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Tumor signatures in the blood
Michael R Speicher & Klaus Pantel
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© 2014 Nature America, Inc. All rights reserved.
The goal of characterizing solid-tumor genomes with nothing more than a blood sample is now within reach.
Taking biopsies to monitor primary tumors
and metastases is often difficult for practical reasons, and cancer researchers have long
searched for proxy measurements that would
eliminate the need for tumor tissue samples1.
Recent findings highlight the potential of three
new approaches, all of which take advantage of
the discovery that tumors shed parts of themselves into the blood. Analysis of circulating
tumor cells (CTCs), circulating tumor DNA
(ctDNA) or tumor-derived exosomes could
allow rapid monitoring of tumor genome evolution from a simple blood draw—an approach
known as a ‘liquid biopsy’1 (Fig. 1). Two papers
in this issue and one in Science Translational
Medicine describe technologies for extracting
molecular information about parent tumors
from CTCs2, ctDNA3 and exosomes4, bringing the prospect of liquid biopsies a few steps
closer to reality.
Thanks to large-scale cancer genome
sequencing projects, we now possess catalogs of
genetic alterations that are present in a variety
of human tumor types5. What we lack, however,
are minimally invasive methods to monitor the
presence of these genetic alterations in cancer
patients. Serial biopsies of metastatic lesions are
typically not practical because the metastatic
tissue is often inaccessible or reachable only
by invasive procedures. Moreover, different
metastases in a single patient may harbor different genetic alterations6, and the purity and
yield of biopsy samples are usually low.
Writing in this issue, Lohr et al.2 sought
to determine whether CTCs in the blood of
prostate cancer patients can be used to analyze
somatic single-nucleotide variants. Such projects
Michael R. Speicher is at the Institute of Human
Genetics, Medical University of Graz, Graz,
Austria, and Klaus Pantel is at the Institute
of Tumor Biology, University Medical Center
Hamburg Eppendorf, Hamburg, Germany.
e-mail: [email protected] or
[email protected]
face several major technical challenges. First,
because a diploid cell has only 6.6 pg of DNA,
which is insufficient for most types of detailed
genomic analysis, the genome of single cells
usually has to be amplified with a wholegenome amplification protocol (Fig. 1a). Lohr
et al.2 achieved this using ‘multiple displacement’ amplification. However, the amplification success rate varied widely among single
cells, as observed in previous studies.
Another problem is that whole-genome
amplification can introduce bias and polymerase errors, undermining the accuracy of
variant calls. To overcome this, the authors
developed a strategy for assessing the quality and uniformity of coverage in CTC whole
genome amplification–derived libraries before
performing in-depth, expensive sequencing
analysis. Using low-pass (0.05× coverage)
whole-genome sequencing and analysis of
the correlation of single-base coverage across
a chromosome, they were able to measure
the level of amplification bias and accurately
predict which single-cell CTC libraries were
likely to yield robust data after in-depth (>100×
coverage) whole-exome sequencing. However,
even the libraries that passed the low-pass test
showed non-uniform coverage after wholeexome sequencing.
To address this issue, Lohr et al.2 combined
data from independent CTC libraries, an
approach called ‘census-based sequencing’
inspired by previous publications7. Indeed,
combining multiple single CTC libraries
markedly reduced the false-positive rate of
called somatic single-nucleotide variants, and
combining libraries from just three cells recovered >80% of the somatic variants previously
identified in matched bulk tumor samples from
the same patient.
Using this optimized technique, the authors
demonstrated that they could detect CTC
mutations present early in tumor evolution
or in the metastatic deposit. These founder or
‘trunk’ mutations are of high clinical relevance
nature biotechnology volume 32 number 5 may 2014
because they represent potential therapeutic
targets. They also identified panels of mutations in CTCs that did not overlap with those
detected in the primary tumor or metastases. These nonoverlapping mutations are of
interest as they may provide novel insights
into the evolution of tumor genomes or
predictive and prognostic biomarkers for
clinical applications.
In Science Translational Medicine, Bettegowda
et al.3 start with cell-free ctDNA rather than
CTCs as a source of information about tumorassociated somatic single-nucleotide variants and structural rearrangements in a large
number of human tumor types (Fig. 1b). They
found that the percentage of patients with
detectable ctDNA varied substantially depending on tumor type, and within a single tumor
type ctDNA was more likely to be detected in
patients with more advanced disease. In patients
with detectable ctDNA, the absolute number
of mutant DNA fragments varied. The reasons
for this variation are currently unknown, but
mutant DNA abundance did correlate inversely
with probability of two-year survival.
