高分辨率熔解曲线分析法检测非小细胞肺癌KRAS和BRAF基因突变

目的探讨高分辨率熔解曲线分析(HRM)法检测非小细胞肺癌(NSCLC)中KRAS和BRAF基因突变用于临床检测的可行性。方法用HRM法检测64例NSCLC患者KRAS基因第2外显子和BRAF基因第15外显子的突变情况,用直接测序法对结果进行验证。结果 HRM法检测结果表明有9例NSCLC患者发生KRAS基因突变(14.06%)、4例发生BRAF基因突变(6.25%),直接测序法证实两法的结果完全一致;共检测出4种KRAS基因突变类型,G12C(GGT>TGT)的突变率最高(44.4%),BRAF基因突变型均为V600E。结论用HRM法检测临床样本KRAS和BRAF基因突变,具有操作简便、结果准确、成本低的优点,适用于临床检测。     

HRM法检测结直肠癌患者循环DNA中KRAS和EGFR基因突变

摘要:目的：探讨高分辨率熔解曲线(HRM) 法检测结直肠癌患者循环DNA中KRAS和EGFR基因突变的可行性。 方法：用HRM法检测70例结直肠癌患者KRAS基因2号和3号外显子以及EGFR基因19和21号外显子的突变情况,用直接测序法验证结果的准确性。 结果：70 例结直肠癌患者血液标本经HRM法、直接测序法分别检测到18例(25.7%)和17例(24.3%)KRAS基因2号外显子突变;均检测到3例EGFR基因19号外显子突变(4.3%);两法均未检测到3号外显子和21号外显子突变。HRM检测与直接测序法结果符合率为99.6% (279/280)。 结论：HRM法检测结直肠癌患者血样本KRAS和EGFR基因突变,具有准确度高、操作简单、检测成本低等优点,适合在临床推广应用。     

某地区结直肠癌患者KRAS基因及BRAF基因突变状态的检测分析

目的 检测某地区结直肠癌患者KRAS基因和BRAF基因的突变状态及其与年龄、性别的关系.方法 由某地区30例结直肠癌患者石蜡组织中提取DNA,通过直接测序法及荧光定量PCR法检测KRAS基因及BRAF基因突变状态.结果 KRAS基因突变率为43.3%,共发现6种突变类型,主要位于12、13密码子,其中以c.38G>A突变率最高(38.5%).单因素及多因素分析均提示KRAS突变与年龄或性别无相关性.BRAF基因突变率为0.结论 某地区结直肠癌患者KRAS基因突变率高,靶向治疗前进行突变状态检测具有重要临床价值.     

焦磷酸测序法检测结直肠癌患者K-ras基因外显子2第12和13密码子点突变

目的 探讨焦磷酸测序法检测结直肠癌患者肿瘤组织K-ras基因外显子2第12和13密码子点突变方法的临床应用价值.方法 以已知K-ras基因突变的结直肠癌细胞株SW480、DLD-1和野生型细胞株HT-29 DNA作为测序模板检验焦磷酸测序法的准确性.对含不同比例(2％、3％、5％、10％、20％、30％和50％)结直肠癌细胞株K-ras基因的DNA混合样本采用焦磷酸测序法进行基因突变率检测,并与Sanger测序结果平行进行Fisher精确检验比较,评价其灵敏度.同时用焦磷酸测序法检测分析30份临床结直肠癌患者石蜡包埋组织中K-ras基因第12和13密码子突变.结果 当混合已知突变类型的结直肠癌细胞株K-ras基因的DNA样本突变DNA比例在5％和10％浓度时,Sanger测序法检出K-ras基因突变率分别为33.3％ (4/12)和58.3％ (7/12),焦磷酸测序法分别为91.7％(11/12)和100％( 12/12),且2种方法检出K-ras基因突变率的差异有统计学意义(P＜0.05).此外,用焦磷酸测序法从30例结直肠癌患者石蜡包埋组织标本中检出K-ras基因外显子2第12和13密码子突变10例,均为杂合型突变,突变率为33.3％ (10/30).最常见的突变类型为G＞A转换[50％(5/1O)]和G＞T颠换[(30％(3/10)].结论 焦磷酸测序法检测结直肠癌K-ras基因外显子2第12和13密码子突变具有敏感、准确的优点,可用于临床个体化治疗中肿瘤基因突变检测.     

