Immunolocalization of epithelial cell adhesion molecule and matrix metalloproteinase-9 in oral epithelial dysplasia and oral squamous cell carcinoma.

INTRODUCTION: Cancers are complex tissues composed of multiple distinct cell types that participate in heterotypic interactions with one another. Physiologically cell-to-cell contacts formed by dense populations of normal cells operate to suppress further cell proliferation. OBJECTIVES: The objective of the study is to evaluate and compare the immunoexpression of matrix metalloproteinase-9 (MMP-9) and epithelial cell adhesion molecule (EpCAM) in oral epithelial dysplasia (OED) and oral squamous cell carcinoma (OSCC) and to hypothesize their role in the progression in varying grades of these lesions. MATERIALS AND METHODS: A total of 60 samples comprising of 30 cases each of OED and OSCC. Three micrometers thin sections were taken and subjected for hematoxylin and eosin stain and immunohistochemical procedure. The sections were incubated with monoclonal anti-EpCAM anti-MMP-9 antibody. The data were analyzed using SPSS software version 19. RESULTS: The results of the study show EpCAM immunoexpression decreased in OSCC when compared to OED. MMP-9 immunoexpression increased in OSCC when compared to OED (statistically significant, P ≤ 0.05). CONCLUSION: Correlation between EpCAM and MMP-9 may help to unravel the signaling cascades involved in the carcinomatous changes, tumor cell invasion, and progression of OSCCs.

Biochemical analysis of cytokeratin fragment 21-1 concentration and expression of cytokeratin 19 in oral potentially malignant disorders.

INTRODUCTION: Cytokeratin fragment 21-1 (CYFRA21-1), a constituent of the intermediate filament protein is known to be elevated in cancer. In vitro cleavage of cytokeratin 19 (CK19) protein results in the release of it's fragments into the supernatants of premalignant cell lines. This study was designed with the aim to investigate the concentrations of CYFRA21-1 in serum and saliva of oral potentially malignant disorders (OPMD), to evaluate CK19 expression in tissues of the same patients and to correlate the levels of CYFRA21-1 concentration in serum and saliva with CK19 expression in OPMDs, and to compare it with oral squamous cell carcinoma (OSCC), which was taken as positive control. MATERIALS AND METHODS: Concentration of CYFRA21-1 was measured in saliva and serum of 30 OPMD cases with five patients having OSCC using ELISA technique and analysis of CK19 protein expression in the tissue of same patients using immunohistochemical technique was done. RESULTS: Concentration of CYFRA21-1 in saliva and serum with regard to CK19 protein expression in tissues was significantly higher in control group than in study groups. CONCLUSION: CYFRA21-1 can be used as a promising diagnostic molecule and as an adjunctive marker for early detection, disease staging, and monitoring.

Immunohistochemical expression of p53 and murine double minute 2 protein in odontogenic keratocyst versus variants of ameloblastoma.

INTRODUCTION: Oncogenes and tumor suppressor genes play a major role in cancer formation, growth, and progression. One of the important findings in this area is that murine double minute 2 (MDM2) oncogene is a negative regulator of wild-type p53. In tumors, expressing wild-type p53, inhibition of MDM2 expression will stabilize p53 and allow it to perform its proapoptotic function, while simultaneously preventing MDM2 from exerting its p53-independent oncogenic effects. The intracellular levels of p53 are tightly regulated by MDM2, as it is a key player in autoregulatory feedback loop under nonstressed conditions. The p53-MDM2 relationship is vital not only for essential functions of the cell, but it also appears to be an integrated part of the complex cellular network which supports the importance of this affair and is a hallmark for its coexistence. SUBJECTS AND METHODS: This study was designed to identify immunohistochemically the expression of p53 and MDM2 gene using monoclonal antibody in 60 cases of formalin-fixed paraffin-embedded tissue blocks, of which 20 cases were of solid multicystic ameloblastoma (SMA), 20 cases were of odontogenic keratocyst (OKC), and 20 cases were of unicystic ameloblastoma (UA). RESULTS: Immunoexpression of p53 and MDM2 was highest in OKC followed by SMA and was minimum in UA. Further results showed positive correlation between both the molecules. CONCLUSION: The studied showed that the relationship has a significant role in cancer etiology and progression and therefore is an important topic for future research which should help in the development of new therapeutic agent against cancer.

