Infection rates of external ventricular drains are reduced by the use of silver-impregnated catheters.

BACKGROUND: External ventricular drainage (EVD) placement for temporary cerebrospinal fluid (CSF) diversion is a frequent therapeutic procedure. Several types of EVD catheters are currently available, some of which have an antibacterial effect. This study compares the rates of CSF infections in patients with different types of EVD catheters. METHODS: This is a retrospective study of 403 patients with a total of 529 implanted EVDs. We analyze the occurrence of EVD-associated infections, microbiological diagnosis, type of EVD catheter (plain polyurethane vs. silver-impregnated), duration of CSF diversion, primary disease, and outcome. RESULTS: There were a total of 29 patients with EVD infections in the whole study group (7.1 %). A pathogen was detected in all cases. Coagulase-negative staphylococci were detected most frequently (20 out of 29 cases, 70 %). The rate of infections by catheter type was 7.6 % (11 of 145) and 13.8 % (4 out of 29) for two different types of non-coated polyurethane catheters. Silver-impregnated polyurethane catheters became infected in 6.1 % (14 out of 228). The differences between non-coated and silver-coated catheters were statistically significant. CONCLUSIONS: This study provides comparative data on EVD infections with regard to the type of catheter. Silver-impregnated catheters showed significantly lower infection rates when compared to non-impregnated catheters. The results are critically discussed and compared with the published literature.

Intervertebral disc replacement for cervical degenerative disease--clinical results and functional outcome at two years in patients implanted with the Bryan cervical disc prosthesis.

BACKGROUND: This is a prospective study of patients with degenerative cervical disease who underwent ventral discectomy and disc replacement with the Bryan((R)) cervical disc prosthesis. The objective was to investigate clinical outcome at 2 years of patients implanted with the Bryan disc and to evaluate function of the implant itself. METHODS: Fifty-four consecutive patients with cervical disc herniation and/or spondylosis with preserved mobility in the affected spinal segments were enrolled. Patients presented clinically with cervical radiculopathy and/or myelopathy with or without neck pain. A standard anterior cervical discectomy was carried out and a Bryan disc was implanted in the affected levels. A total of 59 prosthetic discs were implanted, in 49 patients at a single level and in 5 at two adjacent levels. The neurological status was evaluated pre-operatively and at one and two years thereafter. Plain X-rays, CT, and MRI were used for pre-operative diagnostics. Post-operative follow-up was done by X-rays. FINDINGS: All patients had an excellent or good neurological outcome according to the Odom criteria. Loss of function (motion range <3 degrees) was found in 7 (12%) out of 59 Bryan discs at two years after surgery. Heterotopic ossification (HO) of the McAffee grades 1-4 was seen in a total of 17 (29%) segments. There were no implant dislocations or migrations. CONCLUSIONS: Implantation of the Bryan disc resulted in excellent or good neurological outcome in all patients. The surgical technique was safe and without complications. Twelve percent of the implanted Bryan discs lost mobility at two years, mainly due to HO. A trend was seen towards development of HO in the operated segments. Further investigations with longer follow-up periods and with a control group (e.g. fusion with intervertebral cage) will be necessary for a definitive assessment of the long-term functionality and benefits of artificial cervical discs.

Management of chronic back and leg pain by intrathecal drug delivery.

Intrathecal delivery of analgesic drugs by implantable pump systems has been recognized as a treatment option for patients with chronic pain of benign or malignant origin that is resistant to oral or parenteral medication. Patients with chronic back and leg pain (CBLP), a benign but severely disabling condition of the lumbar spine with multifactorial genesis, have been demonstrated in a number of retrospective and in some prospective clinical studies to benefit from intrathecal delivery of opioid and/or non-opioid substances, either as single drugs or in combinations. In addition, intrathecal therapy for CBLP has been proven safe and less expensive that conventional medical therapy. This chapter summarizes the clinical and experimental evidence and the personal experience of the authors with long-term intrathecal infusion therapy for CBLP. It discusses important clinical issues such as drug selection, drug combinations, and side effects and complications of intrathecal infusion. It is concluded that further clinical research is needed in order to provide stronger evidence for the usefulness of a number of drugs currently used for intrathecal therapy on a mostly empirical basis.

Dual electrode spinal cord stimulation in chronic leg and back pain.

