Mutations in ras oncogenes: Rare events in ultraviolet B radiation-induced mouse skin tumorigenesis

The activation of ras proto-oncogenes by point mutation in a broad spectrum of clinical malignancies and experimentally induced tumors suggests their critical role in cancer induction. To determine whether the activation of ras proto-oncogenes by point mutation also contributes to ultraviolet B radiation (UVB)-induced skin tumorigenesis and whether this event is responsible for the different tumorigenic potentials of UVB radiation in different mouse strains, we analyzed the skin tumors induced by UVB in SKH-1 hairless and C3H mice for specific mutations in the Ha-, Ki-, and N-ras oncogenes. With the same UVB irradiation protocol, the latency period for tumor appearance was longer in C3H mice than in SKH-1 hairless mice. In addition, tumor incidence and multiplicity were also significantly higher (P < 0.001, χ² and Wilcoxon rank sum tests) in SKH-1 hairless mice compared with C3H mice. None of the 30 skin tumor specimens (15 from each mouse strain) analyzed by polymerase chain reaction (PCR) amplification of specific codons followed by dot-blot hybridization with specific probes contained mutation in codons 13 of Ha-ras; 12, 13, and 61 of Ki-ras; or 12 and 13 of N-ras. However, three of the 15 tumors in SKH-1 hairless mice showed either a G³⁵ → A or G³⁵ → T transition at second position of Ha-ras codon 12. Interestingly, one of these tumors (with a G³⁵ → A transition) also harbored an A¹⁸² → G mutation at second position of Ha-ras codon 61. None of the tumors from C3H mice showed mutations in codons 12 or 61 of the Ha-ras oncogene. With regard to codon 61 of the N-ras oncogene, six tumors from SKH-1 hairless mice and 10 tumors from C3H mice showed an A¹⁸³ → T transversion. While G³⁵ → A or G³⁵ → T transition detected by PCR and dot-blot hybridization was confirmed by sequencing, the mutations identified similarly at codon 61 in either the Ha- or N-ras oncogenes could not be verified by sequencing of PCR-amplified products subcloned into plasmid vectors. With the exception of the low incidence of Ha-ras oncogene mutations at codon 12 in SKH-1 hairless mouse skin tumors induced by UVB, the striking absence of mutations in the Ha-, Ki-, and N-ras oncogenes in UVB-induced mouse skin tumors suggests that ras oncogene mutations are rare and thus are not an initiating event in photocarcinogenesis. © 1996 Wiley-Liss, Inc.     

Low incidence of H-, K- and N-ras oncogene mutations in cytological specimens of laryngeal tumours

Laryngeal cancer is a rare type of neoplasia, constituting approximately 2% of all human cancers. Mutations of the ras gene family is one of the main activating mechanisms in human cancer. Their involvement in head and neck cancer has been mainly demonstrated at the level of the overexpression whereas ras mutations in these cancers are rare in the Western world. In the present study we explored the incidence of codon 12-point mutation in the H-, K- and N-ras genes, in 41 laryngeal cytological specimens. These specimens corresponded to 19 benign and 22 malignant lesions of the larynx. Only two specimens carried a codon 12-point mutation in the K-ras gene (4.8%) while no mutation was detected in the H- and N-ras genes. K-ras mutations were detected in one benign and one malignant specimen. These results indicate low incidence of ras oncogene mutations in laryngeal cytological specimens.     

International comparison on ras gene mutations in latent prostate carcinoma

Latent carcinomas of the prostate, discovered at autopsy in men with no prior treatment for prostatic disease, were studied for ras proto-oncogene mutations. Subjects included 21 Japanese, 15 U.S. whites, 15 U.S. blacks, 20 Hawaiian Japanese and 10 Colombians. PCR and sequence-specific oligonucleotide hybridization identified mutations in 5 Japanese, in 1 Hawaiian Japanese, in 1 U.S. black, in 1 U.S. white and in 3 Colombians. The 5 Japanese tumor samples contained 3 point mutations in codon 12 of K-ras and 2 mutations in codon 12 of N-ras respectively. One tumor in a Hawaiian Japanese man also showed a K-ras point mutation at codon 12. Two Colombians and one U.S. black man had tumors with mutations at codon 61 of H-ras, while 1 Colombian showed an N-ras mutation at this codon. The overall frequency of ras gene mutations was low, but point mutations in codon 12 were most common in latent tumors of Japanese, who experienced the lowest incidence and mortality from this tumor. Latent tumors in men from ethnic groups with high mortality and incidence rates showed fewer ras mutations than the Japanese, and these were more likely to involve codon 61. This finding is consistent with prior studies of more aggressive clinical cancers in Japanese men that indicated a higher frequency of mutations at codon 61.     

