Frequent k- ras -2 mutations and p16(INK4A)methylation in hepatocellular carcinomas in workers exposed to vinyl chloride

Vinyl chloride (VC) is a know animal and human carcinogen associated with liver angiosarcomas (LAS) and hepatocellular carcinomas (HCC). In VC-associated LAS mutations of the K- ras -2 gene have been reported; however, no data about the prevalence of such mutations in VC associated HCCs are available. Recent data indicate K- ras -2 mutations induce P16 methylation accompanied by inactivation of the p16 gene. The presence of K- ras -2 mutations was analysed in tissue from 18 patients with VC associated HCCs. As a control group, 20 patients with hepatocellular carcinoma due to hepatitis B (n = 7), hepatitis C (n = 5) and alcoholic liver cirrhosis (n = 8) was used. The specific mutations were determined by direct sequencing of codon 12 and 13 of the K- ras -2 gene in carcinomatous and adjacent non-neoplastic liver tissue after microdissection. The status of p16 was evaluated by methylation-specific PCR (MSP), microsatellite analysis, DNA sequencing and immunohistochemical staining. All patients had a documented chronic quantitated exposure to VC (average 8883 ppmy, average duration: 245 months). K- ras -2 mutations were found in 6 of 18 (33%) examined VC-associated HCCs and in 3 cases of adjacent non-neoplastic liver tissue. There were 3 G --> A point mutations in the tumour tissue. All 3 mutations found in non-neoplastic liver from VC-exposed patients were also G --> A point mutations (codon 12- and codon 13-aspartate mutations). Hypermethylation of the 5' CpG island of the p16 gene was found in 13 of 18 examined carcinomas (72%). Of 6 cancers with K- ras -2 mutations, 5 specimens also showed methylated p16. Within the control group, K- ras -2 mutation were found in 3 of 20 (15%) examined HCC. p16 methylation occurred in 11 out of 20 (55%) patients. K- ras -2 mutations and p16 methylation are frequent events in VC associated HCCs. We observed a K- ras -2 mutation pattern characteristic of chloroethylene oxide, a carcinogenic metabolite of VC. Our results strongly suggest that K- ras -2 mutations play an important role in the pathogenesis of VC-associated HCC.     

Analysis of p53 mutations in histologically normal lung tissues and lung tumors from non‐small cell lung cancer patients

Our previous study showed a characteristic p53 mutational spectrum in lung tumors from lung cancer patients in the Western Pennsylvania region. To further understand the involvement of p53 mutations in lung tumor development, in this study we compared p53 mutational spectra and distribution between tumor cells taken from lung tumor tissue and histologically normal cells taken from tumor‐surrounding tissue obtained from 122 lung cancer patients [67 adenocarcinomas (ACs) and 55 squamous cell carcinomas (SCCs)]. Overall, mutations were detected in exons 5–8 of the p53 gene in cell samples from 39.3% (48/122) of the patients. Twenty‐four mutations were found among the ACs (35.8%, 24/67) and consisted mostly of G to T transversions at codon 248 in either only the tumor tissue (12 cases, 50%), or only the histologically normal tissue (2 cases, 8.3%), or both tissue types (10 cases, 41.7%). Among the SCCs, 24 mutations of both transition and transversion types were detected at multiple codons in either only the tumor tissue (17 cases, 70.8%), or only the histologically normal tissue (3 cases, 12.5%), or both tissues (4 cases, 16.7%). Overall, the distribution of mutations among the tumor tissue and histologically normal tissue was not significantly different between the ACs and SCCs (P > 0.05). In both groups, the mutations in the histologically normal tissue may be identical to or different from those in the tumor tissue. Therefore, p53 mutations are frequent in tumor‐surrounding histologically normal tissue, and some of them might be involved in lung carcinogenesis. © 2008 Wiley‐Liss, Inc.     

