Extracellular vesicle-based liquid biopsy biomarkers and their application in precision immuno-oncology.

While the field of precision oncology is rapidly expanding and more targeted options are revolutionizing cancer treatment paradigms, therapeutic resistance particularly to immunotherapy remains a pressing challenge. This can be largely attributed to the dynamic tumor-stroma interactions that continuously alter the microenvironment. While to date most advancements have been made through examining the clinical utility of tissue-based biomarkers, their invasive nature and lack of a holistic representation of the evolving disease in a real-time manner could result in suboptimal treatment decisions. Thus, using minimally-invasive approaches to identify biomarkers that predict and monitor treatment response as well as alert to the emergence of recurrences is of a critical need. Currently, research efforts are shifting towards developing liquid biopsy-based biomarkers obtained from patients over the course of disease. Liquid biopsy represents a unique opportunity to monitor intercellular communication within the tumor microenvironment which could occur through the exchange of extracellular vesicles (EVs). EVs are lipid bilayer membrane nanoscale vesicles which transfer a plethora of biomolecules that mediate intercellular crosstalk, shape the tumor microenvironment, and modify drug response. The capture of EVs using innovative approaches, such as microfluidics, magnetic beads, and aptamers, allow their analysis via high throughput multi-omics techniques and facilitate their use for biomarker discovery. Artificial intelligence, using machine and deep learning algorithms, is advancing multi-omics analyses to uncover candidate biomarkers and predictive signatures that are key for translation into clinical trials. With the increasing recognition of the role of EVs in mediating immune evasion and as a valuable biomarker source, these real-time snapshots of cellular communication are promising to become an important tool in the field of precision oncology and spur the recognition of strategies to block resistance to immunotherapy. In this review, we discuss the emerging role of EVs in biomarker research describing current advances in their isolation and analysis techniques as well as their function as mediators in the tumor microenvironment. We also highlight recent lung cancer and melanoma studies that point towards their application as predictive biomarkers for immunotherapy and their potential clinical use in precision immuno-oncology.

Fournier's gangrene with dapagliflozin in a rural hospital: a case report.

Sodium-glucose cotransporter 2 (SGLT2) inhibitors, which are used for treatment of type 2 diabetes, are associated with risk of urogenital infections. FDA issued a black box warning about multiple case reports of Fournier's gangrene (FG) observed in patients taking SGLT2 inhibitors. FG is a type of necrotising fasciitis that occurs in the anogenital area. We report a case of a 71-year-old woman with type 2 diabetes on dapagliflozin, presenting with foul-smelling discharge and a large abscess in the perianal area. Her risk factors for FG included her advanced age, obesity, diabetes and trauma to the site. During her stay, dapagliflozin was discontinued and she received procedural debridement, wound care and broad-spectrum intravenous antibiotics. Due to possible association between FG and SGLT2 inhibitors, patients presenting with signs and symptoms of FG who are taking SGLT2 inhibitors should be examined for infection in the urogenital area and treated promptly.

Peptide-Affinity Precipitation of Extracellular Vesicles and Cell-Free DNA Improves Sequencing Performance for the Detection of Pathogenic Mutations in Lung Cancer Patient Plasma.

Liquid biopsy is a minimally-invasive diagnostic method that may improve access to molecular profiling for non-small cell lung cancer (NSCLC) patients. Although cell-free DNA (cf-DNA) isolation from plasma is the standard liquid biopsy method for detecting DNA mutations in cancer patients, the sensitivity can be highly variable. Vn96 is a peptide with an affinity for both extracellular vesicles (EVs) and circulating cf-DNA. In this study, we evaluated whether peptide-affinity (PA) precipitation of EVs and cf-DNA from NSCLC patient plasma improves the sensitivity of single nucleotide variants (SNVs) detection and compared observed SNVs with those reported in the matched tissue biopsy. NSCLC patient plasma was subjected to either PA precipitation or cell-free methods and total nucleic acid (TNA) was extracted; SNVs were then detected by next-generation sequencing (NGS). PA led to increased recovery of DNA as well as an improvement in NGS sequencing parameters when compared to cf-TNA. Reduced concordance with tissue was observed in PA-TNA (62%) compared to cf-TNA (81%), mainly due to identification of SNVs in PA-TNA that were not observed in tissue. EGFR mutations were detected in PA-TNA with 83% sensitivity and 100% specificity. In conclusion, PA-TNA may improve the detection limits of low-abundance alleles using NGS.

