We have established 3 cell lines ORL-48,-115 and-136 from surgically resected specimens obtained from untreated primary human oral squamous cell carcinomas of the oral cavity. The in vitro growth characteristics, epithelial origin, in vitro anchorage independency, human papillomavirus (HPV) infection, microsatellite instability status, karyotype and the status of various cell cycle regulators and gatekeepers of these cell lines were investigated. All 3 cell lines grew as monolayers with doubling times ranging between 26.4 and 40.8 h and were immortal. Karyotyping confirmed that these cell lines were of human origin with multiple random losses and gains of entire chromosomes and regions of chromosomes. Immunohistochemistry staining of cytokeratins confirmed the epithelial origin of these cell lines, and the low degree of anchorage independency expressed by these cell lines suggests non-transformed phenotypes. Genetic analysis identified mutations in the p53 gene in all cell lines and hypermethylation of p16 INK4a in ORL-48 and-136. Analysis of MDM2 and EGFR expression indicated MDM2 overexpression in ORL-48 and EGFR overexpression in ORL-136 in comparison to the protein levels in normal oral keratinocytes. Analysis of the BAT-26 polyadenine repeat sequence and MLH-1 and MSH-2 repair enzymes demonstrated that all 3 cell lines were microsatellite stable. The role of HPV in driving carcinogenesis in these tumours was negated by the absence of HPV. Finally, analysis of the tissues from which these cell lines were derived indicated that the cell lines were genetically representative of the tumours, and, therefore, are useful tools in the understanding of the molecular changes associated with oral cancers.

VPA is a potent inhibitor of proliferation in some HNSCC cell lines, and may be used to treat HNSCC.

Abstract. Inactivation of the retinoblastoma (pRB) pathway is a common event in oral squamous cell carcinoma particularly through the aberrant expression of the components within this pathway. This study examines the alterations of molecules within the pRB pathway by looking at the presence of homozygous deletions in p16INK4A and the expression patterns of pRB, cyclin D1 and CDK4, as well as the presence of human papillomavirus (HPV) in our samples. In our study, 5/20 samples demonstrated deletions of p16 INK4A exon 1α. pRB overexpression was found in 20/20 samples, the expression was mainly observed in all layers of the epithelia, particularly in the basal layer where cells are actively dividing and aberrant pRB expression was found in 12/20 samples. Cyclin D1 and CDK4 overexpression was detected in 6/20 and 2/20 samples respectively in comparison to hyperplasias where both proteins were either not expressed or expressed at minimal levels (<10%). Strikingly, HPV was found to be present in all of our samples, suggesting that HPV plays a significant role in driving oral carcinogenesis. Notably, 17/20 of our samples showed more than one alteration in the pRB pathway, however, we did not find any significant relationship between the presence of HPV, homozygous deletion of p16 INK4A and overexpression of pRB, cyclin D1 and CDK4. Collectively, this data demonstrates that alterations in the pRB pathway are a common event and involve the aberration of more than one molecule within the pathway. Furthermore, the involvement of HPV in all our samples suggests that HPV infection may play an important role in oral carcinogenesis.

Despite the advances in cancer treatment, the 5-year survival rate for oral cancer has not changed significantly for the past 40 years and still remains among the worst of all anatomic sites. Gene expression microarrays have been used successfully in the identification of genetic alterations in cancer development, however, these have hitherto been limited by the need for specimens with good quality intact RNA. Here, we demonstrated the use of formalin-fixed paraffin-embedded tissues in microarray experiments to identify genes differentially expressed between cancerous and normal oral tissues. Forty-three tissue samples were macrodissected and gene expression analyses were conducted using the Illumina DASL assay. We report RNA yield of 2.4 and 0.8 microg/mm(3) from tumour and normal tissues, respectively and this correlated directly with the tissue volume used for RNA extraction. Using unsupervised hierarchical clustering, distinct gene expression profiles for tumour and normal samples could be generated, and differentially expressed genes could be identified. The majority of these genes were involved in regulation of apoptosis and cell cycle, metastasis and cell adhesion including BCL2A1, BIRC5, MMP1, MMP9 and ITGB4. Representative genes were further validated in independent samples suggesting that these genes may be directly associated with oral cancer development. The ability to conduct microarrays on formalin-fixed paraffin-embedded specimens represents a significant advancement that could open up avenues for finding genes that could be used as prognostication and predictive tools for cancer.

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The MDM2 SNP309 has been associated with increased expression of the protein which could suppress p53 function, and has been shown to modulate risk to cancer. We have previously shown that overexpression of MDM2 is a common event in oral cancers. In the present study, we determined the association between the MDM2 SNP309 polymorphism and oral cancer in 207 oral cancer patients and 116 normal subjects. We genotyped the MDM2 SNP309 by PCR-RFLP. Logistic regression was adapted to calculate odds ratios for MDM2 SNP309 polymorphism from univariate and multivariable adjusted models. Our results suggest that MDM2 SNP309 does not confer increased risk to oral cancer (OR=1.55, 95% CI=0.77-3.11). However, the GG/TG genotype was associated with later disease onset in women above 55 years of age. Collectively, our data suggests that MDM2 SNP309 may modulate the risk to oral cancer and is a modifier of the age at oral cancer onset in women above the age of 55 years.

The presence of lymph node metastasis significantly affects the survival of oral squamous cell carcinoma patients and successful detection and removal of positive lymph nodes is crucial in the treatment of this disease. Current evaluation methods still have their limitations in detecting the presence of tumor cells in the lymph nodes, where up to a third of clinically diagnosed metastasis-negative (N0) patients actually have metastasis—positive LNs in the neck. We aimed to develop a molecular signature in the primary tumor that could predict lymph node (LN) metastasis in oral squamous cell carcinoma. The expression levels of 11 proteins was evaluated using immunohistochemical analysis in a tissue microarray (TMA) consisting of 110 specimens from 32 individuals. We used receiver operating characteristic (ROC) curve to identify proteins that could significantly differentiate patients with LN metastasis from those that did not. Unsupervised hierarchical clustering analysis was used to verify the ability of chosen biomarkers in segregating LN positive patients from those with no LN involvement. Kaplan-Meier survival curve and log rank test was utilized to determine the association between LN metastasis and biomarker expression with disease-specific survival. Of the 11 biomarkers, EGFR, HER-2/neu, LAMC2 and RHOC were found to be significantly associated with LN metastasis. Unsupervised hierarchical clustering demonstrated that expression patterns of these 4 proteins could be used to differentiate the positive LN metastasis specimens from specimens that are negative. Collectively, EGFR, HER-2/neu, LAMC2 and RHOC have a specificity of 87.5% and sensitivity of 70% with a prognostic accuracy of 83.4% for LN metastasis. We also demonstrated the LN signature could independently predict disease-specific survival (p = 0.023). In summary, we developed a 4-protein LN signature that could reliably distinguish patients with lymph node metastasis from those who were metastasis free. In addition, this LN signature is also associated with disease-specific survival indicating that would be a useful prognostic tool for the management of oral cancer patients.

Purpose: As a key component of polycomb-repressive complex 2, EZH2 represses target genes through histone methylation and is frequently overexpressed and associated with poor prognosis in common carcinomas. For the first time, we reported EZH2 expression Abstracts / Oral Oncology 47 (2011) S74-S156 S115