Impact of TLR9 and TLR7 gene polymorphisms on prognosis and survival of patients with oral squamous cell carcinoma.

Despite significant efforts in developing new diagnostic and therapeutic modalities, oral squamous cell carcinomas (OSCCs) still exhibit a high recurrence rate, a low five-year survival rate, and an increasing prevalence. Toll-like receptors (TLRs), which initiate and perpetuate immune mechanisms upon activation, have been linked to immune surveillance and the antitumor immune response. The aim of this study was to investigate the association between the polymorphisms of the TLR7 rs3853839 and TLR9 rs187084 genes and OSCC risk, clinicopathological features, and survival. Genotyping was assessed by real-time polymerase chain reaction (PCR) in 95 HPV-negative OSCC patients and 107 age- and sex-matched healthy controls. Patients with lymph node metastases had higher frequencies of the TLR9 rs187084 CC variant genotype compared to the major TT genotype (P = 0.020) and to T-allele carriers (combined TT + CT genotypes, P = 0.015). A higher prevalence of advanced stage III was observed in patients with the TLR9 rs187084 variant CC genotype compared to TT (P = 0.047) and to T-allele carriers (TT + CT, P = 0.037). Kaplan-Meier analysis revealed a lower overall survival rate in patients with the TLR9 rs187084 variant CC genotype compared to TT genotype (P = 0.010, log-rank test) and to T-allele carriers (TT + CT, P = 0.002), though it was not an independent predictor of overall survival. Both TLR9 rs187084 and TLR7 rs3853839 polymorphisms were associated with high alcohol consumption (P = 0.027 and P = 0.001, respectively). The investigated genetic variations were not associated with OSCC susceptibility. The variant CC genotype of the TLR9 rs187084 polymorphism might be a marker of poor survival and tumor progression in OSCC.

Prognostic impact of miR-34b/c DNA methylation, gene expression, and promoter polymorphism in HPV-negative oral squamous cell carcinomas.

Micro RNAs (miRNAs) have a key role in gene expression regulation in cancer. The aim of the current study is to evaluate the prognostic value of miR-34b/c promoter hypermethylation, gene expression, and polymorphism in HPV-negative oral squamous cell carcinomas (OSCC). MiR-34b/c promoter hypermethylation and pre-miR-34b/c polymorphism rs4938723 were evaluated in tumor tissues of 148 patients, and miR-34b expression in 123 HPV-negative OSCC. For risk assessment, the control group was comprised of 175 healthy individuals. MiR-34b/c promoter hypermethylation was determined by methylation-specific PCR. Gene expression, genotyping and HPV screening was assessed by Q-PCR. The data from our hospital cohort indicated that miR-34b/c DNA methylation was associated with nodal status (p = 0.048), and predicted the shorter overall survival of HPV-negative OSCC patients (p = 0.008). Down-regulated miR-34b/c expression was associated with smoking (p = 0.047), alcohol use (p = 0.009), stage (p = 0.025), recurrences (p = 0.000), and a poor survival (p = 0.00029). Median values of miR-34b expression were significantly lower in advanced stages III/IV as opposed to stage I/II, p = 0.006, and in nodal positive vs negative patients (p = 0.045). TCGA data also indicated that tumors with stage I-III expressed significantly higher levels of miR-34b, compared to tumors with stage IV (p = 0.035), Low miR-34b/c expression was associated with poor survival in smokers (p = 0.001) and patients with tongue carcinomas (p = 0.00003), and TCGA analysis confirmed these findings although miR-34b expression and miR-34b/c methylation were not associated with survival outcome in the whole TCGA cohort. A significant negative miR-34b/c expression-methylation correlation was observed in our hospital cohort (p = 0.017) and in TCGA cohort. Pre-miR-34b/c polymorphism was not associated with oral cancer risk. Our findings indicate that miR-34b/c hypermethylation and low miR-34b expression could promote the progression and predict the poor prognosis for HPV-negative OSCC, which suggests miR-34b/c as a promising biomarker and therapeutic target for OSCC in the future.

Pulp Sensitivity: Influence of Sex, Psychosocial Variables, COMT Gene, and Chronic Facial Pain.

