Placebo-Controlled Study of Effects of Low-Energy Shockwave Therapy (LE-ESWT) on Erectile Tissue in a Diabetic Animal Model.

Objective: Low-energy extracorporeal shockwave therapy (LE-ESWT) has been shown to induce organ repair and neo-vascularization. The ability of LE-ESWT to improve erectile function in rodents as measured by improvements in intracavernosal pressure is well-established in various pathological situations. The underlying molecular mechanism are unclear and likely vary between different disorders, making rational drug design for synergetic effects with LE-ESWT difficult, without further research. In this placebo-controlled study, we aim to establish whether LE-ESWT can activate neovascularization biomarkers in diabetic tissues. Material and Methods: Forty Wistar rats, aged 8 weeks, were randomly divided into 4 groups: 8 untreated controls, 12 controls that underwent LE-ESWT treatment, 8 controls with induced diabetes mellitus (DM) and 12 with DM underwent LE-ESWT treatment. DM was induced by streptozotocin. LE-ESWT treatment was performed with a Duolith SD1 machine (Storz), with a total amount of energy of 6.4 J per treatment. The rats received a total of three LE-ESWT treatments with 2-week intervals between treatments. Results: Diabetic rats had significantly elevated blood glucose concentrations compared to control rats (P < 0.001) and experienced significant weight loss compared to controls (P < 0.001). Diabetic rats had elevated creatinine and urea and lower albumin (P < 0.001). Histologic analysis of penile tissue showed significant levels of vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF) expression in the LE-ESWT groups compared to controls (P< 0.01). Conclusion: LE-ESWT induces neo-angiogenesis as expressed by VEGF and FGF in erectile tissue in normal and diabetic rats.

DaPeCa-8: drawing the map of lymphatic drainage in patients with invasive penile cancer - evidence from SPECT/CT and sentinel node surgery.

BACKGROUND: Anatomy of the lymphatic drainage guides the extent of inguinal lymph node dissection in penile cancer. OBJECTIVE: To prospectively assess the lymphatic drainage of penile cancer with single-photon emission computed tomography CT (SPECT-CT) and implications for the extent of inguinal lymph node dissection. METHODS: We assessed the lymphatic drainage of 62 patients with at least unilateral clinical lymph node-negative (cN0) status with SPECT-CT at our tertiary referral centre. We evaluated 122 cN0 inguinal basins and compared them to the histopathological outcome. The inguinal regions were divided into ten different Daseler zones on SPECT-CT. The surgical team filled in a corresponding scheme at sentinel node biopsy and sent lymph nodes from each Daseler zone individually for histopathological examination. RESULTS: SPECT-CT successfully visualized lymphatic drainage in 116 of the 122 cN0 inguinal basins (95.1%). The vast majority of sentinel nodes and all metastatic nodes were located in central and superior inguinal zones, including six metastatic nodes in lateral superior zones. Minimal lymphatic drainage was seen to the inferior Daseler zones and no metastatic deposits were located here. No direct pelvic drainage was observed. CONCLUSIONS: Penile cancer lymphatic drainage is primarily to sentinel in the superior and central zones of Daseler. Colleagues practicing a modified inguinal lymph node dissection as a standard in cN0 patients are encouraged to include all these zones, while the inferior zones can be omitted. This study confirms the absence of lymphatic drainage directly to the pelvic region and supports the practice of omitting pelvic nodes from sentinel node biopsy.

DaPeCa-7: comparative assessment of fluorodeoxyglucose positron emission tomography/computed tomography (CT) and conventional diagnostic CT in diagnosis of lymph node metastases, distant metastases and incidental findings in patients with invasive penile cancer.

