Mesothelioma cancer center

The idea using of gene therapy began with the straight-forward paradigm of treating genetic diseases in which one gene was missing. Although this strategy seems conceptually simple, it has proved to be difficult to implement in practice because the vectors currently available have been unable to provide sufficient levels of gene product over long enough periods of time. Accordingly, most current gene therapy trials are aimed at acquired diseases (such as cancer) in which long-term gene expression is not necessarily required. Recent advances in the understanding of growth factors, molecular oncology, and tumor immunology have provided the rationale for several strategies (Table 1). Many of these approaches have been applied to thoracic malignancies in animal and/or human trials.

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Strategies for Anticancer Gene Therapy

Molecular Chemotherapy

One of the most well-developed approaches to the treatment of cancer using gene therapy is based on the use of "suicide genes" in which DNA that encodes an enzyme capable of generating a toxic metabolite is transferred to tumor cells followed by administration of the nontoxic enzyme substrate.[1] The two systems that are currently most well developed use the bacterial cytosine deaminase gene and the herpes simplex thymidine kinase gene (HSVtk).[2] The gene-encoding cytosine deaminase converts 5-fluorocytosine to the cytotoxic antimetabolite fluorouracil (5-FU). The HSVtk gene converts the normally nontoxic nucleoside analog, ganciclovir (GCV), into a monophosphorylated form that is then converted by the normal mammalian thymidine kinase enzymes to a triphosphorylated form that is extremely toxic and leads to cell death.

Both of these approaches are bolstered significantly by the presence of the "bystander effect,"[1-3] a phenomenon that involves the transfer of toxic metabolites from transduced cells to nontransduced cells allowing for amplification of cytotoxic activity.[1,3,4] Enhanced immune responses to the dying cells may also play a role in vivo. Thus, relatively complete cell killing has been observed with as few as 10% of the tumor cells expressing the HSVtk gene[3,5] or with only 2 to 10% of cells using the cytosine deaminase gene.[2,6]

The use of suicide genes is currently limited to treatment of localized malignancies in which reasonable levels of gene transfer can be expected. A number of approaches have been used to introduce suicide genes into tumors, including injection of "producer" cell lines that secrete retrovirus containing the suicide gene,[7] systemic injection of retroviral vector,[8] direct intratumoral injection of the suicide gene complementary DNA (cDNA),[9] or direct injection of an adenoviral vector containing the suicide gene (see below). Injection of producer cell lines that secrete retrovirus containing the HSVtk gene followed by treatment with GCV has been used successfully in animal models of brain tumor, ovarian cancer, and hepatic carcinoma (reviewed by Moolten[3]). The use of the HSVtk/GCV system has been explored more recently using an adenoviral vector to directly transduce malignant mesothelioma, lung cancer, colon carcinoma, hepatocellular carcinoma, brain tumors, and melanomas.[10-16]

Because of its localized nature, extremely poor prognosis, and lack of effective therapy, malignant mesothelioma has been identified as a reasonable target for localized thoracic cancer gene therapy. Replication-deficient adenovirus efficiently transduced mesothelioma cells both in tissue culture and in animal models,[5,17] and infection with an adenovirus containing the HSVtk gene driven by the Rous sarcoma virus promoter (AdRSVtk) rendered human mesothelioma cells sensitive to doses of GCV that were 2 to 4 logs lower than the doses required to kill cells infected with control virus.[5] Based on these results, the AdRSVtk vector has been used to treat established human mesothelioma tumors and human lung cancers growing within the peritoneal cavities of severe combined immunodeficient (SCID) mice.[10,11] Following GCV therapy, macroscopic tumor was eradicated in 90% of animals and microscopic tumor was undetectable in 80% of animals. Tumor reduction was accompanied by a significant increase in survival. Marked decreases in tumor size have also been seen in a rat model of syngeneic mesothelioma cells growing in the pleural space; however, survival increases have been more modest in this model.[12]

