Abstract
Circulating tumor DNA (ctDNA) analysis is being incorporated into cancer care; notably in profiling patients to guide treatment decisions. Responses to targeted therapies have been observed in patients with actionable mutations detected in plasma DNA at variant allele fractions (VAFs) below 0.5%. Highly sensitive methods are therefore required for optimal clinical use. To enable objective assessment of assay performance, detailed analytical validation is required. We developed the InVisionFirst™ assay, an assay based on enhanced tagged amplicon sequencing (eTAm-Seq™) technology to profile 36 genes commonly mutated in non-small cell lung cancer (NSCLC) and other cancer types for actionable genomic alterations in cell-free DNA. The assay has been developed to detect point mutations, indels, amplifications and gene fusions that commonly occur in NSCLC. For analytical validation, two 10mL blood tubes were collected from NSCLC patients and healthy volunteer donors. In addition, contrived samples were used to represent a wide spectrum of genetic aberrations and VAFs. Samples were analyzed by multiple operators, at different times and using different reagent Lots. Results were compared with digital PCR (dPCR). The InVisionFirst assay demonstrated an excellent limit of detection, with 99.48% sensitivity for SNVs present at VAF range 0.25%-0.33%, 92.46% sensitivity for indels at 0.25% VAF and a high rate of detection at lower frequencies while retaining high specificity (99.9997% per base). The assay also detected ALK and ROS1 gene fusions, and DNA amplifications in ERBB2, FGFR1, MET and EGFR with high sensitivity and specificity. Comparison between the InVisionFirst assay and dPCR in a series of cancer patients showed high concordance. This analytical validation demonstrated that the InVisionFirst assay is highly sensitive, specific and robust, and meets analytical requirements for clinical applications.
Highlights
It has been shown more than 20 years ago that some cancer mutations can be detected noninvasively through analysis of samples including blood plasma, urine, stool and sputum [1,2,3,4,5,6]
To determine the ability of the InVisionFirst assay to call mutations at different allele fractions and its limit of detection (LoD) which we have defined as the point where we would detect a mutation !90% of the time (LoD90), a dilution series was created of sheared Tru-Q7 reference DNA in Tru-Q0
In a recent study of patients treated with osimertinib following detection of an EGFR T790M mutation through Circulating tumor DNA (ctDNA) sequencing, 3 of the 7 best responders had the T790M mutation detected at variant allele fractions (VAFs)
Summary
It has been shown more than 20 years ago that some cancer mutations can be detected noninvasively through analysis of samples including blood plasma, urine, stool and sputum [1,2,3,4,5,6]. The development of methods including digital PCR (dPCR) and its derivatives such as droplet-based digital PCR (ddPCR) subsequently enabled the sensitive and quantitative analysis of ‘hotspot’ mutations or individual mutant alleles [7,8,9]. These more sensitive methods demonstrated the potential of using ctDNA for a range of applications including cancer prognostication, treatment selection, monitoring and even early detection [9,10,11]. They were still limited to assessing just a small number of changes
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