Dendritic cells transfected with heat-shock protein 70 messenger RNA for patients with hepatitis C virus-related hepatocellular carcinoma: a phase 1 dose escalation clinical trial

Abstract

Background: We previously reported overexpression of heat-shock protein (HSP) 70 in hepatitis C virus (HCV)-related hepatocellular carcinoma (HCC) using proteomic profiling and immunohistochemical staining (IHS). This suggested that HSP70 could be a molecular target for treatment of HCC.

Methods: Twelve patients with HCV-related HCC were enrolled in a phase 1 clinical trial. Dendritic cells (DCs) transfected with HSP70 mRNA (HSP70-DCs) induced by electroporation were injected intradermally. Patients were treated three times every 3 weeks. The number of HSP70-DCs injected was 1 × 10(7) as the lowest dose, then 2 × 10(7) as the medium dose, and then 3 × 10(7) as the highest dose. Immunological analyses were performed.

Findings: No adverse effects of grade III/IV, except one grade III liver abscess at the 3 × 10(7) dose, were observed. Thus, we added three more patients to confirm whether 3 × 10(7) is an appropriate dose. Eventually, we chose 3 × 10(7) as the recommended dose of DCs. Complete response (CR) without any recurrence occurred in two patients, stable disease in five, and progression of disease in five. The two patients with CR have had no recurrence for 44 and 33 months, respectively. IHS in one patient who underwent partial hepatectomy showed infiltration of CD8+ T cells and granzyme B in tumors, indicating that the dominant immune effector cells were cytotoxic T lymphocytes with tumor-killing activity.

Interpretation: This study demonstrated that HSP70-DCs therapy is both safe and feasible in patients with HCV-related HCC. Further clinical trials should be considered.

Fig. 1

a Chronological MRI of patient 1. Early phase of dynamic-enhanced EOB-MRI shows patient 1 with CR 1 month after treatment. Upper left image shows the pretreatment state with enhanced lesion. Upper right image shows the state of 1 month after treatment with disappearance of the former lesion. Lower left shows the state 1 year after treatment with the same state as before. The patient has had no liver tumor after 44 months of follow-up. b Chronological CT of patient 4. Early phase of dynamic CT shows patient 4 with CR 3 months after treatment. Upper left CT shows the pretreatment state with enhanced lesion. Upper right CT shows the state 1 month after treatment with a 37.5 % reduction in the former lesion. Lower left CT shows the state 3 months after treatment with complete disappearance of the lesion. Lower right shows the state 2 years after treatment with the same state as before. The patient has had no liver tumors as of a 38-month follow-up

Fig. 2

a Enzyme-linked immunospot analysis. The number of spots is shown. Time points tested were prior to treatment, after the first treatment, after the second treatment, and 1 month after the third treatment. Data are mean ± standard error. b Number of spots in ELISPOT assays at two time points. CR had 39 spots, SD had 8.4 (±7.4, SE), and PD had 7.2(±3.9, SE). c Population kinetics of immune effector cells in patients. Patients with CR and SD tended to have a higher percentage of CD4+/FoxP3+ T cells. In patients with CR and SD, the percentage of CD3−/CD56+ cells, which are supposed to be NK cells, were higher than in PD. For NKG2D-expressing NK cells, the percentages in the patients of CR and SD increased, but not in PD. CD57+ NK cells from the group of CR and SD had the same trend, indicating that activated NK cells were induced by treatment. However, these trends were not statistically significant

Fig. 3

Enhanced IFN-γ production and cytotoxic T lymphocyte activity in patient 12. PBMCs from patient 12 were analyzed by flow cytometry. a and d show the percentage of IFN-γ+/CD3+/CD8+ cells. b and e show the percentage of IFN-γ+/CD3−/CD56+ cells. c and f show the percentage of CD107a+/CD3+/CD8+ cells. a–c show data from responder PBMCs co-cultured with stimulator PBMCs without HSP70-mRNA (mRNA(−)). d–f show data of responder PBMCs co-cultured with stimulator PBMCs encoding HSP70-mRNA (mRNA(+)). The production of IFN-γ and the expression of CD107a were observed in both CTL and NK cells. In CTL, these were relatively highly produced and expressed

Fig. 4

Immunohistochemical expression of HSP70, HLA class 1, CD8, granzyme B, and CD56 in patient 12. The tumor site of the resected specimen expresses HSP70 (a) and HLA class 1 (b) by IHC. At the site of these expressions, dense CD8+ T cells had infiltrated intratumorally and were widely spread (c). These dense infiltrated cells expressed granzyme B as well (d). A few CD56+ cells were detected intratumorally (e)