P53 mutations in colorectal cancer from northern Iran: Relationships with site of tumor origin, microsatellite instability and K-ras mutations

CRC-associated P53 mutations have not been studied extensively in non-Western countries at relatively low CRC risk. We examined, for the first time, 196 paraffin-embedded CRC cases from Northern Iran for mutations in P53 exons 5-8 using PCR-direct sequencing. P53 status and mutation site/type were correlated with nuclear protein accumulation, clinicopathologic variables and data on K-ras mutations and high-level microsatellite instability (MSI-H). We detected 96 P53 mutations in 87 (44.4%) cases and protein accumulation in 84 cases (42.8%). P53 mutations correlated directly with stage and inversely with MSI-H. Distal CRCs were more frequently mutated at major CpG hotspot codons [248 (8/66, 12.1%), 175 (7/66, 10.6%), and 245 (7/66, 10.6%)], while in proximal tumors codon 213, emerged as most frequently mutated (5/28, 17.9% vs. 3/66, 4.5%, P = 0.048). Transitions at CpGs, the most common mutation type, were more frequent in non-mucinous (25% vs. 10.4% in mucinous, P = 0.032), and distal CRC (27% vs. 12.5% in proximal, P = 0.02), and correlated with K-ras transversions. Transitions at non-CpGs, second most common P53 mutation, were more frequent in proximal tumors (15.6% vs. 4.7% in distal, P = 0.01), and correlated with K-ras transitions and MSI-H. Overall frequency and types of mutations and correlations with P53 accumulation, stage and MSI-H were as reported for non-Iranian patients. However P53 mutation site/type and correlations between P53 and K-ras mutation types differed between proximal and distal CRC. The codon 213 P53 mutation that recurred in proximal CRC was previously reported as frequent in esophageal cancer from Northern Iran.     

Inhibition of rat colon tumors by sulindac and sulindac sulfone is independent of K-ras (codon 12) mutation

Nonsteroidal anti-inflammatory drug (NSAID) use reduces the risk of colorectal cancer by 40-50%. Previous studies suggest that effective inhibition of colorectal cancer by NSAIDs may be dependent on the presence or absence of a K-ras mutation. This study was aimed at determining the relationship between inhibition of colorectal cancer by sulindac and sulindac sulfone and the presence of activating K-ras mutations in the 1,2-dimethylhydrazine dihydrochloride rat model. Sulindac (20 mg x kg(-1) x day(-1)), sulindac sulfone (40 mg x kg(-1) x day(-1)), or vehicle was administered orally to male Sprague-Dawley rats for a 4-wk period beginning 20 wk after tumor induction. Tumor number and volume were measured before treatment by laparotomy and colonoscopy and again after treatment. Sulindac and sulindac sulfone treatment significantly reduced the number and volume of colorectal tumors compared with control rats. For K-ras (codon 12) mutation detection, frozen tumor tissue was collected at the endpoint. We found K-ras codon 12 mutations in 11 of 21 (52%) control tumors. The proportion of tumors with K-ras mutations in the sulindac-treated group [5 of 8 (62%); odds ratio = 1.51 (95% confidence interval = 0.29, 8.33)] and the proportion of sulindac sulfone-treated tumors [9 of 14 (64%); odds ratio = 1.63 (95% confidence interval = 0.41, 6.66)] were not significantly different from controls. Tumor inhibition did not correlate with K-ras (codon 12) mutation status, which suggests that the mechanism of inhibition of rat colorectal cancer by sulindac and sulindac sulfone is independent of K-ras mutation.     

Comparison of K-ras gene mutations in tumour and sputum DNA of patients with lung cancer.

Mutations in the K-ras gene are frequently found in lung tumours and are implicated in the development of lung cancer. In order to investigate the clinical usefulness of these mutations in lung cancer, we applied a sensitive method to compare mutations in codon 12 of the K-ras gene in DNA extracted from lung tumours and the matched sputum samples obtained from 22 lung cancer patients. K-ras mutations were identified in the lung tumours of 12 patients (54.5%) and in the sputum samples of 10 patients (45.5%). Nine patients showed an identical mutation in both the tumour and the matched sputum samples. There was a significant association between the presence of a K-ras mutation in a lung tumour and the detection of an identical mutation in the matched sputum sample of the lung cancer patient (kappa = 0.64, 95% confidence interval 0.32-0.95, p <0.01). K-ras mutations were detected in sputum samples from cancer patients with all lung tumour grades, and both in the presence and the absence of lymph node metastasis. Therefore, K-ras mutations may provide useful diagnostic markers for lung cancer.     

