Aims: To investigate circulating tumour DNA (ctDNA) as a biomarker to predict sensitivity and primary or acquired targeted therapy, focusing on advanced breast cancer treated with the CDK4/6 inhibitor palbociclib. CDK4/6 inhibitors are one of the most promising targeted therapies in clinical development with evidence of activity in multiple tumour types. Objectives: 1) Identify genetic aberrations in baseline ctDNA that predict for sensitivity to CDK4/6 inhibition, to select the most appropriate patients for therapy. 2) Assess whether change in abundance of ctDNA following 2 weeks of treatment can identify cancers that are resistant to treatment early. 3) Assess whether analysis of ctDNA can identify the emergence of resistant clones on disease progression. Methodology: The project will utilise samples collected at baseline and day 15 of treatment, then again on progression from two trials treating breast cancer patients with palbociclib, PEARL (350 samples) and PALOMA-3 (500 samples). 1) Identify genetic aberrations in baseline ctDNA that predict for sensitivity to CDK4/6 inhibition by sequencing plasma DNA using a custom targeted Ampliseq panel on the Ion Torrent Personal Genome Machine. Validation will be by using droplet digital polymerase chain reaction assays run on the BioRad QX200 platform. 2) Sequence day 15 plasma, and establish criteria for fractional change in abundance of ctDNA that predict for outcome. 3) Paired plasma DNA samples, baseline and progression, with a high proportion of ctDNA will undergo exploratory whole-exome sequencing. Acquired mutations will be validated, the function of recurrent acquired mutations investigated in vitro. Scientific and medical opportunities: This will be largest cohort of plasma sequenced for ctDNA in the literature, offering an unprecedented opportunity to link prospectively collected clinical data to ctDNA, validating it as a biomarker for targeted therapy potentially for routine clinical practice