Abstract P1-02-05: Identification of HER-2/neu amplification using amplicon based benchtop next generation sequencing

Abstract

Abstract Introduction Aplicon based next generation sequencing (NGS) assays have the potential to incorporate simultaneous assessment of somatic mutations and copy number variation into a single platform. Although not specifically designed for this purpose, we noted numerous examples of apparent HER-2/neu gene amplification in analyzing results from our mutation profiling 46-gene cancer NGS panel (Life Technologies, South San Francisco, CA). Material and Methods Existing amplicon coverage data from the TorrentSuite 2.0 (Life Technologies) pipeline was accessed and used to generate a HER-2/neu coverage proportion (ratio between all HER-2/neu amplicon reads and all other amplicon reads). This ratio was calculated for 170 unique cases of breast carcinoma. A total of 4 cases were eliminated from analysis due to indeterminate IHC/FISH results. Using a total of 166 cases, we performed ROC curve analysis to determine the sensitivity and specificity of NGS based HER-2/neu amplification compared to that of traditional IHC and FISH testing (MedCalc v8.0). Results The NGS based HER-2/neu coverage proportions in the 166 cases showed a clearly non-normal distribution with an outlier cluster of cases with an elevated NGS HER-2/neu ratio (6 cases with ratios >0.045). The distribution of NGS based HER-2/neu ratios for the entire population ranged from 0.01 to a maximum of 0.34. ROC curve analysis of NGS HER-2/neu proportions compared to IHC/FISH data showed a maximal sensitivity of 75.0% and specificity of 100% at an NGS ratio cut off of ≥0.045. In the 166 cases, 6 showed a HER-2/neu coverage proportion of > 0.045 (range 0.045-0.34). Utilizing the 0.045 NGS ratio cut off value, the population frequency of cases positive for HER2 expression was 3.4%. Of the 160 samples with NGS coverage proportion < 0.045, only two cases were positive by IHC/FISH testing. The correlation between NGS HER-2/neu coverage proportion and IHC/FISH testing was highly significant (Spearman rank correlation r = 0.67, p < 0.0001 CI: 0.58-0.75). Conclusions Even utilizing a limited somatic cancer panel that includes less than 200 amplicons in 46 genes, clinically significant HER-2/neu amplification can be readily identified. This is the first study to describe the functional utility of HER-2/neu amplification detection using a NGS based amplicon assay. This study shows that in addition to mutation analysis, amplification data can be simultaneously obtained, which has striking implications for potential clinical utility and HER-2/neu amplification detection outside of traditional IHC and HER2 testing modalities. Furthermore, utilizing this assay as a primary screening modality could limit the additional expense of multiple single gene testing. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P1-02-05.