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

Abstract

Accurate and comparable quantification of somatic mutations is essential for precision oncology, as clinical decision-making increasingly relies on the quantification of molecular biomarkers. Despite major technological advances, inter-laboratory variability and the lack of metrological traceability remain significant barriers to harmonization and confidence in mutation testing results. Reference Measurement Procedures (RMPs) represent a critical framework to address these challenges by anchoring molecular measurements to common quantitative standards. Here, we describe the development and validation of a candidate RMP for the detection and quantification of the clinically relevant NRAS p.Q61R mutation using digital PCR (dPCR). The assay was systematically optimized to maximize specificity and minimize cross-reactivity between wild-type and mutant alleles. Analytical characterization demonstrated excellent linearity across a broad range of variant allele frequencies (vAF), with a limit of detection of 0.1 %. Precision studies performed on commercially available circulating tumour DNA reference materials (RM) showed good repeatability and intermediate precision, while a full measurement uncertainty budget confirmed the robustness of the approach. Comparison with a commercial dPCR assay provided independent support for assay comparability and consistent vAF estimates across the investigated range. Preliminary inter-laboratory assessment supported transferability of the candidate RMP and comparability of the resulting measurements. Overall, this work establishes a metrologically characterized and transferable dPCR-based RMP for NRAS p.Q61R quantification. Its implementation can support the harmonization of molecular measurements, the value assignment of RM, and the alignment of routine and secondary methods, thereby strengthening the reliability of quantitative biomarker assessment in precision oncology.