Analytical validation of the Target Selector ctDNA platform featuring single copy detection sensitivity for clinically actionable EGFR, BRAF, and KRAS mutations

Abstract

BackgroundPersonalized medicine requires accurate molecular profiling for targeted therapy decisions. Insufficient tissue yield or tumor heterogeneity frequently limits the correct tissue biomarker determination. As a noninvasive complement to traditional tissue biopsies, liquid biopsies detect and track cancer driver mutations from biofluids (e.g., blood, urine). Here we present the analytical validation of Target Selector™ ctDNA assays capable of single mutant DNA copy detection.MethodsThe Target Selector ctDNA assay applies a patented Switch-Blocker technology to suppress amplification of background (wild-type) WT alleles, while allowing specific amplification of very low frequency mutant alleles. In contrast to allele specific enrichment technologies like ddPCR, one Switch-Blocker inhibits amplification of a DNA target up to 15 bp in length (e.g., one Switch-Blocker covers all KRAS exon 2, codon 12 and 13 variants). Target enrichment is achieved through a quantitative PCR reaction; subsequent DNA sequencing confirms mutation identity. Analytical validation with cancer cell line DNA was conducted by three independent operators using five instruments across five days.ResultsA total of 3086 samples were tested on EGFR, BRAF and KRAS Target Selector ctDNA assays, with EGFR WT as a reference. All assays showed >99% analytical sensitivity and specificity. Single mutant copy detection is confirmed by experimental data and theoretical estimates. In the presence of 14000 WT DNA copies, limits of detection were: EGFR Del19, 0.01%; EGFR L858R, 0.02%; EGFR T790M, 0.01%; BRAF V600E, 0.01%; KRAS G12C, 0.02%. Inter- and intra-assay analyses showed r2>0.94, suggesting consistent performance among operational variables. Healthy donor samples (100 tests) showed clinical specificity at >99%. Finally, Target Selector clinical experience data of >2200 patient samples is consistent with published tissue mutation prevalence.ConclusionsHighly sensitive Target Selector ctDNA assays with single mutant copy detection and limit of detection at 0.02% or better enable accurate molecular profiling vital for disease management.

Assessment of Circulating Nucleic Acids in Cancer: From Current Status to Future Perspectives and Potential Clinical Applications

“…As these panels are hardly ever customizable, some companies and institutions have decided to design and test their own gene panels. These in-house developed panels, that generally include a smaller number of genes, could enter clinical practice as soon as they are clinically validated, such as Target Selector ctDNA assay [ 29 , 62 , 63 ]. Currently, ultrasensitive deep targeted sequencing is the technology of choice for mutation detection on ctDNA [ 64 ].…”

Nanoparticles: Promising Auxiliary Agents for Diagnosis and Therapy of Thyroid Cancers

“…The most sensitive identification of mutations uses digital PCR, where assays have LODs between 0.0005 and 0.0045% [93]. BRAF V600 and KRAS exon 2 ctDNA assays have a LOD of 0.01% and 0.02%, respectively [94]. Gold NPs were used to increase the sensitivity of mutated BRAF gene detection by immunochemistry [55].…”

Clinical validation of qPCR Target Selector™ assays using highly specific switch-blockers for rare mutation detection

“…The melt profiles shown here are plotted as derivative curves such that peaks become the T m s for the corresponding mutant and wild-type targets. Adapted from our previously published analytical validation study using this same technology 21…”