Radioresistance in oral squamous cell carcinoma with p53 DNA contact mutation

Reliable variables to predict the radiosensitivity of each tumor have not been identified. Recent studies have demonstrated that specific regions of mutations within the core domain of p53 protein correlate with responses to chemotherapy and radiotherapy in some tumor types. In this study, we evaluated the relationship between specific p53 mutations and radiosensitivity in 49 patients with oral squamous cell carcinomas (SCCs) who underwent preoperative radiotherapy. Exons 5 through 8 of the p53 gene were examined by polymerase chain reaction-single-strand conformation polymorphism and direct sequencing. We detected p53 mutations in 27 (55.1%) cases. DNA contact mutations were detected in 11 (40.7%) of these 27 cases in L3 loop, loop-sheet-helix motif, and zinc-binding residues. Tumors containing p53 DNA contact mutations had significantly poorer responses to radiation than the other tumors, although no statistically significant difference between tumors with and without p53 mutations was found. These data indicate that DNA contact mutation of p53 could be a useful marker to predict the radioresistance of oral SCCs.     

p53 mutation and protein accumulation during multistage human esophageal carcinogenesis.

Preinvasive lesions of squamous cell carcinoma are well defined morphologically and provide a model for multistage carcinogenesis. Since alterations in the p53 tumor suppressor gene occur frequently in invasive esophageal squamous cell carcinoma, we examined a set of preinvasive lesions to investigate the timing of p53 mutation. Surgically resected tissues from nine patients with esophageal squamous cell carcinoma contained precursor lesions which had not yet invaded normal tissues. Immunohistochemistry showed high levels of p53 protein in both preinvasive lesions and invasive carcinomas in six cases; sequence analysis of all invasive tumors identified p53 missense mutations in two cases. Preinvasive lesions from both tumors with mutations plus one wild-type tumor were microdissected and sequenced. In one patient there were different mutations in the invasive carcinoma (codon 282, CGGarg > TGGtrp) and a preinvasive lesion (codon 272, GTGval > T/GTGleu/val). In a second case, an invasive carcinoma had a mutation in codon 175 (CGCarg > CAChis), and adjacent preinvasive lesions contained a wild-type sequence. A carcinoma and preinvasive lesion from the third case contained high levels of protein and a wild-type DNA sequence. Therefore, p53 mutation may precede invasion in esophageal carcinogenesis, and multifocal esophageal neoplasms may arise from independent clones of transformed cells. The timing of p53 protein accumulation is favorable for an intermediate biomarker in multistage esophageal carcinogenesis.     

Mutations in the p53 gene in primary human breast cancers.

Twenty-six primary breast tumors were examined for mutations in the p53 tumor suppressor gene by an RNase protection assay and nucleotide sequence analysis of PCR-amplified p53 complementary DNAs. Each method detected p53 mutations in the same three tumors (12%). One tumor contained two mutations in the same allele. Single strand conformation polymorphism analysis of genomic DNA and complementary DNA proved more sensitive in the detection of mutations. Combining this technique with the other two a total of 12 mutations in the p53 gene were demonstrated in 11 tumors (46%), and a polymorphism at codon 213 was detected in another tumor. Loss of heterozygosity on chromosome 17p was detected by Southern blot analysis in 30% of the tumor DNAs. Not all of the tumors containing a point mutation in p53 also had loss of heterozygosity of the remaining allele, suggesting that loss of heterozygosity may represent a later event.     

Aberrations of the p53 tumor suppressor gene in human non-small cell carcinomas of the lung.

Aberrations of the p53 gene in 115 surgical specimens of non-small cell carcinomas of the lung were examined by single-strand conformation polymorphism analysis of polymerase chain reaction products. Structural abnormalities of the p53 gene were observed in 60 tumors (52%), i.e., 8 of 14 large cell carcinomas, 24 of 58 adenocarcinomas, 25 of 37 squamous cell carcinomas, and 3 of 6 adenosquamous carcinomas. Direct sequencing of abnormal DNA fragments revealed 45 single-base substitutions, 9 deletions or insertion of a short nucleotide sequence, and 3 two-base substitutions in 57 tumors. In the other 3 tumors, loss of one of the p53 alleles was observed, with no mutation in the other allele. Allelic loss of the p53 gene was observed in 14 of 43 informative cases (33%), and in 11 of the 14 cases the remaining allele was mutated. The aberrations of the p53 gene were not limited to a particular histological type or clinical stage. Their high frequency suggests that they were involved in the genesis of non-small cell carcinomas of the lung. The mutation frequency (46%) of the p53 gene in tumors carrying mutated ras genes was essentially the same as the overall frequency in lung cancers, suggesting that accumulation of mutations in these two genes in a tumor is a random phenomenon.     

