Microsoft word - posters.rtf

Allelic losses on chromosomes 1p, 19q, 17p and 10q as predictors of survival in oligoastrocytomas
Bissola L, Eoli M, Merciai B, Silvani A, Salsano E, Broggi G, Boiardi A and Finocchiaro G
Istituto Nazionale Neurologico Besta, Milano, Italy.
Oligoastrocytomas are mixed gliomas consisting of varying proportions of astrocytic and oligodedrocytic cells:
genetically they are only partially characterized. Oligodendogliomas often show coincident loss of heterozigosity
(LOH) on chromosome 1p and19q and these marke rs are associated with a good prognosis, possibly related to
sensitivity to chemotherapy. LOH on 17p occurs in approximately one third of malignant astrocytomas, and has
been linked to the formation of secondary glioblastomas. LOH on 10q is present in a fr action of anaplastic
astrocytomas but is highly frequent in glioblastomas. To contribute to the genetic characterization of mixed
gliomas and to look for clinical correlations we have investigated LOH by amplification of different
microsatellites on 1p, 19q, 17p and 10q on lymphocyte and tumor DNA from 34 oligoastrocytomas (9 grade II
OA and 25 grade III AnOA).
In all tumors investigated at least one marker was informative. LOH for 1p was found in 15 cases, LOH for 19q
in12 cases (9 of them were associated with LOH on 19q), LOH for 10 q in 7 cases and LOH for 17 p in 6 cases.
The alterations on 1p, 17p, 10q were never associated. Patients with LOH on 10 q had a shorter survival
compared to all the other patients (p = 0.0024) and the poor prognosis appeared i ndipendent from other clinical
parameters affecting survival. Correlations with losses on 1p and 17q require a longer follow -up for statistical
evaluation, but a trend for longer survival seems associated with 1p and 19q losses. The data support the use of
genetic analysis to help the clinical management of mixed gliomas.
New ribozymes targeting vegf production to inhibit neoangiogenic potential of brain tumor cell lines
Ciafre S.A., Wannenes, F.,and Farace M.G.
Glioblastoma multiforme is one of the most highly vascularized solid neoplasms, with the amount of
neovasculature closely correlated with the degree of malignancy. Among angiogenic factors, vascular
endothelial growth factor (VEGF) plays an important role in the neovascula rization and growth of human
cancers, and particularly in gliomas, where up -regulation of VEGF is a common event. Thus, strategies to down -
regulate VEGF could be applied to reduce the preformed tumor vasculature in established gliomas. We have
designed hammerhead ribozymes against VEGF mRNA, with the aim of strongly down -regulating expression
and secretion of VEGF by glioma cells and inhibiting their proangiogenic potential. Our experimental strategy
can be summarized into the following steps: a) Design of two new ribozymes targeting human VEGF mRNA
specifically cutting its sequence around 5' end, thus yielding short pieces of mRNA completely unavailable to
the translational machinery. b) Cloning of the designed ribozymes into a short region of Adenovirus t ype 2
(Ad2), VAI, owning several features making it an optimal carrier for efficient and specific delivery of ribozymes
into the cytoplasm: VAI is transcribed by the RNA Polymerase III, which drives high levels of expression, its
secondary and tertiary structure confers high stability to the transcribed molecule, and VAI is specifically sorted
into the cytoplasm, where the activity of the ribozyme is required. c) Stable transfection of a human glioma cell
line, U87, with plasmids harboring the anti-VEGF ribozyme-VAI "hybrid" molecules, and selection of single
cell clones. d) ELISA measurement of VEGF secretion from transfected glioma cells. With this assay we have
demonstrated a significant reduction (about 50% of wild type concentration) of VEGF secretion in at least two
transfected clones. We are now looking at VEGF mRNA expression in correlation with ribozyme transcription,
and we will then proceed to functional tests to assess the relative efficiency of the supernatants of!
transfected cell lines in the induction of the proliferation and/or migration of endothelial cells. These in vitro
data are preliminary to further in vivo experiments, where we will study growth and vascularization of
subcutaneously injected tumors in SCID mice, and of experimentally induced intracranial gliomas in rats.
