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

Background

Most breast cancers appear to arise from certain precursors over long periods of time. Enlargement (>50-fold) of normal terminal duct lobular units (TDLUs) by hyperplastic epithelial cells is one of the most common and earliest histologically recognizable alterations with premalignant potential. Understanding how these hyperplastic enlarged lobular units (HELUs) develop and progress could lead to new and effective strategies for breast cancer prevention therapy.

Methods

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.

Results

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 receptor between HELUs and TDLUs. Several fresh human samples of TDLUs and HELUs are in various stages (currently up to generation four) of implantation in mice in attempts to establish stable xenografts.

Conclusions

HELUs are one of the earliest histologically recognizable lesions in the human breast with premalignant potential. They show striking elevations of ER, which may partially explain the hyperplasia leading to their development from TDLUs through increased proliferation and decreased apoptosis, which are both regulated by estrogen. DNA microarrays reveal many additional differences in the expression of genes involved in growth and differentiation. Human xenograft models are under development to support mechanistic studies of these genes to understand their roles in the development and progression of HELUs and how to prevent it.