Abstract
Background: This study evaluated the clinical significance of podoplanin and vascular endothelial growth factor-C (VEGF-C) expression in oral squamous cell carcinoma (OSCC). Patients and Methods: The immunohistochemical expression of podoplanin and VEGF-C were investigated in 42 well-differentiated OSCCs. The Chi-square test was used to analyze the association of biomarker expression with the clinical characteristics, treatment, and outcome, as well as with histopathological microscopic features and histopathological malignancy index. The relationship between podoplanin, VEGF-C and prognosis was also studied. Survival rates were calculated by the Kaplan Meier method. Results: Strong podoplanin expression was associated with male gender (p=0.037) and with early clinical stage (I-II) (p=0.027). Strong podoplanin expression was more frequently found in patients with perineural infiltration. Podoplanin and VEGF-C expression were not significant prognostic factors for patients with OSCC. Conclusion: These results suggest that the strong podoplanin and VEGF-C expression by malignant cells is associated with perineural invasion in patients with OSCC.
Oral squamous cell carcinoma (OSCC) metastasizes via lymphatic channels to cervical lymph nodes and such regional nodal metastasis is the most important prognostic factor, indicating a reduction in the probability of disease control and shortens the survival of these patients (1-4).
In the past, it was thought that lymphatic metastasis was a passive process in which detached tumor cells reached lymph nodes via drainage through pre-existing local lymphatic vessels (4, 5). However, some studies have shown a positive correlation among growth factor expression, peri- and/or intra-tumoral lymphangiogenesis and lymphatic metastasis (6, 7).
The most extensively studied molecular system that signals for tumor lymphangiogenesis and is associated with lymphatic spread from primary cancer is the vascular endothelial growth factor-C/VEGF receptor-3 (VEGF-C/VEGFR-3) signaling axis (4, 5, 8, 9). VEGFR-3 signaling is primarily responsible for the lymphangiogenic response to VEGF-C stimulation and leads to lymphangiogenesis and lymphatic hyperplasia (5, 7).
On the basis of these findings, a range of studies positively correlate with the strong expression of VEGF-C and VEGFR-3 in breast, skin, esophagus, gastric, thyroid and colorectal tumors, and with a higher frequency of metastatic spread to lymph nodes and distant organ metastasis (10-13). Particularly in OSCC, VEGF-C expression was significantly correlated with an elevated peri/intratumoral lymphatic vessel density (14, 15), nodal metastasis (14, 16-20) and poor prognosis (14, 16-20).
At present, the most reliable marker of lymphatic vessels is likely to be podoplanin, a small mucin-type transmembrane glycoprotein, that is expressed on lymphatic but not blood vascular endothelium (8, 9, 21-23). Experimental results have demonstrated that podoplanin mediates a pathway leading to collective cell migration and invasion in vivo and in vitro (22, 24). Clinically, high levels of podoplanin expression, particularly in tumor cells of the invasive front, are associated with lymph node metastasis and predict poor clinical outcomes in patients with OSCC (21).
Although in recent years numerous biological and molecular factors have been proposed as prognosticators in OSCC, these have yet to impact on routine clinical care (25). This study was designed to evaluate the association of podoplanin and VEGF-C expression with clinicopathological parameters in order to determine whether they may be useful markers to predict the presence of nodal metastasis, tumoral invasion and prognosis in patients with OSCC.
Patients and Methods
This study was based on the analysis of 42 patients who underwent surgical treatment for primary OSCC from 1980 to 2000, at the Head and Neck Surgery and Otorhinolaryngology Department of the A.C. Camargo Cancer Hospital, São Paulo, Brazil. The study protocol was approved by the Institutional Ethics Committee (protocol 1022/07).
The inclusion criteria were: (i) primary tumor with the diagnosis of well-differentiated squamous cell carcinoma confirmed by biopsy and located in any area of the mouth (lip, tongue, buccal mucosa, floor of the mouth, palate, gingiva and retromolar area); (ii) no radiotherapy, chemotherapy or other treatment prior to surgery; (iii) no other simultaneous primary tumor, and (iv) tumor tissue available for microscopic analysis.