OBJECTIVES: To evaluate diagnostic accuracy of 18 F-fluorodeoxyglucose positron emission tomography/computed tomography (FDG PET/CT) compared to contrast-enhanced CT in assessment of inguinal lymph node (ILN) metastases, distant metastases and synchronous cancers in patients with penile squamous cell carcinoma (pSCC). PATIENTS AND METHODS: During a 4-year period, patients with pSCC were scheduled for FDG PET/CT prior to surgical treatment at two referral centres that manage all penile cancers in Denmark. The primary endpoint was diagnostic accuracy of FDG PET/CT and of CT alone with histopathology or Response Evaluation Criteria In Solid Tumors (RECIST) as reference. RESULTS: We evaluated 171 patients for distant metastases and synchronous incident cancers and examined 286 groins in 143 patients for LN metastases by FDG PET/CT. Six groins disclosed false negatives. FDG PET/CT sensitivity was 85.4% per patient. In 135 patients (270 groins), CT images were evaluated separately and 22 groins disclosed false negatives. CT sensitivity was 47.5% per patient. FDG PET/CT detected pSCC distant metastases in seven patients. Distant metastases from other cancers were newly detected in three patients. In eight patients, an incidental synchronous cancer was detected. Seven out of the 18 distant malignancies detected depended on FDG PET information. CONCLUSION: This study underlines the increased diagnostic accuracy of FDG PET/CT compared to CT alone in the evaluation of ILN status. In patients with palpable LNs, the advantage of FDG PET/CT over CT is less pronounced. FDG PET/CT may play a role in penile cancer evaluation.

Epigenetic silencing of MEIS2 in prostate cancer recurrence.

BACKGROUND: Current diagnostic and prognostic tools for prostate cancer (PC) are suboptimal, resulting in overdiagnosis and overtreatment of clinically insignificant tumors. Thus, to improve the management of PC, novel biomarkers are urgently needed. RESULTS: In this study, we integrated genome-wide methylome (Illumina 450K DNA methylation array (450K)) and RNA sequencing (RNAseq) data performed in a discovery set of 27 PC and 15 adjacent normal (AN) prostate tissue samples to identify candidate driver genes involved in PC development and/or progression. We found significant enrichment for homeobox genes among the most aberrantly methylated and transcriptionally dysregulated genes in PC. Specifically, homeobox gene MEIS2 (Myeloid Ecotropic viral Insertion Site 2) was significantly hypermethylated (p < 0.0001, Mann-Whitney test) and transcriptionally downregulated (p < 0.0001, Mann-Whitney test) in PC compared to non-malignant prostate tissue in our discovery sample set, which was also confirmed in an independent validation set including > 500 PC and AN tissue samples in total (TCGA cohort analyzed by 450K and RNAseq). Furthermore, in three independent radical prostatectomy (RP) cohorts (n > 700 patients in total), low MEIS2 transcriptional expression was significantly associated with poor biochemical recurrence (BCR) free survival (p = 0.0084, 0.0001, and 0.0191, respectively; log-rank test). Next, we analyzed another RP cohort consisting of > 200 PC, AN, and benign prostatic hyperplasia (BPH) samples by quantitative methylation-specific PCR (qMSP) and found that MEIS2 was significantly hypermethylated (p < 0.0001, Mann-Whitney test) in PC compared to non-malignant prostate tissue samples (AN and BPH) with an AUC > 0.84. Moreover, in this cohort, aberrant MEIS2 hypermethylation was significantly associated with post-operative BCR (p = 0.0068, log-rank test), which was subsequently confirmed (p = 0.0067; log-rank test) in the independent TCGA validation cohort (497 RP patients; 450K data). CONCLUSIONS: To the best of our knowledge, this is the first study to investigate, demonstrate, and independently validate a prognostic biomarker potential for MEIS2 at the transcriptional expression level and at the DNA methylation level in PC.

Elevated miR-615-3p Expression Predicts Adverse Clinical Outcome and Promotes Proliferation and Migration of Prostate Cancer Cells.

miR-615-3p has previously been described as up-regulated in prostate cancer (PC) tissue samples compared with nonmalignant controls; however, its prognostic potential and functional role in PC remain largely unknown. In this study, we investigated the clinical and biological relevance of miR-615-3p in PC. The expression of miR-615-3p was measured in PC tissue specimens from 239 men who underwent radical prostatectomy (RP), and it was investigated if miR-615-3p could predict postoperative biochemical recurrence (BCR). These findings were subsequently validated in three independent RP cohorts (n = 222, n = 273, and n = 387) and functional overexpression studies conducted in PC cells (PC3M). High miR-615-3p expression was significantly associated with BCR in four independent PC patient cohorts (P < 0.05, log-rank test). In addition, high miR-615-3p expression was a significant predictor of PC-specific survival in univariate (hazard ratio, 3.75; P < 0.001) and multivariate (hazard ratio, 2.66; P = 0.008) analysis after adjustment for the Cancer of the Prostate Risk Assessment Post-Surgical (CAPRA-S) nomogram in a merged RP cohort (n = 734). Moreover, overexpression of miR-615-3p in PC cells (PC3M) significantly increased cell viability, proliferation, apoptosis, and migration. Together, our results suggest that miR-615-3p is a significant predictor of postoperative BCR and PC-specific survival and has oncogenic functions in PC cells.

