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

目的 以特异引物双扩增即时PCR技术K-ras检测试剂盒(下称ADx-K-ras即时PCR试剂盒)和Sanger DNA测序法同时检测结直肠癌和肺癌患者K-ras基因,以了解K-ras基因突变频率和突变类型,比较ADx-K-ras即时PCR试剂盒和Sanger DNA测序法用于肿瘤K-ras基因体细胞突变检测的临床价值.方法 收集临床肿瘤病理石蜡切片样品827例,其中肠癌样品583例,肺癌样品244例,提取DNA后,对K-ras基因第12和13密码子进行PCR扩增后,使用ADx-K-ras即时PCR试剂盒进行检测,与此同时,将PCR扩增后的产物进行Sanger DNA测序.两种方法对K-ras基因第12和13密码子的检测结果进一步进行各个突变类型的数目和突变率的统计对比.结果 ADx-K-ras即时PCR试剂盒对827例样品检测都得到了明确的结果,检测成功率为100%,Sanger DNA测序成功检测了677例,成功率为81.9%.583例肠癌样品中ADx-K-ras即时PCR试剂盒检测出突变192例,突变检出率为32.9%,Sanger DNA测序成功的样品533例,检出突变160例,突变检出率为30.0%.244例肺癌样品中ADx-K-ras即时PCR试剂盒检出突变26例,突变检出率为10.7%,Sanger DNA测序成功的样品144例,检出突变12例,突变检出率为8.3%.肠癌中第12密码子第2位的GGT→GAT最常见,占全部突变的35.1%(66/188),其次是第13密码子第2位的GGC→GAC,26.6%(50/188),第12密码子第2位的GGT→GTT,18.6%(35/188),第12密码子第1位的GGT→GCT最少见,1.6%(3/188).肺癌中第12密码子第1位的GGT→GTT最常见,占全部突变的40.9%(9/22),同样第12密码子第1位的GGT→GCT最少见,占全部突变的4.5%(1/22).结论 肠癌中K-ras突变率明显高于肺癌.对于甲醛固定石蜡包埋样品而言,ADx-K-ras即时PCR试剂盒对样品的DNA质量的耐受性较好,检测成功率高于Sanger DNA测序,可替代Sanger DNA测序法成为临床上对肿瘤K-ras基因体细胞突变检测的实用方法.     

结直肠癌原发灶与肝转移灶K-ras基因状态的研究

目的观察K-ras基因在结直肠癌原发灶及肝转移灶的突变状态。方法采用富集PCR配对测序法检测46例（男35例,女11例）结直肠癌肝转移患者原发灶及肝转移灶K-ras基因突变状态,其中结肠癌肝转移21例（45.7%）,直肠癌肝转移25例（54.3%）。结果46例中有18例（39.1%）原发灶K-ras基因为突变型,其中17例对应的肝转移灶K-ras基因为突变型,1例对应的肝转移灶K-ras基因为野生型。28例（60.9%）原发灶K-ras基因为野生型者中,26例对应的肝转移灶K-ras基因为野生型,2例对应的肝转移灶K-ras基因为突变型。统计分析表明,原发灶与肝转移灶K-ras基因突变状况差异无统计学意义（P=1.00）。结论转移性结直肠癌原发灶与肝转移灶的K-ras基因突变状态差异无统计学意义。     

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.     

High-degree tumor budding and podia-formation in sporadic colorectal carcinomas with K-ras gene mutations

In vitro ras activation enhances the epithelial-mesenchymal transition of colorectal carcinoma cells. But ras effects are known to be highly dependent on cell types and the tissue context. Therefore, this study was made to test the hypothesis that in clinical colorectal carcinoma specimens, aggressive invasion phenotypes, specifically tumor budding and podia formation, would correlate with K-ras gene mutations. In a series of 95 clinically sporadic primary colorectal carcinomas collected ad hoc, tumor budding and podia formation were counted using pan-cytokeratin immunohistochemistry, and K-ras gene mutations in codons 12 and 13 were determined. Consistent with the hypothesis, tumor budding and podia formation were observed to be significantly higher in the 32 (34.7%) of the tumors with K-ras gene mutations (29 mutations in codon 12, 3 in codon 13), and this correlation was observed independent of the patterns of invasion (expansive versus infiltrative). Microsatellite status, numbers of losses of heterozygosity, adenomatous polyposis coli and p53 gene mutations, and degree of promoter methylations (CIMP status) were not associated with K-ras gene mutations. Besides their effects on the tumor cell cycles, oncogenic K-ras gene mutations in colorectal carcinomas could be important for aggressive tumor invasion. This may be important in metastasizing disease and could provide a rationale for developing drugs that interrupt ras-signaling cascades.     

