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Published on: April 11, 2016
Analytical validation of the Target Selector ctDNA platform featuring single copy detection sensitivity for
Jason C Poole1, Shan-Fu Wu1, Timothy T Lu1
1Biocept, Inc., San Diego, California, United States of America.
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.
Insights
Target Selector ctDNA assays offer highly sensitive detection of cancer mutations, even at single copy levels. This noninvasive approach aids personalized medicine by enabling accurate molecular profiling from liquid biopsies.
Area of Science:
- Molecular Biology
- Oncology
- Genetics
Background:
- Personalized medicine relies on accurate molecular profiling for targeted therapies.
- Tissue biopsies face limitations due to insufficient yield and tumor heterogeneity.
- Liquid biopsies offer a noninvasive alternative for detecting cancer mutations in biofluids.
Purpose of the Study:
- To present the analytical validation of Target Selector™ ctDNA assays.
- To demonstrate the capability of single mutant DNA copy detection.
- To establish the utility of liquid biopsies for cancer mutation analysis.
Main Methods:
- Utilized patented Switch-Blocker technology to suppress wild-type alleles and amplify mutant alleles.
- Employed quantitative PCR for target enrichment and DNA sequencing for mutation confirmation.
- Conducted analytical validation across multiple operators, instruments, and days using cancer cell line DNA.
Main Results:
- Tested 3086 samples across EGFR, BRAF, and KRAS assays, achieving >99% analytical sensitivity and specificity.
- Demonstrated single mutant copy detection with limits of detection as low as 0.01%.
- Showed high consistency (r2>0.94) and clinical specificity (>99%) in healthy donor samples.
Conclusions:
- Target Selector ctDNA assays provide highly sensitive detection of cancer mutations.
- Single mutant copy detection and low limits of detection enable accurate molecular profiling.
- These assays are vital for effective disease management in personalized medicine.
