EXAMINING THE TECHNIQUE OF ANGIOGENESIS ASSESSMENT IN INVASIVE BREAST-CANCER

The intensity of angiogenesis as measured by the density of microvesse ls has been reported to be associated with a poor prognosis in invasiv e breast cancer in some, but not all, studies. The reasons for these d iscrepancies may be variations in the methodologies used. The monoclon al antibody used to identify the microvessels, the number of high-dens ity areas or 'hotspots' counted and the type of value taken for statis tical analysis (highest count or mean count) have varied between the d ifferent studies. We have assessed which of the three commonly used mo noclonal antibodies provides the best visualization of microvessels in invasive breast cancer and have used methods that give reproducible d ata for the optimum number of 'hotspots' to count for each reagent. Th us, microvessels in formalin-fixed paraffin-embedded specimens from 17 4 primary breast cancers were immunohistochemically stained with monoc lonal antibodies to FVIIIRAg, CD31 and CD34 and ten fields counted at 200 x magnification for each antibody. The highest count and the mean value of the highest of three, five and ten counts were used to examin e the relationship between the density of microvessels and overall sur vival of patients with a median follow-up time of 7.1 years. Antibodie s to CD31 and CD34 identified more vessels than antibodies to FVIIIRAg (median highest count per mm(2): CD31 = 100, CD34 = 100, FVIIIRAg = 8 1). The monoclonal antibody to CD31, however, was the least reliable a ntibody, immunohistochemically staining only 87% of sections compared with 98% for the monoclonal to CD34 and 99% for the monoclonal to FVII IRAg. There was a high degree of correlation between the number of ves sels stained by the different antibodies, though there were some consi derable differences in actual counts for serial sections of the same s pecimen stained by the different antibodies. Patients could be divided into two groups corresponding to those with high microvessel densitie s and those with low microvessel densities. Using Kaplan-Meier surviva l curves, there was a close association for all three antibodies betwe en vessel density and survival whichever method of recording the highe st vessel densities was used. Using log-rank tests and Cox's regressio n analysis, anti-CD34 gave the most significant results of the three a ntibodies, whereas a simple cut-off at the 75th percentile for the hig h and low groups produced the best association with patient survival. For anti-CD34 the highest microvessel density (P = 0.0014) and the mea n value of the highest three microvessel densities (P = 0.004) showed a good correlation with patient death, whereas for anti-CD31 (P = 0.00 8) and anti-FVIIIRAg (P = 0.007) the highest count gave the best corre lation using Cox's regression analysis.