The RBP1-CKAP4 axis activates oncogenic autophagy and promotes cancer progression in oral squamous cell carcinoma.

Retinol-binding protein 1 (RBP1) is involved in several physiological functions, including the regulation of the metabolism and retinol transport. Studies have shown that it plays an important role in the pathogenesis of several types of cancer. However, the role of RBP1 and its correlation with autophagy in oral squamous cell carcinoma (OSCC) pathogenesis remain unknown. In this study, RBP1 was identified as the most significantly upregulated DEPs with a >2-fold change in OSCC samples when compared to normal tissues through iTRAQ-based proteomics analysis coupled with 2D LC-MS/MS. RBP1 overexpression was significantly associated with malignant phenotypes (differentiation, TNM stage, and lymphatic metastasis) of OSCC. In vitro experiments demonstrated that RBP1 was significantly increased in OSCC tissues and cell lines compared with control group. RBP1 overexpression promoted cell growth, migration, and invasion of OSCC cells. Silencing of RBP1 suppressed tumor formation in xenografted mice. We further demonstrated that the RBP1-CKAP4 axis was a critical regulator of the autophagic machinery in OSCC, inactivation of autophagy rescued the RBP1-CKAP4-mediated malignant biological behaviors of OSCC cells. Overall, a mechanistic link was provided by RBP1-CKAP4 between primary oncogenic features and the induction of autophagy, which may provide a potential therapeutic target that warrants further investigation for treatment of OSCC.

Automated oral squamous cell carcinoma identification using shape, texture and color features of whole image strips.

Despite profound knowledge of the incidence of oral cancers and a large body of research beyond it, it continues to beat diagnosis and treatment management. Post physical observation by clinicians, a biopsy is a gold standard for accurate detection of any abnormalities. Towards the application of artificial intelligence as an aid to diagnosis, automated cell nuclei segmentation is the most essential step for the recognition of the cancer cells. In this study, we have extracted the shape, texture and color features from the histopathological images collected indigenously from regional hospitals. A dataset of 42 whole slide slices was used to automatically segment and generate a cell level dataset of 720 nuclei. Next, different classifiers were applied for classification purposes. 99.4 % accuracy using Decision Tree Classifier, 100 % accuracy using both SVM and Logistic regression and 100 % accuracy using SVM, Logistic regression and Linear Discriminant were acquired for shape, textural and color features respectively. The in-depth analysis showed SVM and Linear Discriminant classifier gave the best result for texture and color features respectively. The achieved result can be effectively converted to software as an assistant diagnostic tool.

CircCDR1as upregulates autophagy under hypoxia to promote tumor cell survival via AKT/ERK½/mTOR signaling pathways in oral squamous cell carcinomas.

Autophagy, as an important non-selective degradation mechanism, could promote tumor initiation and progression by maintaining cellular homeostasis and the cell metabolism as well as cell viability. CircCDR1as has been shown to function as an oncogene in cancer progression, however, it remains largely unknown as to how autophagy is regulated by circCDR1as in oral squamous cell carcinoma (OSCC). In this study, we validated the functional roles of circCDR1as in regulation of autophagy in OSCC cells and further investigated how circCDR1as contributed to cell survival via up-regulating autophagy under a hypoxic microenvironment by using combination of human tissue model, in vitro cell experiments and in vivo mice model. We found that hypoxia promoted the expression level of circCDR1as in OSCC cells and elevated autophagy. In addition, circCDR1as further increased hypoxia-mediated autophagy by targeting multiple key regulators of autophagy. We revealed that circCDR1as enhanced autophagy in OSCC cells via inhibition of rapamycin (mTOR) activity and upregulation of AKT and ERK½ pathways. Overexpression of circCDR1as enhanced OSCC cells viability, endoplasmic reticulum (ER) stress, and inhibited cell apoptosis under a hypoxic microenvironment. Moreover, circCDR1as promoted autophagy in OSCC cells by sponging miR-671-5p. Collectively, these results revealed that high expression of circCDR1as enhanced the viability of OSCC cells under a hypoxic microenvironment by promoting autophagy, suggesting a novel treatment strategy involving circCDR1as and the inhibition of autophagy in OSCC cells.

Segmentation and analysis of surface characteristics of oral tissues obtained by scanning electron microscopy to differentiate normal and oral precancerous condition.

