Association of single nucleotide polymorphisms in cell cycle regulatory genes with oral cancer susceptibility.

Alterations in the regulation of the cell cycle are strongly linked to tumorigenesis, so genetic variants in genes critical to control of the cycle are good candidates to have their association with susceptibility to oral cancer assessed. In this hospital-based, case-control study of 445 patients who had been newly-diagnosed with oral cancer and 449 unaffected controls, we used a multigenic approach to examine the associations among a panel of 10 selected polymorphisms in the pathway of the cell cycle that were possibly susceptible to oral cancer. Six of 9 single nucleotide polymorphisms in the cell cycle showed significant risks for oral cancer, the highest risk being evident for p27 (rs34329; Odds ratio 3.05, 95% CI 2.12 to 4.40). A significant risk of oral cancer was also evident for individual polymorphisms of cyclin E (rs1406), cyclin H (rs3093816), cyclin D1-1 (rs647451), cyclin D2 (rs3217901) and Rb1-2 (rs3092904). The risk of oral cancer increased significantly as the number of unfavourable genotypes in the pathway increased, and so the results point to a stronger combined effect of polymorphisms in important cell cycle regulatory genes on predisposition to oral cancer.

Accumulation of inactive p53 protein in oral squamous cell carcinoma: stabilization by protein interaction.

Our previous study showed that p53 protein is accumulated in >60% of cases of oral squamous cell carcinoma (OSCC) and its overexpression is related to poor prognosis. However, the mechanism behind this is still elusive. The present study attempts to dissect p53 alterations at various levels from gene to function in tumor biopsies to understand whether molecular alterations have any relationship with the accumulation of p53 protein in OSCC. Protein-stabilizing mutations were observed in only 13.8% of the samples. Neither p53 polymorphisms nor human papillomavirus (HPV) infection (<2%) has any major role in augmented expression of p53 protein. Analysis of the p53 transcript demonstrated that alteration in the level of p53 mRNA (10%) is not the major mechanistic cause for accumulation of p53 protein, and p21 transcript status showed that the overexpressed p53 protein is functionally inactive. Immunoprecipitation studies demonstrated that the known interactors of p53, such as MDM2 and HSP70, have no significant role in stabilizing p53 and the significant presence of some unknown interactors, sequestering p53, and leading to the aberrant accumulation of p53. Our study suggests that overexpression of inactive p53 protein could be manifested as a result of sequestering from degradation by an unknown interacting protein rather than by gene or transcript level of alteration.

Influence of methylenetetrahydrofolate reductase polymorphisms in oral cancer patients.

BACKGROUND: Methylenetetrahydrofolate reductase (MTHFR) is a key enzyme in folate metabolism. Two common polymorphisms associated with MTHFR gene - C677T and A1298C - influence the thermolabile nature and activity of the enzyme. This study aimed to investigate the role of MTHFR polymorphisms on oral cancer susceptibility and its potential impact on the prognostic outcome. METHODS: Oral cancer cases and controls were genotyped using PCR-RFLP technique for MTHFR C677T and A1298C polymorphisms. Disease susceptibility analysed using regression analysis. The association between clinical outcomes and the polymorphisms were analysed using univariate and multivariate model. RESULTS: The 677CT+TT genotype showed a significant three-fold reduction in oral cancer risk (RR-0.35, p-0.009). 1298CC genotype showed decreased cancer risk when compared to AA+AC genotype (RR-0.55, p-0.062). When prognostic significance of MTHFR polymorphism was evaluated, 677CT+TT patients showed improved survival than the CC individuals (RR = 0.56, P = 0.378). The 1298 CC and AC+CC showed an increased risk for treatment failure and poor survival when compared with the wild AA genotype (HR = 4.27, P = 0.001). CONCLUSION: Here we observed MTHFR C677T to influence oral cancer susceptibility, while A1298C polymorphism associated with patient prognosis. Our data support MTHFR polymorphism to be an independent prognostic marker in oral carcinoma.

Differential roles of p16INK4A and p14ARF genes in prognosis of oral carcinoma.

