Cancer
Controversy and Consensus
Liquid Biopsy: Is There an Advantage to Analyzing
Circulating Exosomal DNA Compared to cfDNA or
Are They the Same?
Christoph Kahlert
Introductory Statement
Cancer is one of the leading causes of death worldwide.
This life-threatening disease requires novel strategies for the
early detection and therapy response prediction. Circulating
DNA was first described 70 years ago. However, only the
recent evolution in the PCR-based sequencing techniques
allow the minimally invasive molecular profiling of circu-
Introduction
Although the cancer death rate has declined by about 1.5%
annually in both men and women for the last decade, it
remains the second leading cause of death worldwide (1). In
the United States, the annual incidence of all types of cancer
accounts for around 1,735,350 new cases, and 609,640 deaths
are estimated to be cancer-related (1). However, there is still a
lack of accurate, efficient and inexpensive screening tests that
could help to diagnose many cancer types at an early stage and
to decrease its mortality significantly. For this reason, there is an
urgent clinical need for additional complementary molecular
biomarkers that can be utilized for early diagnosis or thera-
peutic response prediction. A "liquid biopsy" offers the great
advantage that it is significantly less stressful for the patient and
may also provide much more comprehensive information
about tumor biology. Besides, a "liquid biopsy" can be taken
from the blood at several times and thus help to establish a
molecular profile on the dynamic tumor behavior. Therefore,
blood-based liquid biopsies have come into the spotlight of
translational research as a minimally invasive screening test for
the diagnosis and clinical monitoring of cancer. Two of the
most important biomarker classes in the field of "liquid
biopsy" are cell-free DNA (cfDNA) and exosomes. Exosomes
are a class of extracellular vesicles of endosomal origin with a
size range of 40–150 nm that are released by all cells. They have
a lipid bilayer membrane and are enriched in exosome-related
proteins such as CD9, CD63, CD81 or TSG 101 whereas they
are devoid in markers such as calnexin (CANX; refs. 2–4).
Exosomes derive from the intracellular endosomal compart-
ment in a ceramide-triggered process (5). In contrast,
Department for Visceral, Thoracic and Vascular Surgery at the University
Hospital, Technical University Dresden, Dresden, Germany.
Corresponding Author: Christoph Kahlert, University of Dresden,
Fetscherstraße 74, Dresden 01307, Germany. Phone: 4935-1458-18276;
Fax: 4935-1458-4317; E-mail: christoph.kahlert@uniklinikum-dresden.de
doi: 10.1158/0008-5472.CAN-19-0019
Ó2019 American Association for Cancer Research.
2462 Cancer Res; 79(10) May 15, 2019
Research
lating mutant DNA from small-volume "liquid biopsies"
such as blood, urine, or saliva. In this article, we aim to
summarize the fast-growing evidence for cfDNA and exo-
somal DNA as minimally invasive diagnostic markers in
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solid tumors and to highlight their opposing diagnostic
advantages and disadvantages.
"microvesicles," "ectosomes," or "shed vesicles/particles" have
been thought to originate by direct budding from the plasma
membrane (6). In addition to RNA and proteins, exosomes also
transport DNA that reflects the mutational status of their
parental cells. In this article, we will discuss the complementary
utility of these two fractions of circulating DNA and their
clinical relevance.
The Origin of cfDNA and Exosomal DNA
There are some fundamental differences between the origin and
traits of cfDNA and exosomal DNA. Cell-free DNA can be detected
in healthy individuals as well as in patients with nonmalignant or
malignant disease. Under physiologic conditions, nonmutated
cfDNA might derive from nonmalignant cells undergoing apo-
ptosis or being exposed to natural cell stress. Moreover, elevated
copy numbers of nonmutated cfDNA have been observed in
septic patients, patients suffering from severe trauma or patients
with myocardial infarction (7).
In contrast, mutated cfDNA is predominantly described in
patients with a known malignant disease. In this case, it is
assumed that cell-free DNA results from apoptotic and necrotic
tumor remnants that are insufficiently cleared by infiltrating
phagocytes (8). Another source for mutant cell-free DNA is
discussed as being secreted by circulating tumor cells directly
into the bloodstream. A recent study has investigated the size
distribution of mutated cell-free DNA in comparison with
nonmutated cell-free DNA (9). Mouliere and colleagues have
determined that mutant cfDNA is generally more fragmented
than nonmutant cfDNA, with a maximum enrichment of
tumor cfDNA in fragments between 90 and 150 bp, as well
as enrichment in the size range of 250 to 320 bp (9). Moreover,
the authors of this study could show that size-selected
sequencing could increase the sensitivity for detecting genomic
cancer alterations in cfDNA (9), which might be of particular
significance when translating cfDNA analysis into the clinical
routine.
