dPCR and Cancer Detection
And researchers have adopted dPCR for numerous applications including for analysis of several parameters in cancer patients. In study results published in Clinical Cancer Research last March, researchers from the Royal Marsden Hospital in London described their adaptation of ddPCR to determine the presence of oncogenic amplification through noninvasive analysis of circulating free plasma DNA and exemplify this approach by developing a plasma DNA digital PCR assay for HER2 copy number.
Because HER2 copy number in digital PCR is assessed relative to a reference gene, the investigators used EFTUD2, a gene within the ERBB2 locus found not to co-amplify with HER2 and not subject to normal copy-number variations.
Using the Bio-RAD QX100 ddPCR system, the researchers found that 64% of patients with HER2-amplified cancers were classified as digital PCR HER2-positive and 94% of patients with HER2-nonamplified cancers were classified as HER2-negative by the assay, giving a positive and negative predictive value of 70% and 92%, respectively.
The authors concluded that “digital PCR of plasma DNA has high accuracy in the determination of HER2 status,” and that the approach of analyzing of plasma DNA with digital PCR has the potential to screen for the acquisition of HER2 amplification in metastatic breast cancer. “This approach could potentially be adapted to the analysis of any locus amplified in cancer,” they concluded.
And last September, scientists working at Fred Hutchinson Cancer Research Center, demonstrated that ddPCR technology could be used to precisely and reproducibly quantify microRNA (miRNA) in plasma and serum over the course of different days, potentially allowing further development of miRNA and other nucleic acids as circulating biomarkers.
Under active study as blood-based biomarkers for cancer and other diseases, miRNA measurements in blood samples have been plagued by unacceptably high interday variability, obviating their use as reliable blood-based biomarkers.
“In the field of circulating microRNA diagnostics, droplet digital PCR enables us to finally perform biomarker studies in which the measurements are directly comparable across days within a laboratory and even among different laboratories,” said Muneesh Tewari, M.D., Ph.D., associate member in the Human Biology Division at the Fred Hutchinson Cancer Research Center and lead author of the study.
And Dr. Karlin-Neumann says that ddPCR is “en route to being introduced into clinical practice in a number of areas.” Though, he notes, the “only CLIA lab I know of that currently offers a ddPCR-based test is the University of Washington’s Clinical Laboratory, which offers a ddPCR-based test for detection of chromosomally integrated HHV-6 virus in transplant patients.”
Other labs, he says, that are in the process of developing clinical tests for detection of residual disease in leukemia patients with BCR-ABL translocations include that of Alec Morley, M.D., a pioneer of digital PCR. Dr. Karlin-Neumann also cites the work of Hanlee Ji, M.D., who is measuring copy-number variations by ddPCR in FFPE and cell-free plasma DNA to assess whether gastric and other cancer patients have amplifications in oncogenes that would make them amenable to one of a growing number of targeted therapies.
Importantly, Dr. Karlin-Neumann pointed out that it’s still too early, regardless of the platform used, to be attempting to detect cancer in naïve patients not already known to have cancer since “we do not have the clinical experience to know what changes to look for and what thresholds are meaningful.”
And he notes, until recently, there have not been technologies that allowed us to detect and quantitate below ~1% mutant abundance in either mixed tissue biopsies or in cfDNA in plasma or serum. ddPCR is demonstrating that it is capable of lowering this limit to as low a ~0.01% in a single ddPCR reaction well, and where more material is available, this can be lowered further by use of multiple wells. Similarly, fractional changes in oncogenic amplifications and deletions can be tested with ddPCR in both solid tumors and in cfDNA.
And a team of scientists at the University of California, Berkeley says it has developed a bead-based, microfluidic digital PCR technology and demonstrated its ability to quantitatively measure cancer-related translocation mutations at extremely low levels and subsequently sequence single mutated clones.
The scientists believe that their technology has advantages over commercial emulsion-based droplet digital PCR platforms, such as those offered by Bio-Rad and RainDance Technologies, because it enables downstream sequencing analysis following the digital PCR analysis step.
But is this capability in demand? A RainDance spokesperson told PCR Insider that the company's RainDrop digital PCR system currently does allow for emulsions to be broken following thermal cycling so the amplicons can be rescued and subsequently sequenced. However, RainDance said, it is “just starting to see requests for this kind of thing but it is not a commercial solution on offer at this point.”
But technology will get continue to get piled higher and deeper, as modifications to PCR continue to accrue and scientists figure out how best to use them.
Patricia Fitzpatrick Dimond, Ph.D. ([email protected]), is technical editor at Genetic Engineering & Biotechnology News.
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