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Next-generation sequencing in the clinic


Jason Y Park, Larry J Kricka & Paolo Fortina

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2013 Nature America, Inc. All rights reserved.

Pools of cell lines carrying a variety of known mutations are used to validate the performance of a cancer diagnostic
test based on next-generation sequencing.
As next-generation sequencing (NGS) of tumor
cells becomes more sophisticated, it is likely to
inform all aspects of cancer management, from
diagnostic testing to treatment and drug discovery. In this issue, Frampton et al.1 describe a
comprehensive NGS assay applicable to clinical
samples that identifies single-base substitutions,
copy-number variations and focal amplifications in 287 cancer-related genes, fusion events
involving 19 frequently rearranged genes and
3,549 single-nucleotide polymorphisms in
other locations throughout the genomeall
within a single sequencing run. Validating the
analytic performance of such a test is challenging because it assays so many nucleotide
positions in the genome (~1.5 Mb in total).
The authors therefore use 53 cell lines to create
reference materials for assessing the sensitivity and specificity of variant detection. Finally,
they apply their test to >2,000 clinical cases.
The overall approach of the studyincluding
assay design, creation of reference materials and
validationserves as a model for the development of future clinical NGS tests.
In current clinical practice, mutation analysis of cancer samples is performed to establish diagnoses (e.g., specific translocations
Jason Y. Park is at the Department of Pathology,
University of Texas Southwestern Medical
Center and Childrens Medical Center, Dallas,
Texas, USA, and the Eugene McDermott
Center for Human Growth and Development,
University of Texas Southwestern Medical
Center, Dallas, Texas, USA; Larry J. Kricka is
at the Department of Pathology and Laboratory
Medicine, University of Pennsylvania
Medical Center, Philadelphia, Pennsylvania,
USA; and Paolo Fortina is at the Cancer
Genomics Laboratory, Kimmel Cancer Center,
Department of Cancer Biology, Thomas
Jefferson University, Jefferson Medical College,
Philadelphia, Pennsylvania, USA.
e-mail: jaspar@childrens.com

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in leukemia) or to target therapy (e.g., HER2


amplification in breast carcinomas to determine treatment with Herceptin (trastuzumab,
Roche/Genentech)). But it is unusual to test
for multiple mutations that have not been
previously described in a specific tumor type.
An oncologist in the United States may decide
that a patient will benefit from a US Food and
Drug Administration (FDA)-approved drug,
such as trastuzumab, on a tumor type or
mutation type not approved by the FDA. Such
practice is referred to as off-label use. Of all
chemotherapeutic prescriptions, 3347% are
off-label2.
If a patient has not responded to conventional
therapies, there may be a rationale to perform
NGS testing in order to identify potential drug
targets and then find an appropriate clinical
trial or off-label drug. However, routine implementation of clinical NGS in oncology is still in
its infancy. Current testing methods for cancer
mutations include point mutation assays, exon
sequencing, fluorescence in situ hybridization
and karyotyping. Such tests are available for only
a handful of genetic alterations that can inform
therapy, and the diagnostic yield of any single
test is low. For several reasons, including cost, a
shotgun approach that combines several methods is impractical. The potential advantages of
NGS in achieving high-throughput testing are
clear. Indeed, at a few centers, NGS has been
applied clinically for several years to detect base
substitutions and small deletions in large panels
of genes (tens to thousands), whole exomes and
whole genomes. The study of Frampton et al.1
is important because it shows that NGS can provide superior analytic performance for detecting mutations in oncogenic pathways. Such an
approach may also be lower in cost than conducting multiple non-NGS tests.
Frampton et al.1 improve on previously
reported NGS cancer testing methods by increasing the number and types of genetic alterations
detected as well as demonstrating an extensive
validation with numerous reference materials.