Affinity Captured Urinary Extracellular Vesicles Provide mRNA and miRNA Biomarkers for Improved Accuracy of Prostate Cancer Detection: A Pilot Study.

Serum prostate-specific antigen (sPSA) testing has helped to increase early detection of and decrease mortality from prostate cancer. However, since sPSA lacks specificity, an invasive prostate tissue biopsy is required to confirm cancer diagnosis. Using urinary extracellular vesicles (EVs) as a minimally invasive biomarker source, our goal was to develop a biomarker panel able to distinguish prostate cancer from benign conditions with high accuracy. We enrolled 56 patients in our study, 28 negative and 28 positive for cancer based on tissue biopsy results. Using our Vn96 peptide affinity method, we isolated EVs from post-digital rectal exam urines and used quantitative polymerase chain reaction to measure several mRNA and miRNA targets. We identified a panel of seven mRNA biomarkers whose expression ratio discriminated non-cancer from cancer with an area under the curve (AUC) of 0.825, sensitivity of 75% and specificity of 84%. We also identified two miRNAs whose combined score yielded an AUC of 0.744. A model pairing the seven mRNA and two miRNA panels yielded an AUC of 0.843, sensitivity of 79% and specificity of 89%. Addition of EV-derived PCA3 levels and clinical characteristics to the biomarker model further improved test accuracy. An AUC of 0.955, sensitivity of 86% and specificity of 93% were obtained. Hence, Vn96-isolated urinary EVs are a clinically applicable and minimally invasive source of mRNA and miRNA biomarkers with potential to improve on the accuracy of prostate cancer screening and diagnosis.

A Case of Fournier's Gangrene in a Patient Taking Canagliflozin for the Treatment of Type II Diabetes Mellitus.

BACKGROUND Sodium-glucose cotransporter 2 (SGLT2) inhibitors are a class of antihyperglycemic medications associated with an increased risk of urinary and genital infections due to their glycosuric effects. In 2018, the FDA issued a safety alert warning that multiple cases of Fournier's Gangrene (FG), a severe genital infection, had been reported in patients taking SGLT2 inhibitors. CASE REPORT We present a case of 72-year-old male with type II diabetes mellitus who developed FG while taking the SGLT2 inhibitor canagliflozin. Besides diabetes and canagliflozin use, his other risk factors were his age, gender, and remote history of radiotherapy for prostate cancer. He presented to the Emergency Department (ED) multiple times complaining of rectal pain and was admitted for a possible diagnosis of prostatitis. During his stay, he developed leukocytosis, his pain worsened, and examination of the perianal area was consistent with FG. He was treated with multiple surgical debridement procedures and broad-spectrum antibiotics; the source of infection was determined to be a perianal abscess. He stayed in the hospital for 1 month and was discharged home with outpatient wound care and vacuum dressing changes. Canagliflozin was discontinued during the hospital stay. CONCLUSIONS Due to the possible association of FG with SGLT2 inhibitors, patients who present with signs and symptoms consistent with FG should be examined for possible FG and treated promptly.

Improving patient preparedness for the operating room: A quality improvement study in Winchester District Memorial Hospital - A rural hospital in Ontario.