K-ras codon 12 mutations may be detected in serum of patients suffering from adeno- and large cell lung carcinoma. A preliminary report

We investigated the presence of K-ras mutations in the serum of 40 patients with respectable stages of adeno- and large cell lung carcinomas. Mutations in codon 12 of the K-ras gene were examined by enriched PCR method in DNA extracts from surgical specimens and serum samples. K-ras mutations were detected in 20 (51%) of 39 analyzed tumours, and in 7 (35%) of 20 patients with K-ras gene mutation positive tumours, a mutation was found in the serum DNA. We also found K-ras mutation in two (10.5%) of 19 serum samples obtained from patients whose tumours were not found to harbor mutation and in one serum sample from patient without tumour sample available for investigation. All of the 14 control healthy persons were negative for serum DNA K-ras mutation assay. Although our results are preliminary they show that K-ras mutation may be detected in serum of patients suffering from adeno- and large cell lung carcinomas and confirm the suggestion that at least a part of a free-cell extracellular blood DNA in cancer patients has neoplastic origin and may become a noninvasive target for genetic investigations of lung cancer patients.     

K-ras codon 12 mutations detected with enriched PCR method in operable non-small cell lung cancer

Activated point mutations of the K-ras gene are one of the most common genetic alterations found in human malignancies, including lung cancer, and are largely limited to adenocarcinomas. Using a highly sensitive assay for codon 12 K-ras mutation detection, called enriched PCR, we investigated 130 radically resected stage I-IIIa non-small cell lung cancer (NSCLC). There were statistically less positive results for squamous cell carcinoma (1.3%) than for adenocarcinoma (42.4%) and large cell carcinoma (27.8%). No statistically significant association between results of K-ras mutations and TNM stage of disease was found. In stage I of adenocarcinoma patients the incidence of K-ras mutations was similar to that in stage II or IIIa (40%, 42.9% and 42.9%, respectively). The results of our study showed that K-ras mutations might play an important role in pathogenesis of pulmonary adenocarcinoma due to its high prevalence in the operable tumours.     

Effect of an epidermal growth factor receptor inhibitor in mouse models of lung cancer

Gefitinib (Iressa, ZD1839) is a potent high-affinity competitive tyrosine kinase inhibitor aimed primarily at epidermal growth factor receptor (EGFR). Inhibitors in this class have recently been approved for clinical use in the treatment of advanced non-small cell lung cancer as monotherapy following failure of chemotherapy. We examined the efficacy of gefitinib on lung tumorigenesis in mouse models using both postinitiation and progression protocols. Gefitinib was given at a dose of 200 mg/kg body weight (i.g.) beginning either 2 or 12 weeks following carcinogen initiation. In the postinitiation protocol, gefitinib significantly inhibited both tumor multiplicity (approximately 70%) and tumor load (approximately 90%) in A/J or p53-mutant mice (P < 0.0001). Interestingly, gefitinib was also highly effective against lung carcinogenesis in the progression protocol when individual animals already have multiple preinvasive lesions in the lung. Gefitinib exhibited approximately 60% inhibition of tumor multiplicity and approximately 80% inhibition of tumor load when compared with control mice (both P < 0.0001). These data show that gefitinib is a potent chemopreventive agent in both wild-type and p53-mutant mice and that a delayed administration was still highly effective. Analyses of mutations in the EGFR and K-ras genes in lung tumors from either control or treatment groups showed no mutations in EGFR and consistent mutation in K-ras. Using an oligonucleotide array on control and gefitinib-treated lesions showed that gefitinib treatment failed to alter the activity or the expression level of EGFR. In contrast, gefitinib treatment significantly altered the expression of a series of genes involved in cell cycle, cell proliferation, cell transformation, angiogenesis, DNA synthesis, cell migration, immune responses, and apoptosis. Thus, gefitinib showed highly promising chemopreventive and chemotherapeutic activity in this mouse model of lung carcinogenesis.     

