Ultrasensitive detection of tumor-specific mutations in saliva of patients with oral cavity squamous cell carcinoma.

BACKGROUND: Oral cavity squamous cell carcinoma (OCSCC) is the most common head and neck malignancy. Although the survival rate of patients with advanced-stage disease remains approximately 20% to 60%, when detected at an early stage, the survival rate approaches 80%, posing a pressing need for a well validated profiling method to assess patients who have a high risk of developing OCSCC. Tumor DNA detection in saliva may provide a robust biomarker platform that overcomes the limitations of current diagnostic tests. However, there is no routine saliva-based screening method for patients with OCSCC. METHODS: The authors designed a custom next-generation sequencing panel with unique molecular identifiers that covers coding regions of 7 frequently mutated genes in OCSCC and applied it on DNA extracted from 121 treatment-naive OCSCC tumors and matched preoperative saliva specimens. RESULTS: By using stringent variant-calling criteria, mutations were detected in 106 tumors, consistent with a predicted detection rate ≥88%. Moreover, mutations identified in primary malignancies were also detected in 93% of saliva samples. To ensure that variants are not errors resulting in false-positive calls, a multistep analytical validation of this approach was performed: 1) re-sequencing of 46 saliva samples confirmed 88% of somatic variants; 2) no functionally relevant mutations were detected in saliva samples from 11 healthy individuals without a history of tobacco or alcohol; and 3) using a panel of 7 synthetic loci across 8 sequencing runs, it was confirmed that the platform developed is reproducible and provides sensitivity on par with droplet digital polymerase chain reaction. CONCLUSIONS: The current data highlight the feasibility of somatic mutation identification in driver genes in saliva collected at the time of OCSCC diagnosis.

Elucidating the role of Cyclooxygenase-2 in the pathogenesis of oral lichen planus - an immunohistochemical study with supportive histochemical analysis.

OBJECTIVE: Oral lichen planus (OLP) is a chronic, inflammatory disorder that affects the oral mucous membrane. During an inflammatory response, several chemokines and cytokines are released by the cells of the immune system. Activation of MMPs, along with mast cell-derived chymase and tryptase, degrades the basement membrane structural proteins, resulting in basement membrane breaks. AIM: To investigate the association between the COX-2 expressions, presence of intact or degranulating mast cells within the connective tissue and the extent of basement membrane discontinuity in OLP cases. METHODS: This study included a total of 50 formalin-fixed paraffin-embedded specimens (FFPE) of histologically confirmed cases of idiopathic oral lichen planus. A retrospective cross-sectional analysis was carried out by immunohistochemistry to study the epithelial expression of COX-2 and by the use of special stains such as toluidine blue and periodic acid-Schiff (PAS) to study the mast cell count and basement membrane changes in the oral mucosal tissue, respectively. RESULTS: There was a significant (P = 0.03) association between the COX-2 expressions and mast cell count. As the intensity of COX-2 expression increased from mild to moderate or severe, the number of mast cell count almost doubled. CONCLUSION: Interaction between upregulation of COX-2, mast cell and basement membrane sets a vicious cycle which relates to the chronic nature of the disease. Inhibitors of COX-2 may reduce the inflammatory process preceding the immune dysregulation in OLP.

Exosomes: Mediators of cellular communication in potentially malignant oral lesions and head and neck cancers.

Exosomes are a unique type of extracellular vesicles that contain a plethora of biological cargo such as miRNA, mRNA, long non-coding RNA, DNA, proteins and lipids. Exosomes serve as very effective means of intercellular communication. Due the presence of a lipid bilayer membrane, exosomes are resistant to degradation and are highly stable. This makes them easily identifiable in blood and other bodily fluids such as saliva. The exosomes that are secreted from a parent cell directly release their contents into the cytoplasm of a recipient cell and influence their cellular activity and function. Exosomes can also transfer their content between cancer cells and normal cells and regulate the tumor microenvironment. Exosomes play a vital role in tumor growth, tumor invasion and metastasis. Exosomes provide a multitude of molecular and genetic information and have become valuable indicators of disease activity at the cellular level. This review explores the molecular characteristics of exosomes and the role that exosomes play in the tumorigenesis pathway of potentially malignant oral lesions and head and neck cancers The application of exosomes in the treatment of oral cancers is also envisioned. Exosomes are very small and can easily pass through various biological barriers, making them very good delivery vectors for therapeutic drugs as well as to selectively induce DNA's mRNA and miRNAs into targeted cancer cells.