Abnormal epithelial stratification is a sign of oral dysplasia and hence evaluation of surface characteristics of oral epithelial region can help in detection of cancerous progression. Surface characteristics can be better visualised by Scanning Electron Microscopy (SEM) in comparison to light microscopy. In our study we have developed automated image processing algorithms i.e. Gaussian with median filtering and Gradient filtering, using MATLAB 2016b, to segment the surface characteristics i.e. the ridges and pits in the SEM images of oral tissue of normal (13 samples) and Oral Submucous Fibrosis (OSF) (36 samples) subjects. After segmentation, quantitative measurement of the parameters like area, thickness and textural features like entropy, contrast and range filter of ridges as well as area of pit and the ratio of area of ridge vs. area of pit was done. Statistical significant differences were obtained in between normal and OSF study groups for thickness (p=0.0107), entropy (p<0.00001) and contrast of ridge (p<0.00001) for Gaussian with median filtering and for all the parameters except thickness of the ridge(p=1.386), for Gradient filtering. Thus, computer aided image processing by Gradient filter followed by quantitative measurement of the surface characteristics provided precise differentiation between normal and precancerous oral condition.

Ultrastructural immunolocalization of telomerase and hyaluronate in migrating keratinocytes in a case of oro-pharyngeal squamous cancer.

The ultrastructural immunolocalization of telomerase and hyaluronate has been studied in a case of oropharyngeal squamous carcinoma. Immunofluorescence shows that telomerase immunolabeling is present in the cytoplasm and in nuclei of some keratinocytes during their migration into the underlying connective tissue. The electron microscope shows that the nuclear localization of telomerase mainly occurs in the large nucleoli and in likely Cajal bodies, the sites of assembling and maturation of proteins forming the telomerase complex. Aside ribosomes, the nucleolus has a role in the biosynthesis of this reverse transcriptase during cell proliferation in normal tissues and in tumors. The cytoplasmic labeling for telomerase is frequently associated with an irregular network of keratin bundles but the significance of this observation is unclear. Hyaluronate, detected through ultrastructural immunolocalization of a hyaluronate binding protein, is abundant mostly along the cell membrane of the detaching basal keratinocytes during epithelial mesenchymal transition. A coat of hyaluronate surrounds the free keratinocytes of the squamous epithelium and is present around the connective cells present underneath. The study supports the hypothesis that hyaluronate forms a pathway along which epithelial cells can migrate during epidermal mesenchymal transition and may also shield cancer cells from immune cells.

CerS6 regulates cisplatin resistance in oral squamous cell carcinoma by altering mitochondrial fission and autophagy.

Chemoresistance remains a challenge in the effective treatment of solid tumors, including oral squamous cell carcinoma (OSCC). Mitochondrial dynamics and autophagy have recently been implicated in the chemoresistance of cancer cells. The neutralization of ceramide is also associated with multidrug resistance, and ceramide synthase 6 (CerS6) is known to induce apoptosis. However, whether CerS6 regulates chemoresistance in OSCC is not clearly understood. Therefore, we investigated the role of CerS6 in the susceptibility of OSCC cells to cisplatin. In this study, we observed that cisplatin-resistant OSCC cells process lower levels of fission-state mitochondria and cell apoptosis than cisplatin-sensitive cells, and autophagy was activated in cisplatin-resistant OSCC cells. We found lower CerS6 expression in cisplatin-resistant OSCC cells. Overexpression of CerS6 with lentivirus-encoded CerS6 complementary DNA in cisplatin-resistant OSCC cells increased cisplatin sensitivity. Overexpression of CerS6 enhanced mitochondrial fission and apoptosis and attenuated cisplatin-induced autophagy in cisplatin-resistant OSCC cells. Further investigation indicated that CerS6 might function through altering calpain expression to enhance cisplatin sensitivity. Cisplatin-resistant OSCC cells xenografted onto a nude mouse model confirmed that CerS6 enhanced cisplatin chemotherapy sensitivity to reduce tumor volume. These data indicate that CerS6 could mediate an effective response to cisplatin in chemoresistant OSCC.

The ultrastructural features of the premalignant oral lesions.

