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1 Limitation of Conventional Treatments and Advantage of Immunotherapy for Intermediate to Advanced HCC

Currently, conventional treatments for intermediate or advanced HCC are TACE or chemotherapy, respectively [1–3]. TACE is a procedure to inject an embolic agent with cytotoxic drug, resulting in ischemic necrosis of the tumor. The benefit of TACE in unresectable HCC was demonstrated in two randomized control trials [4, 5]. However, anti-tumor effect of TACE is limited in intrahepatic HCC lesions, and extra-hepatic lesions are not applicable for the TACE. Advanced HCCs with extra-hepatic lesions or vascular invasion in the liver are treated by oral molecular targeting drug, sorafenib. Discontinuation of the sorafenib treatment is often necessary due to the reasons other than progression of HCC, like liver dysfunction, skin disease and fluid retention [6]. Arterial infusion of cytotoxic drug, 5FU and cisplatin, is another option as a treatment for intermediate or advanced HCC [7]. Although the conventional treatments prolong overall survival, those are limited in patients with good hepatic reserve. Immunotherapy should be more suitable for HCC patients with liver dysfunction, because the antigen-specific immunotherapy specifically targets cancer cell, not background hepatocytes. The first DC-based peptide vaccine for castration-resistant prostate cancer patients, sipuleucel-T, approved by Food and Drug Administration (FDA) in United States caused no severe adverse effects [8]. Sipuleucel-T is comprised of DC loaded with prostatic acid phosphatase (PAP) peptide and specifically targets prostate cancers. Sipuleucel-T significantly prolonged overall survival (median survival time; MST 25.8 months) compared with control (MST 21.7 months). The success of sipuleucel-T shed a light in efficiency of immunotherapy and possibility of the therapy to prolong survival in other cancers. Because HCC patients often have impaired hepatic reserve, HCC-specific treatments with little adverse effects are really desired. Thus, HCC-specific immunotherapy is one of the promising treatments.

2 The Current Cell-Based Immunotherapy Trials

Four randomized control trials (RCT) of cell-based immunotherapy for HCC were reported (Table 5.1). All trials were based on infusion of the lymphocytes stimulated with IL-2 in addition to anti-CD3 or other cytokines. Kawata et al. first reported safety of lymphokine-activated killer cells (LAK) which derived from peripheral blood mononuclear cells (PBMC) and then were stimulated with IL-2, but the infusion of LAK didn’t improve both disease-free survival (DFS) and overall survival (OS) [9]. Methods to activate PBMC was then improved by adding various cytokines and anti-CD3, and the activated cells were called cytokine-induced killer cells (CIK) which include activated non-specific αβT cells and CD3+CD56+ NKT cells. CIK treatment first showed clinical benefit in the HCC patients after radical haptic resection in 2000. Takayama et al. reported that lymphocytes generated from PBMC by the stimulation with IL-2 and anti-CD3 significantly prolonged disease-free survival compared with control arm [10]. Hui and colleagues also reported infusion of CIK generated by stimulation with IL-2, anti-CD3, IFN-γ and IL-1α increased DFS rate [11]. Weng et al. also demonstrated clinical benefit of CIK after radical hepatic resection of HCC as the prolonged DFS [12]. However, all 4 RCT failed to prolong OS by infusion of cytokine-stimulated lymphocytes including LAK and CIK. Two meta-analyses of adoptive immunotherapy in postoperative HCC also revealed no benefit in OS although did benefit in DFS [13, 14]. Prolongation of DFS by CIK was only achieved after radical resection of HCC. Kawata’s study targeted advanced HCC by LAK with adriamycin, resulting in no effect in DFS. Benefit of CIK therapy may be limited in prolongation of DFS of patients who undergo radical resection. Novel cell-based immunotherapy which prolongs OS even in advanced HCC, hopefully chemotherapy-resistant HCC, is really desired to develop.

Table 5.1 Randomized control trials of cell-based immunotherapy for HCC
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Clinical trials of immunotherapy using other cells than CIK or LAK are ongoing or were completed according to the databases of clinical trials. According to ClinicalTrial.gov, eight cell-based immunotherapies were conducted as clinical trials so far (Table 5.2). Innate lymphoid cells including NK cells, NKT cells and γδT cells were used as effector cells in 3 trials. No evidence was demonstrated by phase III clinical trials with those innate lymphoid cells so far in any types of cancer, but in theory NK cells, NKT cells or γδT cells can target lack of MHC class I on cancer cells or phosphate antigen induced by abnormal metabolism in the tumor microenvironment. Tumor-infiltrating lymphocytes in 2 trials or dendritic cells in 2 trials were used to target cancers by tumor-specific immune responses.

