|Year : 2015 | Volume
| Issue : 4 | Page : 617-622
Role of podoplanin in potentially malignant disorders and oral squamous cell carcinoma and its correlation with lymphangiogenesis
S Parhar1, H Kaur2, A Vashist3, S Verma2
1 Department of Oral Pathology, Swami Devi Dyal Hospital and Dental College, Barwala, Panchkula, India
2 MM College of Dental Sciences and Reserch Mullana, Ambala, Haryana, India
3 Public Health Dentistry, Shri Sukhmani Dental College and Hospital, Derabassi, Punjab, India
|Date of Web Publication||10-Mar-2016|
Department of Oral Pathology, Swami Devi Dyal Hospital and Dental College, Barwala, Panchkula
Source of Support: None, Conflict of Interest: None
BACKGROUND: Oral squamous cell carcinoma (OSCC) ranks as one of the most common types of cancer and oral potentially malignant lesions (OPMLs) provides with an overall increased risk for development of carcinoma. As podoplanin expression is attracting interest as a marker for cancer diagnosis and prognosis, this study assesses the role of podoplanin expression in such lesions. MATERIALS AND METHODS: Podoplanin expression and lymphatic vessel density (LVD) was determined using D2-40, a marker for podoplanin, in 70 diagnosed cases of potentially malignant lesions and OSCC. RESULTS: Normal epithelium showed negligible podoplanin expression, whereas the expression extended predominantly at the basal layer and the suprabasal layer or above at one or multiple areas in potentially malignant lesions. Podoplanin expression in OSCC showed two different patterns-diffuse and focal. A statistically significant increase in mean LVD was seen from normal epithelium to potentially malignant lesions (P < 0.001) and to OSCC (P < 0.022) while a non-significant increase was seen (P < 0.594) between OPMLs and OSCC. Overall no significant correlation was found between D2-40 epithelial positivity and LVD (P = 0.122). CONCLUSION: This study suggests the utility of podoplanin as a biomarker for cancer risk assessment as it detects the early changes and thus provides an additional value beyond current clinical and histopathological evaluations. Hence, podoplanin is suggested to be a marker of tumor initiation and to a lesser extent of tumor progression.
Keywords: D2-40, lymphatic microvessel density, oral squamous cell carcinoma, podoplanin, potentially malignant lesions
|How to cite this article:|
Parhar S, Kaur H, Vashist A, Verma S. Role of podoplanin in potentially malignant disorders and oral squamous cell carcinoma and its correlation with lymphangiogenesis. Indian J Cancer 2015;52:617-22
|How to cite this URL:|
Parhar S, Kaur H, Vashist A, Verma S. Role of podoplanin in potentially malignant disorders and oral squamous cell carcinoma and its correlation with lymphangiogenesis. Indian J Cancer [serial online] 2015 [cited 2019 Jun 26];52:617-22. Available from: http://www.indianjcancer.com/text.asp?2015/52/4/617/178427
| » Introduction|| |
Several lines of evidence indicate that tumorigenesis in humans is a multistep process. These steps drive the progressive transformation of normal human cells into highly malignant derivatives. Oral potentially malignant lesions (OPMLs) comprise of a multitude of histologically diverse lesions with variable, but overall increased risk for development of oral squamous cell carcinoma (OSCC). OSCC is a complex disease with various origins and ranks as one of the most common types of cancer. So, it is important to understand the biology of squamous cell carcinoma (SCC) and to identify the biological markers that may be able to augment the clinical staging system and may serve as prognostic and predictive markers.
Podoplanin, a 38-kd mucin-like transmembrane glycoprotein, is highly and specifically expressed in lymphatic endothelial cells, but not blood vessel endothelium. Podoplanin expression has been reported in carcinomas of the skin, lung, uterus and esophagus and is highly expressed in oral cancer and some oral pre-malignancies. However, very few studies have been reported until date demonstrating the significance of podoplanin expression and the potential association between its expression patterns and the histopathologic characteristics of oral premalignant lesions  and OSCC. Commercially, available antibody D2-40 specifically recognizes human podoplanin because of its biochemical similarities to M2A antigen  and can therefore, be used for evaluating the podoplanin expression in development of neoplasms and invasion. D2-40 can also been used for immunohistochemical studies of tumor lymphangiogenesis.