Premalignant oral lesions are among the most important risk factors for the development of oral squamocellular carcinoma. Recent population studies indicate a significant rise in the prevalence of leukoplakia, erythroplakia/erythroleukoplakia, actinic cheilitis, submucous fibrosis and erosive lichen planus. Since standard histopathological examination has numerous limitations regarding the accurate appreciation of potential malignant transformation, the present study aims to aid these evaluations using the transmission electron microscopy (TEM) technique, which emphasizes ultrastructural changes pertaining to this pathology. Oral mucosa fragments collected from 43 patients that were clinically and histopathologically diagnosed with leukoplakia, erosive actinic cheilitis and erosive lichen planus have been processed through the classic technique for the examination using TEM and were examined using a Philips CM100 transmission electron microscope. The electron microscopy study has confirmed the histopathological diagnosis of the tissue samples examined using photonic microscopy and has furthermore revealed a series of ultrastructural details that on the one hand indicate the tendency for malignant transformation, and on the other reveal characteristic features of tumor development. All the details furnished by TEM complete the overall picture of morphological changes, specific to these lesions, indicating the importance of using these techniques in establishing both a correct diagnosis and prognosis.

Interactive Visual Exploration of 3D Mass Spectrometry Imaging Data Using Hierarchical Stochastic Neighbor Embedding Reveals Spatiomolecular Structures at Full Data Resolution.

Technological advances in mass spectrometry imaging (MSI) have contributed to growing interest in 3D MSI. However, the large size of 3D MSI data sets has made their efficient analysis and visualization and the identification of informative molecular patterns computationally challenging. Hierarchical stochastic neighbor embedding (HSNE), a nonlinear dimensionality reduction technique that aims at finding hierarchical and multiscale representations of large data sets, is a recent development that enables the analysis of millions of data points, with manageable time and memory complexities. We demonstrate that HSNE can be used to analyze large 3D MSI data sets at full mass spectral and spatial resolution. To benchmark the technique as well as demonstrate its broad applicability, we have analyzed a number of publicly available 3D MSI data sets, recorded from various biological systems and spanning different mass-spectrometry ionization techniques. We demonstrate that HSNE is able to rapidly identify regions of interest within these large high-dimensionality data sets as well as aid the identification of molecular ions that characterize these regions of interest; furthermore, through clearly separating measurement artifacts, the HSNE analysis exhibits a degree of robustness to measurement batch effects, spatially correlated noise, and mass spectral misalignment.

A nomogram for predicting the risk of neck node metastasis in pathologically node-negative oral cavity carcinoma.

OBJECTIVE: To generate a nomogram for predicting the risk of neck node metastasis in pathologically node-negative patients using a combination of variables comprising of protein expression, ultrastructural alterations and clinicopathological parameters. MATERIALS AND METHODS: Surgically removed oral tumours (n = 103) were analysed for the expression of desmosomal and hemidesmosomal assembly proteins by immunohistochemistry and ultrastructural alterations by transmission electron microscopy (TEM). Protein expression, ultrastructural alterations and clinicopathological variables were used to construct nomogram from the training set in 75 patients. Clinical utility of the nomogram was validated in a discrete set of 28 patients. RESULTS: Univariate and multivariate analyses were performed on the training set, and obtained significant variables comprising of integrin ß4 expression (p = .027), number of hemidesmosomes (p = .027)/desmosomes (p = .046), tumour differentiation grade (p = .033) and tumour thickness (p = .024) were used for construction of the nomogram. The area under the curve was calculated for both training 0.821 (95% CI 0.725-0.918) and validation sets 0.880 (95% CI 0.743-1.000). The nomogram demonstrated a predictive accuracy of 73.3% and 78.6% with the sensitivity of 81.4% and 83.3% in the training and validation sets, respectively. CONCLUSIONS: The nomogram constructed on postsurgical tumour samples will be a value addition to histopathology for the detection of neck node metastasis in pathologically node-negative patients.

Diosmin induce apoptosis through modulation of STAT-3 signaling in 7,12 dimethylbenz(a)anthracene induced harmster buccal pouch carcinogenesis.

