Indian Oral Squamous Cell Carcinoma Research Articles

Multi-regional genomic and transcriptomic characterization of a melanoma-associated oral cavity cancer provide evidence for CASP8 alteration-mediated field cancerization

BackgroundPrecancerous and malignant tumours arise within the oral cavity from a predisposed “field” of epithelial cells upon exposure to carcinogenic stimulus. This phenomenon is known as “Field Cancerization”. The molecular genomic and transcriptomic alterations that lead to field cancerization and tumour progression is unknown in Indian Oral squamous cell carcinoma (OSCC) patients.MethodsWe have performed whole exome sequencing, copy-number variation array and whole transcriptome sequencing from five tumours and dysplastic lesions (sampled from distinct anatomical subsites - one each from buccal anterior and posterior alveolus, dorsum of tongue–mucosal melanoma, lip and left buccal mucosa) and blood from a rare OSCC patient with field cancerization.ResultsA missense CASP8 gene mutation (p.S375F) was observed to be the initiating event in oral tumour field development. APOBEC mutation signatures, arm-level copy number alterations, depletion of CD8 + T cells and activated NK cells and enrichment of pro-inflammatory mast cells were features of early-originating tumours. Pharmacological inhibition of CASP8 protein in a CASP8-wild type OSCC cell line showed enhanced levels of cellular migration and viability.ConclusionCASP8 alterations are the earliest driving events in oral field carcinogenesis, whereas additional somatic mutational, copy number and transcriptomic alterations ultimately lead to OSCC tumour formation and progression.

Gene expression analysis of microRNA-1285 in the South Indian oral squamous cell carcinoma population.

Oral squamous cell carcinoma (OSCC) is the most common oral malignant tumor, which has poor prognosis. The traditional investigative modality is invasive biopsy which is the gold standard for diagnosis. In recent years, alternative methods like non-invasive biomarkers have been studied for their potential role in early diagnosis and prognosis. Among them, microRNAs (miRNAs or miRs) are short non-coding RNAs that regulate gene expression in various diseases, including OSCC. Several miRNAs are being researched as non-invasive biomarkers as well as novel therapeutic targets in the treatment of OSCC. MiR expression can be upregulated or downregulated in OSCC. Among the reported miRNAs, miR-1285 is an important miRNA found to be involved in OSCC. The aim of the current study was to quantify the levels of miR-1285 in OSCC samples and to validate their potential role as biomarkers for OSCC detection. Sixteen samples of cancer tissue and normal tissue were evaluated from a total of 25 patients, in the study, conducted in the Department of Oral and Maxillofacial Surgery. The tissues were processed for H&E staining and gene expression analysis of miR-1285. The samples were collected after proper informed consent from the patients. Total RNA isolated was reverse transcribed into cDNA which was used in the gene expression analysis using qRT-PCR. The histopathological examination confirmed the OSCC cases and the gene expression analysis revealed that miR-1285 was significantly downregulated in OSCC tissues. Since miR-1285 showed significant difference between the OSCC and normal tissues it could be postulated as a potential biomarker and therapeutic target for OSCC. Further in-vitro and in-vivo studies could validate their functional role in OSCC.

MicroRNA-34a-3p and its target tumor necrosis factor-α in the regulation of South Indian oral squamous cell carcinoma population.

The most frequent head and neck cancer is oral squamous cell carcinoma (OSCC), the common histological cancer of the oral cavity and is one of the most prevalent forms of cancer globally. It has been known that there are several biomarkers and therapeutic targets that have been discovered for OSCC, but none of them were effective against the progression of OSCC. Interestingly, small non-coding RNAs termed microRNAs (miRNAs) regulate cellular activity by targeting numerous signaling pathways or genes that either promote or repress the progression of diseases. Surprisingly, among the differentially expressed miRNAs, miR-34a was identified to be highly sensitive and specific to OSCC and widely studied for its role in various cancers, including OSCC. The secondary structure of miR-34a-3p was analyzed using bioinformatic analysis and its targets were screened using the TargetScan database. Specimens of 25 OSCC cancer tissues and adjacent normal tissues were collected from the Department of Oral and Maxillofacial Surgery, Saveetha Dental College and Hospitals. The tissues were processed for H&E staining and gene expression analysis of miR-34a-3p and tumor necrosis factor alpha (TNF-α). The minimum free energy for miR-34a-3p was found to be -47.20 kCal which proved the stability of the miRNA. The histopathological examination confirmed the OSCC cases and the gene expression analysis revealed that miR-34a-3p was significantly downregulated in OSCC tissues, whereas TNF-α showed vice versa expression. miR-34a-3p could be postulated as a potential therapeutic target for OSCC.