Molecular Signatures of Tumour and Its Microenvironment for Precise Quantitative Diagnosis of Oral Squamous Cell Carcinoma: An International Multi-Cohort Diagnostic Validation Study.

BACKGROUND: Heterogeneity in oral potentially malignant disorder (OPMD) poses a problem for accurate prognosis that impacts on treatment strategy and patient outcome. A holistic assessment based on gene expression signatures from both the tumour cells and their microenvironment is necessary to provide a more precise prognostic assessment than just tumour cell signatures alone. METHODS: We reformulated our previously established multigene qPCR test, quantitative Malignancy Index Diagnostic System (qMIDS) with new genes involved in matrix/stroma and immune modulation of the tumour microenvironment. An algorithm calculates and converts a panel of 16 gene mRNA expression levels into a qMIDS index to quantify risk of malignancy for each sample. RESULTS: The new qMIDSV2 assay was validated in a UK oral squamous cell carcinoma (OSCC) cohort (n = 282) of margin and tumour core samples demonstrating significantly better diagnostic performance (AUC = 0.945) compared to previous qMIDSV1 (AUC = 0.759). Performance of qMIDSV2 were independently validated in Chinese (n = 35; AUC = 0.928) and Indian (n = 95; AUC = 0.932) OSCC cohorts. Further, 5-year retrospective analysis on an Indian dysplastic lesion cohort (n = 30) showed that qMIDSV2 was able to significantly differentiate between lesions without transformation and those with malignant transformation. CONCLUSIONS: This study validated a novel multi-gene qPCR test on a total of 535 tissue specimens from UK, China and India, demonstrating a rapid minimally invasive method that has a potential application for dysplasia risk stratification. Further study is required to establish if qMIDSV2 could be used to improve OPMD patient management, guide treatment strategy and reduce oral cancer burden.

Orofacial granulomatosis: A case report and a proposal of a diagnostic algorithm for oral granulomatous lesions.

Orofacial granulomatosis (OFG) is an uncommon chronic inflammatory disorder with multifactorial etiology and pathogenesis affecting the orofacial region which appears usually as a persistent and/or recurrent swelling that may involve one or both the lips and/or recurrent ulcers along with other orofacial features. The diagnosis of OFG is challenging and it should be ruled out from other conditions characterized by granulomatous inflammation. This article describes a case of a 30-year-old female patient with upper lip swelling as the main manifestation without any systemic involvement and we have also laid down a proposal of a diagnostic algorithm for differential diagnoses and treatment modalities for OFG which could be helpful for the clinician and dentists alike to rule out other granulomatous disorders thus in effective management of OFG.

MicroRNA's - The vibrant performers in the oral cancer scenario.

MicroRNAs (miRNAs) are a family of small non-coding (18-22 nucleotide) RNA molecules. These molecules regulate gene expression by either inhibiting mRNA translation or by degrading mRNA. A single miRNA can control the expression of target genes, and the expression of a target gene can be regulated by multiple miRNAs. They are key regulators of various biological and pathological processes. These include cell proliferation, development and tumorigenesis. Novel studies have discovered definite signature miRNAs in the initiation and progression of cancers. Interestingly, miRNAs have also been found in fragile genomic sites that are associated with increased cancer risk. These micro RNAs regulate the expression of several genes that play a crucial role in the transition of normal oral mucosa through dysplasia to malignancy. The aim of this review is to recapitulate the current understanding of the many miRNAs that have been identified, the genes that they target and the role that they play in the carcinogenic pathway. The review also highlights the prospective role of miRNAs in the diagnosis, prognosis and treatment of oral cancers.

Association of the co-expression of SOX2 and Podoplanin in the progression of oral squamous cell carcinomas - an immunohistochemical study.

