Development of a multivariable risk model integrating urinary cell DNA methylation and cell-free RNA data for the detection of significant prostate cancer

Background: Prostate cancer exhibits severe clinical heterogeneity and there is a critical need for clinically implementable tools able to precisely and noninvasively identify patients that can either be safely removed from treatment pathways or those requiring further follow up. Our objectives were to develop a multivariable risk prediction model through the integration of clinical, urine-derived cell-free messenger RNA (cf-RNA) and urine cell DNA methylation data capable of noninvasively detecting significant prostate cancer in biopsy naïve patients. Methods: Post-digital rectal examination urine samples previously analyzed separately for both cellular methylation and cf-RNA expression within the Movember GAP1 urine biomarker cohort were selected for a fully integrated analysis (n = 207). A robust feature selection framework, based on bootstrap resampling and permutation, was utilized to find the optimal combination of clinical and urinary markers in a random forest model, deemed ExoMeth. Out-of-bag predictions from ExoMeth were used for diagnostic evaluation in men with a clinical suspicion of prostate cancer (PSA ≥ 4 ng/mL, adverse digital rectal examination, age, or lower urinary tract symptoms). Results: As ExoMeth risk score (range, 0-1) increased, the likelihood of high-grade disease being detected on biopsy was significantly greater (odds ratio = 2.04 per 0.1 ExoMeth increase, 95% confidence interval [CI]: 1.78-2.35). On an initial TRUS biopsy, ExoMeth accurately predicted the presence of Gleason score ≥3 + 4, area under the receiver-operator characteristic curve (AUC) = 0.89 (95% CI: 0.84-0.93) and was additionally capable of detecting any cancer on biopsy, AUC = 0.91 (95% CI: 0.87-0.95). Application of ExoMeth provided a net benefit over current standards of care and has the potential to reduce unnecessary biopsies by 66% when a risk threshold of 0.25 is accepted. Conclusion: Integration of urinary biomarkers across multiple assay methods has greater diagnostic ability than either method in isolation, providing superior predictive ability of biopsy outcomes. ExoMeth represents a more holistic view of urinary biomarkers and has the potential to result in substantial changes to how patients suspected of harboring prostate cancer are diagnosed

epiCaPture: a urine DNA methylation test for early detection of aggressive prostate cancer

Purpose Liquid biopsies that noninvasively detect molecular correlates of aggressive prostate cancer (PCa) could be used to triage patients, reducing the burdens of unnecessary invasive prostate biopsy and enabling early detection of high-risk disease. DNA hypermethylation is among the earliest and most frequent aberrations in PCa. We investigated the accuracy of a six-gene DNA methylation panel (Epigenetic Cancer of the Prostate Test in Urine [epiCaPture]) at detecting PCa, high-grade (Gleason score greater than or equal to 8) and high-risk (D'Amico and Cancer of the Prostate Risk Assessment] PCa from urine. Patients and Methods Prognostic utility of epiCaPture genes was first validated in two independent prostate tissue cohorts. epiCaPture was assessed in a multicenter prospective study of 463 men undergoing prostate biopsy. epiCaPture was performed by quantitative methylation-specific polymerase chain reaction in DNA isolated from prebiopsy urine sediments and evaluated by receiver operating characteristic and decision curves (clinical benefit). The epiCaPture score was developed and validated on a two thirds training set to one third test set. Results Higher methylation of epiCaPture genes was significantly associated with increasing aggressiveness in PCa tissues. In urine, area under the receiver operating characteristic curve was 0.64, 0.86, and 0.83 for detecting PCa, high-grade PCa, and highrisk PCa, respectively. Decision curves revealed a net benefit across relevant threshold probabilities. Independent analysis of two epiCaPture genes in the same clinical cohort provided analytical validation. Parallel epiCaPture analysis in urine and matched biopsy cores showed added value of a liquid biopsy. Conclusion epiCaPture is a urine DNA methylation test for high-risk PCa. Its tumor specificity out-performs that of prostate-specific antigen (greater than 3 ng/mL). Used as an adjunct to prostate-specific antigen, epiCaPture could aid patient stratification to determine need for biopsy

A Role for TLR4 in Clostridium difficile Infection and the Recognition of Surface Layer Proteins

