Keith A. Delman

Effective treatment of pancreatic tumors with two multimutated herpes simplex oncolytic viruses.

Pancreatic cancer is an aggressive, rapidly fatal disease against which current nonsurgical therapy has minimal impact. This study evaluates the efficacy of two novel, replication-competent, multimutated herpes viruses (G207 and NV1020) in an experimental model of pancreatic cancer. Four human pancreatic carcinoma cell lines were exposed to G207 or NV1020, and cell survival and viral progeny production were determined. Flank tumors in athymic mice were subjected to single or multiple injections of 1 X 107 G207 or NV1020, and tumor volume was evaluated over time. For all of the cell lines, G207 and NV1020 produced infection, viral replication, and cell lysis (P <0.05). NV1020 resulted in a higher production of viral progeny compared to G207. The efficacy of viral tumor cell kill was greatest in those cells with the shortest in vitro doubling time. For flank tumors derived from hs766t, single or multiple injections of both viruses were equally effective and significantly reduced flank tumor burden (P <0.05). Complete hs766t flank tumor eradication was achieved in 25% (5 of 20) of animals treated with G207 and 40% (8 of 20) of animals treated with NV1020. In vivo efficacy correlated with in vivo tumor doubling time. There were no adverse effects related to viral administration observed in any animal. NV1020 and G2O7 effectively infect and kill human pancreatic cancer cells in vitro and in vivo. Given the lack of effective nonoperative treatments for pancreatic cancer, oncolytic herpes viruses should be considered for clinical evaluation.

Antitumor efficacy of regional oncolytic viral therapy for peritoneally disseminated cancer

Oncolytic viral therapy is a promising new method of cancer treatment. Peritoneal dissemination of cancer is a common and fatal clinical condition seen in many malignancies, with few effective therapies available. G207, a multimutated replication-competent herpes simplex virus type-1, effectively treats disseminated peritoneal cancer. This study evaluates viral proliferation and subsequent tumoricidal effects in vitro and in vivo after regional viral delivery. In vitro studies demonstrate that G207 efficiently kills five human gastric cancer cell lines, and that permissiveness to viral replication is correlated with cytotoxicity. In a murine xenograft model of human gastric carcinomatosis, peritoneal delivery of G207 effectively kills tumor and prolongs survival. Data from quantitative PCR characterizes peritoneal clearance of virus after intraperitoneal injection, and identifies G207 replication within tumor cells in vivo, similar to in vitro proliferation. Further analysis of various organs confirms that G207 does not replicate within normal tissue after peritoneal delivery. Wild-type KOS viral replication was also demonstrated in vivo, with significant toxicity secondary to dissemination and encephalitis. In vivo viral proliferation of G207 is restricted to tumor cells, is correlated with in vitro assays, and is an important mechanism of anticancer efficacy.

Functional Interaction between Fluorodeoxyuridine-Induced Cellular Alterations and Replication of a Ribonucleotide Reductase-Negative Herpes Simplex Virus

ABSTRACT G207 is an oncolytic herpes simplex virus (HSV) which is attenuated by inactivation of viral ribonucleotide reductase (RR) and deletion of both γ 1 34.5 genes. The cellular counterparts that can functionally substitute for viral RR and the carboxyl-terminal domain of ICP34.5 are cellular RR and the corresponding homologous domain of the growth arrest and DNA damage protein 34 (GADD34), respectively. Because the thymidylate synthetase (TS) inhibitor fluorodeoxyuridine (FUdR) can alter expression of cellular RR and GADD34, we examined the effect of FUdR on G207 bioactivity with the hypothesis that FUdR-induced cellular changes will alter viral proliferation and cytotoxicity. Replication of wild-type HSV-1 was impaired in the presence of 10 nM FUdR, whereas G207 demonstrated increased replication under the same conditions. Combined use of FUdR and G207 resulted in synergistic cytotoxicity. FUdR exposure caused elevation of RR activity at 10 and 100 nM, whereas GADD34 was induced only at 100 nM. The effect of enhanced viral replication by FUdR was suppressed by hydroxyurea, a known inhibitor of RR. These results demonstrate that the growth advantage of G207 in FUdR-treated cells is primarily based on an RR-dependent mechanism. Although our findings show that TS inhibition impairs viral replication, the FUdR-induced RR elevation may overcome this disadvantage, resulting in enhanced replication of G207. These data provide the cellular basis for the combined use of RR-negative HSV mutants and TS inhibitors in the treatment of cancer.

Neoadjuvant treatment of hepatic malignancy: an oncolytic herpes simplex virus expressing IL-12 effectively treats the parent tumor and protects against recurrence-after resection.

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.

Herpes simplex virus (HSV)-mediated ICAM-1 gene transfer abrogates tumorigenicity and induces anti-tumor immunity.

