特异引物双扩增即时PCR与传统测序法检测肠癌、肺癌患者K-ras基因突变的比较

Objective To map out the frequency and types of K-ras gene mutations present incolorectal and lung cancer patients; to evaluate the clinical applicability of a novel real-time double-loop probe PCR using the ADx-K-ras kit, and to compare its performance with the result by using traditional Sanger DNA sequencing in detection of somatic mutations of the tumor genes. Methods A total of 827 formalin-fixed paraffin-embedded (FFPE) blocks including 583 from the colorectal and 244 from the lung cancer patients were assayed. Genomic DNA of the sample tissues was extracted, purified and subjected to PCR amplification of K-ras gene codon 12 and 13 and DNA sequencing was carried on using both the traditional Sanger sequencing method and the ADx's K-ras mutation detection kit, respectively. The mutation rates for K-ras gene at codon 12 and 13, and the mutation frequencies detected by using both methods were analyzed. Results 533 out of 583 (91.4%) colorectal cancer samples and 144 out of 244 lung cancer samples(59.0%) were detected using the traditional Sanger DNA sequencing technique, and 583 out of 583(100.0%) colorectal plus 244 out of 244(100.0%) lung cancers were detected, respectively by using the ADx-K-ras kit. Of the 583 colorectal cancer samples, 192 (32.9%) showed mutations by using the ADx-K-ras kit in comparing with a result of 160 samples (27.4%) with K-ras gene mutation by using the traditional Sanger DNA sequencing technique. Of the 244 lung cancer samples, 26(10.7%) showed K-ras gene mutations by using ADx-K-ras kit, while in 144 samples detected by using the traditional Sanger DNA sequencing technique, only 12 samples (8.3%) showed K-ras gene mutations. In colorectal cancer analyzed, GGT→GAT at codon 12 was the most common event with 35.1% (66/188) mutations, followed by GGC→GAC at codon 13 with 26.6% (50/188) and GGT→GTT at codon 12 with 18.6% (35/188),while GGT→GCT at codon12 was the most rare with only 1.6% (3/188) of the total mutation cases. In patients with lung cancer analyzed,GGT→GTT at codon 12 was the most common mutation, accounting for 40.9% (9/22), and GGT→GCT at codon 12 the most rare with only about 4.5% (1/22) of the total mutation cases. Conclusions K-ras gene mutations were present in colorectal cases, and significantly more frequent than that in lung cancer. There were significant statistical differences between the two methods.ADx-K-ras real-time PCR showed much higher successful detection rates and mutation ratios compared to Sanger sequencing. As a result, the real-time PCR with ADx-K-ras kit proves to have a good clinical applicability and a strong advantage over the traditional Sanger DNA sequencing. It is a effective and reliable tool for clinical screening of somatic gene mutations in tumors.     

子宫内膜癌与K-ras基因突变的研究

目的:分析子宫内膜癌组织中K-ras基因突变状况及其与Ras蛋白表达的相关性,从基因水平探讨K-ras基因在子宫内膜癌发病中的作用机制.方法:诊断明确的子宫内膜癌手术切除标本108例及同期因其他病因行刮宫术的正常子宫内膜.应用免疫组织化学染色法检测K-ras的表达,PCR扩增-直接测序法检测K-ras基因第2外显子12、13密码子突变,分析K-ras基因突变对Ras蛋白表达的影响及临床意义.结果:K-ras蛋白阳性表达率在子宫内膜癌明显高于正常子宫内膜;子宫内膜癌中K-ras基因突变率为33.3%;其中第12位密码子突变占75.0%,突变位点均位于K-ras基因第12位密码子第2位碱基上;第13位密码子突变率为25%,突变位点位于K-ras基因第13位密码子第1、2位碱基上.K-ras基因突变与Ras蛋白表达成正相关.结论:K-ras蛋白的阳性表达率可能是预示子宫内膜癌预后的一个因素,K-ras基因12密码子G→T和G→A突变是主要的突变方式.12密码子的突变可能为Ras蛋白增多的原因.     

