Oral Squamous Cell Carcinoma Biopsies Research Articles

Energy Metabolic Profile in Oral Potentially Malignant Disorders and Oral Squamous Cell Carcinoma: A Preliminary Landscape of Warburg Effect in Oral Cancer.

We hypothesized that cell energy metabolic profiles correlate with normal, dysplastic, and tumor cell/tissue statuses and may be indicators of aggressiveness in oral squamous cell carcinoma (OSCC) cells. The energy-related proteins that were differentially expressed in human OSCC fragments (n = 3) and their adjacent epithelial tissue (TAE) were verified using mass spectrometry (MS). Immunohistochemistry for 4-hydroxynonenal (4-HNE) was performed to evaluate the oxidative stress patterns in OSCC (n = 10), epithelial dysplasia (n = 9), and normal epithelial (n = 4) biopsies. The metabolic energy profile of OSCC aggressiveness was investigated in human OSCC cell lines with different levels of epithelial-mesenchymal transition proteins. The genes associated with the proteins found by MS in this study were analyzed using survival analysis (OS), whereas the genes associated with a poorer prognosis were analyzed using context-specific expression, Gene Ontology (GO) and Cancer Hallmarks for function enrichment analysis. The rationale for all experimental approach was to investigate whether the variation in energy metabolism profile accompanies the different phenotypes (from epithelial to mesenchymal) during the epithelial-mesenchymal transition. All OSCC fragments exhibited an increase in glycolysis-related proteins and a decrease in mitochondrial activity compared to the TAE region (p < 0.05), probably due to the downregulation of pyruvate dehydrogenase and antioxidant proteins. Additionally, the OSCC cell lines with a mesenchymal profile (SCC4, SCC9, and SCC25) had a lower mitochondrial mass and membrane potential and generated lower levels of reactive oxygen and nitrogen species than the TAE region. When we analyzed 4-HNE, the reactive species levels were increased in the epithelial regions of OSCC and potentially malignant lesions. A decrease in the levels of 4-HNE/reactive species was observed in the connective tissue underlying the dysplastic regions and the OSCC invasion zone. Based on this scenario, aggressive OSCC is associated with high glycolytic and oxidative metabolism and low mitochondrial and antioxidant activities, which vary according to the differentiation level of the tumor cells and the stage of carcinogenesis.

Histomorphology based prediction of p16 immunopositivity and p16/HPV DNA co-positivity in oral squamous cell carcinoma

The histomorphological features predictive of p16 and human papilloma virus (HPV DNA) positivity in oropharyngeal carcinoma have been a matter of much debate. However, only few studies have been done on oral squamous cell carcinoma (OSCC) to correlate the histomorphological features with p16 and HPV DNA positivity. Oral squamous cell carcinoma has distinct etiopathogenesis, treatment and prognosis as compared to oropharyngeal carcinomas.A total of 800 oral squamous cell carcinoma biopsy cases were evaluated for features suggestive of HPV infection like basaloid appearance, absence of stromal reaction, nests and lobules of tumor cells with pushing borders, central necrosis, lympho-epithelial morphology, koilocytes, and non-keratinizing or hybrid morphology. Immunohistochemistry was performed for p16 expression (E6H4 clone, CINtec histology, Roche diagnostics). The cases which showed 2+/3+ (from moderate to high intensity) staining with >75 % cells were considered as p16 immunopositive. All the p16 immunopositive cases were subjected to real-time PCR (polymerase chain reaction) for HPV DNA detection to confirm HPV positivity. A total of 139 (17.37 %) OSCC cases were p16 immunopositive and out of these 104 (104/139, 74.8 %) cases showed HPV-DNA PCR positivity. None of the features were found to be predictive on multivariate logistic regression analysis. However, on bivariable analysis, nest/lobule with pushing border was the only histopathological feature which had a significant correlation with p16 immunopositivity (P value = 0.0001) and p16 and HPV DNA copositivity (P value = 0.0001). (Fisher's exact test -two tailed).To conclude-morphology is not really predictive of HPV positivity in OSCC cases. Only one feature- nests and lobule with pushing border is suggestive on bivariable analysis.

