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Published on: August 24, 2017
Analytical Validation and Performance Evaluation of Amplicon-Based Next-Generation Sequencing Assays for Detecting
Ekaterina Olkhov-Mitsel1, Danny Chan1, Kenneth J Craddock1,2
1Precision Diagnostics and Therapeutics Program, Division of Anatomic Pathology, Department of Laboratory Medicine and Molecular Diagnostics, Sunnybrook Health Sciences Centre, Toronto, ON M4N 3M5, Canada.
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.
Insights
Amplicon-based next-generation sequencing (NGS) assays effectively detect gene amplifications in solid tumors but show reduced sensitivity in samples with less than 30% tumor cellularity. Standardized quality control is crucial for reliable results.
Area of Science:
- Oncology
- Genomics
- Molecular Diagnostics
Background:
- Targeted next-generation sequencing (NGS) panels are vital for identifying actionable gene amplifications in solid tumors.
- Gene amplification detection is crucial for guiding targeted cancer therapies.
Purpose of the Study:
- To validate the analytical performance of two amplicon-based NGS assays (Oncomine Comprehensive Panel and Oncomine Focus Assay) for gene amplification detection.
- To assess the impact of tumor cellularity on the sensitivity and specificity of NGS-based amplification detection.
Main Methods:
- Validated two amplicon-based NGS assays: Oncomine Comprehensive Panel (OCAv3) and Oncomine Focus Assay (OFA).
- Assessed OCAv3 performance in 756 formalin-fixed paraffin-embedded (FFPE) tumor samples across various tumor types.
- Analyzed 121 uterine carcinomas for ERBB2 amplification using OCAv3, comparing results with HER2 IHC and FISH.
Main Results:
- NGS assays achieved high positive predictive value with standardized quality control.
- Sensitivity for amplification detection significantly decreased in tumors with <30% cellularity.
- Detection rates and amplification calling were significantly lower in low-cellularity (<30%) tumors compared to higher-cellularity groups.
- A specific threshold for ERBB2 amplification in uterine carcinomas demonstrated 79% sensitivity and 100% specificity, potentially reducing the need for FISH analysis.
Conclusions:
- Amplicon-based NGS assays have demonstrated strengths and limitations for detecting gene amplifications in real-world FFPE samples.
- Tumor cellularity is a critical factor influencing the accuracy of NGS-based amplification detection.
- Optimized analytical thresholds can improve the diagnostic utility of NGS assays for specific gene amplifications.
