目的:本研究的目的是通过RT-qPCR研究晚期肺腺癌中多个驱动基因的突变状态及其意义。
方法:选取2019年4-11月河北医科大学第四医院确诊为肺腺癌的细胞学标本155份。细胞学标本包括浆膜腔积液和细针穿刺活检。在细胞学标本中,108例采用细胞块法(CBM)处理,47例,在DNA/RNA提取前采用一次性膜细胞收集器法(MCM)处理。EGFR的十个驱动基因,ALK,ROS1,BRAF,KRAS,NRAS,HER2RET,PIK3CA,通过扩增难治性突变系统和ABI7500RT-qPCR一步联合检测MET。
结果:使用MCM提取的RNA(p=0.005)和DNA(p=0.001)的纯度均显着高于使用CBM提取的纯度。经过MCM处理的47例新鲜细胞标本均成功进行了多基因检测,而在煤层气处理的108个标本中,6例多基因检测失败。在149个标本中,EGFR单基因突变率,ALK,ROS1,RET,HER2MET,KRAS,NRAS,BRAF,PIK3CA基因突变率为57.71%,6.04%,3.36%,2.68%,2.01%,2.01%,1.34%,0.67%,分别为0%和0%。6例,包括2个共存突变。我们发现突变状态与性别相关(p=0.047),但与年龄(p=0.141)和吸烟状况(p=0.083)无关。我们发现EGFR突变状态与性别相关(p=0.003),年龄(p=0.015)和吸烟习惯(p=0.007),ALK突变状态与年龄相关(p=0.002)。
结论:与CBM相比,MCM可以通过去除杂质和富集肿瘤细胞来提高DNA/RNA提取和PCR扩增的效率。并且我们推测新鲜细胞学标本的成功检出率高于石蜡包埋标本。EGFR,ALK,ROS1突变是晚期肺腺癌患者的主要驱动突变。我们推测EGFR和ALK更容易伴随突变,分别。共存突变患者的靶向治疗需要进一步研究。
OBJECTIVE: The aim of the study was to investigate the mutation status of multiple driver genes by RT-qPCR and their significance in advanced lung adenocarcinoma using cytological specimens.
METHODS: 155 cytological specimens that had been diagnosed with lung adenocarcinoma in the Fourth Hospital of Hebei Medical University were selected from April to November 2019. The cytological specimens included serous cavity effusion and fine-needle aspiration biopsies. Among cytological specimens, 108 cases were processed by using the cell block method (CBM), and 47 cases were processed by the disposable membrane cell collector method (MCM) before DNA/RNA extraction. Ten drive genes of EGFR, ALK, ROS1, BRAF, KRAS, NRAS, HER2, RET, PIK3CA, and MET were combined detected at one step by the amplification refractory mutation system and ABI 7500 RT-qPCR.
RESULTS: The purity of RNA (p = 0.005) and DNA (p = 0.001) extracted by using the MCM was both significantly higher than that extracted by using the CBM. Forty-seven cases of fresh cell specimens processed by the MCM all succeeded in multigene detections, while of 108 specimens processed by the CBM, 6 cases failed in multigene detections. Among 149 specimens, single-gene mutation rates of EGFR, ALK, ROS1, RET, HER2, MET, KRAS, NRAS, BRAF, and PIK3CA mutations were 57.71%, 6.04%, 3.36%, 2.68%, 2.01%, 2.01%, 1.34%, 0.67%, 0% and 0% respectively, and 6 cases including 2 coexistence mutations. We found that mutation status was correlated with gender (p = 0.047), but not correlated with age (p = 0.141) and smoking status (p = 0.083). We found that the EGFR mutation status was correlated with gender (p = 0.003), age (p = 0.015) and smoking habits (p = 0.007), and ALK mutation status was correlated with age (p = 0.002).
CONCLUSIONS: Compared with the CBM, the MCM can improve the efficiency of DNA/RNA extraction and PCR amplification by removing impurities and enriching tumor cells. And we speculate that the successful detection rate of fresh cytological specimens was higher than that of paraffin-embedded specimens. EGFR, ALK, and ROS1 mutations were the main driver mutations in patients with advanced lung adenocarcinoma. We speculate that EGFR and ALK are more prone to concomitant mutations, respectively. Targeted therapies for patients with coexisting mutations need further study.