Comparative Screening of K-ras Mutations in Colorectal Cancer and Lung Cancer Patients Using a Novel Real-Time PCR with ADx-K-ras Kit and Sanger DNA Sequencing

We determined frequency/types of K-ras mutations in colorectal/lung cancer. ADx-K-ras kit (real-time/double-loop probe PCR) was used to detect somatic tumor gene mutations compared with Sanger DNA sequencing using 583 colorectal and 244 lung cancer paraffin-embedded clinical samples. Genomic DNA was used in both methods; mutation rates at codons 12/13 and frequency of each mutation were detected and compared. The data show that 91.4% colorectal and 59.0% lung carcinoma samples were detected conclusively by DNA sequencing, whereas 100% colorectal and lung samples were detected by ADx-K-ras kit. K-ras gene mutations were detected in 32.9–27.4% colorectal samples using kit and sequencing methods, respectively. Whereas 10.6–8.3% lung cancer samples were positively detected by kit and sequencing methods, respectively. Notably, 172/677 showed mutations and 467/677 showed wild type by both methods; 38 samples showed mutations with kit but wild type with sequencing. Mutations in colorectal samples were as follows: GGT → GAT/codon-12 (35.1%); GGC → GAC/codon-13 (26.6%); GGT → GTT/codon-12 (18.2%); and GGT → GCT/codon-12 (1.6%). Mutations in lung samples were as follows: GGT > GTT/codon-12 (40.9%) and GGT > GCT/codon-12 (4.5%). In conclusion, K-ras mutations involved 32.2% colorectal and 10.6% lung samples among this cohort. ADx-K-ras real-time PCR showed higher detection rates (P < 0.05). The kit method has good clinical applicability as it is simple, fast, less prone to contamination and hence can be used effectively and reliably for clinical screening of somatic tumor gene mutations.     

Reduced sensitivity to and ras mutation spectrum of N-ethyl-N-nitrosourea-induced thymic lymphomas in adult C.B-17 scid mice

Scid mice are defective in the ability to repair DNA double strand breaks and, as a consequence, their cells are radiosensitive. Further, they have been shown to be prone to develop thymic lymphomas (TLs) after small doses of ionizing radiation. Little is known, however, on the role of scid mutation in chemical carcinogenesis. To determine if scid mutation increased predisposition to chemical carcinogenesis, we examined both the susceptibility of scid mice to N-ethyl-N-nitrosourea (ENU)-induced lymphomagenesis and the involvement of ras gene activation. Adult female mice at 8 weeks of age were given ENU in their drinking water at 400 ppm for 2-10 weeks. Contrary to expectations, we observed a two to three-fold reduction in TL development in the scid mice. The highest incidence was achieved by ENU treatment for 8 weeks for scid and wild-type C.B-17 mice, of 42 and 85%, respectively (P<0.05). We investigated whether this was attributable to the usage of the ras mutation pathway. There was, however, no significant difference in the frequency and spectrum of K-ras mutation between the scid and wild-type C.B-17 mice. Most of the K-ras mutations were either GGT to GAT transition in codon 12 (11/23: 48%) or CAA to CCA transversion in codon 61 (8/23: 35%) that was independent of scid background. The incidence of N-ras mutation was very low. These results indicate that scid mice are less susceptible to ENU-induced lymphomagenesis and ras gene mutation frequently occurs in both scid and wild-type C.B-17 mice.     

Colonic Paneth cell metaplasia is pre-neoplastic condition of colonic cancer or not?

