Angiogenesis in breast cancer: A comparative study of the observer variability of methods for determining microvessel density

The purpose of this study was to evaluate the reliability of different meth ods for estimating neovascularization in breast cancer and to compare them in terms of observer variability. The microvessel endothelium was stained i mmunohistochemically by antibodies against GD34. The investigated methods i ncluded Chalkley counting, estimation of intratumoral microvessel density ( MVD) by one hot-spot, MVD by the mean value of three hot-spots, and the hig hest value of MVD in three hot-spots. In addition, we applied stereology in the quantification of angiogenesis in the whole tumor section by random an d systematically distributed sampling fields. Each of forty tumors was meas ured with all methods, twice by the same observer and once by another obser ver. Observer variation was analyzed by orthogonal regression, estimating t he slope and intercepts with 95% confidence intervals (CI), and by analysis of agreement using difference plots. Intraobservationally, the methods had variations of the same magnitude (coefficient of variation [CV]similar to 20%). Interobservationally, the stereologic estimate of vessel profiles, Q( A), from the whole tumor section and the Chalkley counting method had the l owest variation (CV similar to 21%), with a small contribution by observers alone (CV 8% to 9%). Interobservationally, the MVD methods had considerabl e variation with a large contribution by observers alone (CV similar to 30% ), which was lowest using the mean of three hot-spots. Correlation slope an d 95% CI of Chalkley were 1.18 (0.95, 1.48), CV 20%; slope of MVD (mean) wa s 1.14 (0.91, 1.43), CV 31%, and slope of MVD (max) was 1.15 (0.92, 1.45), CV 36%. The slope of MVD on one hot-spot was 1.33 (1.08, 1.63); CV 38%. Add itional measurements performed using a conference microscope, eliminating s ubjectivity in hot-spot selection and field sampling, optimized the reprodu cibility: slope was 1.02 (0.99, 1.04); CV of differences, 3.5%. On the othe r hand, reproducibility was not necessarily optimized by choosing the same hot-spot area, because variation in selecting a microscopic field could yie ld different counting numbers. The stereologic estimation of Q(A) based on the whole tumor section had a high reproducibility, with low variation due to observers. The Chalkley and MVD methods had moderate reproducibility, an d the Chalkley method had low variation due to observers alone. For all met hods, the biologic variation among patients was the major contributor to th e total variation. The Chalkley and MVD methods have been published to prov ide significant prognostic estimates in breast cancer, but the Chalkley met hod has less observer variation and may be superior from a methodologic poi nt of view.