Label-free plasma proteomics for the identification of the putative biomarkers of oral squamous cell carcinoma

Mass spectrometry-based label-free proteomics is becoming the analytical tool of interest to identify and quantitate the biomarkers for cancer. The oral squamous cell carcinoma (OSCC) which is one of the leading cancers worldwide, lacks biomarkers for the early prognosis and diagnosis. The present study profiled plasma proteome of the Indian OSCC human patients using a label-free mass spectrometry proteomics approach. The study first time utilized the three most widely used data analysis software MaxQuant (MQ), Proteome discoverer (PD), and Trans proteomic pipeline (TPP) together for label-free quantitation of the proteins. The study identified 16 proteins which can be used as a signature protein panel for OSCC. The pathway analysis showed predominant involvement of the immune system, hemostasis as the major pathways that are indicative of the disease progression. The network analysis showed maximum interaction for the Fibronectin and C-reactive protein. The study demonstrates that plasma proteins contain signatures that can be used as putative biomarkers for OSCC. Based on the label-free quantitation and the mechanistic analysis C-reactive protein, Carbonic anhydrase-1, and Fibronectin are identified as putative biomarkers of OSCC. Once these findings are validated in the large cohorts these protein signatures can be used as a biomarker for OSCC. SignificanceThe oral squamous cell carcinoma (OSCC) is the eighteenth most prevalent malignancy in the world and ranks second in India. There are no biomarkers that could be indicative of the diseased state. Various studies have been carried out on saliva and tumors of OSCC patients in India, but none of the studies have profiled the plasma. We utilized the label-free approach for the first time on the Indian population in generating the panel of plasma proteins which could be indicative of the diseased state. The study identified Carbonic anhydrase 1, C-reactive protein, and Fibronectin proteins as putative biomarkers for the OSCC. The study obtained the signature panel by utilizing the 3 most widely used software for the label-free quanatitation (LFQ) namely MaxQuant, Proteome Discoverer, and Trans proteomic pipeline. The utilization of 3 software for the LFQ reduced the bias and provided a comprehensive list of proteins. All the differential proteins were mechanistically studied for their biological relevance and the pathway and network analysis were carried out. The study helps us in increasing the understanding of the proteins which are involved in the progression of the diseases. Studying the panel of proteins from all biofluids along with plasma in large cohorts of the population will help in understanding the disease in greater depth and help in identifying the relevant biomarkers for the OSCC.

Metabolomic Profiling of an Indian Oral Squamous Cell Carcinoma Subset

Oral cancer (OC) is the sixth most common global malignancy and blazing dilemma all over India. Tobacco and alcohol consumption are the well-established risk factors for the onset of oral cancer. Gujarat is considered to be the high-risk region for oral cancer as it is the tobacco growing belt. Volatile organic compounds (VOCs) have shown a strong correlation with clinicopathological characteristics (CPC) of OC patients. The present study aims to investigate VOCs as potential biomarkers for developing OSCC. Metabolomic profile of 20 OSCC patients (n=20), 10 Healthy individuals (n=10) and 9 Tobacco consumers (n=9) were analysed using Gas chromatography-Mass spectrometry. Principle component analysis (PCA) and partial least squares structure-discriminant analysis (PLS-DA) model has been assembled to compare the metabolomic profile of each group, where highly expressed metabolites were identified for different groups. We observed ten VOCs from oral cancer patients, where propionic acid is found as potential biomarkers end up with vitamin K and propionate metabolism. One-way ANOVA was performed to compare the mean difference between each group and the level of nine metabolites differed significantly in controls vs. oral cancer patients. The present study contributes to the design protocol for metabolomics-based biomarker discovery. Future research requires a more polishing touch to develop new vistas for the better prognosis of cancer patients.

Establishment and characterization of novel human oral squamous cell carcinoma cell lines from advanced‑stage tumors of buccal mucosa.

