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Is there a role for immunotherapy in hepatocellular carcinoma?
Digestive and Liver Disease 38 (2006) 221–225
Review Article
Is there a role for immunotherapy in hepatocellular carcinoma? A. Zerbini, M. Pilli, C. Ferrari, G. Missale ∗ Unit of Infectious Diseases and Hepatology, Azienda Ospedaliero-Universitaria di Parma, Via Gramsci 14, 43100 Parma, Italy Received 5 December 2005; accepted 13 December 2005 Available online 3 February 2006
Abstract Incidence of hepatocellular carcinoma has been rising in the last two decades because of the wide exposure to hepatitis C virus during 1960s and 1970s. Improvement in treatment has been achieved by local ablative therapies, however because of early recurrence and lack of effective chemotherapies, alternative treatments based on stimulation of the anti-tumour immune response could represent new strategies to control hepatocellular carcinoma spread and recurrence. Proof of principle of an effective immunotherapy has been achieved for other solid tumours such as melanoma and several results could be transferred to the immunotherapy of hepatocellular carcinoma. Specific tumour antigens have been identified in hepatocellular carcinoma, such as cancer testis antigens expressed in a large part of hepatocellular carcinomas and alpha-fetoprotein that has been already employed in clinical trials demonstrating immunogenicity without however significant clinical efficacy. Better results have been achieved by non-antigen-specific immunotherapies that demonstrated improvement in recurrence and recurrencefree survival in patients undergoing surgical resection for hepatocellular carcinoma. Passive immunotherapy and targeted therapies blocking tumour cell receptors or enzymatic pathways are already in the clinic for other malignancies and the near future will see these new treatments applied to hepatocellular carcinoma patients along with the development of efficacious active immunotherapies aimed at reducing disease recurrence and improving survival. © 2005 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved. Keywords: HBV; HCC; HCV; Immunotherapy
1. Introduction Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide [1], it is the most common primary liver cancer associated with hepatitis B (HBV) and hepatitis C virus (HCV) chronic infections, alcohol abuse or in developing countries with food contaminated with Aspergillus flavus fungus [2]. Worldwide, it accounts for almost 1 million deaths per year and in the East Asian area its incidence exceeds 30 cases/100,000 per year. The incidence of HCC has been rising in the last two decades in Europe, United States and Japan [3,4]. Approximately 18,000 new cases of HCC are projected to occur in 2005 [5]. This epidemiological change in western countries has been explained by the wide exposure to HBV and HCV during 1960s and 1970s. Therefore, occurrence of ∗
Corresponding author. Tel.: +39 0521 702056; fax: +39 0521 703857. E-mail address: [email protected] (G. Missale).
HCC is expected to continue to rise in the next decades because of the large pool of people infected by HBV and HCV. It is now well accepted that the cytotoxic CD8+ Tcell response can play a central role in cancer control. In melanoma [6] and in advanced ovarian cancer, the intratumoural T-cell infiltrate correlates with clinical outcome [7], and also in HCC the patients with a heavy intra-tumoural T-cell infiltrate are those with a better disease-free survival [8]. HCC would be a very suitable target for testing active immunotherapy for a number of reasons. First, the immune system of patients with HCC will not be affected by concomitant chemotherapy because of no benefit in this tumour type. Second, the progression of HCC seems sufficiently slow to allow priming or boosting of a clinically effective cellular immune response. Third, the liver is a highly vascularised organ that immune effector cells can readily infiltrate. Finally, given the specific attitude of HCC to remain confined within the liver, the tumour burden will be easily quantified during
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the clinical follow-up by standard diagnostic imaging techniques. Surgical therapy offers the opportunity to cure patients with HCC. However, the surgical options are limited by the size and location of the tumour, the presence or absence of vascular invasion, associated vascular thromboses and the characteristics and severity of the underlying liver disease. HCC management is mainly accomplished by local ablative therapies. These techniques determine good results with improvement in survival, although HCC recurrence remains very frequent [9–13]. For these reasons, stimulation of the anti-tumour immune response would represent a novel and auspicable strategy against HCC.
parenchimal organ. These impressive results were obtained by aggressive manipulation of the patient’s immune system that cannot be proposed for routine anti-tumour treatment [23]. Future steps will be to exploit these ideas and experiences to develop a feasible treatment opportunity. HCC is an excellent setting for tumour immunology research and for moving from bench to bedside into the clinic. HCC is not as immunogenic as melanoma; however, it is confined to a single organ in the great part of its natural history, so that it can be easily monitored by radiological techniques, from ultrasound to magnetic resonance and HCC nodules can be reached by US-guided approaches for sampling or intra-tumour injections. Moreover, since no chemotherapy of proven efficacy is available, the patient’s immune system is spared by the toxic effect of first-line aggressive conventional chemotherapy treatments.
