PAF1c links S-phase progression to immune evasion and MYC function in pancreatic carcinoma.

In pancreatic ductal adenocarcinoma (PDAC), endogenous MYC is required for S-phase progression and escape from immune surveillance. Here we show that MYC in PDAC cells is needed for the recruitment of the PAF1c transcription elongation complex to RNA polymerase and that depletion of CTR9, a PAF1c subunit, enables long-term survival of PDAC-bearing mice. PAF1c is largely dispensable for normal proliferation and regulation of MYC target genes. Instead, PAF1c limits DNA damage associated with S-phase progression by being essential for the expression of long genes involved in replication and DNA repair. Surprisingly, the survival benefit conferred by CTR9 depletion is not due to DNA damage, but to T-cell activation and restoration of immune surveillance. This is because CTR9 depletion releases RNA polymerase and elongation factors from the body of long genes and promotes the transcription of short genes, including MHC class I genes. The data argue that functionally distinct gene sets compete for elongation factors and directly link MYC-driven S-phase progression to tumor immune evasion.

Metformin Regulates the miR-205/VEGFA Axis in Renal Cell Carcinoma Cells: Exploring a Clinical Synergism with Tyrosine Kinase Inhibitors.

INTRODUCTION: Metformin (MF) intake could be associated with a favorable outcome in sunitinib (SUT)- and axitinib (AX)-treated clear cell renal cell carcinoma (ccRCC) patients. Functionally, MF induces miR-205, a microRNA serving as a tumor suppressor in several cancers. METHODS: Real-time quantitative PCR, viability assays, and Western blotting analyzed MF and SUT/AX effects in RCC4 and 786-O cells. A tetracycline-inducible overexpression model was used to study the role of miR-205 and its known target gene, VEGFA. We analyzed miR-205 and VEGFA within a public and an in-house ccRCC cohort. Human umbilical vein endothelial cell (HUVEC) sprouting assays examined miR-205 effects on angiogenesis initiation. To determine the influence of the von Hippel-Lindau tumor suppressor (VHL), we examined VHLwt reexpressing RCC4 and 786-O cells. RESULTS: Viability assays confirmed a sensitizing effect of MF toward SUT/AX in RCC4 and 786-O cells. Overexpression of miR-205 diminished VEGFA expression - as did treatment with MF. Tumor tissue displayed a downregulation of miR-205 and an upregulation of VEGFA. Accordingly, miR-205 caused less and shorter vessel sprouts in HUVEC assays. Finally, VHLwt-expressing RCC4 and 786-O cells displayed higher miR-205 and lower VEGFA levels. CONCLUSION: Our results support the protective role of MF in ccRCC and offer functional insights into the clinical synergism with tyrosine kinase inhibitors.

Critical Evaluation of a microRNA-Based Risk Classifier Predicting Cancer-Specific Survival in Renal Cell Carcinoma with Tumor Thrombus of the Inferior Vena Cava.

(1) Background: Clear cell renal cell carcinoma extending into the inferior vena cava (ccRCCIVC) represents a clinical high-risk setting. However, there is substantial heterogeneity within this patient subgroup regarding survival outcomes. Previously, members of our group developed a microRNA(miR)-based risk classifier-containing miR-21-5p, miR-126-3p and miR-221-3p expression-which significantly predicted the cancer-specific survival (CSS) of ccRCCIVC patients. (2) Methods: Examining a single-center cohort of tumor tissue from n = 56 patients with ccRCCIVC, we measured the expression levels of miR-21, miR-126, and miR-221 using qRT-PCR. The prognostic impact of clinicopathological parameters and miR expression were investigated via single-variable and multivariable Cox regression. Referring to the previously established risk classifier, we performed Kaplan-Meier analyses for single miR expression levels and the combined risk classifier. Cut-off values and weights within the risk classifier were taken from the previous study. (3) Results: miR-21 and miR-126 expression were significantly associated with lymphonodal status at the time of surgery, the development of metastasis during follow-up, and cancer-related death. In Kaplan-Meier analyses, miR-21 and miR-126 significantly impacted CSS in our cohort. Moreover, applying the miR-based risk classifier significantly stratified ccRCCIVC according to CSS. (4) Conclusions: In our retrospective analysis, we successfully validated the miR-based risk classifier within an independent ccRCCIVC cohort.

Ki-67, topoisomerase IIα and miR-221 have a limited prostate cancer risk stratification ability on a medium-term follow-up: results of a high-risk radical prostatectomy cohort.

