Roadblocks of Urinary EV Biomarkers: Moving Toward the Clinic

Abstract

Despite remarkable interest in the biomarker potential of urinary extracellular vesicles (uEVs) and the identification of numerous promising candidates, their clinical translation still presents multiple challenges. The opportunities for successful translation are obvious, yet the main roadblocks on the way have hardly been systematically considered and more coordinated approaches are needed to overcome them. In the present review article, we have identified the most relevant roadblocks of clinical translation of urinary EV-based biomarkers and discuss possible solutions to overcome them. These roadblocks are categorized as fundamental and technical but also related to development of novel biomarker assays and clinical acceptance. In addition, hurdles within the regulatory approval process are discussed. It is clear that various roadblocks to clinical translation of urinary EV biomarkers exist; however, they are addressable by promoting rigor and reproducibility as well as collaboration between basic and clinical scientists, clinicians, industry and regulatory bodies. Moreover, knowledge of obstacles for assay development and regulatory requirements should already be considered when developing a new biomarker to maximize the chance of successful translation. This review presents not only a status quo, but also a roadmap for the further development of the field.

Keywords: biomarkers; bladder; exosomes; extracellular vesicles; kidney; liquid biopsy; prostate; translation; urine.

FIGURE 1

Summary of the translational process of uEV biomarkers, roadblocks on the way and potential solutions for overcoming them. Figure created using BioRender.com.

FIGURE 2

Schematic overview of the factors determining urinary extracellular vesicle (uEV) excretion. uEV excretion is the net result of renal EV secretion minus uptake plus contributions from other organs of the genitourinary system. The kidney is the main source of uEVs and therefore nephron mass is an important determinant of the uEV excretion rate. Nephron mass, in turn, is determined by sex and age—men generally have larger kidneys and nephron number decreases with age. Other factors determining uEV secretion are circadian rhythm, dietary intake, medication, water and salt balance and exercise. Although it is clear that cells can take up EVs, the factors influencing this process are less well understood. EVs, extracellular vesicles; uEVs, urinary extracellular vesicles; EPs, extracellular particles; DRE, digital rectal examination. Figure created using BioRender.com.

FIGURE 3

Overview of methodological roadblocks during assay development and clinical validation of uEV‐based biomarkers. Challenges in development of a biomarker product arise from both non‐standardized collection and storage protocols, as well as imperfections of normalization strategies. During further clinical validation, insufficient precision of novel, non‐established EV detection technologies, lack of data integration due to unavailability of well‐curated clinical datasets and discrepancy between clinical standards and experimental EV technology can be problematic. Figure created using BioRender.com.

FIGURE 4

The process of designing the experimental and data integration pipeline involves making decisions about how data will be managed. One important choice is whether to integrate the data before or after reducing its dimensions, and before or after generating a model. The various approaches for integration are explained in the text, and the design should consider the specific goal to be achieved through integration, whether it is a sequential, biological, or model‐based objective. Figure created using BioRender.com.

FIGURE 5

Schematic overview of requirement considerations in uEV assay development and validation. To meet the requirements of everyday clinical use, it has to be assured that the assay under development meets requirements of accuracy as well as analytical specificity and sensitivity. Moreover, reproducibility during repeated measurements and comparability of inter‐laboratory results have to be assured. For quantitative tests, reference intervals and reportable ranges must be determined separately, often in larger cohorts of healthy donors and patient target populations. GCP, Good Clinical Practice; GLP, Good Laboratory Practice; CLSI, Clinical and Laboratory Standards Institute; FDA, Food and Drug Administration; CLIA, Clinical Laboratory Improvement Amendments; ISO, International Organization for Standardization; IVD, In Vitro Diagnostics; LDT, Laboratory Developed Test. Figure created using BioRender.com.

FIGURE 6

Simplified to‐market roadmap, including the regulatory process. Regulatory processes should already be considered from the early phases of diagnostic product development. This assures that the envisioned product passes approval in later phases. Several strategic decisions have to be made: For instance, whether the proposed test should be marketed as an IVD or LDT in‐house assay can severely impact the requirements regarding simplicity of protocols and need of specialized technology. During the clinical validation process, the developing entity (e.g., an academic institution or a company) typically works together with clinical experts to design meaningful clinical trials and conduct them in a GCP‐compliant manner. The partnership with clinicians is also very important during implementation of the new diagnostic test in clinical routine, especially during collecting and reporting of evidence needed to recommend its use in clinical guidelines. Figure created using BioRender.com.

FIGURE 7

Clinical roadblocks of uEV biomarker translation. Once a novel test has completed regulatory approval, several hurdles may occur on the path toward routine clinical use. Depending on the product, additional costs for equipment and training may have to be covered, especially for IVD tests that are offered for decentralized testing. Regardless of the mode of distribution, novel tests often face clinical inertia or even scepticism, since a benefit compared to established standards of care must be scientifically (and often economically) demonstrated and accepted by physicians—which is, for instance, facilitated by incorporating the test into clinical guidelines. This, in turn, requires long‐term educational efforts within the specific field.Figure created using BioRender.com.