Induction of Fenestrae in Human Induced Pluripotent Stem Cell-Derived Endothelial Cells for Disease Modeling.

The endothelial linings of capillaries, such as those in the kidney and small intestines, possess fenestrae that facilitate fluid and exchange of small molecules. Alterations in the size and number of endothelial fenestrae have been implicated in the pathogenesis of various diseases. The re-creation of fenestrated endothelium using human induced pluripotent stem cells (hiPSCs) provides a promising avenue to investigate the involvement of fenestrae in disease mechanisms and pharmacodynamics. In this project, we aim to induce the formation of fenestrae in nonfenestrated hiPSCs-derived endothelial cells (hiPSC-ECs). Vascular endothelial growth factor A (VEGFA) and phorbol myristate acetate (PMA) were used as inducers of fenestrae in hiPSC-ECs. The assessment of fenestrae formation included gene-expression analysis, scanning electron microscopy (SEM), transmission electron microscopy (TEM), and immunofluorescent staining. Endothelial monolayer functionality was evaluated by dextran permeability assays. Stimulation with VEGFA and PMA significantly induced expression of the diaphragmed fenestrae-associated marker, plasmalemmal vesicle-associated protein (PLVAP), in hiPSC-ECs at the mRNA, and protein levels. SEM analysis revealed VEGFA- and PMA-induced fenestrae structures on the cell membrane of hiPSC-ECs. The increased membrane localization of PLVAP visualized by TEM and immunofluorescent staining supported these findings. The induced fenestrated endothelium in hiPSC-ECs demonstrated selective passage of small solutes across a confluent monolayer with intact cell junctions, confirming functional competence. In conclusion, we present a novel methodology for inducing and regulating fenestrated endothelium in hiPSC-ECs. This innovative approach paves the way for the development of fenestrated microvasculature in human organ-on-a-chip systems, enabling complex disease modeling and physiologically relevant investigations of pharmacodynamics.

Effect of Mechanical Stimuli on the Phenotypic Plasticity of Induced Pluripotent Stem-Cell-Derived Vascular Smooth Muscle Cells in a 3D Hydrogel.

Introduction: Vascular smooth muscle cells (VSMCs) play a pivotal role in vascular homeostasis, with dysregulation leading to vascular complications. Human-induced pluripotent stem-cell (hiPSC)-derived VSMCs offer prospects for personalized disease modeling and regenerative strategies. Current research lacks comparative studies on the impact of three-dimensional (3D) substrate properties under cyclic strain on phenotypic adaptation in hiPSC-derived VSMCs. Here, we aim to investigate the impact of intrinsic substrate properties, such as the hydrogel's elastic modulus and cross-linking density in a 3D static and dynamic environment, on the phenotypical adaptation of human mural cells derived from hiPSC-derived organoids (ODMCs), compared to aortic VSMCs. Methods and results: ODMCs were cultured in two-dimensional (2D) conditions with synthetic or contractile differentiation medium or in 3D Gelatin Methacryloyl (GelMa) substrates with varying degrees of functionalization and percentages to modulate Young's modulus and cross-linking density. Cells in 3D substrates were exposed to cyclic, unidirectional strain. Phenotype characterization was conducted using specific markers through immunofluorescence and gene expression analysis. Under static 2D culture, ODMCs derived from hiPSCs exhibited a VSMC phenotype, expressing key mural markers, and demonstrated a level of phenotypic plasticity similar to primary human VSMCs. In static 3D culture, a substrate with a higher Young's modulus and cross-linking density promoted a contractile phenotype in ODMCs and VSMCs. Dynamic stimulation in the 3D substrate promoted a switch toward a contractile phenotype in both cell types. Conclusion: Our study demonstrates phenotypic plasticity of human ODMCs in response to 2D biological and 3D mechanical stimuli that equals that of primary human VSMCs. These findings may contribute to the advancement of tailored approaches for vascular disease modeling and regenerative strategies.

A protocol for the development of core outcome sets for effectiveness trials and clinical audits in renal cell cancer (R-COS).

