Pilot Study of Oncolytic Viral Therapy Using Mutant Herpes Simplex Virus (HF10) Against Recurrent Metastatic Breast Cancer

Background An oncolytic herpes simplex virus type 1 mutant (HF10) has been isolated and evaluated for antitumor efficacy in a syngeneic immunocompetent mouse model, where it was effective against cancer and conferred resistance to rechallenge with tumor cells in all surviving mice. Several studies have shown that HF10 is effective and safe for use against localized or peritoneally disseminated nonneuronal malignant tumors in animals. Methods A pilot study using HF10 was initiated in six patients with cutaneous or subcutaneous metastases from breast cancer. For each patient, .5 mL of HF10 suspension containing various viral doses was injected into one nodule; .5 mL of sterile saline was injected into another. All patients were monitored for local and systemic adverse effects. Nodules were excised 14 days after injection for histopathologic studies. Results All patients tolerated the intratumoral injection of HF10. No adverse effects occurred, and histopathological evaluation revealed 30% to 100% cancer cell death. Conclusions This pilot study found HF10 to be safe and effective against metastatic breast cancer.     

Augmenting major histocompatibility complex class I expression by murine tumors in vivo enhances antitumor immunity induced by an active immunotherapy strategy

OBJECTIVE: Tumors down-regulate major histocompatibility complex class I expression, escaping recognition by the cellular immune response. We hypothesized that augmentation of tumor cell class I expression by interferon-gamma would enhance the cellular antitumor immune response and cure rate of an active immunotherapy strategy. METHODS: B16.F10 tumor cells were exposed to interferon-gamma in culture, and class I expression was quantified using flow cytometry. Syngeneic mice bearing established tumors were injected with interferon-gamma (5000 U, intraperitoneal), and class I expression was assessed using immunohistochemistry. Tumor-specific cytotoxic T lymphocytes were induced in mice by an intratumoral injection of AdCD40L (5 x 10(10) particles), an adenovirus gene transfer vector-based immunotherapy strategy previously demonstrated to augment cellular antitumor immunity. A conjugate-formation assay and the enzyme-linked immunospot assay were used to evaluate the binding and activation of cytotoxic T lymphocytes, respectively. Interferon-gamma was administered to tumor-bearing mice concomitantly with intratumoral AdCD40L. End points measured included the frequencies of cytotoxic T lymphocytes using the enzyme-linked immunospot assay, tumor size, and mouse survival. The role of class I expression was further evaluated by monoclonal antibody blockade in both in vitro and in vivo experiments. RESULTS: B16.F10 cells exposed to interferon-gamma expressed significantly more class I, both in vitro and in vivo, and were able to bind to and activate cytotoxic T lymphocytes more efficiently than untreated cells. Cytotoxic T-lymphocyte frequencies, tumor regression, and the cure rate induced by AdCD40L were augmented by the addition of a single dose of interferon-gamma in tumor-bearing mice. These in vitro and in vivo effects of interferon-gamma were attenuated by class I monoclonal antibody blockade. CONCLUSIONS: Up-regulation of class I expression using interferon-gamma enhances the cellular antitumor immune response and cure rate of AdCD40L, an active immunotherapy strategy. This approach may be useful for human tumors that lack class I expression.     

Cancer gene therapy using a replication-competent herpes simplex virus type 1 vector.

