- Email: [email protected]
Gene Therapy for Liver Tumors
1055-3207 /98 $8.00
CANCER GENE THERAPY
+ .00
GENE THERAPY FOR LIVER TUMORS Nancy M. Carroll, MD, and Kenneth K. Tanabe, MD
The liver remains one of the most common sites of distant metastases for patients with solid tumors as well as a site of primary hepatic malignancies, including hepatocellular carcinoma, peripheral cholangiocarcinoma, angiosarcoma, and hepatoblastoma.11 Most patients with liver metastases also have metastatic disease in other organs. The biology of colon carcinoma is unique in that a significant percentage of patients with metastatic disease harbor disease only in the liver. 41 Certainly, effective regional therapy applied to this group of patients may influence survival. Successful eradication of liver tumors in patients with primary liver cancers also would be expected to improve survival. Surgical resection remains the only potentially curative treatment; however, even patients whose tumors are limited to the liver are rarely candidates for resection.34 Following this logic, many investigators have pursued novel regional hepatic therapies, including cryosurgery, radiofrequency ablation, isolated liver perfusion, hepatic arterial infusion chemotherapy, chemoembolization, hyperthermic hepatic isolated chemoperfusion, ethanol injection, laser ablation, and direct injection of chemotherapy "gels." 5• 27• 31 Gene therapy can be added to this list of experimental approaches for treatment of liver tumors. Some gene therapy approaches are extensions of strategies used to treat diffuse solid tumor metastases, regardless of location (e.g., lung, liver, marrow). Other gene therapy approaches are uniquely suited for tumors that grow in the liver.
This work was supported by the Marshall K. Bartlett Research Fellowship from the Massachusetts General Hospital.
From the Division of Surgical Oncology, Department of Surgery, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts SURGICAL ONCOLOGY CLINICS OF NORTH AMERICA VOLUME 7 • NUMBER 3 • JULY 1998
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At first glance it may appear that efforts devoted to the study of treatment of liver metastases are doomed to failure because of the high prevalence of systemic disease in this patient population. Patients with colon and rectal carcinoma liver metastases, however, serve as model candidates for regional therapy because of the high incidence of "liver-only" metastases. Moreover, liver metastases may serve as a model to test treatment strategies against diffuse disease in which principles of gene therapy can be examined. Information gained in studies on this patient population may be applicable to patients with metastases in other locations. The ability to identify the presence and location of liver tumors continues to improve with new modalities, including magnetic resonance (MR) imaging scans with ferumoxide, position emission tomography (PET) scans, helical computed tomography (CT) scans, radiolabeled monoclonal antibodies, and intraoperative sonography. 24 Nonetheless, the limited sensitivity and limited resolution of these modalities precludes identification of every tumor, especially in patients with solid tumor liver metastases. Accordingly, treatment strategies that require precise tumor localization are much less likely to be successful for patients with several metastases. These include surgical resection, cryoablation, radiofrequency ablation, laser ablation, and ethanol injection. Similarly, gene therapy approaches that rely on direct intratumoral inoculation are unlikely to be effective in the management of liver tumors. Gene therapy approaches that target tumors throughout the liver are much more likely to have a clinically significant impact.
GENE THERAPY STRATEGIES FOR LIVER TUMORS Overview
Strategies to minimize toxicity to normal liver are extremely critical, especially for gene therapy strategies that target diffuse tumors throughout the liver. Specific differences exist between liver tumors and surrounding normal liver, and they can be exploited to target gene therapy to the tumors and reduce toxicity to normal liver. These differences between liver tumor cells and normal liver cells include mitotic activity, expression of tumor-associated antigens, dominant blood supply, expression of cell surface receptors, inactivation of tumor suppressor genes, expression of activated oncogenes, and immunogenicity. 18 Some of these gene therapy strategies already have entered clinical trials.
Vectors
Each of the gene therapy strategies described next requires efficient gene delivery. Several gene delivery vehicles have been developed, and many more are under development. These vehicles have been summarized nicely in other articles in this issue and are not be duplicated herein.
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Mitotic Activity
Hepatocytes in the normal adult liver are quiescent. 20 In contrast, most liver tumors have high mitotic activity, which results in an actively proliferating malignant cell population surrounded by a relatively quiescent normal cell population. These authors have examined a gene therapy approach to liver metastases using herpes simplex virus (HSV) type 1 that attempts to exploit this difference in mitotic activity. 6 HSV is a DNA virus that infects both dividing and nondividing cells, thereby resulting in the production of progeny virions and cell lysis. Although the virus is neurotropic, it can infect most human tissues. The cytolytic property of HSV infection and replication can be used to destroy tumor cells. We have demonstrated that several strains of HSV destroy human colon carcinoma cells efficiently, even when added at a multiplicity of infection (MOI) of less than 1 viral plaque forming unit per 10 tumor cells. The significant antitumor activity of HSV observed in vitro is also observed in vivo. Deletion of an HSV gene required for synthesis of DNA precursors (e.g., viral ribonucleotide reductase) renders the virus replication conditional; the mutant virus can no longer replicate in nondividing cells. The function of the missing viral protein, however, may be replaced by a cellular protein (e.g., cellular ribonucleotide reductase). Accordingly, mitotically active cells provide the DNA precursors and machinery necessary for synthesis of DNA precursors. This cellular complementation of the viral deficiency results in viral replication, production of progeny virions, and host cell death. The HSV ribonucleotide reductase gene encodes an enzyme involved in the production of DNA precursors. We have examined a gene therapy strategy in which an HSV mutant (hrR3) that is deficient in ribonucleotide reductase is introduced via the portal vein into rodents that bear diffuse liver metastases. Because this mutant replicates significantly more efficiently in mitotically active cells, we observed viral gene expression indicative of replication in virtually all of the diffuse liver metastases but not in normal (mitotically inactive) liver. In this model, 96% of liver metastases demonstrated marker gene expression; however, only a small percentage (5%-25%) of the cells in each metastasis expressed the gene. Additional studies are required to determine the effect of repeat treatments in an attempt to infect a larger percentage of the tumor cells in each metastasis. Nonetheless, significant antitumor response can be expected even in the absence of 100% transduction efficiency as a result of bystander killing. 16 Bystander killing refers to the destruction of untransduced tumor cells that are adjacent to transduced tumor cells. The implications of this phenomenon are significant. Gene delivery vehicles are not capable of transducing 100% of tumor cells in any given tumor mass, a limitation that would result in failure of most gene therapy approaches. However, the observation that bystander killing results in destruction of an entire tumor despite successful transduction of only a fraction of the tumor cells certainly holds promise. The mechanisms that lead to bystander killing are currently under investigation.23,30