Lymphangiogenesis is considered to be an important process in the development of tumor metastasis. An increase in the number of lymphatic vessels in the tumor stroma has been shown to correlate with lymph node metastasis. OSCC has three grades of differentiation; well, moderate and poorly differentiated carcinomas, with poorly differentiated tumors showing the worst prognosis. Furthermore, OSCC may arise from OPMLs. However, the stage of tumor growth and invasion at which lymphangiogenesis may begin is still not certain. Therefore, the present study was performed to study the expression of podoplanin in potentially malignant lesions and OSCCs and to study the presence of lymphangiogenesis in OPMLs and OSCC.
| » Materials and Methods|| |
A total of 70 formalin fixed, paraffin embedded blocks of previously diagnosed cases were taken in this retrospective study. The cases were categorized into three main groups. Group I (control) comprising of 10 specimens of normal oral mucosa; Group II including 30 diagnosed cases of OPMLs, which were further subdivided as Group IIa (15 cases of moderate epithelial dysplasia) and Group IIb (15 cases of Severe epithelial dysplasia.); Group III included 30 diagnosed cases of OSCC which were further subdivided as Group IIIa (10 cases of well-differentiated carcinoma), Group IIIb (10 cases of moderately differentiated carcinoma) and Group IIIc (10 cases of poorly differentiated carcinoma). The paraffin-embedded tissue blocks were sliced into 4 µ thick sections for subsequent histologic examinations.
D2-40 primary antibody (DAKO, North America Carpenteria CA, USA), peroxidise labelled streptovidin secondary antibody and diaminobenzidine (DAB) hydrochloride substrate (Biogenx, India) were used. The formalin-fixed, paraffin-embedded tissues were cut into 4 μm sections. The deparaffinised sections were hydrated and antigen retrieval performed using a microwave oven with citrate buffer (pH 6.0). Endogenous peroxidise activity was blocked using hydrogen peroxide and then a power block was used. The slides were then incubated with D2-40 monoclonal antibody for 1 h followed by the secondary antibody, for half an hour and DAB chromogen. The slides were counterstained with hematoxylin. Expression of podoplanin in lymphatic endothelial cells within the stroma served as an internal positive control. Negative controls with an omission of the antiserum from the primary antibody were also included.
Evaluation of stained slides
For evaluation Of podoplanin expression in OPMLs, a scoring system similar to that described by Kawaguchi et al., was used as follows: (0) No expression observed in any part of the epithelium, (1) expression restricted to the basal layer of the epithelium, (2) expression in the basal and suprabasal layers at one area and (3) suprabasal layer expression at two or more areas. Further, on the basis of this score, lesions were classified as podoplanin negative (Score 0-2) and podoplanin positive (Score 2 or higher). In OSCC, podoplanin expression was scored as described by Yuan et al. If 0%; 1-10%; 11-30%; 31-50%; 51-80%; and 81-100% of the tumor cells were positive, quantitative scores from 0 to 5 were assigned respectively. The staining intensity was rated on a scale of 0-3 (0 = negative, 1 = weak, 2 = moderate, and 3 = strong). The raw data were then converted to a German immunoreactive score (IRS) by multiplying the quantitative and staining intensity scores. An IRS score above the median (7 or higher) was considered high reactivity and 0-6 as weak reactivity.
The OSCC cases were also categorized based on the expression pattern of podoplanin in tumor cells. The lesions were categorized as showing diffuse expression of podoplanin in the tumor cells or focal expression in which podoplanin positivity was seen at the proliferating periphery of the tumor cell nests with no expression in the central areas.
Evaluation of lymphatic microvessel density (LMVD) was carried out by counting positive D2-40 lymphatic vessels, with a visible lumen, clearly separated from adjacent microvessels and from other connective tissue components. Packed vessels were assumed as one lymphatic unit. Immunohistochemical D2-40 stained sections were scanned at low magnification to identify the most vascular areas (hot spot areas). Analysis was performed under × 20 objectives and × 10 ocular lenses (×200 magnifications). The average of 10 hot spot fields was taken to calculate LMVD.
