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Molecular Therapies for Colorectal Cancer Metastatic to the Liver
REVIEW
doi:10.1006/mthe.2002.0596, available online at http://www.idealibrary.com on IDEAL
Molecular Therapies for Colorectal Cancer Metastatic to the Liver Philipp Mayer-Kuckuk,1 Debabrata Banerjee,1,* Nancy Kemeny,2 Yuman Fong,3 and Joseph R. Bertino1,*,† 1Program
of Molecular Pharmacology and Therapeutics, 2Department of Medicine, 3Department of Surgery, Memorial Sloan-Kettering Cancer Center, New York, New York 10021, USA *Present address: The Cancer Institute of New Jersey, New Brunswick, New Jersey 08903, USA †To whom correspondence and reprint requests should be addressed. Fax: (732) 235-8181. E-mail: [email protected].
Colorectal cancers are the fourth most commonly diagnosed cancers and will account for over 56,000 deaths in the United States in 2002. A majority of patients with advanced colorectal cancer develop liver metastases during the course of their disease. Treatment of colorectal cancer metastatic to the liver by surgery or chemotherapy is limited and most patients succumb to their disease. Therefore, a broad spectrum of novel treatments, including innovative molecular therapies such as gene and immunotherapy or replication-competent viral therapy, is under preclinical investigation and several clinical trials are in progress. Here we review molecular therapies for colorectal cancer metastatic to the liver. Key Words: colorectal cancer, liver, metastases, molecular therapies, gene therapy, immunotherapy, replication-competent viral therapy, animal model, clinical trial, review
INTRODUCTION Colorectal cancer metastatic to the liver contributes significantly to cancer-related death in the United States. Two-thirds of colorectal cancer patients develop metastases to the liver during the course of their disease. The liver is the primary site of metastases from colorectal cancer and approximately 50,000 cases of hepatic metastases are diagnosed annually in the U.S. Patients with hepatic metastases have a median survival of 9–11 months [1]. The best treatment option for isolated metastases is surgery, which has the potential to cure the disease. However, only 20–25% of the patients who present with hepatic metastases are suitable for resection, and recurrence after surgery is frequent [2]. Following resection, regional chemotherapy using hepatic arterial infusion (HAI) of FUDR (fluorodeoxyuridine, floxuridine, a thymidylate synthase inhibitor precursor that is efficiently extracted by colon tumors in the liver) in combination with dexamethasone plus systemic 5-fluorouracil (5FU)/leucovorin (LV) improved both the local control of hepatic metastasis [3] and, in follow-up data,
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survival as compared with adjuvant systemic chemotherapy with 5FU/LV alone. At 2 years only 10% of the group receiving hepatic arterial infusion had tumor recurrence in the liver. Treatment options for unresectable metastases are further restricted. As reviewed by Koea and Kemeny [4], randomized clinical studies show that a high percentage of patients respond to regional administration of FUDR with either LV or dexamethasone in comparison with systemic therapy. HAI therapy led to up to 50% partial and a subset of complete responses, whereas systemic therapy resulted in 20% partial and no complete response. However, improved survival of patients with unresectable metastases through HAI remains to be proved. In view of the limited current treatment options for colorectal cancer metastatic to the liver, a large number of novel molecular therapies for this disease are under investigation. Molecular therapies to treat hepatic metastases from colorectal cancer are rooted in the development of techniques to specifically design macromolecules such as DNA or antibodies and even viruses or cells (such as dendritic cells) for therapeutic use in vivo. Furthermore, progress in the understanding of the molecular biology of colorectal cancer is key to the development of treatments such as immunotherapy, which is based on the discovery of tumor-specific antigens. In addition, the recently increased insight into development of metastases has resulted in new concepts for the treatment of hepatic metastases, such as anti-angiogenic gene therapy. Finally, novel surgical approaches contribute to treatment strategies based, for example, on regional gene delivery to the hepatic tumor. Although this review is focused on molecular therapies, it is of great interest that many of these therapies have the potential to merge with molecular imaging. With appropriate tracers, this technique will allow specific and noninvasive monitoring of, for example, transgene expression of herpes simplex virus thymidine kinase (HSV1-TK) in living subjects. Recently, Bennett and colleagues [5] used positron emission tomography (PET) to demonstrate in rats the feasibility to monitor infection of primary colorectal flank tumors with an MOLECULAR THERAPY Vol. 5, No. 5, May 2002, Part 1 of 2 Parts Copyright © The American Society of Gene Therapy
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oncolytic virus carrying the HSV1-TK gene. Figure 1 demonstrates the feasibility of imaging a human colorectal tumor expressing HSV1-TK as a fusion protein [6] in the liver of living rats using microPET and the tracer [124I]-2-fluoro-2-deoxy-5-iodouracil--D-arabinofuranoside (P.M.-K. et al., unpublished data). Thus, the fate of macromolecules, viruses, or cells used for molecular therapies may be monitored in individual patients during the course of their treatment. Here we review new approaches to treat hepatic metastases from colorectal cancer in three sections: gene therapy, immunotherapy, and viral therapy. In particular, we review in detail preclinical studies involving animal models of liver metastases from colon cancer, as well as considering past and current clinical studies.
GENE THERAPIES Targeting the Tumor Cell. Most gene therapies require gene transfer directly into the tumor. As described in the following section, gene transfer into the malignant lesion can result in tumor sensitivity to specific drugs, restoration of a tumor suppressor, or enhancement of anti-tumor immunity. It is of special interest that combinations of these approaches result in significantly enhanced and prolonged anti-tumor activity in preclinical animal models. It is also promising that therapies such as HSV1-TK/ganciclovir, cytosine deaminase/5-fluorocytosine, wild-type p53, or the drug Allovectin-7 have already entered human clinical trials. However, the value of these therapies to treat colorectal cancer metastatic to the liver is still unproven. HSV1-TK/Ganciclovir. Expression of HSV1-TK in cancer cells confers sensitivity to the nontoxic prodrug ganciclovir (GCV). The enzyme HSV1-TK selectively converts the nucleotide analog GCV into the monophosphate. Further phosphorylation of GCVmonophosphate by cellular kinases yields the cytotoxic GCV-triphosphate, and incorporation of this artificial nucleotide analog into DNA results in cell death. The first clinical study exploiting HSV1-TK/GCV gene therapy of colorectal cancer metastatic to the liver was conducted recently [7]. Sixteen patients received intratumoral injections of an adenovirus carrying the HSV1TK gene (for gene transfer strategies see Table 1). Patients were treated with virus doses of up to 1 1013 virus particles and subsequently with a fixed dose of GCV. The study showed that up to 1 1013 virus particles were administered safely. Hepatic toxicities were low and the most common side effect was fever. Overall the treatment was well tolerated. Before this clinical study, several preclinical, syngeneic animal models have described the successful application of this therapy to treat colon cancer MOLECULAR THERAPY Vol. 5, No. 5, May 2002, Part 1 of 2 Parts Copyright © The American Society of Gene Therapy
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metastatic to the liver [8–10]. Significant tumor suppression was observed, but in these animal models HSV1-TK/GCV therapy did not result in long-term, disease-free survivals. Thus the strategy of generating an enhanced therapeutic effect using additional immunoresponse-modulating genes was tested. In two studies using mouse models of hepatic metastasis, coexpression of interleukin-2 (IL-2) as well as IL-2 plus granulocyte macrophage colony stimulating factor (GM-CSF) with HSV1-TK significantly decreased tumor volume [9,10]. GM-CSF expression in combination with IL-2 and HSV1-TK/GCV resulted in a long-term survival of 25% of the animals, followed up to 150 days, as compared with a survival of 60 days of animals treated with HSV1-TK/GCV in combination with IL-2 or GM-CSF. The percentage of surviving animals was further enhanced by boosting immunity with irradiated, IL-2-transduced tumor cells. Cytosine Deaminase/5-Fluorocytosine. Cancer cells transduced with the Escherichia coli or yeast cytosine deaminase (CD) selectively convert the nontoxic prodrug 5-fluorocytosine (5FC) into the cytotoxic chemoreagent 5-fluorouracil (5FU). Consequently, transduced cancer cells and, to a variable extent, neighboring nontransduced cells (bystander effect) are killed. A phase I study (NCI–V95-0649; source of clinical trials, Physician Data Query via http://cancernet.nci.nih.gov) of intratumoral delivery of an adenoviral vector containing the CD gene (AdCMV.CD.10) into macroscopic liver metastasis from colon cancer followed by systemic administration of 5FC was conducted [11]. No details of the study have been published yet. However, the feasibility of this approach was shown using an in vivo model of subcutaneously implanted human HT29 tumors in nude mice [12]. Intratumoral injection of a vector (AdCMV.CD) similar to the one used in the clinical study followed by 5FC treatment resulted in a significant decrease of tumor size after 4 weeks compared with controls. Furthermore, hepatic arterial infusion of a retrovirus carrying the CD gene under control of the carcinoembryonic antigen (CEA) promoter was used for regional delivery [13]. Rats bearing hepatic tumors from rat colorectal cancer received 1 107 virus particles followed 48 hours later by daily intraperitoneal injections of 5FC for 7 days. After treatment hepatic tumor volumes were reduced. In contrast to these strategies, which require regional delivery of the CD gene, Block et al. [14] used a syngeneic mouse model of hepatic micrometastasis to study the effect of systemic adenoviral-mediated CD gene transfer in combination with 5FC administration. The adenovirus was injected 48 hours after splenic injection of tumor cells and 24 hours after spleenectomy. Following this procedure, multiple micrometastases in the liver were observed. Specific delivery of the CD gene into the tumor cells was detec-
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TABLE 1: Gene transfer strategies for gene therapies to treat colorectal cancer metastatic to the liver Vector
Promoter
Therapeutic gene
Administration
References
rec. adenovirus
CMV
CD
human, mouse; intratumoral
[11,12]
rec. adenovirus
CMV
p53
rat; intratumoral
[21]
rec. adenovirus
CMV
p53
mouse; peritumoral
[19]
rec. adenovirus
CMV
p53
rat; HAI
[20,21]
rec. adenovirus
CMV
CD
mouse; systemic
[14,35]
rec. adenovirus
CMV
p53
rat; systemic
[21]
rec. adenovirus
CMV
FLT-1
mouse; systemic
[38]
rec. adenovirus
CMV
IL-12
mouse; systemic
[32]
rec. adenovirus
RSV LTR
HSV1-TK, IL-2, GM-CSF
human, mouse; intratumoral
[7,9,10]
rec. adenovirus
RSV LTR
IL-12, 4-1BBL
mouse; intratumoral
[24–26]
rec. adenovirus
RSV
endostatin
mouse; systemic
[37]
rec. adenovirus
CAG
IL-12
mouse; systemic
[23]
rec. adenovirus
n.s.