INTRODUCTION: The purpose of this study was to evaluate the associations of variability in pulp sensitivity with sex, psychosocial variables, the gene that encodes for the enzyme catechol-O-methyltransferase (COMT), and chronic painful conditions (temporomandibular disorders [TMDs]). METHODS: The study was composed of 97 subjects (68 women and 29 men aged 20-44 years). The electric (electric pulp tester) and cold (refrigerant spray) stimuli were performed on mandibular lateral incisors. The results were expressed as pain threshold values for electric pulp stimulation (0-80 units) and as pain intensity scores (visual numeric scale from 0-10) for cold stimulation. The Research Diagnostic Criteria for TMD were used to assess TMD, depression, and somatization. DNA extracted from peripheral blood was genotyped for 3 COMT polymorphisms (rs4680, rs6269, and rs165774) using the real-time TaqMan method. Multivariate linear regression was used to investigate the joint effect of the predictor variables (clinical and genetic) on pulp sensitivity (dependent variables). RESULTS: Threshold responses to electric stimuli were related to female sex (P < .01) and the homozygous GG genotype for the rs165774 polymorphism (P < .05). Pain intensity to cold stimuli was higher in TMD patients (P < .01) and tended to be higher in women. Multivariate linear regression identified sex and the rs165774 COMT polymorphism as the determinants of electric pain sensitivity, whereas TMD accounts for the variability in the cold response. CONCLUSIONS: Our findings indicate that sex/a COMT gene variant and TMD as a chronic painful condition may contribute to individual variation in electric and cold pulp sensitivity, respectively.

miR-183 and miR-21 expression as biomarkers of progression and survival in tongue carcinoma patients.

OBJECTIVES: Micro RNAs (miRNAs) have a major role in human cancerogenesis.The current study investigated the prognostic significance of miR-183 and miR-21 expression in tongue carcinoma patients. MATERIAL AND METHOD: For qPCR of miR-183 and miR-21 expression, total RNA isolated from 60 fresh-frozen tissue of tongue carcinomas was converted into cDNA by TaqMan MicroRNA Reverse Transcription Kit and quantified by TaqMan MicroRNAs Expression Assays. Fold changes in the miRNAs expression, normalized to RNU6B, were determined using 2-ΔΔCt method, and dichotomized into high and low according to cut-off values derived from ROC curve analysis. RESULTS: miR-183 emerged as promising discriminatory biomarker of poor outcome. Tissue over-expression of miR-183, observed in 68.3% of tongue carcinomas, was associated with clinical stage (p = 0.037), tumor size (p = 0.036), and high alcohol intake (p = 0.034).The patients with miR-183 over-expression had significantly shorter overall survival (p = 0.006) and a 5.666 times higher risk of poor outcome (p = 0.005), while miR-21 over-expression carried a tendency towards poorer survival (p = 0.073). However, multivariate analysis revealed that the recurrences were independent adverse prognostic factors, while miR-183 over-expression lost its significance. CONCLUSION: Our results suggests that over-expression of miR-183 in tumor tissue could be a potential marker of clinical stage and a poor survival of tongue carcinoma patients and may be associated with high alcohol consumption. CLINICAL RELEVANCE: Oncogenic miRNAs, such as the investigated miR-183 and miR-21, could be novel prognostic biomarkers of tumor progression and adverse clinical outcome in oral cancer, as well as novel therapeutic targets in cancer.

Prognostic value of the DNMTs mRNA expression and genetic polymorphisms on the clinical outcome in oral cancer patients.

OBJECTIVES: Although the importance of the epigenetic changes in tumors, including oral squamous cell carcinomas (OSCCs), is now becoming apparent, the mechanisms that trigger or cause aberrant DNA methylation in cancer are still unrevealed. DNA methylation is regulated by a family of enzymes, DNA methyltransferases (DNMTs). DNMT gene expression analysis, as well as genetic polymorphisms, has not been previously evaluated in OSCC. MATERIALS AND METHODS: In 65 OSCC patients, SYBR Green real-time PCR method was assessed for relative quantification of DNMT1, DNMT3A, and DNMT3B mRNAs, normalized to TATA-binding protein (TBP) mRNA. The expression levels of all three genes were dichotomized as high or low, with a twofold change of normalized mRNA expression used as the cutoff value. Polymorphisms in DNMT1 (rs2228612) and DNMT3B (rs406193) were analyzed in 99 OSCCs by TaqMan SNPs genotyping assays. RESULTS: DNMT1, DNMT3A, and DNMT3B were overexpressed in 36.9, 26, and 23 % of the OSCC patients, respectively. DNMT1 overexpression was significantly associated with the overall survival, p = 0.029, and relapse-free survival of OSCC patients, p = 0.003. Patients with DNMT1 overexpression, as an independent prognostic factor, had a 2.385 times higher risk to relapse than those with lower expression. The DNMT1 A201G gene polymorphism was associated with a reduced overall survival in OSCC patients, p = 0.036. CONCLUSIONS: Our results suggest that DNMT1 could play an important role in modulating OSCC patient survival. CLINICAL RELEVANCE: DNMT gene expression could be a potential prognostic marker that might lead to an improvement in diagnosis, prognosis, and prospective use of epigenetic-targeted therapy of OSCC.