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。     

APC gene loss of heterozygosity, mutations, E1317Q, and I1307K germ-line variants in sporadic colon cancer in Croatia

Colorectal carcinomas are characterized by multiple genetic aberrations that occur during tumorigenesis. Several tumor suppressor genes associated with colorectal carcinoma have been identified: MCC, APC, p53, nm23-H1, DCC, DPC4. We examined 73 cases of sporadic human colon cancer and corresponding normal tissue samples to evaluate the loss of heterozygosity (LOH) at the APC gene loci. The purpose of this study was also to evaluate whether the LOH at the APC gene is associated with clinicopathological characteristics in sporadic colon cancer. We also investigated presence and the frequency of the most common APC gene mutations and APC E1317Q and I1307K germ-line variants in Croatian colorectal cancer patients. Five markers in all patients were found to be heterozygous and informative for LOH analysis. LOH at the APC locus was detected in 30.1% of tumors were examined. The majority of APC gene LOH was observed in Dukes' B (55.6%) and in the moderately differentiated tumors (42.9%). Only 1309 APC gene mutation was detected in our samples. In one tumor sample, a new sporadic mutation of the APC gene in codon 1374 was detected. APC E1317Q and I1307K germ-line variants were not detected in our population. But APC E1317Q sporadic mutation was found in one tumor sample.     

Germline mutations but not somatic changes at the MYH locus contribute to the pathogenesis of unselected colorectal cancers

MYH-associated polyposis is a recently described, autosomal recessive condition comprising multiple colorectal adenomas and cancer. This disease is caused by germline mutations in the base excision repair (BER) gene MYH. Genes involved in the BER pathway are thus good candidates for involvement in the pathogenesis of sporadic tumors of the large bowel. We have screened a set of 75 sporadic colorectal cancers for mutations in MYH, MTH1, and OGG1. Allelic loss at MYH was also assessed. Selected samples were screened for mutations and allele loss at APC and mutations in p53, K-ras, and beta-catenin. A panel of 35 colorectal cancer cell lines was screened for MYH mRNA and protein expression. One of 75 cancers had bi-allelic germline mutations in MYH and on retrospective analysis of medical records this patient was found to have synchronous multiple small adenomas in addition to carcinoma. No somatic MYH mutations were found and mRNA and protein were expressed in all of our cell lines. There were no clearly pathogenic mutations in MTH1 or OGG1 in any tumor. Bi-allelic germline MYH mutations cause approximately 1 to 3% of unselected colorectal cancers, but appear always to be associated with multiple adenomas. Somatic inactivation of the DNA glycosylases involved in the BER pathway however does not appear to be involved in colorectal tumorigenesis.     

p53 and K-ras gene mutations in carcinoma of the rectum among Finnish women.

AIMS/BACKGROUND: The aim of this study was to identify p53 and K-ras gene mutations in carcinoma of the rectum among Finnish women. Mutation patterns might give clues to aetiological factors when comparisons are made with other human tumours. METHODS: Of 134 women with carcinoma of the rectum, paraffin wax embedded specimens of the tumour tissue were obtained from 118 patients. Genomic DNA was extracted, and exons 4-8 of the p53 gene and codons 12/13 and 61 of the K-ras gene were amplified, and analysed for mutations by single strand conformation polymorphism and direct sequencing. The production of p53 and K-ras proteins was studied by immunohistochemistry. RESULTS: The overall crude frequency for mutations in the p53 gene was 35% but the true frequency appears to be higher (up to 56%). In the K-ras gene, the mutation frequency (15%) was significantly lower than that reported for colon cancer. In the p53 gene, the mutation frequency increased significantly with patient age. In a high proportion of patients (14%) the rectal tumours contained small subclones of tumour cells that displayed extremely rare mutations at codons 110 and 232 of the p53 gene. Hot spot codon 175 mutations were significantly less common in rectal cancer than in cancer of the colon. CONCLUSIONS: Rectal cancer among Finnish women has characteristics in the mutations of the p53 and K-ras genes that are uncommon in other human tumours, including cancer of the colon. A biological explanation of these findings is not clear at present, but might be associated with an unidentified genetic factor in Finland.     

