ER Status Measured by FISH Is a Better Predictor of Prognosis in ER Positive, Postmenopausal Breast Cancer Patients Treated with Adjuvant Tamoxifen Than ER Status Measured by RT-qPCR or IHC.
Authors: Lyng M. , Vang K. , Knoop A. , Ditzel H. , Lykkesfeldt A. , Müller S. , Laenkholm A. 
Published: December 2010

Abstract
Background: The estrogen receptor (ER) is the target for endocrine treatment with tamoxifen. It has been suggested that amplification of ESR1 might be a marker of tamoxifen resistance in post-menopausal ER positive breast cancer patients in the adjuvant setting. In this study we aimed to investigate the correlation between copy number changes in the ESR1 gene, ER gene expression (mRNA) and ER protein status measured by immunohistochemistry (IHC) and the association with recurrence status.Materials and methods: A total of 56 breast cancer patients were included in the study. Both frozen and formalin-fixed paraffin-embedded tumor tissue from the primary tumor were available for analysis. All patients were post-menopausal, high-risk, ER positive breast cancer patients allocated to 1, 2 or 5 years of adjuvant tamoxifen treatment following radical surgery. Twenty-eight patients had recurrence with a median time to recurrence of 2.4 years. The other half of the patients were without recurrence at time of follow-up. The median time of follow-up was 12.0 years. The patients were matched 1-to-1 according to clinico-pathological parameters including duration of treatment. The Dako Histology FISH accessory kit was used for analysis of gene copy number, employing a probe covering the ESR1 gene at 6q25 and a reference probe covering the centromere of chromosome 6. The gene expression was analyzed by a pre-designed real-time PCR assay (ID Hs00174860_m1, Applied Biosystems). ER protein expression was visualized by IHC using the NCL-ER-6F11, 1:100 (Novocastra Ltd) antibody, detected by EnVision Flex+ system (Dako).Results: The gene copy number analysis showed amplification (ratio ESR1/CEN-6 ≥2) in 6/28 (21%) of patients with recurrence and in 1/28 (3%) of patients without recurrence. ESR1 deletion (ratio ESR1/CEN-6 &lt; 0.8) was identified in 3 patients, all without recurrence. A lower median gene expression level was observed in tumors from patients with recurrence compared to patients without recurrence (p=0.07). The percentage positive tumor cells (mean 82%) detected by semi-quantitative bright field microscopy was almost equal in the two groups (p=0.70). Only the gene copy number showed a significant difference (p=0.02) between the patient groups having a significantly higher ESR1/CEN-6 ratio in tumors from patients with recurrence compared to patients without recurrence. A weak positive correlation was observed between ESR1 gene copy number and gene expression (p=0.02), in contrast to a non-significant correlation between gene copy number and protein level (p=0.17).Conclusion: This pilot study shows that measuring the ESR1 gene copy number by FISH seems to be more predictive of tamoxifen resistance compared to measuring the expression of ER by either RT-qPCR or IHC, in ER positive post-menopausal breast cancer.
Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 2004.