The relationship between tumor aggressiveness and cholinergic PET imaging in prostate cancer tissue. A proof-of-concept study.

It was recently shown that high-risk prostate carcinoma (PCa) exhibited parasympathetic neurogenesis. The PET tracer 11C-donepezil is a marker of parasympathetic innervation. Therefore, we studied if parasympathetic nerve density in PCa measured with 11C-donepezil PET correlated with PCa aggressiveness and if metastases could be visualized. Twenty-six patients were included into three groups with varying tumor aggressiveness. Dynamic and static PET scans were performed. Maximal standardized uptake values (SUVmax) were determined in lesions within the prostate, lymph nodes, and bones. SUVmax in primary PCa were compared between groups, and comparisons between SUVmax and Gleason score and Prostate-specific antigen (PSA) were performed. Kinetic modelling was performed and time-activity curves of healthy tissue and tumor tissue fitted and compared. Tumor kinetic parameters were normalized to those of healthy tissue producing ratios of K1 and k2. Median SUVmax in primary PCa was higher in high-grade compared to low-grade PCa (P = 0.052). No correlation was seen between Gleason score and SUVmax (P = 0.28). A trend-level correlation was seen between PSA and SUVmax (P = 0.078). Median SUVmax was 7.7 (4.7-22.5) for suspected lymph node metastases and 8.2 (5.4-14.8) for suspected bone metastases. A significant difference was seen between time-activity tissue curves for low- and high-grade PCa (P = 0.012). Highly significant differences were seen in K1- and k2-ratios between low- and high-grade PCa (P = 0.0006 and P < 0.0001). We showed that 11C-donepezil accumulates in primary PCa and metastases. Simple SUVmax values of the cancer hot spots were higher in high-risk tumors compared to low-risk tumors. Further studies should elucidate the importance of cholinergic neurogenesis for prostate cancer biology.

A novel combined miRNA and methylation marker panel (miMe) for prediction of prostate cancer outcome after radical prostatectomy.

Improved prognostic biomarkers are needed to guide personalized prostate cancer (PC) treatment decisions. Due to the prominent molecular heterogeneity of PC, multimarker panels may be more robust. Here, 25 selected top-candidate miRNA and methylation markers for PC were profiled by qPCR in malignant radical prostatectomy (RP) tissue specimens from 198 PC patients (Cohort 1, training). Using GLMnet, we trained a novel multimarker model (miMe) comprising nine miRNAs and three methylation markers that predicted postoperative biochemical recurrence (BCR) independently of the established clinicopathological CAPRA-S nomogram in Cox multivariate regression analysis in Cohort 1 (HR [95% CI]: 1.53 [1.26-1.84], p < 0.001). This result was successfully validated in two independent RP cohorts (Cohort 2, n = 159: HR [95% CI]: 1.35 [1.06-1.73], p = 0.015. TCGA, n = 350: HR [95% CI]: 1.34 [1.01-1.77], p = 0.04). Notably, in CAPRA-S low-risk patients, a high miMe score was associated with >6 times higher risk of BCR, suggesting that miMe may help identify PC patients at high risk of progression despite favorable clinicopathological factors postsurgery. Finally, miMe was a significant predictor of cancer-specific survival (p = 0.019, log-rank test) in a merged analysis of 357 RP patients. In conclusion, we trained, tested and validated a novel 12-marker panel (miMe) that showed significant independent prognostic value in three RP cohorts. In the future, combining miMe score with existing clinical nomograms may improve PC risk stratification and thus help guide treatment decisions.

5hmC Level Predicts Biochemical Failure Following Radical Prostatectomy in Prostate Cancer Patients with ERG Negative Tumors.

