Sensitive detection of K-ras mutations augments diagnosis of colorectal cancer metastases in the liver.

Postoperative survival of colorectal cancer patients is often delineated by metastases spreading to the liver. Current clinical diagnostic procedures are unable to discover micrometastases in this organ. Our aim was to develop a diagnostic tool for detecting micrometastases that are present at the time of surgery. Therefore, a PCR-RFLP assay was set up tracking point mutations of the K-ras oncogene at codons 12 and 13, based on mismatch primers and restriction enzymes BstXI and XcmI. The detection limit of this assay was one mutant in one million wild-type cells. One hundred forty-two patients with colorectal carcinoma were screened for these mutations in tissue samples from their tumor, proximally adjacent mucosa, and liver. Of these, 67 patients (46%) were positive for a K-ras mutation, of which 58 had codon 12 and 9 had codon 13 mutations. No patient without a K-ras-positive tumor showed a mutation in mucosa, but 11 patients with a K-ras-positive tumor (11 of 58; 19%) were found to bear a K-ras mutation in their mucosa, and in 21 patients (21 of 64; 33%), a K-ras mutation was detected in liver tissue. Sequencing of all mutated samples revealed a 92% confirmation of PCR-RFLP results. In summary, the assay is a useful tool for detecting K-ras codon 12 and 13 mutations and allows early proof of molecular determinants of liver metastases. Such knowledge will improve the staging of colorectal cancer patients and could beneficially influence their prognosis if followed by an effective therapy.     

K-ras and rho A mutations in malignant pleural effusion

Mutations of the Kristen ras (K-ras) gene have been implicated in the pathogenesis of human lung cancer, especially adenocarcinoma, and have been proposed to be a prognostic factor. The K-ras mutation in codon 12 is detectable even in cell-free fluids by using the enriched polymerase chain reaction (PCR) technique. On the other hand, based on experimental results, the rho A mutation in codon 14 is also proposed to be oncogenic as observed in the K-ras mutation. Malignant pleural effusion is a common complication of lung cancer. We studied the point mutation of K-ras codon 12 and rho A codon 14 using enriched PCR in specimens of pleural effusion. Forty patients with pleural effusion were enrolled in this study. The causes of pleural effusion were non-small cell lung cancer (18 cases), small cell lung cancer (6 cases), malignant mesothelioma (2 cases), metastatic lung tumor (5 cases), thymoma (1 case), malignant lymphoma (1 case), and pleuritis tuberculosa (7 cases). The K-ras mutation was detected in 4 of 14 cases with adenocarcinoma, 1 of 3 cases with squamous cell carcinoma, 1 of 1 case with large cell carcinoma, and 1 of 5 cases with metastatic lung tumor, respectively. The rho A mutation was not detected in any pleural effusion examined in this study. Our study demonstrates the usefullness of pleural effusion as a clinical specimen for a search of point mutation of oncogenes. The K-ras codon 12 mutation is readily detected in pleural effusion, and the demonstration of this mutation has potentially important implications for the diagnosis of malignant pleural effusion.     

Detection of human papilloma virus (HPV) and K-ras mutations in human lung carcinomas

The purpose of our study was to assess the prevalence and prognostic significance of HPV infection as well as K-ras codon 12 point mutations in lung cancer. Patients diagnosed with lung carcinoma between 1988 and 1992 (N=99) were selected. HPV detection and typing was performed by PCR from paraffin-embedded tissues, while mutations in codon 12 of K-ras gene were detected using the restriction fragment length polymorphism (RFLP) analysis. The prevalence of HPV infection was 15%, while K-ras codon 12 point mutations were found in 18% of the specimens examined. In 50% of the HPV-positive cases, K-ras gene mutation coexisted. HPV 18 was the most frequent type. No correlation was found between K-ras mutation and HPV infection with sex, age and clinical outcome of the patient, or the histological type and the differentiation grade of the tumor. An association was found between K-ms codon 12 point mutations and the stage of the tumor, occurring more frequently at stage III (p=0.037). Infection with potentially oncogenic HPV types could co-operate with K-ras gene activation in the progression of the disease, since K-ras activation by point mutations seems to be a late event in lung carcinogenesis.     