以测序法为准比较HRM法、ADx-ARMS法和TaqMan探针法检测肺腺癌EGFR基因突变的敏感性

目的采用高分辨率熔解曲线法(HRM法)、Adx-ARMS法、Taq Man探针法和直接测序法检测肺腺癌组织中表皮生长因子受体基因(EGFR)的突变情况,并比较各方法的检测效率和敏感性。方法收集20例肺腺癌石蜡包埋肿瘤组织,其中18例为手术切除标本,2例为穿刺小标本,应用HRM法、ARMS法和Taq Man探针法分别检测EGFR基因突变,对检测结果不一致的样本采用测序法加以验证。结果 20例肺腺癌组织样本中,HRM法、ARMS法和Taq Man法分别在13例、10例和8例组织中检测到EGFR基因突变,差异不显著(P0.05)。差异标本经测序验证后发现,HRM法可检测未知突变,而ARMS法和Taq Man探针法只能检测已知突变。对于2例穿刺小标本,HRM法和ARMS法均检测到突变,而Taq Man探针法未检测出突变,HRM法和ARMs法的检测敏感性高于Taq Man探针法。结论 HRM法、ARMS法、Taq Man探针法和测序法各有优缺点,HRM法敏感性最高,且能检测到少见突变与未知突变,因此HRM法结合测序法能更全面的检测出各种突变类型。     

急性髓系白血病NPM1基因突变检测方法的研究

本研究旨在建立检测急性髓系细胞白血病(AML)患者NPM1基因突变的方法。针对NPM1基因突变集中区域设计引物/探针,建立高分辨熔解曲线方法(HRM)和等位基因特异PCR方法(AS-PCR),通过89份AML标本进行了临床评估,并采用毛细管电泳法(CE)和测序法作为对照进行了验证。结果表明,通过上述4种方法共检出阳性标本17例(19.1%),其中A型突变15例,B型和Nm型突变各1例。上述方法中HRM法检测全面,灵敏度为5%,而AS-PCR法有一定的漏检,但灵敏度高达0.01%。结论:考虑到操作的难易程度,同时也结合临床样品的检测情况,HRM在临床上可法用于NPM1突变筛查,而AS-PCR法可用于后续的微小残留病定量检测。     

运用高灵敏度COLD-PCR法检测胰腺癌和结直肠癌患者KRAS基因突变

目的 评价COLD-PCR法检测胰腺癌和结直肠癌患者KRAS基因突变的价值.方法 采用COLD-PCR/Sanger测序法与普通PCR/Sanger测序法检测KRAS基因野生型结直肠癌细胞系SW116中混有的KRAS基因突变型和结直肠癌细胞系SW480中的KR4S基因突变,确定两种方法 的灵敏度,并采用两种方法 分别检测20例胰腺癌和39例结直肠癌患者石蜡包埋组织的KRAS基因突变,并评价两种方法 的符合率.结果 细胞系检测结果 显示,普通PCR/Sanger测序法和COLD-PCR/Sanger测序法检测KRAS基因突变的灵敏度分别为1∶20和1∶100(突变型:野生型).COLD-PCR/Sanger法检测20例胰腺癌患者KRAS基因突变率[75%(15/20)]高于普通PCR/Sanger测序法[40%(8/20),x2=5.013,P<0.05];COLD-PCR/Sanger测序法检测39例结直肠癌患者的KRAS基因突变率[44%(17/39)]高于普通PCR/Sanger测序法[31%(12/39),x2=1. 372,P=0.174)].两种方法 检测胰腺癌标本的符合率为65%,但一致性较差(Kappa=0.364,P<0.05);而两种方法 检测结直肠癌标本的符合率为87%,且一致性较好(Kappa=0.730,P<0.05).结论 COLD-PCR/Sanger测序法是一种高灵敏性检测胰腺癌和结直肠癌患者KRAS基因突变的方法 .     

