Spatial Distribution of Macrophage and Lymphocyte Subtypes within Tumor Microenvironment to Predict Recurrence of Non-Muscle-Invasive Papillary Urothelial Carcinoma after BCG Immunotherapy.

Non-muscle-invasive papillary urothelial carcinoma (NMIPUC) of the urinary bladder is the most common type of bladder cancer. Intravesical Bacille Calmette-Guerin (BCG) immunotherapy is applied in patients with a high risk of recurrence and progression of NMIPUC to muscle-invasive disease. However, the tumor relapses in about 30% of patients despite the treatment, raising the need for better risk stratification. We explored the potential of spatial distributions of immune cell subtypes (CD20, CD11c, CD163, ICOS, and CD8) within the tumor microenvironment to predict NMIPUC recurrence following BCG immunotherapy. Based on analyses of digital whole-slide images, we assessed the densities of the immune cells in the epithelial-stromal interface zone compartments and their distribution, represented by an epithelial-stromal interface density ratio (IDR). While the densities of any cell type did not predict recurrence, a higher IDR of CD11c (HR: 0.0012, p-value = 0.0002), CD8 (HR: 0.0379, p-value = 0.005), and ICOS (HR: 0.0768, p-value = 0.0388) was associated with longer recurrence-free survival (RFS) based on the univariate Cox regression. The history of positive repeated TUR (re-TUR) (HR: 4.93, p-value = 0.0001) and T1 tumor stage (HR: 2.04, p-value = 0.0159) were associated with shorter RFS, while G3 tumor grade according to the 1973 WHO classification showed borderline significance (HR: 1.83, p-value = 0.0522). In a multivariate analysis, the two models with a concordance index exceeding 0.7 included the CD11c IDR in combination with either a history of positive re-TUR or tumor stage. We conclude that the CD11c IDR is the most informative predictor of NMIPUC recurrence after BCG immunotherapy. Our findings highlight the importance of assessment of the spatial distribution of immune cells in the tumor microenvironment.

Combination of DNA methylation biomarkers with multiparametric magnetic resonance and ultrasound imaging fusion biopsy to detect the local spread of prostate cancer.

BACKGROUND: This study aimed to investigate the extent of field cancerization adjacent to index lesions in prostate cancer (PCa) by measuring DNA methylation of selected tumor suppressor genes in the perifocal tissue of PCa not visible on multiparametric magnetic resonanse imaging (mpMRI) for the safe zone of focal therapy identification. METHODS: A total of 272 patients were enrolled in this study, 44 patients' tissue biosamples were included in the field cancerization research, and 272 urine samples were included in the urine-based test development. Targeted biopsies were performed using the mpMRI/ultrasoundimage fusion system. RESULTS: Quantitative analysis revealed significantly higher DNA methylation levels of RARB, RASSF1, GSTP1 & APC genes in the index lesion compared with perifocal tissue samples 10 mm away from it (p < 0.0001). Notably, the RARB, GSTP1 & APC and RARB, RASSF1, GSTP1 & APC biomarker combinations exhibited the highest sensitivity and specificity comparing the extent of DNA methylation in index lesions and noncancerous prostate tissues 20 mm away (both area under the curve [AUC] = 0.98; p < 0.0001). The analysis of the potential urinary biomarkers showed that the combination of all four DNA methylation biomarkers with prostate-specific antigen (PSA) or PSA density (PSAD) in the blood significantly improves the detection of clinically significant PCa (csPCa). The combination of the four-biomarker test with PSAD allowed the identification of csPCa with ≥90% sensitivity and specificity. CONCLUSION: Thus, this study suggests that for focal therapy by region target hemi-ablation, the safe distance from the index lesion is no less than 10 mm. Noninvasive urine DNA methylation tests in combination with PSAD could be used for further follow-up of the patients, but larger prospective studies with external validation are needed.