Utility of DNA-Specific Stains in Micronuclei Assay as a Marker of Genotoxicity in Oral Potentially Malignant Disorders and Oral Squamous Cell Carcinoma.

BACKGROUND: The term oral potentially malignant disorder (OPMD) was recommended to refer to precancer as it conveys that not all disorders described under this term may transform into cancer. Oral squamous cell carcinoma (OSCC) arises through an accumulation of genetic alterations, deoxy ribonucleic acid (DNA) changes, and epigenetic alterations. Thus, a simple yet a sensitive and specific test for early diagnosis is the need of an hour. The micronuclei (MN) assay in exfoliated epithelial cells is potentially an excellent biomarker to detect chromosome loss or malfunction of mitotic spindle. AIM OF THE STUDY: To compare the frequency of MN in exfoliated cells from oral mucosa exposed to genotoxic agents using different staining procedures and to observe the incidence of micronucleus in potentially malignant and malignant lesions. MATERIALS AND METHODS: The study was undertaken to observe the cytogenetic damage in the exfoliated buccal cells of 75 cases of tobacco-related PMDs, OSCC and control subjects (25 cases from each group) and were evaluated with nonspecific May-Grünwald Giemsa stain and DNA-specific Feulgen stain. The results were statistically determined using SPSS version 17.0. RESULTS: Correlation analyses in the present study depicted that MN frequency was significantly more in oral squamous cell carcinoma than OPMDs and normal group (P < 0.05). Giemsa-stained slides correlated significantly with karyorrhexis, karyolysis, condensed chromatin, and binucleates, whereas no such correlations were found with DNA-specific stains. CONCLUSION: Malignant transformation is accompanied by loss of cell capacity to evolve to death in situations of DNA damage. These findings indicate that nuclear anomalies may be misinterpreted as MN with nonspecific DNA stains and lead to false-positive results in studies with cells of epithelial origin.

Papilliferous Keratoameloblastoma: A Rare Case Report.

Ameloblastoma is true odontogenic tumor of epithelial origin, which is described as locally aggressive with varying chances of recurrence. It is believed to derive from enamel organ, remnants of dental lamina, lining of odontogenic cysts, or basal cells of oral epithelium. Radiologically, it may present as unilocular or multilocular radiolucency commonly. Although conventional ameloblastoma presents typical histological features as described by Vickers and Gorlin, few unusual variants have been reported with different histological patterns. However, the clinical and biological behavior of these lesser known variants has not been established yet due to the scarcity of cases reported. Here, we report an extremely rare case of papilliferous ameloblastoma in a young male patient with 2-year follow-up and presenting with unusual histological presentation than conventional ameloblastoma.

Antioxidant-Oxidant Index as a Biomarker in Oral Potentially Malignant Disorders and Oral Squamous Cell Carcinoma: A Biochemical Study.

INTRODUCTION: Oxidative stress has been implicated in tumourigenesis by affecting the actions of various protein kinases and transcription factors, and resulting in damage to DNA structure by the deamination of nucleotides and the generation of free radicals. Derangements in the antioxidant defence mechanism have been considered in the pathogenesis of oral cancer. AIM: To evaluate and compare the serum levels of nitric oxide and vitamin C in cases of Oral Potentially Malignant Disorders (OPMDs) and Oral Squamous Cell Carcinoma (OSCC) and healthy controls. MATERIALS AND METHODS: Serum from 20 patients with OSCC, 20 patients with OPMDs and 10 healthy subjects were analyzed for levels of nitric oxide and vitamin C using UV spectrophotometry. Antioxidant-Oxidant Index (AOI) was calculated by calculating the ratio between the levels of nitric oxide and vitamin C. RESULTS: The mean nitric oxide levels were elevated in OSCC group, as compared to OPMD and control group, whereas, mean serum vitamin C levels were reduced in OSCC group as compared to OPMDs and control group (statistically significant, p<0.05). There was significant increase in AOI from control group (0.023), OPMDs (0.167) and OSCC group (0.279) (p<0.05). CONCLUSION: Assessment of levels of antioxidant oxidant markers are useful tools in monitoring the progression of OPMDs to OSCC. AOI is an objective tool to assess the oxidative stress in these patients.