To determine the sensitivity and specificity
of ctDNA-based liquid biopsies, Bettegowda
et al.3 focused on mutations in codons 12
and 13 of KRAS. Comparing results obtained
using ctDNA or the primary tumor from the
same patients, they achieved 99.2% specificity
and 87.2% sensitivity with ctDNA. They also
showed that ctDNA-based liquid biopsies can
reveal the acquisition of mutations that likely
contribute to resistance to targeted therapies.
For example, mutations in EGFR pathway
genes were detected in a substantial fraction
of patients after but not before treatment with
EGFR-blocking therapy. Surprisingly, many of
these mutations were not observed in the traditional ‘hotspot region’ of codon 12 of the KRAS
or NRAS gene but rather in codon 61.
Notably, Bettegowda et al.3 sought to compare the quantities of mutant DNA in cell-free
ctDNA and CTCs in the same patients. To this
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a
CTCs
CTC
isolation
WGA
Cell
lysis
WTA
c
Au
Marina Corral Spence/Nature Publishing Group
b
cfDNA
end, they first identified genetic alterations in
primary tumors by whole-genome sequencing.
They then used PCR to detect these alterations in DNA isolated from the cell pellet
(which contains CTCs but also white blood
cells, platelets and other cellular fragments) and
from the supernatant (which contains the ctDNA)
obtained after centrifugation of blood. The analysis was limited to structural rearrangements
because the background level of point mutations in PCR assays is too high to reliably detect
tumor-associated somatic point mutations in
rare CTCs. The results suggested that ctDNA is
more sensitive than CTCs for detecting tumorassociated genetic rearrangements. However,
this conclusion may depend on the method of
isolating CTCs. Because CTCs are rare, constituting as few as 1 cell per 109 normal blood
cells8, detection of tumor-associated genomic
rearrangements from blood cell pellets without
any enrichment for CTCs is challenging even
with advanced quantitative PCR approaches.
Thus, further studies using established CTC
capture and enrichment systems will be needed
to explore the relative sensitivity of assays based
on ctDNA and CTCs.
Also in this issue, Im et al.4 focus on a third
type of material shed from tumors: exosomes
(Fig. 1c). These small (50–100 nm) vesicles
may represent a mobile source of information
about the molecular makeup of the parental
tumor, but it remains difficult to isolate and
analyze them, especially in a high-throughput
manner. Current isolation strategies require
serial differential ultracentrifugation steps,
size-based isolation or affinity purification with
appropriate antibodies (e.g., markers like CD63
or EpCAM). For exosome analysis, traditional
approaches like western blot and enyzme-linked
immunosorbent assays require large amounts
of material, which are often not available, and
extensive processing, which hampers fast highthroughput analysis.
To address these limitations of existing exosome isolation and analytical methods, Im
et al.4 developed a strategy based on surface
plasmon resonance that enables label-free,
high-throughput characterization of proteins in exosomes. The approach uses a chip
called a nano-plasmonic exosome (nPLEX)
sensor, which consists of an array of periodic
nanoholes patterned in a gold film. To enable
specific capture of exosomes, the authors
functionalized each array with affinity ligands
specific for protein markers characteristic of
exosomes (e.g., CD63). Exosome binding to
the array changes the local refractive index of
the nPLEX sensor to an extent proportional to
target protein levels. Spectral shifts or changes
in intensity are monitored to report both the
concentration of exosomes and the abundance
Exosomes
© 2014 Nature America, Inc. All rights reserved.
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Figure 1 Minimally invasive approaches to monitor tumor genome evolution. (a) Circulating
tumor cells (CTCs; blue) are isolated from total blood cells by cell separation systems. Cell lysis yields
pure tumor DNA (gray) and RNA (black); for analysis of tumor-specific mutations, these are subjected
to whole-genome or whole-transcriptome amplification, respectively (WGA, WTA). Proteins in CTCs can
be analyzed by immunohistochemistry and, if viable, CTCs can be subjected to functional analyses8.
(b) Cell-free DNA (cfDNA) is prepared from plasma by several centrifugation and filtration steps.
