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. 2020 Oct;26(4):2577-2585.
doi: 10.1007/s12253-020-00844-w. Epub 2020 Jul 3.

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

Dongfeng Niu  1 , Lei Li  2 , Yang Yu  2 , Wanchun Zang  2 , Zhongwu Li  1 , Lixin Zhou  1 , Ling Jia  1 , Guanhua Rao  2 , Lianju Gao  2 , Gang Cheng  2 , Ke Ji  3 , Dongmei Lin  4
Affiliations

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

Dongfeng Niu et al. Pathol Oncol Res. 2020 Oct.

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.

Keywords: Breast cancer; FISH/IHC; Gastric cancer; HER2 amplification; Next generation sequencing.

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Conflict of interest statement

Lei Li, Wanchun Zang, Lianju Gao, Guanhua Rao, Yun Gao, Gang Cheng and Yang Yu were employee of Novogene Bioinformatics Technology Co., Ltd., Beijing, China. No potential conflicts of interest were disclosed by the other authors. SJK has received consulting/advisory fees from Lilly Oncology, Astellas, Boston Biomedical and Foundation Medicine, Inc. SJK has stock/equity in TP Therapeutics and receives institutional research funding from Merck, Leap therapeutics, and Astellas Pharmaceuticals.

Figures

Fig. 1
Fig. 1
Repeatability of copy number variation detection. NGS-based CNV detections were analyzed using standard materials and replicated libraries
Fig. 2
Fig. 2
The correlation analysis of CNV detection by preparing libraries. a Curve regression analysis between copy number detected by NGS and digital PCR. The copy number detected by digital PCR was used as expected copy number. The copy number detected by NGS was used as detected copy number. b The effect of tumor purity on the detection limit for gene amplifications. Four cell lines each bearing single focal gene amplification (HER2, MET, EGFR and FGFR3) were pooled with their matched normal cell lines in several dilution series. Detection limits were evaluated with dilution series
Fig. 3
Fig. 3
Amplicon-based NGS to detect CNV from breast cancer specimens identified by IHC and FISH. Representative microscopical results of HER2 amplification (positive, negative and equivocal HER2 status) were shown by IHC (a, d, g) and dual color FISH (b, e, h). FFPE resection specimens with identified by FISH were further analyzed by NGS (c, f, i). The y-axis shows log2 copy number ratios of each amplicon from each gene
Fig. 4
Fig. 4
Effect of macrodissection on HER2 detection by NGS in gastric cancer. a Diagram showing macrodissection of FFPE tissue area of HE stained section of gastric cancer. b Serial section from the same specimen for HER2 IHC. c HER2 FISH (red signal, HER2; green signal, CEP17). d CNV detection by NGS after macrodissection. e CNV detection by NGS before macrodissection
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