The statistical significance of differences in D2-40 reactivity levels and LMVD was analyzed by the Mann-Whitney test and Kruskal-Wallis test. Values of P < 0.01 were considered as statistically significant. Correlation between groups was calculated using Spearman's Rho coefficient.
| » Results|| |
Comparison of epithelial podoplanin score
Podoplanin expression in normal gingival epithelium (control) was found to be a minimum [Table 1]. The expression was negligible or restricted to the basal cells that showed mainly membranous and cytoplasmic staining. In the dysplastic epithelium, the expression was variable, i.e. from being predominantly at the basal layer to the expression extending to the suprabasal layer or above at one or multiple areas. Some areas in the dysplastic epithelium showed the D2-40 positive expression in spinous layers also. 23 (76.6%) out of total 30 cases of OPMLs were classified as podoplanin positive and 7 (23.3%) were classified as podoplanin negative. Further, it was found that 9 (60%) of the 15 moderate dysplasia cases and 14 (93.3%) of the 15 severe dysplasia cases were podoplanin positive. The difference in the epithelial scores between normal mucosa and OPMLs was statistically highly significant (P < 0.001). The increase in epithelial score in moderate dysplasia and severe dysplasia, when compared with the normal mucosa was statistically highly significant (P < 0.001). A statistically significant increase in the epithelial score was also seen from moderate to severe dysplasia (P < 0.028).
The epithelial score was highest in OSCC and the difference in the epithelial scores between normal mucosa and OSCC was found to be statistically highly significant (P < 0.001). In our study, OSCC showed two different patterns of podoplanin expression:
- First type showed a diffuse expression of podoplanin in the tumor cells. The membrane of the tumor cells showed a stronger staining than the cytoplasm. This pattern was seen in 3 (30%) of 10 cases of well-differentiated SCC, 6 (60%) of 10 cases of moderately differentiated SCC and 9 (90%) of 10 cases of poorly differentiated SCC [Figure 1]
|Figure 1: Photomicrograph showing diffuse D2-40 epithelial expression (well-differentiated squamous cell carcinoma)|
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- Second type showed focal expression of podoplanin in the peripheral layer of the tumor proliferation area was found. This pattern was seen in 7 (70%) of 10 cases of well-differentiated SCC, 4 (40%) of 10 cases of moderately differentiated SCC and 1 (10%) of 10 cases of poorly differentiated SCC [Figure 2].
|Figure 2: Photomicrograph showing focal D2-40 epithelial expression (well-differentiated squamous cell carcinoma)|
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When OSCCs were further categorized as high and low reactivity on the basis of IRS, we found 17 (56.6%) of 30 cases showed high and 13 (21.6%) of 30 cases showed weak podoplanin expression. On further comparing the three histological type of carcinomas, it was seen that 10 (100%) of 10 cases of well-differentiated carcinoma, 5 (50%) of 10 cases of moderately differentiated carcinoma and 2 (20%) of 10 cases of poorly differentiated carcinoma showed high D2-40 expression [Table 2].
|Table 2: Epithelial podoplanin grading (IRS score) in subgroups of group III (oral squamous cell carcinoma)|
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When D2-40 epithelial expression of OPMLs and SCC were compared with each other, statistically highly significant results were seen (P < 0.001). On the transition of severe dysplasia to well-differentiated SCC, the difference between the mean of the epithelial score came to be statistically highly significant (P < 0.001). Thus, suggesting that podoplanin may help as a marker in diagnosing the early changes occurring in dysplastic lesions to OSCC.
Comparison of LMVD
When LMVD was compared between Control, OPMLs and OSCC, it was that found that the mean LMVD was a minimum for the control group and maximum in OSCC [Table 3]. The difference between control and OPMLs was statistically highly significant (P < 0.001) and that of control and OSCC was statistically significant (P < 0.022). When OSCCs were further categorized based on the histological differentiation, it was seen that mean LMVD in well-differentiated SCC was a minimum, increased in poorly differentiated SCC and maximum mean LMVD was seen in moderately differentiated SCC. The differences in LMVD between the 3 grades of SCC when compared with each other were statistically insignificant.
When LMVD of OPMLs and OSCC were compared with each other, statistically non-significant results were seen (P < 0.594); though, there was an increase in LMVD in OSCC when compared with OPMLs.
In our study, tumor emboli within D2-40 positive lymphatic vessels were present in 11 out of 30 cases of oral SCC. Out of which 2 were of well-differentiated SCC, 3 of moderately differentiated SCC and 6 of poorly differentiated SCC [Figure 3].
|Figure 3: Photomicrograph showing tumor emboli present within lymphatic vessels|
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When we compared overall D2-40 epithelial positivity with the LMVD of all the lesions studied, no significant correlation was found between the two (P = 0.122).
| » Discussion|| |
Though focal expression of podoplanin has been detected on the basal keratinocytes of skin, cervix and esophagus, no positive reaction for podoplanin has been demonstrated in basal cells of morphologically normal oral mucosal epithelium or when inflammation is minimal.
