Selective occurrence of ras mutations in benign and malignant thyroid follicular neoplasms in Taiwan.

Previous studies have demonstrated that point mutations in all three ras genes (H-ras, K-ras, and N-ras) may occur in thyroid neoplasia. However, the overall incidence of ras mutations in thyroid tumors and their frequency in specific histologic types varies widely in different series. Many earlier studies have chosen allele-specific oligonucleotide hybridization approaches to examine ras mutations without further confirmation of the positive samples by DNA sequencing. In this study, mutational hot spots in exon 1 (codons 12/13) and exon 2 (codon 61) of the H-ras, K-ras, and N-ras were polymerase chain reaction (PCR) amplified and sequenced with an automatic sequencer. ras mutations were detected in 4 of 89 (4.5%) benign and malignant thyroid tumors. Three of 8 follicular carcinomas exhibited mutations in codon 61 of H-ras, K-ras, and N-ras, respectively, and mutation at codon 61 of N-ras was found in 1 of 12 follicular adenomas. No mutations were observed in the other tumors, which included 20 nodular goiters, 5 Hürthle cell adenomas, 42 papillary carcinomas, and 2 undifferentiated carcinomas. Our results, obtained by the direct sequencing technique, indicate a lower overall prevalence of ras oncogenes in thyroid tumors than reports in earlier series. However, the frequency of ras mutations in specific histotype of thyroid tumors and their exclusive involvement of codon 61 in our series are similar to those studies utilizing DNA sequencing to detect or to confirm ras gene alterations. The selective occurrence of ras mutations in benign and malignant follicular neoplasms indicates that ras gene alterations have a specific and early role in the development of follicular type of thyroid tumors in Taiwan.     

Infrequent point mutations of ras oncogenes in gastric cancers

Adenocarcinomas of the proximal and distal stomach have significant clinical and biological differences. A study was undertaken to determine if a difference in the incidence of mutated ras oncogenes exists between proximal (gastroesophageal junction or cardia) and distal (body or antrum) gastric tumors, and to assess the overall incidence in gastric cancers from non-Asian patients. Deoxyribonucleic acid from 28 primary gastric adenocarcinomas were analyzed for point mutations in codons 12, 13, and 61 of the Ha-ras, Ki-ras, and N-ras protooncogenes using polymerase-catalyzed chain reaction methodology. Twelve tumors were located at the gastroesophageal junction or cardia, and 16 in the body or antrum. Mutated ras genes were detected in 2 of 28 tumors for an overall incidence of 7%. The mutations occurred in codon 61 of the N-ras gene in a proximal gastric cancer and in codon 12 of the Ki-ras gene in a distal gastric cancer. These data indicate that mutations in ras genes are an uncommon event in primary gastric cancers and that there is no meaningful difference in the incidence of ras mutations in proximal and distal stomach cancers.     

Detection of ras gene mutations of primary hepatic malignant tumors by polymerase chain reaction and direct sequencing method

Ras genes are converted to active oncogenes by point mutations occurring in either codon 12, 13 or 61. We used polymerase chain reaction and direct sequence method for the analysis of these mutations. We examined 13 hepatocellular carcinomas, 8 cholangiocarcinomas, 2 hepatoblastomas and 1 biliary cystadenocarcinoma. Of these tumors, ras gene mutations were detected solely in cholangiocarcinomas. Cholangiocarcinoma showed a high incidence of K-ras gene mutation. Among 8 patients with cholangiocarcinoma, the mutation was detected at codon 12 in 4 and at codon 61 in 1. The incidence of K-ras gene mutation was especially high in the hilar type of cholangiocarcinoma as compared with the peripheral type.     

