Mutations of K-ras oncogene and absence of H-ras mutations in squamous cell carcinomas of the lung

Mutations of the K-ras gene have been implicated in the pathogenesis of human lung adenocarcinomas. In most studies published so far, squamous cell lung cancers harbored ras mutations only exceptionally or no mutations were detected at all. We have examined 141 lung tumor DNA samples for mutations in codons 12, 13, and 61 of K-ras and H-ras oncogenes. A large panel of 118 squamous cell carcinomas was included in the study. For K-ras codon 12, we used a sensitive two-step PCR-restriction fragment length polymorphism method which detects <1% of mutated DNA in the sample. K-ras mutations were found in 17 tumors (12%; 14 in codon 12 and 3 in codon 13). Among 19 adenocarcinomas, mutation was revealed in 7 samples (37%). Of these, one sample harbored two point mutations in codon 12. Nine mutational events were found in squamous cell carcinomas (8%, one adenosquamous carcinoma included, all in codon 12). Of four large cell carcinomas, one contained a mutation. Mutant-enriched PCR products harboring mutations were directly sequenced. Fifteen mutational events were G-->T transversions or G-->A transitions, one was a G-->C transition, and one sample revealed a frameshift deletion of one G from codon 12. Similar mutational spectrum was found in both squamous cell carcinomas and adenocarcinomas, suggesting similar carcinogenic pathways in both histological types of the tumor. The presence of mutations did not correlate with the stage of the disease. Moreover, we analyzed all samples for mutations in codons 12, 13, and 61 of the H-ras gene. We found only one mutation in codon 12. Thus, H-ras mutations apparently play an inferior role in lung carcinogenesis. We conclude that mutations of the K-ras oncogene can play a role in the development of not only lung adenocarcinomas but also of a subset (about 8%) of squamous cell carcinomas.     

Detection of K-ras mutations in lung carcinomas: relationship to prognosis

The K-ras mutation is one of the most common genetic alterations found in human lung cancer. To evaluate the prognostic value of ras gene alterations in lung cancer in a U.S. population, we have screened 173 human lung tumors, which included 127 adenocarcinomas, 37 squamous carcinomas, and 9 adenosquamous carcinomas, for mutations in the K-ras gene using the combination of the PCR and denaturing gradient gel electrophoresis. Forty-three tumors contained K-ras mutations. Of these, 41 were identified among the adenocarcinomas (32%), 1 among the squamous carcinomas (2.7%), and 1 among the adenosquamous carcinomas (11%). Forty of these mutations were found in codon 12 and consisted of 24 G to T transversions, 12 G to A transitions, 2 G to C transversions, and 1 double GG to TT mutation. Two other G to T transversions were found in codon 13, and 1 A to C transversion was found in codon 61. The data showed that gender did not seem to affect the incidence and the types of the K-ras mutations or amino acid changes. Examination of the mutations in adenocarcinomas in relation to overall survival showed no difference in adenocarcinomas with K-ras mutations compared with K-ras-negative adenocarcinomas. However, the substitution of the wild-type GGT (glycine) at codon 12 with a GTT (valine) or a CGT (arginine) showed a strong trend (P = 0.07) toward a poorer prognosis compared with wild-type or other amino acid substitutions. Substitution of the wild-type glycine for aspartate (GAT) showed a strong trend (P = 0.06) for a better outcome than the valine or arginine substitution. Although these trends will require larger patient populations for verification, these data suggest that the prognostic significance of K-ras mutations may depend on the amino acid substitution in the p21(ras) protein.     