Background: Currently, no biomarkers are able to differentiate lethal from relatively indolent prostate cancer (PCa) within high-risk diseases. Nonetheless, several molecules are under investigation. Amongst them, topoisomerase-II-alpha (TOPIIA), Ki67 and miR-221 showed promising results. Our aim was to investigate their prognostic role in the context of biochemical recurrence (BCR), clinical recurrence (CR) and PCa-related death (PcD). Methods: We included 64 consecutive cM0 high-risk PCa [prostate specific antigen (PSA) >20 ng/mL or Gleason Score (GS) >7 or cT >2] undergoing radical prostatectomy (RP). Changes in miR-221 expression and alternative splicing were determined using microarrays. Immunohistochemical determination of Ki67 and TOPIIa were performed using monoclonal antibody MIB-1 and 3F6 respectively. Cox proportional-hazards regression models were used to predict BCR and CR as multivariate analysis. BCR and CR were defined as three consecutive rises in PSA and PSA >0.2 ng/mL and histologically-proven local recurrence or imaging positive for distant metastasis respectively. Results: We included 64 men. Mean pre-operative PSA was 26.53 (range, 1.3-135); all GSs were ≥7 and pT was ≥ T3 in 78.13%. Positive margins and lymph-nodes were present in 42.19% and 32.81% respectively. At a mean follow-up of 5.7 years (range, 1.8-12.5), 42.18% experienced BCR (n=27), 29.68% CR (n=19) and 7.81% PcD (n=5). On univariate analysis positive nodes (<0.01), seminal vesicle invasion (0.02) and miR-221 downregulation (P=0.03), but not Ki67 and TOPIIA (both P>0.5) were associated with BCR whereas only PSA (P<0.01), seminal vesicle invasion (P<0.01) and positive nodes (both P<0.01) were linked to CR. No parameters predicted PcD (all P>0.05) or BCR and CR on multivariate analysis (all P>0.05 - miR-221 HR 0.776; 95% CI: 0.503-1.196 for BCR and HR 0.673; 95% CI: 0.412-1.099 for CR). Limitation of the study include its small sample size and limited follow-up. Conclusions: TOPIIA, Ki-67 and miR-221 may not predict BCR, CR or PcD in high-risk PCa patients who underwent RP at a medium-term follow-up. Longer follow-up and larger cohorts are needed to confirm our findings.

High expression of Sterol-O-Acyl transferase 1 (SOAT1), an enzyme involved in cholesterol metabolism, is associated with earlier biochemical recurrence in high risk prostate cancer.

BACKGROUND: Prostate cancer (PCa) is the most frequent cancer in men. The prognosis of PCa is heterogeneous with many clinically indolent tumors and rare highly aggressive cases. Reliable tissue markers of prognosis are lacking. Active cholesteryl ester synthesis has been associated with prostate cancer aggressiveness. Sterol-O-Acyl transferases (SOAT) 1 and 2 catalyze cholesterol esterification in humans. OBJECTIVE: To investigate the value of SOAT1 and SOAT2 tissue expression as prognostic markers in high risk PCa. PATIENTS AND METHODS: Formalin-fixed paraffin-embedded tissue samples from 305 high risk PCa cases treated with radical prostatectomy were analyzed for SOAT1 and SOAT2 protein expression by semi-quantitative immunohistochemistry. The Kaplan-Meier method and Cox proportional hazards modeling were used to compare outcome. MAIN OUTCOME MEASURE: Biochemical recurrence (BCR) free survival. RESULTS: SOAT1 expression was high in 73 (25%) and low in 219 (75%; not evaluable: 13) tumors. SOAT2 was highly expressed in 40 (14%) and at low levels in 249 (86%) samples (not evaluable: 16). By Kaplan-Meier analysis, we found significantly shorter median BCR free survival of 93 months (95% confidence interval 23.6-123.1) in patients with high SOAT1 vs. 134 months (112.6-220.2, Log-rank p < 0.001) with low SOAT1. SOAT2 expression was not significantly associated with BCR. After adjustment for age, preoperative PSA, tumor stage, Gleason score, resection status, lymph node involvement and year of surgery, high SOAT1 but not SOAT2 expression was associated with shorter BCR free survival with a hazard ratio of 2.40 (95% CI 1.57-3.68, p < 0.001). Time to clinical recurrence and overall survival were not significantly associated with SOAT1 and SOAT2 expression CONCLUSIONS: SOAT1 expression is strongly associated with BCR free survival alone and after multivariable adjustment in high risk PCa. SOAT1 may serve as a histologic marker of prognosis and holds promise as a future treatment target.

Identification of miR-21-5p and miR-210-3p serum levels as biomarkers for patients with papillary renal cell carcinoma: a multicenter analysis.