The authors describe experimental protocols


for handling surgically collected solid tumor
specimens that have been fixed in formalin and
embedded in paraffin. A key step in the assay
is the use of custom-synthesized DNA capture
probes to enrich a sample for specific regions of
the genome. This allows the authors to sequence
their target regions to >500 average depth of
coverage, thereby providing enough information to confidently call mutations. In total, the
capture probes target 4,557 exons from 287
cancer-related genes, 47 introns from 19 genes
frequently rearranged in cancer and 3,549
single-nucleotide polymorphisms.
Frampton et al.1 also improve clinical NGS
protocols by providing a robust analytical validation strategy (Fig. 1). The authors use 53 cell
lines to create three types of reference materials designed to assess the performance of their
method for detecting base substitutions, indels
or copy-number variations. The cell lines are
both of non-tumor origin (HapMap) and
cancer derived. They are pooled into various
admixtures to examine detection both of genetic
alterations and genetic alterations diluted into
normal DNA. Sensitive detection of alterations
in diluted material simulates a surgical specimen
that may have rare tumor cells in a non-tumor
tissue background.
Importantly, the validation approach is consistent with recommendations outlined by the
Next-generation Sequencing: Standardization
of Clinical Testing (Nex-StoCT) workgroup3
convened by the US Centers for Disease
Control and Prevention. These recommendations and others4,5 are intended to ensure that
diagnostic tests based on NGS meet clinical
laboratory regulatory requirements.
Identifying mutations is only the first step.
All clinical NGS tests must also demonstrate
usefulness in improving patient outcomes.
The clinical significance of novel mutations
is difficult to determine, even when they
occur in well-known oncogenic pathways.
Frampton et al.1 say that their test reveals

volume 31 number 11 NOVEMber 2013 nature biotechnology

n e w s and v i e w s

Tumor sample
in paraffin block

Pathology review
DNA extraction
Library preparation

Solution-based target capture


with 23,685 individually synthesized,
biotinylated, 120-bp DNA capture probes

Paired-end sequencing

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2013 Nature America, Inc. All rights reserved.

Map reads to human genome

Variant calling

Base substitutions

Insertions & deletions

20 discrete HapMap cell lines


2 pools of 10 cell lines each
MAF tested from <5% through 100%

28 discrete tumor cell lines


41 pools from tumor cell lines
MAF tested at <10%, 1020% and >20%

Variant type
Reference material
used for validation

Copy-number variations
7 discrete tumor cell lines
42 pools from combinations of tumor
cell lines and normal cell lines
Each tumor cell line tested from 0 to 80%
dilution with normal cell lines

Figure 1 Clinical sequencing test protocol and validation. Patient tumor samples are received as
formalin-fixed, paraffin-embedded blocks, which are from diagnostic surgical pathology. These types
of samples are heterogeneous mixtures of neoplastic and normal cells. Frampton et al.1 design DNA
probes for solution capture of ~1.5 Mb of genomic regions known to be related to tumorigenesis. Variants
are called from the sequencing data using bioinformatic algorithms. The analytical performance of the
assay is demonstrated by manufacturing and testing custom reference materials derived from pooled
cell lines. The pooled cell lines contain genomic DNA alterations that represent a wide spectrum of base
substitutions, insertions and deletions, and copy-number variations. Reference materials for insertions
and deletions and copy-number variations share two cell lines. MAF, mutant allele frequency.

clinically actionable mutations in 76% of tested


tumors. They define a clinically actionable mutation as one that has a clinically available targeted
treatment option or a mechanism-driven clinical
trial. But just having a drug targeted to a specific mutation does not guarantee its efficacy. For
example, included in the list of identified alterations are point mutations and indels in ERBB2

(HER2). Trastuzumab is labeled by the FDA for


use in breast or metastatic gastric or metastatic
gastroesophageal junction adenocarcinomas,
only when HER2 is overexpressed; the label
does not mention point mutations or indels. The
authors note that robust clinical evidence for targeting these alterations must still be generated,
and cite a recent in vitro study of targeted therapy