2. Background Two hundred years ago the first vaccine against smallpox was described [14]. Nowadays, more than 20 infectious diseases are preventable by effective vaccines; however, several other viral, bacterial and parasitic diseases remain a challenge for preventive vaccine and therapeutic immunotherapies. Microbial agents express non-self antigens and molecular structures that are natural activator of the immune system. Instead, tumour-associated antigens (TAAs) are selfproteins modified by genetic alterations or abnormally expressed in a transformed tissue. The idea of cancer immune surveillance goes back to the time Ehrlich formulated the hypothesis of anti-tumour immune response, before lymphocytes were discovered, and to the time (early 1900) Cooley injected sarcomas with Gram-positive bacteria, the so-called ‘Cooley’s toxin’, obtaining tumour size reduction. The antigen was already there, in the tumour, and the ‘Cooley’s toxin’ was the adjuvant; this was the first attempt to exploit the idea of cancer immune surveillance. Attempts to enhance melanoma immunogenicity go back more than 40 years from now [15]. Since 1991, when the first TAA was identified in melanoma [16], several trials of active immunotherapy have been conducted. Experimental evidence that the immune system is able to recognise TAAs as targets and to subsequently destroy the tumour cells has accumulated in the last 10 years; however, limitations in translating successfully this knowledge into clinical applications are still to be overcome. Enhancement of a tumour-specific T-cell response is difficult to achieve because of the tumour immunological tolerance [17,18]. Three main issues have to be addressed by basic, translational and clinical research: identification of immunogenic TAAs, discovery of new adjuvants to employ in the clinic and standardisation of immunotherapeutical protocols to treat patients. Melanoma immunotherapy is the icebreaker of the field. A variety of cancer vaccines have been tested, including the use of immunogenic peptides, recombinant viruses, naked DNA, peptide pulsed dendritic cells (DCs), etc. [19–22]. Proof of principle has been achieved by the evidence of regression of melanoma metastasis from any
3. HCC antigenic targets Several TAAs have been identified, among which antigens of the cancer testis (CT) family are of particular interest, since they are expressed by tumours of different histological types [24,25]. They are silent in normal cells, with the exception of male germline cells that do not express HLA class I molecules and are therefore unable to present antigens to CTL. For these reasons, they are of particular interest in cancer immunotherapy. Melanoma-associated antigens (MAGE), and in particular MAGE-1 and MAGE-3 genes, are expressed in 46–80% of resected HCC tissues and none is detected in the non-tumour liver [26–28]. We have demonstrated the presence of HCC-specific CD8+ T cells, specific for MAGE-1 and MAGE-3 [29], in the tumour and peripheral blood of HCC patients. These results were recently confirmed by the identification of CD8+ T cells specific for two additional CT-antigens: MAGE-10 and SSX-2 [30]. It is relevant to note that ex vivo demonstration of spontaneously arising tumour-specific T cells within the tumour tissue was previously provided only in melanoma; this finding in HCC confirms that tumour-specific T-cell responses are indeed induced in cancer, since in vitro expanded tumour-specific T cells could simply be the result of artefact. These new findings in HCC open the way for pursuing anti-tumour strategies previously employed for melanoma and other solid tumours [31,32] also in the HCC. In almost 50% of HCCs, alpha-fetoprotein (AFP) expression reactivates. The overexpression of this protein, which is the major serum protein in the foetus but is quite low in adults, makes it a possible candidate as an antigenic target for anti-HCC immunotherapy. This possibility is confirmed by the detection of enhanced AFP-specific CD8 cell responses in HCC patients undergoing AFP-peptides vaccination [33] and in mice vaccinated with DNA encoding mouse AFP [34]. These results are promising because they further demonstrate the possibility to break tolerance to a self-protein like AFP.
A. Zerbini et al. / Digestive and Liver Disease 38 (2006) 221–225
HBV and HCV antigens have been considered in the past possible targets for HCC therapy but modified viral proteins specifically expressed in the tumour and not in the adjacent normal liver have never been identified. Oncogenes and modified tumour suppressor genes are also involved in the pathogenesis of HCC and these molecules have been proposed as possible targets of immunotherapy in HCC, as in other tumours. Several other HCC-specific antigens are becoming available through gene array technology studies [35–37]; however, many tumour-specific T-cell responses are probably restricted to individual patients because of the known genetic heterogeneity of HCC. Thus, the identification of dominant TAAs in HCC remains an open challenge.
4. Clinical trials Clinical trials have been conducted in patients with HCC with the aim of enhancing anti-tumour immune responses in order to reduce HCC burden in patients with advanced disease or to reduce the risk of recurrence after curative resection. Different techniques were employed comprising the use of non-specific immunostimulating products, like cytokines, adoptive transfer of autologous-activated lymphocytes, DC-based immunotherapy, AFP-derived peptides vaccination or intra-tumour injection of recombinant adenoviral vectors engineered for the local release of interleukin-12 (IL12) [38]. The largest immunotherapy trial for HCC was conducted in 155 resected patients, who received IL-2 and ␣CD3 in vitro expanded autologous PBMCs to prevent recurrence. This study showed a statistically significant benefit in risk of recurrence, time to recurrence and recurrence-free survival, with a trend also in favour of an improvement in overall survival [39]. The majority of the other trials in HCC were conducted in patients with a high disease burden. Cytokine treatment was performed in 20 stage III and IV inoperable patients; interferon-gamma (IFN-␥) and IL-2 were administered in association with transarterial chemotherapy showing tumour size reduction in 14 patients [40]. A more recent cytokine treatment, based on subcutaneous injection of IFN-␥ and granulocyte–macrophage colony stimulating factor (GMCSF) in advanced HCC, did not show any clinical response [41]. Immature and mature DCs can be easily differentiated in vitro from circulating monocytes or CD34+ precursors; they have been widely employed against cancer in the last 10 years in more than 60 different clinical studies of tumour immunotherapy (http://www.kun.nl/til/dc2003/). Exploiting their unique capacity to internalise antigen, process and present it in the context of HLA-class I and II molecules and provide the optimal co-stimuli to induce antigen-specific CD4 and CD8 T-cell responses, DCs were employed in HCC immunotherapy, like in other tumour settings. DCs
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were pulsed with autologous tumour lysates and injected as immature [42,43] or in vitro matured DCs [44]. Results were quite disappointing with only a few cases of minor or partial responses. A recent trial, based on injection of DCs pulsed with autologous tumour lysates, compared two protocols of vaccination. The first consisted of a weekly series of five vaccinations, and the second was the five-weekly vaccinations followed by a monthly boost. By this second approach a significant improved survival was shown, even though, also in this trial, clinical responses were limited to partial responses or stable disease [45]. Differently from the previously described experiences that were based on multispecific T-cell stimulation by the use of crude tumour protein lysates, vaccination of HCC patients with AFP-derived peptides emulsified in Montanide ISA-51 adjuvant, allowed to monitor the in vivo effect of a well-defined antigen-specific T-cell expansion. The trial conducted in advanced-stage AFPsecreting HCC patients did not report any clinical response but the immunological results were satisfactory, showing that AFP-specific functional T-cell responses could be elicited in these patients even when high serum levels of AFP were present [33].