This study aimed to validate whether 5-hydroxymethylcytosine (5hmC) level in combination with ERG expression is a predictive biomarker for biochemical failure (BF) in men undergoing radical prostatectomy (RP) for prostate cancer (PCa). The study included 592 PCa patients from two consecutive Danish RP cohorts. 5hmC level and ERG expression were analyzed using immunohistochemistry in RP specimens. 5hmC was scored as low or high and ERG was scored as negative or positive. Risk of BF was analyzed using stratified cumulative incidences and multiple cause-specific Cox regression using competing risk assessment. Median follow-up was 10 years (95% CI: 9.5⁻10.2). In total, 246 patients (41.6%) had low and 346 patients (58.4%) had high 5hmC level. No significant association was found between 5hmC level or ERG expression and time to BF (p = 0.2 and p = 1.0, respectively). However, for men with ERG negative tumors, high 5hmC level was associated with increased risk of BF following RP (p = 0.01). In multiple cause-specific Cox regression analyses of ERG negative patients, high 5hmC expression was associated with time to BF (HR: 1.8; 95% CI: 1.2⁻2.7; p = 0.003). In conclusion, high 5hmC level was correlated with time to BF in men with ERG negative PCa, which is in accordance with previous results.

Quantitative Tumor Perfusion Imaging with 82Rb PET/CT in Prostate Cancer: Analytic and Clinical Validation.

The aim of this work was to evaluate 82Rb PET/CT as a diagnostic tool for quantitative tumor blood flow (TBF) imaging in prostate cancer (PCa). Study 1 was performed to evaluate 82Rb as a marker of TBF, using 15O-H2O PET as a reference method. Study 2 investigated the ability of 82Rb uptake measurements to differentiate between PCa and normal prostate. Methods: Study 1: 9 PCa patients scheduled for radical prostatectomy were included. Prostate multiparametric MRI and both cardiac and pelvic 15O-H2O PET and 82Rb PET were performed. PET findings were compared with postprostatectomy Gleason grade group (GGG). Study 2: 15 primary high-risk PCa patients and 12 controls without known prostate disease were included in a clinical drug trial (EudraCT 2016-003185-26). 68Ga-prostate-specific membrane antigen PET/CT scans of PCa patients were available. Pelvic 82Rb PET was performed. Results: Study 1: both 82Rb K1 and 82Rb SUVs correlated strongly with 15O-H2O TBF (ρ = 0.95, P < 0.001, and ρ = 0.77, P = 0.015, respectively). 82Rb SUV and K1 were linearly correlated (r = 0.92, P = 0.001). 82Rb SUV correlated with postprostatectomy GGG (ρ = 0.70, P = 0.03). Study 2: 82Rb SUV in PCa (3.19 ± 0.48) was significantly higher than prostate 82Rb SUV in healthy controls (1.68 ± 0.37) (P < 0.001), with no overlap between groups. Conclusion: Study 1 shows that 82Rb PET/CT can be used for TBF quantification and that TBF can be estimated by simple SUV and suggests that 82Rb SUV is associated with postprostatectomy GGG and, hence, cancer aggressiveness. Study 2 shows that 82Rb uptake is significantly higher in PCa than in normal prostate tissue with no overlap between cohorts, confirming the primary hypothesis of the clinical trial. Consequently, 82Rb PET/CT may have potential as a noninvasive tool for evaluation of tumor aggressiveness and monitoring in nonmetastatic PCa.

Detection of Lymph Node Metastasis in Patients with Bladder Cancer using Maximum Standardised Uptake Value and 18F-fluorodeoxyglucose Positron Emission Tomography/Computed Tomography: Results from a High-volume Centre Including Long-term Follow-up.