OBJECTIVE: The authors investigate the efficacy of hrR3, a viral vector derived from herpes simplex virus type 1 (HSV 1), in destroying colon carcinoma cells in vitro and in vivo. The effect of adding the prodrug ganciclovir in combination with hrR3 infection also is assessed. SUMMARY BACKGROUND DATA: Most cancer gene therapy strategies use viral vectors that are incapable of replication. The HSV 1 vector hrR3 is capable of replication, and its replication is cytotoxic to cells. hrR3 also possesses the HSV-thymidine kinase gene, which converts ganciclovir into a toxic metabolite. Thus, the addition of ganciclovir to hrR3-infected cells may enhance the ability of hrR3 to destroy tumor cells. To increase specificity for tumor cells, hrR3 has a mutated ribonucleotide reductase gene and replicates selectively in cells with high levels of endogenous rbonucleotide reductase. Actively dividing cells such as tumor cells have high levels of endogenous ribonucleotide reductase for synthesis of DNA precursors. The authors are interested in the use of HSV 1 vectors to treat liver metastases from colorectal cancer. METHODS: Ribonucleotide reductase expression in several colon carcinoma cell lines and in primary cultures of human hepatocytes was determined by Western blot analysis. hrR3-mediated cytotoxicity in the colon carcinoma cell lines was determined using an in vitro assay. The human colon carcinoma cell line HT29 was injected into the flanks of nude mice followed by intratumoral injection of hrR3. Tumor growth rate was assessed with and without the addition of intraperitoneal ganciclovir. RESULTS: Ribonucleotide reductase levels in colon carcinoma cell lines are much higher than in primary cultures of human hepatocytes. hrR3 efficiently destroys colon carcinoma cell lines in vitro. A single intratumoral injection of hrR3 into HT29 flank tumors significantly reduces tumor growth rate, and the administration of ganciclovir has no additive effect. CONCLUSIONS: The inherent cytotoxicity of hrR3 replication effectively destroys colon carcinoma cells in vitro and in vivo. This cytotoxicity is not enhanced in vivo by the addition of ganciclovir. In the future, more efficacious and selective HSV 1 vectors may be useful in the treatment of cancer.     

Bortezomib inhibits angiogenesis and reduces tumor burden in a murine model of neuroblastoma

BACKGROUND: Bortezomib is a proteasome inhibitor with pleiotropic antitumor activity. Here we investigate the antiangiogenic and antitumor efficacy of bortezomib against neuroblastoma both in vitro and in a murine model of localized and disseminated disease. METHODS: In vitro activity of bortezomib was assessed by evaluating its effect on cell proliferation and cell cycle status. Localized tumor burden was followed with caliper measurements and total-body bioluminescence in mice with disseminated disease. The antiangiogenic activity was evaluated with immunohistochemistry and human vascular endothelial growth factor (VEGF) enzyme-linked immunosorbent assay on tumor protein extracts. RESULTS: Bortezomib treatment resulted in dose and time-dependent decreases in cell proliferation and resulted in cell cycle arrest. In vivo, bortezomib restricted tumor growth in a model of localized disease and decreased bioluminescence in mice with disseminated disease. That decreased bioluminescence reflected decreased tumor burden was confirmed at necropsy by assessing disease in specific organs. In addition, treatment resulted in a decrease in intratumoral vessel counts and reduced tumor VEGF expression. CONCLUSION: Bortezomib shows significant activity against neuroblastoma in vitro, and it inhibits tumor growth and angiogenesis in vivo. These results suggest that clinical studies of bortezomib are warranted for the treatment of this difficult disease.     

Intratumoral injection of OK-432 in conjunction with fibrinogen greatly enhances antitumor effect on colorectal carcinomas]

We found that antitumor effect of OK-432, a lyophylized preparation of an attenuated strain of streptococcus pyogenes, on colorectal carcinoma was greatly augmented when it was injected intratumorally in conjunction with fibrinogen. Twenty cases of colorectal cancer received intratumoral injection of 5 KE of OK-432 mixed with 80mg of fibrinogen including factor-XIII and 1ml of aprotinin at the time of endoscopic examination. Changes in the shape of the tumors were observed endoscopically within a few days after injection, and in most cases, decrease in the height of tumor margin was noted. Histopathological findings on surgically resected specimens revealed that the fine meshwork of fibrin was formed at the injected site soon after the injection, and a marked infiltration of inflammatory cells including neutrophils, plasma cells, macrophages, eosinophils and lymphocytes. Such granulomatous changes developed over 7 days after injection, and the degradation of tumors were observed. By 14 days after the injection, tumor tissues were largely replaced with granulomas, and shrinkage of tissues were observed. These findings indicated that fibrinogen including factor-XIII and aprotinin has a potential ability to augment the immunoreaction induced by biological response modifiers, and intratumoral injection of OK-432 in conjunction with fibrinogen solution was superior to intratumoral injection of OK-432 alone as the local immunotherapy of colorectal cancer.     