5. Conclusions Thanks to the refinement of treatment protocols and the tremendous investments of the pharmaceutical industry in conventional chemotherapy, improvements in cancer treatment have been relevant in the last decade. However, nowadays it is clear that different approaches for combating cancer are needed for a further step ahead in the field. Biologic therapy of cancer includes the use of new molecules targeted to altered receptors involved in cellular activation, or to key oncogenic signalling pathways as well as the use of monoclonal antibodies in passive immunotherapy against activation receptors, endothelial growth factors and surface molecular markers mediating apoptosis of targeted tumour cells. Active immunotherapy is still in preclinical and clinical trial phase of development but it will become available in the clinics in the near future. The growing knowledge in genomics, proteomics, safe recombinant vectors engineering will provide new tools for treatment of patients with active immunotherapies. Moreover, great innovations are expected for the development of new adjuvant molecules; until now, almost all commercial vaccines are still based only on alum-derived adjuvants, even though immunological mechanisms and receptor involved in triggering immune responses are becoming to be characterised in great detail. Passive immunotherapy of melanoma, by adoptive transfer and manipulation of regulatory Tcell responses, is achieving considerable results. Results in HCC, as in other malignancies will follow in the near future and clinical success will be derived by the combination of new biological treatments with passive and active immunotherapy.
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Conflict of interest statement None declared. [21]
References [22] [1] Bosch FX, Ribes J, Cleries R, Diaz M. Epidemiology of hepatocellular carcinoma. Clin Liver Dis 2005;9:191–211. [2] O’Grady J, Lake JR, Howdle PD. Malignant tumours of the liver. In: O’Grady J, Lake JR, Howdle PD, editors. Comprehensive clinical hepatology. London: Harcourt Publishers; 2000. p. 25.18– 25.21. [3] El-Serag HB, Mason AC. Rising incidence of hepatocellular carcinoma in the United States. N Engl J Med 1999;340:745–50. [4] Hassan MM, Frome A, Patt YZ, El-Serag HB. Rising prevalence of hepatitis C virus infection among patients recently diagnosed with hepatocellular carcinoma in the United States. J Clin Gastroenterol 2002;35:266–9. [5] Jemal A, Murray T, Ward E, Samuels A, Tiwari RC, Ghafoor A, et al. Cancer statistics, 2005. CA Cancer J Clin 2005;55:10–30. [6] Clemente CG, Mihm Jr MC, Bufalino R, Zurrida S, Collini P, Cascinelli N. Prognostic value of tumor infiltrating lymphocytes in the vertical growth phase of primary cutaneous melanoma. Cancer 1996;77:1303–10. [7] Zhang L, Conejo-Garcia JR, Katsaros D, Gimotty PA, Massobrio M, Regnani G, et al. Intratumoral T cells, recurrence, and survival in epithelial ovarian cancer. N Engl J Med 2003;348:203–13. [8] Wada Y, Nakashima O, Kutami R, Yamamoto O, Kojiro M. Clinicopathological study on hepatocellular carcinoma with lymphocytic infiltration. Hepatology 1998;27:407–14. [9] Carr BI. Hepatocellular carcinoma: current management and future trends. Gastroenterology 2004;127(5 Suppl. 1):S218–24. [10] Jansen MC, van Hillegersberg R, Chamuleau RA, van Delden OM, Gouma DJ, van Gulik TM. Outcome of regional and local ablative therapies for hepatocellular carcinoma: a collective review. Eur J Surg Oncol 2005;31:331–47. [11] Mulcahy MF. Management of hepatocellular cancer. Curr Treat Options Oncol 2005;6:423–35. [12] Camma C, Di Marco V, Orlando A, Sandonato L, Casaril A, Parisi P, et al. Treatment of hepatocellular carcinoma in compensated cirrhosis with radio-frequency thermal ablation (RFTA): a prospective study. J Hepatol 2005;42:535–40. [13] Raut CP, Izzo F, Marra P, Ellis LM, Vauthey JN, Cremona F, et al. Significant long-term survival after radiofrequency ablation of unresectable hepatocellular carcinoma in patients with cirrhosis. Ann Surg Oncol 2005;12:616–28. [14] Jenner E. An inquiry into the causes and effects of variolae vaccinae, a disease discovered in some of the western counties of England. London: Sampson Low; 1798. [15] Woodruff MF, Nolan B. Preliminary observations on treatment of advanced cancer by injection of allogeneic spleen cells. Lancet 1963;13:426–9. [16] van der Bruggen P, Traversari C, Chomez P, Lurquin C, De Plaen E, Van den Eynde B, et al. A gene encoding an antigen recognized by cytolytic T lymphocytes on a human melanoma. Science 1991;254:1643–7. [17] Willimsky G, Blankenstein T. Sporadic immunogenic tumours avoid destruction by inducing T-cell tolerance. Nature 2005;437:141– 6. [18] Klein G, Klein E. Surveillance against tumors—is it mainly immunological? Immunol Lett 2005;100:29–33. [19] Parmiani G, Castelli C, Rivoltini L, Casati C, Tully GA, Novellino L, et al. Immunotherapy of melanoma. Semin Cancer Biol 2003;13:391–400. [20] Rosenberg SA, Yang JC, Schwartzentruber DJ, Hwu P, Marincola FM, Topalian SL, et al. Immunologic and therapeutic evaluation of a
