利用二代测序技术检测早期乳腺癌循环肿瘤DNA 75例探索性研究

Exploratory study of circulating tumor DNA detection in early breast cancer：an analysis of 75 next-generation sequencing results

目的 探索利用二代测序技术进行早期乳腺癌循环肿瘤DNA（ctDNA）检测的应用价值.方法 对2015年12月至2016年7月北京大学第一医院乳腺疾病中心收治的早期浸润性乳腺癌患者进行ctDNA检测探索性研究.采集患者血浆标本并提取血浆游离DNA,利用二代测序技术对247个肿瘤相关基因的全外显子或突变热点区域进行测序,分析基因突变结果,并与临床病理信息进行相关性分析.基因测序结果及临床病理相关性采用 χ2检验或Fisher精确概率检验进行验证.结果 共纳入75例早期浸润性乳腺癌患者,均为女性,年龄31~88岁,中位年龄58岁,临床病理资料完整.75例样本中有47例（62.7%）检出突变,共检出18个基因的64种突变.其中10个基因捕获全外显子,分析的3个基因中2个基因的突变位点分布呈现聚集性.基因突变与月经状态、病理类型、T分期、N分期、TNM分期、组织学分级、雌激素受体状态、孕激素受体状态、人类表皮生长因子受体2检测结果、Ki-67及分子分型的相关性分析结果显示,各临床病理特征不同分组间基因突变检出情况差异无统计学意义（P均〉0.05）.结论 二代测序技术检测早期乳腺癌ctDNA对于发现乳腺癌相关基因突变具有应用价值.

Objective To explore the utility of circulating tumor DNA detection in early breast cancer by using next-generation sequencing. Methods This exploratory study of circulating tumor DNA detection is for early invasive breast cancer patients treated in Breast Disease Center, Peking University First Hospital from December 2015 to July 2016. Plasma samples were collected and were used to isolate plasma cell-free DNA. Exons or hotspots of 247 cancer related genes were sequenced by next-generation sequencing. Mutations and their correlation with clinic-pathological factors were analyzed. The correlation between mutations and clinic-pathological factors was evaluated by χ2 test or Fisher′s exact test. Results Seventy-five patients were enrolled in this study. All patients were female and aged from 31 to 88 years with median age of 58 years. All patients′ clinic-pathological records were complete. Sixty-four mutations in 18 genes （ ALK, BCR, ERBB2, ROS1, PDGFRA, EGFR, FGFR2, CYP1B1, CALR, CASP7, BRAF, FGFR1, FGFR3, MET, NRAS, PTEN, KIT, SOD2） were detected in 47 （ 62. 7%） among all 75 patients. Exons were captured in 10 genes, and mutations in 2 of 3 genes analyzed were clustered. Gene mutations were not correlated with menopausal status, histological type, primary tumor （ T） , regional lymph nodes （ N） , TNM stage, histological grade, estrogen receptor status, progesterone receptor status, human epidermal growth factor receptor 2 status, Ki-67 and molecular subtype （ all P＆gt;0. 05） . Conclusion Circulating tumor DNA sequencing by next-generation sequencing was useful for detecting breast cancer-related mutations.