Expression of Cathepsin-D in Odontogenic Cysts and Tumors

Expression of Cathepsin-D in Odontogenic Cysts and TumorsThe view of cathepsin-D in odontogenic cysts and tumours an immunohistochemical concord abstractionistAim Cathepsin-D, a protease, which is an invasion promoter and plays a central fiber in solid tumors including viva cancer. Our aim of the study was to look for their manner pattern in epithelial meander and stroma of odontogenic cysts and tumors and match their vulturineness to the patch book.Methods To straighten the observation patterns of this marker, we examined immunohistochemically on formalin fixed, paraffin embedded sections of 24 odontogenic cysts and 10 odonogenic tumors, which are received for histopathologic examination in the department of vocal pathology, the Oxford alveolar consonant college and hospital, Bangalore.Results The epithelial tissue of granular electric cell ameloblastoma and odontogenic keratocyst showed maximum staining, with departure of stained secular in the conjunction tis sue wall and at the separation of epithelium to capsule in odontogenic keratocyst, compare to other cysts and tumors.Conclusions Cathepsin-D could be one of the enzyme all-important(a) in separation of epithelium and conjugation tissue in odontogenic keratocyst which helps in recurrence and intense expression in granular cell ameloblastoma with spillage into stroma, compare to other odonogenic tumors may explain its hostile behavior, recurrence and metastatic potential. To further validate our findings it is suggested to use to a greater extent sample size of it and monoclonal antibody for cathepsin-D.Key words Cathepsin-D, odontogenic cysts, odontogenic tumors, immunohistochemistry.INTRODUCTIONOdontogenic cysts and tumors constitute an important aspect of oral and maxillofacial pathology. Odontogenic cysts are encountered relatively common in dental intrust and tumors by contrast are uncommon lesions. These lesions are of clinical significance because of their biological beha vior. Various attempts to categorize morphological features to relate the biological application have been made everyplace the years1. It is well established that the cysts of histologenic labeling of odontogenic keratocyst are more rough tending to behave more the like a sub-malignant tumor1-6. It has besides been suggested that cysts other than odontogenic keratocyst showing ceratinization if non more locally aggressive tend to have a pre-disposition to neoplastic change7.There have been attempts to correlate follicle size with aggression in ameloblastoma and morphologically different granular cell variant has been know to be more clinically aggressive, showing metastatic potential8. numerous studies on the enzyme histochemistry of odontogenic cysts and tumors have been conducted over the years for the expression of oxidative enzymes NADH2 and NADPH2, G6PD, glutamate dehydrogenase, acidic phosphates, leucineamino peptidase and ATPase9, 10. The epithelial lining of all the varieties of cysts showed a weak reception for leucineamino peptidase a lysosomal protease, but there was a strong favourableness in the lamina propria of odontogenic keratocyst. Similar studies on follicular ameloblastoma have showed ATPase activity in the peripheral and central cells of the follicle9. Based on these we made an attempt to study the expression of cathepsin-D in odontogenic cysts and tumors, by grouping them into locally aggressive and non-aggressive ground on their clinical and radiographic features.Cathepsin-D is a proteolytic enzyme that belongs to a family known as aspartic proteases. Many homologies in the amino acid sequence have been shown to exist among the members of this group of enzymes, which includes pepsin, gastricin and rennin. Like other enzymes cathepsin-D has been shown to be synthesized in the forerunner form. The enzyme itself is a glycoprotein of approximate molecular weight 52 KD and has an optimum pH of 3.5. Cathepsin-D was present in many o f the normal tissue including epithelium, fibroblast and macrophages11. The physiologic section of cathepsin-D is believed to be involved in self-destruction of senescent or dishonored epithelial cells12. As cathepsin-D is an intracellular lysosomal aspartic protease apart from its role in protein catabolism done the degradation of endocytosed protein. Cathepsin-D has attracted clinical attention because of its over expression in variety of diseases. Increased levels of these enzymes have been reported to be an indicator of aggressive behavior in human tumors including oral squamous cell carcinoma13.MATERIALS AND METHODSTissue used in the study was biopsy material submitted to department of oral pathology, The Oxford Dental College, Hospital and Research centre, Bangalore. Total sample size taken was from 34 patients which comprised of 9 Ameloblastoma (1 plexiform unicystic ameloblastoma), 7 odontogenic keratocyst, 1 adenomatoid odontogenic tumor, 11 Radicular cysts and 6 Dentig erous cysts which were classify into locally aggressive and non aggressive based on their clinical and radiologic features like size and extent of lesion, peripheral cortication, scalloping and basis resorption.