Redefining epithelial characterization in oral submucous fibrosis using morphometric analysis.

BACKGROUND: Oral submucous fibrosis (OSMF) is a chronic oral mucosal disease characterized by progressive deposition of collagen in subepithelial connective tissue and epithelial atrophy. The present study was conducted to evaluate the changes in epithelial thickness and cellular and nuclear morphometric characteristics of parabasal and spinous compartments of the epithelium in various stages of OSMF in comparison to normal epithelium. MATERIALS AND METHODS: Sample included 30 cases of OSMF of various stages and five cases of normal buccal mucosa. Measurements for epithelial thickness and cellular morphology using morphometric analysis were performed on captured digital images using image analysis software. STATISTICAL ANALYSIS: The ANOVA analysis of epithelial thickness and correlation of epithelial thickness with cell contour and cell roundness was done using Karl Pearson's coefficient of correlation. RESULTS: There was a statistically significant decrease in epithelial thickness with increase in severity/stage of OSMF. It was also observed that there is a highly statistically significant correlation (P = 0.000) between the thickness of epithelium and cell morphology in varying stages of OSMF. CONCLUSION: The morphometric parameters and the use of quantitative histological methods in determining the squamous epithelial structures thus made it possible to evaluate that there is reduction in thickness of epithelium or "atrophy" with increasing stages of OSMF which may be due to actual change in cell morphology of the individual cells of specific compartment or all compartments in the epithelium.

Expression of E-cadherin and B-cell lymphoma 2 in oral cancer: A ratio-based planning for targeted therapy.

BACKGROUND: Oral cancer is known to be governed by the antiapototic and loss of cell adhesion properties which dictate its progression. AIM: To study the immunexpression of E-cadherin and Bcl-2 in varying TNM stages and histopathological grades of OSCC. MATERIALS AND METHODS: 11 cases of well differentiated, 10 cases of moderately differentiated and 11 cases of poorly differentiated OSCC were studied immunohistochemically using archival paraffin embedded tissue specimens. STATISTICAL ANALYSIS: Differences between the different variables were analyzed using ANOVA test, Kruskal-Wallis test and post hoc test followed by Bonferroni test. The resulting data was analyzed using SPSS software version 19. RESULTS: The expression of Bcl-2 and E cadherin immunopositivity was associated positively with tumor grade, high T category and Histopathological grades. CONCLUSIONS: The results of this study points to the significance of cell proliferation and invasion as a major determinant of prognosis in OSCC.

E-cadherin-mediated impairment increases anti-apoptotic mechanism through upregulation of Bcl-2: An immunohistochemical study in various patterns of invasion of oral squamous cell carcinoma.

BACKGROUND: Bcl-2 and E-cadherin proteins are known to be involved in the control of apoptotic cell death and invasive potential, respectively, which is an important hallmark of tumor regulation that influences their biologic behavior. AIM: This study investigates the relationship of Bcl-2 and E-cadherin immunoexpression in various Bryne's patterns of invasion. MATERIAL AND METHODS: Immunohistochemical analyses for Bcl-2 and E-cadherin were performed on paraffin-embedded tissue sections on 40 cases (32 cases of Oral squamous cell carcinoma and eight cases of controls) and were scored using qualitative and quantitative (percentage positive) analysis. STATISTICAL ANALYSIS: The resulting data were analyzed using SPSS software version 19. Correlation between patterns of invasion and qualitative scores of Bcl-2 and E-cadherin was calculated using Spearman rho correlation. Difference of mean percentage of positive cells of Bcl-2 and E-cadherin in different patterns of invasion was tested by ANOVA followed by Tukey HSD test. RESULTS: Bcl-2 and E-cadherin immunoreactivity was positively correlated with Bryne's pattern of invasion (P value<.05). An inverse relation was found between Bcl-2 and E-cadherin expression with Bryne's patterns 1-5 of invasion. CONCLUSIONS: The results pointed to the antagonistic role of E-cadherin and Bcl-2 and thus provide the opportunity for cell survival along with increased invasive potential.