Patients with chronic back and leg pain (CBLP) suffer from a disabling spinal condition of multifactorial origin and are often resistant to medical therapy. Spinal cord stimulation (SCS) is a minimally invasive option for treatment of chronic pain in these patients, which involves placement of epidural electrodes close to the midline of the spinal cord. SCS was originally introduced and used for decades with a single electrode. The development of fully implantable dual channel pulse generators connected to dual multicontact electrodes has given pain clinicians a more versatile tool to treat axial low back pain accompanied by radicular neuropathic pain with irregular and asymmetric distribution, a feature which is found in most CBLP patients. It has been hypothesized that using dual electrodes may improve long term outcome for CBLP patients compared with single electrodes. Current evidence however does not lend strong support to this assumption. Given the high cost of treatments for CBLP and of SCS itself, there is an urgent need for high-quality evidence for the effectiveness of dual electrode SCS in relieving pain and/or improving function in patients with CBLP.

Hardware failures in spinal cord stimulation (SCS) for chronic benign pain of spinal origin.

Spinal cord stimulation (SCS) has become an established clinical option for treatment of refractory chronic pain not related to cancer. Current hardware and implantation techniques for SCS are already highly developed and continuously improving, however equipment failures over the course of the long-term treatment are still encountered in a relatively high proportion of treated cases. Percutaneous SCS electrodes seem to be particularly prone to dislocation and insulation failures. This review summarizes the experience of the authors with management of hardware failures and their causes in patients treated with SCS for chronic pain of benign origin. The published literature is critically surveyed and discussed.

Experimental therapies for chronic pain.

Chronic pain, an underestimated but complex medical and social phenomenon, is often resistant to currently used analgesic drugs. The effect of these substances is frequently self-limiting, with increasing level of unwanted side effects caused by increased doses. Moreover, most pharmacological therapies for pain are administered systemically, either via the enteral or the parenteral route, and exert their effects on a multitude of organs and structures in the body regardless of their involvement in chronic pain pathways. Unlike pharmacological agents, biological pain therapies provide a means to target single molecules or specific types of neural cells in spatially limited areas in the central nervous system. Biological therapies utilize externally administered natural or synthetic agents acting at specific receptors on the spinal or supraspinal level, or virus or cell vectors allowing the expression and secretion of such agents in small compartments. By targeting a particular receptor or other specific protein involved in signal transmission, biological approaches to the treatment of chronic pain may provide greater analgesic efficacy without the limitations associated with current pharmacological therapies. This review summarizes published data on the most important of the currently known targets for biological therapy of chronic pain, and focuses on therapeutic approaches for modulation of these targets and on results from preclinical and clinical trials. Biological therapies for chronic pain hold great promise and are rapidly developing, but currently still are in a very early stage and therefore deemed experimental and not suitable for routine clinical use.

Absence of p53 autoantibodies in sera from glioma patients.

Alteration of the tumor suppressor gene p53 is the most frequent genetic feature of human cancer and leads to over-expression and loss of function of the p53 protein in affected cells. Patients with many types of cancer, including breast, lung, and colon carcinoma, were shown to develop auto-immune response against the overexpressed protein and to produce autoantibodies directed to immunodominant epitopes common for both wild type and mutants. The presence of p53 autoantibodies (p53-aAb) seems to be, at least in patients with breast and bronchial tumors, related to an unfavorable prognosis. The present study aimed to investigate the presence of p53-aAb in patients with malignant glioma. Sera from 70 consecutive patients with gliomas graded WHO G III and IV were collected and assayed together with sera from 30 controls. A new photometric sandwich-ELISA was used for semiquantitative analysis of p53-aAb titers. p53 gene and its protein product were examined in formalin-fixed and fresh-frozen tumor tissues using immunohistochemistry, PCR-single-strand conformational polymorphism, and sequencing. Sixty percent of the glioma cases showed immunohistochemically positive cells, thus indicating intracellular accumulation of p53. Sequencing of the hot-spot exons 5-8 revealed mutations in 39% of the tumor cases. In contrast to results in other types of malignant tumors, where up to 40% of patients have high serum titers of p53-aAb, no such antibodies were found in patients with malignant cerebral glioma despite the presence of mutated or alterated p53 protein in the primary tumors. None of the non-cancer control patients had detectable titers of p53-aAb, although sera from five of six lung cancer patients had medium to high titers. The presented data suggest that glial tumors are unusual in the absence of serum antibodies to p53. It is hypothesized that impaired function of most immunocompetent cells invading brain tumors could be the cause for the absence of an autoimmune response.

