Karsten Geletneky

Antitumor Vaccination of Patients With Glioblastoma Multiforme: A Pilot Study to Assess Feasibility, Safety, and Clinical Benefit

PURPOSE Prognosis of patients with glioblastoma is poor. Therefore, in glioblastoma patients, we analyzed whether antitumor vaccination with a virus-modified autologous tumor cell vaccine is feasible and safe. Also, we determined the influence on progression-free survival and overall survival and on vaccination-induced antitumor reactivity. PATIENTS AND METHODS In a nonrandomized study, 23 patients were vaccinated and compared with nonvaccinated controls (n = 87). Vaccine was prepared from patients tumor cell cultures by infection of the cells with Newcastle Disease Virus, followed by gamma-irradiation, and applied up to eight times. Antitumor immune reactivity was determined in skin, blood, and relapsed tumor by delayed-type hypersensitivity skin reaction, ELISPOT assay, and immunohistochemistry, respectively. RESULTS Establishment of tumor cell cultures was successful in approximately 90% of patients. After vaccination, we observed no severe side effects. The median progression-free survival of vaccinated patients was 40 weeks (v 26 weeks in controls; log-rank test, P = .024), and the median overall survival of vaccinated patients was 100 weeks (v 49 weeks in controls; log-rank test, P < .001). Forty-five percent of the controls survived 1 year, 11% survived 2 years, and there were no long-term survivors (> or = 3 years). Ninety-one percent of vaccinated patients survived 1 year, 39% survived 2 years, and 4% were long-term survivors. In the vaccinated group, immune monitoring revealed significant increases of delayed-type hypersensitivity reactivity, numbers of tumor-reactive memory T cells, and numbers of CD8(+) tumor-infiltrating T-lymphocytes in secondary tumors. CONCLUSION Postoperative vaccination with virus-modified autologous tumor cells seems to be feasible and safe and to improve the prognosis of patients with glioblastomas. This could be substantiated by the observed antitumor immune response.

Cytosolic Activation of Cathepsins Mediates Parvovirus H-1-Induced Killing of Cisplatin and TRAIL-Resistant Glioma Cells

ABSTRACT Gliomas are often resistant to the induction of apoptotic cell death as a result of the development of survival mechanisms during astrocyte malignant transformation. In particular, the overexpression of Bcl-2-family members interferes with apoptosis initiation by DNA-damaging agents (e.g., cisplatin) or soluble death ligands (e.g., TRAIL). Using low-passage-number cultures of glioma cells, we have shown that parvovirus H-1 is able to induce death in cells resistant to TRAIL, cisplatin, or both, even when Bcl-2 is overexpressed. Parvovirus H-1 triggers cell death through both the accumulation of lysosomal cathepsins B and L in the cytosol of infected cells and the reduction of the levels of cystatin B and C, two cathepsin inhibitors. The impairment of either of these effects protects glioma cells from the viral lytic effect. In normal human astrocytes, parvovirus H-1 fails to induce a killing mechanism. In vivo, parvovirus H-1 infection of rat glioma cells intracranially implanted into recipient animals triggers cathepsin B activation as well. This report identifies for the first time cellular effectors of the killing activity of parvovirus H-1 against malignant brain cells and opens up a therapeutic approach which circumvents their frequent resistance to other death inducers.

Parvovirus H-1 infection of human glioma cells leads to complete viral replication and efficient cell killing