胰腺癌中EGFR、KRAS、BRAF基因突变状况分析及意义

目的:检测胰腺癌组织EGFR、KRAS、BRAF基因突变状况,为胰腺癌EGFR靶向治疗研究奠定基础.方法:提取胰腺癌及胰腺良性病变石蜡组织切片中基因组DNA,PCR扩增EGFR 18、19、21外显子片段,KRAS2、3外显子片段,BRAF15外显子片段,采用直接测序法检测其突变状况.结果:32例胰腺癌患者中有24例患者存在KRAS基因突变,10例良性病变组织均未发现突变,两者差异具有统计学意义(x2=14.57,P=1.35× 10-4),其中22例12密码子突变(G12D14例,G12V8例);2例61密码子突变(Q61L).所有检测样本中未见BRAF突变.共3例EGFR突变,其中包括1例19外显子突变(de1746-750),2例21外显子突变(L858R),10例良性病变组织未见突变.结论:在胰腺癌中KRAS基因突变可能为EGFR通路失调的主要原因,其次是EGFR突变,BRAF突变未见.     

高分辨率熔解曲线法检测JAK2基因V617F突变及其应用

摘要:目的：探讨实时定量PCR方法和高分辨率熔解曲线法（HRM）在检测JAK2基因V617F突变中的应用。 方法：以JAK2基因V617F发生纯合突变的人红白血病细胞株（HEL）及不含JAK2基因V617F突变的人白血病细胞株（HL60）为阳性对照和阴性对照,优化HRM法检测条件,分别以优化的HRM法和直接测序法对63例临床疑似骨髓增殖性肿瘤（MPN）患者DNA标本进行JAK2基因V617F突变检测,评价2种检测方法结果的一致性。 结果：HRM法可检出系列混合样本中5%的突变型等位基因突变,且重复性较高。以测序法为金标准,HRM法的敏感性和特异性均为100％,两种方法结果一致。 结论：HRM法能够在单一闭管体系中实现对JAK2基因V617F突变的检测,具有较高的敏感性和特异性,可用于JAK2基因V617F突变的临床检测。     

结直肠癌患者BRAF,KRAS,NRAS和PIK3CA基因突变与其病理特征的关系

目的:探讨265例结直肠癌患者BRAF,KRAS,NRAS和PIK3CA基因突变及其病理特征关系。方法:选取2014年12月至2016年12月的265例结直肠癌患者肿瘤组织标本进行回顾性分析,采用PCR扩增–直接测序法检测BR AF基因(15外显子600密码子),KR AS基因(12,13,61密码子突变),NRAS(2号与3号外显子的12密码子、13密码子与61密码子常见的12个突变位点)及PIK3C A(第9,20外显子)基因的突变状态,分析其与结直肠癌临床病理特征的关系。结果:265例患者中存在BRAF基因突变率为6.8%(18/265),KRAS基因突变率为32.1%(85/265),NRAS基因突变率为5.7%(15/265),PIK3CA基因突变率为11.3%(30/265)。NRAS基因和KRAS基因突变与年龄有关(P0.05),与性别、原发部位、组织学类型、分化程度、TNM分期、区域淋巴结转移、远处转移、术后复发转移均无关(P0.05);BRAF,PIK3CA基因在原发部位为右半结肠患者中的突变率明显升高(P0.05),但与年龄、性别、组织学类型、分化程度、TNM分期、区域淋巴结转移、远处转移、术后复发转移均无关(P0.05)。结论:NRAS,PIK3CA基因在中国结直肠癌患者中的突变率较低。KRAS,NRAS基因突变与年龄相关,BRAF,PIK3CA基因与肿瘤原发部位相关,联合检测这些基因的突变情况可以判断疾病的发生发展。     

