Research on Stem Cells

Project location: Italy
Project start date: March 2005 - Project end date: March 2008
Project number: 2005-01
Beneficiary: ATENA Onlus

The following abstracts were published by the Università Cattolica del Sacro Cuore, in September 2005

DR. ANDERSON

Engrafment, migration, differentiation and functional recovery following transplantation of human CNS stem cells into spinal cord injured NOD-scid mice.

Although stem cells are under intense investigation as potential therapeutics for Central Nervous System (CNS) injury and disease, several reports have suggested that stem cells grown as neurospheres and transplanted into an injured environment preferentially differentiate into astrocytes, contributing to glial scar. Additionally, the causal relationship between functional recovery and cell transplantation has not been experimentally  investigated in early studies. Using immunodeficient mice to minimize xenograft rejection, we report that prospectively isolated human fetal CNS-derived stem cells (hCNS-SCs) grown as neurospheres survive, migrate, and express differentiation markers for neurons and
oligodendrocytes after long-term engraftment in spinal cord injured (SCI) NOD-scidmice. Only rarely was GFAP-positive astrocytic differentiation apparent. hCNS-SC engraftment was associated with functional locomotor recovery, an observation that was abolished by
selective ablation of engrafted cells by diphtheria toxin. Finally, hCNS-SC-mediated remyelination was found in both myelin deficient Shiverermice and in SCI models. These data suggest hCNS-SCs may possess therapeutic potential for CNS injury and disease.

Abstract for Advances in Biology and Treatment of Malignant Brain Tumors Meeting in Italy September 23, 2005
Dr.Bigner

Development of Molecularly-Targeted Therapy of Malignant Gliomas with Molecular Genetic Technology.

 Developing molecular-targeted therapy for malignant gliomas requires identification of molecular targets highly expressed in the majority of gliomas, but not expressed in normal brain.  The extracellular molecule, tenascin, was discovered in our laboratory with monoclonal antibody (MAb) technology and now over 300 malignant glioma patients have been treated with our radiolabeled anti-tenascin MAb.  Approximate doubling of survival over conventional therapy in both primary and recurrent glioblastomas (GBMs) has been observed in single arm Phase I and II clinical trials. 

 Cytogenetic technology revealed double minute chromosomes in a majority of GBMs, and quantitative molecular studies revealed up to 100-fold amplification of the wild type epidermal growth factor gene (EGFR).  TP-38, a pseudomonas toxin conjugated to transforming growth factor a, a natural ligand of the EGFR, has been used with convection-enhanced delivery to treat GBM patients.  Several long-term recurrent GBM survivors resulted from TP-38 therapy.  We discovered a tumor-specific epitope called "EGFRvIII" by sequencing around gene deletions of amplified EGFR in GBM.  Pseudomonas immunotoxins, specific for EGFRvIII expressing tumor cells, have been prepared, and will be administered to GBM patients by convection-enhanced delivery. 

 Two methods for genome-wide screens to detect additional molecular targets on malignant brain tumors have been employed.  With Serial Analysis of Gene Expression technology, two molecules, MRP3, a member of the multi-drug resistant family, and glycoprotein neuromedin-B (GPNMB) have been identified as overexpressed in malignant gliomas with little to no expression in normal brain.  MAbs and single fragment chain MAbs have been prepared against MRP3 and GPNMB and therapeutic reagents reactive with them are being developed. 

 Digital karyotyping, another DNA-based genome-wide screen has detected an amplified gene in medulloblastoma, which has served as a molecular marker to predict chemo-susceptibility of anaplastic medulloblastomas. 

 In the future, molecular techniques will be used to type each malignant brain tumor and identify molecules for targeted therapy.  The targeted therapy will be much more specific for tumor and much less damaging to normal brain. 

CONVECTION ENHANCED DELIVERY FOR BRAIN TUMOR THERAPY
Jeffrey Bruce
Columbia University
New York, NY

Mathematical modeling, physiological studies, and increasing numbers of animal and clinical studies have helped to establish convection enhanced delivery (CED) as one of the most promising of future strategies for the treatment of CNS disorders. CED allows versatility for incorporating new chemotherapeutic and immunotherapeutic agents into clinical studies as it has already been validated as a safe treatment strategy in clinical trials for brain tumors. Ongoing and future clinical trials will further elucidate the safety and efficacy of CED and the other local delivery strategies with previously untested agents in the hope of improving the treatment of CNS diseases.

Neural stem cells for neurodegenerative diseases
Elena Cattaneo, Universita' degli Studi di Milano, Dipartimento di Scienze Farmacologiche e Centro di Eccellenza sulle malattie neurodegenerative, (http://www.cattaneolab.it/)

In 1992 Weiss and Reynolds made the landmark discovery that neural stem cells could be maintained in culture via propagation of floating cell clusters termed neurospheres. However, with the notable exception of embryonic stem (ES) cells, it has proven extremely problematic to propagate homogenous cultures of stem cells ex vivo, including those derived from the brain. A major scientific effort is therefore driven toward the identification of conditions that allow the homogenous propagation of neurogenic brain stem cells (from embryonic and somatic sources) for mechanistic studies, disease modeling and experimental transplantations (Rossi and Cattaneo, Nature Reviews Neuroscience, 2002). Toward these aims, I will review the results of an ongoing collaboration with Prof. Austin Smith laboratory (University of Edinburgh) performed in the context of the EuroStemCell Consortium (http://www.eurostemcell.org/).
 

In vitro analysis of mouse Neural Stem/Progenitors Cells genetically modified to express hNGF or hBDNF by a novel multigenic viral expression system.

