“Low-risk” and “High-risk” HPV-infection and K-ras Gene Point Mutations in Human Cervical Cancer: A Study of 31 Cases

To analyze the coexistence of human papilloma virus (HPV) infection and K-ras gene activation in cervical neoplasia, we investigated 31 (seven pre-invasive and 24 invasive) cervical carcinomas for “low-risk” (types 6 and 11) and “high-risk” (types 16 and 18) HPVs and K-ras point mutations using PCR-based technology. “Low-risk” HPVs were not detected in the group investigated; however, 20 of 31 (64%) cases were HPV 16 positive, while HPV 18 was found in only three (9.7%) samples (HPV 6/11 v. HPV 16/18, p < 0.0001; HPV 16 v. HPV 18, p < 0.0001; Fisher's exact test). There was a K-ras codon 12 point mutation in two of 31 (6.4%) neoplasms, with none of the cases showing a K-ras codon 13 point mutation. Two moderately differentiated squamous carcinomas showed K-ras exon 2 gene alterations. Interestingly, none of the pre-invasive cervical carcinomas displayed K-ras gene point mutations. The mean patient age did not differ significantly in the number of HPV-positive and -negative cases. A coexistence of “high-risk” human papillomavirus DNA with K-ras gene alterations was observed in three of 31 (9.7%) neoplasms (one IIA and two IB moderately differentiated cervical carcinomas). Our results suggest that “high-risk” HPVs coexist with K-ras gene alterations in a subset of moderately differentiated carcinomas of the cervix uteri.     

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

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

Detection of K-ras mutation in sputum by mutant-allele-specific amplification (MASA)

From 10 to 30% of lung carcinomas examined to date contain mutant K-ras genes. We report here that the mutant-allele-specific amplification (MASA) method may be useful for detection of the K-ras mutations in cells obtained from the sputum of patients with lung cancer. The PCR product from one of five patients revealed an alteration when mixed oligonucleotides representing variants of the second letter at codon 12 of this gene were used as 5′ primers, and further experiments showed a mutation of GGT (Gly) to GAT (Asp)at codon 12. The MASA system could also be applied to an examination of metastatic lung carcinomas, particularly from adenocarcinomas in colon and pancreas in which frequent K-ras mutations are detected, and to mass-screening for colorectal tumors using DNA isolated from feces as template. © 1993 Wiley-Liss, Inc.     

Duct changes and K-ras mutations in the disease-free pancreas: analysis of type, age relation and spatial distribution

Recent molecular studies have suggested that hyperplastic duct lesions of the pancreas are potential precursors of pancreatic ductal carcinoma. This study examines the type, distribution, age-related incidence and K-ras codon 12 mutation rate of duct lesions in the normal pancreas. Postmortem pancreases from 140 patients were screened for the presence of mucinous cell hypertrophy (MHT), ductal papillary hyperplasia (DPH), adenomatoid ductal hyperplasia (ADH), and squamous metaplasia (SQM). Microdissected cell samples were analyzed for K-ras codon 12 mutations by polymerase chain reaction amplification of exon 1 of the K-ras gene, combined with constant denaturing gel electrophoresis, and analyzed by sequencing. Of the 140 specimens 114 showed duct lesions. The lesions were evenly distributed throughout the pancreas. They were more common beyond the age of 40. MHT was present in 68%, DPH in 36%, ADH in 40%, and SQM in 36% of the cases. K-ras mutations were found in 19 samples from 15 out of 79 pancreases (18%), including all types of duct lesions and a variant of ADH with dense stroma. 67% of the K-ras-positive specimens showed the transition GGT to GAT (8) or GTT (5). Hyperplastic/metaplastic duct changes of the pancreas increase with age, but their distribution pattern in the pancreas differs from that of ductal carcinomas. Received: 16 April 1999/Accepted: 26 May 1999     

K- ras mutation may promote carcinogenesis of endometriosis leading to ovarian clear cell carcinoma