While cfDNA was first described seventy years ago, the
existence of exosomal DNA has long been doubted and has
only been proven for less than a decade. However, nowadays it

is widely accepted that exosomes can contain different types of
DNA including single- and double-stranded genomic DNA as
well as mitochondrial DNA. Balaj and colleagues were one of
the first to show that extracellular vesicles contain single-
stranded DNA (ssDNA), which reflects the genetic status of a
tumor, including amplification of the oncogene c-Myc (10).
Briefly after that, Kahlert and colleagues have shown that
exosomes also contain long fragments of double-stranded DNA
(dsDNA; ref. 11). In this study, exosomes were extracted from
the supernatant of pancreatic carcinoma cell lines and the
serum of healthy donors as well as patients with pancreatic
cancer. Mutations in KRAS and p53 could be detected using
genomic DNA from exosomes derived from pancreatic cancer
cell lines and serum from patients with pancreatic cancer.
Moreover, whole-genome sequencing revealed that serum exo-
somes from patients with pancreatic cancer contain genomic
DNA spanning all chromosomes (11). These data were con-
firmed by Thakur and colleagues, who have validated that the
majority of DNA associated with tumor exosomes is double-
stranded DNA (12). Moreover, this study has revealed that only
a subset of exosomes (10%) contain dsDNA, which is pre-
dominantly located inside the exosomes (12). Besides, the two
studies mentioned above have been able to show that exosomal
double-stranded DNA consists of lager nucleotide fragments
than cfDNA with a size range between 2.5–10 kB (11, 12). This
is another crucial feature that discriminates exosomal DNA
from the short-fragmented cfDNA (9, 11, 12) and should be
taken into account when comparing those two biomarker
classes.
Diagnostic Value of cfDNA and Exosomal
DNA
The analysis of cfDNA has become a very standardized proce-
dure and has a high clinical significance for the minimally invasive
diagnosis of tumors or as a prenatal blood test. Nowadays, the use
of PCR-based assays that can simultaneously assess multiple
regions of driver genes enables screening tests to detect low-
prevalence mutations in cancer patients in a cost-effective and
high throughput procedure (13). A recent study presented a multi-
analyte blood test that primarily targets cell-free DNA from 16
selected driver genes and a panel of 8 selected tumor proteins (13).
This test was applied to examine the blood of 1,005 patients with a
tumor disease including cancers of the ovary, liver, stomach,
pancreas, esophagus, colorectum, lung, or breast. Cohen and
colleagues reported a median sensitivity of 70% among the eight
cancer types evaluated.
Further analysis of subgroups revealed that the results were
strongly dependent on both tumor type and tumor stage. The
highest sensitivity was observed for ovarian cancer (98%), where-
as for breast cancer, the multi-analyte test reached a sensitivity of
33%. Likewise, the sensitivity of stage I cancers amounted to 43%
and increased to 78% in stage III cancers (13).
Regarding these impressive results, one may ask whether the
evaluation of circulating exosomal DNA can provide an addi-
tional clinical value.
Currently, there are only a few studies with conflicting results
that have directly compared the diagnostic significance of
cfDNA with exosomal DNA. A recent study has addressed
this question in patients with melanoma or mastocytosis (14).
For this approach, the authors established a protocol for the
www.aacrjournals.org
Comparison of Exosomal DNA and cfDNA—A Short Opinion
separation of different extracellular vesicle populations includ-
ing oncosomes, microvesicles, and exosomes as well as for the
separation of cfDNA. In human colorectal adenocarcinoma
cells harboring the BRAFV600E mutation, they could prove
that BRAF and BRAFV600E mutations are present in all extra-
cellular vesicle fractions including exosomes as well as in the
cfDNA fraction. However, Klump and colleagues could detect
much higher copy numbers of wild-type and mutant DNA in
the cfDNA fraction in comparison with the extracellular frac-
tion. Next, 2 mL of serum was used from 6 patients with stage
IV melanoma diagnosed positively for the BRAFV600E allele,
and three patients with systemic mastocytosis diagnosed pos-
itively for the cKITD816V allele. The results of this analysis
show that mutant BRAF and cKIT were detected in cfDNA and
exosomal DNA. However, despite a higher yield of total dsDNA
from the exosome fraction, there was an approximately 10-fold
higher amount of copy numbers of the wild-type and mutant
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BRAF and cKIT in the cfDNA fraction (14). These results would
suggest that the analysis of cfDNA is sufficient to determine the
mutational status of a tumor from a liquid biopsy. However,
the authors of this study also note that their observation most
probably cannot be extrapolated to patients with early-stage
disease (14).
Furthermore, there is still a lack of evidence for other cancer
types. The analysis of cell-free DNA often raises the challenge
that the prevalence of mutated cell-free DNA in tumor patients
can be very low. Especially in patients with an early tumor
stage, in which the tumor is potentially resectable and thus
curable, the detection rate of cell-free DNA is often insufficient.