In our study, a significant increase in epithelial score of podoplanin was seen in dysplasia when compared to the normal mucosa and also as the severity of dyplasia increased. Also, more number of cases in severe dysplasia were podoplanin positive than that of moderate dysplasia. Hence, the individuals whose lesions graded as podoplanin negative seemed to be at a lower risk to transform into malignancy than those scored as podoplanin positive. Yuan et al., Raica et al., and Kawaguchi et al., in their studies also showed that podoplanin is expressed in oral dysplastic and hyperplastic lesions with an increased risk of cancer development. In epithelial dysplasia, high podoplanin expression has been associated with an increased risk of progression to invasive cancer, suggesting that podoplanin could serve as a powerful biomarker to predict the risk for oral cancer development in patients with OPMLs. But in contraindication, a decrease in D2-40 expression has been reported in the transition from normality to malignancy. Furthermore, some areas in the dysplastic epithelium showed the podoplanin positive expression in spinous layers also. The upward clonal expansion of the podoplanin expressing cells in the epithelial layers carries a significantly higher risk of oral cancer development. The ability to detect the cells expanding beyond basal layers may allow us to visualize potential clonal expansion, possibly from stem-cell clones, during tumorigenesis. OPMLs with podoplanin expression beyond the basal cell layer may represent truly early neoplastic lesions, enriched in Tumor initiating cells (TIC's) and with a higher risk of progression to invasive cancer. However, Rodrigo et al., in their study found no statistically significant correlation between podoplanin status and the severity of dysplasia.
In our study, we found a significant increase in epithelial podoplanin expression between normal mucosa and OPMLs to OSCC. Longatto-Filho et al., in their study on pre-malignant lesions and carcinomas of the uterine cervix observed a decrease in D2-40 expression in the transition from normality to malignancy, which was in contrast to the results of our study. A gradual decrease in the epithelial score was seen from well to moderate to poorly differentiated SCC. High podoplanin expression in OSCCs could reflect a higher proportion of TIC's, rather than a higher invasive potential as in the present study podoplanin expression is higher in well-differentiated SCC, which are usually less invasive than those of poorly differentiated SCC. OSCC showed two different patterns of podoplanin expression. In diffuse expression of podoplanin in the tumor cells, the membrane of the tumor cells showed a stronger staining than the cytoplasm. This pattern was most prominent in poorly differentiated OSCC and least in well-differentiated tumors. In lesions showing focal expression of podoplanin, both membranous and cytoplasmic positivity was seen in the cells of the peripheral layer of the tumor proliferation area. This could be explained on the basis that the central areas often contained more differentiated cells, mimicking the pattern seen in the dysplastic epithelium. Thus, our results suggest that SCCs with focal expression of podoplanin in the periphery of tumor nests (which is equivalent to the hierarchical distribution pattern) may indicate lower biological aggressiveness. According to this, it is plausible that SCCs showing focal expression pattern represent a well-organized tumor group based on the tumor initiating cells (TICs) concept, whereas SCCs with a diffuse expression pattern could reflect disordered tumors in terms of the developmental hierarchy. This concept is supported by studies conducted by Rodrigo et al. and Shimada et al., Martín-Villar et al., showed that a wild-type human podoplanin promotes a complete EMT in Madin-Darby canine kidney type-II epithelial cells. As in SCC, podoplanin expression is frequently seen in invasive front and in some tumors correlates with down-regulation of the cell to cell adhesion protein E-cadherin.
In the present study, podoplanin expression in well-differentiated tumors, though focal, showed intense cytoplasmic staining, but poorly differentiated tumors showed a switch from cytoplasmic to membranous staining. When epithelial podoplanin expression of OPMLs and oral SCC were compared with each other, statistically highly significant results were seen.
Earlier studies have proposed that podoplanin is able to enhance tumor invasion by enhancing the tumor cell motility. Cancer cell migration and invasion involves active remodeling of the actin cytoskeleton. However, podoplanin does not interact directly with actin, but via ezrin, radixin and moesin (ERM) proteins such as ERM. It has been shown that overexpression of podoplanin leads to an increased phosphorylation of ezrin which links podoplanin to the observed rearrangement of the actin cytoskeleton. Apart from that, podoplanin increases the activities of Rho-family GTPases, mainly RhoA, which is also linked to tumor cell motility.
