Background: The HERmarkBreast Cancer Assay (Monogram Biosciences) is used as an adjunct to HER2 immunohistochemistry (IHC) and HER2 fluorescence in-situ hybridization (FISH) to determine the HER2 status of breast cancer (BC). Based on the high degree of concordance of HERmark with centrally determined HER2 status by IHC/FISH, it is expected that HERmark positive BC patients would realize greater benefit from trastuzumab treatment than HERmark negative BC patients. In the current study, this hypothesis was confirmed using a cohort comprised of: (1) HER2 positive BC patients treated before the availability of trastuzumab, (2) trastuzumab-treated HER2 positive BC patients and (3) HER2 negative BC patients. Methods: HERmark was performed retrospectively on formalin-fixed, paraffin-embedded tumors derived from 305 metastatic breast cancer patients in the Munich Cancer Registry with a median follow up of 10 years. Cases were evenly divided between trastuzumab-untreated HER2 positive (n = 91), trastuzumab-treated HER2 positive (n = 115) and HER2 negative (n = 99) tumors. Cutoffs for HERmark positive and negative status were pre-specified (Huang, Am J Clin Pathol, 134:303 2010). Results: HER2 positive patients treated with trastuzumab had longer overall survival (OS) than those not treated with trastuzumab, as expected (HR = 0.35; p < 0.0001). As reassessed by HERmark, HER2 positive patients treated with trastuzumab experienced longer OS (HR = 0.33; p = 0.0005). In contrast, the benefit of trastuzumab treatment in HERmark negative patients was not statistically significant (HR = 0.55; p = 0.13). Additionally, there was a statistically significant interaction between the degree of trastuzumab benefit and quantitative levels of HER2 expression within the HERmark positive subset (interaction p = 0.032). Potential correlations between additional markers, including p95HER2 and HER3, and treatment outcome are currently being assessed. Conclusions: The HERmark assay identified breast cancer patients who benefited from trastuzumab treatment. Within this group, increased levels of quantitative HER2 expression correlated with increased degree of benefit from trastuzumab therapy.