Genetic alterations in K-ras and p53 cancer genes in lung neoplasms from Swiss (CD-1) male mice exposed transplacentally to AZT

A transplacental carcinogenicity study was conducted by exposing pregnant Swiss (CD-1) mice to 0, 50, 100, 200, or 300 mg of 3'-azido-3'-deoxythymidine (AZT)/kg bw/day, through a 18 to 19-day gestation [National Toxicology Program, NIH Pub. No. 04-4458, 2004]. The incidences of alveolar/bronchiolar adenomas and carcinomas, in the 200 and 300 mg/kg male treatment groups, were significantly greater than that of the controls. In the present study, we evaluated the benign and malignant lung neoplasms from this bioassay for point mutations, in the K-ras and p53 cancer genes that are often mutated in human lung tumors. K-ras and p53 mutations were detected by cycle sequencing of polymerase chain reaction-amplified DNA, isolated from formalin-fixed, paraffin-embedded neoplasms. K-ras mutations were detected in 25 of 38 (66%) of the AZT-induced lung tumors, and the predominant mutations were codon 12 G-->T transversions. p53 mutations were detected in 32 of 38 (84%) of the AZT-induced lung tumors, with the predominant mutations being exon 8, codon 285 A-->T transversions, and exon 6, codon 198 T-->A transversions. No K-ras or p53 mutations were detected in five tumors, examined from control mice. The patterns of mutations identified in the lung tumors suggest that incorporation of AZT or its metabolites into DNA, oxidative stress, and genomic instability may be the contributing factors to the mutation profile and development of lung cancer in these mice.     

K-ras mutations in lung tumors and tumors from other organs are consistent with a common mechanism of ethylene oxide tumorigenesis in the B6C3F1 mouse

Ethylene oxide is a multisite carcinogen in rodents and classified as a human carcinogen by the National Toxicology Program. In 2-year mouse studies, ethylene oxide (EO) induced lung, Harderian gland (HG), and uterine neoplasms. We evaluated representative EO-induced and equivalent spontaneous neoplasms for K-ras mutations in codons 12, 13, and 61. K-ras mutations were identified in 100% (23/23) of the EO-induced lung neoplasms and 25% (27/108) of the spontaneous lung neoplasms. Codon 12 G to T transversions were common in EO-induced lung neoplasms (21/23) but infrequent in spontaneous lung neoplasms (1/108). K-ras mutations were found in 86% (18/21) of the EO-induced HG neoplasms and 7% (2/27) of the spontaneous HG neoplasms. Codon 13 G to C and codon 12 G to T transversions were predominant in the EO-induced HG neoplasms but absent in spontaneous HG neoplasms (0/27). K-ras mutations occurred in 83% (5/6) of the EO-induced uterine carcinomas and all were codon 13 C to T transitions. These data show a strong predilection for development of K-ras mutations in EO-induced lung, Harderian gland, and uterine neoplasms. This suggests that EO specifically targets the K-ras gene in multiple tissue types and that this event is a critical component of EO-induced tumorigenesis.     

High frequency of ras mutations in forestomach and lung tumors of B6C3F1 mice exposed to 1-amino-2,4-dibromoanthraquinone for 2 years

1-Amino-2,4-dibromoanthraquinone (ADBAQ) is an anthraquinone-derived vat dye, and a potent carcinogen in laboratory animals. In a 2-year study with dietary exposure to 10,000 or 20,000 ppm ADBAQ, increased incidence of forestomach and lung tumors were observed in B6C3F1 mice. The present study indentified genetic alterations in H-ras and K-ras proto-oncogenes in ADBAQ-induced tumors. Point mutations in ras proto-oncogenes were identified by restriction fragment length polymorphism, single-stranded conformational polymorphism analysis and cycle sequencing of polymerase chain reaction-amplified DNA isolated from paraffin-embedded squamous cell papillomas and carcinomas in the forestomach, and alveolar/bronchiolar adenomas and carcinomas in the lung. A higher frequency of ras mutations was identified in ADBAQ-induced forestomach (23/32, 72%) and lung tumors (16/23, 70%) than in spontaneous forestomach (4/11, 36%) and lung tumors (26/86, 30%). H-ras codon 61 CTA mutations were detected in (4/8, 50%) ADBAQ-induced forestomach squamous cell papillomas and (10/24, 42%) squamous cell carcinomas, but not in the spontaneous forestomach tumors examined. H-ras codon 61 CGA mutation (6/24, 25%) was also detected in ADBAQ-induced forestomach squamous cell carcinomas. K-ras codon 61 A to T transversions and A to G transitions were prominent in ADBAQ-induced lung alveolar/bronchiolar adenomas and alveolar/bronchiolar carcinomas. The major finding of A to T transversions or A to G transitions in forestomach and lung tumors suggests that ADBAQ or its metabolites target adenine bases in the ras proto-oncogenes and that these mutations play a dominant role in multi-organ     