TIMP-2
mouse; systemic
[30]
retrovirus
CEA
CD
rat; HAI
[13]
DMRIE DOPE
synthetic
CD
mouse; systemic
[15]
vaccina virus
RSV
HLA-B7, -2microglobulin
human; intratumoral
[27]
CD, cytosine deaminase; CAG, cytomegalovirus enhancer plus chicken -actin; CEA, carcinoembryonic antigen; CMV, cytomegalovirus; DMRIE, (2,3-bis-tetradecyloxy) propyl,b-hydroxylethyl, dimethylammoniumbromide; DOPE, dioleoyl phosphatidyl-ethanolamine; GM-CFS, granulocyte macrophage colony-stimulating factor; HAI, hepatic arterial infusion; HSV1-TK, herpes simplex virus 1 thymidine kinase; IL-2, interleukin 2; IL-12, interleukin 12; LTR, long terminal repeat; n.s., not specified; rec., recombinant; TIMP-2, tissue inhibitor of metalloproteinase-2.
ted and after 10 days of 5FC treatment a significant reduction in the number of hepatic metastasis was noted. A systemic, tumor-specific delivery of the CD gene using a vaccina virus vector was reported by Gnant and coworkers [15]. In a model of disseminated liver metastasis in mice, systemic administration of the vector followed by treatment with 5FC resulted in cure rates of up to 30%. An additional observation of interest was made using the CD/5FC strategy. Rats bearing tumors derived from CD-transduced rat colon carcinoma cells were treated with 5FC and subsequently injected with the wild-type rat colon carcinoma cells [16]. Development of wild-type tumors in the animals was not seen. This observation points to a vaccination effect of the CD/5FC therapy. Wild-Type TP53. Approximately 50% of human cancers show mutations in the gene TP53. Therefore, the transfer of wild-type TP53 into cancer cells to restore the tumor-suppressor function of TP53 has been explored. In addition, restoration of wild-type TP53 in tumor cells can result in increasing responses to chemotherapy and/or X-rays. In a phase I study (NCI-V97-1179),
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patients with liver metastasis from colon cancer were treated with a replication-deficient adenovirus containing wild-type TP53 (SCH-58500), via hepatic arterial infusion. Additionally, a phase II trial of SCH-58500 in patients with colorectal cancer metastatic to the liver has commenced. Data on these trials are very limited [17]. A large number of patients with several types of advanced cancers were treated in multicenter phase I and II trials by intratumoral administration of adenovirus encoding wild-type p53 and some partial responses were seen with significant clinical safety [18]. The potential of adenovirus-mediated wild-type p53 gene transfer to inhibit tumor growth of human colon carcinoma cells expressing mutant p53 has been demonstrated previously in vitro and in vivo [19]. Moreover, the feasibility of regional delivery via hepatic arterial infusion of an adenovirus expressing wildtype p53 has been shown in a rat model [20]. In this study, intrahepatic arterial dosing of the virus increased transgene expression in the hepatic tumor tissue and decreased systemic exposure when compared with intravenous (i.v.) dosing. However, this study used a hepatocellular carcinoma in a syngenic rat model. In MOLECULAR THERAPY Vol. 5, No. 5, May 2002, Part 1 of 2 Parts Copyright © The American Society of Gene Therapy
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contrast, a recent report describes very low hepatic tumor transduction following hepatic arterial infusion of an adenovirus in a rat model of human metastatic colorectal carcinoma [21]. No therapeutic effect of adenoviral wild-type p53 transfer was seen. Interleukins. Interleukin-2 (IL-2) and interleukin-12 (IL-12) are growth factors for T cells, which enhance nonspecific immune responses such as natural killer or lymphokine-activated killer cells, as well as the cytotoxic T-cell response. These functions suggest a potential use for IL-2/IL-12 in cancer gene therapy. A phase I/II study is presently underway to evaluate gene therapy with a replication-deficient adenovirus containing the IL-2 gene (TG 1021) in patients with unresectable digestive adenocarcinomas [22]. The adenoviral vector was injected into the liver lesion of a patient with adenocarcinoma metastatic to the liver and lung. However, clinical and preclinical data supporting this approach are limited. The IL-2 gene was also used in a unique manner for adjuvant gene therapy, in which the capability of adenovirus to preferentially infect liver tissue and not tumor tissue was used. In mice, hepatocytes were adenovirally transduced with IL-2, resulting in a temporary inhibition of hepatic metastasis upon injection of tumor cells [23]. Another promising strategy to treat colon cancer metastatic to the liver is based on work by Caruso et al. [24]. Using a model for hepatic metastasis of colon carcinoma in mice, the antitumor efficacy of an adenovirus expressing HSV1-TK and IL-2, in combination with GCV treatment, was compared with an IL-12 expressing virus. The adenovirus-mediated IL-12 gene therapy resulted in increased survival of the animals compared with the HSV1-TK/IL-2 treatment [24]. Additionally, two recent reports have described adenoviral gene therapy strategies for hepatic metastases in mice based on IL-12 in combination with co-stimulation of the T-cell receptor 4-1BB. Binding of 4-1BB to its ligand, presented on antigen-presenting cells, results in expansion of antigen-specific, cytotoxic T cells. In one study [25], the gene encoding the ligand of 4-1BB was transferred into the tumor, and another study used an agonistic anti-4-1BB antibody [26]. Liver tumor regression, a 60–80% long-term survival of at least 3 months (which was 2-fold to 2.5-fold higher than in the non 4-1BB co-stimulated control), and potent long-term antitumor immunity were observed. HLA-B7/-2-Microglobulin. Loss of major histocompatibility (MHC) class I antigens has been reported in several different tumor types including metastasis. HLA histocompatibility antigen (HLA) alterations are detectable in approximately 60% of human tumors. The feasibility and safety of HLA-B7 and -2-microglobulin gene transfer into the liver lesions of patients with MOLECULAR THERAPY Vol. 5, No. 5, May 2002, Part 1 of 2 Parts Copyright © The American Society of Gene Therapy
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colorectal carcinoma have been shown in a phase I study [27]. Both genes were encoded by a single plasmid and formulated with the lipid DMRIE-DOPE to yield the liposomal vector Allovectin-7. Patients received single (10 to 250 mg) or multiple (10 mg) injections of the vector directly into the liver lesion. Gene transfer was detected by PCR in 14 of 15 patients, whereas gene expression was detected by immunohistochemistry only in 5 of 15 patients. There has been no serious toxicity directly attributable to Allovectin-7. Therapeutic effects were not reported. Plasminogen Activator. Plasmin-activating proteinase (tissue plasminogen activator, tPA) is often downregulated in colorectal cancer. One study investigated treatment of hepatic metastases from colorectal cancer using tPA. The survival rates of mice bearing hepatic metastasis from murine colon carcinoma cells retrovirally transduced with tPA or LacZ as a control were compared. Significantly less hepatic tumor growth and prolonged survival of mice with metastasis from tPA-transduced colon cancer cells were observed [28]. Targeting the Liver. This section describes anti-tumor gene therapies based on gene transfer into the liver tissue. The starting point of these strategies was the identification of suitable therapeutic genes, driven by the finding that adenovirus preferentially infects hepatocytes. In general these strategies seem to be less attractive, because of the large number of normal liver cells infected. Moreover, in mouse models expression of adenoviral-mediated therapeutic genes in the liver was shown not to correlate with the injected viral dose [29]. Transduction of hepatocytes was detected after administration of 1 1011 viral particles, whereas lower doses of 1 to 3 1010 particles were taken up efficiently by the hepatic Kupffer cells. Metalloproteinases (MMP) are a family of endopeptidases believed to have a critical role in tumor invasion and metastasis and frequently upregulated in colorectal cancer. A mouse model was used to demonstrate systemic adenoviral delivery and expression of tissue inhibitor of metalloproteinase (TIMP)-2 in the liver [30]. Liver TIMP-2 expressing animals showed a significant reduction in tumor growth of liver micrometastasis of a human colon adenocarcinoma expressing a high level of MMP-2, compared with control animals. Maximum inhibition in tumor growth was seen with TIMP-2 delivery pre- or early post-metastases formation. The group of Melero and Prieto [31] observed a murine strain-dependent difference in liver gene transduction levels by recombinant adenovirus. BALB/c or C57BL/6 mice were injected i.v. with adenovirus carrying a reporter gene (such as -galactosidase or luciferase) under control of the cytomegalovirus (CMV) promoter. A 10- to 100-fold higher transgene expression
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was subsequently measured in the liver of C57BL/6 compared with BALB/c mice. The same group demonstrated, in a syngeneic model of concomitantly subcutaneous and hepatic colon tumors in BALB/c mice, an v3 integrin receptor mediated accumulation of systemically circulating recombinant adenovirus carrying a reporter gene or the IL-12 gene in the tissue surrounding the liver tumor [32]. Liver tumor regression after treatment with adenovirus expressing IL-12 was seen, but a strong synergistic effect with intravenous adoptive T-cell therapy [33] was observed [34]. Antitumor cytotoxic T lymphocytes were generated by injection of the subcutaneous tumors with adenovirus expressing IL-12 and subsequent short-time coculture of isolated lymph node cells together with mitomycin C treated tumor cells. In addition, further experiments suggest that, after adenoviral IL-12 treatment, VCAM-1 (CD 106) is induced in the tumor vasculature followed by T-cell recruitment [32]. Adenovirally CD-transduced hepatocytes have been shown to produce and release 5FU after 5FC treatment in vitro [35], therefore the possibility of inhibiting tumor growth in liver transduced with CD and treated with 5FC was tested. In a model of CD-transduced liver and subsequent colon carcinoma cell injection, tumor growth was suppressed after 15 days of 5FC treatment compared with controls [35]. Targeting the Neovasculature. Tumor anti-angiogenesis, the inhibition of tumor neovascularization, is a novel, highly promising anti-cancer strategy. The following section describes two gene therapy studies using an anti-angiogenic approach for the treatment of hepatic metastases from colorectal cancer. The presented data are encouraging and further studies have to address in detail the specific expression of therapeutically active levels of anti-angiogenic factors as well as the potential consequences of suppression of physiologic angiogenesis by anti-angiogenic gene therapy. Moreover, the application of anti-angiogenic gene therapy as adjuvant therapy or treatment for established tumors has to be assessed. Anti-angiogenic Gene Therapy. The anti-tumor effect of vector-mediated systemic expression of anti-angiogenic proteins, such as soluble endostatin or FLT-1 VEGF receptor was investigated in several studies. Although data cannot be presented here in detail, the observed anti-tumor effects of, for example, endostatin seem to be dependent on the experimental in vivo model. In particular vector type and dose, resulting systemic antiangiogenic protein levels, tumor cell type, as well as the time of tumor cell inoculation compared with start of therapy seem to be critical. Only one study [36] took a comparative approach and observed a superior antitumor effect of FLT-1 VEGF receptor as compared with
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FIG. 1. Noninvasive imaging of an orthotopic human colorectal tumor model in living rats using positron emission tomography (PET) scanning. The cell line C85 was derived from the liver metastases of a colorectal cancer patient. C85 cells were retrovirally transduced to express a double-mutant dihydrofolate reductase fused to the reporter enzyme HSV1-TK. These cells were grown as flank tumors in nude rats and subsequently orthotopically implanted in the rat liver. For non-invasive imaging, rats received the PET tracer [124I]-2-fluoro-2deoxy-5-iodouracil--D-arabinofuranoside ([124I]FIAU) via the penile vein, and 2 hours later animals were anesthetized and subjected to microPET. Shown are three microPET images taken in the transversal, coronal, and sagittal planes. High accumulation of the specific HSV1-TK substrate [124I]FIAU is seen in red, whereas low accumulation appears violet. The hepatic tumor is indicated by a white triangle. Unspecific tracer retention is seen in the bladder (black triangle). Location of tumor mass was verified by magnetic resonance images of the liver, which were taken before microPET imaging (data not shown).
