Antifibrotic effect of silymarin on arecoline-induced fibrosis in primary human buccal fibroblasts: an in silico and in vitro analysis.

BACKGROUND: This study aimed to assess silymarin's anticancer and antifibrotic potential through in silico analysis and investigate its impact on in vitro arecoline-induced fibrosis in primary human buccal fibroblasts (HBF). METHODS & RESULTS: The study utilized iGEMDOCK for molecular docking, evaluating nine bioflavonoids, and identified silymarin and baicalein as the top two compounds with the highest target affinity, followed by subsequent validation through a 100ns Molecular Dynamic Simulation demonstrating silymarin's stable behavior with Transforming Growth Factor Beta. HBF cell lines were developed from tissue samples obtained from patients undergoing third molar extraction. Arecoline, a known etiological factor in oral submucous fibrosis (OSMF), was employed to induce fibrogenesis in these HBFs. The inhibitory concentration (IC50) of arecoline was determined using the MTT assay, revealing dose-dependent cytotoxicity of HBFs to arecoline, with notable cytotoxicity observed at concentrations exceeding 50µM. Subsequently, the cytotoxicity of silymarin was assessed at 24 and 72 h, spanning concentrations from 5µM to 200µM, and an IC50 value of 143µM was determined. Real-time polymerase chain reaction (qPCR) was used to analyze the significant downregulation of key markers including collagen, epithelial-mesenchymal transition (EMT), stem cell, hypoxia, angiogenesis and stress markers in silymarin-treated arecoline-induced primary buccal fibroblast cells. CONCLUSION: Silymarin effectively inhibited fibroblast proliferation and downregulated genes associated with cancer progression and EMT pathway, both of which are implicated in malignant transformation. To our knowledge, this study represents the first exploration of silymarin's potential as a novel therapeutic agent in an in vitro model of OSMF.

Impact of HPV molecular testing with partial genotyping as a feasibility study in cervical cancer community screening program in South India.

Cervical cancer can be eradicated by 2030 by the implementation of a global strategy involving the vaccination of young girls against human papillomavirus (HPV), screening 70% of women in 30-69 years of age and treating 90% of the women with precancerous lesions. For a country with a large population like India, all the three strategies can be a challenge. There is a need for implementation of a high throughput technology that can be scalable. Cobas 4800, a multiplexed assay based on quantitative polymerase chain reaction technology, identifies HPV 16 and HPV 18 along with the concurrent detection of 12 pooled other high-risk HPV infections. This technology was used to test 10 375 women from the South Indian community for the first time as a feasibility program. Upon testing, high-risk HPV was found in 595 (5.73%) women. A total of 127 women (1.2%) were found to be infected with HPV 16, 36 women (0.34%) with HPV 18 and 382 women (3.68%) with the 12 pooled high-risk HPV and multiple mixed infections were found in 50 women (0.48%). It was observed that there was a high prevalence of high-risk HPV in younger women, 30-40 years of age and a second peak was observed at 46-50 years of age. The second peak had higher mixed infections in the 46-50 years of age and this association was statistically significant. We found that 24/50 (48%) of the multiple mixed high-risk HPV infections were in the age group 46-50 years. The current study is the first attempt from India, on a completely automated platform using Cobas 4800 HPV test in a community screening program. This study shows HPV 16 and HPV 18 infections, when differentiated, can be valuable for risk stratification in community screening program. Women in the perimenopausal age (46-50yrs) showed a higher prevalence of multiple mixed infections, signifying a higher risk.

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.

Prognostic relevance of pre-treatment inflammatory biomarkers along with other clinicopathological and treatment factors in oral cavity cancers.

