Influence of type 2 diabetes on immunohistochemical detection of TRAF6, cFos and NFATC1 in the gingiva in cases of chronic periodontitis.

Type 2 diabetes (T2D) and chronic periodontitis (CP) are common diseases worldwide. Although T2D increases the severity of CP and alveolar bone loss, the mechanism of this is not well understood. We investigated using immunohistochemistry the expression of three osteoclast proteins, TRAF6, cFos and NFATc1, in gingival tissues. Gingival tissues were obtained from three groups: HC group, healthy controls; CP group, patients with CP; T2D + CP group, patients with both T2D and CP. Strong immunostaining for TRAF6, cFos and NFATc1 was observed in the gingival epithelium as well as in inflammatory cells in the CP and T2D + CP groups. Immunostaining was most intense in the T2D + CP group. We found strong up-regulation of TRAF6, cFos and NFATC1 in gingiva tissue of subjects with both T2D and CP, which corroborates our hypothesis that T2D potentiates osteoclastogenesis in CP.

Clinicopathological Significance of Cancer Stem Cell Markers (OCT-3/4 and SOX-2) in Oral Submucous Fibrosis and Oral Squamous Cell Carcinoma.

Oral submucous fibrosis (OSMF) is highly prevalent in South East Asia with higher rates of malignant transformation in Indian subcontinent. Numerous biomarkers are now being studied to predict disease prognosis and detect malignant alterations at an early stage. Patients with clinically and biopsy-proven oral submucous fibrosis and oral squamous cell carcinoma were included in the study as the experimental group, while patients without a tobacco or betel nut habit who had their third molars surgically removed were included as the healthy control group. For the immunohistochemistry (IHC) investigation, 5-µm slices from formalin-fixed, paraffin-embedded tissue blocks (FFPE) were obtained. Fresh tissues (n = 45) from all three groups were collected and gene expression was studied using relative quantitation-based qPCR. The protein expression of octamer-binding transcription factor 3/4 (OCT 3/4) and sex-determining region Y-box 2 (SOX 2) was evaluated in the experimental group and compared with healthy controls. The IHC results showed a significant correlation with the expression of OCT 3/4 (p value = 0.000; χ2 = 20.244) and SOX 2 (p value = 0.006; χ2 = 10.101) among OSCC and OSMF patients in comparison to healthy controls. Both OCT 3/4 and SOX 2 showed overexpression of four-fold and three-fold in OSMF when compared to OSCC and healthy controls, respectively. This study shows the significant importance of cancer stem cell markers OCT 3/4 and SOX 2 to assess the disease prognosis in OSMF.

Identification of EGFR as a Biomarker in Saliva and Buccal Cells from Oral Submucous Fibrosis Patients-A Baseline Study.

Oral Submucous Fibrosis (OSMF) is a chronic debilitating disease more frequently encountered in the South-East Asian population. This disease represents a public health priority as it is grouped within oral potentially malignant disorders, with malignant transformation rates of around 7-19%. Hence, early identification of high-risk OSMF patients is of the utmost importance to prevent malignant transformation. Among various biomarkers, EGFR overexpression has an unfavorable clinical outcome, poor prognosis, and low survival rates in Oral Squamous Cell Carcinoma (OSCC). The current study aimed to evaluate the expression of EGFR in saliva and exfoliated buccal cells of OSMF. Immunoexpression of EGFR was observed in healthy controls (n = 11), OSCC (n = 106), and OPMD with dysplasia (n = 56), which showed significant expression with increasing grades of dysplasia and OSCC. EGFR expression was evaluated in saliva and exfoliated buccal cells of healthy controls (n = 15), OSMF (n = 24), and OSCC (n = 10) patients using ELISA, which revealed significant expression in OSMF and OSCC. Validation studies were also performed using real-time PCR (RT-PCR) to compare gene expression in healthy controls (n = 9), OSMF (n = 9), and OSCC (n = 25), which showed significant 18-fold upregulation in OSCC and three-fold upregulation in OSMF when compared to healthy controls. Hence, saliva and exfoliated buccal cells could be considered as potential non-invasive diagnostic samples for the evaluation of high-risk patients of OSMF using EGFR as a biomarker.