Cenciarelli Carlo§, Budoni Manuela§, Mercanti Delio*, Ciotti Teresa*, Aloe Luigi*, Fernandez Eduardo§, Pallini Roberto§, Maira Giulio§, & Casalbore Patrizia*.
§ Istituto di Neurochirurgia-Universita` Cattolica del Sacro Cuore di Roma; ATENA Onlus
*Istituto di Neurobiologia e Medicina Molecolare-CNR Roma

Abstract

Nerve Growth Factor (NGF) and Brain-Derived Neurotrophic Factor (BDNF) play an important role in regulating brain development and behaviour. Neurotrophins protect neurons in animal models of traumatic brain injury, excitotoxicity, aging and in particular NGF prevents the death of basal forebrain cholinergic neurons. In the last years, the utilization of non neural or Neural Stem Cell/Neural Progenitors Cells (NPC/NSCs) genetically modified for long term delivery of neurotrophic molecules and neurogenic transcriptional factors has significantly ameliorated the treatment of diseases with loss or impaired function of neurons. Here, we report the characterization of NPC/NSCs, obtained from postnatal mouse brain, genetically modified to continuously release human recombinant Nerve Growth Factor (hrNGF) or hBDNF. We have constructed a novel viral expression system to efficiently transduce human neurotrophins in neural cells. The viral vector contains a tri-cistronic cassette for coordinated expression of up three coding, we inserted the following genes: i) hrNGF(ß subunit) or hBDNF ii) enhanced Green Fluorescent Protein reporter gene (eGFP) and iii) neomycin resistance gene (NeoR) . NeoR gene was introduced to circumvent variability in infection efficiency of primary cells. In presence of neomycin, we selected neural cell lines that stably expressed both eGFP and hNGF/hBDNF genes under CMV promoter-enhancer transcriptional control. The choice of eGFP gene cassette would offer a valuable tool for in vivo studies. Secreted hrNGF and hBDNF was determined by Enzyme-Linked Immunosorbent Assay (ELISA). Levels of hrNGF were found to be elevated either in proliferation or differentiation conditions in contrast to those of hBDNF, which levels were very similar to control cells. hrNGF bioactivity was assessed by a functional test for neurite outgrowth of PC12 cells. In order to understand whether neurotrophin-expressing cells would have affected their intrinsic properties, these were evaluated regard to neurospheres formation and ability to generate different cell types. After thirty passages in the presence of neomycin in vitro, both cell lines maintained their multipotentiality to differentiate in neurons, astrocytes and oligodendrocytes. Neuronal differentiation was slightly enhanced in hBDNF-secreting cells as shown by ß-tubulin III, MAP2 immunostaining compared to other cell lines. Immunocytochemical analysis of these cells have shown 100% of immunoreactivity to eGFP protein. This study might have implications at two different levels: a) engineered cells could be used to obtain high levels of hNGF in vitro for treatment of varies ophthalmic and skin pathologies, and b) neurotrophin-secreting cells could be utilized in cell therapies for treatment of neurodegenerative diseases and/or of neuropathies affecting the peripheral nervous system.

CANCER STEM CELL TARGETING FOR THE TREATMENT OF GLIOBLASTOMA

Ruggero De Maria

Normal and neoplastic neural tissues derived from a restricted pool of neural stem cells, through the sequential formation of neural progenitors, precursors and mature cells. Normal neural stem cells are capable of self-renewing and regenerating damaged neurons and glia, while neoplastic neural stem cells are believed to represent genetically modified stem cells responsible for the generation of brain tumors. Therefore, understanding the mechanisms that regulate the survival and the proliferation of neural stem and progenitor cells is essential both to exploit their extensive therapeutic potential and to identify the processes leading to neoplastic transformation in the neural tissue. We have isolated and characterized normal and neoplastic primitive neural cells. Absence of caspase-8 and high expression of PED constitute two levels of protection from apoptosis in neural stem and progenitor cells. On the normal side, this double level of protection from apoptosis is likely to allow neural stem cell survival and proliferation in sites of acute neurodegeneration in the presence of inflammatory cytokines and death receptor ligands. On the oncological side, the anti-apoptotic properties of neural stem cells may be exploited by their malignant counterparts to escape physiological tumor suppressor mechanisms and pharmacological therapies. Here, we will discuss recent preclinical evidences that show the ability of malignant gliomas to exploit the epigenetic repression of key proapoptotic genes to counteract the conventional and biological therapeutic procedures proposed so far for glioblastoma treatment.

Genetics and Biology of Glioblastoma
Elizabeth Maher1, 3, Alexander Stegh1, Hyunggee Kim1, 2, Robert Bachoo1, 3, Junying Yuan5, David Louis4, Keith Ligon1, 8, Peter Black9, Patrick Wen1, 3, Lynda Chin1, 7, Ronald DePinho1, 6
1Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA 02115, USA, 2Present address: Cell growth Regulation Laboratory, Division of Bioscience and Technology, College of Life and Environmental Sciences, Korea University, Seoul 136-701, Korea, 3Center for Neuro-Oncology and Department of Neurology, Brigham and Women's Hospital, Boston, 4Department of Pathology, Cancer Center and Neurosurgical Service, Massachusetts General Hospital and Harvard Medical School, Boston, 5Department of Cell Biology, Harvard Medical School, Boston, 6Center for Applied Cancer Science, Dana-Farber Cancer Institute; and Departments of Medicine and Genetics, Harvard Medical School, Boston, 7Department of Dermatology, Harvard Medical School, Boston, 8Department of Pathology, Brigham and Women's Hospital, Harvard Medical School, Boston,
9Department of Neurosurgery, Brigham and Women's Hospital, Harvard Medical School, Boston

Glioblastoma (GBM) is a lethal malignant astrocytic brain tumor characterized by intense chemotherapy (apoptosis) resistance and extensive necrosis.  The genetic underpinnings of these biological properties are incompletely understood and may differ depending on the clinical evolution of the disease.  While several common and distinct signature mutations have been assigned to the clinical subtypes, primary and secondary GBM, the presence of a large number of recurrent chromosomal alterations implies that many genetic events serve to drive their genesis and progression.  Clinically annotated and pathologically validated tumor samples were used to generate a high-resolution subtype-specific view of recurrent copy-number alterations (CNAs) as well as catalog known GBM mutations in these samples.  Consistent with their common phenotypes, the known GBM mutations (e.g. CDK4 and MDM2 amplifications and p16Ink4a and PTEN deletions) were documented and 25 shared novel recurrent CNAs were discovered, several of which harbor cancer-relevant genes.  Strikingly, the majority of recurrent genomic aberrations were subtype-specific and, like the overlap loci, have strong cancer relevance reinforced by the targeting of these loci in other cancer types and/or the presence of known cancer genes. 
From a novel amplicon, a pioneer protein Gained-in-Glioma1 (GIG1) has been identified and was found to possess oncogenic activity and be consistently over-expressed in GBM.  Enforced GIG1 expression confers marked apoptosis resistance in primary cortical astrocytes through a postmitochondrial mechanism of action atypical for anti-apoptotic Bcl-2-related proteins. Its RNAi-mediated knock down sensitizes human glioma cell lines towards apoptosis and reduces tumor explant growth in vivo.  Mechanistically, GIG1 neutralizes effector caspase-7 and caspase-3 activity rather than preserving mitochondrial integrity. The inhibition of post-mitochondrial apoptosis signaling at the level of effector caspase maturation by GIG1 elicits a profound cellular response of necrosis in context of pro-apoptotic stimulation, an activity profile that may underlie a paradoxical feature of GBM - intense apoptosis resistance yet florid cellular necrosis.