Clinical data of the patients were obtained from the medical records and included gender, age, ethnic group, tobacco and alcohol consumption, tumor location, TNM stage, treatment (surgery, postoperative adjuvant radiotherapy), and clinical follow-up (local and/or regional recurrences and death). The well-differentiated OSCC were dived into two groups: with lymph node metastasis (OSCCpN+) and without lymph node metastasis (OSCCpN0) confirmed by histopathological analysis.
A formalin-fixed 3-μm thick section of tumor tissue was taken from the pathology archive for hematoxylin-eosin (HE) staining and immunohistochemistry analyses of podoplanin and VEGF-C expression. The histopathological malignancy grading of the OSCC was established according to Bryne et al. (26) The presence of vascular embolization and perineural, muscular and salivary gland infiltrations in the OSCC were reviewed in the HE-stained tumor sections.
After antigen retrieval using 10 mM citrate buffer, pH 6.0, in a pressure cooker for 4 min, endogenous peroxidase activity was blocked by incubation in 3% H2O2 for 20 min. Each tumor section was incubated overnight at 4°C with a primary monoclonal antibody against podoplanin (C18H5) (Acris Antibodies, DM3500, dilution 1:200, Hiddenhausen, Germany), or VEGF-C (C-20) (Santa Cruz Biotechnology, sc-1881, dilution 1:100, Santa Cruz, CA, USA), in phosphate-buffered saline with bovine serum albumin solution to block non-specific reactions. The podoplanin tumor sections were then incubated with Post Primary Block (NovoLink Max Polymer, RE7260-k; Novocastra, Newcastle-upon-Tyne, UK) followed by the polymer from the same kit. The VEGF-C tumor sections were incubated with secondary rabbit biotinylated anti-goat Ig (Vector, BA-5000; Burlingame, CA, USA) in phosphate-buffered saline 1:500, for 30 min, at 37°C.
Both antibodies were visualized using 3.3’diaminobenzedine tetrahydrochloride (DAB, ref D-5637; Sigma, St. Louis, MO, USA). Sections were counterstained with Harris hematoxylin before being dehydrated and coverslipped. Adjacent normal-appearing lymphatic endothelial cells within the sections served as positive internal control for podoplanin expression. Normal oral mucosa from the surgical margins used as internal control for VEGF-C expression.
Quantitative computer-assisted analysis of 30 fields of invasive tumor front (×400 magnification: 93,992.05 μm2/field) for each tumor sample was performed to evaluate the cytoplasmic and or membranous immunoreactivity for podoplanin and VEGF-C by malignant cells. The images of the invasive tumor front in each section were digitally captured with a camera (Axiocam MR3; Zeiss, Jena, Germany) attached to a light microscope (Axioskop2 Plus; Zeiss) and recorded by a computer program system (Axiovision 4.6; Zeiss). Immunostaining results for podoplanin and VEGF-C expression were evaluated by two investigators without prior knowledge of the tumor's histopathological features and the patient's clinical status. A combined score for podoplanin and VEGF-C expression was based on: (a) the intensity of the immunostaining (0=negative; 1=weak; 2=moderate; 3=strong; 4=very strong) and (b) the percentage of positive cells (0=0% positive cells; 1=<25% positive cells; 2=25-50% positive cells; 3=50-75% positive cells; 4=>75% positive cells), as described by Soini et al. (27). The final immunostaining score was determined by the sum of (a) + (b), ranging from 0 to 8. Final scores with a value greater than 6 were considered as strong expression.
All statistical analyses were performed using SPSS for Windows 13.0 software (Chicago, IL, USA). Associations between podoplanin, VEGF-C expression and the variables studied were verified according to Chi-square test or Fisher's exact test. The survival analysis (overall and disease-free survival) was performed by Kaplan Meier method. Comparisons among survival curves were verified by log-rank test. Values of p<0.05 indicate a significant difference.