[23]
[24]
[25]
[26]
[27]
[28]
[29]
[30]
[31]
[32]
[33]
[34]
[35]
[36]
[37]
[38]
synthetic peptide vaccine for the treatment of patients with metastatic melanoma. Nat Med 1998;4:321–7. Rosenberg SA, Yang JC, Schwartzentruber DJ, Hwu P, Marincola FM, Topalian SL, et al. Impact of cytokine administration on the generation of antitumor reactivity in patients with metastatic melanoma receiving a peptide vaccine. J Immunol 1999;163:1690–5. Scheibenbogen C, Schmittel A, Keilholz U, Allgauer T, Hofmann U, Max R, et al. Phase II trial of vaccination with tyrosinase peptides and granulocyte–macrophage colony stimulating factor in patients with metastatic melanoma. J Immunother 2000;23:275–81. Rosenberg SA, Dudley ME. Cancer regression in patients with metastatic melanoma after the transfer of autologous antitumor lymphocytes. Proc Natl Acad Sci USA 2004;101(Suppl. 2):14639–45. Novellino L, Castelli C, Parmiani G. A listing of human tumor antigens recognized by T cells: March 2004 update. Cancer Immunol Immunother 2005;54:187–207 [Epub 2004, August 7]. Simpson AJ, Caballero OL, Jungbluth A, Chen YT, Old LJ. Cancer/testis antigens, gametogenesis and cancer. Nat Rev Cancer 2005;5:615–25. Luo G, Huang S, Xie X, Stockert E, Chen YT, Kubuschok B, et al. Expression of cancer-testis genes in human hepatocellular carcinomas. Cancer Immun 2002;2:11. Mou DC, Cai SL, Peng JR, Wang Y, Chen HS, Pang XW, et al. Evaluation of MAGE-1 and MAGE-3 as tumour-specific markers to detect blood dissemination of hepatocellular carcinoma cells. Br J Cancer 2002;86:110–6. Kobayashi Y, Higashi T, Nouso K, Nakatsukasa H, Ishizaki M, Kaneyoshi T, et al. Expression of MAGE, GAGE and BAGE genes in human liver diseases: utility as molecular markers for hepatocellular carcinoma. J Hepatol 2000;32:612–7. Zerbini A, Pilli M, Soliani P, Ziegler S, Pelosi G, Orlandini A, et al. Ex vivo characterization of tumor-derived melanoma antigen encoding gene-specific CD8+cells in patients with hepatocellular carcinoma. J Hepatol 2004;40:102–9. Bricard G, Bouzourene H, Martinet O, Rimoldi D, Halkic N, Gillet M, et al. Naturally acquired MAGE-A10- and SSX-2-specific CD8+ T cell responses in patients with hepatocellular carcinoma. J Immunol 2005;174:1709–16. Reynolds SR, Zeleniuch-Jacquotte A, Shapiro RL, Roses DF, Harris MN, Johnston D, et al. Vaccine-induced CD8+ T-cell response to MAGE-3 correlate with clinical outcome in patients with melanoma. Clin Cancer Res 2003;9:657–62. Vantomme V, Dantinne C, Amrani N, Permanne P, Gheysen D, Bruck C, et al. Immunologic analysis of a phase I/II study of vaccination with MAGE-3 protein combined with the AS02B adjuvant in patients with MAGE-3-positive tumors. J Immunother 2004;27:124–35. 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;9:5902–8. Geissler M, Mohr L, Weth R, Kohler G, Grimm CF, Krohne TU, et al. Immunotherapy directed against alpha-fetoprotein results in autoimmune liver disease during liver regeneration in mice. Gastroenterology 2001;121:931–9. Tabor E. Tumor suppressor genes, growth factor genes, and oncogenes in hepatitis B virus-associated hepatocellular carcinoma. J Med Virol 1994;42:357–65. Okabe H, Satoh S, Kato T, Kitahara O, Yanagawa R, Yamaoka Y, et al. Genome-wide analysis of gene expression in human hepatocellular carcinomas using cDNA microarray: identification of genes involved in viral carcinogenesis and tumor progression. Cancer Res 2001;61:2129–37. Wang Y, Han KJ, Pang XW, Vaughan HA, Qu W, Dong XY, et al. Large scale identification of human hepatocellular carcinomaassociated antigens by autoantibodies. J Immunol 2002;169:1102–9. Sangro B, Mazzolini G, Ruiz J, Herraiz M, Quiroga J, Herrero I, et al. Phase I trial of intratumoral injection of an adenovirus
A. Zerbini et al. / Digestive and Liver Disease 38 (2006) 221–225 encoding interleukin-12 for advanced digestive tumors. J Clin Oncol 2004;22:1389–97. [39] 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;356:802–7. [40] Lygidakis NJ, Kosmidis P, Ziras N, Parissis J, Kyparidou E. Combined transarterial targeting locoregional immunotherapy– chemotherapy for patients with unresectable hepatocellular carcinoma: a new alternative for an old problem. J Interferon Cytokine Res 1995;15:467–72. [41] Reinisch W, Holub M, Katz A, Herneth A, Lichtenberger C, Schoniger-Hekele M, et al. Prospective pilot study of recombinant granulocyte macrophage colony-stimulating factor and interferongamma in patients with inoperable hepatocellular carcinoma. J Immunother 2002;25:489–99.
225
[42] 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;17:889–96. [43] 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;52:155–61. [44] Stift A, Friedl J, Dubsky P, Bachleitner-Hofmann T, Schueller G, Zontsich T, et al. Dendritic cell-based vaccination in solid cancer. J Clin Oncol 2003;21:135–42. [45] Lee WC, Wang HC, Hung CF, Huang PF, Lia CR, Chen MF. Vaccination of advanced hepatocellular carcinoma patients with tumor lysate-pulsed dendritic cells: a clinical trial. J Immunother 2005;28:496–504.