BackgroundCostimulatory and cellular adhesion molecules are thought to be essential components of antigen presentation in the immune response to cancer. The current studies examine gene transfer utilizing herpes viral amplicon vectors (HSV) to direct surface expression of adhesion molecules, and specifically evaluate the potential of a tumor-expressing intercellular adhesion molecule-1 (ICAM-1) to elicit an anti-tumor response.Materials and MethodsThe human ICAM-1 (hICAMI) gene was inserted into an HSV amplicon vector and tested in a transplantable rat hepatocellular carcinoma and in a human colorectal cancer cell line. Cell surface ICAM-1 expression was assessed by flow cytometry. Lymphocyte binding to HSV-hICAM1-transduced cells was compared with that to cells transduced with HSV not carrying the ICAM gene. Tumorigenicity of HSV-hICAM1-transduced tumor cells were tested in syngeneic Buffalo rats. Additionally, immunization with irradiated (10,000 rads) HSV-hICAM1-transduced tumor cells was performed to determine its effect on tumor growth.ResultsA 20-min exposure of tumor cells at a multiplicity of infection (MOI) of 1 resulted in high-level cell surface expression of human ICAM in approximately 25% of tumor cells. Transduced rat or human tumor cells exhibited significantly enhanced binding of lymphocytes (p < 0.05). HSV-hICAM1-transduced cells elicited an increase in infiltration by CD4+ lymphocytes in vivo and exhibited decreased tumorigenicity. Immunization with irradiated HSV-hICAM1-transduced cells protected against growth of subsequent injected parental tumor cells.ConclusionsHSV amplicon-mediated gene transfer is an efficient method for modifying the cell surface expression of adhesion molecules. Increased tumor expression of ICAM-1 represents a promising immune anti-cancer strategy.

Effect of murine liver cell proliferation on herpes viral behavior: Implications for oncolytic viral therapy

Replication‐competent herpes simplex oncolytic viruses are promising anticancer agents that partly target increased DNA synthesis in tumor cells. Investigators have proposed that these DNA viruses may be combined with liver resection to enhance killing of liver malignancies. Whether or not the cellular alterations associated with hepatic regeneration affect the efficacy and toxicity of these promising anticancer agents is unknown. This study examined the behavior of two oncolytic viruses, NV1020 and G207, during liver regeneration. When delivered during the peak of liver regeneration, replication and appearance of both G207 and NV1020 in hepatic tissue are enhanced as demonstrated by histochemical staining for the marker gene lac Z, immunohistochemical staining, and quantitative polymerase chain reaction. This increased appearance of virus in liver tissue correlates with increases in cellular ribonucleotide reductase activity and DNA synthesis and is also associated with increased viral binding. However, increased viral presence is transient, and viral detection declines to baseline within 7 days. When these viruses were delivered to animals even as early as 7 days after hepatectomy, there proved to be no measurable viral replication in any organ and no increased morbidity or mortality. In conclusion, the early stages of hepatic regeneration after resection provide an environment suitable for viral replication. Administration of replication‐competent herpes simplex virus during the peak of hepatocyte regeneration (24–48 hours) permits viral productivity in tissue that otherwise does not support viral growth. The increase in hepatotoxicity after hepatectomy is short‐lived and can be predicted by peak hepatocyte DNA synthesis. (HEPATOLOGY 2004;39:1525–1532.)

A quantitative assessment of liver metabolites during jaundice using three dimensional phosphorus chemical shift imaging

Phosphorus metabolites in the jaundiced rat liver were studied by three-dimensional phosphorus chemical shift imaging (CSI). Animals were studied at 1, 2, and 3 weeks post-ligation of the common bile duct. Quantitation of metabolites was performed using an external standard. Metabolite T(1) values were assessed in CSI experiments on normal untreated animals. High-performance liquid chromatography (HPLC) was used to measure adenine nucleotides in a separate group of jaundiced rats. 3D-CSI did not detect significant changes in NTP in jaundiced animals relative to baseline controls. At two and three weeks post bile duct ligation, pH was significantly elevated. HPLC data comparing ATP levels to baseline controls also detected no change except for elevated ATP detected on Day 21. (31)P NMR chemical shift imaging may be used to assess liver metabolites under conditions of stress such as jaundice. However, absolute quantitation requires careful attention to many factors including point spread function, correct T(1) values, and adequate signal-to-noise ratio.

Use of radiolabelled iododeoxyuridine as adjuvant treatment for experimental tumours of the liver

The aim of the study was to determine whether hepatic regeneration stimulates growth of tumour residing within the liver, and whether a difference in the rate of DNA synthesis in liver and tumour may be used to target cancer using the radiolabelled thymidine analogue 5‐iodo‐2′‐deoxyuridine (IUdR).