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.

Figure 1

Microfluidic Array Partitioning (MAP) digital PCR (dPCR) consumable. (A) Photograph of the MAP consumable. The full micro-molded Cyclo Olefin Polymer (COP) is 1″ × 3″ and contains 4 arrays of 20,000 partitions each. (B,C) Scanning Electron Micrograph (SEM) images of the MAP consumable partitions. After the automated reagent loading and digitization step, the arrayed partitions (X = 65 µm, Y = 82 µm, Z = 97 µm in height) are filled with reagent and the thinner connecting channels (10 µm in height, 25 µm in width) are next filled with silicone oil (creating fluidically isolated partitions). (D) A fluorescent image of the raw dPCR experimental results processed from a MAP consumable. PCR reagents containing a FAM-labeled probe and a reference dye (ROX) were loaded into the consumable and then thermal-cycled per user pre-defined settings. The consumable was next imaged in both the ROX and FAM channels. Finally, these two images were then overlaid where the red (ROX channel) represents partitions in which the reagent successfully loaded but no PCR target was present and the green (FAM channel) represents partitions that contained the PCR target.

Figure 2

The prototype of the fully-integrated single dPCR instrument. The photographic image shows the various subassemblies and instrument components of the integrated dPCR instrument. The flowchart below represents the simplified workflow where the instrument carries out the reagent partitioning step, followed by the thermal cycling process and finally the results are acquired and software processes the results in a simple one step workflow.

Figure 3

Principle of digitization in MAP consumable. (A) MAP consumable brightfield microscopy image accompanied by a cross-section depiction of channel heights. (B) The MAP consumable is loaded by applying positive pressure to an inlet well that has been loaded with reagent then overlaid with silicone oil. First the reagent (fluorescent orange) enters the connecting channels adjacent to the partitions. Next the reagent dead-end fills the partitions by outgasing air through the semi gas-permeable thin film. Finally the silicone oil (not visible) enters the connecting channel and fluidically isolates each partition as it passes.

Figure 4

EGFR T790M rare mutant quantification. The reagent was prepared to contain either no template, WT EGFR only, 1% EGFR T790M in a background of WT EGFR or 0.1% EGFR T790M in a background of WT EGFR. The reagent also contained a FAM-labeled EGFR T790M probe along with a HEX-labeled WT EGFR labeled probe. This reagent was loaded into a MAP consumable and processed on the prototype integrated dPCR instrument. A scatter plot of the results show the individual partition fluorescence on the y-axis and partition number on the x-axis (where each data point represents the results from an individual partition). The two tables show the results from measurements in triplicate for the 1.0% and 0.1% EGFR T790M samples.

Figure 5

BCR-ABL1 and ABL1 synthetic gBlock nominal concentration determination. The reagent was prepared to contain either ABL1 gBlock solution as template or BCR-ABL1 gBlock as template. The reagent also contained both a FAM-labeled BCR-ABL1 probe in addition to a HEX-labeled ABL1 probe. This reagent was then loaded into a MAP consumable and an experimental run was carried out on the prototype dPCR instrument. A scatter plot of the results show HEX fluorescence on the y-axis and FAM fluorescence on the x-axis (where each data point represents the results from an individual partition). The two tables contain the averaged results of the measurements in triplicate for the individual gBlock templates.

Figure 6

Quantification of precise BCR-ABL1/ABL1 ratios. Using the nominal gBlock concentrations determined prior, precise combinations of gBlock were generated to contain either 1%, 0.1% or 0.01% BCR-ABL1 gBlock in a background of ABL1 gBlock. The reagent was prepared to contain one of these combinations as the template. The reagent also contained both a FAM-labeled BCR-ABL1 probe in addition to a HEX-labeled ABL1 probe. This reagent was loaded into a MAP consumable and run on the prototype dPCR instrument. A scatter plot of the experimental results show HEX fluorescence on the y-axis and FAM fluorescence on the x-axis (where each data point represents the experimental results from an individual partition). The three tables show averaged results from measurements in triplicate for the individual gBlock combinations: 1%, 0.1% and 0.01% BCR-ABL1.

Figure 7

Personalized dPCR tracking of JMML patient’s complete molecular remission. Biobanked cDNA samples from an individual JMML patient were processed on the prototype dPCR integrated instrument using a FLT3 fusion gene assay designed specifically for this patient. Equal amounts of patient cDNA were used as template for each experimental run. The experimental results indicate that the patient did not respond to the Cytarabine treatment, however the patient showed a dramatic response to Sorafanib which allowed the patient to proceed safely to a hematopoietic stem cell transplant (HSCT). After HSCT all dPCR measurements were true zeros for FLT3 fusion copies/μL showing that the patient had achieved a complete molecular remission.