改良的内切酶突变体富集法检测肺癌标本中EGFR基因突变

背景与目的表皮生长因子受体(epidermal growth factor receptor,EGFR)基因突变是肺癌靶向药物疗效的可靠预测指标,因此基因突变的检测具有非常重要的临床意义。本研究建立使用常规实验仪器、高灵敏度、简便的检测表皮生长因子受体突变的方法,以利于临床中快速的检测EGFR基因突变。方法采用改良的内切酶法富集法检测251例肺腺癌DNA标本中EGFR基因外显子19缺失突变和21(L858R)点突变,并与直接测序进行比较。利用混合突变/野生型EGFR基因的细胞系测定改良方法的灵敏度。结果在251例腺癌标本DNA中使用测序法检测出EGFR外显子19突变46例、外显子21突变26例。采用改良的突变体富集法检另外测出外显子19突变78例、外显子21突变57例,总突变率53.8%。灵敏度检测显示对于外显子19和21,新方法的检测灵敏度达0.5%。结论本方法具有简便、经济、灵敏度高等特点,便于临床快速筛查非小细胞肺癌病理组织中的EGFR基因突变。     

ADx-ARMS方法检测晚期非小细胞肺癌患者胸水标本癌细胞基因突变的临床意义

目的:探讨应用ADx-ARMS方法检测非小细胞肺癌患者胸水标本癌细胞基因突变应用于指导小分子EGFR酪氨酸激酶抑制剂（EGFR-TKIs）治疗的可行性与临床意义。方法:ADx-ARMS检测24例非小细胞肺癌患者胸水标本EGFR基因第19、20和21外显子突变与KRAS基因第2外显子突变。统计分析胸水标本与前期检测过的非小细胞肺癌组织中的EGFR、KRAS突变率差异。结果:24例胸水标本中,EGFR突变与KRAS突变分别为14例（58.3%）和1例（4.2%）。前期检测过的非小细胞肺癌组织EGFR和KRAS突变率分别为47.6%和4.5%。EGFR和KRAS突变率在胸水标本与前期肺癌组织中差异无统计学意义（P>0.05）。结论:对失去手术机会而难以获得组织标本的晚期非小细胞肺癌患者,可应用ADx-ARMS方法选择胸水标本筛查EGFR、KRAS基因突变,从而指导EGFR-TKIs的临床应用。     

非小细胞肺癌中EGFR和K-ras基因突变检测

目的:探讨不同类型非小细胞肺癌的EGFR和K-ras基因突变情况及其与肺癌相关临床病理特征的关系。方法:用厦门艾德ADxARMS试剂盒进行98例非小细胞肺癌患者肿瘤组织中EGFR（18,19,20,21外显子）基因和K-ras（12,13,61密码子）基因突变的检测。所有患者均未接受过吉非替尼的治疗。结果:98例样本中31例发生了EGFR基因突变,突变率为31.6%（31/98）,其中15例为19外显子缺失,13例为21 L858R外显子点突变,3例为20外显子突变,1例为18外显子突变。其中1例既有19外显子缺失突变,又有20外显子突变。腺癌中EGFR基因突变率较鳞癌、腺鳞癌、大细胞癌高。女性患者EGFR基因突变率较男性高。不吸烟患者EGFR基因突变率较吸烟患者高。低分化腺癌患者EGFR基因突变率较中、高分化患者高。21例发生了K-ras基因突变（21.4%）,其中12、13、61密码子均发现突变。突变率腺癌较鳞癌、腺鳞癌、大细胞癌高,与是否吸烟、患者性别、分化程度均无相关性。结论:非小细胞肺癌患者EGFR基因突变检出率较高,K-ras基因突变率较低,且两者不存在同时突变,EGFR基因突变与肺癌组织学类型、分化程度、性别等相关。K-ras基因突变与组织学类型相关。     