BACKGROUND: Oral cancer patients are found to have poor clinical outcome and high disease recurrence rate, in spite of an aggressive treatment regimen. The inactivation of INK4A/ARF loci is reported to be second to p53 inactivation in human cancers. The purpose of this study was to assess the prognostic significance of the molecular aberrations in the INK4A locus for effective identification of aggressive oral carcinoma cases needing alternate therapy. MATERIALS AND METHODS: The study composed of 116 patients freshly diagnosed with oral carcinoma. The genetic and epigenetic status of the p16(INK4A) and p14(ARF) genes was evaluated. The relation between these genic alterations and different treatment end points, such as residual disease (initial response), disease recurrence, and overall survival, along with the standard clinical markers, were analyzed. RESULTS: 62% of the study cases had p16(INK4A) gene abnormalities, with deletion accounting for 33% and methylation for 29%. Alterations in p14(ARF) gene either by deletion (12%) and/or methylation (18%) were observed in 30% of the cases. p16(INK4A) deletion was associated with aggressive tumors, as evidenced by the nodal involvement of the disease. Low or absence of p16(INK4A) protein adversely affected the initial treatment response. Promoter methylation of p16(INK4A) was associated with increased disease recurrence and acts as an independent predictor for worse prognosis. Surprisingly, p14(ARF) methylation associated with lower recurrence rate in oral cancer patients with a good clinical outcome. Overall survival of these patients was associated with tumor size, nodal disease, and p16(INK4A) protein expression pattern. Our results indicate that p16(INK4A) and p14(ARF) alterations constitute a major molecular abnormality in oral cancer cases. CONCLUSION: The molecular profile of INK4A/ARF locus, both at DNA and protein level, could be used as a prognostic biomarker for assessing the aggressiveness of disease in oral carcinoma patients. The study further shows the opposing clinical effect of these two genes, transcribed from the same locus, in oral cancer patients.

CCND1 polymorphisms (A870G and C1722G) modulate its protein expression and survival in oral carcinoma.

Cyclin D1 is an essential regulator of the G1 phase of the cell cycle progression and plays an important role in the transition of the cell from the G1 phase to the S phase of the cell cycle. Overexpression of cyclin D1 is a frequently observed feature of human cancers of diverse histological origin. Recently, we have reported overexpression of cyclin D1 in oral carcinoma. However, the underlying mechanism leading to this aberrant expression remains poorly understood. In this study, we have investigated the role of CCND1 A870G and C1722G polymorphisms on cyclin D1 expression and prognosis in a relatively homogenous population of 178 oral squamous cell carcinoma patients by PCR-SSCP, RFLP, RT-PCR and immunohistochemical methods. Genotype frequencies of both the polymorphisms were conformed to Hardy-Weinberg equilibrium. CCND1 A870G (p=0.004) and C1722G (p=0.012) polymorphisms were significantly associated with cyclin D1 expression. Kaplan-Meier estimates revealed that CCND1 genotypes A870G 'GG' (p=0.012) and C1722G 'CC' (p=0.021) could predict the poor survival of the patients. In multivariate analysis, CCND1 A870G genotype combination (p=0.024, HR - 1.74 (1.08-2.81)) and cyclin D1 expression (p=0.025, HR - 1.72 (1.07-2.77)) were independent predictors of survival in this patient cohort. Our results thus demonstrate, CCND1 polymorphisms stand-in as cis-acting regulatory elements modulating its expression and cyclin D1 genotype and phenotype could provide valuable additional information regarding prognosis of oral cancer patients.

H-Ras mutation modulates the expression of major cell cycle regulatory proteins and disease prognosis in oral carcinoma.