Oral squamous cell carcinoma (OSCC) is a leading cause of mortality in India owing to the high percentage of tobacco chewers, smokers and alcohol consumption. OSCC is highly heterogeneous in nature; therefore poses a challenge in the treatment of the patient. To better understand the heterogeneity of the tumors, an in vitro cell line model is required. However, the efficiency of establishing cell lines from the oral tumors is low. In the present study, three novel cell lines, namely ACOSC3, ACOSC4, and ACOSC16, were isolated and characterized from advanced‑stage treatment‑naive OSCCs originating from the buccal mucosa. The three cell lines exhibited polygonal morphology, which is typical of epithelial cells. Furthermore, immunofluorescence revealed the expression of keratins 8 and 14, thereby confirming the epithelial origin of the cells. DNA content analysis of the three OSCC cell lines revealed aneuploidy. Furthermore, an in vitro orosphere assay revealed the formation of primary orospheres. Notably, the OSCC cell lines were able to give rise to tumors when administered subcutaneously into non‑obese diabetic/severe combined immune deficiency mice. The novelty of the cell lines was also validated by performing short tandem repeat profiling; the STR profiles of the present cell lines did not significantly match with any known established OSCC cell lines present in the DSMZ database, thereby confirming the unique identity of these lines. These cell lines established from tumor samples derived from Indian OSCC patients provide a valuable resource to understand the molecular mechanism involved in tumor resistance and recurrence.

Abstract 3875: Proteome-wide approach to identify novel biomarkers in oral tumorigenesis

Abstract Oral cancer is the most common type of intraoral head and neck cancer in humans, with a worldwide prevalence of more than 400,000 cases. In India, oral cancer ranks in top three in comparison with other cancer types. The majority of oral cancer patients are diagnosed at an advanced disease stage. Although, there have been many advances in oral cancer treatment, the overall survival rate of oral cancer patients only improved marginally over the past 30 years. The treatment regimen of oral cancer is mainly based on the tumor, node and metastasis (TNM) classification and histopathological diagnosis. These methods are subjective and often have low sensitivity to detect the disease in early stages. Hence, there is an urgent need for early diagnosis to develop biomarkers; to identify high risk individuals, to improve cancer detection at early stages, to predict disease outcome and response to therapy. Proteomics have been successfully employed in studies of lung, breast, prostate, gastrointestinal cancer, and head and neck squamous cell carcinoma. Reports are not much available to describe well characterized biomarkers which will aid for early detection of oral cancer. Racial differences were reported in many cancers including its morbidity, mortality, treatment response and survival rates. In Indian patient population, there is a significant knowledge gap exists in utilizing proteomics for biomarker discovery. The aim of this study is to identify novel candidate genes which may be used as prognostic or diagnostic biomarkers for Indian oral squamous cell carcinoma. The tongue tumor and adjacent normal samples were subjected to 2D - Differential in Gel Electrophoresis (2D-DIGE). Differentially expressed proteins in tumor samples were identified using nano-LC-MS/MS and validated by quantitative real time PCR. We found 800 protein spots were differentially expressed and seventy proteins were identified by mass spectrometry. Among them twelve proteins including Myosin isoforms, hemoglobin beta, Annexin A1, Creatinine kinase, Carbonic anhydrase, HSPA8 showed decreased expression while eighteen proteins including Apolipoprotein A1, HSPA5, Alpha 1 antitrypsin, Annexin isoforms, Peptidyl prolyl cis trans isomerase, Serpin isoforms, Tropomyosin, Thioredoxin, Alpha enolase, Calpain, Glutathione S Transferase, Heat shock protein beta 1, S100 isoforms showed increased expression in tongue tumor tissues. Most of the differentially regulated proteins were known to be involved in calcium homeostasis, cell cycle, cell growth and migration. The major outcome of this study was the identification of novel protein molecules which might be used as predictive, prognostic and diagnostic biomarkers for oral cancer. These proteins can also serve as a therapeutic intervention targets for oral cancer progression. This study would be the first study to map the whole proteome profile of the cancerous as well as normal tongue cheek tissue in Indian scenario. Citation Format: Sivagnanam Ananthi, Naga Padma Lakshmi CH, Anbarasu K, Mahalingam Sundarasamy. Proteome-wide approach to identify novel biomarkers in oral tumorigenesis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3875.

Genetic profile of PTEN gene in Indian oral squamous cell carcinoma primary tumors.