Diosmin is naturally found flavanoid in many citrus fruits known to have anti-inflammatory, antihyperglycemic, antioxidant and antimutagenic properties. Effects of Diosmin on IL-6/STAT-3 expression in hamster buccal pouch carcinogenesis remain unclear. Alterations in many genes encode crucial proteins, which regulate cell proliferation, differentiation and apoptosis have been implicated in oral cancer. In the present study, we investigated the effect of dietary Diosmin on IL-6/STAT-3 signaling in the 7,12-dimethylbenz[a]anthracene (DMBA)-induced hamster buccal pouch (HBP) carcinogenesis by examining the protein expression of IL-6/STAT-3 and its related genes. Immunoblotting and immunohistochemical analyses revealed that Diosmin (100mg/kgb.w) supplement inhibits key events in signaling especially STAT-3 phosphorylation and subsequent nuclear translocation. Results revealed that inhibition of proliferation and angiogenesis is associated with regulation of the STAT-3 pathway; where Diosmin prevents phosphorylation of JAK-1 which was ascend by IL-6, thereby inhibiting STAT-3 phosphorylation. Consequently, an imbalance in the Bax/Bcl-2 ratio triggered the caspase cascade in favor of apoptosis. Transmission electron microscopic studies proved the effect of Diosmin on ultrastructural changes. Finally our results provide significant evidence that Diosmin prevents the development and progression of HBP carcinomas through the inhibition of IL-6/STAT-3 signaling and its downstream events. Thus, Diosmin functions as a potent inhibitor of tumor development and progression by targeting IL-6/STAT-3 signaling may be an ideal candidate for cancer chemoprevention.

Performance evaluation of maximal separation techniques in immunohistochemical scoring of tissue images.

This paper presents an automatic scoring method for p53 immunostained tissue images of oral cancer that consist of tissue image segmentation, splitting of clustered nuclei, feature extraction and classification. The tissue images are segmented using entropy thresholding technique in which the optimum threshold value to each color component is obtained by maximizing the global entropy of its gray-level co-occurrence matrix and clustered cells are separated by selectively applying marker-controlled watershed transform. Cell nuclei feature is extracted by maximal separation technique (MS) based on blue component of tissue image and subsequently, each cell is classified into one of four categories using multi-level thresholding. Finally, IHC score of tissue images have been determined using Allred method. A statistical analysis is performed between immuno-score of manual and automatic method, and compared with the scores that have obtained using other MS techniques. According to the performance evaluation, IHC score based on blue component that has high correlation coefficients (CC) of 0.95, low mean difference (MD) of 0.15, and a very close range of 95% confidence interval with manual scores. Therefore, automatic scoring method presented in this paper has high potential to help the pathologist in IHC scoring of tissue images.

Connecting cyto-nano-architectural attributes and epithelial molecular expression in oral submucous fibrosis progression to cancer.

OBJECTIVE: Problems in pre-cancer diagnosis complicate cancer theragnosis as well as life expectancy. There is uncertainty regarding malignant transformation of oral submucous fibrosis (OSF), an oral pre-cancer with dysplastic (OSFWD) and non-dysplastic (OSFWT) subtypes. Understanding the structural, molecular and physical aspects of epithelial homeostasis may be useful. MATERIALS AND METHODS: Histopathological grading of biopsy sections was performed using H&E staining. Alterations in epithelial surface architecture in different groups was evaluated using scanning electron microscopy (SEM). The expression of crucial epithelial genes (p63, CK-5/6, CK-10, E-cadherin and ß-catenin) was studied by immunohistochemistry, Western blot and RT-PCR analysis. RESULTS: SEM observations revealed that the surface epithelial ridge pattern became thick and dense, and pit pattern gradually decreased in OSFWD and oral squamous cell carcinoma (OSCC). p63, ΔNp63 and CK-5/6 were up-regulated in OSFWD and OSCC but down-regulated in OSFWT. CK-10 was down-regulated in OSFWD compared to OSFWT. Cytoplasmic expression of E-cadherin and ß-catenin was elevated in dysplastic and cancerous conditions. Moreover, statistical correlation between SEM features (ridges and pits) and molecular attributes demonstrated a significant positive relationship between the ridge-to-pit ratio and p63 population density (r=0.85) and the ridge-to-pit ratio and CK-5/6 intensity (r=0.63). CONCLUSIONS: Molecular changes related to epithelial progressive maturation and cellular proliferation are correlated with concomitant alteration of epithelial surface architecture which helps to predict the malignant potentiality of OSF.

Influence of selenium on radiogenic collagen destruction and the degree of collagen tissue maturation in stage III oral squamous cell carcinoma patients undergoing therapeutic irradiation.

