A novel digital PCR (dPCR) platform combining off-the-shelf reagents, a micro-molded plastic microfluidic consumable with a fully integrated single dPCR instrument was developed to address the needs for routine clinical diagnostics. This new platform offers a simplified workflow that enables: rapid time-to-answer; low potential for cross contamination; minimal sample waste; all within a single integrated instrument. Here we showcase the capability of this fully integrated platform to detect and quantify non-small cell lung carcinoma (NSCLC) rare genetic mutants (EGFR T790M) with precision cell-free DNA (cfDNA) standards. Next, we validated the platform with an established chronic myeloid leukemia (CML) fusion gene (BCR-ABL1) assay down to 0.01% mutant allele frequency to highlight the platform’s utility for precision cancer monitoring. Thirdly, using a juvenile myelomonocytic leukemia (JMML) patient-specific assay we demonstrate the ability to precisely track an individual cancer patient’s response to therapy and show the patient’s achievement of complete molecular remission. These three applications highlight the flexibility and utility of this novel fully integrated dPCR platform that has the potential to transform personalized medicine for cancer recurrence monitoring.

Digital PCR (dPCR) technology has been proven to be highly sensitive and accurate in detecting copy number variations (CNV). However, a higher-order multiplexing dPCR assay for measuring SMN1 and SMN2 copy numbers in spinal muscular atrophy (SMA) samples has not been reported. Described here is a rapid multiplex SMA dPCR genotyping assay run on a fully integrated dPCR instrument with five optical channels. The hydrolysis probe-based multiplex dPCR assay quantifies SMN1, SMN2, and the total SMN (SMN1 + SMN2) while using RPPH1 gene as an internal reference control. The quadruplex assay was evaluated with characterized control DNA samples and validated with 15 blinded clinical samples from a previously published study. SMN1 and SMN2 copy numbers were completely concordant with previous results for both the control and blinded samples. The dPCR-based SMA copy number determination was accomplished in 90 min with a walk-away workflow identical to real-time quantitative PCR (qPCR). In summary, presented here is a simple higher-order multiplexing solution on a novel digital PCR platform to meet the growing demand for SMA genotyping and prognostics.

Digital PCR (dPCR) has gained popularity in recent years for cancer research such as rare mutation detection, minimal residual disease, treatment selection, and recurrence monitoring. However, currently available technologies suffer from several limitations such as tedious workflow, long time-to-result, poor multiplexity, and inconsistent reagent digitization that severely hindered its broad adoption. We have designed and manufactured a dPCR platform that is capable of consistent sample digitization, thermal cycling and simultaneous interrogation of 20,000 partitions with walk-away workflow. The instrument integrates a loading and digitization manifold, a thermal cycler and an imaging engine with 5 optical channels into a single benchtop box. The Microfluidic Array Partitioning (MAP) consumable is a micro-injection molded, automation-ready plate that contains 16 individual units that can be loaded simultaneously. Each unit partitions an individual sample into 20,000 fluidically isolated partitions while utilizing >90% of inputted sample. We utilized an oncology standard assay, confocal microscopy, and real-time imaging to validate the dynamic range, sample utilization, and digitization consistency of the platform, respectively. Using the European Breast Cancer Standards ERM-AD623 we quantified replicates that were serially diluted from 1 × 105cp/μL down to 10cp/μL. We demonstrated that the dPCR platform can consistently quantify targets over 5-orders-of-magnitude of concentration with high consistency in both quantification and partition numbers. With confocal microscopy, the reagent inside the partitions are visualized to characterize sample utilization. Using conventional qPCR calibration dye ROX and real-time imaging, we show that over 99% of the partitions can be consistently analyzed for high quantification confidence. With the combination of ease-of-use, fast time-to-result and consistent performance, we believe this novel dPCR platform provides unparalleled usability and capability and will allow dPCR to fulfill its promise to impact cancer patient care. Citation Format: Robert Lin, Andrew Zayac, Steve Gallagher, Lingxia Jiang, Felicia Linn, Paul Hung. Fully integrated single instrument imaging-based digital PCR platform [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 43.