Safety, Pharmacokinetic and Pharmacodynamic Evaluation of Teverelix for the Treatment of Hormone-Sensitive Advanced Prostate Cancer: Phase 2 Loading-Dose-Finding Studies.

Background and objectives: Teverelix drug product (DP) is a gonadotropin-releasing hormone antagonist in development for the treatment of patients with prostate cancer in whom androgen deprivation therapy is indicated. The aim of this paper is to present the results of five Phase 2 studies that assessed the pharmacokinetics, pharmacodynamics, efficacy and safety of different loading dose regimens of teverelix DP. Methods: Five single-arm, uncontrolled clinical trials were conducted in patients with advanced prostate cancer. The five different loading dose regimens of teverelix DP tested were (a) a single 90 mg subcutaneous (SC) injection of teverelix DP given on 3 consecutive days (Days 0, 1 and 2); (b) a single 90 mg intramuscular (IM) injection of teverelix DP given 7 days apart (Days 0 and 7); (c) a single 120 mg SC injection of teverelix DP given on 2 consecutive days (Days 0 and 1); (d) 2 × 60 mg SC injections of teverelix DP given on 3 consecutive days (Days 0, 1 and 2), and (e) 2 × 90 mg SC injections of teverelix DP given on 3 consecutive days (Days 0, 1 and 2). The primary efficacy parameter was the duration of action of an initial loading dose regimen in terms of suppression of testosterone to below the castration level (0.5 ng/mL). Results: Eighty-two patients were treated with teverelix DP. Two regimens (90 mg and 180 mg SC on 3 consecutive days) had a mean duration of castration of 55.32 days and 68.95 days with >90% of patients having testosterone levels < 0.5 ng/mL at Day 28. The mean onset of castration for the SC regimens ranged from 1.10 to 1.77 days, while it was slower (2.4 days) with IM administration. The most common adverse event (AE) was injection site reaction. No AEs of severe intensity were reported. Conclusions: Teverelix DP is safe and well tolerated. Castrate levels of testosterone can be rapidly achieved following the subcutaneous injection of teverelix DP on 3 consecutive days. Streamlining of the administration of the loading dose and identifying a suitable maintenance dose will be investigated in future trials.

Germline DNA Damage Response Gene Mutations in Localized Prostate Cancer.

Background and Objectives: Germline DNA damage response (DDR) gene mutations correlate with increased prostate cancer (PCa) risk and a more aggressive form of the disease. DDR mutation testing is recommended for metastatic PCa cases, while eligible information about the mutations' burden in the early-stage localized PCa is still limited. This study is aimed at the prospective detection of DDR pathway mutations in cases with localized PCa and correlation with clinical, histopathological, and radiological data. A comparison to the previously assessed cohort of the advanced PCa was performed. Materials and Methods: Germline DDR gene mutations were assessed prospectively in DNA samples from 139 patients, using a five-gene panel (BRCA1, BRCA2, ATM, CHEK2, and NBN) targeted next-generation sequencing. Results: This study revealed an almost three-fold higher risk of localized PCa among mutation carriers as compared to non-carriers (OR 2.84 and 95% CI: 0.75-20.23, p = 0.16). The prevalence of germline DDR gene mutations in PCa cases was 16.8% (18/107) and they were detected only in cases with PI-RADS 4/5 lesions. BRCA1/BRCA2/ATM mutation carriers were 2.6 times more likely to have a higher (>1) cISUP grade group compared to those with a CHEK2 mutation (p = 0.27). However, the number of cISUP > 1-grade patients with a CHEK2 mutation was significantly higher in advanced PCa than in localized PCa: 66.67% vs. 23.08% (p = 0.047). Conclusions: The results of our study suggest the potential of genetic screening for selected DDR gene mutations for early identification of cases at risk of aggressive PCa.

Urinary microRNAs can predict response to abiraterone acetate in castration resistant prostate cancer: A pilot study.