瘤体内直接注射白细胞介素-7基因抗小鼠乳腺肿瘤免疫效应的研究

目的 观察瘤体内直接注射白细胞介素(IL)-7基因抗小鼠乳腺肿瘤免疫效应.方法 构建IL-7真核表达质粒(pcDNA3-IL-7);建立乳腺癌TM40D细胞BALB/C小鼠移植模型;瘤体内直接注射pcDNA3-IL-7,观察小鼠肿瘤体积变化;酶联免疫吸附试验(ELISA)法检测外周血干扰素(IFN)-γ含量;流式细胞仪检测胞内IFN-γ的分泌量;局部肿瘤经治疗后行常规病理分析.结果 成功构建pcDNA3-IL-7;与对照磷酸盐缓冲液(PBS)组(115.2±11.8) ng/L、pcDNA3组(133.6±9.4) ng/L比较,pcDNA3 -IL-7注射组(242.3±10.1)ng/L外周血IFN-γ明显增高;pcDNA3-IL-7明显抑制肿瘤生长(P＜0.05);流式细胞仪检测显示pcDNA3-IL-7显著促进CD4+T细胞、CD8+T细胞内IFN-γ的分泌量;常规病理显示pcDNA3 -IL-7注射组肿瘤组织大量坏死,炎性细胞和大量淋巴细胞浸润.结论 瘤体内直接注射pcDNA3-IL-7明显抑制小鼠乳腺肿瘤生长,显著促进IFN-γ分泌,增强了小鼠机体抗乳腺肿瘤的免疫效应.     

Cisplatin augments cytotoxic T-lymphocyte-mediated antitumor immunity in poorly immunogenic murine lung cancer

OBJECTIVE: Many tumors are poorly immunogenic and resistant to cytotoxic T-lymphocyte-mediated cell lysis. Because cisplatin has been demonstrated to increase tumor cell Fas receptor expression, we hypothesized that cisplatin will enhance cytotoxic T-lymphocyte tumor cell killing and augment the antitumor effect of an active immunotherapy strategy in a poorly immunogenic murine lung cancer model. METHODS: Lewis lung carcinoma cells were exposed to cisplatin in vitro, and Fas receptor expression and apoptosis in response to an agonistic anti-Fas antibody were quantified using flow cytometry. Wild-type and Fas ligand-deficient mice bearing Lewis lung carcinoma flank tumors were then treated with intraperitoneal cisplatin as well as an intratumoral injection of an adenovirus gene transfer vector encoding CD40 ligand. End points included tumor size, animal survival, and Fas expression (determined using immunofluorescence). Cytotoxicity assays were performed using splenocytes from adenovirus gene transfer vector encoding CD40 ligand-treated animals as effectors and cisplatin-treated Lewis lung carcinoma cells as targets. RESULTS: Cisplatin induced heightened expression of Fas receptor on Lewis lung carcinoma cells in vitro and in vivo and enhanced apoptosis in cells exposed to an agonistic anti-Fas antibody. In vivo, the combination of 1 dose of intraperitoneal cisplatin and intratumoral adenovirus gene transfer vector encoding CD40 ligand inhibited tumor growth and prolonged survival compared with adenovirus gene transfer vector encoding CD40 ligand alone, resulting in a higher cure rate. This effect was lost in Fas ligand-deficient mice. Splenocytes from adenovirus gene transfer vector encoding CD40 ligand-treated wild-type mice lysed cisplatin-treated Lewis lung carcinoma cells more efficiently than untreated Lewis lung carcinoma cells, an effect lost in splenocytes from Fas ligand-deficient mice. CONCLUSION: Cisplatin augments the antitumor effect of a cytotoxic T-lymphocyte-mediated immunotherapy strategy, resulting in a higher cure rate than seen with immunotherapy alone. This effect is associated with the enhanced ability of cytotoxic T lymphocytes to lyse tumor cells that have been exposed to cisplatin through Fas/Fas ligand interactions.     

