Rezvan Ahmadi

BRAF gene duplication constitutes a mechanism of MAPK pathway activation in low-grade astrocytomas

The molecular pathogenesis of pediatric astrocytomas is still poorly understood. To further understand the genetic abnormalities associated with these tumors, we performed a genome-wide analysis of DNA copy number aberrations in pediatric low-grade astrocytomas by using array-based comparative genomic hybridization. Duplication of the BRAF protooncogene was the most frequent genomic aberration, and tumors with BRAF duplication showed significantly increased mRNA levels of BRAF and a downstream target, CCND1, as compared with tumors without duplication. Furthermore, denaturing HPLC showed that activating BRAF mutations were detected in some of the tumors without BRAF duplication. Similarly, a marked proportion of low-grade astrocytomas from adult patients also had BRAF duplication. Both the stable silencing of BRAF through shRNA lentiviral transduction and pharmacological inhibition of MEK1/2, the immediate downstream phosphorylation target of BRAF, blocked the proliferation and arrested the growth of cultured tumor cells derived from low-grade gliomas. Our findings implicate aberrant activation of the MAPK pathway due to gene duplication or mutation of BRAF as a molecular mechanism of pathogenesis in low-grade astrocytomas and suggest inhibition of the MAPK pathway as a potential treatment.

ABCC Drug Efflux Pumps and Organic Anion Uptake Transporters in Human Gliomas and the Blood-Tumor Barrier

Delivery of therapeutic agents to the brain and its neoplasms depends on the presence of membrane transport proteins in the blood-brain barrier and in the target cells. The cellular and subcellular localization of these membrane transporters determines the drug accessibility to the brain and its tumors. We therefore analyzed the expression and localization of six members of the multidrug resistance protein family of ATP-dependent efflux pumps (ABCC1-ABCC6, formerly MRP1-MRP6) and of six organic anion uptake transporters (OATP1A2, OATP1B1, OATP1B3, OATP1C1, OATP2B1, and OATP4A1) in 61 human glioma specimens of different histologic subtypes. Real-time PCRs indicated expressions of ABCC1, ABCC3, ABCC4, and ABCC5. In addition, we detected expressions of the OATP uptake transporter genes SLCO1A2, SLCO1C1, SLCO2B1, and SLCO4A1. At the protein level, however, only OATP1A2 and OATP2B1 were detectable by immunofluorescence microscopy in the luminal membrane of endothelial cells forming the blood-brain barrier and the blood-tumor barrier, but not in the glioma cells. ABCC4 and ABCC5 proteins were the major ABCC subfamily members in gliomas, localized both at the luminal side of the endothelial cells and in the glioma cells of astrocytic tumors and in the astrocytic portions of oligoastrocytomas. These results indicate that expression of ABCC4 and ABCC5 is associated with an astrocytic phenotype, in accordance with their expression in astrocytes and with the higher chemoresistance of astrocytic tumors as compared with oligodendrogliomas. Our data provide a basis for the assessment of the role of uptake transporters and efflux pumps in the accessibility of human gliomas for chemotherapeutic agents.

Differentiation therapy exerts antitumor effects on stem-like glioma cells.

Purpose: Stem-like tumor cells comprise a highly tumorigenic and therapy-resistant tumor subpopulation, which is believed to substantially influence tumor initiation and therapy resistance in glioma. Currently, therapeutic, drug-induced differentiation is considered as a promising approach to eradicate this tumor-driving cell population; retinoic acid is well known as a potent modulator of differentiation and proliferation in normal stem cells. In glioma, knowledge about the efficacy of retinoic acid–induced differentiation to target the stem-like tumor cell pool could have therapeutic implications. Experimental Design: Stem-like glioma cells (SLGC) were differentiated with all-trans retinoic acid–containing medium to study the effect of differentiation on angiogenesis, invasive growth, as well as radioresistance and chemoresistance of SLGCs. In vivo effects were studied using live microscopy in a cranial window model. Results: Our data suggest that in vitro differentiation of SLGCs induces therapy-sensitizing effects, impairs the secretion of angiogenic cytokines, and disrupts SLGCs motility. Further, ex vivo differentiation reduces tumorigenicity of SLGCs. Finally, we show that all-trans retinoic acid treatment alone can induce antitumor effects in vivo. Conclusions: Altogether, these results highlight the potential of differentiation treatment to target the stem-like cell population in glioblastoma. Clin Cancer Res; 16(10); 2715–28. ©2010 AACR.

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.