BACKGROUND: Preoperative staging with 18F-fluorodeoxyglucose (18F-FDG) positron emission tomography (PET) combined with computed tomography (CT) is used for the evaluation of metastatic disease in patients with invasive bladder cancer. The use of quantification with maximum standardized uptake value (SUVmax) of regional lymph nodes (LNs) has been suggested to increase the diagnostic ability for detection of malignancy. OBJECTIVE: Assessment of the utility and clinical relevance of SUVmax in 18F-FDG PET in detecting regional nodal metastases in patients considered for radical cystectomy. DESIGN, SETTING, AND PARTICIPANTS: From 2011 to 2014, we identified a total of 119 patients with urothelial carcinoma who underwent radical cystectomy with extended LN dissection; additionally, 12 patients were identified by preoperative biopsy. All patients underwent 18F-FDG PET/CT before treatment recommendation. Pathological findings were compared with preoperative PET/CT staging and analysed in a regional- or patient-based model according to SUVmax values. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: In total, 2291 LNs were identified in 131 patients; locoregional involvement of 85 LNs were confirmed in 34 patients. RESULTS AND LIMITATIONS: SUVmax >2 analysis: sensitivity±95% confidence interval of 79.4% (62.1-91.3) and specificity 66.5% (55.7-75.3). SUVmax >4 based analysis: sensitivity was 61.8% (43.6-77.8) and specificity was 84.5% (75.8-91.1). Two years of follow-up implied that higher SUVmax is correlated with higher recurrence risk, independent of conventional pathological findings. CONCLUSIONS: 18F-FDG PET/CT using SUVmax of LNs is a useful tool for preoperative evaluation of pelvic LN metastases from invasive bladder cancer and contributes to the selection of patients for personalized treatment. PATIENT SUMMARY: In this report, we establish that it is possible to identify disease from bladder cancer in the lymphatic tissue surrounding the bladder using a scan analysis. This assists in the selection of treatment for patients with bladder cancer and may spare patients from unnecessary procedures.

Non-invasive quantification of tumor blood flow in prostate cancer using 15O-H2O PET/CT.

Tumor blood flow (TBF) measurements in prostate cancer (PCa) provide an integrative index of tumor growth, which could be important for primary diagnosis and therapy response evaluation. 15O-water PET is the non-invasive gold standard but is technically demanding. The aim of this study was to compare the accuracy of three different non-invasive strategies with an invasively measured arterial input function (BSIF): Using image-derived input functions (IDIF) from either 1) a separate heart scan or 2) the pelvic scan or 3) a populations-based input function (PBIF). Nine patients with biopsy-verified PCa scheduled for prostatectomy were included. All patients were characterized with serum levels of PSA (s-PSA), multiparametric magnetic resonance imaging (mpMRI) and post-surgical histopathology Gleason Grade. Dynamic 15O-water was performed of the heart and the pelvic area 15 minutes apart. TBF estimated from both wash-in (K1) and wash-out (k2) constants was calculated using a one-compartmental model. Results: Mean (range) s-PSA was 12 (3-27) ng/mL, Gleason Grade Group was 2.9 (1-5), k2 was 0.44 (0.007-1.2), and K1 was 0.24 (0.07-0.55) mL/mL/min. k2 (BSIF) correlated with s-PSA (r=0.86, P<0.01) and Gleason Grade Group (rho=0.78, P=0.01). BSIF, heart-IDIF and PBIF provided near-identical k2 and K1 (r>0.95, P<0.001) with slopes near unity. The correlations of BSIF and pelvic-IDIF rate constants were good (r>0.95, P<0.001), but individual errors high. In conclusion, non-invasive protocols for 15O-water PET with IDIF or PBIF accurately measures perfusion in prostate cancer and might be useful for evaluation of tumor aggressiveness and treatment response.

Human papillomavirus and squamous cell carcinoma of the urinary bladder: DaBlaCa-10 study.

OBJECTIVE: To evaluate a potential association between Human Papillomavirus (H.P.V.) and squamous cell carcinoma (S.C.C.) urinary bladder cancer (B.C.). Furthermore, the relation between p16INK4a, H.P.V. and B.C. was examined. PATIENTS AND METHODS: Patients were included and divided into three groups based on the histological diagnosis of B.C. SPECIMENS: An extensive exclusion was performed, including accepted casual risk factors for S.C.C. B.C., such as long-term use of catheters, cystolithiasis and Schistosoma hematobium infection. A total of 100 patients were included: 50 with pure S.C.C., 25 with urothelial carcinomas (U.C.) and 25 with squamous differentiation of U.C. (Sq.D.). The patients were operated at one of four major Danish hospitals in the period January 2005 to December 2016. Clinical information was collected from the medical records. Presence of H.P.V. was analyzed using the INNO-LiPA H.P.V. Genotyping Extra II. p16INK4a was analyzed using immunohistochemical (I.H.C.) staining. A p-value <0.05 was considered statistically significant. RESULTS: An overall H.P.V. prevalence of 12/100 (12%) was observed. H.P.V. was demonstrated in 9/50 (18%) of the S.C.C. PATIENTS: Overall, p16INK4a over-expression was observed in 52/100 (52%) patients. However, concomitant H.P.V. positivity and p16INK4a over-expression were observed in only 4/100 (4%) patients. CONCLUSION: The presence of H.P.V. in one fifth of patients with S.C.C. B.C. was demonstrated. H.P.V. infection could have a significant association with S.C.C. B.C. without other known casual risk factors for S.C.C. B.C.