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Another gene therapy strategy that relies on differential mitotic activity between normal hepatocytes and liver tumors is the use of retroviral vectors. Retroviral integration and transgene expression require cellular division. 28 Therefore, retroviruses may allow preferential expression of transgenes in liver tumors rather than normal liver. Unfortunately, retroviruses are relatively labile in vivo compared with other viruses, and in vivo transduction efficiencies using retroviral vectors have been extremely poor. Accordingly, some investigators have examined a gene therapy approach that involves in vivo delivery of retroviral packaging cell lines rather than in vivo delivery of the retroviral vectors themselves. 29 Caruso et aF investigated an approach of direct intratumoral inoculation of retroviral producer cells into isolated liver tumors. The retroviral construct used in these studies contained the HSV thymidine kinase (HSV-tk) gene. Cellular expression of this gene results in the ability to phosphorylate the otherwise nontoxic drug ganciclovir and create a phosphorylated, toxic metabolite. Rats treated with an inoculation of producer cells directly into their liver tumors demonstrated significant tumor regression compared with controls. The mean cancer cell mass was reduced 60%, and some pathologic complete responses were observed. The use of direct intratumoral inoculation in this model, however, poses a serious limitation. As discussed earlier, treatment strategies that require identification of each and every metastasis as well as precise tumor localization are unlikely to be clinically applicable in the treatment of patients with liver metastases. Delivery of vectors that selectively kill metastases diffusely throughout the liver represents a more clinically relevant approach. Accordingly, Hurford et al21 have examined a similar approach designed to target the entire liver. They treated diffuse liver metastases by portal inoculation of retroviral producer cell lines to deliver retroviruses containing the gene for either interleukin (IL)-4 or IL-2. Ninety-two percent of the diffuse hepatic metastases in these animals stained positive for marker gene (Escherichia coli lac Z) expression. This efficiency of transduction was significantly better than that achieved with intraportal delivery of retrovirus particles themselves. Moreover, cytokine gene targeting inhibited metastasis formation and caused significant overall reduction in tumor burden, although no more than 5% to 10% of cells in each tumor were transduced. An inflammatory infiltrate and tumor regression were observed only in tumors treated with the IL-2 and IL-4 retroviral producer cell lines; no inhibition of metastatic growth was identified in tumors treated with NIH3T3 cells that express IL-2 and IL-4. In other words, the observed effect requires retroviral gene transfer. Tumor-Associated Antigens and Virus-Directed Enzyme Prodrug Therapy
Another characteristic trait of many liver tumors is their expression of tumor-associated antigens, such as carcinoembryonic antigen (CEA) and alpha-fetoprotein (AFP). 18 Knowledge of the molecular mechanisms that result in tumor cell expression of these antigens may be combined
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with a therapeutic approach known as virus-directed enzyme prodrug therapy (VDEPT) or gene-directed enzyme prodrug therapy (GDEPT).32 VDEPT and GDEPT involve transduction of tumor cells with a gene that encodes an enzyme that is not normally expressed in the cell. The enzyme typically is capable of converting a relatively nontoxic prodrug into a toxic metabolite. This type of gene is commonly referred to as a suicide gene. Transduction of tumor cells with the suicide gene, followed by administration of the prodrug, results in conversion of the prodrug to a toxic metabolite only in cells that express the suicide gene. This produces high levels of the cytotoxic metabolite in the transduced tumor cells with relatively low systemic levels. Bystander killing of tumor cells neighboring the transduced cells also may take place. The suicide gene most commonly used to date in gene therapy strategies is the HSV-tk gene.7 Unlike mammalian thymidine kinase, HSV-tk can monophosphorylate the prodrug ganciclovir, which is then further phosphorylated into a metabolite that is toxic to dividing cells. Accordingly, transduction of tumor cells with HSV-tk followed by systemic treatment with ganciclovir results in intratumoral conversion of ganciclovir to a toxic compound. As described in the preceding section, intratumoral inoculation of a producing cell line engineered to deliver a retroviral HSV-tk construct results in significant antitumor activity in animals bearing liver tumors.7 This antitumor activity is observed only in animals treated with ganciclovir. Other investigators have delivered the HSV-tk using adenoviral vectors into animals with liver metastases and observed significant antitumor activity after administration of ganciclovir.25 The general approach of VDEPT has been refined using knowledge of transcriptional regulation of genes that encode tumor-associated antigens. For example, the transcriptional regulatory sequence that regulates expression of human CEA has been cloned. When this regulatory sequence is placed upstream from the HSV-tk gene, it limits expression of HSV-tk to cells that express CEA. 38 DiMaio and colleagues 13 used this approach to treat mice bearing human pancreatic carcinoma xenografts. Subsequently, Richards and colleagues32 used transcriptional regulatory sequences of CEA cloned upstream from a gene encoding cytosine deaminase (CD). CD is an effective suicide gene for cancer therapy because it deaminates 5-fluorocytosine to 5-fluorouracil, which has been demonstrated to have significant antitumor activity. CD is expressed in bacteria but is not expressed in mammalian cells. Accordingly, only cells that are transduced with and express CD are sensitive to the otherwise nontoxic prodrug 5-fluorocytosine. One clinical trial that recently opened at Cornell University Medical Center for patients with liver metastases tests an adenoviral vector for transduction of tumor cells with the CD gene. 33 In this trial the vector is inoculated directly into colon carcinoma liver metastases. Radiolabeled flucytosine is used in conjunction with PET scanning to assess viral transduction. Several investigators have used CEA regulatory sequences to limit expression of suicide genes, including HSV-tk and CD, 26 •37 and additional work has been performed examining transcriptional regulatory sequences for other tumor-associated antigens, including alpha-fetoprotein.22,36
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Blood Supply