ABSTRACTS: BACKGROUND: The carcinogenesis of colorectal cancer has been accepted by a model for a cascade of genetic alterations, named the adenoma-carcinoma sequence. In order to elucidate the carcinogenesis of the colorectal cancer more clearly, the genetic abnormalies of the non-neoplastic mucosal epithelium of the colon and rectum should be investigated. It has been speculated that colonic Paneth cell metaplasia (PaM) is one of the pre-neoplastic mucosa of colonic cancer. Therefore, we studied the propria mucosa of the right colon with PaM from the standpoints of the frequency of the K-ras codon 12 mutations (K-ras), which is initial genetic abnormality in colorectal cancer, and the loss of heterozygosity of microsatellite markers (LOH-MS), which has a relationship to development of colorectal cancer. METHODS: Fifty-two regions with PaM histopathologically from 12 surgically resected right colon specimens were studied. DNA extraction of the colonic mucosa with PaM was obtained using a microdissection method, and the frequency of the K-ras of PaM was investigated by enriched polymerase chain reaction-enzyme linked mini-sequence assay, and the frequency of the LOH-MS (D2S123, D17S250 and D5S346) of PaM was examined by high resolution fluorescenced labeled PCR primers. RESULTS: K-ras mutation was detected in fifteen regions among 52 PaM (28.9%). All mutations were a single mutation and GGT changed to AGT in eleven and GAT in four. LOH-MS were detected in twenty-one regions among 52 PaM (40.4%) (D2S123: 35.4%, 17/48 regions, D17S250: 13.7%, 7/51 regions, and D5S346: 0%, 0/52 regions). No K-ras mutations and LOH-MS were detected in the controls (Colorectal mucosa with no PaM). CONCLUSIONS: Colonic mucosa with Paneth cell metaplasia may be one of the pre-neoplastic mucosa in the development of the colonic epithelial neoplasia.     

Point mutations of ras oncogenes are an early event in thyroid tumorigenesis

Identifying the nature of the genetic mutations in thyroid neoplasms and their prevalence in the various tumor phenotypes is critical to understanding their pathogenesis. Mutational activation of ras oncogenes in human tumors occurs predominantly through point mutations in two functional regions of the molecules, codons 12, 13 (GTP-binding domain) or codon 61 (GTPase domain). We examined the prevalence of point mutations in codons 12, 13, and 61 of the oncogenes K-ras, N-ras, and H-ras in benign and malignant human thyroid tumors by hybridization of PCR-amplified tumor DNA with synthetic oligodeoxynucleotide probes. None of the eight normal thyroid tissues harbored point mutations. Four of nineteen nodules from multinodular goiters (21%), 6/24 microfollicular adenomas (25%), 3/14 papillary carcinomas (21%), and 0/3 follicular carcinomas contained ras point mutations. The predominant mutation was a valine for glycine substitution in codon 12 of H-ras. None of the multinodular goiter tumors known to be polyclonal (and thus due to hyperplasia) had point mutations, whereas one of the two monoclonal adenomas arising in nodular glands contained in H-ras codon 12 valine substitution, which was confirmed by sequencing the tumor DNA. These data show that ras activation is about equally prevalent in benign and malignant thyroid neoplasms, and thus may be an early event in the tumorigenic process.     

Nitrative and oxidative DNA damage caused by K-ras mutation in mice

Ras mutation is important for carcinogenesis. Carcinogenesis consists of multi-step process with mutations in several genes. We investigated the role of DNA damage in carcinogenesis initiated by K-ras mutation, using conditional transgenic mice. Immunohistochemical analysis revealed that mutagenic 8-nitroguanine and 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) were apparently formed in adenocarcinoma caused by mutated K-ras. 8-Nitroguanine was co-localized with iNOS, eNOS, NF-κB, IKK, MAPK, MEK, and mutated K-ras, suggesting that oncogenic K-ras causes additional DNA damage via signaling pathway involving these molecules. It is noteworthy that K-ras mutation mediates not only cell over-proliferation but also the accumulation of mutagenic DNA lesions, leading to carcinogenesis.     

