高分辨率熔解曲线分析技术检测血清游离DNA的EGFR基因突变

目的利用高分辨率熔解曲线分析技术(high resolution melting,HRM)检测石蜡包埋组织和血清游离DNA的表皮生长因子受体(epidermal growth factor receptor,EGFR)基因突变,分析两者之间的关系,并探讨其临床应用价值。方法利用HRM技术检测EGFR基因突变的方法,检测200例非小细胞肺癌患者石蜡包埋标本和200例相应的血清游离DNA,并将二者结果进行比较分析。结果HRM法检测非小细胞肺癌患者石蜡包埋组织DNA的EGFR基因突变总检出率为43.5%,血清游离DNA的EGFR基因突变总检出率为25.0%,HRM法检测血清游离DNA的EGFR基因突变与检测石蜡包埋组织DNA的EGFR基因突变相比,敏感性为57.5%,特异性为100%。结论 HRM法检测血清游离DNA的EGFR基因突变为无法获取肿瘤组织标本的患者提供了新的检测机会。     

高分辨率熔解曲线技术检测结直肠癌患者KRAS基因突变

目的探讨高分辨率熔解曲线技术(HRM)检测结直肠癌患者KRAS基因突变的可行性及其临床意义。方法采用HRM技术检测60例结直肠癌患者石蜡包埋组织标本KRAS基因2号外显子12和13位密码子突变,并与Sanger测序法检测结果进行对比分析。结果 HRM法检测结直肠癌患者KRAS基因2号外显子12和13位密码子突变检出率为36.67%(22/60),Sanger测序法检测突变检出率为33.33%(20/60)。HRM法检测突变检出率高于Sanger测序法,HRM法检测敏感性为100%,特异性为95.24%。结论 HRM法检测KRAS基因突变,灵敏度高,敏感性和特异性好,具有操作简便、节约时间、成本低的特点,方法可行。     

高分辨率熔解曲线分析技术在检测非小细胞肺癌组织EGFR基因突变中的应用

目的：探索高分辨率熔解曲线（high resolution melting,HRM）分析技术在检测肺癌组织表皮生长因子受体（epidermal grouthfactor receptor,EGFR）基因突变中的应用。方法：利用HRM技术,进行人肺癌组织EGFR基因外显子19和21突变筛查,并做测序验证。结果：外显子19的HRM分型结果显示,52例样品中有3种类型熔解曲线,45例样品为A型,5例为B型,2例为C型。外显子21的分析结果显示有2种熔解曲线,49例样品为N型,3例样品为M型。测序结果显示,外显子19 A型熔解曲线代表外显子19的野生型,B型熔解曲线代表杂合性缺失delE746-A750（c.2235-2249del）,C型熔解曲线代表杂合性缺失delL747-S752（c.2240-2257del）。外显子21 N型熔解曲线代表野生型,M型熔解曲线代表杂合性L858R（c.2573T-G）,HRM分析结果与测序结果完全一致。结论：本研究表明HRM分析技术能准确检测EGFR基因突变,是一种适合肺癌组织中EGFR基因突变筛查的方法。     

高分辨率熔解曲线分析技术检测BRAF基因V600E突变方法的建立及初步临床应用

目的建立高分辨率熔解曲线分析技术(high resolution melting,HRM)检测BRAF基因V600E突变的方法,并探讨其在临床检测中的应用价值。方法用所建方法检测16例甲状腺乳头状癌患者超声引导下细针穿刺抽吸活检标本,并与测序法结果进行比较分析。结果所建HRM检测方法 Ct值与Tm的CV值均较小,重复性好。HRM法BRAF基因V600E突变检测标本的结果与测序法相比较,突变检出率分别为43.75%和40.00%,敏感性为100.00%,特异性为90.00%。结论成功建立的HRM法检测BRAF基因V600E突变敏感性高,特异性强,重复性好,操作简便,节约时间,成本低,适合细针穿刺抽吸活检标本检测BRAF基因V600E突变。     

HRM法检测肺癌EGFR基因突变

目的：HRM法检测中国不同区域肺癌患者的EGFR基因突变,统计突变类型间比率,为临床EGFR-TKI分子靶向治疗提供依据,并验证HRM法的临床适用性。方法：收集2010年手术切除的肺癌石蜡标本253例,HRM法检测EGFR基因突变情况,并用基因测序法进行验证。结果：在253例肺癌标本中,HRM法检测出EGFR基因突变率为42%,与基因测序法检测出的突变率40%无显著性差异,并检测出2例T790M突变,11例多点突变和2例E18新位点突变。将本实验中收集的东部地区和非东部地区肺癌患者的EGFR基因突变率相比较,无显著性差异。经实验验证,HRM法的灵敏度高于基因测序的灵敏度,且HRM法的特异性为100%。结论：HRM技术具有高灵敏度、高特异性和高准确率等特点,且比基因测序法更简单方便,成本更低,适合在临床开展。明确EGFR突变类型十分必要,可以为临床可否运用EGFR-TKI分子靶向治疗提供重要依据。本实验中收集的肺癌标本EGFR基因突变不存在地域差异。     

