Calibration improves the agreement in grading oral epithelial dysplasia-Findings from a National Workshop in Malaysia.

INTRODUCTION: A major pitfall of many of the established oral epithelial dysplasia (OED) grading criteria is their lack of reproducibility and accuracy to predict malignant transformation. The main objective of this study was to determine whether calibration of practicing oral pathologists on OED grading could improve the reproducibility of the WHO 2017 and the binary OED grading systems. METHODS: A nationwide online exercise was carried out to determine the influence of calibration on the reproducibility of the WHO 2017 and the binary OED grading systems. RESULTS: A significant improvement was observed in the inter-observer agreement for the WHO 2017 OED grading system (K 0.196 vs. 0.448; Kw 0.357 vs. 0.562) after the calibration exercise. The significant difference (p = 0.027) in the level of agreement between those with five or more years and less than 5 years of experience was no more observed (p = 0.426) after the calibration exercise. The percent agreement for binary grading was significantly higher (91.8%) for buccal mucosal lesions as compared to lesions on the tongue after the calibration exercise. CONCLUSION: This study validates the significance of calibration in improving the reproducibility of OED grading. The nationwide exercise resulted in a statistically significant improvement in the inter-observer agreement for the WHO 2017 OED grading system among a large number of oral pathologists. It is highly recommended that similar exercises should be organized periodically by professional bodies responsible for continuing education among oral pathologists to improve the reliability of OED grading for optimal treatment of oral potentially malignant disorders.

A Comprehensive Review of Natural Products as Therapeutic or Chemopreventive Agents against Head and Neck Squamous Cell Carcinoma Cells Using Preclinical Models.

Head and neck squamous cell carcinoma (HNSCC) is a type of cancer that arises from the epithelium lining of the oral cavity, hypopharynx, oropharynx, and larynx. Despite the advancement of current treatments, including surgery, chemotherapy, and radiotherapy, the overall survival rate of patients afflicted with HNSCC remains poor. The reasons for these poor outcomes are due to late diagnoses and patient-acquired resistance to treatment. Natural products have been extensively explored as a safer and more acceptable alternative therapy to the current treatments, with numerous studies displaying their potential against HNSCC. This review highlights preclinical studies in the past 5 years involving natural products against HNSCC and explores the signaling pathways altered by these products. This review also addresses challenges and future directions of natural products as chemotherapeutic and chemoprevention agents against HNSCC.

Dyskeratosis Congenita Links Telomere Attrition to Age-Related Systemic Energetics.

The underlying mechanisms of plasma metabolite signatures of human aging and age-related diseases are not clear but telomere attrition and dysfunction are central to both. Dyskeratosis congenita (DC) is associated with mutations in the telomerase enzyme complex (TERT, TERC, and DKC1) and progressive telomere attrition. We analyzed the effect of telomere attrition on senescence-associated metabolites in fibroblast-conditioned media and DC patient plasma. Samples were analyzed by gas chromatography/mass spectrometry and liquid chromatography/mass spectrometry. We showed extracellular citrate was repressed by canonical telomerase function in vitro and associated with DC leukocyte telomere attrition in vivo, leading to the hypothesis that altered citrate metabolism detects telomere dysfunction. However, elevated citrate and senescence factors only weakly distinguished DC patients from controls, whereas elevated levels of other tricarboxylic acid cycle (TCA) metabolites, lactate, and especially pyruvate distinguished them with high significance. The DC plasma signature most resembled that of patients with loss of function pyruvate dehydrogenase complex mutations and that of older subjects but significantly not those of type 2 diabetes, lactic acidosis, or elevated mitochondrial reactive oxygen species. Additionally, our data are consistent with further metabolism of citrate and lactate in the liver and kidneys. Citrate uptake in certain organs modulates age-related disease in mice and our data have similarities with age-related disease signatures in humans. Our results have implications for the role of telomere dysfunction in human aging in addition to its early diagnosis and the monitoring of anti-senescence therapeutics, especially those designed to improve telomere function.

Extracellular Prostaglandins E1 and E2 and Inflammatory Cytokines Are Regulated by the Senescence Program in Potentially Premalignant Oral Keratinocytes.

Potentially pre-malignant oral lesions (PPOLs) are composed of keratinocytes that are either mortal (MPPOL) or immortal (IPPOL) in vitro. We report here that MPPOL, but not generally IPPOL, keratinocytes upregulate various extracellular tumor-promoting cytokines (interleukins 6 and 8) and prostaglandins E1 (ePGE1) and E2 (ePGE2) relative to normal oral keratinocytes (NOKs). ePGE upregulation in MPPOL was independent of PGE receptor status and was associated with some but not all markers of cellular senescence. Nevertheless, ePGE upregulation was dependent on the senescence program, cyclo-oxygenase 2 (COX2) and p38 mitogen-activated protein kinase and was partially regulated by hydrocortisone. Following senescence in the absence of p16INK4A, ePGEs accumulated in parallel with a subset of tumor promoting cytokine and metalloproteinase (MMP) transcripts, all of which were ablated by ectopic telomerase. Surprisingly, ataxia telangiectasia mutated (ATM) function was not required for ePGE upregulation and was increased in expression in IPPOL keratinocytes in line with its recently reported role in telomerase function. Only ePGE1 was dependent on p53 function, suggesting that ePGEs 1 and 2 are regulated differently in oral keratinocytes. We show here that ePGE2 stimulates IPPOL keratinocyte proliferation in vitro. Therefore, we propose that MPPOL keratinocytes promote the progression of IPPOL to oral SCC in a pre-cancerous field by supplying PGEs, interleukins and MMPs in a paracrine manner. Our results suggest that the therapeutic targeting of COX-2 might be enhanced by strategies that target keratinocyte senescence.