Review Article
Is there a role for immunotherapy in hepatocellular carcinoma? A. Zerbini, M. Pilli, C. Ferrari, G. Missale ∗ Unit of Infectious Diseases and Hepatology, Azienda Ospedaliero-Universitaria di Parma, Via Gramsci 14, 43100 Parma, Italy Received 5 December 2005; accepted 13 December 2005 Available online 3 February 2006
Abstract Incidence of hepatocellular carcinoma has been rising in the last two decades because of the wide exposure to hepatitis C virus during 1960s and 1970s. Improvement in treatment has been achieved by local ablative therapies, however because of early recurrence and lack of effective chemotherapies, alternative treatments based on stimulation of the anti-tumour immune response could represent new strategies to control hepatocellular carcinoma spread and recurrence. Proof of principle of an effective immunotherapy has been achieved for other solid tumours such as melanoma and several results could be transferred to the immunotherapy of hepatocellular carcinoma. Specific tumour antigens have been identified in hepatocellular carcinoma, such as cancer testis antigens expressed in a large part of hepatocellular carcinomas and alpha-fetoprotein that has been already employed in clinical trials demonstrating immunogenicity without however significant clinical efficacy. Better results have been achieved by non-antigen-specific immunotherapies that demonstrated improvement in recurrence and recurrencefree survival in patients undergoing surgical resection for hepatocellular carcinoma. Passive immunotherapy and targeted therapies blocking tumour cell receptors or enzymatic pathways are already in the clinic for other malignancies and the near future will see these new treatments applied to hepatocellular carcinoma patients along with the development of efficacious active immunotherapies aimed at reducing disease recurrence and improving survival. © 2005 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved. Keywords: HBV; HCC; HCV; Immunotherapy
1. Introduction Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide [1], it is the most common primary liver cancer associated with hepatitis B (HBV) and hepatitis C virus (HCV) chronic infections, alcohol abuse or in developing countries with food contaminated with Aspergillus flavus fungus [2]. Worldwide, it accounts for almost 1 million deaths per year and in the East Asian area its incidence exceeds 30 cases/100,000 per year. The incidence of HCC has been rising in the last two decades in Europe, United States and Japan [3,4]. Approximately 18,000 new cases of HCC are projected to occur in 2005 [5]. This epidemiological change in western countries has been explained by the wide exposure to HBV and HCV during 1960s and 1970s. Therefore, occurrence of ∗
Corresponding author. Tel.: +39 0521 702056; fax: +39 0521 703857. E-mail address: [email protected] (G. Missale).
HCC is expected to continue to rise in the next decades because of the large pool of people infected by HBV and HCV. It is now well accepted that the cytotoxic CD8+ Tcell response can play a central role in cancer control. In melanoma [6] and in advanced ovarian cancer, the intratumoural T-cell infiltrate correlates with clinical outcome [7], and also in HCC the patients with a heavy intra-tumoural T-cell infiltrate are those with a better disease-free survival [8]. HCC would be a very suitable target for testing active immunotherapy for a number of reasons. First, the immune system of patients with HCC will not be affected by concomitant chemotherapy because of no benefit in this tumour type. Second, the progression of HCC seems sufficiently slow to allow priming or boosting of a clinically effective cellular immune response. Third, the liver is a highly vascularised organ that immune effector cells can readily infiltrate. Finally, given the specific attitude of HCC to remain confined within the liver, the tumour burden will be easily quantified during
1590-8658/$30 © 2005 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.dld.2005.12.004
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the clinical follow-up by standard diagnostic imaging techniques. Surgical therapy offers the opportunity to cure patients with HCC. However, the surgical options are limited by the size and location of the tumour, the presence or absence of vascular invasion, associated vascular thromboses and the characteristics and severity of the underlying liver disease. HCC management is mainly accomplished by local ablative therapies. These techniques determine good results with improvement in survival, although HCC recurrence remains very frequent [9–13]. For these reasons, stimulation of the anti-tumour immune response would represent a novel and auspicable strategy against HCC.
parenchimal organ. These impressive results were obtained by aggressive manipulation of the patient’s immune system that cannot be proposed for routine anti-tumour treatment [23]. Future steps will be to exploit these ideas and experiences to develop a feasible treatment opportunity. HCC is an excellent setting for tumour immunology research and for moving from bench to bedside into the clinic. HCC is not as immunogenic as melanoma; however, it is confined to a single organ in the great part of its natural history, so that it can be easily monitored by radiological techniques, from ultrasound to magnetic resonance and HCC nodules can be reached by US-guided approaches for sampling or intra-tumour injections. Moreover, since no chemotherapy of proven efficacy is available, the patient’s immune system is spared by the toxic effect of first-line aggressive conventional chemotherapy treatments.