湖南地区肺腺癌EGFR基因突变及突变位点的临床病理特点

目的:研究湖南地区肺腺癌EGFR基因突变及突变位点的临床病理特点。方法:收集中南大学湘雅医院胸外科肺腺癌患者组织标本134例,采用扩增阻滞突变系统(ARMS)检测EGFR外显子18、19、20、21号突变状态。结果:134例肺腺癌患者中EGFR突变68例,突变率50.7%。其中男、女突变率分别为40.7%、58.7%（P=0.039）,吸烟、不吸烟患者突变率分别为30.6%、62.4%（P=0.000）。外显子18、19、20、21号突变分别为2例（2.9%）、32例（47.1%）、3例（4.4%）、31例（45.6%）。19、21号外显子突变在肺癌TNM分期中I-II期与III-IV期比较有差异,21号外显子突变在I-II期多见,P=0.048。结论:湖南地区肺腺癌中女性、不吸烟患者EGFR突变率高,EGFR突变以19、21号外显子为主,19与21号外显子突变相比较,21号外显子突变在肺癌TNM分期中I-II期多见。     

非小细胞肺癌表皮生长因子受体双向基因测序研究

目的 探讨中国人非小细胞肺癌表皮生长因子受体（EGFR）第18、19、21外显子基因突变状态。方法 32例病理证实的非小细胞肺癌组织标本,通过模板DNA提取、定量和Touchdown PCR扩增EGFR exon18、19、21序列,进行正、负链基因测序分析,并与10例非小细胞肺癌患者血液标本对照。结果 32例非小细胞肺癌组织发现7例共9种突变,即已报道的5例19外显子缺失和未见报道的21exonT〉G（L833V）及A〉T（H835L）杂合性突变,另有2例内含子多态改变。中国人非小细胞肺癌突变率为28．1％（9／32）,肺腺癌突变率为31．6％（6／19）。结论 中国人非小细胞肺癌的EGFR突变率与已报道的亚洲人女性肺腺癌的突变率相似,但存在中国人自身新的突变位点（L833V和H835L）及内含子改变,该突变率与国内易瑞沙治疗非小细胞肺癌有效率基本吻合。     

HRM方法检测肺腺癌患者癌性胸水上清液EGFR基因突变的临床意义

目的:探讨应用HRM方法检测肺腺癌患者癌性胸水上清液EGFR基因突变状况及EGFR酪氨酸激酶受体抑制剂治疗的可行性。方法:收集43例肺腺癌患者癌性胸水上清液标本,提取DNA,应用HRM方法检测EGFR基因第18、19、20、21外显子突变状况。统计分析HRM方法与基因测序法检测EGFR突变率的差异。结果:43例肺腺癌患者癌性胸水上清液中,HRM法检测EGFR基因突变共17例,总突变率为39.53%,其中第19外显子突变14例,第21外显子突变3例。基因测序结果显示:EGFR突变14例,总突变率为32.56%,均为第19外显子突变。HRM方法检测第21外显子突变阳性的患者其基因测序结果均为阴性。这可能与HRM方法检测的灵敏度优于测序方法有关。两者突变率差异无统计学意义（P>0.05）。结论:对难以获取组织标本的肺腺癌患者而言,应用HRM方法检测其癌性胸水上清液,是了解其EGFR基因突变状况的可靠途径,对临床筛查靶向治疗药物具有一定的参考价值。     

变性高效液相色谱技术检测非小细胞肺癌患者表皮生长因子受体基因突变的研究

 目的　探讨采用变性高效液相色谱（DHPLC）技术检测表皮生长因子受体（EGFR）基因突变的优势。方法　应用DHPLC技术检测49例非小细胞肺癌（NSCLC）患者EGFR基因第19与21外显子突变情况,并应用DNA直接测序法验证DHPLC检测基因突变的准确性。结果　49例NSCLC患者中,应用DHPLC检测出13例EGFR基因突变;其中第19外显子缺失突变10例（76.92 %）;第21外显子替代突变3例（23.08 %）。DNA直接测序法突变检测结果与DHPLC一致,DHPLC检测EGFR基因突变灵敏度为100 %。结论　DHPLC技术可以快速、准确、大规模筛选EGFR基因突变。     