Gene expression studies demonstrate that the K-ras/Erk MAP kinase signal transduction pathway and other novel pathways contribute to the pathogenesis of cumene-induced lung tumors

National Toxicology Program (NTP) inhalation studies demonstrated that cumene significantly increased the incidence of alveolar/bronchiolar adenomas and carcinomas in B6C3F1 mice. Cumene or isopropylbenzene is a component of crude oil used primarily in the production of phenol and acetone. The authors performed global gene expression analysis to distinguish patterns of gene regulation between cumene-induced tumors and normal lung tissue and to look for patterns based on the presence or absence of K-ras and p53 mutations in the tumors. Principal component analysis segregated the carcinomas into groups with and without K-ras mutations, but failed to separate the tumors based on p53 mutation status. Expression of genes associated with the Erk MAP kinase signaling pathway was significantly altered in carcinomas with K-ras mutations compared to tumors without K-ras mutations or normal lung. Gene expression analysis also suggested that cumene-induced carcinomas with K-ras mutations have greater malignant potential than those without mutations. In addition, significance analysis of function and expression (SAFE) demonstrated expression changes of genes regulated by histone modification in carcinomas with K-ras mutations. The gene expression analysis suggested the formation of alveolar/bronchiolar carcinomas in cumene-exposed mice typically involves mutation of K-ras, which results in increased Erk MAP kinase signaling and modification of histones.     

Molecular analysis of p53 and K-ras in lung carcinomas of coal miners

Thirty-three cases of non-small cell lung cancers (NSCLC) from the archives of National Coal Workers' Autopsy Study were studied for mutational alterations in p53 and K-ras using PCR-SSCP, DNA sequencing and PCR-oligonucleotide probe hybridization techniques. Mutations of the p53 were observed in 4 smokers (19%) and one in a never smoker (8%). Two polymorphisms in smokers were detected at codon 213, a common site for sequence variation. Among the smokers the p53 mutations were in the heavy smokers. In never smokers there was only a single p53 mutation and two K-ras mutations. In never smokers the frequency of K-ras mutations was similar (17%) in smokers, but one never smoker had two K-ras mutations. Mutations of p53 were more frequent in adenocarcinomas (27%) and they were AT-->GC transitions. Four of 11 (36%) adenocarcinomas were found to have K-ras codon 12 mutations, and one adenocarcinoma had two K-ras mutations. There were two large cell undifferentiated carcinomas with p53 mutation and one with a K-ras mutation. Two of the 16 squamous cell carcinomas were positive for p53 mutation, while no K-ras mutations were found in this group. The results of these preliminary studies indicate a moderately different mutational spectrum of p53 and K-ras in coal miners independent of cigarette smoking. The mutational spectrum observed in this study of coal miners with heavy cigarette smoking history suggest a protective effect of coal mine dust in preventing abnormal mutations induced by chemical carcinogens in cigarette smoke or reactive oxygen species. These limited preliminary studies provide insight into the possibility of accurately measuring changes in etiologic markers to unravel the uncertainties of cancer in coal miners.     