Effects of K-ras Gene Mutations in the Development of Lung Lesions Induced by 4-(N- Methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone in A/J Mice

The relationship between the development of peripheral lung lesions induced by tobacco-specific 4-( N -methyl- N -nitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and K- ras gene mutation in A/J mice, and the correlations between histological alterations and the course of lung lesion development after NNK treatment and K- ras gene mutation were investigated. The acquisition of a selective growth advantage by the lung lesions with mutations was also examined using immunohistochemical labeling with bromodeoxyuridine. Thirty female 5 weeks old A/J mice were each injected intraperitoneally with a single dose of NNK (100 mg/kg body weight) and subdivided into 6 groups according to the time after NNK treatment. The lung lesions were characterized histologically as alveolar/bronchiolar hyperplasia, adenoma and adenocarcinoma, and point mutations in codons 12 and 61 of the K- ras gene were detected by polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) and dideoxy sequencing methods. K- ras gene mutations were identified in 7 (58.3%) of 12 hyperplasias, 42 (75.0%) of 56 adenomas and 3 (75.0%) of 4 adenocarcinomas. The most frequent K- ras gene mutation was a G-to-A transition at the second base of codon 12 and this accounted for 86.5% of all the mutations detected. Neither the frequency of activation of this gene nor the specific mutation was affected by the time after NNK treatment and there was no positive correlation between the proliferative activity of lung lesions and the presence of K- ras gene mutations. Thus, K- ras gene mutation is closely associated with the development of NNK-induced peripheral lung lesions in A/J mice, but it plays no role in the selective growth advantage of these lesions.     

EGFR tyrosine kinase domain mutations are detected in histologically normal respiratory epithelium in lung cancer patients

To determine whether EGFR tyrosine kinase domain mutations are early events in the pathogenesis of lung adenocarcinomas, we tested for the presence of EGFR mutations in histologically normal bronchial and bronchiolar epithelia from lung adenocarcinomas bearing the common EGFR mutations. DNA was extracted from microdissected tissue obtained from 21 tumors with known EGFR mutations, 16 tumors without mutation, and 90 sites of normal bronchial and bronchiolar epithelium from the same surgical specimens. With the use of PCR and direct DNA sequencing, EGFR mutations identical to the tumors were detected in the normal respiratory epithelium in 9 of 21 (43%) patients with EGFR mutant adenocarcinomas but none in patients without mutation in the tumors. The finding of mutations being more frequent in normal epithelium within tumor (43%) than in adjacent sites (24%) suggests a localized field effect phenomenon. Our findings indicate that mutation of the tyrosine kinase domain of EGFR is an early event in the pathogenesis of lung adenocarcinomas, and suggest EGFR mutations as an early detection marker and chemoprevention target.     

Differential activation of ras genes by point mutation in human colon cancer with metastases to either lung or liver

To study the possible role of ras oncogene activation in the dissemination of colon cancer, we determined point mutations in codons 12, 13 and 61 in K- and N-ras in 3 groups of tumors: (A) primary tumors of patients who had undergone surgery for Dukes' B (early-stage) colon cancer, (B) primary tumors and metastases from patients undergoing resection of isolated lung metastases and (C) primary tumors and metastases from patients undergoing resection of isolated liver metastases. In 129 samples from 93 patients, 54 (42%) were positive for point mutations in either K- or N-ras. Most mutations (89%) were found in the K-ras gene. In group A (n = 50) ras point mutations were detected in 16 cases (32%) (15 in K-ras and 1 in N-ras). Thirteen out of 23 cases in group B (57%) were positive for a ras point mutation: 10 in K-ras and 3 in N-ras. In group C (n = 20), point mutations in codon 12 of K-ras, but none in H- or N-ras, were found in 10 cases (50%). In 31 cases the primary tumors from the metastases in groups B and C were available for analysis and 15 contained a ras point mutation (48%). Not all mutations were present in both the primary tumor and the metastasis. In 3 instances, a mutation was detected in the metastasis but not in the primary tumor, whereas in 1 case a mutation was found in the primary tumor.     

Detection of ras gene mutations in human lung cancers by single-strand conformation polymorphism analysis of polymerase chain reaction products

A simple, sensitive method of DNA analysis of nucleotide substitutions, namely, single-strand conformation polymorphism analysis of polymerase chain reaction products (PCR-SSCP analysis), was used for detection of mutated ras genes in surgical specimens of human lung cancer. Of a total of 129 tumors analysed, 22 contained a mutated ras gene. Of the 66 adenocarcinomas analysed, 14 contained an activated c-Ki-ras2 gene (the mutations in codon 12 in 6, in codon 13 in 4, in codon 18 in one, and in codon 61 in 3), one contained a c-Ha-ras1 gene with a mutation in codon 61 and 3 contained N-ras genes with mutations (in codon 12 in one and in codon 61 in 2). Mutated rats genes were also found in 2 of 36 squamous cell carcinomas (c-Ha-ras1 genes with mutations in codon 61) and 2 of 14 large cell carcinomas (c-Ki-ras2 genes with mutations in codon 12). No mutation of the ras gene was detected in 8 small cell carcinomas and 5 adenosquamous cell carcinomas. These results indicate that activation of the ras gene was not frequent (17%) in human lung cancers, that among these lung cancers mutation of the ras gene was most frequent in adenocarcinomas (27%) and 73% of the point mutations were in the c-Ki-ras2 gene in codon 12, 13, 18 or 61.     

K-ras activation in premalignant and malignant epithelial lesions of the human uterus

We previously reported (Cancer Res., 50:6139-6145, 1990) a significant frequency of activating point mutations in codon 12 of the K-ras oncogene in endometrial adenocarcinomas of the uterine corpus (series 1). To further define the role of ras activation in the development of endometrial adenocarcinoma, we surveyed cystic, adenomatous, and atypical hyperplasias of uterine endometrium and additional cases of endometrial and cervical carcinoma (series 2) for the presence of activating mutations in cellular protooncogenes of the ras family. Polymerase chain reaction was performed from deparaffinized sections of formalin-fixed paraffin-embedded tissue. We screened for point mutations in codons 12, 13, and 61 of the K-, H-, and N-ras genes by dot blot hybridization analysis with mutation-specific oligomers. Mutations in K-ras were also confirmed by direct genomic DNA sequencing. Of 19 endometrial adenocarcinomas in series 2, point mutations in ras genes were found in 7 tumors. Six contained single-base substitutions, five in codon 12 of K-ras and one in codon 12 of N-ras. The seventh tumor contained two different point mutations in codon 12 of K-ras. In one endometrial adenocarcinoma, tumor cells with point mutations in K-ras were predominantly localized to a portion that had a more aggressive histological pattern. In endometrial hyperplasia, K-ras mutations, one in codon 12 and one in codon 13, were found in 2 of 16 hyperplasias histologically classified as atypical and clinically considered premalignant. None of 6 adenomatous hyperplasias and none of 12 cystic hyperplasias, the latter of which is considered clinically benign, contained any detectable ras mutations. No mutations in H-ras were detected in either carcinomas or hyperplastic tissue.     

Ras-related TC21 is activated by mutation in a breast cancer cell line, but infrequently in breast carcinomas in vivo.

Activating ras mutations are found in many types of human tumour. Mutations in Harvey (H-), Kirsten (K-) and neuronal (N-) ras are, however, rarely found in breast carcinomas. TC21 is a ras family member that shares close homology to H-, K- and N-ras, and activating mutations have been found in ovarian carcinoma and leiomyosarcoma cell lines. We have examined panels of cDNAs from breast, ovarian and cervical cell lines, and primary and metastatic breast tumours for mutations in TC21 using a single-strand conformational polymorphism (SSCP)-based assay. One breast cancer cell line, CAL51, exhibited an altered SSCP pattern, compared with normal tissue, which was due to an A-T base change in codon 72, causing a predicted Gln-Leu activating mutation. Of nine primary and 15 metastatic breast tumour cDNAs analysed, none exhibited an altered pattern by SSCP. The apparently wild-type pattern by SSCP analysis was confirmed by sequence analysis of some of the cDNAs assayed. Thus, we conclude that mutations in TC21 are uncommon in breast carcinomas.     