Mutational Damages in Malignant Lung Tumors

Background: Today, genomic changes are an important cause of the occurrence, growth and progression of cancer. Technological advances in cancer genomic analysis platforms have made it possible to identify genomic alterations that may influence response to lung cancer treatment. Methods: The study examined tumor growth-inhibiting oncogenes and genes responsible for cell growth and division to identify mutations characteristic of malignant lung tumors. The mutations were studied in 400 postoperative samples after amplifying p53 and HRAS fragments and p53, p21Waf1, MDM2 mRNA. p53 or p21Waf1 were expressed in 50% of squamous cell carcinomas and adenocarcinomas of the lung. Results: The study examined tumor growth-inhibiting oncogenes and genes responsible for cell growth and division to identify mutations characteristic of malignant lung tumors. The mutations were studied in 400 postoperative samples after amplifying p53 and HRAS fragments and p53, p21Waf1, MDM2 mRNA. p53 or p21Waf1 were expressed in 50% of squamous cell carcinomas and adenocarcinomas of the lung. HRAS mutations were present in most squamous cell carcinomas and adenocarcinomas of the lung. EcoR1- and Pst1- restriction enzymes destroyed the RT-PCR product of the p53 and p21Waf1 mRNA and increased the level of detected mutations in lung adenocarcinoma to 75% and 50 %, respectively. EGFR mutations were more frequent in lung adenocarcinoma than in lung squamous cell carcinoma. Mutations in EGFR exons 19 and 21 found in 65 of 263 lung tumor samples indicated the tumor sensitivity to EGFR tyrosine kinase inhibitors. EGFR deletions in exon 19 occurred mainly in adenocarcinoma, L858R mutations in EGFR exon 21 were quite common in lung adenocarcinoma. Conclusion: The mutations detected in most squamous cell carcinomas and adenocarcinomas of the lung could be used to diagnose and predict the disease severity and targeted therapy efficacy.     

Mutation of p53 gene codon 63 in saliva as a molecular marker for oral squamous cell carcinomas

The inactivation of tumor suppressor gene (TSG) is important during multistage carcinogenesis. The p53 TSG is frequently mutated in oral squamous cell carcinomas. These mutations can serve as very specific markers for the presence of tumor cells in a background of normal cells. In this study, 10 oral squamous cell carcinoma patients and 27 normal dental students were collected from Chung Shan Medical and Dental College Hospital, Taichung, Taiwan. Extractions of DNA from saliva were obtained. Exon 4 and intron 6 within the p53 gene were amplified with polymerase chain reactions (PCRs) followed by DNA sequence analysis. DNA sequence analysis of PCR products revealed that five of eight (62.5%) tumor saliva samples and five of 27 (18. 52%) healthy saliva samples contained p53 exon 4 codon 63 mutations. These results were significantly different by using Chi-square test (P<0.05). The majority of the base substitutions were C deletions. Probable hot spots for the mutation were identified at exon 4 codon 63, which has not been observed before in head and neck cancers. Our study indicated that mutation of p53 codon 63 in saliva might be a molecular marker for oral squamous cell carcinomas. In addition, the amount of DNA recovered from saliva in most cases is sufficiently large and its quality suitable to enable PCR amplification which could be used in the search for mutations. The protocol described is rapid, cheap, and easy to perform, and may be useful for epidemiological studies for oral carcinogenesis.     