Molecular markers of gliomas: a clinical approach.
M. Eoli Neurological Institute C. Besta, Milan, Italy

Newly developed molecular techniques, such as loss of heterozigosity (LOH) testing, fluorescent in situ hybridization (FISH), DNA sequencing and methylation specific PCR (MSP) are currently being employed for the clinical assessment of gliomas in several laboratories. However  the clinical usefulness of some markers and the context in which  the information obtained should be used are still matter of debate. Therefore  this presentation will focus on the validation and implementation of molecular testing in gliomas, with emphasis on LOH on chromosome 1p, 19 q and 10q and on the O6- Methylguanine-DNA methyltransferase (MGMT) methylation status.
The accurate distinction between  pure oligodendrogliomas (OTs), oligoastrocytomas (OAs) and diffuse astrocytomas (As) has import prognostic and therapeutic implications. Patients with pure oligodendrogliomas are more likely to respond to chemotherapy and have a longer survival than patients with diffuse astrocytomas or oligoastrocytomas  of the same grade.
In OTs lossess of heterozigosity on 1p and 19q are the most frequently detected genetic alterations, being present in more than 80 % of the tumors.. Genetic alterations of OA include LOH on chromosome 1p and/or 19q, observed in approximately 50% of OAs, but also loss of heterozygosity on chromosome 10q and/or 17p, frequently found in astrocytomas and associated with the progression to glioblastoma. Several retrospective  studies suggested that OTS with LOH on 1p and/or 19 q have longer survival and better response to chemotherapy or radiotherapy while the prognostic implications of those markers in OAs are less investigated. We have analysed in 94 OAs (46 grade II and 48 grade III) the patterns of loss of heterozygosity (LOH) of four genomic regions: 1p, 19q, 17p and 10q. LOH 1p was present in 46% of the tumors, LOH 19q in 45%, LOH 17p in 22%; LOH 10q in 16%. LOH 1p and 19q were associated in 32%, other LOH associations were rare (<3%). Progression Free Survival (PFS) was significantly longer in patients with LOH 1p associated or not to LOH on 19q and shorter in those with LOH on 10q.
Because of the clinical relevance of 1p and 19 q deletions in OTs  and OAs, there is a strong interest in the identification of the relevant genes targeted by deletions on these chromosome arms. The assessment of the minimal deleted region in tumors with partial losses of the 1p36 region allowed to pinpoint a 150 kb region including the calmodulin-binding transcription activator 1 (CAMTA1) gene. Another potential candidate on 1p36 is the retinoblastoma protein-interacting zinc finger gene (RIZ1), a member of a nuclear protein-methyltransferase superfamily involved in chromatin-mediated gene expression.
MRI plays an important role in the initial evaluation of patients with gliomas: the identification of clinical and molecular subsets among OTs and OAs raises the question of whether different genetic molecular profiles could be linked with peculiar MRI features. According to Zlatescu and Megyesi , there is a correlation  between the profile of molecular alterations and tumor location. Oligodendrogliomas with LOH  on 1p are preferentially located in the frontal regions and frequently infiltrate widely the parenchyma.
We looked for correlations between MRI features on pre-operatory scan, histo-pathological diagnosis and the molecular genetic profile in 62 patients with grade II or grade III OAs. Tumors with 1p and/or 19q losses showed T1 and T2 inhomogeneous signals less frequently, even when histologically classified as anaplastic oligoastrocytomas.The  temporal location was also less frequent in patients with 1p and /or 19q losses. MRI findings suggestive of high-grade gliomas (undefined margins, not homogeneous T1 and T2 signals, relevant mass effect,  presence of enhancement) were significantly associated with LOH of 10q.
Approximately 40% of gliomas have very low levels of expression of the O6-Methylguanine-DNA Methyltransferase (MGMT), mostly due  to hypermethylation of the MGMT promoter.
MGMT is a DNA repair protein that specifically removes mutagenic, carcinogenic and cytotoxic O6-alkylguanine DNA adducts induced by alkylating agents like nitrosureas. Therefore, MGMT expression is one of the factors implicated in chemoresistance. Some clinical studies suggest that MGMT inactivation by aberrant promoter methylation correlates with a longer PFS following radiation and adjuvant chemotherapy (nitrosureas or temozolomide). However other clinical studies failed to show any relationship between MGMT methylation status and overall survival or response to chemotherapy. This conflicting results could be  in part explained by the fact that heterogeneous groups of patients were analysed.
Using Methylation-Specific PCR (MSP) we investigated the inactivation of the DNA-repair gene MGMT by promoter hypermethylation in 67 GBM obtained from patients subsequently treated by conventional radiotherapy and CDDP + BCNU. We observed that the MGMT gene was methylated in 25 of the 67 tumors (37%). This finding was associated with prolonged overall survival (24 versus 14 months; log-rank test, p=0.0002). Among all GBM tested, secondary GBMs had prolonged overall survival (25 versus 14 months; log-rank test, p=0.0002) than de novo tumors, whereas other prognostic factors were not statistically associated with prognosis. The frequency of MGMT methylation was not associated with age, or other clinical features.

References
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2. Barbashina V, Salazar P, Holland EC, Rosenblum MK, Ladanyi M. Allelic losses at 1p36 and 19q13 in gliomas: correlation with histologic classification, definition of a 150-kb minimal deleted region on 1p36, and evaluation of CAMTA1 as a candidate tumor suppressor gene. Clin Cancer Res 2005;11(3):1119-28.

3. Bauman GS, Ino Y, Ueki K, Zlatescu MC, Fisher BJ, Macdonald DR, Stitt L, Louis DN, Cairncross JG. Allelic loss of chromosome 1p and radiotherapy plus chemotherapy in patients with oligodendrogliomas. Int J Radiat Oncol Biol Phys 2000;48(3):825-30.