Detection of codon 12 K-ras mutations in non-neoplastic mucosa from bronchial carina in patients with lung adenocarcinomas

K-ras activation by point mutation in codon 12 has been reported in lung adenocarcinomas in various models of experimental lung tumours induced by chemical carcinogens. The hypothesis of the presence of cells containing K-ras mutation in non neoplastic bronchial carina, the main site of impaction of airborne contaminants, was investigated by evaluating concurrent lung tumour and non-neoplastic proximal bronchial carinae from 19 patients with lung adenocarcinomas. The restriction fragment length polymorphism enriched PCR method used can detect one mutant allele among 10(3) normal alleles. A mutation was detected in 42% of lung adenocarcinoma samples. No mutation was detected in either tumour or bronchial carinae in nine patients (47%). K-ras mutation was detected in the lung tumour but not in bronchial carinae in four patients (21%), in both the lung tumour and bronchial carinae in four other patients (21%). In two patients (11%), K-ras mutation was detected in at least one bronchial carina, but not in the lung tumour. Mutations of codon 12, confirmed by sequencing analysis of ten samples, were G to T transversion, mostly TGT and GTT in bronchial carinae and lung tumours. Our data show that activated K-ras by point mutation can be present in non-neoplastic bronchial carina mucosa even when no mutation is detected in tumour samples.     

ras gene mutations in non-small cell lung cancers are associated with shortened survival irrespective of treatment intent.

We analyzed 66 non-small cell lung cancer cell lines for mutations at codons 12, 13, and 61 of all three ras genes and correlated the findings with patient survival. We used designed restriction fragment-length polymorphisms to detect mutations after amplification of ras-specific sequences by the polymerase chain reaction. We found 19 mutations of ras genes (29%), and 11 of these 19 (58%) were at codon 12 of the K-ras gene. By univariate analysis, the presence of any ras mutation in cell lines from patients who received curative intent treatment was associated with a shorter survival (P2 = 0.002). For patients who received only palliative treatment, detection of K-ras mutations at codon 12 was associated with a shortened survival (P2 = 0.0103), but this analysis was not statistically significant for the group with any ras mutation (P2 = 0.093). The Cox proportional hazards model also predicted a higher risk for patients with any type of ras mutations. We conclude that ras mutations, present in a subset of non-small cell lung cancers, are independently associated with the shortened survival of patients, irrespective of treatment intent.     

Detection of codon 61 point mutations of the K-ras gene in lung and colorectal cancers by enriched PCR

Mutation of the Kirsten ras (K-ras) gene is one of most common alterations in solid tumors including lung and colorectal cancers. We developed new enriched PCR-RFLP assay to detect mutations of K-ras codon 61 at the 1st and 2nd letters and non-enriched PCR-RFLP assay to detect the 3rd letter mutation. One mutant allele among 10(3) wild-type alleles was detected by enriched PCR-RFLP assay, while one mutant in 10 wild-type alleles was detected by non-enriched PCR-RFLP assay for codon 61 3rd letter. We then examined K-ras codon 12, 13 and 61 mutations in lung and colorectal cancers using these assays. K-ras codon 12 mutation was detected in 10 of 109 (9%) lung cancer and 19 of 83 (23%) colorectal cancer cases. K-ras codon 13 mutation was detected in 2 of 83 (2%) colorectal and 0 of 109 NSCLC cases, respectively. There was no K-ras codon 61 mutation in either type of cancer. Our results demonstrate that enriched PCR-RFLP is a sensitive assay to detect K-ras codon 61 mutation, however, it was extremely rare in lung and colorectal cancers, suggesting organ-specific pathways in mutagenesis of the ras gene family.     