Background: There is inconsistency in outcomes collected in renal cell cancer (RCC) intervention effectiveness studies and variability in their definitions. This makes critical summaries of the evidence base difficult and sub-optimally informative for clinical practice guidelines and decision-making by patients and healthcare professionals. A solution is to develop a core outcome set (COS), an agreed minimum set of outcomes to be reported in all trials in a clinical area. Objectives: To develop three COS for (a) localised, (b) locally advanced and (c) metastatic. RCC study design participants and methods: The methods are the same for each of our three COS and are structured in two phases. Phase 1 identifies potentially relevant outcomes by conducting both a systematic literature review and patient interviews (N ~ 30 patients). Qualitative data will be analysed using framework analysis. In phase 2, all outcomes identified in phase 1 will be entered in a modified eDelphi, whereby patients and healthcare professionals (50 of each) will score each outcome's importance (Likert scale from 1 [not important] to 9 [critically important]). Outcomes scored in the 7-9 range by ≥70% and 1-3 by ≤15% will be regarded as 'consensus in', and the vice versa of this will constitute 'consensus out'. All other combinations will be regarded as equivocal and discussed at consensus meetings (including 10 patients and 10 healthcare professionals) in order to vote on them and ratify the results of the eDelphi. Discussion: The R-COS will reduce outcome reporting heterogeneity and improve the evidence base for RCC. Study registration: The study is registered with the COMET initiative: https://www.comet-initiative.org/studies/details/1406.

The 2022 Updated European Association of Urology Guidelines on the Use of Adjuvant Immune Checkpoint Inhibitor Therapy for Renal Cell Carcinoma.

In KEYNOTE-564, adjuvant pembrolizumab, a PD-1 antibody, significantly improved disease-free survival (DFS) in localised clear-cell renal cell carcinoma (ccRCC) with a high risk of relapse. In 2021, the European Association of Urology RCC Guidelines Panel issued a weak recommendation for adjuvant pembrolizumab for high-risk ccRCC as defined by the trial until final overall survival data and results from other trials were available. Meanwhile, the primary DFS endpoints were not met for adjuvant atezolizumab (PD-L1 inhibitor; IMmotion010), adjuvant nivolumab plus ipilimumab (CheckMate 914), or perioperative nivolumab (PROSPER). Owing to heterogeneity, a meta-analysis is not recommended. Pembrolizumab remains the only immune checkpoint inhibitor currently recommended in this setting. Overall survival data are immature and biomarkers to predict outcome are lacking. Uncertainty exists and overtreatment is occurring. Treatment decisions should be made with caution and with the involvement of each patient. PATIENT SUMMARY: New results from three trials of immunotherapy after surgery for kidney cancer to reduce the risk of recurrence showed no improvement with these treatments. These results are in contrast to an earlier study that showed that the antibody pembrolizumab did extend the time before kidney cancer recurrence, even though it is not yet clear if overall survival is longer. Thus, we cautiously recommend pembrolizumab as additional treatment in high-risk kidney cancer after surgery, but patient preference should be carefully considered and the risk of overtreatment should be discussed.

European Association of Urology Guidelines Panel on Renal Cell Carcinoma Update on the New World Health Organization Classification of Kidney Tumours 2022: The Urologist's Point of View.

The fifth edition of the World Health Organization (WHO) classification of urogenital tumours published in 2022 will be implemented in the European Association of Urology guidelines on renal cell carcinoma for 2023. Here we provide an update summarising changes in the new WHO classification of renal tumours from a clinician perspective.

A Systematic Review of Heterogeneity in Outcome Definition and Reporting in Localised Renal Cancer.