BACKGROUND: Expression of circulating serum microRNAs has not been studied in a cohort of patients with papillary renal cell carcinoma (pRCC) so far. We hypothesized that miRNA deregulation in malignant tissue is reflected in serum and could be used for non-invasive diagnosis of pRCC as well as differentiation between type 1 and type 2 pRCC. METHODS: We selected 11 differentially regulated miRNAs from the Cancer Genome Atlas (TCGA) pRCC data set as potential serum validation candidates. Serum miRNA expression was determined by qRT-PCR in a total of 34 pRCC type 1, 33 pRCC type 2 and 33 control subjects of three german high-volume medical centers. RESULTS: Heatmap and principal component analysis showed that miRNA expression did not cluster the samples into distinct sample groups and that miRNA levels did not significantly discriminate healthy individuals from patients with pRCC, nor between patients with type 1 and type 2 pRCC. However, miR-21-5p levels were significantly increased in patients with advanced pRCC (>pT3, and/or pN+ and/or pM+) in comparison to localized pRCC. Moreover, adding the expression of miR-210-3p, which was significantly down-regulated in localized pRCC sera in comparison to healthy sera, additionally increased diagnostic accuracy in our study cohort. CONCLUSIONS: In our multicenter cohort, we were not able to identify a single miRNA serum marker for pRCC including its subclasses. However, our study revealed that miR-21-5p levels were elevated in advanced disease (with added diagnostic accuracy via addition of miR-210-3p expression), proposing these two miRs as potential biomarkers in pRCC.

miR-221-3p Regulates VEGFR2 Expression in High-Risk Prostate Cancer and Represents an Escape Mechanism from Sunitinib In Vitro.

Downregulation of miR-221-3p expression in prostate cancer (PCa) predicted overall and cancer-specific survival of high-risk PCa patients. Apart from PCa, miR-221-3p expression levels predicted a response to tyrosine kinase inhibitors (TKI) in clear cell renal cell carcinoma (ccRCC) patients. Since this role of miR-221-3p was explained with a specific targeting of VEGFR2, we examined whether miR-221-3p regulated VEGFR2 in PCa. First, we confirmed VEGFR2/KDR as a target gene of miR-221-3p in PCa cells by applying Luciferase reporter assays and Western blotting experiments. Although VEGFR2 was mainly downregulated in the PCa cohort of the TCGA (The Cancer Genome Atlas) database, VEGFR2 was upregulated in our high-risk PCa cohort (n = 142) and predicted clinical progression. In vitro miR-221-3p acted as an escape mechanism from TKI in PC3 cells, as displayed by proliferation and apoptosis assays. Moreover, we confirmed that Sunitinib induced an interferon-related gene signature in PC3 cells by analyzing external microarray data and by demonstrating a significant upregulation of miR-221-3p/miR-222-3p after Sunitinib exposure. Our findings bear a clinical perspective for high-risk PCa patients with low miR-221-3p levels since this could predict a favorable TKI response. Apart from this therapeutic niche, we identified a partially oncogenic function of miR-221-3p as an escape mechanism from VEGFR2 inhibition.

Preoperative Risk-Stratification of High-Risk Prostate Cancer: A Multicenter Analysis.

Background: Cancer-specific survival (CSS) within high-risk non-metastatic prostate cancer varies dramatically. It is likely that within this heterogenous population there are subgroup(s) at extraordinary risk, burdened with an exaptational poor prognosis. Establishing the characteristics of these group(s) would have significant clinical implications since high quality preoperative risk stratification remains the cornerstone of therapeutic decision making to date. Objective: To stratify high-risk prostate cancer based on preoperative characteristics and evaluate cancer specific survival after radical prostatectomy. Method: The EMPaCT multi-center database offers an international population of non-metastatic high-risk prostate cancer. Preoperative characteristics such as age, biopsy Gleason score, PSA and clinical stage were subcategorized. A multivariate analysis was performed using predictors showing significant survival heterogeneity after stratification, as observed by a univariate analysis. Based upon the hazard ratios of this multivariate analysis, a proportional score system was created. The most ideal group distribution was evaluated trough different score cut-off's. The predictive value was tested by the herald C index. Results: An overall 5-years CSS of 94% was noted within the entire high-risk cohort (n = 4,879). Except for age, all preoperative risk factors showed a significantly differing CSS. Multivariate analysis indicated, T4 stage as being the strongest predictor of CSS (HR: 3.31), followed by ISUP grade 5 group (HR 3,05). A score system was created by doubling the hazard ratios of this multivariate analysis and rounding off to the nearest complete number. Multivariate analysis suggested 0, 4, 8, and 12 pts as being the most optimal group distribution (p-value: 0.0015). Five-years CSS of these groups were 97, 93, 87, and 70%, respectively. The calculated Herald C-index of the model was 0.77. Conclusion: An easy-to-use pre-operative model for risk stratification of newly diagnosed high-risk prostate cancer is presented. The heterogeneous CSS of high-risk non-metastatic prostate cancer after radical prostatectomy is illustrated. The model is clinically accessible through an online calculator, presenting cancer specific survival based on individualized patient characteristics.

