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。     

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。     

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。     

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。     

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。     

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。     

ARMS技术联合Taqman探针检测100例非小细胞肺癌EGFR基因突变

背景与目的 表皮生长因子受体(epidermal growth factor receptor,EGFR)基因突变是决定表皮生长因子受体酪氨酸激酶抑制剂(EGFR tyrosine kinase inhibitor,EGFR-TKI)疗效最重要的预测因子,对EGFR基因突变进行检测,对指导患者个体化治疗具有重要意义.EGFR基因突变检测方法有很多,每种方法各有优缺点,本研究拟采用扩增阻滞突变系统(amplification refractory mutation system,ARMS)技术与Taqman探针相结合的方法,建立一种能快速、敏感及特异检测非小细胞肺癌EGFR基因突变的方法.方法 首先,应用Primer Premier 5.0软件在EGFR基因E746 A750和L858R处设计ARMS引物及Taqman水解探针.然后,以包含E746_A750缺失和L858R点突变的质粒为研究对象,进一步分析所建立方法的灵敏度、敏感性以及特异性.最后,用所建立的ARMS-Taqman法检测100例非小细胞肺癌(non-small cell lung cancer,NSCLC)临床标本.结果 在无背景DNA干扰的情况下,ARMS-Taqman法检测灵敏度可达10 copies.对于检测敏感性,在500 copies/μL野生型基因背景下,其敏感性达1％;在5,000 copies/μl野生型基因背景下,其敏感性高达0.1％-0.5％.对于检测特异性,以正常人白细胞DNA为研究对象,21 L858R突变存在一定程度的非特异性扩增,但其最小ΔCt高达14.89,而19 Del未见非特异性扩增.对100份临床标本进行检测,19 Del 21例,21 L858R 18例,总突变率为39.0％.结论 我们所构建的ARMS-Taqman法是一种快速、简便以及具有较高灵敏度和特异性的EGFR基因突变检测方法,值得在临床上进一步推广和验证.     

实时荧光定量PCR和测序法检测非小细胞肺癌EGFR基因突变的比较

背景与目的 以厄洛替尼(Erlotinib)为代表的小分子表皮生长因子受体酪氨酸激酶抑制剂在治疗非小细胞肺癌已经取得很好的疗效.许多研究发现表皮生长因子受体(epidermal growth factor receptor,EGFR)基因突变与酪氨酸激酶抑制剂的疗效密切相关.本研究目的是通过比较荧光定量PCR(Real-time PCR)和DNA测序法检测EGFR基因突变的一致性,建立适用于临床的EGFR基因突变检测方法.方法 应用荧光定量PCR(Real-time PCR)及DNA测序法检测103例非小细胞肺癌标本的EGFR外显子19和21的基因突变,结果比较采用卡方检验.结果 两种方法对EGFR外显子19和21的基因突变的榆测结果无统计学差异,且荧光定量PCR法比测序法更快捷和灵敏.结论 与测序法相比,荧光定量PCR更适用于临床检测.     

Sensitive High-Resolution Melting Analysis for Screening of KRAS and BRAF Mutations in Iranian Human Metastatic Colorectal Cancers

Background: Investigations of methods for detection of mutations have uncovered major weaknesses of direct sequencing and pyrosequencing, with their high costs and low sensitivity in screening for both known and unknown mutations. High resolution melting (HRM) analysis is an alternative tool for the rapid detection of mutations. Here we describe the accuracy of HRM in screening for KRAS and BRAF mutations in metastatic colorectal cancer (mCRCs) samples. Materials and Methods: A total of 1000 mCRC patients in Mehr Hospital, Tehran, Iran, from Feb 2008 to May 2012 were examined for KRAS mutations and 242 of them were selected for further assessment of BRAF mutations by HRM analysis. In order to calculate the sensitivity and specificity, HRM results were checked by pyrosequencing as the golden standard and Dxs Therascreen as a further method. Results: In the total of 1,000 participants, there were 664 (66.4%) with wild type and 336 (33.6%) with mutant codons 12 and/or 13 of the KRAS gene. Among 242 samples randomly checked for the BRAF gene, all were wild type by HRM. Pyrosequencing and Dxs Therascreen results were in line with those of the HRM. In this regard, the sensitivity and specificity of HRM were evaluated as 100%. Conclusion: The findings suggest that the HRM, in comparison with DNA sequencing, is a more appropriate method for precise scanning of KRAS and BRAF mutations. It is also possible to state that HRM may be an attractive technique for the detection of known or unknown somatic mutations in other genes.     

