Evaluation and Comparison of Genomic DNA Extraction Methods and PCR Optimization on Archival Formalin-Fixed and Paraffin-Embedded Tissues of Oral Squamous Cell Carcinoma.

Recovery and amplification of nucleic acids from archived formalin-fixed tissue samples is the most developing field in retrospective genetic studies. We compared different deparaffinization methods and DNA isolation techniques, and intergroup comparisons were performed to evaluate the effectiveness of different storing methods for archival OSCC samples based on obtained mean DNA quantity, quality, and PCR amplification of the P53 gene. The study comprised 75 archival histologically diagnosed OSCC samples which were divided into Group I: Formalin-fixed paraffin-embedded tissue blocks and Group II: Long-term formalin-fixed tissue. A comparison of different deparaffinization methods showed that xylene deparaffinization is an efficient method to obtain suitable DNA. Comparing different DNA isolation techniques illustrated that the conventional phenol-chloroform method gives better integrity to DNA in contrast with the kit method. Comparison between FFPET and long-term FFT samples demonstrated that samples fixed in formalin overnight and embedded in wax yield better quality and quantity DNA in comparison with long-term samples fixed in formalin. To obtain suitable integrity of DNA, tissue samples should be stored by fixing in formalin overnight followed by preparation of paraffin tissue blocks, deparaffinization by xylene, and subjecting them to the conventional phenol-chloroform DNA isolation protocol.

Candidal Prevalence in Diabetics and its Species Identification.

CONTEXT: Candida species is a part of the normal mouth flora. Diabetes mellitus is a predisposing factor for the onset of oral candidiasis. AIM: The objective of this study was to estimate salivary glucose in patient with diabetes and healthy individuals, to determine total candidal counts as well as different candidal species in the saliva of patients with diabetes and nondiabetics. SETTINGS AND DESIGN: A sample size of 80 patients was taken. Out of 80 patients, 30 patients were uncontrolled diabetes patients (Group I), 30 patients were controlled diabetes patients (Group II), and 20 patients were healthy individuals (Group III). SUBJECTS AND METHODS: From all the salivary samples, salivary glucose estimation was done using the glucose oxidase-peroxidase method. Candidal colony-forming units (CFUs) were determined from all the salivary samples. CHROMagar Candida medium was used for the identification of various Candida species. STATISTICAL ANALYSIS USED: One-way ANOVA was used to test for differences between the means of the three groups. Pearson's correlation coefficient test was used to evaluate the relationships between the variables. RESULTS: The mean salivary glucose levels were higher in Group I followed by Group II and Group III. The mean candidal CFU was significantly higher in Group I than in Group II and Group III. CONCLUSION: Candida glabrata was the predominant species found and more so in patients with diabetes and needs further study. Other common species isolated was C. albicans. Species identification can help in better treatment strategies and gain good control over the disease.

Quantification of circulating plasma cell free DNA fragments in patients with oral cancer and precancer.

PURPOSE: Study was aimed to quantify plasma level of total, short and long fragmented cell-free DNA (cfDNA) along with DNA integrity in patients with oral cancer, oral precancer and tobacco users without lesions and normal controls. In addition, study evaluated the correlation of cfDNA with clinicopathologic parameters of oral cancer. METHODOLOGY: Plasma samples were collected preoperatively from 44 patients with oral cancer, 40 patients with oral precancer, 40 tobacco users without any oral lesion and 40 healthy controls without any tobacco habit. cfDNA extraction was carried out from the plasma followed by quantitative and qualitative assessment of extracted DNA. Quantity of short and long fragmented DNA was assessed by using PCR with two different primer sets for the beta-actin gene, amplifying short (102 bp) and long (253 bp) products. The DNA integrity index was measured by calculating the ratio of quantity of long fragmented to short fragmented DNA. All quantitative cfDNA parameters were statistically analyzed to verify their correlation with clinicopathologic parameters. RESULTS: Results showed that total cfDNA level, short and long fragmented cfDNA concentration and DNA integrity was significantly higher in oral cancer group as compare to other (p=0.0001). Study demonstrated that there is no correlation total, short and long cfDNA and DNA integrity with tumor size and histologic type or grading. But positive correlation of total cfDNA was found with nodal metastasis (p=0.001) and clinical stages (p=0.006). CONCLUSION: Quantitative analysis of total cfDNA may be applied as a screening marker for early detection of precancer and cancer as well as for prognostication of oral cancer. Additionally, plasma levels of short and long fragmented cfDNA and DNA integrity index can be applied for early detection of oral cancer.

