Development of an effective predictive screening tool for prostate cancer using the ClarityDX machine learning platform.

The current prostate cancer (PCa) screen test, prostate-specific antigen (PSA), has a high sensitivity for PCa but low specificity for high-risk, clinically significant PCa (csPCa), resulting in overdiagnosis and overtreatment of non-csPCa. Early identification of csPCa while avoiding unnecessary biopsies in men with non-csPCa is challenging. We built an optimized machine learning platform (ClarityDX) and showed its utility in generating models predicting csPCa. Integrating the ClarityDX platform with blood-based biomarkers for clinically significant PCa and clinical biomarker data from a 3448-patient cohort, we developed a test to stratify patients' risk of csPCa; called ClarityDX Prostate. When predicting high risk cancer in the validation cohort, ClarityDX Prostate showed 95% sensitivity, 35% specificity, 54% positive predictive value, and 91% negative predictive value, at a ≥ 25% threshold. Using ClarityDX Prostate at this threshold could avoid up to 35% of unnecessary prostate biopsies. ClarityDX Prostate showed higher accuracy for predicting the risk of csPCa than PSA alone and the tested model-based risk calculators. Using this test as a reflex test in men with elevated PSA levels may help patients and their healthcare providers decide if a prostate biopsy is necessary.

Access to and quality of care for sexual and gender minority women living with HIV in Metro Vancouver, Canada: Results from a longitudinal cohort study.

BACKGROUND: While scarce, literature suggests that women at the intersection of HIV status and gender and/or sexual minority identities experience heightened social and health disparities within health care systems. OBJECTIVES: This study examines the association between sexual and/or gender minority identities and: (1) experiences of poor treatment by health professionals and (2) being unable to access health services among a cohort of women living with HIV in Metro Vancouver, Canada. DESIGN: Data were drawn from a longitudinal community-based cohort of women living with HIV (Sexual Health and HIV/AIDS Women's Longitudinal Needs Assessment). METHODS: We examined associations between sexual and/or gender minority identities and the two outcomes. We drew on explanatory variables to measure sexual minority and gender minority identities independently and a combined variable measuring sexual and/or gender minority identities. The associations between each of these three variables and each outcome were analysed using bivariate and multivariable logistic regression models with generalized estimating equations for repeated measures over time. Adjusted odds ratios and 95% confidence intervals are reported. RESULTS: The study sample included 1460 observations on 315 participants over 4.5 years (September 2014 to February 2019). Overall, 125 (39.7%) reported poor treatment by health professionals and 102 (32.4%) reported being unable to access health care services when needed at least once over the study period. A total of 110 (34.9%) of participants reported sexual and/or gender minority identities, 106 (33.7%) reporting sexual minority identities, with 29 (9.2%) reporting gender minority identities. In multivariable analysis, adjusting for confounders, sexual minority identities, and combined sexual and/or gender minority identities were significantly associated with increased odds of experiencing poor treatment by health professionals (sexual minority adjusted odds ratio = 1.39 (0.94-2.05); sexual and/or gender minority adjusted odds ratio = 1.48 (1.00-2.18)) and being unable to access health services (sexual minority adjusted odds ratio = 1.89 (1.20-2.97); sexual and/or gender minority adjusted odds ratio = 1.91 (1.23-2.98)). In multivariable analysis, gender minority identities were not significantly associated with increased odds of experiencing poor treatment by health professionals (gender minority adjusted odds ratio = 1.38; 95% CI = 0.76-2.52) and being unable to access health services (gender minority adjusted odds ratio = 1.72; 95% CI = 0.89-3.31) possibly due to low sample size among women with gender minority identities. CONCLUSION: Our findings suggest the need for access to inclusive, affirming, trauma-informed health care services tailored specifically for and by women living with HIV with sexual and/or gender minority identities.

Clinical analysis of EV-Fingerprint to predict grade group 3 and above prostate cancer and avoid prostate biopsy.