Orthokeratinized odontogenic cyst masquerading as dentigerous cyst.

The orthokeratinized odontogenic cyst (OOC) is a rare developmental odontogenic cyst that has been considered as a variant of the keratocystic odontogenic tumor until Wright (1981) defined it as a different entity. Recognition of OOC as a unique entity has long been due, yet its inexplicable clinical, radiographic presentation resembling dentigerous cyst due to its association to an impacted tooth, and its histological features makes it rather perplexing. This is the report of a case of OOC in relation to an impacted maxillary canine and its immunohistochemical analysis with Ki-67.

To analyze the concomitant expression of human papillomavirus-16 in the pathogenetic model of p53-dependant pathway in oral squamous cell carcinoma.

BACKGROUND: Oral squamous cell carcinoma (OSCC) accounts for 90% of all primary oral malignancies. Association between human papillomavirus (HPV) as a risk factor of cervical cancer is well known; there is a need to widen the knowledge for its role in oral cancer development. The viral E6 protein of HPV binds to p53 making it nonfunctional. AIMS AND OBJECTIVE: To study mutated/wild type p53 expression using immunohistochemistry and detect HPV-16 presence using polymerase chain reaction (PCR), in OSCC and correlating their expression. MATERIALS AND METHODS: Immunohistochemical staining for p53 molecule in 24 sections of OSCC followed by DNA extraction of the cases using qiagen extraction kit and subsequent HPV-16 detection using PCR technique. STATISTICAL ANALYSIS: The data were analysed using SPSS software version 19. RESULTS: Out of 24 cases of OSCC, twenty cases were positive for P 53 expression and four cases were negative for P 53 expression. Out of the four negative cases, one case was detected positive for HPV-16. CONCLUSION: HPV infection along with p53 expression helps in understanding its exact pathogenesis which further helps in expanding our spectrum of therapeutic modalities.

Characterization and localization of c-kit and epidermal growth factor receptor in different patterns of adenoid cystic carcinoma.

INTRODUCTION: Adenoid cystic carcinoma (ACC) is malignant neoplasm of the salivary glands. It accounts for most cases of minor salivary gland malignancies and a substantial proportion of parotid and submandibular gland malignancies. ACC is associated with a high mortality rate, and it often recurs after prolonged periods of time, and this occurs even when radical excision has been performed. AIMS: The present study was aimed to determine the localization of dual cell population and to analyze the potency of using a system of dual markers (c-kit and epidermal growth factor receptor [EGFR]) in enhancing the characterization of ACC. SUBJECT AND METHOD: Three micrometer thin sections of adenoid cystic carcinoma were obtained. One set of slides was stained by hematoxylin and eosin for reconfirmation of histological diagnosis while the other two sets were stained for c-kit and EGFR using immunohistochemical method. Statistical Analysis Used and Results: Show c-kit expression to be limited to the inner ductal epithelial cells and the EGFR expression mainly to the outer myoepithelial cells in the majority of tubular and cribriform patterns. In solid ACC, c-kit was uniformly positive while EGFR was consistently negative. CONCLUSIONS: C-kit and EGFR biomarkers can be used to enhance the characterization of ACC and to determine the localization of dual cell population which could suggest the dual origin of ACC and provides evidence for the new therapeutic strategy in ACC.

Oral exfoliative cytology as a screening tool for iron overload in ß-thalassemia patients.

BACKGROUND: Increased iron overload is frequent problem in thalassemia patients, and this is monitored by serum ferritin levels or chemical assessment of the iron levels in liver tissue. However, repeated monitoring of serum ferritin levels to assess the iron overload is an invasive procedure associated with practical problems. AIMS: To use Perl's Prussian blue reaction to evaluate the iron overload in beta-thalassemia patients by staining the oral cytosmears. MATERIALS AND METHODS: The study comprised 35 patients diagnosed with beta-thalassemia. Cytosmears were prepared from exfoliated oral epithelial cells, fixed in 70% ethanol and stained with Perl's Prussian blue stain for detection of blue colored granules in the cytoplasm. RESULTS: 29/35 (82.9%) cases showed a positive reaction for Perl's Prussian blue reaction while 6/35 (17%) cases did not show the presence of blue colored granules in the oral cytosmears. The presence of iron detected by Perl's Prussian blue reaction correlated with serum ferritin level (P < 0.05). CONCLUSION: Perl's Prussian blue reaction can be used to evaluate the iron overload in beta-thalassemia patients by staining the oral cytosmears. It is a simple and noninvasive method for assessment of iron overload in such patients.