Table 5.2 Clinical trials of immunotherapy for HCC registered in ClinicalTrial.gov
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In Japan, seven cell-based immunotherapies were conducted as clinical trials so far according to UMIN (Table 5.3). Two trials were γδT cell-based immunotherapy. Four trials were DC-based immunotherapy which intended to induce HCC-specific immune responses. As tumor antigen, tumor lysate was pulsed in DC in 1 trial (UMIN000005820). To load more specific tumor antigen in DC, tumor antigen mRNA-encoding DC was used in 1 trial (UMIN000005836). DC stimulated with OK-432, inactivated streptococcus pyogenes, were used in 1 trial to activate DC (UMIN000001701). Thus, various cell types and techniques were examined in the current clinical trials to enhance anti-tumor immune responses against HCC.

Table 5.3 Clinical trials of immunotherapy for HCC registered on UMIN in Japan
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3 DC-Based Immunotherapy for HCC

Among various types of cell-based immunotherapy, sipuleucel-T is the only therapy which was verified to prolong overall survival of prostate cancer in phase III trial [7]. Sipuleucel-T comprises DC pulsed with tumor-specific antigen, PAP. If tumor-antigen is known, DC-based immunotherapy is a promising therapy to treat HCC according to the success in sipuleucel-T. Palmer reported that phase II trial of tumor lysate-pulsed DC infusion for patients with unresectable advanced HCC achieved disease control rate 28 % (combined partial response and stable disease) [15]. In the trial, hepatocellular carcinoma cell line HepG2 was used for source of tumor lysate. What is the best to load in DC, autologous tumor lysate, cell line-derived lysate, or HCC-specific peptides, is still controversial and to be determined in future.

We activated autologous DC from cancer patients with OK-432 before the infusion [16]. OK432-activated DC expressed high level of co-stimulatory molecules like CD80, CD83, and CD86. OK-432-stimulated DC also highly produced IL-12p40 and IFN-γ and revealed high tumoricidal activity against HCC cell lines like Hep3B and PLC/PRF/5 in in vitro assay. After TAE OK-432-stimulated DC were injected in hepatic artery via catheter. OK432-stimulated DC significantly prolonged recurrence-free survival compared to historical TAE control (P = 0.0017; Fig. 5.1). Although IFN-γ responses with PBMC specific to hTERT which is highly expressed by HCC as tumor antigen didn’t significantly increase after the OK-432-stimulated DC treatment, serum level of TNF, IL-9 and IL-15 was significantly higher in OK-DC group than TAE control patients. In mouse model of OK-DC treatment for HCC, OK-DC clearly induced tumor-specific immune responses against colon cancer cells [17]. It is still controversial that under which mechanism OK-432-stimulated DC exerts anti-tumor effect, tumor-specific immunity or non-specific direct killing. Although the mechanisms underlying the prolonged DFS caused by OK-432-stimulated DC treatment should still be elucidated, DC-based immunotherapy is a promising treatment for HCC.

Fig. 5.1
figure 1

Recurrence-free survival of patients with hepatocellular carcinoma treated with trans-catheter arterial embolization (TAE) with or without infusion of OK-432-stimulated DC. N = 13 in TAE + OK-432-DC group and N = 11 in TAE historical control group

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4 Strategies to Enhance the Effects of DC-Based Cancer Vaccine

In addition to the success in sipuleucel-T for prostate cancer, DC-based vaccines are showing promising results in the clinical trials for other types of cancer. Phase III trial of DCVax-L (Northwest Biotherapeutics), NCT01582672 or NCT01875653 are underway for glioblastoma, renal cell carcinoma or melanoma, respectively [17, 19]. Based on the promising results in DC-based vaccine in various cancers, further strategies to enhance the effect of DC-based therapy are now developing. Combined immunotherapy with DC and CIK for various cancers is also ongoing. Meta-analysis of non-randomized trial demonstrated that chemotherapy + DC + CIK therapy increased the 3-year overall survival rates (RR 11.67, 95 % CI 2.28 to 56.69, P = 0.003) and progression-free survival (RR 0.64, 95 % CI 0.34 to 0.94, P < 0.0001) in patients with non-small cell lung cancer compared with those treated with chemotherapy alone [20]. Infusing more than 2 immune cells might synergistically enhance anti-tumor immune responses. RCT is required to verify the clinical benefit of DC and CIK combination therapy.