Effect of Conventional and Microwave Tissue Processing Technique on DNA Integrity: A Comparative Molecular Analysis.

BACKGROUND: Methods of diagnostic molecular biology are routinely applied on formalin-fixed, paraffin-embedded tissues processed via conventional method. Recently, there has been a growing interest to use microwave technology in histopathology laboratories to overcome the deficiencies of the conventional processing method. Thefore, this study was aimed to compare and analyze the quality and quantity of DNA obtained from tissues processed by conventional and microwave tissue processing techniques and to further ascertain the applicability of the latter for PCR (polymerase chain reaction based research). METHODS: Thirty fresh tissues of oral squamous cell carcinoma (OSCC) were included, and each sample was cut into two equivalent halves. One tissue half was processed by conventional manual method whereas the other half was processed using a domestic microwave oven. DNA was obtained from all the tissues which were then subjected to Polymerase chain reaction (PCR) to evaluate GAPDH (Glyceraldehyde-3-phosphate dehydrogenase) gene expression. RESULTS: The results revealed better DNA yield from microwave processed tissue while the quality of the DNA was alike from both the techniques. CONCLUSION: On the basis of the results obtained, it can be concluded that DNA produced by microwave processed tissues was similar to that obtained by conventional processing technique in terms of quantity and quality. Thus, microwave processed tissue samples can be successfully used for further molecular studies and researches.

Evaluation of role of fixed orthodontics in changing oral ecological flora of opportunistic microbes in children and adolescent.

OBJECTIVE: To determine the prevalence and counts of Streptococcus mutans and Candida species in orthodontic fixed appliance therapy patients and comparison of the efficiency of manual and electronic toothbrushes on minimizing plaque by reducing S. mutans and Candida species in above-mentioned patients. MATERIALS AND METHODS: The study was carried out on total number of sixty patients scheduled for orthodontic treatment (age group: 13-18). Plaque samples were collected to determine oral carriage of S. mutans and Candida of these patients, and readings were noted at T0 (before appliance placement), 2nd and 3rd month after the placement of appliances (T1 and T2). Counts of S. mutans were determined using Dentocult SM kit. Candida was cultured on Sabouraud's dextrose agar. The participants in the study group (orthodontic patients) were then divided into two groups each of 30: Group 1 (manual toothbrush), Group 2 (electronic toothbrush), and plaque samples were taken at the end of 1st, 2nd, and 3rd month to determine the efficacy of manual versus electronic toothbrushes. RESULTS: After orthodontic treatment, colonization of S. mutans and Candida increased dramatically. Results for S. mutans counts and Candida clearly showed superiority of electronic tooth brushing over manual tooth brushing during orthodontic treatment. CONCLUSION: Results clearly showed that orthodontic appliances increase colonization of S. mutans and Candida albicans in oral cavity over the period of treatment time which can be controlled with proper timely brushing. The study also concludes superiority of electronic tooth brushing over manual tooth brushing.

Evaluation of changes in Streptococcus mutans colonies in microflora of the Indian population with fixed orthodontics appliances.

