Replication-competent, attenuated herpes simplex viruses (HSV) have been demonstrated to be effective oncolytic agents in a variety of malignant tumors. Cytokine gene transfer has also been used as immunomodulatory therapy for cancer. To test the utility of combining these two approaches, two oncolytic HSV vectors (NV1034 and NV1042) were designed to express the murine GM-CSF and murine IL-12 genes, respectively. These cytokine-carrying variants were compared with the analogous non-cytokine-carrying control virus (NV1023) in the treatment of murine SCC VII squamous cell carcinoma. All three viruses demonstrated similar infection efficiency, viral replication, and cytotoxicity in vitro. SCC VII cells infected by NV1034 and NV1042 effectively produced GM-CSF and IL-12, respectively. In an SCC VII subcutaneous flank tumor model in immunocompetent C3H/HeJ mice, intratumoral injection with each virus caused a significant reduction in tumor volume compared with saline injections. The NV1042-treated tumors showed a striking reduction in tumor volume compared with the NV1023- and NV1034-treated tumors. On subsequent rechallenge in the contralateral flank with SCC VII cells, 57% of animals treated with NV1042 failed to develop tumors, in comparison with 14% of animals treated with NV1023 or NV1034, and 0% of naive animals. The increased antitumor efficacy seen with NV1042 in comparison with NV1023 and NV1034 was abrogated by CD4(+) and CD8(+) lymphocyte depletion. NV1042 is a novel, attenuated, oncolytic herpesvirus that effectively expresses IL-12 and elicits a T lymphocyte-mediated antitumor immune response against murine squamous cell carcinoma. Such combined oncolytic and immunomodulatory strategies hold promise in the treatment of cancer.

Summary:To determine the outcome of patients with chemosensitive relapsed or primary refractory Hodgkin's disease (HD) or aggressive non-Hodgkin's lymphoma (NHL) whose disease progresses after autologous stem cell transplantation (ASCT), we reviewed the records of 82 patients with HD and 139 patients with NHL transplanted between 1993 and 2000. Disease progression occurred in 25 patients with HD and 66 patients with NHL, with median times to progression (TTP) of 3.8 and 5.1 months, respectively. Median survival times following ASCT failure were 26 and 7.7 months for patients with HD and NHL, respectively. The second-line international prognostic index (sIPI) and the TTP (before or after 3 months from ASCT) independently were predictive of survival for NHL patients. In addition, treatment with rituximab for patients with B cell NHL was associated with improved survival (median 28.6 vs 4.1 months, P ¼ 0.003), independent of the sIPI and TTP. Prognostic factors for patients with HD were not identified. Only two patients, one of whom was among six patients who received second autologous transplants, remain disease-free. The uniformly poor outcome associated with disease progression after ASCT should prompt efforts to assess the feasibility and utility of detecting and treating post transplant residual disease during a minimal disease state, before overt progression.

G207 and NV1020 are two replication -competent, multimutant oncolytic herpes simplex viruses evaluated in the current studies for their anticancer effects in the treatment of gastric cancer. Deletion of both 1 34.5 genes and inactivation of ICP6 ( ribonucleotide reductase ) allows G207 to selectively replicate within tumor cells. NV1020 is another attenuated recombinant herpes virus with deletions of the HSV joint region, with deletion of only one copy of the 1 34.5 gene, and with the ICP6 gene intact. In vitro, both G207 and NV1020 effectively infected, replicated, and killed human gastric cancer cells, with NV1020 being more effective at lower concentrations of virus. In a murine xenograft model of peritoneally disseminated gastric cancer, both NV1020 and G207 reduced tumor burden when given intraperitoneally ( i.p. ) at higher doses. When viral doses were lowered or when advanced tumor was treated, i.p. NV1020 was superior to i.p. G207. In vitro viral replication and cytotoxicity predicted the in vivo antitumor response. Intravenous delivery of either G207 or NV1020 failed to reduce tumor burden, demonstrating the importance of regional therapy as treatment for compartmentalized malignancy. Both agents were safe for use in animals, and immunohistochemistry performed on mouse tissue revealed selective viral targeting of tumor. Oncolytic therapy using genetically engineered HSVs represents a promising strategy for peritoneal malignancies.

ObjectiveTo assess the strategy of combining oncolytic herpes simplex virus (HSV) therapy with immunomodulatory therapy as treatment for experimental colon cancer. The oncolytic HSV recombinant NV1023 and the interleukin 12 (IL-12)-secreting oncolytic NV1042 virus were evaluated in vitro and in vivo with respect to antitumor efficacy. Summary Background DataGenetically engineered, replication-conditional, attenuated HSVs have shown oncolytic activity against a wide variety of solid malignancies. Other strategies for treating cancer have involved immunomodulation and cytokine gene transfer using viral vectors. This study has combined both of these strategies by inserting the murine IL-12 gene into a replication-competent HSV. This approach allows oncolytic therapy to replicate selectively within and lyse tumor cells while providing the host immune system with the cytokine stimulus necessary to recruit and activate inflammatory cells needed to enhance the antitumor effect. MethodsNV1023 is a multimutant HSV based on the wild-type HSV-1 F strain. NV1042 was created by insertion of the mIL-12 gene into NV1023. Cytotoxicity and viral proliferation of both NV1023 and NV1042 within murine CT26 colorectal cancer cells were first shown. Cells infected with NV1042 were then shown to produce significant levels of IL-12. Using an experimental flank model of colon cancer, mice were treated with both high and low doses of NV1023 or NV1042 and were followed up for both cure and reduction in tumor burden.