endostatin in established murine and xenograft tumors. Chen and colleagues [37] showed in a mouse model that systemic delivery of an adenoviral vector that produced secretable murine endostatin resulted in prolonged and elevated levels of circulating endostatin when compared with mice treated with the empty vector. Mice received i.v. a single dose of 2 1011 virus particles, and 13 days later human colon tumor was implanted into the liver. A significantly prolonged survival with 25% complete prevention of tumor growth in the liver was seen in the endostatin-expressing animals in comparison with the controls [37]. Kong et al. [38] investigated the therapeutic effect of adenoviralmediated expression of a secreted form of the FLT-1 VEGF receptor in mice bearing preestablished tumors metastatic to the liver. A reduction of tumor burden was observed in animals expressing FLT-1 compared with controls treated with an empty vector [38]. Both studies show the potential of an anti-angiogenic gene therapy as an adjuvant or acute treatment for colorectal cancer metastatic to the liver.
IMMUNOTHERAPY Immunotherapy of colorectal cancer metastatic to the liver includes several different strategies. Very interesting is the concept of active immunotherapy using ex vivo modified dendritic cells. Data from two vaccination strategies show that this is a very attractive concept as adjuvant therapy. Furthermore, several strategies MOLECULAR THERAPY Vol. 5, No. 5, May 2002, Part 1 of 2 Parts Copyright © The American Society of Gene Therapy
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exploit antibody-based treatments. Dendritic Cells. Dendritic cells (DCs) are antigen-presenting cells capable of priming T cells in an HLArestricted fashion and initiating a T-cell response. In animal models DCs induce T-cell mediated anti-tumor immunity. A phase I/II study is currently investigating active immunotherapy with carcinoembryonic antigen (CEA) RNA transduced DCs in patients with resected hepatic metastasis from adenocarcinoma of the colon (NCI-G98-1456). The feasibility and safety of treatment of patients with cryopreserved DCs loaded with the CEA peptide CAP-1 were shown in a phase I study [39]. Autologous DCs were prepared from peripheral blood mononuclear cells, subsequently loaded with the CAP-1 peptide, and cryopreserved. Pulsed DCs were administered i.v. weekly or biweekly over 4 weeks in escalating dose levels. The same group showed that CEA RNAloaded DCs and CEA peptide-loaded DCs stimulate similar CEA-specific T-cell activity in vitro [40]. An improved approach using DCs loaded with an altered and therefore more potent CEA antigen was reported recently [41]. In this study, 9 of 12 patients with advanced or metastatic colon cancer received a pretreatment with FLT3 ligand, a hematopoietic growth factor, to expand the circulating blood DC population and were subsequently vaccinated with the peptideloaded DCs. Two patients with metastatic colon cancer showed an objective response lasting up to 10 months. Antibodies. Antibodies targeting specific antigens overexpressed in colon cancer cells such as CEA have been extensively investigated and treatment strategies for colorectal cancer have been reviewed [42]. Here we focus on antibody treatments for hepatic metastases from colorectal cancer. Over the past decade several groups reported clinical radioimmunotherapy studies using radioactive iodine labeled anti-CEA, TAG 72, or A33 murine or chimeric antibodies to treat liver metastases from colorectal cancer [43]. In most clinical studies successful tumor targeting and, in some cases, decreased CEA levels or stabilization of disease were observed. However, human anti-mouse antibody (HAMA) response, ineffective dose delivery to the tumor site, and hematologic toxicity resulted in limited efficiency of radioimmunotherapy. The feasibility of radioimmunotherapy was shown by Buchegger et al. [44] in a human colon carcinoma model in nude mice, where complete tumor regression was observed in 8 of 10 mice after treatment with the antibody 131I Mab F(ab)2 fragment. In addition to the studies using iodine isotope, a phase I study using a 90Y-labeled chimeric anti-CEA antibody was reported [45]. One notable but unexplained observation was that patients with extensive hepatic involvement by tumor demonstrated rapid blood clearance and poor tumor targeting of the antibody. Treatment of liver metastases by radioimmunotherapy in MOLECULAR THERAPY Vol. 5, No. 5, May 2002, Part 1 of 2 Parts Copyright © The American Society of Gene Therapy
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combination with radiotherapy has been reviewed [46]. Twenty years ago the anti-tumor activity of murine antibodies directed against the 17-1A (Ep-CAM) receptor on the surface of colon cancer cells was demonstrated in a mouse model [47]. Although HAMA responses, complement-mediated cytolysis, or an anti-idiotype cascade is proposed to be critical for the anti-tumor activity of MoAb 17-1A (edrecolomab), the exact mechanism of action of this antibody is not clear. However, in a clinical phase I/II study (NCI-88-C-64A) at least 15 patients with colorectal cancer metastatic to the liver received the MoAb 17-1A antibody in conjunction with IL-12. Additionally, a study investigating MoAb 17-1A antibody as adjuvant agent (NCI-V85-0072) included colorectal cancer patients with respectable hepatic metastases. Results from these studies are yet not published. Vascular endothelial growth factor (VEGF) is a secreted polypeptide and acts as a strong pro-angiogenic factor in both normal and pathologic angiogenesis, primarily through binding to receptor tyrosine kinases on endothelial cells. Kim et al. [48] demonstrated in an in vivo model suppression of tumor growth by inhibition of VEGF-induced angiogenesis. Tumor growth inhibition was seen in nude mice injected with human sarcoma or glioblastoma cells and subsequently treated with an anti-VEGF antibody. Later, Warren and colleagues [49] reported VEGF gene expression in surgically resected hepatic tumors from colorectal cancer. Furthermore, athymic mice bearing liver metastases from human colorectal cancer were injected with anti-VEGF antibody. Treatment started 24 hours after tumor cell inoculation and was continued for 4 weeks with antibody injections twice a week. Significant reduction in the number and volume of tumors per liver in the treated animals as compared with the control animals was observed [49]. Carcinoembryonic Antigen/B7.1. Vaccination by coexpression of a tumor-associated antigen and the costimulatory molecule B7 has been shown to improve anti-tumor immunity [50]. Two clinical pilot studies have reported a dose-dependent T-cell specific response and stable disease in approximately one of six patients with CEA-expressing metastatic colorectal carcinoma after treatment with ALVAC-CEA B7.1 [51,52]. This vaccine is based on a Canary Pox virus engineered to encode the genes for CEA and B7.1, a T-cell co-stimulatory molecule. The larger study enrolled 39 patients, of which 30 received up to 4.5 108 plaque forming units over 8 weeks intradermally every other week. In the other study the same dose was given intramuscularly (i.m.) to 6 patients, whereas 12 patients received smaller doses. Therapy was well tolerated and biopsies of the vaccine site revealed leukocytic infiltration and CEA expression. A further study assessed the influence of
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prior chemotherapy or administration of granulocyte macrophage colony-stimulating factor (GM-CSF) on ALVA-CEA B7.1 treatment [53]. A negative correlation between the number of chemotherapy regimes received and the generation of T-cell response was seen. GM-CSF given for 5 days before and in the beginning of vaccination significantly increased the number of patients with stable disease, but T-cell precursor frequencies were not elevated. Newcastle Disease Virus. Naturally oncolytic or nonlytic stains of the Newcastle disease virus (NDV) have been explored for cancer therapy, and NDV infection of autologous tumor cells as immunotherapy for patients with advanced colorectal cancer was described [54,55]. In one study, tumor cells from liver metastasis were infected ex vivo with a nonlytic NDV strain in order to modify the tumor cells to stimulate a broad antitumor response after re-injection in the patient [54]. The 23 patients enrolled in the trial well tolerated the vaccination following surgical resection of the liver metastasis. Tumor recurrence after 18 months was reduced by 25% in vaccinated patients compared with patients that had been only surgically treated.
