Indian Journal of Dental Sciences

ORIGINAL ARTICLE
Year
: 2021  |  Volume : 13  |  Issue : 4  |  Page : 224--229

A comparative immunohistochemical study of expression of Syndecan-1 (CD138) and podoplanin in keratocystic odontogenic tumor, orthokeratinized odontogenic cyst and dentigerous cyst


Alka Chahar1, Pooja Narain1, Naveen Chahar2, Jagriti Gupta3, Arpita Kabiraj4,  
1 Department of Oral Pathology and Microbiology, RDCH, Jaipur, India
2 Clinical Practitioner, Jaipur, Rajasthan, India
3 Department of Oral Pathology and Microbiology, Vyas Dental College, Jodhpur, Rajasthan, India
4 Department of Oral Pathology and Microbiology, Index Institute of Dental Sciences, Indore, Madhya Pradesh, India

Correspondence Address:
Arpita Kabiraj
Department of Oral Pathology and Microbiology, Index Institute of Dental Sciences, Indore, Madhya Pradesh
India

Abstract

Background: Odontogenic lesions can bring about diagnostic challenges due to overlapping histology among pathologists. Thus, there are few tumor markers that provide accurate diagnosis. Syndecan-1 (CD-138) and podoplanin (PDPN) are proteoglycans that have been described as substantial diagnostic and prognostic markers in various odontogenic lesions. Aim: This study aims to evaluate and compare the immunohistochemical expression of syndecan-1 (CD-138) and podoplanin in keratocystic odontogenic tumor, orthokeratinized odontogenic cyst (OOC), and dentigerous cyst (DC). Materials and Methods: The formalin-fixed paraffin-embedded tissue blocks of KCOT, OOC, and DCs were retrieved form the archives of department. Three sections each of 3 μm thickness were made using a rotary microtome and they were stained with syndecan-1 (CD-138) and podoplanin (PDPN) using immunohistochemical methods and standard hematoxylin and eosin stain. Results: Immunohistochemical expression of syndecan-1in KCOT was found to be weakly to moderately positive in 6 cases with 2 cases exhibiting positive expression. In OOC, 3 cases displayed negative expression-1 whereas 7 cases were weakly to moderately positive. Immunohistochemical expression of podoplanin in KCOT was observed to be weakly to moderately positive in 4 cases with 5 cases exhibiting strongly positive expression. In OOC, 3 cases displayed negative immunohistochemical expression for podoplanin and 4 cases were strongly positive. Immunoreactivity for podoplanin in DC was negative in 3 cases whereas 5 cases were weakly to moderately positive. Conclusion: The absence of significant correlation between expression of syndecan-1 and podoplanin reinforces the exact role of these proteins in the differentiation of odontogenic lesions which need to be elucidated further.



How to cite this article:
Chahar A, Narain P, Chahar N, Gupta J, Kabiraj A. A comparative immunohistochemical study of expression of Syndecan-1 (CD138) and podoplanin in keratocystic odontogenic tumor, orthokeratinized odontogenic cyst and dentigerous cyst.Indian J Dent Sci 2021;13:224-229


How to cite this URL:
Chahar A, Narain P, Chahar N, Gupta J, Kabiraj A. A comparative immunohistochemical study of expression of Syndecan-1 (CD138) and podoplanin in keratocystic odontogenic tumor, orthokeratinized odontogenic cyst and dentigerous cyst. Indian J Dent Sci [serial online] 2021 [cited 2021 Dec 8 ];13:224-229
Available from: http://www.ijds.in/text.asp?2021/13/4/224/327807


Full Text



 Introduction



Odontogenic cysts and tumors that originate from the same primitive odontogenic epithelium has different pathogenesis and exhibits unusual histologic behavior. Some of the common odontogenic lesions of the jaws are mainly radicular cysts, dentigerous cysts (DCs), and odontogenic keratocysts (OKC).[1] OKC was earlier tabulated under odontogenic cystic lesions comprising of parakeratinized and orthokeratinized histological variants. However, considering the biological behavior and genetic abnormalities, the WHO (2005) has categorized parakeratinized OKC as a benign neoplasm known as keratocystic odontogenic tumor (KCOT) and orthokeratinized variant as orthokeratinized odontogenic cyst (OOC). The potential for neoplastic behavior, aggressive or localized infiltration in KCOT and OOC cannot be determined suitably due to the dearth of evidence. There is no suitable immunohistochemical marker for evaluating both odontogenic cysts and tumors accurately. Odontogenic lesions can bring about diagnostic challenges due to overlapping histology amongst pathologists. Thus, there are few tumor markers that have proven to be useful for their accurate diagnosis.[2]

