Prognostic Nomogram for Lymph-Node Metastasis in Oral Squamous Cell Carcinoma (OSCC) Using Immunohistochemical Marker D2-40.

Introduction: Nomograms are proven in "individualized risk prediction" in sarcomas and breast and prostate cancers. Incorporating immunohistochemical markers and histopathological parameters can enhance accuracy of these graphical representations of statistical predictive models concerning metastasis. D2-40, a monoclonal antibody to podoplanin (regulator of motility expressed in malignant epithelial cells), dually predicts metastatic potential of tumour by estimating the motile tumour phenotype and by detecting lymphatic vessels/density, both essential to metastasis in OSCC. Thus, we propose a model that incorporates D2-40 immunostaining of individual tumour cells (ITC) too with other variables (seen in H+E staining) as a predictive nomogram. Methods: Sixty cases of OSCC were selected with equal number of cases (n=30) of pN0 and pN+ status. Bryne's grading of invasive front of tumour (ITF) was done on H+E-stained slides followed by D2-40 immunostaining for ITCs at ITF and lymphatic vessels. Multivariate regression analysis was used to generate the nomogram of LNM where the predictive contribution of each covariate, namely depth of invasion, D2-40-stained ITCs, gender, histological grade, and worst pattern of invasion (WPOI), was plotted on a scale of 1-100 points. Results: The nomogram showed that the strongest variable in OSCC was the WPOI in H+E-stained section followed by D2-40-positive ITCs and gender. Discussion: Our predictive nomogram for LNM in OSCC surprisingly showed that a tumour with lower score of WPOI (islands vs ITC) showed numerous D2-40-positive ITCs, drastically increasing the probability of metastasis. The concept of "individualized risk prediction" can be used to predict lymph node metastasis using a variety of histopathological criteria that can be visualized in routine and immunohistochemical staining in OSCC with the aid of a nomogram.

Variability in the Epithelial Expression of Ki67 and Polarization Characteristics of Collagen in Odontogenic Cysts and Tumours

ABSTRACT Objective: To assess the proliferation of epithelium (using the Ki67 index) and the polarization pattern of collagen in selected odontogenic cysts and tumours. In addition, an exploratory analysis of the effect of inflammation on the proliferation rate was done. Material and Methods: Following immunohistochemical staining, the labelling/proliferation index of Ki67 was calculated. The thickness and corresponding polarization colour of 100 juxta-epithelial picrosirius red-stained collagen fibers were assessed using linear micrometry with an eyepiece reticule under × 1000 magnification. Inflammation was graded subjectively as mild, moderate, and severe. Results: Overall Ki-67 expression was higher in the radicular cyst, Odontogenic Keratocyst, Ameloblastoma, while suprabasal Ki-67 positivity was maximum in Odontogenic Keratocyst. The stromal collagen fibers in Ameloblastoma showed predominantly green birefringence, whereas Odontogenic Keratocyst had orange birefringence. There was no significant association of inflammation with Ki-67 expression or birefringence patterns. Conclusion: The highest Ki67 expression in the radicular cyst, followed by Odontogenic Keratocyst and Ameloblastoma. Differences in the collagen maturation pattern were noted innately in five lesions studied and were further influenced by inflammatory changes. Epithelial proliferation and concomitant expression of thickness and maturity of the stromal collagen are innate features of the lesion further influenced by inflammation in various odontogenic cysts and tumours and may, in turn, guide the clinical behavior.

Performance of Dental Students in Understanding and Retention of Oral Pathology Concepts: A Comparative Analysis of Traditional versus Live-Field Teaching Methods.

