Digital PCR Platform Research Articles

High Sensitivity Detection and Quantitation of DNA Copy Number and Single Nucleotide Variants with Single Color Droplet Digital PCR

In this study, we present a highly customizable method for quantifying copy number and point mutations utilizing a single-color, droplet digital PCR platform. Droplet digital polymerase chain reaction (ddPCR) is rapidly replacing real-time quantitative PCR (qRT-PCR) as an efficient method of independent DNA quantification. Compared to quantative PCR, ddPCR eliminates the needs for traditional standards; instead, it measures target and reference DNA within the same well. The applications for ddPCR are widespread including targeted quantitation of genetic aberrations, which is commonly achieved with a two-color fluorescent oligonucleotide probe (TaqMan) design. However, the overall cost and need for optimization can be greatly reduced with an alternative method of distinguishing between target and reference products using the nonspecific DNA binding properties of EvaGreen (EG) dye. By manipulating the length of the target and reference amplicons, we can distinguish between their fluorescent signals and quantify each independently. We demonstrate the effectiveness of this method by examining copy number in the proto-oncogene FLT3 and the common V600E point mutation in BRAF. Using a series of well-characterized control samples and cancer cell lines, we confirmed the accuracy of our method in quantifying mutation percentage and integer value copy number changes. As another novel feature, our assay was able to detect a mutation comprising less than 1% of an otherwise wild-type sample, as well as copy number changes from cancers even in the context of significant dilution with normal DNA. This flexible and cost-effective method of independent DNA quantification proves to be a robust alternative to the commercialized TaqMan assay.

NGS-Assisted DNA-Based Digital qPCR Facilitates Stratification Of CML Patients In Long-Term Molecular Remission Based On The Presence Of Detectable BCR-ABL1 DNA

Recent studies indicate that 40% of CML patients on imatinib who achieve complete molecular remission (CMR; equivalent to MR4 or better in current terminology) remain disease-free after drug discontinuation, raising the possibility of an “operational cure”. However, the safe introduction of TKI withdrawal into clinical practice would require a method of evaluating risk of relapse, which is likely to be related to the presence of residual disease undetectable by RT-qPCR. A PCR method based on the use of genomic DNA has been shown to be more sensitive for the detection of residual disease than one that relies on cDNA and may, therefore, help to predict outcome post-withdrawal. However, the former method is arduous requiring a customised patient-specific assay. We describe here a method based on targeted next-generation sequencing (NGS) which allows the rapid identification of BCR-ABL1 breakpoints and the generation of DNA-based qPCR assays. The location of the BCR-ABL1 fusion junction was mapped in disease samples from 32 CML patients using Illumina's MiSeq platform. A custom TruSeq DNA target enrichment system (Illumina) was used to enrich for fragments containing sequences from the BCR and ABL1 genes using probes covering both genes including an additional 50kb of sequence in both 5' and 3' directions. All 32 patients' t(9;22) translocation junctions were successfully mapped using a custom designed bioinformatics algorithm. To date we have designed and validated DNA qPCR assays for 26 of these.On testing consecutive clinical samples from all 26 patients after achievement of MR4 or better (previously defined as CMR; mean 6 samples per patient), the DNA-qPCR assays detected residual disease in 8 out of 26 patients (31%), demonstrating that a DNA-based method is capable of identifying the presence of residual leukaemia in a proportion of patients who would be defined as “disease-free” using conventional RNA-based methodology.One area in which inaccuracy may be introduced into the DNA qPCR technique is in the use of a standard curve generated using serial dilutions of patient's diagnostic material. We therefore sought to further enhance the sensitivity of a DNA-based approach by optimising this technique for use on a digital quantitative PCR (dqPCR) platform, which provides absolute molecular quantification without the need for standard curve. Using DNA dqPCR on a Fludigm BioMark HD instrument, we measured the level of BCR-ABL1 in 48 samples from 6 CML patients after achievement of a sustained response of MR4 or better. At the time point at which patients were first determined to have achieved at least MR4, all samples tested positive by DNA dqPCR. However, in subsequent samples collected two years following achievement of at least an MR4 (the point of entry into ongoing withdrawal trials, at which point all six patients had achieved MR4.5 or better), we found that patients fell into two distinct categories: 1) those whose disease continued to be detectable by DNA dqPCR (n=3), and 2) those whose disease had fallen to a level that was no longer detectable by DNA dqPCR (n=3). In contrast the conventional BCR-ABL1 DNA qPCR method detected disease in only one of these six patients at this time point, suggesting superior sensitivity of a digital PCR-based screening platform.From this pilot study we conclude that NGS-facilitated DNA dqPCR may be used to stratify patients with RT-qPCR-undetectable disease according to residual disease burden and may therefore prove valuable in the identification of patients for whom TKI therapy could be safely reduced or stopped. Disclosures:Milojkovic:BMS: Honoraria; Pfizer: Honoraria; Ariad: Honoraria; Novartis: Honoraria. Apperley:Novartis: Honoraria, Research Funding; Bristol Myers Squibb: Honoraria; Pfizer, Ariad: Honoraria (not direct from company), Honoraria (not direct from company) Other.