BACKGROUND: There is an unmet clinical need for minimally invasive diagnostic tests to improve the detection of grade group (GG) ≥3 prostate cancer relative to prostate antigen-specific risk calculators. We determined the accuracy of the blood-based extracellular vesicle (EV) biomarker assay (EV Fingerprint test) at the point of a prostate biopsy decision to predict GG ≥3 from GG ≤2 and avoid unnecessary biopsies. METHODS: This study analyzed 415 men referred to urology clinics and scheduled for a prostate biopsy, were recruited to the APCaRI 01 prospective cohort study. The EV machine learning analysis platform was used to generate predictive EV models from microflow data. Logistic regression was then used to analyze the combined EV models and patient clinical data and generate the patients' risk score for GG ≥3 prostate cancer. RESULTS: The EV-Fingerprint test was evaluated using the area under the curve (AUC) in discrimination of GG ≥3 from GG ≤2 and benign disease on initial biopsy. EV-Fingerprint identified GG ≥3 cancer patients with high accuracy (0.81 AUC) at 95% sensitivity and 97% negative predictive value. Using a 7.85% probability cutoff, 95% of men with GG ≥3 would have been recommended a biopsy while avoiding 144 unnecessary biopsies (35%) and missing four GG ≥3 cancers (5%). Conversely, a 5% cutoff would have avoided 31 unnecessary biopsies (7%), missing no GG ≥3 cancers (0%). CONCLUSIONS: EV-Fingerprint accurately predicted GG ≥3 prostate cancer and would have significantly reduced unnecessary prostate biopsies.

Antibody titrations are critical for microflow cytometric analysis of extracellular vesicles.

Optimization of flow cytometry assays for extracellular vesicles (EVs) often fail to include appropriate reagent titrations - the most critically antibody titration is either not performed or is incomplete. Using nonoptimal antibody concentration is one of the main sources of error leading to a lack of reproducible data. Antibody titration for the analysis of antigens on the surface of EVs is challenging for a variety of technical reasons. Using platelets as surrogates for cells and platelet-derived particles as surrogates for EV populations, we demonstrate our process for antibody titration, highlighting some of the key analysis parameters that may confound and surprise new researchers moving into the field of EV research. Additional care must be exercised to ensure instrument and reagent controls are utilized appropriately. Complete graphical analysis of positive and negative signal intensities, concentration, and separation or stain index data is highly beneficial when paired with visual analysis of the cytometry data. Using analytical flow cytometry procedures optimized for cells for EV analysis can lead to misleading and nonreproducible results.

Molecular Targeting of the Most Functionally Complex Gene in Precision Oncology: p53.

While chemotherapy is a key treatment strategy for many solid tumors, it is rarely curative, and most tumor cells eventually become resistant. Because of this, there is an unmet need to develop systemic treatments that capitalize on the unique mutational landscape of each patient's tumor. The most frequently mutated protein in cancer, p53, has a role in nearly all cancer subtypes and tumorigenesis stages and therefore is one of the most promising molecular targets for cancer treatment. Unfortunately, drugs targeting p53 have seen little clinical success despite promising preclinical data. Most of these drug compounds target specific aspects of p53 inactivation, such as through inhibiting negative regulation by the mouse double minute (MDM) family of proteins. These treatment strategies fail to address cancer cells' adaptation mechanisms and ignore the impact that p53 loss has on the entire p53 network. However, recent gene therapy successes show that targeting the p53 network and cellular dysfunction caused by p53 inactivation is now possible and may soon translate into successful clinical responses. In this review, we discuss p53 signaling complexities in cancer that have hindered the development and use of p53-targeted drugs. We also describe several current therapeutics reporting promising preclinical and clinical results.

Discovery of Metastatic Regulators using a Rapid and Quantitative Intravital Chick Chorioallantoic Membrane Model.

Recent advances in cancer research has illustrated the highly complex nature of cancer metastasis. Multiple genes or genes networks have been found to be involved in differentially regulating cancer metastatic cascade genes and gene products dependent on the cancer type, tissue, and individual patient characteristics. These represent potentially important targets for genetic therapeutics and personalized medicine approaches. The development of rapid screening platforms is essential for the identification of these genetic targets. The chick chorioallantoic membrane (CAM) is a highly vascularized, collagen rich membrane located under the eggshell that allows for gas exchange in the developing embryo. Due to the location and vascularization of the CAM, we developed it as an intravital human cancer metastasis model that allows for robust human cancer cell xenografting and real-time imaging of cancer cell interactions with the collagen rich matrix and vasculature. Using this model, a quantitative screening platform was designed for the identification of novel drivers or suppressors of cancer metastasis. We transduced a pool of head and neck HEp3 cancer cells with a complete human genome shRNA gene library, then injected the cells, at low density, into the CAM vasculature. The cells proliferated and formed single-tumor cell colonies. Individual colonies that were unable to invade into the CAM tissue were visible as a compact colony phenotype and excised for identification of the transduced shRNA present in the cells. Images of individual colonies were evaluated for their invasiveness. Multiple rounds of selections were performed to decreases the rate of false positives. Individual, isolated cancer cell clones or newly engineered clones that express genes of interest were subjected to primary tumor formation assay or cancer cell vasculature co-option analysis. In summary we present a rapid screening platform that allows for anti-metastatic target identification and intravital analysis of a dynamic and complex cascade of events.