The resulting DNA is a mixture of DNA fragments released from nonmalignant cells (brown) and from
tumor cells (ctDNA; blue). Depending on the tumor stage, and especially during early disease, ctDNA
may represent a minority of all cfDNA. cfDNA is analyzed by next-generation sequencing, which can
reveal somatic copy number changes, single-nucleotide variants and rearrangements. (c) Exosomes can
be efficiently captured from blood using the nPLEX assay and their molecular constituents analyzed;
for example, proteins can be quantified by nPLEX, and RNA can be quantified or sequenced.
of extra- and intravesicular proteins in each
exosome. Although nPLEX and an enzymelinked immunosorbent assay reported similar
expression levels for a panel of extravesicular
markers, the nPLEX sensor was more sensitive, required smaller sample quantities and
was faster.
The nPLEX assay can detect as few as ~3,000
exosomes without labeling. The authors
screened exosomes from different ovarian
cancer cell lines to identify a set of protein
markers capable of specifically detecting exosomes derived from ovarian cancer cells. Out
of a panel of candidate markers, EpCAM and
CD24 distinguished ovarian cancer–derived
exosomes from vesicles originating from nonmalignant cells. In addition, in a small set of
ovarian cancer patient samples, pre- and posttreatment changes in ascites exosomal EpCAM
and CD24 abundance accurately classified
patients as responding or not responding to
therapy. Although Im et al.4 used the nPLEX
assay to monitor exosomal proteins, it is also
possible to release captured exosomes from the
device and analyze their RNA content by quantitative real-time PCR. In this way exosomes
can report on a subset of genomic alterations
in the parental tumor.
The technologies described in the three
papers are important advances in developing
liquid biopsies into a routine diagnostic method.
Several key questions remain to be addressed.
First, to ensure that the appropriate test is
selected for each patient, we need more basic
biological insight into the mechanisms by which
CTCs, ctDNA and exosomes are released from
different tumor types at different stages
of disease. Whether release is altered by
therapeutic interventions is also unknown.
Related to this, many studies focus on
patients with metastatic disease who are
likely to have higher biomarker concentrations than patients with localized disease.
More work is needed to validate the suitability
of these technologies for primary diagnosis of
cancer or for monitoring responses to adjuvant
therapies in early-stage disease9.
Second, amplification bias and sequencing
artifacts are a concern for single-cell analysis
in general. For example, because only approximately 12% of all CTCs studied by Lohr
et al.2 passed the whole-genome amplification
volume 32 number 5 may 2014 nature biotechnology
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© 2014 Nature America, Inc. All rights reserved.
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yield and library quality check, it was necessary
to sequence multiple independent libraries
and combine their results. This poses a
problem for patients with low CTC numbers
and precludes analysis of mutations that are
unique to individual CTCs10. These mutations
could lead to important insights into intrapatient tumor heterogeneity. For example,
we recently performed targeted sequencing of
20 unique CTC mutations and found that 17
of them were also present in small subclones
of the corresponding primary colorectal
cancer or metastatic lesions from the same
patients10.
Third, regardless of the method chosen, it will
be necessary to develop standardized protocols
and to validate their clinical utility through multicenter clinical trials with defined endpoints,
such as overall survival, before their integration
into cancer diagnostics can be recommended.
Independent of applications in cancer, the
technologies reported in these studies may be
useful in fields unrelated to liquid biopsy, such
as minimally invasive prenatal tests to identify
fetal aneuploidy and clinical genetics approaches
for detecting suspected mosaicism. As such,
they could eventually enhance patient care in a
variety of clinical disciplines and further extend
the reach of personalized medicine.
COMPETING FINANCIAL INTERESTS
The authors declare no competing financial interests.
1. Pantel, K. & Alix-Panabieres, C. Cancer Res. 73,
6384–6388 (2013).
2. Lohr, J.G. et al. Nat. Biotechnol. 32, 479–484
(2014).
3. Bettegowda, C. et al. Sci. Transl. Med. 6, 224ra224
(2014).
4.Im, H. et al. Nat. Biotechnol. 32, 488–493 (2014).
5. Lawrence, M.S. et al. Nature 505, 495–501
(2014).
6. Gerlinger, M. et al. N. Engl. J. Med. 366, 883–892
(2012).
7. Hou, Y. et al. Cell 148, 873–885 (2012).
8. Pantel, K., Brakenhoff, R.H. & Brandt, B.
Nat. Rev. Cancer 8, 329–340 (2008).
9. Wan, L., Pantel, K. & Kang, Y. Nat. Med. 19,
1450–1464 (2013).
10.Heitzer, E. et al. Cancer Res. 73, 2965–2975
(2013).