The extremely poor prognosis of malignant gliomas requires the investigation of other than standard therapies, i.e., the application of oncolytic viruses. In our study, we evaluated the effects of the oncosuppressive parvovirus H‐1 on different established glioblastoma cell lines of rat and human origin and on short‐term/low‐passage cultures of human glioblastoma cells. We observed an efficient and dose‐dependent killing of all glioma cell cultures at low multiplicities of infectious particles (MOI) per cell. Southern blot analysis of viral DNA amplification, RT‐PCR analysis of viral RNA expression and Western blot analysis of the expression of viral structural (VP‐1/VP‐2) and nonstructural (NS‐1) proteins demonstrated the biosynthesis of these viral macromolecular components in all of the cultures. Moreover, all the glioma cells were proficient for the production of infectious H‐1 virus particles. The amount of virus production differed between a several fold increase of the input virus titer in most of the short‐term/low‐passage cultures up to 1,000‐fold in one short‐term glioma and in the rat cells. Glioma cells lines and, more importantly, short‐term/low‐passage cultures of human glioblastomas were found to be highly susceptible target cells for H‐1 virus mediated cytotoxicity. The formation of fully infectious progeny particles in infected glioma cells offers the chance for the induction of secondary rounds of infection resulting in an advanced cytotoxic effect. These advantageous characteristics of H‐1 virus infection of glioma cells, combined with the known low toxicity of H‐1 virus in nontransformed cells, make parvovirus H‐1 a promising candidate for oncolytic glioma therapy.

Regression of advanced rat and human gliomas by local or systemic treatment with oncolytic parvovirus H-1 in rat models

Oncolytic virotherapy is a potential treatment modality under investigation for various malignancies including malignant brain tumors. Unlike some other natural or modified viruses that show oncolytic activity against cerebral neoplasms, the rodent parvovirus H-1 (H-1PV) is completely apathogenic in humans. H-1PV efficiently kills a number of tumor cells without harm to corresponding normal ones. In this study, the concept of H-1PV-based virotherapy of glioma was tested for rat (RG-2 cell-derived) and for human (U87 cell-derived) gliomas in immunocompetent and immunodeficient rat models, respectively. Large orthotopic rat and human glioma cell-derived tumors were treated with either single stereotactic intratumoral or multiple intravenous (iv) H-1PV injections. Oncolysis was monitored by magnetic resonance imaging and proven by histology. Virus distribution and replication were determined in brain and organs. In immunocompetent rats bearing RG-2-derived tumors, a single stereotactic intratumoral injection of H-1PV and multiple systemic (iv) applications of the virus were sufficient for remission of advanced and even symptomatic intracranial gliomas without damaging normal brain tissue or other organs. H-1PV therapy resulted in significantly improved survival (Kaplan-Meier analysis) in both the rat and human glioma models. Virus replication in tumors indicated a contribution of secondary infection by progeny virus to the efficiency of oncolysis. Virus replication was restricted to tumors, although H-1PV DNA could be detected transiently in adjacent or remote normal brain tissue and in noncerebral tissues. The results presented here and the innocuousness of H-1PV for humans argue for the use of H-1PV as a powerful means to perform oncolytic therapy of malignant gliomas.

Epstein-Barr virus B95.8 produced in 293 cells shows marked tropism for differentiated primary epithelial cells and reveals interindividual variation in susceptibility to viral infection

Epstein‐Barr virus (EBV), a well‐characterised B‐lymphotropic agent is aetiologically linked to B cell lymphoproliferations, but the spectrum of diseases the virus causes also includes oral hairy leukoplakia, a benign epithelial lesion, as well as carcinomas of the nasopharynx and of the stomach. However, it is still unclear how EBV accesses and transforms primary epithelial cells. Sixteen samples consisting of primary epithelial cells from the sphenoidal sinus or from tonsils were infected with GFP‐tagged recombinant B95.8 EBVs produced in the 293 cell line. The rate of infection was assessed by counting GFP‐positive cells and cells expressing viral proteins. Primary epithelial cells from all samples were found to be sensitive to EBV infection but there was a marked interindividual variation among the tested samples (2–48% positive cells). This suggests heterogeneity in terms of sensitivity to EBV infection in vivo and therefore possibly to EBV‐associated diseases of the epithelium. The virus showed a preferential tropism for differentiated epithelial cells (p63 negative, involucrin positive). In all cases, infected cells expressed EBV lytic proteins but also the LMP1 protein. The viral tropism for differentiated cells and the permissivity of these cells for virus replication reproduced in vitro cardinal features of oral hairy leukoplakia. We have identified a source of EBV that shows unusually strong epitheliotropism for primary epithelial cells that will allow detailed analysis of virus‐cell interactions during virus infection, replication and virus‐mediated transformation.

