occupying only 0.01% of total cell-free DNA (cfDNA)[8]. For exam-
ple, for localized GI tumors [colorectal cancer (CRC), gastric or
gastroesophageal (GE) cancer, and pancreatic cancer], ctDNA can
be detected in 48–73% of patients [9]. However, as tumor burden
increases, deep surveillance of sufficient genomic space enhances
ctDNA detection to nearly 100% [10]. Thus, ctDNA is currently
most applicable to patients with advanced tumor. The traditional
DNA sequencing approaches such as Sanger sequencing or pyrose-
quencing are only able to detect ctDNA in patients with significant
tumor burden. To improve the ability to detect and analyze ctDNA, a
variety of technologies have been developed, including digital PCR,
NGS, BEAMing (beads, emulsion, amplification, and magnetics),
PAP (pyrophosphorolysis-activated polymerization), CAPP-Seq
(cancer personalized profiling by deep sequencing), and TAm-Seq
(tagged-amplicon deep sequencing). The optimal liquid biopsy assay
remains an area of active investigation.
Nonetheless, compared with traditional CTC assays, ctDNA is
arguably more sensitive [11, 12]. For example, in a recent study of
advanced solid tumors, ctDNA was always detected when CTCs
were present [10]. Moreover, ctDNA was often detected when
CTCs were absent. However, it should be noted that this study
did not utilize enrichment methods prior to CTC detection, which
is now the standard step among CTC. In addition to the ongoing
research regarding early detection of malignancy, ctDNA is also
being applied to identify clinically actionable somatic point muta-
tions or deletions, monitor treatment response, and determine
therapeutic resistance to targeted therapeutics and disease progres-
sion. It may also be useful in measuring minimal residual disease.2.3 Circulating
Exosomes
Exosomes are extracellular vesicles containing proteins and nucleic
acids. They are secreted by all cells and circulate in the blood.
Cancer cell-derived exosomes may have unique cell surface pro-
teins, distinguishable from normal exosomes. For example,
glypican-1 (GPC1), a membrane-anchored protein overexpressed
in breast [13] and pancreatic cancer [14], was recently revealed to
be detected exclusively in cancer exosomes. In a validation cohort
of 56 patients with pancreatic ductal adenocarcinoma (PDA) (with
patients from all four stages of cancer including carcinoma in situ),
6 patients with benign pancreatic disease such as chronic pancreati-
tis, and 20 healthy people, GPC1+ circulating exosomes (crExos)
were 100% sensitive and specific for distinguishing patients with
PDAC [15]. This study reveals that exosomes may serve as a
potential noninvasive diagnostic and screening biomarker to detect
early stages of cancer to facilitate possible curative surgical therapy.
More validation studies are necessary to develop exosomes as a
novel cancer biomarker.The Introduction and Clinical Application of Cell-Free Tumor DNA 47