Annals of Surgical Oncology Sign the Guestbook
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Mullen, J. T.
Right arrow Articles by Tanabe, K. K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Mullen, J. T.
Right arrow Articles by Tanabe, K. K.
Annals of Surgical Oncology 10:596-605 (2003)
© 2003 Society of Surgical Oncology

NEW APPROACHES TO THE TREATMENT OF HEPATIC MALIGNANCIES

Viral Oncolysis for Malignant Liver Tumors

John T. Mullen, MD and Kenneth K. Tanabe, MD

From the Division of Surgical Oncology and Department of Surgery, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts.

Correspondence: Address correspondence and reprint requests to: Kenneth Tanabe, MD, Massachusetts General Hospital, 100 Blossom St., Cox Building 626, Boston, MA 02114; Fax: 617-724-3895.

ABSTRACT

Viral oncolysis represents a unique strategy to exploit the natural process of viral replication to kill tumor cells. Although this concept dates back nearly a century, recent advances in the fields of molecular biology and virology have enabled investigators to genetically engineer viruses with greater potency and tumor specificity. In this article we review the general mechanisms by which oncolytic viruses achieve their antineoplastic efficacy and specificity. We focus on the development of several classes of oncolytic viruses for the treatment of malignant liver tumors, including adenoviruses, vaccinia viruses, and herpes simplex viruses, and discuss the results of clinical trials for these viruses. We also describe results from our laboratory research program, which is focused on developing effective, liver tumor–specific Herpes simplex virus 1 mutants.

Key Words: Oncolysis • Replication-conditional viruses • Gene therapy • Clinical trials • Liver tumors

The earliest published reports of using viruses in the treatment of cancer date back to the beginning of this century, when it was noted that patients with various malignancies experienced spontaneous tumor regression after a rabies vaccination or a bout with a viral illness.1,2 Subsequent animal experiments confirmed that viruses are capable of replicating in and lysing experimental murine tumors (oncolysis), and reports followed in the 1950s that demonstrated potent oncolysis of murine tumors by Newcastle disease virus and influenza virus.3 Studies of oncolytic viruses in human cancer patients were initiated around 1950. Perhaps the most recognized of these studies was one performed at the National Cancer Institute in 1956, in which wild-type adenoviruses of different serotypes were injected into patients with cervical carcinomas.4 Many of the patients treated with live virus exhibited tumor regression without evidence of toxicity, followed by tumor progression. This apparent lack of antitumoral efficacy was mirrored in the other human trials of that day,5–7 thereby leading investigators to abandon this mode of therapy. Advances in tumor biology, genetics, and virology in recent decades have provided investigators with the tools necessary to further develop oncolytic viruses for cancer therapy. Viruses that have been examined for their oncolytic potential include adenovirus,8 Herpes simplex virus (HSV),9 reovirus,10 Newcastle disease virus,11 and vaccinia virus.12 In this article, we review the general mechanisms by which oncolytic viruses achieve their antineoplastic efficacy and specificity. In addition, we will focus special attention on the development of several oncolytic viruses for the treatment of malignant liver tumors and track their progress in both preclinical and clinical trials.

MECHANISMS OF ANTITUMORAL EFFICACY
Oncolytic viruses can mediate the destruction of tumor cells by several mechanisms (Table 1, adapted from Kirn13). Viruses directly destroy cells as a result of viral replication, with progeny virion infecting adjacent cells and destroying them by a similar process. Viral replication results in amplification of the original "input dose," which continues until abrogated by an immune response or a lack of susceptible cells. As a second mechanism, some viruses express proteins during their replicative cycle that are cytotoxic to tumor cells. Adenoviruses, for example, express the E3 11.6-kDa death protein and the E4ORF4 protein late in the cell cycle, and both of these proteins are cytotoxic to cells.


View this table:
[in this window]
[in a new window]
  		TABLE 1. Mechanisms of antitumoral efficacy of oncolytic viruses
 
A third mechanism by which oncolytic viruses mediate tumor cell destruction is via the induction of nonspecific and specific antitumor immunity. Tumor cells are inherently weakly immunogenic, in part because they exhibit decreased expression of major histocompatibility complex antigens and stimulatory signals, including cytokines that activate local immune responses. After viral infection of a tumor cell, there is infiltration of lymphocytes and antigen-presenting cells, as well as induction of cytokine release. Infection of tumor cells by a replicating adenovirus, with expression of the E1A protein, leads to increased sensitivity to tumor necrosis factor–mediated killing.14 Also, viral antigens are presented on the cell surface in conjunction with the major histocompatibility complex class I protein, a complex that is then recognized by cytotoxic T lymphocytes that are attracted to the virally transduced tumor. By a mechanism as yet undefined, these cytotoxic T lymphocytes acquire specificity for tumor-specific antigens in the process of viral antigen recognition and cell killing. A systemic, specific antitumor immune response is thereby established in some animal models, as demonstrated with an HSV-1 mutant.15

Oncolytic viruses can sensitize tumor cells to chemotherapy and radiotherapy. For example, the adenovirus E1A gene product is a potent chemosensitizer, particularly in cells with functional p53.16 The E1A gene product induces high levels of p53 in these cells and renders them susceptible to DNA damage from chemotherapy and radiation, whereas normal, nontransformed cells seem to be unaffected by E1A.17

Another mechanism by which oncolytic viruses mediate antineoplastic activity is by expression of therapeutic transgenes inserted into the viral genome. As the virus amplifies itself through successive rounds of replication and infection of neighboring cells, there is a concomitant amplification in transgene expression, which produces an amplified antitumor effect.18 Some researchers have incorporated prodrug-converting enzymes such as viral thymidine kinase (TK) and bacterial cytosine deaminase into replication-conditional adenoviruses to augment tumor cell killing via the "bystander effect."19–21 Other groups have introduced genes that encode biological response modifiers such as interleukins 4 and 12 into oncolytic HSVs in an attempt to augment the antitumor immune response of the host.22,23

MECHANISMS OF ANTITUMORAL SPECIFICITY

Current chemotherapy and radiotherapy are limited by tumor cell resistance to these agents and a relatively narrow therapeutic index. The extent to which dose escalation or combination therapies can be used to overcome resistance or increase tumor cell kill are limited by toxicity to normal tissues. The intent of oncolytic viral therapy is to increase the therapeutic index by restricting viral replication to tumor cells. With each round of viral replication, the viral titer in tumor tissue increases exponentially.