2D-DIGE-Based Proteomic Profiling with Validations Identifies Vimentin as a Secretory Biomarker Useful for Early Detection and Poor Prognosis in Oral Cancers.

Oral tongue squamous cell carcinoma (OTSCC) is an aggressive cancer with high morbidity and mortality rates, despite multimodality management. There are currently no clinically relevant molecular markers that identify patients at higher risk of recurrence and failure. We undertook 2D-DIGE proteomic profiling to study the differentially expressed proteins in OTSCC evaluating their role in prognosis. 2D-DIGE coupled with tandem mass spectrometry was performed on tissues obtained from early staged OTSCC along with its paired apparently adjacent normal tissue samples (n = 10). Top upregulated protein was validated using immunohistochemistry (n = 345), comprising of retrospective early stage OTSCC (n = 150) and prospective series of oral precancers, normal, and oral cancers (n = 195). Saliva samples collected from oral cancer and precancer samples were analyzed by ELISA (n = 146). We found statistically significant differential expression in 151 proteins out of 700 proteins quantified. Top ten differentially regulated proteins were identified using mass spectrometry analysis. We found vimentin, the mesenchymal protein, to be the most upregulated protein in tongue tumor tissues compared to adjacent apparent normal tissues. Vimentin was found to be significantly overexpressed in oral precancers along with cancers compared to normal tissues. The vimentin expression correlated significantly with differentiated states of oral precancers and cancers. Vimentin was also detected at significantly higher levels in saliva collected from oral precancer and cancer patients compared to normal healthy volunteers. Validation of vimentin in an independent series of retrospective early staged OTSCC showed that the vimentin expression is significantly associated with treatment failures and poorer DFS. The vimentin expression is useful as both poor prognostic and early detection marker in oral cancer. Vimentin detection in saliva can be a diagnostic test to detect oral precancers that may have malignant potential, needing closer follow-up, and disease monitoring.

Integrated Proteomics Based on 2D Gel Electrophoresis and Mass Spectrometry with Validations: Identification of a Biomarker Compendium for Oral Submucous Fibrosis-An Indian Study.

Oral Submucous Fibrosis (OSMF) is a chronic debilitating disease more frequently found in the South East Asian population. This disease poses a public health priority, as it is grouped under oral potentially malignant disorders, with malignant transformation rates of around 7 to 13%. Hence, early identification of high-risk OSMF patients is of the utmost importance to prevent malignant transformation. Proteomic expression profiling is a promising method for identifying differentially expressed proteins for disease prognosis and risk stratification in OSMF. In this study, overexpressed proteins in OSMF, OSMF transformed into oral squamous cell carcinoma (OSCC) and normal tissues were evaluated by proteomic analysis using two-dimensional electrophoresis (2DE) and mass spectrometry, which revealed 23 upregulated proteins. Validation was done using immunohistochemistry for three secretory proteins, namely 14-3-3ε (n = 130), carbonic anhydrase 1 (CA 1) (n = 125) and heat shock protein 70 (HSP 70) (n = 117), which showed significant overexpression in OSMF, OSCC compared to normal. The present study is the first of its kind in India to the best of our knowledge, assessing the altered expression of proteins in OSMF and OSMF which has undergone malignant transformation, obtaining a better knowledge of the molecular pathways involved in the disease progression. The current study shows that the biomarkers studied can be potentially useful for risk stratification of OSMF to OSCC serving as novel targets for therapeutic intervention. Clinical validation of the targets can further pave way for precision medicine to improve the quality of life in OSMF patients.

Exome Sequencing with Validations and Expression of p16/CDKN2A Shows no Association with HPV in Oral Cancers.

