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.