Simply better glycoproteins
Bernd Lepenies & Peter H Seeberger
Reducing the glycan heterogeneity of recombinant proteins may improve the
efficacy of biopharmaceuticals.
Glycosylation is integral to more than half
of eukaryotic proteins and influences many
aspects of protein structure and function,
including folding and stability, molecular recognition and immunogenicity1.
Mammalian glycoproteins usually exist
in many different glycoforms, and, in the
context of protein therapeutics, this diversity can adversely affect drug potency and
pharmacokinetics. In this issue, Meuris
et al.2 present an elegant method to reduce
the N-glycan heterogeneity of recombinant
glycoproteins expressed in mammalian cells.
By simplifying the N-glycosylation machinery, the authors expressed glycoproteins with
smaller and more uniform glycan structures.
This strategy opens the way for production of
Bernd Lepenies & Peter H. Seeberger are at the
Max Planck Institute of Colloids and Interfaces,
Department of Biomolecular Systems, Potsdam,
Germany, and Peter H. Seeberger is also at the
Freie Universität Berlin, Institute of Chemistry
and Biochemistry, Berlin, Germany.
e-mail: [email protected] or
[email protected]
therapeutic glycoproteins of higher quality
and efficacy.
Many of the protein drugs on the market
are glycoproteins, including hormones,
cyto­kines and monoclonal antibodies. Two
aspects of glycosylation are particularly
relevant to their therapeutic activity: first,
glycoproteins should ideally display humanlike glycans; and second, glycosylation patterns
should be as uniform as possible.
Humanized glycosylation is desired because
non-human glycans may elicit unwanted
immune reactions. Currently, most therapeutic glycoproteins are produced in mammalian expression systems—mainly Chinese
hamster ovary (CHO) cells. Glycans produced in non-human cell lines can have a
terminal galactose-α-1,3-galactose epitope
not found on human N-glycans. This modification influences the immunogenicity of
glycoproteins and can cause anaphylactic
reactions in patients. In addition, glycoproteins
from CHO cells predominantly contain a
non-human type of sialic acid that induces
antibody responses and accumulates in human
tissues. The issue of uniform glycosylation
nature biotechnology volume 32 number 5 may 2014
patterns on therapeutic glycoproteins is a matter
of regulatory concern because, although some
degree of heterogeneity in biotherapeutics is
often acceptable, it can result in batch-to-batch
variations in efficacy or pharmacokinetics.
Heterogeneity in glycans arises because
glycosylation is not a template-driven process
and is not under direct transcriptional control; instead, it is guided by the amino acid
sequence of the protein and the accessibility
of glycan-processing enzymes, such as glycosyltransferases and glycosidases, in the endoplasmic reticulum and the Golgi apparatus3.
Simply removing N-glycosylation sites from
proteins usually does not solve humanization
or heterogeneity problems because glycans are
often required for correct protein folding and
biological activity.
Several approaches to producing glycoproteins with uniform human-like glycans
have been developed as alternatives to mammalian expression systems. Escherichia coli4,
the yeast Pichia pastoris5, plants6 and insect
cells7 have all been engineered to improve
protein N-glycosylation. For example, expression of mammalian glycoprocessing enzymes
in yeast5 is a promising approach, but further
studies are needed to demonstrate the utility
of a yeast production system for therapeutic
glycoproteins. Glycoprotein expression in
plants offers easy scalability and low production costs. Although there has been some
recent progress toward expressing human-like
glycan structures in plants6, further improvements in these systems are needed. In addition,
chemoenzymatic8 and synthetic strategies from
our own group and others9,10 have been used to
generate N-glycan structures and glycoproteins
displaying humanized N-glycosylation patterns.
A recent example is the chemical synthesis of
the glycosylated hormone erythropoietin9.
However, to be competitive with biological
systems, chemical synthesis methods require
optimization with regard to speed, scalability
and production processes. Therefore, despite
these developments, production of biopharmaceuticals in mammalian cells, with their
established pipelines and demonstrated scalability, remains an attractive option.
The glycoengineering strategy developed
by Meuris et al.2—which the authors call
GlycoDelete—offers an interesting alternative as it achieves a balance between retaining
essential N-glycan functions and reducing
the complexity of N-glycosylation. To accomplish this, the authors manipulated the
glycosylation machinery in the Golgi apparatus responsible for trimming nascent N-glycans
and adding monosaccharide residues. They
started with an existing human embryonic kidney cell line11 that lacks the enzyme
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