Single-Arm, Open-Label, Multicenter Study Of Deep Molecular Response (MR4.5) With Nilotinib In Adult Patients (pts) With Newly Diagnosed Philadelphia Chromosome–Positive Chronic Myeloid Leukemia In Chronic Phase (CML-CP): ENESTnext Study Update

Single-Arm, Open-Label, Multicenter Study Of Deep Molecular Response (MR4.5) With Nilotinib In Adult Patients (pts) With Newly Diagnosed Philadelphia Chromosome–Positive Chronic Myeloid Leukemia In Chronic Phase (CML-CP): ENESTnext Study Update

Sensitivity for detecting PIK3CA mutations in early-stage breast cancer with droplet digital PCR.

11019 Background: PIK3CA is mutated in up to 30% of breast cancers. Classically somatic mutations are identified by Sanger sequencing of the primary tumor specimen. However third generation droplet digital PCR technologies offer a novel platform for quantitative mutation detection with improved sensitivity. Methods: Thirty stage I-III breast cancer patients were consented on an IRB-approved prospective repository study at Johns Hopkins for collection of their primary breast tumor specimen. Formalin-fixed paraffin embedded (FFPE) samples were analyzed by standard sequencing for three PIK3CA hotspot mutations. The DNA from these samples was then analyzed using the RainDrop digital PCR platform with TaqMan probes in a triplex format to simultaneously detect and quantitate hotspot mutations and genome equivalents. Results are expressed as a percentage of mutant to wild-type PIK3CA molecules for each sample. Results: Standard sequencing of all tumors (n=30) identified seven PIK3CA Exon 20 mutations (H1047R) and three Exon 9 mutations (E545K). Samples were scored as PIK3CA mutation positive by digital PCR if the tumor DNA contained at least 5% mutant molecules. All ten mutations identified by sequencing were verified by digital PCR with quantities of mutant molecules ranging from 20.3-55.6% in a given sample. Digital PCR identified additional PIK3CA mutations that were wild type by standard sequencing including three mutant Exon 20 samples, two mutant Exon 9 samples and one sample with an Exon 20 and Exon 9 mutation. Quantities of mutant molecules in these additional samples ranged from 5-28.9%. Conclusions: RainDrop digital PCR offers improved sensitivity and quantification for detecting PIK3CA mutations in FFPE samples using nanograms of DNA. Additional mutations identified by digital PCR may reflect genetic heterogeneity or possibly tissue contamination. The clinical utility of identifying a small proportion of mutations is unknown but may impact eligibility for targeted therapies and clinical trials. Ongoing studies will also address whether the identification of solid tumor mutations in circulating cell-free plasma DNA by digital PCR can improve diagnostics and aid in therapeutic decisions.

Abstract 3180: Mutation detection in circulating free DNA using mutant-enriched PCR and digital PCR

Abstract Mutations in oncogenes such as EGFR, KRAS and PIK3CA, have been shown to have profound effects on pathway signaling and tumor growth. These genetic aberrations are potentially valuable predictors for patient stratification and therapeutic responses to targeted therapies directed against these signaling pathways. Here we evaluate the utility of circulating tumor DNA as a “liquid biopsy” for predictive diagnostics and real-time monitoring of disease. A fundamental challenge with this approach is that, due to the low frequency of the mutant tumor DNA in circulation, a highly sensitive assay is required to detect the single base pair alterations. We have designed and investigated several assay formats, including TaqMan and scorpion-ARM assays for mutation detection using a digital PCR platform. We also employed LNA/PNA blockers and thermostable restriction digestion in PCR reaction to enrich the mutant DNA. The combination of the biased amplification and digital PCR dramatically increased mutation detection 10-100 fold. To assess clinical utility of the technology, data on PIK3CA mutation screening of matched patient tumor and plasma samples from a phase I clinical trial will also be reported. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3180. doi:10.1158/1538-7445.AM2011-3180

