Background: Delayed type hypersensitivity (DTH) skin tests to GP2 were conducted in the randomized, active-controlled, single-blinded, multicenter Phase IIb trial investigating GLSI-100 (GP2+GM-CSF) administered in the adjuvant setting to node-positive and high-risk node-negative breast cancer patients with tumors expressing HER2. Previously it has been reported that subjects with baseline response to GP2 by DTH were more likely to experience a recurrence sooner than those without a positive baseline response. It was of interest to determine if this response was independent of other known prognostic factors. Methods: Patients were randomized and received GLSI-100 (500 mcg GP2: 125 mcg GM-CSF) or control (GM-CSF only) via 6 intradermal injections every 3-4 weeks as part of the Primary Immunization Series (PIS) for 6 months and 4 booster injections every 6 months thereafter. GP2 DTH skin tests were placed at baseline and after the 6th dose. After 48-72 hours, the largest perpendicular diameters of induration were measured and the orthogonal mean was calculated. Induration of over 5mm was considered a positive response. The relationship between prognostic factors such as demographic and disease characteristics and the baseline DTH response were investigated by logistic regression. Results: The study enrolled 180 patients who were HER2 3+ positive and low HER2 expressors (1-2+). After 5 years of follow-up, the Kaplan-Meier estimated 5-year DFS rate in the 46 HER2 3+ patients treated with GLSI-100, if completing the PIS, was 100% versus 89.4% (95% CI:76.2, 95.5%) in the 50 patients treated with GM-CSF (p = 0.0338). Just under 23% of subjects experienced an immune response to GP2 by DTH at baseline. The incidence was similar in patients who were HER2/neu positive and those considered low expressors and was equally distributed across GLSI-100 and placebo-treated subjects. Known prognostic factors such as hormone receptor status, stage at presentation, node status, etc. were evenly distributed across subjects that experienced a baseline DTH response to GP2 and those who did not. Each prognostic factor was investigated in a logistic model to determine if it predicted baseline DTH immune status. The prognostic factors did not predict baseline DTH immune response in the logistic models. There appears to be no correlation between baseline immune response to GP2 and known prognostic factors. Conclusions: Baseline GP2 immune response as measured by the delayed-type hypersensitivity test may be an independent prognostic factor for recurrence. Knowledge of this GP2 immune response may identify a patient with increased risk of rapid recurrence. Clinical trial information: 00524277.