Standardisation of cell-free DNA measurements: An International Study on Comparability of Low Concentration DNA Measurements using cancer variants

ABSTRACT For the impact of genomic testing from liquid biopsies to be maximized, mechanisms to ensure reproducible and comparable test performance will be required. This can be established and maintained through reference measurement procedures and materials with property values that are internationally comparable through traceability to a common standard. To achieve this objective, an interlaboratory study was organised to explore digital PCR (dPCR) for standardisation of cell-free DNA (cfDNA) quantification. Blinded samples of wild-type/variant mixtures of two DNA sequences ( BRAF p.V600E single nucleotide variant or EGFR exon 19 deletion) were provided to 12 laboratories. Laboratories independently designed and applied dPCR assays to determine absolute and relative quantities, with no guidance provided to harmonise the approach. The mean and coefficient of variation (CV) of copy number concentrations for variant sequences were 18 copies/μL (CV 7.2%) ( BRAF variant sample) and 9 copies/μL (CV 25%) ( EGFR variant sample) while the mean variant allele frequencies (vAF) were 8.0% (CV 5.3%) and 0.080% (CV 29%) respectively. This study demonstrated that dPCR was capable of exceptional technical accuracy for variant copy number concentration and vAF, even when different assays and platforms were used. This implies that dPCR offers a unique analytical methodology that can be deployed globally in supporting comparability for cfDNA testing based on the existing framework of the International System of units of measurement.