Two general mechanisms have been used to achieve tumor-selective viral replication: (1) deletion of viral genes that are dispensable on infection of neoplastic cells but are critical for viral replication in nonneoplastic cells (Table 2) and (2) placement of tumor-specific promoters upstream of viral genes that are critical for efficient viral replication. In the first case, one takes advantage of molecular events unique to cancer cells, such as the loss of cell-cycle control by mutations of the tumor suppressor proteins p53 or pRb. One example of this strategy is the oncolytic adenovirus ONYX-015 (formerly dl1520 and now named Ci-1042), which is an attenuated adenovirus with two mutations in the E1B gene that encodes a 55 kDa protein.8 When adenovirus infects a normal cell, p53 levels are upregulated and the cell undergoes either cell-cycle arrest or apoptosis, thereby preventing viral replication. Wild-type adenoviruses evade this "cellular suicide" by expressing the E1B 55-kDa protein, which binds to and inactivates p53 and thus allows viral replication to proceed. ONYX-015 lacks the gene for this protein, so its replication is markedly attenuated in cells with normal p53 function. However, many tumor cell types lack functional p53, and ONYX-015 replicates within and lyses these cells preferentially.8 Although it is presumably the loss of p53 function that accounts for the tumor-selective replication of ONYX-015, several researchers have demonstrated that ONYX-015 efficiently replicates in tumor cells with wild-type p53.24,25 This apparent contradiction was resolved with the identification of p14ARF, a tumor-suppressor gene whose product functionally stabilizes p53.26 Loss of p14ARF permits ONYX-015 replication in tumor cells that retain wild-type p53.27 ONYX-015 seems to replicate in tumors with mutations within the p53 pathway as a whole.


View this table:
[in this window]
[in a new window]
  		TABLE 2. Viral proteins necessary for viral replication and complementing/interacting cellular proteins
 
The second strategy commonly used to achieve tumor-selective viral replication involves regulation of viral genes that are necessary for effective replication, by using tumor-specific or tissue-specific promoters. Rodriguez et al.28 demonstrated this principle by developing an oncolytic adenoviral mutant in which the E1A gene, the expression of which is critical for viral replication, is under the control of the tumor-specific prostate-specific antigen gene promoter-enhancer element. This mutant, CV706 (formerly CN706; Calydon, Inc., Sunnyvale, CA), replicates preferentially in tissues with high-level prostate-specific antigen expression and is currently in a phase I/II trial of intraprostatic injection in patients with locally recurrent prostate carcinoma after definitive radiotherapy.

ONCOLYTIC VIRUSES IN PRECLINICAL AND CLINICAL STUDIES FOR THE TREATMENT OF MALIGNANT LIVER TUMORS

Adenovirus
As mentioned in the introduction to this article, there are two general approaches to achieving tumor-selective viral replication, and both of these approaches have been examined by using mutant adenoviruses to treat malignant liver tumors. The first strategy is to delete gene functions that are critical for viral replication in normal cells but not in tumor cells (Table 2). In the process of viral replication, adenovirus expresses the E1A and E1B gene products, which inactivate p53 and pRB. However, tumor cells very frequently express mutant, nonfunctional forms of pRB and p53, and therefore the E1A and E1B gene products become dispensable in these tumor cells. E1A- or E1B-defective adenoviruses can still replicate in pRb- or p53-defective cells, respectively. In contrast, replication of an E1A- or E1B-defective adenovirus is markedly attenuated in cells with intact pRb and p53 pathways. As described previously, ONYX-015 contains a deletion of the E1Bgene that encodes a 55 kDa protein, and this mutant virus exhibits marked cytopathic effects in cancer cells with a disrupted p53 pathway but only limited cytotoxicity in normal human fibroblasts and endothelial cells with normal p53 pathway function.8 Preclinical studies of ONYX-015 reveal significant tumor regression or growth inhibition associated with viral replication after treatment of p53 mutant tumors.29,30

ONYX-015 is now under study in clinical trials for the treatment of several p53-deficient malignancies, including hepatic malignancies.30–33 Results of a phase I study in patients with unresectable primary and secondary liver tumors demonstrated the safety of ONYX-015 administered intratumorally, intravenously, or intra-arterially up to a dose of 3 x 1011 plaque-forming units. Results of a phase II study in this group of patients revealed that the combination of ONYX-015 and 5-fluorouracil (5-FU), when infused into the hepatic artery, was well tolerated but did not induce significant liver tumor regression, as judged by serial computed tomographic scan measurements.34 However, in another phase I/II dose-escalation trial of intra-arterial ONYX-015 administration to patients with colorectal carcinoma liver metastases, patients who received the highest doses of ONYX-015 experienced better survival than those patients treated during the dose-escalation phase of the study.35 Hepatic artery infusion of ONYX-015 was well tolerated, with no dose-limiting toxicities, and there was evidence of viral infection and replication within the liver tumors.

Use of tumor-specific or tissue-specific promoters to drive the expression of an adenoviral gene that is critical for efficient viral replication has also been examined by using adenoviruses. By replacing the endogenous viral E1A promoter with a heterologous promoter sequence that is more transcriptionally active in tumor cells, viral replication proceeds preferentially in tumor cells compared with normal cells, which lack the proteins necessary to activate this promoter. Hallenbeck et al.36 constructed an oncolytic adenovirus that replicates preferentially in hepatocellular carcinoma (HCC) by placing the E1A gene under the transcriptional control of the tumor-associated alfa fetoprotein (AFP) promoter. This virus, AvE1a04i, replicates preferentially in human AFP-producing HCC cell lines compared with non–AFP-producing cell lines and primary cultures of normal human lung epithelial and endothelial cells. In addition, administration of AvE1a04i into pre-established HCC tumors growing in the flanks of immunodeficient mice results in a >50% long-term survival rate.