Synchronous and metachronous lung carcinomas: Molecular evidence for multicentricity

The present study Is aimed to evaluate the genetic evidence for multicentricity of synchronous and metachronous multiple lung carcinomas. Nineteen cases of synchronous multlple lung carcinomas and 11 cases of metachronous multiple lung carcinomas were analyzed for p53 protein overexpression by lmmunohistochemlstry (DO-7) and for genetic abnormality of the p53 gene by loss of heterozygosity (LOH) at chromosome 17p and by poly-merase chaln reaction (PCR)-singlestrand conformation polymorphism (SSCP) analysis. They were also analyzed for K-ras mutation. DNA from three patients was also sequenced by the dideoxy sequencing method to confirm the presence of mutations and determine the base substitutions. Different spectrums of genetic changes, which were evaluated by a combination of p53 mutation, LOH at chromosome 17p and p53 overexpression, were observed in 11 of 19 cases of synchronous multiple lung carcinomas (57.9%) In the present study. Slmllarly, five of 11 cases of metachronous multiple lung carcinomas (45.4%) showed a different pattern of genetic changes. The present data suggest that some of the multiple carcinomas have dlfferent clonal orlgins, although their histological types are identical, and support the use of genetic markers In the differential diagnosis between metastasis and second primary carcinoma of the lung.     

Predominant K-ras codon 12 G --> A transition in chemically induced lung neoplasms in B6C3F1 mice

Based on long-term toxicity and carcinogenicity studies in B6C3F1 mice conducted by the National Toxicology Program, 2,2-Bis(bromomethyl)-1,3-propanediol (BMP) and tetranitromethane (TNM) have been identified as carcinogens. Following 2 yr of exposure to 312, 625, or 1,250 ppm BMP in feed, or exposure to 0.5 or 2 ppm TNM by inhalation, increased incidences of lung neoplasms were observed in B6C3F1 mice at all exposure concentrations compared to unexposed mice. The present study characterizes genetic alterations in the K-ras protooncogene in BMP- and TNM-induced lung neoplasms, respectively, and compares the findings to spontaneous lung neoplasms from corresponding control mice. The frequencies of the K-ras mutations were 57% (29/51) in BMP-induced lung neoplasms compared to 15% (3/20) in lung neoplasms from dosed feed control mice, and 54% (14/26) in TNM-induced lung neoplasms compared to 60% (3/5) in lung neoplasms from inhalation control mice. G --> A transitions at the second base of the K-ras codon 12 (GGT --> GAT) were the most frequent pattern of K-ras mutations identified in BMP-induced (20/29) and TNM-induced lung neoplasms (13/14), which differed from the mutational patterns identified in the lung neoplasms from unexposed control mice. These results indicate that mutations in the K-ras gene are involved in B6C3F1 lung carcinogenesis following BMP- and TNM-exposure, and the high frequency and specificity of the ras mutation profile in lung neoplasms (G --> A transition) may be due to in vivo genotoxicity by the parent compounds or their metabolites.     

Molecular and immunohistochemical analysis of intraductal papillary neoplasms of the biliary tract

Intraductal papillary neoplasms (IPNs) of the biliary tract are uncommon lesions that may be solitary or may spread extensively along the biliary tree. Some biliary IPNs are histologically and radiologically similar to intraductal papillary mucinous tumors (IPMNs) of the pancreas and present a risk for progression to invasive cholangiocarcinoma. Unlike pancreatic IPMNs, little is known about their molecular pathogenesis. We studied 14 biliary IPNs (including 5 cases with associated invasive cholangiocarcinoma) for genetic alterations in the APC/beta-catenin pathway, K-ras oncogene mutations, p53/chromosome 17p alterations, and Dpc4/18q alterations. Immunohistochemistry was performed for beta-catenin, p53, and Dpc4, and microdissected tissue was analyzed using direct DNA sequencing for exon 1 of K-ras and exon 3 of beta-catenin and allelic loss assays on chromosomes 5q, 17p, and 18q. Activating mutations in codon 12 of the K-ras oncogene were present in 4 of 14 (29%) biliary IPNs. Of these 4 cases, 2 patients had associated invasive cholangiocarcinoma, and identical K-ras mutations were present in both the intraductal and invasive components. Allelic loss on chromosome 18q was present in 4 of 13 informative cases (31%); however, no loss of normal Dpc4 expression was detected by immunohistochemistry. Nuclear accumulation of beta-catenin protein was demonstrated in 3 of 12 cases (25%); however, there were no beta-catenin gene mutations, and allelic loss on 5q was present in only 1 of 10 informative cases (10%). Both immunohistochemistry for p53 and 17p allelic loss assays were negative. Biliary IPNs therefore demonstrate a K-ras gene mutation frequency that is lower than that previously reported for pancreatic IPMNs, but similar to that reported for hepatic cholangiocarcinomas. The presence of K-ras mutations in 2 purely intraductal neoplasms, and identical K-ras mutations in 2 cases with both intraductal and invasive components, suggests that these mutations arise early in tumorigenesis. Finally, the frequency of allelic loss on 18q suggests that a locus on 18q is involved in the molecular pathogenesis of biliary IPNs, but this locus is not DPC4.     

