Lee Peng Karen-Ng

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.

Overexpression of MMP13 is associated with clinical outcomes and poor prognosis in oral squamous cell carcinoma

Matrix metalloproteinase 13 (MMP13) plays a central role in the MMP activation cascade that enables degradation of the extracellular matrix and basement membranes, and it is identified as a potential driver in oral carcinogenesis. Therefore, this study aims to determine the copy number, mRNA, and protein expression of MMP13 in oral squamous cell carcinoma (OSCC) and to associate these expressions with clinicopathological parameters. Copy number, mRNA, and protein expression analysis of MMP13 were determined using real-time quantitative PCR and immunohistochemistry methods in OSCC samples. The correlations between MMP13 expressions and clinicopathological parameters were evaluated, and the significance of MMP13 as a prognostic factor was determined. Despite discrepancies between gene amplification and mRNA and protein overexpression rates, OSCC cases showed high amplification of MMP13 and overexpression of MMP13 at both mRNA and protein levels. High level of MMP13 protein expression showed a significant correlation with lymph node metastasis (P = 0.011) and tumor staging (P = 0.002). Multivariate Cox regression model analysis revealed that high level of mRNA and protein expression of MMP13 were significantly associated with poor prognosis (P < 0.050). Taken together, these observations indicate that the MMP13 protein overexpression could be considered as a prognostic marker of OSCC.

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.

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.

Novel MDM2 splice variants identified from oral squamous cell carcinoma

INTRODUCTION The presence of a variety of MDM2 splice variants has been reported in a range of different tumor types and is associated with poor patient prognosis. Furthermore, several MDM2 variants have been shown to have oncogenic properties. Despite this, MDM2 splice variants have not been comprehensively characterized in oral squamous cell carcinoma (OSCC). MATERIALS AND METHODS MDM2 splice variants were identified by polymerase chain reaction (PCR), using cDNA from 55 OSCC and 20 normal oral mucosa (NOM) tissues. MDM2 amplicons from the polymerase chain reactions were cloned and sequenced. The associations between the presence of MDM2 splice variants as well as the types of MDM2 splice variants with OSCC and patient clinico-pathological data was examined using Fisher Exact and Chi-square tests. RESULTS Thirty-eight MDM2 splice variants were identified from both OSCC and NOM tissues, where the majority (30/38) were exclusively detected in OSCC. Some of these variants were similar to those reported in other cancers whilst 14 novel MDM2 splice variants predicted to code for proteins were also identified. The majority of these variants retained their RING binding domain but had lost the p53 binding site. The presence of MDM2 splice variants was significantly associated with OSCC and increased the risk of OSCC development (OR=9.98; 95% CI=2.94-33.90). CONCLUSION MDM2 splice variants were identified in OSCC at a high frequency and were significantly associated with OSCC development. This suggests that MDM2 splice variants may play an important role in oral carcinogenesis and the functional role of these variants in OSCC should be examined further.

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.

KRT13, FAIM2 And CYP2W1 Mrna Expression In Oral Squamous Cell Carcinoma Patients With Risk Habits

BACKGROUND Expression of KRT13, FAIM2 and CYP2W1 appears to be influenced by risk habits, thus exploring the associations of these genes in oral squamous cell cancer (OSCC) with risk habits, clinico-pathological parameters and patient survival may be beneficial in identifying relevant biomarkers with different oncogenic pathways. MATERIALS AND METHODS cDNAs from 41 OSCC samples with and without risk habits were included in this study. Quantitative real-time PCR was used to analyze KRT13, FAIM2 and CYP2W1 in OSCC. The housekeeping gene (GAPDH) was used as an endogenous control. RESULTS Of the 41 OSCC samples, KRT13 was down-regulated in 40 samples (97.6%), while FAIM2 and CYP2W1 were down-regulated in 61.0% and 48.8%, respectively. Overall, there were no associations between KRT13, FAIM2 and CYP2W1 expression with risk habits, selected socio-demographic and clinico-pathological parameters and patient survival. CONCLUSIONS Although this study was unable to show significance, there were some tendencies in the associations of KRT13, FAIM2 and CYP2W1 expression in OSCC with selected clinic-pathological parameters and survival.

Collagen Triple Helix Repeat Containing-1 (CTHRC1) Expression in Oral Squamous Cell Carcinoma (OSCC): Prognostic Value and Clinico-Pathological Implications.

BACKGROUND: Collagen Triple Helix Repeat Containing 1 (CTHRC1) is a protein often found to be over-expressed in various types of human cancers. However, correlation between CTHRC1 expression level with clinico-pathological characteristics and prognosis in oral cancer remains unclear. Therefore, this study aimed to determine mRNA and protein expression of CTHRC1 in oral squamous cell carcinoma (OSCC) and to evaluate the clinical and prognostic impact of CTHRC1 in OSCC. METHODS: In this study, mRNA and protein expression of CTHRC1 in OSCCs were determined by quantitative PCR and immunohistochemistry, respectively. The association between CTHRC1 and clinico-pathological parameters were evaluated by univariate and multivariate binary logistic regression analyses. Correlation between CTHRC1 protein expressions with survival were analysed using Kaplan-Meier and Cox regression models. RESULTS: Current study demonstrated CTHRC1 was significantly overexpressed at the mRNA level in OSCC. Univariate analyses indicated a high-expression of CTHRC1 that was significantly associated with advanced stage pTNM staging, tumour size ≥ 4 cm and positive lymph node metastasis (LNM). However, only positive LNM remained significant after adjusting with other confounder factors in multivariate logistic regression analyses. Kaplan-Meier survival analyses and Cox model demonstrated that patients with high-expression of CTHRC1 protein were associated with poor prognosis and is an independent prognostic factor in OSCC. CONCLUSION: This study indicated that over-expression of CTHRC1 potentially as an independent predictor for positive LNM and poor prognosis in OSCC.

Distinct pattern of chromosomal alterations and pathways in tongue and cheek squamous cell carcinoma

The purpose of this study was to investigate the cause of behavioral difference between tongue and cheek squamous cell carcinomas (SCCs) by verifying the copy number alterations (CNAs).