Background: Understanding oral aspects of pathology by traditional techniques has always been a paradigm in the field of dental education. Traditional methods of teaching include interaction using black board, projectors, and alternate methods of teaching such as a student-centered approach where live-field demonstrations, audio visual aids, and student interaction are also gaining importance, ultimately promoting active education. The aim of the study was to compare live-field and static-field teaching methods in understanding and retention of the histopathological features in dental students. Methods: This was a cross-sectional analytical study, wherein a uniform cohort of III-year dental students was obtained by randomizing the study subjects. Practical classes were conducted using traditional black board/static pictures and dynamic live-field teaching comprising of microscope connected to an HD screen and projector demonstrating the preferred microscopic field. Alternately, the level of retention of knowledge was measured using customized topic-based tests. The comparison of average scores was done between live-field and static-field teaching groups using the paired t-test. Results: The test scores using the paired t-test were marginally elevated in the conventional mode of teaching; however, it varied with respect to precise topics taken using both the genres of teaching. Conclusion: A balance of both conventional and virtual teaching needs to be achieved to enhance the comprehension in student learning. Nevertheless, in the impending years, advanced research is entailed to see if the virtual mode of teaching could replace the conventional method for the advancement in the study prospects.

Estimation of Age by Evaluating the Occlusal Tooth Wear in Molars: A Study on Dakshina Kannada Population.

PURPOSE: Assessment of teeth to estimate age in the living as well as the dead is well documented in the literature. Though applied in conjunction with other parameters in Gustafson's technique, attrition of teeth solely is an underexplored indicator for age estimation mainly in India. Most of the studies using attrition of teeth have been conducted in bioarchaeology, and in populations where studies are being conducted in forensic contexts, the results are debatable. Thus, the present study aimed to evaluate the accuracy of age estimation using the Average Stage of Attrition (ASA) method by Li and Ji in adults of the Dakshina Kannada region. MATERIALS AND METHODS: This cross-sectional study assessed the degree of attrition in both maxillary and mandibular first and second molars of 136 adults of Dakshina Kannada origin (age range: 20-70 years) according to the 10 stages given by Li and Ji to derive population-specific linear regression equations. RESULTS: Univariate analysis of prediction of age with attrition showed a significant association of attrition score with age, stratified by sex, arch as well as in total (p-value <0.001) with a standard error of estimate of 9.231 years. The estimated age was within ±5 years of the actual age in 44.2% of the females and 50.0% of the males. A comparative review of 23 studies done to estimate age from attrition score clinically is documented in the manuscript. CONCLUSION: The findings of this study suggest that evaluation of tooth wear by the Average Stage of Attrition (ASA) method can be used as a non-invasive means to aid in the determination of age in adults of Dakshina Kannada origin. Although the error increases with age, this method provides a measurable means of seriation into older and younger categories.

Recognition of lysyl oxidase as a potential predictive biomarker for oral squamous cell carcinoma: an immunohistochemical study.

BACKGROUND: Lysyl oxidase (LOX) is a copper amine oxidase which belongs to the LOX multigene family and is normally involved in cross-linking of stromal collagen fibers. LOX expression has been found to be associated with increased episodes of recurrence, metastasis and overall poor prognosis in renal cell carcinomas and melanomas. This study aimed to assess the effects of LOX on the prognosis of oral squamous cell carcinoma (OSCC), which is one of the most common cancers in India. METHODS: The immunohistochemical expression of lysyl oxidase using LOX2 primary antibody was assessed at the tumor proper, invasive tumor front and peritumoral stroma in tissue sections from 40 cases of histologically proven OSCC. RESULTS: LOX expression was elevated in OSCC patients who had lymph node metastasis and in those who died of disease. No significant variation was seen with histological grade. CONCLUSIONS: LOX has a 'pro-neoplastic' effect as it modulates the host stroma to favor increasing tumor mass and worsening prognosis. Increased expression of LOX causes increased collagen fiber cross-linkage that stiffens the stromal matrix. This increases compressive stresses contributing to tissue hypoxia that elevates Rho GTPase-dependent cytoskeletal tension leading to erratic tumor cell morphogenesis that in turn confers motility to these cells resulting in metastasis. Inhibitors of LOX can potentially down-regulate LOX levels in the tumor micro-environment by controlling tissue hypoxia and curtailing the production of hypoxic LOX molecules.

Pattern-Corrected Mitotic Activity Index (PMAI): A Novel Prognosticator of Oral Squamous Cell Carcinoma.

