Abstract
The purpose of this study was to evaluate the immunoexpression of vascular endothelial growth factor C (VEGF-C) and VEGF receptor 3 (VEGFR-3) and their correlation with intratumoural lymphatic density (ILD) and peritumoural lymphatic density (PLD) in metastatic and non-metastatic lower lip squamous cell carcinoma (LLSCC). Twenty-five LLSCC with regional nodal metastasis and 25 LLSCC without metastasis were selected. The percentages of VEGF-C and VEGFR-3 staining in each tumour core and at the deep invasive front were assessed. PLD and ILD were determined using anti-podoplanin antibody. Immunohistochemical findings were correlated with nodal metastasis, clinical staging, local recurrence, clinical outcome, and histological grade. Cytoplasmic immunoexpression of VEGFR-3 in the tumour core was associated with metastasis ( P = 0.009), patient death ( P = 0.008), and histological grade ( P < 0.005). PLD, ILD, and VEGF-C expression showed no significant associations with clinicopathological parameters ( P > 0.05). PLD and ILD were not significantly correlated with the immunoexpression of VEGF-C or VEGFR-3 ( P > 0.05). There was a significant positive correlation between PLD and ILD ( P = 0.004), and between cytoplasmic immunoreactivity of VEGF-C and VEGFR-3 ( P = 0.011). These results suggest an important role for VEGFR-3 in the progression of LLSCC, and highlight the possible influence of its expression on the prognosis of these tumours. ILD and PLD may not be associated with lymph node metastasis in LLSCC.
Lower lip squamous cell carcinoma (LLSCC) is one of the most common tumours of the oral and maxillofacial region, accounting for 25–30% of all cases of oral squamous cell carcinoma. When LLSCC is diagnosed at an early stage, the prognosis is good, with over 90% of patients showing 5-year survival. Nevertheless, 5–20% of patients exhibit cervical lymph node metastasis at diagnosis. In these cases, the 5-year survival can be reduced to only 30%, setting a worse prognosis. The formation of new lymphatic vessels is one of the mechanisms involved in the development of lymph node metastasis in malignant epithelial tumours. In this context, studies have suggested an important role for vascular endothelial growth factor C (VEGF-C) and VEGF receptor 3 (VEGFR-3) proteins.
VEGF-C, a member of the VEGF family proteins, is a crucial molecule in the process of formation of new lymphatic vessels. Through interaction with VEGFR-3, a membrane receptor present on lymphatic endothelial cells, VEGF-C is able to stimulate the proliferation, migration, and survival of lymphatic endothelial cells, thereby promoting lymphangiogenesis. Recent studies have demonstrated that VEGF-C and VEGFR-3 are overexpressed in a variety of human cancers, including head and neck carcinomas.
Studies of oral squamous cell carcinoma have found a positive correlation between VEGF-C expression and lymphatic microvessel density. Moreover, higher levels of VEGF-C expression and lymphatic density in oral squamous cell carcinoma have been associated with the presence of regional lymph node metastasis and advanced clinical stage. In addition to these findings, an association between VEGFR-3 expression and cervical metastasis in oral squamous cell carcinoma has also been found.
Studies investigating the expression of VEGF-C and lymphatic vessel density in lip squamous cell carcinoma are sparse, and their roles in the progression of these tumours are not clearly established. In this context, Oliveira-Neto et al. and Watanabe et al. found no statistically significant relationship between VEGF-C expression and lymphatic vessel density in lip squamous cell carcinoma. Nevertheless, Watanabe et al. suggested that VEGF-C-positive squamous cell carcinoma of the oral region, including the lip, is significantly more likely to result in cervical lymph node metastasis. Thus, the aim of this study was to evaluate VEGF-C and VEGFR-3 immunoexpression and the intra- and peritumoural lymphatic density, in order to determine possible relationships with prognostic parameters in metastatic and non-metastatic LLSCC.
