Prognostic Value of Low-Pass Whole Genome Sequencing of Circulating Tumor DNA in Metastatic Castration-Resistant Prostate Cancer.

BACKGROUND: Multiple treatments are available for metastatic castration-resistant prostate cancer (mCRPC), including androgen receptor signaling inhibitors (ARSI) enzalutamide and abiraterone, but therapy resistance remains a major clinical obstacle. We examined the clinical utility of low-pass whole-genome sequencing (LPWGS) of circulating tumor DNA (ctDNA) for prognostication in mCRPC. METHODS: A total of 200 plasma samples from 143 mCRPC patients collected at the start of first-line ARSI treatment (baseline) and at treatment termination (n = 57, matched) were analyzed by LPWGS (median: 0.50X) to access ctDNA% and copy number alteration (CNA) patterns. The best confirmed prostate specific antigen (PSA) response (≥50% decline [PSA50]), PSA progression-free survival (PFS), and overall survival (OS) were used as endpoints. For external validation, we used plasma LPWGS data from an independent cohort of 70 mCRPC patients receiving first-line ARSI. RESULTS: Baseline ctDNA% ranged from ≤3.0% to 73% (median: 6.6%) and CNA burden from 0% to 82% (median: 13.1%) in the discovery cohort. High ctDNA% and high CNA burden at baseline was associated with poor PSA50 response (P = 0.0123/0.0081), poor PFS (P < 0.0001), and poor OS (P < 0.0001). ctDNA% and CNA burden was higher at PSA progression than at baseline in 32.7% and 42.3% of the patients. High ctDNA% and high CNA burden at baseline was also associated with poor PFS and OS (P ≤ 0.0272) in the validation cohort. CONCLUSIONS: LPWGS of ctDNA provides clinically relevant information about the tumor genome in mCRPC patients. Using LPWGS data, we show that high ctDNA% and CNA burden at baseline is associated with short PFS and OS in 2 independent cohorts.

Independent validation of a pre-specified four-kallikrein marker model for prediction of adverse pathology and biochemical recurrence.

BACKGROUND: Accurate markers for prostate cancer (PC) risk stratification could aid decision-making for initial management strategies. The 4Kscore has an undefined role in predicting outcomes after radical prostatectomy (RP). METHODS: We included 1476 patients with 4Kscore measured prior to RP at two institutions. The 4Kscore was assessed for prediction of adverse pathology at RP and biochemical recurrence (BCR) relative to a clinical model. We pre-specified that all analyses would be assessed in biopsy Grade Group 1 (GG1) or 2 (GG2) PC patients, separately. RESULTS: The 4Kscore increased discrimination for adverse pathology in all patients (delta area under the receiver operative curve (AUC) 0.009, 95% confidence interval (CI) 0.002, 0.016; clinical model AUC 0.767), driven by GG1 (delta AUC 0.040, 95% CI 0.006, 0.073) rather than GG2 patients (delta AUC 0.005, 95% CI -0.012, 0.021). Adding 4Kscore improved prediction of BCR in all patients (delta C-index 0.014, 95% CI 0.007, 0.021; preop-BCR nomogram C-index 0.738), again with larger changes in GG1 than in GG2. CONCLUSIONS: This study validates prior investigations on the use of 4Kscore in men with biopsy-confirmed PC. Men with GG1 PC and a high 4Kscore may benefit from additional testing to guide treatment selection. Further research is warranted regarding the value of the 4Kscore in men with biopsy GG2 PC.

Recurrence rate after radical prostatectomy following primary staging of high-risk prostate cancer with 68Ga-PSMA PET/CT.

