Analytical Validation and Performance Evaluation of Amplicon-Based Next-Generation Sequencing Assays for Detecting ERBB2 and Other Gene Amplifications in Solid Tumors.

BACKGROUND: Targeted next-generation sequencing (NGS) panels are increasingly being utilized to identify actionable gene amplifications (copy number > 4) among solid tumors. METHODS: This study validated the analytical performance of two amplicon-based NGS assays, the Oncomine Comprehensive Panel (OCAv3) and the Oncomine Focus Assay (OFA), for detecting gene amplification in formalin-fixed paraffin-embedded (FFPE) tumors of varying cellularity. OCAv3 was assessed for amplification detection in 756 FFPE samples comprising various tumor types. RESULTS: We demonstrated that with standardized quality control metrics, including median absolute pairwise difference score, these assays can achieve a near-perfect positive predictive value, although their sensitivity for detecting amplifications significantly decreased in tumors with cellularity below 30%. Stratifying tumor cellularity into 10-30%, 31-60%, and 61-95% groups revealed significantly higher gene amplification detection rates in the 31-60% and 61-95% groups versus the 10-30% group (20.6% and 26.7% vs. 9.2%, p < 0.0001). When considering all detected gene amplifications, the average amplification calling per sample was nearly five-fold lower in the 10-30% group versus the 61-95% group (0.11 vs. 0.52; p < 0.0001). To further investigate the analytic performance of OCAv3 in detecting ERBB2 amplification, we analyzed a cohort of 121 uterine carcinomas with confirmed ERBB2 status by HER2 IHC or FISH, in which a threshold incorporating amplifications and tumor cellularity achieved 79% sensitivity and 100% specificity, potentially eliminating the need for FISH analysis in 34% of equivocal cases. In a separate validation cohort, similar analytical performance was observed, with the threshold demonstrating consistent sensitivity and specificity. CONCLUSIONS: This study highlights the strengths and limitations of amplicon-based NGS assays in detecting amplifications using real-world data.