PURPOSE: Despite many studies attributing HPV infection to oropharyngeal tumorigenesis, its involvement in non-oropharyngeal cancers is ambiguous. We have evaluated the mutation profile of p16 along with protein expression and correlated it with the HPV status in oral cancers. METHODS: Somatic mutations in p16 were studied by exome sequencing (n=25) and validated by Sequenom Mass spectrometry (n=50).  Expression of p16 was studied by immunohistochemistry (IHC) and correlated with HPV16/18 status evaluated by PCR, and IHC (n=221) in oral cancers. RESULTS: Out of 25 oral cancer patients' samples sequenced by Exome sequencing, p16 mutations were found in 4 samples (16%). All the p16 mutations were identified in patients with cancers in the site of gingivobuccal complex and not tongue subsite. All the 4 patients with p16 mutations had failed treatment, and showed a significantly poor disease-free survival. Insilico analysis of the types of p16 mutations showed mutated, truncated p16 protein having an increased intrinsic disorder, and all the mutations involved truncation post arginine. Validation of the p16 mutations by mass spectrometry showed 8/50 (16%) of patients harbouring pArg80Ter mutation, of which 7/8 (87.5%) had failed treatment.  Overexpression of p16 in >70% of the tumour cells was found in 21.4% (26/121) OSCC patients, 6.75% (5/74) OPML patients and p16 expression was significantly correlated (p=0.001; χ2 = 25.601) to the grade. All the samples were studied for HPV presence by PCR and IHC. We found that none of the p16 positive tumours showing expression in >70% of the tumour cells harbored HPV both by PCR as well as IHC. CONCLUSION: Our study emphasises the importance of p16 in oral cancers, and shows that oral cancer is not HPV associated and p16 expression is not a surrogate marker for HPV.

Bimodal multispectral imaging system with cloud-based machine learning algorithm for real-time screening and detection of oral potentially malignant lesions and biopsy guidance.

SIGNIFICANCE: Screening and early detection of oral potentially malignant lesions (OPMLs) are of great significance in reducing the mortality rates associated with head and neck malignancies. Intra-oral multispectral optical imaging of tissues in conjunction with cloud-based machine learning (CBML) can be used to detect oral precancers at the point-of-care (POC) and guide the clinician to the most malignant site for biopsy. AIM: Develop a bimodal multispectral imaging system (BMIS) combining tissue autofluorescence and diffuse reflectance (DR) for mapping changes in oxygenated hemoglobin (HbO2) absorption in the oral mucosa, quantifying tissue abnormalities, and guiding biopsies. APPROACH: The hand-held widefield BMIS consisting of LEDs emitting at 405, 545, 575, and 610 nm, 5MPx monochrome camera, and proprietary Windows-based software was developed for image capture, processing, and analytics. The DR image ratio (R610/R545) was compared with pathologic classification to develop a CBML algorithm for real-time assessment of tissue status at the POC. RESULTS: Sensitivity of 97.5% and specificity of 92.5% were achieved for discrimination of OPML from patient normal in 40 sites, whereas 82% sensitivity and 96.6% specificity were obtained for discrimination of abnormal (OPML + SCC) in 89 sites. Site-specific algorithms derived for buccal mucosa (27 sites) showed improved sensitivity and specificity of 96.3% for discrimination of OPML from normal. CONCLUSIONS: Assessment of oral cancer risk is possible by mapping of HbO2 absorption in tissues, and the BMIS system developed appears to be suitable for biopsy guidance and early detection of oral cancers.

Deregulation of extracellular matrix modeling with molecular prognostic markers revealed by transcriptome sequencing and validations in Oral Tongue squamous cell carcinoma.

Oral Tongue Squamous Cell Carcinoma (OTSCC), a distinct sub-group of head and neck cancers, is characteristically aggressive in nature with a higher incidence of recurrence and metastasis. Recent advances in therapeutics have not improved patient survival. The phenomenon of occult node metastasis, even among the purportedly good prognosis group of early-stage and node-negative tongue tumors, leads to a high incidence of locoregional failure in OTSCC which needs to be addressed. In the current study, transcriptome analysis of OTSCC patients identified the key genes and deregulated pathways. A panel of 26 marker genes was shortlisted and validated using real-time PCR in a prospective cohort of 100 patients. The gene expression was correlated with clinicopathological features including occult node metastasis, survival, and therapeutic outcome. The up-regulation of a panel of 6 genes namely, matrix metalloproteinase 9 (MMP9), Laminin subunit Gamma 2 (LAMC2), Desmoglein 2 (DSG2), Plasminogen Activator Urokinase (PLAU), Forkhead Box M1 (FOXM1), and Myosin 1B (MYO1B) was associated with failure of treatment in the early stage (T1, T2). Up-regulation of Tenacin C (TNC) and Podoplanin (PDPN) was significantly correlated with occult node positivity. Immunohistochemical analysis of LAMC2, MMP9, and E-Cadherin (ECAD) confirmed these markers to be indicators of poor prognosis. We propose this panel of valuable prognostic markers can be clinically useful to identify poor prognosis and occult node metastasis in OTSCC patients.

