F-Fluoroestradiol PET Imaging of Activating Estrogen Receptor-α Mutations in Breast Cancer.

The purpose of this study was to determine the effect of estrogen receptor-α gene () mutations at the tyrosine (Y) 537 amino acid residue within the ligand binding domain on F-fluoroestradiol (F-FES) binding and tumor uptake compared with wild-type (WT)-estrogen receptor α (ER). ER-negative MDA-MB-231 breast cancer cells were used to generate stable cell lines that express WT-ER, Y537S, or Y537C mutant ER. Receptor expression and localization were confirmed by Western blot and immunofluorescence, respectively. ER transcriptional function was measured using an estrogen response element-luciferase reporter gene assay and quantitative polymerase chain reaction analysis of ER-regulated endogenous target genes. Saturation binding and competition assays were performed to determine equilibrium dissociation constant (K) and half maximal inhibitory concentration (IC50) values. F-FES uptake was measured in tumor xenografts grown in female athymic nude mice by small-animal PET/CT imaging and tissue biodistribution using 5.55 MBq (150 μCi) of F-FES. A 10-fold-lower injected dose of 0.555 MBq (15 μCi) of F-FES was also used for tissue biodistribution. Statistical significance was determined using ANOVA. Y537S and Y537C mutations resulted in increased ER transcriptional activity in the absence of estrogen compared with WT-ER (11.48 ± 2.42 fold; = 0.0002, and 5.89 ± 0.94 fold; = 0.04, respectively). Constitutive ER activation of two target genes ( and ) in the absence of estrogen was also observed in Y537S- and Y537C-ER cells compared with WT-ER. K values for F-FES were 0.98 ± 0.54 nM for Y537S-ER ( = 0.27) and 0.24 ± 0.03 nM for Y537C-ER ( = 0.95) compared with 0.07 ± 0.03 nM for WT-ER. IC50 values were 0.22 ± 0.09 nM for Y537S-ER ( = 0.97), 0.18 ± 0.09 nM for Y537C-ER ( = 0.99), and 0.19 ± 0.11 nM for WT-ER. Tumor xenografts expressing Y537S-ER (mean percentage injected dose per gram, 1.45 ± 0.06; = 0.77) and Y537C-ER (2.09 ± 0.20; = 0.21) had similar F-FES uptake compared with WT-ER (1.68 ± 0.12). Comparable F-FES uptake between Y537S-, Y537C-, and WT-ER xenografts was also observed using a 10-fold-lower injected dose with the tissue biodistribution assay. Since tumoral uptake of F-FES is not significantly impacted by Y537S-ER or Y537C-ER mutations, the potential diagnostic utility of F-FES PET imaging is expected to be equally valid for patients with or without these activating mutations.