The role of the immune system in the control of... : European Journal of Gastroenterology & Hepatology (original) (raw)
Introduction
Worldwide, hepatocellular carcinoma (HCC) is one of the most common fatal tumours and 1.2 million new cases arise annually. An increase in the number of cases of HCC has been documented in the USA [1] and Europe over the past two decades, and it is believed that the high prevalence of hepatitis C virus (HCV) infection accounts for this increase in the incidence of HCC [2].
Liver resection or transplantation is potentially curative in selected cases [3] but, without therapy, HCC has a poor prognosis and long-term survival is unusual [4]. Spontaneous regression of HCC, defined as a partial or complete involution of tumour in the absence of a specific therapy being applied [5], is rare but around 30 cases have been documented in the literature [6]. The process underlying this phenomenon remains unknown but proposed mechanisms include hormonal influence, withdrawal of agents required for tumour growth, disruption of blood supply and the development of an anti-tumour immune response [7]. Since spontaneous regression of HCC is unusual, well-documented cases deserve analysis, in order to enhance our understanding of this phenomenon. In this issue of European Journal of Gastroenterology & Hepatology, Blondon et al. report two further cases of spontaneous regression of HCC [8]. These cases fulfil the criteria for a partial spontaneous regression and tumour involution was clearly documented by a reduction in number of liver lesions at computed tomography imaging and a fall in serum alpha-fetoprotein (AFP). In both cases, tumour regression followed intraperitoneal spread and the authors propose that dissemination of tumour antigen to the peritoneum induced an anti-tumour immune response. Unfortunately, no studies were undertaken to investigate this hypothesis.
Among the numerous proposed mechanisms of spontaneous regression of HCC, an immunological response may play the most important role. This has stimulated interest in developing immunotherapy as a treatment for HCC [9]. The more promising approaches are beginning clinical trials.
Adoptive immunotherapy of HCC
HCC is an attractive target for immunotherapy because the tumours are often infiltrated with lymphocytes and patients with high levels of tumour infiltrating lymphocytes were found to have a better prognosis after resection [10]. Tumour infiltrating lymphocytes have been isolated and expanded in vitro and subsequently shown to be cytotoxic to HCC targets [11]. Clinical studies utilizing in vitro expanded lymphocytes have shown mixed responses. A randomized, controlled clinical trial showed that disease-free survival after HCC resection was increased by infusion of lymphocytes, non-specifically activated by anti-CD3 and interleukin 2 (IL-2), suggesting a promising role for adoptive T-cell immunotherapy in this setting [12]. Whereas lymphokine activated autologous lymphocytes administered to patients with HCC without surgery induced only two partial remissions, the majority of patients had a decrease in serum AFP, indicating a fall in tumour burden [13,14]. Unfortunately, current methods of isolation and expansion of tumour infiltrating lymphocytes are cumbersome and administration of activated lymphocytes is unlikely to be effective for patients with a large tumour load.
Immunotherapy of HCC using vaccination with a cocktail of Bacillus Calmette–Guérin (BCG) and tumour cells was proposed over 30 years ago [15]. Animal studies demonstrated that this approach can effect eradication of otherwise lethal visceral micrometastases [16,17] but, on occasions, dosing led to enhanced tumour growth. Other investigators have attempted to increase the immunogenicity of HCC by viral and non-viral delivery of immunomodulatory genes such as IL-2 [18], IL-12 [19], and TNF-α [20] to tumour cells. HCC cell lines have also been modified to express molecules such as CD40 ligand and fms-like tyrosine kinase 3 ligand, in an attempt to stimulate the immune response in the local environment of the tumour [21]. Although successful in some circumstances, non-antigen specific techniques such as these are not ideal as the durability of the immune response is unpredictable and a dominant immune response can be generated against the viral vectors.