Phosphatase and tensin homolog (PTEN) is the tumor suppressor gene located on chromosome10q23.3. Genetic variations in the PTEN gene have been found in various sporadic tumors. However, petite is known about the genetic profile of the PTEN gene in oral squamous cell carcinoma (OSCC), which is the eighth most common neoplasm worldwide and leading cancer in India. The purpose of the present study was to determine the frequency of genetic variations in the tyrosine phosphatase domain of the PTEN gene in an Indian OSCC subset. We analyzed tyrosine phosphatase domain encoded by exon 5 of the PTEN gene in 59 OSCC primary tumors using PCR - direct genomic sequencing. We observed one somatic deletion mutation, IVS4-30delT in three OSCC patients; two of them were at an advanced stage of carcinoma. Moreover, we identified one SNP rs 35560700(C>T), in five OSCC patients with the late stage of oral carcinoma. We identified 5% somatic mutational frequency in the intronic region of the tyrosine phosphatase domain of the PTEN gene; however, mutations were found absent in the coding region. Therefore, PTEN gene mutation is not a frequent event in the pathogenesis of OSCC in the targeted Indian cohort.

Interferon Induced Transmembrane Protein-1 Gene Expression as a Biomarker for Early Detection of Invasive Potential of Oral Squamous Cell Carcinomas.

Early detection of malignant transformation with expression biomarkers has significant potential to improve the survival rate of patients as such biomarkers enable prediction of progression and assess sensitivity to chemotherapy. The expression of interferon inducible transmembrane protein 1 (IFITM1) has been associated with early invasion events in several carcinomas, including head and neck cancers, and hence has been proposed as a novel candidate biomarker. As the incidence of oral squamous cell carcinoma (OSCC) is highest in the Indian population, we sought to investigate: 1) the expression pattern of IFITM1 in OSCC tissue samples obtained from Indian patients of Dravidian origin; and 2) the possibility of using IFITM1 expression as a potential biomarker. Total RNA extracted from thirty eight OSCC biopsy samples was subjected to semi-quantitative RT-PCR with IFITM1 and GAPDH specific primers. Of the thirty eight OSCC samples that were analyzed, IFITM1 overexpression was identified in fifteen (39%). Seven expressed a low level, while the remainder expressed high level of IFITM1. The overexpression of IFITM1 in OSCC samples indicates that IFITM1 may be explored for the possibility of use as a high confidence diagnostic biomarker in oral cancers. To the best of our knowledge, this is the first time that IFITM1 overexpression is being reported in Indian OSCC samples.

Prediction of recurrence-free survival using a protein expression-based risk classifier for head and neck cancer

Loco-regional recurrence in 50% of oral squamous cell carcinoma (OSCC) patients poses major challenge for oncologists. Lack of biomarkers that can predict disease aggressiveness and recurrence risk makes the scenario more dismal. On the basis of our earlier global proteomic analyses we identified five differentially expressed proteins in OSCC. This study aimed to develop protein biomarkers-based prognostic risk prediction model for OSCC. Sub-cellular expression of five proteins, S100A7, heterogeneous nuclear ribonucleoproteinK (hnRNPK), prothymosin α (PTMA), 14-3-3ζ and 14-3-3σ was analyzed by immunohistochemistry in test set (282 Indian OSCCs and 209 normal tissues), correlated with clinic–pathological parameters and clinical outcome over 12 years to develop a risk model for prediction of recurrence-free survival. This risk classifier was externally validated in 135 Canadian OSCC and 96 normal tissues. Biomarker signature score based on PTMA, S100A7 and hnRNPK was associated with recurrence free survival of OSCC patients (hazard ratio=1.11; 95% confidence interval 1.08, 1.13, P<0.001, optimism-corrected c-statistic=0.69) independent of clinical parameters. Biomarker signature score stratified OSCC patients into high- and low-risk groups with significant difference for disease recurrence. The high-risk group had median survival 14 months, and 3-year survival rate of 30%, whereas low-risk group survival probability did not reach 50%, and had 3-year survival rate of 71%. As a powerful predictor of 3-year recurrence-free survival in OSCC patients, the newly developed biomarkers panel risk classifier will facilitate patient counseling for personalized treatment.

Prognostic significance of cytoplasmic S100A2 overexpression in oral cancer patients.