Exploring the transcriptome of hormone-naive multifocal prostate cancer and matched lymph node metastases.

BACKGROUND: The current inability to predict whether a primary prostate cancer (PC) will progress to metastatic disease leads to overtreatment of indolent PCs as well as undertreatment of aggressive PCs. Here, we explored the transcriptional changes associated with metastatic progression of multifocal hormone-naive PC. METHODS: Using total RNA-sequencing, we analysed laser micro-dissected primary PC foci (n = 23), adjacent normal prostate tissue samples (n = 23) and lymph node metastases (n = 9) from ten hormone-naive PC patients. Genes important for PC progression were identified using differential gene expression and clustering analysis. From these, two multi-gene-based expression signatures (models) were developed, and their prognostic potential was evaluated using Cox-regression and Kaplan-Meier analyses in three independent radical prostatectomy (RP) cohorts (>650 patients). RESULTS: We identified several novel PC-associated transcripts deregulated during PC progression, and these transcripts were used to develop two novel gene-expression-based prognostic models. The models showed independent prognostic potential in three RP cohorts (n = 405, n = 107 and n = 91), using biochemical recurrence after RP as the primary clinical endpoint. CONCLUSIONS: We identified several transcripts deregulated during PC progression and developed two new prognostic models for PC risk stratification, each of which showed independent prognostic value beyond routine clinicopathological factors in three independent RP cohorts.

Dysregulation and prognostic potential of 5-methylcytosine (5mC), 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC) levels in prostate cancer.

BACKGROUND: Prognostic tools for prostate cancer (PC) are inadequate and new molecular biomarkers may improve risk stratification. The epigenetic mark 5-hydroxymethylcytosine (5hmC) has recently been proposed as a novel candidate prognostic biomarker in several malignancies including PC. 5hmC is an oxidized derivative of 5-methylcytosine (5mC) and can be further oxidized to 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). The present study is the first to investigate the biomarker potential in PC for all four DNA methylation marks in parallel. Thus, we determined 5mC, 5hmC, 5fC, and 5caC levels in non-malignant (NM) and PC tissue samples from a large radical prostatectomy (RP) patient cohort (n = 546) by immunohistochemical (IHC) analysis of serial sections of a tissue microarray. Possible associations between methylation marks, routine clinicopathological parameters, ERG status, and biochemical recurrence (BCR) after RP were investigated. RESULTS: 5mC and 5hmC levels were significantly reduced in PC compared to NM prostate tissue samples (p ≤ 0.027) due to a global loss of both marks specifically in ERG- PCs. 5fC levels were significantly increased in ERG+ PCs (p = 0.004), whereas 5caC levels were elevated in both ERG- and ERG+ PCs compared with NM prostate tissue samples (p ≤ 0.019). Positive correlations were observed between 5mC, 5fC, and 5caC levels in both NM and PC tissues (p < 0.001), while 5hmC levels were only weakly positively correlated to 5mC in the PC subset (p = 0.030). There were no significant associations between 5mC, 5fC, or ERG status and time to BCR in this RP cohort. In contrast, high 5hmC levels were associated with BCR in ERG- PCs (p = 0.043), while high 5caC levels were associated with favorable prognosis in ERG+ PCs (p = 0.011) and were borderline significantly associated with worse prognosis in ERG- PCs (p = 0.058). Moreover, a combined high-5hmC/high-5caC score was a significant adverse predictor of post-operative BCR beyond routine clinicopathological variables in ERG- PCs (hazard ratio 3.18 (1.54-6.56), p = 0.002, multivariate Cox regression). CONCLUSIONS: This is the first comprehensive study of 5mC, 5hmC, 5fC, and 5caC levels in PC and the first report of a significant prognostic potential for 5caC in PC.

Biomarker potential of ST6GALNAC3 and ZNF660 promoter hypermethylation in prostate cancer tissue and liquid biopsies.