Genetic Polymorphisms of Catechol-O-Methyltransferase: Association with Temporomandibular Disorders and Postoperative Pain.

AIMS: To evaluate the association between catechol-O-methyltransferase (COMT) gene polymorphisms and temporomandibular disorders (TMD), TMD pain, psychosocial impairment related to TMD, and postoperative pain. METHODS: A total of 90 patients with a diagnosis of painful TMD and 92 matched controls were investigated for the presence of TMD, TMD pain, and psychosocial variables by the Research Diagnostic Criteria for TMD. In a prospective cohort study of 40 subjects who underwent extraction of at least one fully impacted mandibular third molar, subjects had 6 months post-surgery follow-up of postoperative pain. DNA extracted from peripheral blood was genotyped for three COMT polymorphisms (rs4680, rs6269, and rs165774) by real-time TaqMan method. The association between COMT polymorphisms and clinical variables was determined by calculating odds ratios (OR) and their 95% confidence intervals (CI). RESULTS: Homozygous AA genotype and heterozygous variant A allele carriers (genotype AG/AA) for rs165774 polymorphism were associated with increased risk of TMD compared to wild type (wt) GG genotype (OR = 9.448, P = .006; OR = 2.088, P = .017, respectively). In addition, AA genotype was associated with increased risk of arthralgia (OR = 4.448, P = .011), myofascial pain (OR = 3.543, P = .035), and chronic TMD pain (OR = 6.173, P = .006), compared to wt genotype. AA genotype for rs6269 polymorphism was related to less postoperative chronic TMD pain (P = .025) and lower postoperative acute pain at the extraction site (P = .030). No associations with depression and somatization were observed. CONCLUSION: AA genotype of rs165774 could be a significant risk factor for the development of TMD and TMD pain, while AA genotype of rs6269 presents less postoperative chronic TMD pain and acute pain at a dental extraction site.

Cephalometric analysis of the middle part of the face in patients with mandibular prognathism.

BACKGROUND/AIM: The middle part of the face, that is the maxilla, has always been mentioned as a possible etiologic factor of skeletal Class III. However, the importance of the relationship of maxillary retroposition towards the cranial base is still unclear, although it has been examined many times. The aim of this study was to conduct cephalometric analysis of the morphology of maxilla, including the whole middle part of the face in patients with divergent and convergent facial types of mandibular prognathism, as well as to determine differences betweeen them. METHODS: Lateral cephalometric teleradiograph images of 90 patients were analyzed at the Dental Clinic of the Military Medical Academy, Belgrade, Serbia. All the patients were male, aged 18-35 years, not previously treated orthodontically. On the basis of dentalskeletal relations of jaws and teeth, the patients were divided into three groups: the group P1 (patients with divergent facial type of mandibular prognathism), P2 (patients with convergent facial type of mandibular pragmathism) and the group E (control group or eugnathic patients). A total of 9 cephalometric parameters related to the middle face were measured and analyzed: the length of the hard palate--SnaSnp, the length of the maxillary corpus--AptmPP, the length of the soft palate, the angle between the hard and soft palate--SnaSnpUt, the angle of inclination of the maxillary alveolar process, the angle of inclination of the upper front teeth, the effective maxillary length--CoA, the posterior maxillary alveolar hyperplasia--U6PP and the angle of maxillary prognathism. RESULTS: The obtained results showed that the CoA, AptmPP and SnaSnp were significally shorter in patients with divergent facial type of mandibular prognathism compared to patients with convergent facial type of the mandibular prognathism and also in both experimental groups of patients compared to the control group. SnaSnp was significantly shorter in patients with divergent facial type of mandibular prognathism compared to the control group, whereas SnaSnp was significantly smaller in patients with convergent facial type of mandibular prognathism compared to the control group. Additionally, there was a pronounced incisor dentoalveolar compensation of skeletal discrepancy in both groups of patients with mandibular prognathism manifested in the form of a significant upper front teeth protrusion, but without significant differences among the groups, while the maxillary retrognathism was present in most patients of both experimental groups. A pronounced UGPP was found only in the patients with divergent type of mandibular prognathism. CONCLUSION: The maxilla is certainly one of the key factors which contributes to making the diagnosis, but primarily to making a plan for mandibular prognathism treatment Accurate assessment of the manifestation of abnormality, localization of skeletal problems and understanding of the biological potential are key factors of the stability of/the results of surgical-orthodontic treatment of this abnormality.

