Novel therapies for malignant gliomas: a local affair?

Advances in medical and surgical treatments in the last few decades have resulted in quantum leaps in the overall survival of patients with malignant disease outside the central nervous system, whereas survival of patients with malignant gliomas (World Health Organization Grades III and IV) has remained essentially unchanged. Resection and external-beam fractionated radiotherapy remain the pillars of therapy for malignant gliomas and have shown significant beneficial effects on outcome in many clinical studies. On the other hand, numerous human trials with adjuvant agents, most of them administered systemically and causing serious complications and side effects, have not succeeded in achieving a noteworthy additional extension of survival duration, or have done so only with a considerable deterioration in the quality of life of the treated patients. The concept of local invasiveness of gliomas is not new, but only in the last one to two decades has significant attention been focused on the cell biology and molecular genetics of gliomas. Improved understanding of the fundamental features of tumor cells has resulted in the introduction and increasing clinical use of local therapies in which practitioners opt for spatially defined delivery methods and tumor-selective agents specifically designed to be used in the environment of a brain invaded by glioma. In this review, the authors summarize the key findings in some of the most important clinical studies of locally administered treatments for malignant glioma. A few such therapies have emerged in the last decade, and have shown considerable antitumor activity and a favorable profile of local and systemic side effects. These include biodegradable polymers for interstitial chemotherapy, targeted toxins administered by convection-enhanced delivery, and intra- and peritumorally injected genetically modified viruses conferring glioma-selective toxicity. In addition, areas of possible improvement of these therapies and essential further developments are outlined.

Expression of soluble guanylyl cyclase. Catalytic activity requires two enzyme subunits.

Purified soluble guanylyl cyclase consists of two subunits (70 and 73 kDa) whose primary structures were recently determined. The availability of cDNA clones coding for either subunit allowed to study the question of the functional roles of the two subunits in expression experiments. Enzyme subunits were expressed in COS-7 cells by transfection with expression vectors containing the coding region for the 70 of 73 kDa subunit of the enzyme. No significant elevation in the activity of soluble guanylyl cyclase was observed in cells transfected with cDNA coding for one of the subunits. In contrast, transfection of cells with cDNAs coding for both subunits resulted in a marked increase in activity of soluble guanylyl cyclase. Enzyme activity was stimulated about 50-fold by sodium nitroprusside. The results indicate that formation of cyclic GMP by soluble guanylyl cyclase requires both 70 and 73 kDa subunits.

Combined effects of temozolomide and the ribonucleotide reductase inhibitors didox and trimidox in malignant brain tumor cells.

PURPOSE: Temozolomide (TMZ), an oral alkylating agent with good penetration of the blood-brain barrier, has shown efficacy in the treatment of malignant brain tumors. Ribonucleotide reductase (RR), the rate-limiting enzyme of DNA synthesis, seems to be a complementary target for combination chemotherapy of brain tumors. Trimidox (TX) and didox (DX) are two recently synthesized specific inhibitors of RR. The combinations of TMZ with TX or DX as a basis for synergistic chemotherapy protocols were tested in this study. METHODS: The effects of the single drugs TMZ, DX, and TX, and the combinations TMZ/DX and TMZ/TX were evaluated in the human malignant glioma cell lines U87MG, T98G, LNZ308, and wt1119. In the latter, experiments were carried out in the presence or absence of wild-type p53 protein expressed under the control of a tetracycline-responsive transgene system. Cytotoxicity was evaluated by MTT assays. The isobologram and combination index (CI) method of Chou-Talalay were used to evaluate interactions between drugs. RESULTS: All drugs demonstrated cytotoxicity in brain tumor cells. Synergistic cytotoxic effects (CI<1) for TMZ and TX or DX at different dose levels were demonstrated in most of the examined cell lines. In some instances, however, drug combinations resulted in additive or even antagonistic effects. Toxicity of the single agents and synergy of the combinations did not correlate with wild-type p53 expression in the tumor cells. CONCLUSION: The tumor toxicity of TMZ as a single agent may be modified by combinations with the novel RR inhibitors DX and TX, and is synergistically enhanced in most cases. Depending on the combination ratio, the doses for each drug for a given degree of effect in the combination may be drastically reduced.

