Considering the Case for Extracellular Vesicle-Based Biomarkers

Extracellular Vesicles
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The strong case for extracellular vesicle-based biomarkers.

Extracellular vesicles (EVs) are attractive candidates for diagnostic and prognostic biomarkers; different subpopulations reflect unique physiological processes, they vary with disease state, exist in most or all biofluids, and possess a relatively high level of stability.  

Subsequently, investigations of EV cargo, such as proteins, lipids, DNA and RNA, continue alongside the measurement of physical properties such as EV size and concentration.1,2 With the advent of omics technologies, the exploration of ‘signatures’ has common, i.e., the characterisation of multiple EV-derived analytes.3-5

Unsurprisingly, the EV biomarker space continues to show substantial growth, both in research and commercial settings. This is evident in the increasing number of EV biomarker publications, relevant clinical trials underway, and diagnostic companies focused on progressing EV-based biomarkers to the clinic.  


A note on liquid biopsies: beyond cancer and cells  

It is now common to see the terms ‘extracellular vesicles’ or ‘exosomes’ in the same sentence as ‘liquid biopsy’.6,7 However, this was not always the case. The term ‘liquid biopsy’ appeared in the literature around 2012, in reference to circulating tumour cells (CTCs) as cancer biomarkers.8.9 The idea that cancer cells from solid tumours could be observed in the circulation dates back to work by Australian physician Thomas Ashworth, who in 1869 described the resemblance between circulating cells and tumour cells in the skin of a man with metastatic cancer.10

Since Ashworth’s report, and the rise of liquid biopsy efforts one and a half centuries later, the definition of liquid biopsy seems to have shifted. Now, the term is used to encompass not just blood, cells and cancer – but rather, include a range of biofluids11, biomolecules12 and diseases.13 Despite this, the purpose of liquid biopsies remains the same: to provide non-invasive, molecular insights which match (or beat) what could be obtained with traditional, invasive tissue biopsy. In other cases, EV-based liquid biopsies may provide diagnostic information where tissue biopsies are impractical, such as in Alzheimer’s disease and other neurodegenerative diseases.14

Extracellular vesicle-related biomarkers in screening and diagnostics

The addition of appropriate screening tools may lead to an earlier diagnosis which can have a huge impact on reducing mortality and increasing recovery rate. While diagnostic tests are used to establish the presence or absence of disease in individuals with symptoms or signs of disease, screening tests aim to detect potential disease indicators in asymptomatic individuals, and stratify the population for further investigation.15

To build a foundation for future screening and diagnostic tests, EV-related parameters are being studied across varying disease and physiological states. For example, particular protein signatures derived from EVs in the intrauterine space have been shown to be indicative of the risk of spontaneous preterm delivery and are currently being pursued as risk stratification biomarkers for mothers who have not given birth previously.16

Other proof-of-concept studies include the identification of foetal EV-DNA as a potential screening tool for genetic diseases17, and associations are established between phosphatidylserine-expressing EVs and ovarian cancer18, plasma EV concentration and glioblastoma19, and certain EV-miRNAs and preeclampsia.20 In another example, data-independent acquisition mass spectrometry (DIA-MS) helped establish an association between colorectal cancer (CRC) progression and several proteins potentially associated with EVs.21 Prior to DIA-MS, candidate biomarker proteins were first identified using shotgun proteomics and phosphoproteomics in an assessment of EVs isolated from human CRC cell lines and from the plasma of patients with CRC at different tumour stages.  

Extracellular vesicles as risk stratification and prognostic tools

One prominent example of an EV biomarker being used to stratify disease risk is the ExoDx Prostate Intelliscore (EPI) test, which helps assess the risk of developing prostate cancer. Transrectal ultrasound-guided prostate biopsies are associated with sepsis and antibiotic-resistant bacteria, therefore there is a strong incentive to pursue biopsies only where necessary. Results from the ExoDx Prostate test are used to help guide decisions about whether to proceed to or defer prostate biopsy, thereby avoiding unnecessary biopsies.22The EPI test, launched by Exosome Diagnostics, is a urine-based gene expression assay (ERG, PCA3 and SPDEF) in EVs23 and was granted Breakthrough Device Designation by the U.S. Food and Drug Administration. The status granted by the FDA is an acknowledgement of this clinical need and accelerates efforts to secure FDA clearance; the test has been evaluated in a prospective, observational trial (ClinicalTrials.gov Identifier: NCT03031418) and further investigations are planned (NCT04720599 and NCT04357717) and in progress (NCT03235687).  