Vaccinia Virus
Vaccinia virus is best known as the first widely used vaccine that resulted in the eradication of smallpox and, as such, has the longest track record of use in humans. Several properties of vaccinia virus render it well suited to gene-therapy applications. First, vaccinia virus exhibits tropism for a wide range of mammalian cell types. In addition, its nearly 200-kilobase (kb) genome allows for the delivery of large transgene sequences. Although vaccinia virus administration elicits a vigorous immune response, this immunogenicity can be exploited to augment host immunity against tumor cells. Enhancement of therapeutic immune responses is the principle behind the development of poxvirus vaccines for colon cancer, some of which are now in clinical trials.37 Investigators have generated vaccinia virus mutants that are replication conditional, such that they destroy human cancer cells as a byproduct of viral replication. The strategy most commonly used involves deletion or insertional inactivation of the vaccinia virus TK gene, which inhibits viral replication in normal, nondividing cells but allows viral replication in cells with large intracellular nucleotide pools, such as tumor cells.38 Another strategy involves deletion of the SPI-1 and SPI-2 genes from the vaccinia viral genome. These genes encode viral serine proteases and are required for robust viral replication. However, tumor cells overexpress homologous proteins, and, therefore, vaccinia virus mutants defective in SPI-1 and SPI-2 replicate preferentially in these tumor cells rather than normal cells.39

Puhlmann et al.12 at the National Cancer Institute have nicely demonstrated, in a series of preclinical studies over the last few years, the promise of TK-deleted vaccinia viruses in the treatment of liver metastases. They have shown that a recombinant TK-deleted vaccinia virus vector targets tumor tissue after systemic delivery in mouse models. Moreover, they have shown that by incorporating "suicide genes" into the TK-deleted vector, they can achieve greater antineoplastic efficacy. Systemic administration of the TK-deleted vaccinia virus expressing the suicide gene purine nucleoside phosphorylase in combination with 6-methylpurine deoxyriboside treatment leads to a 50% cure in a mouse model of hepatic metastases.40 Likewise, systemic administration of a TK-deleted vaccinia virus expressing the gene encoding cytosine deaminase in combination with 5-fluorocytosine (5-FC) leads to tumor-specific gene expression and cure rates of up to 30% in mice with established liver metastases.41 Finally, they have extended these findings to a large-animal (rabbit) model of liver metastases, demonstrating tumor-specific gene delivery with a TK-deleted vaccinia virus vector carrying the reporter gene firefly luciferase.42

Herpes Simplex Virus
HSV-1 is an enveloped, double-stranded DNA virus with a genome size of 152 kb. Several features of this virus make it an attractive virus for gene therapy.43 First, as much as 30 kb of the genome may be replaced by transgenes in replication-defective HSV-1 mutants, allowing for delivery of multiple transgenes and use of heterologous promoters. HSV-1 rarely produces severe medical illness in immune-competent adults. Moreover, antiherpetic agents such as acyclovir are available that provide a safety mechanism to shut off viral replication should systemic toxicity ensue. Finally, HSV-1 does not integrate its genome into the cellular genome, as does the retrovirus, for example, so insertional mutagenesis is not a concern.

Our laboratory is interested in the development of oncolytic HSV-1 mutants for the treatment of hepatic malignancies. We will devote the remainder of this article to our own research efforts to design effective, tumor-specific HSV-1 mutants.

GENETIC ENGINEERING, CHARACTERIZATION, AND ANIMAL-MODEL TESTING OF HSV-1 MUTANTS FOR TREATMENT OF LIVER TUMORS

In initial proof-of-principle experiments, we used the HSV-1 mutant hrR3, which has an Escherichia coli ß-galactosidase gene inserted into the UL39 gene.44,45 This genetic disruption inactivates the gene for the large subunit of HSV-1 ribonucleotide reductase (infected cell protein 6; ICP6) and thereby attenuates pathologic virulence while permitting lytic infection of specific cell types—those that are actively dividing and have high levels of cellular ribonucleotide reductase and nucleotide precursors that can complement the absence of viral ribonucleotide reductase. We have demonstrated that cellular ribonucleotide reductase levels are extremely low in normal liver and hepatocytes and high in liver metastases.46 Accordingly, titers of infectious hrR3 recovered after infection of colon carcinoma cells are three log orders greater than those recovered after infection of hepatocytes.46,47 In contrast, wild-type HSV-1 strains replicate equally well in hepatocytes and colon carcinoma cells. In mice with diffuse liver metastases, hrR3 replicates preferentially in liver metastases and not in normal liver after portal venous injections (via the spleen), as evidenced by lacZ expression only in the metastases and not in normal liver.46,47 However, if mice are subjected to a partial hepatectomy before injection of hrR3, compensatory hepatic regeneration in normal liver increases levels of ribonucleotide reductase and nucleotide precursors, thereby promoting hrR3 replication in normal liver.48 These results provide further evidence that the mechanism by which an ICP6-defective HSV-1 mutant such as hrR3 replicates preferentially in liver metastases rather than normal liver is the ability of actively dividing cells to complement the absence of viral ribonucleotide reductase function.

HSV-1 replication in the diffuse liver metastases in this model is oncolytic. After a single intravascular administration of hrR3 to mice bearing diffuse liver metastases, viral replication is observed only in liver metastases and not in any normal tissues, and this produces substantial antineoplastic effects that result in both prolongation of survival and cure.48 The hrR3-mediated tumor inhibition is equivalent in immunocompetent and nude mice, suggesting that the host immune response is not the primary mechanism of tumor destruction. Administration of hrR3 produces significant antitumor activity in both naïve mice and mice that have been previously vaccinated against HSV-1. The presence of neutralizing antibodies to HSV-1 neither attenuates nor augments the antineoplastic response.48 After these initial proof-of-principle experiments, we have broadened our work with three goals in mind: (1) construction and characterization of HSV-1 mutants whose replication is more selective for neoplastic cells, (2) construction and characterization of HSV-1 mutants that have greater antineoplastic efficacy, and (3) development of a toxicology program in preparation for clinical trials.

Regulation of HSV-1 Replication and Oncolysis
Differential expression of cellular ribonucleotide reductase between liver tumors and normal liver serves to drive replication of HSV-1 mutants that are defective in viral ribonucleotide reductase preferentially in the liver metastases. However, several other populations of normal cells exist, such as those in the bone marrow and gut mucosa, that also have high mitotic activity and can functionally complement the absence of viral ribonucleotide reductase. Theoretically, these cell populations can support robust hrR3 replication, which might result in toxicity. We are unable to detect polymerase chain reaction evidence of HSV-1 in various organs 10 days after either subcutaneous or portal venous inoculation of hrR3, whereas polymerase chain reaction evidence of HSV-1 is identified in virtually every organ after inoculation with wild-type HSV-1. Nonetheless, there remains good rationale to examine strategies to further attenuate HSV-1 replication in nonneoplastic cells. Therefore, we are testing the hypothesis that regulation of HSV-1 gene expression by heterologous promoters can further enhance the selectivity of replication in neoplastic cells.