Cerebroventricular infusion of pentosan polysulphate in human variant Creutzfeldt-Jakob disease.

Variant Creutzfeldt-Jakob disease (CJD) is a transmissible spongiform encephalopathy believed to be caused by the bovine spongiform encephalopathy agent, an abnormal isoform of the prion protein (PrP(sc)). At present there is no specific or effective treatment available for any form of CJD. Pentosan polysulphate (PPS), a large polyglycoside molecule with weak heparin-like activity, has been shown to prolong the incubation period of the intracerebral infection when administered to the cerebral ventricles in a rodent scrapie model. PPS also prevents the production of further PrP(sc) in cell culture models. These properties of PPS prompted its cerebroventricular administration in a young man with vCJD. Long-term continuous infusion of PPS at a dose of 11 microg/kg/day for 18 months did not cause drug-related side effects. Follow-up CT scans demonstrated progressive brain atrophy during PPS administration. Further basic and clinical research is needed in order to address the issue of efficacy of PPS in vCJD and in other prion diseases.

A phase III clinical evaluation of herpes simplex virus type 1 thymidine kinase and ganciclovir gene therapy as an adjuvant to surgical resection and radiation in adults with previously untreated glioblastoma multiforme.

Previous uncontrolled clinical trials have shown the in vivo retrovirus (RV)-mediated transduction of glioblastoma cells with the herpes simplex virus thymidine kinase (HSV-tk) gene and subsequent systemic treatment with ganciclovir to be feasible and well tolerated. However, because of continued tumor progression in most patients, the antitumor effect could not be determined using historical controls. Here, we describe a phase III, multicenter, randomized, open-label, parallel-group, controlled trial of the technique in the treatment of 248 patients with newly diagnosed, previously untreated glioblastoma multiforme (GBM). Patients received, in equal numbers, either standard therapy (surgical resection and radiotherapy) or standard therapy plus adjuvant gene therapy during surgery. Progression-free median survival in the gene therapy group was 180 days compared with 183 days in control subjects. Median survival was 365 versus 354 days, and 12-month survival rates were 50 versus 55% in the gene therapy and control groups, respectively. These differences were not significant. Therefore, the adjuvant treatment improved neither time to tumor progression nor overall survival time, although the feasibility and good biosafety profile of this gene therapy strategy were further supported. The failure of this specific protocol may be due mainly to the presumably poor rate of delivery of the HSV-tk gene to tumor cells. In addition, the current mode of manual injection of vector-producing cells with a nonmigratory fibroblast phenotype limits the distribution of these cells and the released replication-deficient RV vectors to the immediate vicinity of the needle track. Further evaluation of the RV-mediated gene therapy strategy must incorporate refinements such as improved delivery of vectors and transgenes to the tumor cells, noninvasive in vivo assessment of transduction rates, and improved delivery of the prodrug across the blood-brain and blood-tumor barrier to the transduced tumor cells.

Helper virus-free herpes simplex virus type 1 amplicon vectors for granulocyte-macrophage colony-stimulating factor-enhanced vaccination therapy for experimental glioma.

Subcutaneous vaccination therapy with glioma cells, which are retrovirally transduced to secrete granulocyte-macrophage colony-stimulating factor (GM-CSF), has previously proven effective in C57BL/6 mice harboring intracerebral GL261 gliomas. However, clinical ex vivo gene therapy for human gliomas would be difficult, as transgene delivery via retroviral vectors occurs only in dividing cells and ex vivo glioma cells have a low growth fraction. To circumvent this problem, a helper virus-free herpes simplex virus type 1 (HSV-1) amplicon vector was used. When primary cultures of human glioblastoma cells were infected with HSV-1 amplicon vectors at an MOI of 1, more than 90% of both dividing and nondividing cells were transduced. When cells were infected with an amplicon vector, HSVGM, bearing the GM-CSF cDNA in the presence of Polybrene, GM-CSF secretion into the medium during the first 24 hr after infection was 1026 ng/10(6) cells, whereas mock-infected cells did not secrete detectable GM-CSF. Subcutaneous vaccination of C57BL/6 mice with 5 x 10(5) irradiated HSVGM-transduced GL261 cells 7 days prior to intracerebral implantation of 10(6) wild-type GL261 cells yielded 60% long-term survivors (>80 days), similar to the 50% long-term survivors obtained by vaccination with retrovirally GM-CSF-transduced GL261 cells. In contrast, animals vaccinated with the same number of nontranduced GL261 cells or with GL261 cells infected with helper virus-free packaged HSV-1 amplicon vectors carrying no transgene showed only 10% long-term survivors. In conclusion, helper virus-free HSV-1 amplicon vectors appear to be effective for cytokine-enhanced vaccination therapy of glioma, with the advantages that both dividing and nondividing tumor cells can be infected, no viral proteins are expressed, and these vectors are safe and compatible with clinical use.

