Abstract 4175: Robust immunohistochemical assay to characterize human cancer tissues for prevalence of vascular endothelial growth factor receptor 3 (VEGFR3)

VEGFR3 plays a key role in the regulation of lymphangiogenesis in adults, and is also important for tumor angiogenesis and metastasis. In order to characterize human cancer tissues for imunohistochemical (IHC) localization and prevalence of VEGFR3 protein, we developed a robust IHC assay in our lab using a mouse monoclonal primary antibody (Millipore). To optimize the various assay parameters, we utilized high-quality VEGFR3 positive (and internal negative) control tissues (Kaposi's sarcoma, angiosarcoma, lymph node). Reagent negative controls were satisfactory. Specificity of the primary anti-VEGFR3-antibody was supported by a discrete band at the expected molecular weight on western blots using a VEGFR3 transfected cell lysate and negative control cell lines. We also demonstrated lack of specific vascular VEGFR3 staining in pre-absorption experiments with VEGFR3 (but not VEGFR1 or 2) recombinant protein, supporting selectivity of the primary antibody for VEGFR3. Using the fully optimized assay, we stained a human multi-tumor screening tissue microarray (TMA) to demonstrate full range of specific vascular VEGFR3 staining in glioblastoma and carcinomas of the colon, breast, ovary, pancreas, lung, larynx, kidney, cervix, bladder, extrahepatic cholangiocarcinoma and malignant melanoma. Specific, unequivocal VEGFR3 immunoreactivity was interpreted qualitatively (VEGFR3 positive/negative) in tumor blood and lymphatic vessels. No tumor cell staining was seen. Some colon cancer tissues in multi-tumor TMA were VEGFR3+, while others were VEGFR3-. The observed variation in VEGFR3 expression and vascular distribution on screening TMA were further evaluated in two independent cohorts of well-characterized human colorectal cancer tissues (organ-specific TMAs) by a Board-certified, subspecialty GI pathologist (AN), with overall VEGFR3 prevalence rates of 62% (36 of 58 cases) and 56% (55 of 98 cases). Using CD34 and D2-40 IHC assays from a CLIA-certified lab, we confirmed IHC localization of VEGFR3 protein both in the stromal blood vessels and lymphatics within the invasive colorectal cancer stroma. In conclusion, following well-established IHC assay development and standardization protocols and efficient workflow paradigm, we have used a technically robust IHC assay to characterize routinely processed archival human cancer tissues and have demonstrated specific VEGFR3 expression patterns, tissue localization and prevalence in human colorectal cancer tissues. Based on i