K-ras mutations and p53 alterations in neoplastic and nonneoplastic lesions associated with longstanding ulcerative colitis.

We have analyzed K-ras mutations and p53 alterations in 39 tumor and nontumor samples taken from nine patients with longstanding ulcerative colitis and colorectal carcinoma. Two of nine invasive carcinomas contained a K-ras mutation. By a combination of immunohistochemistry and single-strand conformation polymorphism analysis, p53 alterations were found in three of nine carcinomas. Five of 13 dysplastic lesions harbored a mutated K-ras gene, even in the absence of detectable changes in associated invasive tumors. One single focus of dysplastic mucosa harbored concomitant K-ras and p53 gene alterations. In two patients, a K-ras mutation was detected in epithelial lesions considered to be devoid of malignant potential (villous regeneration, active colitis). Our results indicate that: 1) the prevalence of K-ras and p53 genetic alterations found in ulcerative colitis-associated colonic carcinomas appears to be lower than in sporadic carcinomas; 2) K-ras mutations can be detected in dysplasia, villous regeneration, and active colitis and affect a subpopulation of the cells composing the lesions; 3) diverse genetic alterations can be detected in the same patient and the dysplastic lesions can exhibit a different genotype than the carcinomas; and 4) at least part of active colitis and villous regeneration lesions should be considered as preneoplastic in ulcerative colitis.     

Loss of heterozygosity of DPC4 tumor suppressor gene in human sporadic colon cancer

We investigated the prevalence of DPC4 loss of heterozygosity in sporadic colorectal cancer. Thirty-six cases of human sporadic colon carcinoma and corresponding normal tissue samples were examined to evaluate loss of heterozygosity at the DPC4 tumor suppressor locus using variable nucleotide tandem repeat (VNTR) analysis and three polymorphic markers. From 36 analyzed samples 35 (97%) were heterozygous or informative. Loss of heterozygosity at the DPC4 locus was detected in 18 (51%) of informative tumor DNAs. The DPC4 LOH was more frequent in smaller tumors (<5 cm) than in larger ones. There was no correlation between DPC4 LOH and age or sex of patients. There was a negative correlation between DPC4 LOH and histological grade or Dukes' stage of tumors, but without statistic significance. Observed results are in agreement with the view that malignant progression is consequence of many genetic changes. It can be concluded that inactivation of the DPC4 gene plays a role in a multistep process of outgrowth and progression of colon cancer.     

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.     

Evaluation of K-ras 12 oncogene mutations in Venezuelan patients with Helicobacter pylori infection

This work evaluated the infection of H. pylori in the different gastric pathologies and its association with the oncogen K-ras 12. Endoscopy was performed in 62 patients and 3 biopsies from the antral region were taked and used for the histological diagnostic, PCR, and point mutations determination. The results showed a high incidence of H. pylori infection in patients with active chronic gastritis (AcCG) 90%, chronic atrophic gastritis (AtCG) 70%, intestinal metaplasia (IM) 67%, dysplasia (D) 83%, and decrease in in gastric cancer (GC) 33%. Evaluation of the oncogen K-ras 12 showed that 68% of the patients presented mutations in the different analyzed amino acids. In the 12 codon of the K-ras gene, we observed simple point mutations and combination in the same sample in different gastric pathologies. In AcCG samples were detected the greater number of mutations. A decrease of the point mutations were observed in the progression stages to gastric cancer. The presence of these specific mutations would be tumor markers and it determine the possible development of gastric tumors.     