Mutations of the p53 and ras genes in childhood t(1;19)-acute lymphoblastic leukemia

We have investigated the alterations of p53 and ras genes including H-, K-, and N-ras genes in 22 acute lymphoblastic leukemia (ALL) cases and five cell lines carrying t(1;19) by use of polymerase chain reaction (PCR)-single-strand conformation polymorphism (SSCP) analysis and direct sequencing. The mutations of the p53 gene were found in 2 of 20 t(1;19)-ALL cases at diagnosis (10%), all of 4 cases at relapse (100%), and 4 of the 5 cell lines (80%). Four of the five patients who died had missense mutations at codons 49, 177, 179, and 248. In cases examined sequentially, one had the same point mutation at codon 179 at both diagnosis and relapse, and another had the same p53 gene mutation at codon 240 both in leukemic cells at relapse and in a cell line derived at that time. The other case had no mutation at diagnosis but had the mutation at codon 177 at relapse and cell lines derived from blast cells at diagnosis, suggesting that a small number of leukemic cells with the p53 gene mutation at diagnosis might have escaped PCR-SSCP analysis. In cell lines, SCMC-L9 had three point mutations in the p53 gene at codons 175, 248, and 358, whereas SCMC-L10 had frame shift at codons 209-211. One case had a rare polymorphism at codon 11. We found only one mutation of the N-ras gene that was a 2-bp substitution of GGT(Gly) to GTC(Val) at codon 13 among 22 t(1;19)-ALL cases and five cell lines. This case showed no mutation of the p53 gene and has had a good course. These results suggest that in t(1;19)-ALL, mutations of the p53 and ras genes are infrequent at diagnosis and that p53 gene alterations may be associated with relapse phase or progression of t(1;19)-ALL.     

Infrequent point mutations in codons 12 and 61 of ras oncogenes in human hepatocellular carcinomas.

DNA from human hepatocellular carcinomas (HCC) were analysed for the presence of mutations in codons 12 and 61 of the K-ras, H-ras and N-ras genes. The relevant ras sequences were amplified in vitro using the polymerase chain reaction and point mutations detected by selective hybridisation using mutation-specific synthetic oligonucleotides. In one of the 19 HCCs a mutation in codon 61 of the K-ras gene was detected, whilst in 3/19 HCCs a mutation was found in codon 61 of the N-ras gene. The mutations were all heterozygous A-T transversions and were found in HCCs arising in patients with underlying cirrhosis. In two of these patients where the corresponding normal tissue was available only the wild-type ras gene was detected, indicating that oncogenic activation of the ras gene was a consequence of somatic mutation. In another patient the same mutation in codon 61 of the N-ras gene was found in cirrhotic liver tissue and in all four patients the same mutation was also detected in formalin-fixed, paraffin-embedded liver biopsy HCC tissue obtained at diagnosis. These results indicate that mutational activation of the ras genes at codon 61 is an infrequent but possibly early event in the development of HCC in Britain.     

Ras mutations are uncommon in sporadic thyroid cancer in children and young adults

Mutations in the ras genes (H-ras, K-ras, and N-ras) occur in 10-15% of all human cancers, and commonly arise from single base substitutions at codons 12, 13, or 61. Although ras mutations have been found in adult thyroid cancers, they were absent from the two studies which examined childhood thyroid cancers. Both studies included only children with radiation induced thyroid cancer, and it remains unclear if ras mutations occur in children without radiation exposure. To answer this question, we examined archival tissue blocks from 31 children with papillary thyroid cancer (PTC) 4 with follicular thyroid cancer (FTC), 2 with medullary thyroid cancer (MTC), and 1 with lymphoma (LYM). Only 1 patient with PTC had previous radiation exposure. Genomic DNA was extracted and used for PCR amplification of the ras genes. The PCR products were analyzed by oligospecific hybridization for mutations at codons 12, 13, and 61. Two of the PTCs (6.5%) contained ras mutations. Both patients had class II disease and no history of previous radiation exposure. One patient subsequently developed bone and lung metastases. The patient with lymphoma also had a ras mutation (N-61), but ras mutations were absent from all FTC and MTC. These results suggest that ras mutations are uncommon in spontaneous childhood thyroid cancer, but occur with a frequency similar to that found in previous reports of adult differentiated thyroid cancers. The number of subjects was too small to determine if ras mutations are more common in patients with aggressive papillary thyroid cancer.     

