Proliferation assessment in breast cancer: a double-staining technique for AgNOR quantification in MIB-1 positive cells especially adapted for image cytometry.

There are two ways of measuring the cell proliferation. The first one consists of quantifying the number of cycling cells with the help of antibodies directed against cells either in G1, S, G2 or M phase. The second way is to assess the cell cycle duration by the quantification of AgNOR proteins. Measuring both the features on the same slide represents an attractive way to tackle the proliferating activity of a cell culture or a tumor. Here, we propose a MIB-1 and AgNOR double staining method especially adapted to image cytometry measurement using MIB-1 antibody coupled to FITC in order to avoid the thresholding problems encountered with such a multilabeling technique. We have applied this new method on a series of 39 breast cancer cases, with at least 4 years follow-up, in order to determine the prognosis significance of this measurement. MIB-1 alone is not linked to prognosis, while the global mean AgNOR area is significantly linked to prognosis in terms of development of visceral metastasis or death. However, the global mean AgNOR area is insufficient to determine the time limit of appearance of metastasis or relapse. Our results clearly demonstrate that a high mean AgNOR area within a cell population having a high MIB-1 index can discern tumors with a high metastatic potential. By multiplying AgNOR area by the percentage of MIB-1 positive cells we calculate the proliferative activity, P, which brings very important information concerning the time limit of relapse.