REPLICATION-COMPETENT VIRAL THERAPY Cancer treatment using adenovirus or herpex simplex virus was developed through genetically engineering cytolytic strains of these DNA viruses to acquire specificity for human cancer cells. Data obtained from studies using these oncolytic viruses to treat colorectal cancer metastatic to the liver show the safety and feasibility of this approach and are promising for efficacy. Future studies will address tumor specificity and viral replication in the liver, anti-virus immune response, and anti-tumor activity. The application of oncolytic Newcastle disease virus strains for cancer treatment was first tested decades ago, but a loss of oncolytic effect due to the patient’s immune response was observed. However, novel strains of NDV exhibit potent anti-tumor activity and are currently under clinical investigation. Adenovirus. ONYX-015 (dl1520) is a replication-selective adenovirus with a deletion in the E1B-55KDa gene region. As the 55-kDa protein is necessary for inhibition of wild-type p53 function, the virus should replicate specifically in tumor cells lacking normal p53 function. However, recent investigations have questioned the p53 status-dependent specificity [56]. ONYX-015 was evaluated in a pilot study involving patients with colon cancer metastatic to the liver. Patients received i.v. infusions of ONYX-015 up to 2 1013 particles without any dose-limiting toxicity, indicating the safety and feasibility of this approach [57]. In addition, results of a phase
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I/II study of intra-arterial administration of ONYX-015 in patients with metastatic colorectal carcinoma were reported recently [58]. Hepatic arterial infusion of the virus was well tolerated at doses of 2 108 to 2 1012 particles and tumor infection was observed. Antitumoral activity was seen in combination with intravenous 5FU and leucovorin. Details of response rates and duration have not been reported. Herpes Simplex Virus 1. A phase I study of intrahepatic administration of the virus NV 1020 in patients with hepatic metastasis from colon cancer refractory to firstline chemotherapy has been initiated (NCI-G01-1920 and Y.F., MSKCC). NV 1020 is a genetically engineered, replication-competent virus derived from the multimutated HSV1 (strain F) and -2 based virus R 7020. Partial deletion of the thymidine kinase gene within the viral genome results in a high specificity of NV 1020 for dividing cancer cells. The ability of NV 1020 to infect and lyse tumor cells has been shown in a syngeneic animal model of bladder cancer [59]. The feasibility of oncolytic therapy using this virus for colon cancer metastatic to the liver has been demonstrated by Yoon and coworkers [60]. The replication-competent virus hrR3, an HSV1 mutant selective for dividing cells through deletion of the ribonucleotide reductase gene, also dramatically reduced the tumor burden in mice bearing diffuse liver metastasis from colon carcinoma after a single i.v. injection. Newcastle Disease Virus. Results from a phase I trial of intravenous administration of the highly purified, replication-competent, and naturally attenuated Newcastle disease virus, PV701, in patients with advanced cancers including colon cancer have been reported recently [61]. Patients were treated with multiple doses of up to 120 billion plaque forming units (pfu)/m2. The most common side effects throughout the trial were flu-like symptoms. In some patients anti-tumor activity was seen. A colon cancer patient treated with an initial dose of 12 109 pfu/m2 PV701 followed by two doses of 96 109 pfu/m2 showed an overall liver metastases regression of greater than 70% after 2 months [61].
CONCLUSIONS The broad spectrum of therapies currently under investigation to treat colorectal cancer metastatic to the liver is very encouraging. The combination of several of the described treatments, with or without conventional therapies such as surgery or chemotherapy, holds significant promise. Treatments seem to be well tolerated and responses in some patients are encouraging. The establishment of animal models for liver metastases provides a solid basis for further preclinical studies. However, most models are syngeneic and use ex vivo manipulated MOLECULAR THERAPY Vol. 5, No. 5, May 2002, Part 1 of 2 Parts Copyright © The American Society of Gene Therapy
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tumors, therefore most of these models may not closely mimic human cancer. A further limitation of several of these therapeutic approaches is the specific and efficient delivery of the therapeutic gene to the tumor site. For example, gene therapy is dependent on efficient gene transfer and expression in specific target tissue, whereas immunotherapies are highly restricted to tumors from patients expressing known antigens. The studies described here from a young and dynamically growing field of research may lead to new and effective clinical treatment of colorectal cancer metastatic to the liver. ACKNOWLEDGEMENTS Supported by NCI Grant CA 61586. P.M.-K. is a postdoctoral fellow of the Dr. Mildred Scheel Foundation for Cancer Research and J.R.B. is an American Cancer Society Professor.
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19. Harris, M. P., et al. (1996). Adenovirus-mediated p53 gene transfer inhibits growth of human tumor cells expressing mutant p53 protein. Cancer Gene Ther. 3: 121–130. 20. Anderson, S. C., et al. (1998). p53 gene therapy in a rat model of hepatocellular carcinoma: intra-arterial delivery of a recombinant adenovirus. Clin. Cancer Res. 4: 1649–1659. 21. Maron, D. J., et al. (2001). Intra-arterial delivery of a recombinant adenovirus does not increase gene transfer to tumor cells in a rat model of metastatic colorectal carcinoma. Mol. Ther. 4: 29–35, doi:10.1006/mthe.2001.0417. 22. Gilly, F. N., et al. (1998). Gene therapy with AdV-IL2 (TG 1021) in unresectable digestive adenocarcinoma. Phase I-II study, first inclusions. Adv. Exp. Med. Biol. 451: 527–530. 23. Shiratori, Y., et al. (1998). Gene therapy for hepatic micrometastasis of murine colon carcinoma. J. Hepatol. 28: 886–895. 24. Caruso, M., et al. (1996). Adenovirus-mediated interleukin-12 gene therapy for metastatic colon carcinoma. Proc. Natl. Acad. Sci. USA 93: 11302–11306. 25. Martinet, O., et al. (2000). Immunomodulatory gene therapy with interleukin 12 and 4-1BB ligand: long- term remission of liver metastases in a mouse model. J. Natl. Cancer Inst. 92: 931–936. 26. Chen, S. H., et al. (2000). Rejection of disseminated metastases of colon carcinoma by synergism of IL-12 gene therapy and 4-1BB costimulation. Mol. Ther. 2: 39–46, doi:10.1006/mthe.2000.0122. 27. Rubin, J., et al. (1997). Phase I study of immunotherapy of hepatic metastases of colorectal carcinoma by direct gene transfer of an allogeneic histocompatibility antigen, HLA-B7. Gene Ther. 4: 419–425. 28. Hayashi, S., et al. (1999). Inhibitory effect on the establishment of hepatic metastasis by transduction of the tissue plasminogen activator gene to murine colon cancer. Cancer Gene Ther. 6: 380–384. 29. Tao, N., et al. (2001). Sequestration of adenoviral vector by Kupffer cells leads to a nonlinear dose response of transduction in liver. Mol. Ther. 3: 28–35, doi:10.1006/mthe.2000.0227. 30. Brand, K., et al. (2000). Treatment of colorectal liver metastases by adenoviral transfer of tissue inhibitor of metalloproteinases-2 into the liver tissue. Cancer Res. 60: 5723–5730. 31. Mazzolini, G., et al. (2001). Genetic heterogeneity in the toxicity to systemic adenoviral gene transfer of interleukin-12. Gene Ther. 8: 259–267. 32. Mazzolini, G., et al. (2001). (v)(3) integrin-mediated adenoviral transfer of interleukin-12 at the periphery of hepatic colon cancer metastases induces VCAM-1 expression and T-cell recruitment. Mol. Ther. 3: 665–672, doi:10.1006/mthe.2001.0317. 33. Yee, C., Riddell, S. R., and Greenberg, P. D. (1997). Prospects for adoptive T cell therapy. Curr. Opin. Immunol. 9: 702–708. 34. Mazzolini, G., et al. (2000). Adenoviral gene transfer of interleukin 12 into tumors synergizes with adoptive T cell therapy both at the induction and effector level. Hum. Gene Ther. 11: 113–125. 35. Topf, N., Worgall, S., Hackett, N. R., and Crystal, R. G. (1998). Regional ‘pro-drug’ gene therapy: intravenous administration of an adenoviral vector expressing the E. coli cytosine deaminase gene and systemic administration of 5-fluorocytosine suppresses growth of hepatic metastasis of colon carcinoma. Gene Ther. 5: 507–513. 36. Kuo, C. J., et al. (2001). Comparative evaluation of the antitumor activity of antiangiogenic proteins delivered by gene transfer. Proc. Natl. Acad. Sci. USA 98: 4605–4610. 37. Chen, C. T., et al. (2000). Antiangiogenic gene therapy for cancer via systemic administration of adenoviral vectors expressing secretable endostatin. Hum. Gene Ther. 11: 1983–1996. 38. Kong, H. L., et al. (1998). Regional suppression of tumor growth by in vivo transfer of a cDNA encoding a secreted form of the extracellular domain of the flt-1 vascular endothelial growth factor receptor. Hum. Gene Ther. 9: 823–833. 39. Morse, M. A., et al. (1999). A Phase I study of active immunotherapy with carcinoembryonic antigen peptide (CAP-1)-pulsed, autologous human cultured dendritic cells in patients with metastatic malignancies expressing carcinoembryonic antigen. Clin. Cancer Res. 5: 1331–1338. 40. Nair, S. K., et al. (1999). Induction of carcinoembryonic antigen (CEA)-specific cytotoxic T-lymphocyte responses in vitro using autologous dendritic cells loaded with CEA peptide or CEA RNA in patients with metastatic malignancies expressing CEA. Int. J. Cancer 82: 121–124. 41. Fong, L., et al. (2001). Altered peptide ligand vaccination with Flt3 ligand expanded dendritic cells for tumor immunotherapy. Proc. Natl. Acad. Sci. USA 98: 8809–8814. 42. Welt, S., and Ritter, G. (1999). Antibodies in the therapy of colon cancer. Semin. Oncol. 26: 683–690. 43. Ychou, M., et al. (1998). Phase-I/II radio-immunotherapy study with Iodine-131labeled anti-CEA monoclonal antibody F6 F(ab’)2 in patients with non-resectable liver metastases from colorectal cancer. Int. J. Cancer 75: 615–619. 44. Buchegger, F., Pelegrin, A., Delaloye, B., Bischof-Delaloye, A., and Mach, J. P. (1990). Iodine-131-labeled MAb F(ab’)2 fragments are more efficient and less toxic than intact anti-CEA antibodies in radioimmunotherapy of large human colon carcinoma grafted in nude mice. J. Nucl. Med. 31: 1035–1044.
