New Candidate Reference Measurement Procedures for MET CNV Detection and Quantification Using Digital PCR.

backgroundCopy number variation (CNV) of the MET gene is a clinically relevant alteration associated with tumorigenesis, disease progression, and therapy resistance in several cancers, particularly non-small cell lung cancer and colorectal cancer. Elevated MET copy number has prognostic value and can predict response to MET inhibitors, underscoring the clinical need for accurate quantification of MET CNV. However, current diagnostic platforms, such as FISH, qPCR, and NGS, suffer from limited reproducibility, lack of sensitivity, and poor metrological traceability. All these factors lead to poor standardization (e.g. in the reporting units and reference intervals), creating a barrier to inter-laboratory comparability and clinical harmonization. Digital PCR (dPCR) has emerged as a powerful alternative, offering absolute quantification, high sensitivity, and robustness. These features make dPCR particularly suitable for the development of Reference Measurement Procedures (RMPs), essential to establish SI (units) traceability and support the production of certified reference materials.

resultsHere, we report the design, optimization, and validation of a duplex droplet dPCR (ddPCR) assay targeting MET and the diploid reference gene RPPH1. Using synthetic constructs, diploid and MET-amplified cell lines, and reference materials, we systematically optimized assay conditions and evaluated analytical performance. The duplex ddPCR showed excellent linearity (R²=0.988), low intra- and inter-run variability (CV≈3.8–3.9%), and reliable quantification across a dynamic range of copy numbers. Measurement uncertainty was rigorously estimated, aligning with metrological standards. Importantly, baseline measurements in diploid cell lines yielded values consistent with the expected two-copy state, confirming accuracy under physiological conditions.

conclusionThis work introduces a rigorously validated candidate RMP for MET CNV quantification. By enabling traceable and reproducible measurements, the method addresses a critical gap in CNV standardization and provides a foundation for certified reference material development. The adoption of this RMP has the potential to harmonize molecular diagnostics across laboratories, improve the comparability of clinical trial results, and strengthen the integration of CNV testing into precision oncology, ultimately enhancing patient outcomes.