New candidate reference measurement procedure for NRAS p.Q61R quantification by digital PCR

Area of Science:

• Molecular Biology
• Genetics
• Biotechnology

Background:

• Precision oncology relies on accurate somatic mutation quantification for clinical decisions.
• Inter-laboratory variability and lack of metrological traceability hinder reliable mutation testing.
• Reference Measurement Procedures (RMPs) are vital for standardizing molecular measurements.

Purpose of the Study:

• To develop and validate a candidate RMP for NRAS p.Q61R mutation detection and quantification.
• To establish metrological traceability for quantitative biomarker assessment.
• To improve harmonization and confidence in mutation testing results.

Main Methods:

• Development of a digital PCR (dPCR) assay for NRAS p.Q61R.
• Systematic optimization for specificity and allele discrimination.
• Analytical characterization including linearity, limit of detection, and precision studies.
• Measurement uncertainty budget estimation and inter-laboratory assessment.

Main Results:

• The dPCR assay demonstrated excellent linearity across a wide variant allele frequency (vAF) range.
• Limit of detection was 0.1%, with good repeatability and intermediate precision.
• Comparison with a commercial dPCR assay confirmed assay comparability and consistent vAF estimates.
• Preliminary assessment supported the transferability and comparability of the candidate RMP.

Conclusions:

• A metrologically characterized and transferable dPCR-based RMP for NRAS p.Q61R quantification was established.
• This RMP can support harmonization of molecular measurements and value assignment of reference materials.
• Implementation of the RMP enhances the reliability of quantitative biomarker assessment in precision oncology.