Precision cancer monitoring using a novel, fully integrated, microfluidic array partitioning digital PCR platform

Abstract

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.

Molecular Testing in CML between Old and New Methods: Are We at a Turning Point?

“…Very recently, a third-generation dPCR chip-based platform was also tested for MRD monitoring in CML. Indeed, the novel microfluidic array partitioning device was capable of precisely quantifying BCR-ABL1 transcripts down to a 0.01% abundance, with high reproducibility across multiple replicates [39].…”

Development and validation of a 4-color multiplexing spinal muscular atrophy (SMA) genotyping assay on a novel integrated digital PCR instrument

“…dPCR has been used to identify rare gene variants as well as differences in copy number of multiple genes associated with pediatric-onset disorders (reviewed in 24 ). We have demonstrated that this system can identify a rare single nucleotide variant ( EGFR(T790M) ) associated with non-small cell lung carcinoma as well as fusion genes ( BCR-ABL1 and CCDC88C-FLT3 ) that are associated with different types of cancer 29 .…”

“…Patient-derived samples (n = 15) were procured from the Motor Neuron Diseases Research Laboratory (Nemours Alfred I. duPont Hospital for Children). Genomic DNA was isolated from patient-derived fibroblast and lymphoblastoid cell lines and the SMN1 and SMN2 copy numbers were measured using the QuantStudio 3D Digital PCR System (Life Technologies, Waltham, MA) as described previously 27 , 29 . Approval for cell line generation was provided by the Nemours Institutional Review Board and this study is registered on ClinicalTrials.gov (NCT01754441 and NCT02532244).…”

“…The Combinati Absolute Q Digital PCR System consists of Microfluidic Array Partitioning (MAP) consumable plates and a fully integrated instrument that automates partitioning of reagents in the plate, PCR thermocycling and 5-color fluorescence image acquisition. The MAP plate has a standard microplate footprint capable of running up to 16 samples per dPCR run 29 . Each unit is loaded with 10 µL of sample mixture and more than 90% of the loaded sample is partitioned and analyzed in ~ 20,000 pico-scale partitions.…”

“…The Combinati (Palo Alto, California) Absolute Q Digital PCR System (Fig. 1 ) is a single benchtop instrument with 5 optical channels and a qPCR-equivalent, walkaway workflow 29 . Instead of generating air and water emulsions, the Absolute Q features rigid micro-injection-molded partitioning consumable plates which ensure highly consistent partition yield and volume.…”