酶切富集联合DHPLC检测肿瘤患者外周血EGFR和KRAS基因突变及其临床应用

目的 建立一种REDE-DHPLC检测外周血肿瘤游离核酸EGFR、KRAS基因突变的方法,并探讨其临床应用价值.方法 采用限制性内切酶Mse Ⅰ、Msc Ⅰ、BstN Ⅰ和BglⅠ分别切断EGFR基因第19、21号外显子和KRAS基因第12、13号密码子的野生型片段以富集突变片段,建立检测血浆EGFR和KRAS基因突变的REDE-DHPLC法,并采用50%、10%、5%、1%和0.1%稀释度的质粒标准品评价REDE-DHPLC法和传统DHPLC法的检测灵敏度.然后,采用REDE-DHPLC法检测120例NSCLC和120例结直肠癌患者血浆和石蜡组织标本中的EGFR和KRAS基因突变.同时,用传统DHPLC法和测序法进行平行检测,以评价REDE-DHPLC法检测NSCLC和结直肠癌患者血浆中2个基因突变的诊断效能.结果 REDE-DHPLC法对EGFR和KRAS基因4个突变位点检测的灵敏度均达0.1%,传统DHPLC法检测灵敏度均为1.0%,REDE-DHPLC法对含0.1%突变的质粒标准品重复2～3次检测均为阳性.REDE-DHPLC法、传统DHPLC法和测序法检测120例NSCLC患者血浆EGFR基因总突变率分别为27.5%、16.7%和12.5%,检测120例结直肠癌患者血浆KRAS基因总突变率分别为38.3%、25.8%和16.7%.REDE-DHPLC法检测EGFR和KRAS基因总突变率均高于传统DHPLC法(x2值分别为4.092、4.301,P均＜0.05)和测序法(x2值分别为8.438、14.127,P均＜0.05);将REDE-DHPLC法检测EGFR和KRAS基因突变与传统DHPLC法相比,敏感度均为100%(20/20,31/31),特异度分别为87.0%(87/100)和83.2%(74/89);与测序法相比,敏感度均为100%(15/15,20/20),特异度分别为82.9%(87/105)和74.0%(74/100).REDE-DHPLC法与传统DHPLC法检测EGFR、KRAS基因突变的符合率分别为89.2%(107/120,Kappa=0.690,P＜0.05)和87.5%(105/120,Kappa=0.718,P＜0.05).REDE-DHPLC检测血浆与组织标本中EGFR和KRAS基因总突变符合率分别为91.7%(33/36,Kappa=0.939,P＜0.05)和90.2%(46/51,Kappa=0.914,P＜0.05).结论 REDE-DHPLC法灵敏度和特异性高,结果易判读,且可有效避免纯合点突变漏检,有望成为临床实验室外周血EGFR和KRAS基因突变检测的推广方法.

Abstract：

Objective To establish a REDE-DHPLC method for detecting the EGFR and KRAS mutations in plasma DNA from tumor patients, and investigate its clinical significance. Methods Restriction endonucleases Mse Ⅰ , Msc Ⅰ , BstN Ⅰ and Bgl Ⅰ were used to digest the wild type fragments of exon 19,exon 21 of EGFR gene and coden 12, 13 of KRAS gene for enriching the mutation fragments, and REDE-DHPLC method was established to detect EGFR and KRAS mutations. The sensitivities of REDE-DHPLC and conventional DHPLC were analyzed by using a series of plasmids containing 50%, 10%, 5%, 1% and 0. 1% mutation genes. Then, Plasma samples and paraffin-embedded tissue samples of 120 NSCLC patients and 120 colorectal cancer patients were detected by REDE-DHPLC. Compared with conventional DHPLC and sequencing, the diagnostic efficiency of REDE-DHPLC method was evaluated by detecting the mutation status of 2 genes in plasma of NSCLC and colorectal cancer patients. Results The sensitivity values of REDE-DHPLC and conventional DHPLC for detecting mutations in 4 loci were 0. 1% and 1%respectively. Plasmid DNA containing 0.1% mutation gene was detected to be positive continually for 2 to 3 times by REDE-DHPLC. EGFR mutation rates of 120 plasma from NSCLC patients detected by REDE-DHPLC, conventional DHPLC and sequencing methods were 27. 5%, 16. 7% and 12.5% respectively, and KRAS mutation rates of 120 plasma from colorectal cancer patients were 38. 3%, 25. 8% and 16. 7%,respectively. The positive rates of EGFR and KRAS mutation detected by REDE-DHPLC were significantly higher than conventional DHPLC(x2 = 4. 092, 4. 301, all P ＜ 0. 05 ) and sequencing method (x2= 8. 438,14. 127,all P ＜ 0. 05 ). In comparison with conventional DHPLC, the sensitivities of REDE-DHPLC for detecting EGFR and KRAS mutation were 100% (20/20,31/31), the specificities were 87. 0% (87/100)and 83. 2% (74/89). In comparison with sequencing method, the sensitivities of REDE-DHPLC were 100%( 15/15,20/20), the specificities were 82.9% (87/105)and 74. 0% (74/100). The coincidence rate of the two methods for detecting EGFR and KRAS mutation were 89. 2% ( 107/120, Kappa = 0. 690, P ＜ 0. 05 ) and 87.5% ( 105/120, Kappa= 0. 718, P ＜ 0. 05 ). The Consistency of EGFR and KRAS mutation status in plasma and tissues detected by REDE-DHPLC were 91.7% (33/36, Kappa =0. 939,P ＜0. 05)and 90. 2 %(46/51, Kappa = 0. 914, P ＜ 0. 05 ), respectively. Conclusions The REDE-DHPLC method is highly sensitive and specific for detecting EGFR and KRAS mutations in plasma DNA from tumor patients. The results are easy to be interpreted without missing homozygous point mutation, which indicate that the detection of EGFR and KRAS mutations in plasma DNA by REDE-DHPLC could therefore extend to be usedin clinical laboratory.     

