Clinical validation of qPCR Target Selector™ assays using highly specific switch-blockers for rare mutation detection
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Updated: Dec 27, 2025
Wild-type Blocking PCR Combined with Direct Sequencing as a Highly Sensitive Method for Detection of Low-Frequency Somatic Mutations
Published on: March 29, 2017
Lyle Arnold1,2, Vassilios Alexiadis1, Tim Watanaskul1
1Research and Development, Biocept Inc, San Diego, California, USA.
Aims:
The identification of actionable DNA mutations associated with a patient's tumour is critical for devising a targeted, personalised cancer treatment strategy. However, these molecular analyses are typically performed using tissue obtained via biopsy, which involves substantial risk and is often not feasible. In addition, biopsied tissue does not always reflect tumour heterogeneity, and sequential biopsies to track disease progression (eg, emergence of drug resistance mutations) are not well tolerated. To overcome these and other biopsy-associated limitations, we have developed non-invasive 'liquid biopsy' technologies to enable the molecular characterisation of a patient's cancer using peripheral blood samples.
Methods:
The Target Selector ctDNA platform uses a real-time PCR-based approach, coupled with DNA sequencing, to identify cancer-associated genetic mutations within circulating tumour DNA. This is accomplished via a patented blocking approach suppressing wild-type DNA amplification, while allowing specific amplification of mutant alleles.
Results:
To promote the clinical uptake of liquid biopsy technologies, it is first critical to demonstrate concordance between results obtained via liquid and traditional biopsy procedures. Here, we focused on three genes frequently mutated in cancer: EGFR (Del19, L858, and T790), BRAF (V600) and KRAS (G12/G13). For each Target Selector assay, we demonstrated extremely high accuracy, sensitivity and specificity compared with results obtained from tissue biopsies. Overall, we found between 93% and 96% concordance to blinded tissue samples across 127 clinical assays.
Conclusions:
The switch-blocker technology reported here offers a highly effective method for non-invasively determining the molecular signatures of patients with cancer.
Insights
Liquid biopsies offer a non-invasive method for cancer mutation detection, showing high concordance with traditional tissue biopsies. This technology enables personalized cancer treatment by analyzing circulating tumor DNA (ctDNA) from blood samples.
Area of Science:
- Oncology
- Molecular Biology
- Genetics
Background:
- Personalized cancer treatment relies on identifying actionable DNA mutations.
- Traditional tissue biopsies carry risks, may not reflect tumor heterogeneity, and are poorly tolerated for sequential monitoring.
- Liquid biopsies using peripheral blood offer a non-invasive alternative for cancer molecular characterization.
Purpose of the Study:
- To develop and validate non-invasive liquid biopsy technologies for cancer molecular profiling.
- To demonstrate the concordance between liquid biopsy results and traditional tissue biopsies.
- To enable personalized cancer treatment strategies through accessible molecular analysis.
Main Methods:
- The Target Selector circulating tumor DNA (ctDNA) platform was employed.
- A real-time PCR-based approach combined with DNA sequencing was used.
- A patented blocking technology was utilized to suppress wild-type DNA amplification, enabling specific amplification of mutant alleles.
Main Results:
- High accuracy, sensitivity, and specificity were demonstrated for the Target Selector assays compared to tissue biopsies.
- Concordance rates between 93% and 96% were observed across 127 clinical assays when compared to blinded tissue samples.
- The study focused on frequently mutated genes in cancer, including EGFR, BRAF, and KRAS.
Conclusions:
- The developed switch-blocker technology provides a highly effective non-invasive method for determining cancer molecular signatures.
- Liquid biopsies represent a promising advancement for cancer diagnostics and treatment planning.
- This technology overcomes limitations associated with traditional biopsy procedures.