"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.     

Mutations of ras protooncogenes and p53 tumor suppressor gene in cardiac hemangiosarcomas from B6C3F1 mice exposed to 1,3-butadiene for 2 years

1,3-Butadiene is a multisite carcinogen in rodents. Incidences of cardiac hemangiosarcomas were significantly increased in male and female B6C3F1 mice that inhaled 1,3-butadiene (BD) for 2 years. Eleven BD-induced cardiac hemangiosarcomas were examined for genetic alterations in ras protooncogenes and in the p53 tumor suppressor gene. Nine of 11 (82%) BD-induced hemangiosarcomas had K-ras mutations and 5 of 11 (46%) had H-ras mutations. All of the K-ras mutations were G-->C transversions (GGC-->CGC) at codon 13; this pattern is consistent with reported results in BD-induced lung neoplasms and lymphomas. Both K-ras codon 13 CGC mutations and H-ras codon 61 CGA mutations were detected in 5 of 9 (56%) hemangiosarcomas. The 11 hemangiosarcomas stained positive for p53 protein by immunohistochemistry and were analyzed for p53 mutations using cycle sequencing of polymerase chain reaction (PCR) amplified DNA isolated from paraffin-embedded sections. Mutations in exons 5 to 8 of the p53 gene were identified in 5 of 11 (46%) hemangiosarcomas, and all of these were from the 200- or 625-ppm exposure groups that also had K-ras codon 13 CGC mutations. Our data indicate that K-ras, H-ras, and p53 mutations in these hemangiosarcomas most likely occurred as a result of the genotoxic effects of BD and that these mutations may play a role in the pathogenesis of BD-induced cardiac hemangiosarcomas in the B6C3F1 mouse.     

Epidermal growth factor receptor gene mutations in atypical adenomatous hyperplasias of the lung.

Activating epidermal growth factor receptor (EGFR) gene mutations are frequently detected in lung adenocarcinomas, especially adenocarcinomas with a nonmucinous bronchioloalveolar carcinoma component. EGFR-mutated lung adenocarcinomas respond well to EGFR tyrosine kinase inhibitors. We previously found that most (88%) pure nonmucinous bronchioloalveolar carcinomas (adenocarcinoma in situ) already harbor EGFR mutations, indicating that the mutations are an early genetic event in the pathogenesis. We examined 54 atypical adenomatous hyperplasias, precursor lesions of lung adenocarcinomas, obtained from 28 Japanese patients for the hotspot mutations of EGFR exons 19 and 21 and K-ras codon 12. EGFR mutations were observed in 17 of the 54 (32%) atypical adenomatous hyperplasias examined: Ten and seven atypical adenomatous hyperplasias had deletion mutations at exon 19 or point mutations (L858R) at exon 21, respectively. We did not observe apparent histological differences between atypical adenomatous hyperplasias with and without EGFR mutations. K-ras mutation (G12S) was detected in only one atypical adenomatous hyperplasia. As EGFR mutational frequency of atypical adenomatous hyperplasias was much lower than that of nonmucinous bronchioloalveolar carcinomas, we surmise that EGFR-mutated atypical adenomatous hyperplasias, but not atypical adenomatous hyperplasias with wild-type EGFR, are likely to progress to nonmucinous bronchioloalveolar carcinomas.     