Another difference between liver metastases and surrounding normal liver is their blood supply; liver metastases derive most of their blood supply from the hepatic artery, whereas normal hepatocytes derive most of their blood supply from the portal vein. Breedis and Young4 studied liver tumor circulation in animal models and autopsy specimens using dye injection. Microscopy revealed dye uptake in tumors after hepatic arterial injection. Portal venous injection resulted in dye uptake at only the periphery of the tumors. They estimated that 80% to 100% of the blood supply of the tumors in the autopsy specimens was derived from the hepatic artery. Infusion of chemotherapeutic agents into the hepatic artery results in higher concentrations of the agents in liver tumors than is achievable with intravenous administration. 15 Hepatic arterial infusion of chemotherapy has been shown to increase the regional efficacy of chemotherapy compared with intravenous or intraportal delivery. This strategy also can be used to augment gene delivery to liver tumors and reduce unwanted transduction of hepatocytes. Blesing and Kerr2 plan a clinical trial of administration of a retroviral vector encoding the CD gene to hepatic metastases of colorectal carcinoma via hepatic arterial infusion. Venook and his colleagues at University of California, San Francisco, are also investigating intrahepatic arterial vector infusion with an adenovirus carrying the p53 gene.33 These studies may show if the theoretical advantage of hepatic arterial vector delivery translates into actual enhanced tumor destruction. Cell Surface Receptors
Colon carcinoma liver metastases also express higher levels of specific cell surface receptors, such as the epidermal growth factor (EGF) receptor.18·35 At least one team of investigators has examined a technique to target tumor cells based on this finding. Cristiano and Roth12 at M.D. Anderson Cancer Center have chemically modified recombinant human EGF to attach it to DNA through use of poly-L-lysine. The complex binds preferentially to cells that overexpress the EGF receptor. Degradation of the DNA after fusion of the endosome with a lysosome is prevented by an endosomal lysis agent, such as a replication-defective adenovirus. Coupling of a replication-defective adenovirus directly to the EGF /DNA complex results in fourfold higher gene expression. Although this technique has been used to destroy lung cancer cells, it is also applicable to colon carcinoma liver metastases, which frequently express high levels of EGF receptor. Oncogenes and Tumor Suppressor Genes
The genetic alterations associated with tumor progression have been characterized in recent years, and some investigators have examined strat-
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egies in which gene therapy can be applied to correct these alterations.39 Mutations in the ras oncogene and p53 tumor suppressor gene are common in the progression of human colon carcinoma. It remains commonly accepted that an accumulation of genetic alterations leads to the development of cancer. Although many of these altered genes have been identified (e.g., AFC, DCC, ras, p53), most of the genetic alterations that lead to cancer have not yet been characterized. Accordingly, replacement of all defective genes to treat cancer is a daunting task. However, quite importantly, it appears that correction of only one of the many genetic defects in a cancer cell may inhibit tumorigenicity, a finding that has dramatic consequences for gene therapy strategies in which oncogene and tumor suppressor gene function is targeted.17 Normal p53 activity is lacking in almost half of primary and metastatic tumors in the liver. 3, 42 Moreover, introduction of the normal (wildtype) p53 gene results in programmed cell death (apoptosis) and inhibits tumor growth. A replication-deficient adenovirus constructed to transduce the wild-type p53 gene has shown significant antitumor activity in preclinical models. This vector is currently being tested at University of California, San Francisco, in patients with primary liver tumors and liver metastases. 33 In this trial, the adenoviral construct is introduced via the hepatic artery. Other studies have demonstrated that introduction of vectors that contain the wild-type p53 gene into livers via the portal vein does not result in toxicity to normal hepatocytes. 14 Another approach used to target tumor cells with mutant p53 has been examined by Bischoff et al. 1 These researchers have constructed a replication-competent adenovirus by deletion of only the adenoviral ElB gene. During adenoviral infection of a cell, the ElB protein normally interacts with wild-type p53. This appears to inactivate p53, thereby allowing efficient viral replication, which results in cell death. Accordingly, adenoviruses that lack ElB can replicate efficiently in (and destroy) cells that are missing functional p53, such as tumor cells. However, because of the absence of ElB, this adenovirus is unable to replicate in (and destroy) cells that possess normal functional p53. The antitumor efficacy of this virus has been demonstrated in preclinical models and is currently being examined in clinical trials. Chemotherapy agents such as cisplatin and 5-fluorouracil appear to augment the antitumor effects achieved with this virus. 19 An alternative approach to introduction of the p53 gene into tumors is injection of plasmid DNA containing the p53 directly into tumors. This therapy is currently being examined in a trial at Hammersmith Hospital in London. 33 lmmunogenicity
Tumor cells express tumor-associated and tumor-specific antigens that can be recognized by the immune system. The ability of the immune system to mount an antitumor response has been demonstrated clearly.40 Researchers have investigated several methods to enhance the host immune system, including use of autologous tumor vaccines, treatment with nonspecific immunostimulatory agents, and transfer of immune effector
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(e.g., tumor infiltrating lymphocytes (TIL)) cells. Several researchers have investigated methods to enhance these therapies by gene transfer. Immunotherapy has been combined with suicide gene therapy in an attempt to augment tumor lysis by several investigators.8- 10 Modulation of the host immune system is a treatment strategy generally applicable to all forms of tumors, including liver metastases. Results of research in this area have been reviewed in other articles. CONCLUSIONS
These studies demonstrate the potential of gene therapy to address the clinical challenge posed by liver metastases. Several gene therapy trials for patients with liver tumors are currently under way. Clearly, numerous problems must be solved for successful clinical application of gene therapy to be achieved. One of the most important areas for future research stems from the overwhelming need for a more efficient gene delivery vehicle. In addition, the problems of tissue specificity and prolonged transgene expression must be overcome before gene therapy strategies will have a significant impact on the treatment of liver tumors. Advances in our understanding of the genetics and biology of liver tumors must be incorporated into future gene therapy strategies. Hopefully, continued research will allow gene therapy to impact significantly the treatment of patients afflicted with liver tumors. References 1. Bischoff JR, Kirn DH, Williams A, et al: An adenovirus mutant that replicates selectively in p53-deficient human tumor cells. Science 274:373, 1996 2. Blesing CH, Kerr DJ: Intra-hepatic arterial drug delivery. Journal of Drug Targeting 3:341, 1996 3. Bookstein R, Demers W, Gregory R, et al: p53 gene therapy in vivo of hepatocellular and liver metastatic colorectal cancer. Semin Oneal 23:66, 1996 4. Breedis C, Young G: The blood supply of neoplasms in the liver. Am J Pathol 30:969, 1954 5. Busch E, Kemeny MM: Colorectal cancer: Hepatic-directed therapy: The role of surgery, regional chemotherapy, and novel modalities. Semin Oncol 22:494, 1995 6. Carroll NM, Chiocca EA, Takahashi K, et al: Enhancement of gene therapy specificity for diffuse colon carcinoma liver metastases with recombinant herpes simplex virus. Ann Surg 224:323, 1996 7. Caruso M, Panis Y, Gagandeep S, et al: Regression of established macroscopic liver metastases after in situ transduction of a suicide gene. Proc Natl Acad Sci US A 90:7024, 1993 8. Caruso M, Pham-Nguyen K, Kwong Y, et al: Adenovirus-mediated interleukin-12 gene therapy for metastatic colon carcinoma. Proc Natl Acad Sci US A 93:11302, 1996 9. Chen S, Chen XHL, Wang Y, et al: Combination gene therapy for liver metastasis of colon carcinoma in vivo. Proc Natl Acad Sci US A 92:2577, 1995 10. Chen S, Kasai K, Xu B, et al: Combination suicide and cytokine gene therapy for hepatic metastases of colon carcinoma: Sustained antitumor immunity prolongs animal survival. Cancer Res 56:3758, 1996 11. Cotran RS, Kumar V, Robbins SL: Robbins' Pathologic Basis of Disease, ed 4. Philadelphia, WB Saunders, 1989, p 958
