The objective of the study was to evaluate the utility of NV1042, a replication competent, oncolytic herpes simplex virus (HSV) containing the interleukin-12 (IL-12) gene, as primary treatment for hepatic tumors and to further assess its ability to reduce tumor recurrence following resection. Resection is the most effective therapy for hepatic malignancies, but is not possible in the majority of the patients. Furthermore, recurrence is common after resection, most often in the remnant liver and likely because of microscopic residual disease in the setting of postoperative host cellular immune dysfunction. We hypothesize that, unlike other gene transfer approaches, direct injection of liver tumors with replication competent, oncolytic HSV expressing IL-12 will not only provide effective control of the parent tumor, but will also elicit an immune response directed at residual tumor cells, thus decreasing the risk of cancer recurrence after resection. Solitary Morris hepatomas, established in Buffalo rat livers, were injected directly with 107 particles of NV1042, NV1023, an oncolytic HSV identical to NV1042 but without the IL-12 gene, or with saline. Following tumor injection, the parent tumors were resected and measured and the animals were challenged with an intraportal injection of 105 tumor cells, recreating the clinical scenario of residual microscopic cancer. In vitro cytotoxicity against Morris hepatoma cells was similar for both viruses at a multiplicity of infection of 1 (MOI, ratio of viral particles to target cells), with >90% tumor cell kill by day 6. NV1042 induced high-level expression of IL-12 in vitro, peaking after 4 days in culture. Furthermore, a single intratumoral injection of NV1042, but not NV1023, induced marked IL-12 and interferon-γ (IFN-γ) expression. Both viruses induced a significant local immune response as evidenced by an increase in the number of intratumoral CD4(+) and CD8(+) lymphocytes, although the peak of CD8(+) infiltration was later with NV1042 compared with NV1023. NV1042 and NV1023 reduced parent tumor volume by 74% (P<.003) and 52% (P<.03), respectively, compared to control animals. Treatment of established tumors with NV1042, but not with NV1023, significantly reduced the number of hepatic tumors after resection of the parent tumor and rechallenge (16.8±11 (median=4) vs. 65.9±15 (median=66) in control animals, P<.025). In conclusion, oncolytic HSV therapy combined with local immune stimulation with IL-12 offers effective control of parent hepatic tumors and also protects against microscopic residual disease after resection. The ease of use of this combined modality approach, which appears to be superior to either approach alone, suggests that it may have clinical relevance, both as primary treatment for patients with unresectable tumors and also as a neoadjuvant strategy for reducing recurrence after resection.
Primary and secondary hepatic malignancies are responsible for over 1 million annual deaths worldwide.1,2,3,4,5 While hepatic resection offers the possibility of long-term survival in some patients, diffuse hepatic involvement by tumor or severe underlying liver dysfunction precludes resection in the majority.6 Furthermore, even when a complete resection is achieved, recurrent cancer is common. After partial hepatectomy, cancer recurrence is most often seen in the liver remnant, apparently the combined result of progression of residual microscopic cancer cells, undetectable at the time of operation, and postoperative immunosuppression.7 Chemotherapy may reduce the incidence of recurrence in some patients after resection and may provide some measure of disease control in those with unresectable tumors,8,9,10 but the overall results of chemotherapy have been disappointing. New and innovative treatment approaches are therefore necessary if mortality related to these cancers is to be further reduced.
One such novel and potentially widely applicable approach involves stimulation of host immune effector mechanisms10,11 using gene transfer techniques to deliver immunostimulatory cytokines to solid tumors.12,13,14 Production of cytokines in proximity to putative tumor antigens has been shown experimentally to result in antitumor immunity that prevents subsequent development of cancer.12,14,15,16,17,18,19,20,21,22 Recent animal studies from this laboratory have shown that neoadjuvant treatment of established, solitary liver tumors with modified, replication incompetent herpes simplex virus (HSV) encoding the interleukin-12 (IL-12) gene resulted in high levels of gene expression within the tumor and significantly reduced the incidence of tumor recurrence after resection.23 However, this study showed no significant response of the parent tumor, which remains a major limitation of this treatment strategy.