Syndecan-1 (CD-138) and podoplanin (PDPN) are proteoglycans that have been described as substantial diagnostic and prognostic markers in various odontogenic lesions. They help in the regulation of cell movement in numerous physiological and pathological conditions. The podoplanin expression accelerates cell motility in vitro and induces cell invasion and metastasis of the malignant epithelial tumor.[3] Hence, the present study was undertaken to evaluate and compare the immunohistochemical expression of syndecan-1 (CD-138) and podoplanin in keratocystic odontogenic tumor, OOC, and DC and also to delineate the role of these molecular markers in the differential diagnosis and local invasiveness of these lesions.

 Materials and Methods



The present retrospective study was carried out in the Department of Oral Pathology and Microbiology, Rajasthan Dental College and Hospital, Jaipur, following clearance from the ethical committee of the institute. A total of 30 samples were considered for the study and were categorized into three groups with 10 cases in each. Group 1, II, III included 10 cases of histopathologically diagnosed KCOT, OOC, and DC each, respectively. The formalin-fixed paraffin-embedded tissue blocks of KCOT, OOC, and DC were retrieved form the archives of department. Three sections each of 3 μm thickness were made using a rotary microtome and they were stained with syndecan-1 (CD-138) and podoplanin (PDPN) using immunohistochemical methods and standard hematoxylin and eosin stain. Normal oral mucosa was taken as external positive control for syndecan-1 (CD-138) and tonsil tissue as external positive control for podoplanin. Compound microscope (Lawrence and Mayo) and Digital Camera (Nikon D5200 with Nicon Lens 18 mm-105 mm) were used for microscopic photographs.

Anti-podoplanin mouse monoclonal antibody and Poly HRP secondary detection system was used for immunohistochemical staining. Immunohistochemical staining was carried out. The area of optimal staining for syndecan-1 and podoplanin was marked using a magnification ×100. The presence of brown-colored end products at the site of target antigen (cytoplasm and cell membrane of the basal and suprabasal layers) was considered as positive immunoreactivity for CD-138 and PDPN. Within the marked area, immunohistochemical reactivity for syndecan-1 and podoplanin was evaluated and graded in three groups ([−] negative; [+] weakly to moderately positive; [++] strongly positive [Figure 1],[Figure 2],[Figure 3],[Figure 4],[Figure 5],[Figure 6]).[3],[4],[5] (Color Plates 1, 2, 3, 4, 5 and 6). The data were assessed by two observers and were subjected to statistical analysis.{Figure 1}{Figure 2}{Figure 3}{Figure 4}{Figure 5}{Figure 6}

 Results



The immunohistochemical reactions for syndecan-1 and podoplanin in tissue sections of respective groups were semi-quantitatively assessed by two observers to remove any possible bias. To minimize the interobserver variability; the scores of two observers were subjected kappa statistics that showed that there was good agreement (0.8) between the two observers. The interobserver variability test showed that there was no significant statistical difference between two observations. Hence, further evaluation was done with respect to the results of observer 1. Immunohistochemical expression of syndecan-1 in keratocystic odontogenic tumor (KCOT) was found to be negative in 2 (20%) cases, whereas the same was observed to be weakly to moderately positive in 6 (60%) cases with 2 (20%) cases exhibiting positive expression. On application of Chi-square test, the same was found to be statistically significant (P < 0.05). In OOC group out of 10 cases, 3 (30%) displayed negative immunohistochemical expression for syndecan-1 whereas 7 (70%) cases were weakly to moderately positive. Immunohistochemical expression for syndecan-1 in OOC was found to be statistically nonsignificant (P > 0.05) on application of Chi-square test. Immunoreactivity for syndecan-1 in DC was negative in 6 (60%) and strongly positive in 4 (40%) cases and was found to be statistically nonsignificant (P > 0.05) [Table 1].{Table 1}

Mean syndecan-1 expression for group I, II, and III was observed to be 2.00 ± 0.67, 1.70 ± 0.48, and 1.80 ± 1.03, respectively. The total mean square of all the three groups was 0.233 with “F” value being 0.401. Comparison of mean expression of syndecan-1 was found to be statistically nonsignificant (P > 0.05), on application of ANOVA test [Table 2]. On application of Tukey honestly significant difference (HSD), comparison of mean syndecan-1 expression between the three study groups was found to be statistically nonsignificant (P > 0.05) [Table 3].{Table 2}{Table 3}