2. Background Two hundred years ago the first vaccine against smallpox was described [14]. Nowadays, more than 20 infectious diseases are preventable by effective vaccines; however, several other viral, bacterial and parasitic diseases remain a challenge for preventive vaccine and therapeutic immunotherapies. Microbial agents express non-self antigens and molecular structures that are natural activator of the immune system. Instead, tumour-associated antigens (TAAs) are selfproteins modified by genetic alterations or abnormally expressed in a transformed tissue. The idea of cancer immune surveillance goes back to the time Ehrlich formulated the hypothesis of anti-tumour immune response, before lymphocytes were discovered, and to the time (early 1900) Cooley injected sarcomas with Gram-positive bacteria, the so-called ‘Cooley’s toxin’, obtaining tumour size reduction. The antigen was already there, in the tumour, and the ‘Cooley’s toxin’ was the adjuvant; this was the first attempt to exploit the idea of cancer immune surveillance. Attempts to enhance melanoma immunogenicity go back more than 40 years from now [15]. Since 1991, when the first TAA was identified in melanoma [16], several trials of active immunotherapy have been conducted. Experimental evidence that the immune system is able to recognise TAAs as targets and to subsequently destroy the tumour cells has accumulated in the last 10 years; however, limitations in translating successfully this knowledge into clinical applications are still to be overcome. Enhancement of a tumour-specific T-cell response is difficult to achieve because of the tumour immunological tolerance [17,18]. Three main issues have to be addressed by basic, translational and clinical research: identification of immunogenic TAAs, discovery of new adjuvants to employ in the clinic and standardisation of immunotherapeutical protocols to treat patients. Melanoma immunotherapy is the icebreaker of the field. A variety of cancer vaccines have been tested, including the use of immunogenic peptides, recombinant viruses, naked DNA, peptide pulsed dendritic cells (DCs), etc. [19–22]. Proof of principle has been achieved by the evidence of regression of melanoma metastasis from any
3. HCC antigenic targets Several TAAs have been identified, among which antigens of the cancer testis (CT) family are of particular interest, since they are expressed by tumours of different histological types [24,25]. They are silent in normal cells, with the exception of male germline cells that do not express HLA class I molecules and are therefore unable to present antigens to CTL. For these reasons, they are of particular interest in cancer immunotherapy. Melanoma-associated antigens (MAGE), and in particular MAGE-1 and MAGE-3 genes, are expressed in 46–80% of resected HCC tissues and none is detected in the non-tumour liver [26–28]. We have demonstrated the presence of HCC-specific CD8+ T cells, specific for MAGE-1 and MAGE-3 [29], in the tumour and peripheral blood of HCC patients. These results were recently confirmed by the identification of CD8+ T cells specific for two additional CT-antigens: MAGE-10 and SSX-2 [30]. It is relevant to note that ex vivo demonstration of spontaneously arising tumour-specific T cells within the tumour tissue was previously provided only in melanoma; this finding in HCC confirms that tumour-specific T-cell responses are indeed induced in cancer, since in vitro expanded tumour-specific T cells could simply be the result of artefact. These new findings in HCC open the way for pursuing anti-tumour strategies previously employed for melanoma and other solid tumours [31,32] also in the HCC. In almost 50% of HCCs, alpha-fetoprotein (AFP) expression reactivates. The overexpression of this protein, which is the major serum protein in the foetus but is quite low in adults, makes it a possible candidate as an antigenic target for anti-HCC immunotherapy. This possibility is confirmed by the detection of enhanced AFP-specific CD8 cell responses in HCC patients undergoing AFP-peptides vaccination [33] and in mice vaccinated with DNA encoding mouse AFP [34]. These results are promising because they further demonstrate the possibility to break tolerance to a self-protein like AFP.
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HBV and HCV antigens have been considered in the past possible targets for HCC therapy but modified viral proteins specifically expressed in the tumour and not in the adjacent normal liver have never been identified. Oncogenes and modified tumour suppressor genes are also involved in the pathogenesis of HCC and these molecules have been proposed as possible targets of immunotherapy in HCC, as in other tumours. Several other HCC-specific antigens are becoming available through gene array technology studies [35–37]; however, many tumour-specific T-cell responses are probably restricted to individual patients because of the known genetic heterogeneity of HCC. Thus, the identification of dominant TAAs in HCC remains an open challenge.
4. Clinical trials Clinical trials have been conducted in patients with HCC with the aim of enhancing anti-tumour immune responses in order to reduce HCC burden in patients with advanced disease or to reduce the risk of recurrence after curative resection. Different techniques were employed comprising the use of non-specific immunostimulating products, like cytokines, adoptive transfer of autologous-activated lymphocytes, DC-based immunotherapy, AFP-derived peptides vaccination or intra-tumour injection of recombinant adenoviral vectors engineered for the local release of interleukin-12 (IL12) [38]. The largest immunotherapy trial for HCC was conducted in 155 resected patients, who received IL-2 and ␣CD3 in vitro expanded autologous PBMCs to prevent recurrence. This study showed a statistically significant benefit in risk of recurrence, time to recurrence and recurrence-free survival, with a trend also in favour of an improvement in overall survival [39]. The majority of the other trials in HCC were conducted in patients with a high disease burden. Cytokine treatment was performed in 20 stage III and IV inoperable patients; interferon-gamma (IFN-␥) and IL-2 were administered in association with transarterial chemotherapy showing tumour size reduction in 14 patients [40]. A more recent cytokine treatment, based on subcutaneous injection of IFN-␥ and granulocyte–macrophage colony stimulating factor (GMCSF) in advanced HCC, did not show any clinical response [41]. Immature and mature DCs can be easily differentiated in vitro from circulating monocytes or CD34+ precursors; they have been widely employed against cancer in the last 10 years in more than 60 different clinical studies of tumour immunotherapy (http://www.kun.nl/til/dc2003/). Exploiting their unique capacity to internalise antigen, process and present it in the context of HLA-class I and II molecules and provide the optimal co-stimuli to induce antigen-specific CD4 and CD8 T-cell responses, DCs were employed in HCC immunotherapy, like in other tumour settings. DCs
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were pulsed with autologous tumour lysates and injected as immature [42,43] or in vitro matured DCs [44]. Results were quite disappointing with only a few cases of minor or partial responses. A recent trial, based on injection of DCs pulsed with autologous tumour lysates, compared two protocols of vaccination. The first consisted of a weekly series of five vaccinations, and the second was the five-weekly vaccinations followed by a monthly boost. By this second approach a significant improved survival was shown, even though, also in this trial, clinical responses were limited to partial responses or stable disease [45]. Differently from the previously described experiences that were based on multispecific T-cell stimulation by the use of crude tumour protein lysates, vaccination of HCC patients with AFP-derived peptides emulsified in Montanide ISA-51 adjuvant, allowed to monitor the in vivo effect of a well-defined antigen-specific T-cell expansion. The trial conducted in advanced-stage AFPsecreting HCC patients did not report any clinical response but the immunological results were satisfactory, showing that AFP-specific functional T-cell responses could be elicited in these patients even when high serum levels of AFP were present [33].
