Droplet Digital PCR Using HER2/EIF2C1 Ratio for Detection of HER2 Amplification in FFPE Breast Cancer Tissues with Low or Equivocal HER2 Expression

Background: HER2 amplification/overexpression is the predictive biomarker for HER2-targeted therapy. The aim of this study is to investigate whether droplet digital PCR (ddPCR) using the HER2/EIF2C1 ratio could be an alternative HER2 detection assay in formalin-fixed, paraffin-embedded (FFPE) BC tissues with low or equivocal HER2 expression. Methods and Patients: We determined HER2 status by ddPCR in 150 FFPE BC tissues previously classified as IHC1+, IHC2+, and IHC3+; 90 of these were previously determined as FISH-negative and FISH-positive. Optimal cutoff thresholds for the HER2/EIF2C1 ratio, determined by the receiver operating characteristics (ROC) curve, were 2.72 (98% sensitivity, 88% specificity) and 2.64 (89.23% sensitivity, 92% specificity) using IHC and FISH as standard methods, respectively. Results: The concordance rate of HER2 status (n=144) determined by IHC/FISH and ddPCR was 89.58% (kappa=0.791, 89.85% sensitivity, 89.33% specificity). The HER2/EIF2C1 ratio in the IHC3+ group was significantly higher than in IHC1+ and IHC2+ groups (P<0.0001). In IHC3+, the concordance between IHC/FISH and ddPCR was 98% (kappa=1.00). In IHC2+ (n=44), the concordance between FISH and ddPCR was 79.54% (kappa=0.579, 65% sensitivity, 91.7% specificity); the HER2/EIF2C1 ratio in FISH-positive cases was significantly higher than in FISH-negative cases (P<0.001). Interestingly, 12% of IHC1+ cases showed HER2 amplification by ddPCR. Conclusion: Thus, ddPCR using the HER2/EIF2C1 ratio is a robust, sensitive, and accurate assay and represents an alternative method to determine HER2 amplification. This technique should be used to clarify HER2 amplification in breast cancer patients with low or equivocal HER2 expression (IHC1+ or IHC2+ with FISH-negative), which may benefit from novel HER2-directed ADCs. The heterogeneity of HER2-expressing cells contributes to discordant results between ddPCR and FISH.