K-ras oncogene mutation in cancer and precancerous lesions of the gallbladder

BACKGROUND AND OBJECTIVES: To determine whether K-ras mutation plays any role in the development and progression of gallbladder cancer, or has any clinical or pathological significance in gallbladder cancer patients, we investigated the presence and incidence of this mutation in the normal mucosa, and precancerous and cancerous lesions of the gallbladder. METHODS: DNA was obtained from normal mucosa, dysplastic mucosa, primary cancer tissues, and metastatic lymph nodes that were identified and microdissected from the paraffin blocks of 20 gallbladder cancer cases. K-ras codon 12 mutations were investigated using a modified two-step polymerase chain reaction and the restriction fragment length polymorphism method, and by direct sequencing with an automated sequencer. RESULTS: K-ras mutations were detected in the tissues of 10 out of the 20 patients. A mutation was present in the dysplastic epithelium associated with the primary carcinoma in 3 out of 12 specimens, in metastatic carcinoma in 1 out of 5 patients, and in primary carcinoma in 8 out of 20 patients. Mutation was found only once in the dysplastic, noncancerous epithelium, and only once in a metastatic tumor although not detectable in the primary cancer. Direct sequencing showed that the mutations were G to C substitutions (GGT-->CGT) at the first site of codon 12, except in two cases (GGT-->TGT). There were no correlations between K-ras mutations and clinicopathological factors. CONCLUSIONS: K-ras mutations were detected in half of the gallbladder cancer cases. We suggest that K-ras mutation may play a role in the development of premalignant lesions or early carcinogenesis in some gallbladder cancers. We were unable to find any evidence that K-ras mutation plays any role in tumor progression or metastasis, or that it has any clinicopathological significance.     

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.     

High frequency of K-ras mutations in normal appearing lung tissues and sputum of patients with lung cancer

To evaluate the possible use of mutant ras as a biomarker for lung cancer, we have analyzed “normal appearing” lung tissue, lung tumor, lung metastases and sputum samples from patients with non-small cell lung cancer (NSCLC). As a control, we used lung tissue and sputum samples from patients without oncological diseases or lung disorders. Our analyses were performed with the aid of enriched PCR (EPCR), a method which enables detection of ras mutation even if present at low incidence. EPCR identified K-ras codon 12 mutations in 10% of lung tissues obtained from patients with no lung diseases, whereas the same mutation was detected in 60% of samples of normal appearing lung tissues obtained from patients with NSCLC, 62% of NSCLC tumors and 80% of metastases. Analysis of sputum samples of patients with NSCLC identified 47% to harbor mutant ras allele, whereas 12.5% of controls diagnosed with non-oncological lung diseases carried this mutation. Most of these mutations were detected with the aid of EPCR only, indicating that a minority of cells in a given sample harbor this mutation. The ability to detect K-ras codon 12 mutation in 60% of lung tissue samples and in 47% of sputum samples taken from patients with lung cancer (as compared with 10% and 12.5% of respective controls) points to the potential use of ras mutation as a biomarker for exposure and possible identification of patients who may be at a higher risk of developing lung cancer. © 1995 Wiley-Liss, Inc.     

K-ras codon 12 and 13 mutations are correlated with differential patterns of tumor cell dissemination in colorectal cancer patients

The aim of this prospective study was to relate the incidences of cytokeratin 20 (CK20) and guanylylcyclase C (GCC) in lymph node, liver, and bone marrow specimens of 245 colorectal cancer (CRC) patients with the K-ras oncogene status of the corresponding primary tumor. Qualitative RT-PCR detection of CK20 and GCC mRNA was used as marker of circulating epithelial cells (CEC). Samples were considered positive for CEC only when both markers were detected concomitantly. For the detection of K-ras mutations, a PCR-RFLP assay was used. In the group with K-ras mutated primary carcinomas (n=92), CEC were detected in 62% of lymph node-, 43% of liver-, and 2% of bone marrow samples. No statistical significance was found when comparing these results with those from patients with K-ras wild-type carcinoma (59%, 46%, and 0%, respectively). In contrast to this combined evaluation, separate analysis of K-ras codons 12 (n=75, 82%) and 13 (n=17, 18%) revealed significantly differing CEC incidences. Lymph node specimens from corresponding K-ras codon 13 mutated carcinomas showed a significantly higher CEC incidence (82%) than the groups with codon 12 mutation (57%, p<0.05) or K-ras wild-type sequence (59%, p<0.05). Unlike these findings in lymph nodes, liver biopsies from corresponding carcinomas with K-ras codon 12 mutation or wild-type sequence were significantly more often positive for CEC (31% and 29%) than specimens from K-ras codon 13 mutated primary CRC (12%, p<0.04, respectively). In conclusion, colorectal carcinomas with K-ras codon 12 mutation showed the same pattern of tumor cell dissemination as their K-ras wild-type counterparts. Since K-ras codon 12 mutations prevailed 4-fold over codon 13 mutations, combined analysis of the two codons showed the same result. However, sub-analysis of patients with K-ras codon 13 mutation revealed that the respective CEC incidence was significantly increased in lymph nodes, but decreased in liver biopsies.     