Microsatellite instability and K-ras mutations in patients with ulcerative colitis

Patients with inflammatory bowel disease (IBD), particularly ulcerative colitis (UC), have an increased incidence of colorectal carcinoma. The underlying mechanism is unknown, but we postulated that microsatellite instability (MSI) might predispose the colonic mucosa of UC patients to mutations, thereby increasing their cancer risk. We also sought to determine the frequency of K-ras mutations, to determine whether MSI predisposed to K-ras mutations and to compare the molecular phenotype of biopsy and resection specimens in the same patient. We also sought to determine whether molecular alterations found in biopsy specimens presaged their presence in subsequent resection specimens. Two hundred fifty-eight specimens from 52 patients were examined for K-ras mutations by direct sequencing. Seventy-one of the specimens were neoplastic. MSI was evaluated after polymerase chain reaction (PCR) amplification using primers directed at 8 microsatellite loci. Of the patients, 18.2% had K-ras mutations, and 30.8% had MSI in at least 1 locus. Of K-ras mutations, 81.8% were G to A substitutions involving the second nucleotide of codons 12 or 13. Only 0.7% of the samples showed a high level of MSI. No relationship existed between MSI and K-ras mutations, even in the 2 samples with high-level MSI. The numbers are small, but it appeared that MSI in biopsies failed to predict its presence in resection specimens. In contrast, K-ras mutations present in biopsy specimens tended to predict their presence in resections. K-ras mutations were found predominantly in neoplastic mucosae, whereas MSI was found predominantly in regenerative mucosae. The lack of any relationship between MSI and K-ras mutations suggests that MSI in the UC replicative mucosa does not predispose to colonic neoplasia via a K-ras-mediated pathway. This is probably related to the fact that the MSI is generally low-level MSI.     

Inverse relationship between microsatellite instability and K-ras and p53 gene alterations in colon cancer

Some studies have shown an inverse relationship between microsatellite instability in colon cancer and mutations in p53 and K-ras, whereas others have not. We therefore evaluated these features in a population-based sample of 496 individuals with colon cancer. Microsatellite instability was determined by a panel of 10 tetranucleotide repeats, the Bethesda consensus panel of mono- and dinucleotide repeats, and coding mononucleotide repeats in transforming growth factor-beta receptor type II, hMSH3, BAX, hMSH6, and insulin-like growth factor receptor type II. Mutations in codons 12 and 13 in K-ras were evaluated by sequencing. p53 overexpression (as detected by immunohistochemistry) was used as an indicator of p53 mutation; this was evaluated in 275 of the tumors. K-ras mutations were present in 33.2% of tumors, p53 overexpression in 51.5%, and microsatellite instability (as determined by the Bethesda consensus panel) in 12.5%. K-ras mutations were significantly less common in unstable tumors than stable tumors (11.8% versus 36.9%, P: < 0.001). p53 overexpression was significantly less common in unstable tumors than stable tumors (20.0% versus 55.7%, P: < 0.001). These inverse relationships between microsatellite instability and ras gene mutations and p53 overexpression were shown to be independent of tumor site in logistic regression analyses. All other measures of instability also showed statistically significant inverse relationships independent of tumor site with alterations in ras and p53, and instability results determined by the panel of 10 tetranucleotide repeats were highly significantly related to those determined by the Bethesda consensus panel. Coding mononucleotide repeat mutations were significantly more common in unstable tumors than stable tumors (85.7% versus 1.0%, P: < 0.001). We conclude that there is an inverse relationship between microsatellite instability and mutations in p53 and K-ras, and that the molecular profile of colon cancers with microsatellite instability is characterized by relatively infrequent mutations in K-ras and p53 and relatively frequent mutations in coding mononucleotide repeats.     

Detection of K-ras mutations in colon cancer by PHA-PHFA (preferential homoduplex formation assay)

The mutations of K-ras gene have been demonstrated at frequencies of about 40% in human colorectal cancer. We applied a developed PCR-preferential homoduplex formation assay (PCR-PHFA) to detect a point mutation of K-ras gene in the surgical specimens from thirty patients with colorectal cancer. This method is based on the strand competition during hybridization between a double labeled amplicon, prepared from biotin and DNP labeled primers, and an unlabeled amplicon. The procedure of this method is simple and speedy, and suitable to detect mutations in a small number of samples. By using this method, the mutations were found in 37% (11/30) and confirmed by sequencing analysis. The results suggest that the PCR-PHFA system may be useful for detecting low frequent mutation of K-ras gene even in the case of an early cancer.     

Intratumor heterogeneity of K-ras2 mutations in colorectal adenocarcinomas: association with degree of DNA aneuploidy.

