When analyzing data from a recent cohort study that is monitoring a set of cancer patients over the course of their treatment, Hanlee Ji feels confident in the results he sees. Dr. Ji and his lab recently developed a single color digital polymerase chain reaction (dPCR) blood test that quickly and easily detects cancer-associated genetic mutations in circulating tumor DNA (ctDNA) in a patient’s blood. The test helps determine, in real time, tumor progression and whether an ongoing treatment is effective by quantifying mutated DNA in a sample. Dr. Ji’s high confidence in his results stems from the robustness of the assay itself.
As a medical oncologist, clinical geneticist, and research director at Stanford University, Dr. Ji has focused for years on developing new approaches to the study of gastrointestinal cancers with clinical and translational applications. In his studies, he has used different methods for detecting and quantifying ctDNA, from next-generation sequencing (NGS) and quantitative PCR (qPCR) to dPCR. Given its unique ability to generate an absolute measurement, dPCR provides the basis for the lab’s latest success.
The dPCR advantage
Digital PCR is a highly precise approach to independent nucleic acid detection and quantification that does not require a standard curve or reference. Each sample is partitioned into thousands of individual reactions that are run through simple endpoint PCR, where partitions containing the target mutation emit a strong fluorescent signal. The partitions are analyzed for the presence or absence of a fluorescent signal to calculate the absolute number of molecules in a sample.
dPCR’s high sensitivity lends itself to quantification applications that have otherwise been difficult to perform. dPCR can detect rare mutations on wild-type backgrounds, haplotypes, and low-abundance targets in the blood (such as circulating tumor cells and cell-free DNA), which permits liquid biopsies that are less invasive, costly, and time-consuming than traditional tissue biopsies. The technology also improves copy number variation analysis by accurately detecting minimal copy changes with fewer replicates than qPCR.
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“Reproducibility is the key to any scientific endeavor, and the research community at large has recognized that there are areas that we can improve on in terms of reproducible results. dPCR is an immense asset in this regard, due to its ability to perform absolute quantification as opposed to relative quantification methods,” says Madhuri Ganta, senior product manager of the digital biology center at Bio-Rad Laboratories.
In a recent study, scientists used dPCR to quantify copy number concentrations and the fractional abundance of KRAS-mutant DNA with a single nucleotide variation among a background of excess wild-type DNA. They demonstrated dPCR’s ability to reliably quantify the concentration of KRAS-mutated DNA across 21 laboratories within a strict margin of error. The study authors concluded that dPCR permits accurate quantification by minimizing variability from common sources of error that can influence qPCR results, including PCR inhibitors that can alter assay efficiency and standard curves that can cause cross experiment variation.
“Refined sample partitioning using droplets combined with endpoint cycling minimizes the effects of PCR inhibitors, allowing for better quantification of rare targets in complex backgrounds where amplification bias might otherwise occur,” explains Ganta.
dPCR requires that the target sequences must first be known, but nonetheless, physicians are routinely obtaining patient-specific sequences through a liquid biopsy over the course of a patient’s work-up. These developments facilitate platforms such as Dr. Ji’s blood test, which can be incorporated as a more rapid and cost-effective approach for serial monitoring of treatment effectiveness and downstream prognosis.
As we learn more about certain diseases and their associated mutations, enabling improved disease detection and monitoring, mutation-specific dPCR kits can offer quicker and more accurate diagnoses. Bio-Rad recently introduced the CE-IVD marked QXDx BCR-ABL %IS Kit for their QX200 ddPCR Dx System, which detects the BCR-ABL fusion gene, a biomarker for chronic myelogenous leukemia (CML). Current tests can detect low MR values, but the precision and accuracy of results are still questionable. “Bio-Rad’s ddPCR technology with the QXDx BCR-ABL kit improves detection at low levels with the accuracy of absolute quantification, enabling the CML community to better manage treatment,” explains Chinmay Sheth, marketing manager of clinical diagnostics at Bio-Rad.
