Demystifying the Lab
FIGURE.
Schematic Comparison of Exome and Genome Sequencing
Panel A shows the targeted nature of exome sequencing, with sequence reads concentrated over the
coding portions of genes. This is in contrast to genome sequencing, shown in Panel B, in which the
sequence reads are nearly randomly distributed over the entire genome. Each approach has advantages
over the other, some of which are listed in the two panels.
only want to know about
one particular mutation
and don’t need to spend
the time or money on a
broader panel.”
A laboratory must have
specialized equipment to
perform NGS, and physicians also must be trained
to interpret the data
and develop appropriate
therapeutic strategies for
a wide range of molecular
subtypes.6,7 For this reason,
“WGS is still primarily in
the realm of research since
we don’t know how to interpret the overwhelming
majority of what we find,”
Dr. Bejar said.
WES: An Unbiased
Approach
“Even if we were to perform WGS on a patient
or that patient’s tumor,
we would likely restrict
our analysis to mutations whose implications
are best understood,” he
added. “These are almost
entirely in protein coding
regions, which are much
better covered by WES.”
WES is an unbiased
way to examine every protein coding region, which
allows hematologists to
locate mutations and polymorphisms that they were
not initially looking for.
Reprinted with permission from The New England Journal of Medicine. (Source: Biesecker LG, Green RC. Diagnostic clinical genome
and exome sequencing. N Engl J Med. 2014;370:2418-25.)
since disruptive mutations can occur
almost anywhere along the length of a
gene,” Dr. Bejar said.
Which Genomic Panel Should
You Choose?
The goal of genomic studies is to improve
diagnostic precision and patient outcomes, but establishing the best circumstances for their use remains a challenge.
“As always, the clinical picture is the
most important factor in determining
which test is appropriate,” Dr. Welch said.
“The physician must understand what
question is being asked and then apply
the best test to answer it.” There are no
cut-and-dried rules for when to apply a
specific test, but the experts interviewed
for this article provided some guidance
about which test to use when.
WGS: Too Much Information to Handle?
WGS is most appropriate when structural
variants or non-coding mutations are suspected, or when there is uncertainty about
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ASH Clinical News
which chromosome position to evaluate.
But it may be an information overload.
“Ideally, we would use WGS all of the
time, as it is intended to capture all of the
genomic DNA information,” Dr. Slager
noted. “However, the current infrastructure is not able to handle such a large
number of samples with a quick enough
turnaround for clinical practice (e.g.,
within 1-2 days for results).”
Dr. Slager also pointed out that,
even when the infrastructure for WGS
is in place, it might not be warranted for
analysis of a specific genomic region
known to be cancer-related, such as a gene
or pathway. “Simpler genotyping panels
would get the information easily and
quickly without the overhead that comes
with managing all the data generated from
WGS technologies.”
Dr. Bejar agreed that a more targeted
approach is useful for quickly detecting
a specific mutation, such as the JAK2
V617F mutation in a patient with polycythemia vera. “In that scenario, we really
SNP Genotyping:
A Happy Medium?
“At the moment, WGS is
not well suited for routine
clinical use. It’s more expensive and often
less sensitive than other approaches.
This may change as sequencing becomes
cheaper and our ability to interpret what
we find grows,” Dr. Bejar said. “Even WES
is overkill in most clinical situations.”
Cost also remains a significant factor
when deciding which genomic test to
use. WGS is the most expensive approach
and not yet cheap enough for routine
clinical use, with SNP genotyping as the
most affordable option. He suggested
that for most diseases, including cancers,
targeted sequencing of specific genes is
the sensible choice for obtaining high
coverage at relatively low cost. “SNP
genotyping arrays that look at SNPs
across the genome remain useful in some
clinical scenarios but are rapidly being
eclipsed by the falling cost of WES and
other targeted sequencing techniques.”
The Present and the Future of Genomics
One challenge with the routine use of
genomic profiling today is that the cost
of computing is not falling as quickly
as the cost of sequence production, so
computing power is not sufficient to deal
with all the raw data being generated by
NGS.5,7 “We are entering a phase when the
analysis costs more than the sequencing
production,” Dr. Welch pointed out.
“Most genomic panels in clinical use
focus on the exons of a small number of
target genes. In cancer, these are typically
oncogenes and tumor suppressor genes
that are recurrently mutated – a subset
of which will have important clinical
implications,” Dr. Bejar said. “As the cost
of targeted sequencing has fallen, this
approach has become increasingly favored
in the clinical setting,” Dr. Bejar said.
Recently, at the 2014 ASH Annual
Meeting, Janine Pichardo, BS, from the
department of Pathology at Memorial
Sloan Kettering Cancer Center in New
York, and researchers demonstrated the
clinical utility of this approach. Using a
commercially available NGS-based gene
panel targeting hundreds of cancer-related
genes, investigators were able to diagnose
a wide spectrum of hematologic malignancies known to be difficult to diagnose,
subclassify, and risk-stratify with conventional methods.9
“Our study shows that a broad sequencing panel targeting single nucleotide variations, insertions, deletions,
copy number alterations, and translocations may improve diagnostic accuracy
in 10 to 15 percent of patients with
hematologic malignancies,” Ms. Pichardo
said. “In our opinion, physicians treating
hematologic malignancies should make
every effort to integrate comprehensive
targeted genomic profiling to the care of
patients with hematologic malignancies.”
—By Amy Dear ●
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