Limb apraxia in multiple sclerosis.

BACKGROUND/AIM: There are almost no studies on apraxia in people with multiple sclerosis. Although the white matter is damaged in MS, it is not the only location in which the pathological changes are present. Demyelinated lesions in the cortex have recently been recognized as important components of multiple sclerosis pathology. The aim of this study was to determine whether apraxia is present among people with MS, and the importance of demographic characteristics and impairment of functional systems at conceptualization and execution of movements. METHODS: The experimental group consisted of 30 patients, mean age 51.34 +/- 7.70 years. The patients in the experimental group were diagnosed with MS according to the McDonald criteria. The control group consisted of 30 healthy subjects, mean age 50.30 +/- 10.47 years. For research purposes, we used the following instruments: Questionnaire for Collecting Demographic Data, Kurtzke Functional Systems Scores, Waterloo-Sunnybrook Apraxia Battery (WatAB). Execution of motion tasks that are a part of the WatAB were incorporated in the System for the Observation and Analysis of Motor Behavior. RESULTS: Our study showed that limb apraxia was common in people with MS. Apraxia was present during pantomime in 26.70% of the patients, and during the imitation of movements in 44.80% of the patients. Gender, age, education level, duration of disease and a form of MS did not determine the quality of conceptualization and execution of movements. The time elapsed from the last exacerbation was a determinant of quality of executed movements. Impairments of functional systems predicted impairments of movement execution. The expanded disability scale score correlated with the severity of apraxia. CONCLUSION: Our study confirm the presence of apraxia in MS. It is necessary to carry out further studies using functional magnetic resonance imaging, as well as the conduct longitudinal studies to determine the precise structure of motor behavior in people with MS.

A cephalometric analysis of the cranial base and frontal part of the face in patients with mandibular prognathism.

BACKGROUND/AIM: The literature suggests different views on the correlation between the cranial base morphology and size and saggital intermaxillary relationships. The aim of this study was to investigate the cranial base morphology, including the frontal facial part in patients with mandibular prognathism, to clarify a certain ambiguities, in opposing viewspoints in the literature. METHODS: Cephalometric radiographies of 60 patients were analyzed at the Dental Clinic of the Military Medical Academy, Belgrade, Serbia. All the patients were male, aged 18-35 years, with no previous orthodontic treatment. On the basis of dental and sceletal relations of jaws and teeth, the patients were divided into two groups: the group P (patients with mandibular prognathism) and the group E (the control group or eugnathic patients). A total of 15 cephalometric parametres related to the cranial base, frontal part of the face and sagittal intermaxillary relationships were measured and analyzed. RESULTS: The results show that cranial base dimensions and the angle do not play a significant role in the development of mandibular prognathism. Interrelationship analysis indicated a statistically significant negative correlation between the cranial base angle (NSAr) and the angles of maxillary (SNA) and mandibular (SNB) prognathism, as well as a positive correlation between the angle of inclination of the ramus to the cranial base (GoArNS) and the angle of sagittal intermaxillary relationships (ANB). Sella turcica dimensions, its width and depth, as well as the nasal bone length were significantly increased in the patients with mandibular prognathism, while the other analyzed frontal part dimensions of the face were not changed by the malocclusion in comparison with the eugnathic patients. CONCLUSION: This study shows that the impact of the cranial base and the frontal part of the face on the development of profile in patients with mandibular prognathism is much smaller, but certainly more complex, so that morphogenetic tests of the maxillomandibular complex should be included in further assessment of this impact.