p53 mutations in primary human lung tumors and their metastases

In a total of 26 primary human lung tumors and 60 metastases derived from them, exons 5–8 of the p53 tumor suppressor gene were analyzed by single-strand conformation polymorphism and subsequent direct DNA sequencing of amplified DNA. Mutational inactivation of the p53 gene was identified in four of five squamous cell carcinomas, three of nine adenocarcinomas, and two of nine small-cell carcinomas, the overall incidence being 35%. Point mutations occurred at a similar incidence in exons 5–8, with a preference for G←T transversions. In seven of nine cases (78%), mutations were identical in the primary tumor and all of its metastases, indicating that in lung tumors, p53 mutations usually precede metastasis and that hematogenic and lymphogenic dissemination of tumor cells to other tissues is not associated with a selection against p53 inactivation. In one case, a kidney metastasis had the same mutation as the primary squamous cell carcinoma, whereas a liver metastasis had no mutation, indicating heterogeneity of the primary lung neoplasm and selective metastasis of mutated and nonmutated tumor cells to kidney and liver, respectively. Only in one liver metastasis was a mutation identified that was neither present in the primary lung tumor nor in a kidney metastasis, suggesting that occasionally p53 mutations occur after metastatic spread. © 1994 Wiley-Liss, Inc.     

The p53 tumor suppressor gene of the marsupial Monodelphis domestica: cloning of exons 4-11 and mutations in exons 5-8 in ultraviolet radiation-induced corneal sarcomas.

Inactivating p53 mutations are found in many ultraviolet radiation (UVR)-induced skin tumors. We examined 12 UVR-induced corneal tumors of the marsupial Monodelphis domestica for mutations in exons 5-8 of p53 and compared their mutational spectrum with that of UVR-induced skin tumors of other species. First we cloned and characterized a cDNA extending from the middle of exon 4 through exon 11 of the Monodelphis p53 gene. Based on the sequence information obtained, primers were designed to amplify introns 4-9 of the gene; intron primers to amplify individually exons 5-8 were subsequently developed. 'Cold' single strand conformational polymorphism analysis followed by reamplification of DNA with altered mobility and cycle sequencing revealed single p53 mutations in four of 12 tumors (33%), including one mutation in exon 5, two identical mutations in exon 7 and one mutation in exon 8. All mutations were at dipyrimidine sites and occurred on the non-transcribed strand. Three of the four were hallmark UVR-induced C-->T alterations. Three of the mutations were found at sites corresponding to human codons 248 and 273, which are mutational hotspots in human and murine UVR-induced squamous cell carcinomas. Our findings suggest that UVR-induced corneal sarcomas in Monodelphis will be valuable in studying mechanisms of p53 mutation in UVR-induced tumors.     

Frequent p53 mutations in head and neck cancer.

Squamous cell carcinomas of the head and neck (SCCHN) are associated strongly with the use of tobacco and alcohol, but little is known about the molecular pathogenesis of these tumors. In the present study, we analyzed SCCHN for mutations in the tumor suppressor gene p53 by immunocytochemistry and complementary DNA sequencing. Overexpression of p53 protein was detected in 13 (100%) of 13 SCCHN cell lines and in tumor cells cultured directly from 10 (77%) of 13 patients with SCCHN. Direct evidence for p53 mutations was obtained by sequencing p53 complementary DNA from eight SCCHN cell lines and two tumor xenografts. The genetic alterations included seven missense mutations resulting in single amino acid substitutions, a mutation encoding a stop codon, one 10-base pair deletion, and one 2-base pair addition. All seven missense mutations were G to T transversions, five of which were clustered at codons 245 and 248. A similar high frequency of G to T transversions predominates in lung cancer, another tobacco-related disease. Mutation of the p53 gene is the most common genetic alteration detected in SCCHN and implicates this gene locus as a critical site of specific damage by mutagenic carcinogens in tobacco, one of the important risk factors in the etiology of this disease.     

