CDK4/6 inhibition with endocrine therapy is now a standard of care for advanced estrogen receptor-positive breast cancer. Mechanisms of CDK4/6 inhibitor resistance have been described preclinically, with limited evidence from clinical samples. We conducted paired baseline and end-of-treatment circulating tumor DNA sequencing from 195 patients in the PALOMA-3 randomized phase III trial of palbociclib plus fulvestrant versus placebo plus fulvestrant. We show that clonal evolution occurs frequently during treatment, reflecting substantial subclonal complexity in breast cancer that has progressed after prior endocrine therapy. RB1 mutations emerged only in the palbociclib plus fulvestrant arm and in a minority of patients (6/127, 4.7%, P = 0.041). New driver mutations emerged in PIK3CA (P = 0.00069) and ESR1 after treatment in both arms, in particular ESR1 Y537S (P = 0.0037). Evolution of driver gene mutations was uncommon in patients progressing early on palbociclib plus fulvestrant but common in patients progressing later on treatment. These findings inform future treatment strategies to address resistance to palbociclib plus fulvestrant.Significance: Acquired mutations from fulvestrant are a major driver of resistance to fulvestrant and palbociclib combination therapy. ESR1 Y537S mutation promotes resistance to fulvestrant. Clonal evolution results in frequent acquisition of driver mutations in patients progressing late on therapy, which suggests that early and late progression have distinct mechanisms of resistance. Cancer Discov; 8(11); 1390-403. ©2018 AACR. See related commentary by Schiff and Jeselsohn, p. 1352 This article is highlighted in the In This Issue feature, p. 1333.

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