Endometriosis shares some features characteristic of malignancy; however, it remains unclear whether endometriosis is a precursor to malignant disease. The objective is to determine the genetic relationship between endometriosis and ovarian clear cell carcinoma (OCCA). Among 37 Japanese patients with OCCA who underwent primary surgery at Showa University Hospital between 1987 and 1999, K-ras mutations were detected in 6. Three of these patients had ectopic endometrial tissue adjacent to the site of carcinoma. These cases demonstrated areas of endometriosis and areas of OCCA bordered by atypical endometriosis. We retrieved cells from regions of endometriosis and atypical endometriosis, as well as OCCA cells, by laser microdissection in each case. K-ras mutations were analyzed in each specimen dissected. DNA analysis of each region revealed that K-ras mutations were detectable in OCCA but not in endometriosis or atypical endometriosis. It is thought that a number of genetic alterations are involved in malignant transformation. It is possible that K-ras mutations are associated with malignant transformation of atypical endometriosis into OCCA, although further research is needed to define this mechanism.     

Intratumor Cellular Heterogeneity and Alterations in ras Oncogene and p53 Tumor Suppressor Gene in Human Prostate Carcinoma

To assess the potential role of ras oncogene activation and P53 tumor suppressor gene mutations in the development of human prostate carcinoma, nine cases of histologically heterogeneous prostate tumors obtained from total prostatectomies were probed for these specific events. Each tumor was divided into 5 to 10 areas according to different growth or histological patterns. Targeted DNA sequences coding for ras and p53 were amplified by the polymerase chain reaction, analyzed by single-strand conformational polymorphisms, and confirmed by direct DNA sequencing. Point mutations of the ras gene were found in three of the nine tumors. Two contained K-ras codon 13 and H-ras codon 61 mutations, found in only one and three areas of each lesion, respectively. The third tumor contained two different point mutations in K-ras codons 13 and 61 in different foci of the sample. Loss of heterozygosity at the polymorphic codon 72 in the p53 gene was detected in two of four informative cases (50%) showing fragment cleavage by restriction fragment length polymorphism analysis. Mutations in p53, missense transversions, single base insertions, and two base deletions were also detected in three tumors. The present results reveal mutated ras and p53 occasionally occurring in small foci of the tumor and that genetic mutations in p53, as opposed to those in ras, are more closely associated with invasive growth of heterogeneous prostate carcinoma.     

Intraductal papillary-mucinous tumours represent a distinct group of pancreatic neoplasms: an investigation of tumour cell differentiation and K-ras,p53 and c-erbB-2 abnormalities in 26 patients

Intraductal papillary growth of mucin producting hypersecreting, columnar cells characterizes a group of rare pancreatic exocrine neoplasms which we propose to call intraductal papillary-mucinous tumors (IPMT). We analysed the histopathology of 26 IPMT in relation to gastro-enteropancreatic marker expression, genetic changes and biology. Four IPMT showing only mild dysplasia were considered to be adenomas. Nine tumours displayed moderate dysplasia and were regarded as borderline. Severe dysplasia-carcinoma in situ changes were found in 13 IPMT which were therefore classified as intraductal carcinomas. Six of these carcinomas were frankly invasive and two of these had lymph node metastases. The invasive component resembled mucinous noncystic carcinoma in all but one tumour which showed a ductal invasion pattern. Immunohistochemically, an intestinal marker type was found in most carcinomas, while gastric type differentiation prevailed among adenomas or borderline tumours. K-ras mutations (seven at codon 12 and one at codon 13) were found in 31% of IPMT (2 adenomas, 1 borderline, 5 carcinomas). Nuclear p53 overexpression was detected in 31% of IPMT (6 carcinomas and 2 borderline IPMT) and correlated with p53 mutations (one at exon 8 and the other at exon 5) in two carcinomas. p53 abnormalities were unrelated to K-ras mutation. c-erbB-2 overexpression was observed in 65% of IPMT, with various grades of dysplasia. Twenty-two of 24 patients are alive and well after a mean post-operative follow-up of 41 months. Only two patients, both with invasive cancer at the time of surgery, died of tumour disease. It is concluded that pancreatic IPMT encompass neoplasms which, in general, have a favorable prognosis, but are heterogeneous in regard to grade of dysplasia and marker expression. Adenoma, borderline tumour, intraductal carcinoma and invasive carcinoma can be differentiated. p53 changes but not K-ras mutation or c-erbB-2 overexpression are related to the grade of malignancy. Most IPMT differ in histological structure, marker expression and behaviour from ductal adenocarcinoma.     