Current diagnostic and prognostic tools for prostate cancer (PC) are suboptimal, leading to overdiagnosis and overtreatment. Aberrant promoter hypermethylation of specific genes has been suggested as novel candidate biomarkers for PC that may improve diagnosis and prognosis. We here analyzed ST6GALNAC3 and ZNF660 promoter methylation in prostate tissues, and ST6GALNAC3, ZNF660, CCDC181, and HAPLN3 promoter methylation in liquid biopsies. First, using four independent patient sample sets, including a total of 110 nonmalignant (NM) and 705 PC tissue samples, analyzed by methylation-specific qPCR or methylation array, we found that hypermethylation of ST6GALNAC3 and ZNF660 was highly cancer-specific with areas under the curve (AUC) of receiver operating characteristic (ROC) curve analysis of 0.917-0.995 and 0.846-0.903, respectively. Furthermore, ZNF660 hypermethylation was significantly associated with biochemical recurrence in two radical prostatectomy (RP) cohorts of 158 and 392 patients and remained significant also in the subsets of patients with Gleason score ≤7 (univariate Cox regression and log-rank tests, P < 0.05), suggesting that ZNF660 methylation analysis can potentially help to stratify low-/intermediate-grade PCs into indolent vs. more aggressive subtypes. Notably, ZNF660 hypermethylation was also significantly associated with poor overall and PC-specific survival in the RP cohort (n = 158) with long clinical follow-up available. Moreover, as proof of principle, we successfully detected highly PC-specific hypermethylated circulating tumor DNA (ctDNA) for ST6GALNAC3, ZNF660, HAPLN3, and CCDC181 in liquid biopsies (serum) from 27 patients with PC vs. 10 patients with BPH, using droplet digital methylation-specific PCR analysis. Finally, we generated a three-gene (ST6GALNAC3/CCDC181/HAPLN3) ctDNA hypermethylation model, which detected PC with 100% specificity and 67% sensitivity. In conclusion, we here for the first time demonstrate diagnostic biomarker potential of ST6GALNAC3 and ZNF660 methylation, as well as prognostic biomarker potential of ZNF660. Furthermore, we show that hypermethylation of four genes can be detected in ctDNA in liquid biopsies (serum) from patients with PC.

Diagnostic and Prognostic MicroRNA Biomarkers for Prostate Cancer in Cell-free Urine.

BACKGROUND: Widespread use of prostate-specific antigen (PSA) testing for prostate cancer (PC) detection has led to extensive overdiagnosis and overtreatment. Urine-based microRNA (miRNA) biomarkers could be useful in PC diagnosis and prognosis. OBJECTIVE: To train and validate urine-based microRNA (miRNA) biomarkers that may assist in PC diagnosis and prognosis. DESIGN, SETTING, AND PARTICIPANTS: We profiled the expression levels of 92 miRNAs via reverse transcriptase-poymerase chain reaction in cell-free urine samples from 29 patients with benign prostatic hyperplasia (BPH) and 215 patients with clinically localized PC (cohort 1). Our findings were validated in an independent cohort of 29 BPH patients and 220 patients with clinically localized PC (cohort 2). RESULTS AND LIMITATIONS: We identified and validated several deregulated miRNAs in urine samples from PC patients. In addition, we trained a novel diagnostic three-miRNA model (miR-222-3p*miR-24-3p/miR-30c-5p) that distinguished BPH and PC patients with an area under the curve (AUC) of 0.95 in cohort 1, and was successfully validated in cohort 2 (AUC 0.89). Furthermore, we trained a novel prognostic three-miRNA model (miR-125b-5p*let-7a-5p/miR-151-5p) that predicted time to biochemical recurrence after radical prostatectomy independently of routine clinicopathological parameters in cohort 1, and was successfully validated in cohort 2. CONCLUSIONS: Future clinical implementation of our novel diagnostic and prognostic three-miRNA signatures could help in primary diagnosis of PC and guide treatment decisions. Further validation studies are warranted. PATIENT SUMMARY: Using two large patient cohorts, we searched for novel prostate cancer biomarkers in urine. We found two new sets of microRNA biomarkers in urine that could accurately predict the presence of prostate cancer and the likelihood of recurrence after prostatectomy. Further studies are needed before an actual clinical test can be developed.

