Dhananjaya Saranath

Concurrent hypermethylation of multiple regulatory genes in chewing tobacco associated oral squamous cell carcinomas and adjacent normal tissues.

The methylation pattern in the promoter region of p16, DAPK, MGMT and GSTP1 genes was investigated in oral cancer tissues and tumor associated adjacent tissues, using methylation specific PCR assay. The samples constituted 60 primary oral tumors and corresponding adjacent clinically and histopathologically normal mucosa, and buccal epithelial scrapings from 20 normal healthy individuals without any tobacco habits. The incidence of hypermethylation in oral tumor and adjacent mucosa for p16 gene was 66.7 and 50%, for DAPK was 68.3 and 60%, and MGMT gene was 51.7 and 26.7%, respectively. The overall hypermethylation in the three genes in the primary tumor was 86.7%, and corresponding adjacent normal mucosa tissues 76.7%. Hypermethylation was not observed in the promoter region of GSTP1 gene in either the primary tumors or the corresponding adjacent normal mucosa. Absence of aberrant methylation in the four genes was noted in buccal scrapings from normal healthy individuals with no tobacco habits. Thus, a high frequency of promoter region hypermethylation was observed in p16, DAPK and MGMT genes in oral cancer tissues as well as in corresponding adjacent normal mucosa. Our results indicate that epigenetic alteration of these genes is a frequent event in oral cancer, and is an early event observed in normal oral mucosa of the patients, indicating the critical importance of the epigenetic alteration in chewing tobacco associated oral carcinogenesis.

Detection of HPV-16 genome in human oral cancers and potentially malignant lesions from India.

The presence of high risk human papilloma virus (HPV) 16 and 18 was examined in 100 oral cancer patients of Indian descent, 80 patients with potentially malignant oral lesions and corresponding clinically normal mucosa from 48 of these patients. Additionally, presence of HPV-33, -6 and -11 was also studied in 86 oral cancers, 50 potentially malignant oral lesions and 30 corresponding normal oral mucosa. All the patients with oral cancer and oral lesions, were long term tobacco-chewers, and a majority of the patients were in Advanced Stages III and IV. The DNA samples were amplified by polymerase chain reaction (PCR) using HPV L1 consensus primers. Typing of HPV was performed by Southern hybridization analysis of the PCR products using HPV-16, -18, -33, -6 and -11 type specific oligonucleotide probes. HPV-16 was detected in 15 out of 100 (15%) oral tumours, 27 out of 80 (34%) potentially malignant lesions and 15 out of 48 (31%) of the corresponding normal mucosa in the patients with oral lesions. HPV-18 was not detected in any of the oral cancers, oral lesions and normal mucosa. HPV-33 and the low-risk HPV-6 and -11 were also not detected in the oral cancers, oral lesions and corresponding normal mucosa. A significantly higher prevalence of HPV-16 was observed in oral lesions (27 out of 80, 34%) as compared to oral cancers (15 out of 100, 15%). The observed difference of 19% (95% confidence interval [CI]: 6%, 31%), between these two proportions was statistically significant at the 5% level of significance. Our data indicates that HPV-16 may play a direct role in a certain proportion of oral cancers; whereas in a subpopulation of oral cancers HPV-16 infection may be vital in the early events associated with development of potentially malignant oral lesions, and the presence of the virus not essential in the progression of the oral lesion to frank malignancy.

Expression of bcl-2 and bax in chewing tobacco-induced oral cancers and oral lesions from India.

Deregulation of oncogenes and tumor suppressor genes involved in apoptosis has been associated with tumor development and progression. To investigate the involvement of apoptosis regulating proteins in oral cancer in Indian patients, primarily associated with chewing tobacco habits, immunohistochemical expression of bcl-2 and bax was examined in 63 oral squamous cell carcinomas, and 31 putative premalignant lesions. Our studies revealed overexpression of tumor specific cytoplasmic bcl-2 in 56% and bax in 43% oral cancers. The oral cancers in the Indian patients are preceded by premalignant oral lesions; hence oral lesions were examined for bcl-2 and bax expression. We observed aberrant expression of bcl-2 in 16% oral lesions comprising leukoplakias and SMF and bax in 55% oral lesions. We have already reported, p53 expression in these oral cancers and lesions. It was noteworthy that 30% oral cancers demonstrated a p53+bcl2+ pattern, and 14% samples exhibited p53+bcl2+bax+ pattern. However, none of the oral lesions showed concurrent deregulation of p53 and bcl-2 or all the three genes. Interestingly 45% oral lesions were p53-bax+ as compared to 18% oral cancers; while 39% oral lesions were bcl2-bax+ as compared to 14% oral cancers, indicating overexpression of bax in oral lesions, in the absence of p53 and bcl-2 proteins. Significant correlation was observed between positive nodal status and bcl2+ (p=0.047) and p53+bcl-2+ (p=0.01) in oral cancers. Kaplan Meier survival analysis showed significantly (p=0.059) higher survival in patients with p53-oral tumors than with p53+ tumors. Our studies thus indicate frequent overexpression of apoptosis regulators bcl-2, bax and p53 proteins in oral cancers, and a subset of oral lesions, representing early events in oral carcinogenesis. The aberrant bcl-2 expression and loss of p53 function observed, may play an important role in the tumorigenesis of oral cancers by allowing escape from apoptosis and enabling additional genetic alterations to accrue.