竞争性等位基因特异性荧光探针聚合酶链法检测肺腺癌组织 EGFR 基因突变

目的：探讨竞争性等位基因特异性荧光探针聚合酶链（castPCR）法检测初治肺腺癌组织表皮生长因子受体（EGFR）基因突变的应用价值,及 EGFR 突变状态与患者临床生物学行为的相关性。方法：收集2010年10月至2014年12月南京鼓楼医院103例初治肺腺癌组织标本及患者的临床病理资料。使用 castPCR 法检测 EGFR 基因突变（外显子19 del 2235-2249和 del 2236-2250、外显子20 T790M、外显子21 L858R）。结果：103例肺腺癌患者肿瘤组织中共有54例检出了存在至少一个位点的 EGFR 基因突变（突变率52.43%）,其中敏感突变（外显子19和/或外显子21）45人（43.69%）,外显子20突变11人（10.68%）。同时发现部分患者存在敏感突变与敏感突变、敏感突变与耐药突变共存的现象,其中外显子19和20共突变2人,外显子19和21共突变4人,外显子20和21共突变1人。结论：通过对103例肺腺癌患者肿瘤组织 EGFR 基因突变状态的检测,初步论证了 castPCR 法在肺癌 EGFR 驱动突变检测方面的临床可行性。     

特异引物双扩增实时PCR法和Sanger DNA测序法检测肺癌组织中表皮生长因子受体基因突变

目的 探讨特异引物双环探针扩增实时PCR技术表皮生长因子受体(EGFR)检测方法(ADx-EGFR实时PCR法)和Sanger DNA测序法在肺癌EGFR基因体细胞突变检测中的临床价值.方法 收集肺癌组织石蜡切片208例,分别采用ADx-EGFR实时PCR法和Sanger DNA测序法检测肺癌组织中EGFR基因外显子18、19、20、21的突变类型,计算其突变率,分析两种检测方法检测EGFR基因突变的一致性.结果 208例肺癌组织中,ADx-EGFR实时PCR法成功检测208例,检出EGFR基因突变40例,突变检出率为19.2％;Sanger DNA测序法成功检测196例,检出突变22例,突变检出率为11.2％.肺癌组织中主要以外显子19缺失和外显子21上的L858R的点突变为主,分别占4.8％ (10/208)和11.6％ (23/208),其余的突变类型少见.结论 对于甲醛固定石蜡包埋组织而言,ADx-EGFR实时PCR法检测EGFR基因成功率和突变检出率均高于Sanger DNA测序法,可成为临床上检测肿瘤EGFR基因突变的方法.     

非小细胞肺癌EGFR基因突变的临床意义研究

  目的   探讨非小细胞肺癌EGFR基因外显子突变与其临床病理特征的关系。   方法   利用ADx-ARMS?EGFR基因突变检测试剂盒,检测214例未接受过Gefitinib治疗的非小细胞肺癌患者组织中EGFR基因外显子18、19、20和21突变。   结果   非小细胞肺癌组织中EGFR基因总突变率为45.8%（98/214）,外显子18、19、20和21的突变率分别为0.93%（2/214）、22.0%（47/214）、2.3%（5/ 214）和20.6%（44/214）。另有2例19和21外显子双重突变。EGFR基因在肺腺癌组织中的总突变率为50.3%（93/185）明显高于肺鳞状细胞癌17.2%（5/29）（P=0.001）。EGFR基因在女性患者中的突变率57.0%（57/100）高于男性36.0%（41/114）（P=0.002）,EGFR基因在NSCLC淋巴结转移患者中的突变率（66.7%）显著高于无淋巴结转移患者（39.5%）（P < 0.05）,但EGFR基因突变率与肺癌患者的年龄、肿瘤分级和临床分期均无显著性差异（P>0.05）。   结论   中国肺癌尤其是肺腺癌患者存在EGFR基因的较高突变率,EGFR外显子19、21突变结合肺癌的临床病理特征有望成为评估TKI治疗非小细胞肺癌疗效的分子标志。      