Screening for mutations of the human thyroid peroxidase gene in patients with congenital hypothyroidism.

While congenital hypothyroidism in 80-90% of the affected individuals is caused by thyroid dysgenesis (athyrosis, ectopy or hypoplasia), hypothyroidism in patients with a thyroid gland of normal position and size can be due to regulatory or enzymatic defects of thyroid hormone biosynthesis. Beside defects of thyroglobulinsynthesis, defects of the sodium-iodide-transporter or the TSH-receptor, a defect of the thyroidperoxidase, the key-enzyme of thyroid hormone biosynthesis, can cause a total iodide organification defect and thereby congenital hypothyroidism. We screened 14 of 103 patients (13.6%) with non familial congenital hypothyroidism and a normally developed thyroid gland detected by the newborn screening program with the PCR-SSCP (single-stranded-conformational-polymorphism) technique for mutations in the exons 2, 8, 9, 10 and 14 of the human thyroperoxidase gene, and in which mutations had been described previously in Dutch and Brazilian families with total organification defects. Most of the previously reported mutations were found in exons 8, 9 and 10 which code for the caralytic part of the enzyme. In two patients a GGCC-duplication in exon 8 was detected leading to a premature stop codon in exon 9. While one patient without neonatal goiter was homozygous for this mutation, the second patient was only heterozygous thus demanding another mutation on the second TPO-allel to explain the phenotype. Since the GGCC duplication is easily demonstrable by a NaeI digestion, because it creates a restriction site for this enzyme, screening for this mutation is indicated since it is easy to perform. In contrast to the perchlorate discharge test molecular genetic studies are less invasive, but as useful in making a definitive diagnosis in the individual patient. Furthermore it is the first feasible step to study the etiology and epidemiology of the so far only putative defects of thyroid hormone biosynthesis leading to congenital hypothyroidism.

Expression and clinical relevance of NY-ESO-1, MAGE-1 and MAGE-3 in neuroblastoma.

Human genes NY-ESO-1, MAGE-1 and MAGE-3 code for antigens which are expressed in malignancies of various histological types but not in normal tissues except testis. These antigens might therefore represent potential targets for specific immunotherapy. We studied the expression of genes NY-ESO-1, MAGE-1 and MAGE-3 in 98 neuroblastoma tumors by reverse transcription-polymerase chain reaction (RT-PCR). MAGE-1 was expressed in 66%, NY-ESO-1 in 36% and MAGE-3 in 33% of the tumors. NY-ESO-1 gene expression was associated with age older than one year (p = 0.017), more differentiated tumor histology (p = 0.044), elevated urinary vanillylmandelic acid (VMA, p = 0.018) and normal serum ferritin levels (p = 0.023). MAGE-1 expression correlated significantly with normal serum ferritin levels (p = 0.009) and absence of MycN amplification (p = 0.007) while MAGE-3 expression was associated with absence of metastasis (p = 0.027). We conclude that approximately 70% of the neuroblastoma tumors express at least one of the genes coding for NY-ESO-1, MAGE-1 or -3, respectively.

Identification of proteins resembling G-protein alpha subunits in locust muscle.

In locust skeletal muscle, FMRFamide-like peptides decrease a K+ conductance. Functional data suggest the involvement of G-proteins. For identification of G-protein alpha-subunits, membranes of locust skeletal muscle were probed with ADP-ribosylating bacterial toxins, the photoreactive GTP analog, [alpha-32P]GTP azidoanilide, and with antibodies against mammalian alpha-subunits. Multiple guanine nucleotide-binding proteins of approximately 24-95 kDa were detected. Pertussis toxin catalyzed the ADP-ribosylation of two proteins comigrating with the ADP-ribosylated alpha-subunits of the mammalian G-proteins Go and Gi. Cholera toxin promoted ADP-ribosylation of a protein comigrating with mammalian cholera toxin substrates (i.e., Gs alpha-subunits). An antibody against mammalian Go alpha-subunits detected a 54-kDa protein. Thus proteins with properties of mammalian G-protein subunits are present in insect muscle.

