Patterns, predictors and prognostic relevance of high-grade hematotoxicity after temozolomide or temozolomide-lomustine in the CeTeG/NOA-09 trial.

PURPOSE: In the randomized phase III trial CeTeG/NOA-09, temozolomide (TMZ)/lomustine (CCNU) combination therapy was superior to TMZ in newly diagnosed MGMT methylated glioblastoma, albeit reporting more frequent hematotoxicity. Here, we analyze high grade hematotoxicity and its prognostic relevance in the trial population. METHODS: Descriptive and comparative analysis of hematotoxicity adverse events ≥ grade 3 (HAE) according to the Common Terminology of Clinical Adverse Events, version 4.0 was performed. The association of HAE with survival was assessed in a landmark analysis. Logistic regression analysis was performed to predict HAE during the concomitant phase of chemotherapy. RESULTS: HAE occurred in 36.4% and 28.6% of patients under CCNU/TMZ and TMZ treatment, respectively. The median onset of the first HAE was during concomitant chemotherapy (i.e. first CCNU/TMZ course or daily TMZ therapy), and 42.9% of patients with HAE receiving further courses experienced repeat HAE. Median HAE duration was similar between treatment arms (CCNU/TMZ 11.5; TMZ 13 days). Chemotherapy was more often discontinued due to HAE in CCNU/TMZ than in TMZ (19.7 vs. 6.3%, p = 0.036). The occurrence of HAE was not associated with survival differences (p = 0.76). Regression analysis confirmed older age (OR 1.08) and female sex (OR 2.47), but not treatment arm, as predictors of HAE. CONCLUSION: Older age and female sex are associated with higher incidence of HAE. Although occurrence of HAE was not associated with shorter survival, reliable prediction of patients at risk might be beneficial to allow optimal management of therapy and allocation of supportive measures. TRIAL REGISTRATION: NCT01149109.

Cross-sectional increase of adherence to multidisciplinary tumor board decisions.

BACKGROUND: Cancer research has made great progress in the recent years. With the increasing number of options in diagnosis and therapy the implementation of tumorboards (TUBs) has become standard procedure in the treatment of cancer patients. Adherence tests on tumor board decisions are intended to enable quality assurance and enhancement for work in tumor boards in order to continuously optimize treatment options for cancer patients. METHODS: Subject of this study was the adherence of the recommendations made in three of 14 tumorboards, which take place weekly in the Center for Integrated Oncology (CIO) at the University Hospital Bonn. In total, therapy recommendations of 3815 patient cases were checked on their implementation. A classification into four groups has been made according to the degree of implementation. A second classification followed regarding the reasons for differences between the recommendation and the therapy which the patient actually received. RESULTS: The study showed that 80.1% of all recommendations in the three TUBs were implemented. 8.3% of all recommendations showed a deviance. Most important reasons for the deviances were patient wish (36.5%), patient death (26%) and doctoral decision, due to the patient's comorbidities or side effects of the treatment (24.1%).Interestingly, deviance in all three tumor boards in total significantly decreased over time. CONCLUSIONS: Aim of the study was to clarify the use of tumor boards and find approaches to make them more efficient. Based on the results efficiency might be optimized by increased consideration of patients` preferences, improved presentation of patient-related data, more detailed documentation and further structuring of the tumor board meetings.

[Treatment of the glioma microenvironment]. / Therapie des Gliommikromilieus.

Therapeutic concepts for malignant gliomas increasingly target the genetically non-transformed tumor stroma rather than the tumor cells themselves. There are two particular compartments of the tumor stroma which are currently tackled: the vascular compartment by using antiangiogenic treatment with the aim of vascular normalization and the immune compartment with the aim of enhancing or inducing anti-tumor immunity. Although the vascular endothelial growth factor (VEGF) A antibody bevacizumab has not been approved for the treatment of malignant glioma in European countries, there is evidence from smaller trials of biological efficacy particularly in recurrent disease and the results of a large European phase III study testing the clinical efficacy are currently expected. Immunotherapies are on the verge of entering the clinical arena with the first randomized phase III clinical trials having already been completed. In these studies, active vaccination and checkpoint inhibitors which are approved for other tumor entities are being tested. This article provides an overview on the current antiangiogenic and immunological therapies for gliomas, summarizes the results of clinical trials and discusses further developments.

Bevacizumab treatment in malignant meningioma with additional radiation necrosis. An MRI diffusion and perfusion case study.

