Expression of vascular endothelial growth factor (VEGF)-C and VEGF-D, and their receptor VEGFR-3, during different stages of cervical carcinogenesis

Cervical carcinogenesis has well-defined stages of disease progression including three grades of pre-invasive lesions--cervical intraepithelial neoplasia grades 1-3 (CIN 1-3)--and invasive cervical cancer. However, the biological properties of CIN lesions prone to develop invasive disease are not well defined. Recent observations suggest that early invasive disease spreads to regional lymph nodes in several tumour types and that growth factors (VEGF-C and VEGF-D) involved in new lymphatic vessel formation may play a crucial role in this process. The present study has assessed the expression of VEGF-C and VEGF-D, and their receptor VEGFR-3, in 152 cervical lesions (33 CIN 1, 33 CIN 2, 37 CIN 3, and 49 squamous cell carcinomas) to determine whether expression of lymphangiogenic factors occurs prior to invasion. The presence of lymphatic vessels was determined using LYVE-1 and podoplanin staining, as well as double immunostaining for LYVE-1/CD34 and podoplanin/CD34. In situ hybridization was performed to determine VEGFR-3 mRNA expression. A significant positive correlation was found between VEGF-C, VEGF-D, and VEGFR-3 expression through the different stages of cervical carcinogenesis. Significant differences in protein expression for VEGF-C, VEGF-D, and VEGFR-3 were found between CIN 1-2 and CIN 3 (p<0.001 for all), but not between CIN 3 and cervical cancer. More than 50% of the CIN 3 lesions showed moderate to strong staining for VEGF-C and VEGF-D, whereas most of the early pre-cancerous lesions (CIN 1 and 2) were negative. In cervical cancer, similar observations to those in CIN 3 were found. VEGFR-3 mRNA expression was found in the cytoplasm of epithelial neoplastic cells and VEGFR3 protein expression was found in more than 50% of CIN 3 lesions and cervical cancers, compared with 15% in CIN 1 and 2. These findings suggest an autocrine growth stimulation pattern via VEGFR-3. Adjacent CIN 3 was present in nine cervical cancers and displayed strong expression for VEGF-C, VEGF-D, and VEGFR-3. These results suggest that in cervical carcinogenesis a switch to the lymphangiogenic phenotype may occur at the stage of CIN 3.