Antitumor Effect of Gemcitabine on Orthotopically Inoculated Human Gallbladder Cancer Cells in Nude Mice

Background The prognosis of gallbladder carcinoma is poor; therefore, investigating the efficacy of new chemotherapy agents is essential for the treatments for this tumor. Recently, several studies have reported clinical trials using gemcitabine as treatment for advanced gallbladder cancers. However, the antitumor effects of gemcitabine on gallbladder carcinoma have not been examined in in vitro and in vivo model systems. Methods We examined the cytotoxicity of gemcitabine in four biliary tract cancer cell lines using the WST-1 assay. In addition, we examined the effect of gemcitabine on gallbladder cancers resulting from orthotopic inoculation of NOZ gallbladder tumor cells into nude mice. One week after transplantation, the mice were randomized into two groups: In Group A, the mice were treated by an intra-peritoneal injection of 0.9% sodium chloride for three weeks after inoculation (control). In Group B, the mice were treated by an intra-peritoneal injection of gemcitabine (125 mg / kg) for three weeks. All mice were sacrificed one week after the end of treatment, and macroscopic and histological findings were evaluated. The expression levels of proliferating-cell nuclear antigen (PCNA) were examined to investigate cellular proliferation activity, and Tunnel assays were performed to determine apoptotic status. Survival duration of the mice after gemcitabine treatment was compared to that of untreated mice. Results The gemcitabine sensitivity of the four biliary tract cancer cell lines was similar in a dose dependent manner. In the in vivo models, the Group A mice showed huge tumors of the gallbladder, with liver invasion and lymph node metastases. However, there were no abdominal tumors in the Group B mice, and microscopic gallbladder cancer could only be detected from histological findings. The mean percent of PCNA-positive tumor cells was significantly higher in tumors from mice in Group A (71.9%) compared to those of Group B (34.7%). The mean percent of Tunnel-positive tumor cells was significantly lower in mice from Group A (2.0%) than those from Group B (5.7%). Survival duration was prolonged significantly in the gemcitabine-treated mice relative to untreated mice. Conclusions Gemcitabine treatment may inhibit tumor progression and prolong survival in gallbladder cancer by inhibiting cell proliferation and inducing apoptosis.     

CL1-SR39: A noninvasive molecular imaging model of prostate cancer suicide gene therapy using positron emission tomography

PURPOSE: We developed a prostate cancer tumor model capable of being noninvasively imaged using positron emission tomography (PET) based on expression of the herpes simplex virus thymidine kinase (HSV1-tk) reporter gene. MATERIALS AND METHODS: The androgen independent, metastatic prostate cancer cell lines CL1 and CL1-GFP were stably transfected with the mutant HSV1-tk gene pcDNA3.1/pCMV-sr39tk, which has increased ability to phosphorylate penciclovir. The presence of the sr39tk gene product was analyzed by Western blot analysis and relative thymidine kinase enzyme activity was assessed by a functional thymidine kinase enzyme activity assay. Subcutaneous and orthotopic CL1 and CL1-SR39 tumor xenografts were established in SCID mice. The ability to image CL1-SR39 was assessed using fluorodeoxyglucose and F-penciclovir ( F-FHBG) micro-PET (a rodent PET scanner). To investigate the systemic distribution of intratumoral sr39tk injections established CL1 tumors were transiently injected with first generation adenoviral vectors carrying the sr39tk gene under control of the strong cytomegalovirus promoter Ad-CMV-HSV1-sr39tk and imaged using micro-PET. RESULTS: Transfection of sr39tk into CL1 cells was successful. CL1-SR39 thymidine kinase enzyme activity was greater than twice the activity of the glioma cell line C6-SR39 control and above the threshold necessary for micro-PET detection. Fluorodeoxyglucose micro-PET in SCID mice was positive for CL1 and CL1-SR39 tumors. Selective micro-PET of subcutaneous CL1-SR39 tumors was done using F-FHBG. Micro-PET imaging after systemic and intratumoral injection of Ad-CMV-HSV1-sr39tk revealed significant systemic transgene leakage with significant hepatic expression of sr39TK protein. CONCLUSIONS: Molecular based imaging of sr39tk transfected prostate cancer tumors and adenoviral delivered HSV1-tk suicide gene therapy based on the selective conversion and intracellular trapping of F-FHBG by sr39tk is feasible. Potential applications include noninvasive monitoring of the location, duration and intensity of gene constructs, which may contribute to the safety of clinical gene therapy protocols, and noninvasive imaging of the prostate cancer xenograft response to experimental therapy.     

