Analysis Of KRAS Gene Mutations Research Articles

Molecular Analysis to Detect Pancreatic Ductal Adenocarcinoma in High-Risk Groups

Screening of high-risk groups for pancreatic cancer has not been adopted because of concerns regarding specificity and sensitivity. Suitability of a combination of 3 novel molecular screening techniques was investigated. Pancreatic juice was extracted from 146 patients with pancreatic ductal adenocarcinoma, chronic pancreatitis, or biliary tract stones. p53 mutations were analyzed by using a modified yeast functional assay, K-ras status was analyzed using mutation-specific real-time PCR and the proportion of p16(INK4a) promoter methylation was estimated using comparative methylation-specific real-time PCR. p53 mutations were detected in 20 of 48 (42%) cancer cases, none of 49 controls, and 2 of 49 (4%) patients with pancreatitis. K-ras mutations were detected in 31 of 57 (54%) cancer patients, 13 of 61 (21%) controls, and 23 of 67 (34%) patients with pancreatitis. Twenty-six of 42 (62%) cancer patients had promoter methylation levels > 12%, compared with 3 of 24 (13%) controls, and 2 of 26 (8%) with pancreatitis. Mutations in p53 or high-level p16(INK4a) promoter methylation occurred in 29 of 36 (80%) patients with cancer, 3 of 24 (13%) controls, and 3 of 22 (13%) with pancreatitis. Three patients (8%) of 36 with cancer; 14 of 24 (58%) controls, and 13 of 22 (59%) patients with pancreatitis had no marker. The gallstone disease patients had a high rate of positive K-ras mutations, possibly reflecting the fact that they were not disease free. Combination molecular analysis increased the discrimination between patients with malignant and benign disease. This level of discrimination would allow patients in high-risk groups to be stratified from negligible risk to over 50% probability of an early cancer.

Single strand conformation polymorphism analysis of K-ras gene mutations by capillary electrophoresis with laser-induced fluorescence (LIF) detector

Mutations of K-ras gene play an important role in neoplastic progression. The capillary electrophoresis–single strand conformation polymorphism (CE–SSCP) technique is available for the detection of gene mutations. Using an automated capillary electrophoresis with short-chain linear polyacrylamide, after denaturation of PCR products, injections were performed at reverse polarity of 5 kV for 15 s and the separations were carried out under a constant voltage of 8 kV. Of 16 specimens of lung cancer tissue, two specimens were found to have abnormal peaks in the electrophoretogram. CE–SSCP is rapid, automated, and has high performance.

Analysis of K-ras gene mutations in rare pancreatic and ampullary tumours.

Mutation of the K-ras oncogene is a frequent event in pancreatic ductal carcinogenesis and it is believed to occur at an early stage in the development of pancreatic cancer. However, little is known of the role of K-ras mutations in rare pancreatic epithelial neoplasms, endocrine tumours or other non-epithelial tumours of the pancreas. Furthermore, limited data are available regarding the role of K-ras mutations in the pathogenesis of ampullary tumours. Using single-strand conformation polymorphism (SSCP) and direct sequencing of polymerase chain reaction (PCR)-amplified fragments, we analysed codons 12 and 13 for the presence of oncogenic mutations of the K-ras oncogene. Tissues were obtained from patients undergoing tumour resection for various rare pancreatic or ampullary neoplasms (number of cases in brackets): ampullary adenoma (1), neuro-endocrine tumour (3), malignant fibrous histiocytoma of the pancreas (1), pancreatic cystadenocarcinoma (1), serous cystadenoma (1), and primary and metastatic adenocarcinoma of the ampulla (5) and pancreas (3). K-ras gene mutations at codon 12 were detected in both pancreatic adenocarcinomas and in the metastatic lesion, whereas two ampullary cancers harboured a point mutation at codon 13: GGC-->GGG and GGC-->GGT. None of the other tumours exhibited a K-ras gene mutation at codons 12 or 13. Pancreatic tumours other than ductal adenocarcinoma of the pancreas do not harbour mutations of the K-ras oncogene. In addition, ampullary adenocarcinomas may present with codon 13 mutations; however, these mutations were not associated with amino acid substitution. Therefore, K-ras gene mutations seem to be a specific genetic alteration contributing to the pathogenesis of pancreatic ductal adenocarcinoma.

Analysis of K-ras gene mutations in human pancreatic cancer cell lines and in bile samples from patients with pancreatic and biliary cancers