BACKGROUND AND PURPOSE: Recently two retrospective cohort studies report efficacy of bevacizumab in patients with recurrent atypical and anaplastic meningioma. Another successful therapeutic option of bevacizumab seems to be treatment of cerebral radiation necrosis. However, the antiangiogenic effects in MRI diffusion and perfusion in meningiomas have not been previously described in detail. The objective of this research was to evaluate the clinical and MR imaging effects of bevacizumab in a malignant meningioma patient harboring additional cerebral radiation necrosis. CASE PRESENTATION: We report the case of an 80-year-old woman who underwent bevacizumab therapy (5 mg/kg every 2 weeks for 2 months) for treatment of a symptomatic radiation necrosis in malignant meningiomatosis of World Health Organization (WHO) grade III. The patient was closely monitored with MRI including diffusion and perfusion studies. Upon bevacizumab therapy, the clinical situation was well stabilized over a period of 4 months until the patient unfortunately died due to pneumonia/septicemia probably unrelated to bevacizumab therapy. Consecutive MRI demonstrated 4 important aspects: (1) considerable decrease of the contrast medium (CM)-enhanced radiation necrosis, (2) mixed response with respect to the meningiomatosis with stable and predominantly growing tumor lesions, (3) a new diffusion-weighted imaging (DWI) lesion in a CM-enhanced tumor as described in gliomas, which we did not interpret as a response to bevacizumab therapy, and (4) new thrombembolic infarcts, which are a known side-effect of bevacizumab treatment. CONCLUSION: Bevacizumab is effective in the treatment of radiation necrosis. We could not confirm the potential antitumor effect of bevacizumab in this patient. However, we could describe several new radiographic effects of bevacizumab therapy in malignant meningioma.

Gliosarcoma with extensive extracranial metastatic spread and familial coincidence: A case report.

Gliosarcoma is a rare histopathological subtype of glioblastoma. Metastatic spreading is unusual. In this report, we illustrate a case of gliosarcoma with extensive extracranial metastases with confirmation of histological and molecular concordance between the primary tumor and a metastatic lesion of the lung. Only the autopsy revealed the extent of metastatic spread and the hematogenous pattern of metastatic dissemination. Moreover, the case bared a familial coincidence of malignant glial tumors as the patient's son was diagnosed with a high-grade glioma shortly after the patient's death. By molecular analysis (Sanger and next generation panel sequencing), we could confirm that both patient's tumors carried mutations in the TP53 gene. Interestingly, the detected mutations were located in different exons. Altogether, this case draws attention to the fact that sudden clinical aggravation could be caused by the rare phenomenon of metastatic spread and should therefore be always taken into consideration, even at an early disease stage. Furthermore, the presented case highlights the contemporary value of autoptic pathological examination.

Ifosfamide, carboplatin and etoposide in recurrent malignant glioma.

After failure of temozolomide, there is no established standard salvage chemotherapy for patients with recurrent glioblastoma (GBM). Two phase II trials combining ifosfamide, carboplatin and etoposide chemotherapy (ICE) showed favorable results. We therefore applied the ICE protocol to 13 patients (10 GBM, 3 anaplastic astrocytomas). Partial or complete remissions were not observed. None of the 13 patients survived progression-free for 6 months. Our retrospective analysis suggests that the ICE regimen is not effective in patients with recurrent high-grade glioma if applied at second or third relapse.

Seizures in patients with low-grade gliomas--incidence, pathogenesis, surgical management, and pharmacotherapy.

Seizures complicate the clinical course of > 80% of patients with low-grade gliomas. Patients with some tumor variants almost always have epilepsy. Diffuse low-grade gliomas (LGG) are believed to cause epilepsy through partial deafferentiation of nearby brain cortex (denervation hypersensitivity). Glioneural tumors may interfere with local neurotransmitter levels and are sometimes associated with structural abnormalities of the brain which may produce seizures. The severity of tumor associated epilepsy varies considerably between patients. Some cases may present with a first seizure. Others suffer from long-standing pharmacoresistant epilepsy. Seizure control rates of > 70-80% can be expected after complete tumor resections. Patients with drug-resistant epilepsy require a comprehensive preoperative epileptological work-up which may include the placement of subdural (and intraparenchymal) electrodes or intraoperative electrocorticography (ECoG) for the delineation of extratumoral seizure foci. Partial and subtotal tumor resections are helpful in selected cases, i.e. for gliomas involving the insula. In one series, 40% of patients presented for surgery with uncontrolled seizures, i.e. medical therapy alone often fails to control tumor-related epilepsy. Use of the newer (second generation) non-enzyme inducing antiepileptic drugs (non-EIAED) is encouraged since they seem to have lesser interactions with other medications (e.g. chemotherapy). Chemotherapy and irradiation may have some minor beneficial effects on the patients' seizure disorder. Overall 60-70% of patients may experience recurrent epilepsy during long-term follow-up. Recurrent seizures (not infrequently heralding tumor recurrence) after surgery continue to pose significant clinical problems.