Specific antitumor effect of lymphokine-activated lymphocytes obtained from tumor-bearing mice after intratumoral injection of interleukin-2 (IL-2)

Specific antitumor effects of lymphokine-activated lymphocytes obtained from tumor-bearing mice after intratumoral injection of IL-2 were studied. Inbred C57BL/6 mice bearing syngeneic tumor (B16,3LL) were used. After intratumoral consecutive injection of recombinant human IL-2 (rhIL-2), the splenocytes of these mice were cultured with rhIL-2 and the effector cells were obtained. Specific antitumor effects of the effector cells against B16 and 3LL were studied in vitro and in vivo. Surface antigens of them were also analysed. The results were as follows: 1) 51Cr-release test under coculture with rhIL-2 showed that cytotoxicity against the host tumor cells with the effector cells became specifically augmented. 2) Winn assay showed specific inhibition of the host tumor growth with the effector cells. 3) Adoptive transfer of te effector cells specifically diminished the size of the host tumor and prolonged the life span of the mice. 4) The lymphokine-activated lymphocytes obtained from normal and non-treated tumor-bearing mice had no specific antitumor effect. 5) The analysis of the surface antigens indicated that Thy1.2+L3T4+ T cells increased in the effector cells as the specific cytotoxicity of them were augmented, while in the effector cells from normal and non-treated tumor-bearing mice, Thy1.2+L3T4+ T cells decreased and Thy1.2+Lyt2+ T cells increased.     

Intratumoral injection of biological response modifier (BRM)

Specific antitumor effects of lymphokine activated lymphocytes obtained from tumor-bearing mice after intratumoral injection of IL-2 were studied. Furthermore, effects of preoperative endoscopic intratumoral injection combined IL-2 and Lentinan or OK-432 were clinically studied against gastric cancer. The results were as follows: After intratumoral consecutive injection of recombinant human IL-2 (rhIL-2), the splenocytes of these mice were cultured with rhIL-2 and the effector cells were obtained. 1) Adoptive transfer of the effector cells specifically diminished the size of the host tumor and prolonged the life span of the mice. 2) The analysis of the surface antigens indicated the Thy1.2, Thy1.2+ L3T4+ cells increased in the effector cells as the specific cytotoxicity of them were augmented. 3) Preoperative endoscopic intratumoral injection combined IL-2 and Lentinan or OK-432 induced immunocytes including antigen-presenting cells in the site of the gastric cancer, and increased the IL-2R and the LAK activity of PBL. This method was considered as effective as an adjuvant treatment of gastric cancer surgery as a in vivo sensitization.     

Convection-enhanced delivery of paclitaxel for the treatment of recurrent malignant glioma: a phase I/II clinical study

OBJECT: A minority of patients with recurrent glioblastomas multiforme (GBMs) responds to systemic chemotherapy. The authors investigated the safety and efficacy of intratumoral convection-enhanced delivery (CED) of paclitaxel in patients harboring histologically confirmed recurrent GBMs and anaplastic astrocytomas. METHODS: Fifteen patients received a total of 20 cycles of intratumoral CED of paclitaxel. The patients were observed daily by performing diffusion-weighted (DW) magnetic resonance (MR) imaging to assess the convective process and routine diagnostic MR imaging to identify the tumor response. Effective convection was determined by the progression of the hyperintense signal within the tumor on DW MR images, which corresponded to a subsequent lytic tumor response displayed on conventional MR images. Of the 15 patients, five complete responses and six partial responses were observed, giving a response rate of 73%. The antitumor effect was confirmed by one biopsy and three en bloc resections of tumors, which showed a complete response, and by one tumor resection, which demonstrated a partial response. Lack of convection and a poor tumor response was associated with leakage of the convected drug into the subarachnoid space, ventricles, and cavities formed by previous resections, and was seen in tumors containing widespread necrosis. Complications included transient chemical meningitis in six patients, infectious complications in three patients, and transient neurological deterioration in four patients (presumably due to increased peritumoral edema). CONCLUSIONS: On the basis of our data we suggest that CED of paclitaxel in patients with recurrent malignant gliomas is associated with a high antitumor response rate, although it is associated with a significant incidence of treatment-associated complications. Diffusion-weighted MR images may be used to predict a response by demonstrating the extent of convection during treatment. Optimization of this therapeutic approach to enhance its efficacy and reduce its toxicity should be explored further.     

