmed 22 ALK+ and 6 ALK- tumors were used as study samples. Unstained sections and scanned images of HE-stained slides from each tumor block were distributed to participating centres. IHC protocols with best signal to noise ratio using the 5A4 (Novocastra) or ALK-1 (Dako) antibodies were developed for various auto-stainers and implemented to suit the existing conditions of the participating centres. A common FISH protocol using the ALK break-apart probe (Abbott Molecular, Chicago, IL) was developed based on published reports. H-score was used to assess IHC. FISH signals were scored in 100 tumor cells/case by 2-3 pre-trained persons. A second round IHC study using newly distributed slides was completed by 8 centres. Results: Independent IHC scores from 12 centres and FISH scores from 11 centres were collected and analysed. The intraclass correlation coefficients (ICC) between centres for IHC and FISH were 0.84 and 0.68, respectively. Following the analysis of initial IHC results, a second round study resulted in improved ICC of 0.94. One of 23 tumors revealed IHC-/FISH+ discrepancy, with the FISH revealing unusual signal configurations that suggested an atypical rearrangement. However, the sensitivity and specificity of FISH results across centres using the 15 aberrant signals cut-off ranged from 86.7-100% and 100%, respectively. Conclusions: Standardization across multiple centres for ALK testing by IHC and FISH can be achieved. IHC detected all FISH+ ALK tumors, except for one discrepant case with atypical FISH finding of unknown clinical implication. The study was supported by a Pfizer Canada grant.

e22159 Background: Recent advances in lung cancer treatment embrace the recognition of molecular pathways implicated in its pathogenicity, paving the path to personalized therapies. We conducted a retrospective analysis to characterize the molecular features of the population treated for non-squamous non-small cell lung cancer (NS-NSCLC) in the province of Quebec. Methods: 622 patients with NS-NSCLC and adequate tumor blocks, treated at the CHUM between 2006 and 2008, were included. All samples were tested for ALK translocations (by IHC and FISH), EGFR classical exon 19 and 21 mutations by PCR (fragment analysis and qPCR) and for KRAS codon 12 and 13 mutations by mismatch PCR-RFLP. Molecular features were matched to demographic characteristics and clinical outcomes. Results: So far, complete results are available for 153 patients. Considering the amount of tumor tissue available, this population is largely represented by patients with local or loco-regional disease (n= 140, 91.5%). A minority of patients (10.3%) was never or light smokers (< 10 pack-yrs). Only 2 patients (1.3%) were of Asian descent. The following table depicts the outcomes of this cohort of patients segregated according to mutation status and extent of disease. Conclusions: ALK rearrangements were not identified in this unselected NS-NSCLC population characterized by localized disease and strong smoking history. ALK translocation prevalence in different populations is likely to be largely influenced by its tumor stage distribution, tobacco exposure and the use of selection criteria for molecular testing. An expanded cohort of patients will be presented at the meeting. [Table: see text]

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

Introduction Fluorescence in situ hybridization (FISH) is currently the standard for diagnosing anaplastic lymphoma kinase (ALK)-rearranged (ALK+) lung cancers for ALK inhibitor therapies. ALK immunohistochemistry (IHC) may serve as a screening and alternative diagnostic method. The Canadian ALK (CALK) study was initiated to implement a multicenter optimization and standardization of laboratory developed ALK IHC and FISH tests across 14 hospitals. Methods Twenty-eight lung adenocarcinomas with known ALK status were used as blinded study samples. Thirteen laboratories performed IHC using locally developed staining protocols for 5A4, ALK1, or D5F3 antibodies; results were assessed by H-score. Twelve centers conducted FISH using protocols based on Vysis' ALK break-apart FISH kit. Initial IHC results were used to optimize local IHC protocols, followed by a repeat IHC study to assess the results of standardization. Three laboratories conducted a prospective parallel IHC and FISH analysis on 411 consecutive clinical samples using post-validation optimized assays. Results Among study samples, FISH demonstrated 22 consensus ALK+ and six ALK wild type tumors. Preoptimization IHC scores from 12 centers with 5A4 and the percent abnormal cells by FISH from 12 centers showed intraclass correlation coefficients of 0.83 and 0.68, respectively. IHC optimization improved the intraclass correlation coefficients to 0.94. Factors affecting FISH scoring and outliers were identified. Post-optimization concurrent IHC/FISH testing in 373 informative cases revealed 100% sensitivity and specificity for IHC versus FISH. Conclusions Multicenter standardization study may accelerate the implementation of ALK testing protocols across a country/region. Our data support the use of an appropriately validated IHC assay to screen for ALK+ lung cancers.

Non-necrotizing epithelioid granulomas have been described in association with many primary tumors. In such cases, they are designated as sarcoid-like reaction. Although it is more seen in carcinomas than in sarcomas, it is very rarely reported in renal carcinoma. Here, we describe a rare association of prominent peritumoral sarcoid-like reaction without intratumoral granulomas and conventional clear cell renal carcinoma in a 62-year-old-male, without clinical or laboratory finding of sarcoidosis. At 30 months follow-up, he had no recurrence. Virtual Slides The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/4054525336657922