miR-221 Augments TRAIL-Mediated Apoptosis in Prostate Cancer Cells by Inducing Endogenous TRAIL Expression and Targeting the Functional Repressors SOCS3 and PIK3R1.

miR-221 is regarded as an oncogene in many malignancies, and miR-221-mediated resistance towards TRAIL was one of the first oncogenic roles shown for this small noncoding RNA. In contrast, miR-221 is downregulated in prostate cancer (PCa), thereby implying a tumour suppressive function. By using proliferation and apoptosis assays, we show a novel feature of miR-221 in PCa cells: instead of inducing TRAIL resistance, miR-221 sensitized cells towards TRAIL-induced proliferation inhibition and apoptosis induction. Partially responsible for this effect was the interferon-mediated gene signature, which among other things contained an endogenous overexpression of the TRAIL encoding gene TNFSF10. This TRAIL-friendly environment was provoked by downregulation of the established miR-221 target gene SOCS3. Moreover, we introduced PIK3R1 as a target gene of miR-221 in PCa cells. Proliferation assays showed that siRNA-mediated downregulation of SOCS3 and PIK3R1 mimicked the effect of miR-221 on TRAIL sensitivity. Finally, Western blotting experiments confirmed lower amounts of phospho-Akt after siRNA-mediated downregulation of PIK3R1 in PC3 cells. Our results further support the tumour suppressing role of miR-221 in PCa, since it sensitises PCa cells towards TRAIL by regulating the expression of the oncogenes SOCS3 and PIK3R1. Given the TRAIL-inhibiting effect of miR-221 in various cancer entities, our results suggest that the influence of miR-221 on TRAIL-mediated apoptosis is highly context- and entity-dependent.

Defining the Most Informative Intermediate Clinical Endpoints for Predicting Overall Survival in Patients Treated with Radical Prostatectomy for High-risk Prostate Cancer.

BACKGROUND: Given the prolonged natural history of clinically localized, high-risk prostate cancer, there is a need for the identification of intermediate clinical endpoints (ICEs) to predict long-term overall survival (OS). OBJECTIVE: To explore the role of novel potential ICEs based on clinical follow-up to predict long-term survival in patients with high-risk prostate cancer. DESIGN, SETTING, AND PARTICIPANTS: Overall, 3507 patients treated at 12 tertiary referral centers between 1988 and 2016 were evaluated. INTERVENTION: Radical prostatectomy (RP) with extended pelvic lymph node dissection. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: The impact of biochemical recurrence (BCR) and clinical recurrence (CR) within 1, 3, 5, and 7yr after surgery on the risk of OS was evaluated in multivariable Cox regression analyses. In patients with BCR, the impact of progression to CR within 6mo and 1, 3, and 5yr on long-term OS was investigated. Discrimination was assessed using Harrell's c index. RESULTS AND LIMITATIONS: Median follow-up for survivors was 76mo. The 5- and 10-yr OS and cancer-specific survival rates were 94% and 81% versus 98% and 95%, respectively. On a time-varying multivariable analysis, BCR (hazard ratio [HR]: 1.02; 95% confidence interval [CI]: 1.00, 1.04) and CR (HR: 1.05; 95% CI: 1.03-1.07) emerged as predictors of OS (p<0.001). The development of CR within 5yr after surgery was the most informative ICE for predicting OS (c index: 0.74). In patients with BCR, progression to CR within 12mo represented the most informative predictor for the subsequent risk of dying from all causes. Patients who developed BCR within 5yr after RP and progressed to CR within 12mo had a 10-yr OS rate of 47%. These results require prospective validation. CONCLUSIONS: When predicting long-term survival in surgically treated high-risk patients, progression to CR within 5yr of RP confers the highest discrimination with respect to other landmark points. In men experiencing BCR, progression to CR within the subsequent 12mo achieved the highest discrimination. Further studies are needed to validate our findings. PATIENT SUMMARY: We investigated the most informative intermediate clinical endpoints for predicting overall survival (OS). Occurrence of clinical recurrence within 5yr after radical prostatectomy confers the highest discrimination to a model predicting OS.

miR-221-5p regulates proliferation and migration in human prostate cancer cells and reduces tumor growth in vivo.