BackgroundOral squamous cell carcinoma (OSCC) patients are at high risk of loco-regional recurrence and 5-year survival rates are about 50%. Identification of patients at high risk of recurrence will enable rigorous personalized post-treatment management. Most novel biomarkers have failed translation for clinical use because of their limited successful validation in external patient cohorts. The aim of this study was to determine the prognostic significance of alterations in sub-cellular expression of S100A2, a pro-tumorigenic calcium binding protein, identified as a candidate biomarker in our proteomic analysis in OSCC and validation of its clinical utility in an external cohort.MethodsIn a retrospective study, immunohistochemical analysis of S100A2 was carried out in 235 Indian OSCC (Test set) and 129 normal oral tissues, correlated with clinicopathological parameters and disease outcome over 122 months for OSCC patients following the REMARK criteria. The findings were validated in an external cohort (Validation set 115 Canadian OSCC and 51 normal tissues) and data analyzed using the R package.ResultsSignificant increase in cytoplasmic and decrease in nuclear S100A2 expression was observed in OSCC in comparison with normal tissues. Cox multivariable regression analysis internally and externally validated cytoplasmic S100A2 association with tumor recurrence. Kaplan Meier analysis of patients stratified to high and low risk groups showed significantly different recurrence free survival (Test set- log rank test, p = 0.005, median survival 16 and 69 months respectively and Validation set - p < 0.00001, median survival 9.4 and 59.9 months respectively); 86% and 81% of patients who had recurrence were correctly stratified into the high risk group. Seventy percent and 81% patients stratified into low risk group did not show cancer recurrence within 1 year in Test and Validation sets.ConclusionsOur study provided clinical evidence for the potential of cytoplasmic S100A2 overexpression as a predictor of recurrence risk in OSCC patients. A unique translational aspect of our study is validation of S100A2 as prognostic marker in two independent cohorts (Canadian and Indian) suggesting this protein is likely to find widespread utility in clinical practice for identifying oral cancer patients at high risk of disease recurrence.

Lack of mutations in protein tyrosine kinase domain coding exons 19 and 21 of the EGFR gene in oral squamous cell carcinomas.

The epidermal growth factor receptor (EGFR) plays a vital role in the activation and inactivation of receptor tyrosine kinases. Mutations in exons 19 and 21 of EGFR are commonly found to be associated with non small cell lung carcinoma and triple negative breast cancer, enhancing sensitivity to EGFR targeting chemotherapeutic agents. Since amplification and prolonged activation of EGFR molecules have been identified in oral squamous cell carcinomas (OSCC), we investigated whether OSCCs carried mutations in exons 19 and 21 of EGFR to their incidence. Tumor chromosomal DNA isolated from forty surgically excised oral squamous cell carcinoma tissues was subjected to PCR amplification with intronic primers flanking exons 19 and 21 of the EGFR gene. The PCR amplicons were subsequently subjected to direct sequencing to elucidate the mutation status. Data analysis of the EGFR exon 19 and 21 coding sequences did not show any mutations in the forty OSCC samples that were analyzed. To the best of our knowledge, this is the first study to have investigated the genetic status of exons 19 and 21 of EGFR in Indian OSCCs and identified that mutation in EGFR exon 19 and 21 may not contribute towards their genesis. The absence of mutations also indicates that oral cancerous lesions may not be as sensitive as other cancers to chemotherapeutic agents targeting EGFR.

Clinico-pathological correlation of E-cadherin expression at the invasive tumor front of Indian oral squamous cell carcinomas: An immunohistochemical study.

Background:Recent studies have indicated that although malignant cells at the invasive tumor front, bare morphological resemblance to the cells at central portion of the tumor, their molecular character differs significantly. E-cadherin is a cell-cell adhesion molecule that connects epithelial cells. This study attempts to correlate the E-cadherin expression at the invasive tumor front with tumor differentiation along with its clinico-pathological parameters.Materials and Methods:Immunohistochemical staining with E-cadherin was carried out on archival cases of primary oral squamous cell carcinomas (n = 30). The E-cadherin expression at the invasive tumor front was analyzed and was linked to clinico-pathological parameters including patient prognosis.Results:The downregulation of E-cadherin expression at the invasive tumor edge when compared with patient's prognosis yielded a significant correlation (P = 0.041) but its correlation with the degree of differentiation determined was not significant (P = 0.27). Also, its association with tumor size and lymph node status was negative.Conclusions:Loss of E-cadherin expression at the invasive tumor front is an important event in the progression of oral squamous cell carcinomas. Tumors with a loss of expression of E-cadherin are those which had a poor prognosis

