细胞学标本表皮生长因子受体基因突变检测的规范化流程

A standard protocol for detection of EGFR mutations in cytologic specimens

目的 探讨细胞学标本表皮生长因子受体（EGFR）基因突变检测的规范化流程.方法 收集北京6家医院287例临床怀疑肺癌的细胞学标本,应用设计的规范化检测流程检测细胞学标本中EGFR基因突变状态.选择40例非小细胞肺癌（NSCLC）患者富于肿瘤细胞胸水标本,比较同一标本采用石蜡包埋和细胞学甩片中刮取癌细胞同时检测EGFR突变的一致性,比较直接测序（Seq）法及扩增受阻突变系统（ARMS）方法检测EGFR基因突变状态的差异.对应用吉非替尼治疗的患者分析其基因突变状态与疗效的关系.结果 287例怀疑肺癌的细胞学标本中,发现肿瘤细胞236例（82.2％,236/287）,其中寡肿瘤细胞标本31例（13.1％,31/236）,富于肿瘤细胞学标本205例（86.9％,205/236）.富于肿瘤细胞学标本中,180例（87.8％,180/205）诊断为NSCLC.Seq法检测富于肿瘤细胞NSCLC标本EGFR基因突变率为27.8％ （50/180）,腺癌标本为28.2％ （50/177）.ARMS方法检测富于肿瘤细胞NSCLC标本EGFR基因突变率为45.6％ （82/180）,腺癌标本为46.3％（82/177）.寡肿瘤细胞标本ARMS方法检测EGFR突变率为38.7％（12/31）,与富于肿瘤细胞标本EGFR突变率差异无统计学意义（P=0.12）.40例富于癌细胞胸水标本中,ARMS检测石蜡包埋细胞学标本和细胞学甩片刮取癌细胞标本的EGFR基因突变率均为37.5％ （15/40）,一致率为100％.吉非替尼治疗ARMS（＋）组和ARMS（-）组患者的无进展生存时间（PFS）分别为12个月和2个月（P ＜0.001）,总生存时间（OS）分别为19个月和7个月（P=0.003）.吉非替尼治疗Seq（＋）组和Seq（-）组患者的PFS分别为12个月和7个月（P =0.227）,OS分别为20个月和15个月（P=0.510）.吉非替尼治疗Seq（＋）ARMS（＋）组和Seq（-）ARMS（＋）组患者的PFS分别为12个月和10个月（P =0.354）,OS分别为20个月和17个月（P=0.334）.结论 细胞学标本EGFR基因突变检测的流程具有可靠性和可行性,病理质控和免疫组化染色在细胞学标本EGFR基因突变检测流程中作用重要,细胞学标本EGFR基因突变检测需采用高灵敏度的检测方法.

Objective The aim of this study was to establish a standard protocol for detection of EGFR mutations in cytologic specimens.Methods 287 cytologic samples were collected from the patients who were suspected of having lung cancer at six hospitals in Beijing.A detection protocol for EGFR mutations was designed.Two comparative experiments were carried out for the coincidence in EGFR mutation rates between direct sequencing （Seq） and amplification refractory mutation system （ARMS） methods,and between 40 matched cytologic samples with formaldehyde-fixed paraffin embedded （FFPE） cytologic blocks and cytospin slides.Results Tumor cells were found in 236 out of 287cases （82.2％,236/287）.Among them,there were 31 cases （13.1％,31/236） of low tumor cell content samples and 205 cases （86.9％,205/236） of high tumor cell content samples.180 cases in the high tumor cell content samples （87.8％,180/205）were diagnosed to be consistent with NSCLC.25 out of 194 cases were ruled out or indefinite to be diagnosed as NSCLC by immunohistochemistry.By direct sequencing,the mutation rate of EGFR was 27.8％ （50/180） in NSCLC samples and 28.2％ （50/177） in adenocarcinoma samples （high tumor content samples）.By ARMS,the mutation rate of EGFR was 45.6％ （82/180） in NSCLC samples and 46.3％ （82/177） in adenocarcinoma samples （high tumor content samples）.The EGFR mutation rate in low tumor content samples was 38.7％ （12/31）,there was no significant difference in EGFR mutation rates between the groups of low tumor cell content samples and high tumor cell content samples （P =0.12）.The concordance rate of EGFR mutation rates was 100％ between scraping tumor cells from slides samples and from FFEP blocks in the 40 matched samples.Forty-eight out of 180 definitive NSCLC patients received Gefitinib therapy.The FPS was 12 months in the gefitinib-treated ARMS＋ group and 2 months in the ARMS-group （P ＜0.001）,and the OS was 19 months in the gefitinib-treated ARMS ＋ group and 7 months in the ARMS-group （P =0.003）,but no significant differences were found in the efficacy （PFS and OS） of Gefitinib between Seq ＋ and Seq-groups （P =0.227,P =0.510,respectively）,and Seq＋/ARMS＋ and Seq-/ARMS＋ groups （P =0.354,P =0.334,respectively）.Conclusions The detection protocol for EGFR mutations in cytological specimens introduced in this study is tested to be reliable and feasible.Pathological evaluation and immunohistochemistry are important in the detection procedure of EGFR mutations in cytologic specimens.High sensitivity methods should be selected for detection of EGFR mutations in cytologic samples.