Prevalence of microsatellite instability,inactivation of mismatch repair genes,p53 mutation,and human papillomavirus infection in Korean oral cancer patients

To determine the etiologic factors of human oral cancer, we examined the prevalence of microsatellite instability (MSI), the inactivation of mismatch repair (MMR) genes, p53 mutation, and human papillomavirus (HPV) infection (HPV-16, -18, and -33) in 86 Korean oral cancer specimens, including 76 squamous cell carcinomas and 10 salivary gland tumors. MSI was observed in 3 of the 76 squamous cell carcinomas (4%) and 2 of 10 salivary gland tumors (20%). As MSI is a hallmark of the inactivation of the MMR genes, the genetic status of hMSH2 and hMLH1, and hypermethylation of the hMLH1 promoter region were investigated in oral cancers displaying MSI. Inactivation of the hMLH1 gene by either mutation or hypermethylation was observed 4 of the 5 MSI oral cancers. Mutation of the p53 gene was found in 11 of 76 squamous cell carcinomas (14.5%) but not in the salivary gland tumors. PCR assay revealed the presence of HPV DNA in 11 of the 76 squamous cell carcinomas (14.5%) and 4 of the 10 salivary gland tumors (40%). Type 18 HPV DNA was predominant in 11 of the HPV-infected squamous cell carcinomas (72.7%) and 4 of the HPV-infected salivary gland tumors (50%). Two squamous cell carcinoma tissues were found both to be HPV-infected and to harbor the p53 mutation. Our results suggest: i) that MSI plays a role in the pathogenesis of Korean oral cancers, squamous cell carcinomas (4%) and salivary gland tumors (20%); ii) that genetic alteration or hypermethylation of the hMLH1 gene may be the principal inactivating mechanism in Korean oral cancer with MSI; and iii) that inactivation of the p53 gene by either mutation or HPV infection is frequent in Korean squamous cell carcinomas (26%) and salivary gland tumors (40%).     

High Incidence of p53 Gene Mutation in Human Ovarian Cancer and Its Association with Nuclear Accumulation of p53 Protein and Tumor DNA Aneuploidy

Using the poylmerase chain reaction and single-strand conformation polymorphism analysis, p53 gene mutations were examined in 24 cases of ovarian tumor including 14 ovarian carcinomas and 2 borderline cases of common epithelial type, 7 germ cell tumors, and one stromal tumor. Abnormal bands indicating mutations were detected in 12 (50%) of the cases examined, being present most frequently in common "epithelial" ovarian carcinoma (71%, 10/14). One case each of squamous cell carcinoma originating in a dermoid cyst and anaplastic dysgerminoma were positive for mutation. Direct sequencing confirmed 12 mutations and revealed G→A and G→C nucleotide changes in 5 and 3 cases (42% and 25%), respectively. The mutation was localized at the CpG site of the gene in 3 cases. Immunohistochemical examination of p53 protein in 21 cases and DNA flow-cytometrical analysis in 17 cases were also performed. Nuclear accumulation of the p53 protein and DNA aneuploidy pattern were detected in 11 (52%) and 9 (53%) cases, respectively. These were significantly correlated with p53 gene mutation ( P <0.01 and P <0.05, respectively; Fisher's exact test). Neither mutation of the p53 gene, nuclear accumulation of p53 protein nor DNA aneuploidy was detected in borderline cases of common "epithelial" type, typical dysgerminoma and immature teratoma. These results suggest that p53 gene mutation, nuclear accumulation of the protein and the DNA aneuploidy pattern are events occurring almost simultaneously in the progression of ovarian tumors, and that p53 abnormalities seem to be correlated with a high grade of malignancy.     

High incidence of p53 gene mutation in human ovarian cancer and its association with nuclear accumulation of p53 protein and tumor DNA aneuploidy.

Using the polymerase chain reaction and single-strand conformation polymorphism analysis, p53 gene mutations were examined in 24 cases of ovarian tumor including 14 ovarian carcinomas and 2 borderline cases of common epithelial type, 7 germ cell tumors, and one stromal tumor. Abnormal bands indicating mutations were detected in 12 (50%) of the cases examined, being present most frequently in common "epithelial" ovarian carcinoma (71%, 10/14). One case each of squamous cell carcinoma originating in a dermoid cyst and anaplastic dysgerminoma were positive for mutation. Direct sequencing confirmed 12 mutations and revealed G-->A and G-->C nucleotide changes in 5 and 3 cases (42% and 25%), respectively. The mutation was localized at the CpG site of the gene in 3 cases. Immunohistochemical examination of p53 protein in 21 cases and DNA flow-cytometrical analysis in 17 cases were also performed. Nuclear accumulation of the p53 protein and DNA aneuploidy pattern were detected in 11 (52%) and 9 (53%) cases, respectively. These were significantly correlated with p53 gene mutation (P < 0.01 and P < 0.05, respectively; Fisher's exact test). Neither mutation of the p53 gene, nuclear accumulation of p53 protein nor DNA aneuploidy was detected in borderline cases of common "epithelial" type, typical dysgerminoma and immature teratoma. These results suggest that p53 gene mutation, nuclear accumulation of the protein and the DNA aneuploidy pattern are events occurring almost simultaneously in the progression of ovarian tumors, and that p53 abnormalities seem to be correlated with a high grade of malignancy.     