4. Breil M, Tortosa A, Verger E, Miguel Gil J, Vinolas N, Villa S, Acebes JJ, Caral L, Pujol T, Ferre I, Ribalta T, Graus F. Prognostic significance of O6 Methyl-guanine-DNA Methyltransferase determined by promoter hypermethylation and immunoistochemical expression in anaplastic glioma. Clin Cancer Res 2005; 11: 5167-5174
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8. Esteller M, Garcia-Foncillas J, Andion E, Goodman SN, Hidalgo OF, VanaclochaV, Baylin SB, Herman JG. Inactivation of the DNA-repair gene MGMT and the clinical response of gliomas to alkylating agents. N Engl J Med. 2000;  343(19):1350-4.

9. Fallon KB, Palmer CA, Roth KA, Nabors LB, Wang W, Carpenter M, Banerjee R, Forsyth P, Rich K, Perry A. Prognostic value of 1p, 19q, 9p, 10q, and EGFR-FISH analyses in recurrent oligodendrogliomas. J Neuropathol Exp Neurol 2004;63(4):314-22.
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11. Hegi ME, Diserens AC, Gorlia T, Hamou MF, de Tribolet N, Weller M, Kros JM, Hainfellner JA, Mason W, Mariani L, Bromberg JE, Hau P, et al. MGMT gene silencing and benefit from temozolomide in glioblastoma. N Engl J Med 2005;352(10):997-1003.
12. Hoang-Xuan K, He J, Huguet S, Mokhtari K, Marie Y, Kujas M, Leuraud P, Capelle L, Delattre JY, Poirier J, Broet P, Sanson M. Molecular heterogeneity of oligodendrogliomas suggests alternative pathways in tumor progression. Neurology 2001;57(7):1278-81.

13. Megyesi JF, Kachur E, Lee DH, Zlatescu MC, Betensky RA, Forsyth PA, Okada Y, Sasaki H,  Mizoguchi M, Louis DN, Cairncross JG. Imaging Correlates of Molecular Signatures in Oligondendrogliomas. Clin Cancer Res 2004; 10: 4303-4306.
14. Zlatescu MC, TehraniYazdi A, Sasaki H, Megyesi JF, Betensky RA, Louis DN, Cairncross JG. Tumor location and growth pattern correlate with genetic signature in oligodendroglial neoplasms. Cancer Res 2001;61(18):6713-5.

Intraoperative visualisation for gliomas resection: the role of functional neuronavigation and 1.5 Tesla MRI
R. Fahlbusch, C. Nimsky, O. Ganslandt
Neurosurgical Department, University Erlangen-Nuremberg, Erlangen, Germany

Intraoperative high-field magnetic resonance imaging (MRI) combined with microscope-based neuronavigation is a safe and reliable technique providing immediate intraoperative quality control in brain tumors.

Methods: A rotating operating table was adapted to a 1.5 Tesla MR scanner (Siemens Sonata Maestro Class), placed in a radiofrequency-shielded operating theatre. Active magnetic shielding of the scanner allows the combination of surgery in the fringe field with high-field MRI. The navigation microscope (Zeiss NC4) placed in the 5 G zone in combination with a ceiling mounted navigation system (BrainLab Vector Vision) enables integrated microscope-based neuronavigation.

Results: In the first 2½ (since 4/2004) years 479 patients were investigated by intraoperative high-field MRI, among them 214 craniotomies, 90 burr hole procedures, and 175 patients that were operated on a transsphenoidal approach. Pituitary tumor, gliomas, and epilepsy surgery were the major indications. Comparing pre- and intraoperative image quality did not show any significant differences, i.e. intraoperative high-field MRI provides intraoperative anatomical images at a high quality that resembles the standard imaging quality of neuroradiological routine. Intraoperative imaging resulted in a modification of the surgical strategy in 29 %; concerning gliomas these rates were even higher with 41 % Glioma resections were more complete in WHO grade I-III, gliomas with less morbidity: Less than 3 % of our patients with tumors in or adjacent to eloquent areas suffered from additional neurological deficits.

Perspectives: The original concept of intraoperative MRI and functional neuronavigation has been expanded by including Proton-spectroscopy and diffusion weighted images (DTI). Proton-spectroscopy data are helpful to expand the tumor resection from anatomical to metabolic mapping in gliomas. Meanwhile DTI allows visualisation of pathways, especially the pyramidal tract, which can be integrated into neuronavigation and intraoperatively re-visualised.

References:
Nimsky, C. ..., R. Fahlbusch. Volumetric assessment of gliomas ... , Neurosurgery 55: 2004, 358
Nimsky, C. ...., R Fahlbusch. Preoperative and intraoperative diffusion tensor imaging ..., Neurosurgery 56: 2004, 130
Telomerase as a possible target in brain tumors therapy

Maria Laura Falchetti1, Antonio Sorrentino1,  Maria Patrizia Mongiardi1, Paolo Fiorenzo1, Nicola Maggiano2, Francesco Pierconti2, Luigi Maria Larocca2, Giulio Maira3, Roberto Pallini3, Andrea Levi1
INeMM CNR1, Institutes of  Human Pathology2 and Neurosurgery3, Catholic University, Rome, Italy.