Ras oncogene mutations in benign and malignant thyroid neoplasms

Current models for tumorigenesis propose that a series of genetic alterations occur during the progression from the normal cell to the malignant phenotype. Mutations in each of the three ras genes (K-ras, H-ras, and N-ras) have been identified in many human neoplasms, including thyroid cancer. In this study we examined genomic DNA from benign and malignant thyroid neoplasms for mutations that are known to activate the ras oncogenes (codons 12, 13, and 61). DNA from frozen surgically excised tissue (n = 8) and from formalin-fixed paraffin-embedded tissue (n = 30) was amplified by the polymerase chain reaction and screened for mutations using oligonucleotide-specific hybridization. No mutations were identified in follicular adenomas (n = 9). In follicular carcinomas, 2 of 14 tumors contained mutations (N-ras 61, Gln to Arg), and both of these patients had bone metastases. One of 15 papillary carcinomas had a ras mutation (H-ras 12, Gly to Ser). In contrast to other studies, we found that ras mutations are relatively uncommon in both benign and malignant thyroid neoplasms. Studies of larger numbers of tumors and comparisons of different patient populations will be required to assess a possible association of mutations in N-ras 61 with clinically aggressive follicular cancer.     

Analysis of N-ras gene mutations in acute myeloid leukemia by allele specific restriction analysis

N-ras gene activation occurs via single base substitutions in codons 12, 13, and 61. We have developed a rapid screening method, termed allele specific restriction analysis (ASRA), for detection of N-ras mutations at these three critical codons in acute myeloid leukemia (AML). Patient DNA samples are amplified by the polymerase chain reaction (PCR) by using primers that induce restriction sites in normal but not mutant N-ras alleles. We have used ASRA to identify 5 point mutations in four out of 19 patients at initial presentation of de novo AML. Three patients had one mutation at codon 12, 13, or 61 respectively, while a fourth patient had concurrent mutations at codons 12 and 13. N-ras mutations were more common in patients over 65 years of age (P less than 0.04), but did not correlate with FAB classification, attainment of complete remission, disease free survival, or overall survival. ASRA can also be used as the first step in a more sensitive approach to the detection of ras mutations. When ASRA was combined with allele specific oligonucleotide (ASO) hybridization the sensitivity and specificity of these assays were increased. This allowed identification of additional low level mutations in two patients. The data presented here constitute the first complete analysis of N-ras mutations in leukemia by ASRA and include the first identification of three concurrent N-ras mutations in a single leukemic patient. By facilitating sensitive sequential studies, ASRA should contribute to our understanding of the role of N-ras mutations in leukemogenesis.     

Detection of N-Ras codon 61 mutations in subpopulations of tumor cells in multiple myeloma at presentation

Activating point mutations in codons 12, 13, or 61 of the K-ras and N-ras genes have been reported to occur in up to 40% of patients with multiple myeloma at presentation. In a study of 34 presentation myeloma cases using a sensitive polymerase chain reaction-restriction fragment length polymorphism strategy on enriched tumor cell populations, the present study detected N-ras codon 61 mutation-positive cells in all patients. Quantitative plaque hybridization using allele-specific oligonucleotide probes showed that in the majority of patients, ras mutation-positive cells comprise only a subpopulation of the total malignant plasma cell compartment (range, 12%-100%). Using clonospecific point mutations in the 5' untranslated region of the BCL6 gene to quantitate clonal B cells in FACS-sorted bone marrow populations from 2 patients, the representation of ras mutation-positive cells was independent of immunophenotype. These observations imply that mutational activation of N-ras codon 61 is a mandatory event in the pathogenesis of multiple myeloma; such mutations provide a marker of intraclonal heterogeneity that may originate at an earlier ontologic stage than immunophenotypic diversification of the malignant B cell clone.     

A method to detect and characterize point mutations in transcribed genes: amplification and overexpression of the mutant c-Ki-ras allele in human tumor cells.