ras Gene mutations and hpv infection are common in human laryngeal carcinoma

To evaluate the role of ras activation and human papillomavirus (HPV) infection in laryngeal carcinoma, we analyzed tumor DNA from 43 cases, including 25 primary laryngeal tumors, 12 lymph-node and one skin metastases, and 5 recurrent laryngeal carcinomas. Thirteen normal laryngeal tissues and 7 benign laryngeal nodule biopsy specimens along with normal tissue surrounding laryngeal carcinoma in 2 cases were also included. The polymerase-chain-reaction technique was used to amplify DNA fragments containing codon 12 and 61 of H-, K- and N-ras, also HPV 16, 18 and 33 DNA, subsequently hybridized with sequence-specific oligonucleotides. DNA samples from 22 patients with laryngeal carcinoma revealed ras mutations (18 in N-ras codon 12, 6 in H-ras codon 61, and 3 in K-ras codon 61). Likewise, HPV DNA was found in 16 cases (HPV 16, 18 and 33 in 3 cases, 14 cases and 1 case respectively). ras mutations were significantly higher in metastatic tumors (10 of 13 cases) than in primary (11 of 25 cases) and recurrent laryngeal carcinomas (1 of 5 cases). HPV DNA was detected in 60% of recurrent, 44% of primary and 15% of metastatic tumors. Only 2 of the 13 normal laryngeal tissues and 1 out of 7 laryngeal nodule specimens were found to contain HPV DNA. These results suggest that ras activation, especially in N-ras codon 12.1 (GGT → AGT) and HPV infection are 2 important factors in (multistage) laryngeal carcinogenesis. The ras mutation may be associated with metastatic ability of the tumor.     

Induction and characterization of cytotoxic T-lymphocytes recognizing a mutated p21ras peptide presented by HLA-A*0201

The ras oncogene is frequently found to be activated in human cancer through point mutations at codons 12, 13 or 61. We explored whether these altered p21 ras protein sequences contain peptide sequences that can activate naive CD8⁺ cytotoxic T lymphocytes (CTL). Several wild-type and mutated p21 ras peptides were identified that carry a binding motif for human leukocyte antigen (HLA)-A*0201. Two peptides were found to bind strongly to this allele. CD8* CTL bulk cultures specifically reacting with one of these peptides could be induced, using processing-defective T2 cells loaded with peptide CLLDILDTAGL as stimulators. The peptide is derived from p21ras, position 51–61, and carries a 61 Gln → Leu mutation. In contrast, a 9-mer peptide CLLDILDTA corresponding to amino acid sequence 51–59 of wild-type p21ras did not yield reactive CTL cultures. T-cell clones with low affinity for the 11-mer peptide were isolated from CLLDILDTAGL-reactive bulk cultures. These T cells did not lyse melanoma cells transfected with 61-Leu N-ras, although lysis was found when these transfectants were pulsed with the 11-mer peptide. Possibly, T cells of higher affinity may be required to demonstrate processed peptide on the cell surface. The combined experiments suggest that a peptide derived from mutated p21ras can be recognized by HLA class 1-restricted CTL, whereas an analogous wild-type p21ras peptide may not be immunogenic. © 1995 Wiley-Liss, Inc.     

Far less frequent mutations in ras genes than in the p53 gene in skin tumors of xeroderma pigmentosum patients

Mutations in Ha-ras, and n-rasNras genes in squamous and basal cell carcinomas in patients with xeroderma Pigmentosum (XP) were examined by the polymerase chain reaction followed by single-strand conformation polymorphism analysis and direct base sequencing. No mutation was detected in codons 12, 13, and 61 of the ras genes in XP skin tumors. This was in contrast with previous findings of a high frequency of mutation in the p53 gene in skin tumors in XP patients. A novel mutation in codon 6 of the ki-ras gene was detected in a squamous cell carcinoma. The mutation was a C→T transtion at a dipyrimidine (5′-CT) sequence and could have been produced by solar ultraviolet light. The mutated ras gene did not have the ability to transform NIH/3T3 cells. In three tumors, multiple base substitutions were detected in exon 1 of the Ki-ras and N-ras genes. These results and our previous work on p53 gene mutations suggest that mutations in ras genes are far less frequent than in the p53 gene in the skin tumors in XP patients and that ras genes are less important in skin tumorigenesis in XP patients that is the p53 gene.     

