Background: Trastuzumab Deruxtecan (TDXd), a novel antibody-drug conjugate (ADC) that combines trastuzumab with a topoisomerase I inhibitor, has recently demonstrated high efficacy in HER2-overexpressing breast cancer after trastuzumab failure. Resistance to TDM1 have recently suggested to be dynamically associated with distinct circulating ctDNA species (Allegretti et al, Mol Cancer 2021). A prospective study aiming to evaluate the feasibility of Liquid Biobsy (LB) in monitoring ctDNA in pts receiving TDXd in the national Expanded Access Program was conducted. Methods: In this prospective study, LB for ctDNA analysis (evaluating ‘per pt’ ctDNA species and Variant Allelic Frequencies, VAF) was performed using 52-gene targeted NGS panel, in patients who progressed after two or more prior anti-HER2-based regimens and candidates to TDXd (3-weekly 5.4 mg/kg). This preliminary analysis reports data referring to Time-0 (T0) and T6 (cycle 6) assays. Results: From April 2021, LBs were collected for 14 pts and to date 8 are evaluable for LB. Median pts’ age was 59 yrs (range 38-72) and median number of previous anti-HER2 lines was 6 (2-11); 4 pts had Pertuzumab/Trastuzumab plus taxane as first-line and all pts received TDM-1. Median cycles of TDXd administered was 7.5 (1-10). At T0, 5/8 pts had at least one detectable ctDNA specie, and the remaining 3 developed at least one ctDNA at T6. ctDNA species and VAFs ranged from 1 to 5, and A0.1% to 68.94%, respectively. Decreases and increases were observed simultaneously in the same pt. The former varied from marginal to drastic, whereas the latter were invariably below 0.5% in VAFs. ctDNA monitoring was possible in 8/8 pts and at T6 ctDNA progression was detectable in 5/8 pts. Two pts displayed multiple HER2 copy number variations. Conclusions: The early results of this study suggest that considerable ctDNA burden, marked clonal complexity, and variable clonal response to TDXd can be found in pretreated HER2 positive patients, who progressed after antiHER2 therapies. Although the very small sample, this complex tumor evolution is surprising in light of the bystander payload effect of TDXd.