In addition to their efficacy as gene transfer agents, attenuated herpes viruses have potent antitumor activity by direct oncolysis.24,25 Unlike HSV vectors used solely for gene transfer, oncolytic HSV are replication competent although modified by deletion of certain strategic viral growth genes.25,26 The protein products of these genes are abundantly expressed in tumor cells compared to normal cells. As a result, tumor cells, unlike normal cells, are capable of supporting viral replication and are therefore specifically targeted for viral-induced cell lysis. Also, since a large number of progeny virus arise from a small number of initially infected cells, injection of a large number of viral particles can be avoided. Previous studies have shown that oncolytic HSV effectively kills a wide range of human tumor cells, both in vitro and in vivo.24,27,28 Furthermore, after intravenous administration of these agents in animals, viral replication is seen only in neoplastic cells, and there is no evident toxicity to normal tissue.14,15,16,19,24,29,30,31,32
More recent generations of oncolytic HSV have been created that contain a variety of immunostimulatory genes, such as GMCSF and IL-12, while maintaining replication competence and oncolytic activity. Prelim-inary work suggests that combined immunostimulatory and oncolytic therapy enhances tumor cell kill.33 In the present experiments, NV1042, a replication competent, oncolytic HSV expressing IL-12, was used to treat established hepatic tumors prior to resection. IL-12 was chosen because of its broad and potent immunostimulatory activity.31,34,35 The ability of NV1042 to reduce the burden of the parent tumor, as well as its potential as a neoadjuvant agent in reducing tumor recurrence following resection, were analyzed.
Materials and methods
Male Buffalo rats were housed in individual cages in a temperature-(22°C) and humidity-controlled room with a 12-h day/night cycle. Animals had free access to food and water. Animal weights were recorded at the beginning of each experiment and then every 3 days thereafter. All animals received care under approved protocols in compliance with Memorial Sloan-Kettering Cancer Center's Institutional Animal Care and Use Committee guidelines.
Rat hepatoma cells (Morris hepatoma McA-R-7777), a syngeneic hepatoma cell line, were obtained from the American Type Culture Collection (ATCC No. CRL 1601, Rockville, MD, USA) and maintained in Dulbecco's modified Eagle's medium (DMEM) culture media supplemented with 4500 g/l glucose, 100 cm3/l of donor horse serum, 5% fetal calf serum (FCS), and 5 mM L-glutamine. Cells were periodically implanted subcutaneously into rat flanks to ensure tumorigenicity. Portal injection of Morris hepatoma (MH) cells, either by direct venipuncture or by intrasplenic injection, is a well-established model and reliably produces 50–100 hepatic tumors within the liver 3 weeks after injection.7
Two attenuated, replication competent, oncolytic HSV were used in this study, NV1023 and NV1042, obtained from Medigene, Inc. (San Diego, CA, USA). These viruses were derived from NV1020, a nonselected clonal derivative of R7020 obtained from B Roizman.36,37 NV1021 and NV1022 are precursors of NV1023 and NV1042 and derived from NV1020. NV1021 contains the E. coli lacZ gene under control of the ICP47 (US12) promoter. The α4-driven thymidine kinase (TK) gene present in NV1020 and NV1021 was deleted from NV1021 to create the TK negative virus NV1022. NV1023 was created by repair of the endogenous HSV-1TK gene and the UL24 promoter in NV1022. NV1042 was created by insertion of the murine IL-12 cDNA into NV1021 and selection of TK-negative recombinants. The endogenous TK -gene and UL24 promoter were repaired to generate stocks of NV1042. The genetic structures of NV 1023 and NV1042 were confirmed by Southern blot analysis. NV1042 is therefore identical to NV1023 except that the former contains the murine m35 and m40 fragments of IL-12 within the HSV2 segment, separated by an internal ribosome entry site (IRES) that allows intracellular translation of the intact, biologically active molecule. The construction of these viruses has previously been described.33,38 The viruses NV1020, NV1023, and NV1042 were propagated on Vero cells and titers measured by standard plaque assay.
In vitro cytotoxicity
The ability of NV1023 and NV1042 to infect and kill MH was assessed by standard cytotoxicity assays. Cells were plated at 2 × 104 cells/well in 12-well plates (Costar, Corning Inc., Corning, NY, USA) and were infected with NV1042 or NV1023 at a multiplicity of infection (MOI, ratio of virus to target cells) of 0.1, 1.0, and 2.0. Control wells were treated with media alone. Cell viability was measured by counting live cells via trypan blue exclusion at 24-h intervals, carried out to 7 days. All assays were performed in triplicate.
