|Year : 2003 | Volume
| Issue : 2 | Page : 40-43
Monoclonal antibody to desmosomal antigen Dsg1: A reliable adjunct to histopathology
Simarpreet Virk1, Alka Kale2
1 Senior lecturer in Oral Pathology, SGRD Inst. of Dental Sciences, Amritsar, India
2 Department of Oral Pathology, KLES Inst. of Dental Sciences and Research, Belgaum, India
883-Circular Road, Amritsar, Punjab
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Desmosomes are cell surface modifications that provide strong adhesion between epithelial cells. The presence of desmosomes in a majority of epithelia make them useful epithelial markers. This paper describes a monoclonal antibody 32-2B to desmosomal glycoprotein 1 (Dsg1) or desmoglein, preserved in routinely processed paraffin embedded sections. This antibody was tested for its ability to stain normal oral epithelium and epithelium derived tumors. It reacted with all oral epithelial specimens tested but showed negative staining in those specimens which lacked desmosomal structures like connective tissue cells and lymph nodes.
These characteristics demonstrate that 32-2B is a reliable epithelial marker that may be useful in diagnostic histopathology.
Keywords: Monoclonal antibody, desmoglein, epithelium, desmosomes, 32-2B
|How to cite this article:|
Virk S, Kale A. Monoclonal antibody to desmosomal antigen Dsg1: A reliable adjunct to histopathology. J Oral Maxillofac Pathol 2003;7:40-3
|How to cite this URL:|
Virk S, Kale A. Monoclonal antibody to desmosomal antigen Dsg1: A reliable adjunct to histopathology. J Oral Maxillofac Pathol [serial online] 2003 [cited 2020 May 27];7:40-3. Available from: http://www.jomfp.in/text.asp?2003/7/2/40/40935
| Introduction|| |
Our present view of the body's cellular organization has a history spanning at least three centuries and like the most scientific advances, it has closely followed the development of technology.
In the recent years, morphological studies of cells and tissues have been replaced by objective molecular markers which are increasingly providing important criteria leading to a better understanding and a biologically meaningful classification system in pathology. An increasing number of cell specific or differentiation specific molecules (protein s, glycoproteins etc) have provided an increasing list of agents which may be used as markers  .
Of the numerous markers of cellular differentiation, certain components of cytoskeleton, such as intermediate filaments have proven to be particularly useful  .
Antibodies to keratins have been widely used to identify epithelial tissues but they represent a multigene family and are differentially expressed in different epithelia necessitating the use of a panel of antisera to guarantee recognition in all epithelial tissues  .
Molecules that come much close to being the universal epithelial constituents are the components of adhesive intercellular junctions which form membrane anchors for keratin intermediate filaments.
These cell surface modifications are called as desmosomes (maculae adherences) and provide a strong adhesion between epithelial cells. Desmosomes occur in all epithelia  so far studied except pigmented retinal epithelium and lens epithelium. The presence of desmosomes in majority of epithelia should enable them to be used as a reliable epithelial marker.
Several ultrastructural morphometrical analyses have investigated the size and number of desmosomes in various situations ranging from normality to disease ,, . However, electron microscopy (EM) is not only time consuming and expensive, but is also susceptible to the well known empirical limitations of such methodology. Such problems and limitations make this method dependent on the quality and experience of the particular pathologist  .
Clearly, in this respect, an immuno-morphologic means of detection at light microscopic level has several advantages over morphological studies based on EM. Monoclonal antibodies and polyclonal antisera raised against desmosomal components have permitted their immunohistochemical visualization at the light microscopic level in frozen section material, unfortunately limiting their use in routine diagnostic histopathology ,,, . Viela et al in 1987 developed a monoclonal antibody 32-2B (MAb 32-2B) against desmosomal glycoprotein 1 (Dsg1) or desmoglein, a transmembrane glycoprotein of mr 165000 present in a wide range of epithelia. The epitope recognized by this antibody is preserved in routinely processed paraffin embedded sections and may therefore serve as a useful marker for epithelial tissues  .
| Materials and Methods|| |
This study involved the use of formalin fixed paraffin embedded tissues of previously diagnosed leukoplakia, verrucous carcinoma and squamous cell carcinoma of the oral cavity from the archival files of K.L.E's Institute of Dental Sciences, Belgaum and Government Medical College, Amritsar. Normal adult mucosa served as positive control. Normal lymph nodes served as negativetissue control (specific negative control). The replacement of primary antibody by a wash buffer, served as an additional negative control (non-specific negative control).
A total number of 15 leukoplakias and 10 verrucous carcinomas and 50 squamous cell carcinomas of variable differentiation were examined.
Immunostaining method ,
Serial sections (4 m) were cut from each block for both routine hematoxylin and eosin staining and for immunostaining using the Envision system (horse radish peroxidase based two step staining method, Dako Corp, Carpenteria, USA).
Subsequent to the antigen retrieval procedure, the sections were immersed in phosphate buffered saline (PBS) bath for 3 minutes. Endogenous peroxidase was blocked by incubation with 0.03% hydrogen peroxide containing sodium azide for 5 (0±1) minutes. The slides were then rinsed gently with distilled water or buffer solution from a wash bottle followed by a PBS bath for 5 minutes. Excess buffer was tapped off and primary antibody (mouse anti bovine desmoglein monoclonal 322B, 1:50 dilution, epithelial morphogenesis group, UK) was applied to cover the specimen for 30 (±1) minutes at room temperature.
After washing the sections in PBS bath for 5 minutes, the sections were then covered for 30 (±1) minutes at room temperature with enough labeled polymer from the Envision System Kit.
