The expression of cathepsin-D in odontogenic cysts and tumors: an immunohistochemical study
Aim: Cathepsin-D, a protease, which can be an invasion promoter and takes on a central role in stable tumors including oral cancer. Our goal of the analysis was to look for their expression routine in epithelium and stroma of odontogenic cysts and tumors and correlate their aggressiveness to the staining level.
Methods: To elucidate the expression patterns of this marker, we evaluated immunohistochemically on formalin fixed, paraffin embedded parts of 24 odontogenic cysts and 10 odonogenic tumors, which are received for histopathologic evaluation in the section of dental pathology, the Oxford Dental care college and hospital, Bangalore.
Results: The epithelium of granular cell ameloblastoma and odontogenic keratocyst showed maximum staining, with spillage of stained material in the connective tissues 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 important in parting of epithelium and connective tissue in odontogenic keratocyst which assists with recurrence and powerful manifestation in granular cell ameloblastoma with spillage into stroma, compare to other odonogenic tumors may explain its aggressive patterns, recurrence and metastatic potential. To further validate our conclusions it is suggested to work with more test size and monoclonal antibody for cathepsin-D.
Key words: Cathepsin-D, odontogenic cysts, odontogenic tumors, immunohistochemistry.
Odontogenic cysts and tumors constitute an important aspect of dental and maxillofacial pathology. Odontogenic cysts are encountered relatively common in dentist and tumors in comparison are uncommon lesions. These lesions are of specialized medical significance because of their biological behavior. Various endeavors to categorize morphological features to relate the natural activity have been made on the years1. It is well established that the cysts of histologenic labeling of odontogenic keratocyst are more aggressive maintaining behave more like a sub-malignant tumor1-6. It has additionally been advised that cysts other than odontogenic keratocyst exhibiting keratinization or even more locally aggressive tend to have a pre-disposition to neoplastic change7. There were efforts to correlate follicle size with hostility in ameloblastoma and morphologically different granular cell variant has been regarded as more clinically hostile, exhibiting metastatic potential8. Numerous studies on the enzyme histochemistry of odontogenic cysts and tumors have been conducted through the years for the expression of oxidative enzymes NADH2 and NADPH2, G6PD, glutamate dehydrogenase, acid phosphates, leucineamino peptidase and ATPase9, 10. The epithelial lining of all types of cysts demonstrated a weak reaction for leucineamino peptidase a lysosomal protease, but there was a strong positivity in the lamina propria of odontogenic keratocyst. Similar studies on follicular ameloblastoma have proved ATPase activity in the peripheral and central skin cells of the follicle9. Predicated on these we made an attempt to study the manifestation of cathepsin-D in odontogenic cysts and tumors, by grouping them into locally aggressive and non-aggressive predicated on their scientific 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 on the list of members of this band of enzymes, which include pepsin, gastricin and rennin. Like other enzymes cathepsin-D has been proven to be synthesized in the precursor form. The enzyme itself is a glycoprotein of approximate molecular weight 52 KD and comes with an optimum pH of 3. 5. Cathepsin-D was within many of the normal cells including epithelium, fibroblast and macrophages11. The physiologic role of cathepsin-D is thought to be involved with self-destruction of senescent or broken epithelial skin cells12. As cathepsin-D is an intracellular lysosomal aspartic protease aside from its role in protein catabolism through the degradation of endocytosed protein. Cathepsin-D has enticed specialized medical attention because of it's over manifestation in variety of diseases. Increased levels of these enzymes have been reported to be an signal of aggressive habit in human being tumors including oral squamous cell carcinoma13.
MATERIALS AND METHODS:
Tissue found in the analysis was biopsy materials submitted to division of oral pathology, The Oxford Oral College, Clinic and Research centre, Bangalore. Total sample size considered 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 Dentigerous cysts which were grouped into locally extreme and non extreme based on their professional medical and radiologic features like size and extent of lesion, peripheral cortication, scalloping and root resorption.
*This particular radicular cyst was an extensive lesion increasing from the maxillary dog to the third molar stretching into and destroying the maxillary sinus and acquired caused root resorption from canine to second molar without causing any bony development. The initial professional medical impression was that of the malignancy arising in the maxillary sinus.
Formalin fixed paraffin embedded sections of odontogenic cysts and tumors were stained by hematoxylin and eosin stain, the serial sections of the same was analyzed by Immuno histochemistry procedure using cathepsin-D and detected under the microscope for the power of cathepsin-D staining manifestation or non- expression. Controls were prepared by omitting primary antibody.
A grading system for level of expression was devised and used.