ras-family gene mutations in neoplasia of the ampulla of vater

Mutations in the first and second exons of Ha-, Ki- and N-ras oncogenes were investigated in 17 epithelial tumors of the ampulla of Vater by single-strand conformation polymorphism analysis and direct sequencing of DNA fragments amplified by polymerase chain reaction. The panel included 12 intestinaltype adenocarcinomas, 3 villous adenomas, I papillary carcinoma and I neuroendocrine carcinoma. Six cases (35%) contained ras mutations, affecting codon 12 of Ki-ras in 2 adenomas and 3 carcinomas, and of N-ras in I adenoma. All mutations were found in adenomas and among cancers with adenomatous areas, whereas none of the cases lacking adenomatous areas contained mutations. This suggested that ampullary cancers represent heterogeneous diseases with respect to the presence or absence of adenomatous areas and, among those with adenomatous areas, with respect to the presence of activated ras genes. Ki-ras mutated cases included 3 of 4 tumors which mainly involved the intraduodenal bile duct, thus suggesting that a proportion of Ki-ras -mutated ampullary cancers might correspond to those originating from the epithelium of the bile duct component of the ampulla.     

p53 Mutations in sporadic adrenocortical tumors

Non-familial human adrenocortical adenomas and carcinomas were screened for mutations in exons 5–8 of the p53 tumor suppressor gene by single-strand-conformation-polymorphism (SSCp) analysis, followed by direct sequencing of PCR-amplified DNA. point mutations in codons 12, 13 and 61 in H-ras, K-ras and N-ras proto-oncogenes were similarly assessed by direct DNA sequencing. Three out of 15 primary adrenocortical carcinomas (20%) contained a mis-sense point mutation in the conserved regions (exons 5 and 8) of the p53 gene. Mutations were located in codon 157 (GTC → TTC; Val → phe), codon 163 (TAC → AAC; Tyr → Asn), and codon 273 (CGT → TGT; Arg → Cys). The mutation in codon 157 was detected in the primary tumor as well as in brain and lymph-node metastases. Among 18 adrenocortical adenomas, there was only a single non-miscoding mutation in codon 295 (CCT → CCC; pro → pro). These data suggest that mutational inactivation of the p53 gene occurs in a minority (20%) of sporadic adrenocortical carcinomas and that these mutations constitute a late event in the multi-step process of malignant transformation. No ras mutations were detected in any of these tumors, suggesting that these genes are not involved in the development of tumors originating from the adrenal cortex.     

Ha-ras mutations in N-nitrosomorpholine-induced lesions and inhibition of hepatocarcinogenesis by antisense sequences in rat liver

To evaluate the application of Ha-ras mRNA antisense oligonucleotide therapy for liver tumors, we examined the frequency and types of mutation in codon 61 of the Ha-ras oncogene in preneoplastic lesions and hepatocellular carcinomas induced by N-nitrosomorpholine (NNM) in rats. Thirty-seven percent of preneoplastic lesions and 50% of hepatocellular carcinomas contained mutations, mostly CAA-CTA and CAA-AAA transversions. We also investigated the effects on NNM-induced lesions of an antisense oligonucleotide directed against a point mutation (CAA-CTA) in codon 61 of Ha-ras mRNA. In this experiment, Sprague-Dawley rats were given free access to water containing NNM for 8 weeks and received twice-weekly i.p. injections of a mutated Ha-ras antisense oligonucleotide with a 5′ phosphorothioate linkage or a sense oligonucleotide in oligonucleotide-liposome complexes. At week 16, rats that had received the mutated Ha-ras antisense oligonucleotides had significantly fewer and smaller preneoplastic lesions positive for glutathione-S-transferase, placental type, and had smaller hepatocellular carcinomas than rats that had received the sense oligonucleotide. Mean cellular fluorescence in the liver was found to increase with higher doses of mutated, fluorescein-isothiocyanate-labeled antisense or sense oligonucleotides. Moreover, mutated Ha-ras antisense oligonucleotide decreased the expression of mutated Ha-ras mRNA (CAA-CTA). Our findings indicate that mutated Ha-ras antisense oligonucleotide significantly inhibits hepatocarcinogenesis in rats and could be an effective therapy against liver tumors. Int. J. Cancer 72:815–820, 1997. © 1997 Wiley-Liss, Inc.     