Materials and methods
Fifty cases of LLSCC were selected for this study. These specimens were divided into two groups: a non-metastatic group consisting of 25 cases of LLSCC without regional nodal metastasis, and a metastatic group consisting of 25 cases of LLSCC with regional nodal metastasis. Information regarding clinical staging (TNM), local recurrence, and clinical outcome (survival data and death) were collected from the medical records. Only cases with a minimum postoperative follow-up of 24 months were included in the study. In cases where records were incomplete, the patients were contacted for additional information. Only cases of LLSCC derived from surgical resections, with paraffin blocks containing sufficient material for analysis of the invasive front of the tumour, were included in the sample. Metastasis in the cervical lymph nodes was confirmed histopathologically in all cases. The tumours of patients who had undergone radiotherapy, chemotherapy, or any other treatment before surgery were excluded. This study was approved by the necessary research ethics committee.
Sections 5 μm thick were obtained from paraffin-embedded tissue blocks, deparaffinized, and stained with haematoxylin and eosin for histological examination. The histological grading of the malignancy was established for each case of LLSCC in a blinded fashion by two observers. The tumours were graded as well-differentiated, moderately differentiated, or poorly differentiated according to the World Health Organization (WHO) criteria . Histopathological grading of the malignancy at the invasive front of the tumour was performed according to the system proposed by Bryne et al. Cases with a total score of ≤8 were classified as a low grade malignancy, whereas those receiving a total score of >8 were classified as a high grade malignancy.
Immunohistochemical methods
For the immunohistochemical study, sections 3 μm thick were obtained from paraffin-embedded tissue blocks. The tissue sections were deparaffinized and immersed in 3% hydrogen peroxide to block endogenous peroxidase activity. The tissue sections were then washed in phosphate-buffered saline (PBS). The antigen retrieval, antibody dilution, and clone type for VEGF-C, VEGFR-3, and podoplanin are shown in Table 1 . After treatment with normal serum, tissue sections were incubated in a moist chamber with primary antibodies. The sections were then washed twice in PBS and treated with a polymer-based complex (ADVANCE HRP; Dako, Carpinteria, CA, USA) at room temperature to bind the primary antibodies. Peroxidase activity was visualized by immersing tissue sections in diaminobenzidine (Liquid DAB+ Substrate; Dako), resulting in a brown reaction product. Finally, the sections were counterstained with Mayer haematoxylin and coverslipped. Sections of lymphangioma were used as positive control for VEGF-C, VEGFR-3, and podoplanin. As negative control, samples were treated as described above, except that the primary antibody was replaced with a solution of bovine serum albumin in PBS.
| Specificity | Clone | Company | Dilution | Antigen retrieval | Incubation |
|---|---|---|---|---|---|
| Podoplanin | D2-40 | Dako | 1:400 | Tris EDTA, pH 9, Pascal, 121 °C, 3 min | Overnight, 4 °C |
| VEGF-C | H-190 | Santa Cruz | 1:400 | Tris EDTA, pH 9, Pascal, 121 °C, 3 min | Overnight, 4 °C |
| VEGFR-3 | C-20 | Santa Cruz | 1:400 | Citrate, pH 6, Pascal, 121 °C, 3 min | 60 min |
Immunostaining assessment and statistical analysis
The immunohistochemical analysis was performed in a blinded fashion by two observers. An Olympus BX41 light microscope (Olympus Co., Tokyo, Japan) was used. The expression of VEGF-C and VEGFR-3 was analyzed only in neoplastic epithelial cells at the deep invasive front and in the tumour core. The expression of podoplanin was analyzed only on lymphatic vessels.
The assessment of lymphatic density was performed quantitatively, using an adaptation of the method described by Kyzas et al. At 40× magnification, the five fields with the highest number of immunostained vessels for podoplanin were identified in each sample, and the vessels were counted at 200× magnification. Lymphatic density was defined as the number of lymphatic vessels per optical field (corresponding to an examination area of 0.7386 mm 2 ). Counts were performed both within the tumour area (intratumoural lymphatic density (ILD)) and within an area of 500 μm from the tumour border (peritumoural lymphatic density (PLD)).