Activating mutations of the Ras is a moderately frequent event in oral carcinogenesis in Indian patients. Ras pathway has essential roles in regulation of various phases of the cell cycle, especially at G1 phase. Despite a large body of in vitro evidence, the multidimensional interaction between mutated Ras pathway and G1 cell cycle regulatory proteins in tumours in vivo is poorly determined. In the present study, DNA samples were screened for mutations in hot spot exons of B-Raf and hot spot codons 12, 13 and 61 of H-, K- and N-Ras by PCR-SSCP. Mutations were confirmed by direct sequencing. Expression of G1 cell cycle regulatory proteins-cyclin D1, CDK4, Rb, p53, p16 and p21 and proliferation marker PCNA was analysed immunohistochemically. The results revealed the absence of B-Raf mutations in oral carcinoma in spite of 12.5% of the samples showing H-Ras mutation. The H-Ras mutant cases showed significantly low cyclin D1 (P=0.027) and CDK4 (P=0.046) expression and overexpression of Rb (P=0.011) and p16 (P=0.026). H-Ras mutant carriers also had significantly high recurrence-free survival (P=0.033). In summary the present study demonstrated an epistatic interaction between H-Ras mutation and G1 cell cycle regulatory proteins in vivo. H-Ras mutation, thus, defines a molecular subtype of oral carcinoma with favourable outcome and unique biology.

Carcinoma of tongue and the buccal mucosa represent different biological subentities of the oral carcinoma.

PURPOSE: Clinico-epidemiological studies show that the behaviour of the tongue cancer is different from the cancer originating at other sites of the oral cavity. However, studies identifying the reason for such difference are lacking in the literature. METHODS: In the present study, we have attempted to see whether any difference existed in the cell cycle regulatory mechanism of these tumours by comparing immunohistochemically the expression of major cell cycle regulatory proteins in 147 buccal and 94 tongue carcinoma (anterior two-third of tongue) prospectively. RESULTS: On comparison of buccal and tongue carcinoma, expression of p16 and p21 showed significant difference. In combined analysis, simultaneous down regulation of p16 and p21 was seen in 47% of tongue cancer cases as against 28% in buccal carcinoma (P=0.004). In univariate analysis, none of the clinico-biological variables studied showed significant association with survival in tongue carcinoma, whereas, some of the clinico-biological variables associated with survival in buccal carcinoma. Among the biological markers, the overexpression of cyclin D1 (P=0.007) and p53, detected using both the clones of antibodies-DO7 (P=0.008) and PAb240 (P=0.014) and the down regulation of p16 (0.033), showed significant association with shorter disease free survival (DFS) in these cases. Whereas in the case of overall survival (OS), overexpression of p53 [DO7 (P=0.031) and PAb240 (P=0.017)] and cyclin D1 (P=0.001) associated with poor survival. In multivariate analysis, the expression pattern of p53 and p16 protein influences the DFS whereas cyclin D1 expression showed independent association with the OS in buccal carcinoma. CONCLUSIONS: Thus, tongue and buccal cancers represent different biological subentities, and such differences should be considered in oral cancer management.

Influence of single nucleotide polymorphisms in H-Ras and cyclin D1 genes on oral cancer susceptibility.

Although tobacco usage and alcohol consumption are the major risk factors for oral cancer, there are individual variations in genetic susceptibility to oral cancer. The Ras pathway plays an important role in oral carcinogenesis. High percentage of Ras mutation in oral carcinoma was reported from India. Cyclin D1, a downstream member of the Ras pathway, was also shown to be overexpressed in the majority of oral cancers and the overexpression was shown to be associated with poor prognosis. In the present study, we have evaluated the association of the single nucleotide polymorphisms in the H-Ras (C81T) and cyclin D1 (A870G and C1722G) genes and oral cancer risk in 176 oral cancer cases and 142 hospital based controls matched by age and sex. All the polymorphisms studied conformed to Hardy-Weinberg equilibrium. The comparison of the CCND1 A870G and C1722G genotype frequencies in cases and controls did not show any significant association with oral cancer risk. In H-Ras C81T polymorphism, TC+CC genotype showed a one and half fold increased risk (OR=1.59) for oral cancer. On stratified analysis, the observed increased risk was more evident among men (OR=2), while such an increased risk was not seen among women. Thus, our data suggests that the variant 'C' allele of the H-Ras (C81T) is associated with a higher risk for oral carcinoma, particularly in male population and thus, this polymorphism could be a low penetrance gene predisposition factor for oral carcinoma.

Phenotypic alterations in Rb pathway have more prognostic influence than p53 pathway proteins in oral carcinoma.

