6 research outputs found
Development of a highly sensitive liquid biopsy platform to detect clinically-relevant cancer mutations at low allele fractions in cell-free DNA
<div><p>Introduction</p><p>Detection and monitoring of circulating tumor DNA (ctDNA) is rapidly becoming a diagnostic, prognostic and predictive tool in cancer patient care. A growing number of gene targets have been identified as diagnostic or actionable, requiring the development of reliable technology that provides analysis of multiple genes in parallel. We have developed the InVision™ liquid biopsy platform which utilizes enhanced TAm-Seq™ (eTAm-Seq™) technology, an amplicon-based next generation sequencing method for the identification of clinically-relevant somatic alterations at low frequency in ctDNA across a panel of 35 cancer-related genes.</p><p>Materials and methods</p><p>We present analytical validation of the eTAm-Seq technology across two laboratories to determine the reproducibility of mutation identification. We assess the quantitative performance of eTAm-Seq technology for analysis of single nucleotide variants in clinically-relevant genes as compared to digital PCR (dPCR), using both established DNA standards and novel full-process control material.</p><p>Results</p><p>The assay detected mutant alleles down to 0.02% AF, with high per-base specificity of 99.9997%. Across two laboratories, analysis of samples with optimal amount of DNA detected 94% mutations at 0.25%-0.33% allele fraction (AF), with 90% of mutations detected for samples with lower amounts of input DNA.</p><p>Conclusions</p><p>These studies demonstrate that eTAm-Seq technology is a robust and reproducible technology for the identification and quantification of somatic mutations in circulating tumor DNA, and support its use in clinical applications for precision medicine.</p></div
Plot showing sensitivity and inter-operator variability of eTAm-Seq technology using low, medium and high input DNA.
<p>Experiments were performed in two laboratories (Laboratory 1 –upper; Laboratory 2 –lower) by different operators, performed on separate days and different NGS runs.</p
Sensitivity of the eTAm-Seq technology with 8000 amplifiable copies of DNA input per sample.
<p>Sensitivity of the eTAm-Seq technology with 8000 amplifiable copies of DNA input per sample.</p
Quantitative agreement of 5% AF and 1% AF reference standard spiked into plasma, and measured by eTAm-Seq technology and dPCR.
<p>Mean mutant AF (%) ± SD are displayed for each technology (n = 5* (5% AF standard); n = 6 (1% AF standard)). By spiking into plasma containing background wild-type DNA, the resulting mix was confirmed to contain lower AFs than the original reference standards (original mutant AF values 5% standard: 5% (<i>EGFR</i>); 6.3% (<i>KRAS</i>, <i>NRAS</i>, <i>PIK3CA</i>); 1% standard: 1% (<i>EGFR</i>), 1.3% (<i>KRAS</i>, <i>NRAS</i>, <i>PIK3CA</i>). (*1 data point omitted due to anomalous extraction efficiency).</p
InVision liquid biopsy tumor profiling panel.
<p>The coverage per gene is indicated, including hotspots, comprehensive or full coverage of coding regions (70%–100% tiling coverage) and CNVs. SNVs = Single Nucleotide Variants; Indels = short insertions or deletions; CNVs = Copy Number Variants.</p
International Interlaboratory Digital PCR Study Demonstrating High Reproducibility for the Measurement of a Rare Sequence Variant
This study tested the claim that
digital PCR (dPCR) can offer highly reproducible quantitative measurements
in disparate laboratories. Twenty-one laboratories measured four blinded
samples containing different quantities of a <i>KRAS</i> fragment encoding G12D, an important genetic marker for guiding
therapy of certain cancers. This marker is challenging to quantify
reproducibly using quantitative PCR (qPCR) or next generation sequencing (NGS) due to the presence
of competing wild type sequences and the need for calibration. Using
dPCR, 18 laboratories were able to quantify the G12D marker within
12% of each other in all samples. Three laboratories appeared to measure
consistently outlying results; however, proper application of a follow-up
analysis recommendation rectified their data. Our findings show that
dPCR has demonstrable reproducibility across a large number of laboratories
without calibration. This could enable the reproducible application
of molecular stratification to guide therapy and, potentially, for
molecular diagnostics