Cohort profile: the Alberta Prostate Cancer Research Initiative (APCaRI) Registry and Biorepository facilitates technology translation to the clinic through the use of linked, longitudinal clinical and patient-reported data and biospecimens from men in Alberta, Canada.

PURPOSE: The Alberta Prostate Cancer Research Initiative (APCaRI) Registry and Biorepository was established in 2014 by the APCaRI to facilitate the collection of clinical and patient-reported data, biospecimen, to measure prostate cancer outcomes and to support the development and clinical translation of innovative technologies to better diagnose and predict outcomes for patients with prostate cancer. PARTICIPANTS: Men suspected with prostate cancer and referred to Urology centres in Alberta were enrolled in the APCaRI 01 study, while men with a prior prostate cancer diagnosis participated in the APCaRI 03 study from 1 July 2014 to 30 June 2019. The APCaRI Registry and Biorepository links biospecimens and data from a wide representation of patients drawn from an Alberta population of more than 4 million. FINDINGS TO DATE: From 1 July 2014 to 30 June 2019, total APCaRI 01 and 03 study recruitment was 3754 men; 142 (4%) of these men withdrew in full, 65 men (2%) withdrew biospecimens and 123 men (3%) died of any cause. Over this same time, 8677 patient-reported outcome measure (PROM) surveys and 7368 biospecimens were collected and are available from the registry and biorepository, respectively. The data entry error rate was 0.8% and 0.95% for critical and non-critical values, respectively, and 1.8% for patient-reported surveys. FUTURE PLANS: The APCaRI Registry and Biorepository will collect longitudinal data and PROM surveys until 2024, patient outcomes up to 25 years after recruitment and biospecimen storage for up to 25 years. The APCaRI cohorts will continue to provide data and samples to researchers conducting retrospective studies. The richness of the data and biospecimens will complement many different research questions, ultimately to improve the quality of care for men with prostate cancer.

Novel therapeutic targets for cancer metastasis.

Introduction: Metastatic cancers are extremely difficult to treat, and account for the vast majority of cancer-related deaths. The dissemination of tumor cells to distant sites is highly dynamic, asynchronous, and involves both tumor and host intrinsic factors. Effective therapeutic targets to block metastasis will need to disrupt key pathways that are required for multiple stages of metastasis.Areas covered: This review discusses the heterogeneity of cancers and metastasis, with an emphasis on motility as a key driver trait of metastasis. Recent metastatic cancer studies that identified either host or cancer cell intrinsic factors important for metastasis, using single gene-deficient animal models or 3D intravital imaging of avian embryo models, are also discussed. Potential metastatic blocking targets are listed as they relate to metastatic cancer therapy.Expert opinion: The development of metastatic disease is a complex interplay of genetic and epigenetic factors from the host and cancer cells acting in a patient-specific manner. Inhibiting key driver traits of metastasis should yield survival benefit at any stage of the disease, and we look forward to the next generation of personalized medicines for cancer therapy that target cancer cell motility for increased therapeutic efficacy.

PROSPeCT: A Predictive Research Online System for Prostate Cancer Tasks.

PURPOSE: An online clinical information system, called Predictive Research Online System Prostate Cancer Tasks (PROSPeCT), was developed to enable users to query the Alberta Prostate Cancer Registry database hosted by the Alberta Prostate Cancer Research Initiative. To deliver high-quality patient treatment, prostate cancer clinicians and researchers require a user-friendly system that offers an easy and efficient way to obtain relevant and accurate information about patients from a robust and expanding database. METHODS: PROSPeCT was designed and implemented to make it easy for users to query the prostate cancer patient database by creating, saving, and reusing simple and complex definitions. We describe its intuitive nature by exemplifying the creation and use of a complex definition to identify a "high-risk" patient cohort. RESULTS: PROSPeCT was made to minimize user error and to maximize efficiency without requiring the user to have programming skills. Thus, it provides tools that allow both novice and expert users to easily identify patient cohorts, manage individual patient care, perform Kaplan Meier estimates, plot aggregate PSA views, compute PSA-doubling time, and visualize results. CONCLUSION: This report provides an overview of PROSPeCT, a system that helps clinicians to identify appropriate patient treatments and researchers to develop prostate cancer hypotheses, with the overarching goal of improving the quality of life of patients with prostate cancer. We have made available the code for the PROSPeCT implementation at https://github.com/max-uhlich/e-PROSPeCT .