In vitro IL-12 production
IL-12 production from cells infected in vitro by NV1023 or NV1042 was measured by ELISA. Supernatants were harvested at 24-h intervals from both viral groups at the different MOIs. IL-12 production was determined by ELISA (R&D systems, Minneapolis, MN, USA), and all assays were performed in triplicate.
All animal work was performed under guidelines established by the Memorial Sloan-Kettering Cancer Center Institutional Animal Care and Use Committee. Buffalo rats were purchased from Charles River Laboratories (Wilmington, MA, USA). Male Buffalo rats (200–250 g) were housed two per cage and allowed food and water ad libitum. For all invasive procedures, the animals were anesthetized with intraperitoneal (i.p.) pentobarbital injection (50 mg/kg) and inhalational xylazine. All operative procedures were performed after satisfactory anesthesia had been established, using an upper midline incision and sterile conditions.
Establishment of solitary hepatic tumors23
Cultured MH cells were trypsinized and implanted subcutaneously into the flanks of Buffalo rats (106 cells). Once the tumors had reached approximately 1–2 cm in diameter, they were harvested, cut into 3 mm pieces using a core biopsy needle, and then implanted under the capsule of the median liver lobe of naïve rats. This was done by making a small incision in the hepatic capsule and inserting the tumor completely. The tumors were allowed to incorporate into the hepatic parenchyma for 1 week before additional investigations were performed, at which time the tumors generally measured 4–5 mm in size.
In vivo cytokine production
IL-12 and interferon-γ (IFN-γ) production were assessed from tumor nodules harvested from Buffalo rat livers. Solitary tumors were established in rat livers, as described above. After 7 days, the tumors were injected with 50 μl of either 1 × 107 plaque-forming units (PFU) of NV1023, NV1042, or PBS (control). The tumor nodules were then harvested and homogenized using T-Per Tissue Protein Extraction Reagent (Pierce, Rockford, IL, USA) at a concentration of 1 ml/0.1 g of tissue. Homogenized tissues samples were centrifuged at 13 000 rpm for 5 min and supernatants were collected. IL-12 and IFN-γ production were quantified by ELISA (R & D systems, Minneapolis, MN, USA). Assays were performed in duplicate.
Treatment of hepatic tumors in rats
As described above, the rats were reopened to expose the hepatic tumor nodules 7 days after tumor implantation. The tumors were then directly injected with 50 μl of either 1 × 107 PFU of NV1023, 1 × 107 PFU of NV1042 or phosphate-buffered saline (PBS, control). The incisions were closed and the animals were returned to their cages. Animal weights, grooming, and food and water intake were followed closely three times per week. After 10 days of direct injection of the tumor nodules, the animals underwent a third laparotomy and the previously implanted tumors were resected with a margin of normal liver. To recreate the clinical scenario of resection of tumor-bearing liver in the face of microscopic residual disease, all animals were injected intrasplenically with 5 × 105 MH cells 20 min before the parent tumor was resected. Hepatic resection was performed by ligating the vascular supply to the median hepatic lobe at its base. The animals were resuscitated with 3 cm3 of 0.9% saline i.p. just prior to closing.
The volume of the resected parent tumor nodules was measured using the following formula: Tumor volume=4/3 (π) (DL3/2) (Ds3/2) (Ds3/2) where DL is the longest diameter and Ds is the shortest diameter.33 Following tumor resection and rechallenge, the animals were returned to their cages and followed closely for 3 weeks. When the weight of the PBS-injected control animals began to decline (approximately day 21 post-tumor inoculation), the experiment was terminated. The animals were killed by CO2 inhalation, the livers were harvested and the tumor nodules in the liver remnant were counted.