Interleukin-12 transduced dendritic cells induce regression of established murine neuroblastoma

BACKGROUND/PURPOSE: Interleukin 12 (IL-12) is a potent proinflammatory cytokine that enhances cytotoxic T lymphocytes (CTL) and natural killer (NK) cell activity. The goal of these experiments was to assess whether adenoviral-mediated IL-12 expression by dendritic cells (DC) could induce an antitumor immune response in a murine model of neuroblastoma. METHODS: Syngeneic A/J mice were inoculated subcutaneously with 1 x 10(6) cells from a murine neuroblastoma-derived cell line (TBJ). Murine DC were transduced in vitro with adCMV-mIL-12, and 1 x 10(6) cells were injected intratumorally. Tumor growth in these mice was compared with control animals injected with enhanced green-filled protein (EGFP) transduced DC or saline. The role of CTL was evaluated through cytotoxicity assays. Immunohistochemical analysis of tumor, spleen, and lymph node was performed to characterize the behavior and fate of various populations of immune effector cells in these tissues. RESULTS: The tumors of mice injected with adIL-12 transduced DC all underwent complete regression over a 3-week period. Splenocytes isolated from mice 7 days after intratumoral injection of adIL-12 DC showed increased cytolytic activity relative to control animals in vitro. Immunohistochemistry of tumor and lymph tissue showed increased amounts of DC and T lymphocyte infiltration and a slight decrease in apoptosis relative to the control groups. CONCLUSIONS: Increased IL-12 production by DC induced a significant antitumor response in a poorly immunogenic murine neuroblastoma model. These results show the vital role of DC in the immunobiology of the disease, and that protection of these cells from tumor induced apoptosis is a critical aspect for immunotherapies treating this aggressive tumor.     

重组腺病毒介导IL-12、IL-2联合基因治疗前列腺癌的实验研究

目的　探讨转移性前列腺癌的治疗方法 ,为前列腺癌免疫基因治疗提供实验依据。　方法　采用重组腺病毒介导IL 12、IL 2联合免疫基因疗法 ,对 6 6只C5 7BL/ 6小鼠前列腺癌模型进行致瘤性和抑瘤性观察。　结果　腺病毒AdmIL 12、AdhIL 2能有效表达目的基因。接种野生型 ,转AdLacZ及转AdmIL 12、AdhIL 2混合RM 1细胞的小鼠致瘤比例分别为 10 / 10、10 / 10及 2 / 10 ;成瘤时间分别为 (12 .3± 1.5 )d、(12 .8± 1.0 )d、(2 2 .5± 2 .1)d ;接种 30d后肿瘤结节直径分别为 (35 .0±2 .0 )mm、(34 .0± 2 .6 )mm、(10 .5± 3.5 )mm。与对照组相比 ,转AdmIL 12、AdhIL 2基因RM 1细胞小鼠致瘤率下降 ,成瘤时间延迟 (P <0 .0 1)、瘤结节小 (P <0 .0 1)。AdmIL 12、AdhIL 2瘤体注射还可抑制肿瘤生长 (P <0 .0 1) ,减少肺转移灶数目 (P <0 .0 1)。　结论　IL 12、IL 2联合基因治疗可诱发前列腺癌小鼠的肿瘤特异性免疫反应。     