Results
The analysis of 42 patients with OSCC revealed a Caucasian male predominance for both groups. Age ranged from 28 to 83 years for OSCCpN0 and 35 to 73 years for OSCCpN+. The use of tobacco and/or alcohol was reported by most of the patients in both groups.
Regarding the tumor location, the gingiva was the primary site in 36.0% of OSCCpN0 patients and the floor of mouth in 58.6% of the OSCCpN+ group. Based on the International Union Against Cancer (UICC) classification of oral cavity carcinomas, most tumors (64.0%) of the OSCCpN0 group were classified as initial clinical stage carcinomas (I-II); in the OSCCpN+ group, 52.9% of the tumors were classified as advanced clinical stage carcinomas (III or IV).
All 42 patients underwent surgical resection of the primary tumor and 100.0% of the OSCCpN+ patients were submitted to elective neck dissection. Only 36.0% of the OSCCpN0 group underwent cervical dissection. Seven (28.0%) OSCCpN0 patients and 82.4% of the OSCCpN+ group had postoperative adjuvant radiotherapy.
Strong expression of podoplanin in the invasive front of well-differentiated oral squamous cell carcinoma in early clinical stage.
Local/regional recurrences occurred in 47.1% of the patients with OSCCpN+ and 12.0% in the OSCCpN0 group. A second primary tumor was more frequently detected in the OSCCpN0 (12.5%) than in the OSCCpN+ (11.8%) group.
A statistically significant difference (p<0.05) was found between the OSCCpN0 and OSCCpN+ groups with regard to N stage, clinical stage, neck dissection, postoperative radiotherapy and local/regional recurrences. Additionally, OSCCpN+ patients presented higher frequency of lymphatic embolization (p<0.001), and perineural (p=0.001) and muscular (p=0.016) infiltration. Surgical margins were negative for malignant epithelial cells in all patients.
Microscopically, most OSCCs exhibited characteristics of well-differentiated carcinoma presenting intense keratinization, discrete nuclear polymorphism, few mitotic figures per high-power field (×400 magnification) and intense-chronic inflammatory infiltrate. According to the histopathological malignant grading described by Bryne et al. (26), 35 OSCCs (83.0%) were classified as more differentiated (5-10 scores) and 7 (17.0%) as less differentiated tumors (11-20 scores). No statistically significant differences were found between the OSCCpN0 and OSCCpN+ groups with regard to histopathological malignant grading.
The analysis of podoplanin expression was performed in 33 OSCCs because there were not enough tumor fragments. Immunohistochemical analysis showed strong cytoplasmic and/or membranous staining for podoplanin in 13 (39.4%) out of the 33 tumors (Figure 1). In surgical margins, containing hyperplastic and dysplastic epithelial areas, podoplanin was expressed in the basal cell layer (Figure 2) and in the endothelial cells of lymphatic vessels.
Transition area between oral squamous cell carcinoma with strong podoplanin expression and surgical margin adjacent to the tumor where podoplanin was highly expressed in the basal cell layer of the hyperplastic epithelium and in the lymphatic vessels.
Strong expression of vascular endothelial growth factor C in the invasive front of the well-differentiated oral squamous cell carcinoma.
Tables I and II display the association between podoplanin expression and clinicopathological factors of these 33 patients. Podoplanin expression by malignant cells correlated with male gender (p=0.037), advanced clinical stage (p=0.027) and glandular infiltration (p=0.003). In contrast, there were no statistically significant correlations regarding the other clinical and pathological parameters and podoplanin expression, but it was observed that patients with strong podoplanin expression presented a higher frequency of local/regional recurrences, perineural infiltration and salivary glandular invasion (Tables I and II).
Correlation of podoplanin, and vascular endothelial growth factor-C (VEGF-C) expression by malignant cells with clinical features in patients with oral squamous cell carcinoma.
Correlation of podoplanin, and vascular endothelial growth factor-C (VEGF-C) expression by malignant cells with histopathological parameters in patients with oral squamous cell carcinoma.