After washing the slides, they were incubated with adequate quantities of prepared DAB substrate chromogen (Dia minobenzidine, Dako Corp, Carpenteria, USA) for 5-10 minutes. The sections were then washed in PBS buffer for 5 minutes and counterstained with Mayer's hematoxylin for 1 minute. Slides were then dipped 10 times into a bath of blueing agent (37 mM ammonia). This was followed by rinsing in a bath of distilled water for 2-5 minutes and dehydration in increasing concentrations of alcohol. The slides were cleared and mounted in DPX.
Negative controls and positive controls were included in each staining batch.
| Results|| |
Normal Epithelium (n=20)
The monoclonal antibody 32-2B showed positive staining in normal oral epithelium whereas non epithelial tissues such as submucosal connective tissue showed no staining.
The staining was strongest in the stratum spinosum. There was no staining in the parakeratinized or orthokeratinized layers and the basal layer stained very weakly.
Neither collagen nor blood vessels stained. The staining pattern revealed punctate and pericellular localization of the antigens, outlining the cell membranes of the keratinocytes [Figure 1]a & b. The negative control sections (specific and non specific) showed no staining.
Verrucous carcinoma (n=10)
All sections of verrucous carcinomas showed extensive pericellular staining for Dsg1 [Figure 3] throughout the nonkeratinized layers of the tumour. The keratin plug as well as the lower surface of basal cells remained unstained. Absence of staining in the keratin plug is consistent with advanced keratinization and the subsequent reduction and loss of desmosomes
The most common finding in leukoplakia was focally preserved, pericellular staining for Dsg1 [Figure 2] with absence of staining in dysplastic areas of non keratinized epithelium. Fully keratinized layers of the epithelium remained unstained. None of the leukoplakias demonstrated positive pericellular localization of Dsg1 throughout the full thickness of transformed or dysplastic epithelium.
Squamous cell carcinoma (n=50)
Labeling patterns varied from one case to another [Figure 4]a, b and sometimes within the same carcinoma tissue and the variations applied both to the number and pattern of cell labeling. Squamous cell carcinoma demonstrated either a focal loss of staining or complete loss of this antigen.
Four different staining patterns were observed. In some sections, there was normal pattern with strong pericellular staining, but weak or negative staining of terminally differentiating cells in the centre of the tumor A second pattern showed diffuse cytoplasmic staining throughout the tumour. Third pattern showed localized juxtanuclear staining and in the fourth pattern there was significant loss of expression.
| Discussion|| |
The monoclonal anti-desmosomal antibody 32-2B reacts reliably with epithelia and epithelial tumors in formalin fixed paraffin embedded tissues which is of value in diagnostic immunohistochemistry as well as in research studies.
In our study, the normal epithelium showed positive staining with MAb 32-2B whereas non-epithelial tissues such as submucosal connective tissue showed no staining. These reactions were as would be expected from the known distribution of desmosomes as demonstrated by ultrastructural studies done by Allen and Potten  .
The staining was strongest in stratum spinosum. There was no staining in parakeratinized surface layers. This is because numerous desmosomes are located on the extensively interfolded membrane in the stratum spinosum. As the cells migrate up in the cornified layers, the desmosomes are replaced by intercellular cementing substance.
The basement membrane did not stain at all. Neither collagen nor blood vessels took any stain ,.
The staining pattern revealed punctate and pericellular localization of the antigen outlining the cell membranes of keratinocytes. The staining reaction is entirely similar to that observed in normal mucosa with MAb 32-2B .
In our study, we noticed that all cases of leukoplakia showed focal loss of Dsg1 marker in dysplastic layers remained unstained.
In our study, we noticed either a focal loss or complete loss of staining in carcinoma specimens. Some sections also showed cytoplasmic staining which was either diffuse or localized to juxtanuclear positions. The presence of diffuse cytoplasmic staining has been noticed before in cultured human keratinocytes maintained at low calcium concentrations (<0.05 mM), where cells don't assemble desmosomes. On transferring the cells to a medium containing a physiological concentration of calcium, desmosomes rapidly assembled between cells and diffuse cytoplasmic staining disappeared , .
It is possible therefore that diffuse staining of desmogleins in tumor cell cytoplasm indicates the presence of unassembled material. Localised juxtanuclear staining for desmosomal antigens most likely represents internalized desmosomes. Desmosome internalization has been reported in electron microscopic studies of many different types of benign and malignant neoplasms of epithelial origin , . Experimentally induced desmosome internalization occurs as a response of cells to loss of intercellular adhesion. An alternate possibility is that such localized juxtanuclear staining represents accumulation of desmosomal antigen in golgi apparatus  .
The potential significance of intracytoplasmic desmosomes can be suggested from the work of Overton. It was found that when epithelial cells which were forming desmosomes at a high rate were aggregated with foreign cells, the epithelial cells formed desmosomes with characteristics of their own cell membrane and frequently ingested these desmosomes. Extension of this suggestion to neoplastic epithelium indicates that there may be significant difference in the plasma membranes of cells within the same neoplasm.
The advent of monoclonal antibody to desmosomes has opened several possibilities to further the accuracy of diagnostic pathology.
First of all, the desmosomal antibody 32-2B has several advantages over cytokeratins. Their distinct pattern of staining punctate and pericellular areas is easily distinguished from the background level of insignificant staining often present in immunohistochemical reactions.
Another important aspect of desmoglein antigen is that Dsg1's are highly conserved in vertebrate evolution and seem to be very similar in all desmosome containing cells.This is not the case with cytokeratins which represent a multigene family showing differential expression of polypeptides in different epithelia and carcinomas.
Therefore it may be argued that desmosomal proteins and structures are particularly reliable markers of an epithelial nature. In addition the reported antibody 32-2B, reacts on routinely processed paraffin wax sections making it even more valuable in routine diagnostic histopathology  .
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[Figure - 1], [Figure - 2], [Figure - 3], [Figure - 4]