Evaluation of Next Generation Sequencing for Detecting HER2 Copy Number in Breast and Gastric Cancers

Dongfeng Niu,Lianju Gao,Lei Li,Ke Ji,Zhongwu Li,Lixin Zhou,Guanhua Rao,Yang Yu,Ling Jia,Wanchun Zang,Gang Cheng,Dongmei Lin

doi:10.1007/s12253-020-00844-w

Abstract

Amplicon-based next generation sequencing (NGS) approaches have been preferentially adopted by the clinical laboratories on the basis of a short turnaround time (TAT) and small DNA input needs. However, little work has been done to assess the amplicon-based NGS methods for copy number variation (CNV) detection in comparison with current standard methods like immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH). The correlation between NGS based CNV detection and the later standard methods has remained unexplored. We developed an amplicon-based panel to detect human epidermal receptor growth factor (HER2) amplification in formalin-fixed paraffin-embedded (FFPE) tumor tissue samples from 280 breast cancer and 50 gastric cancer patients. Assessment by IHC and FISH was conducted in parallel, and descriptive statistics were used to assess the concordance. The copy number detected by NGS was correlated with either the average HER2 copy number (signals/cell) (r = 0.844; p < 0.001) or the HER2/CEP17 ratio (r = 0.815; p < 0.001). We determined a cut-off value for NGS to categorize HER2 amplification status by using 151 HER2 non-amplified FFPE samples. In breast cancer patients, the cut-off value was 2.910, with 95.35%, 98.67% and 97.29% sensitivity, specificity and concordance, respectively. However, this cut-off value displayed low sensitivity in gastric cancer patients (64.71%), and the following macrodissection procedure was not effective for increasing sensitivity (57.14%). Evaluation of HER2 copy number with NGS in our study was comparable with IHC and FISH in breast cancer patients, but concordance in gastric cancer was only moderate. The greater discordance in gastric cancer may reflect the underlying biological mechanisms, and further study is warranted. NGS-based HER2 assessment may decrease the equivocal HER2 determinations in breast cancer patients assessed by FISH/IHC.

Highlights

The reliable identification of clinically actionable genomic alteration method is critical for the precision cancer therapy guidance
We evaluated accuracy and concordance of Next generation sequencing (NGS) detection compared with the gold-standard fluorescence in situ hybridization (FISH)/IHC analysis methodologies
We used a normal pool derived from 14 normal breast cancer patient formalin-fixed paraffin-embedded (FFPE) samples instead of the matched normal sample for a reference of diploid genome comparison

Summary

Introduction

The reliable identification of clinically actionable genomic alteration method is critical for the precision cancer therapy guidance. Generation sequencing (NGS) has the capability to simultaneously assess multiple genes with a limited biopsy material, representing both a cost and tissue-efficient alternative to current single-gene assessment methods [1,2,3]. Prior studies have shown that NGS enables a reliable detection for copy number variations (CNV) from the same assays used to detect sequence alterations, but less information is available on amplicon-based target sequences [4,5,6,7]. Several factors influence CNV detection, including the number of amplicons per gene, average read dept., and tumor purity within the sample [7]. An assay and algorithm shall need to be fully validated before being clinically used

Methods

Results

Conclusion