Chung et al.49 constructed an HSV-1 mutant, designated Myb34.5, in which both endogenous copies of the {gamma}134.5 gene are deleted, and this gene has been reinserted by homologous recombination into the ICP6 locus under the regulation of the B-myb promoter. HSV-1 expression of {gamma}134.5 is required for robust viral replication, because interaction between this viral protein and cellular protein phosphatase 1{alpha} is required to dephosphorylate eIF2{alpha}.50–52 Cells normally phosphorylate eIF2{alpha} in response to viral infection, which leads to a shutdown of protein synthesis required for viral replication. Expression of {gamma}134.5 during HSV-1 infection leads to eIF2{alpha} dephosphorylation, thereby bypassing this cellular defense against viral infection and replication. Accordingly, HSV-1 mutants that are completely defective in {gamma}134.5 expression are significantly attenuated in their ability to generate progeny virion in normal cells. The HSV-1 mutant Myb34.5 is similar to hrR3 in its preferential replication in mitotically active cells by virtue of its defective viral ribonucleotide reductase expression, and, in addition, Myb34.5 replication is further regulated by the B-myb promoter activity. This promoter upregulates gene expression in E2F-deregulated cells and in cycling cells.

We compared the replication of Myb34.5 with that of hrR3 and other HSV-1 mutants both in normal human hepatocytes and in colon carcinoma cells. The added level of viral replication control provided by the B-myb promoter regulation of {gamma}134.5 was readily apparent. Myb34.5 replication in colon carcinoma cells is as robust as that of hrR3, whereas its replication in normal human hepatocytes is significantly more attenuated than that of hrR3.53 Similarly, Myb34.5 replication in mice with diffuse liver metastases after portal venous administration is better restricted to liver metastases compared with hrR3. The median lethal dose of Myb34.5 in mice after intravascular administration is also higher (e.g., less toxic) than that of hrR3. In a model designed for repeated portal venous administrations of virus to mice with diffuse liver metastases, four doses of Myb34.5 markedly prolong survival and cure a subset of mice.

In a separate strategy, we have examined whether tumor-associated promoters (e.g., carcinoembryonic antigen, muc1) can regulate immediate-early viral gene expression in HSV-1 to limit viral replication (and oncolysis) specifically to tumor cells.53a HSV-1 mutants whose lytic replicative cycle is regulated by a tumor-associated promoter have been constructed. Several HSV-1 genes are candidate genes for transcriptional regulation with these promoters. Several HSV-1 mutants have been constructed in which specific tumor-associated promoters regulate the expression of critical HSV-1 genes. Viral gene expression and replication of these mutants, as well as their antineoplastic efficacy, are currently under examination.

Another approach to limiting viral replication to neoplastic cells involves finding nodal points between signal transduction pathways usurped during HSV-1 lytic replication and signal transduction pathways that are central in neoplastic transformation. For example, as discussed previously, HSV-1 expression of {gamma}134.5 is required for robust viral replication, because interaction between this viral protein and cellular protein phosphatase 1{alpha} is required to dephosphorylate eIF2{alpha}. HSV-1 mutants that are completely defective in {gamma}134.5 expression are significantly attenuated in their ability to generate progeny virion in most cells. However, eIF2{alpha} is dephosphorylated in cells with upregulation of the ras signal transduction pathway,10 such that {gamma}134.5 expression may not be required for HSV-1 replication in such cells. Accordingly, HSV-1 mutants that are defective in {gamma}134.5 preferentially replicate in cells with an activated ras pathway. The {gamma}134.5-defective HSV-1 mutants are therefore presently being examined as oncolytic agents for ras-activated tumors. Further studies of interactions between HSV-1 gene function and neoplastic transformation should bring forth alternative strategies for limiting HSV-1 replication to neoplastic cells.

Enhancing the Efficacy of HSV-1 Mutants for Treatment of Liver Tumors
Viral oncolysis has several theoretical advantages compared with chemotherapy or radiotherapy. Nonetheless, viral oncolysis alone may be insufficient to completely eradicate tumors. Consequently, we have also examined several strategies to enhance viral oncolysis. One approach combines viral oncolysis with suicide gene therapy. Transgenes that encode proteins which convert metabolically inactive prodrugs into chemotherapeutically active metabolites can be incorporated into the HSV-1 genome. For example, the yeast cytosine deaminase gene product efficiently converts 5-FC to 5-FU, which is one of the most active chemotherapy agents used against gastrointestinal malignancies. We have inserted this gene into the UL39 locus of HSV-1 in a mutant designated HSV1yCD, thereby enabling HSV1yCD-infected cells to convert 5-FC to 5-FU.19 The combination of HSV-1 replication and intratumoral conversion of 5-FC to 5-FU is more effective than either modality alone in treatment of mice with diffuse liver metastases.

There are several advantages of a strategy that combines HSV-1 lytic replication in tumor cells together with intratumoral prodrug activation. First, the combined effects of two different mechanisms of tumor cell destruction are more effective than either mechanism alone. Second, the antitumor activity observed in prodrug activation models has been partially attributed to "bystander killing," a term that refers to the death of untransduced tumor cells that are adjacent to transduced tumor cells.54–57 The importance of bystander killing lies in the realization that it is unlikely that any gene delivery vehicle will be able to transduce 100% of tumor cells, whereas bystander killing may result in complete destruction of a tumor despite transduction of only a fraction of tumor cells. A third advantage of combined-modality treatment by using therapies with different mechanisms of resistance is the dramatically reduced risk of emergence of resistant clones of tumor cells. Treatment strategies that rely solely on in vivo activation of a single prodrug for treatment of solid tumors would very likely fail because of the emergence of drug-resistant clones. Even the incorporation of a second prodrug activation gene to allow in vivo activation of two prodrugs runs the significant risk of failure due to drug-resistant tumor clones. In support of this notion, patients with solid tumor metastases are presently treated with multiple-agent chemotherapy regimens but are never cured, because of the eventual emergence of drug-resistant tumor cells.58,59 The long history of failures with numerous polychemotherapy regimens in an attempt to cure patients with solid tumor metastases argues strongly that strategies that rely solely on the activation of a single prodrug or even multiple prodrugs are doomed to failure because of the emergence of drug-resistant clones.59 In contrast, a strategy that combines two completely different mechanisms of antitumor activity, such as intratumoral prodrug activation and HSV-1–mediated lytic replication, may reduce the risk of tumor cell resistance. Last but not least, transgene expression is better distributed throughout a tumor after direct intratumoral inoculation of a replication-conditional HSV-1 compared with a replication-defective HSV-1.18

