3.1 Baseline patient characteristics
Patients’ tumor characteristics and treatments by menopausal status are summarized in Table 1, including 4207 pre-menopausal and 4559 post-menopausal women. Compared with pre-menopausal patients, post-menopausal patients had a lower median E2 concentration, an older diagnostic age (91.7% vs.9.5%), and were more often diagnosed with pT2-3 (50.3% vs. 46.2%), pN3 (9.7% vs. 8.3%) and stage Ⅲ disease (15.9% vs.14%). While pre-menopausal patients were more likely to have a significantly higher proportion of Grade Ⅰ (11.8 % vs. 8.4%) and ER-positive (69.8% vs. 46.2%), PR-positive (72.3% vs. 59%) and HER2- tumors (90.8% vs. 87.3%). In addition, pre-menopausal patients were more likely to receive adjuvant chemotherapy, radiotherapy, and endocrine treatment compared with post-menopausal women. During a median follow-up of 65 months (95% CI: 63-67 months), 982 (385 in pre- menopausal and 597 in post-menopausal) progressions, including 612 (203 in pre- menopausal and 409 in post-menopausal) deaths, occurred. The proportion of patients’ characteristics was significantly distinct between pre-menopausal and post-menopausal cases (P<0.001) and the study population was then stratified according to menopausal status.
3.2 Prognostic effects of E2 levels on breast cancer survival
The association of the serum E2 levels with breast cancer survival, including OS and PFS, is presented in Figure 1 according to menopausal status. The ranges of menopausal status-specific quartile groups of the E2 value were as follows: pre-menopause, quartile 1 (Q1): ≤38.83 ng/L, quartile 2 (Q2): 38.84 to 82.94 ng/L, quartile 3 (Q3): 82.95 to 149.9 ng/L and quartile 4 (Q4): >149.9 ng/L and post-menopause, quartile 1 (Q1): ≤5 ng/L, quartile 2 (Q2): 5.1 to 7.49 ng/L, quartile 3 (Q3): 7.50 to 13.94 ng/L and quartile 4 (Q4): >13.94 ng/L. Q3 was used as reference group in further analysis.
Figure 1A shows HRs (95% CI) for OS and PFS in pre-menopausal patients. Patients with E2 <82.95 ng/L or E2 >142.9 ng/L had an increased hazard of OS with the references (Q1: HR = 1.42, 95% CI: 0.76–2.67; Q2: HR = 1.90, 95% CI: 1.02–3.54; Q4: HR = 1.50, 95% CI: 0.78–2.89), the serum E2 quartile group 2 was at a greater risk of death than the other groups (P=0.042); serum E2 quartile groups 2 and 4 had a significantly higher risk for progression than serum E2 quartile group 3 (Q2: HR = 1.62, 95% CI: 1.04–2.52; Q4: HR = 1.79, 95% CI: 1.17–2.75).
Figure 1B shows HRs (95% CI) for OS and PFS in post-menopausal patients. The highest serum E2 level has significantly higher HR for death than the Q3 level (HR=1.35, 95% CI: 1.04-1.74; P=0.023). The highest E2 levels had a non-significantly higher risk for progression (Q1: HR = 1.10, 95% CI: 0.89–1.37; Q2: HR = 1.12, 95% CI: 0.82–1.51; Q4: HR = 1.15, 95% CI: 0.93–1.43).
3.3 Dose-response relationship between E2 and breast cancer survival
The dose-response relationships between E2 and death or progression were analyzed in patients before and after menopause, adjusting for the same confounding factors (Figure2). When E2 level was a continuous variable attempting to account for non-linear association, the relationship between survival endpoints and E2 was significantly nonlinear in pre-menopausal women. E2 levels were prognostic of OS in a J-shaped pattern (nonlinear P=0.039); risk was flat to a threshold (~250ng/L), then increased gradually (Figure2A). E2 levels were prognostic of PFS in a U-shaped pattern (nonlinear P=0.013); risk was higher at the extreme (300 ng/L) but lower at intermediate values (>50 to <150 ng/L) (Figure2B). The plot of the figure2A and 2B showed a J-shaped and U-shaped relationship between log relative hazard and E2, indicating that lower or higher levels of E2 were associated with a higher hazard of death or progression, with the lowest risk of 128.7ng/L and 103.3ng/L, respectively. When the E2 concentration was above 300ng/L, the risk of PFS gradually became flat, whereas OS was still elevated. As for post-menopausal women, there was a linear relationship, and the linearity P values were 0.105 and 0.158, respectively. The plot showed that the risk of death or progression increased gradually with increasing E2 concentration, both tending to be stable after 30ng/L (Figure2C and 2D).
3.4 ER status of patients modified the prognostic effects of E2 levels on breast cancer survival
When we analyzed the estrogen receptor status, both ER-positive and ER-negative demonstrated inconsistent fluctuation curves (Figure3). Patient ER status modified the prognostic association of E2 levels and OS or PFS in the pre-menopausal women. Among ER-positive patients, E2 levels were associated with OS or PFS in a linear pattern (linear OS: P=0.568; PFS: P=0.412), whereas among ER-negative patients, E2 levels were associated with OS or PFS in a U-shaped pattern (nonlinear OS: P=0.0006; PFS: P=0.001) (Figure3A and 3B). Compared with ER-negative patients, the risk of disease progression or death was higher in ER-positive breast cancer patients when E2 concentration was 100ng/L-300ng/L. In post-menopausal women, there was a positive linear correlation between E2 and death or progression in ER-positive and ER-negative breast cancer (Figure 3C and 3D). ER status did not modify the prognostic association between E2 levels with OS or PFS in the post-menopausal women. Within 10 ng/L, the progression or death risk for ER-positive was approximately the same as that for ER-negative. As for above 10ng/L, the risk for ER-positive was higher than ER-negative.