Journal of Pathology J Pathol 2003; 201: 544–554. Published online 26 September 2003 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/path.1467 Original Paper Expression of vascular endothelial growth factor (VEGF)-C and VEGF-D, and their receptor VEGFR-3, during different stages of cervical carcinogenesis Philippe O Van Trappen, 1 * Dawn Steele, 2 David G Lowe, 2 Suhail Baithun, 2 Nigel Beasley, 3 Wilko Thiele, 4 Herbert Weich, 5 Jaya Krishnan, 4 John H Shepherd, 1 Michael S Pepper, 6 David G Jackson, 3 Jonathan P Sleeman 4 and Ian J Jacobs 1 1 Department of Gynaecological Oncology, Cancer Research UK Translational Oncology Laboratory, St Bartholomew’s and the Royal London School of Medicine and Dentistry, Queen Mary University of London, Charterhouse Square, London EC1M 6BQ, UK 2 Academic Department of Histopathology, St Bartholomew’s and the Royal London School of Medicine and Dentistry, Queen Mary University of London, London EC1A 7BE, UK 3 MRC Human Immunology Unit, Weatherall Institute for Molecular Medicine, John Radcliffe Hospital, Headington, Oxford, UK 4 Forschungszentrum Karlsruhe, Institut f¨ ur Toxikologie und Genetik, Postfach 3640, D-76021 Karlsruhe, Germany 5 German Research Centre for Biotechnology (GBF), Mascheroder Weg 1B, D-38124 Braunschweig, Germany 6 Department of Morphology, University Medical Center, Geneva, Switzerland *Correspondence to: Dr Philippe O Van Trappen, Cancer Research UK Translational Oncology Laboratory, John Vane Science Centre, St Bartholomew’s and the Royal London School of Medicine and Dentistry, Queen Mary University of London, Charterhouse Square, London EC1M 6BQ, UK. E-mail: p.o.vantrappen@qmul.ac.uk Received: 19 December 2002 Revised: 29 May 2003 Accepted: 17 July 2003 Abstract 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. Copyright  2003 John Wiley & Sons, Ltd. Keywords: cervical intraepithelial neoplasia; cervical cancer; lymphangiogenesis Introduction Cervical carcinogenesis has well-defined stages of disease progression: pre-invasive lesions including three grades of cervical intraepithelial neoplasia (CIN, grades 1–3) occur prior to developing invasive cer- vical cancer. Certain high-risk human papillomavirus (HPV) types — such as HPV 16 and 18 — are the main aetiological factors for cervical cancer and are required for progression towards malignancy [1,2]. However, only a small percentage of infected women will progress through the different grades of CIN to develop cervical cancer. The biological properties of CIN that is prone to progress towards invasive disease are not completely defined. Two phases can be recognized with regard to neo- vascularization (angiogenesis) during tumour progres- sion: a pre-vascular and a vascular phase. The tran- sition from one phase to the other is referred to as the ‘angiogenic switch’ [3]. The key molecule Copyright  2003 John Wiley & Sons, Ltd.