Quantitative enriched PCR (QEPCR), a highly sensitive method for detection of K-ras oncogene mutation

We have developed a rapid and highly sensitive method for the detection of mutant K-ras codon 12 allele in the presence of 10⁵ copies of the wild-type alleles. This sensitivity is achieved by selective amplification of mutant K-ras sequences, using a two-stage procedure with modified primers. In the first stage, primers consist of K-ras sequences in the 3′ portion and polyomavirus sequence (to minimize homology with human genome) on the 5′ portion. The 3′ portion also consists of mismatch sequence that generates an MvaI site in normal, but not mutant, K-ras codon 12 alleles. Thus, following the first round of 20 cycles, restriction enzyme cleavage is carried out to selectively digest normal K-ras codon 12 alleles. To enrich mutant alleles, a second amplification is performed using tail primers that recognize the polyoma, but not human sequences. This design ensures that in the second amplification only mutant alleles that were pre-amplified in the first round would serve as template for this reaction. Ethidium bromide-stained polyacrylamide gel electrophoresis (PAGE) of second–stage PCR product that has been digested with MvaI is used to monitor the presence of mutant alleles, detected at sensitivity of 1/10⁵. This technique offers high sensitive detection of mutant K-ras alleles using a new concept of tail-primer design and is likely to assist in identifying patients at risk to develop pancreatic, colon, or lung cancer, which harbor high incidence of mutant ras alleles. Hum Mutat 10:322–325, 1997. © 1997 Wiley-Liss, Inc.     

Enhanced expression of EGF receptor and low frequency of ras mutations in X-ray-induced rat thyroid tumours

Radiation is recognized as a carcinogenic factor for the thyroid gland. In this experimental study, oncogene expression was investigated in radiation-induced rat thyroid tumours. Forty 3-month-old Wistar rats received X-ray-irradiation to the neck region; 40 animals were untreated controls. After 14 months, thyroid tumours had developed in 25 of the 29 irradiated animals still alive; 76% of these tumours were considered malignant. No tumours developed in controls. Mutations of codons 12–13 and 59–63 of H-, K- and N-ras were analysed by PCR-SSCP (single-strand conformation polymorphism analysis) and sequencing of DNA from thyroid tissue. SSCP indicated a ras mutation frequency of 8%, but only one K-ras codon 12 (Gly-Cys) mutation was confirmed by sequencing. Protooncogene expression was analysed by mRNA slot blot hybridization analysis and immunohistochemistry. K-ras mRNA expression and EGF receptor mRNA and protein expression were significantly increased in the irradiated animals compared with controls, and in tumours versus nontumour tissue. This study of radiation-induced rat thyroid tumours demonstrates that ras expression may be subject to changes apart from activating mutations. Increased expression of EGF receptor in the tumours parallels the situation in human thyroid cancer. Received: 18 May 1999 / Accepted: 31 May 1999     

Denaturing gradient gel electrophoresis (DGGE) assay for K-ras and N-ras genes: detection of K-ras point mutations in human lung tumour DNA

Point mutations in the ras oncogenes are very common in lungcancers as well as in many of the other solid tumours. To effectivelyexamine the occurrence of these mutations in a large numberof tumour samples, we have applied denaturing gradient gel electrophoresis(DGGE) for the analysis of point mutations of the K-ras andN-ras genes, using GC-clamped, PCR-amplified DNA fragments.Among the 68 tumour DNA samples, we detected 14 mutations inthe K-ras gene. This was 78% of the mutations identified byoligonucleotide hybridization. Altogether, eight of the ninedifferent kinds of base substitutions found in the tumour sampleswere detected by the DGGE assay, representing substitutionsat codons 12, 13, and 61 of the K-ras gene. Six of the detectedmutations were guanine to thymine transversions at codon 12;this was the most common type of alteration. On the basis ofour experience, the present non-radioactive DGGE analysis seemsto be readily applicable for detection of the mutations in theK-ras and N-ras genes. Types of ras gene mutations frequentin adenocarcinomas of the lung are also discussed.     