BACKGROUND: Despite novel agents have been introduced to treat castration resistant prostate cancer (CRPC) during the last decade, up to one-third of CRPC patients face primary resistance to new generation compounds. Therefore, sensitive molecular tools are urgently needed for reliable treatment selection and response prediction. This study aimed to evaluate urinary miRNAs and blood circulating androgen receptor (AR) transcript level as a tool for noninvasive outcome prediction for CRPC patients undergoing abiraterone acetate (AA) therapy. METHODS: Prostate cancer-specific miR-148a, -365, -375, and -429 were analyzed in 129 urine samples collected from 100 CRPC patients before and during AA therapy via quantitative reverse transcription PCR. To test the prognostic value, urinary miRNA levels alone, as well as combined with AR level were associated with progression-free survival (PFS) and overall survival (OS). RESULTS: Level of urinary miR-375 was the highest in CRPC in comparison to noncancerous controls, as well as in combination with miR-429 was predictive for short PFS in AA-treated patients (HR = 2.2, 95% CI: 1.1-4.2, p = 0.023). Especially high prognostic power of all analyzed miRNAs was observed in CRPC cases with high blood AR levels. For PFS prediction a tandem of miR-429 and high AR reached HR of 5.0 (95% CI: 2.2-11.8, p < 0.001), while for prediction of OS the best combination was demonstrated by miR-148a and AR with HR of 3.1 (95% CI: 1.4-7.1, p = 0.006). CONCLUSIONS: Urinary miRNAs could be used as prognostic biomarkers for CRPC patients to predict response to AA therapy, especially for the cases with high blood AR levels.

Evaluation of Renal Function after Partial Nephrectomy and Detection of Clinically Significant Acute Kidney Injury.

Background and Objectives: Consequences of partial nephrectomy (PN), intraoperative hypotension (IOH) and postoperative neutrophil to lymphocyte ratio (NLR) may cause postoperative acute kidney injury (AKI) and in long-term-chronic kidney disease (CKD). Our study aimed to identify the AKI incidence after PN, to find clinically significant postoperative AKI and renal dysfunction, and to determine the predictor factors. Materials and Methods: A prospective observational study consisted of 91 patients who received PN with warm ischemia, and estimated preoperative glomerular filtration rate (eGFR) ≥ 60 mL/min and without abnormal albuminuria. Results: 38 (41.8%) patients experienced postoperative AKI. Twenty-one (24.1%) patients had CKD upstage after 1 year follow-up. Sixty-seven percent of CKD upstage patients had AKI 48 h after surgery and 11% after 2 months. All 15 (16.5%) patients with CKD had postoperative AKI. With IOH, OR 1.07, 95% CI 1.03−1.10 and p < 0.001, postoperative NLR after 48 h (OR 1.50, 95% CI 1.19−1.88, p < 0.001) was the major risk factor of AKI. In multivariate logistic regression analysis, the kidney's resected part volume (OR 1.08, 95% CI 1.03−1.14, p < 0.001) and IOH (OR 1.10, 95% CI 1.04−1.15, p < 0.001) were retained as statistically significant prognostic factors for detecting postoperative renal dysfunction. The independent risk factor for clinically significant postoperative AKI was only IOH (OR, 1.06; p < 0.001). Only AKI with the CKD upstage group has a statistically significant effect (p < 0.0001) on eGFR 6 and 12 months after surgery. Conclusions: The presence of AKI after PN is not rare. IOH and NLR are associated with postoperative AKI. The most important predictive factor of postoperative AKI is an NLR of over 3.5. IOH is an independent risk factor for clinically significant postoperative AKI and together with kidney resected part volume effects postoperative renal dysfunction. Only clinically significant postoperative AKI influences the reduction of postoperative eGFR after 6 and 12 months.

Promoter Methylation of PRKCB, ADAMTS12, and NAALAD2 Is Specific to Prostate Cancer and Predicts Biochemical Disease Recurrence.

