Molecular response to aromatase inhibitor treatment in primary breast cancer

Abstract

Background: Aromatase inhibitors such as anastrozole and letrozole are highly effective suppressants of estrogen synthesis in postmenopausal women and are the most effective endocrine treatments for hormone receptor positive breast cancer in such women. Little is known of the molecular effects of these agents on human breast carcinomas in vivo.

Methods: We randomly assigned primary estrogen receptor positive breast cancer patients to treatment with anastrozole or letrozole for 2 weeks before surgery. Expression profiling using cDNA arrays was conducted on pretreatment and post-treatment biopsies. Sample pairs from 34 patients provided sufficient RNA for analysis.

Results: Profound changes in gene expression were seen with both aromatase inhibitors, including many classical estrogen-dependent genes such as TFF1, CCND1, PDZK1 and AGR2, but also many other genes that are likely to represent secondary responses; decrease in the expression of proliferation-related genes were particularly prominent. Many upregulated genes are involved in extracellular matrix remodelling, including collagens and members of the small leucine-rich proteoglycan family (LUM, DCN, and ASPN). No significant differences were seen between letrozole and anastrozole in terms of molecular effects. The gene changes were integrated into a Global Index of Dependence on Estrogen (GIDE), which enumerates the genes changing by at least twofold with therapy. The GIDE varied markedly between tumours and related significantly to pretreatment levels of HER2 and changes in immunohistochemically detected Ki67.

Conclusion: Our findings identify the transcriptional signatures associated with aromatase inhibitor treatment of primary breast tumours. Larger datasets using this approach should enable identification of estrogen-dependent molecular changes, which are the determinants of benefit or resistance to endocrine therapy.

Figure 1

Heatmap of unsupervised clustering of pretreatment and post-treatment biopsies. A heatmap of the unsupervised clustering of the 34 pretreatment and post-treatment samples (labeled A and B respectively) using 2,418 of the most variable genes is shown. The entire heatmap is shown in miniature on the left. Clusters containing the genes (a) ESR1, (b) MKI67, (c) ERBB2 and (d) TFF1 are shown in detail. Out of 34 pairs of biopsies, 18 co-aggregated at the first or second level in the sample dendrogram.

Figure 2

Comparison of GIDE score and change in Ki67 immunohistochemistry with ESR1 and ERBB2 expression. (a) Significant positive correlation of the Global Index of Dependence on Estrogen (GIDE) scores for each pair of biopsies with percentage decrease in Ki67 immunohistochemistry (IHC) is shown. (b) Significant negative correlation of the GIDE score to the pretreatment expression of ERBB2, as derived from microarray profiling, is shown. Also shown are comparisons of the change in Ki67 immunohistochemistry (% decrease) is shown with pretreatment (c) ESR1 expression and (d) ERBB2 expression. Finally, comparisons of GIDE scores with pretreatment immunohistochemical measurements (Allred scores) are shown for (e) estrogen receptor (ER) and (f) progesterone receptor (PgR).

Figure 3

Expression changes in key index genes in response to AI treatment. Individual log ratio measurements are plotted and joined with a line in each of the paired biopsies. Individual results are shown for the downregulated genes PDZK1, TFF1, AGR2 and CCND1, and the upregulated genes DCN, LUM and ASPN. The percentage decrease in Ki67 immunohistochemistry (IHC) is shown in the bottom left panel for comparison.

Figure 4

Supervised clustering of pre and post treatment biopsies. The 421 genes that best distinguished pretreatment and post-treatment biopsies were used to cluster the samples in the heatmap shown on the left. Three clusters of genes are shown in greater detail on the right: (a) a proliferation cluster representing genes associated with proliferation and cell cycle progression, (b) an estrogen cluster of known highly estrogen-responsive genes and (c) an extracellular matrix (ECM) cluster of genes known to be involved in ECM remodelling.

Figure 5

Vector diagrams of metagenes representing estrogen response, proliferation and ECM remodelling. Metagene values derived from the mean values of all the genes in each of the clusters in Figure 4 are plotted and connected with a line from dots (pretreatment values) to arrowheads (post-treatment values). Estrogen metagene values are compared with the (a) proliferation metagene and (b) the extracellular matrix (ECM). The six biopsies with the lowest pretreatment estrogen metagene are coloured in red. The four biopsies with HER2 amplification and high ERBB2 expression are shown with green dots, and samples with the lowest responses in the proliferation metagene are highlighted in blue.