Validation of the Point-EXACCT method in non-small cell lung carcinomas

K-ras point mutations are often detected in part of the lung carcinomas. For the validation of a highly sensitive and rapid assay for known point mutations, Point-EXACCT (Biochim Biophys Acta 1998; 1379:42-52), we analyzed 89 non-small cell lung carcinomas and compared the results with two sequencing methods. No point mutations were found with double-stranded sequencing. Single-stranded sequencing detected six patients positive for K-ras codon 12. When Point-EXACCT was used, K-ras codon 12 mutations were detected in 8 of 52 patients with squamous cell carcinomas, 10 of 29 patients with adenocarcinomas, and 3 of 8 patients with large cell carcinomas. The finding of K-ras mutations in squamous cell carcinomas is explained by the high sensitivity of the method. Therefore, Point-EXACCT may be applicable to detection of those alterations occurring at a low frequency among an excess of cells with wild-type DNA.     

Rapid detection of K-ras gene mutations in canine lung cancer using single-strand conformational polymorphism analysis

A total of 126 spontaneous lung tumors from pet dogs were examined for K-ras mutations within exon 1 and exon 2 using a non-radioisotope single-strand conformational polymorphism analysis (SSCP) detection method on PCR products. Mutations were confirmed by direct DNA sequencing. Tumors were classified as adenomas (9), bronchioloalveolar carcinomas (59), adenocarcinomas (30), adenosquamous carcinomas (16), squamous cell carcinomas (3) and anaplastic carcinomas (9). Nineteen mutations were detected in the malignant tumors: 18 occurred in exon 1 codon 12 and one in exon 2 codon 61. No mutations were present in the adenomas. The most common mutation was a G-->A transition (11/19) in the second position of codon 12. Based on this study, K-ras mutations occur in canine non-small cell lung carcinomas. The frequency and type of mutation more closely matches tumors from human non-smokers with K-ras mutations than smokers. With the application of screening techniques such as SSCP, large numbers of dog tumors can be examined to provide a large animal model for comparative studies of carcinogenesis.     

K-ras gene point mutations in human endometrial carcinomas: correlation with clinicopathological features and patients' outcome

In order to evaluate the role of K-ras gene point mutations in the progression of endometrial carcinoma, we applied the polymerase chain reaction/restriction-fragment-length polymorphism technique to 57 tumours surgically removed from women of Polish origin. We assessed the relationship between K-ras gene activation and clinicopathological features as well as patients' outcome. Mutational activation in codon 12 of the K-ras gene was detected in 8 out of 57 (14%) endometrial carcinomas, while in codon 13 of the K-ras gene no point mutations were noted. A correlation between the histological type of the tumour and codon 12 K-ras gene mutation was noted (P < 0.05; Fisher exact test). K-ras gene mutation was not related to the patients' age, surgical stage, histological grade or to the depth of myometrial invasion. A trend towards a poorer prognosis was noted during the follow-up of patients whose tumours had shown K-ras codon 12 point mutations, but the difference was not significant (P = 0.06; log-rank test). Our data indicate that point mutations in codon 12 of the K-ras gene are a rare event in human endometrial carcinomas. The lack of correlation between K-ras point mutations and clinicopathological features (except histological type) supports the hypothesis of a random activation of the K-ras gene in human neoplastic endometrium.     

K-ras point mutations are rare events in premalignant forms of Barrett's oesophagus

OBJECTIVE: In Barrett's adenocarcinomas, in contrast to squamous oesophageal carcinomas, K-ras point mutations are thought to be a frequent event. The frequency of K-ras point mutations in premalignant forms of Barrett's oesophagus (metaplasia, dysplasia) leading to adenocarcinoma with increased risk is currently not known. To establish the frequency of K-ras mutations in premalignant forms of Barrett's oesophagus, we investigated oesophageal biopsy specimens with Barrett's metaplastic and dysplastic epithelium for point mutations in the K-ras gene/codons 12, 13. DESIGN: A total of 412 biopsies from patients with Barrett's oesophagus were histologically classified into biopsies with metaplasia (n = 252), dysplasia (n = 105) and adenocarcinoma (n = 11), as well as biopsies distant from disease (normal, n = 37 and hyperplastic squamous epithelium, n = 7). METHODS: DNA from biopsy specimens was amplified by polymerase chain reaction (PCR) with a modified primer for generating a restriction site in the case of wild type in codon 12. Wild-type or point mutations in the K-ras gene/codons 12, 13 were detected by restriction fragment length analysis of the PCR products. RESULTS: Point mutations in K-ras/codon 12 were found in 9 biopsies (n = 1 in metaplasia, n = 4 in dysplasias, n = 4 in adenocarcinomas). All the other biopsies showed the wild type of K-ras/codon 12. No K-ras/codon 13 mutation (GGCgly-->GACasp) was observed. CONCLUSION: Mutations in K-ras/codon 12 were rarely found in premalignant forms of Barrett's oesophagus. Whereas the screening for K-ras point mutations in metaplastic sites of Barrett's epithelium seems not to be of practical value, the screening for mutations in dysplastic lesions might be helpful to estimate the individual risk for progression of Barrett's epithelium to adenocarcinoma. A further evaluation in larger numbers of patients is needed.     