The molecular diversity of prostate cancer (PCa) has been demonstrated by recent genome-wide studies, proposing a significant number of different molecular markers. However, only a few of them have been transferred into clinical practice so far. The present study aimed to identify and validate novel DNA methylation biomarkers for PCa diagnosis and prognosis. Microarray-based methylome data of well-characterized cancerous and noncancerous prostate tissue (NPT) pairs was used for the initial screening. Ten protein-coding genes were selected for validation in a set of 151 PCa, 51 NPT, as well as 17 benign prostatic hyperplasia samples. The Prostate Cancer Dataset (PRAD) of The Cancer Genome Atlas (TCGA) was utilized for independent validation of our findings. Methylation frequencies of ADAMTS12, CCDC181, FILIP1L, NAALAD2, PRKCB, and ZMIZ1 were up to 91% in our study. PCa specific methylation of ADAMTS12, CCDC181, NAALAD2, and PRKCB was demonstrated by qualitative and quantitative means (all p < 0.05). In agreement with PRAD, promoter methylation of these four genes was associated with the transcript down-regulation in the Lithuanian cohort (all p < 0.05). Methylation of ADAMTS12, NAALAD2, and PRKCB was independently predictive for biochemical disease recurrence, while NAALAD2 and PRKCB increased the prognostic power of multivariate models (all p < 0.01). The present study identified methylation of ADAMTS12, NAALAD2, and PRKCB as novel diagnostic and prognostic PCa biomarkers that might guide treatment decisions in clinical practice.

Systematic and MRI-Cognitive Targeted Transperineal Prostate Biopsy Accuracy in Detecting Clinically Significant Prostate Cancer after Previous Negative Biopsy and Persisting Suspicion of Malignancy.

Background and objectives: Overdiagnosis, overtreatment, and the need for repeated procedures caused by transrectal ultrasound guided prostate biopsies and their related complications places a heavy burden on healthcare systems. This was a prospective cohort validating study to access the clinical accuracy of systematic and MRI-cognitive targeted transperineal prostate biopsies in detecting clinically significant prostate cancer after a previous negative biopsy and persistent suspicion of malignancy. The primary goal was to assess the ability of multiparametric magnetic resonance imaging (mpMRI) to detect clinically significant prostate cancer with an additional goal to assess the diagnostic value of systematic and MRI-cognitive transperineal biopsies. Materials and Methods: In total, 200 patients were enrolled who had rising serum prostate specific antigen (PSA) levels for at least 4 months after a previous negative transrectal ultrasound (TRUS) biopsy. All eligible men underwent 1.5T prostate mpMRI, reported using the Prostate Imaging Reporting and Data System version 2 (PI-RADS v2), followed by a 20-region transperineal prostate systematic biopsy and additional targeted biopsies. Results: Systematic 20-core transperineal prostate biopsies (TPBs) were performed for 38 (19%) patients. Systemic 20-core TPB with additional cognitive targeted biopsies were performed for 162 (81%) patients. Clinically significant prostate cancer (csPC) was detected for 31 (15.5%) patients, of which 20 (64.5%) cases of csPC were detected by systematic biopsy, eight (25.8%) cases were detected by targeted biopsy, and three (9.7%) both by systematic and targeted biopsies. Conclusions: Cognitive mpMRI guided transperineal target biopsies increase the detection rate of clinically significant prostate cancer after a previously negative biopsy. However, in a repeat prostate biopsy setting, we recommend applying a cognitive targeted biopsy with the addition of a systematic biopsy.

Analysis of AR-FL and AR-V1 in Whole Blood of Patients with Castration Resistant Prostate Cancer as a Tool for Predicting Response to Abiraterone Acetate.