Effector T-Cell Infiltration Positively Impacts Survival of Glioblastoma Patients and Is Impaired by Tumor-Derived TGF-β

Purpose: In glioma—in contrast to various other cancers—the impact of T-lymphocytes on clinical outcome is not clear. We investigated the clinical relevance and regulation of T-cell infiltration in glioma. Experimental Design: T-cell subpopulations from entire sections of 93 WHO°II–IV gliomas were computationally identified using markers CD3, CD8, and Foxp3; survival analysis was then done on primary glioblastomas (pGBM). Endothelial cells expressing cellular adhesion molecules (CAM) were similarly computationally quantified from the same glioma tissues. Influence of prominent cytokines (as measured by ELISA from 53 WHO°II–IV glioma lysates) on CAM-expression in GBM-isolated endothelial cells was determined using flow cytometry. The functional relevance of the cytokine-mediated CAM regulation was tested in a transmigration assay using GBM-derived endothelial cells and autologous T-cells. Results: Infiltration of all T-cell subsets increased in high-grade tumors. Most strikingly, within pGBM, elevated numbers of intratumoral effector T cells (Teff, cytotoxic and helper) significantly correlated with a better survival; regulatory T cells were infrequently present and not associated with GBM patient outcome. Interestingly, increased infiltration of Teff cells was related to the expression of ICAM-1 on the vessel surface. Transmigration of autologous T cells in vitro was markedly reduced in the presence of CAM-blocking antibodies. We found that TGF-β molecules impeded transmigration and downregulated CAM-expression on GBM-isolated endothelial cells; blocking TGF-β receptor signaling increased transmigration. Conclusions: This study provides comprehensive and novel insights into occurrence and regulation of T-cell infiltration in glioma. Specifically, targeting TGF-β1 and TGF-β2 might improve intratumoral T-cell infiltration and thus enhance effectiveness of immunotherapeutic approaches. Clin Cancer Res; 17(13); 4296–308. ©2011 AACR.

Genomic and Expression Profiling of Glioblastoma Stem Cell-Like Spheroid Cultures Identifies Novel Tumor-Relevant Genes Associated with Survival

Purpose: Glioblastoma spheroid cultures are enriched in tumor stem-like cells and therefore may be more representative of the respective primary tumors than conventional monolayer cultures. We exploited the glioma spheroid culture model to find novel tumor-relevant genes. Experimental Design: We carried out array-based comparative genomic hybridization of spheroid cultures derived from 20 glioblastomas. Microarray-based gene expression analysis was applied to determine genes with differential expression compared with normal brain tissue and to nonneoplastic brain spheroids in glioma spheroid cultures. The protein expression levels of three candidates were determined by immunohistochemistry on tissue microarrays and correlated with clinical outcome. Functional analysis of PDPN was done. Results: Genomic changes in spheroid cultures closely resembled those detected in primary tumors of the corresponding patients. In contrast, genomic changes in serum-grown monolayer cultures established from the same patients did not match well with the respective primary tumors. Microarray-based gene expression analysis of glioblastoma spheroid cultures identified a set of novel candidate genes being upregulated or downregulated relative to normal brain. Quantitative real-time PCR analyses of 8 selected candidate genes in 20 clinical glioblastoma samples validated the microarray findings. Immunohistochemistry on tissue microarrays revealed that expression of AJAP1, EMP3, and PDPN was significantly associated with overall survival of astrocytic glioma patients. Invasive capacity and RhoA activity were decreased in PDPN-silenced spheroids. Conclusion: We identified a set of novel candidate genes that likely play a role in glioblastoma pathogenesis and implicate AJAP1, EMP3, and PDPN as molecular markers associated with the clinical outcome of glioma patients. (Clin Cancer Res 2009;15(21):6541–50)