Direct serum and tissue assay for EGFR mutation in non-small cell lung cancer by high-resolution melting analysis

Biological therapy with epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) have noted promising outcomes for patients with non-small cell lung carcinoma (NSCLC), especially those with mutated EGFR. Tissue EGFR gene mutation testing can predict the benefit of taking a first-line EGFR-TKI, thus, allowing the physician to prescribe the most suitable therapy. Unfortunately, most lung cancer patients, especially NSCLC patients present with advanced disease that is surgically unresectable. The goal of this study was to develop high-resolution melting (HRM) assays to detect EGFR mutations in exons 18 to 21, compare their sensitivity and concordance to direct sequencing, and evaluate the feasibility and reliability of serum as a tissue alternate for routine EGFR mutation screening. EGFR mutations of 126 Formalin-Fixed Paraffin-Embedded (FFPE), 47 fresh frozen tissues and from 47 matched pre-operation serum specimens of NSCLC patients were screened by the HRM assays. EGFR mutations by HRM were confirmed through sequencing. We found 78 EGFR mutations in 70 FFPE tissues, 25 EGFR mutations in 24 fresh frozen tissues, with a mutation rate of 55.56% (70/126) and 51.06% (24/47), respectively. Most mutations were correctly identified by sequencing. EGFR mutations were detected in 22 serum samples from 24 tissue EGFR mutation-positive patients. The concordance rate between serum and tissue in EGFR mutation screening was 91.67%. We conclude that the HRM assay can provide convincing and valuable results both for serum and tissues samples, thus, it is suitable for routine serum EGFR mutation screening for NSCLC patients, especially those surgically unresectable.     

High Resolution Melting Analysis for Epidermal Growth Factor Receptor Mutations in Formalin-fixed Paraffin-embedded Tissue and Plasma Free DNA from Non-small Cell Lung Cancer Patients

Background:The aim of the research was to explore a cost effective, fast, easy to perform, and sensitive methodfor epidermal growth factor receptor (EGFR) mutation testing. Methods: High resolution melting analysis (HRM)was introduced to evaluate the efficacy of the analysis for dectecting EGFR mutations in exons 18 to 21 usingformalin-fixed paraffin-embedded (FFPE) tissues and plasma free DNA from 120 patients. Results: The totalEGFR mutation rate was 37.5% (45/120) detected by direct sequencing. There were 48 mutations in 120 FFPEtissues assessed by HRM. For plasma free DNA, the EGFR mutation rate was 25.8% (31/120). The sensitivityof HRM assays in FFPE samples was 100% by HRM. There was a low false-positive mutation rate but a highfalse-negative rate in plasma free DNA detected by HRM. Conclusions: Our results show that HRM analysis hasthe advantage of small tumor sample need. HRM applied with plasma free DNA showed a high false-negativerate but a low false-positive rate. Further research into appropriate methods and analysis needs to be performedbefore HRM for plasma free DNA could be accepted as an option in diagnostic or screening settings.     

Unmet needs in squamous cell carcinoma of the lung: potential role for immunotherapy

The relationship between epidermal growth factor receptor (EGFR) mutation status and EGFR-tyrosine kinase inhibitors (TKI) efficacy in non-small cell lung cancer (NSCLC) patients has been well established. However, there is no available standard to define the optimal testing method and specimen type required for the detection of EGFR mutations. In this study, we compare results of ADx-amplification refractory mutation system (ARMS) and direct sequencing for the detection of EGFR mutation and prediction of EGFR-TKI efficacy for surgery and biopsy tumor tissues in 158 NSCLC patients. For 71 surgery samples, there were 13 and 17 positive samples detected by direct sequencing and ARMS, respectively. For 87 biopsy samples, direct sequencing and ADx-ARMS found 15 and 32 positive samples, respectively. For surgery samples, sensitivity of direct sequencing and ARMS was 72.2 % (13/18) and 94.4 % (17/18), respectively. For the biopsy samples, sensitivity of direct sequencing and ARMS was 44.1 % (15/34) and 94.1 % (32/34), respectively. For the biopsy and surgery samples, the ORRs for EGFR positive and negative patients detected by direct sequencing were 46.1 versus 16.7 and 66.7 versus 1.1 %, respectively. For ADx-ARMS, the ORR for EGFR positive patients was significantly higher than for negative patients (55.6 vs. 5.6 %). The median progression-free survival time of patients with EGFR wild type detected by direct sequencing (4.2 months) was significantly longer than that of patients with wild type detected by ARMS (1.7 months). ARMS has a higher sensitivity and specificity than direct sequencing for EGFR detection of mutation in both surgical and biopsy samples, and the results from ARMS are more consistent with the efficacy of EGFR-TKIs treatment.     

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。     

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.     

Comparison of High-Resolution Melting (HRM) Analysis with Direct Sequencing for the Detection of DNMT3A Mutations in AML Patients

Objective: Acute myeloid leukemia (AML) is caused by abnormal gene expression following mutations. Many of the mutations in AML lead to gene instability and poor response to treatment. Among these mutations, DNMT3A mutation is exceedingly important due to its major role in methylation and its effect on the expression of other genes. Aberrant methylation due to DNMT3A mutations that mostly occur in exon 23, affects the overall survival (OS) of patients with AML and myelodysplastic syndromes (MDS) showing the importance of identification of these mutations. According to the association of these mutations with short overall survival and disease progression in AML patients, we aimed to investigate DNMT3A gene exon 23 mutations using HRM. Methods: Fifty peripheral blood samples were taken from patients with AML. Mononuclear cells were isolated by ficoll method, and DNA was extracted. Then, mutation detection was detected using the HRM method. Efficacy of the HRM method in mutation detection was compared with direct sequencing method as gold standard. Results: Mutations in codon 23 of the DNMT3A gene were detected in 5 patients (10%). All of the detected mutations were missense type. A comparison between direct sequencing and HRM analysis demonstrated full concordance of mutation detection. Conclusion: According to the full consistency between the HRM and direct sequencing methods, HRM is suggested to be adopted as an alternative for the common time-consuming methods in detecting the gene mutations.