The ras family of oncogenes are the most frequently activated group of dominant transforming genes in both human and experimental cancers. The ras family of genes encode highly similar proteins with molecular weights of 21 kDa which are thought to play a key role in signal transduction. Activation in vivo by point mutations results in the ras p21 protein being maintained in the activated form and stimulating cellular proliferation autonomously. Point mutations at codon 12 of K-ras have been observed in >75% of cases of adenocarcinomas of the exocrine pancreas. The type and frequency of K-ras gene mutations in pancreatic cancer cell lines and in bile samples from patients with cytologically-proven biliary tract malignancies and from patients with non-malignant disorders of the biliary tract were determined. Codons 12, 13 and 61 of the K-ras gene were analysed by using restriction fragment length polymorphisms created through mismatched primers during polymerase chain reaction (PCR) of genomic DNA. A mutation of codon 12 of K-ras was detected in 10 of 13 (77%) human pancreatic cancer cell lines. The amino-acid substitutions were glycine to aspartate (5 samples), arginine (2), valine (2) and cysteine (1). No mutations were found at codons 13 or 61. A mutation at codon 12 of K-ras was detected in 9 of 18 (50%) of bile samples analysed. Eleven bile samples had positive cytology for malignancy of pancreaticobiliary origin, and 4 (36%) of these had a codon 12 mutation. Mutations were detected in 5 of the 7 (71%) cytologically-negative bile samples, although malignancy was subsequently diagnosed in 2 of these patients on further histology, and was suspected in 3 other cases on clinical and radiological criteria. This method provides a rapid determination of K-ras gene mutations in bile samples for patients with pancreatic and biliary tract diseases, which may be useful when considering future therapy directed at inhibition of activated ras-induced signal transduction pathways.

Long-term follow-up of patients with chronic pancreatitis and K-ras gene mutation detected in pancreatic juice

BACKGROUND & AIMS: Conflicting reports have raised the question whether K-ras gene mutations in chronic pancreatitis are related to the development of pancreatic neoplasm. The aim of the present study was to clarify this issue by surveying patients with chronic pancreatitis and K-ras gene mutation over 4 years.METHODS: K-ras point mutations at codon 12 in duodenal juices obtained during exocrine function tests were examined by enriched polymerase chain reaction-restriction fragment length polymorphism and direct DNA sequence analysis, and long- term follow-up of these patients was performed.RESULTS: K-ras gene mutation was found in 20 of 54 patients (37%) with chronic pancreatitis. The types of mutation were GAT in 7, GTT in 11, and TGT in 2 patients. Pancreatic neoplasm occurred in none of the mutation- positive patients over a mean follow-up period of 78 months.CONCLUSIONS: K-ras gene mutation in patients with chronic pencreatitis did not seem to be related to the development of pancreatic neoplasm during the follow-up period, and analysis of K-ras gene mutation seems of little use for diagnosing pancreatic neoplasm in patients with chronic pancreatitis.(Gastroenterology 1997 Aug;113(2):593-8)

Analysis of K-ras gene mutation in hyperplastic duct cells of the pancreas without pancreatic disease

BACKGROUND & AIMS: We and others have previously shown that the mutation of K-ras codon 12 was found in the majority of pancreatic adenocarcinomas. The mutation has also been identified in the pancreatic duct with mucous cell hyperplasia in association with chronic pancreatitis. Ductal hyperplasia is also frequently found in the pancreas free from pancreatic carcinoma or chronic pancreatitis. The aim of this study was to assess the incidence and types of mutations in hyperplastic foci in these cases. METHODS: The nucleotide sequence of the K-ras gene at codon 12 of the DNA extracted from microdissected hyperplastic epithelium of the pancreatic duct obtained at autopsy in patients without pancreatic adenocarcinoma or chronic pancreatitis was analyzed. RESULTS: Of 38 patients with 79 hyperplastic foci, 12 patients (with 19 hyperplastic foci) had mutations. None of the 16 normal ducts in 12 specimens had this mutation. The nucleotide sequence of the codon in 53% of ductal hyperplastic foci was TGT or AGT, both of which were not found in 30 cases of adenocarcinoma. CONCLUSIONS: These results suggest that the ras gene mutation occurs frequently in multifocal hyperplastic foci of pancreatic duct and that the mutations may not have direct relevance to the carcinogenesis of pancreatic cancer. (Gastroenterology 1996 Jan;110(1):227-31)

Analysis of K-ras gene mutations in malignant and nonmalignant endobronchial tissue obtained by fiberoptic bronchoscopy.

Using polymerase chain reaction DNA amplification, we identified K-ras oncogene mutations in bronchial biopsies obtained from patients undergoing bronchoscopy for clinical indications. We hypothesized that these mutations would be found in a field encompassing malignant and nonmalignant tissues in patients with pulmonary carcinomas, and also possibly in tissue from some patients who smoked but did not have pulmonary malignancy. We found K-ras mutations in endobronchial biopsies from nine of 22 patients (41%) with carcinoma of the lung. In five of these patients, mutations were found in both malignant and nonmalignant specimens. In two instances, mutations were found only in the neoplastic tissue, and in two cases mutations were present only in the nonmalignant tissue. Furthermore, we identified two patients with K-ras mutations among seven patients in whom no clinical malignancy was apparent. Our data suggest that some oncogene activations may occur over a broad area in smokers with and without malignancy, and they imply that K-ras mutations may serve as surrogate markers for malignant potential.

Analysis of K-ras gene mutations in resectable non-small cell lung cancer (NSCLC) by polymerase chain reaction (PCR) and oligodeoxynucleotide hybridization

Analysis of K-ras gene mutations in resectable non-small cell lung cancer (NSCLC) by polymerase chain reaction (PCR) and oligodeoxynucleotide hybridization