nature biotechnology volume 31 number 11 NOVEMber 2013

for nonamplification HER2 mutations in breast


cancer6. Although a single in vitro study may
guide potential off-label use, it is not sufficient
evidence for a successful FDA review of a diagnostic test kit intended to guide chemotherapy.
There are scenarios when off-label use of
chemotherapeutics is considered best practice for patient care. However, off-label use
has well-known risks to patients, including
the unknown risk-benefit ratio of administering compounds that frequently have toxic side
effects7. In general, the biology of most cancers
is not understood well enough to confidently
transfer a chemotherapeutic from one clinical setting to another in the absence of clinical
trials. For example, the mutational status of
KRAS in metastatic colon cancer predicts the
efficacy of anti-EGFR therapeutics. A logical
hypothesis would be that KRAS mutational status may also predict the efficacy of anti-EGFR
therapeutics in earlier-stage colon cancer.
However, a recent clinical trial of earlier-stage
colon cancer found that KRAS mutational status does not predict the efficacy of adding antiEGFR therapy8. This is one of many examples
of how chemotherapeutic selection is not a
simple logic equation but a task best guided
by clinical trials.
The challenge of properly prescribing chemotherapeutics off-label will remain even as
the use of NGS in clinical settings becomes
more widespread9. Multiple oncology trials
to evaluate NGS are currently under way10.
In addition to examining the clinical benefits
of sequencing panels of genes, similar to the
panel used by Frampton et al.1, some of these
studies are also exploring mRNA transcript
profiling and sequencing of whole exomes
and whole genomes. To maximize the utility
of such studies, we would argue that patients
who are treated with drugs for off-label indications based on the findings of NGS testing should be encouraged to participate in
registries that document their outcomes.
Frampton et al.1 have established such a registry for patients (http://clinicaltrials.gov/show/
NCT01851213).
Regardless of whether clinical NGS is successful in identifying genetic alterations that
can be treated with existing drugs, Frampton
et al.1 report a notably high frequency of mutations in oncogenic pathways (on average 1.57
mutations per case, with 1,579 distinct alterations in 2,112 cases). If these mutations are all
important in driving tumorigenesis, the high
number will represent a challenge to current
drug development models that target a single
mutation with a single chemotherapeutic.
COMPETING FINANCIAL INTERESTS
The authors declare no competing financial interests.

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n e w s and v i e w s
1. Frampton, G.M. et al. Nat. Biotechnol. 31, 10231031
(2013).
2. Conti, R.M. et al. J. Clin. Oncol. 31, 11341139 (2013).
3. Gargis, A.S. et al. Nat. Biotechnol. 30, 10331036 (2012).
4. Rehm, H.L. et al. Genet. Med. 15, 733747 (2013).
5. CAP Laboratory Accreditation Program. Molecular
Pathology Checklist http://www.cap.org/apps/docs/
laboratory_accreditation/checklists/new/molecular_
pathology_checklist.pdf (College of American
Pathologists, 2013).

6. Bose, R. et al. Cancer Discov. 3, 224237


(2013).
7. Krzyzanowska, M.K. J. Clin. Oncol. 31, 11251127
(2013).
8. Alberts, S.R. et al. J. Am. Med. Assoc. 307,
13831393 (2012).
9. Garber, K. J. Natl. Cancer Inst. 103, 8486
(2011).
10. Simon, R. & Roychowdhury, S. Nat. Rev. Drug Discov.
12, 358369 (2013).

Two views on light sheets


Carl G Ebeling & Erik M Jorgensen

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2013 Nature America, Inc. All rights reserved.

A dual-view light-sheet microscope combines isotropic spatial resolution with


high speed and minimal phototoxicity.
The confocal microscope, long the dominant
imaging technique in cell biology, is approaching obsolescence, soon to be superseded by a
new generation of optical microscopes. The
approach most likely to take its place is selective plane illumination microscopy (SPIM), a
technique that delivers lower doses of light to
the sample and achieves faster image acquisition compared with confocal systems. The
major drawback of conventional SPIM is its
poor axial resolution: whereas optical sections
in confocal microscopes are 800-nm thick,
those in light-sheet microscopes are 26 m.
In this issue, Wu et al.1 describe a new dualview SPIM instrument capable of providing
330-nm resolution, not only along the lateral
axes (x and y planes) but also along the optical axis (z plane). The key insight is to use
two orthogonal objectives that alternate in a
duty cycle between excitation and detection
as they scan through the sample. Light-sheet
microscopy now stands to deliver upon its initial promiserapid imaging of living organisms with low photo-dosage and high spatial
resolution in three dimensions.
In traditional epifluorescence microscopy, a
column of light excites fluorescent molecules
above and below the focal plane in the specimen (Fig. 1a). The out-of-focus light generates
a blurry image without detail, and absorption of
light by biological tissue leads to phototoxicity
Carl G. Ebeling is at the Department of Biology,
Howard Hughes Medical Institute, University
of Utah, Salt Lake City, Utah, USA, and the
Department of Physics, University of Utah, Salt
Lake City, Utah, USA, and Erik M. Jorgensen is
at the Department of Biology, Howard Hughes
Medical Institute, University of Utah, Salt Lake
City, Utah, USA.
e-mail: jorgensen@biology.utah.edu