一种KRAS基因突变检测方法的建立及初步应用

目的建立一种KRAS基因实时荧光定量PCR-Sanger测序突变检测方法,并初步探讨其临床应用价值。方法以KRAS基因热点突变区域12、13密码子为研究位点设计特异性扩增、测序引物,利用已知野生型、突变型样品以TA克隆技术构建相应质粒作为标准品,建立KRAS基因实时荧光定量PCR—Sanger测序突变检测方法,并进行方法学和应用评估。结果成功构建了KRAS基因12、13密码子野生型、突变型质粒。建立了KRAS基因实时荧光定量PCR—Sanger测序突变检测方法,该方法灵敏度高（9．39×10^1copies／uL）,重复性好（实时荧光定量PCR部分批内、批间变异系数分别为1．60％、2．54％）。该法与传统Sanger测序法同时检测40例临床样品,结果符合率为100％。结论本研究成功建立了可用于临床样品检测的KRAS基因实时荧光定量PCR—Sanger测序突变检测方法。     

高分辨率熔解曲线分析法检测急性髓系白血病c-kit基因突变

目的 应用高分辨率熔解(HRM)曲线分析法检测急性髓系白血病(AML)患者c-kit基因常见突变N822K和D816V.方法 对21例t(8;21)阳性AML患者骨髓标本的c-kit基因第17号外显子进行PCR扩增、HRM突变检测及DNA测序验证.结果 HRM分析发现21例患者中6例(28.6%)的c-kit基因扩增产物出现异常熔解曲线,经测序验证1例为D816V杂合子突变,5例为N822K杂合子突变.结论 HRM分析是一种简便、快速、特异和高通量的c-kit基因突变筛查方法.

Abstract：

Objective To detect the common mutations (D816V and N822K) of c-kit gene in acute myeloid leukemia (AML) using high-resolution melting analysis (HRM). Methods HRM analysis was established to screen c-kit mutations in PCR products of c-kit exon 17 in 21 AML patients with t(8;21 ). PCR products were sequenced to confirm the mutation. Results HRM analysis identified an aberrant melting curve in 6 cases (28.6%), which were confirmed by direct DNA sequencing as one D816V mutation and five N822K mutation. Conclusion HRM analysis is a convenient, rapid, specific and high-throughput technique for scanning c-kit gene mutation in AML.     