Clostridium difficile is the etiological agent of antibiotic-associated diarrhoea (AAD) and pseudomembranous colitis in humans. The role of the surface layer proteins (SLPs) in this disease has not yet been fully explored. The aim of this study was to investigate a role for SLPs in the recognition of C. difficile and the subsequent activation of the immune system. Bone marrow derived dendritic cells (DCs) exposed to SLPs were assessed for production of inflammatory cytokines, expression of cell surface markers and their ability to generate T helper (Th) cell responses. DCs isolated from C3H/HeN and C3H/HeJ mice were used in order to examine whether SLPs are recognised by TLR4. The role of TLR4 in infection was examined in TLR4-deficient mice. SLPs induced maturation of DCs characterised by production of IL-12, TNFα and IL-10 and expression of MHC class II, CD40, CD80 and CD86. Furthermore, SLP-activated DCs generated Th cells producing IFNγ and IL-17. SLPs were unable to activate DCs isolated from TLR4-mutant C3H/HeJ mice and failed to induce a subsequent Th cell response. TLR4−/− and Myd88−/−, but not TRIF−/− mice were more susceptible than wild-type mice to C. difficile infection. Furthermore, SLPs activated NFκB, but not IRF3, downstream of TLR4. Our results indicate that SLPs isolated from C. difficile can activate innate and adaptive immunity and that these effects are mediated by TLR4, with TLR4 having a functional role in experimental C. difficile infection. This suggests an important role for SLPs in the recognition of C. difficile by the immune system

Investigating chemopreventive and chemotherapeutic applications of cannabinoids in prostate cancer

Prostate cancer is the 5th leading cause of cancer death in men, responsible for more than 375,000 deaths worldwide in 2020. For patients with localised prostate cancer, the 5-year survival rate is close to 100%. However, metastatic prostate cancer remains a lethal disease, with a 5-year survival rate of just 30%. Therefore, novel therapeutic strategies are urgently needed to improve clinical outcomes for patients with advanced prostate cancer. Cannabinoids, chemical components of the cannabis plant, pose a possible solution. Substantial preclinical evidence demonstrates that cannabinoids can modulate key hallmarks of cancer, including cell death, proliferation, migration and invasion, and angiogenesis. However, few studies have investigated the anti-cancer potential of cannabinoids in prostate cancer. Our overall hypothesis was that plant-derived cannabinoids (phytocannabinoids) have chemotherapeutic and chemopreventive effects in prostate cancer. To test this hypothesis, our specific aims were to assess the effects of cannabinoids on various hallmarks of cancer, to investigate the mechanisms underlying the observed phenotypic effects, and to determine whether phytocannabinoids have antioxidant or chemopreventive effects in non-cancerous prostate cells. First, we used cell line models of prostate cancer to measure the effects of cannabinoid treatment on cell viability, survival, proliferation, apoptosis, and migration and invasion. The non-psychoactive phytocannabinoid GL1a inhibited prostate cancer cell viability and proliferation, with no significant increase in apoptosis. GL1a reduced the invasiveness of highly metastatic PC-3 cells. We also found some evidence that combinations of cannabinoid compounds produce enhanced anti-cancer activity. However, GL1a also reduced viability and induced apoptosis in non-cancerous prostate cells and further investigation into possible off-target effects is warranted. Having shown that GL1a reduced cell proliferation and invasion, our next goal was to investigate the underlying mechanisms. Specifically, we aimed to identify the receptor targets of GL1a in prostate cancer cells and to measure the effects of GL1a treatment on the expression of cell cycle proteins, the phosphorylation of protein kinases, and the expression and secretion of proteins involved in cell invasion. GL1a reduced the expression of the key cell cycle proteins cyclin D3, CDK2, CDK4, and CDK1, and reduced the phosphorylation and activation of the protein kinase AKT. Additionally, we found some evidence that GL1a may increase the expression of E-cadherin, an adhesion protein associated with a non-invasive phenotype. The effects of GL1a on cell viability were not blocked by CB1 or CB2 antagonists, TRPV ion channel blockers, or a GPR55 agonist, suggesting that GL1a acts independently of these targets in prostate cancer cells. Finally, we assessed the antioxidant and cytoprotective potential of low-dose cannabinoids in non-cancerous prostate cells. Hydrogen peroxide was used to induce oxidative stress. Phytocannabinoid treatment at the doses tested produced no significant cytoprotective or antioxidant effects. However, drug exposure times and doses may require further optimisation. This study provides novel insights into the phenotypic effects and mechanisms of action of cannabinoids in prostate cancer cells. GL1a reduces prostate cancer cell proliferation and invasion. The underlying mechanisms include altered expression of key cell cycle regulators and modulation of AKT phosphorylation. Future studies should aim to identify the receptor target(s) of GL1a and further investigate its mechanisms of action, as well as testing the effects of cannabinoid treatment in more biologically relevant experimental models. Ultimately, clinical trials will be needed to determine whether the observed phenotypic effects of GL1a in vitro can translate to therapeutic benefits and improved outcomes in patients with prostate cancer