Survival rates of the patients with oral squamous cell carcinoma according to podoplanin and vascular endothelial growth factor-C (VEGF-C) expression by malignant cells, and histological lymph node status.
Strong cytoplasmic immunostaining for VEGF-C was observed in 52.4% of well-differentiated OSCC (Figure 3). Tables I and II display the association between the clinicopathological factors and VEGF-C expression of the 42 OSCCs. There were no statistically significant correlations regarding the main clinical and pathological parameters and VEGF-C expression by malignant cells, although when the patients were analyzed in sub-groups, it was noted that patients with strong VEGF-C expression presented a higher frequency of being had postoperative radiotherapy, local/regional recurrences, second primary tumor, lymph node involvement, angiolymphatic embolization, as well as perineural and muscular infiltration.
The clinical follow-up for these 42 patients ranged from 0.33 to 229.57 months (mean±SD=85.0±71.69 months). At the end of the follow-up period, 13 patients (31.0%) were alive with no evidence of disease, one (2.4%) was alive with cancer, 10 (23.8%) had died of other causes and 16 (38.1%) had died of primary tumor. Two patients (4.8%) were lost to follow-up.
Podoplanin and VEGF-C expression by malignant cells did not have any prognostic value for OSCC, as summarized in Table III. Positive lymph node metastasis (pN+) represented a significant risk of death for patients with OSCC, influencing the overall (p=0.001) and cancer-specific (p<0.001) survival rates (Table III).
Discussion
The metastatic dissemination of OSCC usually occurs through the lymphatic system (1, 2) and regional nodal metastasis is the major cause of locoregional recurrence, leading to poor prognosis and to high probability of eventual death from disease (3). Current treatment options for neck metastasis in advanced head and neck squamous cell carcinoma (SCCHN) include cervical lymphadenectomy and/or radiation therapy, and these can have considerable morbity. Any molecular therapies that could inhibit the invasive and metastatic potential of early SCCHN could notably improve overall patient morbity and mortality and may be able to offer significant improvements toward local disease control in advanced disease. Molecular therapies could also have a role in the treatment of bulky N+ neck disease, since agents that could slow or reverse any progression of lymphatic metastasis would have great clinical utility in conjunction with surgery and or chemotherapy and radiation (1).
For both groups OSCCpN0 and OSCCpN+, most tumors occurred in Caucasian men with positive history of tobacco and alcohol consumption, reinforcing the notion that this synergism is an important etiological risk factor for OSCC (25). Demographic and clinical data presented in this study (Table I) are similar to previous reports for OSCC (2, 14, 16, 19, 21, 28-30).
On the basis of the UICC criteria of oral cavity carcinomas, most of the OSCCpN0 group were at an initial clinical stage (I and II), while 52.9% of the OSCCpN+ group were at advanced clinical stage (III or IV). These results were expected since it is well-established that positive pathologic nodes (pN+) have the most decisive influence on prognosis in patients with OSCC (2, 25), as demonstrated in the present study.
All 17 OSCCpN+ patients underwent selective neck dissection. Occult lymph node metastases were detected in six (35.3%) of them. This percentage is lower than that of Byers et al. (31), but higher than those of Pimenta-Amaral et al. (2), Okamoto et al. (32) and Faustino et al. (30).
Bryne's grading system (26) was used, in which the morphological features are determined from the most invasive front of the tumor. The invasive tumor front consists of the most aggressive cells, which have the ability to invade surrounding tissue structures, including vessels, and thereby metastasize. The characteristics of the invasive tumor front are of major significance for the prognosis of oral cancer (33).
According to the histopathological malignancy grading, most (83.0%) of the 42 OSCCs were classified as more differentiated tumors, a finding consistent with other published studies (16, 18, 19, 30). No statistically significant differences were found between the OSCCpN0 and OSCCpN+ groups with regard to histopathological malignant grading.
OSCCpN+ patients presented a significantly higher frequency of lymphatic embolization, perineural and muscular infiltrations. These results demonstrated that OSCCpN+ patients had more highly aggressive neoplasms than the OSCCpN0 group and, consequently, poor clinical outcome. Others have reported that perineural and muscular infiltrations are strongly associated with poor prognosis for patients with OSCC (25).