BACKGROUND: Accurate primary staging is one of the most important issues for initial management of prostate cancer (PCa) patients to perform an optimal selection of patients for curative intended treatment. 68Ga-Prostate-Specific-Membrane-Antigen (PSMA) PET/CT was found superior to conventional imaging both for detection of recurrence after curative intended treatment and for primary staging. We studied the recurrence rate after radical prostatectomy in high-risk PCa patients primary staged with 68Ga-PSMA PET/CT compared with conventional imaging. MATERIAL AND METHODS: The study included 247 D'Amico high-risk PCa patients treated with radical prostatectomy (RP) after primary staging with 68Ga-PSMA PET/CT and a reference group of 137 high-risk patients with RP after conventional imaging (99mTc bone scintigraphy and CT). Recurrence rates were assessed by Cox regression and Kaplan-Meier analysis. RESULTS: The 5-year recurrence-free survival rate was 71.1% in the 68Ga-PSMA PET/CT cohort compared with 56.4% in the conventional imaging cohort. Primary staging by 68Ga-PSMA PET/CT reduced biochemical recurrence (BCR) risk by 42% (HR = 0.58 (0.41-0.83), p = .004). CONCLUSION: The present data could indicate a lower recurrence rate after RP following primary staging with 68Ga-PSMA PET/CT compared to conventional imaging, likely due to improved selection of patients for surgery.

Immune cell analyses of the tumor microenvironment in prostate cancer highlight infiltrating regulatory T cells and macrophages as adverse prognostic factors.

Improved risk stratification is needed for patients with localized prostate cancer. This study characterized and assessed the prognostic potential of distinct immune cell infiltration patterns in the prostate tumor microenvironment. Using tissue microarrays, multiplex immunohistochemistry/immunofluorescence, and automated digital pathology, we analyzed radical prostatectomy specimens from two large patient cohorts (training: n = 470; validation: n = 333) to determine infiltration levels of seven immune cell types in malignant versus benign prostate tissue: CD3+ CD8- FoxP3- T helper cells, CD3+ CD8+ FoxP3- cytotoxic T cells (CTLs), CD3+ CD8- FoxP3+ regulatory T cells (Tregs ), CD20+ B cells, CD68+ CD163- M1 macrophages, CD68+ CD163+ M2 macrophages, and tryptase+ mast cells. Results were further validated by cell type enrichment analyses of bulk tumor RNAseq data from a third independent patient cohort (n = 99). Prognostic potential was assessed by Kaplan-Meier and uni-/multi-variate Cox regression analyses. Clinical endpoint was biochemical recurrence. All seven immune cell types were enriched in prostate cancer versus benign stroma, while there was selective enrichment for B cells, Tregs , M1 and M2 macrophages, and depletion of mast cells and CTLs in prostate cancer epithelium. In all three cohorts, high levels of infiltrating Tregs , M1, and M2 macrophages in stroma and/or epithelium were associated with biochemical recurrence (p < 0.05; log-rank test). After adjustment for routine clinical variables, Tregs and M2 macrophages remained significant adverse predictors of biochemical recurrence (p < 0.05; multivariate Cox regression). Our comprehensive analyses of immune cell infiltration patterns in the prostate tumor microenvironment highlight infiltrating Tregs , M1, and M2 macrophages as adverse predictors of prostate cancer outcome. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

The effect of assessing genetic risk of prostate cancer on the use of PSA tests in primary care: A cluster randomized controlled trial.

BACKGROUND: Assessing genetic lifetime risk for prostate cancer has been proposed as a means of risk stratification to identify those for whom prostate-specific antigen (PSA) testing is likely to be most valuable. This project aimed to test the effect of introducing a genetic test for lifetime risk of prostate cancer in general practice on future PSA testing. METHODS AND FINDINGS: We performed a cluster randomized controlled trial with randomization at the level of general practices (73 in each of two arms) in the Central Region (Region Midtjylland) of Denmark. In intervention practices, men were offered a genetic test (based on genotyping of 33 risk-associated single nucleotide polymorphisms) in addition to the standard PSA test that informed them about lifetime genetic risk of prostate cancer and distinguished between "normal" and "high" risk. The primary outcome was the proportion of men having a repeated PSA test within 2 years. A multilevel logistic regression model was used to test the association. After applying the exclusion criteria, 3,558 men were recruited in intervention practices, with 1,235 (34.7%) receiving the genetic test, and 4,242 men were recruited in control practices. Men with high genetic risk had a higher propensity for repeated PSA testing within 2 years than men with normal genetic risk (odds ratio [OR] = 8.94, p < 0.01). The study was conducted in routine practice and had some selection bias, which is evidenced by the relatively large proportion of younger and higher income participants taking the genetic test. CONCLUSIONS: Providing general practitioners (GPs) with access to a genetic test to assess lifetime risk of prostate cancer did not reduce the overall number of future PSA tests. However, among men who had a genetic test, knowledge of genetic risk significantly influenced future PSA testing. TRIAL REGISTRATION: This study is registered with ClinicalTrials.gov, number NCT01739062.

