Wan Mahadzir Wan Mustafa

Micromanipulation of culture niche permits long-term expansion of dental pulp stem cells - an economic and commercial angle

Stem cells isolated from dental pulp possess the capacity for self-renewal and the potential for multi-lineage differentiation. However, dental pulp stem cells have different characteristics in terms of their culture conditions. The success of stem cells culture is governed by its micro-environmental niche. Therefore, we studied the effects of culture niche on long-term expansion of dental pulp stem cells in terms of cell morphology, growth kinetics, senescence pattern, cell surface marker expression differentiation capacity, and seeding plating density of dental pulp stem cells in four different, widely used media composition Among the various basal media tested, α-minimum essential media and knock out-minimum essential media supplemented with 10% fetal bovine serum were found to be the most optimal media composition in preserving the phenotypic characteristics and differentiation potential for prolonged periods as compared with DMEM-F12 and DMEM-LG. Plating density has been shown to affect overall yield. As a conclusion, the adoption of an appropriate culture system significantly improved cell yield, thus enabling the attainment of sufficient yields for therapeutic applications economizing in terms of cost of production and minimizing seeding cell density for maximum yield.

Valproic acid: growth inhibition of head and neck cancer by induction of terminal differentiation and senescence.

There are limited studies on the effects of drugs that modulate epigenetic regulation for head and neck squamous cell carcinoma (HNSCC). This study determined the effect of valproic acid (VPA) on HNSCC.

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.

Genetically-defined novel oral squamous cell carcinoma cell lines for the development of molecular therapies

Emerging biological and translational insights from large sequencing efforts underscore the need for genetically-relevant cell lines to study the relationships between genomic alterations of tumors, and therapeutic dependencies. Here, we report a detailed characterization of a novel panel of clinically annotated oral squamous cell carcinoma (OSCC) cell lines, derived from patients with diverse ethnicity and risk habits. Molecular analysis by RNAseq and copy number alterations (CNA) identified that the cell lines harbour CNA that have been previously reported in OSCC, for example focal amplications in 3q, 7p, 8q, 11q, 20q and deletions in 3p, 5q, 8p, 18q. Similarly, our analysis identified the same cohort of frequently mutated genes previously reported in OSCC including TP53, CDKN2A, EPHA2, FAT1, NOTCH1, CASP8 and PIK3CA. Notably, we identified mutations (MLL4, USP9X, ARID2) in cell lines derived from betel quid users that may be associated with this specific risk factor. Gene expression profiles of the ORL lines also aligned with those reported for OSCC. By focusing on those gene expression signatures that are predictive of chemotherapeutic response, we observed that the ORL lines broadly clustered into three groups (cell cycle, xenobiotic metabolism, others). The ORL lines noted to be enriched in cell cycle genes responded preferentially to the CDK1 inhibitor RO3306, by MTT cell viability assay. Overall, our in-depth characterization of clinically annotated ORL lines provides new insight into the molecular alterations synonymous with OSCC, which can facilitate in the identification of biomarkers that can be used to guide diagnosis, prognosis, and treatment of OSCC.

Genome Wide Analysis of Chromosomal Alterations in Oral Squamous Cell Carcinomas Revealed over Expression of MGAM and ADAM9

Despite the advances in diagnosis and treatment of oral squamous cell carcinoma (OSCC), mortality and morbidity rates have not improved over the past decade. A major drawback in diagnosis and treatment of OSCC is the lack of knowledge relating to how genetic instability in oral cancer genomes affects oral carcinogenesis. Hence, the key aim of this study was to identify copy number alterations (CNAs) that may be cancer associated in OSCC using high-resolution array comparative genomic hybridization (aCGH). To our knowledge this is the first study to use ultra-high density aCGH microarrays to profile a large number of OSCC genomes (n = 46). The most frequently amplified CNAs were located on chromosome 11q11(52%), 2p22.3(52%), 1q21.3–q22(54%), 6p21.32(59%), 20p13(61%), 7q34(52% and 72%),8p11.23–p11.22(80%), 8q11.1–q24.4(54%), 9q13–q34.3(54%), 11q23.3–q25(57%); 14q21.3–q31.1(54%); 14q31.3–q32.33(57%), 20p13–p12.3(54%) and 20q11.21–q13.33(52%). The most frequently deleted chromosome region was located on 3q26.1 (54%). In order to verify the CNAs from aCGH using quantitative polymerase chain reaction (qPCR), the three top most amplified regions and their associated genes, namely ADAM5P (8p11.23–p11.22), MGAM (7q34) and SIRPB1 (20p13.1), were selected in this study. The ADAM5P locus was found to be amplified in 39 samples and deleted in one; MGAM (24 amplifications and 3 deletions); and SIRPB1 (12 amplifications, others undetermined). On the basis of putative cancer-related annotations, two genes, namely ADAM metallopeptidase domain 9 (ADAM9) and maltase-glucoamylase alpha-glucosidase (MGAM), that mapped to CNA regions were selected for further evaluation of their mRNA expression using reverse transcriptase qPCR. The over-expression of MGAM was confirmed with a 6.6 fold increase in expression at the mRNA level whereas the fold change in ADAM9 demonstrated a 1.6 fold increase. This study has identified significant regions in the OSCC genome that were amplified and resulted in consequent over-expression of the MGAM and ADAM9 genes that may be utilized as biological markers for OSCC.