Concurrent mutation in exons 1 and 2 of the K-ras oncogene in colorectal cancer

The K-ras gene is frequently mutated in colorectal cancer and has been associated with tumor initiation and progression; approximately 90% of the activating mutations are found in codons 12 and 13 of exon 1 and just under 5% in codon 61 located in exon 2. These mutations determine single aminoacidic substitutions in the GTPase pocket leading to a block of the GTP hydrolytic activity of the K-ras p21 protein, and therefore to its constitutive activation. Point mutations in sites of the K-ras gene, other than codons 12, 13 and 61, and other types of genetic alterations, may occur in a minority of cases, such as in the less frequent cases of double mutations in the K-ras gene. However, all mutations in this gene, even those which occur in non-canonical sites or double mutations, are relevant oncogenic alterations in colorectal cancer and may underlie K-ras pathway hyperactivation. In the present study, we report the case of a patient with colorectal cancer presenting a concurrent point mutation in exons 1 and 2 of the K-ras gene, a GGT to TGT substitution (Glycine to Cysteine) at codon 12, and a GAC to AAC substitution (Aspartic Acid to Asparagine) at codon 57. In addition, we found in the same patient's sample a silent polymorphism at codon 11 (Ala11Ala) of exon 1.     

A novel activating mutation of the K-ras gene in human primary colon adenocarcinoma

We identified a novel type of point mutation at the 22nd codon of the K-ras gene in a primary colon cancer. The mutation was C to A transversion substituting lysine (AAG) for normal glutamine (CAG) codon. Biological activity of this mutant K-ras gene was tested by expression of full-length cDNA clones in NIH3T3 cells. Most of the K-ras Lys22-transfected cells exhibited an increased saturation density, a lower serum requirement, and transformed morphology reminiscent to the typical K-ras Val12 transformants. However, the tumorigenicity of K-ras Lys22 transformants in nude mice was significantly less potent than that of K-ras Val12 transformants; only a high copy number transformant produced tumors. Even though the activation is incomplete, the finding that the majority of tumor cells in the specimen carried the K-ras Lys22 mutation suggests that this mutation might be advantageous for growth of tumor cells in vivo.     

Characterization of a novel oncogenic K-ras mutation in colon cancer

Activating mutations of RAS are frequently observed in subsets of human cancers, indicating that RAS activation is involved in tumorigenesis. Here, we identified and characterized a novel G to T transversion mutation of the K-ras gene at the third position of codon 19 (TTG) which substituted phenylalanine for leucine in 3 primary colon carcinomas. Biological and biochemical activity was examined using transformed NIH3T3 cells expressing mutant or wild-type K-ras. Transformants harboring the K-ras mutation at codon 19 showed proliferative capacity under serum-starved conditions, less contact inhibition, anchorage-independent growth, tumorigenicity in nude mice and elevation of active Ras-GTP levels. These results indicated that this novel mutation possesses high oncogenic activity.     

Diallyl sulfide enhances azoxymethane-induced preneoplasia in Fischer 344 rat colon

Azoxymethane (AOM) is an indirect-acting colon carcinogen that produces a high incidence of precancerous lesions, referred to as aberrant crypt foci (ACF), in rats. This study was undertaken to determine whether high dose gavage administration of the cytochrome P-450 2E1 (CYP2E1) inhibitor and chemopreventive agent, diallyl sulfide, would reduce the incidence and severity of ACF formation in the distal colons of AOM-treated Fischer 344 rats. Seven-week-old male rats received 150 or 50 mg/kg diallyl sulfide by gavage 24 and 2 h prior to two weekly i.p. injections of AOM (20 mg/kg). Ten weeks after the last injection of AOM the rats were sacrificed and the colons removed and stained with 0.2% methylene blue. ACF were visualized using stereomicroscopy. Rats pretreated with diallyl sulfide exhibited a significant increase in the number of ACF/cm in the distal colon compared with rats receiving AOM alone. This increase in ACF number was seen in ACF of all sizes. To examine the effects of diallyl sulfide on the initiation stage of AOM-induced carcinogenesis, mutations in the K-ras proto-oncogene were also investigated. ACF and normal appearing colonic mucosa (0.2-0.5 mm3) were microdissected for subsequent PCR-RFLP analysis of a codon 12 (GGT-GGA) activating mutation in the K-ras gene. Greater than 90% of ACF from AOM-treated animals, regardless of diallyl sulfide treatment, exhibited activating K-ras mutations. K-ras mutations were also detected in normal appearing mucosa of AOM-treated animals, although at a lesser frequency (15-35%). These studies demonstrate that diallyl sulfide given in large gavage doses enhances AOM-induced preneoplasia in rats and suggests that diallyl sulfide may alter the disposition of AOM intermediates and/or enhance colonic promotional activity in the rat.     