The importance of monitoring and predicting disease severity is illustrated by the current COVID-19 pandemic, where there is a need to assess and prioritise the need for hospitalisation, and identify new therapeutic targets. An exploration of EV protein profiles in COVID-19 patients revealed differences between groups of varying disease severity, demonstrating the potential use of EV markers in this setting.24 Similarly, the use of EVs in assessments of disease severity have been considered for many other conditions including liver disease25, chronic obstructive pulmonary disease26 and pneumonia and sepsis.27

Extracellular vesicles in companion diagnostic tests

In recent years there has been increasing interest in personalised medicine, i.e., the development of more targeted therapies to the individual’s needs. Specifically, personalised medicine seeks to improve stratification and timing of healthcare by utilising biological information and biomarkers on the level of molecular disease pathways, genetics, proteomics as well as metabolomics.28 Targeted treatments are only useful when there is a diagnostic test available that can match individuals to the therapy, i.e., a ‘companion diagnostic’ test – and there is a huge potential for EVs to help fill this space. Ideally, therapies and companion diagnostic tests will be developed alongside each other.29 There are also opportunities for improved diagnostics once the personalised medicine is already on the market, as the proportion of responsive patients is often lower than hoped.30 An EV-related example of this was reported recently, whereby a detection assay for programmed cell death ligand-1 (PD-L1) in EVs may be used to complement an existing companion diagnostic test for lung cancer which relies on assessing PD-L1 expression in tumor tissues.30

Open questions in the extracellular vesicle-diagnostic landscape

EV-based diagnostics have been referred to as the next generation of biomarkers, and their potential is undeniable.31-33 Yet, there are many challenges to be overcome and it remains to be seen exactly how EVs will fit into the diagnostic landscape. How will their potential be fulfilled? Will EV-based diagnostics replace existing tests? Will there be a single parameter, or a signature, which can be used to definitively confirm the presence or absence of disease? Or, will EV parameters only supplement existing tests? Visions for the future include the use of EV-based diagnostics to monitor disease progression over time, and an increasing uptake of omics technologies to obtain a deeper understanding of EVs.34-36

Bridging the gap from discovery to application: what’s it going to take?

Evidently, the diagnostic potential for EVs and their cargo is massive, and there has been an explosion of interest across many fields of basic research. Most studies are aimed at improving our general understanding of EV function in health and disease, and together they lay the foundation for the development of important diagnostic tests. Developing a test that is applicable to real-world settings, however, is immensely challenging, and requires the navigation of many obstacles that are unique to later stages of biomarker development. Clinical translation may be challenging, but it is possible; already, the EPI test is making headway, and many other companies and research institutes are gearing up to establish their own impactful EV-based tests. In the next article of this series, we discuss requirements, challenges and general considerations for taking EV biomarkers to the clinic