Antigen specific immunotherapy of HCC
Many tumours produce proteins that could serve as antigens in the immune response; collectively, these are known as tumour associated antigens (TAAs). Potential TAAs in HCC include AFP [22], representatives of the melanoma antigen gene (MAGE) family [23] and glypican-3 [24]. Although some TAAs are mutated proteins, the majority are ‘self’ proteins where expression has been derepressed or upregulated. ‘Self’ proteins are only weakly immunogenic and, therefore, must be presented in an immunostimulatory context by professional antigen-presenting cells, before they can engender an immune response [25].
Dendritic cells are the most efficient antigen-presenting cells, specializing in the uptake, processing and presentation of antigen [26]. Work in animal models has suggested that a broad and durable anti-tumour response depends on the antigen specific activation of CD8+ (cytotoxic) and CD4+ (T helper) T cells [27]. Dendritic cells can process and present both intracellular and extracellular protein in the form of peptide bound to MHC class 1 and class 2 molecules, making them extremely effective in the stimulation of naive T cells [28]. Dendritic cells loaded with TAA have been evaluated as vaccines in animal models and, although questions remain regarding the optimal method for the production of dendritic cells, antigen loading and maturation, progress has been made with groups using dendritic cells loaded with TAA in clinical trials [29].
A number of animal models of HCC have shown encouraging results for vaccination strategies based on dendritic cells. Vollmer et al. showed that vaccinations with dendritic cells, transduced with an adenovirus encoding AFP, were able to prevent or delay growth of an AFP-producing tumour cell line in mice and this was accompanied by the appearance of AFP specific cytotoxic T lymphocytes [30]. This success led to examination of the protein structure of AFP to identify human MHC class 1 restricted peptide epitopes. Four peptides were identified which, when loaded onto dendritic cells, were able to generate T-cell immune responses in vitro and were naturally processed and presented in association with HLA-A2 molecules [31]. When HLA-A*0201 patients with AFP-positive HCC were immunized with three bi-weekly intradermal vaccinations of the four AFP peptides emulsified in incomplete Freund's adjuvant, all patients generated T-cell responses to the peptides, as measured by direct IFN-γ enzyme-linked immunospot (ELISPOT) and MHC class 1 tetramer assays [32]. Clearly, tolerance to this HCC TAA can be overcome. The disadvantage of inducing an immune response to AFP is that only 40–60% of patients with HCC express this TAA [33]. This has prompted work investigating immune responses against other HCC associated TAAs, such as HCA661 [34]. However, immunotherapy using a monovalent vaccine may allow the clonal expansion of tumour cells negative for that antigen, thus limiting the durability of any response.
A number of approaches have been taken to generate a polyvalent vaccine. By fusing dendritic cells with syngeneic hepatoma cells, Kawada and colleagues generated a fusion cell capable of presenting multiple TAA and these hybrid cells were able to prevent the growth of implanted hepatoma cells and prevent local recurrence after surgical resection in rats [35]. Due to the ability of dendritic cells to take up and present antigen via class 1 and class 2 pathways, pulsing dendritic cells with tumour lysate has been evaluated as a method of loading dendritic cells with multiple TAA [36]. Two recently published reports demonstrated that vaccination with dendritic cells, generated ex vivo from monocytes and loaded with autologous tumour lysate, is feasible in patients with HCC and there was no reported toxicity [37,38]. Immune responses to tumour lysate are difficult to measure due to the undefined nature of the antigens present, but Iwashita et al. were able to demonstrate delayed-type hypersensitivity reactions to the neo-antigen keyhole limpet haemocyanin in 70% of patients after vaccination with dendritic cells that had been pulsed with tumour lysate and keyhole limpet haemocyanin [38]. The clinical utility of tumour lysate is, however, limited by the need for large amounts of tumour tissue. In addition, there are potential concerns of the possibility of inducing autoimmunity to ‘housekeeping’ proteins.
An alternative method of loading dendritic cells with multiple TAA is by the use of total tumour RNA, which can be amplified from a small amount of starting material, even as small as a single tumour cell [39]. TAA can also be introduced into dendritic cells by electroporation of mRNA. This approach has limited toxicity to dendritic cells and TAAs introduced in this way are processed and presented as peptides on the cell surface to lymphocytes [40].