Cell-based delivery of cytokines allows for the differentiation of a doxycycline inducible oligodendrocyte
precursor cell line in vitro
Chen U, Elmshauser C, Motta I, and Beck E
We report here partial characteriz aton of a "tet-on± glial O2A precursor cell line established from rtTA -
SV40Tag double transgenic mice. In culture, withdrawal of doxycycline prevents proliferation and the cell line
undergoes apoptosis. Importantly, differentiation into type -2-astrocytes and oligodendrocytes can be induced
when the cell line is cultured, in the absence of doxycycline, with epithelial stem cell lines secreting hIL3 or
hIL6. In contrast, no maturation into progeny was observed when a hCNTF -secreting cell line was used as the
co-culture partner under the same condition. This glial O2A precursor cell line may provide a tool to develop cell
therapy for damaged CNS.
Muth H, Elmshauser C, Broad S, Schipke C, Kettenman H, Beck E, Kann M, Motta I, and Chen U. "Cell -based
delivery of cytokines allows for the differentiation of a doxycycline inducible oligodendrocyte precursor cell line
in vitro± Journal Gene Medicine, in press (2001).
Treatment of recurrent glioblastoma with HSVtk and IL -2 gene intratumoural implantation followed by
intravenous Ganciclovir administration.
Colombo F, Zanusso M, Casentini L, Mazzetto M, Volpin L, Cervellini P, De Luca P.
Recurrent malignant gliomas have been treated in our Department with a gene therapy protocol that combines
immune stimulation with methabolic activation of Ganciclovir (GCV). 10 patients affected by glioblastoma
recurrences after standard treatment underwent multiple (6 -8),single session, imaged guided, stereotactic
intratumoral injections of murine therapeutic retrovir al vector producer cells. The vector was a newly designed
Moloney murine leukemia virus (LXSN prototype) expressing the human interleukin 2 (IL -2) gene and the
herpes simplex virus Type 1 (HSV-1) thimidine kinase (tk) gene.We didn't observe any surgical co mplication or
adverse reaction to the procedure. 7 days after implantation, the patients have been treated with ganciclovir IV
for 14 days and followed by magnetic resonance imaging (MRI) before and after gene surgery.2 patients affected
by a large tumoral recurrence died within two months. 4 patients survived from 5 to 12 months.In a seven
months ago t!
reated woman, the disease is starting again. Since 4-6 months 3 other patients are followed after therapy. Follow-
up by MRI and CT demonstrated areas of ne crosis around the site of injection in two patients. In one patient a
decrease in solid tumor volume was observed. In two cases the whole brain underwent a complete
neuropathological study including an analysis for the presence of therapeutic genes. With t he aim of ascertaing
"in vivo" the efficency of transduction, two patients underwent stereotactic biopsy sampling of the site of the
vector producer cells implantation, immediately before GCV treatment. Samples were studied with PCR,
molecular analysis and with immunehistochemistry. Treated tumors showed a low level of "in vivo" transfer of
the tk gene . On the other side, MRI follw-up demonstrated a GCV induced tumor necrotizing effect around the
site of injection that has been roughly estimated to involve a volume of about 2000 cumm per injection. In view
of the low trans duction efficiency it is likely that bystander mechanism contribute to this. In fact our study
provides evidence of IL-2 elicited immune-inflammatory cells response around the site of inj ection. The short
range extension of the therapeutic effect suggest an an employ of this technique in cases of early, well
demarcated, small dimensions recurrences.
Development of adenoviral vectors specifically replicating in glial cells.
de Leeuw B, Hoeben R, Brenner M and Sillivis Smitt P.
Efficacy of HSV-TK suicide treatment of malignant glioblastoma using non-replicating adenoviral vectors has
proven not sufficient for tumor treatment. To take advantage of the higher efficiencies of replicating viruses,
while reducing the containment risk involved, we chose a strategy in which adenoviral vectors are developed that
replicate selectively in glial cells. As candidate promoters to drive the adenoviral E1 region we investigated
derivatives of the promoter of the human Glial Fibrillary Acidic Protein (GFAP) gene (gfa2 promoter) with
respect to expression levels and glial specificity. The derivative promoters contain the GFAP enhancer elements
(the respective B and ABD regions) in triplicate.
Plasmids containing the nuclear beta galactosidase gene following the respective promoters were transiently
transfected into glioblastoma cell lines U251 (>95% of cells GFAP positive), T98G (< 5% of cells GFAP
positive), retinal cell line 911 (<5% of cells GFAP positive) and lung carcinoma cell line A549 (GFAP negative).