The majority of human cancers express high levels of telomerase activity that allows repeated cell duplications and may also promote cell growth and reduce apoptosis. Telomerase is thus a potential target for treatment of neoplasia although the outcome of telomerase inhibition may strongly depend on the repertoire of genetic alterations in transformed cells. Grade IV astrocytomas, also called glioblastoma multiforme (GBM), are very aggressive neuroepithelial tumors with extremely poor prognosis. All GBMs express the mRNA for the catalytic subunit of telomerase (hTERT) as opposed to grade I and grade II astrocytomas that are telomerase positive in about 30% of cases, and grade III astrocytoma that are positive in about 80% of cases. It is therefore suspected that telomerase might contribute to the aggressive behavior of GBMs.  In addition to glioma tumor cells, hTERT mRNA is also expressed by the endothelial cells of the tumor vasculature, and this expression correlates with the up regulation of cMYC mRNA.  Since hTERT expression is not detected in other instances of pathological angiogenesis, it is possible that telomerase might specifically allow the endothelial cells to proliferate within the stressing microenvironment created by a growing neoplasm. To get insights into the role of telomerase in GBM growth, we used RNA interference to down regulate the expression of telomerase in two established cell lines of GBM, TB10 (secondary GBM) and U87MG (de novo GBM), and investigated whether telomerase inhibition affects the growth of the tumor cells both in vitro and in xenografted nude mice. To address the function of telomerase in endothelial cells survival within a growing tumor, we cografted GBM cells and primary human vascular endothelial cells (HUVEC), which have previously been engineered so that telomerase expression was either prevented or up regulated. Here, we show the following, i) telomerase inhibition has no effect on GBM growth in vitro but strongly reduces growth of subcutaneously or intracranially grafted GBM cells, ii) this effect does not seem to require substantial telomere attrition,   iii) telomerase inhibition appears to affect the malignant phenotype of xenografted GBM so that necrosis, one main histological landmark for glioblastoma, is remarkably reduced,   iv) over expression of telomerase allows long term survival of HUVEC cells subcutaneously engrafted in nude mice. Usefulness of telomerase inhibition in cancer therapy was challenged by the concern that lack of telomerase may result in dysfunctional telomeres and thus may promote genomic instability and cancer progression. Our data suggest, however, that the reduction of telomerase activity might impair tumor growth within a few population doublings that is before any substantial telomere shortening.  Antitelomerase drugs may also decrease angiogenesis by targeting the vascular endothelial cells of the tumor vasculature that do not bear transforming mutations and that might be quite sensitive to anti-telomerase treatments.  In addition, the location of the vascular endothelial cells in the abluminal portion of the tumor vessels, right in contact with the bloodstream, makes this target extremely suitable for perfusion chemotherapy.

DENDRITIC CELL VACCINATION FOR GLIOBLASTOMA MULTIFORME AND TUMOR NEUROSPHERES AS A POTENTIAL TARGET FOR IMMUNOTHERAPY

Pellegatta S, Tunici P, Cusimano M, Suarez-Merino B and Finocchiaro G.Unit of Experimental Neuro-Oncology, Istituto Nazionale Neurologico Besta, Milano, Italy.

Immunotherapy plays a central role in the search for new treatments for glioblastoma multiforme (GBM). In particular, several phase I studies have been performed using dendritic cells (DC) pulsed by syngeneic tumor homogenates as a vaccine for relapsing GBM (Yu et al, 2004). As a first step in the preparation of appropriate conditions for a clinical evaluation trial we have performed pre-clinical experiments on immune-competent mice injected intra-cerebrally (ic) with syngeneic GL261 GBM cells and treated subcutaneously (sc) with DC loaded with a GL261 homogenate. Murine DC were prepared from the bone marrow of C57Bl6/J.EGFP transgenic mice (H-2b). Experiments were based on vaccination with DC pulsed with GL261 tumor extracts. Survival rates of vaccinated mice was significantly higher than that of mice treated with PBS (p<0.0005), tumor extract (p=0.001), unpulsed-DC (p=0.05). Mice injected with DCs pulsed with tumor extract (n=8) or with tumor extracts only (n=8) were also investigated by MRI, JAM assay and histological analysis. The JAM assay was performed by examining the ability of splenocytes (including CD8+ T cells) to lyse autologous tumor cells in vitro. In mice vaccinated with pulsed DC, MRI showed no evidence of tumor: robust cytotoxicity was demonstred with JAM assay, and a significant presence of CD3+/CD8+ cell infiltrates into the tumor was evaluated histologically. We also injected C57BL6J mice with GL261 GBM cells on day 0 and treated on day 7, 14 and 21 by injection of: PBS, tumor extracts and DCs pulsed with tumor extract. Survival rates of vaccinated mice was higher than that of mice treated with PBS (p<0.05), or tumor extract alone (p=0.2) These results support the development of DC-based clinical trials for GBM patients that do not respond to standard therapies.
At the same time more specific targets for this immunotherapy approach should be considered . Recently it has been proposed that only a subset of GBM cells has the potential to perpetuate the tumor. These cancer stem-like cells (CSC) can self-renew as neurospheres.
We have obtained neurospheres from three adult patients with GBM and found that they can express glial and neuronal markers in vitro and generate tumors in the brain of CD1 nu/nu mice. Although these tumors were highly infiltrating, they did not completely recapitulate the features of the original GBM. Cells from two GBM were cloned and we found a positive relationship between the fraction of  clone-originating cells and their proliferation in vitro. Adherent cells were also grown in the absence of EGF and b-FGF and when injected into mouse brains they also caused large infiltrating gliomas, but later and less frequently than neurospheres (p<0.02 on Kaplan Meier analysis, log rank test). These observations suggest that EGF-bFGF may facilitate the growth of tumor initiating cells with cancer stem-like properties: thus, tumor neurospheres could be considered as a novel target for DC vaccination in GBM.

Eric C. Holland
Memorial Sloan Kettering Cancer Center
New York, NY. 

Unregulated PDGF expression in neural progenitors, caused by retroviral-mediated gene transfer, leads to the formation of gliomas with an oligodendroglioma phenotype.  The grade of these tumors can be elevated by either elevation in the expression of PDGF, or the addition of mutations in INK4a-Arf.  Previous work has also indicated that the expression of PDGF appears to be non-uniform in these tumors where some cells express large amounts of the PDGF while other cells show PDGF expression below the level of detection.  The data imply a possibility that some of the tumor cells may not be derived from the originally infected cells but rather recruited from the surrounding brain.  To investigate this possibility, we generated an RCAS vector that expresses both PDGF at moderate levels inducing low-grade gliomas in a wild type background, and high levels of GFP allowing the determination of progeny of the originally infected cells from recruited cells.   Additional mutations in ink4a, arf or pten lead to elevated grade of gliomas generated by this vector.  We find that in all grades of these gliomas the tumors are composed of both progeny (GFP+) and recruited cells (GFP-), and that both cell types contribute to the secondary structures of Scherer.  The loss of tumor suppressor genes, particularly arf and pten, promotes the recrutability of cells into high-grade glioma features such and pseudopalisading necrosis.  Furthermore, the recruited cells with deletions in arf and pten expresses many of the markers of CNS stem and progenitor cells.  The data imply substantial complexity and heterogeniety in gliomas, and potentially multiple sources for stem-like cells in these tumors.
Neuroectodermal tumors as diseases of development

Dr. Iavarone

Institute for Cancer Genetics, Department of Neurology and Department of Pathology, Columbia University Medical Center, New York, USA

Proliferation of progenitor cells and their decision to differentiate must be tightly controlled to avoid tumor formation. The Id family includes a crucial group of proteins, widely expressed during development and responsible for the timing of cell cycle exit and differentiation. Our work discovered that genetic oncogenic changes converge on the activation of Id2 to implement multiple hallmarks of embryonal neuroectodermal tumors, such as uncontrolled cell proliferation, anaplasia and neoangiogenesis.