EGFR mutation analysis in sputum of lung cancer patients: A multitechnique study

Objectives

Epidermal growth factor receptor (EGFR) mutations have been identified in lung adenocarcinomas and are associated with high response chance to EGFR tyrosine kinase inhibitors. EGFR mutations can be detected in tumour tissue, cytology specimens and blood from lung cancer patients. Thus far, EGFR mutation analysis has not been systematically demonstrated for sputum samples. The aim of the present study was to determine whether EGFR mutation analysis is attainable on sputum samples, employing different assays in a multicenter study.

Materials and methods

Sputum DNA from 10 lung cancer patients with confirmed EGFR mutation in their tumour tissue, 10 lung cancer patients without evidence of an EGFR mutation, and 10 patients with chronic obstructive pulmonary disease (COPD) was used for mutation analysis by Cycleave PCR, COLD-PCR, PangaeaBiotech SL Technology (PST), and High Resolution Melting, respectively. Targeted resequencing (TruSeq Amplicon Cancer Panel) and droplet digital PCR were additionally performed on the 10 samples with EGFR mutation.

Results

Dependent on the assay, EGFR mutations could be detected in 30–50% of the sputum samples of patients with EGFR mutations. The different techniques revealed consistent results, with slightly higher sensitivity for PST. Neither the lung cancer patients without EGFR mutation nor the COPD controls tested positive for EGFR mutations in their sputum samples, indicating high clinical specificity of all assays.

Conclusion

EGFR mutations can be detected in sputum samples from patients with EGFR-mutated non-small cell lung cancer, which may replace biopsy procedure for some patients.     

Occurrence of mutations in the epidermal growth factor receptor gene in X-ray-induced rat lung tumors

Epidermal growth factor receptor ( EGFR ) gene alterations have been found in human lung cancers. However, there is no information on the factors inducing EGFR mutations. In rodents, K- ras mutations are frequently found in many lung carcinogenesis models, but hitherto, Egfr mutations have not been reported. Their presence was therefore investigated in representative lung carcinogenesis models with 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), N -nitrosobis(2-hydroxypropyl)amine (BHP), 2-amino-3,8-dimethylimidazo[4,5- f ]quinoxaline (MeIQx) and ethyl carbamate (urethane), as well as X-ray irradiation. With the chemical carcinogenesis models, no mutations were detected in Egfr , which is in clear contrast to the high rates observed in either codon 12 or 61 of K- ras (21/23 of the lung tumors induced with NNK, 4/5 with MeIQx, 1/4 with urethane and 7/18 with BHP). However, in the X-ray-induced lung tumors, Egfr mutations with amino acid substitution were observed in exons 18 and 21 (4/12, 33%), but no activating mutation of K- ras was detected. In addition, one and four silent mutations were identified in K- ras (exon 1) and Egfr (exons 18, 20 and 21), respectively. Most mutations in both Egfr and K- ras were G/C→A/T transitions (7/8, 88% and 31/34, 91%, respectively). Although, the mutational patterns in equivalent human lesions were not completely coincident, this first report of Egfr mutations in an experimental lung tumor model suggests that X-rays or other factors producing oxygen radicals could cause EGFR mutations in some proportion of lung cancers in humans. ( Cancer Sci 2008; 99: 241–245)     

Relevance of Ki-67 antigen expression and K-ras mutation in colorectal liver metastases.

AIMS: The liver is a frequent site of metastases from colorectal cancer. While these lesions are potentially amenable to surgical resection, they are usually very aggressive, and recurrence is frequent. Mutations of the proto-oncogene K- ras are thought to impart a strong growth signal to tumour cells and are closely associated with the development of malignancies of the colon and rectum. Hepatic metastases from colorectal cancer have notably elevated proliferative rates. The present study was performed to investigate the relationship between proliferation or K- ras mutation and prognosis following curative resection of colorectal liver metastases. METHODS: Colorectal liver metastases from 41 patients undergoing curative hepatic resection were examined for proliferation status and presence of K- ras mutations. The proliferative activity was assessed by Ki-67 immunohistochemistry. DNA from the same tissue samples was screened for point mutations in codon 12 of the K- ras gene using a novel microplate-based allelic-specific hybridization assay. Ki-67 scores and K- ras status were then related with patient survival as determined through retrospective analysis. RESULTS: Median survival was 40 months. Patients with high Ki-67 scores (> or = 50%) had significantly shorter median survival compared with those with low scores (30 vs 44 months, log-rank P=0.02). A high Ki-67 score was an independent negative prognostic factor by multivariate regression analysis (relative risk=3.04, P=0.036). K- ras point mutations were detected in 6/41 patients (15%), but mutational status did not correlate with Ki-67 score or survival. CONCLUSIONS: These findings suggest that the tumour proliferative index is a useful predictor of aggressive tumour behaviour and an indicator of patient survival. The presence of K- ras mutations does not appear to correlate with tumour proliferation status or patient survival. Copyright Harcourt Publishers Limited.     

Incidence and possible clinical significance of K-ras oncogene activation in adenocarcinoma of the human lung

47 tumor samples, 45 of which were obtained at thoracotomy for non-small cell lung cancer were examined for mutational activation of the oncogenes H-ras, K-ras, and N-ras. A novel, highly sensitive assay based on oligonucleotide hybridization following an in vitro amplification step was employed. ras gene mutations were present in nine of 35 adenocarcinomas of the lung (all K-ras), in two of two lung metastases of colorectal adenocarcinomas (1 x K-ras, 1 x N-ras) and in one adenocarcinoma sample obtained at autopsy (H-ras). All K-ras and H-ras mutations were in either position 1 or 2 of codon 12, while the N-ras mutation was in position 2 of codon 61. The potential clinical significance of K-ras activation was analyzed using the combined results of this and of our earlier study (S. Rodenhuis et al., New Engl. J. Med., 317: 929-935, 1987). Lung adenocarcinomas with K-ras mutations tended to be smaller and were less likely to have spread to regional lymph nodes at presentation. With a median follow up of 10 months, survival data are still immature. None of six adenocarcinomas of nonsmokers had a K-ras mutation and only one of four who had stopped smoking more than 5 years before. We conclude that mutational K-ras activation is present in about a third of adenocarcinomas of the lung and that the mutational event may be a direct result of one or more carcinogenic ingredients of tobacco smoke. Studies involving larger numbers of patients are required to confirm the association of K-ras activation with smoking and the inverse relation with tumor progression.     

Demonstration of ras and p53 Gene Mutations in Carcinomas in the Forestomach and Intestine and Soft Tissue Sarcomas Induced by N-Methyl-N-nitrosourea in the Rat

The presence of ras family and p53 gene mutations in rat forestomach, intestine and liver tumors and soft tissue sarcomas induced by N methyl- N -nitrosourea (MNU) was examined using polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) followed by direct sequencing analysis. In the forestomach squamous cell carcinomas (SCC), Ha- ros and p53 mutations were detected in 2 (40%) and 4 (80%) of 5 cases, respectively. The figures for Ki- ras and p53 gene mutations in adenocarcinomas of the large and small intestines were 3 (18.8%) and 5 (31.3%) of 16 cases. Soft tissue sarcomas in different sites were found to have mutations of Ki- ras in 7 (23.3%)and of p53 in 9 (30%) of 30 cases. One forestomach SCC and 2 soft tissue sarcomas had double p53 mutations in different exons. Single cases of forestomach SCC and intestinal adenocarcinoma had mutations in both Ki- ras and p53 genes. No mutations were found in counterpart benign tumors or hepatocellular adenomas. The p53 mutation spectrum revealed preferential clustering within exon 8 for the forestomach SCCs, and exons 5 and 8 for the intestinal adenocarcinomas, whereas the distribution was evenly spread through exons 5 to 8 in soft tissue sarcomas. All the detected ras or p53 mutations were G:C to A:T transitions. These results indicate firstly that specific Ki- ras , Ha- ras and p53 gene mutations in MNU-induced lesions are related to particular alkylation sites (G:C to A:T transitions) and secondly, although not essential, Ki- ras , Ha- ras or p53 gene mutations may be involved in the progression stage of forestomach, intestine and soft tissue neoplasms induced by MNU.     

Expression and alteration of ras and p53 proteins in patients with lung carcinoma accompanied by idiopathic pulmonary fibrosis

BACKGROUND: The ras oncogene and the p53 tumor suppressor gene play important roles in the carcinogenic process of lung carcinoma. The authors evaluated whether alterations of the ras and p53 proteins may contribute to the development of lung carcinoma in patients with idiopathic pulmonary fibrosis (IPF) and whether such alterations may explain the high incidence of lung carcinoma among patients with IPF. METHODS: Lung tissues were obtained from 35 patients who had IPF without complications of lung carcinoma and from 36 patients who had IPF with complications of lung carcinoma. Altered expression of ras and p53 proteins was evaluated by immunohistochemistry, and mutations of both genes were evaluated by polymerase chain reaction-single strand conformation polymorphism and sequencing analyses. RESULTS: The frequency of expression of ras protein in type II alveolar pneumocytes was significantly greater in lung tissues from patients with IPF who had lung carcinoma compared with lung tissues from patients with IPF who did not have lung carcinoma (75% vs. 40%, respectively; P < 0.01). K-ras point mutation in codon 12 (GGT to GTT transversion) was detected in lung tissue with interstitial pneumonia, in which ras protein was overexpressed in type II alveolar pneumocytes obtained from 2 of 41 patients with IPF complicated by lung carcinoma, causing amino acid substitution (Gly to Val) in both patients. A p53 mutation was detected in three of six lung tissue samples from patients who had IPF lung with positive p53 immunoreactivity, and multiple mutations were detected in two samples. CONCLUSIONS: Expression of ras protein in type II alveolar pneumocytes and mutation in the codon 12 of K-ras gene in lung tissue may contribute to the induction of lung carcinoma in patients with IPF. Furthermore, the presence of multiple mutations in the p53 gene may explain the high incidence lung carcinoma in patients with IPF.     

中国肺腺癌患者上皮生长因子受体基因突变的研究

目的:分析我国肺腺癌患者上皮生长因子受体(EGFR)基因突变的发生率和突变类型。方法:在上海、杭州和昆明等地收集61例肺腺癌及其正常肺组织,采用PCR扩增和基因测序方法对组织DNA中EGFR外显子19~21基因突变进行分析。结果:正常肺组织中EGFR基因均为野生型,肺腺癌组织中EGFR基因突变检测率为47.5%(29/61),其中外显子19和21突变分别占突变总数的55.2%(16/29)和44.8%(13/29),外显子20未检测到突变。外显子19突变发生在第746~752位密码子,均为碱基缺失突变,有6种不同类型。外显子21突变全部是第858位密码子碱基替换突变。EGFR基因突变与患者性别和年龄无显著相关性。但昆明和上海等地患者的基因突变存在明显差异。结论:EGFR基因突变是一种肿瘤特异性的体细胞遗传改变,突变发生率约占肺腺癌总数的一半,其中以外显子19和21突变为主。我国EGFR基因突变存在地域差异。     

Detection of activating mutations in the ras family genes in cytological specimens from lung-tumors

Mutations in the ras family genes (K-ras mainly) represent a common event in lung tumorigenesis which is frequently associated with poor clinical outcome. In order to investigate whether K-ras mutations are detectable in cytological material obtained from patients with lung cancer, 37 cytological specimens (16 fine needle aspiration and 21 bronchoscopy) were assessed for codon 12 point mutations in the H-, K- and N-ras genes by combined polymerase chain reaction-restriction fragment length polymorphism. K-ras codon 12 point mutations were found in 8 out of 37 (22%) specimens while no mutations were found in the H-ras and N-ras genes. Mutations were found in 27% (3 out of 11) of adenocarcinomas while in squamous cell carcinomas the incidence of mutations was 18% (3 out of 17). In addition, a K-ras codon 12 point mutation was found in one (12%) among 8 small cell carcinomas and in the only Hodgkin's lymphoma with metastasis in the lung. Our results are in agreement with previous results that recognise high incidence of K-ras activation in lung carcinomas, and indicate that detection of mutant ras alleles is possible in cytological material.     

Mutations of ras genes distinguish a subset of non-small-cell lung cancer cell lines from small-cell lung cancer cell lines.

We screened a panel of 103 human lung cancer cell lines for the presence of point mutations at codons 12, 13 or 61 of the human K-, H- and N-ras genes, using restriction fragment length polymorphisms (RFLP), created through mismatched primers during polymerase chain reaction (PCR) of genomic DNA. We found ras mutations in 22/61 (36%) non-small-cell lung cancer (NSCLC) cell lines, predominantly in K-ras codon 12. Identical mutations were present in uncultured tumor materials corresponding to 11 cell lines containing mutated ras genes. ras mutations were found not only in adenocarcinoma cell lines (9/32, 28%), but also in cell lines derived from other types of NSCLC (13/29, 45%). In contrast, none of 37 small-cell lung cancer (SCLC) cell lines and five extra-pulmonary small-cell cancer cell lines had ras mutations. ras mutations were not correlated with sex of the patients, tumor extent, prior therapy status or in vitro culture time. G to T or A to T transversions were the most common base substitutions, occurring in codons 12 and 61 respectively. We conclude that ras mutations play a role in the pathogenesis of a subset of NSCLC but are not involved in SCLC.     

K-ras activation in non-small cell lung cancer in the dog.

To investigate the role of K-ras mutations in canine non-small cell lung cancer, we first determined the nucleotide sequence of the normal canine K-ras gene and then examined 21 canine lung tumors for activating K-ras mutations. Canine K-ras was analyzed by direct sequencing of polymerase chain reaction products generated with oligonucleotide primers derived from the human K-ras sequence. Four nucleotide differences were found between the canine and human K-ras sequence from position 5 to 211. The deduced amino acid sequence of the canine gene was identical to that of the human. Activated K-ras alleles were detected in 5 of the 21 canine lung tumors examined. The activating lesions were point mutations, predominantly in codon 12. Of the 14 adenocarcinomas examined, 2 (14%) had K-ras mutations. Two of 5 (40%) adenosquamous carcinomas and the only large cell carcinoma also contained activated alleles. The overall frequency of K-ras point mutation in non-small cell lung cancer (25%) is similar to that reported in human non-small cell lung cancer. We conclude that K-ras activation by point mutation is associated with, but not necessary for, non-small cell lung cancer development in the dog.     

176例非小细胞肺癌的EGFR基因突变分析

目的 分析非小细胞肺癌（NSCLC）中上皮生长因子受体（EGFR）基因突变的发生率和突变类型。方法 收集123例正常肺组织和176例肺癌组织,采用PCR扩增和基因测序方法,对组织DNA中EGFR外显子19～21基因突变进行分析。结果 正常肺组织中EGFR基因均为野生型,肺癌组织中EGFR基因突变检测率为32．4％（57／176例）,其中,外显子19和21突变分别占突变总数的64．9％（37／57例）和31．6％（18／57例）,外显子20突变少见,仅占3．5％（2／57例）。外显子19突变发生在第746～753位密码子,均为碱基缺失突变,有7种不同类型。外显子20突变发生在第789—793位密码子,为碱基替换突变。外显子21突变全部是第858位密码子碱基替换突变。EGFR基因突变多见于女性,肺腺癌和腺鳞癌。结论 EGFR基因突变是一种肿瘤特异性的体细胞遗传改变,突变发生率约占肺癌总数的1／3,其中以外显子19和21为主。女性、肺腺癌和腺鳞癌中突变多见。