SOX2 is a transcription factor related to the maintenance of stem cells in a pluripotent state. Podoplanin is a type of transmembrane sialoglycoprotein, which plays an important role in tumor progression and metastasis. This study aims to determine association of SOX2 and podoplanin expression in the progression of oral squamous cell carcinomas and to elucidate the association between two proteins. Label="METHODOLOGY">The immunohistochemical expression of SOX2 and podoplanin were evaluated in 60 cases of primary oral squamous cell carcinomas. The correlation between the SOX2 and podoplanin expression and the clinicopathological features of the tumors and the patient outcomes were assessed. RESULTS The expression of SOX2 was seen in 38/60 (63%) of the cases and the expression for podoplanin was seen in 45/60 (75%) cases. There was a significant inverse correlation between the expression of SOX2 and podoplanin with the tumor grade (p=0.002 and p=0.017, respectively). There was a high expression of SOX2 in 9/13 cases that presented with disease free survival. Survival analysis showed that a high expression of SOX2 correlated positively (p=0.043) with the disease-free survival. There was a significant positive association between the pattern of SOX2 and podoplanin expression (p=0.002). CONCLUSION A high expression of SOX2 was associated with better disease-free survival. The expression of podoplanin was associated with the degree of differentiation of the tumors. Analysis of these biomarkers can aid in the prognosis and treatment of oral squamous cell carcinomas.

Osteoid osteoma of the mandible: A case report with review of the literature.

Osteoid osteoma is a benign skeletal neoplasm most frequently observed in young individuals. The tumor most commonly occurs in the femur, the tibia, and the phalanges; however, jaw lesions are very rare. Herein, we report a rare case of osteoid osteoma that presented in the mandible of a 20-year-old boy. This report also reviews the cases of osteoid osteomas of the jaws that have been reported in the English literature so far.

The prognostic implication of the expression of EGFR, p53, cyclin D1, Bcl-2 and p16 in primary locally advanced oral squamous cell carcinoma cases: a tissue microarray study.

Oral squamous cell carcinomas comprise a heterogeneous tumor cell population with varied molecular characteristics, which makes prognostication of these tumors a complex and challenging issue. Thus, molecular profiling of these tumors is advantageous for an accurate prognostication and treatment planning. This is a retrospective study on a cohort of primary locally advanced oral squamous cell carcinomas (n = 178) of an Indian rural population. The expression of EGFR, p53, cyclin D1, Bcl-2 and p16 in a cohort of primary locally advanced oral squamous cell carcinomas was evaluated. A potential biomarker that can predict the tumor response to treatment was identified. Formalin-fixed paraffin-embedded tumor blocks of (n = 178) of histopathologically diagnosed cases of locally advanced oral squamous cell carcinomas were selected. Tissue microarray blocks were constructed with 2 cores of 2 mm diameter from each tumor block. Four-micron-thick sections were cut from these tissue microarray blocks. These tissue microarray sections were immunohistochemically stained for EGFR, p53, Bcl-2, cyclin D1 and p16. In this cohort, EGFR was the most frequently expressed 150/178 (84%) biomarker of the cases. Kaplan-Meier analysis showed a significant association (p = 0.038) between expression of p53 and a poor prognosis. A Poisson regression analysis showed that tumors that expressed p53 had a two times greater chance of recurrence (unadjusted IRR-95% CI 2.08 (1.03, 4.5), adjusted IRR-2.29 (1.08, 4.8) compared with the tumors that did not express this biomarker. Molecular profiling of oral squamous cell carcinomas will enable us to categorize our patients into more realistic risk groups. With biologically guided tumor characterization, personalized treatment protocols can be designed for individual patients, which will improve the quality of life of these patients.

Cyclooxygenase-2--An Imperative Prognostic Biomarker in Oral Squamous Cell Carcinoma--An Immunohistochemical Study.

Oral squamous cell carcinoma (OSCC) is the most common head and neck squamous cell carcinoma (HNSCC) with metastasis and tumor recurrence resulting in 90 % of cancer associated mortality. COX-2, an inflammatory biomarker, has been shown to play a significant role in tumorigenesis of OSCC. To study the expression of COX-2 in OSCC by immunohistochemistry and investigate its association with the clinicopathological parameters including patient survival. A cross sectional study was carried out in 75 histologically confirmed cases of OSCC. COX-2 expression was evaluated by indirect streptavidin biotin method. The expression was semi-quantitatively assessed using established criteria. The expression profile of COX-2 was correlated with the clinicopathological details like tumor size, regional lymphnode metastasis, distant metastasis, clinical stage, local recurrence of tumor, histological grade, and survival of patient. Chi square and Kaplan Meier statistical tests were applied for assessing this association. COX-2 expression was absent in normal oral mucosa. Over expression of COX-2 was seen in 58 out of 75 specimens of OSCC. Overexpression of COX-2 was significantly associated with the lymphnode involvement, histological grade, local recurrence of tumor and patient survival. COX-2 expression represents an important biomarker of prognostic significance that may be used to identify a subset of patients at high risk and to predict patient survival.

