Mutation screening is becoming a standard for evaluating treatment options of patients diagnosed with cancer. Notably, certain hotspot mutations can give valuable insight into efficacy of response to various treatments. For example, mutations such as EGFR p.T790M and KRAS p.G12D, p.G12V and p.G13D indicate potential reduced responsiveness to Tyrosine Kinase Inhibitors (TKIs), whereas PIK3CA mutations such as p.H1047R indicate positive response to PI3K/AKT/mTOR signaling pathway inhibitors. With its unparalleled precision and sensitivity, digital PCR is ideally suited for liquid biopsy applications in which low amounts of relevant mutations exist in the sample.
The Combinati Absolute Q, a novel one-step dPCR technology, was used with the Applied Biosystems™ TaqMan™ Liquid Biopsy dPCR Assays to perform rare target detection for 5 hot-spot cancer mutations in the KRAS, EGFR, and PIK3CA genes. MAFs down to 0.1% were detected among a high background wild-type concentration.
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Workflow and Methods
Titration Series Preparation
Five hot-spot cancer mutations were selected for this study: KRAS p.G12D, KRAS p.G12V, KRAS p.G13D, EGFR p.T790M, and PIK3CA p.H1047R. A series of DNA mixtures were prepared by titrating mutation-bearing plasmids into a high background of wild-type normal gDNA for each of the mutations selected. Each point along the titration series contained 15 nanograms of gDNA, and mutation plasmid targeting final mutation allele frequencies (MAFs) of 10%, 1%%, 0.1%. and 0%.
Figure 1. Absolute Q workflow.
Absolute Q Workflow
Using the Absolute Q’s simple workflow, PCR mix was prepared using the Combinati 5X MasterMix and loaded into the microfluidic array partitioning (MAP) plate. Subsequently, 10µL of Combinati Isolation Buffer was overlaid into each well used and gaskets applied across all units of the plate. Finally, the plate was loaded onto the Absolute Q where partitioning, thermal cycling, and data collection were completed on the instrument in approximately 90 minutes.
The thermal cycling parameters for use of the TaqMan Liquid Biopsy dPCR Assays on the Absolute Q are listed in Table 1. For select performance evaluation studies, the extension temperature and thermal dwell times were modified. Modifications are indicated when applicable.
Table 1. Thermal Cycling Parameters on the Combinati Absolute Q.
Rare Mutation Allele Detection
Detecting circulating tumor DNA for liquid biopsy applications is challenging because the molecules bearing the target of interest are only a small fraction of the total circulating cell free DNA collected in the sample. Liquid biopsy assays must be able to accurately quantify rare, single nucleotide polymorphisms, among high levels of wild-type background DNA with outstanding sensitivity and precision. For this study, five cancer-relevant mutations were selected to demonstrate the high precision and sensitivity on the Absolute Q (Workflow and Methods).
For each assay, the total number of mutation molecules and observed mutation allele fraction (MAF) were calculated for each point of the titration series (10%, 1%, 0.1% and 0%). Figure 2a illustrates 2-dimensional digital PCR data generated on the Absolute Q from one replicate of each titration point of the KRAS p.G12V assay test. As expected, the number of total mutation (FAM channel, denoted in blue) positive partitions decreases with each successive point, reflecting a reduction in KRAS pG12V mutation molecules overall while the wild-type (VIC channel, denoted in red) partitions remain constant. Figure 2b shows the linear relationship observed between the expected and observed MAF across all titration series assays. Using a Pearson correlation test, the total number of observed mutation molecules and calculated MAF for each of the titration series were highly correlated with the expectations for all five assays (R = 1.0, p<0.001).
Figure 2A. Two dimensional scatter-plot representation of individual partition amplitudes in the VIC and FAM channels of the Combinati Absolute Q for the KRAS p.G12V specific assay.
Figure 2B Results of absolute quantifitacion of mutation molecules across a titration mutation bearing plasmid spiked into a background of 15 nanograms of wild-type human genomic DNA. Titration series points were targeted to be 10%, 1% and 0.1% mutation allele frequency (MAF).
5X Digital PCR Mix for Low Concentration Samples
A major challenge for many liquid biopsy applications is detection of ultra-rare variants which exist at an extremely low concentration. Sample input volume alongside workflow dead-volume can inhibit the detection of these rare variants by limiting the total number of accessible molecules possible for input. To address this, Combinati offers a 5X dPCR MasterMix for use with liquid biopsy assays. Use of this formulation enables users to add up to 66% more sample volume to each dPCR reaction than when using a 2X formulation. We compared the performance of the 5X MasterMix against a standard 2X PCR MasterMix – testing each assay’s full titration series. Both formulations demonstrate comparable quantification results across each titration series (Figure 3).
Figure 3. Comparison of 5X Combinati mastermix (purple) and 2Xmastermix (orange) mutation molecule quantification for 5 liquid biopsy assays.
Best-in-class Reagent Partitioning with the MAP16
The Combinati Absolute Q utilizes a novel method of reaction isolation on fixed microfluidic array partitioning (MAP) plates. Without a reliance on emulsion based reagent partitioning, over 90% of the loaded sample is analysed in each unit, improving confidence in rare molecule detection. The MAP16 plate consistently generates approximately 20,000 of uniformly filled micro-reactions with no user interaction. The total number of viable partitions across 5 plates are shown in Figure 4. With the exception of one, each unit had over 20,300 viable partitions with an average of 20,457 (±98) accepted partitions per unit.
Figure 4. Total accepted or viable partitions of each of the 16 units per plate for 5 MAP plates used for the Liquid Biopsy assay testing.
Figure 5. Comparison of ultra-fast and standard dPCR thermal protocols on Absolute Q.
Short Dwell Times Maintain High Specificity
The Absolute Q dPCR platform paired with the MAP plate enables targeted thermal points to be reached much quicker, due to the low reagent volume sequestered in each partition. Using this feature, the dwell times at denaturation and annealing extension were set to 0 seconds, thereby reducing the overall time to complete PCR. To demonstrate heightened precision possible using ultra-fast PCR, we compared our dPCR thermal protocol listed in Table 1 (0 second denature, 0 second annealing and extension) to the current dPCR protocol recommendation of 15 seconds denature, 30 seconds anneal/extend. We performed this test using the PIK3CA p.H1047R titration. Figure 4 shows consistent quantification performance for low MAF targets between the two thermal profiles.
Digital PCR (dPCR) enables rare target detection even among a high amount of background non-target presence. The Combinati Absolute Q dPCR platform consists of a familiar microtiter-plate formatted consumable and a fully integrated instrument. The platform offers a simplified one-step workflow identical to traditional quantitative PCR (qPCR). The Absolute Q was used with the Applied Biosystems TaqMan Liquid Biopsy dPCR Assays to perform rare target detection for 5 hot-spot cancer mutations. A sensitivity of 0.1% MAF was demonstrated for each assay showcasing the Absolute Q’s cross-assay robustness for clinically relevant rare target detection.