Long-term survival in a rodent model of disseminated brain tumors by combined intrathecal delivery of herpes vectors and ganciclovir treatment.

Brain tumors that have disseminated into cerebrospinal fluid (CSF) pathways are an unresolved therapeutic problem, especially in pediatric neurooncology. Here a gene therapy approach using the herpes simplex virus type 1 thymidine kinase (HSV-TK)/ganciclovir (GCV) paradigm was tested using an HSV vector in a rodent model of disseminated central nervous system tumors. 9L-gliosarcoma cells were implanted simultaneously into the brain and the CSF of syngeneic rats. Five days later, resulting intracerebral and leptomeningeal tumors were treated by intrathecal injection of a replication-conditional HSV vector. This vector was defective for the ribonucleotide reductase gene, but contained an intact HSV-tk gene. Systemic GCV treatment was started 2 days after vector application and continued for 14 days. Tumor-free, long-term survival (LTS) was achieved in 90% of the animals treated with this combined therapeutic approach, whereas only 30% LTS was found in animals that had received the vector alone and 10% LTS in untreated animals. This therapeutic response probably involves oncolytic, on-site replication of the vector, activation of GCV by a HSV-TK, and a strong immune response both to the vector and to 9L cells. Apparent vector-related mortality was observed in 20% of animals without subsequent GCV therapy, but no vector-related mortality was found when the animals were treated with GCV after vector application. Given the successful outcome of this experimental treatment and the apparent potential of GCV to control HSV-related toxicity, intrathecal application of HSV vectors combined with GCV treatment may be a promising approach for treatment of disseminated brain tumors.

New prodrug activation gene therapy for cancer using cytochrome P450 4B1 and 2-aminoanthracene/4-ipomeanol.

Vector-mediated transfer of prodrug-activating genes provides a promising means of cancer gene therapy. In a search for more selective and more potent bioactivating enzymes for gene therapy of malignant brain tumors, the toxicity-generating capacity of the rabbit cytochrome P450 isozyme CYP4B1 was investigated. Rabbit CYP4B1, but not rat or human isozymes, efficiently converts the inert prodrugs, 2-aminoanthracene (2-AA) and 4-ipomeanol (4-IM), into highly toxic alkylating metabolites. Toxicity of these two prodrugs was evaluated in culture in parental and genetically modified rodent (9L) and human (U87) glioma cell lines stably expressing CYP4B1, and in vivo in a subcutaneous 9L tumor model in nude mice. The most sensitive CYP4B1-expressing glioma clone, 9L4B1-60, displayed an LD50 of 2.5 microM for 2-AA and 4-IM after 48 h of prodrug incubation, whereas 20 times higher prodrug concentrations did not cause any significant toxicity to control cells. Substantial killing of control tumor cells by 2-AA was achieved by co-culturing these cells with CYP4B1-expressing cells at a ratio of 100:1, and toxic metabolites could be transferred through medium. In both CYP4B1-expressing cells and co-cultured control cells, prodrug bioactivation was associated with DNA fragmentation, as assayed by fluorescent TUNEL assays and by annexin V staining. Alkaline elution of cellular DNA after exposure to 4-IM revealed extensive protein-DNA crosslinking with single-strand breakage. Growth of 9L-4B1 tumors in nude mice was inhibited by intraperitoneal injection of 4-IM with minimal side effects. Potential advantages of the CYP4B1 gene therapy paradigm include: the low concentrations of prodrug needed to kill sensitized tumor cells; low prodrug conversion by human isozymes, thus reducing toxicity to normal cells; a tumor-killing bystander effect that can occur even without cell-to-cell contact; and the utilization of lipophilic prodrugs that can penetrate the blood-brain barrier.