The two well-defined pathways that are shown to be prominently altered in a variety of cancers are the cell cycle regulatory pathways led by either p53 or Rb genes. The present study is undertaken to find the pathway that is more altered in oral carcinoma at protein level, with special emphasis on its prognostic significance. The expression pattern of key molecules of the Rb and p53 pathways, such as Rb, cyclin D1, CDK4, p16, p53, p21 and Bcl-2 and the proliferative marker PCNA were analysed in 348 oral carcinoma specimens by immunohistochemical technique. The expression index of these molecules and various clinicopathological factors were statistically correlated with treatment end points to assess its prognostic efficacy after following up these patients up to a maximum of 48 months with a median of 23 months. Rb pathway proteins, Rb (P=0.016), cyclin D1 (P=0.0001) and p16 (P=0.012) showed significant association with disease-free survival, and p16 (P=0.041) and cyclin D1 (P=<0.0001) with the overall survival. Among p53 pathway proteins studied, only p53 expression index showed association with both disease-free survival and overall survival. Multivariate analyses confirmed that the biological variables, cyclin D1 and p16 and the clinical variable, 'stage of disease' were independent predictors of disease-free survival and overall survival. Subgrouping of the patients on the basis of p16 and cyclin D1 expression revealed that the subgroup having downregulation of p16 and overexpression of cyclin D1 exhibited the worst disease-free survival and overall survival compared to the other subgroups. The present data showed that disabling of the Rb and p53 pathways were frequent events in oral carcinoma. The study also demonstrated that the Rb pathway proteins are comparatively more important than p53 pathway proteins for the prognostication of oral carcinoma patients. The combined evaluation of p16 and cyclin D1 in oral carcinoma could identify a group of patients with the worst survival who might therefore need alternate or more intense treatment strategies.

p53, p16 and cyclin D1: molecular determinants of radiotherapy treatment response in oral carcinoma.

Management of oral cancer by radiotherapy has witnessed promising advances in the past few years, with patient-tailored radio fractionation regimens. Different fractionation schedules, conventional and altered regimes, have been used in curative radiotherapy. Although contribution of biological markers on radio response has been evaluated, its unique influence on various radio fractionation schemes has not been accounted so far. Our study analyses a set of proteins that previously demonstrated radio response influence for their possible prognostic value in decision-making process between the respective fractionation schemes. Expression patterns of regulatory proteins such as p53, cyclin D1, p16, Cdk4, p21, Rb, bcl-2 and PCNA were determined by immunohistochemistry utilizing monoclonal antibodies in 125 patients who received curative radiotherapy dose. Among these 125 patients, 90 (72%) received altered fractionation, whereas 35 (28%) received conventional fractionation. p53 over-expression correlated with local treatment failure among the patients treated with conventional fractionation whereas cyclin D1 over-expression and p16 underexpression were associated with local treatment failure as well as overall survival in altered fractionation treated cases. Our findings suggest that wild-type p53 status may be an important parameter for achieving high local control in those patients undergoing conventional fractionation, where as intact p16 and cyclin D1 status may be beneficial for effective local control in patients who are treated with altered fractionation. Furthermore, it can be assumed that conventional fractionation employs p53-mediated apoptosis, whereas altered fractionation activates the functional G1 cell-cycle checkpoint for tumor growth suppression.

Genetic polymorphism of CYP1A1, GSTM1 and GSTT1 genes in Indian oral cancer.