Detection of Genetic Alterations in Oral Squamous Cell Carcinoma Using Multiplex LigationDependent Probe Amplification (MLPA)

@#Deletions and amplifications of genes often occur during multistep progression from oral precancer, seen as oral epithelial dysplasia (OED) to cancerous stage. These genetic alterations could be used as markers to aid in detection of oral squamous cell carcinomas (OSCC). This study explored the use of multiplex ligation-dependent probe amplification (MLPA) technique in detecting OSCC and OED specific genetic alterations. MLPA was used to detect gains and losses of 106 genes in DNA extracted from frozen tissue samples of 10 OSCC and 10 noncancer patients. Two biopsies of OED were analyzed to explore the alterations in oral potentially malignant disorders. There were significant differences (p<0.001) in the number of alterations in OSCC and dysplasia compared to non-cancer samples respectively. The most frequently altered genes in OSCC were PTP4A3, RECQL4, ATM, and KLK3 (60%). Five genes (MYC, SLA, TNFRSF1A, MESDC1, MIF) were altered in 50% of OSCC samples. These nine genes were specific to OSCC samples (p<0.05). Some genes, including MYB, MET, CASP2, SLA and PTEN occurred in 50% of OED samples. MLPA was able to detect genetic alterations, that are present only in the OSCC samples and showed potential to be used as an adjunctive tool in early diagnosis of OSCC.

The Extracellular Metabolome Stratifies Low and High Risk Potentially Premalignant Oral Keratinocytes and Identifies Citrate as a Potential Non-Invasive Marker of Tumour Progression.

Premalignant oral lesions (PPOLs) which bypass senescence (IPPOL) have a much greater probability of progressing to malignancy, but pre-cancerous fields also contain mortal PPOL keratinocytes (MPPOL) that possess tumour-promoting properties. To identify metabolites that could potentially separate IPPOL, MPPOL and normal oral keratinocytes non-invasively in vivo, we conducted an unbiased screen of their conditioned medium. MPPOL keratinocytes showed elevated levels of branch-chain amino acid, lipid, prostaglandin, and glutathione metabolites, some of which could potentially be converted into volatile compounds by oral bacteria and detected in breath analysis. Extracellular metabolites were generally depleted in IPPOL, and only six were elevated, but some metabolites distinguishing IPPOL from MPPOL have been associated with progression to oral squamous cell carcinoma (OSCC) in vivo. One of the metabolites elevated in IPPOL relative to the other groups, citrate, was confirmed by targeted metabolomics and, interestingly, has been implicated in cancer growth and metastasis. Although our investigation is preliminary, some of the metabolites described here are detectable in the saliva of oral cancer patients, albeit at a more advanced stage, and could eventually help detect oral cancer development earlier.

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.

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.

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.

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.

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

BACKGROUND: 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). METHODS: Array comparative genomic hybridization (aCGH) was used to profile unique deletions and amplifications that are involved with tongue and cheek SCC, respectively. This was followed by pathway analysis relating to CNA genes from both sites. RESULTS: The most frequently amplified regions in tongue SCC were 4p16.3, 11q13.4, and 13q34; whereas the most frequently deleted region was 19p12. For cheek SCC, the most frequently amplified region was identified on chromosome 9p24.1-9p23; whereas the most common deleted region was located on chromosome 8p23.1. Further analysis revealed that the most significant unique pathway related to tongue and cheek SCCs was the cytoskeleton remodeling and immune response effect on the macrophage differentiation pathway. CONCLUSION: This study has showed the different genetic profiles and biological pathways between tongue and cheek SCCs. © 2013 Wiley Periodicals, Inc. Head Neck 36: 1268-1278, 2014.

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.

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.

Combined effects of isothiocyanate intake, glutathione S-transferase polymorphisms and risk habits for age of oral squamous cell carcinoma development.

Dietary isothiocyanates (ITCs) found in cruciferous vegetables (Brassica spp.) has been reported to reduce cancer risk by inducing phase II conjugating enzymes, in particular glutathione S-transferases (GSTs). This case-control study was aimed at determining associations between dietary ITCs, GSTs polymorphisms and risk habits (cigarette smoking, alcohol drinking and betel-quid chewing) with oral cancer in 115 cases and 116 controls. Information on dietary ITC intake from cruciferous vegetables was collected via a semi-quantitative food frequency questionnaire (FFQ). Peripheral blood lymphocytes were obtained for genotyping of GSTM1, GSTT1 and GSTP1 using PCR multiplex and PCR-RFLP. Chi-square and logistic regression were performed to determine the association of ITC and GSTs polymorphism and risk of oral cancer. When dietary ITC was categorized into high (greater than/equal to median) and low (less than median) intake, there was no significant difference between cases and control group. Logistic regression yielding odd ratios resulted in no significant association between dietary ITC intake, GSTM1, GSTT1 or GSTP1 genotypes with oral cancer risk overall. However, GSTP1 wild-type genotype was associated with later disease onset in women above 55 years of age (p= 0.017). Among the men above 45 years of age, there was clinical significant difference of 17 years in the age of onset of oral cancer between GSTP1 wild-type + low ITC intake and GSTP1 polymorphism + high ITC intake (p= 0.001). Similar conditions were also seen among men above 45 years of age with risk habits like drinking and chewing as the earlier disease onset associated with GSTP1 polymorphism and high ITC intake (p< 0.001). This study suggests that combination effects between dietary ITCs, GSTP1 polymorphism and risk habits may be associated with the risk of oral cancer and modulate the age of disease onset.