Abstract
PurposeDroplet digital PCR (ddPCR) is a highly accurate method to determine DNA concentration and detect copy number variations. We developed an approach to assess HER2 gene amplification status using ddPCR with two sequences of TFF3 as reference probes. Experimental design.76 templates of carcinoma DNA were prepared from formalin-fixed paraffin-embedded (FFPE) tissues. Digital PCR assay of the copy number of HER2 and TFF3 DNA was performed on the samples. The results were compared to prior fluorescent in-situ hybridation (FISH) assays performed on the same samples. ResultsThe ddPCR assay had high concordance with the conventionally used immunohistochemistry (IHC) and FISH methods. The ddPCR method returned fewer indeterminate results than IHC. Concordance between a ddPCR plus FISH method and IHC plus FISH can rise to 98.7% (75/76) after validation is carried out. ConclusionIt's potentially possible to improve the sensitivity and specifity of HER2 ddPCR assays using reference sequences not co-localized with HER2 on chromosome 17, and combining results from multiple sequences. Adopting an approach based on ddPCR HER2 assays plus FISH could lead to reduced costs, labour, and time consumption compared to current IHC plus FISH standard, while not losing precision.
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