Cowpea mosaic virus nanoparticles for cancer imaging and therapy.

Nanoparticle platforms are particularly attractive for theranostic applications due to their capacity for multifunctionality and multivalency. Some of the most promising nano-scale scaffold systems have been co-opted from nature including plant viruses such as cowpea mosaic virus (CPMV). The use of plant viruses like CPMV as viral nanoparticles is advantageous for many reasons; they are non-infectious and nontoxic to humans and safe for use in intravital imaging and drug delivery. The CPMV capsid icosahedral shape allows for enhanced multifunctional group display and the ability to carry specific cargoes. The native tropism of CPMV for cell-surface displayed vimentin and the enhanced permeability and retention effect allow them to preferentially extravasate from tumor neovasculature and efficiently penetrate tumors. Furthermore, CPMVs can be engineered via several straightforward chemistries to display targeting and imaging moieties on external, addressable residues and they can be loaded internally with therapeutic drug cargoes. These qualities make them highly effective as biocompatible platforms for tumor targeting, intravital imaging and cancer therapy.

Interleukin-32 promotes detachment and activation of human Langerhans cells in a human skin explant model.

BACKGROUND: Cross-talk between skin keratinocytes (KCs) and Langerhans cells (LCs) plays a fundamental role in the body's first line of immunological defences. However, the mechanism behind the interaction between these two major epidermal cells is unknown. Interleukin (IL)-32 is produced in inflammatory skin disorders. We questioned the role of IL-32 in the epidermis. OBJECTIVES: We aimed to determine the role of IL-32 produced by KCs on surrounding LCs. METHODS: We used an ex vivo human explant model from healthy donors and investigated the role of IL-32 on LC activation using imaging, flow cytometry, reverse transcriptase quantitative polymerase chain reaction and small interfering (si)RNA treatment. RESULTS: Modified vaccinia virus ankara (MVA) infection induced KC death alongside the early production of the proinflammatory cytokine IL-32. We demonstrated that IL-32 produced by MVA-infected KCs induced modest but significant morphological changes in LCs and downregulation of adhesion molecules, such as epithelial cell adhesion molecule and very late antigen-4, and CXCL10 production. The treatment of KCs with IL-32-specific siRNA, and anti-IL-32 blocking antibody significantly inhibited LC activation, demonstrating the role of IL-32 in LC activation. We also found that some Toll-like receptor ligands induced a very high level of IL-32 production by KCs, which initiated LC activation. CONCLUSIONS: We propose, for the first time, that IL-32 is a molecular link between KCs and LCs in healthy skin, provoking LC migration from the epidermis to the dermis prior to their migration to the draining lymph nodes.

Intravital imaging tumor screen used to identify novel metastasis-blocking therapeutic targets.

Cancer cell motility is a key driver of metastasis. Although the intravasation of cancer cells into the blood stream is highly dependent on their motility and metastatic dissemination is the primary cause of cancer related deaths, current therapeutic strategies do not target the genes and proteins that are essential for cell motility. A primary reason for this is because the identification of cell motility-related genes that are relevant in vivo requires the visualization of metastatic lesions forming in an appropriate in vivo model. The cancer research community has lacked an in vivo and intravital metastatic cancer model that could be imaged as motility developed, in real-time. To address this, we developed a novel quantitative in vivo screening platform based on intravital imaging in shell-less ex ovo chick embryos. We applied this imaging approach to screen a human genome-wide short hairpin RNA library (shRNA) versus the highly motile head and neck cancer cells (HEp3 cell line) introduced into the chorioallantoic membrane (CAM) of chick embryos and identified multiple novel in vivo cancer cell motility-associated genes. When the expression of several of the identified genes was inhibited in the HEp3 tumors, we observed a nearly total block of spontaneous cancer metastasis.

Identification of Nitrogen Use Efficiency Genes in Barley: Searching for QTLs Controlling Complex Physiological Traits.