Immunohistochemical staining for intratumoral CD4(+) and CD8(+) lymphocytes
Solitary hepatic tumors were established as described above and injected with 1 × 107 PFU of NV1023 (IL-12 negative) or NV1042 (IL-12 positive) and harvested at 0, 1, 3, and 7 days after injection. The tumors were snap frozen, embedded in Tissue-Tek and stored at −20°C. The slides were then sectioned (8 μm thick) and used for immunoperoxidase staining for CD4(+) and CD8(+) lymphocytes. Sections of rat spleens served as positive controls. Tumors at day 0 were stained prior to injection of any virus and served as the negative controls. The anti-CD4 monoclonal antibody (Serotec, Raleigh, NC, USA) was used at a dilution of 1 : 20,000; and the anti-CD8 monoclonal antibody (Caltag, Burlingame, CA, USA) was used at a dilution of 1 : 2000. Briefly, the slides were washed in running water for 2–5 min. Endogenous peroxidase was quenched with a 5-min incubation with 3% hydrogen peroxide and then washed in distilled water followed by PBS, pH=7.2. The slides were then placed in 0.05% bovine serum albumin (BSA) in PBS for 1 min followed by 2% BSA/PBS for 10 min. Diluted primary antibody (150 μl) was then added and left overnight at 4°C. The primary antibody was then removed and the slides washed three times in PBS for a total of 30 min. A 1 : 500 dilution of the secondary antibody (biotinylated anti-mouse, rat adsorbed, Vector Corp.) in 1% BSA/PBS was then added for 40 min at room temperature. After three washes in PBS (10 min each), a 1 : 500 dilution of peroxidase-conjugated streptavidin (Dako Corp., Carpinteria, CA, USA) in 1% BSA/PBS was added for 30 min at room temperature. After further washing as above, the slides were transferred to a bath of diaminobenzidine (DAB) in PBS for 15 min. The sections were then counterstained using Harris modified hematoxylin (Fisher Scientific, Atlanta, GA, USA), decolorized, dehydrated and permanently mounted. The final stained sections were reviewed by a pathologist (DK) blinded to the experimental protocol. The population of intratumoral CD4(+) and CD8(+) lymphocytes were scored on a scale of 1+ (lowest) to 4+ (highest).
Results are expressed as the mean±SE of the mean, unless otherwise indicated. Categorical variables were compared using χ2 analysis and continuous variables were compared using the Student's t-test.
In vitro cytotoxicity
In order to determine the cytotoxicity of the viruses used, cell survival was measured in vitro by trypan blue exclusion. At an MOI of 1 and 2, both NV0123 and NV1042 killed >90% of the cells by day 6 (P<.04 compared to control). At an MOI of 0.1, NV1023 showed little cytotoxicity at 7 days after infection, while the MH cells infected with NV1042 at the same MOI were static in their growth, with nearly an equal number of cells present at day 7 as were present at the start of the assay (3.3 × 104 vs. 2 × l04 cells, respectively). There was no statistical difference between the in vitro cytotoxic potential of NV1023 and NV1042 at an MOI of 1 or 2. Representative results are shown in Figure 1.
In vitro IL-12 production
Supernatants from the cytotoxicity assay at an MOI of 2 were used to assay for IL-12 production. The NV1023 group showed no IL-12 production, as expected, while the NV1042 group produced high levels. Peak IL-12 production in the NV1042-treated cells, normalized to cell count, were seen at 96 h and then returned back to baseline. Figure 2 shows the results of a representative experiment assessing IL-12 production per day normalized to 1 × 106 cells at an MOI of 2.
In vivo cytokine production
NV 1042-treated tumors exhibited peak levels of IL-12 24 h after injection, which then diminished over time, while tumors injected NV1023 produced no IL-12 at any time (Fig 3). NV1042 induced a similar increase in IFN-γ production, which increased in parallel with IL-12 production, peaking at 24 h then declining somewhat more gradually to baseline by 3 days. There was modest expression of IFN-γ after injection of PBS and NV1023, although this was well below the peak expression and somewhat later than that seen with NV1042 (Fig 3).
Viral treatment of established hepatic tumors
These experiments assessed the ability of NV1023 and NV1042 to treat established liver tumors. Solitary hepatomas were injected with 1 × 107 PFU (in 50 μl) of either NV1023, NV1042, or 50 μl of PBS (n=8 animals/treatment arm). After 10 days, the animals were reopened, the parent tumor nodules resected and the animals were rechallenged with an intraportal injection of 5 × 105 MH cells. The mean volume of the resected parent tumors injected with PBS was 1031±186 mm3. By contrast, the tumors treated with NV1023 measured 493±106 mm3 (P<.03 compared to controls) and those treated with NV1042 measured 272±114 mm3 (P<.003 versus controls). Figure 4 shows representative results from one of three separate experiments.