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12. Cristiano RJ, Roth JA: Epidermal growth factor mediated DNA delivery into lung cancer cells via the epidermal growth factor receptor. Cancer Gene Therapy 3:4, 1996 13. DiMaio JD, Clary BM, Via DF, et al: Directed enzyme pro-drug gene therapy for pancreatic cancer in vivo. Surgery 116:205, 1994 14. Drazan KE, Shen XD, Csete ME, et al: In vivo adenoviral-mediated human p53 tumor suppressor gene transfer and expression in rat liver after resection. Surgery 116:197, 1994 15. Ensminger WD, Rosowsky A, Raso V, et al: A clinical-pharmacological evaluation of hepatic arterial infusions of 5-fluoro-2-deoxyuridine and 5-fluorouracil. Cancer Res 38:3784, 1978 16. Freeman SM, Abboud CN, Whartenby KA, et al: The "bystander effect": Tumor regression when a fraction of the tumor mass is genetically modified. Cancer Res 53:5274, 1993 17. Goyette MC, Cho K, Fasching CL, et al: Progression of colorectal cancer is associated with multiple tumor suppressor gene defects but inhibition of tumorigenicity is accomplished by correction of any single defect via chromosome transfer. Mol Cell Biol 12:1387, 1992 18. Gutman M, Fidler IJ: Biology of human colon cancer metastasis. World J Surg 19:226, 1995 19. Heise C, Sampson-Johannes A, Williams A, et al: ONYX-015, an ElB gene-attenuated adenovirus, causes tumor-specific cytolysis and antitumoral efficacy that can be augmented by standard chemotherapeutic agents. Nature Medicine 3:639, 1997 20. Hoffman AL, Rosen HR, Ljubimova JU, et al: Hepatic regeneration: Current concepts and clinical implications. Semin Liver Dis 14:190, 1994 21. Hurford RK, Dranoff G, Mulligan RC, et al: Gene therapy of metastatic cancer by in vivo retroviral gene targeting. Nat Genet 10:430, 1995 22. Ido A, Nakata K, Kato Y, et al: Gene therapy for hepatoma cells using a retrovirus vector carrying herpes simplex virus thymidine kinase gene under the control of human alphafetoprotein gene promoter. Cancer Res 55:3105, 1995 23. Ishii-Morita H, Agbaria R, Mullen CA, et al: Mechanism of "bystander effect" killing in the herpes simplex thymidine kinase gene therapy model of cancer treatment. Gene Therapy 4:244, 1997 24. Kruskal JB, Kane RA: Imaging of primary and metastatic liver tumors. Surgical Oncology Clinics of North America 5:231, 1996 25. Kwong Y, Chen S, Kasai K, et al: Adenoviral-mediated suicide gene therapy for hepatic metastases of breast cancer. Cancer Gene Therapy 3:339, 1996 26. Lan K, Kanai F, Shiratori Y, et al: Tumor-specific gene expression in carcinoembryonic antigen-producing gastric cancer cells using adenovirus vectors. Gastroenterology 111:1241, 1996 27. Liu CL, Fan ST: Nonresectional therapies for hepatocellular carcinoma. Am J Surg 173:358, 1997 28. Miller D, Adam M, Miller A: Gene transfer by retrovirus vectors occurs only in cells that are actively replicating at the time of infection. Mol Cell Biol 10:4239, 1990 29. Rainov NG, Kramm CM, Aboody-Guterman K, et al: Retrovirus-mediated gene therapy of experimental brain neoplasms using the herpes simplex virus-thymidine kinase/ ganciclovir paradigm. Cancer Gene Therapy 3:99, 1996 30. Ramesh R, Marrogi AJ, Munshi A, et al: In vivo analysis of the "bystander effect": A cytokine cascade. Exp Hematol 24:829, 1996 31. Ravikumar TS: Interstitial therapies for liver tumors. Surgical Oncology Clinics of North America 5:365, 1996 32. Richards CA, Austin EA, Huber BE: Transcriptional regulatory sequences of carcinoembryonic antigen: Identification and use with cytosine deaminase for tumor specific gene therapy. Hum Gene Ther 6:881, 1995 33. Roth JA, Cristiano RJ: Gene therapy for cancer: What have we done and where are we going? J Natl Cancer Inst 88:21, 1997 34. Scheele J, Stangl R, Altendorf-Hofmann A: Hepatic metastases from colorectal carcinoma: Impact of surgical resection on the natural history. Br J Surg 77:1241, 1990 35. Singh RK, Tsan R, Radinsky: Influence of the host microenvironment on the clonal selection of human colon carcinoma cells during primary tumor growth and metastasis. Clin Exp Metastasis 15:140, 1997 36. Su H, Chang Jc; Xu SM, et al: Selective killing of AFP-positive hepatocellular carcinoma cells by adeno-associated virus transfer of the herpes simplex virus thymidine kinase gene. Hum Gene Ther 7:463, 1996
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37. Tanaka T, Kanai F, Lan K, et al: Adenovirus-mediated gene therapy of gastric carcinoma using cancer specific gene expression in vivo. Biochem Biophys Res Commun 231:775, 1997 38. Tanaka T, Kanai F, Okabe S, et al: Adenovirus-mediated prodrug gene therapy for carcinoembryonic antigen-producing human gastric carcinoma cells in vitro. Cancer Res 56:1341, 1996 39. Tomlinson I, Ilyas M, Novelli M: Molecular genetics of colon cancer. Cancer Metastasis Rev 16:67, 1997 40. Tuting T, Storkus WJ, Lotze MT: Gene-based strategies for the immunotherapy of cancer. Journal of Molecular Medicine 75:478, 1997 41. Weiss L, Grundmann E, Torhost J, et al: Haematogenous metastatic patterns in colonic carcinoma: An analysis of 1541 necropsies. J Pathol 150:195, 1986 42. Xu GW, Sun ZT, Forrester K, et al: Tissue-specific growth suppression and chemosensitivity promotion in human hepatocellular carcinoma cells by retroviral-mediated transfer of the wild-type p53 gene. Hepatology 24:1264, 1996
Address reprint requests to Kenneth K. Tanabe, MD Division of Surgical Oncology, Cox 626 Massachusetts General Hospital Boston, MA 02114
CANCER GENE THERAPY
+ .00
GENE THERAPY FOR LIVER TUMORS Nancy M. Carroll, MD, and Kenneth K. Tanabe, MD
The liver remains one of the most common sites of distant metastases for patients with solid tumors as well as a site of primary hepatic malignancies, including hepatocellular carcinoma, peripheral cholangiocarcinoma, angiosarcoma, and hepatoblastoma.11 Most patients with liver metastases also have metastatic disease in other organs. The biology of colon carcinoma is unique in that a significant percentage of patients with metastatic disease harbor disease only in the liver. 41 Certainly, effective regional therapy applied to this group of patients may influence survival. Successful eradication of liver tumors in patients with primary liver cancers also would be expected to improve survival. Surgical resection remains the only potentially curative treatment; however, even patients whose tumors are limited to the liver are rarely candidates for resection.34 Following this logic, many investigators have pursued novel regional hepatic therapies, including cryosurgery, radiofrequency ablation, isolated liver perfusion, hepatic arterial infusion chemotherapy, chemoembolization, hyperthermic hepatic isolated chemoperfusion, ethanol injection, laser ablation, and direct injection of chemotherapy "gels." 5• 27• 31 Gene therapy can be added to this list of experimental approaches for treatment of liver tumors. Some gene therapy approaches are extensions of strategies used to treat diffuse solid tumor metastases, regardless of location (e.g., lung, liver, marrow). Other gene therapy approaches are uniquely suited for tumors that grow in the liver.