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.

A five-microRNA model (pCaP) for predicting prostate cancer aggressiveness using cell-free urine.

Improved biomarkers for prostate cancer (PC) risk stratification are urgently needed. Here, we aimed to develop a novel multimarker model for prediction of biochemical recurrence (BCR) after curatively intended radical prostatectomy (RP), based on minimally invasive sampling of blood and urine. We initially measured the levels of 45 selected miRNAs by RT-qPCR in exosome enriched cell-free urine samples collected prior to RP from 215 PC patients (Cohort 1, training). We trained a novel logistic regression model (pCaP), comprising five urine miRNAs (miR-151a-5p, miR-204-5p, miR-222-3p, miR-23b-3p and miR-331-3p) and serum prostate-specific antigen (PSA), which significantly predicted time to BCR in Cohort 1 (univariate Cox regression analysis: HR = 3.12, p < 0.001). Next, using the same exact numeric cutoff for dichotomization as trained in Cohort 1, we tested and successfully validated the prognostic potential of pCaP in two additional cohorts, including 199 (Cohort 2, HR = 2.24, p = 0.002) and 205 (Cohort 3, HR = 2.15, p = 0.004) RP patients, respectively. pCaP remained a significant predictor of BCR, also after adjustment for pathological T-stage, surgical margin status and Gleason grade group (p < 0.05 in multivariate Cox regression analysis: HR = 2.72, 1.94 and 1.83 for Cohorts 1, 2 and 3, respectively). Additionally, pCaP scores correlated positively with the established clinical risk stratification nomogram CAPRA in all three PC cohorts (Pearson's rho: 0.45, 0.39 and 0.44). Together, our results suggest that the minimally invasive pCaP model could potentially be used in the future to improve PC risk stratification and to guide more personalized treatment decisions. Further clinical validation studies are warranted.

Independent Validation of a Diagnostic Noninvasive 3-MicroRNA Ratio Model (uCaP) for Prostate Cancer in Cell-Free Urine.

BACKGROUND: Detection of prostate cancer (PC) based on serum prostate-specific antigen (PSA) testing leads to many unnecessary prostate biopsies, overdiagnosis, and overtreatment of clinically insignificant tumors. Thus, novel and more accurate molecular biomarkers are required. METHODS: Using reverse transcription quantitative PCR, we measured the concentrations of 45 preselected microRNAs (miRNAs) in extracellular vesicle-enriched cell-free urine samples from 4 independent patient cohorts from Spain and Denmark, including 758 patients with clinically localized PC, 289 noncancer controls with benign prostatic hyperplasia (BPH), and 233 patients undergoing initial transrectal ultrasound (TRUS)-guided prostate biopsy owing to PC suspicion (101 with benign and 132 with malignant outcome). Diagnostic potential was assessed by ROC and decision curve analysis. RESULTS: We identified and successfully validated 8 upregulated and 21 downregulated miRNAs in urine from PC patients. Furthermore, we validated a previously identified 3-miRNA diagnostic ratio model, uCaP (miR-222-3p*miR-24-3p/miR-30c-5p). High uCaP scores were distinctive of PC in urine samples from BPH vs PC patients in 3 independent cohorts [area under the curve (AUC) = 0.84, 0.71, 0.72]. Additionally, uCaP predicted TRUS biopsy results with greater accuracy than PSA (AUC uCaP = 0.644; AUC PSA = 0.527) for patients within the diagnostic gray zone (PSA ≤ 10 ng/mL). CONCLUSIONS: We successfully validated a urine-based diagnostic 3-miRNA signature for PC (uCaP) in 3 independent patient cohorts from 2 countries. In the future, the simple and noninvasive uCaP test may be used to help more accurately select patients for prostate biopsy. Prospective clinical validation is warranted.

Aberrant DOCK2, GRASP, HIF3A and PKFP Hypermethylation has Potential as a Prognostic Biomarker for Prostate Cancer.

Prostate cancer (PCa) is a clinically heterogeneous disease and currently, accurate diagnostic and prognostic molecular biomarkers are lacking. This study aimed to identify novel DNA hypermethylation markers for PCa with future potential for blood-based testing. Accordingly, to search for genes specifically hypermethylated in PCa tissue samples and not in blood cells or other cancer tissue types, we performed a systematic analysis of genome-wide DNA methylation data (Infinium 450K array) available in the Marmal-aid database for 4072 malignant/normal tissue samples of various types. We identified eight top candidate markers (cg12799885, DOCK2, FBXO30, GRASP, HIF3A, MOB3B, PFKP, and TPM4) that were specifically hypermethylated in PCa tissue samples and hypomethylated in other benign and malignant tissue types, including in peripheral blood cells. Potential as diagnostic and prognostic biomarkers was further assessed by the quantitative methylation specific PCR (qMSP) analysis of 37 nonmalignant and 197 PCa tissue samples from an independent population. Here, all eight hypermethylated candidates showed high sensitivity (75⁻94%) and specificity (84⁻100%) for PCa. Furthermore, DOCK2, GRASP, HIF3A and PKFP hypermethylation was significantly associated with biochemical recurrence (BCR) after radical prostatectomy (RP; 197 patients), independent of the routine clinicopathological variables. DOCK2 is the most promising single candidate marker (hazard ratio (HR) (95% confidence interval (CI)): 1.96 (1.24⁻3.10), adjusted p = 0.016; multivariate cox regression). Further validation studies are warranted and should investigate the potential value of these hypermethylation candidate markers for blood-based testing also.

Top2 and Sgs1-Top3 Act Redundantly to Ensure rDNA Replication Termination.

Faithful DNA replication with correct termination is essential for genome stability and transmission of genetic information. Here we have investigated the potential roles of Topoisomerase II (Top2) and the RecQ helicase Sgs1 during late stages of replication. We find that cells lacking Top2 and Sgs1 (or Top3) display two different characteristics during late S/G2 phase, checkpoint activation and accumulation of asymmetric X-structures, which are both independent of homologous recombination. Our data demonstrate that checkpoint activation is caused by a DNA structure formed at the strongest rDNA replication fork barrier (RFB) during replication termination, and consistently, checkpoint activation is dependent on the RFB binding protein, Fob1. In contrast, asymmetric X-structures are formed independent of Fob1 at less strong rDNA replication fork barriers. However, both checkpoint activation and formation of asymmetric X-structures are sensitive to conditions, which facilitate fork merging and progression of replication forks through replication fork barriers. Our data are consistent with a redundant role of Top2 and Sgs1 together with Top3 (Sgs1-Top3) in replication fork merging at rDNA barriers. At RFB either Top2 or Sgs1-Top3 is essential to prevent formation of a checkpoint activating DNA structure during termination, but at less strong rDNA barriers absence of the enzymes merely delays replication fork merging, causing an accumulation of asymmetric termination structures, which are solved over time.

DNA Topoisomerases maintain promoters in a state competent for transcriptional activation in Saccharomyces cerevisiae.

To investigate the role of DNA topoisomerases in transcription, we have studied global gene expression in Saccharomyces cerevisiae cells deficient for topoisomerases I and II and performed single-gene analyses to support our findings. The genome-wide studies show a general transcriptional down-regulation upon lack of the enzymes, which correlates with gene activity but not gene length. Furthermore, our data reveal a distinct subclass of genes with a strong requirement for topoisomerases. These genes are characterized by high transcriptional plasticity, chromatin regulation, TATA box presence, and enrichment of a nucleosome at a critical position in the promoter region, in line with a repressible/inducible mode of regulation. Single-gene studies with a range of genes belonging to this group demonstrate that topoisomerases play an important role during activation of these genes. Subsequent in-depth analysis of the inducible PHO5 gene reveals that topoisomerases are essential for binding of the Pho4p transcription factor to the PHO5 promoter, which is required for promoter nucleosome removal during activation. In contrast, topoisomerases are dispensable for constitutive transcription initiation and elongation of PHO5, as well as the nuclear entrance of Pho4p. Finally, we provide evidence that topoisomerases are required to maintain the PHO5 promoter in a superhelical state, which is competent for proper activation. In conclusion, our results reveal a hitherto unknown function of topoisomerases during transcriptional activation of genes with a repressible/inducible mode of regulation.

Stroma-specific gene expression signature identifies prostate cancer subtype with high recurrence risk.

Current prognostic tools cannot clearly distinguish indolent and aggressive prostate cancer (PC). We hypothesized that analyzing individual contributions of epithelial and stromal components in localized PC (LPC) could improve risk stratification, as stromal subtypes may have been overlooked due to the emphasis on malignant epithelial cells. Hence, we derived molecular subtypes of PC using gene expression analysis of LPC samples from prostatectomy patients (cohort 1, n = 127) and validated these subtypes in two independent prostatectomy cohorts (cohort 2, n = 406, cohort 3, n = 126). Stroma and epithelium-specific signatures were established from laser-capture microdissection data and non-negative matrix factorization was used to identify subtypes based on these signatures. Subtypes were functionally characterized by gene set and cell type enrichment analyses, and survival analysis was conducted. Three epithelial (E1-E3) and three stromal (S1-S3) PC subtypes were identified. While subtyping based on epithelial signatures showed inconsistent associations to biochemical recurrence (BCR), subtyping by stromal signatures was significantly associated with BCR in all three cohorts, with subtype S3 indicating high BCR risk. Subtype S3 exhibited distinct features, including significantly decreased cell-polarity and myogenesis, significantly increased infiltration of M2-polarized macrophages and CD8 + T-cells compared to subtype S1. For patients clinically classified as CAPRA-S intermediate risk, S3 improved prediction of BCR. This study demonstrates the potential of stromal signatures in identification of clinically relevant PC subtypes, and further indicated that stromal characterization may enhance risk stratification in LPC and may be particularly promising in cases with high prognostic ambiguity based on clinical parameters.

DNA hairpins as temperature switches, thermometers and ionic detectors.

Temperature is of major importance in most branches of science and technology as well as in everyday life, and with the miniaturization of electronic devices and the increasing ability to make research into small-scale systems, a specific need for very small thermostats and thermometers has been created. Here we describe how DNA molecules can be used as nanoscale sensors to meet these requirements. We illustrate how the hybridization kinetics between bases in DNA molecules combined with conformational changes of the DNA backbone can be exploited in the construction of simple but versatile temperature switches and thermometers, which can be built into electronic systems. DNA based sensors are at the same time applicable as ion detectors to monitor the chemical environment of a specific system.

Results from the PRIMA Trial: Comparison of the STHLM3 Test and Prostate-specific Antigen in General Practice for Detection of Prostate Cancer in a Biopsy-naïve Population.

BACKGROUND: Current management of prostate cancer (PC) lacks biomarker tests and diagnostic procedures that can accurately distinguish clinically significant and clinically insignificant PCs at an early stage of the disease. OBJECTIVE: To compare the Stockholm 3 (STHLM3) test and prostate-specific antigen (PSA) as entry tests for magnetic resonance imaging (MRI) in a prospective study of PC diagnosis in general practice. DESIGN, SETTING, AND PARTICIPANTS: Participants were biopsy-naïve men aged 50-69 yr who had a PSA test in general practice. Participants with PSA 1-10 ng/ml also had an STHLM3 test and were referred for MRI if the STHLM311 test was positive (risk ≥11%) and/or PSA ≥3 ng/ml, and to targeted MRI-guided biopsy (MRGB) if their Prostate Imaging-Reporting and Data System (PI-RADS) score was ≥3. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: The primary outcome was the number of International Society of Urological Pathology grade group ≥2 (GG ≥2) cases detected with a positive STHLM311 test versus PSA ≥3 ng/ml. Post hoc analysis was performed using a higher STHLM3 test cutoff (risk ≥15%; positive STHLM315 test). RESULTS AND LIMITATIONS: Between January 2018 and December 2021, we recruited 1905 men. The STHLM3 test was performed in 1134 participants. Of these, 437 underwent MRI and 117 underwent MRGB, which detected 38 (32.5%) GG ≥2 and 52 (44.4%) with GG 1 cases. In comparison to PSA ≥3 ng/ml, a positive STHLM311 test increased detection of GG ≥2 from 30 to 37 cases (23.3%, 95% confidence interval [CI] 5.6-52.2%) and detection of GG 1 from 37 to 50 cases (35.1%, 95%CI 11.6-66.7%). STHLM315 positivity did not differ from PSA ≥3 ng/ml regarding detection of GG ≥2 PC (30 vs 32; 6.6%, 95% CI -8.1% to 25.9%), GG 1 PC (37 vs 37; 0.0%, 95% CI -19.6% to 25.0%), or MRGB use (88 vs 83; -5.7%, 95% CI -17.9% to 7.4%), but reduced MRI scans from 320 to 236 (-26.2%, 95% CI -33.1% to -18.9%). CONCLUSIONS: The STHLM311 test improved sensitivity but not specificity for detection of GG ≥2 PC in the clinical setting of nonsystematic PC testing in general practice. Further studies are needed to validate a possible benefit of using a higher cutoff for STHLM3 positivity as an entry test for MRI. PATIENT SUMMARY: We used a test called STHLM3 for detection of prostate cancer in general practice and compared its performance to the conventional PSA (prostate-specific antigen) test. We found that STHLM3 test results of 11% or above were not better at selecting men for MRI (magnetic resonance imaging) scans than the PSA test with a cutoff of 3 ng/ml or above. Analysis suggested that a higher cutoff for a positive STHLM3 test may improve selection of men for MRI scans, but further validation is needed.

Danish Prostate Cancer Consortium Study 1 (DPCC-1) protocol: Multicentre prospective validation of the urine-based three-microRNA biomarker model uCaP.

INTRODUCTION: The primary objective of the Danish Prostate Cancer Consortium Study 1 (DPCC-1) is to provide validation for a novel urine-based microRNA biomarker, called uCaP, for a diagnosis of prostate cancer. METHODS AND ANALYSIS: Eligible participants are biopsy naïve men aged ≥18 years with prostate-specific antigen (PSA) levels ≥3 ng/mL, who are referred to prostate MRI due to suspicion of PC at one of the following three major urology/uroradiology centers: Aarhus University Hospital, Herlev & Gentofte University Hospital, or Odense University Hospital, where MRI and targeted biopsy are implemented in clinical use. Exclusion criteria include previous diagnosis of urogenital cancer, contraindication to MRI, gender reassignment treatment or PSA level >20 ng/mL. The participants will be asked to donate a urine sample in connection with their MRI. The study is observational, uses a diagnostic accuracy testing setup and will integrate into the current diagnostic pathway.We will measure the levels of the three microRNAs in the uCaP model (miR-222-3 p, miR-24-3 p and miR-30c-5p) in extracellular vesicle-enriched cell-free urine samples, to assess if uCaP can improve specificity and retain sensitivity for International Society of Urological Pathology Grade Group ≥2 PC, when used as a reflex test to PSA ≥3 ng/mL. We hypothesise that uCaP can improve selection for prostate MRI and reduce the number of unnecessary scans and biopsies. ETHICS AND DISSEMINATION: This study is approved by the Central Denmark Region Committee on Health Research Ethics (reference number: 1-10-72-85-22). All participants will provide written informed consent. Study results will be published in peer-reviewed journals and presented in scientific meetings. TRIAL REGISTRATION NUMBER: NCT05767307 at clinicaltrials.gov.

Cost-Effectiveness Analysis of Stockholm 3 Testing Compared to PSA as the Primary Blood Test in the Prostate Cancer Diagnostic Pathway: A Decision Tree Approach.

OBJECTIVE: This study evaluated the cost effectiveness of using Stockholm 3 (STHLM3) testing compared to the prostate-specific antigen (PSA) test in the diagnostic pathway for prostate cancer. METHODS: We created a decision tree model for PSA (current standard) and STHLM3 (new alternative). Cost effectiveness was evaluated in a hypothetical cohort of male individuals aged 50-69 years. The study applied a Danish hospital perspective with a time frame restricted to the prostate cancer diagnostic pathway, beginning with the initial PSA/STHLM3 test, and ending with biopsy and histopathological diagnosis. Estimated values from the decision-analytical model were used to calculate the incremental cost-effectiveness ratio. Deterministic and probabilistic sensitivity analyses were conducted to test the robustness of the base-case analysis. RESULTS: The model-based analysis revealed that STHLM3 testing was more effective than the PSA, but also more costly, with an incremental cost-effectiveness ratio of €511.7 (95% credible interval, 359.9-674.3) for each additional correctly classified individual. In the deterministic sensitivity analysis, variations in the cost of STHLM3 had the greatest influence on the incremental cost-effectiveness ratio. In the probabilistic sensitivity analysis, all iterations were positioned in the north-east quadrant of the incremental cost-effectiveness scatterplot. At a willingness to pay of €700 for an additional correctly classified individual, STHLM3 had a 100% probability of being cost effective. CONCLUSIONS: Compared to the PSA test as the initial testing modality in the prostate cancer diagnostic workup, STHLM3 testing showed improved incremental effectiveness, however, at additional costs. The results were sensitive to the cost of the STHLM3 test; therefore, a lower cost of the STHLM3 test would improve its cost effectiveness compared with PSA tests.

Microbiota of the prostate tumor environment investigated by whole-transcriptome profiling.

BACKGROUND: With over 350,000 estimated deaths worldwide in 2018, prostate cancer (PCa) continues to be a major health concern and a significant cause of cancer-associated mortality among men. While cancer in general is considered a disease of the human genome, there is a growing body of evidence suggesting that changes to the healthy microbiota could play a vital role in cancer development, progression, and/or treatment outcome. METHODS: Using a metatranscriptomic approach, we annotated the microbial reads obtained from total RNA sequencing of 106 prostate tissue samples from 94 PCa patients (discovery cohort). We investigated microbial dysbiosis associated with PCa by systematically comparing the microbiomes between benign and malignant tissue samples, between less vs. more-aggressive PCa, and between patients who had biochemical recurrence as opposed to those who did not. We further performed differential gene expression and cell type enrichment analysis to explore the host transcriptomic and cellular responses to selected microbial genera. A public dataset (GSE115414) of total RNA sequencing reads from 24 prostate tissue samples (8 benign and 16 malignant) served as the validation cohort. RESULTS: We observed decreased species diversity and significant under-representation of Staphylococcus saprophyticus and Vibrio parahaemolyticus, as well as significant over-abundance of Shewanella in malignant as compared to benign prostate tissue samples in both the discovery (p < 0.01) and validation (p < 0.05) cohorts. In addition, we identified Microbacterium species (p < 0.01) to be significantly over-abundant in pathologically advanced T3 tumors compared to T2 in the discovery cohort. Malignant samples having high vs. low Shewanella counts were associated with downregulated Toll-like receptor signaling pathways and decreased enrichment of dendritic cells. Malignant samples having low vs. high V. parahaemolyticus counts were enriched for olfactory transduction and drug metabolism pathways. Finally, malignant samples were enriched for M1 and M2 macrophages as compared to benign tissue samples. CONCLUSIONS: The results from this exploratory study support the existence of an important biological link between the prostate microbiota and PCa development/progression. Our results highlight Shewanella, V. parahaemolyticus, and Microbacterium sp. as interesting candidates for further investigation of their association with PCa.

Predicting Grade group 2 or higher cancer at prostate biopsy by 4Kscore in blood and uCaP microRNA model in urine.