[Neurological complications of neurooncological therapy]. / Neurologische Komplikationen der neuroonkologischen Therapie.

Neurological complications of therapeutic procedures for brain tumors are increasingly being recognized. These encompass the classic types of central and peripheral neurotoxicity, such as radiotherapy-induced leukoencephalopathy and platinum-induced neuropathy. However, the advent of novel protocols and targeted therapeutics has expanded the spectrum of neurological complications. A problem of considerable importance is pseudoprogression after radiochemotherapy with temozolomide. Among the new targeted drugs complications of therapy with bevacizumab are the subject of intense discussion. In this review article the neurotoxic potential of intrathecal chemotherapy, kinase inhibitors, immunological strategies and local therapies are summarized. Knowledge about neurological complications of brain tumor therapy procedures is important for risk assessment and patient information.

Nimustine (ACNU) plus teniposide (VM26) in recurrent glioblastoma.

BACKGROUND: In a previous trial (NOA-01), the combination of nimustine and teniposide showed efficacy in previously untreated glioblastoma (GBM). After establishing temozolomide as standard first-line therapy in GBM patients, the nimustine (ACNU)/teniposide (VM-26) combination has been employed as salvage chemotherapy for recurrent GBM. However, data on the toxicity and efficacy of this regimen in recurrent GBM are lacking. PATIENTS AND METHODS: In two neurooncological centers, all patients with recurrent GBM treated with nimustine (90 mg/m(2), day 1/42) and teniposide (45-70 mg/m(2), days 1-3/42) were analyzed retrospectively for progression-free survival (PFS), overall survival (OS) and toxicity. RESULTS: Thirty-five patients (median age 51 years, range 25-71 years) were identified. Six months after chemotherapy initiation, PFS was 29% and the median OS 6 months; 23% of patients were alive > or = 1 year after initiation of nimustine-teniposide chemotherapy. Grade 4 hematotoxicity was observed in 12 of 35 patients (34%) and in 14 of 83 evaluable chemotherapy courses (17%). CONCLUSIONS: The benefit of the nimustine-teniposide combination is moderate in patients with recurrent GBM. The data support the efficacy of the nimustine-teniposide chemotherapy, but the rate of high-grade hematotoxicity is increased.

DNA methylation, transcriptome and genetic copy number signatures of diffuse cerebral WHO grade II/III gliomas resolve cancer heterogeneity and development.

BACKGROUND: Diffuse lower WHO grade II and III gliomas (LGG) are slowly progressing brain tumors, many of which eventually transform into a more aggressive type. LGG is characterized by widespread genetic and transcriptional heterogeneity, yet little is known about the heterogeneity of the DNA methylome, its function in tumor biology, coupling with the transcriptome and tumor microenvironment and its possible impact for tumor development. METHODS: We here present novel DNA methylation data of an LGG-cohort collected in the German Glioma Network containing about 85% isocitrate dehydrogenase (IDH) mutated tumors and performed a combined bioinformatics analysis using patient-matched genome and transcriptome data. RESULTS: Stratification of LGG based on gene expression and DNA-methylation provided four consensus subtypes. We characterized them in terms of genetic alterations, functional context, cellular composition, tumor microenvironment and their possible impact for treatment resistance and prognosis. Glioma with astrocytoma-resembling phenotypes constitute the largest fraction of nearly 60%. They revealed largest diversity and were divided into four expression and three methylation groups which only partly match each other thus reflecting largely decoupled expression and methylation patterns. We identified a novel G-protein coupled receptor and a cancer-related 'keratinization' methylation signature in in addition to the glioma-CpG island methylator phenotype (G-CIMP) signature. These different signatures overlap and combine in various ways giving rise to diverse methylation and expression patterns that shape the glioma phenotypes. The decrease of global methylation in astrocytoma-like LGG associates with higher WHO grade, age at diagnosis and inferior prognosis. We found analogies between astrocytoma-like LGG with grade IV IDH-wild type tumors regarding possible worsening of treatment resistance along a proneural-to-mesenchymal axis. Using gene signature-based inference we elucidated the impact of cellular composition of the tumors including immune cell bystanders such as macrophages. CONCLUSIONS: Genomic, epigenomic and transcriptomic factors act in concert but partly also in a decoupled fashion what underpins the need for integrative, multidimensional stratification of LGG by combining these data on gene and cellular levels to delineate mechanisms of gene (de-)regulation and to enable better patient stratification and individualization of treatment.