A Feedback Loop between MicroRNA 155 (miR-155), Programmed Cell Death 4, and Activation Protein 1 Modulates the Expression of miR-155 and Tumorigenesis in Tongue Cancer.

MicroRNA 155 (miR-155) is an oncomir, generated as a noncoding RNA from the BIC gene whose promoter activity is mainly controlled via activation protein 1 (AP-1) and NF-κB transcription factors. We found that the expression levels of miR-155 and programmed cell death 4 (Pdcd4) exhibit inverse relationships in tongue cancer cells (SAS and AWL) and tumor tissues compared to their relationships in normal FBM cells and normal tongue tissues, respectively. In silico and in vitro studies with the 3' untranslated region (UTR) of Pdcd4 via luciferase reporter assays, quantitative PCR (qPCR), and Western blotting showed that miR-155 directly targets Pdcd4 mRNA and blocks its expression. Ectopic expression of Pdcd4 or knockdown of miR-155 in tongue cancer cells predominantly reduces AP-1-dependent transcriptional activity of the BIC promoter and decreases miR-155 expression. In this study, we demonstrate that miR-155 expression is modulated by a feedback loop between Pdcd4, AP-1, and miR-155 which results in enhanced expression of miR-155 with a consequent progression of tongue tumorigenesis. Further, miR-155 knockdown increases apoptosis, arrests the cell cycle, regresses tumor size in xenograft nude mice, and reduces cell viability and colony formation in soft-agar and clonogenic assays. Thus, the restoration of Pdcd4 levels by the use of molecular manipulation such as using a miR-155 sponge has an essential role in the therapeutic intervention of cancers, including tongue cancer.

A facile gold nanoparticle-based ELISA system for detection of osteopontin in saliva: Towards oral cancer diagnostics.

In the current study, we emphasize that osteopontin is overexpressed in oral squamous cell carcinoma. Overexpression of osteopontin levels was confirmed by mRNA quantification studies and immunohistochemistry analysis. Based on this, a gold nanoparticle-based ELISA system was developed for non-invasive osteopontin detection. The incorporation of AuNRs (Gold nanorods) or AuNSs (Gold nanospheres) in the conventional ELISA improved the sensitivity of analyses. A considerably lowered detection limit in case of AuNR (detection limit: 0.02ngmL-1) and AuNS (detection limit: 0.03ngmL-1) modified assay was obtained as compared to commercially available OPN ELISA kit (detection limit: 0.14ngmL-1). The modified ELISA had a wide linear detection range (0.31-20ngmL-1), good reproducibility, and specificity against the tested interferents in the saliva. Finally, the nanoELISA was validated with osteopontin spiked in artificial and normal saliva samples and observed to show good recovery (95.4-97.85%), which indicates the application potential of the developed kit for real sample analysis.

Erratum: "Quick chip assay using locked nucleic acid modified epithelial cell adhesion molecule and nucleolin aptamers for the capture of circulating tumor cells" [Biomicrofluidics 9(5), 054110 (2015)].

[This corrects the article DOI: 10.1063/1.4930983.].

Oral gingival metastasis: A diagnostic dilemma.

Oral cavity is a rare target for metastasis with an incidence of 1% among all oral cancers. In 24% of such cases, oral metastasis is the first indication of an undiagnosed primary. Metastatic oral malignancies have been reported in the mandible, tongue, and gingiva. Although gingival metastasis has been reported from lung, prostate, rectal carcinoma in men and carcinoma of breast, adrenal glands, and genitalia in females, gingival metastasis from carcinoma of the penis has not been reported. Herein, a case of metastatic gingival carcinoma that developed after extraction of teeth from primary carcinoma of the penis is presented. An extensive literature search revealed no such similar case reports.