This work was supported by the Marshall K. Bartlett Research Fellowship from the Massachusetts General Hospital.
From the Division of Surgical Oncology, Department of Surgery, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts SURGICAL ONCOLOGY CLINICS OF NORTH AMERICA VOLUME 7 • NUMBER 3 • JULY 1998
617
618
CARROLL & TANABE
At first glance it may appear that efforts devoted to the study of treatment of liver metastases are doomed to failure because of the high prevalence of systemic disease in this patient population. Patients with colon and rectal carcinoma liver metastases, however, serve as model candidates for regional therapy because of the high incidence of "liver-only" metastases. Moreover, liver metastases may serve as a model to test treatment strategies against diffuse disease in which principles of gene therapy can be examined. Information gained in studies on this patient population may be applicable to patients with metastases in other locations. The ability to identify the presence and location of liver tumors continues to improve with new modalities, including magnetic resonance (MR) imaging scans with ferumoxide, position emission tomography (PET) scans, helical computed tomography (CT) scans, radiolabeled monoclonal antibodies, and intraoperative sonography. 24 Nonetheless, the limited sensitivity and limited resolution of these modalities precludes identification of every tumor, especially in patients with solid tumor liver metastases. Accordingly, treatment strategies that require precise tumor localization are much less likely to be successful for patients with several metastases. These include surgical resection, cryoablation, radiofrequency ablation, laser ablation, and ethanol injection. Similarly, gene therapy approaches that rely on direct intratumoral inoculation are unlikely to be effective in the management of liver tumors. Gene therapy approaches that target tumors throughout the liver are much more likely to have a clinically significant impact.
GENE THERAPY STRATEGIES FOR LIVER TUMORS Overview
Strategies to minimize toxicity to normal liver are extremely critical, especially for gene therapy strategies that target diffuse tumors throughout the liver. Specific differences exist between liver tumors and surrounding normal liver, and they can be exploited to target gene therapy to the tumors and reduce toxicity to normal liver. These differences between liver tumor cells and normal liver cells include mitotic activity, expression of tumor-associated antigens, dominant blood supply, expression of cell surface receptors, inactivation of tumor suppressor genes, expression of activated oncogenes, and immunogenicity. 18 Some of these gene therapy strategies already have entered clinical trials.
Vectors
Each of the gene therapy strategies described next requires efficient gene delivery. Several gene delivery vehicles have been developed, and many more are under development. These vehicles have been summarized nicely in other articles in this issue and are not be duplicated herein.
GENE THERAPY FOR LIVER TUMORS
619
Mitotic Activity
Hepatocytes in the normal adult liver are quiescent. 20 In contrast, most liver tumors have high mitotic activity, which results in an actively proliferating malignant cell population surrounded by a relatively quiescent normal cell population. These authors have examined a gene therapy approach to liver metastases using herpes simplex virus (HSV) type 1 that attempts to exploit this difference in mitotic activity. 6 HSV is a DNA virus that infects both dividing and nondividing cells, thereby resulting in the production of progeny virions and cell lysis. Although the virus is neurotropic, it can infect most human tissues. The cytolytic property of HSV infection and replication can be used to destroy tumor cells. We have demonstrated that several strains of HSV destroy human colon carcinoma cells efficiently, even when added at a multiplicity of infection (MOI) of less than 1 viral plaque forming unit per 10 tumor cells. The significant antitumor activity of HSV observed in vitro is also observed in vivo. Deletion of an HSV gene required for synthesis of DNA precursors (e.g., viral ribonucleotide reductase) renders the virus replication conditional; the mutant virus can no longer replicate in nondividing cells. The function of the missing viral protein, however, may be replaced by a cellular protein (e.g., cellular ribonucleotide reductase). Accordingly, mitotically active cells provide the DNA precursors and machinery necessary for synthesis of DNA precursors. This cellular complementation of the viral deficiency results in viral replication, production of progeny virions, and host cell death. The HSV ribonucleotide reductase gene encodes an enzyme involved in the production of DNA precursors. We have examined a gene therapy strategy in which an HSV mutant (hrR3) that is deficient in ribonucleotide reductase is introduced via the portal vein into rodents that bear diffuse liver metastases. Because this mutant replicates significantly more efficiently in mitotically active cells, we observed viral gene expression indicative of replication in virtually all of the diffuse liver metastases but not in normal (mitotically inactive) liver. In this model, 96% of liver metastases demonstrated marker gene expression; however, only a small percentage (5%-25%) of the cells in each metastasis expressed the gene. Additional studies are required to determine the effect of repeat treatments in an attempt to infect a larger percentage of the tumor cells in each metastasis. Nonetheless, significant antitumor response can be expected even in the absence of 100% transduction efficiency as a result of bystander killing. 16 Bystander killing refers to the destruction of untransduced tumor cells that are adjacent to transduced tumor cells. The implications of this phenomenon are significant. Gene delivery vehicles are not capable of transducing 100% of tumor cells in any given tumor mass, a limitation that would result in failure of most gene therapy approaches. However, the observation that bystander killing results in destruction of an entire tumor despite successful transduction of only a fraction of the tumor cells certainly holds promise. The mechanisms that lead to bystander killing are currently under investigation.23,30