p53 status in spontaneous and dimethylnitrosamine—induced renal cell tumors from rats

Rats carrying the Eker tumor–susceptibility mutation (Eker rats) are predisposed to developing renal cell carcinoma. Rats heterozygous for the Eker mutation develop spontaneous multiple bilateral renal cell tumors by the age of 1 yr. In a previous study, Eker-mutation carrier and noncarrier rats were exposed to the renal carcinogen dimethylnitrosamine (DMN), and male rats carrying the Eker mutation exhibited a 70-fold increase in the induction of renal adenomas and carcinomas when compared with noncarrier rats. In this study, spontaneous and DMN-induced rat renal cell tumors (adenomas and carcinomas) were analyzed for mutations of the p53 gene by direct sequencing of cDNA polymerase chain reaction products. There were no mutations in p53 cDNA derived from renal tumors from six untreated rats. Mutations were found in one of 15 of the DMN-induced tumors: a transition at codon 140, CCT → CTT, in a renal adenoma. Additionally, seven cell lines derived from spontaneous renal cell tumors did not contain mutations in p53. The low frequency of p53 mutations (one of 21 renal cell tumors and none of seven cell lines derived from renal cell tumors) indicates that the development of both spontaneous and carcinogen-induced renal tumors involved a non–p53-dependent pathway. As p53 is infrequently mutated in human renal cell carcinomas and in rat renal mesenchymal tumors, it is likely that a tumor suppressor gene or genes other than p53 are involved in the development of renal cancer. © 1995 Wiley-Liss Inc.     

P53 expression is associated with a high-degree of tumor DNA aneuploidy and incidence of p53 gene mutation, and is localized to the aneuploid component in colorectal carcinomas

p53 protein expression was studied by immunoblotting in 34 colorectal carcinomas and 28 of the corresponding normal mucosas, and correlated with tumor DNA ploidy as measured by flow cytometry. p53 protein was detected in 35% (12/34) of the tumors; the normal mucosas were negative. Fifty-five percent (12/22) of the tumors examined for mutations within the four hotspots (exons 5-8) of the p53 gene had point mutations. p53 expression correlated significantly with the presence of p53 gene mutations; 67% (8/12) of the tumors with mutations expressed p53, whereas only one of 10 tumors where no mutations were detected expressed the protein (p=0.01). Four tumors with p53 gene mutations did not express p53. Fifty-nine percent (20/34) of the tumors were aneuploid. p53 expression correlated significantly with aneuploidy; a total of 55% (11/20) of the aneuploid tumors were positive for p53 compared to 7% (1/14) of the diploid tumors (p=0.009). All of the 11 highly aneuploid tumors (1.31 less-than-or-equal-to DNA index (DI); less-than-or-equal-to 1.86) expressed p53, whereas all of the 9 moderately aneuploid tumors (1.11 less-than-or-equal-to DI less-than-or-equal-to 1.29) were p53-negative. Flow cytometry was also used to resolve cell cycle- and ploidy specific p53 expression in nuclei in 4 aneuploid tumors. p53 expression in these tumors was confined to the aneuploid component, whereas the diploid component was negative. p53 was seen in nuclei in all phases of the cell cycle of proliferating aneuploid cells. Neither p53 expression nor tumor DNA ploidy were correlated with Dukes' stage (p=1.00 and 0.72, respectively). The data suggest that high levels of mutant p53 may play a causative role in the generation of highly aneuploid tumors.     

Mutations in the p53 tumor suppressor gene in human cutaneous squamous cell carcinomas.