Molecular Portrait of Oral Tongue Squamous Cell Carcinoma Shown by Integrative Meta-Analysis of Expression Profiles with Validations.

Oral Tongue Squamous cell carcinoma (OTSCC), the most frequently affected oral cancer sub-site, is associated with a poor therapeutic outcome and survival despite aggressive multi- modality management. Till date, there are no established biomarkers to indicate prognosis and outcome in patients presenting with tongue cancer. There is an urgent need for reliable molecular prognostic factors to enable identification of patients with high risk of recurrence and treatment failure in OTSCC management. In the current study, we present the meta-analysis of OTSCC microarray based gene expression profiles, deriving a comprehensive molecular portrait of tongue cancer biology, showing the relevant genes and pathways which can be pursued further to derive novel, tailored therapeutics as well as for prognostication. We have studied 5 gene expression profiling data sets available on exclusively oral tongue subsite comprising of sample size; n = 190, consisting of 111 tumors and 79 normals. The meta- analysis results showed 2405 genes differentially regulated comparing OTSCC tumor and normal. The top up regulated genes were found to be involved in Extracellular matrix degradation (ECM) and Epithelial to mesenchymal transition (EMT) pathways. The top down regulated genes were found to be involved in detoxication pathways. We validated the results in clinical samples (n = 206), comprising of histologically normals (n = 10), prospective (n = 29) and retrospective (n = 167) OTSCC by evaluating MMP9 and E-cadherin gene expression by qPCR and immunohistochemistry. Consistent with meta-analysis results, MMP9 mRNA expression was significantly up regulated in OTSCC primary tumors compared to normals. MMP9 protein over expression was found to be a significant predictor of poor prognosis, disease recurrence and poor Disease Free Survival (DFS) in OTSCC patients. Analysis by univariate and multivariate Cox proportional hazard model showed patients with loss of E-cadherin expression in OTSCC tumors having a poorer DFS (HR = 1.566; P value = 0.045) and poorer Overall Survival (OS) (HR = 1.224; P value = 0.003) respectively. Combined over-expression of MMP9 and loss of E-cadherin membrane positivity in the invasive tumor front (ITF) of OTSCC had a significant association with poorer DFS (Log Rank = 16.040; P value = 0.001). These results suggest that along with known clinical indicators of prognosis like occult node positivity, assessment of MMP9 and E-cadherin expression at ITF can be useful to identify patients at high risk and requiring a more intensive treatment strategy for OTSCC. Meta-analysis study of gene expression profiles indicates that OTSCC is a disease of ECM degradation leading to activated EMT processes implying the aggressive nature of the disease. The triggers for these processes should be studied further. Newer clinical application with agents that can inhibit the mediators of ECM degradation may be a key to achieving clinical control of invasion and metastasis of OTSCC.

p53 Expression Helps Identify High Risk Oral Tongue Pre- malignant Lesions and Correlates with Patterns of Invasive Tumour Front and Tumour Depth in Oral Tongue Squamous Cell Carcinoma Cases.

Oral tongue squamous cell carcinoma (OTSCC) is the most common oral cancer subtype with a maximum propensity for regional spread. Our objective was to study if p53 expression might have any correlation with aggressive patterns of invasion within oral tongue cancers as well as with the histologically identified degree of oral tongue dysplasia. p53 immunoexpression was studied using immunohistochemistry in early staged OTSCCs (n=155), oral tongue dysplasias, (n=29) and oral tongue normal specimens (n=10) and evaluated for correlations with histological and clinicopathological parameters. Our study (n=194) showed a pattern of p53 expression increasing with different grades of tongue dysplasia to different grades of invasive OTSCC (p=0.000). Among the OTSCC tumours, positive p53 expression was seen in 43.2% (67/155) and a higher p53 labelling index was significantly associated with increased Bryne's grade of the tumour invasive front (p=0.039) and increased tumour depth (p=0.018). Among the OTSCC patients with tobacco habits, (n=91), a higher p53 labelling index was significantly associated with increased risk of local recurrence (p=0.025) and with lymphovascular space involvement (p=0.014). Evaluation of p53 through varying degrees of dysplasia to oral tongue cancer indicates that p53 expression is linked to aggressive features of oral tongue cancers and tongue precancers entailing a closer monitoring in positive cases. Among the OTSCCs, p53 expression is associated with tumour aggressiveness correlating with increased grading of invasive tumour front and tumour depth.