TMD in class III patients referred for orthognathic surgery: psychological and dentition-related aspects.

OBJECTIVE: To investigate temporomandibular disorders (TMD), psychosocial, and occlusal variables in class III orthognathic surgery patients with respect to the control subjects, and to compare psychosocial and occlusal features in class III patients with different Research Diagnostic Criteria for TMD (RDC/TMD) diagnoses. MATERIALS AND METHODS: The study enrolled 44 class III patients referred for orthognathic surgery and 44 individuals without a malocclusion. TMD, depression and somatization were assessed by RDC/TMD. Occlusal analysis included Helkimo's Occlusal Index items, overjet and overbite. RESULTS: In the controls, patients with class III deformities had higher prevalence of myogenic TMD, increased grade of chronic pain, and more occlusal deviations. Within the study group, TMD patients reported higher depression score (P < 0.01), myofascial pain was related to higher depression and somatization grades (P < 0.01, P < 0.05 respectively), and disc displacement showed relation with RCP-ICP slide interferences (P < 0.05). CONCLUSION: With respect to subjects without a malocclusion, TMD in class III dentofacial deformities is similar in prevalence, but differs in clinical appearance. Occlusal, but not psychosocial features deviate from those in the controls. While psychosocial variables accompanied TMD and myofascial pain, increased RCP-ICP slide was related to disc displacement in class III patients.

Health-related quality of life assessment in Serbian schoolchildren hospitalized for malignant disease.

BACKGROUND/AIM: Although long-term survival of childhood cancer patients is significantly improved, prolonged treatment and hospitalization might have negative impacts on child development. The aim of this study was to verify profile of health-related quality of life parameters in population of schoolchildren during hospitalization and treatment for malignant disease. METHODS: The Serbian version of Pediatric Quality of Life Inventory Version 4.0 (PedsQLTM4.0) Generic Core Scales was applied. A total of 120 schoolchildren were analyzed: 60 patients hospitalized for prolonged malignant disease treatment and 60 healthy schoolchildren from public schools. The study was done at the Institute for Oncology and Radiology of Serbia, as well as in four schools. RESULTS: Generally, schoolchildren hospitalized for cancer treatment demonstrated lower scores on physical, emotional, social and school functioning when compared to healthy schoolchildren from regular public schools. Significant differences were observed for all the 8 items of the Physical Health Scale, in 2 out of 5 items of the Emotional Functioning Scale, in 4 out of 5 items of the Social Functioning Scale, and 3 out of 5 items of the School Functioning Scale. CONCLUSIONS: The Serbian version of PedsQL 4.0 Generic Core Scales could be successfully used to evaluate physical, emotional, social and school functioning of hospitalized children and adolescent. Schoolchildren hospitalized for prolonged tumor treatment have poorer HRQOL scores compared to general healthy population, however the level of remaining physical, emotional and social parameters should provide solid foundation for their potential rehabilitation, education and inclusion.

Temporomandibular disorders after orthognathic surgery in patients with mandibular prognathism with depression as a risk factor.

OBJECTIVE: To examine the prevalence of temporomandibular disorders (TMD) after orthodontic-surgical treatment in patients with mandibular prognathism and analyze psychosocial variables related to TMD. MATERIALS AND METHODS: The case-control study comprised 40 patients with mandibular prognathism who underwent combined orthodontic-surgical treatment (orthognathic surgery group). Forty-two patients with untreated mandibular prognathism served as a control group. Research diagnostic criteria for temporomandibular disorders was used in order to assess the clinical diagnosis of TMD (Axis I) and to estimate depression, somatization and patient's disability related to chronic pain (Axis II). RESULTS: The overall prevalence of TMD was not significantly different between the groups. Myofascial pain was significantly higher, while arthralgia, arthritis and arthrosis was significantly lower in the orthognathic group compared with the controls (90.5% vs 50.0%, 0.0% vs 27.8%, respectively) (p < 0.05). Females in orthognathic surgery group showed higher prevalence of TMD (p < 0.05) and myofascial pain (p < 0.01) and increased level of chronic pain (p < 0.05) in comparison with post-operative males. No significant difference in chronic pain, somatization and depression scores was found between investigated groups. With respect to presence of TMD within the groups depression was higher in untreated subjects with dysfunction (p < 0.05). CONCLUSION: Prevalence of TMD immediately after completion of orthodontic-surgical treatment for mandibular prognathism is similar to frequency of dysfunction in untreated subjects, is significantly higher in females and is most commonly myogenic. Furthermore, females show an increased level of chronic pain post-operatively. Somatization and depression levels do not differ between patients with corrected prognathism and untreated prognathic patients.

