SUMMARY – PROJECT 3 By delaying surgery until after neoadjuvant chemotherapy (NAC), it is possible to assess the impact of therapy on breast tumors in real time. Women with aggressive disease who respond well to NAC have a better prognosis than women who fail to respond. There needs to be a way to identify high risk non-responders early in treatment and determine what alternative or additional treatments may be of benefit to them. Prior molecular profiling studies have identified markers that are predictive of chemotherapy response or outcome using pre- treatment biopsies. However, treatment induces significant changes in tumor biology; and some of these changes are associated with response to therapy. Thus, we hypothesize that interrogating the dynamic molecular changes over the course of treatment will enable us to better characterize resistance mechanisms and improve our ability to predict non-response (or sensitivity). In this Project, we will use (or generate as needed) next generation sequencing (mRNA and DNA) and reverse phase protein arrays (protein/phospho- protein) to perform comprehensive molecular characterization of serial biopsies from I-SPY 2 patients treated with graduated experimental agents. In Aim 1 we will evaluate druggable/actionable pathways in residual disease of non-responding patients undergoing treatment with standard or targeted agents/combinations. In Aim 2 we will incorporate early treatment-induced changes to improve predictive models of treatment resistance/sensitivity. In Aim 3 we will leverage this data compendium to develop a dynamic portrait of how key molecular pathways evolve as patients move from pre-treatment through to surgery in the context of treatment and receptor subtype. Our goal is to determine whether the residual disease characteristics identified in Aim 1 are evident at the early treatment or pre-treatment time points, and to assess the stability of early predictive/resistance signals. These efforts will inform both mid-treatment assessments of patient response to therapy and the development of a molecularly rationalized strategy for switching predicted non-responding patients to more effective treatment, developed as part of other Projects within the overall Program Project.