Development and characterization of nucleosomal DNA-based reference materials for the epidermal growth factor receptor gene liquid biopsy.

Liquid biopsy has emerged as a valuable tool in cancer research and diagnosis; however, its clinical utility relies heavily on the accurate detection of low variant allele frequencies (VAFs). Evaluating such sensitivity demands reference materials (RMs) that biologically resemble cell-free DNA (cfDNA) in addition to confirmed basic attributes. Here, we report the development and characterization of a pilot batch of nucleosomal DNA (nDNA)-based EGFR cfDNA RMs. These RMs harbor clinically relevant mutations (T790M, L858R, G719S, and exon 19 deletion (E746_A750del; COSM6223)) at four nominal VAF levels (0%, 0.2%, 1%, and 5%). Analytical validation using digital PCR (dPCR) confirmed assay specificity with limits of detection determined to be below 0.2% VAF. The pilot batch exhibited acceptable homogeneity across bottles and maintained stability under simulated transport conditions and after long-term storage for three years at -70 °C. Orthogonal validation via amplicon sequencing confirmed the reference values with high concordance to dPCR. These findings provide evidence that nDNA-based RMs can serve as a biologically representative and reliable standard for validating assays at low VAFs, expanding to large-scale RM production and interlaboratory evaluation.