Role of CYP2E1genetic polymorphism in the development of oral leukoplakia among tobacco users in North Indian population.

AIM: The aim of the study to find out role of CYP2E1 genetic polymorphism in development of oral leukoplakia among tobacco users in North Indian population, this study was carried out at Department of Oral and Maxillofacial Pathology, King George’s Medical University, Lucknow, UP. STUDY DESIGN: Study include a total of 105 leukoplakia patients were genotyped for CYP2E1 polymorphism (93 males and 12 females; mean age ± SD: 47.5 ± 10.6) and 96 unrelated healthy controls (85 males and 11 females; mean age ± SD: 49 ± 11.1). All the patients had either reported for treatment of leukoplakia or were diagnosed with leukoplakia during routine oral examination. RESULTS: A total of 105 leukoplakia patients and 96 controls were included in the study. The mean age of leukoplakia patients and control were 47 ± 10 and 51 ± 10 years respectively. The exclusive smokers comprised 62 (59%) leukoplakia patients and 53 (53%) controls. The exclusive smokeless tobacco users were 16 (15%) in leukoplakia patients and 27 (28%) in controls groups, while 27 (26%) leukoplakia patients and 16 (17%) controls have both types (smoking as well as smoke less) of tobacco habits simultaneously. Range of life time smoking exposure in leukoplakia and controls were (5‑80 PY in both groups) but the mean smoking exposure in both groups were (leukoplakia: 28 ± 21.8 PY, control: 27: ±17 PY). But the mean smokeless tobacco dose in two groups were (leukoplakia: 150 ± 175 CY, controls: 137 ± 110 CY). CONCLUSION: All the results demonstrate an association between CYP2E1 genetic polymorphism and leukoplakia risk, premalignant lesion. It indicates that the CYP2E1 polymorphism, singly showed a protection towards the oral leukoplakia. Independent confirmation of this finding is required, and additional examination of the joint effect of CYP2E1genotype and other non‑tobacco‑related exposures is needed before more conclusive interpretation of our results can be made. This study demonstrates the importance of genetic variations in CYP2E1genes in susceptibility towards oral leukoplakia and it is conceivable that these variants will interact with environmental carcinogens and possibly some combinations of these genotypes will be at a high risk to oral leukoplakia.

Association between polymorphisms at N-acetyltransferase 1 (NAT1) & risk of oral leukoplakia & cancer.

Background & objectives: N-acetyltransferases 1 and 2 (NAT1 and NAT2) are important enzymes for metabolism of tobacco carcinogens. Due to polymorphisms, improper activities of these enzymes might lead to the formation of DNA adducts that may modulate risk of tobacco related oral precancer and cancer. Previously, it was shown that NAT2 polymorphisms did not modulate the risk of oral precancer and cancer. We undertook this study to check whether polymorphisms at NAT1 can modulate the risk of oral leukoplakia and cancer either alone or in combination with NAT2. Methods: Genotypes at four SNPs on NAT1 were determined by TaqMan method in 389 controls, 224 leukoplakia and 310 cancer patients. Genotype data were analyzed to know haplotypes and acetylation status of individuals and, then to estimate the risk of diseases. Using our previously published NAT2 data, combination of NAT1 and NAT2 acetylation genotypes of patients and controls were also analyzed to estimate the risk of diseases. Results: Analysis of NAT1 genotype data revealed that 1088T and 1095C alleles exist in strong linkage disequilibrium (r2=0.97, P<0.0001) and SNPs are in Hardy-Weinberg Equilibrium (P=0.1). Wild type or normal acetylating and variant or rapid acetylating alleles were two major alleles (frequencies 0.62 and 0.36, respectively) present in the control population. NAT1 rapid acetylation could not modulate the risk of leukoplakia and cancer (OR=0.9, 95% CI: 0.6-1.3; OR=1.0, 95% CI: 0.7-1.4, respectively). Analysis of combined NAT1 and NAT2 acetylating data also showed no significant enhancement of the risk of diseases. Interpretation & conclusions: NAT1 rapid acetylation alone as well as combination of NAT1 rapid-NAT2 slow acetylation did not modulate the risk of oral precancer and cancer in our patient population. So, NAT1/NAT2 metabolized carcinogen products may not be involved in tobacco related oral precancer and cancer. It may be interpreted that large sample size as well as combination of polymorphisms at other candidate loci may be important to estimate the risk of a complex disease like oral cancer.

Coexpression of p53 and Ki 67 and lack of c-erbB2 expression in oral leukoplakias in India

Oral cancer is commonly preceded by premalignant lesions and conditions. The clinician's ability to identify lesions at an increased risk of cancer development is critical for its control. The purpose of this study was to compare the expression of tumor suppressor gene p53, proliferation marker Ki-67, and oncogene c-erbB2 and to evaluate the relevance of their co-expression in the diagnosis of, and prognosis for, oral leukoplakia. In the present study, the expression of biomarkers was studied immunohistochemically in 55 cases of leukoplakia (26 without dysplasia, 29 with dysplasia) and 10 cases of normal epithelia. The Labeling Indices (LI) of p53 and Ki-67 were found to increase significantly with an increase in the grade of dysplasia. A significant correlation was also found between the LI of p53 and that of Ki-67. It was also observed that c-erbB2 expression was only cytoplasmic, indicating incomplete receptor degradation. Hence, it can be concluded from the present study that the increased expression of p53 and Ki-67 with an increase in the grade of dysplasia suggests that their co-expression may be used for the identification of high-risk lesions. Also, c-erbB2 has no pathogenetic role in early carcinogenesis in the studied population, although incomplete receptor degradation, as evidenced by cytoplasmic staining, may indicate an early change.