Selective uptake of viral and monocrystalline particles delivered intra-arterially to experimental brain neoplasms.

In this study we investigated the intra-arterial delivery of viral and nonviral particles to experimental brain tumors. A herpes simplex virus (HSV) vector and monocrystalline iron oxide nanoparticles (MION) were injected into the internal carotid artery of Fisher 344 rats harboring intracerebral 9L gliosarcomas, using bradykinin to disrupt the blood-tumor barrier. Brain and internal organs were stained both for virus-mediated gene expression and for iron. Quantitative comparisons of gene expression and MION uptake with and without blood-tumor barrier disruption were performed in the center and at the periphery of the tumor mass, as well as in normal brain. In addition, MION distribution was traced in vivo by MR imaging. Delivery of HSV into 9L gliosarcoma cells was greatly enhanced by intra-carotid bradykinin infusion. Virus-mediated expression of the HSV-thymidine kinase (TK) and beta-galactosidase gene products was highest at the tumor periphery as compared to the tumor center. Selective HSV infection of multiple tumor foci was achieved in both hemispheres without affecting normal brain. MION uptake was high at the tumor periphery even without blood-tumor barrier disruption. Bradykinin increased MION uptake predominantly in the center of the tumor with virtually no effect at the periphery. These findings show that selective blood-tumor barrier disruption by bradykinin can be used to enhance HSV-mediated gene delivery to tumor cells in the periphery of brain tumors. A crucial aspect in the treatment of malignant brain tumors is the eradication of tumor cells infiltrating the brain; bradykinin may facilitate access of vectors to these areas by selective disruption of their neovasculature.

Herpes vector-mediated delivery of marker genes to disseminated central nervous system tumors.

The present study investigated the ability of a recombinant herpes simplex virus type 1 (HSV) vector to deliver genes into disseminated brain tumor foci through intrathecal injection of the vector. The animal model was designed to simulate brain tumors with cerebrospinal fluid (CSF) metastases, which are found especially in the pediatric population. 9L gliosarcoma cells were injected both into the right frontal lobe and in through the cisterna magna of adult rats. The HSV vector, hrR3, was inoculated intrathecally 5 days later. This vector is defective in the gene for ribonucleotide reductase, and, therefore, replicates preferentially in dividing cells; it retains an intact HSV-thymidine kinase gene (HSV-tk). Two days after injection of the vector, immunohistochemical staining for HSV thymidine kinase (HSV-TK) revealed expression in frontal tumors, as well as in leptomeningeal tumor foci along the entire neuroaxis. HSV-TK-immunopositive cells were most frequent in small tumors contacting the CSF pathways. Frontal lobe tumors showed the highest density of HSV-TK-immunopositive cells around their periphery with little expression in central parts. Some paraventricular neurons temporarily showed HSV-TK-immunolabeling at this early time point. The number of HSV-TK-immunopositive tumor cells markedly decreased 5 days after injection of the HSV vector. In all animals, some toxicity was observed in the first 2-4 days after virus injection with extensive leptomeningeal inflammation. In conclusion, intrathecal application of HSV vectors can mediate widespread transfer of the therapeutic HSV-tk gene into disseminated tumors throughout the brain and CSF pathways. Although there was marked toxicity associated with intrathecal injection of this vector, this mode of gene delivery offers a promising approach for treatment of CSF-metastases in conjunction with development of less toxic vectors.

Intraarterial delivery of adenovirus vectors and liposome-DNA complexes to experimental brain neoplasms.