Abstract 4892: A novel droplet digital PCR platform accurately detects and quantifies molecular targets exceeding performance of other methods by orders of magnitude

Abstract Accurate detection and quantification of molecular targets is a challenge that arises across a range of oncology applications. In particular, existing techniques are faced with difficulties when trying to quantify copy number alterations or when attempting to detect single point mutations in the presence of high background of wildtype DNA. This work assesses the performance of a new droplet digitial PCR (ddPCRTM) platform in addressing these challenges. The ddPCR system partitions the sample into an emulsion of 20,000 stable mono-dispersed nanoliter droplets, such that some droplets have the target of interest and some do not. The emulsion is then thermocycled where each droplet serves as an independent reactor for PCR. After PCR, every droplet is read with a two-color fluorescence detector to assess whether the targets were amplified. The number of positive and negative droplets is used to compute an absolute concentration of the target in the sample with high precision and accuracy. The simple workflow uses standard 96-well plate processing enabling the system to generate data for millions of PCR replicates within a matter of hours. Many genomic regions undergo copy number alterations in human cancers and are associated with clinical features. Existing methods (including FISH, CGH, real-time PCR) suffer from various shortcomings, such as cumbersome workflow, limited sensitivity, and inability to discriminate better than 1.5 to 2 fold differences. Here we show that ddPCR provides accurate and reproducible copy number estimates with precision better than 10%. Specifically, using several systems of germline copy number variation we show CNV estimates that are highly reproducible, cluster tightly near integer values, and span 0 to 13 copies – so that a sample with 13 copies can be reliably distinguished from one with 12. Detection and quantification of rare mutations in the presence of highly homologous wild-type background is important in many oncology applications including analyses of heterogeneous tumors, monitoring response to therapy, and the analyses of cell free DNA in plasma. Conventional sequencing and real time PCR typically fail to detect the mutant when it's present at less than 10% frequency relative to the wildtype background. Here, we use a spike-in series with EGFR mutations to show that ddPCR can readily quantify targets down to the level of 0.01% mutant relative to the wild-type DNA. The system can be used for single molecule detection in the presence of tens of thousands of highly homologous molecules. In sum, we show that droplet digital PCR system produces highly accurate and reproducible quantification of molecular targets, with orders of magnitude performance improvements over conventional techniques for copy number estimation and rare event detection. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4892. doi:10.1158/1538-7445.AM2011-4892

Microfluidics Digital PCR Reveals a Higher than Expected Fraction of Fetal DNA in Maternal Plasma

The precise measurement of cell-free fetal DNA in maternal plasma facilitates noninvasive prenatal diagnosis of fetal chromosomal aneuploidies and other applications. We tested the hypothesis that microfluidics digital PCR, in which individual fetal-DNA molecules are counted, could enhance the precision of measuring circulating fetal DNA. We first determined whether microfluidics digital PCR, real-time PCR, and mass spectrometry produced different estimates of male-DNA concentrations in artificial mixtures of male and female DNA. We then focused on comparing the imprecision of microfluidics digital PCR with that of a well-established nondigital PCR assay for measuring male fetal DNA in maternal plasma. Of the tested platforms, microfluidics digital PCR demonstrated the least quantitative bias for measuring the fractional concentration of male DNA. This assay had a lower imprecision and higher clinical sensitivity compared with nondigital real-time PCR. With the ZFY/ZFX assay on the microfluidics digital PCR platform, the median fractional concentration of fetal DNA in maternal plasma was > or =2 times higher for all 3 trimesters of pregnancy than previously reported. Microfluidics digital PCR represents an improvement over previous methods for quantifying fetal DNA in maternal plasma, enabling diagnostic and research applications requiring precise quantification. This approach may also impact other diagnostic applications of plasma nucleic acids, e.g., in oncology and transplantation.