Detection of K-ras point mutations in sputum from patients with adenocarcinoma of the lung by point-EXACCT

PURPOSE: Kirsten ras (K-ras) point mutations are found in 30% to 56% of pulmonary adenocarcinomas by means of highly sensitive techniques. Recently, the Point-EXACCT (point mutation detection using exonuclease amplification coupled capture technique) method was described, which detected one cell with a mutation in 15,000 normal cells. The aim of this study was to examine whether K-ras point mutations could be found with this rapid method in the sputum of patients with adenocarcinoma of the lung. PATIENTS AND METHODS: DNA from paraffin-embedded adenocarcinoma and corresponding sputum samples were analyzed for mutations of the K-ras gene. Twenty-eight biopsy specimens and 54 sputum samples of 22 patients were used for amplification and K-ras codon 12 point mutation detection. RESULTS: In 11 of 22 patients (50%), a mutation in K-ras codon 12 was shown in the tumor sample. In five of 11 patients (45%) with a K-ras mutation in the tumor, the same type of mutation was identified in at least one sputum sample. A mutation could not be detected in any of the sputum samples from patients with a K-ras-negative tumor. Time between K-ras point mutation detection in sputum and clinical diagnosis of lung cancer varied from 1 month to almost 4 years. In two of the five patients with K-ras-positive sputum specimens, malignant cells were found with cytologic examination. CONCLUSION: Point-EXACCT is suitable for the detection of K-ras point mutations in sputum samples of patients with adenocarcinoma of the lung. This approach may be an important adjunct to cytology in the early diagnosis of lung cancer.     

Augmentation of the affinity of HLA class I-binding peptides lacking primary anchor residues by manipulation of the secondary anchor residues

The p53 tumor suppressor gene has been found to be altered in almost all human solid tumors, whereas K-ras gene mutations have been observed in a limited number of human cancers (adenocarcinoma of colon, pancreas, and lung). Studies of mutational inactivation for both genes in the same patient's sample on non-small-cell lung cancer have been limited. In an effort to perform such an analysis, we developed and compared methods (for the mutational detection of p53 and K-ras gene) that represent a modified and universal protocol, in terms of DNA extraction, polymerase chain reaction (PCR) amplification, and nonradioisotopic PCR-single-strand conformation polymorphism (PCR-SSCP) analysis, which is readily applicable to either formalin-fixed, paraffin-embedded tissues or frozen tumor specimens. We applied this method to the evaluation of p53 (exons 5-8) and K-ras (codon 12 and 13) gene mutations in 55 cases of non-small-cell lung cancer. The mutational status in the p53 gene was evaluated by radioisotopic PCR-SSCP and compared with PCR-SSCP utilizing our standardized nonradioisotopic detection system using a single 6-microns tissue section. The mutational patterns observed by PCR-SSCP were subsequently confirmed by PCR-DNA sequencing. The mutational status in the K-ras gene was similarly evaluated by PCR-SSCP, and the specific mutation was confirmed by Southern slot-blot hybridization using 32P-labeled sequence-specific oligonucleotide probes for codons 12 and 13. Mutational changes in K-ras (codon 12) were found in 10 of 55 (18%) of non-small-cell lung cancers. Whereas adenocarcinoma showed K-ras mutation in 33% of the cases at codon 12, only one mutation was found at codon 13. As expected, squamous cell carcinoma samples (25 cases) did not show K-ras mutations. Mutations at exons 5-8 of the p53 gene were documented in 19 of 55 (34.5%) cases. Ten of the 19 mutations were single nucleotide point mutations, leading to amino acid substitution. Six showed insertional mutation, and three showed deletion mutations. Only three samples showed mutations of both K-ras and p53 genes. We conclude that although K-ras and p53 gene mutations are frequent in non-small-cell lung cancer, mutations of both genes in the same patient's samples are not common. We also conclude that this universal nonradioisotopic method is superior to other similar methods and is readily applicable to the rapid screening of large numbers of formalin-fixed, paraffin-embedded or frozen samples for the mutational analysis of multiple genes.     