The analysis of the immunoexpression of VEGF-C and VEGFR-3 was performed using the method of Warburton et al., with some modifications. The five fields in the tumour core (defined as the most inner region of the tumour, accounting for up to 50% of the tumour area) and at the deep invasive front (defined as the most invasive area at the tumour/host interface) that contained the largest numbers of immunostained cells were identified by light microscopy. Digital images of these five microscopic fields (400× magnification) were acquired with an Olympus EVOLT E-330 digital camera (Olympus Co.) and transferred to ImageJ software (National Institutes of Health, Bethesda, MD, USA). The numbers of positive and negative cells were determined in each field and the percentage of cells exhibiting cytoplasmic staining for VEGF-C and cytoplasmic staining and membrane staining for VEGFR-3 was calculated for each case.
The results were submitted to statistical analysis using SPSS version 17.0 software (SPSS Inc., Chicago, IL, USA). For clinical staging, stages I and II were combined into one group and stages III and IV into another group. The ILD, PLD, and percentage of stained cells for VEGF-C and VEGFR-3 at the deep invasive front and in the tumour core were compared by non-parametric Mann–Whitney test and Kruskal–Wallis test. Spearman’s correlation test was performed to verify possible correlations between lymphatic density and the percentage of VEGF-C- and VEGFR-3-immunopositive cells. For all tests, the significance level was set at 5% ( P < 0.05).
Results
Clinical and morphological analyses
Regarding clinical staging, four (8%) cases were classified as stage I, 18 (36%) as stage II, 15 (30%) as stage III, and 13 (26%) as stage IV. Local recurrence was not evident for the majority of both metastatic and non-metastatic tumours (76%). Analysis of the clinical outcome revealed a higher frequency of tumour remission (92%) in non-metastatic LLSCC when compared to metastatic tumours (56%).
Regarding the morphological analysis, there was a predominance of high-grade malignancy (68%) and moderately differentiated tumours (64%) in metastatic LLSCC. In contrast, most non-metastatic LLSCC were classified as low-grade malignancies (84%) and well-differentiated tumours (60%) ( Table 2 ).
| Characteristics | Non-metastatic LLSCC ( n = 25), n (%) | Metastatic LLSCC ( n = 25), n (%) |
|---|---|---|
| Local recurrence | ||
| Yes | 6 (24) | 6 (24) |
| No | 19 (76) | 19 (76) |
| Clinical outcome | ||
| Remission | 23 (92) | 14 (56) |
| Death | 2 (8) | 11 (44) |
| Histological grading (invasive front) | ||
| Low grade | 21 (84) | 8 (32) |
| High grade | 4 (16) | 17 (68) |
| Histological grading (WHO) | ||
| Well-differentiated | 15 (60) | 3 (12) |
| Moderately differentiated | 8 (32) | 16 (64) |
| Poorly differentiated | 2 (8) | 6 (24) |
Immunohistochemical analysis
Lymphatic density
Intra- and peritumoural lymphatic vessels were strongly immunopositive for podoplanin. Neoplastic cells also presented cytoplasmic/membrane positivity for podoplanin, with intense immunoexpression at the periphery and weak immunostaining at the centre of the tumour islands ( Fig. 1 A and B ).
