METHODS IN LABORATORY INVESTIGATION - HETEROGENEITY AND REPRODUCIBILITY OF MICROVESSEL COUNTS IN BREAST-CANCER

BACKGROUND: Microvessel counting has proven to be of prognostic value in breast cancer, as shown in different retrospective studies. However , methodology has not been studied widely, and this must be done befor e the method can become clinically applicable. The aim of this study w as to determine the degree of heterogeneity and reproducibility of mic rovessel counts (MC) in breast cancer. EXPERIMENTAL DESIGN: In 10 case s of breast cancer, the available blocks (2-4 blocks) containing invas ive tumor parts were selected, and four sections (4 mu m) were cut wit h interdistances of 100 mu m. In each section, two or three invasive a reas (0.5 x 0.5 cm) were demarcated. Microvessels, visualized by immun ohistochemistry (CD31 Ab), were counted by one observer in 10 systemat ically selected fields of vision (400 x magnification). Furthermore, m icrovessels were counted in four fields with the highest microvessel d ensity (''hot spots''). Coefficients of variation (CV) were calculated for the different sampling levels. RESULTS: Repeated MC yielded high intra- and interobserver reproducibility (correlation coefficients > 0 .92). For the systematic counting method, CV between MC from different areas within one section was on average 17.1% (0.7-52.1). When compar ing MC from corresponding areas in different sections from the same bl ock, CV was on average 14.7% (0.5-41.9), and for MC from different blo cks of the same tumor, CV was on average 25.8% (9.9-44.6). Nested ANOV A showed an approximately equal contribution to the total variance of the different sampling levels, except for the variation between sectio ns (not significant). For the hot spot MC, CV for different sections f rom the same block was on average 11.1% (0.7-29.5) and for different b locks from the same tumor, 24.2% (5.7-54.9). Nested ANOVA showed that variation between different blocks from the same tumor contributed mos t to the total variance. CONCLUSIONS: There is a noteworthy heterogene ity in MC between different areas from the same section, between corre sponding areas in different sections from the same block, and between different blocks from the same tumor. Consequently, one must carefully scan all the available tumor material in each case for the best hot s pot. The hot spot approach is efficient and reproducible, but only a c omparative prognostic evaluation can show whether it is clinically mor e useful than systematic counts.