Droplet digital PCR using HER2/EIF2C1 ratio for detection of HER2 amplification in FFPE breast cancer tissues with low or equivocal HER2 expression

Abstract

HER2 amplification/overexpression is the predictive biomarker for HER2-targeted therapy. However, there are technical limitations to HER2 detection by fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC). Recently, novel HER2-directed antibody-drug conjugates (ADCs) have shown benefits in HER2-negative breast cancer (BC) with low HER2 expression (i.e., IHC1+ or IHC2+ with FISH-negative). We investigated 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. 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. 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. Thus, ddPCR using the HER2/EIF2C1 ratio should be an alternative HER2 detection assay in FFPE BC tissues, even with low or equivocal HER2 expression.