High susceptibility of transgenic rats carrying the human c-Ha-ras proto-oncogene to chemically-induced mammary carcinogenesis

A rat line carrying three copies of the human c-Ha-ras proto-oncogenes, including its own promoter region, was established and designated as Hras128. Expression of the transgene was detected in all organs by Northern blot analysis. To examine its influence on susceptibility to mammary carcinogenesis, female rats were treated with N-methyl-N-nitrosourea (MNU) or 7,12-dimethylbenz[a]anthracene (DMBA) at 50 days of age. With MNU, all the transgenic rats rapidly developed multiple mammary carcinomas within as short as 8 weeks (14.1 tumors/rat), in contrast to 0.46 tumors/rat in non-transgenic rats. PCR-RFLP analysis and direct sequencing for the transgene indicated that the large majority of carcinomas (38/44, 86.4%) contained cells with mutations at codon 12 in exon 1. However, comparison of the signal densities of the mutated band to dilution scale bands revealed that the cells with the mutated transgene were not in the majority. By PCR-SSCP analysis for codons 12 and 61 of the rat endogenous c-Ha-ras gene, no mutations were detected. Similarly, with DMBA, almost all (13/14, 92.9%) the transgenic rats developed multiple mammary carcinomas (9.39 tumors/rat) within 16 weeks, and 4 out of 12 (33.3%) non-transgenic rats had only small tumors (0.83 tumors/rat). A lower incidence of mutation of the transgene was found in codon 12 (5/25, 25%) than in MNU-induced tumors, but mutations were detected in codon 61 (7/20, 35%). No mutations were detected in the rat endogenous gene. No mutation was found in the rat endogenous c-Ha-ras gene in non-transgenic rats. As observed in both the MNU- and DMBA-induced tumor cases, the population of cells with the mutated transgene were in the minority. The results thus indicate that rats carrying the transduced human c-Ha-ras proto-oncogene are highly susceptible to MNU- and DMBA-induced mammary carcinogenesis and that this is not primarily due to mutations of the transgene or endogenous c-Ha-ras gene. Furthermore, irrespective of the mechanism of enhanced susceptibility, the Hras128 transgenic rats can be utilized for the screening of mammary carcinogens.     

Detection of K-ras and p53 mutations in sputum samples of lung cancer patients using laser capture microdissection microscope and mutation analysis

Mutations in the p53 tumor suppressor gene and the K-ras oncogene have been frequently found in sputum and bronchoalveolar lavage (BAL) samples of lung cancer patients and other patients prior to presenting clinical symptoms of lung cancer, suggesting that they may provide useful biomarkers for early lung cancer diagnosis. However, the detection of these gene mutations in sputum and BAL samples has been complicated by the fact that they often occur in only a small fraction of epithelial cells among sputum cells and, in the case of p53 gene, at many codons. In this study, sputum cells were collected on a filter membrane by sputum cytocentrifugation and morphologically analyzed. Epithelial cells were selectively taken by using a laser capture microdissection microscope and analyzed by polymerase chain reaction (PCR) and single-stranded conformational polymorphism (SSCP) for p53 mutations and by PCR and denaturing gradient gel electrophoresis (DGGE) for K-ras mutations. This method was used to analyze sputum of 15 Chinese women with lung cancer from Xuan Wei County, China and detected mutations in sputum of 7 (46.7%) patients, including 5 patients with p53 mutations, 1 patient with a K-ras mutation, and 1 patient with K-ras and p53 mutations. For comparison, only two of the mutations were detected by conventional methods. Therefore, the laser capture/mutation analysis method is sensitive and facilitates the detection of low-fraction mutations occurring throughout the p53 and K-ras genes in sputum of lung cancer patients. This method may be applicable to the analysis of epithelial cells from clinically normal sputum or BAL samples from individuals with a high risk for developing lung cancer.