BACKGROUND: Orthodontic therapy has oral ecological changes causing increased numbers of mutans streptococci in saliva and plaque. The purpose of this study was to estimate counts and colonization pattern of Streptococcus mutans after application of fixed orthodontic appliances. MATERIALS AND METHODS: Plaque samples of randomly selected sixty patients were collected before placement of orthodontic appliances from buccal and labial aspects of the anterior teeth and four first molars and readings were recorded as T0. After placement of appliances (0.22 MBT preadjusted Gemini), i.e., 2(nd) and 3(rd) month, the plaque samples were collected again from same site and readings were recorded as T1 and T2, respectively. Counts of S. mutans in these patients were determined by using DM Strips (Orion Diagnostic, Espoo, Finland). Kruskal-Wallis test and Mann-Whitney U-test were used to find out significant differences between different time interval for Dentocult score for S. mutans in orthodontic patients (P < 0.001). RESULTS: Prior to the treatment, 46 patients (76%) showed mild and 14 patients (24%) showed moderate colonization of S. mutans. After treatment, the severity of colonization increased showing fifty patients (84%) moderate and six patients (10%) showing severe colonization of S. mutans at T1, which further increased in severity at T2 with 54 patients (90%) showing severe colonization with S. mutans. CONCLUSION: Results showed that fixed orthodontic appliance increases colonization of S. mutans during orthodontic treatment.

Exploratory Study to Evaluate Changes in Serum Lipid Levels as Early Diagnostic and/or Prognostic Indicators for Oral Submucous Fibrosis and Cancer among Gutkha Consumers in India.

BACKGROUND: In India smokeless tobacco users account for 25.9% of total tobacco use, Gutkha being the most common form. This study evaluated the association between serum lipid profile and Gutkha use as an early diagnostic and/or prognostic indicator for oral submucous fibrosis (OSMF) and oral cancer (Oral Ca) in non-smokers and non-alcohol consumers. MATERIALS AND METHODS: A total of 163 participants were recruited from two sites in India. Participants were divided into four groups: individuals without any Gutkha chewing habit and without any oral lesions (control group), individuals with Gutkha chewing habit but without any oral lesions (GWL), patients with a confirmed clinical diagnosis of OSMF, and patients with a confirmed diagnosis of Oral Ca. Mixed linear modelling (MLM) was conducted to detect the change in mean serum lipid levels among four groups. RESULTS: The sample consisted of 69% males and 31% females. Results of MLM show an inverse relationship between serum lipid levels in OSMF, and Oral Ca groups when compared to the control group. CONCLUSIONS: The results of our study demonstrated that GWL, OSMF and Oral Ca patients had lower serum lipid levels. Low serum lipid levels could be an indicator of initial neoplastic changes in oral cancer.

Vinegar as a disinfectant of extracted human teeth for dental educational use.

CONTEXT: Extracted human teeth are routinely used in dentistry to learn technical and preclinical skills. Centers for Disease Control and Prevention (CDC) has adopted guidelines for infection control of extracted teeth used for research and teaching, requiring that teeth be sterilized before use. Many of the proposed disinfection methods starting from use of formalin, sodium hypochlorite and to autoclaving have their own drawbacks and may not be practical. AIM: To assess the effectiveness of vinegar for disinfection/sterilization of extracted human teeth. MATERIALS AND METHODS: A total of 80 extracted non-carious human teeth were kept in seven disinfectant media-10% formalin, 3% hydrogen peroxide, 5.25% sodium hypochlorite, 70% alcohol, vinegar, neem extract and normal saline. Ten samples were placed in each disinfectant individually for a period of 7 days, at room temperature. In all, 10 teeth were treated with microwave irradiation at 650 W for 3 min. Later, teeth from each group were placed individually in separate test tubes containing 10 ml of tryptic soy broth at 37°C for 48 h to observe the evidence of growth of microorganisms. Semiquantitative analysis of all the samples was done in Clade agar at 37°C for 48 h. STATISTICAL ANALYSIS USED: The number of teeth disinfected in each group was compared using Chi square test. RESULTS: 10% formalin, 3% hydrogen peroxide and vinegar were totally effective. The result was statistically significant with a Chi square value of 61.414 and P < 0.001. CONCLUSIONS: Vinegar can be used as an effective disinfectant medium for extracted human teeth.

Assessment of tissue eosinophilia as a prognosticator in oral epithelial dysplasia and oral squamous cell carcinoma-an image analysis study.