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45. Wong, J. Y. C., et al. (2000). A phase I radioimmunotherapy trial evaluating 90yttrium-labeled anti-carcinoembryonic antigen (CEA) chimeric T84.66 in patients with metastatic CEA-producing malignancies. Clin. Cancer Res. 6: 3855–3863. 46. Buchegger, F., et al. (2000). Radioimmunotherapy of colorectal cancer liver metastases: combination with radiotherapy. Ann. N Y Acad. Sci. 910: 263–269; discussion 269–270. 47. Herlyn, D. M., Steplewski, Z., Herlyn, M. F., and Koprowski, H. (1980). Inhibition of growth of colorectal carcinoma in nude mice by monoclonal antibody. Cancer Res. 40: 717–721. 48. Kim, K. J., et al. (1993). Inhibition of vascular endothelial growth factor-induced angiogenesis suppresses tumour growth in vivo. Nature 362: 841–844. 49. Warren, R. S., et al. (1995). Regulation by vascular endothelial growth factor of human colon cancer tumorigenesis in a mouse model of experimental liver metastasis. J. Clin. Invest. 95: 1789–1797. 50. Tartaglia, J., Bonnet, M. C., Berinstein, N., Barber, B., Klein, M. and Moingeon, P. (2001). Therapeutic vaccines against melanoma and colorectal cancer. Vaccine 19: 2571–2575. 51. Horig, H., et al. (2000). Phase I clinical trial of a recombinant canarypoxvirus (ALVAC) vaccine expressing human carcinoembryonic antigen and the B7.1 co-stimulatory molecule. Cancer Immunol. Immunother. 49: 504–514. 52. von Mehren, M., et al. (2000). Pilot study of a dual gene recombinant avipox vaccine containing both carcinoembryonic antigen (CEA) and B7.1 transgenes in patients with recurrent CEA-expressing adenocarcinomas. Clin. Cancer Res. 6: 2219–2228. 53. von Mehren, M., et al. (2001). The influence of granulocyte macrophage colony-
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stimulating factor and prior chemotherapy on the immunological response to a vaccine (ALVAC-CEA B7.1) in patients with metastatic carcinoma. Clin. Cancer Res. 7: 1181–1191. 54. Schlag, P., et al. (1992). Active specific immunotherapy with Newcastle-disease-virusmodified autologous tumor cells following resection of liver metastases in colorectal cancer. First evaluation of clinical response of a phase II-trial. Cancer Immunol. Immunother. 35: 325–330. 55. Ockert, D., et al. (1996). Newcastle disease virus-infected intact autologous tumor cell vaccine for adjuvant active specific immunotherapy of resected colorectal carcinoma. Clin. Cancer Res. 2: 21–28. 56. McCormick, F. (2000). ONYX-015 selectivity and the p14ARF pathway. Oncogene 19: 6670–6672. 57. Nemunaitis, J., et al. (2001). Intravenous infusion of a replication-selective adenovirus (ONYX-015) in cancer patients: safety, feasibility and biological activity. Gene Ther. 8: 746–759. 58. Reid, T., et al. (2000). Phase I/II study of intra-arterial administration of a replicationselective adenovirus CI-1042 (Onyx-015) in patients with metastatic colorectal cancer. Proc. Amer. Soc. Clin. Oncol. 6: 519. 59. Cozzi, P. J., et al. (2001). Intravesical oncolytic viral therapy using attenuated, replication-competent herpes simplex viruses G207 and Nv1020 is effective in the treatment of bladder cancer in an orthotopic syngeneic model. Faseb J. 15: 1306–1308. 60. Yoon, S. S., et al. (2000). An oncolytic herpes simplex virus type 1 selectively destroys diffuse liver metastases from colon carcinoma. Faseb J. 14: 301–311. 61. Pecora, A. L., et al. Phase I trial of intravenous administration of PV701, an oncolytic virus, in patients with advanced solid cancers. J. Clin. Oncol. (in press).
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Molecular Therapies for Colorectal Cancer Metastatic to the Liver Philipp Mayer-Kuckuk,1 Debabrata Banerjee,1,* Nancy Kemeny,2 Yuman Fong,3 and Joseph R. Bertino1,*,† 1Program
of Molecular Pharmacology and Therapeutics, 2Department of Medicine, 3Department of Surgery, Memorial Sloan-Kettering Cancer Center, New York, New York 10021, USA *Present address: The Cancer Institute of New Jersey, New Brunswick, New Jersey 08903, USA †To whom correspondence and reprint requests should be addressed. Fax: (732) 235-8181. E-mail: [email protected].
Colorectal cancers are the fourth most commonly diagnosed cancers and will account for over 56,000 deaths in the United States in 2002. A majority of patients with advanced colorectal cancer develop liver metastases during the course of their disease. Treatment of colorectal cancer metastatic to the liver by surgery or chemotherapy is limited and most patients succumb to their disease. Therefore, a broad spectrum of novel treatments, including innovative molecular therapies such as gene and immunotherapy or replication-competent viral therapy, is under preclinical investigation and several clinical trials are in progress. Here we review molecular therapies for colorectal cancer metastatic to the liver. Key Words: colorectal cancer, liver, metastases, molecular therapies, gene therapy, immunotherapy, replication-competent viral therapy, animal model, clinical trial, review
INTRODUCTION Colorectal cancer metastatic to the liver contributes significantly to cancer-related death in the United States. Two-thirds of colorectal cancer patients develop metastases to the liver during the course of their disease. The liver is the primary site of metastases from colorectal cancer and approximately 50,000 cases of hepatic metastases are diagnosed annually in the U.S. Patients with hepatic metastases have a median survival of 9–11 months [1]. The best treatment option for isolated metastases is surgery, which has the potential to cure the disease. However, only 20–25% of the patients who present with hepatic metastases are suitable for resection, and recurrence after surgery is frequent [2]. Following resection, regional chemotherapy using hepatic arterial infusion (HAI) of FUDR (fluorodeoxyuridine, floxuridine, a thymidylate synthase inhibitor precursor that is efficiently extracted by colon tumors in the liver) in combination with dexamethasone plus systemic 5-fluorouracil (5FU)/leucovorin (LV) improved both the local control of hepatic metastasis [3] and, in follow-up data,
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survival as compared with adjuvant systemic chemotherapy with 5FU/LV alone. At 2 years only 10% of the group receiving hepatic arterial infusion had tumor recurrence in the liver. Treatment options for unresectable metastases are further restricted. As reviewed by Koea and Kemeny [4], randomized clinical studies show that a high percentage of patients respond to regional administration of FUDR with either LV or dexamethasone in comparison with systemic therapy. HAI therapy led to up to 50% partial and a subset of complete responses, whereas systemic therapy resulted in 20% partial and no complete response. However, improved survival of patients with unresectable metastases through HAI remains to be proved. In view of the limited current treatment options for colorectal cancer metastatic to the liver, a large number of novel molecular therapies for this disease are under investigation. Molecular therapies to treat hepatic metastases from colorectal cancer are rooted in the development of techniques to specifically design macromolecules such as DNA or antibodies and even viruses or cells (such as dendritic cells) for therapeutic use in vivo. Furthermore, progress in the understanding of the molecular biology of colorectal cancer is key to the development of treatments such as immunotherapy, which is based on the discovery of tumor-specific antigens. In addition, the recently increased insight into development of metastases has resulted in new concepts for the treatment of hepatic metastases, such as anti-angiogenic gene therapy. Finally, novel surgical approaches contribute to treatment strategies based, for example, on regional gene delivery to the hepatic tumor. Although this review is focused on molecular therapies, it is of great interest that many of these therapies have the potential to merge with molecular imaging. With appropriate tracers, this technique will allow specific and noninvasive monitoring of, for example, transgene expression of herpes simplex virus thymidine kinase (HSV1-TK) in living subjects. Recently, Bennett and colleagues [5] used positron emission tomography (PET) to demonstrate in rats the feasibility to monitor infection of primary colorectal flank tumors with an MOLECULAR THERAPY Vol. 5, No. 5, May 2002, Part 1 of 2 Parts Copyright © The American Society of Gene Therapy
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oncolytic virus carrying the HSV1-TK gene. Figure 1 demonstrates the feasibility of imaging a human colorectal tumor expressing HSV1-TK as a fusion protein [6] in the liver of living rats using microPET and the tracer [124I]-2-fluoro-2-deoxy-5-iodouracil--D-arabinofuranoside (P.M.-K. et al., unpublished data). Thus, the fate of macromolecules, viruses, or cells used for molecular therapies may be monitored in individual patients during the course of their treatment. Here we review new approaches to treat hepatic metastases from colorectal cancer in three sections: gene therapy, immunotherapy, and viral therapy. In particular, we review in detail preclinical studies involving animal models of liver metastases from colon cancer, as well as considering past and current clinical studies.