Dendritic cell therapy of primary brain tumors.

BACKGROUND: Although current treatment modalities for malignant gliomas, such as surgery, radiation and chemotherapy, have been improved markedly in the past two decades, the prognosis of these neoplasms remains poor, the two year survival rate being approximately 5%. Therefore, alternative treatment options, such as gene therapy and immunotherapy are rapidly gaining momentum. One of the most promising immunotherapeutic approaches for the treatment of cancer is the vaccination of cancer patients with dendritic cells (DC) pulsed with tumor antigens. Immunotherapy with DC seems to be able to overcome, at least partially, the immunosuppressive state associated with primary malignant gliomas. DC therapy proved to be safe in both animal models and clinical trials. No serious side effects and no evidence of autoimmune toxicity occurred. Most studies used DC pulsed with an array of tumor-associated antigens rather than single peptides, to allow for presentation of unknown tumor-specific antigens to DC. Routes of administration either were subcutaneous, intradermal or intraperitoneal, with multiple injections of DC to enhance antitumor immunity. DC therapy as an adjuvant treatment for patients with malignant glioma seems to be biologically safe. Further clinical studies are warranted.

G12 and G13 alpha-subunits are immunochemically detectable in most membranes of various mammalian cells and tissues.

The cDNAs of two putatively pertussis toxin-insensitive G-protein alpha-subunits, alpha 12 and alpha 13, were recently cloned. mRNA analyses based on the reverse transcriptase polymerase chain reaction indicated a widespread distribution of both mRNAs [Strathmann, M. P., and Simon, M. I. (1991) Proc. Natl. Acad. Sci. USA 88, 5582-5586]. Generating specific antibodies directed against internal and C-terminal peptide sequences, we identified alpha 12 protein in all and alpha 13 protein in most tissues and cell lines tested. No species differences were observed, indicating a high degree of identity between mammalian species. Strong immunoreactive signals of both proteins were obtained in neuronal cell membranes of various species. Our results support the hypothesis that G12 and G13 are involved in pertussis toxin-insensitive pathways of signal transduction common to most tissues.

Immunogene therapy of recurrent glioblastoma multiforme with a liposomally encapsulated replication-incompetent Semliki forest virus vector carrying the human interleukin-12 gene--a phase I/II clinical protocol.

Glioblastoma multiforme (GBM) is an incurable brain tumor resistant to standard treatment modalities such as surgery, radiation, and chemotherapy. Since recurrent GBM tends to develop predominantly within the infiltrative rim surrounding the primary tumor focus, novel therapy strategies need in addition to focal tumor destruction to target this somewhat diffuse area. This is a phase I/II clinical study in adult patients with recurrent GBM which is aimed at evaluating biological safety, maximum tolerated dose, and antitumor efficacy of a genetically modified replication-disabled Semliki forest virus vector (SFV) carrying the human interleukin 12 (IL-12) gene and encapsulated in cationic liposomes (LSFV-IL12). The vector will be administered in doses of 1 x 10(7)-1 x 10(9) infectious particles by continuous intratumoral infusion, thus exploiting the advantages of convection-enhanced drug delivery in the brain. The present protocol is also designed to investigate systemic and local immune response and to identify factors predicting tumor response to LSFV-IL12 therapy, such as volume of extracellular space of the tumor, volume of contrast enhancing lesion, and immune status of the patients. SFV, an insect alphavirus, infects mitotic and non-mitotic cells and triggers apoptosis in tumor cells within 48-72 h. Preclinical work with the LSFV-IL12 vector in breast and prostate cancer animal models demonstrated its biosafety and some antitumor efficacy. An ongoing phase I clinical study in patients with melanoma and renal cell carcinoma seems also to confirm the biosafety of intravenously administered vectors. This protocol will be the first study of SFV-IL12 therapy of human recurrent GBM.