One of the most exciting advances in recent cancer immunotherapy is the success of breaking immune checkpoints to prolong survival of cancer-bearing patients. Development of antibodies against negative regulators of T cell, CTLA-4 and PD-1, induced high clinical responses in various cancers [21–23]. Breaking tolerogenic aspects of tumor-infiltrating T cells might enhance effect of DC-based vaccine to induce tumor-specific cytotoxic T cell populations. Combining DC immunotherapy with anti-CTLA-4 antibody was feasible and well tolerated in advanced melanoma patients and induced high clinical responses compared to either treatment alone [24]. Blockade of PD-1 as well as inhibitory cytokine IL-10 or TGF-β may augment anti-tumor effect of DC-vaccine therapy [25].

Because massive cell death of the cancer releases damage-associated molecular pattern (DAMP) and subsequent acute inflammation accompanied with the production of cytokines and chemokines, it activates tumor-infiltrating DC [26]. This provides a rationale for combining DC-based therapy with radio-frequency ablation (RFA) or TACE which destroy large part of cancer tissues. As support of the idea, we reported that activation of endogenous DC population and subsequent anti-tumor T cell responses are induced by injection of ECI301, an active variant of CCL3, only if the injection was combined with RFA treatment of liver cancer [27]. Moreover, infusion of OK-432-stimulated DC prolonged DFS in combination with TAE in the clinical trial [16] and OS in combination with RFA in murine model [17]. Conventional chemotherapy also augments anti-tumor immune effects by innate cells activated by gut commensal bacteria and probably pathogen-associated molecular pattern (PAMP) [28]. Radiation increases release of lipopolysaccharide from gut to blood, resulting in activation of DC and the enhanced cytotoxicity of adoptively transferred T cells [29]. Thus, combination of DC-based vaccine and conventional cancer therapy including RFA, TACE, chemotherapy and radiation will be tested to see clinical benefit.

5 Future Perspective

Although DC-based immunotherapy is the only cell-based immunotherapy which has been approved by FDA so far, whether the infused DC induce effective anti-tumor immunity depends on immunosuppressive background of the host and strategies to break the tolerant environment [30]. Adoptive transfer of tumor-specific T cells will target cancer independently of immunosuppressive milieu in cancer-bearing hosts if enough number of the T cells is infused [31]. Genetic engineering enabled to induce large number of tumor-specific TCR-transgenic T-cell ex vivo [32–34]. The transfer of tumor antigen-specific TCR-transgenic T cells eradicated some metastatic solid tumors like melanoma in clinical trials. Complete responses or partial responses were observed with gp100-targeting T-cell transfer in 6.3 or 12.5 % of metastatic melanoma patients, respectively [34]. Partial responses were achieved by NY-ESO-1-targeting T-cell transfer in 67 % of synovial cell sarcoma patients, and 27.2 % melanoma patients accompanied with 18 % complete responses [33]. TCR targeting HCC-specific antigen is required for establishment of efficient T-cell transfer therapy for HCC. The emerging development of induced pluripotent stem (iPS) cell technology might further enable to produce large number of tumor-specific TCR-bearing T cells by generating iPS from tumor-infiltrating lymphocytes [35].

Even if “good” tumor-specific antigens are used, DC or tumor-specific T-cell-based immunotherapy cannot overcome heterogeneity in cancer tissues. Because a cancer tissue includes heterogeneous cancer cell populations, some populations should be resistant to tumor-specific immunotherapy. One possible method to overcome the heterogeneity is targeting cancer stem cells (CSC) in the tissue [36]. If antigens specific to CSC are known, DC or TCR-transgenic T-cell therapy might regress whole cancer tissues including heterogeneous populations. In leukemia model, Wilms Tumor 1 (WT1) and cancer testis antigen termed PRAME are potential antigens related to cancer stem cells, and the vaccine using those antigens killed leukemic stem cells [36]. Because CSC-specific antigens are still unknown in HCC, further examination of the antigens is required.