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Development of a nine-variant reference material panel to standardize cell-free DNA detection
Chunyan Niu1, Jiejie Zhang1,2, Yan Fang1,2
1Center for Advanced Measurement Science, National Institute of Metrology, Beijing, 100013, China.
Analytical and Bioanalytical Chemistry
|May 23, 2024
Summary
Researchers developed a novel reference material panel for cell-free DNA (cfDNA) mutation detection. This panel aids in validating liquid biopsy assays for cancer biomarker analysis.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- Cell-free DNA (cfDNA) analysis is a promising cancer biomarker, offering an alternative to tissue biopsies.
- Accurate performance evaluation of cfDNA detection assays is hindered by the absence of standardized reference materials.
Purpose of the Study:
- To develop a comprehensive reference material panel for cfDNA mutation detection.
- To support the validation and quality control of liquid biopsy assays.
Main Methods:
- Generated reference materials with nine hotspot mutations (KRAS/BRAF/EGFR/PIK3CA) at varying variant allele frequencies (VAFs).
- Mimicked cfDNA by enzymatically digesting cell-line DNA into 154-173 bp fragments.
- Precisely quantified VAFs using validated digital PCR assays.
- Confirmed panel reliability with two independent next-generation sequencing (NGS) assays.
Main Results:
- Developed a reference material panel covering nine key cancer-associated mutations.
- Established precise VAFs for each mutation, ranging from 0.33% to 23.9%.
- Demonstrated high accuracy in VAF determination via digital PCR.
- Achieved concordant results when validating the panel with independent NGS assays.
Conclusions:
- The novel cfDNA reference material panel is suitable for validating liquid biopsy assays.
- This panel can enhance the reliability and quality control of cfDNA-based cancer biomarker detection.
- Facilitates accurate performance evaluation of various cfDNA detection methodologies.