BACKGROUND: Despite latest advances in prostate cancer (PCa) therapy, PCa remains the third-leading cause of cancer-related death in European men. Dysregulation of microRNAs (miRNAs), small non-coding RNA molecules with gene expression regulatory function, has been reported in all types of epithelial and haematological cancers. In particular, miR-221-5p alterations have been reported in PCa. METHODS: miRNA expression data was retrieved from a comprehensive publicly available dataset of 218 PCa patients (GSE21036) and miR-221-5p expression levels were analysed. The functional role of miR-221-5p was characterised in androgen- dependent and androgen- independent PCa cell line models (C4-2 and PC-3M-Pro4 cells) by miR-221-5p overexpression and knock-down experiments. The metastatic potential of highly aggressive PC-3M-Pro4 cells overexpressing miR-221-5p was determined by studying extravasation in a zebrafish model. Finally, the effect of miR-221-5p overexpression on the growth of PC-3M-Pro4luc2 cells in vivo was studied by orthotopic implantation in male Balb/cByJ nude mice and assessment of tumor growth. RESULTS: Analysis of microRNA expression dataset for human primary and metastatic PCa samples and control normal adjacent benign prostate revealed miR-221-5p to be significantly downregulated in PCa compared to normal prostate tissue and in metastasis compared to primary PCa. Our in vitro data suggest that miR-221-5p overexpression reduced PCa cell proliferation and colony formation. Furthermore, miR-221-5p overexpression dramatically reduced migration of PCa cells, which was associated with differential expression of selected EMT markers. The functional changes of miR-221-5p overexpression were reversible by the loss of miR-221-5p levels, indicating that the tumor suppressive effects were specific to miR-221-5p. Additionally, miR-221-5p overexpression significantly reduced PC-3M-Pro4 cell extravasation and metastasis formation in a zebrafish model and decreased tumor burden in an orthotopic mouse model of PCa. CONCLUSIONS: Together these data strongly support a tumor suppressive role of miR-221-5p in the context of PCa and its potential as therapeutic target.

The EMPaCT Classifier: A Validated Tool to Predict Postoperative Prostate Cancer-related Death Using Competing-risk Analysis.

BACKGROUND: Accurate prediction of survival after radical prostatectomy (RP) is important for making decisions regarding multimodal therapies. There is a lack of tools to predict prostate cancer-related death (PCRD) in patients with high-risk features. OBJECTIVE: To develop and validate a prognostic model that predicts PCRD combining pathologic features and using competing-risks analysis. DESIGN, SETTING, AND PARTICIPANTS: This was a retrospective multi-institutional observational cohort study of 5876 patients affected by high-risk prostate cancer. Patients were treated using RP and pelvic lymph node dissection (PLND) in a multimodal setting, with median follow-up of 49 mo. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: For PCRD prediction, a multivariate model with correction for competing risks was constructed to evaluate pathologic high-risk features (pT3b-4, Gleason score ≥8, and pN1) as predictors of mortality. All possible associations of the predictors were combined, and then subgroups with similar risk of PCRD were collapsed to obtain a simplified model encoding subgroups with significantly differing risk. Eightfold cross-validation of the model was performed. RESULTS AND LIMITATIONS: After applying exclusion criteria, 2823 subjects were identified. pT3b-4, Gleason score ≥8, and pN1 were all independent predictors of PCRD. The simplified model included the following prognostic groups: good prognosis, pN0 with 0-1 additional predictors; intermediate prognosis, pN1 with 0-1 additional predictors; poor prognosis, any pN with two additional predictors. The cross-validation yielded excellent median model accuracy of 88%. The retrospective design and the short follow-up could limit our findings. CONCLUSIONS: We developed and validated a novel and easy-to-use prognostic instrument to predict PCRD after RP+PLND. This model may allow clinicians to correctly counsel patients regarding the intensity of follow-up and to tailor adjuvant treatments. PATIENT SUMMARY: Prediction of mortality after primary surgery for prostate cancer is important for subsequent treatment plans. We present an accurate postoperative model to predict cancer mortality after radical prostatectomy for high-risk prostate cancer.

Impact of Lymph Node Burden on Survival of High-risk Prostate Cancer Patients Following Radical Prostatectomy and Pelvic Lymph Node Dissection.

