ORIGINAL ARTICLE Head and neck squamous cell carcinoma lymphatic spread and survival: Relevance of vascular endothelial growth factor family for tumor evaluation Elen Alves de Sousa, MSc, 1 Silvia Vanessa Lourenc¸o, PhD, 2 Fl  avia Paiva Prudente de Moraes, 1 Jos  e Guilherme Vartanian, MD, PhD, 3 Jo ~ ao Gonc¸alves–Filho, MD, PhD, 3 Luiz Paulo Kowalski, MD, PhD, 3 Fernando Augusto Soares, MD, PhD, 1,2 Cl  audia Malheiros Coutinho–Camillo, PhD 1 * 1 Department of Pathology, AC Camargo Cancer Center, S~ ao Paulo, Brazil, 2 Department of General Pathology, Dental School, University of S~ ao Paulo, Brazil, 3 Department of Head and Neck Surgery and Otorhinolaryngology, AC Camargo Cancer Center, S~ ao Paulo, Brazil. Accepted 10 May 2014 Published online 00 Month 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/hed.23765 ABSTRACT: Background. Head and neck squamous cell carcinoma (HNSCC) is primarily a locoregional disease in which the cervical lymph nodes are the chief site of metastasis. The purpose of this study was to examine the relationship between lymphangiogenesis and clinicopatho- logical aspects of HNSCC and its metastasis. Methods. Fifty-two patients with HNSCC and metastatic lymph nodes from 21 of these subjects were analyzed by immunohistochemistry. Results. The HNSCC samples were predominantly negative for vascular endothelial growth factor (VEGF)-C, VEGF-D, and vascular endothelial growth factor receptor (VEGFR)3. There was an association between the density of lymph vessels (measured by D2-40 staining) in the lymph nodes and advanced-stage tumors. There was no link between the expression of these proteins and survival rates. Conclusion. Although lymphatic spread is a significant event in the pro- gression of HNSCC, the expression of VEGF-C, VEGF-D, and VEGFR3 does not correlate with clinicopathological characteristics, suggesting that other signaling pathways mediate lymphangiogenesis in HNSCC. V C 2014 Wiley Periodicals, Inc. Head Neck 00: 000–000, 2014 KEY WORDS: lymphangiogenesis, head and neck squamous cell car- cinoma, immunohistochemistry, vascular endothelial growth factor (VEGF) family, D2–40 INTRODUCTION Head and neck squamous cell carcinoma (HNSCC) is the fifth most common tumor worldwide. 1–3 Many clinical and pathological factors have been associated with local recur- rence of HNSCC, such as stage, lymph node metastasis, and perineural invasion. 4,5 Metastatic spread of HNSCC usually occurs via the lymphatic system. The presence of lymph node metastasis is the most significant prognostic factor and significantly decreases survival rates. 6–10 Lymphangiogenesis is the growth of new lymphatic vessels through a similar multistep process as in angio- genesis, comprising endothelial cell migration, prolifera- tion, and rearrangement that is associated with the degradation, reconstruction, and production of the extrac- ellular matrix. However, the mechanism by which the lymph proliferation is initiated is unknown. 10–12 Several studies have demonstrated that vascular endothelial growth factors (VEGFs) in tumor cells stimulate endothe- lial cells to grow and generate new lymphatic vessels. 10,11 On activation of vascular endothelial growth factor recep- tor (VEGFR)3 through its binding to VEGF-C and VEGF-D, a signaling cascade is activated, effecting the migration and growth of lymphatic endothelial cells and protecting them from apoptosis. 10–17 Lymphangiogenesis has been examined by immunohisto- chemistry in various cancers, including breast, pancreatic, renal cell, liver, prostate, and head and neck, but the findings are controversial. 10,18 Some studies have examined the func- tion of VEGF family members in tumor progression and lymph node metastasis in HNSCC, but the results remain debated. 13,19–21 One of the most reliable markers that is used to determine the presence of lymphatic vessels is podoplanin. Podoplanin is a 38-kDa membrane glycoprotein that is recognized by the monoclonal antibody D2-40, which has been used in research and clinical practice to distinguish lymphatic endo- thelium from endothelial cells of blood vessels (the latter does not express podoplanin). 14,18,22,23 Patients with a high lymphatic vessel density (LVD) are at increased risk for lymph node metastasis, supporting the hypothesis that LVD mediates lymphatic spread in oral carcinoma. 17,21,22,24 Based on findings that metastatic spread of head and neck tumors usually occurs via the lymphatic system and because lymphangiogenesis studies in various cancers have generated contradictory results, we measured the expression of lymphangiogenesis markers in HNSCC by immunohistochemistry. The purpose of this study was to determine the impact of potential lymphangiogenesis on the diagnostic and prognostic parameters of these tumors. MATERIALS AND METHODS Tissue samples Paraffin-embedded tissue samples from 52 HNSCC cases and metastatic lymph nodes from 21 of these *Corresponding author: C. M. Coutinho–Camillo, AC Camargo Cancer Center, Centro Internacional de Pesquisa, Rua Tagu a, 440, S~ ao Paulo, Brazil, 01508- 010. E-mail: claumcc@terra.com.br HEAD & NECK—DOI 10.1002/HED MONTH 2014 1