K-ras gene mutations: an unfavorable prognostic marker in stage I lung adenocarcinoma

Activation of K-ras gene by point mutations, a common finding in lung adenocarcinomas, has been suggested to decrease patient survival. We investigated 109 lung adenocarcinomas, mostly small, peripheral, stage I tumours (81/109) for presence of K-ras gene mutations at codons 12 and 13. Mutations were detected by denaturing gradient gel electrophoresis analysis of specific sequences amplified by polymerase chain reaction from DNA extracted from archival pathological material. Thirty-three of 109 (30.3%) tumours showed mutations at codon 12 (28/33, 84.8%) or 13 (5/33, 15.2%) of the gene. Mutations and type of nucleotide substitutions were differently distributed among cytological subtypes, being more prevalent among less differentiated (G2 and G3) tumours and among bronchial than bronchiolo-alveolar type adenocarcinomas. Survival analysis showed an adverse effect of K-ras mutation on survival, restricted to stage I tumours. Median survival for 81 stage I patients was 30 months for non-mutated tumours versus 20 months for mutated tumours (p = 0.016). Multivariate analysis showed that age of patient (p = 0.001) and K-ras mutation status (p = 0.04) were the only independent factors influencing survival significantly. These data strengthen the hypothesis that K-ras gene mutations may be useful in identifying a subgroup of patients with poor outcome.     

Detection and clinical correlations of ras gene mutations in human ovarian tumors

In epithelial ovarian neoplasms K-ras codon 12 gene mutations show a wide variation fluctuating between 4-39% in invasive carcinomas and 20-48% in borderline malignant tumors. In this study, we showed the pattern of point mutations in codon 12 of the K-ras, H-ras and N-ras genes, using polymerase chain reaction restriction fragment length polymorphism analysis in 74 tissue specimens of Greek patients with epithelial ovarian tumors. K-ras and H-ras gene mutations were detected in 11/48 (23%) and 3/48 (6%) cases with primary invasive ovarian carcinomas, respectively, while N-ras gene mutations were not found. No mutation of K-, H- and N-ras genes was detected in 23 ovarian cystadenomas. In 1 out of 3 borderline ovarian tumors (33%) we found an H-ras gene mutation. The prevalence of mutations in K-ras gene was 1/8 (13%) in mucinous, 7/29 (24%) in serous, 1/3 (33%) in endometrioid and 2/8 (25%) in clear-cell adenocarcinomas and in H-ras gene 1/8 (13%) in mucinous and 2/29 (7%) in serous adenocarcinomas. Analysis of the results revealed no significant correlation between ras gene mutations and clinicopathological parameters or clinical outcome of this primary invasive ovarian carcinoma population. Our present data suggest that ras gene mutations in invasive ovarian carcinomas occur in 29% of Greek patients and are not associated with the differentiation of the epithelial cells or the response of patients to adjuvant platinum-based chemotherapy.     

