Estrogen receptor alpha gene ESR1 amplification may predict endocrine therapy responsiveness in breast cancer patients

Abstract

Estrogen receptor (ER) alpha plays a crucial role in normal breast development and has also been linked to mammary carcinogenesis and clinical outcome in breast cancer patients. However, the molecular mechanisms controlling the expression of ERalpha are as yet not fully understood. Gene amplification is one of the important factors regulating protein expression. Recent studies on the amplification of the ESR1 gene, which encodes ERalpha, have presented conflicting data. Using fluorescence in situ hybridization and real-time quantitative polymerase chain reaction analysis, we examined the ESR1 status in a series of breast cancer tissues and analyzed its clinical importance. ESR1 gene amplification and gain were found in 22.6 and 11.3% of samples, respectively, as determined by three-dimensional fluorescence in situ hybridization assay. Moreover, ESR1 amplification and amplification plus gain were significantly negatively correlated with tumor size, number of positive lymph nodes, negative ERalpha, and positive human epidermal growth factor receptor 2 status. It has also been shown that ESR1 amplification strongly correlates with higher expression levels of ER protein and that patients with ESR1 amplification in their tumors apparently experience longer disease-free survival than those without. Our data suggest that ESR1 amplification might prove to be helpful in selecting patients who may potentially benefit from endocrine therapy.

Figure 1

Kaplan–Meier analysis of the effect on estrogen receptor (ER) status and ESR1 amplification on disease‐free survival for three groups of cancers: group 1, tumors with ESR1 amplification; group 2, ER‐positive tumors (Allred scores of 3–8) lacking ESR1 amplification; and group 3, ER‐negative tumors (Allred scores of 0–2).

Figure 2

Frequency distribution of ESR1 amplification ratios in 147 breast cancers analyzed by real‐time quantitative polymerase chain reaction. ESR1 gene status was defined by the ratio of ESR1 versus RNase P, as follows: >2.0 indicated amplification, between 1.5 and 2.0 indicated gain. The majority of cases had an amplification ratio <1.5, indicating normal copy number of ESR1. Two cases were considered to be amplified by real‐time quantitative polymerase chain reaction.

Figure 3

Image restoration and analyses of three‐dimensional images of the ESR1 locus. (a) A series of images was taken through the z‐axis. (b) Localization of the ESR1 gene (green) and centromere 6 (red) is shown in the two fluorescence in situ hybridization images (z1, z2) and projected one. In a single‐plane image, the ESR1 gene could not be detected, as indicated by an arrowhead in z1 and by an arrow in z2, because of the small size of the amplicon.

Figure 4

Examples of fluorescence in situ hybridization findings in breast cancers with (a) normal ESR1 copy number and (b–d) ESR1 amplification. The ESR1 gene probe is labeled in green, the centromere 6 reference probe in red. (a) Two copies of ESR1 per nucleus. (b) Tiny confluent ESR1 gene clusters. (c) Small cluster with three to nine clearly distinguishable ESR1 gene copies. (d) Large clusters of more than 10 gene signals.