High frequency and low specificity of ras gene mutations in rat Zymbal's gland tumors induced by 2-amino-3-methylimidazo[4, 5-f]quinoline

The activating mutations of all three ras genes in rat Zymbal'sgland tumors induced by a food mutagen, 2-amino-3-methyl-imidazo[4,5-f) inoline (IQ) were analyzed. DNA fragments of the Ha-ras,Ki-ras and N-ras oncogenes were amplified from formalin-fixedand paraffin-embedded tissues by the polymerase chain reaction(PCR) and analyzed for activating mutations involving codons12, 13 and 61 by oligoncleotide differential hybridization.All nine Zymbal's gland tumors examined, including three papillomas,were found to contain either an Ha-ras or Ki-ras mutation. Thesemutations were located in either codon 13 or 61 of Ha-ras, andin either codon 12 or 13 of Ki-ras. Of the nine mutations, threewere G     

Effects of pseudofype retrovirus containing human N-RAS antisense gene on the growth of human liver cancer LTNM4 transplanted in nude mice

An amphotropic pseudotype retrovirus containing human N-ras antisense gene was constructed and packaged with helper cells. It has been previously demonstrated that the virus did inhibit the growth of human hepatocarcinoma cell line PLC/PRF/5in vitro accompanied with the blockage of p21 expression. Based on these results, further study was carried on to examine the effect of these viruses on the growth of human hepatoma transplanted LTNM4 in nude mice. It has been shown that the retrovirus containing human antisense N-ras gene could inhibit the hepatoma in nude mice at a rate of 78% (P<0.05) as compared with saline control. No inhibition was observed in group treated with retrovirus which contained no N-ras sequence. These resultsin vivo lend further support that human N-ras antisense gene mediated by retrovirus could block the expression of the relevant oncogene and lead to the inhibition of cancer growth. It also provided the basis for further approaches of gene therapy for human cancer.     

C-erbB-2, p53, N-ras expression in hepatocellular carcinoma

To assess the relationship between C-erbB-2, p53, N-ras status at a premalignant stage and in HCC, the authors studied the imunohistological expression of these genes in HCC, liver cirrhosis and in the adjacent normal resected liver tissue, using monoclonal antibody to mutated p53 and activated C-erbB-2, N-ras. C-erbB-2 was expressed in 97.1% (35/36) of HCC and 100% (18/18) of hepatic cirrhosis, low level C-erbB-2 expression was observed in 2/14 (14.3%) of normal liver specimens; The positive incidence of overexpression of mutant p53 protein in HCC and hepatic cirrhosis were 55.6% (20/36) and 66.7% (10/18) respectively; 29 (76.5%) specimens of HCC and 16 (88.9%) of hepatic cirrhosis were positive for N-ras protein. The overexpression of the three oncogene proteins were significantly higher than that of normal liver tissues (P<0.01). These results indicated that activated C-erbB-2, N-ras and altered p53 genes may have a role in human HCC pathogenesis through promiting the development of HCC from hepatic cirrhosis and the progression of HCC.     

Point Mutations of c-ras Genes in Human Bladder Cancer and Kidney Cancer

Point mutations of c-ras genes at codons 12, 13 and 61 were analyzed in 26 cases of bladder cancer and 16 cases of kidney cancer. DNA prepared from either frozen tissues or 10% formalin-fixed, paraffin-embedded tissues were amplified by means of polymerase chain reaction methods, and mutations were analyzed by dot blot hybridization assays with oligonucleotide probes. In three cases of bladder cancer c-ras mutations were found, at codons 13 and 61 of c-Ha-ras and at codon 61 of c-Ki-ras, while no mutation was found in kidney cancer. No mutation was found in normal bladder epithelial tissues from the same patients. Our findings, taken together, may indicate relative scarcity of c-ras mutations in these types of human cancer. The results of dot blot hybridization assays and DNA sequencing showed a G-to-C transition of the first nucleotide at codon 13 of c-Ha-ras. This is the first time that such a point mutation has been detected in human cancer tissues.     

Association of H-ras mutations with adenocarcinomas of the parotid gland

We have examined 17 adenocarcinomas and 2 mixed tumors of the salivary glands for mutational activation of the oncogenes H-ras, K-ras and N-ras. The presence of mutations was determined by in vitro amplification of gene fragments spanning codons 12, 13 and 61 and the use of mutation-specific oligonucleotide hybridization. ras mutations were present in 3 of 13 adenocarcinomas (23%) of the parotid gland. The mutations were confirmed and characterized by means of cycle sequencing of polymerase chain reaction (PCR) fragments. In all 3 cases, the mutation was an A:T to G.C transition at the second position of codon 61 of the H-ras gene. This rather unusual ras mutation could provide a clue to identify one or more carcinogens involved in the pathogenesis of parotid cancer. © 1994 Wiley-Liss, Inc.     

