A unique case of epithelioid sarcoma involving the mandibular alveolar mucosa.

Epithelioid sarcoma (ES) is an infrequent, malignant, mesenchymal, soft-tissue neoplasm of uncertain histogenesis characterized by epithelioid cytomorphology. The lack of SMARCB1/INI1 (Integrase interactor 1) expression typifies this pathology. It usually presents as a painless, indolent, slowly enlarging lesion or rarely as an aggressive growth. Epithelioid sarcoma affects mostly pediatric patients or young adults. It recurs quite frequently and possesses a high degree of metastatic potential. Two predominant histomorphologic patterns include classic/conventional type and proximal types. Epithelioid sarcoma immunohistochemically shows vimentin, cytokeratin, epithelial membrane antigen, CD34 positivity, and negative staining with S100 and desmin. Management is generally multimodal, including wide surgical resection, radiotherapy, and chemotherapy. To date, to our knowledge, only 11 instances of head and neck ES have been reported in the literature. Our case deals with the diagnosis of intraoral ES in a 55-year-old female patient, probably the first one to involve the mandibular mucosa, based on relevant clinical-radiologic-pathologic-immunohistochemical findings.

MiRNAs as non-invasive biomarkers in the serum of Oral Squamous Cell Carcinoma (OSCC) and Oral Potentially Malignant Disorder (OPMD) patients.

OBJECTIVE: Cell-free microRNAs have shown differential levels in the serum of individuals under disease conditions suggesting its potential to act as biomarkers. A population specific miRNA signature in oral cancer is reported in different studies. We aim to identify a set of serum specific miRNAs that may differentiate oral cancer, oral pre-malignant conditions from the healthy individuals. DESIGN: We investigated the levels of 24 miRNAs in the serum of 47 Oral squamous cell carcinoma (OSCC) patients, 20 patients with Oral potentially malignant disorders (OPMD) and 42 healthy controls from Eastern India. Small RNAs were isolated from serum samples followed by cDNA synthesis. Levels of miRNAs were determined using qRT-PCR. The sources of serum specific miRNAs were evaluated using GTEx-RNAseq and TCGA-HNSCC database. RESULTS: Five miRNAs, miR-483-5p, miR-31-5p, Let-7b-5p, miR-486-5p and miR-30e-5p showed significant elevation in OSCC patients. An Elastic-Net model with 4 miRNAs classified OSCC from healthy controls with 80 % sensitivity, 64.3 % specificity, and 72.4 % accuracy. Mir-483-5p and miR-31-5p was significantly overexpressed in OSCC tissues as well as significantly higher in the serum of Leukoplakia and Verrucous carcinoma patients suggesting their potential as early disease markers. MiR-483-5p showed a consistent elevated level in the serum/plasma of oral cancer patients across different population and was found to be tumour specific while, the rest of the miRNAs showed variable results across different studies. CONCLUSIONS: Our study suggested that the serum miRNAs in oral cancer and pre-malignant disorder conditions can be used as a non-invasive marker for screening of these oral conditions.

Effect of Lysyl Oxidase G473 A Polymorphism on Lysyl Oxidase and Total Soluble Collagen Expression in Oral Submucous Fibrosis.

BACKGROUND: Oral submucous fibrosis (OSF) is a debilitating collagen-metabolic disorder leading to submucosal fibrosis and trismus. Lysyl oxidase (LOX), a critical collagen biosynthetic enzyme, is up-regulated in OSF. Polymorphisms in the Lysyl oxidase gene have been associated with increased risk of OSF and might affect normal collagen synthesis, accumulation, or degradation, crucial in determining fibrosis severity. METHODS: One hundred OSF cases and 100 controls were genotyped for LOX G473A(Arg158Gln) polymorphism by polymerase chain reaction-restriction fragment length polymorphism. The expression of LOX was estimated both by quantitative mRNA analysis and western blot. Total soluble collagen was evaluated from mucosal tissue obtained from OSF cases. Immunohistochemical (IHC) localization of type 1 collagen was performed in mucosal tissue obtained from patients carrying various genotypes. RESULTS: Heterozygous G473A genotype was significantly higher in OSF cases [2.063(95% CI =1.059-4.016)], among 26-40 years age-group [4.375(95% CI=1.323-14.267),p=0.029] and in male patients [2.38 (95% CI= 1.107-5.121), p= 0.042]. LOX expression was significantly higher in cases of the heterozygous or homozygous carrier (p <0.001). We found the total soluble collagen level significantly (p <0.001) higher among patients carrying GA or AA genotype. IHC revealed focal deposition of type1 collagen in the submucosal tissue; comparatively higher deposition was evident in mucosal tissue of OSF patients carrying AA genotype. CONCLUSIONS: These findings suggest LOX G473A polymorphism confers an increased risk of OSF and may affect collagen accumulation in OSF cases.