卵巢浆液性交界及恶性肿瘤K-ras基因突变分析

目的探讨K-ras基因在卵巢浆液性交界及恶性肿瘤发生发展过程中的作用。方法收集51例卵巢浆液性肿瘤标本,包括经典型交界性浆液性肿瘤18例,微乳头型交界性浆液性肿瘤11例,浸润性微乳头型浆液性癌12例,经典型浆液性癌10例。采用显微切割技术获取肿瘤细胞后,提取基因组DNA、PCR技术扩增K—ras基因第一外显子,通过直接测序的方法鉴定K—ras基因第12、13密码子的突变情况。结果1例经典型交界性浆液性肿瘤K—ras基因第12密码子发生突变,突变类型为GGT→GTT即甘氨酸→缬氨酸,余50例标本未见突变;所有标本K—ras基因第13密码子均为野生型。结论K—ras基因第12、13密码子在被检患者中卵巢浆液性交界及恶性肿瘤中的突变频率很低,其在该肿瘤发生发展过程中可能不起主要作用。     

中国人大肠癌K-ras基因突变的研究

目的：检测K－ras基因在国人散发性大肠癌中的突变情况,探讨K-ras基因突变在中国人大肠癌中的特点以及与临床病理参数的关系。方法：微解剖取正常粘膜组织15例、癌组织35例、PCR扩增、DNA测序、检测K－ras第12、13和61密码子的突变情况。结果：癌组织K－ras突变率为14．3％（5／35）,均发生在第12密码子（GGT→GAT）。第13和61密码子无突变,正常粘组织无K－ras突变。伴有12密码子突变的大肠癌患者年龄较大,大体以隆起型为主,均分布在C和D期,有较强的侵袭性。结论：中国人大肠癌K-ras突变率为14．3％,低于欧美国家且仅发生在第一外显子的12密码子的第二碱基。大肠正常粘膜无K－ras突变。K-ras突变与患者的年龄、肿瘤的大体形态和Dukes分期等临床病理参数有关。     

胃癌组织中HER-2基因扩增和K-ras基因突变的关系

目的探讨胃癌组织中HER-2蛋白表达和基因扩增与K-ras基因突变的关系及其意义。方法采用免疫组化、FISH和焦磷酸测序技术对67例胃癌组织中HER-2蛋白表达、HER-2基因扩增与K-ras基因的突变率进行了检测。结果 HER-2蛋白阳性率为40.3%(27/67),其中HER-2蛋白3+者9.0%(6/67),HER-2蛋白2+者13.4%(9/67),HER-2蛋白1+者17.9%(12/67)。FISH检测HER-2基因扩增率为18.5%(5/27),HER-2基因拷贝数增加和基因扩增者共48.1%(13/27)。K-ras基因突变定量检测为7.5%(5/67),均为K-ras基因第12密码子突变,其中低于10%低丰度突变2例,高于10%高丰度突变3例(突变数值分别为:17、29、30)。除1例为GGT→GAT突变型外,其它均为GGT→GTT突变型。本组K-ras基因突变5例中除1例既有K-ras基因突变,又有HER-2基因扩增,另外4例HER-2基因均无扩增。结论检测胃癌中HER-2扩增时选用抗肿瘤药物治疗的靶点曲妥珠单抗,同时可选用K-ras基因突变的抗肿瘤药物治疗的靶点西妥昔单抗;联合检测胃癌组织中HER-2基因扩增和K-ras基因突变为靶向抗肿瘤药物治疗过程中受益提供参考指标。     