Immunology of HCC and cirrhosis
The immunological events occurring during the evolution of HCC are incompletely understood. Despite expressing high levels of proteins, such as AFP, that can act as antigens and the presence of T cells capable of responding to these antigens HCC continue to grow. This may be due to the general immunosuppression that accompanies malignant disease or local immunosuppressive factors secreted by the tumour itself. Reactive T cells may circulate in the periphery yet fail to infiltrate tumours due to lack of expression of the correct adhesion molecules. Dendritic cells in cirrhosis [41] and patients with hepatitis C and B viral infection [42] may be functionally impaired, with decreased allostimulatory capacity and an immature phenotype. Recent evidence suggests that, in HCC, the tumour produces an environment hostile to the development of dendritic cells as CD83-positive dendritic cells were not found in tumour nodules in HCC and the numbers of CD83-positive dendritic cells in liver were significantly lower in patients with HCC compared to those with cirrhosis alone [43].
Conclusion
New therapies are urgently needed for HCC. The experimental and circumstantial evidence that immune mechanisms can cause regression of HCC opens up exciting possibilities for the immunotherapy of HCC. Therapies based on dendritic cells hold much promise but fundamental questions regarding the optimal method of harvesting, loading and maturing dendritic cells still need to be answered. The best route of administration of dendritic cell-based vaccines is still unknown and adjuvant therapies to target dendritic cells or lymphocytes to tumours may be needed. The ultimate role of tumour vaccines may be in preventing disease in high-risk patients, such as those with cirrhosis.
References
1. El-Serag HB, Mason AC. Rising incidence of hepatocellular carcinoma in the United States. N Engl J Med 1999; 340:745–750.
2. El-Serag HB, Mason AC. Risk factors for the rising rates of primary liver cancer in the United States. Arch Intern Med 2000; 160:3227–3230.
3. Marin-Hargreaves G, Azoulay D, Bismuth H. Hepatocellular carcinoma: surgical indications and results. Crit Rev Oncol Hematol 2003; 47: 13–27.
4. Okuda K, Ohtsuki T, Obata H, Tomimatsu M, Okazaki N, Hasegawa H, et al. Natural history of hepatocellular carcinoma and prognosis in relation to treatment. Study of 850 patients. Cancer 1985; 56:918–928.
5. Everson TC, Cole WH. Spontaneous regression of malignant disease. J Am Med Assoc 1959; 169:1758–1759.
6. Okada S, Abo T. Spontaneous regression of hepatocellular carcinoma. J Gastroenterol Hepatol 2000; 15:965–966.
7. Cole WH. Efforts to explain spontaneous regression of cancer. J Surg Oncol 1981; 17:201–209.
8. Blondon H, Fritsch L, Cherqui D. Two cases of spontaneous regression of multicentric hepatocellular carcinoma after intraperitoneal rupture. Eur J Gastroenterol Hepatol 2004; 16:1355–1360.
9. Geissler M, Mohr L, Ali MY, Grimm CF, Ritter M, Blum HE. Immunobiology and gene-based immunotherapy of hepatocellular carcinoma. Z Gastroenterol 2003; 41:1101–1110.
10. Kawata A, Une Y, Hosokawa M, Uchino J, Kobayashi H. Tumor-infiltrating lymphocytes and prognosis of hepatocellular carcinoma. Jpn J Clin Oncol 1992; 22:256–263.
11. Aruga A, Yamauchi K, Takasaki K, Furukawa T, Hanyu F. Induction of autologous tumor-specific cytotoxic T cells in patients with liver cancer. Characterizations and clinical utilization. Int J Cancer 1991; 49:19–24.
12. Takayama T, Sekine T, Makuuchi M, Yamasaki S, Kosuge T, Yamamoto J, et al. Adoptive immunotherapy to lower postsurgical recurrence rates of hepatocellular carcinoma: a randomised trial_. Lancet_ 2000; 356: 802–807.
