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

Megan E Dueck,Sammy S Datwani,Alexander K Chao,Steve Gallagher,Robert Lin,Paul Hung,Lingxia Jiang,Andrew Zayac,Elliot Stieglitz

doi:10.1038/s41598-019-55872-7

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.

Highlights

It is becoming increasingly accepted that while Generation Sequencing (NGS) is an invaluable tool for broad based sequence characterization, its precision in tracking rare mutant events is low
The novel Digital Polymerase Chain Reaction (dPCR) platform consists of a micro-molded plastic Microfluidic Array Partitioning (MAP) consumable device (Fig. 1) and a fully integrated single instrument that combines pneumatic consumable sample loading, thermal cycling, 3-color fluorescence image acquisition with control and analysis software (Fig. 2, Supplemental Fig. 1)
Since the micro-molded dPCR partitions are shallow (X = 65 μm, Y = 82 μm, Z = 97 μm in height) and located directly above the thermal-cycling heating element, sample results can be obtained using

Summary

Introduction

It is becoming increasingly accepted that while Generation Sequencing (NGS) is an invaluable tool for broad based sequence characterization, its precision in tracking rare mutant events is low. The advent of microfluidics has greatly aided the advancement of dPCR; current available partitioning technologies suffer from limitations in three critical areas – low partition volume consistency, low partition number consistency and high dead volume of loaded sample. Unanalyzed sample via a high dead volume in the consumable introduces subsampling error and reduces the overall accuracy of the results. The combination of rare targets with high dead volume synergistically lowers dPCR accuracy and broader based applicability for clinical applications. We present a novel Microfluidic Array Partitioning (MAP) consumable device that utilizes precisely manufactured high volume injection molded arrays combined with precise fluidic control to overcome current dPCR limitations. Coupled with a novel precise fluidic loading process, the MAP consumable device utilizes >95% of input sample and significantly reduces subsampling error compared to other currently available technologies that utilize

Methods

Results

Conclusion