Through the use of restriction landmark genome scanning (RLGS), we recently examined gene-associated CpG island methylation in Juvenile Pilocytic Astrocytoma (JPA) to identify candidate tumor suppressor genes in this childhood brain tumor. Compared with normal pediatric cerebellum, several genes were aberrantly methylated in regulatory regions. Some of these genes have important functions for development and patterning of the cerebellum. Reversal of methylation by treatment of glioma cell lines with 5-azacytidine restored expression of the genes that are methylated in JPA. 

The nature of the events targeted by Id2 during development and neural cancer and the functional role of methylation in the pathogenesis of JPA will be discussed.
Cellular therapies for human sci: the olfactory mucosa experience:

Lima, C*; Vital, JP;Escada, P;Peduzzi, J.
Hospital de Egas Moniz, Portugal

BACKGROUND: Olfactory mucosa is a readily accessible source of olfactory ensheathing and stem-like/progenitor cells for neural repair.

AIM and METHODS: To determine the safety and feasibility of transplanting olfactory mucosa autografts into patients with traumatically injured spinal cords, experimental animal studies and a human pilot trial were conducted. Experimental animal studies consisted of olfactory mucosa transplants in guinea pigs with subacute transected spinal cords and in rats with chronic, severe contusive spinal cord injury. In the human studies, 7 patients ranging from 18-32 years of age (ASIA class A or B) were treated at 6 months-6.5 years post-injury. Olfactory mucosa autografts were transplanted into lesions ranging from 1-6 cm that were present at C3-T8 vertebral levels. Operations were performed between July 2001 and March 2003. MRI, EMG, ASIA neurological and otolaryngological evaluations were performed before and after surgery.

RESULTS: Animal experiments demonstrated graft integration and functional improvement. In human studies, MRI studies revealed moderate to complete filling of the lesion sites. No significant adverse events were observed clinically, except for a slight sensory decrease in one patient . On a 18 months follow-up almost every patient improved in their ASIA neurological scores. Using parametric statistics, there was a significant improvement in the sensory light touch, sensory pinprick, and motor legs component of the ASIA scores using both parametric and non-parametric statistics. Olfaction returned completely within 3 months. From March 2003 to present 43 additional patients performed the surgery with variable levels of recovery and similar and/or better outcomes. Results from these patients with long follow-up are being updated.

CONCLUSION: This study demonstrates that olfactory mucosa autograft transplantation into the human injured spinal cord is feasible, safe, and potentially beneficial.. Long-term patient monitoring is however necessary to rule out any delayed side effects and assess any further improvements.

NEURAL STEM CELLS FOR CLINICAL APPLICATION

Stephen Minger

Neural cell replacement has been shown to provide therapeutic benefit to patients with neurodegenerative disorders.  However, the reliance on relatively large numbers of early gestational human embryos, particularly for Parkinson's disease, makes it unlikely that this will benefit more than a small number of individuals.  One alternative is to isolate neural stem cells of therapeutic interest from specific regions the developing brain and to expand these cells ex vivo to the requisite numbers required for transplantation.  To date, it has not been possible to maintain the multipotentcy of neural stem cells in long-term culture, and thus provide the types of neural cells required for clinical applications.  Embryonic stem cells, derived from the inner cell mass of the early blastocyst, have the developmental capacity to develop into every type of cell and tissue of the body, and thus may serve as an alternative source population of neural cells for CNS cellular replacement.  This presentation will focus on attempts to derive specific sets of neural stem/precursor cells from human embryonic stem cells that may have therapeutic benefit in neurodegenerative disease, spinal cord damage and traumatic brain injury.
LOCOREGIONAL PRE-TARGETED 90Y-BIOTIN RADIOIMMUNOTHERAPY (PAGRIT®) IN GLIOBLASTOMA PATIENTS
Giovanni Paganelli, Divisione di Medicina Nucleare, Istituto Europeo di Oncologia, Milano

In an attempt to overcome the low uptake of radiolabeled monoclonal antibodies by the tumour and improve the tumour-to-blood ratio, various studies have examined the concept of tumour pre-targeting based on the separate administration of MoAbs and radiolabeled isotopes. Among the tumour pre-targeting strategies investigated, a 3-step method was studied and applied with encouraging results by our group: this model is based on the avidin-biotin system.
Pre-targeted antibody guided radioimmunotherapy (PAGRIT®), as systemic or locoregional application has the potential to become a well tolerated therapeutic option in the management of High Grade Gliomas (HGG), complementing traditional regiments. HGG are apt targets for evaluating the therapeutic efficacy of new pre-targeting strategies since: a) the prognosis is poor and patients' life expectancy is less than a year; b) a suitable marker, the glycoprotein tenascin, is abundant in the stroma of HGG, but not in normal cerebral tissues.
In a previous phase I-II study, the safety profile and anti-tumor efficacy of pre-targeting locoregional radioimmunotherapy (LR-RIT), based on the "3 step" method, was assessed in 24 high-grade glioma patients (pts). The encouraging results in terms of low toxicity and objective response rate (25%) prompted us to continue our study.
METHODS: 73 pts with hystologically confirmed glioblastoma (GBM) were treated with the "3 step" 90Y-biotin based LR-RIT. All pts had a catheter implanted at 2nd surgery and underwent at least two cycles of LR-RIT (range 2-7) with two months interval. Thirty-five out of 73 pts were also treated with Temozolomide (TMZ). Two cycles of TMZ (200 mg/m2/day, for 5 /28 days) were administered in between each course of LR-RIT. Overall survival (OS), and progression free survival (PFS) were calculated.
RESULTS: Objective response was achieved in 12.3% of pts, SD in 63%, while 24.7% progressed. In the 38 pts treated with LR-RIT alone, median OS and PFS were respectively 17.5 months (95%CI=[17-20]) and 5 months (95%CI=[4-8]), while in the 35 treated with the combined treatment (LR-RIT+TMZ) respective values were 25 months (95%CI=[23-30]) and 10 months (95%CI=[9-18], (p<0.01). The addition of TMZ to LR-RIT did not increase neurological toxicity, and no major hematological toxicity was observed.
CONCLUSIONS: These results confirm the safety and the efficacy of 90Y LR-RIT in recurrent GBM pts. The poor prognosis of this neoplasm justifies catheter implant at first surgical debulking.

