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


Autoři: Jason C. Poole aff001;  Shan-Fu Wu aff001;  Timothy T. Lu aff001;  Cecile Rose T. Vibat aff001;  Anh Pham aff001;  Errin Samuelsz aff001;  Manisha Patel aff001;  Jeffrey Chen aff001;  Tony Daher aff001;  Veena M. Singh aff001;  Lyle J. Arnold aff001
Působiště autorů: Biocept, Inc., San Diego, California, United States of America aff001;  Aegea Biotechnologies, Inc., San Diego, California, United States of America aff002
Vyšlo v časopise: PLoS ONE 14(10)
Kategorie: Research Article
doi: 10.1371/journal.pone.0223112

Souhrn

Background

Personalized 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.

Methods

The 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.

Results

A 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.

Conclusions

Highly 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.

Klíčová slova:

Biomarkers – Biopsy – Blood – Cancer treatment – Mutation – Mutation detection – Polymerase chain reaction – Circulating tumor DNA


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Článek vyšel v časopise

PLOS One


2019 Číslo 10

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