10053 Background: Neoangiogenesis and lymphangiogenesis represent prognostic factors in human malignancies. Determination of microvessel density (MVD) by immunohistology is labor-intensive and subject to interobserver variability. We evaluated real time PCR to quantify MVD in primary tumor samples from patients with esophageal cancer. Methods: We performed real-time PCR analyzes of endothel-specific (VE-Cadherin, P1H12, VEGFR-2, tie-2), lymphendothel-specific (Prox, LYVE, VEGFR-3) antigens and of angiogenic growth factors (VEGF-A, VEGF-C, VEGF-D, Ang-1, Ang-2) in primary esophageal carcinoma tissue of 54 patients. These results were compared to MVD determined immunohistochemically by CD31 staining. Results: For validation, MNC samples spiked with HUVECs were analyzed by qPCR for VE-CAD and P1H12 yielding a linear correlation (r=0.99 and 0.96 respectively). Expression of endothelial markers was highly correlated in tumor samples, e.g. CD144 with CD146 τb=0.451, CD144 with VEGFR-2 τb =0.744 and CD144 with tie-2 τb=0.684 (p for all comparisons < 0.0001). QPCR results were compared to MVD determination by CD31 staining in a subgroup of 33 patients. The highest association between both methods was found for CD144 (τb=0.258, p=0.0379) and VEGFR-2 (τb=0.222, p=0.0745) indicating that immunohistology and qPCR yield comparable results. MVD was significantly linked to the expression of VEGF-A, -C,-D and Ang1 and Ang2 (p for all comparisons <0.0001). We analyzed expression of lymphendothelial cell antigens Prox, LYVE and VEGFR-3 for quantification of lymphatic vessels. A close association between the expression of different lymphendothelial factors was seen (LYVE with Prox τb=0.334, p=0.0021, LYVE with VEGFR-3 τb=0.450, p=0.0150). MVD and lymphvessel density was not linked. Lymph node metastases detected on surgical specimen were associated with MVD determined immunohistologically (p=0.003)or by qPCR (p=0.048) and to VEGF-C expression (p=0.04). Conclusions: QPCR analysis of CD144 and VEGFR2 represents a novel tool for quantification of MVD in tumor samples. Expression of VEGFR2 and VEGF-C is associated with lymph node metastasis in patients with esophageal carcinoma. No significant financial relationships to disclose.
The role of microvessel density, lymph node metastasis, and tumor size as prognostic factors of distant metastasis in colorectal cancer