PURPOSE: Reliable molecular diagnostic tools are still unavailable for making informed treatment decisions and monitoring the response in patients with castration resistant prostate cancer. We evaluated the significance of whole blood circulating androgen receptor transcripts of full length (AR-FL) and splice variants (AR-V1, AR-V3 and AR-V7) as biomarkers of abiraterone acetate treatment resistance in patients with castration resistant prostate cancer. MATERIALS AND METHODS: After retrospective analysis in 112 prostate specimens AR-FL, AR-V1, AR-V3 and AR-V7 were evaluated in 185 serial blood samples, prospectively collected from 102 patients with castration resistant prostate cancer before and during abiraterone acetate therapy via reverse transcription quantitative polymerase chain reaction. RESULTS: AR-FL was present in all samples while AR-V1, AR-V3, AR-V7 and at least 1 of them was detected in 17%, 55%, 65% and 81% of castration resistant prostate cancer blood samples, respectively. The highest amount of AR-V1 was found in blood of patients whose response time was short and medium in comparison to extended. Patients with a higher level of AR-FL and/or AR-V1 had the shortest progression-free survival and overall survival (p <0.0001). CONCLUSIONS: Blood circulating AR-FL or AR-V1 can serve as blood based biomarkers for identification of the primary resistance to abiraterone acetate and the tool to monitor de novo resistance development during abiraterone acetate treatment.

The Impact of Prostate Cancer Upgrading and Upstaging on Biochemical Recurrence and Cancer-Specific Survival.

Background and Objectives: Significant numbers of prostate cancer (PCa) patients experience tumour upgrading and upstaging between prostate biopsy and radical prostatectomy (RP) specimens. The aim of our study was to investigate the role of grade and stage increase on surgical and oncological outcomes. Materials and Methods: Upgrading and upstaging rates were analysed in 676 treatment-naïve PCa patients who underwent RP with subsequent follow-up. Positive surgical margin (PSM), biochemical recurrence (BCR), metastasis-free survival (MFS), overall (OS) and cancer specific survival (CSS) were analysed according to upgrading and upstaging. Results: Upgrading was observed in 29% and upstaging in 22% of PCa patients. Patients undergoing upgrading or upstaging were 1.5 times more likely to have a PSM on RP pathology. Both upgrading and upstaging were associated with increased risk for BCR: 1.8 and 2.1 times, respectively. Mean time to BCR after RP was 2.1 years in upgraded cases and 2.7 years in patients with no upgrading (p <0.001), while mean time to BCR was 1.9 years in upstaged and 2.8 years in non-upstaged cases (p <0.001). Grade and stage increase after RP were associated with inferior MFS rates and ten-year CSS: 89% vs. 98% for upgrading (p = 0.039) and 87% vs. 98% for upstaging (p = 0.008). Conclusions: Currently used risk stratification models are associated with substantial misdiagnosis. Pathological upgrading and upstaging have been associated with inferior surgical results, substantial higher risk of BCR and inferior rates of important oncological outcomes, which should be considered when counselling PCa patients at the time of diagnosis or after definitive therapy.

Clinical significance of miRNA host gene promoter methylation in prostate cancer.

Only a part of prostate cancer (PCa) patients has aggressive malignancy requiring adjuvant treatment after radical prostatectomy (RP). Biomarkers capable to predict biochemical PCa recurrence (BCR) after RP would significantly improve preoperative risk stratification and treatment decisions. MicroRNA (miRNA) deregulation has recently emerged as an important phenomenon in tumor development and progression, however, the mechanisms remain largely unstudied. In the present study, based on microarray profiling of DNA methylation in 9 pairs of PCa and noncancerous prostate tissues (NPT), host genes of miR-155-5p, miR-152-3p, miR-137, miR-31-5p, and miR-642a, -b were analyzed for promoter methylation in 129 PCa, 35 NPT, and 17 benign prostatic hyperplasia samples (BPH) and compared to the expression of mature miRNAs and their selected targets (DNMT1, KDM1A, and KDM5B). The Cancer Genome Atlas dataset was utilized for validation. Methylation of mir-155, mir-152, and mir-137 host genes was PCa-specific, and downregulation of miR-155-5p significantly correlated with promoter methylation. Higher KDM5B expression was observed in samples with methylated mir-155 or mir-137 promoters, whereas upregulation of KDM1A and DNMT1 was associated with mir-155 and mir-152 methylation status, respectively. Promoter methylation of mir-155, mir-152, and mir-31 was predictive of BCR-free survival in various Cox models and increased the prognostic value of clinicopathologic factors. In conclusion, methylated mir-155, mir-152, mir-137, and mir-31 host genes are promising diagnostic and/or prognostic biomarkers of PCa. Methylation status of particular miRNA host genes as independent variables or in combinations might assist physicians in identifying poor prognosis PCa patients preoperatively.