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Another gene therapy strategy that relies on differential mitotic activity between normal hepatocytes and liver tumors is the use of retroviral vectors. Retroviral integration and transgene expression require cellular division. 28 Therefore, retroviruses may allow preferential expression of transgenes in liver tumors rather than normal liver. Unfortunately, retroviruses are relatively labile in vivo compared with other viruses, and in vivo transduction efficiencies using retroviral vectors have been extremely poor. Accordingly, some investigators have examined a gene therapy approach that involves in vivo delivery of retroviral packaging cell lines rather than in vivo delivery of the retroviral vectors themselves. 29 Caruso et aF investigated an approach of direct intratumoral inoculation of retroviral producer cells into isolated liver tumors. The retroviral construct used in these studies contained the HSV thymidine kinase (HSV-tk) gene. Cellular expression of this gene results in the ability to phosphorylate the otherwise nontoxic drug ganciclovir and create a phosphorylated, toxic metabolite. Rats treated with an inoculation of producer cells directly into their liver tumors demonstrated significant tumor regression compared with controls. The mean cancer cell mass was reduced 60%, and some pathologic complete responses were observed. The use of direct intratumoral inoculation in this model, however, poses a serious limitation. As discussed earlier, treatment strategies that require identification of each and every metastasis as well as precise tumor localization are unlikely to be clinically applicable in the treatment of patients with liver metastases. Delivery of vectors that selectively kill metastases diffusely throughout the liver represents a more clinically relevant approach. Accordingly, Hurford et al21 have examined a similar approach designed to target the entire liver. They treated diffuse liver metastases by portal inoculation of retroviral producer cell lines to deliver retroviruses containing the gene for either interleukin (IL)-4 or IL-2. Ninety-two percent of the diffuse hepatic metastases in these animals stained positive for marker gene (Escherichia coli lac Z) expression. This efficiency of transduction was significantly better than that achieved with intraportal delivery of retrovirus particles themselves. Moreover, cytokine gene targeting inhibited metastasis formation and caused significant overall reduction in tumor burden, although no more than 5% to 10% of cells in each tumor were transduced. An inflammatory infiltrate and tumor regression were observed only in tumors treated with the IL-2 and IL-4 retroviral producer cell lines; no inhibition of metastatic growth was identified in tumors treated with NIH3T3 cells that express IL-2 and IL-4. In other words, the observed effect requires retroviral gene transfer. Tumor-Associated Antigens and Virus-Directed Enzyme Prodrug Therapy
Another characteristic trait of many liver tumors is their expression of tumor-associated antigens, such as carcinoembryonic antigen (CEA) and alpha-fetoprotein (AFP). 18 Knowledge of the molecular mechanisms that result in tumor cell expression of these antigens may be combined
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with a therapeutic approach known as virus-directed enzyme prodrug therapy (VDEPT) or gene-directed enzyme prodrug therapy (GDEPT).32 VDEPT and GDEPT involve transduction of tumor cells with a gene that encodes an enzyme that is not normally expressed in the cell. The enzyme typically is capable of converting a relatively nontoxic prodrug into a toxic metabolite. This type of gene is commonly referred to as a suicide gene. Transduction of tumor cells with the suicide gene, followed by administration of the prodrug, results in conversion of the prodrug to a toxic metabolite only in cells that express the suicide gene. This produces high levels of the cytotoxic metabolite in the transduced tumor cells with relatively low systemic levels. Bystander killing of tumor cells neighboring the transduced cells also may take place. The suicide gene most commonly used to date in gene therapy strategies is the HSV-tk gene.7 Unlike mammalian thymidine kinase, HSV-tk can monophosphorylate the prodrug ganciclovir, which is then further phosphorylated into a metabolite that is toxic to dividing cells. Accordingly, transduction of tumor cells with HSV-tk followed by systemic treatment with ganciclovir results in intratumoral conversion of ganciclovir to a toxic compound. As described in the preceding section, intratumoral inoculation of a producing cell line engineered to deliver a retroviral HSV-tk construct results in significant antitumor activity in animals bearing liver tumors.7 This antitumor activity is observed only in animals treated with ganciclovir. Other investigators have delivered the HSV-tk using adenoviral vectors into animals with liver metastases and observed significant antitumor activity after administration of ganciclovir.25 The general approach of VDEPT has been refined using knowledge of transcriptional regulation of genes that encode tumor-associated antigens. For example, the transcriptional regulatory sequence that regulates expression of human CEA has been cloned. When this regulatory sequence is placed upstream from the HSV-tk gene, it limits expression of HSV-tk to cells that express CEA. 38 DiMaio and colleagues 13 used this approach to treat mice bearing human pancreatic carcinoma xenografts. Subsequently, Richards and colleagues32 used transcriptional regulatory sequences of CEA cloned upstream from a gene encoding cytosine deaminase (CD). CD is an effective suicide gene for cancer therapy because it deaminates 5-fluorocytosine to 5-fluorouracil, which has been demonstrated to have significant antitumor activity. CD is expressed in bacteria but is not expressed in mammalian cells. Accordingly, only cells that are transduced with and express CD are sensitive to the otherwise nontoxic prodrug 5-fluorocytosine. One clinical trial that recently opened at Cornell University Medical Center for patients with liver metastases tests an adenoviral vector for transduction of tumor cells with the CD gene. 33 In this trial the vector is inoculated directly into colon carcinoma liver metastases. Radiolabeled flucytosine is used in conjunction with PET scanning to assess viral transduction. Several investigators have used CEA regulatory sequences to limit expression of suicide genes, including HSV-tk and CD, 26 •37 and additional work has been performed examining transcriptional regulatory sequences for other tumor-associated antigens, including alpha-fetoprotein.22,36
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Blood Supply