5. Conclusions Thanks to the refinement of treatment protocols and the tremendous investments of the pharmaceutical industry in conventional chemotherapy, improvements in cancer treatment have been relevant in the last decade. However, nowadays it is clear that different approaches for combating cancer are needed for a further step ahead in the field. Biologic therapy of cancer includes the use of new molecules targeted to altered receptors involved in cellular activation, or to key oncogenic signalling pathways as well as the use of monoclonal antibodies in passive immunotherapy against activation receptors, endothelial growth factors and surface molecular markers mediating apoptosis of targeted tumour cells. Active immunotherapy is still in preclinical and clinical trial phase of development but it will become available in the clinics in the near future. The growing knowledge in genomics, proteomics, safe recombinant vectors engineering will provide new tools for treatment of patients with active immunotherapies. Moreover, great innovations are expected for the development of new adjuvant molecules; until now, almost all commercial vaccines are still based only on alum-derived adjuvants, even though immunological mechanisms and receptor involved in triggering immune responses are becoming to be characterised in great detail. Passive immunotherapy of melanoma, by adoptive transfer and manipulation of regulatory Tcell responses, is achieving considerable results. Results in HCC, as in other malignancies will follow in the near future and clinical success will be derived by the combination of new biological treatments with passive and active immunotherapy.
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Conflict of interest statement None declared. [21]
References [22] [1] Bosch FX, Ribes J, Cleries R, Diaz M. Epidemiology of hepatocellular carcinoma. Clin Liver Dis 2005;9:191–211. [2] O’Grady J, Lake JR, Howdle PD. Malignant tumours of the liver. In: O’Grady J, Lake JR, Howdle PD, editors. Comprehensive clinical hepatology. London: Harcourt Publishers; 2000. p. 25.18– 25.21. [3] El-Serag HB, Mason AC. Rising incidence of hepatocellular carcinoma in the United States. N Engl J Med 1999;340:745–50. [4] Hassan MM, Frome A, Patt YZ, El-Serag HB. Rising prevalence of hepatitis C virus infection among patients recently diagnosed with hepatocellular carcinoma in the United States. J Clin Gastroenterol 2002;35:266–9. [5] Jemal A, Murray T, Ward E, Samuels A, Tiwari RC, Ghafoor A, et al. Cancer statistics, 2005. CA Cancer J Clin 2005;55:10–30. [6] Clemente CG, Mihm Jr MC, Bufalino R, Zurrida S, Collini P, Cascinelli N. Prognostic value of tumor infiltrating lymphocytes in the vertical growth phase of primary cutaneous melanoma. Cancer 1996;77:1303–10. [7] Zhang L, Conejo-Garcia JR, Katsaros D, Gimotty PA, Massobrio M, Regnani G, et al. Intratumoral T cells, recurrence, and survival in epithelial ovarian cancer. N Engl J Med 2003;348:203–13. [8] Wada Y, Nakashima O, Kutami R, Yamamoto O, Kojiro M. Clinicopathological study on hepatocellular carcinoma with lymphocytic infiltration. Hepatology 1998;27:407–14. [9] Carr BI. Hepatocellular carcinoma: current management and future trends. Gastroenterology 2004;127(5 Suppl. 1):S218–24. [10] Jansen MC, van Hillegersberg R, Chamuleau RA, van Delden OM, Gouma DJ, van Gulik TM. Outcome of regional and local ablative therapies for hepatocellular carcinoma: a collective review. Eur J Surg Oncol 2005;31:331–47. [11] Mulcahy MF. Management of hepatocellular cancer. Curr Treat Options Oncol 2005;6:423–35. [12] Camma C, Di Marco V, Orlando A, Sandonato L, Casaril A, Parisi P, et al. Treatment of hepatocellular carcinoma in compensated cirrhosis with radio-frequency thermal ablation (RFTA): a prospective study. J Hepatol 2005;42:535–40. [13] Raut CP, Izzo F, Marra P, Ellis LM, Vauthey JN, Cremona F, et al. Significant long-term survival after radiofrequency ablation of unresectable hepatocellular carcinoma in patients with cirrhosis. Ann Surg Oncol 2005;12:616–28. [14] Jenner E. An inquiry into the causes and effects of variolae vaccinae, a disease discovered in some of the western counties of England. London: Sampson Low; 1798. [15] Woodruff MF, Nolan B. Preliminary observations on treatment of advanced cancer by injection of allogeneic spleen cells. Lancet 1963;13:426–9. [16] van der Bruggen P, Traversari C, Chomez P, Lurquin C, De Plaen E, Van den Eynde B, et al. A gene encoding an antigen recognized by cytolytic T lymphocytes on a human melanoma. Science 1991;254:1643–7. [17] Willimsky G, Blankenstein T. Sporadic immunogenic tumours avoid destruction by inducing T-cell tolerance. Nature 2005;437:141– 6. [18] Klein G, Klein E. Surveillance against tumors—is it mainly immunological? Immunol Lett 2005;100:29–33. [19] Parmiani G, Castelli C, Rivoltini L, Casati C, Tully GA, Novellino L, et al. Immunotherapy of melanoma. Semin Cancer Biol 2003;13:391–400. [20] Rosenberg SA, Yang JC, Schwartzentruber DJ, Hwu P, Marincola FM, Topalian SL, et al. Immunologic and therapeutic evaluation of a