We observed that in normal oral mucosal tissue, the lymphatic vessels were relatively small and evenly distributed in the underlying superficial lamina propria [Figure 4]. A significant increase was found in LMVD from the control group to OPMLs. Our results herein reported, strongly suggest that lymphatic vessel proliferation occurs early in precancerous lesions. Similar results were reported by Ali in his study. This suggests that the molecular armamentarium of proliferating lymphatic neovascularization begins early in the intraepithelial lesions. Earlier reports also indicated an increased density of lymphatic vessels associated with severe dysplasia/carcinoma in situ and suggested an essential role of lymphangiogenesis in the progression non-invasive to invasive lesions in cervical carcinogenesis.,
In the present study, LMVD were significantly higher in OSCCs than normal mucosa whereas, an insignificant increase in LMVDs was seen in OSCC when compared to OPMLs. Sedivy et al. Xie et al., and Longatto-Filho et al., also reported a significant increase in LMVD in SCC from control groups.
In the present study, the lymphatic vessels at the invading edge of OSCCs were enlarged and dilated, these may depict the mechanism leading to lymphatic spread and metastasis. Gombos et al., in their study on lymphatic vessel density (LVD) in SCC of the uterine cervix concluded that high peritumoral LVD may be one of the mechanisms leading to lymphatic invasion and metastatic spread. Franchi et al., in their study found that Lymphatic vessels were significantly numerous and larger in the peritumoral area and the number and relative area of intratumoral and peritumoral lymphatics was significantly higher in HNSCC cases with lymph node metastasis. This was further supported by areas showing the presence of tumor cells within the lymphatic vessels in 11 out of 30 cases of oral SCC in the present study, out of which 2 were of well-differentiated SCC, 3 of moderately differentiated SCC and 6 of poorly differentiated SCC. Thus, the presence of tumor emboli suggests a higher rate of metastasis associated with poorly differentiated SCC than the other two grades of SCC. Furthermore, the tumor emboli in the lymphatic vessels in the present study were found to be negative for podoplanin in all of the 11 cases, even when the main tumor cells showed positivity. Similar expression of tumor emboli have been reported earlier. This higher proportion of poorly differentiated SCC showing tumor emboli, when compared to well and moderately differentiated tumors, shows the poorer prognosis of these cases. The podoplanin expression of the tumor cells in these cases was membranous and not cytoplasmic. This could suggest that loss of differentiation of tumor cells can be associated with switch from cytoplasmic to membranous expression of podoplanin and also with increased metastatic potential.
On comparing LMVD between the three grades of SCC, the results were statistically non-significant indicating that histopathological grading may not be the only factor influencing tumor lymphangiogenesis. Furthermore, Miyahara et al. and Franchi et al. had reported earlier that no correlation existed between LMVD and tumor grade from their study.
When LMVD of oral premalignant lesions and SCC were compared with each other, statistically non-significant results were seen; though, there is an increase in LMVD in SCC when compared with premalignant lesions.
When we compared overall epithelial podoplanin expression with the LMVD of all the lesions studied, no significant correlation was found between the two. Furthermore, when the individual lesions were compared for the correlation, no positive correlation could be derived for Group I, Group II and Group III. Thus, it could be suggested that no association is observed between podoplanin expression in tumor cells and LVD of the tumor.
| » Conclusion|| |
Thus, podoplanin positivity may be a strong factor for oral cancer development. The presence of podoplanin positive cells, specifically at the invasive front of the tumor nests, suggests an important role of podoplanin in the tumor initiation rather than its progression. The switch of podoplanin expression from cytoplasmic to membranous in PDSCC and the associated presence of tumor emboli in these cases suggest that reduced podoplanin expression may be associated with poor prognosis. Early lymphatic vessel proliferation occurring in precancerous lesions suggests that the molecular armamentarium of proliferating lymphatic neovascularization begins early in the intraepithelial lesions. Significant increase in LMVD in OSCC suggests that tumoral lymphangiogenesis may be an indicator of the risk of lymph node metastasis in patients with OSCC.
Thus, this study suggests the utility of podoplanin as a biomarker for cancer risk assessment, as it detects the early changes and thus providing additional value beyond current clinical and histopathological evaluations.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4]
[Table 1], [Table 2], [Table 3]