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Page 14 of 18 Happel et al. J Cancer Metastasis Treat 2020;6:32 I http://dx.doi.org/10.20517/2394-4722.2020.71

CHALLENGES OF EXRNA IN LIQUID BIOPSY
Although exRNAs are more sensitive and specific biomarkers than proteins, and better reflect the cell
dynamic than DNA does, there are limitations in the use of exRNA as biomarkers. EV heterogeneity and
the complexity of its exRNA cargo, are sources of variability in exRNA profiling within and across studies, which
[69]
has been a significant hinderance. To address the lack of consistency and reproducibility, Srinivasan et al.
demonstrated that exRNA sequencing reproducibility varies across isolation methods and that the
performance of exRNA isolation methods can vary across biofluids and RNA species. To stimulate exRNA
biomarker development, they developed miRDaR (https://exrna.shinyapps.io/mirdar/), an interactive web-
based application to help investigators select the optimal exRNA isolation method for their studies based
on the biofluid of interest. The development of standardized sample isolation and analysis procedures
would allow a more meaningful comparison and integration of data from different studies, which may
facilitate the development of exRNA based clinical applications.

EVs are heterogeneous in nature and technical challenges remain in EV isolation. Current methods for
isolating EVs from complex biofluids cannot clearly identify EV cellular origins within a pool of highly
abundant vesicles. As such, there is no way to clearly differentiate cancer-derived EVs from healthy
host cell-derived EVs in biofluids. However, ERCC2 efforts should be able to address this pressing
challenge. A recent report described a process for EV enrichment by identifying cancer cell membrane
proteins compared with healthy cell membrane proteins using TCGA Human Protein Atlas and GTEx,
[70]
and presented isolation of tumour derived EVs from animal serum . This finding is encouraging to
pursue exRNA biomarker research for detecting cancer at a very early stage. Better characterization of
the differences between exRNA profiles of diseased and healthy individuals will allow the diagnostic and
prognostic utility of exRNA-based profiling to increasingly becoming a reality [18,50,71] .

CURRENT STATUS OF EXRNA AS BIOMARKER
It is conceivable that EVs, exosomes, and exRNA are important resources for developing cancer
biomarkers. In this regard, a growing number of scientific reports suggest exRNA as a reliable non-invasive
alternative to the invasive approaches for diagnosis, treatment and prognosis of cancer. Recently, a U.S.A.-
based diagnostics company utilized exRNA as a predictive marker for prostate cancer, and developed a
urine exosome gene expression assay to identify higher-grade prostate cancer among patients with elevated
PSA levels [50,58] . U.S. FDA granted Bio-Techne Breakthrough Device Designation to this test (ExoDx
Prostate IntelliScore, EPI), which is the first exosome-based liquid biopsy test to receive this Designation.
The National Comprehensive Cancer Center Network included EPI as a recommended test in their Clinical
Practice Guidelines for Oncology for Prostate Cancer Early Detection (Version 1.2019). While this is a
significant step forward in exosome/exRNA-based test development, advancement in this technology is
required to address all types of cancers.

The explosion of technological advancements including sophisticated bioinformatics and availability of
better tools offer a wide spectrum of opportunities to explore exosomes/exRNA for developing reliable
biomarker tests using liquid biopsy samples to accelerate real-time cancer diagnosis and molecularly guided
therapy.

However, there are challenges to isolate tumour-specific exRNA and use as biomarkers for clinical oncology
due to inadequate separation technology and heterogeneity of exRNA carriers. Current methods for
isolating EVs from complex biofluids does not clearly define the cell-of-origin or target cell of exRNA cargo
and, therefore, are unable to determine with certainty the tissue of origin. This warrants improvement in
EV separation technology, and better understanding of EV targeting and cargo release.