K-ras gene mutations and loss of heterozygosity at the p53 gene locus relative to histological characteristics of mucin-producing tumors of the pancreas

Mucin-producing tumors (MPTs) of the pancreas accompanied by carcinomas usually include various grades of dysplasia in the ductal epithelium, and invasive areas are histologically similar to those of common invasive ductal carcinomas, suggesting that MPTs provide a good tool to investigate early stages of pancreatic carcinogenesis. Thus, to clarify genetic alterations in the early stage of pancreatic carcinogenesis, we analyzed K-ras gene mutations and loss of heterozygosity (LOH) at the p53 gene locus using 37 cases of MPTs harboring dysplastic epithelium. Further, we conducted an extended, multifocal microdissection analysis focusing on the histological features of ductal epithelium and the distribution of genetic alterations for 3 cases of MPT positive for LOH of the p53 gene to determine the relation to tumor progression. K-ras gene mutations were detected with high frequency in 50% or more cases of the adenomas (14 of 19), borderline tumors (4 of 7), and carcinomas (8 of 11), whereas LOH of the p53 gene was limited to carcinomas (3 of 5 informative cases, 60%) and always accompanied by K-ras gene mutation. Investigation of a total of 126 microdissection sites from 3 cases showed the presence of K-ras gene mutations in mild dysplasia and all (100%) regions of moderate or more marked dysplasia, whereas LOH of the p53 gene showed more gradual tendency to increase with grade from moderate dysplasia. In addition, the multifocal genetic analysis showed K-ras gene mutations to be widely distributed throughout tumors, whereas LOH of the p53 gene was localized to 1 or a few areas. Further, topographically delimited areas with the same histology in the same tumor did not always show the same genetic alteration. In conclusion, we could confirm that both the K-ras and p53 gene alterations occur in the intraductal stage of MPT, and the latter is superimposed on the former during the course of tumor progression. However, the pattern of association of histological features with genetic alteration differs from tumor to tumor.     

P53 Gene Mutations in Pituitary Carcinomas

Although p53 overexpression detected by immunohistochemistry has been reported in pituitary adenomas and carcinomas, genetic mutations in the p53 gene have not been previously detected in these tumors. We analyzed a series of eight pituitary adenomas and six pituitary carcinomas by immunohistochemistry, polymerase chain reaction amplification, and sequencing of p53 exon 5 through exon 8 for genetic mutations. Three carcinomas showed more than 20% expression of p53 protein in the tumor cells. One of these tumors with 60% overexpression of p53 protein had a mutation in codon 248, a common “hot spot” for p53 mutation, while the other carcinoma with 90% overexpression of p53 protein had a mutation in codon 135. All adenomas were negative for p53 mutations and had 15% of the cells expressing the p53 protein. Analysis of control tumors including four lung carcinomas with proven p53 mutations also had greater than 85% of the tumor cells overexpressing p53 protein. Two breast carcinoma cell lines with known p53 mutations, MBA-MD 231 and MBA-MD-486, also showed greater than 85% of the tumor cells overexpressing p53. These results show that p53 mutations are present in a subset of pituitary carcinomas and are usually associated with a high percentage of tumor cells overexpressing the p53 protein.     

APC, K-ras codon 12 mutations and p53 gene expression in carcinoma and adenoma of the gall-bladder suggest two genetic pathways in gall-bladder carcinogenesis

Currant histopathoiogical evidence suggests that gall-bladder cancer has two main morphological pathways for Its development: de novo ( ab Inltlo ) origin and adenoma-carcinoma sequence. In order to investigate the genetic difference between them, APC mutations were examined by RNase protection analysis, K-ras mutations by nested porymerase chain reaction-restriction fragment length polymorphism analysis, and p53 gene overexpression by immunoriisto-chemical analysis in both tumors and benign lesions of the gall-bladder. Overexpression of the p53 gene was detected in 105 of 164 (64%) de novo carcinomas regardless of size and depth of Invasion, but not in 16 tumors of carcinoma-ln-pyloric-gland-type adenoma, or In 51 adenomas (47 pyloric gland-type and 4 Intestinal-type). K-ras codon 12 mutation was detected in 4 of 40 (10%) de novo carcinomas, ail four being associated with p53 gene overexpression, but not in 12 tumors of carcinoma in adenoma or 16 adenomas (14 pyloric gland-type and 2 intestinal-type). APC mutation was not found in 16 de novo carcinomas or the one pyloric gland-type adenoma examined. These results suggest that there are two distinct genetic pathways in gall-bladder carcinogenesis; that is, de novo carcinoma develops from a predominant p53 alteration with low K-ras mutation, and carclnoma-in-pylortc-gland-type adenoma develops from p53 -, K-ras -, and APC -gene-unrelated, as yet unknown, alteration.     