Use of an oncolytic virus secreting GM-CSF as combined oncolytic and immunotherapy for treatment of colorectal and hepatic adenocarcinomas

BACKGROUND: 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 multimutated oncolytic HSV carrying the gene for murine GM-CSF that attempts to combine these 2 anticancer strategies. The purpose of this study was to compare NV1034 to NV1023, the parent HSV mutants lacking GM-CSF, to determine if such combined oncolytic and immunotherapy using a single vector has advantages over oncolytic therapy alone. METHODS: Expression GM-CSF in vitro did not alter the infectivity, cytotoxicity, or replication of NV1034 compared to the noncytokine-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 and NV1023 each demonstrated potent antitumor activity. RESULTS: Treatment with NV1034 had significantly better antitumor effect compared to treatment with NV1023. Furthermore, there was no difference in the antitumor efficacy of these viruses in mice depleted of CD4+ and CD8+ T lymphocytes. CONCLUSIONS: Viral vectors combining oncolytic and immunotherapy are promising agents in treatment of colorectal carcinoma and hepatoma.     

Interleukin 12 secretion enhances antitumor efficacy of oncolytic herpes simplex viral therapy for colorectal cancer

OBJECTIVE: To 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 DATA: Genetically 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. METHODS: NV1023 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. RESULTS: Both viruses could replicate within and kill CT26 cells in vitro, with 100% cytotoxicity achieved after infection by either virus. Only NV1042 could produce mIL-12. Therapy using high viral doses to treat animals in vivo showed equal efficacy between NV1023 and NV1042, with five of seven cures for each virus. When viral doses were lowered, only the cytokine-producing NV1042 virus could reduce tumor burden and cure animals of their disease. CONCLUSIONS: Both NV1023 and NV1042 have the oncolytic potential to kill colon cancer cells at higher doses. Cytokine production by NV1042 may allow lower doses of viral therapy to be used without losing antitumor efficacy. The combination of oncolytic viral therapy and immunomodulatory strategies should be further investigated as treatment for colon cancer.     

Clinical study on the effect of intratumoral administration of OK-432 on breast cancer--anti-tumor immune response at the site of local injection]

To elucidate the mechanism of tumor reduction and local immune response after administration of OK-432 into the lesions of breast cancer, patients were given intratumoral injection of OK-432 before surgery. And precise identification of infiltrative lymphocytes was performed. The subjects were 25 patients with primary breast cancer. These patients were randomly divided into the following 3 groups: 10 patients given intratumoral injection of OK-432, 5 patients given intratumoral injection of physiological saline and 10 patients give no treatment. After operation frozen specimens of each tumor were subjected to simple immunohistochemical staining with Leu 2a, Leu 3a and Leu 7, and to immunohistochemical double staining with combination of Leu 2a x Leu 15 and Leu 3a x Leu-DR. Examination of infiltrative lymphocytes in the tumor revealed the positive rate was 100% for Leu 2a + cells (strongly positive, 70%), 90% for Leu 3a + cells (strongly positive, 70%) and 90% for Leu 7 + cells (strongly positive, 0%) in patients given intratumoral injection of OK-432. Double staining showed cytotoxic T cells and helper T cells were predominant among Leu 2a + cells and Leu3a + cells, respectively. Cytotoxic T cells, helper T cells and NK cells were induced at the tumor site after intratumoral injection of OK-432, suggesting the antitumor immune response of these cells is involved in tumor reduction following local administration of OK-432.     

Radio-guided occult lesion localization in patients undergoing breast-conserving surgery and sentinel node biopsy

BACKGROUND: The objective of the study was to evaluate the feasibility of radio-guided occult lesion localization (ROLL) in breast-conserving surgery for nonpalpable breast cancer. METHODS: Altogether 215 breast cancer patients were included in a prospective study. Ultrasonographically guided intratumoral injection of radioactive tracer was used for tumor localization in 64 patients with nonpalpable tumors, visible in breast ultrasonography (the ROLL group). Nonpalpable tumors, visible only in mammography, were localized with a help of a guidewire in 14 patients (the WGR group). The remaining 137 patients had palpable tumors. RESULTS: The proportion of specimens with close or involved margins (0 to 3 mm) was the same in the ROLL group (6%) and among cases with palpable tumors (5%). In addition the median length of the closest margin did not differ significantly between the groups. CONCLUSIONS: The results of ROLL for nonpalpable breast cancer are comparable with those for resection of palpable tumors.     