Association of tissue eosinophilia with oral squamous cell carcinoma has shown variable results ranging from favourable to unfavourable or even having no influence on prognosis. Also, very few studies have been done to know the role of eosinophils in premalignancy. So the present study investigated role of eosinophilic infiltration in oral precancer and cancer and its possible use as a prognosticator. 60 histopathologically proven cases (20 cases each of metastatic and nonmetastatic oral squamous cell carcinoma and oral leukoplakia with dysplasia of various grades) were included. Congo red is used as a special stain for eosinophils. Each specimen slide was viewed under high power in 10 consecutive microscopic fields for counting of eosinophils. As a result, a significant increase in eosinophil count was found in oral carcinomas compared to dysplasia. Nonmetastatic cases showed higher counts than metastatic carcinomas. So, it is concluded that eosinophilia is a favourable histopathological prognostic factor in oral cancer. Moreover, higher eosinophil counts in carcinoma group compared to dysplasia group proved that they might have a role in stromal invasion thus suggesting that quantitative assessment of tissue eosinophilia should become a part of the routine histopathological diagnosis for oral precancer and OSCC.

Stromal myofibroblasts in nonmetastatic and metastatic oral squamous cell carcinoma: An immunohistochemical study.

BACKGROUND AND AIMS: Myofibroblasts are one of the important components of the tumor microenvironment which could possibly play an important role in tumor progression. The purpose of this study was to compare the presence of α-smooth muscle actin (α-SMA) and CD34 positive fibroblasts in nonmetastatic and metastatic oral squamous cell carcinoma and to evaluate their role in tumor metastasis. MATERIALS AND METHODS: Ten cases each of histologically proven metastatic and nonmetastatic oral squamous cell carcinoma formed the study group. The tissue sections were stained immunohistochemically for α-SMA and CD34. The stromal spindle cells positive for these markers in the study groups were counted and compared. RESULTS: α-SMA positive cases were more in the metastatic group and CD34 positive cases were found to be more in the nonmetastatic tumors. CONCLUSIONS: Though difference in the staining pattern was statistically nonsignificant, the inverse relationship between α-SMA and CD34 positive cells is indicative of dynamic nature and the influence of tumor stroma in tumor progression and metastasis.

Cancer Therapy: A Continuance of Health Burden.

BACKGROUND: Cancer diagnosis coupled with emotional impact converge to create one of the most difficult physical and emotional periods of life. Cancer treatment causes plethora of short and long term complications which can be so debilitating that patient may interrupt treatment. Pretreatment oral assessment and supportive oral care during and after cancer therapy can increase quality of life and supportive care costs. METHODS: Study was conducted on 189 patients (86: head and neck cancer cases, group I and 103: other than head and neck cancer cases, group II) receiving cancer therapy. Patients were subjected to clinical assessment and findings were recorded in specially designed proforma and complete oral (objective and subjective) and constitutional findings were recorded. RESULTS: Among the patients undergoing chemotherapy in both groups, prevalence of oral findings was found to be highest with methotrexate whereas constitutional symptoms was found to be highest with doxyrubicin. Whereas in radiotherapy patients subjective and objective oral symptoms increased from 10th - 30th fractionated dose of radiations and then subsequently decreased and constitutional symptoms were found to be consistent in all fractionated dosages with lowest at 50th fraction. Under combined chemo and radiotherapy patients, constitutional symptoms were highest than the oral findings. CONCLUSIONS: Cancer therapy can greatly damage the normal tissues and diminish patients quality of life and often leads to serious clinical sequelae. Therefore, therapy induced damage should be anticipated and prevented whenever possible and managed early.

Osteoid osteoma of mandible.

Osteomas are benign, slow-growing osteogenic tumours rarely occurring in the craniofacial bones. Osteomas are characterised by the proliferation of compact and/or cancellous bone. It can be of a central, peripheral, or extra skeletal type. The peripheral type arises from the periosteum and is rarely seen in the mandible. The lingual surface and lower border of the body are the most common locations of these lesions. They are usually asymptomatic and can be discovered in routine clinical and radiographic examination. In this paper, the authors present a large solitary peripheral osteoma located in the inferior surface of the left angle of mandible and causing facial deformity in a 14-year-old boy. Radiographic examination by diagnostic radiographs revealed mixed appearance (radiopaque- radiolucent), well-circumscribed, pedunculated mass approximately 3.5 cm in size. The osteoma was removed surgically, and no recurrence has been observed.