Radiotherapy: Patients with liver metastasis, portal vein tumor thrombosis and pulmonary metastasis were treated with precise radiotherapy [planning gross target volume (PGTV), ten fractions at 5 Gy each; planning target volume (PTV), ten fractions at 2.5 Gy each] combined with PPV-DC-CTL; patients with bone metastasis were treated with precise radiotherapy of bone (PGTV, ten fractions at 4 Gy each; PTV, ten fractions at 3 Gy each) combined with PPV-DC-CTL; patients with peritoneal metastasis were treated with precise TomoTherapy of the peritoneum (PTV, two fractions at 0.5 Gy each, BID) combined with PPV-DC-CTL.
Selection of PPV peptides: First, peptide candidate library, including mutated peptides and highly expressed peptides, was established according to the gene mutation and expression spectra of HCC and previous studies about PPV. Peptides for vaccination of every specific patient were selected from the peptide candidate library with the consideration of the preexisting immunity of the host before vaccination (Figure 1). The detailed procedure was described in references[7,9] and the personalized peptides for these nine patients are as follows:
P1: CORE-18, MUC-12, KRAS-A02-G13D1, PSCA-76
P2: PI3KCA-A02-H1047L-1, CORE-35, WTP53-149, AFP-137
P3: EGFR-800, KRAS-A11-G13D, CYPB-84, CTNNB1-A11-S45F
P4: KRAS11-12C, EGFR-54, AFP-403, Survivin28-80
P5: AFP-357, VEGFR2-169, KRAS-A11-12C, MRP3-1293
P6: KRAS-A11-12D, CTNNB1-A11-41A, CTNNB1-A11-S45F, KRAS-A11-12R
P7: SART3-109, CORE-18, PSCA-7, hTERT-540
P8: AFP-357, KRAS-A11-12D, VEGFR2-169, PSCA-776
P9: CTNNB1-A11-S45F, CTNNB11-41A, CTNNB11-45P, EGFR-54
Collection of peripheral blood mononuclear cells (PBMC) and transfusion of DC-CTL: PPV peptides were load on DC (D0), which were sorted from PBMC and then these DC were infused back to patients after culture for 7 d in vitro (D7). To obtain the CTL, we used PPV and PPV-DC to stimulate the T cells. In 12 to 15 days, these CTL were transfused to patients (D12-15) (Figure 2). The above steps were defined as one cycle and it was repeated every 21 d.
Figure 2 Time schedule of collection of peripheral blood mononuclear cells and transfusion of DC-CTL.
PPV: Personalized peptide vaccination; DC: dendritic cells; CTL: Cytotoxic lymphocytes; PBMC: Peripheral blood mononuclear cells.