Prostate cancer solitary metastasis to anal canal: case report and review of literature.

BACKGROUND: Here we present the first cases of prostate cancer solitary metastasis to anal canal. CASE PRESENTATION: A 67-year-old male patient underwent radical prostatectomy with ilio-obturator lymphonodectomy in 2016 due to poorly differentiated ductal adenocarcinoma (Gleason 4 + 5(40%) = 9) pT3bN0. Two months later increasing PSA rate was noted and the patient started adjuvant intermittent androgen deprivation therapy combined with radiotherapy. Year after patient was admitted to the hospital complaining of dyschezia, pain in anal canal, and bloody stool. Digital rectal examination revealed an anal fissure with ulceration. A biopsy from ulcerated area showed poorly differentiated ductal adenocarcinoma of the prostate. Because there was no evidence of distant metastases on abdominal computed tomography (CT) scan and pelvic magnetic nuclear resonance imaging (MRI) and the only metastasis was in anal canal patient underwent laparoscopic abdominoperineal resection (APR). Postoperative course was uneventful and patient was discharged at postoperative day 7. CONCLUSIONS: Our presented case is the first to describe prostate cancer solitary metastasis to anal canal and we always have to be aware of possible rare disease while assessing the patient with rectal bleeding. Biopsy most of the time is the only and the most reliable test to differentiate between the diseases.

The utility of urine-circulating miRNAs for detection of prostate cancer.

BACKGROUND: In this paper, the utility of urine-circulating microRNAs (miRNAs) as the potential biomarker of prostate cancer (PCa), the second most prevalent male cancer worldwide, was evaluated. METHODS: Cancerous (N=56) and non-cancerous (N=16) prostate tissues were analysed on TaqMan Low Density Array, with the initial screening of 754 miRNAs in a subset of the samples. The abundance of selected miRNAs was analysed in urine specimens from two independent cohorts of patients with PCa (N=215 overall), benign prostatic hyperplasia (BPH; N=23), and asymptomatic controls (ASC; N=62) by means of quantitative reverse transcription PCR. RESULTS: Over 100 miRNAs were found deregulated in PCa as compared with non-cancerous prostate tissue. After thorough validation, four miRNAs were selected for the analysis in urine specimens. The abundance of miR-148a and miR-375 in urine was identified as specific biomarkers of PCa in both cohorts. Combined analysis of urine-circulating miR-148a and miR-375 was highly sensitive and specific for PCa in both cohorts (AUC=0.79 and 0.84) and strongly improved the diagnostic power of the PSA test (AUC=0.85, cohort PCa1), including the grey diagnostic zone (AUC=0.90). CONCLUSIONS: Quantitative measurement of urine-circulating miR-148a and miR-375 can serve as the non-invasive tool for sensitive and specific detection of PCa.

Detection of miRNAs in urine of prostate cancer patients.