GENE THERAPIES Targeting the Tumor Cell. Most gene therapies require gene transfer directly into the tumor. As described in the following section, gene transfer into the malignant lesion can result in tumor sensitivity to specific drugs, restoration of a tumor suppressor, or enhancement of anti-tumor immunity. It is of special interest that combinations of these approaches result in significantly enhanced and prolonged anti-tumor activity in preclinical animal models. It is also promising that therapies such as HSV1-TK/ganciclovir, cytosine deaminase/5-fluorocytosine, wild-type p53, or the drug Allovectin-7 have already entered human clinical trials. However, the value of these therapies to treat colorectal cancer metastatic to the liver is still unproven. HSV1-TK/Ganciclovir. Expression of HSV1-TK in cancer cells confers sensitivity to the nontoxic prodrug ganciclovir (GCV). The enzyme HSV1-TK selectively converts the nucleotide analog GCV into the monophosphate. Further phosphorylation of GCVmonophosphate by cellular kinases yields the cytotoxic GCV-triphosphate, and incorporation of this artificial nucleotide analog into DNA results in cell death. The first clinical study exploiting HSV1-TK/GCV gene therapy of colorectal cancer metastatic to the liver was conducted recently [7]. Sixteen patients received intratumoral injections of an adenovirus carrying the HSV1TK gene (for gene transfer strategies see Table 1). Patients were treated with virus doses of up to 1 1013 virus particles and subsequently with a fixed dose of GCV. The study showed that up to 1 1013 virus particles were administered safely. Hepatic toxicities were low and the most common side effect was fever. Overall the treatment was well tolerated. Before this clinical study, several preclinical, syngeneic animal models have described the successful application of this therapy to treat colon cancer MOLECULAR THERAPY Vol. 5, No. 5, May 2002, Part 1 of 2 Parts Copyright © The American Society of Gene Therapy
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metastatic to the liver [8–10]. Significant tumor suppression was observed, but in these animal models HSV1-TK/GCV therapy did not result in long-term, disease-free survivals. Thus the strategy of generating an enhanced therapeutic effect using additional immunoresponse-modulating genes was tested. In two studies using mouse models of hepatic metastasis, coexpression of interleukin-2 (IL-2) as well as IL-2 plus granulocyte macrophage colony stimulating factor (GM-CSF) with HSV1-TK significantly decreased tumor volume [9,10]. GM-CSF expression in combination with IL-2 and HSV1-TK/GCV resulted in a long-term survival of 25% of the animals, followed up to 150 days, as compared with a survival of 60 days of animals treated with HSV1-TK/GCV in combination with IL-2 or GM-CSF. The percentage of surviving animals was further enhanced by boosting immunity with irradiated, IL-2-transduced tumor cells. Cytosine Deaminase/5-Fluorocytosine. Cancer cells transduced with the Escherichia coli or yeast cytosine deaminase (CD) selectively convert the nontoxic prodrug 5-fluorocytosine (5FC) into the cytotoxic chemoreagent 5-fluorouracil (5FU). Consequently, transduced cancer cells and, to a variable extent, neighboring nontransduced cells (bystander effect) are killed. A phase I study (NCI–V95-0649; source of clinical trials, Physician Data Query via http://cancernet.nci.nih.gov) of intratumoral delivery of an adenoviral vector containing the CD gene (AdCMV.CD.10) into macroscopic liver metastasis from colon cancer followed by systemic administration of 5FC was conducted [11]. No details of the study have been published yet. However, the feasibility of this approach was shown using an in vivo model of subcutaneously implanted human HT29 tumors in nude mice [12]. Intratumoral injection of a vector (AdCMV.CD) similar to the one used in the clinical study followed by 5FC treatment resulted in a significant decrease of tumor size after 4 weeks compared with controls. Furthermore, hepatic arterial infusion of a retrovirus carrying the CD gene under control of the carcinoembryonic antigen (CEA) promoter was used for regional delivery [13]. Rats bearing hepatic tumors from rat colorectal cancer received 1 107 virus particles followed 48 hours later by daily intraperitoneal injections of 5FC for 7 days. After treatment hepatic tumor volumes were reduced. In contrast to these strategies, which require regional delivery of the CD gene, Block et al. [14] used a syngeneic mouse model of hepatic micrometastasis to study the effect of systemic adenoviral-mediated CD gene transfer in combination with 5FC administration. The adenovirus was injected 48 hours after splenic injection of tumor cells and 24 hours after spleenectomy. Following this procedure, multiple micrometastases in the liver were observed. Specific delivery of the CD gene into the tumor cells was detec-
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TABLE 1: Gene transfer strategies for gene therapies to treat colorectal cancer metastatic to the liver Vector
Promoter
Therapeutic gene
Administration
References
rec. adenovirus
CMV
CD
human, mouse; intratumoral
[11,12]
rec. adenovirus
CMV
p53
rat; intratumoral
[21]
rec. adenovirus
CMV
p53
mouse; peritumoral
[19]
rec. adenovirus
CMV
p53
rat; HAI
[20,21]
rec. adenovirus
CMV
CD
mouse; systemic
[14,35]
rec. adenovirus
CMV
p53
rat; systemic
[21]
rec. adenovirus
CMV
FLT-1
mouse; systemic
[38]
rec. adenovirus
CMV
IL-12
mouse; systemic
[32]
rec. adenovirus
RSV LTR
HSV1-TK, IL-2, GM-CSF
human, mouse; intratumoral
[7,9,10]
rec. adenovirus
RSV LTR
IL-12, 4-1BBL
mouse; intratumoral
[24–26]
rec. adenovirus
RSV
endostatin
mouse; systemic
[37]
rec. adenovirus
CAG
IL-12
mouse; systemic
[23]
rec. adenovirus
n.s.
TIMP-2
mouse; systemic
[30]
retrovirus
CEA
CD
rat; HAI
[13]
DMRIE DOPE
synthetic
CD
mouse; systemic
[15]
vaccina virus
RSV
HLA-B7, -2microglobulin
human; intratumoral
[27]
CD, cytosine deaminase; CAG, cytomegalovirus enhancer plus chicken -actin; CEA, carcinoembryonic antigen; CMV, cytomegalovirus; DMRIE, (2,3-bis-tetradecyloxy) propyl,b-hydroxylethyl, dimethylammoniumbromide; DOPE, dioleoyl phosphatidyl-ethanolamine; GM-CFS, granulocyte macrophage colony-stimulating factor; HAI, hepatic arterial infusion; HSV1-TK, herpes simplex virus 1 thymidine kinase; IL-2, interleukin 2; IL-12, interleukin 12; LTR, long terminal repeat; n.s., not specified; rec., recombinant; TIMP-2, tissue inhibitor of metalloproteinase-2.
ted and after 10 days of 5FC treatment a significant reduction in the number of hepatic metastasis was noted. A systemic, tumor-specific delivery of the CD gene using a vaccina virus vector was reported by Gnant and coworkers [15]. In a model of disseminated liver metastasis in mice, systemic administration of the vector followed by treatment with 5FC resulted in cure rates of up to 30%. An additional observation of interest was made using the CD/5FC strategy. Rats bearing tumors derived from CD-transduced rat colon carcinoma cells were treated with 5FC and subsequently injected with the wild-type rat colon carcinoma cells [16]. Development of wild-type tumors in the animals was not seen. This observation points to a vaccination effect of the CD/5FC therapy. Wild-Type TP53. Approximately 50% of human cancers show mutations in the gene TP53. Therefore, the transfer of wild-type TP53 into cancer cells to restore the tumor-suppressor function of TP53 has been explored. In addition, restoration of wild-type TP53 in tumor cells can result in increasing responses to chemotherapy and/or X-rays. In a phase I study (NCI-V97-1179),
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patients with liver metastasis from colon cancer were treated with a replication-deficient adenovirus containing wild-type TP53 (SCH-58500), via hepatic arterial infusion. Additionally, a phase II trial of SCH-58500 in patients with colorectal cancer metastatic to the liver has commenced. Data on these trials are very limited [17]. A large number of patients with several types of advanced cancers were treated in multicenter phase I and II trials by intratumoral administration of adenovirus encoding wild-type p53 and some partial responses were seen with significant clinical safety [18]. The potential of adenovirus-mediated wild-type p53 gene transfer to inhibit tumor growth of human colon carcinoma cells expressing mutant p53 has been demonstrated previously in vitro and in vivo [19]. Moreover, the feasibility of regional delivery via hepatic arterial infusion of an adenovirus expressing wildtype p53 has been shown in a rat model [20]. In this study, intrahepatic arterial dosing of the virus increased transgene expression in the hepatic tumor tissue and decreased systemic exposure when compared with intravenous (i.v.) dosing. However, this study used a hepatocellular carcinoma in a syngenic rat model. In MOLECULAR THERAPY Vol. 5, No. 5, May 2002, Part 1 of 2 Parts Copyright © The American Society of Gene Therapy