Optimized allele-specific real-time PCR assays for the detection of common mutations in KRAS and BRAF

Mutations in the oncogenes KRAS and BRAF have been identified as prognostic factors in patients with colorectal diseases and as predictors of negative outcome in epidermal growth factor receptor-targeted therapies. Therefore, accurate mutation detection in both genes, KRAS and BRAF, is of increasing clinical relevance. We aimed at optimizing allele-specific real-time PCR assays for the detection of common mutations in KRAS and the BRAF Val600Glu mutation using allele-specific PCR primers for allelic discrimination and probes (TaqMan) for quantification. Each reaction mix contains a co-amplified internal control to exclude false-negative results. Allele-specific real-time PCR assays were evaluated on plasmid model systems providing a mutation detection limit of 10 copies of mutant DNA in proportions as low as 1% of the total DNA. Furthermore, we analyzed 125 DNA samples prepared from archived, formalin-fixed, paraffin-embedded colorectal carcinomas and compared results with those obtained from direct-sequence analysis. All mutations determined by sequence analysis could be recovered by allele-specific PCR assays. In addition, allele-specific PCR assays clearly identified three additional samples affected by a mutation. We propose these allele-specific real-time PCR assays as a low-cost and fast diagnostic tool for accurate detection of KRAS and BRAF mutations that can be applied to clinical samples.     

A PIK3CA pyrosequencing-based assay that excludes pseudogene interference

Phosphatidylinositol 3'-kinase gene (PIK3CA) encodes a lipid kinase that regulates signaling pathways downstream of epidermal growth factor receptor and is mutated in 10% to 30% of colorectal cancers. Activating mutations in this gene up-regulates the AKT signaling pathway, making it a potentially useful therapeutic target. Mutations in PIK3CA are not exclusive of mutations in KRAS, BRAF, or NRAS. We designed a pyrosequencing assay to detect mutations in all three positions of codons 542 and 545 in exon 9 and codon 1047 in exon 20 of this gene. The exon 9 reverse PCR primer was designed to avoid amplifying a pseudogene in chromosome 22 that has >95% homology with exons 9 through 13 in PIK3CA. Two hundred colorectal cancers from FFPE tissue previously characterized for KRAS mutation status were evaluated for PIK3CA mutations. In KRAS-mutated samples, 20% had an additional mutation in PIK3CA. The mutation rate in KRAS wild-type samples was 7.5%. When using our assay, pseudogene was not observed in any of these samples. In addition, pseudogene- and gene-specific amplification was performed on DNA from 40 additional colorectal cancers. Sequencing of these PCR products yielded the expected gene or pseudogene sequence in all cases. Thus, we have developed a PIK3CA pyrosequencing assay capable of detecting mutations in all three positions in the three hot spot codons with no pseudogene interference.     

KRAS and BRAF mutation analysis in routine molecular diagnostics: comparison of three testing methods on formalin-fixed, paraffin-embedded tumor-derived DNA

Accurate mutation detection assays are strongly needed for use in routine molecular pathology analyses to aid in the selection of patients with cancer for targeted therapy. The high-resolution melting (HRM) assay is an ideal prescreening tool, and SNaPshot analysis offers a straightforward genotyping system. Our present study was determined to compare these mutation testing methods on formalin-fixed, paraffin-embedded (FFPE) tumor-derived DNA. We compared the performance of HRM, followed by cycle sequencing (HRM-sequencing); multiplex PCR assay, followed by SNaPshot analysis (multiplex mutation assay); and a successor assay using HRM, followed by SNaPshot (HRM-SNaPshot) for mutation analysis of both KRAS (codon 12/13/61) and BRAF (codon 600/601). In a series of 195 FFPE-derived DNA specimens, a high genotypic concordance between HRM-sequencing and multiplex mutation assay was found (κ, 0.98; 95% CI, 0.94 to 1), underlining the potential of a combined HRM-SNaPshot approach. In reconstruction experiments, the analytical sensitivity of HRM-SNaPshot was twofold to fourfold higher than HRM-sequencing and multiplex mutation assay, respectively. In addition, HRM-SNaPshot had a good performance rate (99%) on FFPE tumor-derived DNA, and mutation detection was highly concordant with the predecessor assays (κ for both, 0.98). The occurrence of BRAF and KRAS mutations is mutually exclusive. HRM-SNaPshot is an attractive method for mutation analysis in pathology, given its good performance rate on FFPE-derived DNA, high analytical sensitivity, and prescreening approach.