- Polyclonal rabbit anti-human key cathepsin-D, 7ml ready to use (DAKO Corporation N1625). Denmark
- Biotinylated anti-mouse, anti-rabbit, anti-goat Igs, Website link/secondary antibody, 15 ml prepared to use. (DAKO LSAB+ system, K0679).
- Streptavidin conjugated to horseradish peroxidase. (DAKO LSAB+ system, K0679).
- Liquid Diamino benzidine chromogen.
OBSERVATION AND RESULTS:
All odontogenic cysts and tumors were witnessed for level of cathepsin-D stain in epithelium and stroma/ connective muscle capsule by grouped into mild, moderate and noticeable staining. Statistical analysis was done using students T test. Stand 1 shows number of instances where cathepsin-D shows slight, moderate and noticeable staining in a variety of epithelial tiers and stroma. Table 2 shows statistical connection of staining depth of cathepsin-D in each part and stroma/capsular wall structure between each odontogenic cysts. Stand 3 shows statistical relation of staining depth of cathepsin-D in each part and connective cells stroma between each odontogenic tumors.
The idea of immunohistochemistry staining for a lysosomal protease cathepsin-D in odontogenic cysts and tumors of differing biological behavior design was with the expectation that it could contribute to a better knowledge of metabolic operations that are responsible for that behavior. Typically we've always centered on the epithelium in odontogenic cysts and epithelial tumors. Much like the mesmerizing effect of giant cells in massive cell lesions, the epithelium in odontogenic cysts and epithelial tumors has performed a magnetic quality for researchers. The epithelial aspect dictates the medical diagnosis, but the role of connective structure wall and the stromal cells in tumors hasn't always been given due account. The epithelium is not necessarily at the evolving front of the lesions as is especially seen in case of cysts. In this study as well as the epithelium we also looked at the expressivity of cathepsin-D in the connective tissue and stromal cells.
In granular cell ameloblastoma we discovered marked staining pattern in the cytoplasm of the granular cells, often spilling into the connective tissue which may donate to the aggressive aspect 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) exhibited less strong staining structure and the staining was restricted to cytoplasm of these epithelial cells with reduced stromal staining. Apart from the granular cell ameloblastoma we're able to not derive any correlation between clinical behaviour and cathepsin-D appearance. One of the 3 cyst types we found a quality epithelial staining structure in odontogenic keratocyst in comparison to radicular and dentigerous cysts. Among 7 odontogenic keratocyst only 1 case revealed superficial granular staining of the epithelial cells with no parting of epithelium from connective structure. In every other cases we witnessed granular staining through the entire thickness of the epithelium, more in the basal and supra-basal levels, with strong/marked staining at the region of parting of epithelium from connective tissue with granular staining design in separation area (fig 3a &3b).
In dentigerous cysts there was only superficial staining of epithelium. The radicular cysts demonstrated homogeneous staining in the entire length of epithelium (fig4). In the main one radicular cyst that was clinically more intense; a similar design of staining was seen. Though the epithelial staining in radicular cysts was almost similar to that seen in odontogenic keratocysts we didn't find any regions of cleavage between epithelium and connective muscle. Within the odontogenic keratocyst the staining structure though like the radicular cysts, in the area of split the staining was very strong, plus some stained material was seen in the space between your epithelium and the connective tissue resulting in the speculation that the increased manifestation may donate to the split, which might have prognostic results in conditions of recurrence using cleaving of epithelium at the time of attempted enucleation or biopsies.
In addition to variations in staining habits of the epithelial coating of different types of cysts, their walls showed variant in staining from the epithelial end to the bony end. All of the cyst types confirmed 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 intermediate area confirmed relatively scanty appearance. This design of increasing expression appeared to correlate with increasing hostility. The one radicular cyst grouped in the list of aggressive lesion demonstrated extreme staining in the most peripheral areas similar compared to that observed in the odontogenic keratocyst. All the inflammatory cells observed in connective tissue wall and keratin of the surface part and granules of granular coating of odontogenic cysts exhibited extreme staining.
To the best of our knowledge this is actually the first study on appearance of cathepsin-D in odontogenic cysts and tumors although studies on several other lysosomal enzymes like leucineaminopeptidase etc have been publicized. Hence it might be presumptuous on our part to make claims on the role of cathepsin-D in competitive behavior of odontogenic cysts and tumors, however that there surely is perceptible variance in expression would suggest that additional initiatives in the region may help to understand the metabolic functions that lead to intense behaviour. Another area wide open for exploration is precystic epithelium as in the case of periapical granulomas and the role of these enzymes in cystogenesis.
Acknowledgements: Dr. Srivasta MDS (for statistical research).
Professor, Sri Rajiv Gandhi Teeth College and medical center, R T Nagar, Bangalore-94.