31 Background: Therapeutic vaccine is emerging as a potentially efficacious and safe treatment for cancer patients. However, markers are not available to identify patients more likely to benefit from this treatment. Aim of this retrospective study was to analyze before treatment immune subsets that correlated with clinical outcome in metastatic breast cancer patients treated with docetaxel alone or in combination with vaccine. Methods: We applied multi-color flow cytometry analysis of PBMCs harvested prior to treatment from patients (n=43) enrolled in a small randomized phase II study of docetaxel alone (n=20) or in combination with PANVAC-V (Vaccinia) and PANVAC-F (Fowlpox) encoding for the tumor-associated antigens CEA and MUC-1, along with a TRIad of COstimulatory Molecules (B7-1, ICAM-1, and LFA-3; called TRICOM) (n=23). Frequency of more than 200 immune sub-populations before treatment was measured by flow cytometry. Each of the resulting subsets was ranked in tertiles. For immune subsets that correlated directly with PFS, 2 points were assigned if the frequency fell in the highest tertile, 1 point if in the middle, and 0 if in the lowest tertile. For subsets that correlated inversely with PFS, the points were assigned in the opposite order. An immunoscore was calculated based on the sum of points assigned to each subset. Log-Rank analysis, with the cutoff based on the median of the immunoscores, was performed to evaluate differences in PFS between patients with a low and high immunoscore. Results: In vaccine plus docetaxel arm, 10 immune subsets from PBMCs before treatment correlated with PFS and were used for the calculation of the immunoscore. Patients with an immunoscore above the median showed a statistically significant longer PFS compared to those with lower score in vaccine plus docetaxel arm (p<0.001, HR=0.1466, 95% CI= 0.0478-0.4375) but not in docetaxel alone arm (p=0.097, HR=0.418, 95% CI=0.147-1.172). Conclusions: Calculation of an immunoscore from PBMCs before treatment based on flow cytometry screening of immune subsets may identify patients that will most likely benefit from vaccine combination immunotherapy.