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.     

Implications and prognostic value of K-ras mutation for early-stage lung cancer in women

BACKGROUND: Because there is no clear consensus as to the predictive value of K-ras gene mutation for survival in patients with lung cancer, we examined the occurrence of K-ras mutations in a large, prospective case series of non-small-cell lung cancer (NSCLC). Our goals were to define the patient characteristics associated with K-ras mutation and to determine whether mutation of this gene might be a biomarker of patient prognosis. METHODS: Consecutive, newly diagnosed patients with lung cancer treated with potentially curative resection over a 4-year period were recruited for study. The mutation status of K-ras codon 12 in each patient's tumor DNA was determined by means of polymerase chain reaction-restriction fragment length polymorphism analysis of archived pathology specimens. Analyses were restricted to adenocarcinoma. RESULTS: There was a statistically significant association between female sex and K-ras mutation after adjustment for carcinogen exposures (odds ratio = 3.3; 95% confidence interval [CI] = 1.3-7.9); mutations were found only in smokers. Comparison of Kaplan-Meier curves indicated a strong association between K-ras mutation and decreased patient survival (two-sided P =.009); analysis stratified by pathologic staging groups revealed that this association was statistically significant only for stage I tumors (two-sided P =.002). Cox proportional hazards modeling indicated that K-ras codon 12 mutation was a statistically significant predictor of patient survival, after adjustment for the effects of age, sex, and stage (risk ratio = 1.8; 95% CI = 1.1-3.1). CONCLUSIONS: After adjustment for environmental exposures, non-small-cell lung tumors in women appear to be more likely than those in men to harbor K-ras mutations, suggesting a possible role of estrogen exposure in either the initiation or the selection of K-ras mutant clones in adenocarcinoma. In addition, our data suggest that K-ras codon 12 mutation is a marker of aggressive NSCLC, as evidenced by its association with decreased patient survival, particularly for early-stage disease.     

K-ras mutations in lung carcinomas from nonsmoking women exposed to unvented coal smoke in China

Lung cancer mortality rate in nonsmoking women in Xuan Wei (XW) County is the highest in China. The XW lung cancer rate is associated with exposure to coal smoke, containing high concentrations of polycyclic aromatic hydrocarbons (PAHs), in unvented homes. Here we investigated codon 12 K-ras mutations in lung tumors or sputum samples from 102 XW lung cancer patients (41 nonsmoking women and 61 smoking men). In addition, we analyzed specimens from 50 lung cancer patients (14 nonsmoking women, 33 smoking men and three nonsmoking men), from Beijing and Henan (B&H), where natural gas is the main domestic fuel. K-ras mutations were found in nine women (21.9%) and 14 men (22.9%) from XW, with G to T transversions accounting for 66.7 and 85.7%, respectively. Among B&H patients, one woman (7.1%) and six men (16.7%) had K-ras mutations, with G to T transversions accounting for 66.7% of the mutations in the men. Therefore, the frequency and type of K-ras mutations in XW nonsmoking women are similar to those of K-ras mutations found in both XW and B&H smoking men. On the other hand, the mutation frequency in XW women is higher than, although not statistically significant from, that in the B&H nonsmoking women (P=0.28, two-sided Fisher's Exact Test). These results suggest an association between exposure to coal smoke and the increased K-ras mutation frequency in XW nonsmoking female lung cancer patients. They also suggest that the mutagens and/or mechanisms of mutations in these nonsmoking women are similar to those responsible for K-ras mutations in cigarette smoking lung cancer patients, which are probably induced largely by chemicals such as PAHs.     

