Activation and dramatically increased cytolytic activity of tumor specific T lymphocytes after radio-frequency ablation in patients with hepatocellular carcinoma and colorectal liver metastases

Abstract

Aim: To assess if a specific cytotoxic T cell response can be induced in patients with malignant liver tumors treated with radio-frequency ablation (RFA).

Methods: Six Patients with liver metastases of colorectal cancer and 6 with hepatocellular carcinoma (HCC) underwent RFA. Blood was sampled before, 4 and 8 wk after RFA. Test antigens were autologous liver and tumor lysate obtained from each patient by biopsy. Peripheral T cell activation was assessed by an interferon gamma (IFNgamma) secretion assay and flow cytometry. T cells were double-stained for CD4/CD8 and IFNgamma to detect cytotoxic T cells. The ratio of IFNgamma positive and IFNgamma negative T cells was determined as the stimulation index (SI). To assess cytolytic activity, T cells were co-incubated with human CaCo colorectal cancer and HepG2 HCC cells and release of cytosolic adenylate kinase was measured by a luciferase assay.

Results: Before RFA SI was 0.021 (+/- 0.006) for CD4(+) and 0.022 (+/- 0.004) for CD8(+) T cells against nonmalignant liver tissue and 0.018 (+/- 0.005) for CD4(+) and 0.021 (+/- 0.004) for CD8(+) cells against autologous tumor tissue. Four weeks after RFA SI against tumor tissue increased to 0.109 (+/- 0.005) for CD4(+) and 0.11 (+/- 0.012) for CD8(+) T cells against HCC, and to 0.115 (+/- 0.031) for CD4(+) and 0.15 (+/- 0.02) for CD8(+) cells for colorectal metastases (P < 0.0001). No increased SI was observed with nonmalignant tumor tissue at all time points. Before RFA cytolytic activity against the respective cancer cells was low with 2.62 (+/- 0.37) relative luminescence units (RLU), but rose more than 100 fold 4 and 8 wk after RFA. Spontaneous release was < 2% of maximum release in all experiments.

Conclusion: Patients with primary and secondary tumors of the liver show a significant tumor-specific cytotoxic T-cell stimulation with a dramatically increased tumor specific cytolytic activity of CD8(+) T cells after RFA.

Figure 1

Stimulation indices of CD8⁺ T cells. The Box-Whisker-plots show the ratio of IFNγ secretion of stimulated vs unstimulated CD8⁺ T cells as determined by a cytokine secretion and capture assay before, 4 and 8 wk after RFA. White boxes represent the patients with HCC and grey boxes the patients with colorectal cancer metastases. Outlayers are marked with "a", extremes with "b". For significances refer to table 1.

Figure 2

Stimulation indices of CD4⁺ T cells. The Box-Whisker-plots show the ratio of IFNγ secretion of stimulated vs unstimulated CD4⁺ T cells as determined by a cytokine secretion and capture assay before, 4 and 8 wk after RFA. White boxes represent the patients with HCC and grey boxes the patients with colorectal cancer metastases. Outlayers are marked with o, extremes with *. For significances refer to table 1.

Figure 3

Adenylate kinase (AK) release of cultured tumor cells. Release of adenylate kinase (AK) from the human hepatoma cell line HepG2 and the hman colorectal carcinoma cell line CaCo co-incubated with PBMC isolated before, 4 and 8 wk after RFA. The release of AK correlates with the cytotoxic and NK T cell mediated damage to the cultured cells. HepG2 cells were used for PBMC from the patients with HCC and CaCo cells for the patients with metastasis of colorectal carcinoma.