Short-term complications for percutaneous ultrasound-guided biopsy of renal masses in adult outpatients.

Background Ultrasound-guided percutaneous kidney tumor biopsy (UGPKB) plays an important role in the diagnosis of renal tumor but there are no consensuses with respect to the length and the extend of the post-biopsy observation period. Purpose To assess the short-term complication rate after UGPKB and to evaluate whether the onset of complications allows for the procedure to be performed in an outpatient setting with same-day discharge. Material and Methods Between March 2012 and March 2014, a total of 287 UGPKB were performed in an outpatient setting at a Danish university referral center. All patient records were retrospectively reviewed and post-biopsy complications as well as biochemical parameters were registered. Results The overall complication rate was 3.8% (11 patients). Major complications occurred in 1.0% of all cases (three patients); one patient with ongoing bleeding that required intervention and two patients with septicemia. Minor complications occurred in 2.8% of cases (eight patients); six patients with self-limiting gross hematuria, one patient with small asymptomatic subcapsular hematoma, and one patient with vasovagal syncope. The timing of both minor and major complication onset ranged from the time of biopsy and up to four days after discharge. Conclusion UGPKB of indeterminate renal masses in adult patients in an outpatient setting appears to be a safe procedure with a very low rate of major complications. Same-day discharge after renal mass biopsy seems feasible.

Prostate cancer: in-bore magnetic resonance guided biopsies at active surveillance inclusion improve selection of patients for active treatment.

Background Active surveillance (AS) of low-risk prostate cancer (PCa) is an accepted alternative to active treatment. However, the conventional diagnostic trans-rectal ultrasound guided biopsies (TRUS-bx) underestimate PCa aggressiveness in almost half of the cases, when compared with the surgical specimen. Purpose To investigate if additional multi-parametric magnetic resonance imaging (mpMRI) of the prostate and MRI-guided in-bore biopsies (MRGB) at AS inclusion would improve selection of patients for active treatment. Material and Methods All patients enrolled in AS programs at two Danish centers, from October 2014 to January 2016, were offered an mpMRI 8-12 weeks after the initial diagnostic TRUS-bx. Candidates had low-risk disease (PSA < 10 ng/mL, 6 or GS 6 (3 + 3) lesions with ≥ 6 mm maximal cancer core length (MCCL). Results A total of 78 patients were included and in 21 patients a total of 22 PIRADS-score 4 or 5 lesions were detected. MRGB pathology revealed that 17 (81%) of these and 22% of the entire AS population harbored significant cancers at AS inclusion. In eight (38%) cases, the GS was upgraded. Also, nine patients (43%) had GS 6 (3 + 3) foci with MCCL ≥ 6 mm. Conclusion In an AS cohort based on TRUS and TRUS-bx diagnostic strategies, supplemental mpMRI and in-bore MRGB were able to efficiently reclassify a substantial number of patients as candidates for immediate active treatment.

Monitoring Treatment Response and Metastatic Relapse in Advanced Bladder Cancer by Liquid Biopsy Analysis.

Of the patients undergoing radical cystectomy, 20-80% experience relapse. Minimally invasive methods for early detection of metastatic relapse after cystectomy and for monitoring ongoing therapy are urgently needed to improve individualised follow-up and treatment. Therefore, we evaluated the use of circulating tumour DNA (ctDNA) in plasma and urine to detect metastatic relapse after cystectomy and measure treatment efficacy. We exome sequenced tumour and germline DNA from patients with muscle-invasive bladder cancer and monitored ctDNA in 370 liquid biopsies throughout the disease courses by 84 personalised digital droplet polymerase chain reaction assays targeting 61 genes. Patients were prospectively recruited between 2013 and 2017. Patients with metastatic relapse had significantly higher ctDNA levels compared with disease-free patients (p<0.001). The median positive lead time between ctDNA detection in plasma and diagnosis of relapse was 101 d after cystectomy (range 0-932 d). Early detection of metastatic relapse and treatment response using liquid biopsies represents a novel, highly sensitive tool for monitoring patients, supporting clinicians, and guiding treatment decisions. PATIENT SUMMARY: Measurement of tumour-specific mutations in plasma and urine may be a powerful tool to monitor response during treatment and identify early signs of metastatic disease.