A similar gene driven by a CMV promoter was transfected as a control. The percentage of positive cells was
determined using a LacZ assay whereas expression levels per positive cell were estimated usi ng an ONPG assay.
The triple B enhancer-type of GFAP promoter proved to be the strongest in our assay, with levels comparable to
the CMV promoter (911: B3 = factor 5 lower; U251: factor 3 lower; A549: no GFAP expression) after correction
for differences in percentage of LacZ positive cells (911: 30 -15%; U251: 6-15% and A549 <1% ), which is a
combination between transformation efficiency and CMV/GFAP expression. The normal gfa2 promoter had a
clearly lower expression level per cell (factor 40 -300 lower than CMV for 911 resp. U251).
Adenoviruses were prepared with the same GFAP promoter -LacZ constructs in the E1 region. Although these
vectors will not be replicative, they will pinpoint to the cell types permissible for replication in case E1 would be
placed under control of these promoters. These will among others be used to infect human glioblastoma sferoids.
Combined treatment of brain tumors with gene directed enzyme prodrug therapy and ionizing radiation
Desaknai S(1), Hamada H(2), Safrany G(1) (1):National Research Institute for Radiobiology and Radiohygiene,
Budapest, Hungary, (2):Biomedical Research Center, Sapporo Medical University, Sapporo, Japan
We have studied the combined therapeutic effect of gene directed enzyme prodrug therapy (GDEPT) and local
irradiation for the treatment of brain tumors. Murine glioma 261 (Gl261) cells were transduced with an
adenoviral vector encoding both the uracil phosphorybosil transferase (UPRT) and thymidine kinase ( TK) genes
that sensitizes cells to 5-fluorouracil (5-FU) and ganciclovir, respectively. Under in vitro conditions the
combined treatment with 5-FU and ganciclovir resulted in an additive cytotoxic effect. The combination of 5 -FU
and ganciclovir treatments with irradiation increased cytotoxicity by three orders of magnitude.
During in vivo experiments, either subcutaneous or intracranial tumors were established in C57Bl mice by
transplanting drug-sensitizing gene containing Gl261 cells. In subcutaneous tumors combined 5-FU and
ganciclovir treatments significantly slowed down tumor progression compared either to untreated controls or to
single agent treatments. The survival of brain tumor bearing mice was also significantly improved (60 -80% after
double agent treatment). Combination with local irradiation increased further the therapeutic effect (90 -100%
survival). The combined modality treatment was also effective when only part of the tumor cells carried the
drug-sensitizing genes. When 20% of the tumor cells were transduced 60-70% of the animals survived, at 10%
rate the survival was still 40-50%.
In conclusion the combination of GDEPT with ionizing radiation resulted in decreased tumor growth and
enhanced survival.
Thalidomide as an anti-angiogenic agent in newly diagnosed glioblastomas: a randomised trial
Eoli M, Silvani A, Salmaggi A, Milanesi I, Fariselli L, Boiardi A
Thalidomide, (alpha-phtalimidoglutarimide), a synthetic derivate of glutamic acid, has been shown to inhibit
endothelial cell proliferation in vitro and angiogenesis in a rabbit cornea micropcket models (Moreira AL, 1999).
For this property the drug is under investigation as antitumor agent. In patients with recurrent high grade gliomas
the drug seems to be effective in achieving stabilization of disease according to recently conducted phase II trials
(Fine H, 2000, Short SC, 2001).
To evaluate safety and potential effectiveness of thalidomide, we conducted a randomized phase II trial in newly
diagnosed glioblastoma patients. Preliminary results regards 24 patients, age 18-65, all patients underwent
surgery with diagnosis of glioblastoma Within three weeks after surgery, before starting radiotherapy, patients
started chemotherapy with CDDP 100 mg/m2 + BCNU 160 mg /m2 every seex weeks (a rm A) or chemotherapy
with CDDP 100 mg/m2 + BCNU 160 mg /m2 every seex weeks and Thalidomide at 400 mg/day p.o.
continuously (arm B). All patients received radiotherapy according to the same protocol. Response was assessed
at 8 weeks intervals and disease progression was defined as radiological evidence of increased tumor size
according to Mc Donald criteria. No difference in Time to Tumor progression was observed: TTP 11 months in
arm A and 10 months in arm B. Thalidomide was generally well tolerated. No adjunctive acute radiotherapy
related toxicities were observed in patients receiving the drug.