AIM: To determine the impact of the extent of lymph node invasion (LNI) on long-term oncological outcomes after radical prostatectomy (RP). MATERIAL AND METHODS: In this retrospective study, we examined the data of 1,249 high-risk, non-metastatic PCa patients treated with RP and pelvic lymph node dissection (PLND) between 1989 and 2011 at eight different tertiary institutions. We fitted univariate and multivariate Cox models to assess independent predictors of cancer-specific survival (CSS) and overall survival (OS). The number of positive lymph node (LN) was dichotomized according to the most informative cutoff predicting CSS. Kaplan-Meier curves assessed CSS and OS rates. Only patients with at least 10 LNs removed at PLND were included. This cutoff was chosen as a surrogate for a well performed PNLD. RESULTS: Mean age was 65 years (median: 66, IQR 60-70). Positive surgical margins were present in 53.7% (n = 671). Final Gleason score (GS) was 2-6 in 12.7% (n = 158), 7 in 52% (n = 649), and 8-10 in 35.4% (n = 442). The median number of LNs removed during PLND was 15 (IQR 12-17). Of all patients, 1,128 (90.3%) had 0-3 positive LNs, while 126 (9.7%) had ≥4 positive LNs. Patients with 0-3 positive LNs had significantly better CSS outcome at 10-year follow-up compared to patients with ≥4 positive LNs (87 vs. 50%; p < 0.0001). Similar results were obtained for OS, with a 72 vs. 37% (p < 0.0001) survival at 10 years for patients with 0-3 vs. ≥4 positive LNs, respectively. At multivariate analysis, final GS of 8-10, salvage ADT therapy, and ≥4 (vs. <4) positive LNs were predictors of worse CSS and OS. Pathological stage pT4 was an additional predictor of worse CSS. CONCLUSION: Four or more positive LNs, pathological stage pT4, and final GS of 8-10 represent independent predictors for worse CSS in patients with high-risk PCa. Primary tumor biology remains a strong driver of tumor progression and patients having ≥4 positive LNs could be considered an enriched patient group in which novel treatment strategies should be studied.

Very long-term survival patterns of young patients treated with radical prostatectomy for high-risk prostate cancer.

OBJECTIVE: In patients with a long life expectancy with high-risk (HR) prostate cancer (PCa), the chance to die from PCa is not negligible and may change significantly according to the time elapsed from surgery. The aim of this study was to evaluate long-term survival patterns in young patients treated with radical prostatectomy (RP) for HRPCa. MATERIALS AND METHODS: Within a multiinstitutional cohort, 600 young patients (≤59 years) treated with RP between 1987 and 2012 for HRPCa (defined as at least one of the following adverse characteristics: prostate specific antigen>20, cT3 or higher, biopsy Gleason sum 8-10) were identified. Smoothed cumulative incidence plot was performed to assess cancer-specific mortality (CSM) and other cause mortality (OCM) rates at 10, 15, and 20 years after RP. The same analyses were performed to assess the 5-year probability of CSM and OCM in patients who survived 5, 10, and 15 years after RP. A multivariable competing risk regression model was fitted to identify predictors of CSM and OCM. RESULTS: The 10-, 15- and 20-year CSM and OCM rates were 11.6% and 5.5% vs. 15.5% and 13.5% vs. 18.4% and 19.3%, respectively. The 5-year probability of CSM and OCM rates among patients who survived at 5, 10, and 15 years after RP, were 6.4% and 2.7% vs. 4.6% and 9.6% vs. 4.2% and 8.2%, respectively. Year of surgery, pathological stage and Gleason score, surgical margin status and lymph node invasion were the major determinants of CSM (all P≤0.03). Conversely, none of the covariates was significantly associated with OCM (all P≥ 0.09). CONCLUSIONS: Very long-term cancer control in young high-risk patients after RP is highly satisfactory. The probability of dying from PCa in young patients is the leading cause of death during the first 10 years of survivorship after RP. Thereafter, mortality not related to PCa became the main cause of death. Consequently, surgery should be consider among young patients with high-risk disease and strict PCa follow-up should enforce during the first 10 years of survivorship after RP.

Metformin-Derived Growth Inhibition in Renal Cell Carcinoma Depends on miR-21-Mediated PTEN Expression.

PURPOSE: Metformin (MF) acts as a tumour-suppressor in renal cell carcinoma (RCC) by inhibiting the AKT/mTOR pathway via AMPK activation. Here, we explore the influence of miR-21 and its target gene PTEN on MF effects in CAKI-1 and CAKI-2 cells. METHODS: Proliferation assays (MTS) and qRT-PCR after transient transfection with pre- and anti-miR-21 and MF treatment were conducted. AMPK-dependency was assessed via transfection of siAMPK. The expression of PTEN, AKT and miR-21 after transient pre-miR-21 transfection and MF treatment was analysed. RESULTS: We demonstrate that CAKI-1 cells, which were found to be less sensitive towards MF, showed a significant higher miR-21 and lower PTEN expression than CAKI-2. This was confirmed in a primary RCC collective (n = 28): miR-21 and PTEN expression correlated negatively. MF treatment lowered miR-21 AMPK-dependently and increased PTEN expression in the cell lines. Ectopic miR-21 regulation modulated MF sensitivity. Western blot analysis showed that pre-miR-21 transfection and MF treatment regulated PTEN expression with impact on pAKT levels in the cells. CONCLUSIONS: We show that differing MF sensitivity in RCC cells is associated with and mediated through the regulation of miR-21/PTEN expression with an impact on subsequent AKT signalling. This provides imaginable clinical implications regarding MF therapy of RCC patients for the future.