Whole-Exome Sequencing Analysis of Oral Squamous Cell Carcinoma Delineated by Tobacco Usage Habits.

Oral squamous cell carcinoma (OSCC) is a common cancer of the oral cavity in India. Cigarette smoking and chewing tobacco are known risk factors associated with OSCC. However, genomic alterations in OSCC with varied tobacco consumption history are not well-characterized. In this study, we carried out whole-exome sequencing to characterize the mutational landscape of OSCC tumors from subjects with different tobacco consumption habits. We identified several frequently mutated genes, including TP53, NOTCH1, CASP8, RYR2, LRP2, CDKN2A, and ATM. TP53 and HRAS exhibited mutually exclusive mutation patterns. We identified recurrent amplifications in the 1q31, 7q35, 14q11, 22q11, and 22q13 regions and observed amplification of EGFR in 25% of samples with tobacco consumption history. We observed genomic alterations in several genes associated with PTK6 signaling. We observed alterations in clinically actionable targets including ERBB4, HRAS, EGFR, NOTCH1, NOTCH4, and NOTCH3. We observed enrichment of signature 29 in 40% of OSCC samples from tobacco chewers. Signature 15 associated with defective DNA mismatch repair was enriched in 80% of OSCC samples. NOTCH1 was mutated in 36% of samples and harbored truncating as well as missense variants. We observed copy number alterations in 67% of OSCC samples. Several genes associated with non-receptor tyrosine kinase signaling were affected in OSCC. These molecules can serve as potential candidates for therapeutic targeting in OSCC.

Molecular alterations in oral cancer between tobacco chewers and smokers using serum proteomics.

BACKGROUND: Tobacco exposure (through smoking or chewing) is one of the predominant risk factors associated with the development of oral squamous cell carcinoma (OSCC). Despite the growing number of patients diagnosed with OSCC, there are few circulating biomarkers for identifying individuals at a higher risk of developing the disease. Successful identification of candidate molecular markers for risk assessment could aid in the early detection of oral lesions and potentially be used for community screening of high-risk populations. OBJECTIVE: Identification of differentially expressed proteins in the serum of oral cancer patients which can serve as biomarkers for the diagnosis of the onset of oral cancer among tobacco users. METHODS: We employed a tandem mass tag (TMT)-based quantitative proteomics approach to study alterations in the serum proteomes of OSCC patients based on their tobacco exposure habits (chewing and smoking) compared to healthy individuals with no history of using any form of tobacco or any symptoms of the disease. RESULTS: Mass spectrometry-based analysis resulted in the identification of distinct signatures in the serum of OSCC patients who either chewed or smoked tobacco. Pathway analysis revealed opposing effects of dysregulated proteins enriched in the complement-coagulation signaling cascades with a high expression of the Serpin family of proteins observed in OSCC patients who chewed tobacco compared to healthy individuals whereas these proteins showed decreased levels in OSCC patients who smoked. ELISA-based validation further confirmed our findings revealing higher expression of SERPINA6 and SERPINF1 across serum of OSCC patients who chewed tobacco compared to healthy individuals. CONCLUSIONS: This study serves as a benchmark for the identification of serum-based protein markers that may aid in the identification of high-risk patients who either chew tobacco or smoke tobacco.

Proteomic Alterations Associated with Oral Cancer Patients with Tobacco Using Habits.