References

  1. Amintas S, Vendrely V, Dupin C, et al. Next-generation cancer biomarkers: extracellular vesicle DNA as a circulating surrogate of tumor DNA. Frontiers in Cell and Developmental Biology. 2021;8. doi:10.3389/fcell.2020.622048
  2. Veziroglu EM, Mias GI. Characterizing extracellular vesicles and their diverse RNA contents. Frontiers in Genetics. 2020;11. doi:10.3389/fgene.2020.00700
  3. Rontogianni S, Synadaki E, Li B, et al. Proteomic profiling of extracellular vesicles allows for human breast cancer subtyping. Communications Biology. 2019;2(1):1-13. doi:10.1038/s42003-019-0570-8
  4. Budden CF, Gearing LJ, Kaiser R, Standke L, Hertzog PJ, Latz E. Inflammasome‐induced extracellular vesicles harbour distinct RNA signatures and alter bystander macrophage responses. Journal of Extracellular Vesicles. 2021;10(10). doi:10.1002/jev2.12127
  5. Jakubec M, Maple-Grødem J, Akbari S, Nesse S, Halskau Ø, Mork-Jansson AE. Plasma-derived exosome-like vesicles are enriched in lyso-phospholipids and pass the blood-brain barrier. Ramos JW, ed. PLOS ONE. 2020;15(9):e0232442. doi:10.1371/journal.pone.0232442
  6. Zhou B, Xu K, Zheng X, et al. Application of exosomes as liquid biopsy in clinical diagnosis. Signal Transduction and Targeted Therapy. 2020;5(1):1-14. doi:10.1038/s41392-020-00258-9
  7. García-Silva S, Gallardo M, Peinado H. DNA-loaded extracellular vesicles in liquid biopsy: tiny players with big potential? Frontiers in Cell and Developmental Biology. 2021;8. doi:10.3389/fcell.2020.622579
  8. O’Flaherty JD, Gray S, Richard D, et al. Circulating tumour cells, their role in metastasis and their clinical utility in lung cancer. Lung Cancer. 2012;76(1):19-25. doi:10.1016/j.lungcan.2011.10.018
  9. de Wit S, van Dalum G, Terstappen LWMM. Detection of circulating tumor cells. Scientifica. 2014;2014:1-11. doi:10.1155/2014/819362
  10. Ashworth T. A case of cancer in which cells similar to those in the tumours were seen in the blood after death. Aust Med J. 2021;14:146. Accessed October 19, 2021. https://ci.nii.ac.jp/naid/10027663080/
  11. Satyal U, Srivastava A, Abbosh PH. Urine Biopsy—Liquid gold for molecular detection and surveillance of bladder cancer. Frontiers in Oncology. 2019;9:1266. doi:10.3389/fonc.2019.01266
  12. Wu X, Li J, Gassa A, et al. Circulating tumor DNA as an emerging liquid biopsy biomarker for early diagnosis and therapeutic monitoring in hepatocellular carcinoma. International Journal of Biological Sciences. 2020;16(9):1551-1562. doi:10.7150/ijbs.44024
  13. Veiga G, Alves B, Perez M, et al. NGAL and SMAD1 gene expression in the early detection of diabetic nephropathy by liquid biopsy. Journal of Clinical Pathology. 2020;73(11):713-721. doi:10.1136/jclinpath-2020-206494
  14. Kim KM, Meng Q, Perez de Acha O, et al. Mitochondrial RNA in Alzheimer’s disease circulating extracellular vesicles. Frontiers in Cell and Developmental Biology. 2020;8:581882. doi:10.3389/fcell.2020.581882
  15. Gilbert R, et al. Assessing diagnostic and screening tests: Part 1. Concepts. Western Journal of Medicine. 2001;174(6):405-409. doi:10.1136/ewjm.174.6.405
  16. McElrath TF, Cantonwine DE, Jeyabalan A, et al. Circulating microparticle proteins obtained in the late first trimester predict spontaneous preterm birth at less than 35 weeks’ gestation: a panel validation with specific characterization by parity. American Journal of Obstetrics & Gynecology. 2019;220(5):488.e1-488.e11. doi:10.1016/j.ajog.2019.01.220
  17. Zhang W, Lu S, Pu D, et al. Detection of fetal trisomy and single gene disease by massively parallel sequencing of extracellular vesicle DNA in maternal plasma: a proof-of-concept validation. BMC Medical Genomics. 2019;12(1). doi:10.1186/s12920-019-0590-8
  18. Lea J, Sharma R, Yang F, Zhu H, Ward ES, Schroit AJ. Detection of phosphatidylserine-positive exosomes as a diagnostic marker for ovarian malignancies: a proof of concept study. Oncotarget. 2017;8(9):14395-14407. doi:10.18632/oncotarget.14795