992

and photobleaching. Confocal microscopy


eliminates out-of-focus light by masking it with
a pinhole before detection, greatly improving
the axial resolution of the image. However,
laser light is absorbed throughout the sample,
causing phototoxicity and photobleaching.
The basic problem is that the excitation beam
overlaps the detection path. In addition, the
slow acquisition rates preclude imaging of fast
events in living specimens.
In SPIM, by contrast, the specimen is illuminated by an orthogonal light sheet, thereby
eliminating out-of-focus light in the detection
path (Fig. 1b)2. The resulting image is sharp,
and the specimen does not absorb light outside the focal plane. But light-sheet microscopy is hampered by poor axial resolution.
Creating the orthogonal light path requires
multiple objectives in close proximity to the
sample and hence to each other. High numerical aperture objectives would generate thinner
light sheets on the excitation side and better
resolution on the detection side, but because
of their bulky diameters and short working
distances, it is not feasible to use them. Lower
numerical aperture objectives with longer
working distances are used instead. This
weakness has led to a search for improved
SPIM microscope designs and the creation of
many SPIM spin-offs.
A common approach to overcome poor
axial resolution is to image the sample from
multiple viewpoints and then computationally
fuse the data into a single composite image.
This concept was first implemented by rotating the sample so that the z axis becomes the
new x axis3. To mitigate scattering effects and
to increase simultaneous coverage of the sample, a second strategy has been to add another
set of excitation and detection pathways4,5.
While still rotating the sample, opposing

excitation objectives illuminate the sample


from opposite sides, and two detection objectives orthogonal to the excitation plane collect the fluorescence emission. However, the
downside to this technique is increased light
exposure and phototoxicity.
Rotating the sample has its own drawbacks,
such as prolonging acquisition time, but the
advantages of imaging the same volume from
multiple vantage points is an increase in resolution. With the proper post-analysis software,
the best spatial information from each image
is preserved, whereby the poor axial resolution
in one orientation is replaced with the higher
lateral resolution from the second orientation.
In short, multiview acquisition begins to solve
the axial resolution problem. Previous implementations of multiple viewpoint acquisition systems35 require anywhere from 4 to
36 different acquisitions of the same volume,
unfortunately offsetting the inherent advantages of the low photo-dosage of conventional
light-sheet microscopy. Moreover, multiview
methods have not yet demonstrated true isotropic resolution (i.e., the same resolution in
all spatial directions).
Wu et al.1 offer a solution to many of the
issues plaguing light-sheet microscopy. They
show that their dual-view, inverted SPIM
(diSPIM) setup achieves isotropic resolution
with minimal phototoxicity while keeping the
sample immobile and mounted by conventional methods. The enabling innovation is the
use of a duty cycle whereby the two objectives
alternate in rapid sequence between excitation
and detection (Fig. 1c). Each volume is therefore imaged only twice, and from orthogonal
vantage points. The two volumes are then combined computationally into a single, isotropic
image by a fast, joint-deconvolution algorithm
(Fig. 1d). Together with fast, scientific-grade,
complementary metal oxide semiconductor
cameras, an imaging readout speed of 200
Hz is achieved, yielding a volumetric acquisition time of 2 Hz, which is about 10 times
faster than what has been demonstrated with
the fastest comparable methods. The method
also makes selective plane illumination more
practical because standard sample preparations, namely a specimen on a coverslip, can
be used, as opposed to the time-consuming
agar-immersion methods of sample preparation that SPIM instruments generally require.
This has the further advantage of limiting the
bench-to-instrument time, which is critical in
studies looking at rapid dynamic processes.
The authors show that the high acquisition
speed, low phototoxicity and isotropic resolution of their diSPIM setup allow the imaging
of biological processes that would have been
difficult to observe with more conventional

volume 31 number 11 NOVEMber 2013 nature biotechnology

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