Association of VEGF-A genetic polymorphisms with cancer risk and survival in advanced-stage oral squamous cell carcinoma patients.

BACKGROUND: Vascular endothelial growth factor (VEGF), a key mediator of angiogenesis, is overexpressed in a wide variety of human cancers, including oral squamous cell carcinoma (OSCC). In this study, we examined whether individual polymorphisms within VEGF-A gene, rs699947 (-2578C/A), rs1570360 (-1154G/A), rs2010963 (-634G/C), rs3025039 (+936C/T) or their haplotypes are associated with an oral cancer risk and survival. METHODS: A case-control study was conducted on 114 OSCC patients and control group of 126 individuals without a previous cancer history, all the Caucasian race and the same ethnicity, matched by age and gender. VEGF-A genotypes were analyzed using TaqMan SNP Genotyping Assays, Applied Biosystems. RESULTS: The -1154 GG genotype was significantly associated with the decreased overall survival in OSCC patients (p = 0.010, log rank test). Stratified analysis revealed that in patients with nodal metastases and stage III, -1154 GG genotype was related to worse survival, p = 0.009, p = 0.013, respectively. The multivariate analysis revealed that -1154 GG genotype is an independent adverse factor for survival in the OSCC (HR = 1.899, [1.138-3.168], p = 0.014). The +936 CC genotype was associated with advanced staged OSCC (p = 0.050). The three polymorphisms, -2578, -1154 and -634 were in linkage disequilibrium (LD). The CAG haplotype could be associated with an increased oral cancer risk, OR = 7.967, [1.730-36.689], p = 0.008, while CGG haplotype could be associated with a decreased oral cancer risk, OR = 0.561, [0.326-0.964], p = 0.036. CONCLUSIONS: VEGF-A -1154 GG genotype could be considered as a prognostic marker of poor survival in advanced-stage OSCC patients. Haplotypes of VEGF-A gene may be associated with susceptibility to OSCC.

Vitamin D receptor, CYP27B1 and CYP24A1 genes polymorphisms association with oral cancer risk and survival.

BACKGROUND: Genetic polymorphisms of vitamin D receptor gene (VDR) and genes involved in vitamin D metabolism pathway, CYP27B1 and CYP24B1, may affect individual susceptibility to oral squamous cell carcinoma. The purpose of this study was to evaluate the associations between VDR, CYP27B1 and CYP24A1 gene polymorphisms with oral cancer risk and survival. METHODS: Study cohort consisted of 110 patients with oral cancer and 122 healthy controls. The genotypes of the analysed genes were determined by PCR-RFLP or real-time PCR method. RESULTS: The significant decrease of oral cancer risk was observed in individuals with heterozygote genotype of CYP24A1 gene (rs2296241) (odds ratio 0.281, P = 0.000) in comparison with wild type. Patients with VDR FokI ff wild type genotype had significantly worse overall survival (P = 0.012, log rank) compared with heterozygous and mutated genotype combined. A stratified analysis by the lymph node involvement and tumour stage showed that ff is associated with poor survival in groups with and without lymph node involvement (P = 0.025, P = 0.040, respectively) and in stage III tumours (P = 0.026). Multivariate Cox's regression analysis revealed that VDR FokI could be considered an independent prognostic factor. CONCLUSION: Our findings indicate that CYP24A1 gene polymorphism might have an influence on the susceptibility to oral cancer. VDR FokI polymorphism was associated with worse survival and could be considered as an independent prognostic marker.

Prognostic significance of tumor-related genes hypermethylation detected in cancer-free surgical margins of oral squamous cell carcinomas.