Keywords: radioimmunotherapy, temozolomide, glioblastoma, pre-targeting, monoclonal antibodies, avidin, 90Y-biotin.

Transplantation of Neural Stem Cells onto the Injured Spinal Cord.
Experimental Studies.

Roberto Pallini, Lucia Ricci Vitiani *, Patrizia Casalbore °, Carlo Cenciarelli, Francesco Facchiano *, Liverana Lauretti, Luigi Maria Larocca ç, Maria Laura Falchetti °, Eduardo Fernandez, Ruggero De Maria *, Giulio Maira, Eugenio Parati §.

Laboratory for Neural Stem Cells, Center for Research on Regeneration of the Nervous System, Department of Neurosurgery, Catholic University School of Medicine, Rome,   ° Institute of Neurobiology and Molecular Medicine, CNR, Rome, and  § Laboratory of Neurobiology, Department of Neurobiology and Neurorestorative Therapies, National Neurological Institute C. Besta, Milan;    ç Institute of Pathology, Catholic University School of Medicine, Rome;  * Department of Hematology, Oncology and Molecular Medicine, Istituto Superiore di Sanità, Rome, Italy.

Neural stem cells (NSCs) have been shown to proliferate as undifferentiated spheroids and to differentiate into the major phenotypes of the adult CNS in vitro.  These features candidate NSCs for transplantation in cases of neural trauma or degenerative diseases.  Here, murine NSCs were engrafted onto the injured spinal cord (SC) of rodents in order to assess their potential to survive and differentiate following traumatic injury.  Homologous NSCs survived as long as 9 months after grafting, migrated from the grafting site with a tropysm towards the lesion, and either remained undifferentiated or differentiated into the astrocytic phenotype without neuronal or oligodendrocytic differentiation.  The tips of severed descending axonal paths, which were labeled using anterograde axonal tracers, came adjacent to NSCs without forming synapse-like structures. Grafting with NSCs partly improved motor behaviour on foot-print analysis of ambulation.  When genetically engineered to overexpress the vascular endothelial growth factor (VEGF), the grafted NSCs increased vessel density in the injured area.   Irrespective of the use of immunosuppression,  xenografts of murine NSCs elicited an immune response in the rat SC that resulted in their rejection.  In the traumatically injured SC, differentiation of engrafted NSCs is restricted exclusively towards the astrocytic phenotype.  The project of repairing severed axon systems with NCSc that would receive afferent impulses and project on denervated targets still requires a more accurate knowledge of molecular mechanisms underlying glial scarring and axonal regeneration.  However, the use of genetically modified NSCs that express trophic factors appears an actracting and immediate tool in SC transplantation research. 

Neural stem cells and multiple sclerosis
Stefano Pluchino, Neuroimmunology Unit-HSR, Milano, Italy
Intravenously injected syngeneic adult neural progenitor cells (aNPC) promote multifocal remyelination and functional recovery in mice affected by a chronic-progressive (CP) form of experimental autoimmune encephalomyelitis (EAE) (Pluchino et al., 2003). To further explore the role of adult neural precursor cells (aNPCs) in protecting mice from chronic neurodegeneration during experimental autoimmune encephalomyelitis (EAE), we have injected intravenously (i.v.) syngenic aNPCs into mice with a relapsing-remitting form of the disease (R-EAE). SJL mice were immunized subcutaneously with proteolipid protein (PLP)139-151 and i.v. injected with 1x106 millions of syngenic aNPCs either at the disease onset (13 days post-immunization [dpi]) or after the occurrence of the first clinical relapse (31 dpi). After transplantation, i.v.-injected aNPCs entered both the brain and the spinal cord, selectively reached inflamed CNS areas, and showed to be able to survive for up to 106 days after transplantation. Three months after immunization, mice transplanted at disease onset or after the first relapse showed a significant (p<0.05) decrease of the disease score, the cumulative score and of the number of relapses. A significant decrease of the extent of demyelination and axonal loss was also found. Interestingly, the majority of the transplanted aNPCs persisting and surviving over time within the CNS around inflamed deep blood vessels maintained an mainly undifferentiated phenotype. Interestingly, transplanted cells accumulated selectively around brain post-capillary venules where glio- and neuro-genic regulators (e.g. BMP-4, Noggin) - possibly contributing to the maintenance of aNPCs in a quiescent state - were co-localized. Thus, our data indicate that aNPCs - once i.v. transplanted - may survive within the CNS for long periods of time and protect from relapsing-remitting EAE, owing to their ability to selectively reach perivenular inflamed area where stem cell regulators are selectively expressed.

-Pluchino S, Quattrini A, Brambilla E, Gritti A, Salani G, Dina G, et al. Injection of adult neurospheres induces recovery in a chronic model of multiple sclerosis. Nature 2003; 422: 688-94.

Molecular Genetics in the Assessment of Treatment Response in Childhood Gliomas, and  Applications for Novel Therapies.
 Ian F. Pollack.  University of Pittsburgh, Pittsburgh, PA, USA.