The genetically engineered, replication-conditional HSV-1 mutant rRp450 is engineered such that it is defective in expression of the large subunit of viral ribonucleotide reductase and expresses the rat cytochrome P450 2B1 transgene. This gene can be used as a suicide gene because it encodes an enzyme responsible for the bioactivation of prodrugs including cyclophosphamide, ifosfamide, and procarbazine, thus providing a means for potential intratumoral generation of alkylating metabolites.60,61 Treatment of mice harboring diffuse HCC with the combination of rRp450 and systemic cyclophosphamide leads to marked tumor reduction and prolongation of survival.62

We have examined interactions between prodrug bioactivation and HSV-1 replication by using different prodrugs. Specific transgenes that encode enzymes responsible for chemotherapeutic prodrug bioactivation may attenuate viral replication, thereby reducing the efficacy of HSV-1–mediated oncolysis. Other transgenes achieve their intended antineoplastic effects without inhibiting viral replication. For example, yeast cytosine deaminase bioactivation of 5-FC to 5-FU enhances antineoplastic efficacy without significantly attenuating HSV-1 replication,19 and cytochrome P450 2B1–mediated bioactivation of cyclophosphamide does not significantly impair HSV-1 replication but, rather, enhances the overall antineoplastic efficacy.62 In contrast, HSV-1 TK bioactivation of ganciclovir significantly impairs viral replication.19,62 Similarly, expression of murine endostatin adds to the antineoplastic efficacy of HSV-1–mediated oncolysis without simultaneously impairing viral replication (K.K.T., unpublished data, 2001). Understanding this mechanism of interaction between cellular responses to bioactivated prodrugs and viral replication is critical for both clinical trial design and vector design.

Preclinical Toxicology in Nonhuman Primates of HSV-1 Mutants for Liver Tumor Therapy
A key requirement of an oncolytic HSV-1 vector is that it should be safe when administered intravascularly to humans. The necessary preclinical safety testing requires animal models; however, no animal species accurately models the diversity and severity of herpetic diseases in humans. In addition, significant differences exist between species in their susceptibility to HSV-1 infection. Mice, guinea pigs, and rabbits can be infected with HSV-1; however, these species are not suitable species for HSV-1 safety testing. After administration of HSV-1 to these animals, they usually succumb to paralysis and death caused by neurotropic spread of the virus to the central nervous system. This pathogenesis is quite distinct from that observed in humans. In addition, recurrent lesions arising from virus reactivation are uncommon in mice and rabbits. Guinea pigs exhibit spontaneous reactivation after genital inoculation; however, unlike human lesions, these recurrent lesions are frequently sterile. The value of this animal model lies more in its utility for the study of genital infections.63–66

Among old-world primates, the gibbon is susceptible to experimental infection with HSV-166,67; however, significantly more experimental data have been published on experimental and spontaneous HSV-1 infections of a new-world primate, Aotus monkeys (A. trivirgatus and A. nancymae). Of all of the models described in the literature, only Aotus monkeys exposed to HSV-1 by natural routes of infection exhibit features resembling those in human infections, including latency and reactivation.68–71 The owl monkey serves as an excellent model for studies of several viruses and presently serves as the most accepted nonhuman primate species for examination of HSV-1 infection.69,71,72 We are presently involved in the production of clinical-grade rRp450 and Myb34.5 for preclinical toxicology studies in Aotus monkeys before clinical trials in patients with liver tumors.

NV1020 (formerly R7020; MediGene, Inc., San Diego, CA) is another genetically engineered oncolytic HSV-1 that is being actively studied in clinical trials. This virus is now in a phase I trial to evaluate its safety as an oncolytic virus for the treatment of patients with colorectal carcinoma liver metastases. NV1020 was originally designed as a candidate for human immunization against infections with HSV-1 and HSV-2.73 It contains a 700-bp deletion in the endogenous HSV-1 TK gene, as well as a 15-kb deletion across the joint region of the long and short components of the HSV-1 genome. The long/short junction of NV1020 contains a 5.2-kb fragment of HSV-2 DNA inserted for previous vaccine studies and an exogenous copy of the HSV-1 TK gene under the control of the powerful HSV-1 {alpha}4 promoter. Thus, NV1020 has only one copy of {gamma}134.5 deleted and maintains sensitivity to acyclovir and ganciclovir. The virus has demonstrated genetic stability and safety in extensive rodent and primate studies, as well as in limited human vaccine trials.69,73 NV1020 treatment of rodents with pancreatic carcinoma,74 transitional cell carcinoma,75 hormone-resistant prostate carcinoma,76 and chemotherapy-/radiotherapy-resistant epidermoid carcinoma77 caused rapid regression of the flank tumor xenografts. Tumor destruction was accelerated with the addition of ionizing radiation to therapy with NV1020.78 On the basis of these encouraging preclinical results, a phase I trial of intrahepatic arterial injection of NV1020 in patients with colorectal carcinoma liver metastases is currently under way.

CONCLUSIONS

Viral oncolysis represents a unique strategy to exploit the natural process of viral replication to destroy tumors. Progress in the fields of virology and molecular biology have enabled investigators to pursue strategies to genetically engineer viruses to augment their replication in tumor cells and to attenuate their replication in normal cells. Although it is too early to predict the ultimate clinical utility of this strategy, early results confirm the potential of viral oncolysis to effectively treat cancer.

ACKNOWLEDGMENTS

Supported by National Institutes of Health Grants CA71345 (JTM) and CA76183 (KKT) and the Claude E. Welch Research Fellowship (JTM).

The acknowledgments are available online at www.annalssurgicaloncology.org.

FOOTNOTES

This article reviews the general mechanisms by which oncolytic viruses achieve their antineoplastic efficacy and specificity, results of preclinical studies of viral oncolysis, and results of clinical trials.

Received for publication July 18, 2002. Accepted for publication November 24, 2002.