Association of the co-expression of SOX2 and Podoplanin in the progression of oral squamous cell carcinomas - an immunohistochemical study

Abstract SOX2 is a transcription factor related to the maintenance of stem cells in a pluripotent state. Podoplanin is a type of transmembrane sialoglycoprotein, which plays an important role in tumor progression and metastasis. This study aims to determine association of SOX2 and podoplanin expression in the progression of oral squamous cell carcinomas and to elucidate the association between two proteins. Methodology: The immunohistochemical expression of SOX2 and podoplanin were evaluated in 60 cases of primary oral squamous cell carcinomas. The correlation between the SOX2 and podoplanin expression and the clinicopathological features of the tumors and the patient outcomes were assessed. Results: The expression of SOX2 was seen in 38/60 (63%) of the cases and the expression for podoplanin was seen in 45/60 (75%) cases. There was a significant inverse correlation between the expression of SOX2 and podoplanin with the tumor grade (p=0.002 and p=0.017, respectively). There was a high expression of SOX2 in 9/13 cases that presented with disease free survival. Survival analysis showed that a high expression of SOX2 correlated positively (p=0.043) with the disease-free survival. There was a significant positive association between the pattern of SOX2 and podoplanin expression (p=0.002). Conclusion: A high expression of SOX2 was associated with better disease-free survival. The expression of podoplanin was associated with the degree of differentiation of the tumors. Analysis of these biomarkers can aid in the prognosis and treatment of oral squamous cell carcinomas.

Clinico-pathological correlation of E-cadherin expression at the invasive tumor front of Indian oral squamous cell carcinomas: An immunohistochemical study.

BACKGROUND: Recent studies have indicated that although malignant cells at the invasive tumor front, bare morphological resemblance to the cells at central portion of the tumor, their molecular character differs significantly. E-cadherin is a cell-cell adhesion molecule that connects epithelial cells. This study attempts to correlate the E-cadherin expression at the invasive tumor front with tumor differentiation along with its clinico-pathological parameters. MATERIALS AND METHODS: Immunohistochemical staining with E-cadherin was carried out on archival cases of primary oral squamous cell carcinomas (n = 30). The E-cadherin expression at the invasive tumor front was analyzed and was linked to clinico-pathological parameters including patient prognosis. RESULTS: The downregulation of E-cadherin expression at the invasive tumor edge when compared with patient's prognosis yielded a significant correlation (P = 0.041) but its correlation with the degree of differentiation determined was not significant (P = 0.27). Also, its association with tumor size and lymph node status was negative. CONCLUSIONS: Loss of E-cadherin expression at the invasive tumor front is an important event in the progression of oral squamous cell carcinomas. Tumors with a loss of expression of E-cadherin are those which had a poor prognosis.

Cytomorphological analysis of keratinocytes in oral smears from tobacco users and oral squamous cell carcinoma lesions--a histochemical approach.

AIM: To analyse the cytomorphological features of keratinocytes in smears obtained from the oral mucosa of tobacco users and from oral squamous cell carcinoma lesions. METHODOLOGY: Oral smears were obtained from clinically, normal appearing mucosa of oral squamous cell carcinoma patients (n=20) and from the mucosa of smokers (n=20), and apparently healthy individuals (n=20) were used as controls. The smears were histochemically stained and cytomorphological assessment of the keratinocytes was carried out. One-way ANOVA (Analysis of Variance) was used for comparing the parameters among multiple groups and Tukey-HSD test was used to compare the mean values between groups. RESULTS: The mean nuclear area of keratinocytes from the mucosa of tobacco users was 46 +/- 2.57 and that of the oral squamous cell carcinoma lesion was 81.54 +/- 4.31. While there was a significant (P = 0.001) reduction in the cellular area of keratinocytes from oral squamous cell carcinoma lesion when compared with those from oral smears of tobacco users. CONCLUSION: Cytomorphometric analysis of keratinocytes can serve as a useful adjunct in the early diagnosis of oral squamous cell carcinomas.