OBJECTIVE: The main aim was to assess the efficiency of the Mitotic Activity Index (MAI) and a novel index devised by us, the Pattern-Corrected Mitotic Activity Index (PMAI) in prognostication of Oral Squamous Cell Carcinoma in terms of lymph node involvement, margin, recurrence and survival status. MATERIAL AND METHOD: The study group consisted of 60 cases of histologically-proven Oral Squamous Cell Carcinoma with known status of prognostic indicators. Hematoxylin and eosin stained sections of the tumor proper were utilized for assessment of mitotic activity and pattern of invasion. The Mitotic Activity Index and Pattern-Corrected Mitotic Activity Index were then calculated and correlated with the prognosticators. RESULTS: Mitotic Activity Index was higher in patients who had better survival and low recurrence rates. Pattern-Corrected Mitotic Activity Index showed the greatest percentage increase in relation to lymph node involvement as compared to the other indices. Kaplan Meier survival analysis showed that a higher Pattern-Corrected Mitotic Activity Index ( > 1.45) was associated with poorer survival (37.19 months). CONCLUSION: Lack of significant association of the Mitotic Activity Index in relation to prognosticators could be attributed to a tumor having a migratory phenotype rather than a proliferative phenotype as seen in late-stage tumors. Late-stage tumors have more of a poorer pattern of invasion which is reflected best by Pattern-Corrected Mitotic Activity Index by correlating with poorer survival and lymph node involvement.

Expression of α-smooth muscle actin in benign and malignant salivary gland tumors: An immunohistochemical study.

BACKGROUND AND OBJECTIVES: Myoepithelial cells (ME) are known to contribute in the patterning of salivary gland neoplasms (SGN) and possess cytoplasmic smooth muscle actin (SMA) revealed by alpha SMA (α-SMA). The present study aimed to assess the expression of α-SMA in selected benign and malignant SGN (pleomorphic adenoma [PA], mucoepidermoid carcinoma (MEC), adenoid cystic carcinoma (ACC), and polymorphous low-grade adenocarcinoma (PLGA). MATERIALS AND METHODS: The intensity and pattern of expression of α-SMA were studied in 25 cases of SGN's ACC (n = 7), MEC (n = 8), PA (n = 8), and PLGA (n = 2), and correlated with the histological patterns. RESULTS: Maximum expression of α-SMA in the epithelial compartment was seen in ACC, followed by PA, whereas MEC and PLGA showed completely negative staining. The connective tissue expression was mild in ACC and MEC. The myxoid stroma of PA with "melting" pattern was weakly positive for α-SMA. The stroma in PLGA showed complete negativity. In ACC, α-SMA-positive cells were lining the cribriform spaces, small islands, and dispersed within large islands. Small nests showed complete positivity for α-SMA. INTERPRETATION AND CONCLUSION: In ACC, α-SMA expression supports the involvement of ME in epithelial organization explaining the histological patterns seen. In PA, the expression correlates with the predominantly secretory nature of ME. The absence of epithelial positivity in MEC and PLGA suggest that ME has less role to play in their histogenesis. The weak stromal positivity observed in MEC and ACC may be attributed to the positive immunoreactivity of myofibroblasts playing a role in modulating the course of SGN's.

A correlation of immunohistochemical expression of TP53 and CDKN1A in oral epithelial dysplasia and oral squamous cell carcinoma.