INTRODUCTION: We set out to assess whether selenium, an antioxidant mineral could influence radiogenic collagen maturation. MATERIALS AND METHODS: The study comprise of normal (Group I), untreated oral carcinoma cases (Group II) (n = 20), cases who underwent radiotherapy (Group IIa) n = 10 and cases supplemented with selenium along with radiotherapy (Group IIb) n = 10. RESULTS: Spectrophotometric estimation and luminescence spectral assignment of collagen showed improved collagen maturation status. Measurement of the mature collagen cross-links hydroxylysylpyridinoline and lysylpyridinoline by high-performance liquid chromatography on irradiated tissues showed a considerable decrease in the selenium Group IIb (P < 0.05) indicating a decrease in collagen fragments. Electron microscopic studies showed significant morphological alteration in the selenium group. The micro nucleus frequency, decreased in radiation group (P < 0.05) compared with untreated (P < 0.05). While much more decrease observed in the selenium group (P < 0.05). DISCUSSION: The results represent the effect of selenium treatment with a bearing on carcinogenic process to curtail it, thus enhancing the maturity of collagen.

High resolution live cell Raman imaging using subcellular organelle-targeting SERS-sensitive gold nanoparticles with highly narrow intra-nanogap.

We report a method to achieve high speed and high resolution live cell Raman images using small spherical gold nanoparticles with highly narrow intra-nanogap structures responding to NIR excitation (785 nm) and high-speed confocal Raman microscopy. The three different Raman-active molecules placed in the narrow intra-nanogap showed a strong and uniform Raman intensity in solution even under transient exposure time (10 ms) and low input power of incident laser (200 µW), which lead to obtain high-resolution single cell image within 30 s without inducing significant cell damage. The high resolution Raman image showed the distributions of gold nanoparticles for their targeted sites such as cytoplasm, mitochondria, or nucleus. The high speed Raman-based live cell imaging allowed us to monitor rapidly changing cell morphologies during cell death induced by the addition of highly toxic KCN solution to cells. These results strongly suggest that the use of SERS-active nanoparticle can greatly improve the current temporal resolution and image quality of Raman-based cell images enough to obtain the detailed cell dynamics and/or the responses of cells to potential drug molecules.

Effects of TiO2 nano glass ionomer cements against normal and cancer oral cells.

BACKGROUND/AIM: Incorporation of nanoparticles (NPs) into the glass ionomer cements (GICs) is known to improve their mechanical and antibacterial properties. The present study aimed to investigate the possible cytotoxicity and pro-inflammation effect of three different powdered GICs (base, core build and restorative) prepared with and without titanium dioxide (TiO2) nanoparticles. MATERIALS AND METHODS: Each GIC was blended with TiO2 nanopowder, anatase phase, particle size <25 nm at 3% and 5% (w/w), and the GIC blocks of cements were prepared in a metal mold. The GICs/TiO2 nanoparticles cements were smashed up with a mortar and pestle to a fine powder, and then subjected to the sterilization by autoclaving. Human oral squamous cell carcinoma cell lines (HCS-2, HSC-3, HSC-4, Ca9-22) and human normal oral cells [gingival fibroblast (HGF), pulp (HPC) and periodontal ligament fibroblast (HPLF)] were incubated with different concentrations of GICs in the presence or absence of TiO2 nanoparticles, and the viable cell number was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method. Prostaglandin E2 was quantified by enzyme-linked immunosorbent assay (ELISA). Changes in fine cell structure were assessed by transmission electron microscopy. RESULTS: Cancer cells exhibited moderate cytotoxicity after 48 h of incubation, regardless of the type of GIC and the presence or absence of TiO2 NPs. GICs induced much lower cytotoxicity against normal cells, but induced prostaglandin E2 production, in a synergistic wanner with interleukin-1ß. CONCLUSION: The present study shows acceptable to moderate biocompatibility of GICs impregnated with TiO2 nanoparticles, as well as its pro-inflammatory effects at higher concentrations.

Prognostic factors in oral and oropharyngeal cancer based on ultrastructural analysis and DNA methylation of the tumor and surgical margin.

