LGC eBook - 2017 - 15

Accelerating Science Using Integrated PCR Tools * PCR Shows Off Its Clinical Chops

"Current qPCR technology depends on realtime fluorescence accumulation as the PCR is
occurring, which can be an effective means of
detecting and quantifying DNA targets in
nondegraded samples," commented Dawne
Shelton, Ph.D., staff scientist, Digital Biology
Center, Applications Development Group,
Bio-Rad Laboratories. "Amplification efficiency
is critical; if that amplification efficiency
changes because of sample quality, it is
hidden in the qPCR methodology."
"In ddPCR, that is a big red flag," Dr. Shelton
continued. "It changes the format of how the
data looks immediately so you know the amount
of inhibition and which samples are too
inhibited to use."
Tissue types vary and contain different degrees
of fat or other content that can also act as PCR
inhibitors. In blood monitoring, the small circulating fragments of DNA are extremely degraded;
in addition, food, supplements, or other compounds ingested by the patient may have an
inhibitory effect.
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Clinical labs test for these variabilities and clean
the blood, but remnant PCR inhibitors can remain.
In ddPCR, a single template is partitioned into a
droplet. If the droplet contains a good template,
it produces a signal; otherwise, it does not-a
simple yes or no answer.
"Even if there is no PCR inhibition, most clinical
samples yield very small amounts of nucleic acid,"
Dr. Shelton added. "To make a secure decision
using qPCR is difficult because you are in a gray
zone at the very end of its linear range. ddPCR
operates best with small sample amounts and
provides good statistics for confidence in your
results."
Currently, at least a half-dozen clinical trials worldwide are using digital PCR, and half of them are
using the Bio-Rad QX200 Droplet Digital PCR
system. Examples of studies include: examining
BCR-ABL monitoring in patients with chronic myelogenous leukemia (CML), identifying activating
mutations in epidermal growth factor receptor
(EGFR) for first-line therapy of new drugs in patients with lung cancer, and the monitoring of

resistance mutations such as EFGR T790M in
patients with non-small cell lung cancer (NSCLC).
Clovis Oncology used a technology called BEAMing
(Beads, Emulsions, Amplification, and Magnetics),
a type of digital PCR for blood-based molecular
testing, to perform EGFR testing on almost 250
patients in clinical trials. In BEAMing, individual
EGFR gene copies from plasma are separated into
individual water droplets in a water-in-oil emulsion.
The gene copies are then amplified by PCR on
magnetic beads.
The beads are counted by flow cytometry
using fluorescently labeled probes to distinguish
mutant beads from wild-type. Because each bead
can be traced to an individual EGFR molecule in
the patient's plasma, the method is highly
quantitative.
"BEAMing is particularly well-suited for the
detection of known mutations in circulating
tumor DNA. In this circumstance, the mutation of
interest often occurs at low levels, perhaps only
1-2 copies per milliliter or even less, and in a high

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