Infrequent loss of heterozygosity at adenomatous polyposis coli gene locus in Indian oral cancers.

We have investigated loss of heterozygosity (LOH) of the adenomatous polyposis coli (APC) tumor suppressor gene using polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) in 86 untreated oral cancer patients, using matched oral cancer tissue and corresponding peripheral blood cell (PBC) DNA samples. PBC from 70 normal healthy individuals, were also analyzed for allelic distribution of APC gene. A 133 bp fragment, spanning exon 11 of the APC gene was amplified, and RsaI digestion of the PCR product defined the alleles as either homozygous 133 bp (Rsa(-/-)) or 87 and 46 bp (Rsa(+/+)) fragments, and heterozygous (Rsa(+/-)) exhibiting the three fragments. Distribution of the three alleles, Rsa(-/-), Rsa(+/+), and Rsa(+/-) in the oral cancer patients was observed as 10.5, 51.1 and 38.4%; whereas normal healthy individuals showed 11.4, 37.1 and 51.4%, respectively. In the informative heterozygous (Rsa(+/-)) oral cancer patients, LOH was infrequent, demonstrated in two of 33 (6%) samples. Thus, the APC gene was infrequently altered by LOH at the polymorphic RsaI locus in exon 11 in the tobacco associated Indian oral cancer, unlike the smoking tobacco/alcohol associated oral cancers from Western countries.

p53 inactivation in chewing tobacco-induced oral cancers and leukoplakias from India.

The inactivation of p53 tumour suppressor gene vis-á-vis point mutation, overexpression and degradation due to Human Papilloma virus (HPV) 16/18 infection, was examined in chewing tobacco-associated oral cancers and oral leukoplakias from India. The analysis of mutations was assessed by polymerase chain reaction (PCR) with single strand conformation polymorphism (PCR-SSCP) of exons 5-9 on DNA from 83 oral cancer cases, and the mutations confirmed by direct nucleotide sequencing of the PCR products. p53 protein expression was evaluated by immunohistochemical analysis on paraffin-embedded sections of 62 representative oral cancer biopsies and 22 leukoplakias, using p53-specific monoclonal antibody DO-7. The presence of HPV16/18 was detected in the 83 oral cancer cases by PCR analysis using HPV L1 consensus sequences, followed by Southern hybridization with type-specific oligonucleotide probes. Forty-six per cent (38/83) of oral cancer tumours showed p53 alterations, with 17% (14/83) showing point mutations, 37% (23/62) with overexpression and 25% (21/83) with presence of HPV16 wherein the E6 HPV16 protein degrades p53. HPV18 was not detected in any of the samples. Ninety-two per cent concordance was observed between missense point mutations and overexpression of p53 protein. A significant correlation was not observed between p53 alterations in oral cancer and clinico-pathological profile of the patients. Twenty-seven per cent (6/22) of oral leukoplakias showed p53 overexpression. The overall p53 alterations in oral cancer tissues and oral lesions are comparable to data from the oral cancers reported in the Western countries with smoking and alcohol-associated oral cancers, and suggest a critical role for p53 gene in a significant proportion of oral cancers from India. The overexpression of p53 protein in leukoplakias may serve as a valuable biomarker for identifying individuals at high risk of transformation to malignant phenotype.

H-ras-1 restriction fragment length polymorphism in normal individuals and oral cancer patients in India.

Restriction fragment length polymorphism (RFLP) of the human H-ras-1 gene has been indicated as a marker for detection of individuals at high risk of cancer. We have investigated the association of RFLP at the H-ras-1 locus and susceptibility to oral cancer by Southern hybridization analysis in 77 primary oral tumors and 99 healthy donors. The frequency distribution of the BamHI fragments of H-ras-1 revealed homozygous or heterozygous alleles in the two subpopulations. The heterozygous genotype occurred more frequently in the normal subjects (53%) as compared to the cancer patients (36%). Four common alleles-C1 to C4, were noted predominantly in both groups, with rare alleles detected at a lower frequency. The common allele with 7.6 kb BamHI fragment was significantly higher in normals (10%) than in the tumor population (4%) (P < 0.05). However, a similar distribution of rare alleles in both groups indicated that the presence of rare alleles is not indicative of predisposition to oral cancer.

Amplification and overexpression of epidermal growth factor receptor gene in human oropharyngeal cancer.