Evaluation of DNA damage in oral precancerous and squamous cell carcinoma patients by single cell gel electrophoresis.

Context: Single cell gel electrophoresis (SCGE) or "comet assay" is a rapid and very sensitive fluorescent microscopic method for detecting various forms of DNA damage at individual cell level. Aims: The aim of the present study was to detect the extent of DNA damage in oral cancer, oral submucous fibrosis (OSMF) and leukoplakia in comparison to normal individual. Settings and Design: A total of 44 consecutive patients with oral cancer (n=26), leukoplakia (n=12) and OSMF (n=6) and 10 healthy normal volunteers with normal oral epithelia (controls) were recruited from Dr. R. Ahmed Dental College and Hospital and were assessed for the extent of DNA damage using SCGE following clinical diagnosis. Materials and Methods: Peripheral blood was collected by venepuncture and comet assay was performed using SCGE. Mean tail length was compared between diagnostic groups and between different oral habit groups using t-tests and analysis of variance (ANOVA). Pearson's product moment correlation was used to examine the linear association between the extent of DNA damage and oral habit pack-years. Scheffe's pair-wise test was employed to adjust for multiple comparisons. Results: None of the controls were associated with any oral habits. Mean (±SD) tail lengths (in mm) for cancer (24.95 ± 5.09) and leukoplakia (12.96 ± 2.68) were significantly greater than in controls (8.54 ± 2.55, P<0.05). After adjustment, well-, moderately, and poorly differentiated carcinomas had significantly greater tail length than controls. Whereas the extent of DNA damage in cancer cases was significantly greater in leukoplakia than in compared to OSMF (11.03 ± 5.92), the DNA damage in latter was not different from controls. DNA damage for people with any oral habit (19.78 ± 7.77) was significantly greater than those with no habits (8.54 ± 2.55; P<0.0001). Conclusions: DNA damage measured by SCGE is greater in leukoplakia and squamous cell carcinoma, but not in OSMF. Deleterious oral habits are also associated with greater DNA damage.

[ expression of P53 in leukoplakia, squamous cell carciboma and normal oral epithelium using immunohistochemical staining method]

Leukoplakia is the most common premalignant or potentially malignant lesion of the oral mucosa and its potentiality for malignant transformation is unpredictable. The aim of the present study was to evaluate p53 expression in normal oral epithelium, leukoplakia and oral squamous cell carcinoma. The standard Biotin streptavaidin peroxidase immunohistochemical staining method was used to study the expression of p53 on formalin fixed, paraffin embedded blocks of 8 cases of squamous cell carcinoma, 20 cases of leukoplakia and 10 cases of normal oral epithelium. There was no significant difference between immunostaining of leukoplakia and normal epithelium groups in the expression of P53, but the distribution patterns of p53 was mainly localized in the basal layer in the group of normal oral mucosa, while it extended into the suprabasal cell layer in leukoplakia group. P53 expression in squamous cell carcinoma group was higher than other groups. Considering the findings the expression of p53 in suprabasal cell layers in leukoplakia might show poor clinical outcome and alterations of p53 might be an important factor in the development of oral cancer

A qualitative and quantitative estimation of AgNORS in dysplastic and non-dysplastic leukoplakias.

Using a silver staining technique, nucleolar organizer region--associated proteins (AgNORS) were studied in formalin, fixed paraffin embedded tissue blocks of normal oral buccal mucosa epithelium, leukoplakia with dysplasias and leukoplakia without dysplasias. Fifty cases, that comprised of 10 cases of normal oral buccal mucosa epithelium and 40 cases, of leukoplakia without dysplasia and with mild, moderate and severe leukoplakia were examined with respect to the relationship between AgNOR counts and histologic grading. The mean AgNOR count per nucleus increased from normal oral buccal mucosa epithelium to leukoplakia without dysplasia to leukoplakia with dysplasia. Higher counts, wider scatter and smaller size of AgNOR in the nuclei were seen as the grading of dysplasia increased from mild to severe. It is suggested that this method has potential in distinguishing between dysplastic and non dysplastic leukoplakias and for early diagnosis, prognosis and treatment planning of dysplastic lesions.

Loss of p53 gene as a biomarker of high risk oral leukoplakias.

We have investigated loss of heterozygosity of p53 tumor suppressor gene in Indian oral cancer patients, individuals with premalignant leukoplakia lesions, and corresponding normal mucosa, to study the status of p53 alleles in oral cancer pathogenesis. Fifty oral cancers, and 42 oral leukoplakia lesions and corresponding clinically normal oral mucosa from 18 individuals, were analysed. Peripheral blood cells (PBCs) from all the individuals and 47 normal healthy volunteers were also included in the study. Polymerase chain reaction(PCR) of p53 Exon4, followed by restriction enzyme digestion with AccII due to the enzyme polymorphic site at Exon4 codon72, was used to detect homozygosity/heterozygosity of p53 alleles, and compared with the allelic pattern in the corresponding PBC. The PCR product subjected to AccII digestion detected 259 bp, 160/99 bp fragments indicating heterozygosity of p53 alleles in 69% of the 139 individuals. On comparison of the p53 allelic distribution in the lesions or tumour tissues, and corresponding PBC, LOH was observed in 20.5% oral tumors and 22% leukoplakias. However, there was no evidence of LOH in the clinically normal mucosa available from 16 individuals with leukoplakia. Our studies demonstrated LOH of p53 allele in early and advanced stages of oral cancers, as well as leukoplakias, perhaps indicating p53 LOH as one of the early events in oral carcinogenesis. Thus, p53 LOH may be useful as a biomarker in defining a certain population of high risk leukoplakias that may progress to oral cancer.