Lymphangiogenesis in metastatic lymph nodes of oral squamous cell carcinoma

Abstract

Background: Regional lymph node metastasis is a crucial negative prognostic factor in oral squamous cell carcinoma (OSCC) which significantly reduces survival rates in patients affected. Whilst angiogenic and lymphangiogenic factors have been extensively investigated in primary OSCC, their expression in metastatic lymph nodes remains uncertain.

Objective: To investigate the expression of markers associated with lymphangiogenesis (vascular endothelial growth factor (VEGF)-C, D, VEGF receptor 3 (VEGFR3) and prospero homeobox 1 protein (PROX1)) in cervical lymph nodes from OSCC patients with and without metastatic deposits. Method: Formalin-fixed paraffin -embedded (FFPE) blocks were accessioned from the Oral Cancer Research Coordinating Centre (OCRCC), University of Malaya, Malaysia. Samples were divided into two groups; Group A comprised cervical lymph nodes with histologically confirmed metastatic deposits from primary OSCC (n=17) and Group B, cervical lymph nodes from patients with primary OSCC without metastatic deposits, (n=17). Immunohistochemistry (IHC) was undertaken with antibodies against VEGF- C, VEGF-D, VEGFR3 and PROX1. Quantitative analysis using ImageJ was used to delineate the extent of positivity (proportion and intensity) and lymphatic vessel density (LVD). Three samples from each group were subsequently selected for gene expression analysis of the lymphangiogenic markers (VEGFC, VEGFD, VEGFR3 and PROX) using qPCR. Result: IHC showed significantly greater VEGFC expression in Group A compared with Group B (p=0.0002). Significant positive correlation was found between VEGFC and TNM stage (p=0.004). No statistically significant differences were observed in the protein and gene expression level of the other tested markers. Conclusion: This is the first study demonstrating significant overexpression of VEGFC in positive lymph nodes and suggests that VEGFC is an important growth factor involved in OSCC lymph node metastasis.