Abstract
Currently, there are no recommended alternative assays for HER2 cases deemed equivocal by immunohistochemistry and fluorescent in situ hybridization. Digital PCR (ddPCR), a highly accurate method to determine DNA copy number, could be a robust alternative for clinical HER2 diagnostics. HER2 and CEP17 copy numbers were quantified using two ddPCR platforms (QX200 and RainDrop) in 102 samples of invasive breast cancers. Compared to routine assays, ddPCR gave a sensitivity and specificity of 82.8% and 97.3% respectively, with a kappa value of 0.833 (p < 0.001). Moreover, the method proved to be robust as the results from two platforms was highly correlated (R2 = 0.91; Concordance rate = 97%; κ = 0.923, P < 0.001). Its performance was further tested on 114 HER2 equivocal cases in an independent validation cohort. 75% (21/28) of cases with HER2 amplification and 95% (82/86) of HER2 non-amplified case were classified as positive and negative by ddPCR respectively (κ = 0.709, P < 0.001). Notably, in the HER2 amplified cases, a lower percentage of HER2 positive cells could be related to the discordant results. Altogether, ddPCR is a robust alternative for clinical HER2 diagnostics. However, intratumoral heterogeneity of HER2 status still pose a challenge for HER2 analysis by ddPCR.
Highlights
Human epidermal growth factor receptor 2 (HER2) has been used as a predictive and prognostic marker in breast cancers
When HER2 status were defined with Droplet Digital PCR (ddPCR) and fluorescence in situ hybridization (FISH), the ddPCR HER2:CEP17 ratio in patients with HER2 amplified tumor was significantly elevated compared to HER2 non-amplified cases (Fig. 1B)
Reliable and timely HER2 testing is of paramount importance in guiding decision regarding HER2 targeted therapy in breast cancer patients
Summary
Human epidermal growth factor receptor 2 (HER2) has been used as a predictive and prognostic marker in breast cancers. One of the underlying reasons is that they are affected by variations in testing conditions (such as fixation, reagent and assay protocol used2) compromising their reproducibility and accuracy They are labor intensive and time consuming, especially for FISH. It is subjected to inter-observer variability, in IHC which is done qualitatively rather than quantitatively[5, 6] Another challenge is, in some cases, the test results can be indeterminate, either due to technical issues or the inherent features of the tumor. Its performance in comparison to the standard techniques has not been well established In these studies, only limited number of samples was included, precluding a more detailed and systematic analysis, on HER2 equivocal cases. We have examined the usefulness of dPCR to determine HER2 amplification in IHC 2+ cases
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