摘要目的 通过对晚期肺癌初治及耐药患者的血浆循环肿瘤DNA( ctDNA)标本应用高通量二代测序(NGS)技术进行基因检测分析来评估基于ctDNA的NGS技术的临床应用价值.方法共纳入381例2017年3月至2018年5月期间在中国医学科学院肿瘤医院就诊的肺部恶性肿瘤患者的血液标本,采用基于杂交捕获法的NGS技术,检测患者血浆ctDNA中与肺癌相关的10个驱动基因的变异状态.通过对比分析39例血浆-组织/细胞学配对标本,分析基于NGS方法检测ctDNA中驱动基因变异的灵敏度及特异度.另外分析了一代及三代表皮生长因子受体( EGFR)酪氨酸激酶抑制剂(TKI)靶向耐药患者的ctDNA检测基因变异的耐药机制.结果 该研究中39例血液标本有抽血2周内的组织/细胞学配对标本,均来自非小细胞肺癌患者,其中21例为女性,18例男性;年龄最小者29岁,最大者82岁,平均年龄59岁.血浆与组织NGS检测一致率为84. 62%(33/39);39例配对标本中有34例为Ⅲ～Ⅳ期的晚期肺癌患者,晚期病例血浆与组织NGS检测一致率为88. 24%(30/34).未接受过靶向治疗的基线检测患者231例,其中10例为肺鳞癌,未检出驱动基因突变;221例肺腺癌中EGFR基因突变最为常见(32. 58%,72/221),其次为ALK基因变异(7. 69%,17/221)及KRAS基因突变(5. 81%,13/221).接受过一代TKI治疗并已耐药的病例133例,其中39. 10%(52/133)的患者检出T790M突变;另外有3. 01%(4/133)的患者检出耐药原因为旁路激活.在能够检测出原敏感突变的耐药患者中,约53. 06%(52/98)的患者能检测出T790M突变.接受过三代TKI治疗并已耐药的病例17例,其中4例患者检出耐药原因为出现与原T790M突变位点顺式的C797S错义突变.结论虽然基于血浆ctDNA的二代测序检测存在一定的假阴性率,但其与组织基因突变检测的一致性较高. EGFR-TKI耐药肺癌患者中约有近一半可通过血浆ctDNA二代测序检测找到耐药原因,为基于二代测序的液体活检技术在临床的应用提供了直接证据.

abstractsObjective Next-generation sequencing ( NGS) was performed on circulating tumor DNA (ctDNA) samples from tyrosine kinase inhibitor (TKI)-na?ve non-small cell lung cancers (NSCLC) and TKI-relapsed NSCLC to investigate the clinical value. Methods A total of 381 plasma samples from patients who were diagnosed with lung cancer in Cancer Hospital Chinese Academy of Medical Sciences from March 2017 to May 2018 were enrolled in the study. NGS was performed using a custom-designed panel that covers 10 lung cancer-related driven genes. Paired plasma-tissue samples from 39 patients were collected to analyses the sensitivity and specificity of detecting driver gene mutations using ctDNA. NGS was also performed on plasma samples from TKI-relapsed patients to identify TKI resistance mechanisms. Results Thirty-nine plasma samples collected from 39 NSCLC patients ( including 21 female and 18 male) with corresponding tissue biopsies were analyzed for the sensitivity and specificity. The average age was 56 years (range 29 to 82 years). A high concordance of 84. 62%(33/39) was observed between ctDNA and tissue biopsies. Compared with tissue biopsies, NGS sensitivity for ctDNA was 82. 14% and specificity was 90. 91%. Among these 39 patients, 34 were advanced stage patients ( III-IV stage) . The concordance, sensitivity, and specificity for ctDNA among the advanced stage patients were 88. 24%(30/34), 86. 36%(29/34) and 91. 67%(31/34), respectively. Among the 381 plasma samples [including 231 TKI-na?ve patients and 150 epithelial growth factor receptor( EGFR)-TKI relapsed patients], EGFR mutation was the most common driver gene among the 221 TKI-na?ve lung adenocarcinoma patients (32. 58%, 72/221). For 133 patients who progressed after first-generation EGFR-TKI, T790M was found to be the most frequent resistant mechanism ( 39. 10%, 52/133), as well as bypass activation ( 3. 01%, 4/133; such as MET amplification and ERBB2 amplification) . Among those first-generation EGFR-TKI relapsed patients with EGFR sensitive mutations, T790M was detected in 53. 06%( 52/98) . For the 17 patients who progressed after third-generation EGFR-TKI, C797S was found to be the most common resistant mechanism ( 4/17) . Conclusions The concordance, sensitivity and specificity between ctDNA and tissue biopsies are acceptable. ctDNA analysis provides valuable information for lung cancer patients' targeted treatment, especially for patients not fitted for biopsies.