Aberrant Promoter Methylation of PTEN Gene among Indian Patients with Oral Squamous Cell Carcinoma

Oral cancer is the second most common cancer in men and accounts for 50%-70% of the total cancer mortality in India. PTEN is a tumor suppressor gene that plays a critical role in controlling cell growth and survival. Promoter hypermethylation of the PTEN gene has been reported in many tumors. However, little is known about the association between promoter methylation and oral squamous cell carcinoma (OSCC). Therefore, we aimed to detect the role of PTEN hypermethylation in OSCC patients in the Indian population. Genomic DNA was isolated from 100 fresh oral tumor specimens and was subjected to bisulfite conversion. Methylation-specific PCR was employed on the converted DNA to investigate the methylation status. Of the total cases examined for PTEN promoter methylation we found that 35% were positive and 65% were negative. When evaluated in connection with tumor differentiation it was found that 82% of poorly differentiated, 24% of moderately differentiated and 32% of well differentiated OSCC samples were methylated. Methylation was further correlated with patient age, sex and tumor grade. Interestingly, we found that patient age and grade of differentiation were significantly associated with PTEN promoter methylation (p=0.05 and 0.0019, respectively) while sex was not (p=0.9). The present study underlines the importance of PTEN hypermethylation among Indian OSCC patients.

PTEN-mediated AKT activation contributes to the reduced apoptosis among Indian oral squamous cell carcinoma patients

The tumor suppressor gene PTEN negatively regulates Akt, a downstream mediator phosphoinositol 3-kinase. Several studies have reported the role of PTEN gene in Akt downregulation and apoptosis induction in different cancers and cell lines. However, the role of loss of PTEN expression in Akt activation and spontaneous apoptosis in oral squamous cell carcinoma clinical specimens is not well established. We investigated the expression of PTEN and phospho-Akt in 146 formalin-fixed (archived) paraffin-embedded oral squamous cell carcinoma tissue sections through immunohistochemical analysis. Programmed cell death (apoptosis) was determined by Terminal deoxynucleotidyl Transferase Biotin-dUTP Nick End Labeling assay. Sixty-one percent loss of PTEN expression and 68.5% Akt activation was observed in oral squamous cell carcinoma. A significant correlation was found between loss of PTEN expression and Akt activation. Loss of PTEN expression and Akt activation were further correlated with different clinical parameters and found to be significantly correlated with tumor stage. Apoptotic index was estimated and correlated with PTEN expression and Akt activation. The percentage of apoptotic cells varied from 0.2 to 14.1%. Low apoptotic index was observed in 105 (72%) of samples, and it was found to be significantly related with loss of PTEN expression and phospho-Akt The present study confirms the contribution of loss of PTEN expression in Akt phosphorylation and spontaneous apoptosis suppression in the specimens of oral cancer. Both PTEN and phospho-Akt are likely to be concerned with oral cancer progression and reduced incidence of spontaneous apoptosis.

Promoter hypermethylation in Indian primary oral squamous cell carcinoma

We evaluated promoter hypermethylation of a panel of tumor suppressor genes as a means to detect epigenetic alterations in oral squamous cell carcinomas (OSCC) of Indian-origin and compare with North-American head and neck squamous cell carcinomas (HNSCC). Quantitative-methylation-specific PCR was used to investigate the promoter methylation status of DCC, EDNRB, p16(INK4a) and KIF1A in 92 OSCC, and compared to 48 paired normal tissues and 30 saliva and sera samples from healthy control subjects. Aberrant methylation of at-least one of these genes was detected in 74/92 (80.4%) OSCC; 72.8% at EDNRB, 71.7% at KIF1A, 47.8% at p16(INK4a) and 58.7% at DCC; and in 5 of 48 (10.4%) normal oral tissues. None of the saliva and sera samples from controls exhibited DNA methylation in these four target genes. Thirty-two of 72 node positive cases harbored p16(INK4a) and DCC hypermethylation (p = 0.005). Thus, promoter hypermethylation in genes analyzed herein is a common event in Indian OSCC and may represent promising markers for the molecular staging of OSCC patients. We found higher frequency of p16(INK4a) methylation (47.8%) in this Indian cohort in comparison with a North-American cohort (37.5%). In conclusion, aberrant methylation of EDNRB, KIF1A, DCC and p16(INK4a) genes is a common event in Indian OSCC, suggesting that epigenetic alterations of these genes warrant validation in larger studies for their potential use as biomarkers.