Oral squamous cell carcinoma (OSCC) is characterized by high mortality rates. High incidence of local recurrences in the normal-appearing surgical margins of OSCC patients indicates the existence molecular alterations, including DNA methylation, which could not be detectable by histopathologic analysis. The objective of our study was to determine correlation of tumor-related genes hypermethylation detected in histopathologically negative surgical margins with clinical and prognostic parameters. The genes selected for methylation analysis covered a wide range cellular processes including cell cycle control (p16), apoptosis (DAPK and RASSF1A), Wnt signaling (APC, WIF1 and RUNX3), cell-cell adhesion (E-cad), and DNA repair (MGMT and hMLH1). All of 47 patients had histologically confirmed negative surgical margins. For each of patient, samples from primary malignant tissue and the two consecutive surgical margins were taken at the time of surgery. DNA methylation was determined by multiplex nested methylation-specific PCR. Twenty-seven patients were margin-positive for promoter hypermethylation of at least one gene under study. The presence of DAPK promoter hypermethylation detected in surgical margins was associated with the decreased overall survival (p=0.004, log rank test). Multivariate analysis revealed that DAPK promoter hypermethylation in surgical margins is an independent prognostic factor for overall survival, HR=4.105 (1.458-11.555, 95% CI, p=0.007). Hypermethylation of other tumor-related genes under study did not have prognostic significance. These results demonstrate that DNA hypermethylation in histologically negative surgical margins is a frequent event. Promoter hypermethylation of DAPK gene detected in surgical margins may be a useful molecular marker for the poor survival in OSCC patients. Further investigation into the therapeutic potential of these findings in OSCC is warranted.

Genetic polymorphisms of ADH1C and CYP2E1 and risk of oral squamous cell carcinoma.

OBJECTIVE: Several studies have suggested that the metabolism of alcohol is modulated by the polymorphisms in genes encoding ethanol-metabolizing enzymes, including alcohol dehydrogenase 1C, ADH1C, and cytochrome P450-dependent monooxygenase, CYP2E1. Genetic polymorphisms of ethanol-metabolizing enzymes may affect individual susceptibility to oral cancer. The purpose of this study was to investigate the associations between ADH1C and CYP2E1 gene polymorphisms with oral squamous cell carcinoma in an ethnically homogeneous Caucasian population. DESIGN: Case-control study. SETTING: Serbian national general hospital. SUBJECTS AND METHODS: The study was conducted on 123 oral cancer patients and a control group of 177 individuals of the Caucasian race and the same ethnicity, matched in age and gender, without previous cancer history. The control group consisted of 120 population-based and 57 hospital-based controls of heavy-drinking individuals. Genetic polymorphisms of ADH1C SspI, ADH1C HaeIII, CYP2E1 RsaI, and CYP2E1 Ins were determined by the polymerase chain reaction and restriction fragment length polymorphisms. RESULTS: After adjustment by potential confounders, the significant increase of oral cancer risk, independent of alcohol drinking, was observed in individuals with the variant ADH1C SspI*2/*2 genotype (odds ratio, 3.029; P = .014) and in combined ADH1C SspI*1/*2 and ADH1C SspI*2/*2 genotypes (odds ratio, 2.605; P = .002), compared to the ADH1C*1/1* wild type. The association of other polymorphisms under study was not observed. CONCLUSION: This study suggested that the ADH1C SspI polymorphism could play a significant role in the etiology of oral cancer, whereas ADH1C HaeIII, CYP2E1 RsaI, and CYP2E1 Ins could have minor influence.

Gene hypermethylation in tumor tissue of advanced oral squamous cell carcinoma patients.

Oral squamous cell carcinoma (OSCC) is characterized by high mortality rate and rising incidence. The aim of this study was to examine the methylation of particular tumor suppressor genes promoters in OSCC and to correlate the methylation status with the tumor-host features and patients survival. The genes selected for our investigation are involved in key cellular processes of malignant transformation, including cell cycle control (p16), apoptosis (Death Associated Protein Kinase, DAPK), Wnt signaling (Adenomatous Polyposis Coli, APC), cell-cell adhesion (E-cadherin, E-cad), and DNA repair (O(6)-methylguanine-DNA methyltransferase, MGMT, Werner syndrome gene, WRN). In 77 patients with OSCC, hypermethylation was determined by nested methylation-specific PCR method. Methylation of p16 gene promoter was detected in 58.4% of samples, E-cad in 42.9%, DAPK in 36.8%, MGMT in 33.8%, WRN in 23.8%, and APC in 18.2% of OSCC samples. Patients with E-cad promoter methylation had significantly worse overall survival (p=0.039, log-rank test), while hypermethylation of other genes did not have prognostic significance. Stratified analysis by the lymph node involvement and tumor stage showed that E-cad promoter methylation is associated to poor survival in N positive (p=0.024), and stage III tumors (p=0.027), as an opposite to N0 and stage II tumors, where E-cad methylation did not have prognostic significance (p=0.849, p=0.794, respectively). Our findings indicate that multiple genes are frequently aberrantly methylated in OSCC, and that E-cad promoter methylation should be considered as a potential molecular marker for the poor survival in advanced OSCC.