MDM2 SNP309 does not confer an increased risk to oral squamous cell carcinoma but may modulate the age of disease onset

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.

Genome-wide analysis of oral squamous cell carcinomas revealed over expression of ISG15, Nestin and WNT11

BACKGROUND Multistep pathways and mechanisms are involved in the development of oral cancer. Chromosomal alterations are one of such key mechanisms implicated oral carcinogenesis. Therefore, this study aims to determine the genomic copy number alterations (CNAs) in oral squamous cell carcinoma (OSCC) using array comparative genomic hybridization (aCGH) and in addition attempt to correlate CNAs with modified gene expression. MATERIALS AND METHODS Genome-wide screening was performed on 15 OSCCs using high-density aCGH. On the basis of pathway analysis, three genes (ISG15, Nestin and WNT11) which mapped to CNA regions were selected for further evaluation of their mRNA expression using quantitative reverse transcriptase PCR (qRT-PCR). RESULTS Copy number alterations were observed on multiple genomic regions, including amplifications on 1p, 3q, 5p, 6p, 7p, 8q, 9q, 11q, 12q, 16p, 18p and deletions on 3p, 7q, 8p, 11q, 19q and 20q. Among the three selected genes, ISG15 had the highest mRNA expression level with a 22.5-fold increase, followed by Nestin with a 4.5-fold increase and WNT11 with a 2.5-fold increase. CONCLUSIONS This study has identified several major CNAs in oral cancer genomes and indicated that this correlates with over expression of the ISG15, WNT11, and Nestin genes.

A prospective study on elderly patients with facial fractures in a developing country

BACKGROUND This paper describes the incidence, aetiology, treatment and complications of facial fractures seen among the elderly in a developing country. METHODS A prospective study evaluated 85 patients over 60 years of age who were diagnosed with facial fractures over a period of 12 months in 23 public hospitals nationwide. RESULTS The elderly accounted for 4.5% of the total number of patients seen with facial fractures during the study period. Elderly men outnumbered women by a ratio of 4.31:1. Of the elderly patients, 35.3% had at least one medical condition, the commonest of which was hypertension. Road traffic accidents were the main cause of injury. The fractures were treated in only 26.2% of cases. Complications were uncommon. CONCLUSIONS With a low incidence, and conservative treatment often being practised, the healthcare burden of treating facial fractures among the elderly in Malaysia is at present still low.

Genetic Alterations of Chromosome 8 Genes in Oral Cancer

The clinical relevance of DNA copy number alterations in chromosome 8 were investigated in oral cancers. The copy numbers of 30 selected genes in 33 OSCC patients were detected using the multiplex ligation-dependent probe amplification (MLPA) technique. Amplifications of the EIF3E gene were found in 27.3% of the patients, MYC in 18.2%, RECQL4 in 15.2% and MYBL1 in 12.1% of patients. The most frequent gene losses found were the GATA4 gene (24.2%), FGFR1 gene (24.2%), MSRA (21.2) and CSGALNACT1 (12.1%). The co-amplification of EIF3E and RECQL4 was found in 9% of patients and showed significant association with alcohol drinkers. There was a significant association between the amplification of EIF3E gene with non-betel quid chewers and the negative lymph node status. EIF3E amplifications did not show prognostic significance on survival. Our results suggest that EIF3E may have a role in the carcinogenesis of OSCC in non-betel quid chewers.

Co-Expression of TWIST1 and ZEB2 in Oral Squamous Cell Carcinoma Is Associated with Poor Survival.

Oral squamous cell carcinoma (OSCC) is an aggressive disease accounting for more than 260,000 cancer cases diagnosed and 128,000 deaths worldwide. A large majority of cancer deaths result from cancers that have metastasized beyond the primary tumor. The relationship between genetic changes and clinical outcome can reflect the biological events that promote cancer’s aggressive behavior, and these can serve as molecular markers for improved patient management and survival. To this end, epithelial-mesenchymal transition (EMT) is a major process that promotes tumor invasion and metastasis, making EMT-related proteins attractive diagnostic biomarkers and therapeutic targets. In this study, we used immunohistochemistry to study the expression of a panel of transcription factors (TWIST1, SNAI1/2, ZEB1 and ZEB2) and other genes intimately related to EMT (CDH1 and LAMC2) at the invasive tumor front of OSCC tissues. The association between the expression of these proteins and clinico-pathological parameters were examined with Pearson Chi-square and correlation with survival was analyzed using Kaplan Meier analysis. Our results demonstrate that there was a significant differential expression of CDH1, LAMC2, SNAI1/2 and TWIST1 between OSCC and normal oral mucosa (NOM). Specifically, CDH1 loss was significantly associated with Broder’s grading, while diffused LAMC2 was similarly associated with non-cohesive pattern of invasion. Notably, co-expression of TWIST1 and ZEB2 in OSCC was significantly associated with poorer overall survival, particularly in patients without detectable lymph node metastasis. This study demonstrates that EMT-related proteins are differentially expressed in OSCC and that the co-expression of TWIST1 and ZEB2 could be of clinical value in identifying patients with poor survival for appropriate patient management.