Evaluation of denaturing high-performance liquid chromatography as a rapid detection method for identification of epidermal growth factor receptor mutations in nonsmall-cell lung cancer

BACKGROUND: Somatic mutations of the epidermal growth factor receptor (EGFR) gene in nonsmall-cell lung cancer (NSCLC) may predict responsiveness to tyrosine kinase inhibitors. These mutations are commonly identified using DNA sequencing methods. Although considered the gold standard, this approach is time-consuming. In addition, this approach requires large diagnostic specimens and a high ratio of tumor-to-normal-tissue DNA for optimal results. The use of denaturing high-performance liquid chromatography (dHPLC) as a method to screen for the 2 predominant EGFR mutations is reported. METHODS: Clinical specimens from 104 NSCLC patients were analyzed for EGFR mutations in exons 19 and 21. After DNA extraction and polymerase chain reaction (PCR), both direct sequencing and dHPLC were performed and the results were compared. RESULTS: Sequencing revealed a total of 7 mutations: 3 deletion mutations in exon 19 and 4 missense mutations in exon 21. dHPLC showed the presence of genomic alterations in 23 samples, including the 7 identified by sequencing plus 16 additional samples (10 in exon 19 and 1 in exon 21). dHPLC fractions were isolated, reamplified, and sequenced to confirm the results. In serial dilution studies, dHPLC was able to detect mutations in samples containing as little as 1.6% to 6.25% mutated DNA, whereas direct sequencing required at least 30%. CONCLUSIONS: dHPLC is an efficient and more sensitive method for screening for genomic alterations in exons 19 and 21 of the EGFR gene compared with direct sequence analysis. These data suggest that dHPLC should be implemented as a screening tool for detection of EGFR mutations.     

Detection and comparison of peptide nucleic acid-mediated real-time polymerase chain reaction clamping and direct gene sequencing for epidermal growth factor receptor mutations in patients with non-small cell lung cancer

EGFR tyrosine kinase inhibitors (EGFR-TKIs) are recommended as first-line therapy in patients with advanced, recurrent, or metastatic non-squamous non-small cell lung cancer (NSCLC) that have active EGFR mutations. The importance of rapid and sensitive methods for the detection of EGFR mutations is emphasized. The aim of this study is to examine the EGFR mutational status by both direct DNA sequencing and peptide nucleic acid (PNA)-mediated real-time PCR clamping and to evaluate the correlation between the EGFR mutational status and the clinical response to EGFR-tyrosine kinase inhibitors. Clinical specimens from 240 NSCLC patients were analyzed for EGFR mutations in exons 18, 19, 20 and 21. All clinical data and tumor specimens were obtained from 8 centers of the Korean Molecular Lung Cancer Group (KMLCG). After genomic DNA was extracted from paraffin-embedded tissue specimens, we performed PNA-mediated real-time PCR clamping and direct DNA sequencing for the detection of EGFR mutations. Of 240 tumor samples, PNA-mediated PCR clamping was used to detect genomic alterations in 83 (34.6%) samples, including 61 identified by sequencing and 22 additional samples (10 in exon 19, 9 in exon 21, and 3 in both exons); direct DNA sequencing was used to identify a total of 63 (26.3%) mutations that contained 40 deletion mutations in exon 19 (63.5%) and 18 substitution mutations (28.6%) in exon 21. PNA-mediated PCR clamping was used to identify more mutations than clinical direct sequencing, whereas clinical outcomes were not significantly different between the groups harboring activating mutations detected by each method. These data suggest that PNA-mediated real-time PCR clamping exhibits high sensitivity and is a simple procedure relative to direct DNA sequencing that is a useful screening tool for the detection of EGFR mutations in clinical settings.     