*This particular radicular cyst was an lengthened lesion extending from the maxilla canine to the third molar extending into and destroying the maxillary sinus and had caused root resorption from canine to second molar without ca development any bony expansion. The initial clinical impression was that of a malignancy arising in the maxillary sinus.METHODOLOGYFormalin fixed paraffin embedded sections of odontogenic cysts and tumors were stained by hematoxylin and eosin stain, the serial sections of the same was studied by Immuno histochemistry procedure using cathepsin-D and observed under the microscope for the gaudiness of cathepsin-D staining expression or non- expression. Controls were vigilant by omitting primary antibody.A grading system for intensity of expressio n was devised and used.Antibody usedPolyclonal rabbit anti-human primary cathepsin-D, 7ml ready to use (DAKO Corporation N1625). DenmarkBiotinylated anti-mouse, anti-rabbit, anti-goat Igs, interrelate/secondary antibody, 15 ml ready to use. (DAKO LSAB+ system, K0679).Streptavidin conjugated to horseradish peroxidase. (DAKO LSAB+ system, K0679). luculent Diamino benzidine chromogen.OBSERVATION AND RESULTSAll odontogenic cysts and tumors were observed for intensity of cathepsin-D stain in epithelium and stroma/ connective tissue capsule by categorized into mild, moderate and mark staining. Statistical analysis was done using students T test. Table 1 shows number of cases in which cathepsin-D shows mild, moderate and mark staining in mixed epithelial layers and stroma. Table 2 shows statistical relation of staining intensity of cathepsin-D in from each one layer and stroma/capsular wall between each odontogenic cysts. Table 3 shows statistical relation of staining intensity of cath epsin-D in each layer and connective tissue stroma between each odontogenic tumors. railleryThe idea of immunohistochemistry staining for a lysosomal protease cathepsin-D in odontogenic cysts and tumors of varying biological behavior pattern was with the hope that it could contribute to a better fellow feeling of metabolic processes that are responsible for that behavior. Traditionally we have always pore on the epithelium in odontogenic cysts and epithelial tumors. Much like the hyp nonic effect of giant cells in giant cell lesions, the epithelium in odontogenic cysts and epithelial tumors has held a magnetic quality for research workers. The epithelial circumstances dictates the diagnosis, but the role of connective tissue wall and the stromal cells in tumors has not always been given due consideration. The epithelium is not always at the advancing front of these lesions as is especially seen in case of cysts. In this study in addition to the epithelium we also looked at the e xpressivity of cathepsin-D in the connective tissue and stromal cells.In granular cell ameloblastoma we observed attach staining pattern in the cytol of the granular cells, often spilling into the connective tissue which may contribute to the aggressive nature of the lesion and its propensity for metastasis (Fig 1a 1b). As compared to the granular cell ameloblastoma other odontogenic tumor types such as follicular, unicystic, plexiform ameloblastoma and adenomatoid odontogenic tumor (Fig 2a 2b) showed less intense staining pattern and the staining was restricted to cytoplasm of these epithelial cells with minimal stromal staining. Apart from the granular cell ameloblastoma we could not hail any correlation between clinical behaviour and cathepsin-D expression. Among the 3 cyst types we found a characteristic epithelial staining pattern in odontogenic keratocyst in comparison to radicular and dentigerous cysts. Among 7 odontogenic keratocyst only one case showed little granular staining of the epithelial cells with no separation of epithelium from connective tissue. In all other cases we observed granular staining through the full thickness of the epithelium, more in the basal and supra-basal layers, with intense/marked staining at the region of separation of epithelium from connective tissue with granular staining pattern in separation zone (fig 3a 3b).In dentigerous cysts there was only superficial staining of epithelium. The radicular cysts showed uniform staining in the entire length of epithelium (fig4). In the one radicular cyst which was clinically more aggressive a similar pattern of staining was observed. though the epithelial staining in radicular cysts was almost similar to that seen in odontogenic keratocysts we did not find any areas of cleavage between epithelium and connective tissue. In the odontogenic keratocyst the staining pattern though similar to the radicular cysts, in the area of stick the staining was very intense, and some staine d material was noticed in the space between the epithelium and the connective tissue leading to the venture that the increased expression may contribute to the split, which may have type consequences in terms of recurrence by way of cleaving of epithelium at the time of attempted enucleation or biopsies .In addition to variations in staining patterns of the epithelial lining of the different types of cysts, their walls showed variation in staining from the epithelial end to the bony end .All the cyst types showed expressivity in the immediate sub-epithelial region as well as the bony end of the cyst wall. The intensity of staining progressively increased from the dentigerous cyst through the radicular cyst to the odontogenic keratocyst. The mediocre zone showed relatively scanty expression. This pattern of increasing expression seemed to correlate with increasing aggression. The one radicular cyst grouped in the list of aggressive lesion showed intense staining in the most periph eral areas similar to that seen in the odontogenic keratocyst. All the inflammatory cells seen in connective tissue wall and keratin of the surface layer and granules of granular layer of odontogenic cysts showed intense staining.To the best of our intimacy this is the first study on expression of cathepsin-D in odontogenic cysts and tumors although studies on unhomogeneous other lysosomal enzymes like leucineaminopeptidase etc have been published. Hence it may be assuming on our part to make claims on the role of cathepsin-D in aggressive behaviour of odontogenic cysts and tumors, however that there is perceptible variation in expression would suggest that additional efforts in the area may help to view the metabolic processes that lead to aggressive behaviour. Another area open for geographic expedition is precystic epithelium as in the case of periapical granulomas and the role of these enzymes in cystogenesis.Acknowledgements Dr. Srivasta MDS (for statistical analysis).Prof essor, Sri Rajiv Gandhi Dental College and hospital, R T Nagar, Bangalore-94.