Tumor challenge after partial hepatectomy
These experiments assessed the ability of neoadjuvant treatment of hepatic tumors with NV1023 and NV1042 to eradicate microscopic residual cancer cells after hepatic resection. The livers were harvested and tumor nodules counted, 3 weeks after resection of the parent tumor and intraportal injection of tumor cells. Figure 5 shows representative results from one of three separate experiments (n=8 animals/treatment arm). Animals treated with NV1042 before partial hepatectomy and rechallenge developed 16.8±11 tumor nodules (median=4), which was significantly lower than control animals treated with saline (65.9±15 tumors, median=66, P<.025). Animals treated with NV1023 showed some reduction in the number of hepatic tumors, but this was not significantly different from control animals (29.9±10 tumors, median=30, P<.08 versus control). A total of 50% (4/8) of 14 the animals in the NV1042 group had fewer than five tumors compared to only 12.5% (1/8) of animals in the NV1023 and control groups, respectively. When the results of all three experiments were combined, similar overall results were observed (n=70 animals). Animals treated with NV1042 developed 14.9±9 tumors (median=0) compared to 50±15 (median=25) in the control animals and 41±17 (median=7) in the NV1023 group.
The results of the intratumoral lymphocyte staining are shown in Table 1. CD4(+) lymphocytes increased progressively after injection of both NV1023 and NV1042, from 1+ at day 0 to 4+ at day 7. Likewise, CD8(+) lymphocytes increased after injection of both viruses, although the peak of cellular infiltration was somewhat later with NV1042.
For selected patients with primary and secondary hepatic malignancies, resection is the most appropriate therapy and offers the best opportunity for cure or prolonged disease-free survival.1,2,3,4,5 Cancer recurrence after resection remains a major problem, however, occurring in 60–80% of patients, with the liver remnant being the most common site. Furthermore, in the majority of patients, resection is not an option. Frequently, the extent of cancer within the liver precludes complete resection with preservation of adequate parenchyma. In others, particularly those with hepatocellular carcinoma, chronic underlying liver disease and hepatic dysfunction render even a limited hepatic resection dangerous. Systemic chemo-therapy is largely ineffective against primary hepatic cancers, and although more active agents are available for some metastatic tumors, complete responses and cures with chemotherapy alone are extraordinary. Ablative treatment, such as hepatic artery embolization, cryoablation, or radiofrequency ablation, may be possible in some patients, but these modalities are inappropriate for many, they have never been proven to be superior to best supportive care alone and their role remains ill-defined. Thus, improved therapy, both as an adjuvant to hepatic resection and as primary treatment for the majority of patients who are not candidates for resection, is needed.
Gene therapy is perhaps the most widely investigated novel treatment approach, fueled in large measure by the limitations of the currently available options. Preliminary studies in humans have shown that gene delivery with viral vectors can be done safely, given either intra-arterially or by direct tumor injection.39,40,41 Most gene therapy strategies seek to redirect the host immune response against malignant cells by delivering immuno-stimulatory agents in proximity to putative tumor antigens.23,42,43 Many approaches attempt to induce expression of such molecules directly within the target tumor using replication incompetent viral vectors. These vectors are capable of delivering the gene of interest to a small fraction of tumor cells, and it is not surprising that they have had limited success against established tumors. On the other hand, strategies that target microscopic disease, either as adjuvant treatment after resection or as neoadjuvant treatment before resection, would seem to have a greater likelihood of success. We have shown previously that neoadjuvant delivery of the gene encoding IL-12, using a replication incompetent HSV-1 vector, resulted in high levels of gene expression within established hepatic tumors and increased intratumoral infiltration with CD4(+) and CD8(+) lymphocytes.23 This experimental strategy protected animals against microscopic residual tumor cells and cancer recurrence after subsequent resection of the established lesion, but had little impact on the parent tumor itself. While such gene transfer approaches may have a role in the minority of patients who go on to resection, they remain largely ineffective as primary therapy for patients who will not be submitted to operation.