Oral cancer ranks first among all cancers in males and is the third most common among females in India. Tobacco-derived carcinogens are involved in the development of oral cancer. Environment-gene interaction in oral carcinogenesis is well demonstrated by phase I and II enzymes that are involved in the metabolism of carcinogens. This study looked at the significance of genetic polymorphisms in CYP1A1, GSTM1 and GSTT1 genes in patients with oral cancer. The study included 98 oral cancer patients and 60 age and sex matched healthy controls. Genotypes of CYP1A1, GSTM1 and GSTT1 were determined by PCR-RFLP. GSTM1 null deletion was observed in 49% of oral cancer cases and 33% of control subjects. For GSTT1, 18% of carcinomas and 8% of controls had the null genotype. In the case of CYP1A1 m2 allele, 51% of oral cancers and 17% of normal controls, respectively, had one or both alleles with the isoleucine-->valine substitution. Digestion of the PCR products with enzyme Nco1 revealed polymorphism for CYP1A1 m2 with bands at 263 bp. There was no association between genotypes with tumor size, stage, grade, and age. Since null genotype individuals may possibly be poor detoxifiers with reduced ability to neutralise the reactive carcinogenic intermediates, they may be a high risk category. The frequency distribution of CYP1A1 m2 (Ile/val) genotypes among oral cancer patients was significantly different that from normal controls. The risk of CYP1A1 can be supported by the functional difference between presence of valine and isoleucine; valine type has higher catalytic and mutagenic activity towards benzo[a] pyrene than the isoleucine type. In conclusion, our results suggest that polymorphism in CYP1A1 m2 gene and/or GSTM1 and GSTT1 null genotype may confer an increased risk for oral cancer.

De novo programmed cell death in oral cancer.

AIM: The importance of programmed cell death or apoptosis in the maintenance of tissue homoeostasis and the pathogenesis of oral cancer was analysed in relation to apoptosis regulatory proteins, tissue proliferation and tumour histology. METHODS AND RESULTS: The extent of apoptosis was defined by morphological criteria and the TUNEL (terminal deoxy nucleotidyl transferase-mediated dUTP biotin nick end labelling) assay. p53, bax, bcl-2 and cyclin D1 expression was evaluated by immunocytochemistry. The presence of mutant p53 was analysed using a mutant p53-specific ELISA. An inverse correlation was observed between TUNEL reactivity and histology of the lesion (r = -0.555, P = 0.0001). There was also correlation between TUNEL reactivity and immunoreactivity of apoptosis regulatory proteins. p53 (r = 0.641, P = 0.00023), bcl-2 (r = -0.642, P = 0.00014) and bax (r = 0.651, P = 0.00002). The presence of mutant p53 protein showed an inverse correlation to the extent of apoptosis (r = - 0.301, P = 0.00063). Significant correlation was evident between the bax/bcl-2 ratio and TUNEL (r = 0.652, P = 0.00001) as well as between cyclin D1 and TUNEL reactivity (r = 0.577, P = 0.00001). CONCLUSIONS: Results from this study suggest that apoptosis decreases as histological abnormality increases. Apoptotic regulatory proteins are also altered in a histologically dependent manner. Deregulated proliferation occurs simultaneously with decreased apoptosis during tumour progression in the oral mucosa.

Angiogenesis during tumor progression in the oral cavity is related to reduced apoptosis and high tumor cell proliferation.

Angiogenesis, the growth of new blood vessels, is believed to aid tumor progression and metastasis. Tumor progression is also influenced by the extent of proliferation and apoptosis. This study, therefore, analyzed in lesions of the oral cavity, the significance of angiogenesis in relation to apoptosis, expression of apoptosis regulatory p53, bax and bcl-2 proteins as well as tissue proliferation defined by cyclin D1 expression. Results from this study suggest that angiogenesis increases as histological abnormality increases in the oral mucosa. The expression of apoptosis regulatory proteins also appears to be altered in a histologically dependent manner. The correlation seen between CD34 expression, cyclin D1 and TUNEL reactive cells suggests that increased angiogenesis, decreased apoptosis and deregulated proliferation occur simultaneously during tumor progression in the oral mucosa. Presence of a mutant p53, increased bcl-2 expression and altered bax expression are also involved in this complex process.

Serum levels of copper, zinc, iron and ceruplasmin in oral leukoplakia and squamous cell carcinoma.