13. Onishi S, Saibara T, Fujikawa M, Sakaeda H, Matsuura Y, Matsunaga Y, Yamamoto Y. Adoptive immunotherapy with lymphokine-activated killer cells plus recombinant interleukin 2 in patients with unresectable hepatocellular carcinoma. Hepatology 1989; 10:349–353.
14. Ichida T, Higuchi K, Arakawa K, Ohta H, Sugiyama K, Miyagiwa M, et al. Treatment of hepatocellular carcinoma utilizing lymphokine-activated killer cells and interleukin-2. Cancer Chemother Pharmacol 1989; 23 (suppl):S45–S48.
15. Zbar B, Bernstein I, Tanaka T, Rapp HJ. Tumor immunity produced by the intradermal inoculation of living tumor cells and living Mycobacterium bovis (strain BCG). Science 1970; 170:1217–1218.
16. Rella W, Chaput B. Immunotherapy with tumor cells and BCG in the guinea pig, studied by immunological in vivo and in vitro experiments. Oncology 1978; 35:136–142.
17. Hanna MG Jr, Peters LC. Immunotherapy of established micrometastases with Bacillus Calmette–Guerin tumor cell vaccine. Cancer Res 1978; 38:204–209.
18. He P, Tang ZY, Ye SL, Liu BB, Liu YK. The targeted expression of interleukin-2 in human hepatocellular carcinoma cells. J Exp Clin Cancer Res 2000; 19:183–187.
19. Yamashita YI, Shimada M, Hasegawa H, Minagawa R, Rikimaru T, Hamatsu T, et al. Electroporation-mediated interleukin-12 gene therapy for hepatocellular carcinoma in the mice model. Cancer Res 2001; 61:1005–1012.
20. Cao G, Kuriyama S, Du P, Sakamoto T, Kong X, Masui K, Qi Z. Complete regression of established murine hepatocellular carcinoma by in vivo tumor necrosis factor alpha gene transfer. Gastroenterology 1997; 112:501–510.
21. Yanagi K, Nagayama Y, Nakao K, Saeki A, Matsumoto K, Ichikawa T, et al. Immuno-gene therapy with adenoviruses expressing fms-like tyrosine kinase 3 ligand and CD40 ligand for mouse hepatoma cells in vivo. Int J Oncol 2003; 22:345–351.
22. Bei R, Budillon A, Reale MG, Capuano G, Pomponi D, Budillon G, et al. Cryptic epitopes on alpha-fetoprotein induce spontaneous immune responses in hepatocellular carcinoma, liver cirrhosis, and chronic hepatitis patients. Cancer Res 1999; 59:5471–5474.
23. Luo G, Huang S, Xie X, Stockert E, Chen YT, Kubuschok B, Pfreundschuh M. Expression of cancer-testis genes in human hepatocellular carcinomas. Cancer Immun 2002; 2:11.
24. Capurro M, Wanless IR, Sherman M, Deboer G, Shi W, Miyoshi E, Filmus J. Glypican-3: a novel serum and histochemical marker for hepatocellular carcinoma. Gastroenterology 2003; 125:89–97.
25. Knight SC, Burke F, Bedford PA. Dendritic cells, antigen distribution and the initiation of primary immune responses to self and non-self antigens. Semin Cancer Biol 2002; 12:301–308.
26. Banchereau J, Briere F, Caux C, Davoust J, Lebecque S, Liu YJ, et al. Immunobiology of dendritic cells. Annu Rev Immunol 2000; 18: 767–811.
27. Morse MA, Lyerly HK. Immunotherapy of cancer using dendritic cells. Cytokines Cell Mol Ther 1998; 4:35–44.
28. Di Nicola M, Lemoli RM. Dendritic cells: specialized antigen presenting cells. Haematologica 2000; 85:202–207.
29. Cerundolo V, Hermans IF, Salio M. Dendritic cells: a journey from laboratory to clinic. Nat Immunol 2004; 5:7–10.
30. Vollmer CM Jr, Eilber FC, Butterfield LH, Ribas A, Dissette VB, Koh A, et al. Alpha-fetoprotein-specific genetic immunotherapy for hepatocellular carcinoma. Cancer Res 1999; 59:3064–3067.