HRM法检测肺癌EGFR基因突变

目的:HRM法检测中国不同区域肺癌患者的EGFR基因突变,统计突变类型间比率,为临床EGFR-TKI分子靶向治疗提供依据,并验证HRM法的临床适用性。方法:收集2010年手术切除的肺癌石蜡标本253例,HRM法检测EGFR基因突变情况,并用基因测序法进行验证。结果:在253例肺癌标本中,HRM法检测出EGFR基因突变率为42%,与基因测序法检测出的突变率40%无显著性差异,并检测出2例T790M突变,11例多点突变和2例E18新位点突变。将本实验中收集的东部地区和非东部地区肺癌患者的EGFR基因突变率相比较,无显著性差异。经实验验证,HRM法的灵敏度高于基因测序的灵敏度,且HRM法的特异性为100%。结论:HRM技术具有高灵敏度、高特异性和高准确率等特点,且比基因测序法更简单方便,成本更低,适合在临床开展。明确EGFR突变类型十分必要,可以为临床可否运用EGFR-TKI分子靶向治疗提供重要依据。本实验中收集的肺癌标本EGFR基因突变不存在地域差异。     

高分辨率熔解曲线技术检测非小细胞肺癌患者胸水中EGFR基因突变

目的探讨高分辨率熔解曲线(HRM)技术对非小细胞肺癌(NSCLC)患者胸水标本EGFR基因突变进行检测的可行性及临床意义。方法采用高分辨率熔解曲线技术检测30例非小细胞肺癌患者胸水标本EGFR基因18-21外显子基因突变,并与Sanger测序法检测结果进行对比分析。结果HRM法检测胸水中EGFR基因18-21外显子突变总检出率为26.67%(8/30),Sanger测序法检测突变总检出率为23.33%(7/30)。HRM法与Sanger测序法相比,敏感性为100%,特异性为95.83%,阳性预测值为88.89%,阴性预测值为100%。结论运用HRM技术对非小细胞肺癌患者胸水进行EGFR基因突变检测方法可行,适宜推广。     

qPCR-HRM法检测NSCLC组织EGFR突变

目的比较qPCR-HRM法和测序法检测非小细胞肺癌(NSCLC)组织表皮生长因子受体(EGFR)基因突变的差异。方法收集北京协和医院胸外科2010年6月至2011年7月收治的42例非小细胞肺癌患者的石蜡标本,分别用qPCR-HRM法和测序法检测肿瘤组织EGFR基因突变,使用McNemar检验比较其差别。结果 42例标本qPCR-HRM法检测EGFR突变率为33.3%(14/42),测序法为28.6%(13/42),统计学计算未见差异(P=0.5)。结论 qPCR-HRM法检测NSCLC组织EGFR基因突变是一种灵敏、可靠的检测方法,可用于体细胞EGFR突变的检测和测序前突变的筛选。     

维吾尔族肺腺癌患者的EGFR基因突变分析

背景与目的 表皮生长因子受体(epidermal growth factor receptor,EGFR)是一种跨细胞膜糖蛋白,属于受体型酪氨酸激酶家族.以吉非替尼为代表的EGFR酪氨酸激酶抑制剂对于EGFR突变的肺癌患者显示出良好的治疗效果,然而EGFR的突变率在不同民族和不同种族的人群中表现出较大差异.本研究旨在分析维吾尔族中肺腺癌患者肿瘤组织的EGFR基因突变情况,同时比较维吾尔族与汉族肺腺癌患者肿瘤组织EGFR基因突变率的差异性.方法 收集临床肺腺癌患者石蜡包埋组织标本138例,包括68例维吾尔族和70例汉族肺腺癌的样本,采用ARMS(amplification refractory mutation system,ARMS)PCR扩增方法检测EGFR基因外显子18、19、20及21的突变,x2分析对比维吾尔族和汉族肺腺癌EGFR基因突变差异.结果 138例肺腺癌患者中有43例EGFR基因突变,总突变率为31.2％,其中维吾尔族突变11例,突变率为16.2％,汉族突变32例,突变率为45.7％,维吾尔族肺腺癌EGFR突变率与汉族肺腺癌EGFR突变率比较有明显差异(P＜0.001),突变以外显子19-del和L858R为主要突变点.结论 维吾尔族中肺腺癌EGFR基因突变率为16.2％,汉族中EGFR基因突变率为45.7％,维吾尔族肺腺癌EGFR突变率明显低于我国汉族EGFR基因突变.     

