Wolf F. Wieland

The MicroRNA Profile of Prostate Carcinoma Obtained by Deep Sequencing

Prostate cancer is a leading cause of tumor mortality. To characterize the underlying molecular mechanisms, we have compared the microRNA (miRNA) profile of primary prostate cancers and noncancer prostate tissues using deep sequencing. MiRNAs are small noncoding RNAs of 21 to 25 nucleotides that regulate gene expression through the inhibition of protein synthesis. We find that 33 miRNAs were upregulated or downregulated >1.5-fold. The deregulation of selected miRNAs was confirmed by both Northern blotting and quantitative reverse transcription-PCR in established prostate cancer cell lines and clinical tissue samples. A computational search indicated the 3′-untranslated region (UTR) of the mRNA for myosin VI (MYO6) as a potential target for both miR-143 and miR-145, the expression of which was reduced in the tumor tissues. Upregulation of myosin VI in prostate cancer was previously shown by immunohistochemistry. The level of MYO6 mRNA was significantly induced in all primary tumor tissues compared with the nontumor tissue from the same patient. This finding was matched to the upregulation of myosin VI in established prostate cancer cell lines. In luciferase reporter analysis, we find a significant negative regulatory effect on the MYO6 3′UTR by both miR-143 and miR-145. Mutation of the potential binding sites for miR-143 and miR-145 in the MYO6 3′UTR resulted in a loss of responsiveness to the corresponding miRNA. Our data indicate that miR-143 and miR-145 are involved in the regulation of MYO6 expression and possibly in the development of prostate cancer. Mol Cancer Res; 8(4); 529–38. ©2010 AACR.

Prediction of Progression of Non-Muscle-Invasive Bladder Cancer by WHO 1973 and 2004 Grading and by FGFR3 Mutation Status: A Prospective Study

OBJECTIVES The clinical management of non-muscle-invasive urothelial cell carcinoma of the bladder (UCC) is challenging, as it has a marked tendency to recur and to progress. Aim of this study was to investigate the prognostic value of the WHO 1973 and 2004 grading systems and biomarkers FGFR3, CK20 and Ki-67. METHODS In a prospective study, tumours from 221 patients were studied for the expression of CK20 and Ki-67 by immunohistochemistry, and FGFR3 status by SNaPshot mutation detection. Staging and grading were performed according to the WHO classification systems of 1973 and 2004. RESULTS : Median follow-up was 35 mo. Recurrence occurred in 72 of 221 patients. None of the parameters was able to predict disease recurrence. CK20, Ki-67, FGFR3 mutation, molecular grade using FGFR3 mutation analysis and Ki-67, and histological grading and staging were significantly associated with disease progression in stage. In multivariable analyses, WHO 1973 and 2004 grading systems remained statistically significant and independent predictors of progression, with p=0.005 for WHO 1973 and p=0.004 for 2004. FGFR3 status was able to discriminate progressors from nonprogressors in a subset of patients with high-grade UCC (p=0.009). CONCLUSIONS This is the first prospective study comparing the WHO 1973 and 2004 grading systems. We show that both grading systems contribute valuable independent information. Therefore, it should be considered whether a better grading system could be developed that incorporates essential elements from both. The combination of WHO 2004 grading with FGFR3 status allows a better risk stratification for patients with high-grade non-muscle-invasive UCC.

MicroRNA profiles of prostate carcinoma detected by multiplatform microRNA screening.

MicroRNAs (miRNAs) are small RNA molecules that regulate gene expression via posttranscriptional inhibition of protein synthesis. They play a vital role in tumorigenesis. To characterize the diagnostic potential of miRNAs in prostate cancer, a leading cause of cancer mortality, we performed screening of miRNA expression profiles. We used commercially available microarrays to establish miRNA expression profiles from a cohort of 20 cancer samples. The expression of selected miRNAs was analyzed by quantitative real‐time PCR and the identity of miRNA expressing cells was determined by miRNA in situ hybridization. We identified 25 miRNAs that showed a significant differential expression in cancer samples. The comparison with previously published data generated by deep sequencing of cDNA libraries of small RNA molecules revealed a concordance rate of 47% among miRNAs identified with both techniques. The differential expression of miRNAs miR‐375, miR‐143 and miR‐145 was validated by quantitative PCR. MiRNA in situ hybridization revealed that the differential expression is cancer‐cell associated. A combination of three miRNAs correctly classified tissue samples with an accuracy of 77.6% with an area under the receiver–operator characteristic curve of 0.810. Our data extend the knowledge about the deregulation of miRNAs in prostate cancer. The differential expression of several miRNAs is highly consistent using independent cohorts of tumor samples, different tissue preservation methods and different experimental methods. Our results indicate that combinations of miRNAs are promising biomarkers for the diagnosis of prostate cancer.

Eight years' experience with high-intensity focused ultrasonography for treatment of localized prostate cancer.