INTRODUCTION: Full completion of the pre-operative checklist is important for proper preparation of patients before they enter the operating room (OR), thus increasing OR efficiency. It is also critical for patient safety and successful outcomes. According to various literature, full completion of pre-operative checklists varies widely between institutions and occurs anywhere between 21% and 92% of cases.[1],[2] Our pre-project audits revealed a suboptimal patient preparedness for the Winchester District Memorial Hospital (WDMH) OR, since only 25% of cases arriving at the OR had their pre-operative checklist completed in its entirety, with no omissions. METHODS: WDMH performed a 12-month long quality improvement (QI) study to improve patient preparedness for the OR. Multiple QI initiatives were used to induce behavioural change by incorporating process mapping, enabling communication, adjusting the pre-operative checklist based on qualitative staff feedback and implementing a staff education plan. Interventions also included two post-implementation audits. RESULTS: Remarkably, completion of the pre-operative checklist increased from 25% to 67% and finally to 94%. Furthermore, the previous chart's presence and completion of pre-operative orders improved from 87% to 100% and from 82% to 99%, respectively. Another significantly important secondary outcome was improvement in interdepartmental relationships and collaboration. With better communication and checklist completion rates, there came increased patient preparedness and improved efficiency. CONCLUSIONS: Multiple significant improvements and many additional minor improvements strongly suggest that the approaches were used were effective at improving patient preparedness.

Introduction au résumé: Il importe de remplir complètement la liste de vérification préopératoire afin de bien préparer les patients avant leur entrée dans la salle d'opération, ce qui favorise l'efficacité dans la salle d'opération. Cela est aussi essentiel à la sécurité des patients et à l'obtention de résultats positifs chez eux. Selon diverses publications, la réalisation complète de la liste de vérification préopératoire varie considérablement d'un établissement à l'autre, soit de 21 à 92 % des cas[1],[2]. Nos vérifications d'avant-projet ont révélé que les patients du bloc opératoire du Winchester District Memorial Hospital (WDMH) étaient préparés de façon sous-optimale, puisque à peine 25 % des listes de vérification préopératoire étaient complètes et sans omissions à l'arrivée des patients à la salle d'opération. Méthodes: L'hôpital WDMH a réalisé une étude d'amélioration de la qualité (AQ) de 12 mois dans le but d'améliorer la préparation des patients pour la salle d'opération. De nombreuses initiatives d'AQ ont été mises de l'avant pour changer les comportements, soit incorporer la schématisation du processus, favoriser la communication, ajuster la liste de vérification préopératoire en fonction des commentaires qualitatifs du personnel et mettre à exécution un plan d'éducation du personnel. Les interventions comptaient aussi deux vérifications après exécution. Résultats: Remarquablement, la réalisation complète de la liste de vérification préopératoire est passée de 25 à 67% et finalement à 94%. En outre, la présence du dossier et l'exécution des ordonnances préopératoires se sont améliorées, pour passer de 87 à 100 % et de 82 à 99 %, respectivement. L'amélioration des relations et de la collaboration entre services était un autre paramètre d'évaluation secondaire significativement important. La meilleure préparation des patients et une meilleure efficacité ont suivi l'amélioration des communications et des taux de réalisation de la liste de vérification. Conclusions: Les nombreuses améliorations significatives et mineures pointent fortement vers l'efficacité de l'approche utilisée pour améliorer la préparation des patients. Mots-clés: Liste de vérification, efficacité, Ontario, préopératoire, amélioration de la qualité, hôpital rural, service de chirurgie.

Inhibition of Transient Receptor Potential Vanilloid 6 channel, elevated in human ovarian cancers, reduces tumour growth in a xenograft model.