PURPOSE: Oral epithelial dysplasia (OED) occurs on exposure of epithelial cells to carcinogens and genetic alteration. Once the reversible cell damage is surpassed, cells either undergo apoptosis or transform into malignancy, chiefly oral squamous cell carcinoma (OSCC). Progressive accumulation of genetic errors (including mutations in TP53 and CDKN1A) is associated with the initiation and progression of potentially malignant oral lesions toward frank malignancy. The present study attempted to correlate the immunohistochemical expression of CDKN1A and TP53 with increasing severity of OED along with increased aggressiveness of OSCC as reflected in the clinicopathologic variables. MATERIALS AND METHODS: Tissue sections from forty biopsy-proven cases of OED and OSCC were stained with anti-TP53 and anti-CDKN1A mouse monoclonal antibodies. One hundred cells in each case were counted under high power magnification. RESULTS: Poorly differentiated OSCC showed the highest TP53 expression (mean = 70.285), with least expression seen in mild dysplasia (mean = 22.125) (P < 0.001). Higher TP53 count was seen in cases with margin involvement, without recurrence and lymph node involvement and in cases which died of disease. CDKN1A expression was seen only in five cases and that too focally in the cytoplasm, thereby warranting removal of analysis of CDKN1A positivity from the study. CONCLUSION: The expression of TP53 in OED highlights its role in initial carcinogenesis. Although the role of CDKN1A in the cell cycle has been documented, its relationship to various clinical and pathological variables of OSCC and its different treatment modalities could not be adequately assessed.

Expression of Laminin in Oral Squamous Cell Carcinomas

Background and objectives: Laminin is a significant basement membrane (BM) glycoprotein, the expression of which reflects BM integrity more precisely than do other ECM proteins. The present study aimed to evaluate laminin expression in oral squamous cell carcinomas OSCC and to determine any associations with clinico-pathological parameters (surgical margin status, lymph node involvement, survival and recurrence). Methods: Laminin expression was evaluated in 31 cases of biopsy-proven OSCC by immunohistochemical staining and its association with prognosticators and the Brynes grading system was determined by appropriate statistical analysis. Results: We observed a significant increase in linear staining pattern (p<0.001) at the tumour-host interface in well-differentiated OSCC cases, in contrast to poorly differentiated lesions which exhibited intense cytoplasmic expression within tumour cells. Higher cytoplasmic laminin expression was seen in 33.3% of cases with involved surgical margins and 69.2% of cases with lymph node metastasis (along with weak/absent staining of laminin around the tumour-host interface ­ Basement membrane around tumour islands). Similarly, in 60% of the cases who died and in 81.8% of cases with tumour recurrence, moderately intense cytoplasmic laminin expression was seen within tumour cells. On comparing variables of the Brynes grading system, significant cytoplasmic expression of laminin was linked with mild inflammation (p<0.0016) and increased mitotic activity (p<0.008). Conclusion: Based on these observations, immunohistochemical expression of laminin might be useful to evaluate histological differentiation and aggressiveness of OSCCs.

Understanding patterns of invasion: a novel approach to assessment of podoplanin expression in the prediction of lymph node metastasis in oral squamous cell carcinoma.

AIMS: Cellular motility is considered to be central to the process of metastasis, and podoplanin expression can be explored as a prospective link, owing to its ability to modulate the actin cytoskeleton. We aimed to evaluate the tumoral expression of D2-40 (monoclonal antibody against podoplanin) in pathologically neck-node-negative/positive cases (pN0/N+) to characterise the pattern of invasion, potentially explaining the role of various patterns of invasion in causing tumour metastasis. METHODS AND RESULTS: Paraffin-embedded tissue blocks of 60 oral squamous cell carcinoma cases of known nodal status were selected for immunohistochemical staining of tumour (invasive front) by D2-40 along with routine staining by haematoxylin and eosin. Various staining patterns were assessed and evaluated for D2-40 expression, and correlated with nodal status. Tumoral D2-40 expression correspondingly increased with nodal metastasis (P = 0.261). Furthermore, D2-40 staining was more efficient in detecting individual tumour cells, and also characterised the motility factor irrespective of the pattern of invasion (P = 0.001). The pattern of D2-40 staining did not show a significant association with tumour grade, indicating that motility is an overlooked, albeit important, component of the pattern of invasion in routine histological grading. CONCLUSIONS: D2-40 expression successfully identifies the motility profile of tumour, irrespective of the pattern of invasion. The presence of larger motile islands in the tumour cohort supports the concept of 'collective cell migration'. Podoplanin also aids evasion of immune responses by inducing platelet aggregation over tumour cells, thereby favouring distant metastasis. A multivariate model using immunohistochemical staining with D2-40 provides greater sensitivity for the prediction of lymph node metastasis.