A significant percentage of human tumors contain activated ras oncogenes that have acquired oncogenic potential as a result of somatic point mutations at codon 12 or 61 of the encoded ras gene product. We report here a method to detect and characterize mutations in ras genes that is based on the ability of pancreatic ribonuclease (RNase A; EC 3.1.27.5) to cleave RNA heteroduplexes containing single-base mismatches. Using this method, we show that certain human tumor cells contain mutant c-Ki-ras genes, and we define the nature and position of these mutations. At the same time, we describe the presence and estimate the expression of both normal and mutant c-Ki-ras alleles in the same tumor cells. This method should be useful for the diagnostic detection and characterization of single point mutations in expressed genes.     

Analysis of ras gene mutations in childhood myeloid leukaemia

Previous studies have shown that approximately 30% of adult acute myeloid leukaemias and 20% of adult acute lymphoid leukaemias contain point mutated ras oncogenes. In order to assess whether ras oncogenes are also involved in childhood leukaemias, we have used polymerase chain reaction (PCR) amplification and synthetic oligonucleotide probes to study the nature and frequency of ras gene mutations in childhood leukaemias, concentrating largely on the acute myeloid leukaemias (AML). Thirty-four childhood presentation AML DNAs were screened for mutations in and around codons 12, 61 and 117 of N-, K- and H-ras. Eight of these samples (24%) contained ras mutations. As in the adult disease, the gene predominantly involved was N-ras (6/8), with occasional activation of K-ras (2/6). The most common base change was a G----A transition at codon 12 or 13 (4/8). Of the patients with mutant ras, 4/8 were diagnosed as AML FAB subtype M5. Five of the 34 childhood AMLs analysed displayed abnormalities of chromosome 7. However, none of these cases contained a mutant ras gene. One AML patient was studied at relapse, 14 months after initial presentation. The presentation mutation (N61p3) was not detectable, although a new mutation (N13Cys) was readily identified. This observation extends our original finding with presentation and relapse samples of adult AML, in which it was uncommon for the relapse sample to contain the same ras mutation as the presentation DNA. In addition, two out of five patients diagnosed as juvenile CML, were found to harbour mutant ras.     

Frequency and clinicopathology associations of K-ras mutations in colorectal cancer in a northeast Mexican population

Activation of the ras family gene has been implicated in colorectal tumorigenesis, K-ras being the most frequently altered gene. The frequency of K-ras codon 12, 13 and 61 point mutations in patients with colorectal neoplasias was examined. We employed a polymerase chain reaction-restriction fragment length polymorphism assay and single-strand conformational polymorphism to detect mutations. We found that point mutations at codons 12 and 13 were present in 53% and 39% of the tumors, respectively, but none at codon 61. These results agree with previous reports. Point mutations were more frequent in adenomas than in carcinomas, with villous adenomas presenting a higher incidence of mutations than other adenomas. The association between clinical and histopathological parameters was investigated. Our study is the beginning of a new research line in molecular epidemiology of colorectal cancer and is the first to be carried out in one part of the Mexican population.     

Ras oncogene and p53 gene hotspot mutations in colorectal cancers

Abstract Ras oncogene and p53 gene mutations are frequently observed in colorectal cancers. The role of co-operation between these two genes in the tumorigenesis of colorectal cancer was evaluated. Point mutations in K-ras oncogene and hotspot codons of p53 gene of colorectal cancers were evaluated by naturally created or amplified created restriction site method. Nine of 42 cases (21.4%) of colorectal cancer showed K-ras oncogene mutations. Six of 42 cases (14.3%) of colorectal cancer showed p53 gene hotspot point mutations. The low frequency of p53 gene mutation in this series may be due to racial difference or different hotspot codons. When six cases with mutated p53 gene were examined, only one (16.7%) showed concurrent K-ras oncogene codon 12 and p53 gene codon 248 mutations. We concluded that the co-operation between ras oncogene and p53 gene hotspot point mutations in the tumorigenesis of colorectal cancer in Chinese was not common. Other factors such as adenomatous polyposis coli gene mutations, oncogene activation or tumour suppression gene inactivation may be involved.     

Mutations in ras genes in cells cultured from mouse skin tumors induced by ultraviolet irradiation.