Primary intraocular lymphoma: an International Primary Central Nervous System Lymphoma Collaborative Group Report.

BACKGROUND: Primary intraocular lymphoma (PIOL) is an uncommon subset of primary central nervous system lymphoma. Because it is rare and difficult to diagnose, the natural history and optimal management are unknown. PATIENTS AND METHODS: A retrospective study of 83 HIV negative, immunocompetent PIOL patients was assembled from 16 centers in seven countries. RESULTS: Median age at diagnosis was 65. Median ECOG performance status was 0. Presenting symptoms included blurred vision, decreased visual acuity, and floaters. Median time to diagnosis was 6 months. Diagnosis was made by vitrectomy (74), choroidal/retinal biopsy (6) and ophthalmic exam (3). Eleven percent had positive CSF cytology. Initial treatment was categorized as focal in 23 (intra-ocular methotrexate, ocular radiotherapy) or extensive in 53 (systemic chemotherapy, whole brain radiotherapy). Six received none; details are unknown in one. Forty-seven relapsed: brain 47%, eyes 30%, brain and eyes 15%, and systemic 8%. Median time to relapse was 19 months. Focal therapy alone did not increase risk of brain relapse. Median progression free (PFS) and overall survival (OS) were 29.6 and 58 months, respectively, and unaffected by treatment type. CONCLUSION: Treatment type did not affect relapse pattern, median PFS or OS. Focal therapy may minimize treatment toxicity without compromising disease control.

Progressive multifocal leukoencephalopathy: value of diffusion-weighted and contrast-enhanced magnetic resonance imaging for diagnosis and treatment control.

Progressive multifocal leukoencephalopathy (PML) is caused by the replication of JC virus in oligodendrocytes of immunocompromised patients. Diagnosis usually relies on the polymerase chain reaction (PCR)-based demonstration of JC virus DNA in the cerebrospinal fluid. As previous reports have suggested that some patients may benefit from antiviral therapy, non-invasive early diagnosis is highly desirable. Repetitive magnetic resonance imaging (MRI) examinations (two to nine) were obtained in seven patients (aged 40-67 years, six males, one female) with classical clinical and imaging findings of PML. Five patients had underlying hematological disorders and two acquired immune deficiency syndrome. PCR of the cerebrospinal fluid (CSF) specimen was positive for JC virus DNA in six patients. MRI sequences included T2-, T1- and diffusion-weighted (DW) images in all patients and diffusion-tensor imaging (DTI) in four cases. DTI was once performed at 3T, in the remaining patients at 1.5T. All patients received antiviral treatment with cidofovir in addition to the treatment of the underlying disorder. MRI showed areas of T2 hyperintensity with involvement of the subcortical U-fibers and restricted diffusion in all patients. Areas of diffusion abnormality correlated with disease progress. Contrast enhancement was encountered once after successful treatment and heralded clinical remission with virus elimination from the CSF. Hence, MRI including DW and contrast-enhanced images may be used to evaluate disease activity. Contrast enhancement may indicate an inflammatory response and thus herald immunologic virus elimination.

Positron emission tomography-based imaging of transgene expression mediated by replication-conditional, oncolytic herpes simplex virus type 1 mutant vectors in vivo.