Background: Inflammation has traditionally been considered to be one of the hallmarks of cancer, and systemic inflammatory responses have a prognostic value in many solid cancers. The use of inflammation-based prognostic markers along with traditional clinicopathological prognostic markers in oral cavity cancers has not been studied well. Materials and Methods: This is a retrospective study from a prospectively maintained database of patients with oral cancers who were managed in a regional cancer center in south India. The study included patients with squamous cell carcinoma of the oral cavity who were treated with curative intent from January to December 2016. Results and Discussion: Three hundred sixty-one patients met the eligibility criteria and were included in the study. The median age of our patient cohort was 45 years; the male-to-female ratio was 3.7:1. All of the patients underwent curative treatments after a multi-disciplinary board concurrence. Advanced T stage, patients with buccal mucosal cancers and patients who received upfront non-surgical treatments have poorer survival outcomes. The clinicopathological variables that predicted a poorer overall survival in the cohort of patients treated with upfront surgery were advanced T Stage, higher grade, presence of perineural invasion, a higher inflammatory maker, and combination of platelet and neutrophil lymphocyte ratio (COP-NLR). Conclusion: Our unique study of oral cavity cancer patients with a primary aim of exploring the prognostic significance of the pre-treatment inflammatory markers gave very interesting results. The prognostic significance of COP-NLR and other inflammatory markers in oral cancers need to be further explored. More importantly, our study has clearly reiterated that meaningful long-term survival outcomes in oral cavity cancers can only be achieved with the incorporation of upfront surgery.

Preparation and Characterization of Silymarin Gel: A Novel Topical Mucoadhesive Formulation for Potential Applicability in Oral Pathologies.

Silybum marianum has been used for centuries by herbalists and physicians to treat different forms of liver diseases. It contains flavonoid, which has antioxidant, anti-inflammatory, antifibrotic and anticancer properties. The objective of this research was to develop a silymarin-based mucoadhesive gel for prolonged release in oral mucosa and to evaluate the same by using in vitro drug release kinetic models and ex vivo methods for drug permeation using chicken buccal mucosa. The mucoadhesive gel was formulated in different trials by varying the concentration of silymarin and polymer. Out of 10 formulation trials, the F10 optimized trial was characterized for in vitro physicochemical parameters such as pH, homogeneity, viscosity, stability, drug content, in vitro drug release, in vitro antioxidant assay and ex vivo permeation study. Trial 10 was chosen as the best trial formulation among the other trials and was marked as an optimal trial. The physicochemical properties observed were pH to be 6.4 ± 0.01, the gel free of lumps, spreadability of 23.75 ± 0.03 and drug content of 32.77 ± 0.20 mg/g. It had no physiological changes such as color shift or fluid exudate segregation after 6 months of storage at room temperature. In vitro drug release established the presence of a non-fickian mechanism and demonstrated dose-dependent antioxidant activity. Ex vivo findings indicated 21.97 ± 0.18% release, proving that the gel can permeate through the oral mucosal membrane. Our future research will concentrate on expanding the therapeutic scope by developing the formulation trial F10 to a nanoformulation and conducting clinical trials for its potential use in various oral diseases.

Time intervals and patient-level factors in oral cancer diagnostic pathways: An application of the WHO framework in India.

BACKGROUND: Oral cancer, a leading cancer-site in India, is often detected at advanced stages. We evaluated the time intervals from first symptom to help-seeking and diagnosis among oral cancer patients. METHODOLOGY: In this cross-sectional study, we recruited 226 consecutive oral cancer patients (mean age ( ± SD) 51.9 years ( ± 10.9); 81.9% men; 70.3% advanced stage) registered for diagnosis and treatment, between 2019 and 2021 at a cancer care centre in South India. We used WHO framework and previously standardized tools to record time intervals (appraisal, help-seeking and diagnostic) and baseline characteristics. We utilized multivariable logistic regression models to test the associations between 'prolonged (i.e., over 1 month) time intervals') and patient-level factors to estimate odds ratios (ORs) with 95% confidence intervals (CIs). RESULTS: Over a half of patients presented with prolonged appraisal (60%) and help-seeking intervals (57%), and a third (34%) reported prolonged diagnostic interval. Patients with no formal education, no routine healthcare visits, no self-reported risk factors, and those who did not perceive initial symptoms to be serious were 2-4 times more likely to have prolonged appraisal and help-seeking than the rest. High travel costs and self-decision for visiting healthcare facility prolonged help-seeking. Diagnostic interval was prolonged only among women OR= 2.7 (95% CI: 1.2-6.1)) and in patients whose first doctor's opinion was 'nothing to worry' OR (=7.3 (95% CI: 2.6-20.5)). 'Correct knowledge of cancer' shortened appraisal and help-seeking intervals and 'incorrect knowledge and negative beliefs' prolonged diagnostic interval. CONCLUSION: Our findings highlight that interventions targeting sociocultural and economic determinants, symptom awareness, sensitizing persons at risk (especially women) and primary care providers might reduce overall time to diagnosis. Further, patients without any known risk factors for oral cancer might be at-risk for prolonged appraisal interval. These might help inform 'pull' strategies for cancer control in India and similar settings.

