Gene expression in histologically normal epithelium from breast cancer patients and from cancer-free prophylactic mastectomy patients shares a similar profile

Abstract

Introduction: We hypothesised that gene expression in histologically normal (HN) epithelium (NlEpi) would differ between breast cancer patients and usual-risk controls undergoing reduction mammoplasty (RM), and that gene expression in NlEpi from cancer-free prophylactic mastectomy (PM) samples from high-risk women would resemble HN gene expression.

Methods: We analysed gene expression in 73 NlEpi samples microdissected from frozen tissue. In 42 samples, we used microarrays to compare gene expression between 18 RM patients and 18 age-matched HN (9 oestrogen receptor (ER)+, 9 ER-) and 6 PM patients. Data were analysed using a Bayesian approach (BADGE), and validated with quantitative real-time PCR (qPCR) in 31 independent NlEpi samples from 8 RM, 17 HN, and 6 PM patients.

Results: A total of 98 probe sets (86 genes) were differentially expressed between RM and HN samples. Performing hierarchical analysis with these 98 probe sets, PM and HN samples clustered together, away from RM samples. qPCR validation of independent samples was high (84%) and uniform in RM compared with HN patients, and lower (58%), but more heterogeneous, in RM compared with PM patients. The 86 genes were implicated in many processes including transcription and the MAPK pathway.

Conclusion: Gene expression differs between the NlEpi of breast cancer cases and controls. The profile of cancer cases can be discerned in high-risk NlEpi from cancer-free breasts. This suggests that the profile is not an effect of the tumour, but may mark increased risk and reveal the earliest genomic changes of breast cancer.

Figure 1

Representative histologically normal epithelium. Representative examples of 10 μm H&E-stained guide sections showing histologically normal epithelial cells identified for microdissection.

Figure 2

qPCR validation of microarray data. (A) depicts results using RNA from original samples (technical validation) and (B) depicts results using RNA from independent samples (prospective validation). Each panel shows qPCR results for six test genes. Test genes are listed on the x axis, and the mean fold change in expression in each sample group (HN or PM), compared with expression in the reference group (RM), is shown on the y axis. Fold changes were calculated using the ddCT method, in which fold change data are represented as 2^−ddCT. Error bars depict the standard error of the mean dCT values. Significant differences (P<0.05) are denoted with an asterisk.

Figure 3

Clustering of RM, HN, and PM samples based on gene expression. Hierarchical clustering of RM, HN, and PM samples using 98 probe sets identified as differentially expressed between 18 RM and 18 HN samples. The relative abundance of each transcript for each sample is represented as a coloured block, with green representing fold changes >2 and red representing probe sets with fold changes <2.

Figure 4

Gene list analysis. Genes identified as participating in the MAPK pathway, MAPK activating functions, and functions induced by MAPK, as annotated in DAVID analysis. Genes are arranged according to the p38 MAPK pathway, with the processes activating the pathway in boxes at the top of the figure, and processes affected by the MAPK pathway in boxes at the bottom of the figure.