¹⁸F-Fluoroestradiol PET Imaging of Activating Estrogen Receptor-α Mutations in Breast Cancer.

Abstract

The purpose of this study was to determine the effect of estrogen receptor-α gene (<i>ESR1</i>) mutations at the tyrosine (Y) 537 amino acid residue within the ligand binding domain on ¹⁸F-fluoroestradiol (¹⁸F-FES) binding and <i>in vivo</i> tumor uptake compared with wild-type (WT)-estrogen receptor α (ER). <b>Methods:</b> 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_d) and half maximal inhibitory concentration (IC50) values. ¹⁸F-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 ¹⁸F-FES. A 10-fold-lower injected dose of 0.555 MBq (15 μCi) of ¹⁸F-FES was also used for tissue biodistribution. Statistical significance was determined using ANOVA. <b>Results:</b> Y537S and Y537C mutations resulted in increased ER transcriptional activity in the absence of estrogen compared with WT-ER (11.48 ± 2.42 fold; <i>P</i> = 0.0002, and 5.89 ± 0.94 fold; <i>P</i> = 0.04, respectively). Constitutive ER activation of two target genes (<i>PGR</i> and <i>TFF1</i>) in the absence of estrogen was also observed in Y537S- and Y537C-ER cells compared with WT-ER. K_d values for ¹⁸F-FES were 0.98 ± 0.54 nM for Y537S-ER (<i>P</i> = 0.27) and 0.24 ± 0.03 nM for Y537C-ER (<i>P</i> = 0.95) compared with 0.07 ± 0.03 nM for WT-ER. IC50 values were 0.22 ± 0.09 nM for Y537S-ER (<i>P</i> = 0.97), 0.18 ± 0.09 nM for Y537C-ER (<i>P</i> = 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; <i>P</i> = 0.77) and Y537C-ER (2.09 ± 0.20; <i>P</i> = 0.21) had similar ¹⁸F-FES uptake compared with WT-ER (1.68 ± 0.12). Comparable ¹⁸F-FES uptake between Y537S-, Y537C-, and WT-ER xenografts was also observed using a 10-fold-lower injected dose with the tissue biodistribution assay. <b>Conclusion:</b> Since tumoral uptake of ¹⁸F-FES is not significantly impacted by Y537S-ER or Y537C-ER mutations, the potential diagnostic utility of ¹⁸F-FES PET imaging is expected to be equally valid for patients with or without these activating <i>ESR1</i> mutations.