Background: Angiogenesis (formation of new blood vessels) is associated with tumor growth and metastasis in patients with solid tumors, including those of the head and neck. Nitric oxide (NO) production may contribute to these processes. We assessed the role of the NO pathway in angiogenesis and tumor progression in patients with head and neck cancer. Methods: Biochemical assays were used to measure NO synthase (NOS) activity and cyclic guanosine monophosphate (cGMP) levels in specimens of tumor and normal mucosa obtained from 27 patients. Microvessels in tumor specimens were identified by CD-31-specific immunohistochemical staining. Associations between microvessel densities, levels of NOS, and cGMP were examined by use of two-sided statistical tests. Tumor specimens and human squamous carcinoma A-431 cells were grown as explants on the corneas of rabbits, and the effect of the NOS inhibitor Nv-nitro-L-arginine-methyl ester (L-NAME) was tested. Results: Levels of total NOS, inducible NOS, and cGMP were higher in tumor specimens than in specimens of normal mucosa (all P<.0001). Tumor specimens from patients with lymph node metastases presented a higher total NOS activity (P = .005) and were markedly more vascularized than tumor specimens from patients with no lymph node involvement (P = .0002). Microvessel density at the tumor edge was an independent predictor of metastasis for this series of patients (odds ratio = 1.19; 95% confidence interval = 1.07–2.89; P = .04). A-431 cells and tumor specimens exhibiting high levels of NOS activity induced angiogenesis in the rabbit cornea assay; when NO production was blocked, tumor angiogenesis and growth were repressed. Conclusions: The NO pathway appears to play a key role in tumor angiogenesis and spread in patients with head and neck cancer.

We evaluated the role of COX-2 pathway in 35 head and neck cancers (HNCs) by analyzing COX-2 expression and prostaglandin E2 (PGE2) production in relation to tumor angiogenesis and lymph node metastasis. COX-2 activity was also correlated to vascular endothelial growth factor (VEGF) mRNA and protein expression. COX-2 mRNA and protein expression was higher in tumor samples than in normal mucosa. PGE2 levels were higher in the tumor front zone in comparison with tumor core and normal mucosa (P<.0001). Specimens from patients with lymph node metastasis exhibited higher COX-2 protein expression (P=.0074), PGE2 levels (P=.0011) and microvessel density (P<.0001) than specimens from patients without metastasis. A significant correlation between COX-2 and tumor vascularization (r(s)=0.450, P=.007) as well as between COX-2 and microvessel density with VEGF expression in tumor tissues was found (r(s)=0.450, P=.007; r(s)=0.620, P=.0001, respectively). The induction of COX-2 mRNA and PGE2 synthesis by EGF and Escherichia coli lipopolysaccharide (LPS) in A-431 and SCC-9 cell lines, resulted in an increase in VEGF mRNA and protein production. Indomethacin and celecoxib reversed the EGF- and LPS-dependent COX-2, VEGF, and PGE2 increases. This study suggests a central role of COX-2 pathway in HNC angiogenesis by modulating VEGF production and indicates that COX-2 inhibitors may be useful in HNC treatment.

BACKGROUND: Tumor metastasis to regional lymph nodes via the lymphatic system represents the first step of dissemination in head and neck squamous cell carcinoma (HNSCC) and serves as a major prognostic indicator for disease progression and as a guide for therapeutic strategies. In the current study, the authors investigated whether tumor lymphangiogenesis may be related to the risk of lymph node metastasis and to clinical outcome in patients with HNSCC.METHODS: Immunostaining for the lymphatic marker D2‐40 was used, and lymphangiogenesis was quantified within the tumor and in the peritumoral area in 52 HNSCC specimens using computer‐assisted morphometric analysis.RESULTS: Lymphatic vessels were found to be significantly more numerous and larger in the peritumoral area compared with within the tumor, and the number and relative area of intratumoral and peritumoral lymphatics was significantly higher in HNSCC cases with lymph node metastasis. Multivariate analysis demonstrated that high peritumoral lymphangiogenesis (above the median value) was associated with an increased risk of developing lymph node metastasis. No correlation was found between tumor lymphangiogenesis and the disease‐free or overall survival in the current series.CONCLUSIONS: The results indicate that peritumoral lymphangiogenesis may be an indicator of the risk of lymph node metastasis in patients with HNSCC. Cancer 2004. © 2004 American Cancer Society.

BACKGROUND: Numerous reports have documented a direct involvement of matrix metalloproteinase (MMP) overexpression in the development and progression of head and neck squamous cell carcinoma (HNSCC). In this study, the authors examined whether the expression of MMPs in HNSCC is correlated with other steps involved in tumor growth and metastasis, like angiogenesis, activation the nitric oxide (NO) pathway, and alteration of the p53 tumor suppressor gene.METHODS: MMP‐1, MMP‐2, and MMP‐9 expression levels were examined immunohistochemically in samples from 43 patients with HNSCC. Microvessel density (MVD) was determined by immunostaining of endothelial cells with anti‐CD31 monoclonal antibody. Inducible nitric oxide synthase (iNOS) activity and cyclic guanosine monophosphatate (cGMP) levels were assessed in fresh tumor samples, whereas exons 5–9 of the p53 gene were analyzed by reverse transcriptase‐polymerase chain reaction, single‐strand conformation polymorphism analysis and were sequenced.RESULTS: MMP‐1 overexpression (>10% of tumor cells) was identified in 32 tumors (74.5%), whereas elevated levels of MMP‐2 and MMP‐9 were detected in 17 tumors (39.5%) each. Tumors with MMP‐9 overexpression were characterized by significantly higher MVD (P = 0.05) and significantly higher iNOS activity and cGMP levels (P = 0.005 and P = 0.02, respectively). Moreover, p53 mutation was associated strongly with MMP‐9 overexpression (P = 0.004). Conversely, no correlation was found between MMP‐1 and MMP‐2 expression, angiogenesis, iNOS activity, cGMP levels, and p53 mutation in this series.CONCLUSIONS: This study documents the existence of a correlation between MMP‐9 expression, activity of the iNOS pathway, p53 status, and angiogenesis in patients with HNSCC. This raises the possibility that p53 mutation, which frequently is present in HNSCC, may result in increased angiogenesis and invasiveness related to increased nitric oxide and MMP production by tumor cells, ultimately contributing to tumor progression. Cancer 2002;95:1902–10. © 2002 American Cancer Society.DOI 10.1002/cncr.10916