摘要
目的建立基于肿瘤特异性蛋白70(SP70)的靶向免疫磁珠胸腔积液循环肿瘤细胞(CTC)富集技术,并通过富集CTC二代测序(NGS)分析,评估其在非小细胞肺癌(NSCLC)精准治疗中应用价值。方法将单克隆抗体NJ001共价偶联至磁珠表面,建立基于SP70靶向磁珠富集技术,并进行了检出限、符合率、干扰实验、回收率及临床性能评估。对南京医科大学第一附属医院2016年3月至2017年8月43例NSCLC患者胸腔积液采用靶向富集阳性细胞后NGS分析,并与未富集处理样本NGS做比较(χ^(2)检验或Fisher精确检验)。结果基于SP70靶向免疫磁珠富集CTC技术,可以特异性富集肿瘤细胞。检出限为104个人肺腺癌细胞(SPC-A1)/L,阴阳性符合率、敏感度和特异度均为100%(3/3)。内源性干扰物质(红细胞、血红蛋白、白细胞、上皮细胞和三酰甘油)与外源性干扰物质(乙二胺四乙酸二钾、头孢西丁、卡铂和紫杉醇)对本方法均无干扰作用,回收率为56.0%(56000/100000)。SP70靶向富集43份样本中共检出30种基因的65个位点突变,本方法的表皮生长因子受体(EGFR)突变检出率为95%(19/20),高于未富集处理样本NGS的65%(13/20,χ^(2)=5.625,P=0.044)。结论本研究建立了新的基于SP70靶向免疫磁珠富集CTC液体活检方法,较未富集处理样本提高了NGS检测敏感度和特异度。
Objective To establish tumor specific protein(SP70)targeted tumor cell enrichment technology and to assess applicational value of next-generation sequencing(NGS)analysis for enriched circulating tumor cell(CTC)in precision medicines of non-small cell lung cancer(NSCLC).Methods The monoclonal antibody NJ001 was covalently coupled to the surface of magnetic beads to build targeted magnetic bead enrichment technology based on SP70.The limit of detection,coincidence rate,interference experiment,recovery test and clinical performance were evaluated.From March 2016 to August 2017,NGS analysis with or without pre-treatment of targeted enrichment for serous fluids of 43 NSCLC in the First Affiliated Hospital with Nanjing Medical University were compared(Kappa or Fisher exact test).Results The CTC enrichment technology based on SP70 targeted immunomagnetic beads can specifically enrich tumor cells.The limit of detection was 104 SPC-A1 cells/L,and the coincidence rate,sensitivity and specificity were 100%(3/3).The endogenous interfering substances such as red blood cells,hemoglobin,white blood cells,epithelial cells and triglycerides had no interfering effects,as well as the exogenous interfering substances such as EDTA-K2,cefoxitin,carboplatin and paclitaxel.The recovery rate was 56.0%(56000/100000).A total of 30 gene mutations including 65 loci were found in 43 NSCLC under SP70 targeted enrichment,with a higher detection rate compared with unenrichment method[95.0%(19/20)vs 65.0%(13/20),χ^(2)=5.625,P=0.044].Conclusion In this study,SP70-targeted enriched CTC liquid biopsy method was established,with higher sensitivity and specificity of NGS detection than unenrichment method.
作者
张丽霞
周禹同
杨淑娴
朱琼
徐建
穆原
顾春荣
巨桓宇
张伟
潘世扬
Zhang Lixia;Zhou Yutong;Yang Shuxian;Zhu Qiong;Xu Jian;Mu Yuan;Gu Chunrong;Ju Huanyu;Zhang Wei;Pan Shiyang(Department of Laboratory Medicine,the First Affiliated Hospital With Nanjing Medical University/Branch of National Clinical Research Center for Laboratory Medicine,Nanjing 210029,China)
出处
《中华检验医学杂志》
北大核心
2025年第6期737-742,共6页
Chinese Journal of Laboratory Medicine
基金
国家自然科学基金(82272401)
江苏省医学重点学科/实验室(ZDXK202239)。
关键词
液体活检
循环肿瘤细胞
二代测序
肿瘤特异性蛋白70
Liquid biopsy
Circulating tumor cell
Next generation sequencing
Tumor specific protein 70
作者简介
通信作者:潘世扬,Email:sypan@njmu.edu.cn。