肺癌脱落细胞端粒酶活性检测的临床意义

目的：探讨肺癌脱落细胞端粒酶活性检测的临床意义。方法：收集63例肺癌患者和31例非肺癌肺疾病患者的支气管肺泡灌洗液，同时行刷片和灌洗液细胞学检查；34例肺癌患者痰液，31例非肺癌肺疾病患者痰液；彩PCR－TRAP银染法检测端粒酶活性。结果：肺癌和非肺癌肺疾病支气管肺泡灌洗液端粒酶活性阳性率分别为76．2％（48／63）和6．5％（2／31），P＜0．01；肺癌支气管肺泡灌洗液端粒酶活性阳性率高于刷片细胞学阳性率（58．7％，37／63），P＞0．05；高于灌洗液细胞学阳性率（14．7％，9／63），P＜0．01。肺癌痰液和非肺癌肺疾病痰液端粒酶活性阳性率分别为29．4％（10／34），3．2％（1／31），P＜0．01。结论：肺癌端粒酶活性检测有较高的特异性和敏感性，可应用于肺癌的临床诊断。支气管肺泡灌洗液和痰液端粒酶检测能提高肺癌检出率。     

Activating mutations in the k-ras gene in ulcerative-colitis and crohns-disease

Patients with ulcerative colitis (UC) and Crohn's disease have an increased risk for developing cancer of the colon. Mutations in the K-ras gene are relatively frequent in specimens from patients with sporadic colon cancer, but less frequent in cases of cancer complicating ulcerative colitis. In order to study the problem further we used the polymerase chain reaction (PCR) technique followed by a restriction fragment length polymorphism (RFLP) assay, to detect mutations at codon 12 of K-ras in biopsy specimens from patients with UC or Crohn's disease. Six among 27 patients (22.2%) with UC and 2 of the 19 patients (10.5%) with Crohn's disease examined, carried a mutation at codon 12 of K-ras. Our results indicate that mutations in K-ras may be a genetic marker that would reveal the predisposition to colon cancer among this group of patients.     

Point mutation of K-ras gene in cisplatin-induced lung tumours in A/J mice

The risks of secondary lung cancer in patients with early stage non-small and small cell lung cancers are estimated to be 1-2% and 2-10% per patient per year, respectively. Surprisingly, the incidence of second primary cancer in locally advanced non-small cell lung cancer at 10 years, following cisplatin-based chemotherapy with concurrent radiotherapy, increases to 61%. Those patients, on the road to being cured, cannot overlook the possibility of developing a second primary cancer. We developed a second primary lung cancer model using cisplatin as a carcinogen in A/J mice to screen for chemopreventive agents for a second malignancy. In the primary lung tumour model, 4-(methyl-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK), benzo(a)pyrene (BaP), urethane induces specific K-ras mutations in codon 12, codon 12, and codon 61, respectively, in the A/J mice. In this study, we investigated the mechanisms of carcinogenicity by cisplatin in the A/J mice. In the cisplatin-induced tumours, we found no K-ras codon 12 mutation, which is the major mutation induced by NNK or BaP. K-ras gene mutations in codon 13 and codon 61 were found in one tumour (4%) and five tumours (17.8%), respectively. These findings suggest that cisplatin is partially related to K-ras codon 61 mutations, and that the mechanism of carcinogenicity by cisplatin is different from that by NNK or BaP.     

Detection of circulating cancer cells with K-ras oncogene using membrane array

K-ras oncogene is frequently found in human cancers and thus may serve as a potential diagnostic marker for cancer cells in circulation. So far, there is no reliable method for detecting cancer cells with K-ras oncogene in peripheral blood. The objective of this study was to develop a diagnostic membrane array using activated K-ras oncogene-associated molecules as detection targets. In our previous study, cDNA microarray analysis showed that there were 94 genes differentially expressed in K-ras mutant stably transfected adrenocortical cells. In the present study, we obtained 22 up-regulated genes in the closest relation to K-ras oncogene through bioinformatic analysis. At first, we carried out membrane array analysis by using in vitro culture cells. We demonstrated that this diagnostic technique was feasible and highly sensitive. A number as low as 5 cancer cells bearing K-ras oncogene in 1 ml of blood could be distinctively detected. Then, we collected blood specimens from 76 cancer patients. Direct sequencing analysis of these 76 samples showed that K-ras mutation was present in 43 patients with mutation sites mainly at codons 13, 15 and 61, which have been commonly established to be activated sites. We subsequently analyzed these 76 specimens with our diagnostic membrane array. Thirty-nine specimens were detected as positive for activated K-ras oncogene. Eighty percent (12/15) of mutations occurred at codon 13, 72.7% (8/11) at codon 61, and 88.9% (8/9) at codon 15 were accurately detected by our diagnostic membrane. Finally, through a series of biostatistical analyses, the sensitivity, specificity and accuracy of the diagnostic membrane array were 83.7, 90.9 and 86.8%, respectively. These findings suggest that the K-ras oncogene membrane array has a great potential for further investigation and clinical application.     