Alpha-2-Macroglobulin Levels in Gingival Crevicular Fluid Pre- and Post-scaling and Root Planing with Adjunctive Tetracycline Fibers in Chronic Periodontitis: A Randomized Controlled Trial.

BACKGROUND: This split-mouth clinical study aimed to investigate levels of alpha-2-macroglobulin (a2M) in gingival crevicular fluid (GCF) of chronic periodontitis patients pre- and post-scaling and root planing (SRP) with or without adjunctive use of tetracycline fibers. MATERIALS AND METHODS: In 30 patients of chronic periodontitis, samples of GCF were collected from the gingival sulcus before SRP. Recording of clinical parameters was conducted. This was followed by local drug delivery (LDD) of tetracycline fibers in test sites. In control sites, no LDD was done. Second samples of GCF were taken 90 days after treatment. Samples of crevicular fluid were analyzed to determine the levels of a2m. RESULTS: A gain of clinical attachment (CAL) of 3.30 mm for SRP and LDD and for SRP alone was 1.62 mm (p < 0.001). The pocket probing depth was significantly decreased by 2.43 mm for SRP and LDD and for SRP alone was 1.61 mm (p < 0.001) after 90 days. Alpha-2-macroglobulin was significantly reduced in GCF by SRP and SRP and LDD after 90 days (p < 0.001). CONCLUSION: Clinical and biochemical variables showed a more favorable outcome when SRP was combined with LDD of tetracycline fibers in management of patients suffering from chronic periodontitis.

Neurilemmoma of Retromolar Region in the Oral Cavity.

Neurilemmoma also known as schwannoma is benign nerve sheath tumor rarely occurring in the oral cavity. Only 1% of all extracranial schwannomas show that intraoral occurrence with tongue is the commonest site and retromolar region is the least common site. It presents as encapsulated, slow growing, solitary, smooth-surfaced, usually asymptomatic tumor. We report a case of 70-year-old male with well-defined mass on left retromolar region which was painless and slow growing. Diagnosis is made by histological examination and immunohistochemistry analysis to confirm the neural tissue origin of the lesion. The treatment is complete surgical excision of the lesion without recurrence.

Peripheral ameloblastoma of gingiva with cytokeratin 19 analysis.

A 42-year-old woman presented with a gingival mass in the lingual vestibule of the mandibular incisor premolar region. On intraoral examination, the swelling was non-tender and firm. Surgical excision was carried out and subsequent histopathological examination revealed areas resembling ameloblastoma-like and basaloid areas with atypical features suggestive of basal cell carcinoma. Immunohistochemical analysis was carried out to ascertain the origin of the lesion.

Relevance of tumor angiogenesis patterns as a diagnostic value and prognostic indicator in oral precancer and cancer.

Tumor angiogenesis occurs by recruitment of endothelial cell precursors or by sprouting of existing capillaries, which differ from the normal vasculature by having an altered morphology that can be exploited for diagnosis and as a prognostic indicator. Improved technologies have propelled diagnosis into a new era. These technologies have to be used with great precision. The diagnosis of a dysplastic premalignant lesion of the oral mucosa cannot be based solely on clinical findings. Therefore histologic evaluation of a representative biopsy specimen is necessary. Accurate judgment of the proper site for biopsy is essential for reaching a correct diagnosis. The aim of this report is to analyze the vascular patterns with the help of direct oral microscopy and the technique of stereo-optical microscopy in the oral cavity to select biopsy sites, and compare the outcome of a directed biopsy with that of biopsy specimens obtained from sites selected solely on the basis of clinical criteria. The study sample comprised 50 oral mucosal lesions. A statistically significant difference was noted between samples judged to be microscopically representative sites. We conclude that this method would aid in early and better diagnosis and treatment planning of oral premalignant and malignant lesions by assessing the various vascular patterns in the mucosa.