A Comparison of Clinicopathological Differences in Oral Squamous Cell Carcinoma in Patients Below and Above 40 Years of Age.

INTRODUCTION: Recent times have revealed an increase in incidence of Oral Squamous Cell Carcinoma (OSCC) in young adults including those who lack association with typical risk factors such as tobacco. There are reported variations in clinical behaviour of tumours in young and older individuals. AIM: Present study evaluated differences in clinicopathological characteristics between two groups of OSCC, below and above 40 years of age. MATERIALS AND METHODS: An analytical study was performed on two groups of OSCC patients, below and above 40 years of age. Clinicopathological parameters of site distribution, type of habit, histological grade, nodal metastasis, margin status, mitotic index and Argyrophilic Nucleolar Organizing Regions (AgNOR) count were compared. Chi-square test and Students t- test were applied for statistical analysis. RESULTS: Present study revealed that mean AgNOR count was significantly higher in older group (6.38) than younger group (4.27). However, no significant differences were noted in site distribution, tobacco habit, histological grade, mitotic index, nodal metastasis and status of resected surgical margins between the two age groups. A trend for increased metastasis and poor histological differentiation was also observed in the older and younger age group respectively. Most common site was buccal mucosa followed by tongue in both groups. CONCLUSION: Reasons for documented variability in tumour characteristics between young and older patients are currently unclear. Difference in AgNOR count found in present study is suggestive of variability in proliferative and ploidy characteristics between different age groups and supports the hypothesis of genetic and epigenetic influences in development of oral cancer.

Odontogenic Cyst with Verrucous Proliferation Exhibiting Melanin Pigmentation.

Verrucous proliferation arising from odontogenic cysts is a rare entity. We report an unusual case of an infected odontogenic cyst with verrucous proliferation and melanin pigmentation in a 13-year-old male patient who presented with an intraoral swelling in relation to impacted teeth 26 and 27. The enucleated lesion was diagnosed as an odontogenic keratocyst and the patient died within two years of presentation due to multiple recurrences. The clinical, radiological, and microscopic features of the lesion are presented with an attempt to discuss the etiopathogenesis. The case hereby reported is uncommon with only eight cases reported in the literature.

Dysgenetic Polycystic Disease of Minor Salivary Gland: A Rare Case Report and Review of the Literature.

Polycystic (dysgenetic) disease of the salivary glands is a rare entity that has only recently been described in the literature. The disease is more commonly seen in females and majority of the cases have presented as bilateral parotid gland swellings. This case presenting in a 21-year-old male is the first of this unusual entity involving solely the minor salivary gland on the lower lip. This case report highlights the importance for the clinician to be aware of this differential diagnosis, when treating an innocuous lesion like a mucocele.

Salivary Amylase as a Marker of Salivary Gland Function in Patients Undergoing Radiotherapy for Oral Cancer.

BACKGROUND: The aim of this study was to evaluate salivary amylase in patients with primary oral cancer undergoing radiotherapy as the main modality of treatment. MATERIALS/METHODS: The study was conducted on ten histologically proven cases of oral cancer undergoing radiotherapy. Stimulated whole saliva was collected at three stages of radiotherapy-0, 3, and 6 weeks. Salivary amylase was estimated using Henry-Chiamori method and comparison was made with appropriate age- and gender-matched controls. RESULTS: Salivary amylase levels showed significant decrease in healthy subjects when compared to oral cancer patients (P < 0.001). The latter group also showed changing trend with initial decrease from 0 to 3 weeks followed by increase from 3 to 6 weeks following radiotherapy (P < 0.0528). CONCLUSIONS: The trend in changes in the levels of salivary amylase could be used as a surrogate marker of salivary gland function in patients with oral cancer undergoing radiotherapy as primary treatment.

Oncocytes in Mucoepidermoid Carcinoma of the Palate: Diagnostic Challenges.