In this study we demonstrate precision quantification of Seraseq ctDNA EGFR T790M mutation mix at AF0.1% (mutant/wild-type ratio) using a novel one-step digital PCR (dPCR) platform. This novel yet simple workflow has the potential to make cancer liquid biopsy a clinical application. EGFR is an important drug target for the treatment of non-small cell lung carcinoma (NSCLC). During the treatment of NSCLC with tyrosine kinase inhibitors (TKIs), there is typically a significant response initially, followed by a secondary mutation as the carcinoma develops resistance. Early detection of cancer can better inform patient treatment and guide drug selection. One key EGFR mutation that leads to TKI resistance is the T790M mutation. Only a few clinical assays have been approved as companion diagnostics in patient biopsies (FFPE or plasma), while a larger number of laboratory developed assays (LDTs) under CLIA/CAP guidance are finding routine use in cancer disease diagnosis or treatment monitoring. Herein we describe the validation of a novel, fully integrated dPCR platform for the detection and absolute quantification of EGFR T790M. The integrated dPCR platform consists of a patented micro-molded plastic consumable and a fully-integrated instrument combining consumable sample loading, thermal cycling and 5-color fluorescence detection. The platform was designed to have a simplified, single-step workflow and provide results in less than one hour. In addition, developments in the micro-molded plastic consumable allow for near-zero dead volume. The novel integrated instrument is 100% dry and contamination-free, making it attractive for the transition to clinical applications. Seraseq ctDNA EGFR T790M mutation mix AF1% and AF0.1% reference standards in combination with a commercially available EGFR T790M dPCR assay were used to validate the novel integrated platform. In conclusion, we highlight the capability to precisely quantify samples as low as 0.1% T790M EGFR in a background of wild-type EGFR with high reproducibility and high accuracy. Citation Format: Megan E. Dueck, Robert Lin, Andrew Anfora, Andrew Zayac, Steve Gallagher, Omo Clement, Dana Ruminsky-Lowe, Paul Hung. Validation of a novel one-step digital PCR platform with precision circulating cell-free DNA standards [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2296.

In this study we present a single-step, one-color quantification of mutant versus wild-type allele using a novel integrated, clinic-optimized digital PCR (dPCR) platform. Allelic variants in DNA or RNA can be distinguished and quantified using Polymerase Chain Reaction (PCR) in concert with intelligent primer and probe design. Most commonly, a researcher or clinician will use unlabeled forward and reverse primers that bind to conserved regions outside of the allelic variation in addition to two differently labeled hydrolysis probes that bind at the region of allelic variation. Here we describe a novel method that leverages the precision of digital PCR (dPCR) technology and requires only one-color fluorescence utilizing probe-free, intercalating dye chemistry. The expense in developing or purchasing a complete PCR assay is normally embedded in the cost of the fluorescently labeled probe. The method described here eliminates the need for the fluorescently labeled probe, thereby reducing chemistry cost. Furthermore, technical complexity is reduced by requiring only one-color fluorescence. In our assay, we use a common unlabeled reverse primer that binds downstream of the allelic variation. Two unlabeled, allele-specific forward primers bind preferentially to either the wild-type or mutant allele at the site of allelic variation. The mutant forward primer contains a non-annealing tail (ie: stretch of the DNA primer that does not anneal to the initial DNA template) that will cause the ultimate mutant PCR product to be longer than the wild-type product. Digital PCR is characterized by dividing up a bulk PCR reaction volume into thousands of fluidically isolated partitions or droplets. In the presence of an intercalating dye, a longer amplicon will result in a higher endpoint fluorescence in that specific dPCR partition. As such, using our novel assay, dPCR partitions containing mutant versus wild-type amplicons can be distinguished by differing levels of end-point partition fluorescence. In this study we used the BRAF gene and the clinically relevant BRAF V600E mutation to benchmark this assay using a novel integrated one-step dPCR platform. This dPCR platform consists of a single, fully-integrated instrument along with a novel micro-injection molded plastic consumable providing a simple, single-step workflow. The instrument is dry and contamination-free, making it an attractive instrument for easy transition to the clinic. Parameters including thermal-cycling time and primer concentration were serially optimized for the novel integrated dPCR platform. This work highlights that we can accurately and precisely quantify samples containing varying ratios of wild-type versus V600E BRAF down to 0.1% mutant/wild-type ratio using one color of fluorescence and a novel integrated single-step walkaway dPCR workflow. Citation Format: Megan E. Dueck, Christina Wood-Bouwens, Andrew Zayac, Robert Lin, Steve Gallagher, Hanlee Ji, Paul Hung. Single-color accurate and precise quantification of wild-type versus mutant allele using a novel integrated digital PCR platform [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2455.