BACKGROUND AND AIM: Prostate cancer (PCa) is the second most prevalent oncologic disease among men worldwide. Expression of various transcripts, including miRNAs, is markedly deregulated in cancerous prostate tissue. This study aimed at identifying a PCa-specific expression profile of miRNAs for subsequent use in noninvasive diagnostics. MATERIALS AND METHODS: MiRNA expression was profiled in 13 PCa tissues using human miRNA microarrays. Highly expressed miRNAs were selected for the analysis in urine of patients with PCa (N=143) and benign prostate hyperplasia (BPH; N=23) by means of real time PCR, while miRNAs showing the expression differences in relation to clinical variables were further analyzed in 52 PCa and 12 noncancerous prostate tissues (NPT) on TaqMan Low Density Arrays (TLDA). RESULTS: Analysis of miRNA expression in prostate tissue linked miR-95 to aggressive form of PCa. This miRNA was up-regulated in high grade (P=0.041), the TMPRSS2-ERG fusion-positive tumors (P=0.026), and in patients with subsequently developed biochemical recurrence (BCR; P=0.054) after radical prostatectomy. MiRNAs highly expressed in PCa tissues were also detectable in urine from PCa patients. Moreover, the urinary levels of miR-21 had significant discriminatory power (P=0.010) to separate PCa patients from BPH, while the combined analysis of urinary miR-19a and miR-19b was prognostic for BCR. In PCa, the diagnostic potential of urinary miRNA panel (miR-21, miR-19a, and miR-19b) was higher than that of the PSA test (AUC=0.738 vs. AUC=0.514). CONCLUSIONS: Measurement of urinary levels of PCa-specific miRNAs could assist in more specific detection of PCa and prediction of BCR.

Frequent down-regulation of ABC transporter genes in prostate cancer.

BACKGROUND: ATP-binding cassette (ABC) transporters are transmembrane proteins responsible for the efflux of a wide variety of substrates, including steroid metabolites, through the cellular membranes. For better characterization of the role of ABC transporters in prostate cancer (PCa) development, the profile of ABC transporter gene expression was analyzed in PCa and noncancerous prostate tissues (NPT). METHODS: TaqMan Low Density Array (TLDA) human ABC transporter plates were used for the gene expression profiling in 10 PCa and 6 NPT specimens. ABCB1 transcript level was evaluated in a larger set of PCa cases (N = 78) and NPT (N = 15) by real-time PCR, the same PCa cases were assessed for the gene promoter hypermethylation by methylation-specific PCR. RESULTS: Expression of eight ABC transporter genes (ABCA8, ABCB1, ABCC6, ABCC9, ABCC10, ABCD2, ABCG2, and ABCG4) was significantly down-regulated in PCa as compared to NPT, and only two genes (ABCC4 and ABCG1) were up-regulated. Down-regulation of ABC transporter genes was prevalent in the TMPRSS2-ERG-negative cases. A detailed analysis of ABCB1 expression confirmed TLDA results: a reduced level of the transcript was identified in PCa in comparison to NPT (p = 0.048). Moreover, the TMPRSS2-ERG-negative PCa cases showed significantly lower expression of ABCB1 in comparison to NPT (p = 0.003) or the fusion-positive tumors (p = 0.002). Promoter methylation of ABCB1 predominantly occurred in PCa and was rarely detected in NPT (p < 0.001). CONCLUSIONS: The study suggests frequent down-regulation of the ABC transporter genes in PCa, especially in the TMPRSS2-ERG-negative tumors.

Epigenetic markers of prostate cancer in plasma circulating DNA.

Epigenetic differences are a common feature of many diseases, including cancer, and disease-associated changes have even been detected in bodily fluids. DNA modification studies in circulating DNA (cirDNA) may lead to the development of specific non-invasive biomarkers. To test this hypothesis, we investigated cirDNA modifications in prostate cancer patients with locally confined disease (n = 19), in patients with benign prostate hyperplasias (n = 20) and in men without any known prostate disease (n = 20). This initial discovery screen identified 39 disease-associated changes in cirDNA modification, and seven of these were validated using the sodium bisulfite-based mapping of modified cytosines in both the discovery cohort and an independent 38-patient validation cohort. In particular, we showed that the DNA modification of regions adjacent to the gene encoding ring finger protein 219 distinguished prostate cancer from benign hyperplasias with good sensitivity (61%) and specificity (71%). We also showed that repetitive sequences detected in this study were meaningful, as they indicated a highly statistically significant loss of DNA at the pericentromeric region of chromosome 10 in prostate cancer patients (p = 1.8 × 10(-6)). Based on these strong univariate results, we applied machine-learning techniques to develop a multi-locus biomarker that correctly distinguished prostate cancer samples from unaffected controls with 72% accuracy. Lastly, we used systems biology techniques to integrate our data with publicly available DNA modification and transcriptomic data from primary prostate tumors, thereby prioritizing genes for further studies. These data suggest that cirDNA epigenomics are promising source for non-invasive biomarkers.