The presence of epidermal growth factor receptor (EGF-R) gene was determined in 84 patients with squamous cell carcinoma (SCC) of the oropharyngeal region--a highly prevalent, chewing-tobacco associated malignancy in India, using Southern hybridisation analysis of DNA extracted from primary tumor tissues. We observed a 3- to 8-fold amplification of EGF-R gene in 19/66 (29%) of the SCCs of the oral cavity, and about 30-fold EGF-R amplification in 2/18 (11%) hypopharyngeal cancers. Dot blot analysis of total RNA from several tumour tissues, revealed overexpression of the EGF-R gene in the examined patients, with the EGF-R gene amplified. 4 patients with single copy EGF-R gene, did not exhibit overexpression of the gene. Within our sample set, no correlation was evident between EGF-R gene amplification and clinico-pathological parameters of the malignancy. The amplification and overexpression of the EGF-R gene observed in the primary tumour tissues of 25% (21/84) of the human oropharyngeal cancers, indicate possible involvement of the gene in the pathogenesis of these cancers.

Loss of allelic heterozygosity at the harvey ras locus in human oral carcinomas.

The Harvey ras locus was examined for restriction fragment polymorphism and loss of allelic heterozygosity in 62 oral cancer patients. Southern blot analysis on BamHI digests of the tumour tissue DNA, revealed 23 patients with H-ras-1 heterozygosity. The probes used to study the polymorphism were the BamHI 6.6-kb fragment encoding the complete H-ras-1 sequence plus the variable tandem repeat (VTR) region, and the 1-kb MspI fragment encoding the VTR region. The allelic heterozygosity was better resolved by PvuII and further confirmed by TaqI. In addition, TaqI digestion demonstrated a unique VTR rearrangement indicated by 2.1-kb, 0.9-kb and 0.6-kb fragments, implying additional TaqI sites, in three of the patients. Further analysis of matched tumor tissue and peripheral blood cell DNA from the same patient demonstrated tumor-associated loss of one of the allelic fragments in 7/23 (30%) of the patients with H-ras-1 heterozygosity. However, the loss was not significantly correlated to clinicopathological parameters staging the disease. Thus, our data showing loss of H-ras-1 alleles and VTR rearrangement, with relatively high incidence (9/23; 39%) in the oral cancer patients at various stages of the disease, implies H-ras-1 involvement as an early event in the process of oral carcinogenesis.

Restriction fragment length polymorphism of the L-myc gene in oral cancer patients.

Restriction fragment length polymorphism (RFLP) of the L-myc gene was examined in DNAs from primary tumour tissues and peripheral blood cells (PBC) of 76 Indian patients with squamous cell carcinoma of the oral cavity, and PBC from 101 normal healthy volunteers. The patients and the normal healthy volunteers were classified into three genetic types according to the polymorphic patterns defined by the two alleles (6.6 kb, S fragment; and 10.0 kb, L fragment). DNA isolated from the PBC of each patient always exhibited the same pattern of L-myc alleles as that observed for the corresponding tumour DNA. However, a striking correlation was found between the RFLP pattern and the stage of differentiation of the tumours, as well as the size of the tumour. Thus, a preponderance of the S fragment was observed in the poor to moderately differentiated tumours and the larger (greater than 4 cm) sized tumours. Further, analysis of L-myc RFLP with the clinical pattern of the malignancy showed no significant correlation with nodal metastasis, TNM staging or recurrence of the tumour. The relative ratios of the three genotypes (L-L, L-S, S-S) in the oral cancer patients were not significantly different from those seen in the healthy Indians, implying no predisposition to oral cancer by either allele. However, our results showed that oral cancer patients with a genotype including an S fragment are more likely to develop a poor to moderately differentiated tumour or a larger tumour than a patient without an S fragment. The L and S alleles were equally distributed in the population, with the frequency of each allele being 0.50, consistent with Hardy-Weinberg's law.

Oncogene amplification in squamous cell carcinoma of the oral cavity.

We have determined the prevalence of amplification of c-myc, N-myc, L-myc, H-ras, Ki-ras, and N-ras oncogenes in 23 cases of squamous cell carcinoma of the oral cavity, using Southern hybridization analysis of DNA extracted from the primary tumor tissues. Nick-translated oncogene probes and oncogene inserts labeled to high specific activities were used. We observed a 5- to 10-fold amplification of one or more of c-myc, N-myc, Ki-ras and N-ras oncogenes in 56% of the tumor tissue samples, with these oncogenes not being amplified in the peripheral blood cells of the same patients. L-myc and H-ras were not amplified in any of our samples. The oncogene amplifications seemed to be associated with advanced stages of squamous cell carcinomas, with the ras and myc family oncogenes being amplified in stages 3 and 4. Hybridization with N-myc detected an additional 2.3 kb EcoRI fragment, along with the normal 2.1 kb fragment. Our data also demonstrated amplification of multiple oncogenes in the same tumor tissue sample. About 60% of the samples with amplified oncogenes showed simultaneous amplification of 2 or more oncogenes. The results showing different oncogene amplifications in similar tumors, as well as multiple oncogene amplifications in the same tumor, suggest that these oncogenes may be alternatively or simultaneously activated in oral carcinogenesis.