Quick chip assay using locked nucleic acid modified epithelial cell adhesion molecule and nucleolin aptamers for the capture of circulating tumor cells.

The role of circulating tumor cells (CTCs) in disease diagnosis, prognosis, monitoring of the therapeutic efficacy, and clinical decision making is immense and has attracted tremendous focus in the last decade. We designed and fabricated simple, flat channel microfluidic devices polydimethylsiloxane (PDMS based) functionalized with locked nucleic acid (LNA) modified aptamers (targeting epithelial cell adhesion molecule (EpCAM) and nucleolin expression) for quick and efficient capture of CTCs and cancer cells. With optimized flow rates (10 µl/min), it was revealed that the aptamer modified devices offered reusability for up to six times while retaining optimal capture efficiency (>90%) and specificity. High capture sensitivity (92%) and specificity (100%) was observed in whole blood samples spiked with Caco-2 cells (10-100 cells/ml). Analysis of blood samples obtained from 25 head and neck cancer patients on the EpCAM LNA aptamer functionalized chip revealed that an average count of 5 ± 3 CTCs/ml of blood were captured from 22/25 samples (88%). EpCAM intracellular domain (EpICD) immunohistochemistry on 9 oral squamous cell carcinomas showed the EpICD positivity in the tumor cells, confirming the EpCAM expression in CTCs from head and neck cancers. These microfluidic devices also maintained viability for in vitro culture and characterization. Use of LNA modified aptamers provided added benefits in terms of cost effectiveness due to increased reusability and sustainability of the devices. Our results present a robust, quick, and efficient CTC capture platform with the use of simple PDMS based devices that are easy to fabricate at low cost and have an immense potential in cancer diagnosis, prognosis, and therapeutic planning.

Risk stratification of early stage oral tongue cancers based on HPV status and p16 immunoexpression.

BACKGROUND: Recent epidemiological data have implicated human papilloma virus (HPV) infection in the pathogenesis of head and neck cancers, especially oropharyngeal cancers. Although, HPV has been detected in varied amounts in persons with oral dysplasia, leukoplakias and malignancies, its involvement in oral tongue carcinogenesis remains ambiguous. MATERIALS AND METHODS: HPV DNA prevalence was assessed by PCR with formalin fixed paraffin embedded sections (n=167) of oral tongue squamous cell carcinoma patients and the physical status of the HPV16 DNA was assessed by qPCR. Immunohistochemistry was conducted for p16 evaluation. RESULTS: We found the HPV prevalence in tongue cancers to be 51.2%, HPV 16 being present in 85.2% of the positive cases. A notable finding was a very poor concordance between HPV 16 DNA and p16 IHC findings (kappa<0.2). Further molecular classification of patients based on HPV16 DNA prevalence and p16 overexpression showed that patients with tumours showing p16 overexpression had increased hazard of death (HR=2.395; p=0.005) and disease recurrence (HR=2.581; p=0.002) irrespective of their HPV 16 DNA status. CONCLUSIONS: Our study has brought out several key facets which can potentially redefine our understanding of tongue cancer tumorigenesis. It has emphatically shown p16 overexpression to be a single important prognostic variable in defining a high risk group and depicting a poorer prognosis, thus highlighting the need for its routine assessment in tongue cancers. Another significant finding was a very poor concordance between p16 expression and HPV infection suggesting that p16 expression should possibly not be used as a surrogate marker for HPV infection in tongue cancers. Interestingly, the prognostic significance of p16 overexpression is different from that reported in oropharyngeal cancers. The mechanism of HPV independent p16 over expression in oral tongue cancers is possibly a distinct entity and needs to be further studied.