Allele-Specific Digital PCR Enhances Precision and Sensitivity in the Detection and Quantification of Copy Number...

While genetic analyses are vital in cancer diagnostics, measuring copy number (CN) alterations remains challenging, especially in heterogenous samples such as (liquid) biopsies. Using digital PCR, CN alterations are classically studied by comparing the abundances of the target of interest to a stable genomic reference. Alternatively, CNs may be quantified based on the allelic (im)balance of a heterozygous germline single-nucleotide polymorphism (SNP) present in the target locus.

Classic and SNP-based approaches were evaluated in silico using a novel R library, and in vitro by analyzing 2 types of control samples (with known CN values, mixed at various ratios) and biological specimens (primary uveal melanomas from 5 patients as well as 2 formalin-fixed paraffin-embedded [FFPE] samples) in duplex and multiplex experimental setups. All samples were selected based on heterozygosity for common SNPs on chromosome 3p and 8q. The precision and sensitivity of the two approaches were compared.

Both approaches resulted in CN values close to expected as long as a stable reference was identified (classic approach) and the allelic imbalance was caused by the loss/gain of only one of both variants (SNP-based approach). Under typical experimental conditions, the SNP-based approach had higher precision and sensitivity for CN values < 4.6. For example, a CN value of 2.1 was detected in approximately 40% of the classic approach experiments compared to approximately 75% with the SNP-based approach. Additionally, the SNP-based approach performed better than the classic approach for FFPE specimens.

Using digital PCR, the limits of detecting CN alterations in heterogeneous DNA can be improved by using a SNP-based approach.