Telomeric repeat-length alterations in colorectal carcinoma are associated with loss of heterozygosity and point mutation in p53 gene

The telomere, the terminal region of eukaryotic chromosomes, plays an important role in the stability of DNA replication and in the protection of chromosomal ends. To investigate whether the two-stage mechanism of cellular aging and immortalization in vitro is involved in the carcinogenesis and immortalization of human colorectal carcinomas, we examined for genetic alterations in the telomeres and in the p53, Rb, and K-ras genes. Based on our results, we discuss the effects that these genetic changes might have on mechanisms, such as the mortality stage 1 (M1), that normally prevent immortalization in carcinoma cells. Telomeric repeat-lengths (TRL) were measured by Southern blot analysis, and p53 Rb and K-ras gene variants were detected by PCR based assays. Thirty-six primary colorectal carcinomas were examined. Telomere alterations were found in 19 of 36 (52.8%) cases, while mutations of the p53 gene were observed in 15 of 36 (41.7%) cases and LOH involving p53 observed in 14 of 36 (38.9%) cases. Twelve of 15 (80%) cases with p53 mutations also showed altered TRL, so that p53 mutations were positively associated with TRL alterations (p=0.008). Ten of 14 (71.4%) cases with LOH of p53 also showed alteration in TRL, also revealing a positive association with TRL (p=0.09). The six cases with both p53 mutation and LOH all showed altered TRL. K-ras gene mutations and LOH involving the Rb gene were not associated with alterations in TRL. These results suggest that inactivation of p53 is one of the factors that promotes immortalization and overcomes M1 in colorectal carcinoma.     

吸烟与肺癌p53及K-ras基因突变关系的研究进展

p53和K ras基因突变常见于肺癌,吸烟是引起p53和K ras基因突变的主要危险因素。吸烟 相关性肺癌基因突变以G→T为主,这在其它肿瘤及不吸烟者肺癌很少见。p53基因突变常发生于第157、 156、245、248、273密码子,K ras基因突变常发生于第12密码子,他们都是烟草致癌物的强结合位点。对吸烟 与p53、K ras基因突变关系的研究,在临床早期诊断及判断预后等方面均有应用价值。     

Lung tumours from non-smoking subjects: A p53-related genetic instability in a subset of cases.

Tumours developed in non-smoking patients represent about 10% of all lung neoplasms and show peculiar morphologic and genetic features. In a previous study, we found a constant association between p53 gene alterations and loss of heterozygosity (LOH) at the FHIT locus in a subset of non-smoking tumours (7 cases out of 35 analyzed), so we hypothesized that a genomic instability associated with p53 mutations could be the cause of FHIT LOH in these neoplasms. To test this hypothesis, in the same panel of tumours, we investigated the presence of LOH at 7 other microsatellite loci located on different chromosomes. Interestingly we found that all of the tumours with p53 alterations and LOH at the FHIT locus showed loss of heterozygosity at all of the informative tested loci. This association was statistically significant (p=0.0001). Our data indicate the presence of a generalized genomic instability in this subset of lung tumours. Since p53 alterations were mostly G:C --> A:T transitions and frameshift deletions, we are tempted to hypothesize that the genomic instability observed in non-smoking patients could be caused by particular p53 alterations. In fact, other kind of p53 mutations (G:C --> T:A transversions), frequently found in a series of 35 tumours of smokers used as control, were not associated with LOH at microsatellites loci. However, we cannot exclude that p53 alterations are a consequence and not the cause of the genomic instability. In this case, we have to admit that a gene(s), upstream of p53, is implicated in genome destabilisation in a subset of lung adenocarcinomas developed in non-smoking patients.     