REFERENCES

  1. Dock G. Rabies virus vaccination in a patient with cervical carcinoma. Am J Med Sci 1904; 127: 563.
  2. DePace N. Sulla scomparsa di un enorme cancro vegetante del collo dell’utero senza cura chirurgica. Ginecologia 1912; 9: 82–9.
  3. Sinkovics J, Horvath J. New developments in the virus therapy of cancer: a historical review. Intervirology 1993; 36: 193–214.[Medline]
  4. Smith R, Huebner RJ, Rowe WP, Schatten WE, Thomas LB. Studies on the use of viruses in the treatment of carcinoma of the cervix. Cancer 1956; 9: 1211–8.[CrossRef][Medline]
  5. Newman W, Southam CM. Virus treatment in advanced cancer. Cancer 1954; 7: 106–18.
  6. Southam CM, Hilleman MR, Werner JH. Pathogenicity and oncolytic capacity of RI virus strain RI-67 in man. Trans N Y Acad Sci 1960; 22: 657–73.
  7. Cassel WA, Garrett RE. Newcastle disease virus as an antineoplastic agent. Cancer 1965; 18: 863–8.[CrossRef][Medline]
  8. Bischoff JR, Kirn DH, Williams A, et al. An adenovirus mutant that replicates selectively in p53-deficient human tumor cells. Science 1996; 274: 373–6.[Abstract/Free Full Text]
  9. Martuza RL, Malick A, Markert JM, Ruffner KL, Coen DM. Experimental therapy of human glioma by means of a genetically engineered virus mutant. Science 1991; 252: 854–6.[Abstract/Free Full Text]
  10. Coffey MC, Strong JE, Forsyth PA, Lee PWK. Reovirus therapy of tumors with activated ras pathway. Science 1998; 282: 1332–4.[Abstract/Free Full Text]
  11. Lorence RM, Reichard KW, Katubig BB, et al. Complete regression of human neuroblastoma xenografts in athymic mice after local Newcastle disease virus therapy. J Natl Cancer Inst 1994; 86: 1228–33.[Abstract/Free Full Text]
  12. Puhlmann M, Brown CK, Gnant M, et al. Vaccinia as a vector for tumor-directed gene therapy: biodistribution of a thymidine kinase-deleted mutant. Cancer Gene Ther 2000; 7: 66–73.[CrossRef][Medline]
  13. Kirn DH. Replicating oncolytic viruses: an overview. Exp Opin Invest Drugs 1996; 5: 753–62.
  14. Gooding LR. Regulation of TNF-mediated cell death and inflammation by human adenoviruses. Infect Agents Dis 1994; 3: 106–15.[Medline]
  15. Toda M, Rabkin SD, Kojima H, Martuza RL. Herpes simplex virus as an in situ cancer vaccine for the induction of specific anti-tumor immunity. Hum Gene Ther 1999; 10: 385–93.[CrossRef][Medline]
  16. Lowe SW, Bodis S, McClatchey A, et al. p53 status and the efficacy of cancer therapy in vivo. Science 1994; 266: 807–10.[Abstract/Free Full Text]
  17. Ganly I, Eckhardt SG, Rodriguez GI, et al. A phase I study of Onyx-015, an E1B attenuated adenovirus, administered intratumorally to patients with recurrent head and neck cancer. Clin Cancer Res 2000; 6: 798–806.[Abstract/Free Full Text]
  18. Ichikawa T, Chiocca EA. Comparative analyses of transgene delivery and expression in tumors inoculated with a replication-conditional or -defective viral vector. Cancer Res 2001; 61: 5336–9.[Abstract/Free Full Text]
  19. Nakamura H, Mullen JT, Chandrasekhar S, Pawlik TM, Yoon SS, Tanabe KK. Multimodality therapy with a replication-conditional herpes simplex virus 1 mutant that expresses yeast cytosine deaminase for intratumoral conversion of 5-fluorocytosine to 5-fluorouracil. Cancer Res 2001; 61: 5447–52.[Abstract/Free Full Text]
  20. Wildner O, Blaese RM, Morris JC. Therapy of colon cancer with oncolytic adenovirus is enhanced by the addition of herpes simplex virus-thymidine kinase. Cancer Res 1999; 59: 410–3.[Abstract/Free Full Text]
  21. Freytag SO, Rogulski KR, Paielli DL, Gilbert JD, Kim JH. A novel three-pronged approach to kill cancer cells selectively: concomitant viral, double suicide gene, and radiotherapy. Hum Gene Ther 1998; 9: 1323–33.[Medline]
  22. Andreansky S, He B, van Cott J, et al. Treatment of intracranial gliomas in immunocompetent mice using herpes simplex viruses that express murine interleukins. Gene Ther 1998; 5: 121–30.[CrossRef][Medline]
  23. Parker JN, Gillespie GY, Love CE, Randall S, Whitley RJ, Markert JM. Engineered herpes simplex virus expressing IL-12 in the treatment of experimental murine brain tumors. Proc Natl Acad Sci U S A 2000; 97: 2208–13.[Abstract/Free Full Text]
  24. Goodrum FD, Ornelles DA. p53 status does not determine outcome of E1B 55-kilodalton mutant adenovirus lytic infection. J Virol 1998; 72: 9479–90.[Abstract/Free Full Text]
  25. Rothmann T, Hengstermann A, Whitaker NJ, Scheffner M, zur Hausen H. Replication of Onyx-015, a potential anticancer adenovirus, is independent of p53 status in tumor cells. J Virol 1998; 72: 9470–8.[Abstract/Free Full Text]
  26. Quelle DE, Zindy F, Ashmun RA, Sherr CJ. Alternative reading frames of the INK4a tumor suppressor gene encode two unrelated proteins capable of inducing cell cycle arrest. Cell 1995; 83: 993–1000.[CrossRef][Medline]
  27. Ries SJ, Brandts CH, Chung AS, et al. Loss of p14ARF in tumor cells facilitates replication of the adenovirus mutant dl1520 (ONYX-015). Nat Med 2000; 6: 1128–33.[CrossRef][Medline]
  28. Rodriguez R, Schuur ER, Lim HY, Henderson GA, Simons JW, Henderson DR. Prostate attenuated replication competent adenovirus (ARCA) CN706: a selective cytotoxic for prostate-specific antigen-positive prostate cancer cells. Cancer Res 1997; 57: 2559–63.[Abstract/Free Full Text]
  29. Heise CC, Williams AM, Xue S, Propst M, Kirn DH. Intravenous administration of ONYX-015, a selectively replicating adenovirus, induces antitumoral efficacy. Cancer Res 1999; 59: 2623–8.[Abstract/Free Full Text]