Detection of K-ras oncogene activation in human lung cancer and its possible clinical application

By using a modified polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) technique, we detected K-ras codon 12 mutation in 102 formalin-fixed, paraffin-embedded tissues from surgical samples of lung cancer patients. The X2 test was used to determine the statistical significance of difference, according to the presence or absence of mutation in codon 12 of K-ras oncogene. We found 25 cases (24%) positive for mutation of K-ras 12 codon. Mutation occurred in 6 of the 40 cases (15%) of squamous cell carcinoma, 18 of 37 adenocarcinoma cases (49%), 1 of 2 adenosquamous cases, 0 of 1 carcinoid patient, but no K-ras activation was found in small cell carcinoma (0/22) cases. Analysis of the clinical and pathological features of 37 adenocarcinoma cases showed no apparent associations between the K-ras codon 12 mutation and sex, disease stage, tumor size (T), metastatic status (M) and the degree of differentiation (all P values greater than 0.05), but the nodes (N) of K-ras-positive adenocarcinoma tended to be more than the K-ras-negative ones (P < 0.01). From 26 male cases of adenocarcinoma mutation in codon 12 of K-ras occur more frequently in adenocarcinoma from smokers than non-smokers (P < 0.05), suggesting that smoking is an important factor in the induction of the mutation. Among 37 adenocarcinoma cases, only 25 cases can be traced the recurrence rate in 1-year. The 1-year recurrence rates were 85% (11/13) in K-ras mutational patients, more than 25% (3/12) in K-ras negative ones (P < 0.01), whereas there was no relationship between recurrence and differentiation in these 25 cases. The findings suggest the K-ras gene mutation may be one of the prognostic markers for human lung adenocarcinoma.     

The presence of K-12 ras mutations in duodenal adenocarcinomas and the absence of ras mutations in other small bowel adenocarcinomas and carcinoid tumors

BACKGROUND: Adenocarcinomas and carcinoid tumors are the most common malignant tumors of the small intestine. K-ras oncogene mutations at codon 12 are common in gastric, pancreatic, and colon carcinomas, with an incidence of 35-88%. K-ras mutations have not been extensively studied in either adenocarcinomas or carcinoid tumors of the small bowel. The purpose of this study was to determine whether ras mutations play an important role in the formation of these tumors. METHODS: Archival tissues from 28 adenocarcinomas and 22 carcinoid tumors of the small bowel were studied, along with archival tissues from 32 adenocarcinomas of the large bowel, which were used as controls. DNA from the small intestine tumors was analyzed for K-ras, H-ras, and N-ras oncogene mutations at codons 12, 13, and 61, using polymerase chain reaction and sequence specific oligonucleotide hybridization techniques. Large bowel adenocarcinomas were analyzed for K-ras mutations at codons 12 and 13. RESULTS: A point mutation of K-ras at codon 12 was detected in 4 of 28 (14.3%) of the small bowel adenocarcinomas, in 12 of 32 (37.5%) of the large bowel adenocarcinomas, and in 0 of 22 small intestine carcinoid tumors. No other K-ras, H-ras, or N-ras mutations were detected in any of the small bowel tumors. Each small intestine K-ras mutation was found in a duodenal adenocarcinoma (4 of 12 cases, 33%), whereas none occurred in 16 other jejunal or ileal adenocarcinomas. CONCLUSIONS: K-ras mutations appear to play a significant role in the pathogenesis of duodenal adenocarcinomas, but they do not appear to be important in the development of jejunal or ileal adenocarcinomas or of carcinoid tumors of the small intestine.     