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contrast, a recent report describes very low hepatic tumor transduction following hepatic arterial infusion of an adenovirus in a rat model of human metastatic colorectal carcinoma [21]. No therapeutic effect of adenoviral wild-type p53 transfer was seen. Interleukins. Interleukin-2 (IL-2) and interleukin-12 (IL-12) are growth factors for T cells, which enhance nonspecific immune responses such as natural killer or lymphokine-activated killer cells, as well as the cytotoxic T-cell response. These functions suggest a potential use for IL-2/IL-12 in cancer gene therapy. A phase I/II study is presently underway to evaluate gene therapy with a replication-deficient adenovirus containing the IL-2 gene (TG 1021) in patients with unresectable digestive adenocarcinomas [22]. The adenoviral vector was injected into the liver lesion of a patient with adenocarcinoma metastatic to the liver and lung. However, clinical and preclinical data supporting this approach are limited. The IL-2 gene was also used in a unique manner for adjuvant gene therapy, in which the capability of adenovirus to preferentially infect liver tissue and not tumor tissue was used. In mice, hepatocytes were adenovirally transduced with IL-2, resulting in a temporary inhibition of hepatic metastasis upon injection of tumor cells [23]. Another promising strategy to treat colon cancer metastatic to the liver is based on work by Caruso et al. [24]. Using a model for hepatic metastasis of colon carcinoma in mice, the antitumor efficacy of an adenovirus expressing HSV1-TK and IL-2, in combination with GCV treatment, was compared with an IL-12 expressing virus. The adenovirus-mediated IL-12 gene therapy resulted in increased survival of the animals compared with the HSV1-TK/IL-2 treatment [24]. Additionally, two recent reports have described adenoviral gene therapy strategies for hepatic metastases in mice based on IL-12 in combination with co-stimulation of the T-cell receptor 4-1BB. Binding of 4-1BB to its ligand, presented on antigen-presenting cells, results in expansion of antigen-specific, cytotoxic T cells. In one study [25], the gene encoding the ligand of 4-1BB was transferred into the tumor, and another study used an agonistic anti-4-1BB antibody [26]. Liver tumor regression, a 60–80% long-term survival of at least 3 months (which was 2-fold to 2.5-fold higher than in the non 4-1BB co-stimulated control), and potent long-term antitumor immunity were observed. HLA-B7/-2-Microglobulin. Loss of major histocompatibility (MHC) class I antigens has been reported in several different tumor types including metastasis. HLA histocompatibility antigen (HLA) alterations are detectable in approximately 60% of human tumors. The feasibility and safety of HLA-B7 and -2-microglobulin gene transfer into the liver lesions of patients with MOLECULAR THERAPY Vol. 5, No. 5, May 2002, Part 1 of 2 Parts Copyright © The American Society of Gene Therapy
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colorectal carcinoma have been shown in a phase I study [27]. Both genes were encoded by a single plasmid and formulated with the lipid DMRIE-DOPE to yield the liposomal vector Allovectin-7. Patients received single (10 to 250 mg) or multiple (10 mg) injections of the vector directly into the liver lesion. Gene transfer was detected by PCR in 14 of 15 patients, whereas gene expression was detected by immunohistochemistry only in 5 of 15 patients. There has been no serious toxicity directly attributable to Allovectin-7. Therapeutic effects were not reported. Plasminogen Activator. Plasmin-activating proteinase (tissue plasminogen activator, tPA) is often downregulated in colorectal cancer. One study investigated treatment of hepatic metastases from colorectal cancer using tPA. The survival rates of mice bearing hepatic metastasis from murine colon carcinoma cells retrovirally transduced with tPA or LacZ as a control were compared. Significantly less hepatic tumor growth and prolonged survival of mice with metastasis from tPA-transduced colon cancer cells were observed [28]. Targeting the Liver. This section describes anti-tumor gene therapies based on gene transfer into the liver tissue. The starting point of these strategies was the identification of suitable therapeutic genes, driven by the finding that adenovirus preferentially infects hepatocytes. In general these strategies seem to be less attractive, because of the large number of normal liver cells infected. Moreover, in mouse models expression of adenoviral-mediated therapeutic genes in the liver was shown not to correlate with the injected viral dose [29]. Transduction of hepatocytes was detected after administration of 1 1011 viral particles, whereas lower doses of 1 to 3 1010 particles were taken up efficiently by the hepatic Kupffer cells. Metalloproteinases (MMP) are a family of endopeptidases believed to have a critical role in tumor invasion and metastasis and frequently upregulated in colorectal cancer. A mouse model was used to demonstrate systemic adenoviral delivery and expression of tissue inhibitor of metalloproteinase (TIMP)-2 in the liver [30]. Liver TIMP-2 expressing animals showed a significant reduction in tumor growth of liver micrometastasis of a human colon adenocarcinoma expressing a high level of MMP-2, compared with control animals. Maximum inhibition in tumor growth was seen with TIMP-2 delivery pre- or early post-metastases formation. The group of Melero and Prieto [31] observed a murine strain-dependent difference in liver gene transduction levels by recombinant adenovirus. BALB/c or C57BL/6 mice were injected i.v. with adenovirus carrying a reporter gene (such as -galactosidase or luciferase) under control of the cytomegalovirus (CMV) promoter. A 10- to 100-fold higher transgene expression
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was subsequently measured in the liver of C57BL/6 compared with BALB/c mice. The same group demonstrated, in a syngeneic model of concomitantly subcutaneous and hepatic colon tumors in BALB/c mice, an v3 integrin receptor mediated accumulation of systemically circulating recombinant adenovirus carrying a reporter gene or the IL-12 gene in the tissue surrounding the liver tumor [32]. Liver tumor regression after treatment with adenovirus expressing IL-12 was seen, but a strong synergistic effect with intravenous adoptive T-cell therapy [33] was observed [34]. Antitumor cytotoxic T lymphocytes were generated by injection of the subcutaneous tumors with adenovirus expressing IL-12 and subsequent short-time coculture of isolated lymph node cells together with mitomycin C treated tumor cells. In addition, further experiments suggest that, after adenoviral IL-12 treatment, VCAM-1 (CD 106) is induced in the tumor vasculature followed by T-cell recruitment [32]. Adenovirally CD-transduced hepatocytes have been shown to produce and release 5FU after 5FC treatment in vitro [35], therefore the possibility of inhibiting tumor growth in liver transduced with CD and treated with 5FC was tested. In a model of CD-transduced liver and subsequent colon carcinoma cell injection, tumor growth was suppressed after 15 days of 5FC treatment compared with controls [35]. Targeting the Neovasculature. Tumor anti-angiogenesis, the inhibition of tumor neovascularization, is a novel, highly promising anti-cancer strategy. The following section describes two gene therapy studies using an anti-angiogenic approach for the treatment of hepatic metastases from colorectal cancer. The presented data are encouraging and further studies have to address in detail the specific expression of therapeutically active levels of anti-angiogenic factors as well as the potential consequences of suppression of physiologic angiogenesis by anti-angiogenic gene therapy. Moreover, the application of anti-angiogenic gene therapy as adjuvant therapy or treatment for established tumors has to be assessed. Anti-angiogenic Gene Therapy. The anti-tumor effect of vector-mediated systemic expression of anti-angiogenic proteins, such as soluble endostatin or FLT-1 VEGF receptor was investigated in several studies. Although data cannot be presented here in detail, the observed anti-tumor effects of, for example, endostatin seem to be dependent on the experimental in vivo model. In particular vector type and dose, resulting systemic antiangiogenic protein levels, tumor cell type, as well as the time of tumor cell inoculation compared with start of therapy seem to be critical. Only one study [36] took a comparative approach and observed a superior antitumor effect of FLT-1 VEGF receptor as compared with
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FIG. 1. Noninvasive imaging of an orthotopic human colorectal tumor model in living rats using positron emission tomography (PET) scanning. The cell line C85 was derived from the liver metastases of a colorectal cancer patient. C85 cells were retrovirally transduced to express a double-mutant dihydrofolate reductase fused to the reporter enzyme HSV1-TK. These cells were grown as flank tumors in nude rats and subsequently orthotopically implanted in the rat liver. For non-invasive imaging, rats received the PET tracer [124I]-2-fluoro-2deoxy-5-iodouracil--D-arabinofuranoside ([124I]FIAU) via the penile vein, and 2 hours later animals were anesthetized and subjected to microPET. Shown are three microPET images taken in the transversal, coronal, and sagittal planes. High accumulation of the specific HSV1-TK substrate [124I]FIAU is seen in red, whereas low accumulation appears violet. The hepatic tumor is indicated by a white triangle. Unspecific tracer retention is seen in the bladder (black triangle). Location of tumor mass was verified by magnetic resonance images of the liver, which were taken before microPET imaging (data not shown).
endostatin in established murine and xenograft tumors. Chen and colleagues [37] showed in a mouse model that systemic delivery of an adenoviral vector that produced secretable murine endostatin resulted in prolonged and elevated levels of circulating endostatin when compared with mice treated with the empty vector. Mice received i.v. a single dose of 2 1011 virus particles, and 13 days later human colon tumor was implanted into the liver. A significantly prolonged survival with 25% complete prevention of tumor growth in the liver was seen in the endostatin-expressing animals in comparison with the controls [37]. Kong et al. [38] investigated the therapeutic effect of adenoviralmediated expression of a secreted form of the FLT-1 VEGF receptor in mice bearing preestablished tumors metastatic to the liver. A reduction of tumor burden was observed in animals expressing FLT-1 compared with controls treated with an empty vector [38]. Both studies show the potential of an anti-angiogenic gene therapy as an adjuvant or acute treatment for colorectal cancer metastatic to the liver.