  19. Sabbagh Q, Andre-Gregoire G, Guevel L, Gavard J. Vesiclemia: counting on extracellular vesicles for glioblastoma patients. Oncogene. 2020;39(38):6043-6052. doi:10.1038/s41388-020-01420-x
  20. Salomon C, Guanzon D, Scholz-Romero K, et al. Placental exosomes as early biomarker of preeclampsia: potential Role of Exosomal MicroRNAs Across Gestation. The Journal of Clinical Endocrinology & Metabolism. 2017;102(9):3182-3194. doi:10.1210/jc.2017-00672
  21. Zheng X, Xu K, Zhou B, et al. A circulating extracellular vesicles-based novel screening tool for colorectal cancer revealed by shotgun and data-independent acquisition mass spectrometry. Journal of Extracellular Vesicles. 2020;9(1):1750202. doi:10.1080/20013078.2020.1750202
  22. Tutrone R, Donovan MJ, Torkler P, et al. Clinical utility of the exosome based ExoDx Prostate(IntelliScore) EPI test in men presenting for initial Biopsy with a PSA 2–10 ng/mL. Prostate Cancer and Prostatic Diseases. 2020;23(4):607-614. doi:10.1038/s41391-020-0237-z
  23. Raja N, Russell CM, George AK. Urinary markers aiding in the detection and risk stratification of prostate cancer. Translational Andrology and Urology. 2018;7(S4):S436-S442. doi:10.21037/tau.2018.07.01
  24. Krishnamachary B, Cook C, Kumar A, Spikes L, Chalise P, Dhillon NK. Extracellular vesicle‐mediated endothelial apoptosis and EV‐associated proteins correlate with COVID‐19 disease severity. Journal of Extracellular Vesicles. 2021;10(9). doi:10.1002/jev2.12117
  25. Thietart S, Rautou P-E. Extracellular vesicles as biomarkers in liver diseases: a clinician’s point of view. Journal of Hepatology. 2020;73(6). doi:10.1016/j.jhep.2020.07.014
  26. Soni S, Garner JL, O’Dea KP, et al. Intra-alveolar neutrophil-derived microvesicles are associated with disease severity in COPD. American Journal of Physiology-Lung Cellular and Molecular Physiology. 2021;320(1):L73-L83. doi:10.1152/ajplung.00099.2020
  27. Hermann S, Brandes F, Kirchner B, et al. Diagnostic potential of circulating cell-free microRNAs for community-acquired pneumonia and pneumonia-related sepsis. Journal of Cellular and Molecular Medicine. 2020;24(20):12054-12064. doi:10.1111/jcmm.15837
  28. Schleidgen S, Klingler C, Bertram T, Rogowski WH, Marckmann G. What is personalized medicine: sharpening a vague term based on a systematic literature review. BMC Medical Ethics. 2013;14(1). doi:10.1186/1472-6939-14-55
  29. Health C for D and R. Companion Diagnostics. FDA. Published online July 3, 2019. Accessed September 6, 2021. https://www.fda.gov/medical-devices/in-vitro-diagnostics/companion-diagnostics
  30. Wu F, Gu Y, Kang B, et al. PD-L1 detection on circulating tumor-derived extracellular vesicles (T-EVs) from patients with lung cancer. Translational Lung Cancer Research. 2021;10(6). doi:10.21037/tlcr-20-1277
  31. Pang B, Zhu Y, Ni J, et al. Extracellular vesicles: the next generation of biomarkers for liquid biopsy-based prostate cancer diagnosis. Theranostics. 2020;10(5):2309-2326. doi:10.7150/thno.39486
  32. Palazzolo S, Memeo L, Hadla M, et al. Cancer Extracellular Vesicles: Next-Generation Diagnostic and Drug Delivery Nanotools. Cancers. 2020;12(11):3165. doi:10.3390/cancers12113165
  33. Sandim V, Monteiro RQ. Extracellular vesicle fingerprinting: the next generation for cancer diagnosis? Signal Transduction and Targeted Therapy. 2020;5(1):1-3. doi:10.1038/s41392-020-00385-3
  34. Hussey GS, Pineda Molina C, Cramer MC, et al. Lipidomics and RNA sequencing reveal a novel subpopulation of nanovesicle within extracellular matrix biomaterials. Science Advances. 2020;6(12):eaay4361. doi:10.1126/sciadv.aay4361
  35. Kreimer S, Belov AM, Ghiran I, Murthy SK, Frank DA, Ivanov AR. Mass-Spectrometry-Based Molecular Characterization of Extracellular Vesicles: Lipidomics and Proteomics. Journal of Proteome Research. 2015;14(6):2367-2384. doi:10.1021/pr501279t
  36. Whitham M, Febbraio MA. Redefining Tissue Crosstalk via Shotgun Proteomic Analyses of Plasma Extracellular Vesicles. PROTEOMICS. 2018;19(1-2):1800154. doi:10.1002/pmic.201800154

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