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Page 2 of 11 Gasparello et al. J Cancer Metastasis Treat 2019;5:52 I http://dx.doi.org/10.20517/2394-4722.2019.17

LIQUID BIOPSY: A NEW FRONTIER FOR CANCER DIAGNOSTICS
In the field of cancer diagnosis and treatment, liquid biopsy is a new diagnostic tool that investigates
circulating tumor cells (CTC) and/or cell-free nucleic acids in the peripheral blood [Figure 1A]. Liquid
biopsy is considered one of the most advanced non-invasive diagnostic systems. It provides key molecular
information relevant to important clinical decisions and, being “longitudinal” (it can be repeated as many
times as needed), it fits the idea of precision medicine possibly more than other “static” techniques based
[1-5]
on the analysis of tissue nucleic acids . Diagnostic actions made possible by liquid biopsy include, but
are not limited to, early diagnosis, staging, prognosis, prediction of therapy response and follow up during
therapeutic intervention [6-11] .

In addition to the use of CTCs [12-15] and circulating tumor DNA (ctDNA) [16-18] , other important targets for
liquid biopsy are circulating microRNAs (miRNAs) [19-24] , a family of small (19 to 25 nucleotides in length)
noncoding RNAs playing important roles in controlling post-transcriptional gene expression. Regulatory
miRNAs reduce protein synthesis through selective interactions with complementary sequences of target
messenger RNAs (mRNAs) [25-27] . Single or multiple mRNAs can be targeted at their 3'-UTR, CDS, 5'-UTR
sequences, and it is calculated that more than 60% of human mRNAs are microRNA targets . Dysregulation
[26]
of microRNAs has been associated with a variety of human pathologies, including cancer [28-31] . In this case
miRNAs behave both as tumor promoters (oncomiRNAs and metastamiRNAs) and tumor suppressor
molecules , depending on their mRNA targets (oncosuppressor mRNAs or mRNA coding oncoproteins,
[29]
respectively) with opposing activity on cancer cells. Based on this, it is not surprising that circulating cell-
free miRNAs have been actively investigated as liquid biopsy analytes. OncomiRNAs are abundant in
several extracellular body fluids, where they are protected and stabilized by exosome-like structures and
small intraluminal vesicles produced by a variety of cells (including cancer cells) [32-36] . Hence, elevated levels
of several miRNAs (including miR-221, miR-222, miR-141, miR-92a, miR-21, miR-155, miR- 506, miR-4316,
miR-4772-3p, and miR-29a) are present in the blood from patients with colorectal carcinomas (CRC) and
may contribute to diagnosis and prognosis [21,37-42] . Furthermore, is well established that miRNAs may help
in monitoring therapeutic approaches. For instance, Ogata-Kawata et al. reported that serum exosomal
[22]
miRNA levels (let-7a, miR-1229, miR-1246, miR-150, miR-21, miR-223, and miR-23a) were higher in CRC
patients than controls, were already detectable at early disease stages, and that they were significantly down-
regulated after surgical resection.

TECHNOLOGIES FOR MICRORNA ANALYSIS
In order to quantify miRNAs in the plasma and other body fluids isolated from cancer patients, several
types of technologies for RNA analysis have been proposed [43-51] . Quantitative real-time PCR (RT-qPCR) ,
[52]
[53]
[55]
[54]
NGS RNA sequencing , miRNA microarray analysis , and digital PCR are the most used [Table 1] and
can be employed not only for tissue or cells but also for highly diluted samples, such as body fluids. One
of the major limits of RT-qPCR and ddPCR is the limited number of miRNAs that can be quantified for
single run. This problem was partially solved by introduction of TaqMan low density arrays, that allows to
[56]
quantify the content of a significant number of miRNAs (about 700 miRNAs) using PCR-based methods .
In addition to these methodologies, other technologies have been described for direct miRNA detection
from serum samples. For example, Chapin et al. proposed rolling circle amplification (RCA) based on
[57]
the use of a universal adapter ligated to the targets captured on encoded gel microparticles. The system
allows the multiplexed profiling of miRNA at sub-femtomolar concentration. Interestingly, Williams et al. [58]
proposed a miRNA detection technique able to amplify miRNAs directly in body fluids, avoiding upstream
sample preparation. The technique, based on isothermal target amplification, has a sensitivity positioned in
the femtomolar range. Other conventional technologies normally proposed for miRNA detection in cells
or tissues, such as northern blotting , are not suitable for miRNA detection in body fluids, due to the low
[59]
sensitivity of the technology, requiring therefore large amounts of RNA. Other unconventional miRNA