结直肠粘膜隐窝异常病灶K-ras、APC基因突变的研究

目的　分析隐窝异常病灶（aberrant crypt foci,ACF）的基因水平变化特点及其作为结直肠癌最早期形态变化的分子依据,探讨ACF与腺瘤之间的关系。方法　经微解剖分离提取15例正常粘膜腺体、34例ACF、15例腺瘤和35例结直肠癌组织进行DNA测序,检测K-ras第12、13、61密码子和APC第15外显子“突变密集区”的突变。结果　正常粘膜无K-ras和APC基因突变,ACF、腺瘤和癌组织K-ras突变率分别为17.6%（6/34）、13.3%（2/15）和14.3%（5/35）,3者突变率相近。4例ACF、腺瘤和癌组织的突变位于12密码子的第2碱基（GGT→GAT）,2例ACF突变发生在13密码子的第2碱基（GGC→GAC）。ACF中K-ras的突变特点与同来源的癌组织保持一致,即患者年龄较大,大体以隆起型为主,所有组织未发现有61密码子的突变。癌和腺瘤APC基因突变率相近,癌为22.9%（8/35）,腺瘤为26.7%（4/15）明显高于ACF的2.9%（1/34,P<0.05),癌组织APC与患者的年龄、肿瘤位置、大体类型、组织分化程度无关。结论　ACF可能是结直肠癌最早期的形态改变,ACF的形态学变化、部分基因的改变均有别于腺瘤,提示（1）ACF有可能是腺瘤前的形态改变;（2）结直肠癌的发生可能存在“正常上皮→ACF→癌变”这一途径。     

K-ras基因状态与结直肠癌临床病理特征的关系

目的观察结直肠癌原发灶K-ras基因的突变,探讨其与临床病理特征的关系。方法运用实时荧光定量PCR法检测230例结直肠癌组织K-ras基因12、13密码子的突变,利用χ2检验分析其与临床病理特征的相关性。结果 230例结直肠癌患者中,84例K-ras基因发生突变,突变率为36.5%,其中12密码子突变65例(28.2%)、13密码子突变19例(8.3%)。结直肠癌肺转移患者K-ras基因突变率较无肺转移患者高(P=0.022),12、13单密码子突变与临床病理特征(患者年龄、性别、肿瘤部位、病理分型、TNM分期、Dukes分期、区域淋巴结及肝肺转移)无关(P>0.05)。结论结直肠癌K-ras基因突变可能与肺转移存在相关性,检测K-ras基因突变对结直肠癌患者临床个体化治疗具有指导意义。     

结直肠癌原发灶、门静脉血和肝转移灶K-ras基因突变的研究

目的:观察结直肠癌患者门静脉血液、原发肿瘤组织及相应肝转移灶K-ras基因突变情况,分析三者的一致性,探讨结直肠癌患者门静脉血K-ras基因突变与肝转移关系。方法:实时荧光定量PCR技术和基因测序技术检测59例结直肠癌患者门静脉血液、原发肿瘤组织及15例肝转移灶K-ras基因突变,结合其临床资料分析。结果:59例结直肠癌组织中20例(33.9%)发现K-ras基因突变,18例(30.5%)结直肠癌患者的门静脉血中也发现K-ras基因突变,15例肝转移灶中8例(53.3%)发现K-ras基因突变,与原发癌组织的基因突变率差异不明显(P0.05)。18例门静脉血存在K-ras基因突变者,其相应的肿瘤组织中均发现K-ras突变。结直肠癌组织中无K-ras基因突变者,患者门静脉血未发现基因突变。8例肝转移灶发现K-ras基因突变者门静脉血亦均有K-ras基因突变,7例肝转移灶无K-ras突变者门静脉血也无K-ras突变。原发肿瘤组织、相应门静脉血和5例同时性、2例异时性肝转移灶的K-ras基因突变类型基本一致(即K-ras基因12密码子GGT突变为GAT或GTT),1例异时性肝转移灶K-ras基因突变类型为13密码子GGC突变为GAC。原发癌组织与门静脉血K-ras基因突变一致率为96.6%(57/59),肝转移灶与门静脉血K-ras基因突变情况基本一致,但突变类型有不同。结论:结直肠癌的原发灶、门静脉血及肝转移灶的K-ras基因突变较为一致,原发癌组织和门静脉血均有K-ras基因的突变,预示着肿瘤可能通过血行转移至肝脏。     