Context: Outcomes in renal cell carcinoma (RCC) are reported inconsistently, with variability in definitions and measurement. Hence, it is difficult to compare intervention effectiveness and synthesise outcomes for systematic reviews and to create clinical practice guidelines. This uncertainty in the evidence makes it difficult to guide patient-clinician decision-making. One solution is a core outcome set (COS): an agreed minimum set of outcomes. Objective: To describe outcome reporting, definitions, and measurement heterogeneity as the first stage in co-creating a COS for localised renal cancer. Evidence acquisition: We systematically reviewed outcome reporting heterogeneity in effectiveness trials and observational studies in localised RCC. In total, 2822 studies (randomised controlled trials, cohort studies, case-control studies, systematic reviews) up to June 2020 meeting our inclusion criteria were identified. Abstracts and full texts were screened independently by two reviewers; in cases of disagreement, a third reviewer arbitrated. Data extractions were double-checked. Evidence synthesis: We included 149 studies and found that there was inconsistency in which outcomes were reported across studies and variability in the definitions used for outcomes that were conceptually the same. We structured our analysis using the outcome classification taxonomy proposed by Dodd et al. Outcomes linked to adverse events (eg, bleeding, outcomes linked to surgery) and renal injury outcomes (reduced renal function) were reported most commonly. Outcomes related to deaths from any cause and from cancer were reported in 44% and 25% of studies, respectively, although the time point for measurement and the analysis methods were inconsistent. Outcomes linked to life impact (eg, global quality of life) were reported least often. Clinician-reported outcomes are more frequently reported than patient-reported outcomes in the renal cancer literature. Conclusions: This systematic review underscores the heterogeneity of outcome reporting, definitions, and measurement in research on localised renal cancer. It catalogues the variety of outcomes and serves as a first step towards the development of a COS for localised renal cancer. Patient summary: We reviewed studies on localised kidney cancer and found that multiple terms and definitions have been used to describe outcomes. These are not defined consistently, and often not defined at all. Our review is the first phase in developing a core outcome set to allow better comparisons of studies to improve medical care.

A Renewal of the TNM Staging System for Patients with Renal Cancer To Comply with Current Decision-making: Proposal from the European Association of Urology Guidelines Panel.

Over the past decade, only minor changes have been introduced in the TNM staging system for renal cancer. Conversely, many milestones and modifications in management of the disease have been achieved, especially for patients with locally advanced and metastatic cancers. The European Association of Urology guidelines panel proposes a new TNM classification scheme for staging of renal cell carcinoma to reflect these breakthrough clinical improvements.

An interdisciplinary consensus on the management of brain metastases in patients with renal cell carcinoma.

Brain metastases are a challenging manifestation of renal cell carcinoma. We have a limited understanding of brain metastasis tumor and immune biology, drivers of resistance to systemic treatment, and their overall poor prognosis. Current data support a multimodal treatment strategy with radiation treatment and/or surgery. Nonetheless, the optimal approach for the management of brain metastases from renal cell carcinoma remains unclear. To improve patient care, the authors sought to standardize practical management strategies. They performed an unstructured literature review and elaborated on the current management strategies through an international group of experts from different disciplines assembled via the network of the International Kidney Cancer Coalition. Experts from different disciplines were administered a survey to answer questions related to current challenges and unmet patient needs. On the basis of the integrated approach of literature review and survey study results, the authors built algorithms for the management of single and multiple brain metastases in patients with renal cell carcinoma. The literature review, consensus statements, and algorithms presented in this report can serve as a framework guiding treatment decisions for patients. CA Cancer J Clin. 2022;72:454-489.

Multidisciplinary integrated care pathway for von Hippel-Lindau disease.

BACKGROUND: Clinical pathways are care plans established to describe essential steps in the care of patients with a specific clinical problem. They translate (inter)national guidelines into local applicable protocols and clinical practice. The purpose of this article is to establish a multidisciplinary integrated care pathway for specialists and allied health care professionals in caring for individuals with von Hippel-Lindau (VHL) disease. METHODS: Using a modified Delphi consensus-making process, a multidisciplinary panel from 5 Dutch University Medical Centers produced an integrated care pathway relating to the provision of care for patients with VHL by medical specialists, specialized nurses, and associated health care professionals. Patient representatives cocreated the pathway and contributed quality criteria from the patients' perspective. RESULTS: The panel agreed on recommendations for the optimal quality of care for individuals with a VHL gene mutation. These items were the starting point for the development of a patient care pathway. With international medical guidelines addressing the different VHL-related disorders, this article presents a patient care pathway as a flowchart that can be incorporated into VHL expertise clinics or nonacademic treatment clinics. CONCLUSIONS: Medical specialists (internists, urologists, neurosurgeons, ophthalmologists, geneticists, medical oncologists, neurologists, gastroenterologists, pediatricians, and ear-nose-throat specialists) together with specialized nurses play a vital role alongside health care professionals in providing care to people affected by VHL and their families. This article presents a set of consensus recommendations, supported by organ-specific guidelines, for the roles of these practitioners in order to provide optimal VHL care. This care pathway can form the basis for the development of comprehensive, integrated pathways for multiple neoplasia syndromes.