To evaluate the efficiency of gene delivery in gene therapy strategies for malignant brain tumors, it is important to determine the distribution and magnitude of transgene expression in target tumor cells over time. Here, we assess the time- and vector dose-dependent kinetics of recombinant herpes simplex virus (HSV)-1 vector-mediated gene expression and vector replication in culture and in vivo by a recently developed radiotracer method for noninvasive imaging of gene expression (J. G. Tjuvajev et al., Cancer Res., 55: 6126-6132, 1995). The kinetics of viral infection of rat 9L gliosarcoma cells by the replication-conditional HSV-1 vector, hrR3, was studied by measuring the accumulation rate of 2-[14C]-fluoro-5-iodo-1-beta-D-arabinofuranosyl-uracil (FIAU), a selective substrate for viral thymidine kinase (TK). The level of viral TK activity in 9L cells was monitored by the radiotracer assay to assess various vector doses and infection times, allowing vector replication and spread. In parallel, viral yields and levels of Escherichia coli beta-galactosidase activity were assessed quantitatively. To study vector replication, spread and HSV-1-tk and lacZ gene coexpression in vivo, first- or second-generation recombinant HSV-1 vectors (hrR3 or MGH-1) were injected into s.c. growing rat 9L or human U87 deltaEGFR gliomas in nude rats at various times (8 h to 8 days) and at various vector doses [1 x 10(6) to 2 x 10(9) plaque-forming units (PFUs)] prior to imaging. For noninvasive assessment of HSV-1-tk gene expression (124I-labeled FIAU % dose/g), 0.15 mCi of 124I-labeled FIAU was injected i.v. 8 h after the last vector administration, and FIAU positron emission tomography (PET) was performed 48 h later. For the assessment of HSV-1-tk and lacZ gene coexpression, 0.2 mCi of 131I-labeled FIAU was injected i.v. 24 h after the last vector administration. Forty-eight h later, animals were killed, and tumors were dissected for quantitative autoradiographical and histochemical assessment of regional distribution of radioactivity (TK expression measured as 131I-labeled FIAU % dose/g) and coexpressed lacZ gene activity. The rates of FIAU accumulation (Ki) in hrR3-infected 9L cells in culture, which reflect the levels of HSV-1-tk gene expression, ranged between 0.12 and 3.4 ml/g/min. They increased in a vector dose- and infection time-dependent manner and correlated with the virus yield (PFUs/ml), where the PFUs:Ki ratios remained relatively constant over time. Moreover, a linear relationship was observed between lacZ gene expression and FIAU accumulation 5-40 h after infection of 9L cells with a multiplicity of infection of 1.5. At later times (> 52 h postinjection), high vector doses (multiplicity of infection, 1.5) led to a decrease of FIAU accumulation rates, viral yield, and cell pellet weights, indicating vector-mediated cell toxicity. Various levels of HSV-1-tk gene expression could be assessed by FIAU-PET after in vivo infection of s.c. tumors. The levels of FIAU accumulation were comparatively low (approximately ranging from 0.00013 to 0.003% injected dose/g) and were spatially localized; this may reflect viral-induced cytolysis of infected tumor cells and limited lateral spread of the virus. Image coregistration of tumor histology, HSV-1-tk related radioactivity (assessed by autoradiography), and lacZ gene expression (assessed by beta-galactosidase staining) demonstrated a characteristic pattern of gene expression around the injection sites. A rim of lacZ gene expression immediately adjacent to necrotic tumor areas was observed, and this zone was surrounded by a narrow band of HSV-1-tk-related radioactivity, primarily in viable-appearing tumor tissue. These results demonstrate that recombinant HSV-1 vector-mediated HSV-1-tk gene expression can be monitored noninvasively by PET, where the areas of FIAU-derived radioactivity identify the viable portion of infected tumor tissue that retains FIAU accumulation ability, and that the accumulation rate of FIAU in culture, Ki, reflects the number of HSV-1 viral particles in the infected tumor cell population [4.1 +/- 0.6 x 10(6) PFUs/Ki unit (PFUs divided by ml/min/g)]. Moreover, time-dependent and spatial relationships of HSV-1-tk and lacZ gene coexpression in culture and in vivo indicate the potential for indirect in vivo imaging of therapeutic gene expression in tumor tissue infected with any recombinant HSV-1 vector where a therapeutic gene is substituted for the lacZ gene.

Therapy of leptomeningeal metastasis in solid tumors.

Leptomeningeal metastasis (LM), i.e. the seeding of tumor cells to the cerebrospinal fluid (CSF) and the leptomeninges, is a devastating and mostly late-stage complication of various solid tumors. Clinical signs and symptoms may include cranial nerve palsies, radicular symptoms, signs of increased intracranial pressure such as headache, nausea and vomiting, and cognitive dysfunction. In cases of suspected LM, the highest diagnostic sensitivity is provided by the combination of CSF cytology and contrast-enhanced MRI (cranial as well as complete spine). The therapeutic spectrum includes radiotherapy of the clinically involved region as well as systemic and intrathecal chemotherapy. The choice of treatment modalities depends on the type of LM (non-adherent tumor cells in the CSF vs. nodular contrast-enhancing tumor growth), additional systemic involvement (uncontrolled vs. controlled systemic disease) and additional involvement of the CNS parenchyma (LM as the only CNS involvement vs. LM+parenchymal CNS metastases). Larger contrast-enhancing nodular LM or symptomatic lesions of the spine may be treated with radiotherapy. In case of uncontrolled systemic disease, the treatment regimen should include systemic chemotherapy. The choice of systemic treatment should take into account the histology of the primary tumor. Intrathecal chemotherapy is most important in cases of LM of the non-adherent type. There are three substances for routine use for intrathecal chemotherapy: methotrexate, cytarabine, and thiotepa. Liposomal cytarabine shows advantages in terms of longer injection intervals, a sufficient distribution in the entire subarachnoid space after lumbar administration and improved quality-of-life. The role of new agents (e.g. rituximab and trastuzumab) for intrathecal therapy is still unclear.

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.

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.