Carcinogen—induced diploid hepatocytes: Sensitive target cells for transformation by mutated c-Ha-ras oncogene

Sequential treatment of partially (two-thirds) hepatectomized rats with diethylnitrosamine and 2-acetylaminofluorene induces the emergence of diploid hepatocytes in rat liver. These carcinogen-induced diploid cell populations are thought to contain the progenitors of hepatocellular carcinoma (HCC), i.e., initiated, cells. In the study presented here, we addressed the question of whether putative mutations in carcinogen-induced diploid hepatocytes can cooperate with activated oncogenes in the process of transformation in vitro. Both carcinogenesis in vivo and transformation in vitro have been shown to be multistep processes requiring at least two independent transforming events. Diploid and polyploid rat hepatocytes were isolated by centrifugal elutriation. The purity of the elutriated fractions was 88 ± 3% in the diploid fraction and 84 ± 3% in the polyploid fraction. Hepatocytes from both the elutriated cell fractions and, for comparison, hepatocytes from untreated rats were transfected by electroporation with oncogene expression vectors containing the mutated human T24 c-Ha-ras gene and of the N-myc gene. Transient expression of transfected DNA was similar in both hepatocyte populations. No cell lines could be established by using the N-myc vector. In contrast, the carcinogen-induced diploid hepatocytes, but not polyploid hepatocytes, could be converted by transfection with the ras vector into permanent anchorage-independent growing cell lines with hepatocyte-like morphology and differentiation. These cell lines expressed the myc proto-oncogene and transforming growth factor-α constitutively. Thus, carcinogeninduced diploid hepatocytes are sensitive to tranformation by the ras oncogene, suggesting cooperation between putative preexisting mutations in the diploid cells and the ras oncogene product in hepatocellular transformation.     

Activated N-ras oncogene in a transformant derived from a rat small intestinal adenocarcinoma induced by 2-aminodipyrido[1,2-a:3',2'-d]imidazole

DNAs from five intestinal adenocarcinomas induced by 2-aminodipyrido[1,2-a:3',2'-d]imidazole,which is present in broiled fish, were subjected to transfectionassay using NIH3T3 cells as recipients. The DNA from only oneadenocarcinoma induced a morphologically transformed focus.Rat N-ras sequences were detected in the primary transformantand in three tested secondary transformants. In the activatedN-ras oncogene, a G—T transversion at the first letterof codon 12 was detected. The original tumor DNA did not hybridizewith the oligonucleotide representing the mutated allele, butdid hybridize with the one representing the normal allele. Fromthese data we concluded either that the activation of the N-rasoncogene had occurred during the transfection or that the activatedN-ras oncogene had been present in a minor population of cellsin the original tumor.     

Assessment of mutations in ki-ras and p53 in colon cancers from azoxymethane- and dimethylhydrazine-treated rats

Mutations in the Ki-ras oncogene and the p53 tumor suppressor gene are known to occur at high frequencies in human colon cancers. We measured the frequency of mutations in these two genes in colon adenocarcinomas obtained from a widely used experimental model of human colon carcinogenesis: F344 rats treated with the carcinogens azoxymethane (AOM) or dimethylhydrazine (DMH). We detected codon 12 mutations in Ki-ras in approximately 60% of colon adenocarcinomas induced by either carcinogen. We characterized the rat p53 intron-exon junctions to construct primers for polymerase chain reaction amplification of this gene. We discovered that the rat p53 gene was structurally different from the human p53 gene, as the rat gene was missing one intron between exons 6 and 7. Both single-stranded DNA conformational polymorphism analysis and direct DNA sequencing of the highly conserved regions of rat exons 5–7 were conducted because the corresponding human regions (exons 5–8) have been reported as being mutated most frequently in human colon cancers. Using these methods, we were unable to identify any p53 mutations in the highly conserved regions of exons 5–7 in either AOM- or DMH-induced colon adenocarcinomas. These data confirm that Ki-ras was mutated in most colon cancers in AOM- or DMH-treated rats but indicate that molecular alterations in the p53 gene, if they occur in this animal model, are different from most p53 mutations in human colon cancers. Mol. Carcinog. 19:137–144, 1997. © 1997 Wiley-Liss, Inc.     