Clinical significance of vascular endothelial growth factor expression in patients with carcinoma of the mouth floor and tongue.

BACKGROUND/AIM: Although there are several types of malignant oral cancers, more than 90% of all diagnosed oral cancers are squamous cell carcinoma (OSCC). Angiogenesis is a cascade-like mechanism which is essential for tumor growth and metastasis. Therefore, vascular endothelial growth factor (VEGF) expression in OSCC and its effect on clinicopathological characteristics and prognosis is of major interest. So far researches have shown that increased expression of this gene, in other words enhanced sinthesis of this protein (VEGF), independently on other factors, increases a chance for local relapse, and distant metastasis. Consequently, patients with OSCC have poor disease-free survival, as well as poor overall survival. The aim of the study was to determine clinical significance of VEGF expression in patients with stage II and III OSCC. METHODS: This retrospective study analysed 40 patients who had been operated for OSCC of their tongue and the mouth floor. Of these patients, some had stage II and III OSCC with histological grade, G1-G3 and nuclear grade Ng1-Ng3. Two high quality tissue samples were obtained and immunohistochemical expression of VEGF was quantitatively determined by using high microscope amplification. The value of VEGF expression of 20% was rated as significant expression, whereas tumor cells reactivation less than 20% was considered very low or no expression at all. The patients were followed up for a 3-year period. RESULTS: The obtained results showed that 11 (17.5%) patients had VEGF expression less than 20% and 29 (82.5%) above 20%. A statistical significance was immanent with positive nodal status (p < 0.05) and disease stage (p < 0.05). No statistical correlation was found between the level of VEGF expression and histological and nuclear grade, tumor size, disease relapse or patients overall survival. CONCLUSION: Inspite the controversy about the prognostic relevance of VEGF our results as well as the results of previous studies, suggest that the expression of VEGF is not reliable as a clinical parameter for the prognosis and disease outcome but it is one of the important factors for the disease progression.

[Radiographic cephalometry analysis of dimensions of condylar processus in persons with mandibular prognathism].

INTRODUCTION/AIM: There are numerous factors that influence the formation of condylar processus: the growth and development of cranial base, growth and development of the jaws and alveolar extensions, teething, the way of intercuspidation, the overlap of incisors, functions of masticatory muscles, etc. Considering the fact that the above-mentioned factors significantly differ in persons with different morphological set of the face, we set a hypothesis that dimensions of condylar processus and the mandibular ramus considerably differ in persons with mandibular prognathism compared to eugnatic persons. The aim of this study was to establish the differences in dimensions of condylar processus between the above-mentioned groups. METHODS: Six parameters representing the dimensions of the condylar processus were measured on profile teleradiographs of 30 eugnatic persons and 30 paersons with mandibular prognathism: the height of condylar processus, the height of head of the mandible, width of the head, width of the neck, height of the ramus without the condylar processus and the overall height of the ramus. RESULTS: A considerable difference in the values of the parameters was found, as well as the distribution toward the values of reference. It was found that the height of the condylar processus was significantly greater in persons with mandibular prognathism, whereas the width of the head of the mandible, the width of the neck and the height of the ramus without the condylar processus was considerably decreased within the same group. The height of the head of the mandible and the overall height of the ramus was not significantly changed. CONCLUSION: In persons with mandibular prognathism, morphological features of the condylar processus are changed. The condylar processus lengthens on account of shortening of the lower part of the ramus, and the mentioned lengthening is the most prominent in its condylar neck area which is also the centre of its most intense growth.