Synergy between approaches
dPCR can deliver answers rapidly, with high sensitivity and precision, and at a relatively low cost per sample. Christie Fekete, digital PCR product manager at Thermo Fisher Scientific, comments that dPCR works as a complementary technique that can support NGS or qPCR, with applications evolving over time. Depending on the phase of the study, dPCR can be used to quantify libraries and to perform routine monitoring for mutations identified using NGS. Dr. Ji adds that after initial biomarker discovery, dPCR can be used to screen or validate results and to perform independent validation in larger populations at a fraction of the cost of NGS.
Synergy between dPCR and qPCR can enable scientists to answer their research questions with higher confidence. “qPCR works well for gene expression, genotyping, and miRNA analysis with a low cost per sample and a fast, simple workflow, while dPCR is best for precise and absolute quantification, particularly for rare events, and for analyzing samples containing PCR inhibitors,” explains Fekete.
As the complexity of analysis intensifies with a greater library of gene targets, it will become increasingly difficult to distinguish rare events from artefacts. Thermo Fisher recently released the Applied Biosystems TaqMan Liquid Biopsy digital PCR assays to provide a sensitive, wet-lab validated solution for the accurate detection of low-frequency mutant alleles in cancer research. Optimized for standardized dPCR protocols on the Applied Biosystems QuantStudio 3D and the Bio-Rad QX100/200 systems, the assays are formulated with the wild-type and mutant alleles in a single tube.
dPCR is pushing the boundaries of what can be achieved with other technologies.
"dPCR is pushing the boundaries of what can be achieved with other technologies. Absolute quantification can now be achieved more easily, without a standard curve or reference standard. Small differences in gene expression and copy number variation can be confidently detected. Rare mutations can be detected and quantified. Researchers can detect cancer recurrence and pathogens earlier. The ability to get absolute answers more quickly and easily is a great advantage that dPCR brings to the laboratory, and we see it used across a wide spectrum of applications,” adds Fekete.
Image: dPCR workflow from Thermo Fisher Scientific
A closer look at absolutes
Working in absolutes is a tough practice in biology. Experiments tend to yield relative results that are compared to a measured standard within that experiment. However, when it comes to translating information from one experiment to another, or from one lab to a collaborator, these relative measurements become meaningless.
Moves to create universal standards have helped improve the correlation of results between experiments. But while standardization is making it easier to generalize experimental protocols, relative results are still a centerpiece of many technologies in the laboratory. “Quantitative PCR provides comparably less precise methods of quantification, requiring standard curves or reference genes that make data analysis and results interpretation more time-consuming and tedious,” explains Tom Parks, scientist at Advanced Biotechnologies, Inc.
While dPCR can improve nucleic acid quantification across a wide range of applications, other technologies can still utilize the power of dPCR through digitally qualified standards that can more accurately qualify a standard curve. “Typical standards development involves incorporation of data from several different labs to assign a median value. dPCR enables absolute copy number assignment, with the same value given now and a year from now,“ says Parks.
It has become imperative to standardize absolute measurements in preclinical or translational research environments. Fekete notes Europe’s In Vitro Diagnostics Directive and ISO 17511, which outline the need to derive absolute values to establish real measurements. dPCR supports the routine calibration of quantitative reference standards because of its repeatability and reproducibility as an absolute measurement. Parks adds that a new product line from Advanced Biotechnologies expands standards to include RNA such as HIV-1 (IIIB Strain), RSV-A, and Yellow Fever Virus Quantitated Viral RNA, which can be used for viral load determination or other quantitative experiments.
Advanced Biotechnologies offers both dPCR-quantified DNA and RNA standards. They provide different concentration levels in quantities to accommodate any experimental or commercial need depending on whether a limited standard curve is needed, or a large linear range standard curve better fits an experiment. Data generated using these digitally quantitated standards can be accurately shared between labs or assays without further normalization. The development of standardization guidelines has become a possibility with dPCR and absolute copy number, which will ultimately improve lab-to-lab reproducibility of results.