Trace elements have been extensively studied in recent years to assess whether they have any modifying effects in the etiology of cancer. In the present study serum levels of copper, zinc, ceruloplasmin, total iron and total protein were estimated in 92 patients with oral leukoplakia and squamous cell carcinoma and 45 age and sex matched controls. Copper was significantly increased in oral leukoplakia and cancer in both sexes. The level of zinc decreased significantly only in male patients with leukoplakia and cancer. Copper zinc ratio was found significantly elevated in oral leukoplakia and cancer. A marked increase in serum ceruloplasmin was seen in oral leukoplakia and cancer in both sexes. A significant decrease of serum total iron and proteins were observed only in carcinomas. These findings suggest that reduction in the serum total iron and total protein may be due to malnutrition caused, on its turn, by tumour burden. Serum ceruloplasmin level and copper-zinc ratio demonstrated significant differences in the patients of both sexes from those of controls, while, comparing leukoplakia with cancer, a significant difference is only observed in male patients. Thus, the present study shows that these factors have diagnostic value only in differentiating malignancies from normal and a little value as biomarkers of disease progression. However, the exact mechanism responsible for the alteration of these factors in oral lesions is still not clear and a detailed study on large sample size is therefore needed.

Telomerase activity in premalignant and malignant lesions of human oral mucosa.

Recent studies have demonstrated a strong association between carcinogenesis and re-activation of telomerase in various human tumors. In the present study, we have analyzed the telomerase activity in 105 oral mucosal samples, including normal mucosa and premalignant and malignant lesions, by using the telomeric repeat amplification protocol assay. The telomerase activity was detected in normal oral squamous epithelium and in 75% of the oral leukoplakias and oral carcinomas. Although the telomerase activity was observed in normal and hyperplastic squamous epithelium, it showed some relationship with certain clinico-pathological factors in malignant lesions. Telomerase activity was found to have a relationship with the grade of tumor differentiation. Of 34 well-differentiated squamous cell carcinomas, only 10 (30%) exhibited high telomerase activity, whereas in moderately or poorly differentiated squamous cell carcinomas, all seven (100%) tumors displayed high activity. In addition, the level of telomerase activity had an inverse correlation with the treatment response in the early-stage tumors, and the activity differed significantly between the tumors in the following intraoral sites: nonkeratinizing mucosa (buccal mucosa, alveolus, and floor of mouth) and tongue. This preliminary result shows that telomerase activity is present in normal oral squamous epithelium, as it is in normal hematopoietic cells and in carcinomas, and that telomerase activity has a relationship with degree of tumor differentiation and treatment response. Thus, assessing the telomerase activity may be a useful prognostic marker in oral squamous cell carcinomas.

Expression of programmed cell death regulatory p53 and bcl-2 proteins in oral lesions.

With the ultimate goal of characterizing the molecular pathogenesis of oral cancer, the most predominant malignancy in India, immunocytochemical evaluation of p53 and bcl-2 proteins was carried out in hypeplastic oral mucosa, dysplastic oral mucosa and invasive oral cancer. All subjects gave a similar and almost uniform history of prolonged use of betal quid and tobacco. Expression of p53 was insignificant while bcl-2 was absent in hyperplastic leukoplakia lesions. Both proteins were however expressed in leukoplakia with apparent dysplasia. Almost all invasive cancer lesions showed high levels of both p53 and bcl-2. Good correlation was therefore evident between expression of these two proteins and increasing histologic abnormality. Moreover relative risk evaluation revealed that lesions expressing p53 and bcl-2 had a high probability of having a histology of dysplasia or worse. Since it has been previously shown that wild type p53 regulates the expression of bcl-2, it may be presumed that the protein detected in the dysplastic and malignant oral tissue is of the mutant type. It is also known that p53 is a positive regulator of programmed cell death or apoptosis while bcl-2 is an anti-apoptotic protein. This suggests the possibility that alterations in p53 followed by over-expression of bcl-2 occur early in oral carcinogenesis resulting in defective apoptosis and subsequent tumor progression.

Ultrastructural analysis of the adjacent epithelium of oral squamous cell carcinoma.

Fifteen biopsies of the immediate adjacent epithelium of oral squamous cell carcinoma were examined under light and electron microscopy. Light microscopic examination of one micron thick sections revealed that the majority of lesions (67%) had hyperplastic or mildly dysplastic epithelium while the remaining (33%) had moderate to severe dysplasia. Ultrastructural observations showed that all these lesions had subcellular alterations similar to those seen in frank malignant oral tissue, particularly in the lower half of the epithelium. Important ultrastructural changes observed included bizarre nuclei of basal and lower spinal cells, enlarged and multiple nucleoli, presence of interchromatin and perichromatin granules, loss of desmosomes and marked spongiosis as well as disturbed cellular maturation sequences in the keratinocytes evidenced by abnormal and irregular distribution of maturation markers such as keratohyalin granules and tonofilaments. The present study thus shows the value of electron microscopy in the detection of malignant changes in the adjacent epithelium of oral squamous cell carcinoma.