In this study, we analyzed 10 human squamous cell carcinomas (SCCs) for alterations in the p53 tumor suppressor gene in exons 4 through 9 by single-strand conformation polymorphism (SSCP) analysis. We found that 2 of 10 SCCs displayed unusual SSCP alleles at exon 7 of the p53 gene. Subsequent cloning and sequencing of PCR-amplified exon 7 DNA from these two tumors revealed that one had a G----A transition at the first position of codon 244, predicting a glycine-to-serine amino acid change, while the other tumor exhibited a G----T base change at the second nucleotide of codon 248, predicting an arginine-to-leucine substitution. Because the mutations in the p53 tumor suppressor gene in both tumors were located opposite potential pyrimidine dimer sites (C-C), it is consistent with these mutations having been induced by the ultraviolet radiation present in sunlight. These studies demonstrate that inactivation of the p53 tumor suppressor gene, as well as activation of ras oncogenes, may be involved in the pathogenesis of some human skin cancers.     

Altered expression of wild-type p53 tumor suppressor gene during murine epithelial cell transformation.

An epidermal cell model in which initiated, benign tumor-producing and carcinoma stages were derived from a cloned parental cell strain was used to examine p53 expression during multistage epithelial carcinogenesis. Increased steady-state levels of p53 RNA were detected in squamous cell carcinomas compared to papilloma and normal epidermal cells. Nontumorigenic initiated cell precursors of the carcinomas exhibited normal p53 expression, localizing altered p53 regulation to the malignant conversion stage. Immunoprecipitation and Western immunoblot analyses demonstrated elevated levels of p53 protein in the moderately differentiated carcinoma compared to normal cells, and negligible levels of p53 in the poorly differentiated carcinoma cells. Sequence analysis of p53 complementary DNA from normal and carcinoma cells revealed no mutations in the coding or 5'- and 3'-untranslated regions, suggesting a novel mechanism of p53 inactivation.     

Occurrence of p53 gene abnormalities in gastric carcinoma tumors and cell lines.

We explored the state of the p53 gene in gastric cancer. Using one or more methods, we examined 15 specimens from primary carcinomas (14 tumors, one cell line), five cell lines derived from metastases, and seven paired samples of nonmalignant gastric mucosa. Sequence analyses of complementary DNA containing the entire p53 gene open reading frame demonstrated abnormalities in one of five samples from primary tumors and in all five samples from metastases. The single cell line derived from a primary carcinoma had no abnormality of the gene. The six abnormalities included four point mutations, one base-pair deletion resulting in a frame shift, and a 24 base-pair deletion caused by an intronic point mutation (as determined by sequence analysis of genomic DNA). Four of the six mutations mapped to regions highly conserved among species or involved in simian virus 40 T-antigen binding. Restriction fragment length polymorphism studies confirmed that chromosome 17p allelic deletions occur only in a minority of primary tumors, but that they may occur more frequently in metastases. Northern blotting and ribonuclease protection assays detected only a fraction of the p53 gene abnormalities detected by sequencing. Our findings indicate that mutations of the p53 gene are relatively rare in primary gastric tumors but appear to be relatively frequent in cell lines derived from metastatic lesions. Our results may help in understanding the molecular events associated with progression and metastasis in gastric carcinoma.     

p53 gene mutations in esophageal squamous cell carcinoma and their relevance to etiology and pathogenesis: results in Japan and comparisons with other countries

Esophageal squamous cell carcinoma is a form of cancer that has varying incidence rates among different countries, distinct geographic areas and different ethnic groups. According to previous reports, p53 gene mutations have been identified in 20-80% of these tumors, and these mutations have occurred at an early stage. These findings suggest that such mutations play an important role in esophageal carcinogenesis, and highlight the importance of mutagens, which cause sequence alterations in the p53 gene. In order to clarify the environmental factors and the molecular mechanisms that may be responsible for the occurrence and prevention of a specific mutation in the process of esophageal carcinogenesis, we analyzed p53 gene mutations in 95 samples of esophageal squamous cell carcinoma. We further reviewed published reports investigating the frequency of p53 gene mutations in esophageal cancer from high-risk areas to normal-risk areas and compared these findings to our results in Japan. The frequency of p53 gene mutations in Japanese esophageal cancer is 47.4% and there are three prominent features: (1) a predominance of transversions, in particular the G:C to T:A transversion; (2) a relatively low frequency of transitions; and (3) a relatively high percentage of frameshift mutations. These results indicate the possible importance of the benzo[a]pyrene metabolite and oxidative DNA damage in esophageal carcinogenesis and scarcely correlate with DNA replication errors or alkylation in comparison to other gastrointestinal cancers. In addition, we observed a peculiar sequence of frameshift mutations. Taken together, these data suggest that this tumor suppressor gene plays a critical role in the multistep carcinogenesis process for esophageal squamous cell cancer.     