Adenocarcinoma of the pancreas: detection of occult metastases in regional lymph nodes by a polymerase chain reaction-based assay

BACKGROUND: Stage I (T1-2NOM0) adenocarcinoma of the pancreas is associated with a 5-year survival rate of 15-25%. Despite apparently curative resection and pathologic staging indicating localized disease, these cancers recur. The authors hypothesized that there exists microscopic regional disease that is not detected by surgical exploration or routine histopathology. METHODS: Because 90-95% of pancreatic cancers exhibit codon 12 K-ras mutations, the authors examined regional lymph nodes for mutated K-ras as a marker of metastasis. DNA was extracted from paraffin embedded archival specimens (primary tumors and histologically negative lymph nodes) of patients with Stage I pancreatic adenocarcinoma. The target region of K-ras was amplified by polymerase chain reaction (PCR) and tested for codon 12 mutation by BstN1 restriction digestion (restriction fragment length polymorphism [RFLP]) that recognized normal but not mutated sequences. Cell lines that harbored normal or mutated K-ras and resected jejunum or gallbladder were used as controls. The regional lymph nodes of 22 patients whose tumors harbored mutated K-ras were tested. RESULTS: Dilution experiments with normal and mutant control cell line DNA demonstrated an assay sensitivity for mutated K-ras of 0.1%. Mutated K-ras was found in at least 1 regional lymph node in 16 (73%) of 22 patients with pathologic Stage I pancreatic adenocarcinoma, which suggested metastases not detected by routine histopathology. DNA sequence analysis was performed in four patients and confirmed identical point mutations in the primary tumor and accompanying PCR/RFLP positive lymph nodes. CONCLUSIONS: Pathologic examination of regional lymph nodes in pancreatic adenocarcinoma specimens fails to detect metastases in many patients. Lymph node micrometastasis is one reason for the poor survival rates observed among patients with Stage I cancers. PCR/RFLP may have a role in staging early pancreatic cancers.     

Selective mutation of K-ras by N-ethylnitrosourea shifts from codon 12 to codon 61 during fetal mouse lung maturation

Fetal mouse lung before gestation day 17 shows unique sensitivity to causation of rapidly growing tumors by N-ethylnitrosourea (ENU). Since mouse lung tumors present a mutated K-ras oncogene, we hypothesized that this special susceptibility might reflect an unusual vulnerability of the K-ras gene. Of the lung tumors caused by ENU exposure of BALB/c mice on gestation day 14, 8/25 had a codon 12 mutation in K-ras, vs 4/25 in codon 61. Of 15 tumors after day 16 exposure, three had codon 12 and four codon 61 changes. Tumors from day 18 exposure had only codon 61 mutations (11/16), all A:T to G:C changes (CGA). By contrast, codon 12 (GGT) changes included G:C to T:A, to A:T, and to C:G. These results show significant (P<0.01) shift in the sensitivity of particular K-ras codons to ENU mutation, during fetal mouse lung maturation. In a test of a possible relationship to expression of K-ras, K-ras p21 was measured in lungs of fetal mice, and found to increase markedly on day 18 in comparison to days 14 and 16. Both alkylation of DNA and base damage due to reactive oxygen species are postulated as mechanisms for mutation by ENU, whose efficacies vary with state of fetal lung maturation and K-ras expression.     

Can K-ras codon 12 mutations be used to distinguish benign bile duct proliferations from metastases in the liver? A molecular analysis of 101 liver lesions from 93 patients

It can be difficult to distinguish benign bile duct proliferations (BDPs) from well-differentiated metastatic peripancreatic adenocarcinomas on histological grounds alone. Most peripancreatic carcinomas harbor activating point mutations in codon 12 of the K-ras oncogene, suggesting that K-ras mutational status may provide a molecular basis for distinguishing BDPs from liver metastases. The ability of tests for mutations in codon 12 of K-ras to make this distinction was examined in a two-part study. In the first part we determined the K-ras mutational status of 56 liver lesions and 48 primary peripancreatic adenocarcinomas obtained from 48 patients. In the second part of this study an additional 45 liver lesions were studied. In the first 48 patients, activating point mutations in codon 12 of K-ras were detected in 28 (61%) of the 46 primary carcinomas, in 8 (100%) of 8 liver metastases, in 2 (6.5%) of 31 BDPs, and in none (0%) of 14 liver granulomas. Three BDPs and two primary carcinomas did not amplify. To further estimate the prevalence of K-ras mutations in BDPs we analyzed an additional series of 45 mostly incidental BDPs for K-ras mutations. Three (6.7%) of these 45 harbored K-ras mutations. These results suggest that K-ras mutations may be useful in distinguishing BDPs from metastases in the liver; however, there is some overlap in the mutational spectra of BDPs and pancreatic carcinomas.     

