Background: Everolimus, which inhibits mTOR, is used to treat metastatic ER-positive breast cancer after aromatase inhibitor failure. Because not all patients benefit and toxicity can be prohibitive, it is critical to identify biomarkers of sensitivity to this treatment strategy. Methods: We studied 36 tumors from patients enrolled on protocol NCT00570921 using fulvestrant and everolimus for metastatic ER-positive breast cancer. There were 15 pretreatment tumors from 11 patients who experienced benefit and 14 from 10 patients who did not. DNA was extracted from FFPE tumor tissue and subjected to next-generation-based comprehensive genomic profiling using the FoundationOne assay and alterations were then compared. Results: 9 out of 11 responders (82%) had at least one genomic alteration in the PI3K/AKT/mTOR pathway vs. 7 out of 10 nonresponders (70%). Mutations detected in the responder group were 7 PIK3CA and 1 AKT1, and 1 PTEN truncation, while in nonresponders there were 5 PIK3CA and 2 AKT1 mutations, and 1 RICTOR amplification (in association with a PIK3CA mutation). Interestingly, none of the responders had more than one alteration in individual components of this pathway, while 2 of the nonresponders had more than one alteration observed (one with a concomitant PIK3CA mutation and AKT3 amplification, and one with a PIK3CA mutation and RICTOR amplification). Other notable alterations include mutations in TP53 and GATA3, and amplification of CCND1, with no specific association with everolimus benefit. Additional analysis and frequency of other alterations will be presented in greater detail. Conclusions: In this small study, we did not identify predictors for lack of benefit from everolimus, although it appears that the presence of multiple aberrations in the PI3K/AKT/mTOR pathway may promote de novo resistance. It is possible that genomic alterations alone may not adequately explain breast cancer response to this strategy without functional signaling analysis. Alternatively, it is possible that everolimus benefit in endocrine resistance may be independent of PI3K/mTOR signaling altogether and its mechanism of action is yet to be fully determined.