Genome-wide disease association study in chewing tobacco associated oral cancers

With a view to identify genomic risk variants in chewing-tobacco associated oral cancer patients, a genome-wide association study was conducted in patients of Indian ethnicity with long term tobacco chewing habit. We analyzed 55 oral cancer patients and 92 healthy controls for single nucleotide polymorphisms, using high throughput microarray Illumina Infinium II Assay platform and Human CNV370k-bead chip containing 370,000 single nucleotide polymorphisms. The PLINK software platform defined 298 SNPs with minor allele frequency of several genes significantly increased in oral cancer patients as compared to the controls (p<0.001). Illumina Genome Viewer Software Version 3.2.9, further delineated 93 SNPs with p-values ranging from 9.3×10(-4) to 1.38×10(-5) and Odds ratio of 2.18-8.48, associated with 70 genes. Analysis using Kyoto Encyclopedia of Genes and Genome Pathway database, indicated SNP association with several genes including GRIK2, RASGRP3, CAMK4, SYK, RAPTOR, FHIT, DCC, active in signal transduction; MMP2, CNTNAP2, PTPRJ associated with tumor cell migration; and apoptotic gene IRAK3. The data indicates an inherent role for the genetic constitution of individuals in oral carcinogenesis, with the genomic variants contributing to increased risk or susceptibility to oral cancer.

Clinical implications of epigenetic regulation in oral cancer

Oral cancer is a high incidence cancer which is of major public health concern in India being the most common cancer in males and fifth most common cancer in females in India, contributing to 26% of the global oral cancer burden. The major risk factors of oral cancer are tobacco, alcohol and high risk Human Papilloma Virus type 16/18. However, only 3-12% of the high risk individuals with dysplasia develop oral cancer. Thus, individual genomic variants representing the genomic constitution and epigenetic alterations play a critical role in the development of oral cancer. Extensive epigenetic studies on the molecular lesions including oncogenes, tumor suppressor genes, genes associated with apoptosis, DNA damage repair have been reported. The current review highlights epigenetic regulation with a focus on molecular biomarkers and epidrug therapy in oral cancer. Epigenetic regulation by hypermethylation, histone modifications and specific microRNAs are often associated with early events and advanced stages in oral cancer, and thus indicate epidrug therapy for intervention. The presence of epigenetic marks in oral lesions, cancers and tumor associated mucosa emphasizes indications as biomarkers and epidrugs with therapeutic potential for better patient management.

Multiple single nucleotide polymorphism analysis and association of specific genotypes in FHIT, SAMD4A, and ANKRD17 in Indian patients with oral cancer

Oral cancer has a high incidence primarily because of tobacco chewing habits. However, a small proportion of habitués develop oral cancer, implying a role for genomic variants in its susceptibility.

Molecular pathology of oral cancer: Clinical implications

Purpose: The reconstruction of the ear following resection of part of it especially the helix, scapha and anti-helix is a confronting problem for plastic surgery. Many techniques had been described to approach this issue and to minimize the complications raised from resection of tumors as well. We present our experience with ear reconstruction using chondrocutaneous flaps and a modified Antia-Buch technique in order to obtain a more realistic result with least complications.

Single nucleotide polymorphisms in an Indian cohort and association of CNTN4, MMP2 and SNTB1 variants with oral cancer

Oral cancer is a high incidence cancer in India primarily due to the prevalent tobacco/areca nut chewing habits and hence a major health concern. India constitutes 26% of the global oral cancer burden. Besides the well-established risk factors, the genomic constitution of an individual plays a role in oral cancer. The aim of the current study was to analyse genomic variants represented as single nucleotide polymorphisms (SNPs), analyse their prevalence and investigate risk association of allelotypes/genotypes to oral cancers. Eleven SNPs in genes associated with biological functions were analysed in an Indian cohort (n = 1000) comprising 500 oral cancer patients and 500 long term tobacco habitués as controls, using Allelic discrimination Real-Time PCR assay with SYBR Green dye. Fishers exact test and Odds Ratio were used for statistical analysis. Increased risk was observed for rs9849237 CC [P = 0.008; OR 1.412 (1.09-1.82)] and rs243865 CT [P = 0.004; OR 1.469 (1.13-1.90)] genotypes, whereas rs9849237 CT [P = 0.034; OR 0.755 (0.58-0.97)], rs243865 CC [P = 0.002; OR 0.669 (0.51-0.86)] and rs10090787 CC [P = 0.049; OR 0.774 (0.60-0.99)] genotypes indicated decreased risk to oral cancer. The other SNPs showed equidistribution in both groups. Our data indicated genotypes and alleles in specific SNPs rs9849237, rs243865 and rs10090787 with increased/decreased risk to oral cancer.