Detection of c-Ki-ras gene mutation in paraffin sections of adenocarcinoma and atypical bronchioloalveolar cell hyperplasia of human lung

DNA of minute specimens taken from formalin-fixed and paraffin-embedded tissue was used as a template for the polymerase chain reaction (PCR) to examine whether the c-Ki-ras gene is activated in atypical bronchioloalveolar cell hyperplasia (ABH) of human lung. The c-Ki-ras gene was successfully amplified on 131 samples from 29 cases of lung adenocarcinoma (87.3% of all samples used as templates) by the nested PCR method. Point mutations at codon 12 of the c-Ki-ras gene could be detected in carcinoma tissue of 6 cases (20.7% of all cases), also detected in ABHs showing moderate to severe atypia of 2 cases. PCR amplification is a useful technique for studying pin-point pathological lesions in a routine paraffin section at the molecular level.     

THE PATTERN OF K-rasMUTATION IN PULMONARY ADENOCARCINOMA DEFINES A NEW PATHWAY OF TUMOUR DEVELOPMENT IN THE HUMAN LUNG

Codon 12 of the K-ras oncogene was screened for mutations in 65 surgically-resected primary pulmonary adenocarcinomas and in 32 tissue foci of alveolar atypical hyperplasia (AAH) by a polymerase chain reaction (PCR)-based method. Mutations in either position 1 or position 2 of codon 12 were detected in 16 tumours (25 per cent). When analysed by site of origin, mutations were seen in 9/26 (35 per cent) parenchymal and in 0/12 bronchial adenocarcinomas (P<0·02). K-ras mutations were seen in five AAH lesions from four patients. DNA sequencing showed that the great majority of mutations in both adenocarcinomas and AAH were G–T transversions. These findings provide support for the classification of pulmonary adenocarcinomas into bronchial and parenchymal subtypes and also provide molecular evidence to support the importance of AAH in the development of parenchymal cancers. © 1997 by John Wiley & Sons, Ltd.     

Mutation of the transforming growth factor-β type II receptor gene in right-sided colorectal cancer: relationship to clinicopathological features and genetic alterations

The presence of inactivating mutations in the transforming growth factor-β (TGF-β) type II receptor (RII) gene in colon cancer suggests that it may behave like a tumour suppressor gene. RII is mutated in the majority of colon tumours exhibiting widespread microsatellite instability, a characteristic generally referred to as the replication error phenotype (RER+). We investigated the association between RII mutations and various clinicopathological variables and genetic alterations in a large series of sporadic adenocarcinomas arising in the proximal colon. RII mutations were found in 17 per cent (36/210) of right-sided tumours and in 86 per cent (32/37) of those displaying RER+. They were associated with the absence of lymph node invasion (P=0·04), poor histological differentiation (P=0·006), and with a trend for improved patient survival. Tumours with an RII mutation also showed non-significant trends for a lower incidence of p53 protein overexpression and of p53, K-ras, and APC gene mutation compared with tumours with normal RII. These results indicate that right-sided colorectal tumours containing RII mutations resemble those with the RER+ phenotype in terms of their clinicopathological features and genetic alterations. © 1998 John Wiley & Sons, Ltd.     

Malignant transformation of endometriosis: application of laser microdissection for analysis of genetic alterations according to pathological changes

Endometriosis is considered to be a possible precancerous disease. Pathologically, we observed malignant transformation of endometriosis to clear cell carcinoma or endometriotic carcinoma of the ovary, via the step of atypical endometriosis. Pathological changes reflected genetic alterations. In cases of clear cell carcinoma, we assessed K-ras mutations in regions of normal endometriosis, atypical endometriosis, and clear cell carcinoma, and obtained tissue samples by laser microdissection. We found that K-ras mutations were associated with malignant transformation of clear cell carcinoma. The present results indicate that laser microdissection can be used to combine assessment of genetic and pathological changes.     