Moreover, the detection sensitivity of cfDNA depends on the
signal-to-noise ratio owing to fragments of DNA from cancer
cells (mutation) mixed with DNA from normal cells (no
mutation; ref. 15). Many different factors can influence this
signal-to-noise ratio, such as the administration of chemother-
apy, which can induce a direct tumor breakdown before blood
collection, the cell lysis after blood collection, or the technical
processing of the blood samples. Taking these challenges into
consideration, the analysis of exosomal DNA can likely offer an
additional clinical advantage. Exosomes are very robust due to
their bilayered lipid membrane that protects the encapsulated
cargo against degradation (5). Even a cycle of freezing and
thawing of exosomes hardly affects the integrity of exosomes.
Likewise, exosomes can be stored at 4 C for 48–96 hours or at
70 C for a long time without losing their biological activity
(5, 16). These features may also help to increase the detection
rate of mutated tumor DNA from the blood as shown by two
recent studies.
When comparing exosome-derived DNA to cfDNA in liquid
biopsies of patients with pancreatic ductal adenocarcinoma
(PDAC), Allenson and colleagues could observe a higher detec-
tion rate of KRAS mutations in exosomal DNA than in cfDNA by
droplet digital PCR (ddPCR; ref. 17). Especially in patients with
locally advanced or metastatic pancreatic cancer, the KRAS mutant
call rate from exosomal DNA was much higher (80% and 85%,
respectively) than the KRAS-mutant call rate from cfDNA (30.8%
and 57.9%, respectively). In a complementary study, Bernard and
colleagues have evaluated the clinical utility of cfDNA and exo-
somal DNA in serial blood samples from patients with localized
and metastatic pancreatic cancer (18). They report that the KRAS
mutation detection rate for localized and metastatic pancreatic
cancer was nearly equivalent when profiling cfDNA and exosomal
Cancer Res; 79(10) May 15, 2019 2463
 Kahlert
DNA. However, concordance among tissue from 22 surgically
resected primary pancreatic tumors was 95.5% for exosomal DNA
and only 68.2% for cfDNA. Likewise, concordance from 12
samples derived by fine-needle aspirates was 83.3% for exosomal
DNA and only 66.8% for cfDNA. Moreover, only exosomal DNA
level after neoadjuvant therapy was significantly associated with
disease progression whereas cfDNA did not show correlations
with outcomes in 34 patients with potentially resectable pancre-
atic cancer (18).
Eventually, the selective accumulation of tumor-derived
exosomes by tumor-specific surface markers might improve
the signal-to-noise ratio by depleting the fraction of exosomes
and cfDNA from nonmalignant cells. The subsequent analysis
of purified mutant DNA could lead to a more detailed insight
into the mutation profile of the primary tumor. Melo and
colleagues have described this approach in their study when
they have demonstrated, that the proteoglycan "glypican-1
(GPC1)" is predominantly expressed on the surface of exo-
somes of pancreatic carcinomas both in vitro and in vivo (19).
One of the key findings of this study was that the authors
divided exosomes into a GPC1-positive and GPC1-negative
fraction using FACS sorting. Subsequently, mutant KRAS was
analyzed at the mRNA level. In this analysis, the investigators
were able to detect the mutated KRAS mRNA exclusively in the
GPC1-positive fraction. In contrast, no mutated KRAS mRNA
was detectable in the GPC1-negative fraction (19). This
approach could be very promising in the future to increase
the detection rate of mutated nucleic acids in the blood. In fact,
nowadays, there are active efforts to develop "lab-on-a-chip"
solutions to capture tumor-specific exosomes by cancer-asso-
ciated surface markers such as glypican-1 from a small sample
of blood (20). Soon, these medical devices might facilitate the
applicability of exosomes in the clinic, for example, by enrich-
ing tumor exosomes from a high background of nontumor-
derived exosomes and directly subjecting them to further bio-
analysis. Also, the exosome-selected enrichment of mutated
DNA based on tumor-specific markers could help to minimize
false-positive results in healthy individuals without a detect-
able tumor disease.
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2464 Cancer Res; 79(10) May 15, 2019
Conclusion
Both cfDNA and circulating exosomal DNA are powerful diag-
nostic tools to unravel the mutational landscape of cancer from a
liquid biopsy by a minimally invasive method. So far, it remains
elusive whether the DNA profiling of one of those two fractions is
superior to the other one. On the one hand, the analysis of cfDNA
offers the advantage that the method has already been established
much longer and might already be used in routine clinical
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that directly compare the clinical significance of exosomal DNA
and cfDNA in translational studies.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Acknowledgments
C. Kahlert received DFG KA 3511/3-1. This work was supported in part by the
Roland Ernst Stiftung f€
ur Gesundheitswesen (05/15).
Received January 4, 2019; revised February 6, 2019; accepted March 4, 2019;
published first May 1, 2019.
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Cancer Res; 79(10) May 15, 2019 2465