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relevant and important in the context of precision cancer therapy, since this approach will result in non-
invasive procedures using body fluids as test samples, and essentially eliminate unnecessary repeat biopsies
[19]
for diagnosis and monitoring effectiveness of a therapy . ExRNAs are now being evaluated as biomarkers
in a variety of cancers and this review provides an understanding of the present status of exosome/exRNA-
based cancer biomarker research, acknowledges challenges, and addresses the need to identify, develop,
and validate clinically relevant exosomal exRNAs as cancer biomarkers.

LIQUID BIOPSY
Liquid biopsy is a term generally used to describe the collection of a body fluid to test for diagnostic
information that will guide patient management. Various biological fluids can be used for liquid biopsies,
[20]
but blood is one of the most accessible fluids along with urine and saliva . The ultimate goal of liquid
biopsies in cancer patients is to be informative about the underlying tumour biology and establish
biomarker clinical utility with clear prognostic value. Non-invasive measurement of cancer biomarkers
using liquid biopsy allows for patient stratification, screening, monitoring treatment response, and
detecting minimal residual disease following therapy/surgery and recurrence. The emergence of sensitive
nucleic acid and protein biomarkers detection technologies have enabled the development of liquid biopsies
with clinical applications in oncology. Currently, tumour biopsy is the preferred diagnostic tool available
to clinicians to detect and monitor treatment for cancer. Since many tissues are difficult or impossible to
biopsy or resect, and biopsies cannot provide information on treatment efficacy in real-time, RNA-based
biomarkers are being developed to address these issues. A liquid biopsy platform that enables non-invasive
real-time detection of cancer biomarkers may significantly reduce the need for tissue biopsy. Advancements
in liquid biopsies are a key objective of precision oncology, with the goal of improving the diagnosis and
treatment of cancer .
[21]

Tumour derived liquid biopsy analytes in the blood include circulating tumour cells (CTC), circulating
[21]
tumour DNA (ctDNA), exRNA, exosomes, and EVs . CTCs and ctDNA are the two analytes that have
more reported utility than others as biomarkers in precision oncology. CTCs are tumour cells that have
presumably been shed from the primary tumour and/or metastatic lesions into the bloodstream. CtDNA
can be detected in the blood as part of the total cell-free DNA (cfDNA) pool, but is specifically derived
[22]
from cancerous cells . Clinical applications for CTCs and cfDNA include prediction of cancer prognosis,
selection and monitoring of therapeutic regimens, and drug target applications .
[21]
Current challenges in cancer diagnostics using liquid biopsy
While liquid biopsies are increasingly being used for molecular diagnostics in oncology, challenges remain.
One limitation in using CTCs for clinical applications is the scarcity of CTCs in the blood. The abundance
9
of CTCs in the blood is low (approximately 1 cell per 1 × 10 blood cells in patients with metastatic cancer),
and only a limited number of CTCs can be isolated from a single blood sample [23-25] . Similarly, ctDNA
concentration can vary from 0.01% to 90% of total cfDNA and, in general, the amount of ctDNA increases
with tumour burden [26,27] . These extreme low concentrations can make detection and analysis challenging.
While CTCs can be analysed at the DNA, RNA, and protein levels, and provide information on functional
cellular characteristics, analyses of CTCs provide limited information on tumour heterogeneity [28,29] .

CtDNA provides a more comprehensive view of the tumour genome as it reflects DNA released from
multiple tumour regions or different tumour foci to capture tumour heterogeneity [30-32] . However, due to the
high fragmentation rate and low abundance of ctDNA, and high background levels of wild-type DNA in
blood, the analysis is particularly challenging. Whole genome sequencing of cfDNA suggests both cfDNA
and ctDNA are likely derived from apoptotic cells . While CTCs are shed from a tumour once it reaches
[33]
a certain stage in development and ctDNA is released from dying cells, exRNA secretion (biogenesis) is a
normal cellular process. This makes exRNA and EVs better candidates to provide insight into early stage
cancers where cell death is not yet occurring.