Related Experiment Video
Updated: Mar 21, 2026
Employing Digital Droplet PCR to Detect BRAF V600E Mutations in Formalin-fixed Paraffin-embedded Reference Standard Cell Lines
Published on: October 8, 2015
Screening for circulating RAS/RAF mutations by multiplex digital PCR
Rikke Fredslund Andersen1, Anders Jakobsen2
1Department of Clinical Immunology and Biochemistry, Vejle Hospital, Vejle, Denmark.
Abstract:
Recent years have shown a large interest in the application of liquid biopsies in cancer management. Circulating tumor DNA (ctDNA) has been investigated for potential use in treatment selection, monitoring of treatment response, and early detection of recurrence. Advances have been hampered by technical challenges primarily due to the low levels of ctDNA in patients with localized disease and in patients responding to therapy. The approach presented here is a multiplex digital PCR method of screening for 31 mutations in the KRAS, NRAS, BRAF, and PIK3CA genes in the plasma. The upper level of the limit of blank, which defines the specificity of the multiplexes, was 0.006%-0.06%. Mutations found by multiplex analyses were identified and quantified by duplex analyses. The method was tested on samples from cholangiocarcinoma patients with known tumor mutational status. Mutations found in the tumor were also found in plasma samples in all cases with analyses for all other mutations being negative. There was a perfect agreement as to wild type status in tumor and plasma. The method combines a high sensitivity with the ability to analyze for several mutations at a time and could be a step towards routine clinical application of liquid biopsies.
Insights
This study introduces a sensitive multiplex digital PCR method for detecting multiple cancer gene mutations in plasma. This liquid biopsy approach shows promise for routine clinical use in cancer management.
Area of Science:
- Oncology
- Molecular Biology
- Genetics
Background:
- Liquid biopsies are gaining interest for cancer management, including treatment selection and recurrence monitoring.
- Detecting circulating tumor DNA (ctDNA) is challenging due to low levels in early-stage disease or treatment response.
- Technical hurdles limit the widespread clinical application of ctDNA analysis.
Purpose of the Study:
- To develop and validate a sensitive multiplex digital PCR method for screening multiple cancer-associated mutations in plasma.
- To assess the clinical utility of this method in cholangiocarcinoma patients.
Main Methods:
- A multiplex digital PCR assay was developed to screen for 31 mutations in KRAS, NRAS, BRAF, and PIK3CA genes in plasma.
- The specificity was determined by the limit of blank (0.006%-0.06%).
- Mutations detected by multiplex analysis were confirmed and quantified using duplex analyses.
Main Results:
- The method demonstrated high sensitivity and specificity for detecting mutations in plasma.
- In cholangiocarcinoma patients, all tumor-detected mutations were also found in plasma samples.
- Perfect agreement was observed for wild-type status between tumor and plasma, with all other mutations testing negative.
Conclusions:
- The developed multiplex digital PCR method offers a sensitive and simultaneous analysis of multiple gene mutations from plasma.
- This approach represents a significant step towards the routine clinical application of liquid biopsies in cancer management.
- The method's high accuracy in detecting mutations and wild-type status supports its potential for clinical decision-making.