With the exception of certain rare childhood glioma variants, pediatric gliomas are histologically similar to lesions that arise in adults.  However, they have distinct molecular features, suggesting age-related pathways of tumorigenesis.  Whereas high-grade gliomas in adults, particularly grade IV lesions, typically have amplification of EGFR and mutations of PTEN, our results in the multi-institutional CCG-945 cohort demonstrated that such changes are rare in pediatric high-grade gliomas.  In contrast, approximately 40% of pediatric malignant gliomas have p53 mutations, which were noted to constitute an adverse prognostic factor.  P16 or Rb deletions are observed in approximately half of tumors, and correlate with p53 mutations.  Unlike the situation in adults, chromosome 1p and 19q deletions, although present in a subset of gliomas, is not associated with a favorable prognosis.  Studies are in progress to determine whether prospective categorization of tumors by these molecular features identifies prognostically distinct tumor subgroups.  Our recent studies indicate that categorization of pediatric malignant gliomas by their expression of drug resistance markers, such as alkylguanyl-DNA alkyltransferase, may also help to predict response to alkylator-based chemotherapeutic agents, such as lomustine and temozolomide.  In parallel with these analyses, studies are in progress to test new therapeutic approaches for these tumors that capitalize on information gained about relevant molecular targets. 
Molecular markers and biological behaviour of gliomas

Guido Reifenberger

Department of Neuropathology, Heinrich-Heine-University, Düsseldorf, Germany

Glioma classification is based on the histological assessment of the tumor tissue according to the WHO classification system. However, response to therapy and prognosis may be highly variable from patient to patient, even among patients with histologically identical gliomas. Therefore, it is of great importance to identify novel molecular markers that improve the prediction of response to therapy and survival time. We investigated different types of human gliomas for aberrations at the chromosome, gene and mRNA levels using focussed genetic and epigenetic analyses of candidate genes and chromosomes, as well as microarray-based approaches for global expression and genomic profiling. For the latter purpose, we devised a genomic microarray for array-based comparative genomic hybridization (array-CGH) analysis of primary brain tumors. Application of this microarray for the genomic profiling of different types of gliomas revealed that molecular classification of these tumors based on their genomic profiles was largely congruent with histological classification, with some notable exceptions. Correlation of molecular genetic data with patient survival revealed significant correlations of certain genetic alterations with overall survival of glioma patients, including combined losses of 1p and 19q as well as other changes. Taken together, our findings indicate that molecular markers provide clinically useful information that can improve the diagnostic assessment of gliomas.

Enumerating neural stem cell frequency in the embryonic and adult CNS

Brent A. Reynolds
Queensland Brain Institute, University of Queensland, Brisbane, Australia

The Neurosphere Assay (NSA) is widely used to isolated, propagate and enumerate stem cells derived from the CNS. A central tenet of the NSA is the premise that all neurospheres are derived from a stem cell and hence, quantifying the number of spheres represents an accurate predictor of stem cell frequency.  This premise, however, is based on the unfounded assumption that since a stem cell can form a neurosphere that all neurospheres must be derived from a stem cell. Analysis of long-term serial passage indicates that the NSA overestimates stem cells by an order of magnitude. Hence, a need exists for an assay that is able to provide an accurate and meaningful read-out of stem and progenitor cell numbers in the embryonic and adult CNS.

To more correctly calculate stem and progenitor cell frequency, we have developed two new assays; i) a single-step culture assay called the Neural Colony Forming Cell (NCFC) assay, and ii) a mathematical model that uses long-term serial passage date to estimate stem and progenitor cell numbers. Functional analysis of the NCFC assay and validation of the mathematical model using embryonic and adult precursors indicates that both assays provide a meaningful and accurate calculation of stem and progenitor cell frequency. The ability to correctly measure and distinguish between these two populations will allow us to better understand the genetic and epigenetic regulation of stem and progenitor cells during development, in the adult CNS and access their contribution to tumor formation.

ERK1/2 EXPRESSION IN GLIOBLASTOMA AND PERITUMORAL TISSUE.
G. Lama, A. Mangiola°, C. Anile°, S. De Silvestri, G. Sabatino°, B. Pettorini°, L. Lauriola°°, C. Giannitelli, G. La Torre°°°, G. Maira°, G. Sica.
Istituto di Istologia ed Embriologia, °Istituto di Neurochirurgia, °°Istituto di Anatomia Patologica, °°°Unità di Epidemiologia e Biostatistica, Facoltà di Medicina e Chirurgia, U.C.S.C., Roma.
The extracellular signal-regulated kinase (ERK1/2) belongs to the mitogen activated protein kinase (MAPK) family and is generally involved in cell proliferation and differentiation. The activity is expressed in neurons due to physiological stimuli, while in glial cells is present in case of subacute or chronic lesion. Activation of ERK1/2 has been reported in human glioma cells, but scarce information derives from literature concerning its presence in tissue surrounding the tumor.
The aim of the present work was to investigate, by immunohistochemistry, the presence of both total (t) and phosphorylated (p) ERK1/2 expression in the peripheral areas surrounding glioblastoma multiforme (GBM). This study involved 26 patients with supratentorial primary GBM treated by surgery between October 2002 and October 2004.
Twenty-six tissue specimens were obtained from the tumor (first area), 21 from a second area (<1 cm from the edge of the tumor) and 17 from a third area (from 1 cm up to 3.5 cm from the edge of the tumor).
In peripheral areas both apparently normal and reactive astrocytes were present, whereas tumor cells were in a different percentage or absent.
Immunohistochemistry was performed on tissue sections obtained from  formalin-fixed paraffin-embedded samples using a polyclonal antibody anti-p44/42 MAPK (tERK1/2) and a monoclonal antibody anti-phospho-p44/42 MAPK (pERK1/2). Detection was achieved with the ABC method using DAB as the chromogen.
The percentage of t and pERK 1/2 labeled cells was evaluated from randomly selected fields.
Total ERK1/2 expression was detected in all areas considered and was uniformly distributed. Immunostaining was mostly observed in the cytoplasm.
The activity appeared to be higher in the peripheral areas than in the tumor (p<0.006 Wilcoxon Signed Rank test). The difference is small and has not a clear biological meaning.
Phosphorylated ERK1/2, almost located in the nuclei, was heterogeneously distributed. It was expressed in neoplastic cells and, in peripheral areas, was found in both reactive and apparently normal astrocytes. No statistically significant difference in the expression was found between the tumor and the peripheral areas. In 5 out of the 12 cases where tumor cells did not appear in tissue surrounding the tumor, pERK1/2 was less expressed in the third area than in the second (p<0.04 Wilcoxon Signed Rank test).
Cox proportional hazards survival regression analysis showed that patients with a percentage of pERK1/2 stained cells >33.28% and age =65 years had about 4 times greater risk of death than those with a percentage of pERK1/2 stained cells ?33.28% and age <65 years.
Our findings are in agreement with data from literature regarding pERK1/2 expression in GBM. Further investigation is required to clarify the meaning of the expression in apparently normal glial cells.
Supported by FIRB 2001