EGFR第一内含子（CA）n双核苷酸重复多态性和基因突变与肺癌分子靶向治疗的反应

目的：对肺癌组织EGFR基因突变和第一内含子（CA）n双核苷酸重复多态性与分子靶向药物治疗反应相关性进行初步研究。方法：用基因测序的方法检测了观察组共116例肺癌体细胞标本;对照组20例肺癌患者血液标本。对全部标本的EGFR基因18、19、21外显子,以及其中48例第一内含子（CA）n重复序列进行基因测序分析并随访了45例肺癌患者分子靶向药物（易瑞沙）治疗情况。结果：观察组116例标本中共发现20例（17．25％）EGFR基因突变,其中17例为外显子突变,3例为内含子杂合突变。对照组20例肺癌患者血液标本均未发现突变。肺腺癌的突变率为21．62％,比鳞癌（8．33％）、其它类型癌（11．11％）略高,但无统计学差异。EGFR基因突变在女性患者中（7／36,19．45％）所占比例比男性患者（13／80,16．25％）略高,但无统计学差异。观察组中,随访外显子突变患者眼用易瑞沙的有效率（包5％）显著高于未突变者（0％）（P〈0．01）,疾病控制率（100％）高于未突变组（44．4％）（P〈0．05）.外显子突变患者服用易瑞沙治疗的疾病控制率（100％）高于未服用者（40％）（P〈0．05）。内含子杂合的3例中有2例服用易瑞沙,有效率为0％,疾病控制率50％。观察组与对照组有23例（CA）n短重复（CA≤16）（47．9％）,25例（CA）n长重复（CA〉16）（52．1％）。EGFR第一内含子（CA）n重复片段长短与基因突变无明显相关,OR（OddsRatio）〈2。观察组中,16例突变患者与18例未突变患者（CA）n重复片段的长短与分子靶向药物治疗有效率、疾病控制率均无统计学差异（P〉0．05）。结论：EGFR基因突变组患者分子靶向药物治疗有效率（62．5％vs0）、疾病控制率（100％VS44．4％）要明显优于无突变组。在肺癌中,EGFR基因体细胞突变是决定表皮生长因子受体酪氨酸激酶抑制剂敏感性的最重要的因素。（CA）n重复片段长短与EGFR基因突变无明显相关。分析突变因素关联的（CA）n短重复患者与（CA）n长重复患者的分子靶向药物治疗有效率、疾病控制率无统计学差异。由于部分未突变肺癌患者使用易瑞沙出现一定的疾病控制率,有关分子靶向药物疗效与（CA）n重复的关系有待进一步单因素研究。     

Relationship between Epidermal Growth Factor Receptor Gene Mutations and Clinicopathological Features in Patients with Non-Small Cell Lung Cancer in Western Turkey

Background: To investigate epidermal growth factor receptor (EGFR) gene mutations in patients with nonsmallcell lung cancer (NSCLC) and to analyze any relationship with clinicopathological features and prognosis.Materials and Methods: EGFR gene exons 18-21 in 48 specimens of paraffin-embedded tumor tissue fromNSCLC patients were amplified by PCR, followed by direct sequencing and analysis of links to clinicopathologicalfeatures and prognosis. Results: EGFR mutations were detected in 18 of 48 (42.6%) patients with NSCLC. Therewere 9 cases of mutations in exon 20, 7 in exon 19 and 2 in exon 21. Mutations were more frequently observedin women (5/7 pts, 71.4%) than in men (13/41 pts, 31.7%) (p=0.086) and in non-smokers (5/5 pts, 100%) thansmokers (13/43 pts, 30.2%). There was negative correlation of EGFR mutations with smoking status (p=0.005).EGFR mutations were more frequently observed with adenocarcinoma histology (13/32 pts, 40.6%) than in othertypes (5/16 pts, 31.3%) (p=0.527). The patients with EGFR mutations had better survival than those with wildtypeEGFR (p=0.08). There was no association of EGFR mutations with metastatic spread. Conclusions: EGFRmutations in NSCLC were here demonstrated more frequently in females, non-smokers and adenocarcinomahistology in the western region of Turkey. Patients with EGFR mutations have a better prognosis.     