Telomerase inhibition and impairment of TRF2 function as a possible approach to GBM gene therapy.
Falchetti ML*, Levi A*, Larocca LM°, Maira Gò and Pallini Rò.
*Istituto di Neurobiologia e Medicina Molecolare, CNR, Roma.
Istituti di °Anatomia Patologica e òNeurochirurgia, Univ. Cattolica del Sacro Cuore, Roma.
Telomerase reactivation is a key event in tumorigenesis and it has been detected in a high percentag e of human
tumors. Glioblastoma multiforme (GBM) is the most aggressive neuroepithelial tumor and its prognosis remains
poor even after appropriate treatment. In previous works, we demonstrated that the vast majority of GBM
reactivate telomerase (1), and that all GBM express the mRNA for the catalytic subunit of telomerase enzyme,
hTERT (2). Moreover, we found that not only the neoplastic cells of GBM but even the proliferating
endothelial cells of the tumor vessels express hTERT mRNA (3). These data ca ndidate GBM to a gene therapy
targeted to an anti-telomerase strategy.
We are now evaluating the possibility of a parallel approach based on the expression of a dominant negative
(DN) version of the telomere binding protein TRF2. The role of TRF2 is to pro tect human telomeres from end-
to-end fusions, preventing chromosomal rearrangements. TRF2 function is crucial to maintain telomere integrity.
It has recently been described that, in mouse models, telomere dysfunction impairs DNA repair and enhances
sensitivity to ionizing radiation (4). Preliminary results will be presented on the expression of a regulatable form
of DNTRF2, which is inducible at the post-translational level.
The development of gene therapy techniques which associate inhibition of telomerase activity and impairment of
TRF2 function through a DNTRF2 is likely to have great potential in treatment of GBM tumors.
LEONARDI, H MAEKE and G PAGANELLI - European Institute Of Oncology, Italy #Vanderbilt University, Nashville, TN (USA): Department of Radiology and Radiological Sciences °Nuclear Medicine Department, University of Basel, Switzerland. *Dipartimento di Ingegneria Nucleare, Politecnico di Milano, Italy Locoregional (LR) administration of 90Y-conjugates after surgical debulking is a promising treatment for gliomas. LR protocol with the somatostatin analogue DOTATOC labeled with 90Y was developed. Its potentiality was investigated in 10 patients (pts) by evaluating the absorbed dose delivered to brain adjacent tissue (BAT). Escalating activities of 370-740 MBq were injected into the surgical resection cavity (SRC) via an appropriate catheter. Scintigraphic images were acquired at 1 and 16 h, along with blood and urine sampling up to 48 h post injection. A dosimetric model based on Monte Carlo simulations combined to the MIRD formalism was developed to evaluate the residence time in S RC (•SRC) and absorbed doses in BAT. The activity (A) in the SRC was considered equal to the total A injected less A in the blood and less the cumulative A eliminated in the urine. BAT was divided in adjacent shells 1 mm thick to evaluate the dose distribu tion around the SCR. The cavity radius (r) and the depth of diffusion of the radioconjugate through the BAT (d) were varied in order to adapt the model to different patients' situations. Based on the most likely diffusion of the DOTATOC through the tissue the parameter d ranged from 0 to 3 mm. Patients were divided in two groups - group I (4 pts): SCR connected to ventricula, group II (6 pts): SRC not connected to ventricula. Higher cumulative %IA in the urine (30% to 50% at 50h) and irregular biodistrib ution were found in group I, with liquor and kidney uptake. In group II, clearance from SRC was slow as confirmed by pharmacokinetics: the blood curve reached low %IA and a slight slope and cumulative %IA in the urine ranged between 10% and 30% at 50%. In group II, mean •SRC was 60 쳌 10 h: in case of no diffusion, absorbed doses to shell II resulted 560 and 60 Gy/GBq for r=12 and 25 mm ; in case of r=12 mm and slight diffusion (d=1 mm), absorbed dose to shells I, III and VI were 1300, 2500, and 270 Gy/GBq. Mean dose to normal brain resulted 15 mGy/GBq. These results indicates that LR 90Y-DOTATOC therapy allows to deliver very high doses to target tissue sparing normal brain. Further developments of the mod el will allow to improve the dosimetric evaluations in normal organs when SRC is connected to ventricula. Keywords: Locoregional treatment; dosimetry; Y-90-DOTATOC, glioma.