Tobacco abuse is a major risk factor associated with the development of oral squamous cell carcinoma. Differences in molecular aberrations induced by tobacco exposure by chewing or smoking form are not well studied in case of oral cancer. We used tandem mass tag-based quantitative proteomic approach to delineate proteomic alterations in oral cancer patients based on their history of tobacco using habits (patients who chewed tobacco, patients who smoked tobacco, and those with no history of tobacco consumption). Our data identified distinct dysregulation of biological processes and pathways in each patient cohort. Bioinformatics analysis of dysregulated proteins identified in our proteomic study revealed dysregulation of collagen formation and antigen processing/presentation pathway in oral cancer patients who smoked tobacco, whereas proteins associated with the process of keratinization showed enrichment in patients who chewed tobacco. In addition, we identified overexpression of proteins involved in immune pathways and downregulation of muscle contraction-mediated signaling events in all three cohorts, irrespective of tobacco using habits. This study lays the groundwork for identification of protein markers that may aid in identification of high-risk patients for cancer development based on the history of tobacco exposure habits.

Molecular alterations in oral cancer using high-throughput proteomic analysis of formalin-fixed paraffin-embedded tissue.

Loss of cell differentiation is a hallmark for the progression of oral squamous cell carcinoma (OSCC). Archival Formalin-Fixed Paraffin-Embedded (FFPE) tissues constitute a valuable resource for studying the differentiation of OSCC and can offer valuable insights into the process of tumor progression. In the current study, we performed LC-MS/MS-based quantitative proteomics of FFPE specimens from pathologically-confirmed well-differentiated, moderately-differentiated, and poorly-differentiated OSCC cases. The data were analyzed in four technical replicates, resulting in the identification of 2376 proteins. Of these, 141 and 109 were differentially expressed in moderately-differentiated and poorly differentiated OSCC cases, respectively, compared to well-differentiated OSCC. The data revealed significant metabolic reprogramming with respect to lipid metabolism and glycolysis with proteins belonging to both these processes downregulated in moderately-differentiated OSCC when compared to well-differentiated OSCC. Signaling pathway analysis indicated the alteration of extracellular matrix organization, muscle contraction, and glucose metabolism pathways across tumor grades. The extracellular matrix organization pathway was upregulated in moderately-differentiated OSCC and downregulated in poorly differentiated OSCC, compared to well-differentiated OSCC. PADI4, an epigenetic enzyme transcriptional regulator, and its transcriptional target HIST1H1B were both found to be upregulated in moderately differentiated and poorly differentiated OSCC, indicating epigenetic events underlying tumor differentiation. In conclusion, the findings support the advantage of using high-resolution mass spectrometry-based FFPE archival blocks for clinical and translational research. The candidate signaling pathways identified in the study could be used to develop potential therapeutic targets for OSCC.

Dysbiosis of Oral Microbiota During Oral Squamous Cell Carcinoma Development.

Infection with specific pathogens and alterations in tissue commensal microbial composition are intricately associated with the development of many human cancers. Likewise, dysbiosis of oral microbiome was also shown to play critical role in the initiation as well as progression of oral cancer. However, there are no reports portraying changes in oral microbial community in the patients of Indian subcontinent, which has the highest incidence of oral cancer per year, globally. To establish the association of bacterial dysbiosis and oral squamous cell carcinoma (OSCC) among the Indian population, malignant lesions and anatomically matched adjacent normal tissues were obtained from fifty well-differentiated OSCC patients and analyzed using 16S rRNA V3-V4 amplicon based sequencing on the MiSeq platform. Interestingly, in contrast to the previous studies, a significantly lower bacterial diversity was observed in the malignant samples as compared to the normal counterpart. Overall our study identified Prevotella, Corynebacterium, Pseudomonas, Deinococcus and Noviherbaspirillum as significantly enriched genera, whereas genera including Actinomyces, Sutterella, Stenotrophomonas, Anoxybacillus, and Serratia were notably decreased in the OSCC lesions. Moreover, we demonstrated HPV-16 but not HPV-18 was significantly associated with the OSCC development. In future, with additional validation, this panel could directly be applied into clinical diagnostic and prognostic workflows for OSCC in Indian scenario.