Mutations in ras oncogenes were detected in cultured cells of mouse skin tumors induced by near-UV irradiation. DNA extracted from the UV-induced tumor cells was transfected to golden hamster embryo cells, and focus-forming ability was confirmed in 22 of 26 cell strains, 15 of which had the repetitive mouse sequence. Mouse ras genes were detected in 10 of these 22 cell strains. Point mutations in the ras genes were at Ha-ras codon 13 (GGC-->GTC in two strains, GGC-->AGC in one strain), Ki-ras codon 61 (CAA-->GAA in two strains), and N-ras codon 61 (CAA-->CAT in two strains, CAA-->AAA in two strains). In one tumor cell strain no base change was directed. Most mutations occurred at dipyrimidine sites. Pyrimidine dimers or pyrimidine(6-4)pyrimidone photoproducts are the likely cause of the skin cancers. The base change occurred preferentially at G.C base pairs, and transversions predominated.     

Relating aromatic hydrocarbon-induced DNA adducts and c-H-ras mutations in mouse skin papillomas: the role of apurinic sites.

Mouse skin tumors contain activated c-H-ras oncogenes, often caused by point mutations at codons 12 and 13 in exon 1 and codons 59 and 61 in exon 2. Mutagenesis by the noncoding apurinic sites can produce G-->T and A-->T transversions by DNA misreplication with more frequent insertion of deoxyadenosine opposite the apurinic site. Papillomas were induced in mouse skin by several aromatic hydrocarbons, and mutations in the c-H-ras gene were determined to elucidate the relationship among DNA adducts, apurinic sites, and ras oncogene mutations. Dibenzo[a,l]pyrene (DB[a,l]P), DB[a,l]P-11,12-dihydrodiol, anti-DB[a,l]P-11,12-diol-13,14-epoxide, DB[a,l]P-8,9-dihydrodiol, 7,12-dimethylbenz[a]anthracene (DMBA), and 1,2,3,4-tetrahydro-DMBA consistently induced a CAA-->CTA mutation in codon 61 of the c-H-ras oncogene. Benzo[a]pyrene induced a GGC-->GTC mutation in codon 13 in 54% of tumors and a CAA-->CTA mutation in codon 61 in 15%. The pattern of mutations induced by each hydrocarbon correlated with its profile of DNA adducts. For example, both DB[a,l]P and DMBA primarily form DNA adducts at the N-3 and/or N-7 of deoxyadenosine that are lost from the DNA by depurination, generating apurinic sites. Thus, these results support the hypothesis that misreplication of unrepaired apurinic sites generated by loss of hydrocarbon-DNA adducts is responsible for transforming mutations leading to papillomas in mouse skin.     

Ras mutations in human pituitary tumors.

The cellular basis for pituitary neoplasia is poorly understood. Mutations that activate the ras protooncogenes have been identified in a number of different types of human cancers and potentially represent one of the genetic alterations that occur in pituitary tumors. In this study we examined 19 pituitary tumors for the occurrence of ras mutations. The tumor types included 11 nonfunctioning adenomas, 6 somatotroph adenomas, and 2 prolactinomas. Each of the three ras genes (K-ras, N-ras, and H-ras) was amplified from pituitary tumor DNA using the polymerase chain reaction. Oligonucleotide-specific hybridization was used to screen for mutations that inhibit GTPase activity and cause activation of the ras oncogene. No ras mutations were observed in 18 of the pituitary adenomas. However, a mutation was identified in codon 12 of the H-ras gene (Gly to Val) in a recurrent prolactinoma that was highly invasive and ultimately proved to be fatal. We conclude that ras mutations are uncommon in pituitary adenomas, but may provide a marker for highly invasive tumors.     

Frequency of molecular alterations affecting ras protooncogenes in human urinary tract tumors.