p53基因第8外显子在喉癌中的缺失和点突变

目的 探讨抑癌基因p53第8外显子突变在喉鳞癌分子发病机理中所起的作用。方法 应用聚合酶链反应－单链构象多态性（PCR－SSCP）银染技术和DNA直接测序法,检测喉鳞癌新鲜组织中抑癌基因p53基因第8外显子的突变情况。结果 60例喉鳞癌组织中,15例发生迁移率异常的SSCP区带。在SSCP阳性样本中随机抽出4例进行测序分析,发现两个标本在288位密码子缺失一个A,两个标本在292位密码子上缺失一个A。285、287密码子上共发生3次G→T的颠换,286密码子第3位碱基发生A→T的颠换。结论 喉鳞癌p53基因点突变与碱基缺失常常同时并存。p53基因第8外显子突变在喉癌的发生中可能起着重要作用。     

xTAG液相芯片技术用于肺癌 EGFR基因突变检测的临床适用性

目的：分析肺癌EGFR基因第18~21号外显子突变,以Sanger测序法为参照,探讨xTAG液相芯片用于临床样本检测的适用性。方法随机选择1139例Ⅰ~Ⅳ期肺癌组织标本,提取DNA,分别采用xTAG液相芯片法和Sanger测序法检测EG-FR基因第18~21号外显子突变情况,评价xTAG液相芯片法检测的敏感性和特异性。结果液相芯片法：共1134例患者获得基因突变检测结果,Sanger测序法：共1105例患者获得基因突变检测结果,检测成功率分别为99．56%和97．01%。以San-ger测序法为参照,xTAG液相芯片法检测的敏感性和特异性分别为99．59%和94．54%,两种方法均检出少数样本存在双外显子突变。两种方法检测出的突变型别完全吻合。结论采用xTAG液相芯片法可有效检测肺癌EGFR基因突变,实现突变分型,且相对测序法更方便、高效,适合临床推广应用。     

K-ras基因突变在内蒙古地区结直肠癌患者中研究

目的检测内蒙地区结直肠癌K-ras基因突变情况,并结合临床病理资料加以分析。方法提取15例结直肠癌患者结直肠癌手术切除标本组织的DNA,对产物进行基因序列癌组织的DNA聚合酶链反应（PCR）扩增、DNA直接测序分析。结果 K-ras基因突变率为0%,几种分化型的结直肠癌均未发现K-ras基因突变类型,包括12密码子（GGT）、13密码子（GGC）。结论我院结直肠癌患者k-ras基因突变率为0%,转移性结直肠癌患者原发肿瘤与转移灶肿瘤k-ras基因型均相同;结直肠癌患者k-ras基因突变与否与年龄、性别、肿瘤浸润深度、肿瘤组织学类型无关。     

K-ras mutation detection in colorectal cancer using the Pyrosequencing technique

The identification of gene mutations is a critical goal for the assessment of diagnosis and prognosis in cancer disease, particularly by direct sequencing. Pyrosequencing is a straightforward, non-electrophoretic DNA sequencing method using the luciferase-luciferin light release as a signal for nucleotide incorporation into a PCR template DNA. In this study, we aimed to investigate mutations in the K-ras gene using Pyrosequencing technology, because its reliable chemistry and robust detection mechanism allow for rapid, real-time detection of sequencing events. For the simultaneous detection of the predominant K-ras codons 12 and 13 mutations, we established a sequencing protocol based on the design of a single PCR primer pair and a single sequencing primer. The assay has been validated with DNA from 65 colorectal carcinomas. Furthermore, analysis of the rare K-ras codon 61 mutation was included. In 29% (19/65) of the patients, the K-ras gene was found to be mutated, whereas codons 12 and 13 were most frequently affected (18/65, 27.7%). Mutations with the highest frequency were G-->A transitions (12/19, 63%), followed by G-->T transversions (5/19, 26%). Overall survival was significantly shorter in patients with a tumor containing K-ras codon 12 mutations than in those without K-ras codon 12 mutations (p=0.024). In conclusion, we found Pyrosequencing to be a suitable technology for fast detection of hot-spot mutations in the K-ras oncogene. We demonstrated an important relationship between K-ras codon 12 mutations and overall survival in colorectal cancer patients.     