K-Ras and microsatellite marker analysis of fine-needle aspirates from intraductal papillary mucinous neoplasms of the pancreas

Preoperative diagnosis of pancreatic cystic lesions is difficult despite the combination of cytomorphology, radiographic imaging characteristics, and fluid tumor markers such as carcinoembryonic antigen. Intraductal papillary mucinous neoplasms (IPMNs) represent a subset of preinvasive pancreatic cystic neoplasms and are associated with accumulated genetic mutations, especially K-ras and tumor suppressor genes such as p53. Application of molecular techniques to cyst fluid obtained by endoscopic ultrasound guided fine-needle aspiration (EUSFNA) may contribute to preoperative assessment.Sixteen patients with pancreatic cystic lesions had cyst fluid obtained by preoperative pancreatic EUSFNA or intraoperative aspiration. All patients subsequently underwent surgical resection of the pancreas and IPMN was documented in all (6 adenomas, 6 borderline tumors, and 4 carcinomas). DNA was extracted from the cyst fluids and mutational analysis for K-ras point mutations and loss of heterozygosity (LOH) analysis using a preselected panel of genomic loci were performed. LOH was observed in 3 of 4 carcinomas as compared to 4 of 11 adenomas and borderline lesions (1 was QNS). LOH and K-ras mutations were both acquired in 2 of 4 carcinomas and in 1 of 12 adenoma/borderline lesions.Although the study is small, molecular analysis for LOH and K-ras mutations is useful in the preoperative evaluation of cystic pancreatic lesions. Increasing degree of neoplasia appears to correlate with increased genetic abnormality using a panel of selected genomic markers.     

Alteration of the p53 gene of lung carcinomas with sarcomatous transformation (spindle cell carcinoma): analysis of four cases

Lung carcinoma with sarcomatous transformation (LCST) is highly aggressive and characterized by local invasion and/or distant metastasis, which leads to a shorter survival than ordinary lung carcinomas. Therefore, to elucidate whether the malignant potential of the spindle cell element in LCST is associated with the alteration of the p53 gene, four cases were examined by analyses of overexpression of the p53 oncoprotein, mutation of the p53 gene and loss-of-heterozygosity (LOH) at chromosome 17p. In two cases overexpression of the p53 oncoprotein of the spindle cell component showed a higher degree of staining than that of the carcinoma component; LOH was identified in both carcinoma and sarcomatous components in one case, while in contrast, another case showed LOH in the sarcomatous component only. Mutations were clearly detected in two cases; one showed a CTT to CGT transversion in codon 194 of exon 6 in both components, whereas the other showed a CTG to CAG transversion in codon 265 of exon 8 in the sarcomatous component only. On the basis of these observations, it suggested that the sarcomatous component shows a higher frequency of p53 gene abnormalities in comparison to the carcinoma component. These results also suggested that the acquisition of malignant potential in the sarcomatous component, or the morphological alteration of carcinoma cells, is correlated with abnormalities associated with the p53 gene.     

K-ras codon 12 and p53 mutations in biopsy specimens and bile from biliary tract cancers

To evaluate whether it is useful for diagnosis to detect K-ras and p53 mutations in biopsy specimens and bile of biliary tract lesions, 12 cholangiocarcinomas (CC), eight cases of cholangitis, seven gallbladder carcinomas (GBC), seven gallbladder cholesterol polyps, four cases of adenomyomatosis of the gallbladder and five cases of cholecystitis were examined. K-ras and p53 mutations in bile were detected by a two-step polymerase chain reaction (PCR) and nested PCR-single-strand conformation polymorphism (SSCP) analysis. In addition, p53 protein expression in biopsy specimens from CC were examined by immunostaining. K-ras mutations at codon 12 were detected in 50% of CC and 57.1% of GBC in both biopsy specimens and bile. The incidence of p53 mutations was 33.3% in CC and 42.9% in GBC. p53 protein overexpression was observed in 60% CC biopsy specimens. In contrast, K-ras and p53 abnormalities were not detected in any non-neoplastic biliary tract lesion. K-ras and p53 mutations in biliary tract cancers showed the same mutation patterns in spite of differences in the collection methods used between bile and biopsy specimens or surgically resected tissue. Genetic analysis of K-ras and p53 mutations in biopsy specimens and bile may be useful for the diagnosis of biliary tract cancers, although it may be effectively limited to patients with advanced disease.