Members of the ras gene family are activated as oncogenes in many different human cancers. To systematically determine the frequency at which such genes might be involved in the neoplastic process affecting a specific target tissue, urothelial cells, we surveyed a large series of urinary tract tumors for ras oncogenes by DNA transfection and by molecular genetic analysis. Harvey (Ha)-ras oncogenes were detected in 2 of 38 tumors by transfection, molecularly cloned in biologically active form, and shown to contain single base changes at codon 61 leading to substitutions of arginine and leucine, respectively, for glutamine at this position. One additional Ha-ras oncogene was identified in a bladder carcinoma by restriction polymorphisms at codon 12. In one of 21 tumors, we observed a 40-fold amplification of the Kirsten (Ki)-ras gene. No amplification of other ras genes was detected in any of the tumors analyzed. Our findings strengthen the conclusion that codons 12 and 61 are the major "hot spots" of ras oncogene activation and suggest that quantitative alterations in expression due to gene amplification may provide an alternative mechanism for ras gene activation in primary human tumors.     

Ras oncogene mutations are rare late stage events in chronic myelogenous leukemia

DNA from bone marrow and peripheral blood samples of 44 chronic myelogenous leukemia (CML) patients were analyzed for the presence of mutations of codons 12, 13 or 61 of the N-ras, H-ras, or K-ras genes. In seven patients, samples were available from both their chronic phase and blast crisis. A total of 29 samples examined were at chronic phase and 22 were at blast crisis (eight lymphoid, eight myeloid, and six undifferentiated). No mutations were identified in N-ras or H-ras. Two patients in myeloid blast crisis had K-ras mutations, one patient at codon 12, the other at codon 13. In the former patient the mutation was not present and the latter patient was not tested in chronic phase. Our findings indicate ras mutations are an infrequent late stage event in CML that occur in myeloid blast crisis.     

Prevalence of Ras mutations in thyroid neoplasia

OBJECTIVE: Mutations at codons 12, 13 or 61 of ras which result in constitutive activation occur frequently in human malignancies. There have been varied reports on their prevalence and hence their likely significance in the pathogenesis of primary thyroid neoplasia. To address this, we have examined a large series of benign and malignant thyroid tumours for ras mutations. DESIGN: Genomic DNA was analysed for the presence of mutations at codons 12, 13 and 61 of H-ras, K-ras and N-ras by allele-specific oligonucleotide hybridization. Direct DNA sequencing was used to confirm the mutations. PATIENTS: A total of 90 samples with benign (66) and malignant (24) thyroid disease were investigated. RESULTS: A total of 14/90 (15.5%) samples had a ras mutation. All mutations were at codon 61 of either N-ras or K-ras. The positive cases were 1/25 (4%) nodular goitre, 7/38 (18%) follicular adenoma, 4/9 (44%) follicular carcinoma, 1/1 anaplastic carcinoma, 1/1 follicular variant of papillary carcinoma, and 1 metastatic follicular carcinoma in which the primary tumour had the same mutation. CONCLUSIONS: Our data demonstrate a relatively low overall prevalence of ras mutations in thyroid neoplasia, with a predominance in follicular neoplasms. Their presence in follicular adenomas suggests that they may have an early aetiological role in the development of thyroid neoplasia.     

Mutational activation of N- and K-ras oncogenes in plasma cell dyscrasias

The frequency of N- and K-ras oncogene mutations was investigated in plasma cell dyscrasias. Genomic DNAs from 128 patients were selected for this study: 30 monoclonal gammopathies of undetermined significance, 8 solitary plasmacytomas, 77 multiple myelomas (MM), and 13 plasma cell leukemias (PCL). A two-step experimental approach was devised. All samples were screened for mutations by single-strand conformation polymorphism analysis. DNA fragments displaying an altered electrophoretic mobility were further studied by direct sequencing to confirm and characterize the nature of the mutations. Ras mutations are not randomly distributed because they are detectable only in MM (9%) and PCL (30.7%). N-ras codons 12, 13, and 61 and K-ras codon 12 were found to be mutated, but N-ras codon 61 mutation was the most frequent finding (63.6%). In conclusion, ras mutations were found in PCL, and in a subset of MM characterized by advanced-stage disease and adverse prognostic parameters. Furthermore, based on our findings, it is possible to speculate that ras mutations represent a late molecular lesion in the process of multistep carcinogenesis.