This study investigated the intraarterial delivery of genetically engineered replication-deficient adenovirus vectors (AVs) and cationic liposome-plasmid DNA complexes (lipoDNA) to experimental brain tumors. Adenovirus or lipoDNA was injected into the internal carotid artery (ICA) of F344 rats harboring intracerebral 9L gliosarcomas, using bradykinin (BK) to selectively permeabilize the blood-tumor barrier (BTB). Brain and internal organs of the animals were collected 48 hr after vector injection and stained for expression of the marker gene product, beta-galactosidase (beta-Gal). Intracarotid delivery of AV to 9L rat gliosarcoma without BTB disruption resulted in transgene expression in 3-10% of tumor cells distributed throughout the tumor. Virus-mediated expression of beta-gal gene products in this tumor model was particularly high in small foci (< or = 0.5 mm), which had invaded the normal brain tissue surrounding the main tumor mass. In these foci more than 50% of tumor cells were transduced. BK infusion increased the amount of transgene-expressing cells in larger tumor foci to 15-30%. In the brain parenchyma only a few endothelial cells expressed beta-gal owing to AV-mediated gene transfer. Intracarotid delivery of lipoDNA bearing a cytoplasmic expression cassette rendered more than 30% of the tumor cells positive for the marker gene without BTB disruption. The pattern of distribution was in general homogeneous throughout the tumor. BK infusion was able to increase further the number of transduced tumor cells to more than 50%. Although lipoDNA-mediated gene transfer showed increased efficacy as compared with AV-mediated gene transfer, it had less specificity since a larger number of endothelial and glial cells also expressed the transgene. AV and lipoDNA injections, in the absence and presence of BK, also resulted in transduction of peripheral organs. AV showed its known predilection for liver and lung. In the case of lipoDNA, parenchymal organs such as liver, lung, testes, lymphatic nodes, and especially spleen, were transduced. These findings indicate that intracarotid application of AV and lipoDNA vectors can effectively transduce tumor cells in the brain, and that BTB modulation by BK infusion can further increase the number of transgene-expressing tumor cells.

Therapeutic efficiency and safety of a second-generation replication-conditional HSV1 vector for brain tumor gene therapy.

A second-generation replication-conditional herpes simplex virus type 1 (HSV) vector defective for both ribonucleotide reductase (RR) and the neurovirulence factor gamma34.5 was generated and tested for therapeutic safety and efficiency in two different experimental brain tumor models. In culture, cytotoxic activity of this double mutant HSV vector, MGH-1, for 9L gliosarcoma cells was similar to that of the HSV mutant, R3616, which is defective only for gamma34.5, but was significantly weaker than that of the HSV mutant hrR3, which is defective only for RR. The diminished tumoricidal effect of the gamma34.5 mutants could be accounted for by their reduced ability to replicate in 9L cells. The MGH-1 vector did not achieve significant prolongation of survival in vivo in the syngeneic 9L rat gliosarcoma model for either single brain tumor focus or multiple intracerebral and leptomeningeal tumors, when the vector was applied intratumorally or intrathecally, respectively, and with or without subsequent ganciclovir (GCV) treatment. In identical 9L brain tumor models with single and multiple foci, application of hrR3 with or without GCV was previously shown to result in marked long-term survival. Contrary to the findings with intrathecal injection of hrR3, no vector-related mortality was observed in any animals treated with MGH-1. Thus, in these rat brain tumor models, the double mutant, replication-conditional HSV vector MGH-1 showed a higher therapeutic safety than the RR-minus vector, hrR3, but had clearly decreased therapeutic efficiency compared to hrR3. The development of new HSV vectors for brain tumor gene therapy will require a balance between maximizing therapeutic efficacy and minimizing toxicity to the brain. Standardized application in brain tumor models as presented here will help to screen new HSV vectors for these requirements.

Characterization of a canine glioma cell line as related to established experimental brain tumor models.

A large animal tumor model for anaplastic glioma has been recently developed using immunotolerant allogeneic Beagle dogs and an established canine glioma cell line, J3T. This model offers advantages in terms of tumor morphology and similarity to human anaplastic glioma. The present study was aimed at evaluating the biological characteristics of the J3T canine glioma cell line as related to experimental gene therapy studies. Furthermore, development and morphology of canine brain tumors in a xenogeneic immunodeficient SCID mouse model was investigated. It was demonstrated that cultured J3T cells can be efficiently infected by adenovirus (AV), herpes-simplex type I (HSV), or retrovirus (RV) vectors, as well as by non-virus vectors such as cationic liposome/DNA complexes. Thus, in terms of infectability and transfectability, J3T cells seem to be closer to human glioma than the 9L rodent gliosarcoma. Cytotoxicity of selection antibiotics such as G418, puromycin, and hygromycin on J3T cells essentially resemble cytotoxicity seen with other established glioma lines, for example, 9L, U87, or U343. RV-mediated HSV-TK/GCV gene therapy demonstrated comparable LD50 for TK-expressing and control (non-expressing) J3T and 9L cells treated with Ganciclovir. Further, it was proven that J3T cells are tumorigenic and may grow heterotopically and orthotopically in a xenogeneic immunodeficient host, the SCID mouse, although morphology and growth pattern of these xenogeneic tumors differ from the demonstrated invasive phenotype in the Beagle dog.

