Analytical Validation and Performance Evaluation of Amplicon-Based Next-Generation Sequencing Assays for Detecting ERBB2 and Other Gene Amplifications in Solid Tumors
by
, ,
Ekaterina Olkhov-Mitsel
1,†, 
Danny Chan
1,†,
Kenneth J. Craddock
1,2,
August Lin
3,4,
Grace Luk
1,
Rashmi S. Goswami
1,2,
Hong Wang
5,
Anna Plotkin
1,2,
Sharon Nofech-Mozes
1,2,
David M. Hwang
1,2 and
Weei-Yuarn Huang
1,2,* 1
Precision Diagnostics and Therapeutics Program, Division of Anatomic Pathology, Department of Laboratory Medicine and Molecular Diagnostics, Sunnybrook Health Sciences Centre, Toronto, ON M4N 3M5, Canada
2
Department of Laboratory Medicine and Pathobiology, Temerty Faculty of Medicine, University of Toronto, Toronto, ON M5S 1A8, Canada
3
Biological Sciences Platform, Sunnybrook Research Institute, Sunnybrook Health Sciences Centre, Toronto, ON M4N 3M5, Canada
4
Department of Pathology and Laboratory Medicine, University of Western Ontario, London, ON N6A 3K7, Canada
5
Genetics Program, North York General Hospital, Toronto, ON M2K 1E1, Canada
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Cancers 2024, 16(23), 3927; https://doi.org/10.3390/cancers16233927
Submission received: 21 October 2024
/
Revised: 13 November 2024
/
Accepted: 21 November 2024
/
Published: 23 November 2024
(This article belongs to the Special Issue Application of Genomic Testing in Precision Oncology)
Simple Summary
One way cancer develops is through gene amplification, where copies of certain genes increase. Traditionally, this has been detected using a technique called fluorescence in situ hybridization (FISH). However, newer methods like amplicon-based next-generation sequencing (NGS) are increasingly being used to detect DNA mutations and can also identify changes in gene copy numbers. However, NGS has not yet been validated for routine clinical use for detecting gene amplification. This study evaluated NGS as a less resource-intensive alternative to traditional methods like FISH. The findings highlight the strengths and limitations of using amplicon-based NGS assays for detecting gene amplification in preserved pathology tissue samples. By modifying the criteria for measuring gene copy numbers and considering tumor characteristics, this study aimed to enhance the accuracy of amplicon-based NGS in detecting gene amplifications with a specific focus on ERBB2 gene amplification. This could potentially decrease the reliance on FISH testing and help improve testing strategies in clinical settings, ultimately guiding treatment decisions.
Abstract
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.
