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Exploring Droplet Digital PCR Technology
Industry Insight

Exploring Droplet Digital PCR Technology

Exploring Droplet Digital PCR Technology
Industry Insight

Exploring Droplet Digital PCR Technology


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Droplet digital PCR (ddPCR) technology is a digital form of the PCR method that utilizes a water-oil emulsion system. This technique partitions nucleic acid samples into thousands of nanoliter-sized droplets. Then, PCR amplification is conducted within each droplet. This approach requires less sample than other PCR approaches that are available, which is advantageous in many settings. Recently, we interviewed George Karlin-Neumann, Clinical Director, Digital Biology Group at Bio-Rad Laboratories to learn more about ddPCR and its applications.

Molly Campbell (MC) What is droplet digital PCR and what are its main clinical applications?

George Karlin-Neumann (GKN):
Digital PCR (dPCR) is a method for detecting and quantifying nucleic acid target sequences by subdividing a sample into many small partitions, or compartments, each of which either contains or doesn’t contain the target of interest. Positive partitions (i.e. strongly fluorescent) are counted after running a typical Taqman reaction to endpoint.

Droplet digital PCR (ddPCR) is an implementation of dPCR that uses dynamically generated droplets as sample partitions as opposed to pre-formed nanochambers. In ddPCR, droplets are read single-file in a droplet flow cytometer to score the number of positive and negative droplets in a sample, which enables scientists to determine the concentration of target molecules present in the sample. ddPCR is a technology that can detect and precisely quantify various types of DNA and RNA alterations with a high degree of reliability and using either a very small or large amount of sample (i.e. where only a few or >100,000 copies of a desired target are present). This ability makes it useful for applications such as liquid biopsy which uses blood samples and is rapidly being adopted by labs. ddPCR is often employed by cancer researchers and oncologists to track disease progression and determine therapy response. One such test for patients with non-small cell lung cancer uses blood samples to test for DNA mutations and returns results to oncologists within 72 hours, weeks or months before an imaging result may be known.

A clinical assay based on ddPCR technology, the QXDx CR-ABL %IS Kit, has been both CE-IVD-marked and FDA cleared. Physicians can use these systems and tests to detect minimal residual disease in patients with chronic myeloid leukemia to make treatment decisions with greater certainty. Bio-Rad’s QX200 ddPCR system has also shown promise in other applications, both clinical and non-clinical. Clinical applications include monitoring the effectiveness of kidney, liver and lung transplants and detecting fetal aneuploidy and mendelian diseases in non-invasive prenatal tests, pathogens, and neonatal genetic diseases such as spinal muscular atrophy. In the research laboratory, ddPCR can be used to
detect and measure single nucleotide variations, indels, copy number variations, gene rearrangements, genome structure, methylated DNA, and gene transcript and miRNAs levels, especially as applied to cancer liquid biopsy, infectious disease research and clinical testing. Finally, ddPCR can also be used for environmental testing, such as measuring water quality and food contamination by genetic modification.

MC: Why is it advantageous to use ddPCR and NGS together in liquid biopsies, rather than separately?

GKN:
ddPCR and NGS serve two critical, but distinctive roles in liquid biopsy-based cancer diagnostics. With a liquid biopsy, clinicians can collect cell-free tumor DNA from the blood or other body fluids such as urine and detect and quantify known mutations to prescribe targeted therapies or track patient response and disease progression based on the tumor’s genetic profile. NGS is the gold standard technique for identifying novel mutations and rearrangements in ctDNA that will ultimately impact cancer progression. Through gene panels or whole genome sequencing, NGS yields a broad inventory of the mutations in a tumor. But it is cost prohibitive to use NGS to track these mutations over time. It is also labor-intensive and has a much longer turnaround time to return results when compared to ddPCR, which typically has greater precision, sensitivity and specificity for known alterations. Combining ddPCR and NGS, however, can leverage the benefits and overcome the limitations of both techniques. Once NGS is used to detect the full set of mutations in ctDNA, researchers can rely on the sensitivity and precision of ddPCR to monitor a smaller set of actionable gene markers and track tumor progression and treatment response in near real time. Droplet digital PCR’s advantages in speed and affordability over NGS make it more attractive for serial monitoring. ddPCR also enables reporting of absolute copies of a mutation per milliliter of blood, which may be a more robust metric than mutant allele frequency (MAF) when precise quantitative measurements are needed to make clinical decisions.

