Abstract P2-05-03: Intra-tumor heterogeneity of the estrogen receptor predicts less benefit from tamoxifen therapy and poor long-term breast cancer patient survival – Retrospective analyses of the STO-3 randomized trial
Authors: Lindström LS , Yau C , Czene K , Thompson CK , van't Veer LJ , Nordenskjöld B , Stål O , Fornander T , Benz CC , Borowsky AD , Esserman LJ 
Published: March 2017

Abstract
Background
We and others have shown that the clinically used breast cancer markers alter their expression throughout tumor progression, influencing patient survival (Lindström et al, JCO 2012). What are the likely explanations to our findings? Here, we aimed to determine whether breast cancer intra-tumor heterogeneity of the estrogen receptor (ER) is a marker of tumor aggressiveness and benefit of tamoxifen therapy in a large randomized trial.
Material and methods
The Stockholm Tamoxifen (STO-3) trial enrolled postmenopausal lymph node negative breast cancer patients with a tumor size of less than 30 mm, between 1976 and 1990, to be randomized to receive adjuvant tamoxifen versus not. From the original randomized trial cohort approximately half of the patients (778 patients) had primary tumor formalin-fixed paraffin-embedded blocks available and were included in our study. No significant differences in age and period of diagnosis, type of surgery received, receptor status, tumor grade and size were observed between the treatment arms.
All tumor slides were immunostained in a central laboratory using the SP1 antibody. ER slides were scored by two independent breast cancer pathologists assessing the fraction of cancer cells for each ER intensity level (0, +1, +2 or +3) compared to established standards. The resulting distribution of ER stained tumor cells defines intra-tumor heterogeneity of ER (Rao's quadratic entropy (QE),Potts et al, Lab Invest 2012). Intra-tumor heterogeneity was categorized using the third tertile as cut-off for high heterogeneity (726 patients).
Analyses of long-term breast cancer specific survival (25 years) by intra-tumor heterogeneity of ER were performed using univariate Kaplan-Meier and multivariate Cox proportional hazard modeling adjusting for treatment arm, age and period of diagnoses, ER, progesterone receptor (PR), HER2, Ki-67, tumor grade, and tumor size. Further, a test of correlation was performed to investigate whether intra-tumor heterogeneity of ER was correlated to the percentage of ER positive cells, the H-Score or the Luminal A and B subtype (PAM50).
Results
In the univariate Kaplan-Meier analyses, a statistically significant difference in long-term survival by intra-tumor heterogeneity of ER was seen for all patients (log rank, P=0.018), tamoxifen treated arm (log rank, P=0.0033), but not untreated arm (log rank, P=0.19). However in the multivariate analysis, patients with high intra-tumor heterogeneity of ER in the treated arm as well as in the untreated arm had an almost two-fold increased long-term risk of fatal breast cancer disease as compared to patients with low or intermediate heterogeneity (Treated arm: HR, 2.06; 95% CI, 1.04-4.07 and Untreated arm: HR, 1.71; 95% CI, 1.01-2.87).
No significant correlation of intra-tumor heterogeneity to the tested variables was seen.
Conclusions
Patients with high intra-tumor heterogeneity of ER had less benefit from tamoxifen therapy and an increased long-term risk of fatal breast cancer disease. Our findings should be clinically relevant since therapy benefit was evaluated in a randomized trial with long-term follow-up.
Citation Format: Lindström LS, Yau C, Czene K, Thompson CK, van't Veer LJ, Nordenskjöld B, Stål O, Fornander T, Benz CC, Borowsky AD, Esserman LJ. Intra-tumor heterogeneity of the estrogen receptor predicts less benefit from tamoxifen therapy and poor long-term breast cancer patient survival – Retrospective analyses of the STO-3 randomized trial [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P2-05-03.