Local Intra-arterial Fibrinolysis of Thromboemboli Occurring During Neuroendovascular Procedures With Recombinant Tissue Plasminogen Activator

Background and Purpose— There is a lack of systematic data regarding local intra-arterial fibrinolysis (LIF) of thromboemboli occurring during neuroendovascular procedures with the use of recombinant tissue plasminogen activator (rtPA). We report our technique for treating LIF of intracerebral thromboemboli occurring during neuroendovascular procedures. Methods— Nine of 723 patients (1.2%) who underwent neuroendovascular procedures during the period from January 1997 to September 2002 suffered thromboembolic complications. These patients were treated by LIF with a maximum dose of 0.9 mg rtPA per kilogram body weight. Recanalization was categorized as successful (Thrombolysis in Myocardial Infarction [TIMI] grade 2 or 3) versus unsuccessful (TIMI grade 0 or 1), and clinical outcome was categorized as independent (Rankin Scale score 0 to 2) versus dependent or dead (Rankin Scale score 3 to 6). Results— The minimum time between thrombus detection and beginning of LIF was 10 minutes, and the maximum time was 90 minutes. Successful recanalization was achieved in 4 of 9 patients (44%). All 9 patients suffered cerebral ischemic infarctions, and none of the patients sustained intracerebral hemorrhage. Two patients (22%) died from malignant brain infarctions. Four patients (44%) remained moderately disabled, and 3 patients (33%) were severely disabled 3 months after LIF. Conclusions— Although we used relatively high doses of rtPA, the recanalization rates and clinical outcome of LIF in our patients were not satisfactory. Strategies for the prevention of thromboemboli during neuroendovascular procedures must be improved, and novel fibrinolytic or thrombolytic techniques should be developed.

Sero-epidemiological analysis of the risk of virus infections for childhood leukaemia

Virus infections have been thought to be involved in the development of childhood leukaemia. In order to address this issue we determined, in a case‐control study, the prevalence of antibodies to viruses infecting blood or bone‐marrow cells [Epstein‐Barr virus (EBV), human herpes virus type 6 (HHV‐6), parvovirus B19] as well as to the human virus known for its tumour‐suppressive properties, the adeno‐associated virus type 2 (AAV‐2), in the sera of 121 children with leukaemia in Germany, and in 197 control individuals, hospitalized for other reasons, and matched for age and gender to the cases. In addition, we developed a questionnaire to be answered by the childrens parents, in order to gain information on previous infections of the children as well as to calculate for factors which may influence serological findings. Comparative determination of the prevalence of antibodies against AAV‐2, B‐19 or HHV‐6 revealed no significant differences in cases and controls. However, antibodies to EBV were more frequently found in children with leukaemia younger than 6 years of age (age at the time of diagnosis of leukaemia) than in controls. Apparently, infection with AAV‐2 has no protective effect in childhood leukaemia, in contrast to results observed for other malignancies. Similarly, and in accordance with results on leukaemia in adults, we found no indication of a protective effect of infection with the parvovirus B‐19. The data suggest that EBV, which is known to be involved in various lymphomas, may play a role in the development of childhood leukaemia in young children.