MC: Why is ddPCR more cost-efficient than NGS?  

GKN:
Unlike NGS, which provides a top-down view of the genome, ddPCR focuses on precise and reproducible detection and quantification of known targets with phenotypic significance. ddPCR also requires minimal manipulation of the sample before running a test; it does not require library construction, as in NGS. Additionally, ddPCR is robust, reproducible, and tolerant to inhibitory substances, making failed reactions rarer.  Good dynamic range and sensitivity, as well as multiplexing more than 2 targets to be quantified per ddPCR reaction will enable only one or a few wells to be run for most biological measurements.

MC: How can ddPCR be used to predict cancer reoccurrence following surgery?

GKN:
Using ddPCR technology to evaluate liquid biopsy samples enables physicians to predict very early during treatment how a patient will respond. We’ve seen in numerous cancer types that patients’ circulating tumor DNA (ctDNA) levels correlate with their treatment outcome. For example, at the American Association of Cancer Research conference this year, researchers showed in a proof-of-principle study that ddPCR can predict whether patients with malignant pleural mesothelioma (MLM) respond positively to surgery. The scientists compared genetic differences between tumor and normal tissue in 11 patients with MPM and developed a ddPCR assay that could detect MPM-specific ctDNA. When they tested patient blood samples collected before surgery, they found that patients with MPM-specific ctDNA had a worse prognosis after surgery. In another study presented at the American Society of Clinical Oncologists conference this year, researchers used ddPCR to track genetic rearrangements and copy number variations in ctDNA in patients with pediatric osteosarcoma. After tracking these patients before and after surgery or chemotherapy, they discovered that the presence of rearrangement markers predicted treatment outcome in 85 percent of patients and correlated with the likelihood of reoccurrence. 

MC: Are there any limitations to using ddPCR in a clinical environment?

GKN:
A ddPCR-based liquid biopsy is not effective for every cancer patient. ddPCR can only examine a small number of mutations and a small number of gene targets at a time. In cancers that require the analysis of a large number of candidate mutations, NGS would be a better approach. Finally, since liquid biopsies in general rely on ctDNA, neither ddPCR or NGS are suited for monitoring tumors that do not release DNA into the blood, such as brain tumors. For cancers like this, traditional techniques such as imaging may be more effective.

MC: How can ddPCR techniques continue to be improved going forward?

GKN:
ddPCR technology can already be used to reliably monitor patients’ responses to therapy with a short turnaround time. Bio-Rad is working on increasing the capacity of ddPCR for higher levels of multiplexing where limited sample is available and maximal sensitivity is desired for multiple targets. Implementing more powerful scanning methods in ddPCR can allow us to screen segments of DNA for alterations at multiple positions in a specified region of the genome.

For the future, Bio-Rad is working on increasing the capacity of ddPCR for higher levels of multiplexing where limited sample is available and maximal sensitivity is desired for multiple targets. We are also developing ways for ddPCR to better interface with NGS-profiled samples to improve the ability of physicians to monitor patients’ responses to therapy and disease progression.

Beyond that, it’s a matter of learning more about our own biology. ddPCR has shown promise not only in cancer, but also other conditions including HIV, organ transplantation, and Type I diabetes. As we learn more about the role of genetics and epigenetics in disease, researchers will develop new ddPCR-based assays for more conditions, enabling the greater use of ddPCR technology in both the laboratory and the clinic.

George Karlin-Neumann, Clinical Director, Digital Biology Group, Bio-Rad Laboratories, was speaking with Molly Campbell, Science Writer, Technology Networks.

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Molly Campbell
Molly Campbell
Science Writer
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