Another difference between liver metastases and surrounding normal liver is their blood supply; liver metastases derive most of their blood supply from the hepatic artery, whereas normal hepatocytes derive most of their blood supply from the portal vein. Breedis and Young4 studied liver tumor circulation in animal models and autopsy specimens using dye injection. Microscopy revealed dye uptake in tumors after hepatic arterial injection. Portal venous injection resulted in dye uptake at only the periphery of the tumors. They estimated that 80% to 100% of the blood supply of the tumors in the autopsy specimens was derived from the hepatic artery. Infusion of chemotherapeutic agents into the hepatic artery results in higher concentrations of the agents in liver tumors than is achievable with intravenous administration. 15 Hepatic arterial infusion of chemotherapy has been shown to increase the regional efficacy of chemotherapy compared with intravenous or intraportal delivery. This strategy also can be used to augment gene delivery to liver tumors and reduce unwanted transduction of hepatocytes. Blesing and Kerr2 plan a clinical trial of administration of a retroviral vector encoding the CD gene to hepatic metastases of colorectal carcinoma via hepatic arterial infusion. Venook and his colleagues at University of California, San Francisco, are also investigating intrahepatic arterial vector infusion with an adenovirus carrying the p53 gene.33 These studies may show if the theoretical advantage of hepatic arterial vector delivery translates into actual enhanced tumor destruction. Cell Surface Receptors
Colon carcinoma liver metastases also express higher levels of specific cell surface receptors, such as the epidermal growth factor (EGF) receptor.18·35 At least one team of investigators has examined a technique to target tumor cells based on this finding. Cristiano and Roth12 at M.D. Anderson Cancer Center have chemically modified recombinant human EGF to attach it to DNA through use of poly-L-lysine. The complex binds preferentially to cells that overexpress the EGF receptor. Degradation of the DNA after fusion of the endosome with a lysosome is prevented by an endosomal lysis agent, such as a replication-defective adenovirus. Coupling of a replication-defective adenovirus directly to the EGF /DNA complex results in fourfold higher gene expression. Although this technique has been used to destroy lung cancer cells, it is also applicable to colon carcinoma liver metastases, which frequently express high levels of EGF receptor. Oncogenes and Tumor Suppressor Genes
The genetic alterations associated with tumor progression have been characterized in recent years, and some investigators have examined strat-
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egies in which gene therapy can be applied to correct these alterations.39 Mutations in the ras oncogene and p53 tumor suppressor gene are common in the progression of human colon carcinoma. It remains commonly accepted that an accumulation of genetic alterations leads to the development of cancer. Although many of these altered genes have been identified (e.g., AFC, DCC, ras, p53), most of the genetic alterations that lead to cancer have not yet been characterized. Accordingly, replacement of all defective genes to treat cancer is a daunting task. However, quite importantly, it appears that correction of only one of the many genetic defects in a cancer cell may inhibit tumorigenicity, a finding that has dramatic consequences for gene therapy strategies in which oncogene and tumor suppressor gene function is targeted.17 Normal p53 activity is lacking in almost half of primary and metastatic tumors in the liver. 3, 42 Moreover, introduction of the normal (wildtype) p53 gene results in programmed cell death (apoptosis) and inhibits tumor growth. A replication-deficient adenovirus constructed to transduce the wild-type p53 gene has shown significant antitumor activity in preclinical models. This vector is currently being tested at University of California, San Francisco, in patients with primary liver tumors and liver metastases. 33 In this trial, the adenoviral construct is introduced via the hepatic artery. Other studies have demonstrated that introduction of vectors that contain the wild-type p53 gene into livers via the portal vein does not result in toxicity to normal hepatocytes. 14 Another approach used to target tumor cells with mutant p53 has been examined by Bischoff et al. 1 These researchers have constructed a replication-competent adenovirus by deletion of only the adenoviral ElB gene. During adenoviral infection of a cell, the ElB protein normally interacts with wild-type p53. This appears to inactivate p53, thereby allowing efficient viral replication, which results in cell death. Accordingly, adenoviruses that lack ElB can replicate efficiently in (and destroy) cells that are missing functional p53, such as tumor cells. However, because of the absence of ElB, this adenovirus is unable to replicate in (and destroy) cells that possess normal functional p53. The antitumor efficacy of this virus has been demonstrated in preclinical models and is currently being examined in clinical trials. Chemotherapy agents such as cisplatin and 5-fluorouracil appear to augment the antitumor effects achieved with this virus. 19 An alternative approach to introduction of the p53 gene into tumors is injection of plasmid DNA containing the p53 directly into tumors. This therapy is currently being examined in a trial at Hammersmith Hospital in London. 33 lmmunogenicity
Tumor cells express tumor-associated and tumor-specific antigens that can be recognized by the immune system. The ability of the immune system to mount an antitumor response has been demonstrated clearly.40 Researchers have investigated several methods to enhance the host immune system, including use of autologous tumor vaccines, treatment with nonspecific immunostimulatory agents, and transfer of immune effector
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(e.g., tumor infiltrating lymphocytes (TIL)) cells. Several researchers have investigated methods to enhance these therapies by gene transfer. Immunotherapy has been combined with suicide gene therapy in an attempt to augment tumor lysis by several investigators.8- 10 Modulation of the host immune system is a treatment strategy generally applicable to all forms of tumors, including liver metastases. Results of research in this area have been reviewed in other articles. CONCLUSIONS
These studies demonstrate the potential of gene therapy to address the clinical challenge posed by liver metastases. Several gene therapy trials for patients with liver tumors are currently under way. Clearly, numerous problems must be solved for successful clinical application of gene therapy to be achieved. One of the most important areas for future research stems from the overwhelming need for a more efficient gene delivery vehicle. In addition, the problems of tissue specificity and prolonged transgene expression must be overcome before gene therapy strategies will have a significant impact on the treatment of liver tumors. Advances in our understanding of the genetics and biology of liver tumors must be incorporated into future gene therapy strategies. Hopefully, continued research will allow gene therapy to impact significantly the treatment of patients afflicted with liver tumors. References 1. Bischoff JR, Kirn DH, Williams A, et al: An adenovirus mutant that replicates selectively in p53-deficient human tumor cells. Science 274:373, 1996 2. Blesing CH, Kerr DJ: Intra-hepatic