Role of glutathione, glutathione S-transferases and multidrug resistance-related proteins in cisplatin sensitivity of head and neck cancer cell lines

Spontaneous proliferative liver lesions were found in 15 (13 males and 2 females) of 244 (122 of each sex) transgenic (Tg) mice carrying the human prototype c-H-ras gene (rasH2). The liver lesions included 3 foci of cellular alteration, 1 hepatocellular adenoma, 5 hepatocellular carcinomas, and 4 hepatic hemangiosarcomas in the males and 1 focus of cellular alteration and 1 hepatocellular carcinoma in the females. The mutation patterns of the human and endogenous mouse c-H-ras codon 61 in these proliferative liver lesions were analyzed by DNA amplification using polymerase chain reaction, single-strand conformation polymorphism (PCR-SSCP), and oligonucleotide dot blot hybridization. The hepatocellular carcinomas in 4 males and 1 female contained a point mutation in the mouse c-H-ras gene: 3, 1, and 1 carcinomas had a CAA to AAA transversion at the first base of codon 61, a CAA to CTA transversions, and a CAA to CGA transition at the second base of codon 61, respectively. No point mutations in the human c-H-ras transgene were detected in any hepatocellular carcinoma. All 4 hepatic hemangiosarcomas had a CAG to CTG transversion at codon 61 of the human c-H-ras gene, but no point mutations were detected in codon 61 of the mouse c-H-ras gene. No mutations in human or mouse c-H-ras codon 61 were detected in altered cell foci or hepatocellular adenoma. These results indicate that spontaneous liver tumors in rasH2 Tg mice contain different mutation patterns depending on the histologic type or cell origin of the tumors (i.e., hepatocellular carcinomas or hepatic hemangiosacomas). The absence of similar mutations in foci of cellular alteration and the hepatocellular adenoma suggests that the occurrence of codon 61 point mutations is a late event in the progression of hepatocellular neoplasia in rasH2 Tg mice.     

Prevalence of G-to-T transversions among K-ras oncogene mutations in human colorectal tumors in Yugoslavia

Human colorectal carcinoma tissue sampled from 37 patients, routinely graded into Dukes' stages A, B and C and histologically examined for the level of differentiation, were analyzed for the presence of point mutations in the K-ras oncogene. Seventeen cases out of the 37 analyzed were found to have a mutation in either the 12th or the 13th codon of the K-ras gene, giving an overall frequency of mutation of 46%. The incidence of mutations in Dukes' stages A, B and C was 33, 46 and 58% respectively. Although the frequency of mutation appears to be similar to that reported for the USA population, the spectrum of point mutations in codons 12 and 13 of the K-ras gene in the Yugoslav population appears to differ significantly. G-to-T transversions make up 77% of all mutations present, with the distribution as follows: 18% at the first base and 59% at the second base of codons 12 and 13. G-to-A transitions at the second base is the only other mutation identified, occurring mainly in codon 13 in colorectal tumors of all 3 stages.     