DNA Methylation-Guided Prediction of Clinical Failure in High-Risk Prostate Cancer.

BACKGROUND: Prostate cancer (PCa) is a very heterogeneous disease with respect to clinical outcome. This study explored differential DNA methylation in a priori selected genes to diagnose PCa and predict clinical failure (CF) in high-risk patients. METHODS: A quantitative multiplex, methylation-specific PCR assay was developed to assess promoter methylation of the APC, CCND2, GSTP1, PTGS2 and RARB genes in formalin-fixed, paraffin-embedded tissue samples from 42 patients with benign prostatic hyperplasia and radical prostatectomy specimens of patients with high-risk PCa, encompassing training and validation cohorts of 147 and 71 patients, respectively. Log-rank tests, univariate and multivariate Cox models were used to investigate the prognostic value of the DNA methylation. RESULTS: Hypermethylation of APC, CCND2, GSTP1, PTGS2 and RARB was highly cancer-specific. However, only GSTP1 methylation was significantly associated with CF in both independent high-risk PCa cohorts. Importantly, trichotomization into low, moderate and high GSTP1 methylation level subgroups was highly predictive for CF. Patients with either a low or high GSTP1 methylation level, as compared to the moderate methylation groups, were at a higher risk for CF in both the training (Hazard ratio [HR], 3.65; 95% CI, 1.65 to 8.07) and validation sets (HR, 4.27; 95% CI, 1.03 to 17.72) as well as in the combined cohort (HR, 2.74; 95% CI, 1.42 to 5.27) in multivariate analysis. CONCLUSIONS: Classification of primary high-risk tumors into three subtypes based on DNA methylation can be combined with clinico-pathological parameters for a more informative risk-stratification of these PCa patients.

An anti-ubiquitin antibody response in transitional cell carcinoma of the urinary bladder.

BACKGROUND: To use combinatorial epitope mapping ("fingerprinting") of the antibody response to identify targets of the humoral immune response in patients with transitional cell carcinoma (TCC) of the bladder. METHODS: A combinatorial random peptide library was screened on the circulating pool of immunoglobulins purified from an index patient with a high risk TCC (pTa high grade plus carcinoma in situ) to identify corresponding target antigens. A patient cohort was investigated for antibody titers against ubiquitin. RESULTS: We selected, isolated, and validated an immunogenic peptide motif from ubiquitin as a dominant epitope of the humoral response. Patients with TCC had significantly higher antibody titers against ubiquitin than healthy donors (p<0.007), prostate cancer patients (p<0.0007), and all patients without TCC taken together (p<0.0001). Titers from superficial tumors were not significantly different from muscle invasive tumors (p = 0.0929). For antibody response against ubiquitin, sensitivity for detection of TCC was 0.44, specificity 0.96, positive predictive value 0.96 and negative predictive value 0.41. No significant titer changes were observed during the standard BCG induction immunotherapy. CONCLUSIONS: This is the first report to demonstrate an anti-ubiquitin antibody response in patients with TCC. Although sensitivity of antibody production was low, a high specificity and positive predictive value make ubiquitin an interesting candidate for further diagnostic and possibly immune modulating studies.

Natural history of surgically treated high-risk prostate cancer.

BACKGROUND: No data exist on the patterns of biochemical recurrence (BCR) and their effect on survival in patients with high-risk prostate cancer (PCa) treated with surgery. The aim of our investigation was to evaluate the natural history of PCa in patients treated with radical prostatectomy (RP) alone. MATERIALS AND METHODS: Overall, 2,065 patients with high-risk PCa treated with RP at 7 tertiary referral centers between 1991 and 2011 were identified. First, we calculated the probability of experiencing BCR after surgery. Particularly, we relied on conditional survival estimates for BCR after RP. Competing-risks regression analyses were then used to evaluate the effect of time to BCR on the risk of cancer-specific mortality (CSM). RESULTS: Median follow-up was 70 months. Overall, the 5-year BCR-free survival rate was 55.2%. Given the BCR-free survivorship at 1, 2, 3, 4, and 5 years, the BCR-free survival rates improved by+7.6%,+4.1%,+4.8%,+3.2%, and+3.7%, respectively. Overall, the 10-year CSM rate was 14.8%. When patients were stratified according to time to BCR, patients experiencing BCR within 36 months from surgery had higher 10-year CSM rates compared with those experiencing late BCR (19.1% vs. 4.4%; P<0.001). At multivariate analyses, time to BCR represented an independent predictor of CSM (P<0.001). CONCLUSIONS: Increasing time from surgery is associated with a reduction of the risk of subsequent BCR. Additionally, time to BCR represents a predictor of CSM in these patients. These results might help provide clinicians with better follow-up strategies and more aggressive treatments for early BCR.

