Automated immunofluorescence analysis defines microvessel area as a prognostic parameter in clear cell renal cell cancer.

Microvessel density (MVD) has been reported to have prognostic relevance for clear cell renal cell carcinoma (ccRCC). However, this finding is controversial because of the difficulty of MVD evaluation in this complex vascularized tumor type. The present study evaluates the use of an automated quantitative analysis (AQUA) system for objective and reproducible determination of tumor vascularization in clear cell renal cell carcinoma (ccRCC). The AQUA system was applied to tissue microarrays with 284 primary ccRCC tumors. To determine angiogenesis in ccRCC, we created an epithelial/stromal mask consisting of CD10, epithelial membrane antigen, and vimentin to distinguish epithelial tumor cells from CD34-positive endothelial cells. Using immunofluorescence and computer-aided quantification of CD34 expression, we measured the relative microvessel area (MVA) and compared the MVA to the manually counted MVD. The MVA determined by AQUA in a test set with 209 ccRCCs ranged from 0\% to 30.3\% (mean +/- SD, 10.1\% +/- 6.3\%). The manually determined MVD ranged from 6 to 987 vessels/mm(2) (416.8 +/- 252.8 vessels/mm(2)). MVA and MVD were significantly correlated (P < .001). A larger MVA was associated with histologic grade (P < .001), tumor stage (P =.008), presence of metastasis (P = .005), presence of sarcomatoid areas (P < .001), and tumor-specific survival (P < .001). Using MVA as defined in the test set, all associations with clinical and pathologic parameters were confirmed in a second independent validation set. MVA determination by AQUA is an objective and reliable method to quantify tumor vascularization in ccRCC. A large MVA correlates with a high MVD and is associated with better patient prognosis.