A herpes simplex virus type 1 mutant with gamma 34.5 and LAT deletions effectively oncolyses human U87 glioblastomas in nude mice

OBJECTIVE: We previously constructed a novel recombinant herpes simplex virus with deletions in the gamma 34.5 and LAT genes. LAT was replaced by the gene for green fluorescent protein, to allow monitoring of viral transduction in vitro and in vivo. We previously confirmed that this virus, designated DM33, retains its oncolytic properties in vitro and is inhibited with respect to spontaneous reactivation. The objective of this study was to demonstrate the therapeutic efficiency of this virus in the treatment of human gliomas in nude mice. METHODS: Thirty BALB/c nude mice underwent stereotactic implantation of U-87 MG gliomas in the right corpus striatum. Subsequently, mice received intratumoral inoculations of either DM33 (n = 20) or virus-free medium (n = 10). Ten mice given injections of DM33 were also treated intraperitoneally with ganciclovir. RESULTS: Intratumoral administration of DM33 to nude mice bearing intracranial U-87 MG human gliomas prolonged survival times, compared with saline-treated control animals (P < 0.05). Histological analyses of treated tumors demonstrated decreased tumor size and tumor cell lysis. Control tumors averaged 7.05 +/- 0.83 mm(2) (mean +/- standard error), whereas the average for the DM33 group was 4.61 +/- 1.57 mm(2) and that for the DM33 plus ganciclovir group was 2.49 +/- 1.32 mm(2). The difference in tumor sizes between the control group and the DM33 plus ganciclovir group was statistically significant (P = 0.044). Viral infection was limited to the tumors, and replication was not observed in normal neurons or glia. CONCLUSION: The efficacy of this virus in the treatment of experimental gliomas, its safety (as confirmed by its inability to reactivate), and its attenuated neurovirulence make DM33 a promising oncolytic agent for glioma therapy.     

Intratumoral rIL2-based immunotherapy in B16 melanoma.

Limiting factors in systemic recombinant interleukin-2 (rIL2) therapy may be overcome by intratumoral (IT) administration. A series of experiments was conducted to assess the efficacy of IT rIL2 alone and in combination with LAK cells and IFN-gamma. C57BL/6 mice bearing B16-F10 subcutaneous tumors were randomly assigned to treatment groups including: noninjected controls, IT placebo (NaCl, D5W), IT bovine serum albumin (BSA), IT rIL2 (centrally and peripherally), IT rIL2/LAK, IT rIL2/IFN-gamma, and intraperitoneal (IP) rIL2. A tumor size-dependent dose of cytokine was injected daily and LAK cells were given weekly. Systemic immune response was assessed by splenocyte mitogenesis and T-cell subset distribution using thymidine radioassay and flow cytometry, respectively. In terms of survival and tumor growth rate, IT rIL2 was superior to noninjected control, IT placebo, IT BSA, and IP rIL2 (P less than 0.05). The addition of IT LAK cells conferred no therapeutic advantage. The combination of rIL2 and gamma IFN-gamma had a slight survival benefit over rIL2 alone (30.8 days vs 20.4 days). Histologic analysis demonstrated an increase presence of intratumoral macrophages in the IT rIL2-treated tumors (P less than 0.05). Lymphocyte mitogenesis and L3T4+ subset were not altered by any treatment. In vitro thymidine uptake by tumor cells was not affected by rIL2 nor IFN-gamma alone but the combination of rIL2 and IFN-gamma resulted in significant tumor cell growth inhibition. Spontaneous lung metastases were more prevalent following central IT rIL2 (75% vs 29%, P = 0.07) not accountable by needle trauma but avoidable by the use of peritumoral injection.(ABSTRACT TRUNCATED AT 250 WORDS)