二代测序技术在肺癌组织相关基因突变检测中的临床应用价值

目的基于二代测序技术分析肺癌相关基因突变及位点分布状况，探讨突变基因与临床病理特征之间的关系及临床应用价值。 方法收集唐都医院呼吸与危重症医学科2017年12月至2018年6月行支气管镜活检和手术切除的肺癌组织样本53例，采用Illumina二代测序平台分别检测22个基因共103个突变热点区域，描述检测结果，统计分析高频突变基因与临床病理特征之间的关系。 结果22个肺癌相关基因中共检测到19个基因突变；103个肺癌相关突变位点中共检测到71个突变，其中肿瘤蛋白P53(TP53)基因的突变率为45.07%(32/71)，表皮生长因子受体(EGFR)基因突变率为9.86%(7/71)，细胞周期蛋白依赖性激酶抑制因子2A(CDKN2A)基因的突变率为8.45%(6/71)，大肠癌K-鼠肉瘤病毒癌基因(KRAS)突变率为5.63%(4/71)，肿瘤组织腺瘤样息肉病基因(APC)的突变率为4.23%(3/71)，磷酸酰肌醇-3-激酶催化亚基(PIK3CA)、ERBB4、神经营养性酪氨酸激酶受体1(NTRK1)、SMO、KIT基因的突变率均为2.82%(2/71)，GNAQ、CTNNB1、MAP2K1、ATM、成纤维细胞生长因子受体(FGFR3)、NOTCH1、上皮钙黏蛋白编码基因(CDH1)、成视网膜细胞瘤1(RB1)、HRAS基因的突变率均为1.41%(1/71)。TP53突变者共有29例，其中3例患者具有双位点突变，共有32个突变位点。EGFR突变者共有6例，其中1例患者具有双位点突变，共有7个突变位点。CDKN2A突变均为单位点突变。TP53的5号外显子Val173Glu错义突变、8号外显子Arg273Leu错义突变、9号外显子Lys320Ter无义突变的突变率均为6.25%(2/32)，其他位点突变率均为3.13%(1/32)。EGFR的19号外显子Glu746Ala750del缺失突变、21号外显子Arg868Leu错义突变的突变率分别为42.86%(3/7)、28.57%(2/7)，其他位点突变率均为14.29%(1/7)。CDKN2A所有突变位点的突变率均为16.67%(1/6)。TP53突变频率在低、中分化肺癌患者中较高，差异具有统计学意义(χ²＝7.58，P＝0.023)。EGFR突变在病理类型(χ²＝7.45，P＝0.024)和分化程度(χ²＝14.51，P＝0.001)方面的差异具有统计学意义；临床各分期间比较，差异无统计学意义(χ²＝6.37，P＝0.081)。 结论Illumina二代测序平台可作为分析肺癌多基因突变的检测手段，TP53、EGFR和CDKN2A突变频率较高，对肺癌的临床诊疗具有一定的指导意义。

Clinicopathological significance of next-generation sequencing technology for detection of gene mutations in lung cancer tissues

ObjectiveBased on the next-generation sequencing technology (NGS), to explore the mutations and site distribution of lung cancer related genes, the relationship between mutant genes and clinicopathological features, and its clinical application value. MethodsA total of 53 lung cancer tissue samples from bronchoscopy biopsy and surgical resection were collected from the department of Respiratory and Critical Care Medicine of Tangdu Hospital from December 2017 to June 2018. The Illumina next-generation sequencing platform was used to detect a total of 103 mutation hotspots in 22 genes, describing the test results and analyzing the relationship between high frequency mutant genes and clinicopathological features. ResultsA total of 19 gene mutations were detected in 22 lung cancer-related genes, 71 mutations were detected in 103 lung cancer-associated mutation sites, of which the mutation rates of TP53, EGFR, CDKN2A, KRAS, APC genes were 45.07% (32/71), 9.86%(7/71), 8.45% (6/71), 5.63% (4/71), 4.23% (3/71), the mutation rates of PIK3CA, ERBB4, NTRK1, SMO and KIT were all 2.82% (2/71), and the mutation rates of GNAQ, CTNNB1, MAP2K1, ATM, FGFR3, NOTCH1, CDH1, RB1, HRAS were all 1.41% (1/71). There were 29 cases of TP53 mutations, 3 of which had double site mutations, so TP53 had 32 mutation sites. There were 6 cases of EGFR mutations, 1 of which had a double site mutation, so EGFR had 7 mutation sites. The CDKN2A mutations were all single point mutations. The mutation rates of Val173Glu missense mutation of exon 5 of TP53, Arg273Leu missense mutation of exon 8 and Lys320Ter nonsense mutation of exon 9 were all 6.25%(2/32), and the mutation rates of other sites were 3.13%(1/32). The mutation rates of the Glu746 and Ala750del deletion mutation of exon 19 and the Arg868Leu missense mutation of exon 21 were 42.86%(3/7) and 28.57%(2/7), respectively. Also, the mutation rates of other sites were 14.29%(1/7). Additionally, the mutation rates of all mutation sites of CDKN2A were 16.67%(1/6). The frequency of TP53 mutation was higher in patients with poorly and moderately differentiated lung cancer, and the difference was statistically significant (χ²=7.58, P=0.023). The differences in EGFR mutations between the pathological type (χ²=7.45, P=0.024) and the degree of differentiation (χ²=14.51, P=0.001) were statistically significant. There was no statistically significant difference in different stage (χ²=6.37, P=0.081). ConclusionsIllumina next-generation sequencing platform can be used as a detection method for multi-gene mutations in lung cancer. The high frequency of TP53, EGFR and CDKN2A mutations has certain guiding significance for the clinical diagnosis and treatment of lung cancer, but further confirmation of large sample data is needed.