Thirty-nine and 30 patients received RFA 1 and 2-4 times, respectively. After treatments, HCC recurrence was evaluated with dynamic CT or MRI every 3-4 months. All patients gave written informed consent to participate in the study in accordance with the Helsinki declaration, and this study was approved by the regional ethics committee (Medical Ethics Committee of Kanazawa University). Blood samples were tested for hepatitis B surface antigen and hepatitis C virus (HCV) antibody using commercial immunoassays (Fuji Rebio, Tokyo, Japan). The patients with HCV antibody were tested for serum HCV RNA by real-time PCR (Roche, Tokyo, Japan), and 49 of Epigenetics inhibitor 52 patients with HCV antibody were HCV RNA–positive.

HLA-based typing of PBMCs from patients and normal blood donors was performed using reverse sequence-specific oligonucleotide analysis with polymerase chain reaction (PCR-RSSO). The serum alpha-fetoprotein (AFP) level was measured via enzyme immunoassay (AxSYM AFP, Abbott Japan, Tokyo, Japan), and the pathological grading of tumor cell differentiation was assessed according to the Daporinad cost general rules for the clinical and pathological study of primary liver cancer.8

The severity of liver disease was evaluated according to the criteria of Desmet et al. using biopsy specimens of liver tissue, where F4 was defined as cirrhosis.9 Fifty-five patients who participated in the present study received liver biopsy with RFA. Another 14 patients received liver biopsy 1-3 years before RFA. Eleven peptides that we

previously identified as being useful for analysis of immune response in HLA-A24–positive HCC patients were selected.10-13 Human immunodeficiency virus (HIV) envelope-derived peptide (HIVenv584)14 and cytomegalovirus (CMV) pp65-derived peptide (CMVpp65328)15 were also selected as control peptides. Peptides were synthesized at Sumitomo Pharmaceuticals MCE (Osaka, Japan). They were identified using mass spectrometry, and their purities were determined to be >90% by analytical high-performance liquid chromatography. PBMCs were isolated before and 2-4 weeks after HCC treatments as described.11 In the patients who received RFA 2-4 times, PBMCs were obtained 2-4 weeks after the final treatment. In some patients, PBMCs were also obtained 24 weeks after RFA. PBMCs were resuspended in Roswell Park Memorial Institute 1640 medium containing 80% fetal calf serum and 10% dimethyl sulfoxide and cryopreserved until use. Interferon-γ (IFN-γ) ELISPOT assays were performed as described.11 Negative controls consisted of an HIV envelope–derived peptide (HIVenv584).14 Positive controls consisted of 10 ng/mL phorbol 12-myristate 13-acetate (PMA, Sigma) or a CMVpp65-derived peptide (CMVpp65328).15 The colored spots were counted with a KS ELISpot Reader (Zeiss, Tokyo, Japan). The number of specific spots was determined by subtracting the number of spots in the absence of an antigen from the number in its presence.