Oncolytic cancer therapy using herpes simplex viruses (HSV) that have direct tumoricidal effects and cancer immunotherapy using the cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) have each been effective in preclinical testing. NV1034 is a multi-mutated oncolytic HSV carrying the gene for murine GM-CSF that attempts to combine these two anticancer strategies. The purpose of this study was to compare NV1034 to NV1023, the parent HSV mutants lacking GM-CSF, in order to determine if such combined oncolytic and immunotherapy using a single vector has advantages over oncolytic therapy alone. In vitro, expression GM-CSF did not alter the infectivity, in vitro cytotoxicity, or replication of NV1034 compared to the non-cytokine secreting control. Tumors infected with NV1034 produced GM-CSF in picogram quantities. In vivo efficacy of the viruses against murine colorectal carcinoma CT26 and murine hepatoma Hepa l-6 was then tested in subcutaneous tumors in syngeneic Balb/c and C57 L/J mice respectively. In these immune competent models, NV1034 or NV1023 each demonstrated potent antitumor activity. Treatment with NV1034 had significantly better antitumor effect compared to treatment with NV1023. Furthermore, in mice depleted of CD4+ and CD8+ T-lymphocytes, there was no difference in the antitumor efficacy of these viruses. Viral vectors combining oncolytic and immunotherapy are promising agents in treatment of colorectal carcinoma and hepatoma.

Attenuated, replication‐competent herpes simplex virus mutants are attracting interest because of their ability to replicate within and kill tumor cells while remaining of low pathogenicity to normal tissues. In this study we investigated the ability of two oncolytic candidates, G207 and NV1020, to infect and lyse human and murine transitional cell carcinoma (MBT‐2) cells in vitro and their in vivo efficacy in a well‐established immunocompetent animal model of bladder cancer. Both viruses were effective at infecting, replicating within, and achieving subsequent cell lysis for all four human bladder cancer cell lines and MBT‐2. We found a strong correlation between infection efficiency and subsequent cell death. In vivo studies demonstrated that these viruses were effective with a single intravesical instillation and even more effective with multiple instillations at reducing tumor burden and achieving cures of orthotopic bladder cancer in syngeneic C3h/Hej mice. Immunohistochemistry and histological studies demonstrated that viral replication and cell death were restricted to bladder cancer cells. These results suggest that both G207 and NV1020 hold particular promise for intravesical treatment of human bladder cancer and that ease of intravesical instillation facilitates efficient delivery of virus to tumor cells.

In the present study we applied proton-decoupled 31 P magnetic resonance spectroscopic imaging (MRSI) to noninvasively assess liver metabolism in patients who had undergone a partial hepatectomy (PH). Proton-decoupled 31 P chemical shift imaging was performed in 47 patients 2-28 days following major hepatectomy, and the results were compared with those from eight control subjects. All studies were performed on a 1.5T MR imager (General Electric, Milwaukee, WI) equipped with a stand-alone proton decoupler. A 31 P-1 H resonator pair was used for data acquisition, and 31 P data were obtained in 34 min. Liver regeneration was characterized by increases in phosphoethanolamine (PE), and decreases in nucleoside triphosphates (NTP), glycerophosphoethanolamine (GPE), and glycerophosphocholine (GPC). These alterations were most marked 48 -72 hr after hepatectomy and returned to baseline within 3 weeks. The level of PE measured by MRSI was also found to depend on the percentage of liver that was removed, while changes in levels of cellular high energy phosphates were independent of the size of liver resection. Implementation of proton-decoupling was critical for assessing individual phosphomonoester and phosphodiester components. This study demonstrates that 31 P MRSI can be used to assess metabolic changes in humans during liver regeneration, and may be useful for assessing derangement of the regenerative process or guiding adjuvant chemotherapies. The regenerative capacity of the liver is the underlying basis of the approximately 50000 liver resections performed yearly in this country to treat benign and malignant diseases (1). Resections of up to 90% of functional liver parenchyma are now performed at many major centers around the world, and mortality rates are routinely less than 3% (2-4). Almost all knowledge of the cellular physiology of the hepatic regenerative process has been derived from animal studies, particularly in rodents (5). It is known that Ͼ85% of the rat hepatocytes proceed into S-phase within 48 hr after partial hepatectomy (PH), and that cellular stores of high-energy phosphates decrease and then normalize within 5-7 days (6 -8). Until recently, studies of liver regeneration in humans have been restricted to volumetric assessments of regeneration by cross-sectional imaging, or to measurements of hepatocyte-synthetic capacity (9,10). Both of these parameters usually recover late in the course of liver regeneration. There is also great variability in the data regarding the time course of liver volume recovery, with estimates ranging from 3 weeks to several months (9,11).In the present study we utilized proton-decoupled 31 P magnetic resonance spectroscopic imaging (MRSI) to noninvasively assess liver metabolism in patients following major hepatectomy. This technique permits the measurement of cellular high-energy phosphates, individual cell membrane phospholipid metabolites (phosphoethanolamine (PE), phosphocholine (PC), glycerophosphoethanolamine (GPE), and glycerophosphocholine (GPC)), and intracellul...

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 10 7 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 10 5 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 490% 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-g (IFN-g) 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% (Po.003) and 52% (Po.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 (16.8711 (median ¼ 4) vs. 65.9715 (median ¼ 66) in control animals, Po.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.