非小细胞肺癌胸腔积液与配对肿瘤组织中EGFR基因突变检测的比较

目的:探讨非小细胞肺癌胸腔积液与配对肿瘤组织标本中EGFR基因突变检测结果的一致性,评价胸腔积液标本检测EGFR基因突变的应用价值.方法:收集非小细胞肺癌患者胸腔积液与配对肿瘤组织样本72例,采用ARMS方法,检测样本中EGFR基因第18~21外显子突变情况.结果:细胞学样本和组织学样本中EGFR基因突变阳性率分别为48.61%和51.39%,两者差异无统计学意义(P>0.05),二者一致率为92.11%,不一致率为7.89%.结论:二者的一致率较高,恶性胸水可以作为无法获得肿瘤组织的晚期非小细胞肺癌EGFR基因检测的有效样本.     

非小细胞肺癌中表皮生长因子受体基因突变ADx-ARMS分析及其与临床病理特征的相关性

目的:回顾性分析非小细胞肺癌(NSCLC)中采用ADx-ARMS法检测的表皮生长因子受体(EGFR)的基因突变率、突变分布特征及其与临床病理特征的相关性。方法:收集NSCLC标本共139例,其中包括手术切除样本 83 例、穿刺活检样本27 例、胸腔积液样本 18例及血液样本11例。所有样本均采用ADx-ARMS法检测EGFR基因酪氨酸激酶编码区第19至21号外显子的突变。 结果:在139例NSCLC标本中共检测出EGFR基因突变53例,突变率为38.1%;第19至21号外显子的突变率分别为43.4%(23/53)、0(0/53) 和56.6%(30/53);年龄大于等于中位年龄(60岁)与小于中位年龄的患者EGFR基因突变率分别为35.6%(26/73) 和40.9%(27/66),差异无统计学意义(P>0.05);女性患者中EGFR基因突变率(49.0%,25/51) 高于男性患者基因突变率(31.8%,28/88,P<0.05);腺癌中的基因突变率(41.8%,46/110)显著高于鳞癌(15.0%,3/20,P<0.01),而与未能分型的患者(37.5%,3/8)差异无统计学意义(P>0.05)。在手术切除、穿刺活检、胸腔积液以及血液样本中EGFR基因突变检出率分别为42.2%(35/83)、37.0%(10/27)、38.9%(7/18)和9.1%(1/11)。 结论:ADx-ARMS法是检测NSCLC中EGFR基因突变的快速有效方法,但不能检测未知突变类型。EGFR基因的总突变率与年龄无显著相关性;EGFR基因突变在女性和腺癌中多见;EGFR基因突变检出率与样本类型密切相关,对无法取得切除样本的患者,穿刺活检及胸腔积液样本是检测EGFR基因突变的有效样本。     

中国肺腺癌患者上皮生长因子受体基因突变的研究

目的:分析我国肺腺癌患者上皮生长因子受体(EGFR)基因突变的发生率和突变类型。方法:在上海、杭州和昆明等地收集61例肺腺癌及其正常肺组织,采用PCR扩增和基因测序方法对组织DNA中EGFR外显子19~21基因突变进行分析。结果:正常肺组织中EGFR基因均为野生型,肺腺癌组织中EGFR基因突变检测率为47.5%(29/61),其中外显子19和21突变分别占突变总数的55.2%(16/29)和44.8%(13/29),外显子20未检测到突变。外显子19突变发生在第746~752位密码子,均为碱基缺失突变,有6种不同类型。外显子21突变全部是第858位密码子碱基替换突变。EGFR基因突变与患者性别和年龄无显著相关性。但昆明和上海等地患者的基因突变存在明显差异。结论:EGFR基因突变是一种肿瘤特异性的体细胞遗传改变,突变发生率约占肺腺癌总数的一半,其中以外显子19和21突变为主。我国EGFR基因突变存在地域差异。     