The origin of a salivary gland tumour is attributed to cells at various levels of differentiation which present histologically as diverse tissues and cellular patterns. Mitochondria-rich, eosinophilic oncocytes are cells commonly encountered in salivary gland neoplasms. We report a case of mucoepidermoid carcinoma (MEC) in the palate of a 43-year-old female that exhibited a prominent oncocytic component. While the parotid and submandibular glands have been reported as predominant sites for oncocytic MEC (OMEC), the palate and minor salivary glands are rare sites for occurrence. Also, most of the reported cases of OMEC have been histologically of low-grade mucoepidermoid carcinoma with large cystic spaces and good prognosis. In this article, we discuss the differential diagnosis and diagnostic workup of an MEC presenting with oncocytes.

Tumour-Associated Tissue Eosinophilia in Oral Squamous Cell Carcinoma- A Boon or a Bane?

INTRODUCTION: The infiltration of tumour stroma by eosinophils, Tumour-Associated Tissue Eosinophilia (TATE) is known to modulate the evolution of Oral Squamous Cell Carcinoma (OSCC). Identification of eosinophils in the inflammatory stroma has been proven to be an important factor in prognostication of malignant tumours including cancers of mouth, oesophagus, larynx, pharynx, breast, lung, intestine and genitourinary tract. AIM: Our study aimed to assess the role of TATE as a prognosticator in OSCC as visualized by Haematoxylin and Eosin (H&E) and congo red staining. MATERIALS AND METHODS: Thirty histologically-proven cases of OSCC were retrieved from the archives of Department of Oral Pathology, Manipal College of Dental Sciences, Mangalore, Manipal University, Karnataka, India. Two serial sections of 4µm thickness were made and subjected to routine staining with H&E and modified congo red staining, where eosinophil granules stained red and nuclei stained blue. In 40x magnification, 10 HPF at invasive tumour front were assessed for counting eosinophils by placing a 49 square grid (measuring 0.0289 sq mm). STATISTICAL ANALYSIS: The TATE was compared with the prognosticators using Mann-Whitney U-test. The grades of carcinoma were correlated with TATE using Kruskal-Wallis test followed by Post-hoc Bonferronis correction. Agreement of the number of eosinophils counted in the two staining techniques (H&E and Congo red) in OSCC was achieved using interclass correlation coefficient, and Friedman's test. A value of p< 0.05 was considered statistically significant. RESULTS: Our results showed that tissue eosinophil counts were higher in well-differentiated cases of OSCC, cases with lymph node involvement, decreased survival, without margin involvement and in cases that did not recur. H&E stain showed significantly better visualization of eosinophils resulting in higher eosinophil counts than when seen with Congo red (p=0.008). CONCLUSION: Thus, TATE can be used as a surrogate marker in prediction of survival and recurrence in OSCC. H&E proved to be a better stain for evaluation of eosinophils.

Computer-based method of bite mark analysis: A benchmark in forensic dentistry?

AIM: The study aimed to determine the technique with maximum accuracy in production of bite mark overlay. MATERIALS AND METHODS: Thirty subjects (10 males and 20 females; all aged 20-30 years) with complete set of natural upper and lower anterior teeth were selected for this study after obtaining approval from the Institutional Ethical Committee. The upper and lower alginate impressions were taken and die stone models were obtained from each impression; overlays were produced from the biting surfaces of six upper and six lower anterior teeth by hand tracing from study casts, hand tracing from wax impressions of the bite surface, radiopaque wax impression method, and xerographic method. These were compared with the original overlay produced digitally. RESULTS: Xerographic method was the most accurate of the four techniques, with the highest reproducibility for bite mark analysis. The methods of wax impression were better for producing overlay of tooth away from the occlusal plane. CONCLUSIONS: Various techniques are used in bite mark analysis and the choice of technique depends largely on personal preference. No single technique has been shown to be better than the others and very little research has been carried out to compare different methods. This study evaluated the accuracy of direct comparisons between suspect's models and bite marks with indirect comparisons in the form of conventional traced overlays of suspects and found the xerographic technique to be the best.

The post-analytical phase of histopathology practice: Storage, retention and use of human tissue specimens.