31. Butterfield LH, Meng WS, Koh A, Vollmer CM, Ribas A, Dissette VB, et al. T cell responses to HLA-A*0201-restricted peptides derived from human alpha fetoprotein. J Immunol 2001; 166:5300–5308.
32. Butterfield LH, Ribas A, Meng WS, Dissette VB, Amarnani S, Vu HT, et al. T-cell responses to HLA-A*0201 immunodominant peptides derived from alpha-fetoprotein in patients with hepatocellular cancer. Clin Cancer Res 2003; 9(16 pt 1):5902–5908.
33. Cancer of the Liver Italian Program (CLIP) Investigators. A new prognostic system for hepatocellular carcinoma: a retrospective study of 435 patients. Hepatology 1998; 28:751–755.
34. Chan RC, Pang XW, Wang YD, Chen WF, Xie Y. Transduction of dendritic cells with recombinant adenovirus encoding HCA661 activates autologous cytotoxic T lymphocytes to target hepatoma cells. Br J Cancer 2004; 90:1636–1643.
35. Kawada M, Ikeda H, Takahashi T, Ishizu A, Ishikura H, Katoh H, Yoshiki T. Vaccination of fusion cells of rat dendritic and carcinoma cells prevents tumor growth in vivo. Int J Cancer 2003; 105:520–526.
36. Shibolet O, Alper R, Zlotogarov L, Thalenfeld B, Engelhardt D, Rabbani E, Ilan Y. NKT and CD8 lymphocytes mediate suppression of hepatocellular carcinoma growth via tumor antigen-pulsed dendritic cells. Int J Cancer 2003; 106:236–243.
37. Ladhams A, Schmidt C, Sing G, Butterworth L, Fielding G, Tesar P, et al. Treatment of non-resectable hepatocellular carcinoma with autologous tumor-pulsed dendritic cells. J Gastroenterol Hepatol 2002; 17: 889–896.
38. Iwashita Y, Tahara K, Goto S, Sasaki A, Kai S, Seike M, et al. A phase I study of autologous dendritic cell-based immunotherapy for patients with unresectable primary liver cancer. Cancer Immunol Immunother 2003; 52:155–161.
39. Heiser A, Maurice MA, Yancey DR, Wu NZ, Dahm P, Pruitt SK, et al. Induction of polyclonal prostate cancer-specific CTL using dendritic cells transfected with amplified tumor RNA. J Immunol 2001; 166: 2953–2960.
40. Eppler E, Horig H, Kaufman HL, Groscurth P, Filgueira L. Carcinoembryonic antigen (CEA) presentation and specific T cell-priming by human dendritic cells transfected with CEA-mRNA. Eur J Cancer 2002; 38:184–193.
41. Ninomiya T, Akbar SM, Masumoto T, Horiike N, Onji M. Dendritic cells with immature phenotype and defective function in the peripheral blood from patients with hepatocellular carcinoma. J Hepatol 1999; 31: 323–331.
42. Kakumu S, Ito S, Ishikawa T, Mita Y, Tagaya T, Fukuzawa Y, Yoshioka K. Decreased function of peripheral blood dendritic cells in patients with hepatocellular carcinoma with hepatitis B and C virus infection. J Gastroenterol Hepatol 2000; 15:431–436.
43. Chen S, Akbar SM, Tanimoto K, Ninomiya T, Iuchi H, Michitaka K, et al. Absence of CD83-positive mature and activated dendritic cells at cancer nodules from patients with hepatocellular carcinoma: relevance to hepatocarcinogenesis. Cancer Lett 2000; 148:49–57.
Keywords:
hepatocellular carcinoma; spontaneous regression; dendritic cells; immunotherapy
© 2004 Lippincott Williams & Wilkins, Inc.