Different angiogenic phenotypes in primary and secondary glioblastomas

Primary and secondary glioblastomas (pGBM, sGBM) are supposed to evolve through different genetic pathways, including EGF receptor and PDGF and its receptor and thus genes that are involved in tumor‐induced angiogenesis. However, whether other angiogenic cytokines are also differentially expressed in these glioblastoma subtypes is not known so far, but this knowledge might be important to optimize an antiangiogenic therapy. Therefore, we studied the expression of several angiogenic cytokines, including VEGF‐A, HGF, bFGF, PDGF‐AB, PDGF‐BB, G‐CSF and GM‐CSF in pGBMs and sGBMs as well as in gliomas WHO III, the precursor lesions of sGBMs. In tumor tissues, expression of all cytokines was observed albeit with marked differences concerning intensity and distribution pattern. Quantification of the cytokines in the supernatant of 30 tissue‐corresponding glioma cultures revealed a predominant expression of VEGF‐A in pGBMs and significantly higher expression levels of PDGF‐AB in sGBMs. HGF and bFGF were determined in nearly all tumor cultures but with no GBM subtype or malignancy‐related differences. Interestingly, GM‐CSF and especially G‐CSF were produced less frequently by tumor cells. However, GM‐CSF secretion occurred together with an increased number of simultaneously secreted cytokines and correlated with a worse patient prognosis and may thus represent a more aggressive angiogenic phenotype. Finally, we confirmed an independent contribution of each tumor‐derived cytokine analyzed to tumor‐induced vascularization. Our data indicate that an optimal antiangiogenic therapy may require targeting of multiple angiogenic pathways that seem to differ markedly in pGBMs and sGBMs.

Differentiating primary central nervous system lymphoma from glioma in humans using localised proton magnetic resonance spectroscopy.

In order to characterise primary central nervous system lymphomas (PCNSL) and to evaluate if 1H spectroscopy improves the preoperative differential diagnosis of PCNSL and glioma, seven immunocompetent patients with PCNSL and 21 patients with glioma were examined using single voxel, short echo time magnetic resonance spectroscopy (MRS; 1.5 T, STEAM 1500/20). All PCNSL demonstrated massively elevated lipid resonances and markedly elevated choline. Similarly increased lipid resonances were only found in seven necrotic glioblastomas, PCNSL differing from all solid astrocytomas by massively elevated lipid resonances. Additionally, PCNSL had higher Cho/Cr ratios than all grades of astrocytoma. In conclusion, we found that massively elevated lipid resonances are a hallmark of PCNSL in immunocompetent patients. Together with a markedly elevated Cho/Cr ratio, MRS provides metabolic information which may improve the preoperative differentiation of PCNSL and glioma.

Long-term outcome and survival of surgically treated supratentorial low-grade glioma in adult patients

PurposeThe appropriate management of low-grade gliomas is still a matter of debate. So far, there are no randomized studies that analyze the impact of surgical resection on patient outcome. The value of the data obtained from the few retrospective reports available is often limited.Patients and methodsIn the present study, we performed an analysis on data of 130 adult low-grade glioma patients. Extent of the resection was evaluated in correlation to the overall survival (OS) and progression-free survival (PFS) using Cox regression multivariate analysis.ResultsExtended surgery was shown to prolong OS and PFS significantly. Re-surgery in the case of a tumor relapse has a significant impact on OS and PFS, too.ConclusionsIn summary, we could retrospectively evaluate a large case series of well-defined low-grade gliomas patients with a long follow-up period showing that extended surgery would be the most effective therapy for low-grade glioma patients even in recurrent diseases.PURPOSE The appropriate management of low-grade gliomas is still a matter of debate. So far, there are no randomized studies that analyze the impact of surgical resection on patient outcome. The value of the data obtained from the few retrospective reports available is often limited. PATIENTS AND METHODS In the present study, we performed an analysis on data of 130 adult low-grade glioma patients. Extent of the resection was evaluated in correlation to the overall survival (OS) and progression-free survival (PFS) using Cox regression multivariate analysis. RESULTS Extended surgery was shown to prolong OS and PFS significantly. Re-surgery in the case of a tumor relapse has a significant impact on OS and PFS, too. CONCLUSIONS In summary, we could retrospectively evaluate a large case series of well-defined low-grade gliomas patients with a long follow-up period showing that extended surgery would be the most effective therapy for low-grade glioma patients even in recurrent diseases.

Critical care management of potential organ donors: our current standard

Abstract: Caring for a brain dead potential organ donor requires a shift in critical care from the extensive treatment of increased intracranial pressure towards strategies to maintain donor organ function. Suboptimal, unstandardized critical care management of organ donors, however, is one of the main reasons for insufficient organ procurement. The pathophysiological changes following brain death entail a high incidence of complications including hemodynamic instability, endocrine and metabolic disturbances, and disruption of internal homeostasis that jeopardize potentially transplantable organs. Strategies for the management of organ donors exist and consist of the normalization of donor physiology. This has resulted in standardized efforts to improve the critical care delivered to potential organ donors, increasing not only the number, but also the quality of suitable organs and aiming at an optimal outcome for the recipients. In this review, we discuss the pathophysiological changes associated with brain death and present the current guidelines at our department, which are optimized based on available literature.