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Limited data are available on estradiol (E2) levels during fulvestrant treatment in women with hormone receptor-positive breast cancer. We measured plasma E2 levels in women receiving fulvestrant using liquid chromatography-tandem mass spectrometry. Patient characteristics and treatment efficacy were assessed in relation to E2 levels. A cutoff of 2.72 pg/mL was used because it defines E2 suppression and postmenopausal status. A total of 69 women were enrolled, with a median age of 48 years. The median duration of fulvestrant treatment was 11.6 months. The median E2 level across the cohort was 3.60 pg/mL, with considerable interindividual variability (range: 1.11-526.13 pg/mL), and 49 women (71.0%) had E2 levels above 2.72 pg/mL. Eleven women (15.9%) had premenopausal E2 levels (> 10 pg/mL). During a median follow-up period of 8.4 months, there was no statistically significant difference in progression-free survival (PFS) between women with E2 levels >2.72 pg/mL and those with E2 levels ≤ 2.72 pg/mL (p = 0.391). However, among women who benefited from first- or second-line fulvestrant therapy (PFS > 6 months), those with E2 levels > 2.72 pg/mL exhibited significantly poorer PFS compared to those with E2 levels ≤ 2.72 pg/mL (p = 0.043). These findings support the need for E2 monitoring in women receiving fulvestrant therapy to better assess E2 status and its association with treatment efficacy.