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.

The impact of prehistoric human dispersals on the presence of tobacco-related oral cancer in Northeast India.

BACKGROUND: Northeast (NE) India is a subject of debate for predicting its involvement in prehistoric anatomically modern human (AMH) dispersal. The unique lifestyle and genetic characteristics of native ethnic groups in this region are believed to be responsible for their susceptibility to tobacco-related oral cancer (TrOC). The present study assessed mitochondrial macro-haplogroup (mHG) diversity and TrOC susceptibility autosomal loci to evaluate the impact of prehistoric AMH dispersal on the present day's high TrOC prevalence in major NE Indian ethnics. METHODS: We considered 175 unrelated individuals from 35 ethnic groups and previously published 374 sequences for sequencing-based assessment of mtDNA-based marker by subsequent analyses like haplogroup diversity, phylogenetic, genetic structure by AMOVA, and MDS, descriptive statistics of demographic parameters, and migration analysis. Besides, we selected prolonged tobacco-chewing 124 case-control individuals from similar ethnic backgrounds for genotyping 115 autosomal loci in Sequenom iPLEX MassARRAY™ platform and mined 1000genome data (n = 398) for consequent global admixture and ancestry-specific allele frequencies-based analyses. RESULTS: Our mtDNA-based findings suggested that NE populations were distinct from other Indian populations, owing to the first wave of migration from ancient southern China (∼54kya) and two successive spatial expansion events at âˆ¼45kya and âˆ¼43kya. Consequently, it probably acted as another source for prehistoric AMH dispersal in N/NE Asia. Besides, the second wave of back-migration from SE Asia (∼40kya) probably replaced the mitochondrial footprints of survivors from the first migrants and introduced the TrOC susceptibility traits in this region. Afterward, the autosomal marker-based observations on the transition of the disease-associated admixture component 'K6' from SE Asia reconfirmed these results. Moreover, we also observed that the mitochondrial mHG 'R' is significantly associated with the risk of TrOC (OR > 9.5) in NE India. Furthermore, the possible onset of the phenotypic expression of those traits was predicted at âˆ¼4kya, thus, contributing to present-day's TrOC prevalence. CONCLUSIONS: This study reflects its uniqueness by revealing an updated AMH dispersal route for the peopling in and out of NE India, which probably introduced the disease-causing traits in the ancestral NE Indian population. Those traits were then imprinted in their genome to get transferred through their respective generations, forming the present-day's TrOC-prevalent NE Indian population.

Prevalence of Human Papillomavirus (HPV) 16 and 18 in Oral Malignant and Potentially Malignant Disorders: A Polymerase Chain Reaction Analysis - A Comparative Study.