Large scale genotype- and phenotype-driven machine learning in Von Hippel-Lindau disease.

Von Hippel-Lindau (VHL) disease is a hereditary cancer syndrome where individuals are predisposed to tumor development in the brain, adrenal gland, kidney, and other organs. It is caused by pathogenic variants in the VHL tumor suppressor gene. Standardized disease information has been difficult to collect due to the rarity and diversity of VHL patients. Over 4100 unique articles published until October 2019 were screened for germline genotype-phenotype data. Patient data were translated into standardized descriptions using Human Genome Variation Society gene variant nomenclature and Human Phenotype Ontology terms and has been manually curated into an open-access knowledgebase called Clinical Interpretation of Variants in Cancer. In total, 634 unique VHL variants, 2882 patients, and 1991 families from 427 papers were captured. We identified relationship trends between phenotype and genotype data using classic statistical methods and spectral clustering unsupervised learning. Our analyses reveal earlier onset of pheochromocytoma/paraganglioma and retinal angiomas, phenotype co-occurrences and genotype-phenotype correlations including hotspots. It confirms existing VHL associations and can be used to identify new patterns and associations in VHL disease. Our database serves as an aggregate knowledge translation tool to facilitate sharing information about the pathogenicity of VHL variants.

Patient-reported Experience of Diagnosis, Management, and Burden of Renal Cell Carcinomas: Results from a Global Patient Survey in 43 Countries.

The International Kidney Cancer Coalition (IKCC) is a federation of 46 affiliated patient organisations representing 1.2 million patients worldwide that is committed to reducing the global burden of kidney cancer. A large-scale global survey of patients with renal cell carcinoma (RCC) to capture real-world experiences has never been undertaken. The 35-question survey was designed to identify geographic variations in patient education, experience, awareness, access to care, best practices, quality of life, and unmet psychosocial needs. A total of 1983 responses were recorded from 43 countries in 14 languages. Analysis revealed key findings. (1) At diagnosis, 43% of all respondents had no understanding of their RCC subtype. (2) Shared decision-making remains aspirational: globally, 29% of all patients reported no involvement in their treatment decision, responding "My doctor decided for me". (3) While 96% of respondents reported psychosocial impacts, surprisingly, only 50% disclosed them to their health care team. (4) Lastly, 70% of patients were not asked to participate in a clinical trial, although 90% indicated they would be interested. The survey reflects patient perspectives from diverse clinical scenarios in which different treatment options are available. The data point to actionable deficits in the fields of clinical trials, psychosocial support, and shared decision-making. PATIENT SUMMARY: In this brief report, we highlight the key results from the first large-scale global survey of patients with kidney cancer to capture real-world experiences. This survey reflects patient perspectives from diverse clinical scenarios in which different treatment options are available. We conclude that there is a need for improvement in the fields of clinical trials, psychosocial support, and shared decision-making.

Coronavirus Host Genetics South Africa (COHG-SA) database-a variant database for gene regions associated with SARS-CoV-2 outcomes.

The SARS-CoV-2 virus is responsible for the COVID-19 global public health emergency, and the disease it causes is highly variable in its clinical presentation. Clinical phenotypes are heterogeneous both in terms of presentation of symptoms in the host and response to therapy. Several studies and initiatives have been established to analyse and review host genetic epidemiology associated with COVID-19. Our research group curated these articles into a web-based database using the python application-server framework Django. The database provides a searchable research tool describing current literature surrounding COVID-19 host genetic factors associated with disease outcome. This paper describes the COHG-SA database and provides an overview of the analyses that can be derived from these data.