Trimodality treatment in Stage III nonsmall cell lung carcinoma: prrognostic impact of K-ras mutations after neoadjuvant therapy

BACKGROUND: In a trimodality treatment approach for Stage III nonsmall cell lung carcinoma (NSCLC), the prognostic impact of the ras mutation status in resection specimens was evaluated. METHODS: Forty patients with Stage III NSCLC underwent tumor resection after neoadjuvant treatment with two cycles of chemotherapy (ifosfamide, carboplatin, and etoposide) and subsequent twice-daily radiotherapy (45 grays [Gy]; 2 x 1.5 Gy/day) with concurrent carboplatin and vindesine. Assessment of K-ras codon 12 mutation status was performed in the paraffin embedded resection specimens by a two-step polymerase chain reaction followed by restriction fragment length polymorphism analysis. RESULTS: K-ras mutation status could be assessed in 28 cases. A K-ras codon 12 point mutation was found in 13 of 28 resection specimens (46%). The mutation was found independently of gender, age, tumor stage, and clinical response status and occurred more frequently in adenocarcinomas. Even after complete resection, the presence of a K-ras mutation was a significant predictor for a poor progression free survival (P = 0.005). CONCLUSIONS: These data suggest that further evaluation of the K-ras codon 12 mutation status in trials on neoadjuvant and adjuvant therapy is warranted. This may contribute to the identification of stratification variables for future treatment approaches.     

The balanced induction of K-ras codon 12 and 13 mutations in mucosa differs from their ratio in neoplastic tissues

The aim of this study was to compare the ratio of K-ras codon 12 and 13 mutations in various tissues of colorectal cancer patients. Multiple samples of inconspicuous mucosa and a sample of carcinoma tissue were taken from 36 colorectal cancer patients (group I) and these results were compared with those from polyp and carcinoma tissues of another 48 colorectal cancer patients (group II). A polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay was used to detect the respective point mutations. The results of this assay were complemented by sequencing the K-ras mutations. In mucosa tissue, the ratio of codon 12 and 13 mutations was nearly equal (0.9:1) whereas the respective ratio in tumour tissue showed a strong preponderance of K-ras codon 12 mutations (14:1, p=0.004). In polyp tissue of patients from group II, the ratio was 2.7:1 and that in carcinomas was 19:1 (p=0.053). The prevalence of both types of mutation was 14.6% in all mucosa samples, corresponding to 30.6% of group I patients. The K-ras mutation rate in carcinoma tissue of the same patients was 38.9%. Similarly, 33.4% of all polyp and 41.7% of all carcinoma samples from group II harboured K-ras codon 12 and/or 13 mutations. Sequencing confirmed 59 of 60 K-ras codon 12 mutations, but due to the detection limit for sequencing (1:10(4)) only 10 of 20 K-ras codon 13 mutations were confirmed. It is concluded that after balanced induction K-ras codon 12 mutations increase in frequency relative to K-ras codon 13 mutations during tumour progression.     

Detection of k-ras mutations in nonsmall cell lung-carcinoma

Forty-five non-small cell lung cancers (NSCLC) were examined for the presence of K-ras mutations in codon 12 using RFLP (restriction fragment length polymorphism) and ARMS (amplification refractory mutation system) assays. The RFLP analysis consisted of a PCR and subsequent digestion of the product with BstNI. Three adenocarcinomas and one adenosquamous carcinoma were shown to have mutations at codon 12. All of these samples were also examined using the ARMS assay for mutations at codon 12 and second base G to A transitions at codon 13 of the K-ras gene. The same four samples were confirmed to have a single base change in codon 12. No G to A transitions were found at codon 13. The four mutations were: one G to C transversion, one G to A transition and two G to T transversions. All mutations occurred at the second position of codon 12 as shown by the ARMS assay. Both of these techniques are rapid and reproducible for the identification of mutations in the K-ras gene and have potential for use in cancer diagnosis.