The origins of multiple squamous cell carcinomas in the aerodigestive tract

BACKGROUND: Chemoprevention and cessation of smoking and alcohol may prevent development of multiple tumors (MTs) in the aerodigestive tract if new MTs arise independently, but they are of no benefit if MTs are due to migration of an already transformed clone of tumor cells. This issue was addressed in this study by investigation of the clonality among MTs. METHODS: Mutation analysis of the entire coding region of p53 and loss of heterozygosity (LOH) pattern analysis of microsatellite markers on chromosome arms 3p, 9p, and 17p are promising for the investigation of clonality. In the first part of this study, the authors established the variability and stability of these clonal markers by comparing primary head and neck squamous cell carcinomas (HNSCCs) with their metastases. In the second part of this study, the authors evaluated nine patients with multiple HNSCCs using these markers. In the final part, the authors illustrate the use of these clonal markers in 11 patients for whom there was diagnostic uncertainty as to whether their second squamous cell carcinoma was either a new primary tumor, a metastasis, or a recurrence. RESULTS: Both p53 gene mutations and LOH patterns were stable during tumor progression. Furthermore, the variability of p53 gene mutations was high. More than 90% of the tumors contained a p53 mutation. A particular mutation never occurred more than twice in a total of 69 primary HNSCCs. Five of 69 cases presented a common mutation. In contrast, LOH patterns showed less variability; they were identical in 5 of 16 cases. The metachronous HNSCCs from nine patients all showed different p53 mutations, and in the three cases that were subjected to LOH analysis different patterns were observed. All 11 patients for whom there was diagnostic uncertainty about the origin of their second squamous cell carcinoma could be categorized as having multiple primary tumors, disseminated disease, or recurrent disease. CONCLUSIONS: Metachronous HNSCCs at different locations are not clonally related and thus have not developed from the migration of tumor cells.     

Reversibility of proliferative lesions and induction of non-papillary tumors in rat urinary bladder treated with phenylethyl isothiocyanate

Phenylethyl isothiocyanate (PEITC), generally thought to be a chemopreventive agent for various kinds of genotoxic carcinogens, has been found to induce rat urinary bladder carcinomas in our laboratory. To cast light on the underlying mechanisms, the reversibility of urothelial proliferative lesions and the frequencies of H-ras and p53 mutations in the induced rat urinary bladder tumors were investigated. F344 male rats were given diet containing 0.1% PEITC for 48 weeks and then killed, or for 32 weeks and then returned to normal diet without supplement for 1, 3, 7 days or 16 weeks before death. At 7 days after withdrawal of PEITC treatment, carcinomas were observed in only two of 24 rats but a high incidence of dysplasias was evident. Furthermore, 16 weeks after withdrawal, seven of 12 (58.3%) rats had carcinomas. In addition, carcinomas were induced in 11 of 12 (91.7%) rats continuously receiving PEITC for 48 weeks. Most of the carcinomas were characterized as of non-papillary transitional cell type with occasional squamous cell differentiation and/or glandular components. Bromodeoxyuridine labeling indices (LIs) were increased by PEITC administration even in normal-looking epithelium. After withdrawal of treatment, LIs in simple and papillary or nodular (PN) hyperplasias were markedly decreased and these lesions gradually disappeared, while values for dysplasias and carcinomas, which persisted, were only slightly decreased. A silent point mutation was found in H-ras in one of 12 tumor samples (8.3%), whereas seven (58.3%) had mutations in p53. These results indicate that PEITC itself is a carcinogen for the rat urinary bladder, and that while the simple and PN hyperplasia induced by PEITC are reversible, dysplasia is irreversible with the potential to give rise to non-papillary carcinomas with frequent p53 mutations.