In the current study, podoplanin and VEGF-C expression by malignant cells of the OSCC was evaluated according to the semi-quantitative system described by Soini et al. (27). According to Faustino et al., use of this objective methodology minimizes the subjectivity and facilitates comparison of the results with those of future studies (30), an essential step for establishment of the real influence of biomarker expression on the clinical and biological behavior of OSCC.
In our series, no statistically significant correlations were found regarding clinicopathological parameters and the VEGF-C expression by malignant cells of the OSCC (Tables I and II). However, it was noted that OSCCpN+ patients with strong VEGF-C expression had a higher frequency of postoperative radiotherapy, local/regional recurrences, second primary tumor, lymph node involvement and angiolymphatic embolization, as well as perineural and muscular infiltrations. Concerning recurrence, our results are similar to those found by Tanigaki et al. who reported that VEGF-C expression was associated with locoregional recurrence in tongue squamous cell carcinoma (19).
In recent years, evidence is emerging to correlate strong VEGF-C expression in tumor cells with an increased likelihood of lymph node metastasis (14, 16-20). In the present study aswell as in others (28-30), no statistically significant correlation was found between lymph node metastasis of OSCC and strong VEGF-C expression. These findings indicate that clinical significance and isolated immunobiomarker analysis can be difficult to interpret due to the diversity of clinical and biological tumor behavior.
Histologically, in our patient groups, podoplanin-positive cells were located at the basal layer of hyperplastic epithelial areas (Figure 2) and in the endothelial cells of lymphatic vessels. In OSCC, podoplanin was expressed at the periphery of the tumor cell nests at the invasive edge of the tumor. This immunoexpression pattern of podoplanin was described by others in oral leukoplakia (34), and squamous cell carcinomas of the oral cavity (21, 24, 28), lung (35), uterine cervix (36) and skin (37).
Experimental results have demonstrated that podoplanin mediates a pathway leading to collective cell migration and invasion in vivo and in vitro (22). During this so-called epithelial–mesenchymal transition, cells lose epithelial markers, such as E-cadherin, and gain the expression of mesenchymal markers, such as N-cadherin and vimentin, as well as podoplanin.
Regarding clinicopathological parameters, strong podoplanin expression was more frequently detected in males and in those with early clinical stage I and II tumors. These results confirm the observation that tumor cells of OSCC, in the early stage of development, express podoplanin (21, 34).
In our series, patients with strong podoplanin expression had a higher frequency of local/regional recurrences and perineural infiltration (Tables I and II). Despite there being no statistically significant difference, these data along with the one of significant glandular invasion reinforce the idea that podoplanin may serve as a predictor of lymph node metastasis in OSCC (21).
In contrast with literature regarding oral cancer (14, 16, 19, 21, 38), podoplanin and VEGF-C expression did not influence the overall and cancer-specific survival of patients with OSCC, as shown in Table III. The only significant risk of death for patients with OSCC was positive lymph node metastasis (pN+), influencing the overall (p=0.001) and cancer-specific (p<0.001) survival rates (Table III).
Conclusion
Although prognosis was not significantly influenced by podoplanin or VEGF-C expression, the tendency of strong podoplanin and VEGF-C expression to occur in patients with OSCC more frequently presenting local/regional recurrences and tumor invasion is of clinical importance. Such strong biomarker expression, associated with positive lymph node involvement, may be useful to identify OSCC patients at risk of a more unfavorable clinical outcome. Further studies are clearly necessary to define the true role of podoplanin and VEGF-C in oral cancer progression, and particularly their affect on patient outcome and prognosis.
Acknowledgements
This study was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq - grant #131847/2007-4) and by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP – grants 2005/04577-4 and 2007/04907-0).
- Received June 26, 2013.
- Revision received July 15, 2013.
- Accepted July 16, 2013.
- Copyright© 2013 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved
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