MR appearance of an intracranial dural arteriovenous fistula leading to cervical myelopathy

Objective: To report the MRI, myelographic, and angiographic findings as well as the clinical and radiologic time course of an intracranial dural arteriovenous fistula (DAVF) leading to cervical myelopathy; and to review the pertinent literature. Background: Cervical myelopathy from an intracranial DAVF draining into spinal medullary veins is extremely uncommon. However, knowledge about the MR features of these lesions is important because an improper diagnosis might result in delayed or incorrect treatment. Methods: In a patient with progressive cervical myelopathy, T2- and proton density (PD)-weighted MRI, contrast-enhanced T1-weighted images, and a contrast-enhanced MR angiogram of the cervical spinal cord were acquired. Additionally, intraarterial digital substraction angiography (DSA) of the right and left common carotid arteries was performed. Results: MRI findings included swelling of the cervical spinal cord, hyperintensity of the cervical cord on T2- and PD-weighted MRI, and an enlarged vessel at the ventral surface of the cord on MR angiography. No parenchymal contrast enhancement of the spinal cord was noted on T1-weighted MRI. DSA revealed an intracranial DAVF fed by four branches of the left external carotid artery and draining into spinal medullary veins. The fistula was treated with endovascular embolization, leading to considerable clinical improvement of the patient. Conclusions: To avoid an improper diagnosis or a delayed or incorrect treatment of myelopathy resulting from an intracranial DAVF, cerebral intraarterial angiography may be indicated in cases of otherwise unexplainable cervical myelopathy.

Tumor Selectivity of Oncolytic Parvoviruses: From in vitro and Animal Models to Cancer Patients

Oncolytic virotherapy of cancer is among the innovative modalities being under development and especially promising for targeting tumors, which are resistant to conventional treatments. Presently, at least a dozen of viruses, belonging to nine different virus families, are being tested within the frames of various clinical studies in cancer patients. Continuously growing preclinical evidence showing that the autonomous rat parvovirus H-1 (H-1PV) is able to kill tumor cells that resist conventional treatments and to achieve a complete cure of various human tumors in animal models argues for its inclusion in the arsenal of oncolytic viruses with an especially promising bench to bedside translation potential. Oncolytic parvovirus safe administration to humans relies on the intrinsic preference of these agents for quickly proliferating, metabolically, and biochemically disturbed tumor versus normal cells (tumor selectivity or oncotropism). The present review summarizes and discusses (i) preclinical evidence of H-1PV innocuousness for normal cells and healthy tissues in vitro and in animals, respectively, (ii) toxicological assessments of H-1PV mono- or combined therapy in tumor-bearing virus-permissive animal models, as well as (iii) historical results of experimental infection of human cancer patients with H-1PV. Altogether, these data argue against a risk of H-1PV inducing significant toxic effects in human patients. This highly favorable safety profile allowed the translation of H-1PV preclinical research into a Phase I/IIa clinical trial being currently in progress.

Oncolytic H-1 Parvovirus Shows Safety and Signs of Immunogenic Activity in a First Phase I/IIa Glioblastoma Trial

Oncolytic virotherapy may be a means of improving the dismal prognosis of malignant brain tumors. The rat H-1 parvovirus (H-1PV) suppresses tumors in preclinical glioma models, through both direct oncolysis and stimulation of anticancer immune responses. This was the basis of ParvOryx01, the first phase I/IIa clinical trial of an oncolytic parvovirus in recurrent glioblastoma patients. H-1PV (escalating dose) was administered via intratumoral or intravenous injection. Tumors were resected 9 days after treatment, and virus was re-administered around the resection cavity. Primary endpoints were safety and tolerability, virus distribution, and maximum tolerated dose (MTD). Progression-free and overall survival and levels of viral and immunological markers in the tumor and peripheral blood were also investigated. H-1PV treatment was safe and well tolerated, and no MTD was reached. The virus could cross the blood-brain/tumor barrier and spread widely through the tumor. It showed favorable pharmacokinetics, induced antibody formation in a dose-dependent manner, and triggered specific T cell responses. Markers of virus replication, microglia/macrophage activation, and cytotoxic T cell infiltration were detected in infected tumors, suggesting that H-1PV may trigger an immunogenic stimulus. Median survival was extended in comparison with recent meta-analyses. Altogether, ParvOryx01 results provide an impetus for further H-1PV clinical development.