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。     

含有少量腺癌成分的肺鳞状细胞癌（SQCC-mGC）基因突变分析

目的:检测SQCC-mGC中的致癌基因突变,以期指导肺鳞癌靶向治疗患者的筛选。方法:用HRM技术检测100例病理确诊的肺鳞癌标本中肺癌驱动基因。结果:与单纯SQCC组相比,SQCC-mGC组带有已知致癌基因突变的比例更高（23.3% vs 4.3%）,差异具有统计学意义(P＜0.001)。结论:EGFR、ALK或KRAS基因突变在SQCC-mGC中出现频率较高,在治疗前对SQCC-mGC患者进行致癌基因突变检测,有助于筛选靶向治疗的潜在受益人群。     

Identification of EGFR mutations in esophageal cancer

Background

It is well known that the prognosis for esophageal cancer is worse than for other digestive cancers in spite of multimodality treatment, and there is an urgent need to improve this situation. The epidermal growth factor receptor (EGFR) inhibitor, gefitinib, was approved in Japan to treat advanced non-small cell lung cancer patients and several papers have since reported that the successfully treated patients had genetic mutations in EGFR.

Purpose

The aim of this study was to investigate the existence of EGFR mutations in esophageal cancer cell lines and primary lesions, and also to explore the possibility of treating esophageal cancer using gefitinib.

Materials and Methods

Nineteen esophageal cancer cell lines were cultured and DNA was extracted using an ultracentrifugation method. Fifty cases of primary cancer and corresponding normal tissue samples were obtained and DNA was extracted using the same protocol. Nested PCR and DNA sequencing targeting exons 18, 19, 20 and 21 of EGFR were performed to investigate the presence of mutations in esophageal cancer cell lines and primary tumors.

Results

Three of the 19 cell lines had the same silent mutation at nucleotide 2607, a G-to-A substitution in exon 20. One of the 50 patients had an EGFR mutation in codon 719, resulting in an amino acid substitution from glycine to aspartic acid.

Conclusion

EGFR mutations in esophageal carcinoma are rare but do exist, and thus gefitinib could be included in esophageal cancer treatment regimens by selecting those patients who possess such mutations.     

Frequent EGFR mutations in brain metastases of lung adenocarcinoma

Lung adenocarcinomas often metastasize to the brain, and the prognosis of patients with brain metastases is still very poor. The epidermal growth factor receptor (EGFR) gene is mutated in a considerable fraction of primary lung adenocarcinomas, in particular those with drastic response to EGFR tyrosine kinase inhibitors. The present study was designed to elucidate the prevalence of EGFR mutations in brain metastases and the timing of their occurrence during cancer progression. EGFR mutations were detected in 12 of 19 metastatic lung adenocarcinomas to the brain (63%). This frequency was higher than those in previous studies for EGFR mutations at various stages of lung adenocarcinoma in East Asia, including Japan (i.e., 20-55%). In 6 cases with EGFR mutations, the corresponding primary lung tumors were also examined for the mutations, and in all of them, the same types of EGFR mutations were detected also in the primary tumors. In 2 of them, second metastatic brain tumors in addition to the first ones were also available for analysis, and the same types of EGFR mutations were detected in both the first and second ones in both cases. These results indicate that EGFR mutations are present frequently in brain metastases and occur preceding brain metastasis. These findings will be highly informative for treatment of metastatic lung adenocarcinoma to the brain.     

Clinical outcomes and secondary epidermal growth factor receptor (EGFR) T790M mutation among first-line gefitinib,erlotinib and afatinib-treated non-small cell lung cancer patients with activating EGFR mutations

Gefitinib, erlotinib and afatinib are approved for first-line treatment of advanced non-small cell lung cancer (NSCLC) bearing an activating epidermal growth factor receptor (EGFR) mutation. However, the clinical outcomes among the three EGFR tyrosine kinase inhibitors (TKIs) are still controversial. We aimed to evaluate clinical outcomes and secondary EGFR T790M mutation among the three EGFR TKIs. From May 2014 to January 2016, a total of 301 patients received treatment with gefitinib, erlotinib or afatinib, for first-line treatment of advanced NSCLC with an activating EGFR mutation, based on their clinicians’ choice. The median overall survival (OS) was 37.0 months. Although the baseline characteristics of patients were unequal, progression-free survival and OS did not differ among the 3 groups. Multivariate analysis found that gefitinib (adjusted odds ratio [aOR] 3.29, 95% confidence interval [CI], 1.15–9.46, p = 0.027), EGFR TKI treatment duration more than 13 months (aOR 3.16, 95% CI, 1.20–8.33, p = 0.020), male (aOR 3.25, 95% CI, 1.10–9.66, p = 0.034), initial liver metastasis (aOR 4.97, 95% CI 1.18–20.96, p = 0.029) and uncommon EGFR mutation (aOR 0.14, 95% CI, 0.02–0.97, compared to EGFR deletion 19, p = 0.047) were independent factors for secondary T790M mutation. In real-world practice, choosing first line EGFR TKI based on the patients’ clinical characteristics yielded good clinical outcomes. First-line gefitinib, longer EGFR TKI treatment duration, male, initial liver metastasis and uncommon EGFR mutations may be independent factors for secondary EGFR T790M mutation.