  30. Heise C, Ganly I, Kim YT, Sampson-Johannes A, Brown R, Kirn D. Efficacy of a replication-selective adenovirus against ovarian carcinomatosis is dependent on tumor burden, viral replication and p53 status. Gene Ther 2000; 7: 1925–9.[CrossRef][Medline]
  31. Mulvihill S, Warren R, Venook A, et al. Safety and feasibility of injection with an E1B-55 kDa gene-deleted replication-selective adenovirus (ONYX-015) into primary carcinomas of the pancreas: a phase I trial. Gene Ther 2001; 8: 308–15.[CrossRef][Medline]
  32. Lamont JP, Nemunaitis J, Kuhn JA, Landers SA, McCarty TM. A prospective phase II trial of ONYX-015 adenovirus and chemotherapy in recurrent squamous cell carcinoma of the head and neck (the Baylor experience). Ann Surg Oncol 2000; 7: 588–92.[Abstract]
  33. Nemunaitis J, Khuri F, Ganly I, et al. Phase II trial of intratumoral administration of ONYX-015, a replication-selective adenovirus, in patients with refractory head and neck cancer. J Clin Oncol 2001; 19: 289–98.[Abstract/Free Full Text]
  34. Habib NA, Sarraf CE, Mitry RR, et al. E1B-deleted adenovirus (dl 1520) gene therapy for patients with primary and secondary liver tumors. Hum Gene Ther 2001; 12: 219–26.[CrossRef][Medline]
  35. Reid TR, Galanis E, Abbruzzese J, et al. Intra-arterial administration of a replication-selective adenovirus Ci-1042 (Onyx-015) in patients with colorectal carcinoma metastatic to the liver: safety, feasibility and biological activity (abstract). Proc Am Soc Clin Oncol 20; 549a: 2001.
  36. Hallenbeck PL, Chang YN, Hay C, et al. A novel tumor-specific replication-restricted adenoviral vector for gene therapy of hepatocellular carcinoma. Hum Gene Ther 1999; 1: 1721–33.
  37. Schlom J, Tsang KY, Kantor JA, et al. Strategies in the development of recombinant vaccines for colon cancer. Semin Oncol 1999; 26: 672–82.[Medline]
  38. Buller RM, Smith GL, Cremer K, Notkins AL, Moss B. Decreased virulence of recombinant vaccinia virus expression vectors is associated with a thymidine kinase-negative phenotype. Nature 1985; 317: 813–5.[CrossRef][Medline]
  39. Naik AM, Xu H, Alexander HR, Bartlett DL. A mutant vaccinia virus with improved tumor selectivity. 54th Annual Society of Surgical Oncology Cancer Symposium 2001: 40a.
  40. Puhlmann M, Gnant M, Brown CK, Alexander HR, Bartlett DL. Thymidine kinase-deleted vaccinia virus expressing purine nucleoside phosphorylase as a vector for tumor-directed gene therapy. Hum Gene Ther 1999; 10: 649–57.[CrossRef][Medline]
  41. Gnant MFX, Puhlmann M, Alexander HR, Bartlett DL. Systemic administration of a recombinant vaccinia virus expressing the cytosine deaminase gene and subsequent treatment with 5-fluorocytosine leads to tumor-specific gene expression and prolongation of survival in mice. Cancer Res 1999; 59: 3396–403.[Abstract/Free Full Text]
  42. Gnant MFX, Noll LA, Irvine KR, et al. Tumor-specific gene delivery using recombinant vaccinia virus in a rabbit model of liver metastases. J Natl Cancer Inst 1999; 91: 1744–50.[Abstract/Free Full Text]
  43. Martuza RL. Conditionally replicating herpes vectors for cancer therapy. J Clin Invest 2000; 105: 841–6.[Medline]
  44. Goldstein DJ, Weller SK. Factor(s) present in herpes simplex virus type 1 infected cells can compensate for the loss of the large unit of the viral ribonucleotide reductase: characterization of an ICP6 deletion mutant. Virology 1988; 166: 41–51.[CrossRef][Medline]
  45. Goldstein DJ, Weller SK. Herpes simplex virus type 1-induced ribonucleotide reductase activity is dispensable for virus growth and DNA synthesis: isolation and characterization of an ICP6 lacZ insertion mutant. J Virol 1988; 62: 196–205.[Abstract/Free Full Text]
  46. Carroll NM, Chiocca EA, Takahashi K, Tanabe KK. Enhancement of gene therapy specificity for diffuse colon carcinoma liver metastases with recombinant herpes simplex virus. Ann Surg 1996; 224: 323–30.[CrossRef][Medline]
  47. Yoon SS, Carroll NM, Chiocca EA, Tanabe KK. Cancer gene therapy using replication-competent herpes simplex virus type 1. Ann Surg 1998; 228: 366–74.[CrossRef][Medline]
  48. Yoon SS, Nakamura H, Carroll NM, Bode BP, Chiocca EA, Tanabe KK. An oncolytic herpes simplex virus type 1 selectively destroys diffuse liver metastases from colon carcinoma. FASEB J 2000; 14: 301–11.[Abstract/Free Full Text]
  49. Chung RY, Saeki Y, Chiocca EA. B-myb promoter retargeting of herpes simplex virus gamma 34.5 gene-mediated virulence toward tumor and cycling cells. J Virol 1999; 73: 7556–64.[Abstract/Free Full Text]
  50. Chou J, Kern ER, Whitley RJ, Roizman B. Mapping of herpes simplex virus-1 neurovirulence to gamma 134.5, a gene nonessential for growth in culture. Science 1990; 250: 1262–6.[Abstract/Free Full Text]
  51. Chou J, Roizman B. The gamma 1(34.5) gene of herpes simplex virus 1 precludes neuroblastoma cells from triggering total shutoff of protein synthesis characteristic of programed cell death in neuronal cells. Proc Natl Acad Sci U S A 1992; 89: 3266–70.[Abstract/Free Full Text]
  52. He B, Gross M, Roizman B. The gamma(1)34.5 protein of herpes simplex virus 1 complexes with protein phosphatase 1alpha to dephosphorylate the alpha subunit of the eukaryotic translation initiation factor 2 and preclude the shutoff of protein synthesis by double-stranded RNA-activated protein kinase. Proc Natl Acad Sci U S A 1997; 94: 843–8.[Abstract/Free Full Text]
  53. Nakamura H, Kasuya H, Mullen JT, et al. Regulation of herpes simplex virus {gamma}134.5 expression and oncolysis of diffuse liver metastases by Myb34.5. J Clin Invest 2002; 109: 871–82.[CrossRef][Medline]
  54. Mullen JT, Kasuya H, Yoon SS, et al. Regulation of herpes simplex virus 1 replication using tumor-associated promoters. Ann Surg 2002; 236: 502–12.[CrossRef][Medline]