Elevated prostate-specific antigen (PSA) levels often lead to unnecessary and possibly harmful transrectal ultrasound guided biopsy, e.g. when the biopsy is negative or contains only low-grade insignificant cancer, unlikely to become symptomatic in the man's normal lifespan. A model based on four-kallikrein markers in blood (commercialized as 4Kscore) predicts risk of Grade group 2 or higher prostate cancer at biopsy, reducing unnecessary biopsies. We assessed whether these results extend to a single institution prostate biopsy cohort of Danish men and are enhanced by three microRNAs from urine (referred to as uCaP). The 4Kscore measured in cryopreserved blood from 234 men referred for 10+ core biopsy to Aarhus University Hospital, 29 with PSA > 25 ng/ml. We explored uCaP in urine from 157 of these men. Combined with age and DRE findings, both 4Kscore and uCaP could accurately predict Grade group 2 or higher prostate cancer (all patients: AUC = 0.802 and 0.797; PSA ≤ 25: AUC = 0.763 and 0.759). There was no additive effect when combining the 4Kscore and uCaP. Limitations include a study cohort with higher risk than commonly reported for biopsy cohorts. Our findings further support the clinical use of the 4Kscore to predict Grade group 2 or higher cancers in men being considered for biopsy.

The transcriptional landscape and biomarker potential of circular RNAs in prostate cancer.

BACKGROUND: Circular RNAs (circRNAs) constitute a largely unexplored source for biomarker discovery in prostate cancer (PC). Here, we characterize the biomarker potential of circRNAs in PC, where the need for novel diagnostic and prognostic tools to facilitate more personalized management is pressing. METHODS: We profiled the transcriptomic landscape of circRNAs in PC by total RNA sequencing of 31 adjacent-normal and 143 tumor samples from localized (radical prostatectomy (RP)) and metastatic PC patients (cohort 1, training). Diagnostic and prognostic potential was evaluated in cohort 1, and 39 top circRNA candidates were selected for validation in two additional PC cohorts (cohort 2, n = 111; RP cohort 3, n = 191) by NanoString-based expression analysis. Biochemical recurrence (BCR)-free survival was assessed using Kaplan-Meier, univariate, and multivariate Cox regression analyses. The circRNA candidates were further detected in extracellular vesicle (EV)-enriched plasma samples from PC patients and controls (cohort 4, n = 54). RESULTS: Expression of circABCC4, circFAT3, circATRNL1, and circITGA7 was highly cancer-specific (area under the curve 0.71-0.86), while low circITGA7 expression was significantly (P < 0.05) associated with BCR in univariate analysis in two RP cohorts. Moreover, we successfully trained and validated a novel 5-circRNA prognostic signature (circKMD1A/circTULP4/circZNF532/circSUMF1/circMKLN1) significantly associated with BCR beyond routine clinicopathological variables (RP cohort 1: P = 0.02, hazard ratio = 2.1; RP cohort 3: P < 0.001, hazard ratio = 2.1). Lastly, we provide proof-of-principle for detection of candidate circRNAs in EV-enriched plasma samples from PC patients. CONCLUSIONS: circRNAs hold great biomarker potential in PC and display both high cancer specificity and association to disease progression.

A genome-wide CRISPR-Cas9 knockout screen identifies novel PARP inhibitor resistance genes in prostate cancer.

DNA repair gene mutations are frequent in castration-resistant prostate cancer (CRPC), suggesting eligibility for poly(ADP-ribose) polymerase inhibitor (PARPi) treatment. However, therapy resistance is a major clinical challenge and genes contributing to PARPi resistance are poorly understood. Using a genome-wide CRISPR-Cas9 knockout screen, this study aimed at identifying genes involved in PARPi resistance in CRPC. Based on the screen, we identified PARP1, and six novel candidates associated with olaparib resistance upon knockout. For validation, we generated multiple knockout populations/clones per gene in C4 and/or LNCaP CRPC cells, which confirmed that loss of PARP1, ARH3, YWHAE, or UBR5 caused olaparib resistance. PARP1 or ARH3 knockout caused cross-resistance to other PARPis (veliparib and niraparib). Furthermore, PARP1 or ARH3 knockout led to reduced autophagy, while pharmacological induction of autophagy partially reverted their PARPi resistant phenotype. Tumor RNA sequencing of 126 prostate cancer patients identified low ARH3 expression as an independent predictor of recurrence. Our results advance the understanding of PARPi response by identifying four novel genes that contribute to PARPi sensitivity in CRPC and suggest a new model of PARPi resistance through decreased autophagy.