Genetic heterogeneity in sporadic colorectal adenomas

The majority of colorectal cancers develop from adenomatous polyps under the influence of factors that are still poorly understood. Tumourigenesis is generally considered a multistep process in which multiple genetic alterations occur, eventually reflected in abnormalities of the cellular DNA content. Macroscopical features such as tumour size and tumour architecture (tubular, tubulovillous, or villous) are correlated wit the chance of malignancy in the lesion. Grade of dysplasia can be considered an indicator for the level of progression of the adenoma towards invasive carcinoma. These characteristics were correlated with the presence or absence of K-ras mutations and the DNA ploidy in a prospective study performed on 46 large sporadic colorectal adenomas resected by endoscopy. DNA ploidy and K-ras mutations were analysed in two samples taken at distant sites in the adenomas. Aneuploidy was present in 12 adenomas (26 per cent) and K-ras mutations occurred in 26 (57 per cent). A highly significant correlation was found between aneuploidy and adenoma size, architecture, and grade of dysplasia. The presence of K-ras mutations was significantly correlated only with the size of the adenomas. The proportion of adenomas with aneuploidy and/or a K-ras mutation increased when two samples were analysed instead of one. This observation suggests that the prevalence of genetic mutations and of aneuploidy is probably underestimated, as generally only one sample is investigated. No correlation was observed between K-ras mutations and ploidy. This study demonstrates the presence of genetic heterogeneity in colorectal adenomas and supports the notion that K-ras mutation is an early event, while aneuploidy is a late event in the adenoma–carcinoma sequence. © 1997 John Wiley & Sons, Ltd.     

NIH3T3 transfectant containing human K-ras oncogene shows enhanced metastatic activity after in vivo tumor growth or co-culture with fibroblasts

A clone of NIH3T3 transformant (H-3), obtained by transfecting genomic DNA of a human colon carcinoma cell line, contains human K-ras oncogene and yields metastatic pulmonary nodules after intravenous injection of the cells into nude mice. This metastatic ability was enhanced remarkably after in vivo tumor growth (subcutaneous tumor formation in nude mice) accompanied by increased mRNA expression and gene amplification of the human-derived K-ras oncogene, while it declined gradually as the passage number increased in vitro, with corresponding decreases of gene amplification and mRNA expression. Six subclones were randomly selected from H-3 cells which had been subcultured to passage 22. All of the clones in culture showed almost the same low level of metastatic ability and exhibited little K-ras oncogene amplification with correspondingly low mRNA expression. However, after they formed tumors in nude mice, every clone acquired high metastatic ability and the gene amplification increased, with elevated mRNA expression. These experimental facts indicated that acquisition of metastatic ability coupled with the function of K-ras oncogene was conditional in nature, being strongly affected by in vivo tumor circumstances. The low metastatic and G-418-resistant H-3 cells were co-cultured with BALB/c3T3 fibroblasts for 2–4 weeks. After removal of fibroblasts by exposure to G-418, the tumor cells exhibited increased metastatic ability and human K-ras oncogene mRNA, suggesting an intimate interaction between H-3 cells and fibroblasts influencing the function of transfected human K-ras oncogene. Fibroblasts of the host animal may thus have an important role in generating enhanced metastatic activity of H-3 cells.     

K-ras is an essential gene in the mouse with partial functional overlap with N-ras

Mammalian ras genes are thought to be critical in the regulation of cellular proliferation and differentiation and are mutated in ~30% of all human tumors. However, N-ras and H-ras are nonessential for mouse development. To characterize the normal role of K-ras in growth and development, we have mutated it by gene targeting in the mouse. On an inbred genetic background, embryos homozygous for this mutation die between 12 and 14 days of gestation, with fetal liver defects and evidence of anemia. Thus, K-ras is the only member of the ras gene family essential for mouse embryogenesis. We have also investigated the effect of multiple mutations within the ras gene family. Most animals lacking N-ras function and heterozygous for the K-ras mutation exhibit abnormal hematopoietic development and die between days 10 and 12 of embryogenesis. Thus, partial functional overlap appears to occur within the ras gene family, but K-ras provides a unique and essential function.     