arterial drug delivery. Journal of Drug Targeting 3:341, 1996 3. Bookstein R, Demers W, Gregory R, et al: p53 gene therapy in vivo of hepatocellular and liver metastatic colorectal cancer. Semin Oneal 23:66, 1996 4. Breedis C, Young G: The blood supply of neoplasms in the liver. Am J Pathol 30:969, 1954 5. Busch E, Kemeny MM: Colorectal cancer: Hepatic-directed therapy: The role of surgery, regional chemotherapy, and novel modalities. Semin Oncol 22:494, 1995 6. Carroll NM, Chiocca EA, Takahashi K, et al: Enhancement of gene therapy specificity for diffuse colon carcinoma liver metastases with recombinant herpes simplex virus. Ann Surg 224:323, 1996 7. Caruso M, Panis Y, Gagandeep S, et al: Regression of established macroscopic liver metastases after in situ transduction of a suicide gene. Proc Natl Acad Sci US A 90:7024, 1993 8. Caruso M, Pham-Nguyen K, Kwong Y, et al: Adenovirus-mediated interleukin-12 gene therapy for metastatic colon carcinoma. Proc Natl Acad Sci US A 93:11302, 1996 9. Chen S, Chen XHL, Wang Y, et al: Combination gene therapy for liver metastasis of colon carcinoma in vivo. Proc Natl Acad Sci US A 92:2577, 1995 10. Chen S, Kasai K, Xu B, et al: Combination suicide and cytokine gene therapy for hepatic metastases of colon carcinoma: Sustained antitumor immunity prolongs animal survival. Cancer Res 56:3758, 1996 11. Cotran RS, Kumar V, Robbins SL: Robbins' Pathologic Basis of Disease, ed 4. Philadelphia, WB Saunders, 1989, p 958
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12. Cristiano RJ, Roth JA: Epidermal growth factor mediated DNA delivery into lung cancer cells via the epidermal growth factor receptor. Cancer Gene Therapy 3:4, 1996 13. DiMaio JD, Clary BM, Via DF, et al: Directed enzyme pro-drug gene therapy for pancreatic cancer in vivo. Surgery 116:205, 1994 14. Drazan KE, Shen XD, Csete ME, et al: In vivo adenoviral-mediated human p53 tumor suppressor gene transfer and expression in rat liver after resection. Surgery 116:197, 1994 15. Ensminger WD, Rosowsky A, Raso V, et al: A clinical-pharmacological evaluation of hepatic arterial infusions of 5-fluoro-2-deoxyuridine and 5-fluorouracil. Cancer Res 38:3784, 1978 16. Freeman SM, Abboud CN, Whartenby KA, et al: The "bystander effect": Tumor regression when a fraction of the tumor mass is genetically modified. Cancer Res 53:5274, 1993 17. Goyette MC, Cho K, Fasching CL, et al: Progression of colorectal cancer is associated with multiple tumor suppressor gene defects but inhibition of tumorigenicity is accomplished by correction of any single defect via chromosome transfer. Mol Cell Biol 12:1387, 1992 18. Gutman M, Fidler IJ: Biology of human colon cancer metastasis. World J Surg 19:226, 1995 19. Heise C, Sampson-Johannes A, Williams A, et al: ONYX-015, an ElB gene-attenuated adenovirus, causes tumor-specific cytolysis and antitumoral efficacy that can be augmented by standard chemotherapeutic agents. Nature Medicine 3:639, 1997 20. Hoffman AL, Rosen HR, Ljubimova JU, et al: Hepatic regeneration: Current concepts and clinical implications. Semin Liver Dis 14:190, 1994 21. Hurford RK, Dranoff G, Mulligan RC, et al: Gene therapy of metastatic cancer by in vivo retroviral gene targeting. Nat Genet 10:430, 1995 22. Ido A, Nakata K, Kato Y, et al: Gene therapy for hepatoma cells using a retrovirus vector carrying herpes simplex virus thymidine kinase gene under the control of human alphafetoprotein gene promoter. Cancer Res 55:3105, 1995 23. Ishii-Morita H, Agbaria R, Mullen CA, et al: Mechanism of "bystander effect" killing in the herpes simplex thymidine kinase gene therapy model of cancer treatment. Gene Therapy 4:244, 1997 24. Kruskal JB, Kane RA: Imaging of primary and metastatic liver tumors. Surgical Oncology Clinics of North America 5:231, 1996 25. Kwong Y, Chen S, Kasai K, et al: Adenoviral-mediated suicide gene therapy for hepatic metastases of breast cancer. Cancer Gene Therapy 3:339, 1996 26. Lan K, Kanai F, Shiratori Y, et al: Tumor-specific gene expression in carcinoembryonic antigen-producing gastric cancer cells using adenovirus vectors. Gastroenterology 111:1241, 1996 27. Liu CL, Fan ST: Nonresectional therapies for hepatocellular carcinoma. Am J Surg 173:358, 1997 28. Miller D, Adam M, Miller A: Gene transfer by retrovirus vectors occurs only in cells that are actively replicating at the time of infection. Mol Cell Biol 10:4239, 1990 29. Rainov NG, Kramm CM, Aboody-Guterman K, et al: Retrovirus-mediated gene therapy of experimental brain neoplasms using the herpes simplex virus-thymidine kinase/ ganciclovir paradigm. Cancer Gene Therapy 3:99, 1996 30. Ramesh R, Marrogi AJ, Munshi A, et al: In vivo analysis of the "bystander effect": A cytokine cascade. Exp Hematol 24:829, 1996 31. Ravikumar TS: Interstitial therapies for liver tumors. Surgical Oncology Clinics of North America 5:365, 1996 32. Richards CA, Austin EA, Huber BE: Transcriptional regulatory sequences of carcinoembryonic antigen: Identification and use with cytosine deaminase for tumor specific gene therapy. Hum Gene Ther 6:881, 1995 33. Roth JA, Cristiano RJ: Gene therapy for cancer: What have we done and where are we going? J Natl Cancer Inst 88:21, 1997 34. Scheele J, Stangl R, Altendorf-Hofmann A: Hepatic metastases from colorectal carcinoma: Impact of surgical resection on the natural history. Br J Surg 77:1241, 1990 35. Singh RK, Tsan R, Radinsky: Influence of the host microenvironment on the clonal selection of human colon carcinoma cells during primary tumor growth and metastasis. Clin Exp Metastasis 15:140, 1997 36. Su H, Chang Jc; Xu SM, et al: Selective killing of AFP-positive hepatocellular carcinoma cells by adeno-associated virus transfer of the herpes simplex virus thymidine kinase gene. Hum Gene Ther 7:463, 1996
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37. Tanaka T, Kanai F, Lan K, et al: Adenovirus-mediated gene therapy of gastric carcinoma using cancer specific gene expression in vivo. Biochem Biophys Res Commun 231:775, 1997 38. Tanaka T, Kanai F, Okabe S, et al: Adenovirus-mediated prodrug gene therapy for carcinoembryonic antigen-producing human gastric carcinoma cells in vitro. Cancer Res 56:1341, 1996 39. Tomlinson I, Ilyas M, Novelli M: Molecular genetics of colon cancer. Cancer Metastasis Rev 16:67, 1997 40. Tuting T, Storkus WJ, Lotze MT: Gene-based strategies for the immunotherapy of cancer. Journal of Molecular Medicine 75:478, 1997 41. Weiss L, Grundmann E, Torhost J, et al: Haematogenous metastatic patterns in colonic carcinoma: An analysis of 1541 necropsies. J Pathol 150:195, 1986 42. Xu GW, Sun ZT, Forrester K, et al: Tissue-specific growth suppression and chemosensitivity promotion in human hepatocellular carcinoma cells by retroviral-mediated transfer of the wild-type p53 gene. Hepatology 24:1264, 1996
Address reprint requests to Kenneth K. Tanabe, MD Division of Surgical Oncology, Cox 626 Massachusetts General Hospital Boston, MA 02114