European Association of Urology Guidelines on Renal Cell Carcinoma: The 2022 Update.

CONTEXT: The European Association of Urology (EAU) Renal Cell Carcinoma (RCC) Guideline Panel has prepared evidence-based guidelines and recommendations for the management of RCC. OBJECTIVE: To present a summary of the 2022 RCC guideline, which is based on a standardised methodology including systematic reviews (SRs) and provides transparent and reliable evidence for the management of RCC. EVIDENCE ACQUISITION: For the 2022 update, a new literature search was carried out with a cutoff date of May 28, 2021, covering the Medline, EMBASE, and Cochrane databases. The data search focused on randomised controlled trials (RCTs) and retrospective or controlled comparator-arm studies, SRs, and meta-analyses. Evidence synthesis was conducted using modified GRADE criteria as outlined for all the EAU guidelines. EVIDENCE SYNTHESIS: All chapters of the RCC guideline were updated on the basis of a structured literature assessment, and clinical practice recommendations were developed. The majority of the studies included were retrospective with matched or unmatched cohorts and were based on single- or multi-institution data or national registries. The exception was systemic treatment of metastatic RCC, for which there are several large RCTs, resulting in recommendations that are based on higher levels of evidence. CONCLUSIONS: The 2022 RCC guidelines have been updated by a multidisciplinary panel of experts using the highest methodological standards. These guidelines provide the most reliable contemporary evidence base for the management of RCC in 2022. PATIENT SUMMARY: The European Association of Urology panel for guidelines on kidney cancer has thoroughly evaluated the research data available to establish up-to-date international standards for the care of patients with kidney cancer.

Co-creating with patients an impact framework across the medicine's life cycle: a qualitative study exploring patients' experiences of involvement in and perceptions of impact measures.

BACKGROUND: The biopharmaceutical industry is challenged with efficiently delivering medicines that patients truly value. This can be addressed by engaging patients and caregivers throughout a medicine's life cycle, ensuring that products meet the needs and expectations of those who take them. While isolated best practice examples of patient engagement exist, they remain relatively ad hoc and not fully embedded within Research & Development (R&D) practices. To encourage more patient engagement, the 'impact' of patient engagement projects (PEP) must be objectively measured and demonstrated. Some frameworks have been proposed; however, there is no evidence of widespread adoption, nor have patients' perspectives been robustly explored. The objective of this qualitative study was therefore to understand patients' perspectives of impact measurement that can be systematically applied within a biopharmaceutical company. METHODS: Semi-structured interviews were conducted with 13 patient organisation (PO) representatives exploring their experiences of engagement and reflections on 23 candidate patient engagement impact measures categorised into five groups: Medicines R&D Priorities; Clinical Trial Design; Regulatory & Market Access Submissions; Product Support & Information; and Disease Support & Information. Thematic analysis was undertaken and impact measures revised in line with interview participant feedback. Emerging themes and revisions to impact measures were validated at a joint workshop with 4 patient advisors representing 4 POs. RESULTS: The study revealed that PO representatives feel a deep sense of accomplishment and ownership when collaborating on PEPs with biopharmaceutical companies. They largely conceptualise 'impact' as positive, tangible and useful outcomes. The revisions made to the pre-defined patient engagement impact measures fell into three broad categories: (1) a requirement for greater context; (2) capturing the nature of patient influence; and (3) terminology changes. The greatest number of revisions concerned 'requiring greater context', for example, including additional descriptions, patient quotes, and satisfaction. CONCLUSIONS: This study sheds light on how patient advocates view 'impact'. Typically this means delivering 'value' important for them. Therefore, the authors of this paper created the term 'value-impact' to comprehensively characterise this conceptualisation, and propose a value-impact measurement plan, incorporating longitudinal data. Through this understanding and in light of other recently published work, wide-scale adoption and implementation of the measurement of value-impact across the biopharmaceutical industry can be realised.

