HER2 Amplification by Next-Generation Sequencing in Lung Carcinoma: A Comparison of NGS Amplified and Non-amplified Cases by Immunohistochemistry and In Situ Hybridization

Background: Although the role of HER2 amplification and its evaluation methods are well known in breast carcinoma, methods for detection of HER2 amplification in non-small cell lung carcinoma are unclear. Next-generation sequencing is widely used in searching multiple therapeutic targets, and it is possible to evaluate copy number variation of genes by next-generation sequencing. Aims: To re-evaluate the HER2 status of non-small cell lung carcinoma cases detected as HER2 amplified and non-amplified by next-generation sequencing via the most commonly used HER2 investigation methods in routine pathology practice, namely immunohistochemistry and in situ hybridization. Study Design: Retrospective cross-sectional study. Methods: Among the 256 patients whose mutation profiles were examined by next-generation sequencing, HER2 amplified (13 cases) and non-HER2-amplified (13 cases) were determined as study and control groups, respectively, by next-generation sequencing. HER2 next-generation sequencing amplified tumors were investigated for HER2 expression and amplification using immunohistochemistry and silver in situ hybridization. Results: From a group of 256 non-small cell lung carcinoma, 33 tumors (12.8%) showed HER2 amplification with next-generation sequencing. Although we observed more frequent HER2 positivity by immunohistochemistry in next-generation sequencing-amplified cases, when compared to non-amplified cases (50% and 23% respectively), the difference was not significant (P = .221). Within the HER2 amplified group, inter-method-agreement was very good between next-generation sequencing results amplification and in situ hybridization status. Next-generation sequencing results showed a strong interclass correlation coefficient with HER2/cell (P = .009, r = 0.777) and HER2/CEP17 ratio (P = .001, r = 0.805). The median HER2/CEP17 ratio was higher in the next-generation sequencing amplified group (P = .013); however, three cases were found to be amplified by silver in situ hybridization among the next-generation sequencing non-amplified cases. EGFR and FGFR1 amplification were more frequent in HER2 next-generation sequencing amplified group than next-generation sequencing non-amplified group (P < .001). Conclusion: Until the effects of HER2 amplification on the HER2 protein are well understood and pulmonary carcinoma algorithms are defined, non-small cell lung carcinomas found to be amplified by next generation sequencing should be verified by additional methods.