Background: AA treatment may suppress androgens and estrogens that stimulate BCa growth. We conducted a biomarker analysis of circulating tumor cells (CTCs) and formalin-fixed paraffin-embedded tissues (FFPETs) from a phase 2 study of the efficacy and safety of AA in postmenopausal ER+ BCa pts to analyze serum steroid concentrations and identify subgroups with AA sensitivity. Methods: Treatments: AA (1 g/d) + P (5 mg/d) vs AA + P + E (25 mg/d) vs E alone. FFPETs from diagnosis and blood samples collected at baseline, during treatment, and at the end of treatment were used for molecular characterization and serum endocrine marker analysis. Expression of androgen receptor (AR), estrogen receptor (ER), Ki-67, CYP17, CYP19, and other biomarkers was evaluated in CTCs and/or FFPETs. Cox regression analysis stratified by number of prior therapies and type of therapy was used to evaluate biomarker associations with progression-free survival (PFS). Statistical comparisons were performed for treatment effect in biomarker-positive and -negative subgroups using Cox regression to identify those with better PFS. Results: Serum testosterone, estradiol, and estrone were decreased by AA treatment. An increase in progesterone was observed in most pts (AA + P and AA + P + E) but was not associated with PFS. 104 pts had ≥ 3 CTCs (median age 62; prior NSAI in metastatic setting 66%). Individually, baseline expression of AR or ER in CTCs or FFPETs was not associated with PFS. However, there was a positive association for the combination of AR and ER expression in CTCs with PFS in favor of AA + P + E vs E (HR 0.41 [0.16-1.07], p = 0.070). In recent FFPETs obtained < 1 yr prior to first dose (67 pts), AR expression suggested positive association with PFS (HR 0.56 [0.24-1.33], p = 0.191) for AA + P + E vs E. Conclusions: A positive pharmacodynamic effect of AA was shown by the decrease in serum estrogen and androgen and potentially by the increase in progesterone in most pts. Baseline AR and ER expression in CTCs and AR expression in recent FFPETs may predict improved PFS. Clinical trial information: NCT01381874.