To further validate the prognostic value of the RB-LOH signature, we evaluated pre-treatment baseline plasma samples from a second independent cohort of 357 plasma samples, hereafter Plasma-2 cohort, which included 121 patients with advanced HR+/HER2- breast cancer treated with endocrine therapy and a CDK4/6 inhibitor at Hospital Clínic of Barcelona and the Vall d’Hebron Institute of Oncology (VHIO) and assayed using our ctDNA WGS-assay. This evidence concerns the gene CDK4 and breast carcinoma.