Association of Wilms' tumor with primary brain tumor in siblings.

The cases of two young male siblings independently developing unilateral Wilms' tumors and brain tumors are reported. The renal tumors were resected; the first child was treated with chemotherapy and the second child was given additional radiotherapy. Five years after treatment, both children developed a second primary neuroectodermal tumor. All four tumors showed a high proliferative activity, and rapidly progressing disease led to the death of the first child. Histopathological and molecular studies were carried out on all four neoplasms. No functionally relevant mutation was found in selected exons of the p53, K-ras and WT1 gene loci of tumor and germ line DNA. Since additional family members had developed brain tumors and carcinomas, this peculiar association of neoplasms may be due to germ line mutation of a hitherto unidentified oncogene acting in a recessive or weakly dominant fashion.

Gene therapy for brain tumors.

Gene therapy has opened new doors for treatment of neoplastic diseases. This new approach seems very attractive, especially for glioblastomas, since treatment of these brain tumors has failed using conventional therapy regimens. Many different modes of gene therapy for brain tumors have been tested in culture and in vivo. Many of these approaches are based on previously established anti-neoplastic principles, like prodrug activating enzymes, inhibition of tumor neovascularization, and enhancement of the normally weak anti-tumor immune response. Delivery of genes to tumor cells has been mediated by a number of viral and synthetic vectors. The most widely used paradigm is based on the activation of ganciclovir to a cytotoxic compound by a viral enzyme, thymidine kinase, which is expressed by tumor cells, after the gene has been introduced by a retroviral vector. This paradigm has proven to be a potent therapy with minimal side effects in several rodent brain tumor models, and has proceeded to phase 1 clinical trials. In this review, current gene therapy strategies and vector systems for treatment of brain tumors will be described and discussed in light of further developments needed to make this new treatment modality clinically efficacious.

Rabbit cytochrome P450 4B1: A novel prodrug activating gene for pharmacogene therapy of hepatocellular carcinoma.

Gene therapy using vector-mediated transfer of prodrug activating genes is a promising treatment approach for malignant tumors. As demonstrated recently, the novel prodrug activating gene coding for rabbit cytochrome P450 4B1 (CYP4B1) is able to induce tumor cell death at low micromolar concentrations in glioblastoma cells after treatment with the prodrug 4-ipomeanol (4-IM) in vitro and in vivo. The rabbit CYP4B1 converts this prodrug and other furane analogs and aromatic amines, such as 2-aminoanthracene, to highly toxic alkylating metabolites, whereas the human isoenzyme exhibits only minimal enzymatic activity. In the present study, the cDNA encoding rabbit CYP4B1 was used for pharmacogene therapy of hepatocellular carcinoma (HCC). Cell clones derived from the human HCC cell lines Hep3B, HuH-7, and HepG2 and stably expressing the chimeric protein CYP4B1-EGFP (the CYP4B1 coding sequence fused to the enhanced green fluorescent protein (EGFP) gene) were selected. HCC clones expressing EGFP served as controls. 4-IM rapidly induced tumor cell death in CYP4B1-EGFP-expressing clones at low concentrations (a 50% lethal dose of between 0.5 and 2 microg/mL). No signs of toxicity were found in control cells expressing EGFP even at high prodrug concentrations (20 microg/mL). Cell death occurred by apoptosis and was independent of functional p53. A pronounced direct bystander effect was observed in Hep3B cells, whereas bystander HepG2 and HuH-7 cells were highly resistant to toxic 4-IM metabolites. These results demonstrate that the CYP4B1/4-1M system efficiently and rapidly induces cell death in HCC cells, and that a cell line-specific mechanism may exist that limits the extent of the bystander effect of this novel prodrug activating system.