Over the past half century, the use of nitrogen (N) fertilizers has markedly increased crop yields, but with considerable negative effects on the environment and human health. Consequently, there has been a strong push to reduce the amount of N fertilizer used by maximizing the nitrogen use efficiency (NUE) of crops. One approach would be to use classical genetics to improve the NUE of a crop plant. This involves both conventional breeding and quantitative trait loci (QTL) mapping in combination with marker-assisted selection (MAS) to track key regions of the chromosome that segregate for NUE. To achieve this goal, one of initial steps is to characterize the NUE-associated genes, then use the profiles of specific genes to combine plant physiology and genetics to improve plant performance. In this study, on the basis of genetic homology and expression analysis, barley candidate genes from a variety of families that exhibited potential roles in enhancing NUE were identified and mapped. We then performed an analysis of QTLs associated with NUE in field trials and further analyzed their map-location data to narrow the search for these candidate genes. These results provide a novel insight on the identification of NUE genes and for the future prospects, will lead to a more thorough understanding of physiological significances of the diverse gene families that may be associated with NUE in barley.

Understanding Plant Nitrogen Metabolism through Metabolomics and Computational Approaches.

A comprehensive understanding of plant metabolism could provide a direct mechanism for improving nitrogen use efficiency (NUE) in crops. One of the major barriers to achieving this outcome is our poor understanding of the complex metabolic networks, physiological factors, and signaling mechanisms that affect NUE in agricultural settings. However, an exciting collection of computational and experimental approaches has begun to elucidate whole-plant nitrogen usage and provides an avenue for connecting nitrogen-related phenotypes to genes. Herein, we describe how metabolomics, computational models of metabolism, and flux balance analysis have been harnessed to advance our understanding of plant nitrogen metabolism. We introduce a model describing the complex flow of nitrogen through crops in a real-world agricultural setting and describe how experimental metabolomics data, such as isotope labeling rates and analyses of nutrient uptake, can be used to refine these models. In summary, the metabolomics/computational approach offers an exciting mechanism for understanding NUE that may ultimately lead to more effective crop management and engineered plants with higher yields.

The Genetics of Nitrogen Use Efficiency in Crop Plants.

In the past 50 years, the application of synthetic nitrogen (N) fertilizer to farmland resulted in a dramatic increase in crop yields but with considerable negative impacts on the environment. New solutions are therefore needed to simultaneously increase yields while maintaining, or preferably decreasing, applied N to maximize the nitrogen use efficiency (NUE) of crops. In this review, we outline the definition of NUE, the selection and development of NUE crops, and the factors that interact with NUE. In particular, we emphasize the challenges of developing crop plants with enhanced NUE, using more classical genetic approaches based on utilizing existing allelic variation for NUE traits. The challenges of phenotyping, mapping quantitative trait loci (QTLs), and selecting candidate genes for NUE improvement are described. In addition, we highlight the importance of different factors that lead to changes in the NUE components of nitrogen uptake efficiency (NUpE) and nitrogen utilization efficiency (NUtE).

Neurologic Disorders in Immunocompetent Patients with Autochthonous Acute Hepatitis E.

Neurologic disorders, mainly Guillain-Barré syndrome and Parsonage­Turner syndrome (PTS), have been described in patients with hepatitis E virus (HEV) infection in industrialized and developing countries. We report a wider range of neurologic disorders in nonimmunocompromised patients with acute HEV infection. Data from 15 French immunocompetent patients with acute HEV infection and neurologic disorders were retrospectively recorded from January 2006 through June 2013. The disorders could be divided into 4 main entities: mononeuritis multiplex, PTS, meningoradiculitis, and acute demyelinating neuropathy. HEV infection was treated with ribavirin in 3 patients (for PTS or mononeuritis multiplex). One patient was treated with corticosteroids (for mononeuropathy multiplex), and 5 others received intravenous immunoglobulin (for PTS, meningoradiculitis, Guillain-Barré syndrome, or Miller Fisher syndrome). We conclude that pleiotropic neurologic disorders are seen in HEV-infected immunocompetent patients. Patients with acute neurologic manifestations and aminotransferase abnormalities should be screened for HEV infection.

Droplets move over viscoelastic substrates by surfing a ridge.

Liquid drops on soft solids generate strong deformations below the contact line, resulting from a balance of capillary and elastic forces. The movement of these drops may cause strong, potentially singular dissipation in the soft solid. Here we show that a drop on a soft substrate moves by surfing a ridge: the initially flat solid surface is deformed into a sharp ridge whose orientation angle depends on the contact line velocity. We measure this angle for water on a silicone gel and develop a theory based on the substrate rheology. We quantitatively recover the dynamic contact angle and provide a mechanism for stick-slip motion when a drop is forced strongly: the contact line depins and slides down the wetting ridge, forming a new one after a transient. We anticipate that our theory will have implications in problems such as self-organization of cell tissues or the design of capillarity-based microrheometers.