Plant-derived cannabinoids as anticancer agents.

Substantial preclinical evidence demonstrates the antiproliferative, cytotoxic, and antimetastatic properties of plant-derived cannabinoids (phytocannabinoids) such as cannabidiol and tetrahydrocannabinol. The cumulative body of research into the intracellular mechanisms and phenotypic effects of these compounds supports a logical, judicious progression to large-scale phase II/III clinical trials in certain cancer types to truly assess the efficacy of phytocannabinoids as anticancer agents

Powerful turbidity currents driven by dense basal layers

Seafloor sediment flows (turbidity currents) are among the volumetrically most important yet least documented sediment transport processes on Earth. A scarcity of direct observations means that basic characteristics, such as whether flows are entirely dilute or driven by a dense basal layer, remain equivocal. Here we present the most detailed direct observations yet from oceanic turbidity currents. These powerful events in Monterey Canyon have frontal speeds of up to 7.2 m s−1, and carry heavy (800 kg) objects at speeds of ≥4 m s−1. We infer they consist of fast and dense near-bed layers, caused by remobilization of the seafloor, overlain by dilute clouds that outrun the dense layer. Seabed remobilization probably results from disturbance and liquefaction of loose-packed canyon-floor sand. Surprisingly, not all flows correlate with major perturbations such as storms, floods or earthquakes. We therefore provide a new view of sediment transport through submarine canyons into the deep-sea

Factors associated with COVID-19-related death in people with rheumatic diseases: results from the COVID-19 Global Rheumatology Alliance physician-reported registry

Objectives: To determine factors associated with COVID-19-related death in people with rheumatic diseases. Methods: Physician-reported registry of adults with rheumatic disease and confirmed or presumptive COVID-19 (from 24 March to 1 July 2020). The primary outcome was COVID-19-related death. Age, sex, smoking status, comorbidities, rheumatic disease diagnosis, disease activity and medications were included as covariates in multivariable logistic regression models. Analyses were further stratified according to rheumatic disease category. Results: Of 3729 patients (mean age 57 years, 68% female), 390 (10.5%) died. Independent factors associated with COVID-19-related death were age (66-75 years: OR 3.00, 95% CI 2.13 to 4.22; >75 years: 6.18, 4.47 to 8.53; both vs ≤65 years), male sex (1.46, 1.11 to 1.91), hypertension combined with cardiovascular disease (1.89, 1.31 to 2.73), chronic lung disease (1.68, 1.26 to 2.25) and prednisolone-equivalent dosage >10 mg/day (1.69, 1.18 to 2.41; vs no glucocorticoid intake). Moderate/high disease activity (vs remission/low disease activity) was associated with higher odds of death (1.87, 1.27 to 2.77). Rituximab (4.04, 2.32 to 7.03), sulfasalazine (3.60, 1.66 to 7.78), immunosuppressants (azathioprine, cyclophosphamide, ciclosporin, mycophenolate or tacrolimus: 2.22, 1.43 to 3.46) and not receiving any disease-modifying anti-rheumatic drug (DMARD) (2.11, 1.48 to 3.01) were associated with higher odds of death, compared with methotrexate monotherapy. Other synthetic/biological DMARDs were not associated with COVID-19-related death. Conclusion: Among people with rheumatic disease, COVID-19-related death was associated with known general factors (older age, male sex and specific comorbidities) and disease-specific factors (disease activity and specific medications). The association with moderate/high disease activity highlights the importance of adequate disease control with DMARDs, preferably without increasing glucocorticoid dosages. Caution may be required with rituximab, sulfasalazine and some immunosuppressants