非小细胞肺癌患者呼出气冷凝液和肿瘤组织中p16基因突变的研究

Study on p16 gene mutations in tumor tissues and exhaled breath condensate of patients with non-small cell lung cancer

目的研究呼出气冷凝液（EBC）和肺癌组织中p16基因突变，探讨EBC中检测的可行性和临床意义。方法收集30例非小细胞肺癌（NSCLC）患者的肺癌组织和EBC标本，同期20名健康体检者的EBC标本作为对照。提取NSCLC患者手术切除的肺癌组织中的DNA，对β-actin基因片段扩增阳性的EBC标本和已提取的肺癌组织DNA进行p16基因1、2、3号外显子PCR扩增，并进行DNA基因测序，用DNASTAR软件进行突变比对，结果进行统计学分析。结果①30例NSCLC患者的EBC中β-actin基因片段扩增阳性26例，26例中有9例检出p16基因突变，突变率为34．6％；EBC中检出p16基因突变患者，其肺癌组织中均发现p16基因突变。②30例NSCＬC癌组织中检测到p16基因突变15例，突变率为50．0％；癌旁组织均未检测到p16基因突变。③9例NSCLC患者EBC中p16基因突变的外显子为1号外显子3例，2号外显子5例，3号外显子1例；15例NSCLC患者肿瘤组织中p16基因突变的外湿子为1号外显子4例，2号外显子8例，3号外显子3例。④26例NSCLC患者EBC中G－actin基因扩增阳性，Ⅰ期、Ⅱ期和Ⅲ期患者p16基因突变率分别为25．0％（3／12）、28．6％（2／7）和57．1％（4／7）（P〉0．05）；鳞癌和腺癌患者的p16基因突变率分别为42．90．4（6／14）和25．0％（3／12）（P〉0．05）。⑤同一患者EBC与肺癌组织中p16基因突变的外显子种类、突变方式、突变类型和密码子均相同。结论肺癌患者EBC和癌组织中均能检测到p16基因突变，并有高度的一致性。EBC中p16基因突变检测可作为一种简便、快速的肺癌诊断方法。

Objective To study the clinical significance and the feasibility of detection in exhaled breath condensate （EBC） by study on p16 gene mutation in EBC and lung cancer tissue. Methods The lung cancer tissue and EBC specimens of 30 cases of non-small cell lung cancer （NSCLC） were collected, the EBC specimens of 20 cases of physical health were colleted at the same time. DNA of lung cancer tissue was extracted from resecting of operation in NSCLC patients, the β-actin gene fragment of EBC specimens was amplified positive and DNA of lung cancer tissue was extracted and exon 1,2,3 of p16 genewere amplified by PCR amplification and DNA gene sequencing method, using DNASTAR software for alignment of mutations. The results were statistically analyzed. Results ①In the EBC of 30 cases of NSCLC patients, β-actin gene fragment of 26 cases were amplified positive, plG gene mutations were detected in nine cases, mutation rate was 34.6%. p16 gene mutations of patients were detected in the EBC and lung cancer tissue. ②pl6 gene mutations were detected in 15 of 30 cases of NSCLC tissues, mutation rate was 50.0%. p16 gene mutation was not detected in adjacent cancer tissues. ③In 9 cases ofNSCLC patients, p16 gene mutations were exon 1 of three cases, exon 2 of five cases, exon 3 of one case in EBC. In 15 cases of NSCLC patients, p16 gene mutations were exon 1 of four cases, exon 2 of eight cases, exon 3 of three cases in tumor tissues. ④In 26 cases of NSCI.C patients,β-actin gene was amplifiedpositive in EBC. Mutation rate of p16 gene was 25.0% （3/12） in Ⅰ stage, mutation rate of p16 gene was 28.6M （2/7）in Ⅱ stage, mutation rate of p16 gene was 57.1%（4 /7）in Ⅲ stage （ P 〉0.05）. Mutation rate of p16 gene was 42.9% （6/14） in squamous carcinoma, mutation rate of p16 gene was 25.0%（3/12） in adenocarcinoma （ P 〉0.05）. ⑤In EBC and tissue of lung cancer of the same patients, the exon type,mutation way and mutation type and codon of p16 gene mutations were identical. Conclusions The mutation of p16 gene is detected in EBC and tissue of NSCLC patients. EBC and tissue has a high degree of consistency. Detection of mutations p16 gene in EBC can be used as a simple, rapid method for the diagnosis of lung cancer.