Detection of K-ras mutation in sputum by mutant-allele-specific amplification (MASA)

From 10 to 30% of lung carcinomas examined to date contain mutant K-ras genes. We report here that the mutant-allele-specific amplification (MASA) method may be useful for detection of the K-ras mutations in cells obtained from the sputum of patients with lung cancer. The PCR product from one of five patients revealed an alteration when mixed oligonucleotides representing variants of the second letter at codon 12 of this gene were used as 5′ primers, and further experiments showed a mutation of GGT (Gly) to GAT (Asp)at codon 12. The MASA system could also be applied to an examination of metastatic lung carcinomas, particularly from adenocarcinomas in colon and pancreas in which frequent K-ras mutations are detected, and to mass-screening for colorectal tumors using DNA isolated from feces as template. © 1993 Wiley-Liss, Inc.     

Genetic alterations in K-ras and p53 cancer genes in lung neoplasms from B6C3F1 mice exposed to cumene

The incidences of alveolar/bronchiolar adenomas and carcinomas in cumene-treated B6C3F1 mice were significantly greater than those of the control animals. We evaluated these lung neoplasms for point mutations in the K-ras and p53 genes that are often mutated in humans. K-ras and p53 mutations were detected by cycle sequencing of PCR-amplified DNA isolated from paraffin-embedded neoplasms. K-ras mutations were detected in 87% of cumene-induced lung neoplasms, and the predominant mutations were exon 1 codon 12 G to T transversions and exon 2 codon 61 A to G transitions. P53 protein expression was detected by immunohistochemistry in 56% of cumene-induced neoplasms, and mutations were detected in 52% of neoplasms. The predominant mutations were exon 5, codon 155 G to A transitions, and codon 133 C to T transitions. No p53 mutations and one of seven (14%) K-ras mutations were detected in spontaneous neoplasms. Cumene-induced lung carcinomas showed loss of heterozygosity (LOH) on chromosome 4 near the p16 gene (13%) and on chromosome 6 near the K-ras gene (12%). No LOH was observed in spontaneous carcinomas or normal lung tissues examined. The pattern of mutations identified in the lung tumors suggests that DNA damage and genomic instability may be contributing factors to the mutation profile and development of lung cancer in mice exposed to cumene.     

Four new mutations in the DNA mismatch repair gene MLH1 in colorectal cancers with microsatellite instability

Hereditary nonpolyposis colorectal cancer (HNPCC) is frequently associated with inherited mutation in one of four DNA mismatch repair genes. Somatic mutations in the same genes are also found in a subset of sporadic colorectal cancers. A defect in DNA mismatch repair results in an RER (replication error) tumor phenotype. We screened 110 archival and 11 prospectively acquired colorectal cancers for the RER phenotype. A total of 22 cancers were RER-positive. RER-positive tumors were investigated for mutations in the DNA mismatch repair gene MLH1 using single-strand-conformation-polymorphism (SSCP) analysis. We identified four previously undescribed mutations in four different samples. Three mutations were exonic: a point mutation at codon 69 (AGG→AAG [arg→lys]); a single base pair deletion at codon 42/43 (GCAAAATCC→GCAAATCC) leading to a new stop codon downstream; and a point mutation at codon 757 (TAA→TAT) [termination→tyr] which extend the MLH1 peptide by 36 amino acids. The fourth mutation was a 1 base pair insertion six base pairs 5′ to the start of exon 14 (tttgtttt→tttggtttt). The mutations were not seen in the patients' constitutional DNA. The somatic MLH1 mutations identified appear to be causally associated with the RER phenotype. Hum Mutat 12:73, 1998. © 1998 Wiley-Liss, Inc.     

