Droplet digital PCR measurement of HER2 copy number alteration in formalin-fixed paraffin-embedded breast carcinoma tissue.

BACKGROUND Human epidermal growth factor receptor 2 (HER2) testing is routinely performed by immunohistochemistry (IHC) and/or fluorescence in situ hybridization (FISH) analyses for all new cases of invasive breast carcinoma. IHC is easier to perform, but analysis can be subjective and variable. FISH offers better diagnostic accuracy and added confidence, particularly when it is used to supplement weak IHC signals, but it is more labor intensive and costly than IHC. We examined the performance of droplet digital PCR (ddPCR) as a more precise and less subjective alternative for quantifying HER2 DNA amplification. METHODS Thirty-nine cases of invasive breast carcinoma containing ≥30% tumor were classified as positive or negative for HER2 by IHC, FISH, or both. DNA templates for these cases were prepared from formalin-fixed paraffin-embedded (FFPE) tissues to determine the HER2 copy number by ddPCR. ddPCR involved emulsifying hydrolysis probe-based PCR reaction mixtures containing the ERBB2 [v-erb-b2 erythroblastic leukemia viral oncogene homolog 2, neuro/glioblastoma derived oncogene homolog (avian); also known as HER2] gene and chromosome 17 centromere assays into nanoliter-sized droplets for thermal cycling and analysis. RESULTS ddPCR distinguished, through differences in the level of HER2 amplification, the 10 HER2-positive samples from the 29 HER2-negative samples with 100% concordance to HER2 status obtained by FISH and IHC analysis. ddPCR results agreed with the FISH results for the 6 cases that were equivocal by IHC analyses, confirming 2 of these samples as positive for HER2 and the other 4 as negative. CONCLUSIONS ddPCR can be used as a molecular-analysis tool to precisely measure copy number alterations in FFPE samples of heterogeneous breast tumor tissue.