Oral submucous fibrosis: A global challenge. Rising incidence, risk factors, management, and research priorities.

Oral submucous fibrosis is a potentially malignant disorder of the oral cavity, with a high rate of malignant transformation. It is very common among habitual areca nut chewers. The pathogenesis of oral submucous fibrosis is not well established, but it is believed to be a disease of multifactorial origin, including areca nut chewing, ingestion of chilies, genetic factors, immunologic processes and nutritional deficiencies. Genetically susceptible individuals when exposed to areca nut chewing develop this disease over a variable period of time. Oral submucous fibrosis is considered to be a disease of collagen metabolism. Several genetic factors are reported but there is no consensus about the exact mechanism of disease initiation. Variations in histopathological presentation are noted among oral submucous fibrosis patients with habitual areca nut chewing in different forms and other additive agents, eg betel quid, pan masala and gutkha, together with a variety of tobacco habits. The role of epigenetic modifications, such as miRNA regulation, and DNA methylation is also being reported as part of the pathogenesis of oral submucous fibrosis. A combined approach, including analysis of genetic and epigenetic regulations with different habits, might be helpful to better understand the contributory factors and pathogenesis of this serious disorder.

Liquid biopsy: miRNA as a potential biomarker in oral cancer.

Oral cancer is one of the leading cancers in South-Asian countries. Despite the easy access of the oral cavity, the detection and five year survival rates of OSCC patients are dismal. Identification of non-invasive biomarkers to determine the progression and recurrence of OSCC could be of immense help to patients. Recent studies on oral cancer suggest the importance of non-invasive biomarker development. Micro-RNAs (miRNAs) are one of the important components of the cell-free nucleic acids available in different body fluids. Here, we have reviewed the current understanding of circulating miRNAs as non-invasive biomarkers in different body fluids of oral cancer patients. A number of circulating miRNAs are found to be common in the body fluids of OSCC patients, while many of these are study specific, the possible sources of this variability could be due to differences in sample processing, assay procedure, clinical stage of the disease, oral habit and environmental factors. The prognostic and therapeutic significance of these circulating miRNAs are suggested by several studies. Mir-371, mir-150, mir-21 and mir-7d were found to be potential prognostic markers, while mir-134, mir-146a, mir-338 and mir-371 were associated with metastases. The prognostic markers, mir-21 and mir-7d were also found to be significantly correlated with resistance to chemotherapy, while mir-375, mir-196 and mir-125b were significantly correlated with sensitivity to radiotherapy. Despite the promising roles of circulating miRNAs, challenges still remain in unravelling the exact regulation of these miRNAs before using them for targeted therapy.

Identification of potential biomarkers of head and neck squamous cell carcinoma using iTRAQ based quantitative proteomic approach.

Head and neck squamous cell carcinoma (HNSCC) is one of the most common cancers in India. Despite improvements in treatment strategy, the survival rates of HNSCC patients remain poor. Thus, it is necessary to identify biomarkers that can be used for early detection of disease. In this study, we employed iTRAQ-based quantitative mass spectrometry analysis to identify dysregulated proteins from a panel of head and neck squamous cell carcinoma (HNSCC) cell lines. We identified 2468 proteins, of which 496 proteins were found to be dysregulated in at least two out of three HNSCC cell lines compared to immortalized normal oral keratinocytes. We detected increased expression of replication protein A1 (RPA1) and heat shock protein family H (Hsp110) member 1 (HSPH1), in HNSCC cell lines compared to control. The differentially expressed proteins were further validated using parallel reaction monitoring (PRM) and western blot analysis in HNSCC cell lines. Immunohistochemistry-based validation using HNSCC tissue microarrays revealed overexpression of RPA1 and HSPH1 in 15.7% and 32.2% of the tested cases, respectively. Our study illustrates quantitative proteomics as a robust approach for identification of potential HNSCC biomarkers. The proteomic data has been submitted to ProteomeXchange Consortium (http://www.proteomecentral.proteomexchange.org) via the PRIDE public data repository accessible using the data identifier - PXD009241.

Oral squamous cell carcinoma profile in North-Eastern regions of India from habits to histopathology: A hospital-based study.

BACKGROUND: Head and neck cancers constitute about 5%-8% of total body cancers in Europe, America, but in India, this figure is somewhat higher. The aim of this study is to evaluate the current burden of oral cancers in India, particularly North-East India. MATERIALS AND METHODS: A full-length study starting from patient counseling to clinical and histopathological examination and grading was planned. The study was conducted under the guidance of clinician, oral surgeon, oral pathologists, and statistician. RESULTS: In the 3 years study, all the patients with oral lesions are examined clinically, out of them suspected oral cancer patients were histopathologically confirmed as oral squamous cell carcinoma patient. The socioeconomic profile of oral cancer patients in relation to all examined patients was summarized, and results are drawn. CONCLUSION: The studied population is heavily indulgent tobacco consumption. Education for cancer prevention, early detection, and treatment is needed.

Identification of over expressed proteins in oral submucous fibrosis by proteomic analysis.

Early detection and identification of oral pre-malignancy or malignancy help in management of the disease and improve survival rates. Oral submucous fibrosis (OSMF) is a major threat to public health worldwide and especially in Southeast Asian countries. Identification of biomarkers is a necessary step toward early diagnosis and treatment. In this study, differentially expressed proteins between oral submucous fibrotic tissue and normal control tissues were recorded by proteomic analysis using two dimensional electrophoresis (2DE) and MALDI TOF mass spectrometry. By proteomic analysis, 15 proteins were found to be upregulated and 10 proteins downregulated in the OSMF tissues than the control tissues; among these identified proteins, Hsp-70 1B, Calreticulin, and Lumican variant exhibited higher expression in OSMF tissues compared to the control tissues. Immunohistochemical analysis also showed elevated expression of these in OSMF tissues. Further validation was done by real time quantitative RT-PCR analysis; gene expression of Hsp-70 1B, Calreticulin, and Lumican variant were significantly increased (6.2-, 3.3-, 2.8- fold, respectively), whereas Enolase 1 was decreased by 0.5 fold in the OSMF tissues, consistent with proteomic results. The expression of proteins indicates that various cellular signaling pathways must be involved in the processes of fibrosis and suggests that expressed protein molecules play an important role in the pathogenesis of OSMF. These identified proteins may be potentially used in future studies of OSMF enabling to determine diagnostic marker or therapeutic targets of this precancerous condition of oral cavity.

Surgical management of chronic hyperplastic candidiasis refractory to systemic antifungal treatment.

Chronic hyperplastic candidiasis (CHC), earlier known as candidal leukoplakia, is a variant of oral candidiasis that classically presents as a white patch on the commissures of the oral mucosa and it is mostly caused by Candida albicans. Clinically, the lesions are usually asymptomatic and regress after appropriate antifungal therapy and correction of the underlying cause. If the lesions are untreated, a small portion may develop dysplasia and later progress into carcinoma. The purpose of this article is to report a case of CHC in a 57-year-old male patient with a significant smoking habit, who presented with a thick, nonscrapable, brownish-white coating on the dorsum of the tongue for 9 years. This case is of particular importance and concern because of the high risk for malignant transformation in CHC. The role of biopsy and histopathology is also stressed through this case report in arriving at a definitive diagnosis and treatment planning. Further, this case is interesting because it was refractory to local and systemic antifungal treatment and so, surgery was chosen as an alternative treatment modality considering the side effects of the prolonged use of antifungal drugs.

Genome-wide DNA methylation profile identified a unique set of differentially methylated immune genes in oral squamous cell carcinoma patients in India.

BACKGROUND: Oral squamous cell carcinoma (OSCC) is one of the common malignancies in Southeast Asia. Epigenetic changes, mainly the altered DNA methylation, have been implicated in many cancers. Considering the varied environmental and genotoxic exposures among the Indian population, we conducted a genome-wide DNA methylation study on paired tumor and adjacent normal tissues of ten well-differentiated OSCC patients and validated in an additional 53 well-differentiated OSCC and adjacent normal samples. RESULTS: Genome-wide DNA methylation analysis identified several novel differentially methylated regions associated with OSCC. Hypermethylation is primarily enriched in the CpG-rich regions, while hypomethylation is mainly in the open sea. Distinct epigenetic drifts for hypo- and hypermethylation across CpG islands suggested independent mechanisms of hypo- and hypermethylation in OSCC development. Aberrant DNA methylation in the promoter regions are concomitant with gene expression. Hypomethylation of immune genes reflect the lymphocyte infiltration into the tumor microenvironment. Comparison of methylome data with 312 TCGA HNSCC samples identified a unique set of hypomethylated promoters among the OSCC patients in India. Pathway analysis of unique hypomethylated promoters indicated that the OSCC patients in India induce an anti-tumor T cell response, with mobilization of T lymphocytes in the neoplastic environment. Survival analysis of these epigenetically regulated immune genes suggested their prominent role in OSCC progression. CONCLUSIONS: Our study identified a unique set of hypomethylated regions, enriched in the promoters of immune response genes, and indicated the presence of a strong immune component in the tumor microenvironment. These methylation changes may serve as potential molecular markers to define risk and to monitor the prognosis of OSCC patients in India.

Quantitative assessment of silver-stained nucleolar organizer region in odontogenic cysts to correlate the growth and malignant potentiality.

CONTEXT: The most common and important odontogenic cyst involving jaws is the odontogenic keratocyst (OKC) or primordial cyst, the dentigerous cyst and the radicular cyst. These cysts all though do not show similar behavior, they all have the potentiality to recur. Silver nitrate staining of the nucleolar organizer regions (AgNORs) of the benign and malignant lesions is becoming very useful as a diagnostic indicator. Thus, the aim of this study is to assess the diagnostic potential of AgNORs in the cystic epithelium of common odontogenic cysts. MATERIALS AND METHODS: Archived specimens of odontogenic cysts were stained with hematoxylin and eosin stain and AgNOR stain. RESULTS: The comparative evaluation of the AgNOR counts was done among the three varieties of odontogenic cysts, i.e., radicular cysts, dentigerous cysts and OKC and were observed that the mean for OKC was significantly higher than that of radicular cyst. CONCLUSION: Therefore, AgNor could be used as an efficient tool for comparative evaluation of microscopic features such as epithelial thickness, surface keratinization and mural proliferation in dentigerous cyst to that of the AgNOR count.

Molecular Pathology of Malignant Transformation of Oral Submucous Fibrosis.

Oral submucous fibrosis (OSF) is prevalent mostly in Southeast Asia, particularly in the Indian subcontinent. Chewing betel nuts and betel leaves, with or without tobacco, has been associated with OSF. Betel quid contents including guvacine, arecoline, guvacoline, arecaidine, and chavibetol are considered to play an important part in the occurrence of OSF. Transformation of OSF to squamous cell carcinoma (SCC) is variable, but up to 13% conversion of OSF to SCC has been reported. Various genetic and molecular mechanisms impact the malignant transformation of OSF, causing changes in the cell cycle, DNA, keratinocytes, and keratin; tumor-cell proliferation and survival; angiogenesis; fibrosis through epithelial-mesenchymal transitions (EMTs), and tissue hypoxia. All are reviewed here, including potential biomarkers for malignant transformation of OSF. These interactions are not fully understood, but a critical mass of knowledge is building up to ultimately allow the understanding of all mechanisms involved.

Embryonal rhabdomyosarcoma: A rare oral tumor.

Rhabdomyosarcoma is the malignant neoplasm of striated muscle and a relatively uncommon tumor of the oral cavity. Embryonal variety is the most common subtype, observed in children below 10 years of age but occasionally seen in adolescents and young adults. The present report describes a case of embryonal rhabdomyosarcoma in the left posterior buccal mucosa, with extension in the adjacent alveolus, soft palate, oropharynx and nasopharynx of a 17-year-old female.