Analysis of ILD and PLD showed a higher median number of lymphatic vessels in non-metastatic tumours than in metastatic tumours, but the difference was not statistically significant ( Table 3 ; P > 0.05). In addition, no statistically significant differences were observed for ILD and PLD regarding clinical staging, local recurrence, clinical outcome, and histological grading of the malignancy ( Tables 4 and 5 ; P > 0.05). Spearman’s correlation test showed a weak positive correlation between ILD and PLD ( r = 0.405, P = 0.004).
| Characteristics | Non-metastatic LLSCC median (range) | Metastatic LLSCC median (range) | P -value |
|---|---|---|---|
| Lymphatic density | |||
| ILD | 8.39 (0.27–27.35) | 6.50 (0.00–28.70) | 0.218 |
| PLD | 7.31 (2.70–30.05) | 5.41 (1.98–19.76) | 0.077 |
| VEGFR-3 (membrane) | |||
| Tumour core | 3.00 (0.00–83.61) | 3.20 (0.00–76.45) | 0.703 |
| Invasive front | 7.29 (0.00–71.00) | 3.92 (1.04–48.36) | 0.764 |
| VEGFR-3 (cytoplasm) | |||
| Tumour core | 92.35 (2.20–99.59) | 98.00 (55.17–100.00) | 0.009 |
| Invasive front | 90.61 (69.95–99.70) | 91.58 (65.65–100.00) | 0.273 |
| VEGF-C | |||
| Tumour core | 96.34 (43.66–100.00) | 96.64 (13.39–100.00) | 0.553 |
| Invasive front | 90.08 (24.34–100.00) | 92.68 (0.78–100.00) | 0.483 |
| Characteristics | ILD | VEGF-C | VEGFR-3 (membrane) | VEGFR-3 (cytoplasm) | ||||
|---|---|---|---|---|---|---|---|---|
| Median | P -value | Median | P -value | Median | P -value | Median | P -value | |
| Clinical staging | ||||||||
| Stages I/II | 8.12 | 0.584 | 96.34 | 0.830 | 3.25 | 0.708 | 94.44 | 0.278 |
| Stages III/IV | 6.77 | 96.45 | 3.20 | 96.48 | ||||
| Local recurrence | ||||||||
| Yes | 6.49 | 0.649 | 96.64 | 0.909 | 5.69 | 0.180 | 94.47 | 0.593 |
| No | 7.44 | 96.34 | 0.85 | 95.50 | ||||
| Clinical outcome | ||||||||
| Remission | 6.50 | 0.147 | 95.61 | 0.113 | 3.20 | 0.876 | 93.96 | 0.008 |
| Death | 9.21 | 98.84 | 2.00 | 98.00 | ||||
| Histological grading | ||||||||
| Well-differentiated | 6.77 | 0.794 | 96.72 | 0.622 | 0.50 | 0.533 | 88.75 | 0.003 |
| Moderately differentiated | 8.12 | 96.45 | 3.85 | 98.05 | ||||
| Poorly differentiated | 8.12 | 93.84 | 4.43 | 98.00 | ||||
| Characteristics | PLD | VEGF-C | VEGFR-3 (membrane) | VEGFR-3 (cytoplasm) | ||||
|---|---|---|---|---|---|---|---|---|
| Median | P -value | Median | P -value | Median | P -value | Median | P -value | |
| Clinical staging | ||||||||
| Stages I/II | 7.17 | 0.190 | 91.31 | 0.702 | 6.31 | 1.000 | 90.86 | 0.681 |
| Stages III/IV | 6.35 | 91.38 | 4.35 | 91.50 | ||||
| Local recurrence | ||||||||
| Yes | 6.62 | 0.699 | 86.15 | 0.294 | 2.89 | 0.102 | 88.45 | 0.266 |
| No | 6.63 | 92.61 | 6.84 | 91.59 | ||||
| Clinical outcome | ||||||||
| Remission | 6.76 | 0.740 | 90.08 | 0.292 | 6.17 | 0.650 | 90.61 | 0.394 |
| Death | 6.49 | 93.52 | 8.31 | 92.58 | ||||
| Histological grading | ||||||||
| Low grade | 7.31 | 0.292 | 92.54 | 0.867 | 7.29 | 0.814 | 89.34 | 0.191 |
| High grade | 5.41 | 90.08 | 3.92 | 92.80 | ||||
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