Immunohistochemical expression of podoplanin in keratocystic odontogenic tumor (KCOT) was found to be negative in 1 (10%) case, whereas the same was observed to be weakly to moderately positive in 4 (40%) cases with 5 (50%) cases exhibiting strongly positive expression. On application of Chi-square test, the same was found to be statistically nonsignificant (P > 0.05) In OOC group out of 10 cases, 3 (30%) displayed negative immunohistochemical expression for podoplanin whereas 3 (30%) cases were weakly to moderately positive and 4 (40%) cases were strongly positive. Immunohistochemical expression for podoplanin in OOC was found to be statistically nonsignificant (P > 0.05) on the application of Chi-square test. Immunoreactivity for podoplanin in DC was negative in 3 (30%), whereas 5 (50%) cases were weakly to moderately positive and strongly positive in 2 (20%) cases and were found to be statistically nonsignificant (P > 0.05) [Table 4].{Table 4}

Mean podoplanin expression for Group I, II, and III was observed to be 2.4 ± 0.7, 2.1 ± 0.88, and 1.9 ± 0.74, respectively. The total mean square of all the three groups was 0.633 with 'F' value being 1.056. Comparison of mean expression of podoplanin was found to be statistically nonsignificant (P > 0.05) on application of ANOVA test [Table 5]. On application of Tukey HSD, comparison of mean podoplanin expression between the three study groups was found to be statistically nonsignificant (P > 0.05) [Table 6]. On application of Pearson Correlation test, the correlation between the mean syndecan-1 and podoplanin expression recorded was found to be statistically nonsignificant (P > 0.05) in all the three study groups [Table 7] and [Graph 1].{Table 5}{Table 6}{Table 7}[INLINE:1]

 Discussion



Odontogenic cysts and tumors originate from the epithelial, ectomesenchymal or mesenchymal components of the tooth forming apparatus or their remnants entrapped either within the jawbones or into the adjacent soft tissues.[3],[6] The factors that led to the re-classification of OKC included locally destructive and highly recurrent nature of the lesion; the budding basal layer of the OKC into connective tissue consisting of high mitotic figures usually in the suprabasal layers and mutation in the patched-1 gene.[3] However, evidence-based current clinical parameters cannot predict the potential for neoplastic behavior, or aggressive and localized infiltration, in KCOT and OOC. The most perplexing aspect of OOC is its characteristic attachment to the neck of the tooth, quite similar to a DC.[1] Several studies have been carried out using specific markers to underline the differences in their origin and pathogenesis with significant variances seen at the molecular level in KCOT, OOC, DC suggesting the difference in pathophysiological behavior of these lesions. In most recent times, proteoglycans (syndecan-1 and podoplanin) are thought to direct the cell movement in numerous physiological and pathological conditions, right from early embryonic development to tumor invasion and metastasis.[7]

Syndecans are members of the transmembrane heparan sulfate proteoglycan family and are expressed on the surface of all adherent and nonadherent cells. They act as an anchor to stabilize the morphology of epithelial sheets by connecting the extracellular and intracellular cytoskeleton. These help to bind several ligands and matrix components such as growth factors particularly basic-fibroblast growth factor, heparin binding factors, degradative enzymes, protease inhibitors, and some serum proteins.[8]

Podoplanin (also known as Aggrus, M2A, E11, GP38, PDPN) is a 40 kDa O-linked sialoglycoprotein, a molecule expressed in lymphatic endothelial cells and is considered to be related with tooth development and tumor invasion.[2] PDPN has been shown to have an increased expression in various malignant tumors, signifying its possible role in tumor progression.[9] Podoplanin increases the activities of Rho GTPases, mainly RhoA, contributing to cytoskeletal reorganization, suggesting an important role of podoplanin in tumor invasion and metastasis.[10]