Human papillomaviruses in 91 oral cancers from Indian betel quid chewers--high prevalence and multiplicity of infections.

India has one of the world's highest incidences of oral cancer. The habit of chewing betel quid is widespread and is suspected to play a role in the etiology of this disease. Studies in many other countries have also pointed to a role for human papilloma-viruses (HPVs) in the etiology of some oral cancers. In this study we analyzed biopsies from 91 Indian oral cancer patients, most of whom were betel quid chewers, by PCR amplification and direct DNA sequencing. HPV DNA was detected in 74% of these lesions, of which 41% had multiple HPV infections. Among the lesions from different oral sites, lesions of the tongue had the highest rate (9 of 11) of HPV infection. These HPV prevalences are among the highest ever reported in oral cancers. As to individual HPV types, prevalences of HPV-6, HPV-11, HPV-16 and HPV-18 were 13%, 20%, 42% and 47%, respectively. No additional known or novel HPV types were detected. To understand the unexpectedly high prevalences of the "low-risk" types HPV-6 and HPV-11, we compared the subtypes and variants that were found in oral cancers against those from benign genital warts from the same patient population but found no differences. The high prevalence of HPV in the oral cancers of these Indian patients suggests that viral infection is an important etiological component, with betel quid probably causing additional mutagenic steps in the carcinogenic process.

Scanning electron microscopy of different types of oral leukoplakia: comparison with normal and malignant oral mucosa.

The present study analysed surface architecture of normal, premalignant and malignant oral mucosa using scanning electron microscopy to evaluate its role in early diagnosis of potentially malignant oral lesions. The surface ultrastructure of the buccal mucosa in tobacco chewers showed variations from that of non-chewers. Homogenous leukoplakia demonstrated well-defined intercellular junctions and the microrugal surface pattern as seen in normal mucosa. In verrucous leukoplakia, the surface layer consisted of characteristically-shrunken desquamated hyperkeratotic cells. Erosive leukoplakia had a discontinuous superficial layer along with complete loss of intercellular ridges. Speckled leukoplakia also showed marked abnormalities such as thickened irregular protrusions and evidence of a villus-like pattern. These villus-like structures were comparatively prominent in leukoplakia showing dysplasia. Oral carcinoma showed marked altered surface ultrastructure and had a pattern similar to dysplastic lesions. The irregular swollen elongated protrusions with villous-like structures that were observed in carcinoma and dysplastic lesions can, therefore, be considered as surface markers for potentially malignant leukoplakia.

Expression of galactoside-specific endogenous lectins and their ligands in human oral squamous cell carcinoma.

Human endogenous lectins have a wide spectrum of biological functions. The present study analyses the expression of beta-galactoside specific and N-acetyl-D-galactosamine specific endogenous lectins in oral squamous cell carcinomas using biotinylated neoglycoproteins. The expression pattern of beta-galactosyl-containing glycoconjugates or ligands of beta-galactoside specific lectins in these tissues was also studied using an endogenous biotinylated lectin, the human 14-kDa lectin. For comparison a galactoside specific plant lectin from mistletoe, Viscum album was also employed. The results demonstrate that oral squamous cell carcinomas mainly express accessible binding sites for lactosylated neoglycoprotein (90%) while few carcinomas expressed mild amount of N-acetyl-D-galactosamine specific binding sites (40%). There was no difference in the binding patterns of these probes between well and less differentiated carcinomas. Expression of these neoglycoprotein binding sites were mostly concentrated in immature basaloid cells, indicating a possible association with cell proliferation. The binding pattern of D-galactosyl specific lectins (human 14-kDa and mistletoe lectins) showed conspicuous differences. This feature emphasizes the caution that needs to be exercised in interpreting the biological significance of results attained using plant lectins on human tissue.