Prognostic value of RASSF1 promoter methylation in prostate cancer.

PURPOSE: Patients with prostate cancer who have biochemical recurrence after curative therapy are at higher risk for distant metastasis and cancer specific death. Assessment of aberrant DNA methylation in urine might complement currently used clinical prognostic factors and serve as a noninvasive tool for early prediction of biochemical recurrence after radical prostatectomy. MATERIALS AND METHODS: Promoter methylation of 7 genes was evaluated by methylation sensitive polymerase chain reaction in 149 prostate cancer tissues, 37 noncancerous prostate tissues and 17 benign prostatic hyperplasia samples. Quantitative polymerase chain reaction was used for DNA methylation analysis of the urine of 253 patients with prostate cancer and 32 with benign prostatic hyperplasia. RESULTS: In prostate cancer tissue the most frequently methylated genes were RASSF1, GSTP1 and RARB, which combined were positively identified in 85% of cases. These genes were also methylated in the urine of 60% of patients with prostate cancer. RASSF1 was methylated in 45% of prostate cancer urine samples with methylation intensity significantly higher in prostate cancer than in benign prostatic hyperplasia cases (p = 0.018). In a univariate model RASSF1 methylation and the total number of methylated genes in prostate cancer tissue were predictive of time to biochemical recurrence (p = 0.019 and 0.043, respectively). On multivariate analysis RASSF1 methylation together with pathological stage was the most significant predictor of biochemical recurrence in patients with Gleason score 6 tumors when analyzed in tissue and urine (p ≤0.001). CONCLUSIONS: Hypermethylation of RASSF1 in cancerous tissue and urine from patients with prostate cancer correlated with biochemical recurrence after radical prostatectomy. The prognostic potential of this biomarker deserves further investigation.

Prediction of Non-Muscle Invasive Papillary Urothelial Carcinoma Relapse from Hematoxylin-Eosin Images Using Deep Multiple Instance Learning in Patients Treated with Bacille Calmette-Guérin Immunotherapy.

The limited reproducibility of the grading of non-muscle invasive papillary urothelial carcinoma (NMIPUC) necessitates the search for more robust image-based predictive factors. In a cohort of 157 NMIPUC patients treated with Bacille Calmette-Guérin (BCG) immunotherapy, we explored the multiple instance learning (MIL)-based classification approach for the prediction of 2-year and 5-year relapse-free survival and the multiple instance survival learning (MISL) framework for survival regression. We used features extracted from image patches sampled from whole slide images of hematoxylin-eosin-stained transurethral resection (TUR) NPMIPUC specimens and tested several patch sampling and feature extraction network variations to optimize the model performance. We selected the model showing the best patient survival stratification for further testing in the context of clinical and pathological variables. MISL with the multiresolution patch sampling technique achieved the best patient risk stratification (concordance index = 0.574, p = 0.010), followed by a 2-year MIL classification. The best-selected model revealed an independent prognostic value in the context of other clinical and pathologic variables (tumor stage, grade, and presence of tumor on the repeated TUR) with statistically significant patient risk stratification. Our findings suggest that MISL-based predictions can improve NMIPUC patient risk stratification, while validation studies are needed to test the generalizability of our models.