Detailed information about intratumor K-ras2 mutations in colorectal adenocarcinomas and a possible association with DNA content heterogeneity is still lacking. DNA diploid and aneuploid subclones, detected among multiple histologically selected primary sectors (57 superficial and 40 deep) and 9 lymph node metastases, were flow cytometrically sorted and separately submitted to codons 12-13 K-ras2 mutation spectrum analysis. DNA aneuploidy was absent among 20 near and 20 distant mucosa sites and present in 7/9 lymph node metastases and in 17/19 primary tumors (90%). Primary intratumor DNA multiclonality was approximately 50%. Degree of DNA aneuploidy (DNA Index) distribution was nonrandom and showed peaks at approximate mean DNA Index values 1.2, 1.5, and 1.8. K-ras2 mutations were detected in 0/20 mucosa cases, in 2/9 lymph node metastases, and in 9/19 adenocarcinomas (47%). No more than one mutation type per tumor was detected. Intratumor distribution of K-ras2 mutations was homogeneous in 6 and heterogeneous in 3 cases. Homogeneous distribution was associated with DNA near-diploid aneuploidy. K-ras2 mutations were strongly associated with DNA Index in the near-diploid region (83%) and almost absent (5%) among DNA near-triploid subclones (P = 0.0001). K-ras2 mutation intratumor heterogeneity indicates that sampling of the tumor may be a critical step and suggests that K-ras2 activation may be a late event in a subgroup of tumors. Our data also suggest the existence of an early process of the colorectal carcinogenesis that favors both K-ras2 mutations and DNA near-diploid aneuploidy. Onset of DNA near-triploid subclones appears, instead, to be independent from K-ras2 activation.     

Mutation analysis of K-ras and beta-catenin genes related to O6-methylguanin-DNA methyltransferase and mismatch repair protein status in human gallbladder carcinoma

O6-methylguanin-DNA methyltransferase (MGMT) is a DNA repair enzyme that transfers methyl groups from O6-methylguanine to itself. Alkylation of DNA at the O6 position of guanine is the first step by alkylating agents in inducing DNA mutations in an organism. When MGMT and the mismatch repair (MMR) system are impaired, O6-methylguanine mispairs with thymine during DNA replication, resulting in a G:C right curved arrow A:T transitional mutation in DNA. We obtained cancer lesions by manual micro-dissection (MMD) from 26 paraffin-embedded formalin-fixed gallbladder carcinoma and Laser Capture Micro-dissection (LCM) method from 10 fresh frozen specimens. Mutation analysis was performed on the micro-dissected samples for K-ras and beta-catenin genes. At codon 12 of the K-ras gene, the MMD and LCM methods detected mutations in 3 (11.5%) and 1 (10%) case, respectively. In exon 3 of beta-catenin gene, only 1 (3.8%) case revealed a mutation in MMD cancer foci. Two cases without MGMT or MMR expression revealed a G right curved arrow A transition mutation in the K-ras gene. The findings suggested that negative MGMT and MMR status contributed to a G:C right curved arrow A:T transitional mutation in the K-ras gene. However, K-ras and beta-catenin mutations were actually rare in GB carcinoma. Other gene mutations frequently occurring in gallbladder carcinoma might be affected by this negative MGMT and MMR status.     

Evidence for a common molecular pathogenesis in colorectal, gastric, and pancreatic cancer.

We examined tissue extracted from 19 gastric, 7 pancreatic, and 23 colorectal carcinoma specimens to determine the comparative incidence of allele loss on chromosomes 5, 17, and 18 and that of KRAS2 point mutations. Chromosome 5 allele loss occurred at the same frequency in all three gastrointestinal tumors (approximately 30%), whereas chromosome 17 and 18 allele losses were seen at a significantly lower frequency in gastric (20%) and pancreatic (0%) malignancies than in colorectal cancer (57%). Point mutations in KRAS2 were seen in 83% of pancreatic and 52% of colon cancers, but not in gastric cancer specimens. In pancreatic tumors, these mutations were always found in the second nucleotide of codon 12. In colorectal cancer, the distribution was more variable, involving the second nucleotide of codon 13 and both the first and second nucleotides of codon 12. These results suggest that inactivation of the adenomatous polyposis coli gene on chromosome 5 may be an initiating step for carcinomas of the stomach and pancreas as well as of the colon, but that the genes involved in tumor progression events may be tissue- or tumor-specific.