S C Cheong

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.

Dysregulation of miR-31 and miR-375 expression is associated with clinical outcomes in oral carcinoma.

OBJECTIVES To identify differentially expressed miRNA between oral squamous cell carcinoma (OSCC) and non-cancer (NC) and to associate these with clinico-pathological parameters. MATERIALS AND METHODS miRNA microarray profiling was utilized to obtain the expression profile of miRNAs in four OSCC and four NC samples. The expression of miR-31 and miR-375 was further validated in 26 OSCC and three NC samples using real-time-PCR. The association between miRNA expression and clinico-pathological parameters was tested by univariate and multivariate analyses. RESULTS Microarray profiling demonstrated that 15 and four miRNAs were up-regulated and down-regulated, respectively, in OSCC as compared with NC. miR-31 and miR-375 were validated as up- and down-regulated miRNAs, respectively. In univariate analyses, expression of miR-31 was significantly elevated in early stage, tumours with no metastatic nodes and those from the buccal mucosa. By contrast, low miR-375 expression was significantly associated with late stage disease, larger tumour size and the non-cohesive type of pattern of invasion in OSCC. The association between miR-31 expression with tumour staging and site and miR-375 with tumour staging remained significant in multivariate analyses. CONCLUSIONS This study has identified 19 miRNAs significantly associated with OSCC, and expressions of miR-31 and miR-375 were significantly related with clinico-pathological parameters suggesting they could be important in driving oral tumourigenesis.

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.

Common Oncogenic Mutations Are Infrequent in Oral Squamous Cell Carcinoma of Asian Origin

Objectives The frequency of common oncogenic mutations and TP53 was determined in Asian oral squamous cell carcinoma (OSCC). Materials and Methods The OncoCarta™ panel v1.0 assay was used to characterize oncogenic mutations. In addition, exons 4-11 of the TP53 gene were sequenced. Statistical analyses were conducted to identify associations between mutations and selected clinico-pathological characteristics and risk habits. Results Oncogenic mutations were detected in PIK3CA (5.7%) and HRAS (2.4%). Mutations in TP53 were observed in 27.7% (31/112) of the OSCC specimens. Oncogenic mutations were found more frequently in non-smokers (p = 0.049) and TP53 truncating mutations were more common in patients with no risk habits (p = 0.019). Patients with mutations had worse overall survival compared to those with absence of mutations; and patients who harbored DNA binding domain (DBD) and L2/L3/LSH mutations showed a worse survival probability compared to those patients with wild type TP53. The majority of the oncogenic and TP53 mutations were G:C > A:T and A:T > G:C base transitions, regardless of the different risk habits. Conclusion Hotspot oncogenic mutations which are frequently present in common solid tumors are exceedingly rare in OSCC. Despite differences in risk habit exposure, the mutation frequency of PIK3CA and HRAS in Asian OSCC were similar to that reported in OSCC among Caucasians, whereas TP53 mutations rates were significantly lower. The lack of actionable hotspot mutations argue strongly for the need to comprehensively characterize gene mutations associated with OSCC for the development of new diagnostic and therapeutic tools.

Establishing and managing a periodontal biobank for research: the sharing of experience.

Periodontal bio-repositories, which allow banking of clinically validated human data and biological samples, provide an opportunity to derive biomarkers for periodontal diagnosis, prognosis and therapeutic activities which are expected to improve patient management. This article presents the establishing of the Malaysian Periodontal Database and Biobank System (MPDBS) which was initiated in 2011 with the aim to facilitate periodontal research. Partnerships were established with collaborating centres. Policies on specimen access, authorship and acknowledgement policies were agreed upon by all participating centres before the initiation of the periodontal biobank. Ethical approval for the collection of samples and data were obtained from institutional ethics review boards. A broad-based approach for informed consent was used, which covered areas related to quality of life impacts, genetics and molecular aspects of periodontal disease. Sample collection and processing was performed using a standardized protocol. Biobanking resources such as equipment and freezers were shared with the Malaysian Oral Cancer Database and Tissue Bank System (MOCDTBS). In the development of the MPDBS, challenges that were previously faced by the MOCDTBS were considered. Future challenges in terms of ethical and legal issues will be faced when international collaborations necessitate the transportation of specimens across borders.

