Amyza Saleh

Gene expression in human oral squamous cell carcinoma is influenced by risk factor exposure.

Oral squamous cell carcinoma (OSCC) is a world health problem and is associated with exposure to different risk factors. In the west, smoking and alcohol consumption are considered to be the main risk factors whilst in India and southeast Asia, betel quid (BQ) chewing is predominant. In this study, we compared the gene expression patterns of oral cancers associated with BQ chewing to those caused by smoking using Affymetrix microarrays. We found that 281 genes were differentially expressed between OSCC and normal oral mucosa regardless of aetiological factors including MMP1, PLAU, MAGE-D4, GNA12, IFITM3 and NMU. Further, we identified 168 genes that were differentially expressed between the BQ and smoking groups including CXCL-9, TMPRSS2, CA12 and RNF24. The expression of these genes was validated using qPCR using independent tissue samples. The results demonstrate that whilst common genes/pathways contribute to the development of oral cancer, there are also other gene expression changes that are specific to certain risk factors. The findings suggest that different carcinogens activate or inhibit specific pathways during cancer development and progression. These unique gene expression profiles should be taken into consideration when developing biomarkers for future use in prognostic or therapeutic applications.

The ATM tumour suppressor gene is down-regulated in EBV-associated nasopharyngeal carcinoma

A micro‐array analysis using biopsies from patients with EBV‐positive undifferentiated nasopharyngeal carcinoma (NPC) and from cancer‐free controls revealed down‐regulation of tumour suppressor genes (TSG) not previously associated with this disease; one such gene was the ataxia telangiectasia mutated (ATM) gene. Q‐PCR confirmed down‐regulation of ATM mRNA and ATM protein expression in tumour cells was weak or absent in almost all cases. In NPC cell lines, however, ATM was down‐regulated only in the EBV‐positive line, C666.1, and in none of five EBV‐negative lines. In vitro infection of EBV‐negative NPC cell lines with a recombinant EBV was followed by the down‐regulation of ATM mRNA and protein, and only EBV‐positive cells showed a defective DNA damage response following γ‐irradiation. Our data suggest that loss of ATM function could be an important step in the pathogenesis of NPC, and may have implications for the treatment of this disease. Copyright

Transcriptional profiling of oral squamous cell carcinoma using formalin-fixed paraffin-embedded samples.

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.

Promoting oral cancer awareness and early detection using a mass media approach

BACKGROUND AND AIM Less than 50% of oral cancer cases are diagnosed at early stages of the disease and this is in part due to poor awareness and lack of knowledge on the signs and symptoms of oral cancer. This study sought to measure the baseline awareness of oral cancer in Malaysia and aimed to increase public awareness and knowledge of oral cancer using a mass media campaign. METHODS Baseline awareness and impact of the campaign was measured using self-administered questionnaires sent via email to individuals. The campaign was aired on two national television channels and the reach was monitored through an independent programme monitoring system. RESULTS 78.2% of respondents had heard of oral cancer, and this increased significantly after the campaign. However, the ability to recognize signs and symptoms remains unchanged. We found that the level of awareness differed between the distinct ethnic subgroups and the reach of the campaign was not uniform across all ethnicities. CONCLUSION This substantial study to measure the oral cancer awareness in Malaysia provides important baseline data for the planning of public health policies. Despite encouraging evidence that a mass media campaign could increase the awareness of oral cancer, further research is required to address the acceptability, comprehensiveness and effectiveness. Furthermore, different campaign approaches may be required for specific ethnic groups in a multi-ethnic country such as Malaysia.

Oral cancer screening in private dental practices in a developing country: opportunities and challenges

OBJECTIVES Private dental practitioners constitute approximately 40% of all registered dentists in Malaysia, and this group affords an avenue for prevention and early detection of oral cancer. However, such activities are still limited. This study investigated the feasibility of incorporating opportunistic screening of oral cancer in the private dental setting. METHODS Dentists were recruited through two main dental associations in Malaysia and attended a 1-day training session on recognizing abnormalities within the oral cavity. Following the training, the dentists conducted screening and provided risk habits cessation advice at their respective clinics for 6 months. The impact of the program was evaluated by determining the number of patients who were screened and/or provided with risk habits cessation advice. RESULTS Twenty-six dentists took part in the program and conducted opportunistic screening on a total of 2603 individuals. On average, they screened about 23.0% of their patients and 5.1% were given risk habits cessation advice. Notably, dentists who had lower patient load were more likely to conduct opportunistic screening. CONCLUSIONS While the participating private dentists state that they have a role in performing opportunistic screening and providing risk habits cessation advice, these activities are still not a priority area in the private clinics, strongly suggesting that strategies to motivate dentists in this setting are urgently needed.

Four-protein signature accurately predicts lymph node metastasis and survival in oral squamous cell carcinoma

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.

Determination of the global gene expression profile of oral cancers using paraffin-embedded tissues

Introduction: Oral cancer is a common disease in Asia. However, our ability to deliver effective and targeted therapy remains limited by our lack of understanding of the molecular pathogenesis of oral carcinogenesis. One method to understand the nature of genes and gene products whose aberrant expression promote malignant transformation is by using microarrays. However, such studies have been limited by the availability of specimens with intact RNA and adequate clinical data to enable the identi?cation and validation biomarkers either as predictive or therapeutic tools. Objective: We have determined the global gene expression pro?les of oral squamous cell carcinoma (OSCC) of the buccal mucosa using formalin ?xed paraf?n-embedded specimens. Materials and Methods: Gene expression analyses using the DASL Assay were performed on 34 paraf?n embedded tissues, of which 22 samples were OSCC of the buccal mucosa and 12 samples were normal surface epithelium of reactive lesions such as the ?broepithelial polyp from matched site. We used the Illumina Beadstudio Software to compare the expression pro?les of normal and OSCC samples. We selected genes which were differentially expressed by at least two-fold (with the detection p-value <0.01) for further analysis. Results: A total of 47 genes were differentially expressed by at least two-fold between normal and OSCC. Deregulated genes included genes which were involved in cell signaling, adhesion and invasion, such as ITGB4, MMP1, MMP10, MMP7, CXCL9, and ALOX12. We have validated the over-expression of MMP1, ITGB4 and MMP10, and down-regulation of ALOX12 by quantitative real-time PCR. Conclusion: We have successfully conducted global gene expression studies using paraf?n-embedded buccal mucosa specimens. This approach has enabled the identi?cation of genes whose expression can help explain the aggressive nature of oral cancers arising from the buccal mucosa.