INTRODUCTION: Human papillomavirus (HPV) are now being increasingly associated as a cause of oral squamous cell carcinomas (OSCC). This study was designed to evaluate the prevalence of HPV in Pelizaeus-Merzbacher disease (PMD) and OSCC using polymerase chain reaction that might help in better understanding of the role played by this virus in the oncogenic process even from its evolution stage. MATERIALS AND METHODS: Formalin-fixed paraffin-embedded tissue samples (n = 40) of OSCC and mild, moderate, and severe dysplasia were used for this study. DNA was quantified and checked for purity spectrophotometrically. Statistical analysis was performed using SPSS software and statistical significance was assessed using Fischer's exact test (p < 0.05 was considered significant). RESULTS: High-risk (HR)-HPV-16 was found to be positive in 35% of OSCC cases which showed a statistically significant association of HPV 16 with OSCC. Verrucous carcinoma had predominant HPV 16 infection (60%), followed by SCC with 40%. However, this association was not statistically significant. None of the OSCC samples were infected with HPV 18. Among the PMD, we found only 5% showing HR-HPV 16 infection which was not significant. DISCUSSION: Although OSCC is attributed to tobacco and alcohol consumption, a significant proportion of OSCC cases have been demonstrated to contain HPV types. The high-risk HPV type 16 tends to be the most predominant type detected in cases of OSCC.

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.

Current Status and Future Perspectives of Molecular Prevention Strategies for Cervical Cancers.

Cervical cancer continues to be a global health problem; despite the potential for prevention through organised screening programmes that can detect and treat pre-cancerous lesions and also more recently, the availability of HPV (Human Papilloma Virus) vaccines. While routine screening with Pap smear testing has reduced the burden of cervical cancer in the high-income countries, the implementation of organised Pap-based screening programmes has not been found feasible in low-resource settings due to a lack of health care delivery infrastructure and limited health budgets. The well-established causal relationship between cervical cancer development and high-risk-HPV (HR-HPV) infection and the subsequent appreciation of the greater sensitivity of HPV testing over Pap smear cytology eventually lead to HPV testing being incorporated in the primary cervical cancer prevention programmes. An organised cervical cancer screening programme incorporating HR-HPV testing and HPV vaccine administration are currently considered to be the two major interventions for a comprehensive cervical cancer control programme worldwide. However, there are concerns that the requirement of a sophisticated infrastructure with its associated costs may make cervical cancer screening using molecular prevention by HPV testing impracticable to be implemented, especially in resource-poor, low-income countries. Visual Inspection with Acetic acid (VIA) represents one of the alternative methods for cervical cancer screening proposed for the countries with low- to middle-income resources and has gained popularity in India following the successful completion of two randomised controlled trials, but this method but has low sensitivity to detect cervical pre-cancers. More recently, the cost-effectiveness analysis of many studies including randomised controlled trials, even from the low-resource settings, has found that HPV testing is followed by treatment for HPV-positive women to be an effective and cost-effective screening strategy as compared to other screening methods including VIA. The incorporation of self-sampling and HPV testing by partial genotyping has the potential to significantly add to the effectiveness and the cost-effectiveness. The current status and future perspectives of molecular prevention strategies for cervical cancer prevention is further discussed.

Association of DFNA5, SYK, and NELL1 variants along with HPV infection in oral cancer among the prolonged tobacco-chewers.