IMMUNOTHERAPY Immunotherapy of colorectal cancer metastatic to the liver includes several different strategies. Very interesting is the concept of active immunotherapy using ex vivo modified dendritic cells. Data from two vaccination strategies show that this is a very attractive concept as adjuvant therapy. Furthermore, several strategies MOLECULAR THERAPY Vol. 5, No. 5, May 2002, Part 1 of 2 Parts Copyright © The American Society of Gene Therapy
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exploit antibody-based treatments. Dendritic Cells. Dendritic cells (DCs) are antigen-presenting cells capable of priming T cells in an HLArestricted fashion and initiating a T-cell response. In animal models DCs induce T-cell mediated anti-tumor immunity. A phase I/II study is currently investigating active immunotherapy with carcinoembryonic antigen (CEA) RNA transduced DCs in patients with resected hepatic metastasis from adenocarcinoma of the colon (NCI-G98-1456). The feasibility and safety of treatment of patients with cryopreserved DCs loaded with the CEA peptide CAP-1 were shown in a phase I study [39]. Autologous DCs were prepared from peripheral blood mononuclear cells, subsequently loaded with the CAP-1 peptide, and cryopreserved. Pulsed DCs were administered i.v. weekly or biweekly over 4 weeks in escalating dose levels. The same group showed that CEA RNAloaded DCs and CEA peptide-loaded DCs stimulate similar CEA-specific T-cell activity in vitro [40]. An improved approach using DCs loaded with an altered and therefore more potent CEA antigen was reported recently [41]. In this study, 9 of 12 patients with advanced or metastatic colon cancer received a pretreatment with FLT3 ligand, a hematopoietic growth factor, to expand the circulating blood DC population and were subsequently vaccinated with the peptideloaded DCs. Two patients with metastatic colon cancer showed an objective response lasting up to 10 months. Antibodies. Antibodies targeting specific antigens overexpressed in colon cancer cells such as CEA have been extensively investigated and treatment strategies for colorectal cancer have been reviewed [42]. Here we focus on antibody treatments for hepatic metastases from colorectal cancer. Over the past decade several groups reported clinical radioimmunotherapy studies using radioactive iodine labeled anti-CEA, TAG 72, or A33 murine or chimeric antibodies to treat liver metastases from colorectal cancer [43]. In most clinical studies successful tumor targeting and, in some cases, decreased CEA levels or stabilization of disease were observed. However, human anti-mouse antibody (HAMA) response, ineffective dose delivery to the tumor site, and hematologic toxicity resulted in limited efficiency of radioimmunotherapy. The feasibility of radioimmunotherapy was shown by Buchegger et al. [44] in a human colon carcinoma model in nude mice, where complete tumor regression was observed in 8 of 10 mice after treatment with the antibody 131I Mab F(ab)2 fragment. In addition to the studies using iodine isotope, a phase I study using a 90Y-labeled chimeric anti-CEA antibody was reported [45]. One notable but unexplained observation was that patients with extensive hepatic involvement by tumor demonstrated rapid blood clearance and poor tumor targeting of the antibody. Treatment of liver metastases by radioimmunotherapy in MOLECULAR THERAPY Vol. 5, No. 5, May 2002, Part 1 of 2 Parts Copyright © The American Society of Gene Therapy
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combination with radiotherapy has been reviewed [46]. Twenty years ago the anti-tumor activity of murine antibodies directed against the 17-1A (Ep-CAM) receptor on the surface of colon cancer cells was demonstrated in a mouse model [47]. Although HAMA responses, complement-mediated cytolysis, or an anti-idiotype cascade is proposed to be critical for the anti-tumor activity of MoAb 17-1A (edrecolomab), the exact mechanism of action of this antibody is not clear. However, in a clinical phase I/II study (NCI-88-C-64A) at least 15 patients with colorectal cancer metastatic to the liver received the MoAb 17-1A antibody in conjunction with IL-12. Additionally, a study investigating MoAb 17-1A antibody as adjuvant agent (NCI-V85-0072) included colorectal cancer patients with respectable hepatic metastases. Results from these studies are yet not published. Vascular endothelial growth factor (VEGF) is a secreted polypeptide and acts as a strong pro-angiogenic factor in both normal and pathologic angiogenesis, primarily through binding to receptor tyrosine kinases on endothelial cells. Kim et al. [48] demonstrated in an in vivo model suppression of tumor growth by inhibition of VEGF-induced angiogenesis. Tumor growth inhibition was seen in nude mice injected with human sarcoma or glioblastoma cells and subsequently treated with an anti-VEGF antibody. Later, Warren and colleagues [49] reported VEGF gene expression in surgically resected hepatic tumors from colorectal cancer. Furthermore, athymic mice bearing liver metastases from human colorectal cancer were injected with anti-VEGF antibody. Treatment started 24 hours after tumor cell inoculation and was continued for 4 weeks with antibody injections twice a week. Significant reduction in the number and volume of tumors per liver in the treated animals as compared with the control animals was observed [49]. Carcinoembryonic Antigen/B7.1. Vaccination by coexpression of a tumor-associated antigen and the costimulatory molecule B7 has been shown to improve anti-tumor immunity [50]. Two clinical pilot studies have reported a dose-dependent T-cell specific response and stable disease in approximately one of six patients with CEA-expressing metastatic colorectal carcinoma after treatment with ALVAC-CEA B7.1 [51,52]. This vaccine is based on a Canary Pox virus engineered to encode the genes for CEA and B7.1, a T-cell co-stimulatory molecule. The larger study enrolled 39 patients, of which 30 received up to 4.5 108 plaque forming units over 8 weeks intradermally every other week. In the other study the same dose was given intramuscularly (i.m.) to 6 patients, whereas 12 patients received smaller doses. Therapy was well tolerated and biopsies of the vaccine site revealed leukocytic infiltration and CEA expression. A further study assessed the influence of
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prior chemotherapy or administration of granulocyte macrophage colony-stimulating factor (GM-CSF) on ALVA-CEA B7.1 treatment [53]. A negative correlation between the number of chemotherapy regimes received and the generation of T-cell response was seen. GM-CSF given for 5 days before and in the beginning of vaccination significantly increased the number of patients with stable disease, but T-cell precursor frequencies were not elevated. Newcastle Disease Virus. Naturally oncolytic or nonlytic stains of the Newcastle disease virus (NDV) have been explored for cancer therapy, and NDV infection of autologous tumor cells as immunotherapy for patients with advanced colorectal cancer was described [54,55]. In one study, tumor cells from liver metastasis were infected ex vivo with a nonlytic NDV strain in order to modify the tumor cells to stimulate a broad antitumor response after re-injection in the patient [54]. The 23 patients enrolled in the trial well tolerated the vaccination following surgical resection of the liver metastasis. Tumor recurrence after 18 months was reduced by 25% in vaccinated patients compared with patients that had been only surgically treated.
REPLICATION-COMPETENT VIRAL THERAPY Cancer treatment using adenovirus or herpex simplex virus was developed through genetically engineering cytolytic strains of these DNA viruses to acquire specificity for human cancer cells. Data obtained from studies using these oncolytic viruses to treat colorectal cancer metastatic to the liver show the safety and feasibility of this approach and are promising for efficacy. Future studies will address tumor specificity and viral replication in the liver, anti-virus immune response, and anti-tumor activity. The application of oncolytic Newcastle disease virus strains for cancer treatment was first tested decades ago, but a loss of oncolytic effect due to the patient’s immune response was observed. However, novel strains of NDV exhibit potent anti-tumor activity and are currently under clinical investigation. Adenovirus. ONYX-015 (dl1520) is a replication-selective adenovirus with a deletion in the E1B-55KDa gene region. As the 55-kDa protein is necessary for inhibition of wild-type p53 function, the virus should replicate specifically in tumor cells lacking normal p53 function. However, recent investigations have questioned the p53 status-dependent specificity [56]. ONYX-015 was evaluated in a pilot study involving patients with colon cancer metastatic to the liver. Patients received i.v. infusions of ONYX-015 up to 2 1013 particles without any dose-limiting toxicity, indicating the safety and feasibility of this approach [57]. In addition, results of a phase
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I/II study of intra-arterial administration of ONYX-015 in patients with metastatic colorectal carcinoma were reported recently [58]. Hepatic arterial infusion of the virus was well tolerated at doses of 2 108 to 2 1012 particles and tumor infection was observed. Antitumoral activity was seen in combination with intravenous 5FU and leucovorin. Details of response rates and duration have not been reported. Herpes Simplex Virus 1. A phase I study of intrahepatic administration of the virus NV 1020 in patients with hepatic metastasis from colon cancer refractory to firstline chemotherapy has been initiated (NCI-G01-1920 and Y.F., MSKCC). NV 1020 is a genetically engineered, replication-competent virus derived from the multimutated HSV1 (strain F) and -2 based virus R 7020. Partial deletion of the thymidine kinase gene within the viral genome results in a high specificity of NV 1020 for dividing cancer cells. The ability of NV 1020 to infect and lyse tumor cells has been shown in a syngeneic animal model of bladder cancer [59]. The feasibility of oncolytic therapy using this virus for colon cancer metastatic to the liver has been demonstrated by Yoon and coworkers [60]. The replication-competent virus hrR3, an HSV1 mutant selective for dividing cells through deletion of the ribonucleotide reductase gene, also dramatically reduced the tumor burden in mice bearing diffuse liver metastasis from colon carcinoma after a single i.v. injection. Newcastle Disease Virus. Results from a phase I trial of intravenous administration of the highly purified, replication-competent, and naturally attenuated Newcastle disease virus, PV701, in patients with advanced cancers including colon cancer have been reported recently [61]. Patients were treated with multiple doses of up to 120 billion plaque forming units (pfu)/m2. The most common side effects throughout the trial were flu-like symptoms. In some patients anti-tumor activity was seen. A colon cancer patient treated with an initial dose of 12 109 pfu/m2 PV701 followed by two doses of 96 109 pfu/m2 showed an overall liver metastases regression of greater than 70% after 2 months [61].
CONCLUSIONS The broad spectrum of therapies currently under investigation to treat colorectal cancer metastatic to the liver is very encouraging. The combination of several of the described treatments, with or without conventional therapies such as surgery or chemotherapy, holds significant promise. Treatments seem to be well tolerated and responses in some patients are encouraging. The establishment of animal models for liver metastases provides a solid basis for further preclinical studies. However, most models are syngeneic and use ex vivo manipulated MOLECULAR THERAPY Vol. 5, No. 5, May 2002, Part 1 of 2 Parts Copyright © The American Society of Gene Therapy
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tumors, therefore most of these models may not closely mimic human cancer. A further limitation of several of these therapeutic approaches is the specific and efficient delivery of the therapeutic gene to the tumor site. For example, gene therapy is dependent on efficient gene transfer and expression in specific target tissue, whereas immunotherapies are highly restricted to tumors from patients expressing known antigens. The studies described here from a young and dynamically growing field of research may lead to new and effective clinical treatment of colorectal cancer metastatic to the liver. ACKNOWLEDGEMENTS Supported by NCI Grant CA 61586. P.M.-K. is a postdoctoral fellow of the Dr. Mildred Scheel Foundation for Cancer Research and J.R.B. is an American Cancer Society Professor.
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MOLECULAR THERAPY Vol. 5, No. 5, May 2002, Part 1 of 2 Parts Copyright © The American Society of Gene Therapy