Abstract P4-08-01: ESR1 Gene Aberrations Correlate with ER Protein Levels Measured by DCC and IHC
Authors: Lænkholm A-V , Nielsen KV , Knoop AS , Müller S , Rasmussen BB , Ejlertsen B. 
Published: April 2011

Abstract
Background: The Estrogen receptor alfa (ER) is an established biomarker that has been studied in details on the protein and RNA level. Recently, the existence of amplifications and deletions of the ESR1 gene have been documented (1-3), although the frequency of the aberrations has been extensively debated. Here we hypothesize that a positive correlation exists between ESR1 gene copy number and ER protein content measured by both a biochemical ligand assay, dextran coated charcoal (DCC), and immunohistochemistry (IHC).
Material and Methods: From 289 primary high-risk breast cancer patients, randomized in the DBCG 77C trial between August 1977 and November 1982, ER data from DCC analyses was available. An ER positive tumor was defined as ≥10 fmol ER/mg protein (4). Archival tumor tissue was available from 257 patients. ESR1 copy number was analyzed with Dako
Histology FISH Accessory Kit (K5599, DAKO, Glostrup, Denmark) using a probe covering the ESR1 gene at 6q25 and a centromere 6 reference probe (3). IHC analysis for ER was applied on archival paraffin embedded tissue using the antibody ER1D5 (DAKO), 1:200 with a positive cut off value of 10% ER positive tumor cells.
Results: ESR1 FISH analysis was performed successfully in 215 (84%) patients. Amplification (ratio ESR1/CEN-6≥2) was observed in 47 of 215 patients (22%) and ESR1 deletion (ratio ESR1/CEN-6&lt;0.8) was observed in 69 (32%). A positive correlation of ER-DCC with both FISH ESR1 and ER-IHC was found (P&lt;0.0001). The ESR1 amplified tumors had higher average ER-DDC values compared to ESR1 normal tumors (ratio ESR1/ CEN6: 0,80-1,29) and tumors with ESR1 gain (ratio ESR1/CEN6: 1,30-1,99), while deleted tumors had lower ER-DCC values as illustrated in Figure 1.
A significant difference p=0.005 was found for the ESR1 deleted tumors compared to the ESR1 amplified tumors.
Fig. 1 Box plut showing the ER content according to ESR1/CEN-6 status
Discussion: Amplification of the ESR1 gene is associated with higher ER protein content by ER-DCC and more intense immunoreactivity by IHC while ESR1 deletions are associated with decreased content and immunoreactivity compared to tumors with normal ESR1 gene copy numbers. Major variations in ER content and immunoreactivity are however observed within tumors with a normal ESR1 copy number, and other mechanisms than gene aberrations seem to contribute. References:
1. Holst et al. (2007) Estrogen receptor alpha (ESR1) gene amplification is frequent in breast cancer Nat Genet 39: 655
2. Tomita et al. (2009) Estrogen receptor alpha gene ESR1 amplification may predict endocrine therapy responsiveness in breast cancer patients. Cancer Sci 100:1012
3. Nielsen et al. (2010) Amplification of ESR1 may predict resistance to adjuvant tamoxifen in postmenopausal patients with hormone receptor positive breast cancer. Breast cancer Res Treat, June 17 Epub
4. Thorpe et al. (1993) Short recurrence-free survival associated with high oestrogen receptor levels in the natural history of postmenopausal, primary breast cancer. Eur J Cancer 29A:971.

Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P4-08-01.