Niu Dongfeng, Li Lei, Yu Yang, Zang Wanchun, Li Zhongwu, Zhou Lixin, Jia Ling, Rao Guanhua, Gao Lianju, Cheng Gang, Ji Ke, Lin Dongmei
Department of Pathology, Key laboratory of Carcinogenesis and Translational Research (Ministry of Education), Peking University Cancer Hospital & Institute, Beijing, China.
Beijing Novogene Bioinformatics Technology Co., Ltd, Beijing, China.
Pathol Oncol Res. 2020 Oct;26(4):2577-2585. doi: 10.1007/s12253-020-00844-w. Epub 2020 Jul 3.
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.
基于扩增子的下一代测序（NGS）方法因周转时间短（TAT）和所需DNA输入量小而被临床实验室优先采用。然而，与免疫组织化学（IHC）和荧光原位杂交（FISH）等当前标准方法相比，评估基于扩增子的NGS方法用于检测拷贝数变异（CNV）的研究较少。基于NGS的CNV检测与后一种标准方法之间的相关性仍未得到探索。我们开发了一种基于扩增子的检测板，用于检测来自280例乳腺癌和50例胃癌患者的福尔马林固定石蜡包埋（FFPE）肿瘤组织样本中的人表皮生长因子受体（HER2）扩增。同时进行了IHC和FISH评估，并使用描述性统计来评估一致性。NGS检测到的拷贝数与平均HER2拷贝数（信号/细胞）（r = 0.844；p < 0.001）或HER2/CEP17比值（r = 0.815；p < 0.001）相关。我们使用151个HER2未扩增的FFPE样本确定了NGS用于分类HER2扩增状态的临界值。在乳腺癌患者中，临界值为2.910，敏感性、特异性和一致性分别为95.35%、98.67%和97.29%。然而，该临界值在胃癌患者中显示出低敏感性（64.71%），并且随后的宏观解剖程序对提高敏感性无效（57.14%）。在我们的研究中，用NGS评估HER2拷贝数在乳腺癌患者中与IHC和FISH相当，但在胃癌中的一致性仅为中等。胃癌中较大的不一致性可能反映了潜在的生物学机制，值得进一步研究。基于NGS的HER2评估可能会减少通过FISH/IHC评估的乳腺癌患者中HER2测定结果不明确的情况。
