Abstract Background: The primary objective of this phase IIIb, single arm trial is to study circulating tumor DNA (ctDNA) alterations, their evolution during treatment and association with clinical outcome in patients receiving ribociclib and letrozole as first-line therapy for aBC. Here we report baseline ctDNA mutational status and its correlation with clinical-pathological characteristics and response to treatment on the basis of first imaging evaluation. Methods: From February to December 2018, 287 post-menopausal patients (pts) with hormone receptor positive (HR+) and HER2 negative aBC were enrolled in 47 Italian Centers; 271 pts were suitable for biomarker analysis (Biomarker Analysis Set- BAS). Data on first imaging evaluation were available for 225 pts. Baseline liquid biopsies were analyzed both for Copy Number Variation (CNV) by Oncomine Pan-Cancer Cell-Free Assay (Thermo Fisher Scientific) and for Single Nucleotide Variant (SNV) using 533 amplicon custom AmpliSeq HD panel optimized for the detection of low abundance events in ctDNA. Variants were analyzed as both individual genes and as pre-defined pathways (n=10). Results: At BAS, median age was 66 years (range 47-86). At study entry, 60% (109) and 40% (162) of pts had recurrent and de-novo disease, respectively, with 43% (117) of pts with visceral and 24% (64) with bone-only involvement. At least one CNV alteration or a hotspot mutation was detected in 51% of patients. The most frequently altered genes were PIK3CA (22.14%), TP53 (15.50%), FGFR1 (6.64%), CCND1 (4.80%), CCND2 (4.80%), KMT2C (4.43%), MYC (4.06%), CDK4 (3.69%) AKT1 (3.32%), PTEN (3.32%), ERBB2 (2.58%), CCND3 (2.58%), APC (2.21%) and MAP2K4 (2.21%). More than one alteration was observed in 28% pts (either CNV or hotspots). Mutation in KTM2C or alterations in genes belonging to the “Estrogen Receptor nuclear function” (ERnf) pathway (i.e. KTM2C, ESR1, GATA3 and MYC) were more frequent in patients with recurrent vs. de novo disease (Odds Ratio [OR] =7.69, p=0.0499 for KTM2C; OR=2.70, p=0.0381 for ERnf). MYC copy number gain or alterations in the ERnf genes were more frequent in Estrogen Receptor positive (ER+)/Progesteron Receptor negative (PgR-) compared to ER+/PgR+ tumors (OR= 4.07, p=0.0361 for MYC; OR=3.36, p=0.0073 for ERnf). Copy number gain of FGFRs (FGFR1, 2 and 3) were more frequent in patients with visceral versus bone-only disease (OR=5.26, p=0.0144 for FGFRs) and in pts with more than three metastatic sites (OR=8.05, p=0.0052). At first imaging, the Clinical Benefit Rate (CBR, defined as CR + PR + SD) was 90%, with only 10% of pts showing early disease progression (PD). Thirty pts (11%) had at least one alteration in genes belonging to the CDK4/6 pathway (CCND1, RB1, CDK4 and 6, CDKN2A). These alterations were more frequent in the group of patients with early PD (OR=3.23, p=0.0272). MYC gain, TP53 mutations and alterations in the HER family genes (EGFR, ERBB2, 3 and 4) were also more frequent in pts with early PD compared with pts with CBR (OR 5.16, p=0.0280, OR 3.87, p=0.0058 and OR 4.40, p=0.0426, respectively). Conclusion: At the present analysis, alterations in KTM2C and ERnf seem to characterize recurrent vs de novo aBC while FGFRs are prevalent in patients with more aggressive disease. Interestingly, MYC gain, TP53 mutations, alterations in genes of the HER family and CDK4/6 pathway were more likely detected in patients with early PD suggesting these as potential markers of intrinsic resistance to first-line treatment with ribociclib and letrozole. Further analyses for biomarker dynamics and pharmacogenomics are ongoing. Citation Format: Michelino De Laurentiis, Luca Malorni, Giampaolo Bianchini, Roberta Caputo, Mario Giuliano, Alberto Zambelli, Fabio Puglisi, Lucia Del Mastro, Marco Colleoni, Filippo Montemurro, Giulia Bianchi, Ida Paris, Giacomo Allegrini, Laura Amaducci, Marina Elena Cazzaniga, Michele Orditura, Claudio Zamagni, Vincenzo Adamo, Valentina Guarnieri, Nicola Battelli, Maria Rosaria Valerio, Saverio Cinieri, Sara Bianchetti, Donatella Grasso, Daniela Castelletti, Diletta Valsecchi, Ettore Cotroneo, Grazia Arpino. Bioitalee - Biomarker analysis on liquid biopsies of patients treated with ribociclib and letrozole as first-line therapy for advanced breast cancer (aBC) (NCT03439046) [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P5-01-07.