TP53 and EGFR Mutational Status in Thymoma: A Genetic Sequencing Study

Background and objective: Thymoma is a rare malignant tumor that usually with an indolent presentation, which was falsely assumed to be benign previously. The tumor suppressor P53 (TP53) and EGFR gene mutate most frequently in human cancers. We tried to investigate the presence of TP53 and EGFR mutations among thymoma patients referred to an Indonesian referral respiratory hospital and to discuss its potential role in thymoma management and prognosis. Material and methods: Surgically resected tumor tissues were collected from thymoma patients and then underwent genomic analysis. PCR was performed on the extracted Paraffinized  DNA to amplify exon 6 of TP53 and exons 18, 19, and 21 of EGFR. The evaluation of mutational status was done using direct sequencing and sequence analysis of purified PCR products. Results: Among 27 collected samples, TP53 exon 6 mutation, namely  missense mutation and nonsense mutation, was observed in two samples (7.4%). EGFR exon 18 mutation, namely E709K and nonsense mutation, was found in 2 samples (7.4%). An intronic mutation in EGFR exon 19 (3.7%) and exon 21 (3.7%) was observed in one sample. Conclusion: TP53 and EGFR mutations were not most frequent, so it seems that these genes are not involved in the pathogenesis of thymoma in Indonesian patients. Nevertheless, we found two samples with a significant mutation in p53 and EGFR genes, suggesting further research on thymoma prognostification and targeted therapy.     

Mutations of the epidermal growth factor receptor gene in lung cancer: biological and clinical implications

Recently it has been reported that mutations in the tyrosine kinase domain of the epidermal growth factor receptor(EGFR) gene occur in a subset of patients with lung cancer showing a dramatic response to EGFR tyrosine kinase inhibitors. To gain further insights in the role of EGFR in lung carcinogenesis, we sequenced exons 18-21 of the tyrosine kinase domain using total RNA extracted from unselected 277 patients with lung cancer who underwent surgical resection and correlated the results with clinical and pathologic features. EGFR mutations were present in 111 patients (40%). Fifty-two were in-frame deletions around codons 746-750 in exon 19, 54 were point mutations including 49 at codon 858 in exon 21 and 4 at codon 719 in exon 18, and 5 were duplications/insertions mainly in exon 20. They were significantly more frequent in female (P < 0.001), adenocarcinomas (P = 0.0013), and in never-smokers (P < 0.001). Multivariate analysis suggested EGFR mutations were independently associated with adenocarcinoma histology (P = 0.0012) and smoking status (P < 0.001), but not with female gender (P = 0.9917). In adenocarcinomas, EGFR mutations were more frequent in well to moderately differentiated tumors (P < 0.001) but were independent of patient age, disease stages, or patient survival. KRAS and TP53 mutations were present in 13 and 41%, respectively. EGFR mutations never occurred in tumors with KRAS mutations, whereas EGFR mutations were independent of TP53 mutations. EGFR mutations define a distinct subset of pulmonary adenocarcinoma without KRAS mutations, which is not caused by tobacco carcinogens.     

176例非小细胞肺癌的EGFR基因突变分析

目的 分析非小细胞肺癌（NSCLC）中上皮生长因子受体（EGFR）基因突变的发生率和突变类型。方法 收集123例正常肺组织和176例肺癌组织,采用PCR扩增和基因测序方法,对组织DNA中EGFR外显子19～21基因突变进行分析。结果 正常肺组织中EGFR基因均为野生型,肺癌组织中EGFR基因突变检测率为32．4％（57／176例）,其中,外显子19和21突变分别占突变总数的64．9％（37／57例）和31．6％（18／57例）,外显子20突变少见,仅占3．5％（2／57例）。外显子19突变发生在第746～753位密码子,均为碱基缺失突变,有7种不同类型。外显子20突变发生在第789—793位密码子,为碱基替换突变。外显子21突变全部是第858位密码子碱基替换突变。EGFR基因突变多见于女性,肺腺癌和腺鳞癌。结论 EGFR基因突变是一种肿瘤特异性的体细胞遗传改变,突变发生率约占肺癌总数的1／3,其中以外显子19和21为主。女性、肺腺癌和腺鳞癌中突变多见。