OBJECTIVES To report on the long-term results of high-intensity focused ultrasonography (HIFU) in the treatment of localized prostate cancer. METHODS Patients with clinical Stage T1-T2N0M0, biopsy-proven, localized prostate cancer, with a serum prostate-specific antigen (PSA) level of <or=20 ng/mL, Gleason score of <or=7, and with no previous curative prostate cancer treatment, were included. All patients underwent HIFU using the Ablatherm device and were required to have a minimal follow-up of 3 years after the last HIFU session to be included in this analysis. Follow-up included PSA measurement and biopsy performed 3-6 months after treatment and in conjunction with an increasing PSA level. Biochemical failure was defined according to the Phoenix definition (PSA nadir + 2 ng/mL). In determining the disease-free survival rate, treatment was considered to have failed if any of the following occurred: biochemical failure, positive biopsy findings, or the initiation of salvage treatment. RESULTS The study included 163 patients. Within the 4.8 +/- 1.2 years of follow-up, no patient died of prostate cancer. Of the 163 patients, 86.4% achieved a PSA nadir of <1 ng/mL and 92.7% had negative post-treatment biopsy findings. The actuarial biochemical survival rate at 5 years was 75%. The actuarial disease-free survival rate at 5 years was 66%, with salvage treatment initiated for 12% of the patients. On multivariate analysis, the pretreatment PSA level was the only statistically significant predictive factor of recurrence (P = .005). CONCLUSIONS The results after long-term follow-up have indicated that HIFU is an efficient and safe treatment for patients with localized prostate cancer.

Sleep Apnea is an Independent Correlate of Erectile and Sexual Dysfunction

INTRODUCTION Obstructive sleep apnea (OSA) has been linked with erectile dysfunction (ED), but it is unknown whether this association is maintained in the presence of other risk factors for ED. AIM The aim of this study was to evaluate the relationship between ED/sexual dysfunction and polysomnographic measures of sleep apnea in patients with known risk factors for ED. METHODS Prospective cross-sectional analysis of 401 male patients undergoing in-lab polysomnography for suspected OSA. Erectile (EF) and sexual function were assessed by the 15-item International Index of Erectile Function (IIEF-15) questionnaire. MAIN OUTCOME MEASURES Severity of OSA via apnea-hypopnea index (AHI) and mean/lowest nocturnal oxygen saturation (SaO(2)). The IIEF-15 including the sexual domains: EF, intercourse satisfaction, orgasmic function, sexual desire, and overall satisfaction. RESULTS OSA (AHI > 5/h) was diagnosed in 92% of patients. ED (EF subdomain < or = 25) was present in 69% of patients with, and 34% of patients without OSA (P < 0.001). Multivariate stepwise regression analyses including known risk factors for ED, such as age, obesity, coronary heart disease, peripheral occlusive disease, hypertension, diabetes, prostate surgery, and beta-blocker treatment, and measures of sleep apnea identified mean nocturnal SaO(2) as independently associated with ED (P = 0.002; mean [95% CI] normalized slope 0.126 [0.047; 0.205]). Age (P < 0.001), peripheral occlusive disease (P = 0.001), prostate surgery (P = 0.018), and hypertension (P = 0.021) were confirmed as risk factors for ED, but did not abolish the sleep apnea-associated risk. Similar results were obtained for sexual dysfunction. Logistic regression analysis using the diagnosis of ED (EF subdomain < or = 25) as binary dependent variable confirmed that mean nocturnal SaO(2) (P = 0.012), as well as age (P < 0.001) were independently associated with ED. CONCLUSIONS ED and overall sexual dysfunction were highly prevalent in patients with suspected OSA. Irrespective of known risk factors, mean nocturnal SaO(2) was an additional, independent correlate of these dysfunctions, suggesting that OSA-related intermittent nocturnal hypoxemia specifically contributes to their development.

Topographical Anatomy of Periprostatic and Capsular Nerves: Quantification and Computerised Planimetry

BACKGROUND The exact distribution of periprostatic autonomic nerves is under debate. OBJECTIVE To study the topographical anatomy of autonomic nerves of the periprostatic tissue and the capsule of the prostate (CAP). DESIGN, SETTING, AND PARTICIPANTS Whole-mount sections of 30 prostates from patients having undergone non-nerve-sparing radical prostatectomy were investigated after immunohistochemical nerve staining. Sections from the base, the middle, and the apex were evaluated. All sections were divided into 12 sectors, which were combined into the following regions: ventral, ventrolateral, dorsolateral, and dorsal. MEASUREMENTS Quantification of periprostatic and capsular nerves was performed within the sectors. Computerised planimetry of the total periprostatic nerve surface area of each region was performed (Image-J software, Wayne Rasband, National Institute of Health, USA). RESULTS AND LIMITATIONS A total of 3514, 3860, and 3902 periprostatic nerves was counted at the base, the middle, and the apex, respectively (p=0.068). The ratio of periprostatic nerves to capsular nerves was 3.6, 2.1, and 1.9 at the base, the middle, and the apex, respectively (p=0.004). Computerised planimetry revealed a significant decrease in total nerve surface area from the base over the middle towards the apex, with 241.79, 133.64, and 89.50mm(2) (p=0.004). The percentage of total nerve surface area was highest dorsolaterally (84.1%, 75.1%, and 74.5% at base, middle, and apex, respectively) but variable: Up to 39.9% of nerve surface area was found ventrolaterally and up to 45.5% in the dorsal position. The study is limited by the fact that autonomic nerve distribution was only investigated from the base to the apex of the prostate. CONCLUSIONS Periprostatic nerve distribution is variable, with a high percentage of nerves in the ventrolateral and dorsal positions. Total periprostatic nerve surface area decreases from the base towards the apex due to nerves leaving the NVB branching into the prostate. This can only be discovered by nerve planimetry, not by quantification.

