Applications for liquid biopsy cancer testing Genotyping patients – screening for first line therapy for non-small-cell lung cancer (NSCLC). The need to genotype cancer patients prior to treatment is fundamentally important for selecting the appropriate therapy and drugs. Several cancers exhibit specific genetic mutations that effect how they respond to treatment, either increasing the cancer’s sensitivity to the drug, or more frequently seen, increasing the cancer’s resistance to the drug. In lung cancer, one of the most frequently occurring mutations is the exon 19 deletion mutation in EGFR, and it is associated with sensitivity to EGFR tyrosine kinase inhibitors, the first-line treatment for non-small-cell lung cancer (NSCLC). Several EGFR mutation detection assays are already EU- and FDA-approved and used in the management of lung cancer patients for initial genotyping, monitoring drug-resistant clones, and evaluating the response to treatment 4 . However, these assays require DNA extraction prior to qPCR analysis, which not only increases the complexity and cost of the assay, but the potential for contamination and carryover inhibitors which can impact the accuracy of the assay. In order to optimize the workflow, manufacturers of EGFR mutation detection assays have developed specific DNA sample preparation kits as a separate test component 5 . Ideally, the need for DNA extraction and purification could be avoided altogether and qPCR detection of EGFR mutations could be carried out directly on the sample. In the following study, Meridian’s Air-Dryable ™ Direct DNA qPCR Blood mix was examined for its ability to detect single copies of the EGFR exon-19 mutation (Fig. 1) . Known copy numbers of synthetic EGFR Exon 19 Deletion and an EGFR Exon 19 wildtype (negative control), was used in the presence of plasma extract with a standard reaction protocol (95°C for 3 minutes, followed by 50 cycles of 95°C for 10 seconds and 60°C for 25 seconds) using wet or lyophilized (see MDX092 User Guide) Air-Dryable ™ Direct DNA qPCR Blood mix. For EGFR mutation analysis, plasma is often the preferred sample type as hemolysis can occur in whole blood samples and cause interference with many types of laboratory tests. However, plasma samples must be stabilized with anticoagulants to prevent clotting and these additives can cause inhibition in a PCR reaction. Meridian’s
Figure 1. Extraction-free qPCR reduces complexity and increases sensitivity
Figure 1: Known copy numbers of synthetic EGFR Exon 19 Deletion and an EGFR Exon 19 wildtype (negative control), was used in the presence of plasma extract with a standard reaction protocol (95°C for 3 minutes, followed by 50 cycles of 95°C for 10 seconds and 60°C for 25 seconds) using wet or lyophilized (see MDX092 User Guide) Air-Dryable ™ Direct DNA qPCR Blood mix. The advantage of drying down the Air-Dryable ™ Direct DNA qPCR Blood is that more sample can be added to the reaction. As illustrated in (A) when the amount of eluted sample is increased from 35% (orange) to 100% (brown), the sensitivity increases by almost 10-fold. This increase in sensitivity enables the detection of single copies of the E19del-EGFR to be detectable (B, light blue) within a 20 µ L reaction (gDNA standards, EGFR Exon 19 wildtype (Negative Control – brown)).
Air-Dryable ™ Direct DNA qPCR Blood (MDX092) mix was tested for its inhibitor- tolerance to the anticoagulant heparin in a qPCR study detecting an Exon19 mutation. Specifically, a ratio of 0.1% of E19del-EGFR to 99.9% wild type DNA was spiked into a sample containing 20% heparin-plasma. Air-Dryable ™ Direct DNA qPCR Blood was tested alongside KAPA Probe Force and TaqMan ™ Universal Master Mix for its ability to amplify the mutation. Across both concentrations of mutant DNA (10 copies and 1,000 copies), Air-Dryable ™ Direct DNA
qPCR Blood was the only mix to produce an amplification curve, demonstrating its ability to perform in the presence of up to 20% heparin. In contrast, the other mixes were unable to generate a result. Using specimen-specific mixes such as Air- Dryable ™ Direct DNA qPCR Blood confers several advantages over universal mixes, most notably, a greater ability to perform in the presence of inhibitors, and an increased sensitivity enabling extraction-free qPCR analysis.
Figure 2. The effect of 20% heparin-plasma on qPCR sensitivity
Figure 2: Detection of epidermal growth factor receptor (EGFR) mutations in cell-free DNA (cfDNA) is an auxiliary tool for the molecular diagnosis of non-small cell lung cancer (NSCLC), especially when an adequate tumor tissue specimen cannot be obtained. Here a deletion in Exon 19 known to be sensitive to current chemotherapy was analysed, by spiking a known amount containing 0.1% of E19del-EGFR from 99.9% wild type DNA into 20% heparin-plasma. We compared the sensitivity of Air-Dryable ™ Direct DNA qPCR Blood (MDX092) (red) to detect this mutant against A) KAPA Probe Force (blue) and B) TaqMan ™ Universal Master Mix (green). The results illustrate the greater tolerance and so better sensitivity of MDX092 to plasma and heparin anti-coagulant, allowing down to 10 copies of the mutation to be detected.
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