Southeast Asia, especially India, is well known for the highest use of smokeless tobacco. These products are known to induce oral squamous cell carcinoma. However, not all long-term tobacco-chewers develop oral squamous cell carcinoma. In addition, germline variants play a crucial role in susceptibility, prognosis, development, and progression of the disease. These prompted us to study the genetic susceptibility to oral squamous cell carcinoma among the long-term tobacco-chewers. Here, we presented a retrospective study on prolonged tobacco-chewers of Northeast India to identify the potential protective or risk-associated germline variants in tobacco-related oral squamous cell carcinoma along with HPV infection. Targeted re-sequencing (n = 60) of 170 genetic regions from 75 genes was carried out in Ion-PGM™ and validation (n = 116) of the observed variants was done using Sequenom iPLEX MassARRAY™ platform followed by polymerase chain reaction-based HPV genotyping and p16-immunohistochemistry study. Subsequently, estimation of population structure, different statistical and in silico approaches were undertaken. We identified one nonsense-mediated mRNA decay transcript variant in the DFNA5 region (rs2237306), associated with Benzo(a)pyrene, as a protective factor (odds ratio = 0.33; p = 0.009) and four harmful (odds ratio > 2.5; p < 0.05) intronic variants, rs182361, rs290974, and rs169724 in SYK and rs1670661 in NELL1 region, involved in genetic susceptibility to tobacco- and HPV-mediated oral oncogenesis. Among the oral squamous cell carcinoma patients, 12.6% (11/87) were HPV positive, out of which 45.5% (5/11) were HPV16-infected, 27.3% (3/11) were HPV18-infected, and 27.3% (3/11) had an infection of both subtypes. Multifactor dimensionality reduction analysis showed that the interactions among HPV and NELL1 variant rs1670661 with age and gender augmented the risk of both non-tobacco- and tobacco-related oral squamous cell carcinoma, respectively. These suggest that HPV infection may be one of the important risk factors for oral squamous cell carcinoma in this population. Finally, we newly report a DFNA5 variant probably conferring protection via nonsense-mediated mRNA decay pathway against tobacco-related oral squamous cell carcinoma. Thus, the analytical approach used here can be useful in predicting the population-specific significant variants associated with oral squamous cell carcinoma in any heterogeneous population.

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.].

Snail-Modulated MicroRNA 493 Forms a Negative Feedback Loop with the Insulin-Like Growth Factor 1 Receptor Pathway and Blocks Tumorigenesis.

In this study, we have identified one microRNA, microRNA 493 (miR-493), which could simultaneously and directly regulate multiple genes downstream of the insulin-like growth factor 1 receptor (IGF1R) pathway, including IGF1R, by binding with complementary sequences in the 3' untranslated region (UTR) of mRNAs of IGF1R, insulin receptor substrate 1 (IRS1), and mitogen-activated protein kinase 1 (MAPK1), thereby potentiating their inhibitory function at multiple levels in development and progression of cancers. This binding was further confirmed by pulldown of miR with AGO-2 antibody. Further, results from head and neck samples showed that miR-493 levels were significantly downregulated in tumors, with a concomitant increase in the expression of IGF1R and key downstream effectors. Functional studies from miR-493 overexpression cells and nude-mouse models revealed the tumor suppressor functions of miR-493. Regulation studies revealed that Snail binds to the miR-493 promoter and represses it. We found the existence of a dynamic negative feedback loop in the regulation of IGF1R and miR-493 mediated via Snail. Our study showed that nicotine treatment significantly decreases the levels of miR-493-with a concomitant increase in the levels of Snail-an indication of progression of cells toward tumorigenesis, reestablishing the role of tobacco as a major risk factor for head and neck cancers and elucidating the mechanism behind nicotine-mediated tumorigenesis.

Recent Advances in Our Understanding of Well-Differentiated Thyroid Cancers.

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The diagnostic and therapeutic challenges in the management of a double giant parathyroid adenoma.

Primary hyperparathyroidism is a common endocrine disorder of the parathyroid gland. It is mostly seen as a single gland adenoma in up to 85% of the cases with the excess production of parathyroid hormone. Double adenomas although reported are very rare and double giant parathyroid adenomas are even rarer. We possibly report the second case in the literature of primary hyperparathyroidism caused by double giant parathyroid adenomas, presenting with severe symptomatic hypercalcemia and review the diagnostic and therapeutic challenges in its management. The presentation with severe hypercalcemia and the presence of atypia in one of the adenomas added to its uniqueness. A combination of the neck ultrasound and a parathyroid scintigraphy should be used for preoperative localization and selection of the right surgical approach for patients undergoing parathyroidectomy. The parathyroid scintigraphic protocols keep getting refined; it is hence vitally important and practical to adapt the diagnostic algorithms in accordance with local availability and expertise.