K-<Emphasis Type="Italic">ras</Emphasis> mutation may promote carcinogenesis of endometriosis leading to ovarian clear cell carcinoma

Endometriosis shares some features characteristic of malignancy; however, it remains unclear whether endometriosis is a precursor to malignant disease. The objective is to determine the genetic relationship between endometriosis and ovarian clear cell carcinoma (OCCA). Among 37 Japanese patients with OCCA who underwent primary surgery at Showa University Hospital between 1987 and 1999, K-ras mutations were detected in 6. Three of these patients had ectopic endometrial tissue adjacent to the site of carcinoma. These cases demonstrated areas of endometriosis and areas of OCCA bordered by atypical endometriosis. We retrieved cells from regions of endometriosis and atypical endometriosis, as well as OCCA cells, by laser microdissection in each case. K-ras mutations were analyzed in each specimen dissected. DNA analysis of each region revealed that K-ras mutations were detectable in OCCA but not in endometriosis or atypical endometriosis. It is thought that a number of genetic alterations are involved in malignant transformation. It is possible that K-ras mutations are associated with malignant transformation of atypical endometriosis into OCCA, although further research is needed to define this mechanism.     

Hydrogel-microsphere-enhanced surface plasmon resonance for the detection of a K-ras point mutation employing peptide nucleic acid

Highly-sensitive detection of a K-ras point mutation in codon 12, frequently found in pancreatic cancer, based on DNA-carrying hydrogel microspheres as a response enhancer for surface plasmon resonance (SPR), is described. Acrylamide-based microspheres with carboxyl groups were conjugated with DNA probes. Use of the DNA-carrying microsphere in the sandwich method, that is, binding of the microspheres with target DNAs at the sensor surface, enhanced the SPR response as a combined result of increased dielectric constant by the DNA-carrying microspheres. Microspheres lead to response enhancement, as shown by a 100-fold increase in sensitivity compared to that of non-amplified DNA target hybridization. In addition, the advantage of peptide nucleic acid (PNA) in the detection of a K-ras point mutation at the sensor surface by increasing temperature and flow rate is discussed. Results illustrate that the sandwich method through DNA-carrying microspheres for a SPR sensor is a promising approach for ultrasensitive DNA detection.     

ACB‐PCR measurement of spontaneous and furan‐induced H‐ras Codon 61 CAA to CTA and CAA to AAA mutation in B6C3F1 mouse liver

Furan is a rodent liver carcinogen, but the mode of action for furan hepatocarcinogenicity is unclear. H‐ras codon 61 mutations have been detected in spontaneous liver tumors of B6C3F1 mice, and the fraction of liver tumors carrying H‐ras codon 61 CAA to AAA mutation increased in furan‐treated mice. Allele‐specific competitive blocker PCR (ACB‐PCR) has been used previously to quantify early, carcinogen‐induced increases in tumor‐associated mutations. The present pilot study investigated whether furan drives clonal expansion of pre‐existing H‐ras mutant cells in B6C3F1 mouse liver. H‐ras codon 61 CAA to CTA and CAA to AAA mutations were measured in DNA isolated from liver tissue of female mice treated with 0, 1, 2, 4, or 8 mg furan/kg body weight, five days per week for three weeks, using five mice per treatment group. Spontaneous levels of mutation were low, with two of five control mice having an H‐ras codon 61 CTA or AAA mutant fraction (MF) greater than 10^?5. Several furan‐treated mice had H‐ras codon 61 AAA or CTA MFs greater than those measured in control mice and lower bound estimates of induced MF were calculated. However, no statistically‐significant differences were observed between treatment groups. Therefore, while sustained exposure to furan is carcinogenic, at the early stage of carcinogenesis examined in this study (three weeks), there was not a significant expansion of H‐ras mutant cells. Environ. Mol. Mutagen. 54:659–667, 2013. © 2013 Wiley Periodicals, Inc.