Based on these studies, a phase I clinical trial for patients with mesothelioma began in November 1995 at the University of Pennsylvania Medical Center in conjunction with Penn's Institute for Human Gene Therapy. The purpose of the phase I trial was to determine the maximal tolerated dose of AdRSVtk virus instilled into the pleural space, to evaluate the biological effects of therapy, and to evaluate, in preliminary fashion, any response rate. The eligibility criteria for this trial are detailed in Table 2. The protocol is summarized in Table 3. Patients with a presumed diagnosis of malignant mesothelioma had a chest tube placed and the virus instilled into the pleural space. Three days after viral instillation, another biopsy specimen was obtained and IV GCV therapy was started for 14 days. The patients were carefully evaluated for evidence of toxic reactions, viral shedding, immune responses to the virus, and radiographic evidence of tumor response.

Table 2 -- Inclusion Criteria for Penn Phase I Trial of Gene Therapy for Mesothelioma

Criteria

1. All patients must have histologically proved malignant

mesothelioma of pleural origin that cannot be surgically

resected. 2. Patients must have measurable or evaluable disease. 3. Patients must have an Eastern Cooperative Oncology Group

performance status of 1 or 2. 4. Patients must have a WBC count of [greater than or equal to]

4,000/[mm.sup.3] and platelet

count of [greater than or equal to] 100,000/[m.sup.3]. 5. Patients must have a BUN [is less than] 30 mg/dL, creatinine

[is less than] 1.3 mg/

dL, and bilirubin [is less than] 2.0 mg/dL. 6. Patients may not have received prior chemotherapy or gene

therapy. 7. Patients may not have received prior radiotherapy. 8. Patients may not have undergone treatment of a pleural

effusion with placement of a chest tube and sclerosis. 9. Patients must give written informed consent. 10. Pretreatment [FEV.sub.1] must be [is greater than] 2.0 L and patient must be medically able to undergo pulmonary resection. 11. Patients with a history of another malignancy within the last 5 years, other than basal or squamous cell carcinoma of the skin, or cervical in situ carcinoma will be excluded. 12. Patients with brain metastasis at time of study are ineligible. 13. Patients with a serious active infection or other serious illness are ineligible. 14. Patients who are pregnant or lactating or fertile men or women who are not using effective contraception are ineligible. 15. Patients with HIV infection are ineligible.

Table 3 -- Clinical Protocol for Penn Phase I Trial of Gene Therapy for Mesothelioma

Protocol

Day 1. Patients with a presumed diagnosis of malignant

mesothelioma are admitted to the Clinical Research Center and

have a video thoracoscopy during which a chest tube is placed

and biopsy specimens are taken to confirm the diagnosis of

mesothelioma and to assess disease extent. Day 2. Adenovirus containing the HSVtk gene is instilled via the

chest tube and the tube removed. Day 5. Patients undergo a second video thoracoscopy in which

biopsy specimens are taken to evaluate the degree of

inflammation and the efficiency and extent of gene transfer. Days 6-20. IV GCV therapy is begun at a dose of 5 mg/kg bid for

14 days. Day 21 and onward. The patients are discharged from the hospital,

followed up closely as outpatients with clinical examinations,

hematologic and biochemical blood testing, chest radiographs,

and CT scans.

Currently, eight patients have been treated: three patients at the initial dose of virus (1 X [10.sup.9] plaque-forming units [pfu]), the next two at 1 X [10.sup.9.5] Pfu, and the last three at 1 X [10.sup.10] pfu. Although analysis is still ongoing, clinical toxicity has been limited to fever (maximum temperature of 39.4. [degrees] C) approximately 12 h after instillation of the virus and asymptomatic and transient increases in levels from liver function tests. Gene transfer has been visualized by in situ hybridization in a small percentage of cells in one of the patients receiving the high dose of virus. High titers of neutralizing antibodies and [CD4.sup.+]-T-cell proliferative responses to adenoviral structural proteins have been observed within 1 to 2 weeks of viral administration in both blood and pleural fluid. Clinical observation is still ongoing; however, there have been no deaths or evidence of tumor progression noted so far.

Genetic Immunopotentiation