Project Description Endocrine therapy provides significant improvement in long-term outcomes for patients with estrogen receptor- positive (ER+) breast cancer (BC), also known as luminal BC. However, therapy-resistant metastatic disease develops in 20-25% of patients. Upregulation of the HER2 growth factor receptor represents a common escape strategy used by cancer cells to survive and continue to proliferate in an ER-independent manner. To reduce mortality from BC, more accurate diagnostic tools are needed to guide tailored supplementary first line therapy for patients at elevated risk of progression. Clinicians need accurate tumor tests to guide administration of supplementary agents selectively to likely responders and avoid toxicity in non-responders. This project will explore innovative automated histo-cytometric techniques to analyze expression of targetable, therapy resistance-relevant protein HER2 in tumors pre- and post-neoadjuvant endocrine therapy. The strategy is centered on developing the most sensitive metric of HER2 protein expression in ER+/HER2- BC to identify tumors that undergo HER2 upregulation in response to endocrine therapy, as well as identify HER2- associated proliferative ‘hotspots’. We will use multiplex immunofluorescence labeling of histological tumor sections to detect and analyze cellular levels and patterns of HER2 protein expression alone or in conjunction with Ki67 within the tumor tissue. The metrics explored will include marginal and spatial distributions of cellular HER2 signals to capture heterogeneity of expression. Pre- and post-neoadjuvant tumor biopsies and surgical resections from our recently completed neoadjuvant endocrine therapy clinical trial will support the proposed histo-cytometric analyses. We further seek to identify tumor-selective HER2-driven endocrine resistance in BC by identifying the metric of HER2 signal in therapy-naïve tumor biopsies that is most predictive of subsequent upregulation of HER2 or HER2-associated proliferative hot-spots in response to endocrine therapy. The proposed research is expected to yield new diagnostic metrics for rational recruitment of patients into a planned follow-on randomized clinical trial of tailored supplementary HER2-targeted treatment for aggressive ER+ BC. The work has direct potential to positively impact BC mortality.