Allele loss and point mutation in codons 12 and 61 of the c-Ha-ras oncogene in carcinogen-transformed human breast epithelial cells

There is significant evidence that the ras oncogene plays a role in experimental mammary carcinogenesis; the evidence in human breast cancer, however, is more limited. We induced the expression of transformation phenotypes in the human breast epithelial cell line MCF-10F with the chemical carcinogens 7,12-dimethylbenz[a]-anthracene, N-methyl-N-nitrosourea, N-methyl-N-nitro-N′-nitrosoguanidine, and benzo[a]pyrene. This work was designed to clarify whether chemically induced neoplastic transformation correlates with alterations in the ras gene. MCF-10F cells have two c-Ha-ras alleles, identified by 1.0-kb and 1.2-kb restriction fragments. Treatment with carcinogens resulted in the loss of one of the alleles (1.0 kb). Polymerase chain reaction–amplified DNA from all carcinogen-treated cells was analyzed for point mutations in c-Ha-ras at codons 12 and 61. All of the carcinogens induced a mutation of the remaining allele at the first position of codon 12 (GGCÀC). Another frequent mutation occurred at the first position of codon 61 (CAG←GAG). The changes in c-Ha-ras were associated with the emergence of colony formation in agar-methocel, but no specific changes in this gene correlated with the emergence of invasiveness or tumorigenesis, indicating that other genes may be involved in the process. © 1994 Wiley-Liss, Inc.     

K-ras mutations in human adenocarcinoma of the lung: Association with smoking and occupational exposure to asbestos

We investigated point mutational activation of the ras genes (K-ras codons 12, 13 and 61; N-ras codons 12, 13 and 61; H-ras codons 12 and 61) in primary, resected lung cancer by dot blotting and oligonucleotide hybridization. K-ras mutations were found in 14 (29%) of the 48 lung tumour specimens examined, but no N-ras or H-ras mutations were found. The highest frequency of K-ras mutation was observed in adenocarcinoma: 12 of the 21 samples studied (57%) had a mutation, which is one of the highest frequencies reported for lung adenocarcinoma. The commonest type of mutation in these lung tumour samples consisted of transversions: we observed 11, of which 8 (57% of all mutations) were G to T transversions. Most of the 48 patients studied had a history of heavy smoking, either with or without evidence of occupational exposure to asbestos. Statistical analysis revealed-in addition to the highly significant association between the adenocarcinoma type of lung cancer and K-ras mutation-a clear association of K-ras mutations with heavy life-time smoking (≥50 pack-years of cigarette smoking; odds ratio (OR) 4.9, 90% CI 1.2 - 19.5, multivariate analysis). In addition, occupational asbestos exposure showed an elevated, but non-significant, OR of 2.2 (90% CI 0.6 - 8.7) with the presence of K-ras mutation. We conclude that the occurrence of K-ras mutations in adenocarcinoma of the lung is frequent, and that such mutations are associated with heavy life-time exposure to tobacco smoke, possibly in combination with occupational exposure to asbestos fibres.     

A one-step DGGE scanning method for detection of mutations in the K-, N-, and H-ras oncogenes: Mutations at codons 12, 13 and 61 are rare in B-cell non-Hodgkin's lymphoma

Mutations in the N-, K-, and H-ras genes are key events in the process of carcinogenesis of many human cancers and may serve as important targets for therapeutic intervention. We developed a simple diagnostic method that in one step and within 5 hr determines the mutational status of any of the 3 ras genes in a given tumor sample. The method combines polymerase chain reaction (PCR) with denaturing gradient gel electrophoresis (DGGE) and allows simultaneous mutation scanning of 6 regions covering “hot-spot” codons 12, 13 and 61 of the 3 ras genes. The sensitivity of the assay was demonstrated by the analysis of control mutations, either naturally occurring or created by site-directed mutagenesis. We further demonstrate that unambiguous identification of ras mutations can be achieved by heteroduplex analysis in denaturing gradient gels, circumventing sequence analysis. We applied the method to establish the mutational status of all 3 ras genes in 123 samples of B-cell non-Hodgkin's lymphoma. Altogether, one diffuse large B-cell lymphoma and one B-cell chronic lymphocytic leukemia (B-CLL) harbored a mutation (G12S and G12A, respectively) in the K-ras gene, and one B-CLL harbored a mutation (Q61R) in the N-ras gene. We therefore conclude that ras mutations only contribute rarely, if at all, to carcinogenesis in B-cell non-Hodgkin's lymphoma. Int. J. Cancer 71:364-369, 1997. © 1997 Wiley-Liss Inc.