K-ras gene mutations in normal colorectal tissues from K-ras mutation-positive colorectal cancer patients

K-ras gene mutations have been reported as early events in colorectal tumorigenesis, but their role in tumor initiation and development is still unclear. To analyze and compare K-ras mutational patterns between colorectal tissues at different stages of tumor progression in individual patients, 65 colorectal tissue samples, including carcinoma, adenoma, histologically normal mucosa, submucosal muscularis propria, and histologically normal mucosa distant from tumor, were obtained from 13 patients with colorectal cancer. In addition, normal mucosal tissues obtained from four normal individuals were analyzed. Each of the 13 tumors was shown previously to harbor a mutation in either codon 12 or 13 of the K-ras gene by direct sequencing. These tissues were reanalyzed, using the recently established mutant allele enrichment + denaturing gradient gel electrophoresis method, which can detect one mutant allele in 10(4)-10(5) normal alleles, thus allowing for the analysis of infrequent cells bearing mutations against the background of wild-type cells. No K-ras codon 12 mutation was detected by this method in the histologically normal mucosal tissues sampled at the margin of resection distant from the tumor or in those obtained from four normal individuals. On the other hand, these mutations were detected in 9 of 10 adenoma and 6 of 10 mucosa samples from 10 patients with known K-ras codon 12 mutations, and also in 2 of 3 carcinoma, 2 of 3 adenoma, and 1 of 3 mucosa samples obtained from 3 patients with known K-ras codon 13 mutations. Thus, K-ras codon 12 mutations were found to occur with a high frequency (53.8%) in histologically normal mucosa adjacent to tumors of patients with K-ras mutation-positive colorectal cancer, suggesting that they may be useful biomarkers for early detection of colorectal cancer. Furthermore, multiple K-ras mutations were found in tissues of nearly half of the 13 patients, indicating that distinct evolutionary subclones may be involved in the development of tumor in some patients with colorectal cancer.     

Detection of K-ras point mutations at codon 12 in pancreatic juice for the diagnosis of pancreatic cancer by hybridization protection assay: a simple method for the determination of the types of point mutation

The present study was undertaken to detect K-ras oncogene point mutations at codon 12 in pure pancreatic juice (PPJ) by the hybridization protection assay (HPA) method for the diagnosis of pancreatic cancer (PC). This assay can be carried out within 30 min and can determine not only the presence of a mutation, but also the mutational type of K-ras at codon 12. The minimal ratio of mutant DNA detectable by the HPA was 5-10% of the total DNA. PPJ was collected through a cannula under duodenal fiberscope control from 20 patients with PC and 20 patients with chronic pancreatitis (CP). Analysis of PPJ by the HPA revealed that the incidence of K-ras point mutations at codon 12 was 55% (11/20) in patients with PC and 0% (0/20) in those with CP. Mutational types of K-ras at codon 12 in PC were aspartic acid (Asp) in nine cases, both Asp and cysteine in one case, and arginine in one case. Analysis of K-ras point mutations at codon 12 in PPJ using the HPA method seems promising as a new genetic test for the diagnosis of PC, because the HPA method is simple, and can easily determine the mutational type.     

Coexistent minocycline-induced systemic lupus erythematosus and autoimmune hepatitis

BACKGROUND AND OBJECTIVES: The K-ras gene is one of the most extensively investigated oncogenes in a wide variety of human tumors, but has rarely been studied in distal bile duct carcinoma (DBDC). We sought to investigate the diagnostic and prognostic value of K-ras codon 12 mutations in this type of tumor. METHODS: Forty-seven patients who had undergone resection for DBDC were analyzed to reveal the incidence of K-ras codon 12 mutations, the locus most frequently involved. A rapid and simple two-step, semi-nested polymerase chain reaction (PCR) technique was used to detect mutations in paraffin-embedded tumor samples. RESULTS: The PCR mismatch amplification technique demonstrated that 35 (75%) of the 47 tumors harbored a point mutation in codon 12 of the K-ras oncogene. Patients with mutated tumors had no statistically different survival time compared to those patients without a mutation in the tumor. In contrast, negative microscopic margins proved to be a significant prognosticator. CONCLUSIONS: K-ras codon 12 mutations are common in DBDC and may be useful in the diagnosis and early detection of these tumors. However, no prognostic value of these mutations could be identified in this analysis. The results of this study also suggest that negative surgical margins remain the mainstay of prognostication in resectable DBDC. However, due to the small number of patients included in this study, the results obtained should be interpreted with care.