e12518 Background: Despite its purported prognostic significance in breast cancer, Ki67 index assessment remains poorly standardized and features high discordance rates amongst pathologists. Moreover, its clinical utility is presently confined to low stage, ER+/HER2- tumors in determining the advisability of adjuvant chemotherapy. Unfortunately, established cut-offs limit its utility to tumors with either high or low Ki67 index extremes. While Ki67 is a marker of actively dividing cells, it fails to capture the detail of cell cycle phase and dynamics which could be informative in terms of prognosis and tumor sensitivity to specific therapies (e.g. CDK4/6 inhibitors). This study therefore developed two novel indices as surrogates of (i) Ki67 index and (ii) quiescent cell population load (QPL). Methods: Breast cancer (comprising evenly distributed hormone receptor/HER2 status cases) H&E slides ( n = 79 cases/108 slides) were digitized on an Aperio DT3 scanner, and used to extract surrogate Ki67 and QPL indices. Sections were also stained for Ki67 by immunohistochemistry (IHC) using a clinically validated assay and digitized. Whole slide image (WSI) tumor Ki67 counts were performed on QuPath and used to validate the surrogate Ki67 index (Cohen’s kappa score). Indices (i) and (ii) were related to progression free survival (PFS). Survival analyses were performed using Kaplan-Meier (KM; with median cut-off) and Cox Proportional Hazards (as a continuous variable) models. Results: The surrogate Ki67 index showed good concordance with IHC scores (kappa = 0.76; 95%CI 0.61-0.91; P< 0.00001). However, this surrogate index performed better as a prognostic indicator of PFS compared to conventional Ki67 IHC (KM P = 0.048; HR = 1.35, P = 0.015 vs. KM P = 0.70; HR = 1.23, P = 0.08). Prognostically, the QPL index outperformed both Ki67 indices (KM P = 0.03; HR = 3.22, P = 0.001). Conclusions: We have developed two novel surrogate indices of Ki67 and QPL that can be readily automated to analyze H&E breast cancer WSIs. Our results show that both outperformed conventional Ki67 IHC evaluation in terms of prognostication, applied across molecular subtypes, improved informativeness of mid-range Ki67 index calls, and could potentially have predictive merit in selecting patients for cell cycle targeted therapies such as CDK4/6 inhibitors.

e12517 Background: HER2 amplification or overexpression is negative prognostic factor that occurs in circa 15% of primary invasive breast cancers, with positive results opening up eligibility for HER2 targeted (e.g. trastuzumab) combination therapy. However, HER2 intratumoral heterogeneity has been proposed as an explanation for the development of resistance to anti-HER2 targeted therapy. The aim of this retrospective study was to test the prognostic value of a HER2 heterogeneity index on ER+ breast cancer H&E whole slide images (WSIs). Methods: ER+ breast cancers slides ( n=60 cases) of known HER2 status (immunohistochemically assessed over the 0 to 3+ range, with HER2 2+ borderline cases validated by fluorescence in situ hybridization) were digitized on an Aperio AT3 scanner. A subset of these patients ( n=29) was accounted for by HER2 amplified/borderline cases. A HER2 heterogeneity index was tested and compared to standard clinical HER2 status in these cases and related to progression free survival (PFS). Survival analyses were performed using Kaplan-Meier (KM; with median cut-off) and Cox Proportional Hazards (as a continuous variable) models. Results: The HER2 heterogeneity index performed better in terms of prognostication than clinical HER2 status alone in relation to PFS (KM P=0.019; HR 8.06, P=0.01 vs. KM P=0.02; HR 6.75, P=0.048). These differences were more pronounced in the HER2 amplified/borderline tumor subset, where the HER2 heterogeneity index outperformed clinical HER2 status (KM P=0.04; HR 6.38, P=0.001 vs. KM P=0.10; HR 0.64, P=0.20). Conclusions: These findings highlight the merit of identifying HER2 heterogeneity from H&E slides alone and establish this index as a robust prognostic factor in determining PFS in ER+ breast tumors. Future work will extend this investigation into ER- tumors and evaluate patient-specific tumor responses to anti-HER2 targeted therapy.