Background: Wilms’ tumor gene (WT1) is overexpressed in almost all malignancies including PDAC, and is supposed to be most promising tumor-associated antigen in cancer immunotherapies. To investigate superiority of WT1 vaccine in combination with gemcitabine (GEMWT1) compared to GEM alone (GEM) for patients (pts) with advanced PDAC, we designed the randomized phase II trial. Methods: HLA-A*02:01 and/or A*24:02-positive pts with locally advanced or metastatic PDAC without prior treatment, or recurrence after surgery were enrolled. Pts were randomly assigned at a ratio of 1:1 to GEMWT1 or GEM. Pts in GEM were allowed to receive GEM + WT1 vaccine after disease progression. The primary end point was overall survival (OS) rate at 1 year. Secondary end points were progression free survival (PFS), WT1-specific immunogenic response, and safety. WT1 vaccine, which was composed of HLA-restricted 9-mer WT1 peptide (3mg/body) and Montanide ISA51 adjuvant, was intradermally injected on days 1 and 15, and GEM was administrated at 1,000 mg/m² on days 1, 8, and 15 in a 28-day cycle until disease progression. Results: A total of 91 pts were enrolled and 85 evaluable finally (GEMWT1, n = 42; GEM, n = 43). GEMWT1 improved 1-yr OS rate and prolonged PFS compared to GEM (Table). These were remarkable in pts with metastatic PDAC. WT1-specific immunity was assessed with delayed-typed hypersensitivity (DTH) to WT1 peptide and tetramer assay of WT1-specific cytotoxic lymphocytes (WT1-CTLs). Median PFS was 195, 102, and 100 days for DTH(+), DTH(-), and GEM, respectively [HR (90% CI), 0.51 (0.31 to 0.85), 0.80 (0.50 to 1.28), and 1]. WT1-CTLs increased in almost all DTH(+) pts. Conclusions: GEM + WT1 vaccine prolonged PFS in pts with advanced PDAC. Clinical efficacy of this combination therapy was associated with the induction of WT1-CTL responses. Clinical trial information: UMIN000005248.