The WHO classification of 1973 is more suitable than the WHO classification of 2004 for predicting survival in pT1 urothelial bladder cancer

Study Type – Prognosis (systematic review)
 Level of Evidence 2a

Downregulation of Sec23A protein by miRNA-375 in prostate carcinoma

Prostate carcinoma (CaP) is a leading cause of cancer-related death in men. We have previously determined the microRNA (miRNA) profile of primary CaP in comparison with nontumor prostate tissue. miRNAs are small, noncoding RNAs that inhibit protein synthesis on a posttranscriptional level by binding to the 3′-untranslated region (3′-UTR) of their target genes. In primary CaP tissue, we have previously found by miRNA sequencing that miR-375 and miR-200c were upregulated 9.1- and 4.5-fold, respectively. A computational analysis predicted the 3′-UTR of the SEC23A gene as a potential target for both miR-375 and miR-200c. Here, we show that the 3′-UTR of SEC23A mRNA is indeed a target for miR-375 and miR-200c and that both miRNAs downregulate Sec23A protein expression when ectopically expressed in human 293T cells. In primary samples of CaP, we found a direct correlation between reduction of SEC23A mRNA and overexpression of miR-375 but not of miR-200c. The reduced levels of Sec23A protein were inversely correlated to the increased amount of miR-375 in the LNCaP and DU145 CaP cell lines when compared with normal prostate fibroblasts. In primary CaP, we also detected decreased amounts of Sec23A protein when compared with corresponding normal prostate tissue. Ectopically overexpressed Sec23A in LNCaP and DU145 CaP cells significantly reduced the growth properties, indicating that Sec23A might play a role in the induction or growth of prostate carcinoma. Sec23A overexpression reduced cell growth but did not induce apoptosis, whereas inhibition of Sec23A stimulated cell proliferation. Mol Cancer Res; 9(6); 791–800. ©2011 AACR.

Fourteen‐year oncological and functional outcomes of high‐intensity focused ultrasound in localized prostate cancer

High‐intensity focused ultrasound (HIFU) is an alternative treatment option for localized prostate cancer (PCa), which is applied for over 15 years. There are conflicting recommendations for HIFU among urological societies, which can be explained by the lack of prospective controlled studies, reports on preselected patient populations and limited follow‐up providing little information on overall and cancer‐specific survival. We report on a large, unselected consecutive patient series of patients who have undergone primary HIFU for clinically localized PCa with the longest follow‐up in current literature. Our results improve the understanding of the oncological efficacy, morbidity and side effects of primary HIFU.

Lymph Node Density Affects Cancer-Specific Survival in Patients with Lymph Node–Positive Urothelial Bladder Cancer Following Radical Cystectomy

BACKGROUND The prognosis for patients with lymph node (LN)-positive bladder cancer (BCa) is likely affected by the extent of lymphadenectomy in radical cystectomy (RC) cases. Specifically, the prognostic significance of the LN density (ratio of positive LNs to the total number removed) has been demonstrated. OBJECTIVE To evaluate the prognostic signature of lymphadenectomy variables, including the LN density, for a large, multicentre cohort of RC patients with LN-positive BCa. DESIGN, SETTING, AND PARTICIPANTS The clinical and histopathologic data from 477 patients with LN-positive urothelial BCa (pN1-2) were analysed. The median follow-up period for all living patients was 28 mo. MEASUREMENTS Multivariable Cox regression analysis was used to test the effect of various pelvic lymph node dissection (PLND) variables on cancer-specific survival (CSS) based on colinearity in various models. RESULTS AND LIMITATIONS The median number of LNs removed was 12 (range: 1-66), and the median number of positive LNs was 2 (range: 1-25). Two hundred ninety (60.8%) of the patients presented with stage pN2 disease. The median and mean LN density was 17.6% and 29% (range: 2.3-100), respectively, where 268 (56.2%) and 209 (43.8%) patients exhibited am LN density of ≤20% and >20%, respectively. In separate multivariable Cox regression models adjusted for age, sex, pTN stage, grade, associated Tis, and adjuvant chemotherapy, the interval-scaled LN density (hazard ratio [HR]: 1.01; p=0.002) and the LN density, ordinal-scaled by 20% (HR: 1.65; p<0.001) exhibit independent effects on CSS. In addition, an independent contribution appears from the pT but not the pN stage. Limitations include surgeon selection bias when determining the extent of lymphadenectomy. CONCLUSIONS Our results support the prognostic relevance of LN density in patients with LN-positive BCa, where a threshold value of 20% stratifies the population into two prognostically distinct groups. Before LN density is integrated into the clinical decision-making process, these results should be validated by prospective studies with defined LN templates and standardised histopathologic methods.