[23]
[24]
[25]
[26]
[27]
[28]
[29]
[30]
[31]
[32]
[33]
[34]
[35]
[36]
[37]
[38]
synthetic peptide vaccine for the treatment of patients with metastatic melanoma. Nat Med 1998;4:321–7. Rosenberg SA, Yang JC, Schwartzentruber DJ, Hwu P, Marincola FM, Topalian SL, et al. Impact of cytokine administration on the generation of antitumor reactivity in patients with metastatic melanoma receiving a peptide vaccine. J Immunol 1999;163:1690–5. Scheibenbogen C, Schmittel A, Keilholz U, Allgauer T, Hofmann U, Max R, et al. Phase II trial of vaccination with tyrosinase peptides and granulocyte–macrophage colony stimulating factor in patients with metastatic melanoma. J Immunother 2000;23:275–81. Rosenberg SA, Dudley ME. Cancer regression in patients with metastatic melanoma after the transfer of autologous antitumor lymphocytes. Proc Natl Acad Sci USA 2004;101(Suppl. 2):14639–45. Novellino L, Castelli C, Parmiani G. A listing of human tumor antigens recognized by T cells: March 2004 update. Cancer Immunol Immunother 2005;54:187–207 [Epub 2004, August 7]. Simpson AJ, Caballero OL, Jungbluth A, Chen YT, Old LJ. Cancer/testis antigens, gametogenesis and cancer. Nat Rev Cancer 2005;5:615–25. Luo G, Huang S, Xie X, Stockert E, Chen YT, Kubuschok B, et al. Expression of cancer-testis genes in human hepatocellular carcinomas. Cancer Immun 2002;2:11. Mou DC, Cai SL, Peng JR, Wang Y, Chen HS, Pang XW, et al. Evaluation of MAGE-1 and MAGE-3 as tumour-specific markers to detect blood dissemination of hepatocellular carcinoma cells. Br J Cancer 2002;86:110–6. Kobayashi Y, Higashi T, Nouso K, Nakatsukasa H, Ishizaki M, Kaneyoshi T, et al. Expression of MAGE, GAGE and BAGE genes in human liver diseases: utility as molecular markers for hepatocellular carcinoma. J Hepatol 2000;32:612–7. Zerbini A, Pilli M, Soliani P, Ziegler S, Pelosi G, Orlandini A, et al. Ex vivo characterization of tumor-derived melanoma antigen encoding gene-specific CD8+cells in patients with hepatocellular carcinoma. J Hepatol 2004;40:102–9. Bricard G, Bouzourene H, Martinet O, Rimoldi D, Halkic N, Gillet M, et al. Naturally acquired MAGE-A10- and SSX-2-specific CD8+ T cell responses in patients with hepatocellular carcinoma. J Immunol 2005;174:1709–16. Reynolds SR, Zeleniuch-Jacquotte A, Shapiro RL, Roses DF, Harris MN, Johnston D, et al. Vaccine-induced CD8+ T-cell response to MAGE-3 correlate with clinical outcome in patients with melanoma. Clin Cancer Res 2003;9:657–62. Vantomme V, Dantinne C, Amrani N, Permanne P, Gheysen D, Bruck C, et al. Immunologic analysis of a phase I/II study of vaccination with MAGE-3 protein combined with the AS02B adjuvant in patients with MAGE-3-positive tumors. J Immunother 2004;27:124–35. 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;9:5902–8. Geissler M, Mohr L, Weth R, Kohler G, Grimm CF, Krohne TU, et al. Immunotherapy directed against alpha-fetoprotein results in autoimmune liver disease during liver regeneration in mice. Gastroenterology 2001;121:931–9. Tabor E. Tumor suppressor genes, growth factor genes, and oncogenes in hepatitis B virus-associated hepatocellular carcinoma. J Med Virol 1994;42:357–65. Okabe H, Satoh S, Kato T, Kitahara O, Yanagawa R, Yamaoka Y, et al. Genome-wide analysis of gene expression in human hepatocellular carcinomas using cDNA microarray: identification of genes involved in viral carcinogenesis and tumor progression. Cancer Res 2001;61:2129–37. Wang Y, Han KJ, Pang XW, Vaughan HA, Qu W, Dong XY, et al. Large scale identification of human hepatocellular carcinomaassociated antigens by autoantibodies. J Immunol 2002;169:1102–9. Sangro B, Mazzolini G, Ruiz J, Herraiz M, Quiroga J, Herrero I, et al. Phase I trial of intratumoral injection of an adenovirus
A. Zerbini et al. / Digestive and Liver Disease 38 (2006) 221–225 encoding interleukin-12 for advanced digestive tumors. J Clin Oncol 2004;22:1389–97. [39] 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;356:802–7. [40] Lygidakis NJ, Kosmidis P, Ziras N, Parissis J, Kyparidou E. Combined transarterial targeting locoregional immunotherapy– chemotherapy for patients with unresectable hepatocellular carcinoma: a new alternative for an old problem. J Interferon Cytokine Res 1995;15:467–72. [41] Reinisch W, Holub M, Katz A, Herneth A, Lichtenberger C, Schoniger-Hekele M, et al. Prospective pilot study of recombinant granulocyte macrophage colony-stimulating factor and interferongamma in patients with inoperable hepatocellular carcinoma. J Immunother 2002;25:489–99.
225
[42] 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;17:889–96. [43] 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;52:155–61. [44] Stift A, Friedl J, Dubsky P, Bachleitner-Hofmann T, Schueller G, Zontsich T, et al. Dendritic cell-based vaccination in solid cancer. J Clin Oncol 2003;21:135–42. [45] Lee WC, Wang HC, Hung CF, Huang PF, Lia CR, Chen MF. Vaccination of advanced hepatocellular carcinoma patients with tumor lysate-pulsed dendritic cells: a clinical trial. J Immunother 2005;28:496–504.