Loss of Heterozygosity (LOH) on Chromosomes 2q, 3p and 21q in Indian Oral Squamous Cell Carcinoma

Around the world, 200,000 people a year are affected by oral cancer, and the incidence of this disease is 10 times higher in India than Japan, mainly due to the custom of chewing tobacco. Loss of heterozygosity (LOH) on the long arm of chromosome 2 (2q), the short arm of chromosome 3 (3p) and the long arm of chromosome 21 (21q) are observed in several human cancers. We identified novel tumor suppressor loci on these regions in primary oral squamous cell carcinomas (OSCCs) in Japanese. However, there has been no detailed analysis of LOH on these chromosomes in Indians. In the present study, we investigated LOH at 2q, 3p and 21q using 9 microsatellite markers in 26 Indian OSCCs. LOH was detected in 25 (96.2%) out of 26 informative samples at one or more of the loci examined. On the basis of the results, two commonly deleted regions were identified and a detailed deletion map was constructed. In the first region, a high frequency of LOH was observed at the D3S1007 locus (53.8%) on 3p25, which is located in the region neighboring the VHL (von Hippel-Lindau) gene. In the second region, LOH was concentrated at the D3S966 locus (50.0%) on 3p21.3, suggesting the presence of a putative tumor suppressor gene (TSG) associated with OSCCs. These results strongly suggest that there are at least two candidate TSGs located on chromosome 3p, and that alteration in them is associated with the tumorigenesis of OSCCs.

Mutational analysis of the candidate tumor suppressor gene ING1 in Indian oral squamous cell carcinoma.

ING1, a recently identified candidate tumor suppressor gene, involved in the p53 signaling pathway is mapped at chromosome 13q34. Since loss of heterozygosity at 13q34 has been reported in squamous cell carcinoma of head and neck, we screened for mutations in ING1 by polymerase chain reaction-single strand conformation polymorphism in 71 oral squamous cell carcinomas (OSCC) from India, 15 of which were known to harbor p53 mutations. A single polymorphism (G to A) was detected in 14 (19.7%) of the tumors analyzed. No mutation was observed in any of the 71 OSCCs analyzed. These results suggest that ING1 is not a target for mutational inactivation in OSCC of Indians.

The p16INK4alpha/p19ARF gene mutations are infrequent and are mutually exclusive to p53 mutations in Indian oral squamous cell carcinomas.

Eighty-seven untreated primary oral squamous cell carcinomas (SCCs) associated with betel quid and tobacco chewing from Indian patients were analysed for the presence of mutations in the commonly shared exon 2 of p16INK4alpha/p19ARF genes. Polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) and sequencing analysis were used to detect mutations. SSCP analysis indicated that only 9% (8/87) of the tumours had mutation in p16INK4alpha/p19ARF genes. Seventy-two tumours studied here were previously analysed for p53 mutations and 21% (15/72) of them were found to have mutations in p53 gene. Only one tumour was found to have mutation at both p53 and p16INK4alpha/p19ARF genes. Thus, the mutation rates observed were 21% for p53, 9% for p16INK4alpha/p19ARF, and 1% for both. Sequencing analysis revealed two types of mutations; i) G to C (GCAG to CCAG) transversion type mutation at intron 1-exon 2 splice junction and ii) another C to T transition type mutation resulting in CGA to TGA changing arginine to a termination codon at p16INK4alpha gene codon 80 and the same mutation will alter codon 94 of p19ARF gene from CCG to CTG (proline to leucine). These results suggest that p16INK4alpha/p19ARF mutations are less frequent than p53 mutations in Indian oral SCCs. The p53 and p16INK4alpha/p19ARF mutational events are independent and are mutually exclusive suggesting that mutational inactivation of either p53 or p16INK4alpha/p19ARF may alleviate the need for the inactivation of the other gene.

Expression of bcl-2 oncoprotein in Indian oral squamous cell carcinomas.

Thirty-nine oral squamous cell carcinomas were assessed for bcl-2 protein expression by immunostaining of tumour sections. Twenty-three per cent of these tumours showed strong nuclear staining for bcl-2 protein. Tumours of the cheek and tongue together accounted for 77% of overexpression of this protein. When bcl-2 expression was compared with p53 expression, they were found to be non-overlapping. These results suggest that overexpression of either of these genes may substitute each other in the development of oral carcinomas of Indians.