Background: Transient Receptor Potential Vanilloid 6 (TRPV6), a non-voltage gated calcium channel, is implicated in malignancies and correlates with Gleason scores in prostate cancer and with poor prognosis in breast cancer. Data on the TRPV6 status of ovarian malignancies has not received significant attention. The effect of inhibiting TRPV6 activity on ovarian tumour growth has never been reported. Methods: We quantified TRPV6 mRNA and protein in biopsies of five types of ovarian cancer at different stages and grades by quantitative PCR and immunohistochemistry respectively. We verified the presence of TRPV6 in SKOV-3 cells and xenografts by Western Blotting. NOD/SCID mice bearing xenografted ovarian tumours derived from SKOV-3 were treated daily with TRPV6-antagonistic peptides (SOR-C13 and SOR-C27) at 400, 600 and 800 mg/kg delivered intraperitoneally (i.p.) over 12 days. Data from qPCR and tumour growth experiments were compared with a Student's t-test. Immunohistochemical ranking of staining were compared with Kruskall-Wallace one-way ANOVA and Dunn's Multiple Comparison post-test. Results: TRPV6 mRNA and protein are significantly elevated at all stages and grades of 5 ovarian cancer types over normal tissue. Overall qPCR log2 values (n, mean, ± SEM) for mRNA in tumour (n = 165, 5.06 ± 0.16) were greater (p < 0.05) than normal tissues (n = 26, 0.45 ± 0.41). All stages and grades included in the biopsy arrays were significantly greater than normal tissues. Immunohistochemical staining of TRPV6 was ranked >2 (faint in most cells) in 80.5% of tumours (123) while 92% of normal tissues (23) ranked ≤ 2. Daily i.p. injection with SOR-C13 (400, 600 and 800 mg/kg) over 12 days inhibits tumour growth (59%) at the highest dose compared to non-treated controls. SOR-C27 at 800 mg/kg SOR-C27 inhibited tumour growth 55% after 12 days. Results of daily and intermittent dosing (Days 1, 2, 3 and 8, 9, 10) with SOR-C13 were indistinguishable. Conclusion: TRPV6 mRNA and protein are elevated in biopsies of ovarian cancers compared to normal tissue. Inhibition of TRPV6 activity significantly reduces ovarian tumour growth providing evidence that TRPV6 is a feasible oncology target in ovarian cancers.

Rapid isolation of extracellular vesicles from cell culture and biological fluids using a synthetic peptide with specific affinity for heat shock proteins.

Recent studies indicate that extracellular vesicles are an important source material for many clinical applications, including minimally-invasive disease diagnosis. However, challenges for rapid and simple extracellular vesicle collection have hindered their application. We have developed and validated a novel class of peptides (which we named venceremin, or Vn) that exhibit nucleotide-independent specific affinity for canonical heat shock proteins. The Vn peptides were validated to specifically and efficiently capture HSP-containing extracellular vesicles from cell culture growth media, plasma, and urine by electron microscopy, atomic force microscopy, sequencing of nucleic acid cargo, proteomic profiling, immunoblotting, and nanoparticle tracking analysis. All of these analyses confirmed the material captured by the Vn peptides was comparable to those purified by the standard ultracentrifugation method. We show that the Vn peptides are a useful tool for the rapid isolation of extracellular vesicles using standard laboratory equipment. Moreover, the Vn peptides are adaptable to diverse platforms and therefore represent an excellent solution to the challenge of extracellular vesicle isolation for research and clinical applications.

Multi-gene biomarker panel for reference free prostate cancer diagnosis: determination and independent validation.

Identification of biomarkers that can accurately and reliably diagnose prostate cancer is clinically highly desirable. A novel classification method, K-closest resemblance was applied to several high-quality transcriptomic datasets of prostate cancer leading to the discovery of a panel of eight gene biomarkers that can detect prostate cancer with over 96% specificity and sensitivity in leave-one-out cross-validation. Independent validation on clinical samples confirmed the discriminatory power of this gene panel, yielding over 95% accuracy of diagnosis based on receiver-operating characteristic curve analyses. Different levels of validation of the proposed biomarker panel have shown that it allows extremely accurate diagnosis of prostate cancer. Application of this panel can possibly add a fast and objective tool to the pathologist's arsenal following further clinical testing.