Ras gene activation in malignant cells of human ovarian carcinoma peritoneal fluids

In this study, we showed, for the first time, the pattern of point mutations at codon 12 of the K-ras, H-ras and N-ras genes, using polymerase chain reaction and restriction fragment length polymorphism analysis in 47 malignant cytologic specimens of ovarian adenocarcinoma peritoneal fluids. Forty-seven % of the samples were found to carry a point mutation at codon 12 of K-ras gene. Also, 21 cystadenoma peritoneal fluids were used as control specimens for the detection of ras mutations. Fourteen % of these samples were found to carry a point mutation at codon 12 of the K-ras gene. The prevalence of K-ras gene mutations were statistically correlated with FIGO and surgical stage of the malignant specimens. Our data demonstrates that the K-ras gene mutations are mainly affected (47%) in the malignant cells of the peritoneal washings or ascites of women with ovarian adenocarcinomas and may have value for the early diagnosis and monitoring of these neoplasms.     

K-ras point mutations in routinely processed tissues: non-radioactive screening by single strand conformational polymorphism analysis.

AIMS--To develop a non-radioactive method to screen routinely fixed, paraffin wax embedded specimens for the occurrence of point mutations; to evaluate the single strand conformational polymorphism (SSCP) analysis technique for the detection of K-ras point mutations as a result of electrophoretic mobility shifts. METHODS--DNA was extracted from archival specimens of colon cancer and from established colon cancer cell lines with known point mutations. A K-ras gene fragment containing codons 12 and 13 of exon 1 was amplified with the polymerase chain reaction (PCR). Denatured DNA fragments were run on 10% polyacrylamide gels under non-denaturing conditions. After electrophoresis DNA was blotted and the single stranded DNA was detected using a digoxigenin labelled ras probe. The nature of the detected point mutations was identified and confirmed by sequencing and hybridisation with oligonucleotides using 32P labelling. RESULTS--Wild type and aberrant alleles were detected caused by mobility shifts after electrophoresis of the PCR products. Commonly occurring mutations in the K-ras gene--in the first two positions of codon 12--could easily be detected in DNA from archival paraffin wax embedded colon cancer tissue. In all the colon tumour samples studied wild type gene alleles were also found, presumably derived from normal cells in the specimen. CONCLUSIONS--The SSCP method permits rapid non-radioactive screening of adenomas or carcinomas for the occurrence of point mutations in the K-ras gene. But if a mutation is detected by an electrophoretic mobility shift, its identification requires confirmation by sequencing or oligonucleotide hybridisation.     

Analytical performance of a PCR assay for the detection of KRAS mutations (codons 12/13 and 61) in formalin-fixed paraffin-embedded tissue samples of colorectal carcinoma

KRAS mutation testing is mandatory before prescribing anti-epidermal growth factor monoclonal antibodies in the treatment of advanced colorectal cancer. We describe the performance of a TaqMelt polymerase chain reaction (PCR) assay—the cobas? KRAS Mutation Test—designed to detect 19 mutations in codons 12, 13, and 61. The limit of detection was determined using DNA blends from cell lines, plasmids, and formalin-fixed paraffin-embedded tissue specimens. Assay performance was compared to Sanger sequencing using a panel of 188 specimens. Discordant specimens were subjected to next generation pyrosequencing (454). Assay repeatability was assessed using a panel of six specimens. A >95% correct mutation call rate was obtained in all specimen types with ~5% mutant alleles at DNA inputs of 0.8–6.3 ng per PCR reaction; 100% detection rate was observed at the recommended DNA input of 50 ng. The positive percent agreement with Sanger was 97.5% (79/81) for codons 12/13 and 85.7% (6/7) for codon 61. Negative percent agreement was 94.4% (101/107) for codon 12/13 and 99.4% (180/181) for codon 61. Nine of 10 discordant specimens yielded 454 results consistent with the cobas? results. With repeated testing, the assay showed a correct call rate of 100% (192/192) for all operators, instruments, reagent lots, and days tested. The cobas? test detects KRAS mutations in codons 12, 13, and 61 at a limit of detection of <5%. The PCR assay was more sensitive and specific than Sanger sequencing, and performance was highly reproducible. Test performance was not influenced by various endogenous interfering substances or common gut microbes.