Chromatin remodeling-driven autophagy activation induces cisplatin resistance in oral squamous cell carcinoma

It is still challenging to predict the efficacy of cisplatin-based therapy, particularly in relation to the activation of macroautophagy/autophagy in oral squamous cell carcinoma (OSCC). We studied the effect of selected chromatin remodeling genes on the cisplatin resistance and their interplay with autophagy in 3-dimensional tumor model and xenografts. We analyzed gene expression patterns in the cisplatin-sensitive UMSCC1, and a paired cisplatin-resistant UM-Cis cells. Many histone protein gene clusters involved in nucleosome assembly showed significant difference of expression. Gain- and loss-of-function analyses revealed an inverse correlation between cisplatin resistance and HIST1H3D expression, while a positive correlation was observed with HIST3H2A or HIST3H2B expression. In UM-Cis, HIST3H2A- and HIST3H2B-mediated chromatin remodeling upregulates autophagy status, which results in cisplatin resistance. Additionally, knockdown of HIST3H2A or HIST3H2B downregulated autophagy-activating genes via chromatin compaction of their promoter regions. MiTF, one of the key autophagy regulators upregulated in UM-Cis, negatively regulated transcription of HIST1H3D, suggesting an interplay between chromatin remodeling-dependent cisplatin resistance and autophagy. On comparing the staining intensity between cisplatin-sensitive and –insensitive tissues from OSCC patients, protein expression pattern of the selected histone protein genes were matched with the in vitro data. By examining the relationship between autophagy and chromatin remodeling genes, we identified a set of candidate genes with potential use as markers predicting chemoresistance in OSCC biopsy samples.

Assessment of Turnaround Time (TAT) for Oral Squamous Cell Carcinoma Biopsies: A Single-Institution Experience.

Oral squamous cell carcinoma (OSCC) is one of the most common cancers worldwide. A delay in the diagnosis of OSCC can have a drastic impact on management and patient outcomes. One of the most crucial elements in oral management is the timely histopathological final diagnosis. Turnaround time (TAT) is regarded as the most important component of the quality performance evaluation. Many labs have struggled to improve their TATs despite advancements in computerization, transport systems, and analytical technologies. Aim: This study aimed to assess the TAT of OSCC cases, assess the mean TAT period, evaluate any TAT delays, and explore the reasons behind the TAT delays. Materials and methods: OSCC reports in Saveetha Dental College and Hospitals, Chennai, for one year from January 1, 2022, to December 31, 2022, were retrieved from the Dental Information Archival Software (DIAS), and the mean TAT was noted. Further, the number of cases with delay in TAT was also observed, and the reason for their delay was listed. Descriptive statisticsand graphical representation were performed utilizing IBM SPSS Statistics for Windows, V. 23.0 (IBM Corp., Armonk, NY, USA). One-way ANOVA was performed with a significance set at a p-value less than 0.05. 230 OSCC cases were retrieved and included in the TAT evaluation for this study. Among 230 cases, 161 (70%) were incisional and 69 (30%) were excisional biopsies. Only seven (4%) incisional cases and seven (10%) excisional biopsies showed a delay in TAT. The most common reason for the delay in TAT was the requirement for deeper sections and decalcification of bone specimens. Out of 161 incisional cases, only 48 (29%) have undergone excision and further treatment. Twenty-one out of 69 (30%) excisional cases were found to be referral cases from other private institutions. The overall average TAT for 12 months was 3.24 ± 0.41 days for incisional biopsies and 11.88 ± 2.07 days for excisional biopsies. One-way ANOVA revealed a statistically significant p-value of less than 0.00001. Our study sheds light on specific challenges in TAT delay and opportunities for the improvement of TAT. This can result in faster TAT of OSCC reports, further improve patient care, and enable prompt treatment. This study quantified the TAT for OSCC cases and identified critical areas for process improvement. The findings can inform strategies to streamline diagnostic workflows, reduce delays, and ultimately improve the timely delivery of care to patients with OSCC.

Spatial subsetting enables integrative modeling of oral squamous cell carcinoma multiplex imaging data

Oral squamous cell carcinoma (OSCC), a prevalent and aggressive neoplasm, poses a significant challenge due to poor prognosis and limited prognostic biomarkers. Leveraging highly multiplexed imaging mass cytometry, we investigated the tumor immune microenvironment (TIME) in OSCC biopsies, characterizing immune cell distribution and signaling activity at the tumor-invasive front. Our spatial subsetting approach standardized cellular populations by tissue zone, improving feature reproducibility and revealing TIME patterns accompanying loss-of-differentiation. Employing a machine-learning pipeline combining reliable feature selection with multivariable modeling, we achieved accurate histological grade classification (AUC= 0.88). Three model features correlated with clinical outcomes in an independent cohort: granulocyte MAPKAPK2 signaling at the tumor front, stromal CD4+ memory Tcell size, and the distance of fibroblasts from the tumor border. This study establishes a robust modeling framework for distilling complex imaging data, uncovering sentinel characteristics of the OSCC TIME to facilitate prognostic biomarkers discovery for recurrence risk stratification and immunomodulatory therapy development.

Epstein-Barr virus predominance and immunological abnormalities in oral squamous cell carcinoma

BACKGROUND: Both benign and harmful head and neck disorders have been associated with the Epstein-Barr virus (EBV). Many studies have connected EBV to oral squamous cell carcinoma (OSCC). Oral squamous cell carcinoma is the most prevalent type of cancer. Fresh tissue samples from patients with OSCC were tested for the presence of Epstein-Barr virus. OBJECTIVE: To determine the frequency of EBV and IL10 expression in OSCC patients. MATERIALS AND METHODS: Fifty individuals with OSCC and 25 with clinically healthy oral mucosa were studied. In situ hybridization was used for the detection of EBV. Serum IL-10 levels were also evaluated in patients and controls using a human IL-10 ELISA Kit. RESULTS: EBV was detected in 4 healthy patients, 6 with moderately differentiated OSCC, 10 with poorly differentiated OSCC, and 19 with undifferentiated OSCC. These differences were statistically significant (p&lt;0.05). IL-10 expression was more common in OSCC diagnostic groups than healthy controls, and the difference in blood IL-10 levels between patients and controls was statistically significant (p&lt;0.01). CONCLUSION: The prevalence of EBV in OSCC suggests its possible role in oral cavity malignancy. On the other hand, IL-10 is expressed at higher levels in OSCC biopsies; such elevated concentrations may promote viral spread.

Abstract 4530: Single-cell analysis of oral squamous cell carcinoma (OSCC) from smoker vs. non-smoker patients highlights two groups of tumors with distinct immune microenvironments

Abstract Background and Rationale: OSCC is the most frequent head and neck cancer. While it is mainly affecting patients with a smoking and/or alcohol history (SD), 10% to 15% of OSCC are diagnosed in patients with not known risk factors (NSND). We and others have shown that OSCC from NSND patients are characterized by an enrichment in interferon gamma (IFNγ) response and PD-1 signaling pathways together with a higher intratumor T-cell infiltrate. Herein, we seek to gain more insights into the immune and non-immune tumor microenvironment (TME) of OSCC in SD vs. NSND patients. Material and Methods: We made use of single-cell RNA seq (10x Genomics) from non-sorted and CD45 negative enriched cell suspensions of OSCC biopsies from 11 treatment naïve patients, human papillomavirus negative, including 6 SD and 5 NSND patients. Results: All samples were characterized by a strong immune cell infiltrate. Consistent with our previous results, the analysis of 51,629 immune cells confirmed that NSND patients’ samples displayed a significantly higher proportion of T cells (p = 0.005) compared to SD patients. NK cells were also increased, with 2% in SD patients versus 6% in NSND patients (p = 0.008). T cells strongly expressed IFNγ and remarkably, both myeloid cells and tumor cells from NSND patients had an increased enrichment in IFNγ response pathway in NSND as compared to SD patients. The TME of SD patients was characterized by the presence of 15% of plasma cells vs. 3% in NSND patients (p = 0.003). Thus, tumors from NSND versus SD patients represent two distinct groups of tumors characterized by a different immune TME. Moreover, tumors from SD patients displayed 2% vs. 4% Cancer Associated Fibroblasts (CAFs) (p = 0.01). In addition, among CAFs subpopulations, myofibroblast CAFs, defined as the subpopulation producing extracellular matrix proteins were found only in tumors from SD patients, suggesting that distinct CAFs subpopulations may shape the immune TME by affecting the recruitment and function of adaptive immune cells in SD versus NSND patients. Finally, we analyzed tumor cells using non-negative matrix factorization to find Meta Programs (MP), which are set of genes coordinately upregulated. We identified 5 MP associated with different biological functions: epithelial differentiation, stress, cell cycle (G2/M), partial epithelial-to-mesenchymal transition, and epithelial senescence. While the epithelial differentiation MP was found in all patients, the remaining ones were more frequently found in SD patients. Conclusion: Our results show that the TME of OSCC from NSND vs. SD differs in terms of T, NK, and plasma cells composition, CAFs, and tumor cells MP. Integration with spatial transcriptomics is ongoing. Those biological differences may represent an opportunity to refine the therapeutic approaches in the two populations of patients. Citation Format: Yannick Le Meitour, Béatrice Vanbervliet, Sonia Canjura-Rodriguez, Cyril Degletagne, Laurie Tonon, Lucas Michon, Jebrane Bouaoud, Philippe Zrounba, Aude Excoffier, Karène Mahtouk, Pierre Saintigny. Single-cell analysis of oral squamous cell carcinoma (OSCC) from smoker vs. non-smoker patients highlights two groups of tumors with distinct immune microenvironments. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4530.

Inverse correlation of CD3 & vascular endothelial growth factor in incisional oral squamous cell carcinoma biopsies predicts nodal metastasis & poor survival of patients.

Oral squamous cell carcinoma (OSCC) is widely prevalent in the Indian subcontinent mainly due to habit-associated aetiologies. Immune regulation and angiogenesis are the part of tumourigenesis that play a crucial role in metastasis and survival. However, the concurrent expression of vascular endothelial growth factor (VEGF) and CD3 (immune regulator receptor on T-lymphocyte) in the same OSCC tissue samples has not been reported in the Indian population. The present study evaluated the expression of CD3+ T-cells and VEGF in OSCC tissue samples and studied the clinicopathological correlation and survival analysis in an Indian population. This was a retrospective study conducted on 30 formalin-fixed and paraffin embedded sections which were histologically diagnosed as OSCC cases comprising of 15 metastatic OSCC and 15 non-metastatic OSCC with available clinical data and survival status. Reduced expression of CD3+ T-cells and increased VEGF expression were observed in metastatic OSCC samples. The correlation of expression of CD3+ T-cells and VEGF with clinicopathological parameters showed a significant association between these markers with age, nodal status, site of the lesion and survival. Reduced expression of CD3+ T-cells in OSCC was found to be associated with a significantly poor survival. VEGF was found to be over expressed in metastatic OSCC as compared to that in non-metastatic OSCC. The study findings suggest that the evaluation of CD3 and VEGF in incisional OSCC biopsies can be considered for predicting the survival outcome and metastasis.

Histopathologic Oral Cancer Prediction Using Oral Squamous Cell Carcinoma Biopsy Empowered with Transfer Learning.

Oral cancer is a dangerous and extensive cancer with a high death ratio. Oral cancer is the most usual cancer in the world, with more than 300,335 deaths every year. The cancerous tumor appears in the neck, oral glands, face, and mouth. To overcome this dangerous cancer, there are many ways to detect like a biopsy, in which small chunks of tissues are taken from the mouth and tested under a secure and hygienic microscope. However, microscope results of tissues to detect oral cancer are not up to the mark, a microscope cannot easily identify the cancerous cells and normal cells. Detection of cancerous cells using microscopic biopsy images helps in allaying and predicting the issues and gives better results if biologically approaches apply accurately for the prediction of cancerous cells, but during the physical examinations microscopic biopsy images for cancer detection there are major chances for human error and mistake. So, with the development of technology deep learning algorithms plays a major role in medical image diagnosing. Deep learning algorithms are efficiently developed to predict breast cancer, oral cancer, lung cancer, or any other type of medical image. In this study, the proposed model of transfer learning model using AlexNet in the convolutional neural network to extract rank features from oral squamous cell carcinoma (OSCC) biopsy images to train the model. Simulation results have shown that the proposed model achieved higher classification accuracy 97.66% and 90.06% of training and testing, respectively.

Multistage classification of oral histopathological images using improved residual network.

Oral cancer is a prevalent disease happening in the head and neck region. Due to the high occurrence rate and serious consequences of oral cancer, an accurate diagnosis of malignant oral tumors is a major priority. Thus, early diagnosis is very effective to give the patient a prompt response to treatment. The most efficient way for diagnosing oral cancer is from histopathological imaging, which provides a detailed view of inside cells. Accurate and automatic classification of oral histopathological images remains a difficult task due to the complex nature of cell images, staining methods, and imaging conditions. The use of deep learning in imaging techniques and computational diagnostics can assist doctors and physicians in automatically analysing Oral Squamous Cell Carcinoma biopsy images in a timely and efficient manner. Thus, it reduces the operational workload of the pathologist and enhance patient management. Training deeper neural networks takes considerable time and requires a lot of computing resources, due to the complexity of the network and the gradient diffusion problem. With this motivation and inspired by ResNet's significant successes to handle the gradient diffusion problem, in this study we suggest the novel improved ResNet-based model for the automated multistage classification of oral histopathology images. Three prospective candidate model blocks are presented, analyzed, and the best candidate model is chosen as the optimal one which can efficiently classify the oral lesions into well-differentiated, moderately-differentiated and poorly-differentiated in significantly reduced time, with 97.59% accuracy.

Automated classification of cells into multiple classes in epithelial tissue of oral squamous cell carcinoma using transfer learning and convolutional neural network

The analysis of tissue of a tumor in the oral cavity is essential for the pathologist to ascertain its grading. Recent studies using biopsy images reveal computer-aided diagnosis for oral sub-mucous fibrosis (OSF) carried out using machine learning algorithms, but no research has yet been outlined for multi-class grading of oral squamous cell carcinoma (OSCC). Pertinently, with the advent of deep learning in digital imaging and computational aid in the diagnosis, multi-class classification of OSCC biopsy images can help in timely and effective prognosis and multi-modal treatment protocols for oral cancer patients, thus reducing the operational workload of pathologists while enhancing management of the disease. With this motivation, this study attempts to classify OSCC into its four classes as per the Broder’s system of histological grading. The study is conducted on oral biopsy images applying two methods: (i) through the application of transfer learning using pre-trained deep convolutional neural network (CNN) wherein four candidate pre-trained models, namely Alexnet, VGG-16, VGG-19 and Resnet-50, were chosen to find the most suitable model for our classification problem, and (ii) by a proposed CNN model. Although the highest classification accuracy of 92.15% is achieved by Resnet-50 model, the experimental findings highlight that the proposed CNN model outperformed the transfer learning approaches displaying accuracy of 97.5%. It can be concluded that the proposed CNN based multi-class grading method of OSCC could be used for diagnosis of patients with OSCC.

Polarization gating technique extracts depth resolved fluorescence redox ratio in oral cancer diagnostics.

Mortality of oral cancer is often due to late diagnosis. Effective non-invasive diagnostic techniques may increase the survival rate based on an earlier diagnosis.. We report on the application of the polarization gating technique for isolating weakly scattered and highly scattered components of fluorescence emission from the superficial and deeper layers of tissue due to intrinsic fluorophores NADH and FAD. The fluorescence polarization spectra were collected from 21 normal and 67 oral squamous cell carcinoma biopsy tissues. The tissues were excited at 350 nm and the fluorescence emission had two peaks corresponding to NADH, and FAD respectively. The spectra were analyzed using the polarization gating technique along with the spectral deconvolution method to derive the optical redox ratio from different layers of tissue. The fractional change in redox ratio between superficial and deeper layers of tissue exhibits excellent statistical significance (p<10-3) which may be due to a shift in the metabolic pathway from oxidative phosphorylation to glycolysis in the cancer cell. Further, variation in collagen intensity in deeper layers of tissue is observed which may be attributed to the breakdown of collagen fibers in the stroma. Linear discriminant analysis showed that oral cancer tissue is discriminated with a better accuracy using polarization gating technique than that of conventional fluorescence spectroscopy.

NOTCH1 signaling in oral squamous cell carcinoma via a TEL2/SERPINE1 axis.

Inactivating mutations in the EGF-like ligand binding domain of NOTCH1 are a prominent feature of the mutational landscape of oral squamous cell carcinoma (OSCC). In this study, we investigated NOTCH1 mutations in keratinocyte lines derived from OSCC biopsies that had been subjected to whole exome sequencing. One line, SJG6, was found to have truncating mutations in both NOTCH1 alleles, resulting in loss of NOTCH1 expression. Overexpression of the NOTCH1 intracellular domain (NICD) in SJG6 cells promoted cell adhesion and differentiation, while suppressing proliferation, migration and clonal growth, consistent with the previously reported tumour suppressive function of NOTCH1 in OSCC. Comparative gene expression profiling identified SERPINE1 as being downregulated on NICD overexpression and predicted an interaction between SERPINE1 and genes involved in cell proliferation and migration. Mechanistically, overexpression of NICD resulted in upregulation of ETV7/TEL2, which negatively regulates SERPINE1 expression. Knockdown of SERPINE1 phenocopied the effects of NICD overexpression in culture. Consistent with previous studies and our in vitro findings, there were inverse correlations between ETV7 and SERPINE1 expression and survival in OSCC primary tumours. Our results suggest that the tumour suppressive role of NOTCH1 in OSCC is mediated, at least in part, by inhibition of SERPINE1 via ETV7.

Interleukin-1beta and Caspase-3 expression serve as independent prognostic markers for metastasis and survival in oral squamous cell carcinoma.

The Indian subcontinent has the highest incidence and prevalence of oral squamous cell carcinoma (OSCC). Inflammation and apoptosis are two emerging hall marks of cancer that might play a significant role in tumorigenesis and metastasis. Concurrent expression of proinflammatory cytokine (IL-1β) and executioner caspase (Caspase-3) in same OSCC tissue samples has not been reported in an Indian population. The present study aimed to evaluate the immunohistochemical expression of IL-1β and Caspase-3 in same OSCC tissue samples with clinicopathological correlation and survival analysis in Indian population. A retrospective study was conducted utilizing 40 formalin fixed paraffin embedded histologically diagnosed cases of OSCC comprising of 20 metastatic OSCC and 20 non-metastatic OSCC. Increased expression of IL-1β and Caspase-3 were observed in metastatic OSCC. Correlation of expression of IL-1β and Caspase-3 with clinicopathological parameters revealed a significant association between these markers and staging, nodal status and site of the lesion. Over expression of IL-1β and Caspase-3 was associated with advanced stage and poor survival of the patient. IL-1β overexpression showed significantly lower disease-free survival and disease specific survival as well. Overexpression of IL-1β and Caspase-3 in incisional OSCC biopsies could be considered for predicting metastasis and survival outcome.

BLOOD VESSEL DENSITY AS A PREDICTOR IN ORAL SQUAMOUS CELL CARCINOMA USING CD34

INTRODUCTION: Oral squamous cell carcinoma (OSCC) accounts for more than 90% of all oral cancers. Despite advances in the approach to locally advanced disease, about 50% of the tumors will recur. Fifty percent of patients present nodal involvement that is detectable during diagnosis. Of this group, less than 40% survive after five years, compared with a 90% survival rate for patients without metastasis. If the lesion is detected at an early stage and treated effectively, the survival rate could exceed 50%. Many retrospective studies have shown that microvessel density (MVD) could represent a valid independent prognostic factor for the overall survival. The blood vessel density may constitute a relevant parameter for determining the prognosis and guiding biological treatments for aggressive OSCC OBJECTIVES: To assess the MVD using CD34 in lymph node positive and negative cases and to correlate the results with the lymph node status. MATERIALS AND METHODS: MVD was calculated in 22 surgical specimens taken from OSCC patients. Biopsies were taken from the primary tumor of eleven cases proved to have positive lymph node and eleven cases with negative lymph nodes. Immunohistochemical (IHC) staining was performed using a Labeled Strept-Avidin Biotin complex method (LSAB), using the CD34 antibody. RESULTS: OSCC biopsies showed immunoreactivity to CD34, the difference between the mean MVD and lymph node status was significantly correlated. No significant relation with the histological grade site was found. CONCLUSIONS: MVD was significantly correlated with lymph node status and it could be used as an indicator for progression and early metastasis of OSCC. But, no correlation was found between the MVD and the histological grading.

The Optimal Sequencing Depth of Tumor Biopsies for Identifying Clonal Cell Populations

The tumor content of a biopsy and the average depth of coverage are two essential aspects when performing DNA sequencing using next-generation sequencing technologies. The heterogeneous nature of cancer necessitates the identification of distinct clonal cell populations to better understand and treat cancer. Deep sequencing enables researchers to identify these populations, but no consensus on an optimal depth exists for identifying clonal populations. Data from eight deep-sequenced oral squamous cell carcinoma biopsies obtained from three stage IV patients, with various degrees of tumor content, were used to randomly down sample the depth before being subjected to cluster analysis. An increase in coverage resulted in an increase in resolution for clusters of mutations, enabling the identification of distinct clonal cell populations and clonal events. From a depth of 800×, limited gain in resolution can be achieved; and from a depth of 1200×, the resolution stabilizes. Overall, researchers should aim for an average depth of 1000× to 1200× when performing deep sequencing. The tumor content will, however, dictate the resolution and fidelity of the analysis, as an increase in tumor complexity increases the need for higher tumor content.

EXPRESSION OF Ki-67 AND CD105 MARKERS IN ORAL SQUAMOUS CELL CARCINOMA

INTRODUCTION: Oral squamous cell carcinoma (OSCC) is the commonest malignancy in the oral cavity. It is influenced by genetic modifications; as well as numerous environmental factors, such as smoking, tobacco chewing, alcohol consumption, infection by oncogenic viruses, and low intake of fruits and vegetables. CD105 has been introduced as a marker for angiogenesis which stains vessels that are in the proliferating stage. Ki-67is a nuclear protein that is recognized as a nuclear antigen present in proliferating cells, but absent in resting cells. OBJECTIVES: To evaluate and correlate the expression of Ki-67 and CD105 in different histopathological grades of OSCC. MATERIALS AND METHODS: Immunohistochemical (IHC) study using the Ki-67 and CD105 antibody was done on 20 surgical specimens and 5 normal mucosal tissues taken from OSCC patients and healthy individuals respectively. RESULTS: Remarkable improvement in the histology and the ultrastructure of the alveolar bone of rats in Group III was observed. Moreover, hematological values revealed significant decrease in the inflammatory condition of rats with induced RA after fish oil treatment. CONCLUSIONS: Ki-67 and CD105 are expressed in OSCC biopsies. Additionally, the fraction of Ki-67positive tumor cells (the Ki-67 labeling index) is often correlated with the clinical course of OSCC. CD105 microvessel density can be a useful factor for predicting the course of oral squamous cell carcinoma

Synchronous Luminescence Spectroscopy as a Tool in the Discrimination and Characterization of Oral Cancer Tissue.

High incidence of oral cancer is primarily due to ongoing tobacco epidemic. In this work, synchronous luminescence spectroscopy (SLS) has been used to characterize and discriminate oral cancer tissue. Spectral deconvolution method is employed to compute the fluorescence intensity, peak wavelength, and full width half maxima for different endogenous fluorophores. The fluorescence measurements were made on 21 normal and 88 oral squamous cell carcinoma biopsy tissues. Besides, variations in relative concentration of collagen, NADH, and FAD, peak shifts and broadening of peaks are observed for tryptophan, NADH, and FAD, in oral cancer tissues indicating both biochemical and micro environmental changes at cellular level. Linear discriminant analysis showed that oral cancer tissue is discriminated with a sensitivity and specificity of 100% and 95.2% respectively.

IMMUNOHISTOCHEMICAL EXPRESSION OF TLR-4 AND ITS CORRELATION TO VARIOUS GRADES OF ORAL SQUAMOUS CELL CARCINOMA

INTRODUCTION: Oral cancer is a major health problem, causing high morbidity and mortality rates. Oral squamous cell carcinoma (OSCC) accounts for 90-95% of all oral malignancies. During the last decade, significant evidence has suggested that inflammation plays an important role in tumorigenesis, and microenvironment-derived signals which are important constituents in all tumors. Toll-like receptors (TLRs) are classical signals that play a key role in the innate immune system. They drive the inflammatory response and control the cellular proliferation and survival by stimulating the immune cells and promoting integrated processes of inflammation and tissue repair. This can predict that TLR4 may be of great significance in tumors. Hence, this study is to evaluate and correlate the expression of TLR-4 in various grades of oral squamous cell carcinoma (OSCC). OBJECTIVES: To evaluate and correlate the expression of TLR-4 in different histopathological grades of OSCC MATERIALS AND METHODS: An immunohistochemical (IHC) study using the TLR-4 antibody were done on 50 surgical specimens and 10 normal mucosal tissues taken from OSCC patients and healthy individuals, respectively. The staining were performed using a Labeled Strept-Avidin Biotin complex method (LSAB) RESULTS: TLR-4 was expressed in human OSCC biopsies and the expression level was correlated with the tumor differentiation. Higher expression was detected in the well and the moderately differentiated squamous cell carcinomas than the poorly differentiated ones. CONCLUSIONS: TLR-4 expression could be a prognostic factor in oral squamous cell carcinoma cases as expression was detected in the well and moderately differentiated higher than poorly differentiated squamous cell carcinomas.

Predicting Tumor Sensitivity to Chemotherapeutic Drugs in Oral Squamous Cell Carcinoma Patients

Oral Squamous Cell Carcinoma (OSCC) patients respond poorly to chemotherapy. We analyzed the expression of 11 drug response-related genes in 31 OSCC biopsies, collected prior to any treatment, using custom-designed PCR array. Further, we investigated the drug response pattern of selected anticancer drugs by BH3 (Bcl2 Homology-3) profiling in the primary cells isolated from OSCC tissues. Then, we correlated the results of drug-response gene expression pattern with apoptotic priming to predict tumor response to chemotherapy. The best performing drug (BPD) and response differences (RD) between the drugs were identified using statistical methods to select the best choice of drug in a personalized manner. Based on the correlation, we classified OSCC tumors as sensitive (13 tumors), moderately responsive (16 tumors) or resistant (2 tumors) to chemotherapy. We found that up-regulation of genes linked with drug resistance facilitates survival of tumor samples, which was revealed by the percentage of apoptotic priming. Moreover, we found that paclitaxel-induced 40–45% apoptotic priming compared to other drugs. Average response difference (RD) analysis showed that 80% of tumors responded well to paclitaxel as compared to other drugs studied. Therefore, gene expression analysis with BH3 profiling reveals drug sensitivity that could be translated for drug selection before treatment.