Biological features and xenograft models of a very early human premalignant breast lesion

The estrogen receptor (ER) and proliferation (Ki67) were evaluated and compared in TDLUs and HELUs in the same breasts (n = 250) by immunohistochemistry. Apoptosis was also assessed by the TUNEL assay. The rate of ER expression in proliferating cells was assessed by dual-labeled immunofluorescence. Comprehensive gene expression profiling was performed in a subset of samples (currently six matched pairs of TDLUs and HELUs) using RNA isolated from microdissected formalin-fixed paraffin-embedded breast tissue samples and Affymetrix U133-X3P microarrays analyzed by dCHIP software. Xenografts of human TDLUs and HELUs were prepared by implanting isolated epithelial cells into cleared mammary fat pads of estrogen-stimulated immune-compromised mice 'humanized' by prior local injection of immortalized (h-tert transfected) human mammary fibroblasts.The average ER expression was significantly elevated in HELUs compared with adjacent TDLUs (85% vs 30% positive cells, respectively; P < 0.0001). Proliferation was significantly higher (6% vs 2%; P < 0.0001) and apoptosis was significantly lower (0.6% vs 0.2%; P < 0.001) in HELUs than TDLUs. There was a large increase in the proportion of ER-positive proliferating cells in HELUs compared with TDLUs (35% vs 4%; P < 0.0001). In preliminary analysis of the microarray results, HELUs and TDLUs segregated perfectly in unsupervised hierarchical comparisons. In supervised comparisons, many (n = 74) genes showed >3-fold (P < 0.05) differences in expression, with 45 relatively up (from 3.5-fold to 9.5-fold) and 29 relatively down (from 3.5-fold to 12.5-fold) in HELUs versus TDLUs. Especially prominent elevations in HELUs included several genes involved in G-protein signaling, the retinoic acid pathway, and detoxification. Prominent decreases included genes involved in cell cycle inhibition, apoptosis, differentiation, and water transport. Differences were also noted in the expression of genes for ligands of the epidermal growth factor recept