Background: GP2 is a HER2 derived, HLA-A2⁺-restricted immunogenic peptide designed to stimulate CD8⁺T cells to recognize tumor cells with any level of HER2 expression (IHC 1-3+). Accrual to a prospective, randomized, multi-center, phase II trial of the GP2 vaccine for prevention of breast cancer recurrence has completed. Here, the planned primary analysis of disease-free survival (DFS) is presented. Methods: HLA-A2⁺ node positive or high-risk node negative breast cancer patients (pts) with any level of HER2 expression rendered disease-free by standard of care therapy (to include trastuzumab where appropriate) were randomized to receive GP2+GM-CSF (VG) or GM-CSF (CG) alone. Pts received 6 monthly inoculations (primary vaccine series = PVS) followed by 4 boosters administered every 6 months. The Kaplan Meier method was used for statistical analysis. The intention-to-treat (ITT) population is defined as the entire randomly assigned population. The per-treatment (PT) group excluded pts who recurred during the PVS or developed a second malignancy. A pre-specified subgroup analysis was performed based on HER2 expression level. HER2 overexpression (OE) is defined as IHC 3⁺or FISH >2.2. Results: With 89 VG and 91 CG pts enrolled and vaccinated, there are no differences between groups with respect to age, node positivity, tumor size, grade, ER/PR status, and HER2 expression (p>0.05). The vaccine has been well tolerated with toxicities comparable between the VG and CG. Only one grade 3 local and systemic toxicity reaction has been reported in the VG. At 34 (1-60) month median follow-up, DFS was compared in the ITT (85% VG v 81% CG, p = 0.57) and PT (94% VG v 85% CG, p = 0.17) populations. In OE patients (51 VG and 50 CG) DFS was 94% VG v 89% CG, p = 0.86 (ITT) and 100% VG v 89% CG, p = 0.08 (PT). Conclusions: GP2+GM-CSF is a novel vaccine that is safe and well tolerated. This phase II trial demonstrates potentially greater benefit in pts with HER2 OE tumors, in whom there have been no recurrences in the PT group. This may be due to synergism with trastuzumab therapy, thus justifying a phase III trial evaluating GP2 administered in the adjuvant setting to a HER2 OE population. Clinical trial information: NCT00524277.