Immunohistochemical expression for syndecan-1

Immunohistochemical expression of syndecan-1 in KCOT was found to be negative in 20% cases; weakly to moderately positive in 60% cases and 20% cases exhibiting positive expression. Our results were in accordance with the study by Nadalin et al.[4] who also observed positive expression in all KCOT cases. In OOC, 30% cases displayed negative expression for syndecan-1 whereas 70% cases were weakly to moderately positive. Immunoreactivity for syndecan-1 in DC was negative in 60% and strongly positive in 40% cases (statistically nonsignificant). The results of our study were not in agreement with the studies by Nadalin et al.,[4] Al-Otaibi et al.[8] and Özcan et al.[11] who had observed strong expression for syndecan-1. In DC, the epithelium with intense inflammatory reaction exhibited reduced or absent syndecan-1 expression. It is possible that the alterations caused by the inflammatory cells in the epithelial lining contributed to decreased expression of protein. Whereas the typical epithelial lining in KCOT, high and intense expression was maintained even in the presence of inflammatory cells. However, in areas of epithelial hyperplasia secondary to inflammatory reaction, the expression was diminished or absent. This finding was in accordance with that of Nadalin et al.[4] There is evidence that syndecan-1 participates in various processes involved in the modulation of inflammation, mainly in interactions between leukocytes and endothelial cells, which explains its expression in the inflammatory cells.[4] Therefore, in DC, the decreased expression of syndecan-1 could indicate another pathway of action of this proteoglycan in this lesion.[2] Comparison of expression of syndecan-1 in all study groups as well as comparison between the three study groups was found to be statistically nonsignificant. This is in accordance with the results of studies by Nadalin et al.,[4] Al-Otaibi et al.,[8] and also Özcan et al.[11] Thus, syndecan-1 expression does not prove to be a determinant factor for the histopathological features and biological behavior of the cystic lesions. However, syndecan-1 may be a marker for the aggressiveness and the biological behavior of the KCOT.

Immunohistochemical expression of podoplanin

Immunohistochemical expression of podoplanin in KCOT was found to be negative in 10% cases; weakly to moderately positive in 40% cases and 50% cases showed strongly positive expression. The results were in accordance with the observations of Caetano Ados et al.[12] who reported the expression of podoplanin in invasive front of ameloblastomas, KCOT, adenomatoid odontogenic tumors, and calcifying epithelial odontogenic tumors. Immunohistochemically, KCOT cases revealed a strong membranous and cytoplasmic reaction for podoplanin of the basal cell layers as was stated by Friedrich et al.[13] Most of the podoplanin positive cells were restricted to the basal layer of the epithelial lining, with a little expression toward the parabasal or lower middle layers. In our study, the upper layers were negative for this protein as was also seen by Zhang et al.[14] The possible contribution of podoplanin in the local invasiveness and the neoplastic nature of the KCOT may be attributed to the important role, it plays in mediating cellular contractile properties and cytoskeletal reorganization as suggested by Schacht et al.[15] In OOC group, 30% cases displayed negative immunohistochemical expression for podoplanin whereas 30% cases were weakly to moderately positive and 40% cases were strongly positive. Results of our study demonstrated that the expression of podoplanin was evidently higher in KCOT than in OOC, probably because KCOT has more of a neoplastic character, with progression and local invasiveness These findings are comparable to the results of the study conducted by González-Alva et al.[16] Areas with inflammatory changes showed increased expression of podoplanin in the basal layer compared to areas where inflammatory changes were absent as also observed by González-Alva et al.[16] A study by Caetano Ados et al.[12] also showed a statistically significant PDPN expression in KCOT and OOC. Immunoreactivity for PDPN in DC was negative in 30% cases; whereas 50% cases were weakly to moderately positive and strongly positive in 20% cases. Studies have shown that podoplanin expression is required when morphologic changes (regeneration, reparative, or even neoplastic) take place especially when epithelium is associated with inflammatory infiltrate.[12]

Immunohistochemical expression of syndecan-1 versus podoplanin

The present study showed absence of significant correlation between the expression of syndecan-1 and podoplanin, thus, reinforcing that the exact role of these proteins in the differentiation of odontogenic cystic lesions and odontogenic tumors need to be elucidated more elaborately.

 Conclusion



Even though KCOT expressed significant syndecan-1 expression, it does not seem to be a determinant factor of the distinct histopathological features and biological behavior. Moreover, the prognostic significance of syndecan-1 in the aforementioned cystic lesions cannot be addressed here, owing to the limited number of cases included in the study. Although the biological functions of podoplanin have not yet been fully recognized, its expression in KCOT suggests the possible contribution of podoplanin in the local invasiveness and the neoplastic nature of the lesion. Due to the limited sample size, it is thus necessary to reinforce and elucidate the exact role of these proteins in the differentiation of cystic odontogenic lesions and tumors.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

Ethical clearance

The study was approved from the ethical committee of the institution. RDCH/Ethical/2016/2851-C.

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