Comparative proteomics analysis of oral cancer cell lines: identification of cancer associated proteins

BackgroundA limiting factor in performing proteomics analysis on cancerous cells is the difficulty in obtaining sufficient amounts of starting material. Cell lines can be used as a simplified model system for studying changes that accompany tumorigenesis. This study used two-dimensional gel electrophoresis (2DE) to compare the whole cell proteome of oral cancer cell lines vs normal cells in an attempt to identify cancer associated proteins.ResultsThree primary cell cultures of normal cells with a limited lifespan without hTERT immortalization have been successfully established. 2DE was used to compare the whole cell proteome of these cells with that of three oral cancer cell lines. Twenty four protein spots were found to have changed in abundance. MALDI TOF/TOF was then used to determine the identity of these proteins. Identified proteins were classified into seven functional categories – structural proteins, enzymes, regulatory proteins, chaperones and others. IPA core analysis predicted that 18 proteins were related to cancer with involvements in hyperplasia, metastasis, invasion, growth and tumorigenesis. The mRNA expressions of two proteins – 14-3-3 protein sigma and Stress-induced-phosphoprotein 1 – were found to correlate with the corresponding proteins’ abundance.ConclusionsThe outcome of this analysis demonstrated that a comparative study of whole cell proteome of cancer versus normal cell lines can be used to identify cancer associated proteins.

GENIPAC: A Genomic Information Portal for Head and Neck Cancer Cell Systems

Head and neck cancer (HNC)–derived cell lines represent fundamental models for studying the biological mechanisms underlying cancer development and precision therapies. However, mining the genomic information of HNC cells from available databases requires knowledge on bioinformatics and computational skill sets. Here, we developed a user-friendly web resource for exploring, visualizing, and analyzing genomics information of commonly used HNC cell lines. We populated the current version of GENIPAC with 44 HNC cell lines from 3 studies: ORL Series, OPC-22, and H Series. Specifically, the mRNA expressions for all the 3 studies were derived with RNA-seq. The copy number alterations analysis of ORL Series was performed on the Genome Wide Human Cytoscan HD array, while copy number alterations for OPC-22 were derived from whole exome sequencing. Mutations from ORL Series and H Series were derived from RNA-seq information, while OPC-22 was based on whole exome sequencing. All genomic information was preprocessed with customized scripts and underwent data validation and correction through data set validator tools provided by cBioPortal. The clinical and genomic information of 44 HNC cell lines are easily assessable in GENIPAC. The functional utility of GENIPAC was demonstrated with some of the genomic alterations that are commonly reported in HNC, such as TP53, EGFR, CCND1, and PIK3CA. We showed that these genomic alterations as reported in The Cancer Genome Atlas database were recapitulated in the HNC cell lines in GENIPAC. Importantly, genomic alterations within pathways could be simultaneously visualized. We developed GENIPAC to create access to genomic information on HNC cell lines. This cancer omics initiative will help the research community to accelerate better understanding of HNC and the development of new precision therapeutic options for HNC treatment. GENIPAC is freely available at http://genipac.cancerresearch.my/.

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.

Immunohistochemical localization of hTERT protein in oral carcinogenesis

Introduction: Human telomerase reverse transcriptase (hTERT) the catalytic subunit of telomerase is associated with cellular immortalization and tumorigenesis .In situ detection of hTERT using immunohistochemistry (IHC) allows localizing the hTERT- positive cells and also overcomes the limitation of requirement of fresh tissues for molecular techniques such as Telomeric Repeat Amplification Protocol (TRAP) for telomerase/ hTERT detection. The objectives of this study are to detect hTERT protein expression using immunohistochemistry in different histological stages in oral carcinogenesis with paraffin embedded tissue samples and cultured cells and to relate the hTERT expression with different histological stages in oral carcinogenesis. Materials and Methods: A total of 41 parafin-embedded tissue samples with histological diagnoses of normal oral mucosa (n=5), epithelial hyperplasia (n=5), mild epithelial dysplasia (n=9), moderate and severe epithelial dysplasia (n=8) and oral squamous cell carcinoma (OSCC, n=14) were analysed for hTERT protein using IHC. TRAP assays were performed on cultured cells namely fibroblasts (immortalized with hTERT) and keratinocytes (OSC-20, OSCC cell line, H S R R B, Japan) as positive controls. The hTERT- labelling index was determined following previously published criteria. Results: All the tissues showed Grades 3 & 4 staining (>10% positive staining nuclei) in basal and parabasal cell layers which are the normal proliferative progenitor compartments where hTERT expression is considered normal. hTERT expression in the superficial layers of the epithelia was elevated in moderate and severe dysplasia (75%) with Grades 3 & 4 staining, reduced in mild dysplasia (33.3%) and with no expression in epithelial hyperplasia and normal oral mucosa. hTERT protein expression correlated with positive telomerase activity in the cultured cells. Conclusion: There seems to be an up-regulation of hTERT expression with the progression of oral cancer and therefore indicates the feasibility of IHC detection of hTERT protein as a potential prognostic marker in oral carcinogenesis.