There are several aspects to a histopathology practice besides the acquisition of biopsy specimens and histopathological diagnosis. Pathology Departments are home to an abundant source of knowledge in the form of stored specimens and slides. We attempt to highlight the importance of regulation of storage, retention, and appropriate use of human tissue material in research and ownership rights to the same. We also discuss requirement and waiver of informed consent for scientific work involving the use of such tissues, which in the absence of defined laws come under the purview of Institution Review Boards. Pathology Departments, under the binding of the parent institution, are conceded the responsibility of maintenance and retention of pathology specimens. This communication highlights some of the important aspects in human tissue material handling and research, underscoring the necessity for established regulations regarding the same.

Nuclear Fractal Dimensions as a Tool for Prognostication of Oral Squamous Cell Carcinoma.

BACKGROUND: Carcinogenesis follows complex molecular alterations, which are triggered by subtle chromatin architectural changes that are imperceptible to the human eye. As the treatment decisions in Oral Squamous Cell Carcinoma (OSCC) are hindered by the imprecise clinical stage determination and inter-observer variability in histological grading, focus in recent years has shifted to discovering identifiers related to neoplastic cell morphology studied through computer-aided image analysis. One such approach is the assessment of fractal geometry, a technique first described by Mandelbrot, which aids in precise assessment of architecture of natural objects. Assessment and quantification of degree of complexity of these fractal objects (self-similarities in structural complexity at different magnifying scales) is described as fractal dimension (FD). AIM: To evaluate the nuclear fractal dimension (NFD) in OSCC using computer-aided image analysis. MATERIALS AND METHODS: Histological sections of 14 selected cases of Oral Squamous Cell Carcinoma (OSCC) and 6 samples of normal buccal mucosa (as control) were stained with Haematoxylin-Eosin and Feulgen stain for histopathological examination and evaluation of nuclear complexity respectively. Fifteen HPF at Invasive Tumour Front (ITF) and Tumour Proper (TP) of Feulgen-stained sections were selected and photographed in test and control samples. At ITF, TP and normal buccal mucosa 200 nuclei each were selected and analyzed using Image J software to quantify FD. The test and control groups were compared statistically using Independent sample t-test and One-way ANOVA. RESULTS: Nuclear FD increased progressively towards worst tumour staging as compared to normal buccal mucosa. CONCLUSION: Nuclear FD can be considered for quantification of nuclear architectural changes as a prognostic indicator in OSCC.

Driving the Mineral out Faster: Simple Modifications of the Decalcification Technique.

INTRODUCTION: Quicker decalcification is essential for faster diagnosis of hard tissue pathology. Heat and mechanical agitation are known to hasten decalcification. AIM: To compare the rate of decalcification, cellular and staining characteristics of decalcified specimens of bone and teeth by using the conventional method (10% formal formic acid), heating to 45(o)C and by physical agitation with magnetic stirrer. MATERIALS AND METHODS: Weight-matched samples of caprine-origin bone (n=15) and teeth (n=15) were decalcified using three methods namely: a) Gooding and Stewart's fluid; b) Gooding and Stewart's fluid heated to 45(o)C for 6 hours daily; and c) Gooding and Stewart's fluid agitated using a magnetic stirrer for 6 hours daily. Non-lesional skin tissue samples were placed along with each specimen. End point of decalcification (chemical test) was noted; 4 micron sections were taken and stained with H&E. STATISTICAL ANALYSIS: Differences in rate of decalcification and staining characteristics were assessed by Kruskal Wallis test and chi-square test respectively. RESULTS: Hard tissues decalcified faster with stirring and heating methods. The amount of osteocyte retraction noted in bone was significantly reduced in the stirring method. In tooth specimens, modified techniques resulted in poorer nuclear-cytoplasmic contrast of pulp cells. Heating affected the odontoblast layer. Soft tissues exhibited higher eosinophilia in stirring and conventional methods, whereas nuclear-cytoplasmic contrast and chromatin staining was poorest in heating and conventional methods. CONCLUSION: Physical agitation of decalcifying fluid may be recommended while maintaining satisfactory quality of tissue morphology and staining.