Developing medicines involves multiple stages, all essential for pharmaceutical companies. However, this approach is becoming increasingly costly and patients are rarely involved in defining treatment outcomes. Patient insight is important for everyone involved in medicinal development and required by authorities reviewing new treatments. To identify best practices, the difference ('impact') made by engaging with patients must be measured continuously throughout the treatment development process. Possible impact measures have been proposed (for example, the PARADIGM initiative), but problems remain.This study aimed to work with patients and adapt standard measuring approaches that can be used to provide ongoing insight into the impact of patient engagement activities for companies and patients. Researchers interviewed 13 patient organisation (PO) representatives to explore their experiences of impact measures. Thematic analysis was conducted, and data used to adapt 23 potential impact measures in collaboration with patients.This study identified that PO representatives view impact as being positive, tangible, and useful outcomes, achieving a deep sense of accomplishment and ownership for those patients and representatives involved. The authors created the term 'value-impact' to describe how patients perceive such achievements. Furthermore, measures should be adapted to: 1) include greater context such as descriptions, patients quotes and satisfaction; 2) capture patient influence; and 3) use language that is understandable for all parties. Examples and suggestions for engagement at different stages and revised impact measures are presented in this report. It is our hope that this study will encourage more frequent and effective use of patient engagement strategies as medicines are developed.

Pseudouridine synthase 7 is an opportunistic enzyme that binds and modifies substrates with diverse sequences and structures.

Pseudouridine (Ψ) is a ubiquitous RNA modification incorporated by pseudouridine synthase (Pus) enzymes into hundreds of noncoding and protein-coding RNA substrates. Here, we determined the contributions of substrate structure and protein sequence to binding and catalysis by pseudouridine synthase 7 (Pus7), one of the principal messenger RNA (mRNA) modifying enzymes. Pus7 is distinct among the eukaryotic Pus proteins because it modifies a wider variety of substrates and shares limited homology with other Pus family members. We solved the crystal structure of Saccharomyces cerevisiae Pus7, detailing the architecture of the eukaryotic-specific insertions thought to be responsible for the expanded substrate scope of Pus7. Additionally, we identified an insertion domain in the protein that fine-tunes Pus7 activity both in vitro and in cells. These data demonstrate that Pus7 preferentially binds substrates possessing the previously identified UGUAR (R = purine) consensus sequence and that RNA secondary structure is not a strong requirement for Pus7-binding. In contrast, the rate constants and extent of Ψ incorporation are more influenced by RNA structure, with Pus7 modifying UGUAR sequences in less-structured contexts more efficiently both in vitro and in cells. Although less-structured substrates were preferred, Pus7 fully modified every transfer RNA, mRNA, and nonnatural RNA containing the consensus recognition sequence that we tested. Our findings suggest that Pus7 is a promiscuous enzyme and lead us to propose that factors beyond inherent enzyme properties (e.g., enzyme localization, RNA structure, and competition with other RNA-binding proteins) largely dictate Pus7 substrate selection.

Is There a Role for Biomarkers in Surveillance of Pancreatic Neuroendocrine Neoplasms in Von Hippel-Lindau Disease?

Von Hippel-Lindau (VHL) disease is an autosomal dominant disorder characterized by the development of multi-organ neoplasms. Among the manifestations of VHL are pancreatic neuroendocrine neoplasms (panNENs). In order to detect these lesions in a timely manner, patients are enrolled in a surveillance program, in accordance with the several existing VHL guidelines. However, these guidelines remain unclear about the role of biomarkers in diagnosing panNENs, despite the benefits a biomarker may offer regarding early detection of new lesions, thereby possibly limiting radiation exposure, and improving quality of life. The aim is to determine which biomarkers might be available in VHL patients and to assess their clinical relevance in diagnosing panNENs in VHL patients. We searched the databases of PubMed/Medline, Embase, and Web of Science to identify relevant articles. Seven studies assessing the diagnostic or prognostic value of biomarkers were included. The results from these studies were conflicting. Since no evident association between VHL-related panNENs and biomarkers was established in studies with larger study populations, currently biomarkers do not play a significant role in early detection or follow-up for panNENs in VHL patients. The absence of evidence underscores the need for specific research to address this unmet need.