More effective treatment of established tumors is observed using replication competent oncolytic viruses. These agents overcome the problem of adequate delivery to the target tumor by generating a large number of progeny virus from a small number of initially infected cells, each of which can go on to infect and kill additional tumor cells. Additionally, with their ability to infect a greater proportion of cells, appropriately engineered replication competent viruses may effect greater gene delivery and expression of immunostimulatory agents. Relative specificity is ensured by strategic deletion of genes required for viral replication that are expressed abundantly in malignant cells, but at only low levels in normal cells, thereby limiting toxicity. Several studies have shown that oncolytic herpes viruses can infect and kill a wide range of human tumor cell lines in vitro and effectively treat established tumors in vivo.24,27,28
The current study investigates a combined therapeutic approach consisting of neoadjuvant immunostimulatory gene therapy and oncolytic viral therapy in a clinically relevant animal model of hepatic cancer. The agents used in this study are second generation, multimutated herpes viruses clonally derived from R7020 and have been described previously.27,36,37 Both NV1023 and NV1042 are restored for UL24, which enhances viral replication and both have the lacZ marker gene inserted into the ICP47 gene. The ICP47 gene product inhibits MHC class I peptide presentation by virally infected human cells, inactivation of which allows MHC class I antigen expression and should promote immune recognition of HSV-infected tumor cells. Both viruses used in this study are therefore identical with respect to the genetic manipulations aimed at enhancing oncolytic activity. NV1042 has, in addition, the gene encoding IL-12, a potent immune modulator that plays a key role in the development of an antitumor response. IL-12 mediates several important immunologic processes, including stimulation of INF-γ synthesis and activation of natural killer cell function and cytotoxic T-lymphocyte effector activity.31,34,35 NV1042 therefore has the same capacity for direct tumor lysis as NV1023, but, in addition, has the ability to induce intratumoral expression of IL-12, thereby adding to its potential as an anticancer agent by recruiting and activating immune cells in an environment rich in putative tumor antigens.
The results of the present study show that a strategy combining tumor oncolysis with local delivery of immunostimulatory IL-12 effectively treats the parent hepatic tumor, causing significant tumor regression, and also offers protection against hepatic cancer recurrence after the parent tumor is excised. Since both NV1023 and NV1042 were equivalent in their ability to infect and lyse tumor cells, the differences observed in vivo appear to be the result of NV1042's ability to induce intratumoral IL-12 and IFN-γ expression. Indeed, injection of NV1042 markedly increased the population of CD4(+) and CD8(+) lymphocytes within the tumor, as has been observed in previous studies.39,41 Furthermore, in prior work with these viruses in a model of squamous cell carcinoma, depletion of CD4(+) and CD8(+) lymphocytes abrogated the enhanced antitumor efficacy of NV1042 compared with NV1023.39 Not surprisingly, NV1023 injection reduced the parent tumor volume compared to control, although this reduction was less than that effected by NV1042. Additionally, however, NV1023 did offer some protection against cancer recurrence after resection and rechallenge, although the difference was not significantly different from control animals and was not as striking as that seen with NV1042. A possible explanation for this is that NV1023 also induced a nonspecific intratumoral lymphocytic infiltration related to tumor lysis alone. The modest increase in IFN-γ production after intratumoral injection of NV 1023 would support this. The enhanced in vivo antitumor activity of NV 1042 compared to NV1023 cannot therefore be attributed solely to the recruitment of immune effector cells, but likely also relates to other actions of IL-12, which were not directly measured in this study.
In summary, the present study demonstrates the efficacy of oncolytic HSV viral therapy in a clinically relevant model of liver cancer. Oncolytic viral therapy alone effectively treats the parent tumor. Antitumor activity is enhanced when oncolysis is combined with the immunostimulatory effects of intratumoral IL-12 expression. Such a combined approach thus offers improved control of the parent tumor and, when used in the neoadjuvant setting, also offers protection against microscopic residual disease after resection. Owing to its ease of use and apparent efficacy, this approach may have clinical relevance, and the results encourage further work toward applying such a strategy in conjunction with hepatic resection or as primary therapy for unresectable hepatic malignancy in humans.
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Supported in part by training Grant T32 CA 09501 (ME) and US Public Health Service grants RO1CA75416, RO1CA72632, and RO1CA61524 from the National Institutes of Health and MBC-99366 from the American Cancer Society (YF).
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Jarnagin, W., Zager, J., Klimstra, D. et al. Neoadjuvant treatment of hepatic malignancy: an oncolytic herpes simplex virus expressing IL-12 effectively treats the parent tumor and protects against recurrence-after resection. Cancer Gene Ther 10, 215–223 (2003). https://doi.org/10.1038/sj.cgt.7700558
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