Oral and oropharyngeal cancers are characterized by relatively low 5- year survival rates due to many factors, including local recurrence. The identification of new molecular markers may serve for the estimation of prognosis and thus augment treatment decisions and affect therapy outcome. The aim of this study was to describe the morphological characteristics and the DNA methylation status of the CDKN2A,CDH1, ATM, FHIT and RAR- genes in the central and peripheral part of the tumor and the surgical margin and evaluate their prognostic significance. 53 patients with oral and oropharyngeal cancer were enrolled to the prospective study, and had been primarily treated surgically. Correlations between morphological data, hypermethylation status and clinicopathological data, as well as prognosis, were assessed. Nuclei polymorphism highly correlated with T stage (p < 0.0001), N stage (p < 0.046), and metastases to the lymph nodes pN (p < 0.004 ). Also, the number of cells in irregular mitosis correlated with T stage (p < 0.004), and highly with pN (p < 0.009). The significance of CDKN2A hypermethylation as a good prognostic factor was also established in the Kaplan-Meir test. The ultrastructural analysis showed that none of the examined tumors had homogenous texture and that resection margin specimens clean in HE stained tissue samples frequently contained single tumor cells or few cells in groups surrounded by connective tissue. This indicates the superiority of electron microscopy over standard histopathological analysis. Thus, a combination of such morphological examination with epigenetic parameters described herein could result in the discovery of promising new prognostic markers of the disease.

Human oral cancer cells with increasing tumorigenic abilities exhibit higher effective membrane capacitance.

OBJECTIVE: Although cells with tumorigenic/stem cell-like properties have been identified in many cancers, including oral squamous cell carcinoma (OSCC), their isolation and characterisation is still at early stages. The aim of this study is to characterise the electrophysiological properties of OSCC cells with different tumorigenic properties in order to establish if a correlation exists between tumorigenicity and cellular electrical characteristics. MATERIALS AND METHODS: Rapid adherence to collagen IV was used as a non-invasive, functional method to isolate subsets of cells with different tumorigenic abilities from one oral dysplastic and three OSCC-derived cell lines. The cell subsets identified and isolated using this method were further investigated using dielectrophoresis, a label-free method to determine their electrophysiological parameters. Cell membrane morphology was investigated using scanning electron microscopy (SEM) and modulated by use of 4-methylumbelliferone (4-MU). RESULTS: Rapid adherent cells (RAC) to collagen IV, enriched for increased tumorigenic ability, had significantly higher effective membrane capacitance than middle (MAC) and late (LAC) adherent cells. SEM showed that, in contrast to MAC and LAC, RAC displayed a rough surface, extremely rich in cellular protrusions. Treatment with 4-MU dramatically altered RAC membrane morphology by causing loss of filopodia, and significantly decreased their membrane capacitance, indicating that the highest membrane capacitance found in RAC was due to their cell membrane morphology. CONCLUSION: This is the first study showing that OSCC cells with higher tumour formation ability exhibit higher effective membrane capacitance than cells that are less tumorigenic. OSSC cells with different tumorigenic ability possessed different electrophysiological properties mostly due to their differences in the cell membrane morphology. These results suggest that dielectrophoresis could potentially used in the future for reliable, label-free isolation of putative tumorigenic cells.

Epithelial maturation pattern of dysplastic epithelium and normal oral epithelium exposed to tobacco and alcohol: a scanning electron microscopic study.

BACKGROUND: The detection of oral cancer at an early stage is an optimal strategy and is the most effective approach for preventing further progression. The rationale of the study was to evaluate the epithelial maturation pattern in oral mucosa exposed to tobacco/alcohol and on dysplastic oral mucosa using the scanning electron microscope. METHODS: Fifteen subjects were selected based on clinical examination and divided into three groups: group 1-patients with apparently normal oral mucosa; group 2-patients with oral mucosa exposed to tobacco/alcohol; group 3-patients with clinical diagnosis of leukoplakia. An incisional biopsy was performed from the buccal mucosa. One part of the specimen was prepared for light microscopy and the other part was prepared for scanning electron microscopy. RESULTS: Light microscopy revealed nonkeratinized stratified squamous epithelium in group 1, while group 2 demonstrated hyperparakeratinized stratified squamous epithelium with mild cytological atypia, and group 3 showed architectural and cytological changes. Scanning electron microscopy demonstrated flat-surfaced cells with equidistant parallel microridges in group 1, while group 2 showed irregular and widened microridges with numerous pits and absence of honeycomb pattern. Group 3 showed irregularly arranged broad and swollen cells with numerous pits and irregular microvilli projecting over the surface. CONCLUSION: The present study establishes the relationship of the surface abnormalities to the tendency of the cells to become malignant and thus serves as a tool in early detection of squamous cell carcinoma. It also emphasizes the need of routine follow-up in these high-risk patients for progression of carcinoma.