Abstract: A time course study was designed to investigate the dynamics of copy number aberrations in tumor DNA during treatment of breast cancer patients. A phase II randomized clinical trial of Her2 negative breast cancer patients was conducted, with patients being treated with neoadjuvant chemotherapy (FEC and taxane), with or without bevacizumab. Serial biopsies were obtained at time of diagnosis, at 12 weeks after treatment with FEC +/- bevacizumab, and at 25 weeks after treatment with taxane +/- bevacizumab. Tumor DNA alterations and tumor percentage were studied over time, and substantial differences were observed with some tumors changing mainly between diagnosis and 12 weeks (after the FEC cycle), others between 12 and 25 weeks (taxanes), and still others changing in both time periods. In both treatment arms, good responders (GR) and non-responders (NR) displayed significant difference in genomic instability index (GII) at time of diagnosis. In the Combination arm at time of diagnosis, 25 loci harbored copy number alterations, which were significantly different between the GR and NR. An inverse aberration pattern was observed between the two extreme response groups at 6p22-p12 for patients in the Combination arm. In most cases, tumors that retained aberrations at all time points did not decrease in size. Signs of subclonal reduction were observed, with some aberrations disappearing and others being retained during treatment. In both treatment arms an increase in subclonal amplification was observed at 6p21.1, the locus which contains the VEGFA gene targeted by bevacizumab and was associated with good response. Significant decrease of frequency of subclones carrying gains at 17q21.32-q22 was observed at 12 week, with the peak occurring at TMEM100, an ALK1 receptor signaling-dependent gene essential for vasculogenesis. This implies that cells bearing amplifications of TMEM100 are particularly sensitive to the treatment regime. Taken together, these results suggest that heterogeneity and subclonal architecture influence the response to targeted treatment in combination with chemotherapy, with possible implications for clinical decision making and monitoring of treatment efficacy.