Pretreatment tables predicting pathologic stage of locally advanced prostate cancer.

BACKGROUND: Pretreatment tables for the prediction of pathologic stage have been published and validated for localized prostate cancer (PCa). No such tables are available for locally advanced (cT3a) PCa. OBJECTIVE: To construct tables predicting pathologic outcome after radical prostatectomy (RP) for patients with cT3a PCa with the aim to help guide treatment decisions in clinical practice. DESIGN, SETTING, AND PARTICIPANTS: This was a multicenter retrospective cohort study including 759 consecutive patients with cT3a PCa treated with RP between 1987 and 2010. INTERVENTION: Retropubic RP and pelvic lymphadenectomy. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Patients were divided into pretreatment prostate-specific antigen (PSA) and biopsy Gleason score (GS) subgroups. These parameters were used to construct tables predicting pathologic outcome and the presence of positive lymph nodes (LNs) after RP for cT3a PCa using ordinal logistic regression. RESULTS AND LIMITATIONS: In the model predicting pathologic outcome, the main effects of biopsy GS and pretreatment PSA were significant. A higher GS and/or higher PSA level was associated with a more unfavorable pathologic outcome. The validation procedure, using a repeated split-sample method, showed good predictive ability. Regression analysis also showed an increasing probability of positive LNs with increasing PSA levels and/or higher GS. Limitations of the study are the retrospective design and the long study period. CONCLUSIONS: These novel tables predict pathologic stage after RP for patients with cT3a PCa based on pretreatment PSA level and biopsy GS. They can be used to guide decision making in men with locally advanced PCa. PATIENT SUMMARY: Our study might provide physicians with a useful tool to predict pathologic stage in locally advanced prostate cancer that might help select patients who may need multimodal treatment.

Stratification of high-risk prostate cancer into prognostic categories: a European multi-institutional study.

BACKGROUND: High-risk prostate cancer (PCa) is an extremely heterogeneous disease. A clear definition of prognostic subgroups is mandatory. OBJECTIVE: To develop a pretreatment prognostic model for PCa-specific survival (PCSS) in high-risk PCa based on combinations of unfavorable risk factors. DESIGN, SETTING, AND PARTICIPANTS: We conducted a retrospective multicenter cohort study including 1360 consecutive patients with high-risk PCa treated at eight European high-volume centers. INTERVENTION: Retropubic radical prostatectomy with pelvic lymphadenectomy. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Two Cox multivariable regression models were constructed to predict PCSS as a function of dichotomization of clinical stage (< cT3 vs cT3-4), Gleason score (GS) (2-7 vs 8-10), and prostate-specific antigen (PSA; ≤ 20 ng/ml vs > 20 ng/ml). The first "extended" model includes all seven possible combinations; the second "simplified" model includes three subgroups: a good prognosis subgroup (one single high-risk factor); an intermediate prognosis subgroup (PSA >20 ng/ml and stage cT3-4); and a poor prognosis subgroup (GS 8-10 in combination with at least one other high-risk factor). The predictive accuracy of the models was summarized and compared. Survival estimates and clinical and pathologic outcomes were compared between the three subgroups. RESULTS AND LIMITATIONS: The simplified model yielded an R(2) of 33% with a 5-yr area under the curve (AUC) of 0.70 with no significant loss of predictive accuracy compared with the extended model (R(2): 34%; AUC: 0.71). The 5- and 10-yr PCSS rates were 98.7% and 95.4%, 96.5% and 88.3%, 88.8% and 79.7%, for the good, intermediate, and poor prognosis subgroups, respectively (p = 0.0003). Overall survival, clinical progression-free survival, and histopathologic outcomes significantly worsened in a stepwise fashion from the good to the poor prognosis subgroups. Limitations of the study are the retrospective design and the long study period. CONCLUSIONS: This study presents an intuitive and easy-to-use stratification of high-risk PCa into three prognostic subgroups. The model is useful for counseling and decision making in the pretreatment setting.