  55. 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 1993; 53: 5274–83.[Abstract/Free Full Text]
  56. Chen CY, Chang YN, Ryan P, Linscott M, McGarrity GJ, Chiang YL. Effect of herpes simplex virus thymidine kinase expression levels on ganciclovir-mediated cytotoxicity and the "bystander effect". Hum Gene Ther 1995; 6: 1467–76.[Medline]
  57. Bi WL, Parysek LM, Warnick R, Stambrook PJ. In vitro evidence that metabolic cooperation is responsible for the bystander effect observed with HSV tk retroviral gene therapy. Hum Gene Ther 1993; 4: 725–31.[Medline]
  58. Colombo BM, Benedetti S, Ottolenghi S, et al. The "bystander effect": association of U87 cell death with ganciclovir-mediated apoptosis of nearby cells and the lack of effect in athymic mice. Hum Gene Ther 1995; 6: 763–72.[Medline]
  59. Kaufman D, Chabner BA. Clinical strategies for cancer treatment: the role of drugs. In: Chabner BA, Longo DL, eds. Cancer Chemotherapy and Biotherapy. Philadelphia: Lippincott-Raven, 1996: 1–16.
  60. DeVita VT. Principles of chemotherapy. In: DeVita VTJ, Hellman S, Rosenberg SA, eds. Cancer: Principles and Practice of Oncology. Philadelphia: JB Lippincott Co, 1993: 276–92.
  61. Chase M, Chung R, Chiocca EA. An oncolytic viral mutant that delivers the CYP2B1 transgene and augments cyclophosphamide chemotherapy. Nat Biotech 1998; 16: 444–8.[CrossRef][Medline]
  62. Clarke L, Waxman DJ. Oxidative metabolism of cyclophosphamide: identification of the hepatic monooxygenase catalysts of drug activation. Cancer Res 1989; 49: 2344–50.[Abstract/Free Full Text]
  63. Pawlik TM, Nakamura H, Yoon SS, et al. Oncolysis of diffuse hepatocellular carcinoma by intravascular administration of a replication-competent, genetically engineered herpesvirus. Cancer Res 2000; 60: 2790–5.[Abstract/Free Full Text]
  64. Stanberry LR, Kern ER, Richards JT, Abbot TM, Overall JCJ. Genital herpes in guinea pigs: pathogenesis of the primary infection and description of recurrent disease. J Infect Dis 1982; 146: 397–404.[Medline]
  65. Scriba M. Herpes simplex virus infection in guinea pigs: an animal model for studying latent and recurrent herpes simplex virus infection. Infect Immun 1975; 12: 162–5.[Abstract/Free Full Text]
  66. Hsiung GD, Mayo DR, Lucia HL, Landry ML. Genital herpes: pathogenesis and chemotherapy in the guinea pig model. Rev Infect Dis 1984; 6: 33–50.[Medline]
  67. Emmons RW, Lennette WH. Natural herpesvirus hominis infection of a gibbon (Hylobates lar). Arch Gesamte Virusforsch 1970; 31: 215–8.[CrossRef][Medline]
  68. Smith PC, Yuill TM, Buchanan RD, Stanton JS, Chaicumpa V. The gibbon (Hylobates lar): a new primate host for Herpesvirus hominis. I. A natural epizootic in a laboratory colony. J Infect Dis 1969; 120: 292–7.[Medline]
  69. Melendez LV, Espana C, Hunt RD, Daniel MD, Garcia FG. Natural herpes simplex infection in the owl monkey (Aotus trivirgatus). Lab Anim Care 1969; 19: 38–45.[Medline]
  70. Meignier B, Martin B, Whitley RJ, Roizman B. In vivo behavior of genetically engineered herpes simplex viruses R7017 and R7020. II. Studies in immunocompetent and immunosuppressed owl monkeys (Aotus trivirgatus). J Infect Dis 1990; 162: 313–21.[Medline]
  71. Katzin DS, Connor JD, Wilson LA, Sexton RS. Experimental herpes simplex infection in the owl monkey. Proc Soc Exp Biol Med 1967; 125: 391–8.[Medline]
  72. Hunter WD, Martuza RL, Feigenbaum F, et al. Attenuated, replication-competent herpes simplex virus type 1 mutant G207: safety evaluation of intracerebral injection in nonhuman primates. J Virol 1999; 73: 6319–26.[Abstract/Free Full Text]
  73. Barahona H, Melendez LV, Hunt RD, Daniel MD. The owl monkey (Aotus trivirgatus) as an animal model for viral diseases and oncologic studies. Lab Anim Sci 1976; 26: 1104–12.[Medline]
  74. Meignier B, Longnecker R, Roizman B. In vivo behavior of genetically engineered herpes simplex viruses R7017 and R7020: construction and evaluation in rodents. J Infect Dis 1988; 158: 602–14.[Medline]
  75. McAuliffe PF, Jarnagin WR, Johnson P, Delman KA, Federoff Y. Effective treatment of pancreatic tumors with two multimutated herpes simplex oncolytic viruses. J Gastrointest Surg 2000; 4: 580–8.[CrossRef][Medline]
  76. Cozzi PJ, Malhotra S, McAuliffe P, et al. 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 2001; 15: 1306–8.[Abstract/Free Full Text]
  77. Ghosh J, Myers CE. Inhibition of arachidonate 5-lipoxygenase triggers massive apoptosis in human prostate cancer cells. Proc Natl Acad Sci U S A 1998; 95: 13182–7.[Abstract/Free Full Text]
  78. Advani SJ, Chung SM, Yan SY, et al. Replication-competent, nonneuroinvasive genetically engineered herpes virus is highly effective in the treatment of therapy-resistant experimental human tumors. Cancer Res 1999; 59: 2055–8.[Abstract/Free Full Text]
  79. Advani SJ, Sibley GS, Song PY, et al. Enhancement of replication of genetically engineered herpes simplex viruses by ionizing radiation: a new paradigm for destruction of therapeutically intractable tumors. Gene Ther 1998; 5: 160–5.[CrossRef][Medline]



This article has been cited by other articles:


Home page
 Clin Med ResHome page
D. Cross and J. K. Burmester
Gene therapy for cancer treatment: past, present and future.
Clin. Med. Res., September 1, 2006; 4(3): 218 - 227.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Mullen, J. T.
Right arrow Articles by Tanabe, K. K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Mullen, J. T.
Right arrow Articles by Tanabe, K. K.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS