Background: The novel HLA-A*2402-binding peptides derived from KIF20A (RAB6KIFL) belong to the kinesin superfamily of motor proteins, which play critical roles in the trafficking of molecules and organelles during the growth of pancreatic cancer. A phase I cancer vaccination trial using KIF20A determined its safety and immunogenicity in advanced pancreatic cancer (PC) patients (J Immunother 2014;37:36-42). We further performed a phase II trial using not only KIF20A but also an antiangiogenic cancer vaccine targeting VEGFR1 (vascular endothelial growth factor receptor 1) and VEGFR2. This revealed the safety and immunogenicity in advanced colorectal cancer as presented at the 2011 ASCO Oral Abstract Session (J Transl Med. 2014; 12:63). We try to evaluate the benefit of the cancer vaccination in combination with gemcitabine (GEM) as first-line therapy in advanced PC patients. Methods: Sixty-eight chemotherapy naïve PC patients were enrolled to evaluate primarily the one year survival rate, and secondarily overall survival. Each of the three peptides was mixed with 1ml of Incomplete Freund’s adjuvant and subcutaneously administered weekly. GEM was administered at a dose of 1000 mg/m² on days 1, 8, and 15 in a 28-day cycle. All enrolled patients received the therapy without knowing the HLA-A status, and the HLA genotypes were revealed at analysis point and then, the endpoints were evaluated between the HLA-A*2402 positive and HLA-A*2402 negative group. Results: Between June 2012 and May 2013, a total of 68 patients were enrolled in this study. No severe adverse effects of grade 3 or higher related to these three peptides were observed. The one year survival rate was 30.3%. Median survival time was 9.2months. Response rate was 11.8% and disease control rate was 73.5%, respectively. The HLA genotype will be revealed in March 2015 and the endpoints compared between HLA-A*2402 positive and negative group will be presented at this meeting. Conclusions: These results suggest that this combination therapy will be feasible and promising for the treatment of advanced pancreatic cancer. Clinical trial information: UMIN000008082.