Emx2 in Adult Neural Precursor Cells
Gangemi RMR, Daga A, Marubbi D, Rosatt o N, Capra MC, Corte G.
Emx2 is a vertebrate homeobox gene involved in the control of the central nervous system development. In the
forming cerebral cortex Emx2 expression is restricted mainly to the germinal ventricular zone fading away in the
first postmitotic neurons. This expression pattern and the severe impairment of cortex organization and size in
mutant mice suggest a role of Emx2 in the control of proliferation and migration of neural precursor cells. The
observed persistence of Emx2 expression in adult neurogenic areas in vivo is here confirmed at later stages. We
find also that Emx2 is expressed at high levels in adult neural stem cells in vitro and is down modulated upon
Overexpression of Emx2 gene in adult neural stem cells has an antiproliferative effect but it does not influence a
particular differentiation pathway.
Our results suggest that Emx2 may act promoting an asymmetric mode of cell division increasing the size of a
transit amplifying population.
A Preliminary Study of Angiogenesis in Pediatric Glioblastoma Multiforme and its Correlation with
Germano A, Caffo M, Caruso G, Galatioto S., Tomasello F Dip Neurochirurgia, Universit a di Messina, Italy.
Over the last few decades multiple retrospective studies hav e been undertaken to assess survival in patients with
glioblastoma (GM). There is a distinct challenge in bringing modern insights into glial tumour genesis to bear on
improved outcomes for patients. Insights provided by neuroncological, neuroradiological, neuropathological, and
neurosurgical investigations may offer significant advantages in the management of paediatric GBM. Although
the prognosis for patients with glioblastoma multiforme is different between adult and children, to date there is
no specific evidence in the literature to show that angiogenesis can predict the prognosis pediatric GM. The goal
of this study was evaluate angiogenesis as potential indicator of survival in pediatric glioblastoma multiforme.
Angiogenesis was evaluated in six cases of paediatric GBM with multiple criteria, including contrast
enhancement on preoperative CT scan, histological vascular hyperplasia (VH) and endothelial proliferation (EP)
and immunohistochemical tenascin-C (TN-C) expression. We employed a semiquantitative scale, ranging from
non-detected (zero) to marked (+3), for each investigational parameter. We evaluated the influence of
angiogenesis on survival in each case. Results obtained in these cases were compared to early and late
postoperative outcome and survival length. Although based on a limited number of patients, in this preliminary
study, angiogenesis provided information that correlated with survival. As we gain better understanding of the
molecular biology of brain tumours, with the multitude of genet ic alterations and growth factors, new therapeutic
approaches may emerge which may hold the promise for cure.
Role of bcl-2-expression in medulloblastoma and possible relevance to therapy
Giordana MT, Duo D, Gasverde S, Milanese C, Trevisan E
Department of Neuroscience, University of Turin, Turin, Italy.
Modulating the expression of key molecular components of the cell death machinery is an alternative strategy for
the treatment of brain tumors. Bcl-2 overexpression retards the normal course of apop tosis; a decrease of bcl-2
levels sensitizes cells to apoptotic death. Bcl-2 levels in tumors may influence the individual response to
apoptotic insults, including radiation-induced DNA damage. As a suppressor of apoptosis, bcl -2 is active during
maturation of primitive stem cells; therefore, bcl-2 expression in medulloblastoma may alternatively indicate that
tumor cells are differentiating. Aim of the present study was to investigate the role of bcl -2 expression in
medulloblastoma by an immunohistochemical study. Bcl-2 mAb (Dako, 1:50) was applied to the biopsies of 80
medulloblastomas and the results evaluated in relation to patients age (> vs < 16 years), location, type of tumor
(classic vs desmoplastic), MIB-1 immunostaining and labelling index, p53 and MDM2 stat
us and expression, overall postoperative survival. The semiquantitative evaluation demonstrated that bcl -2
expression was more frequent in medulloblastomas of adult patients (76% of cases) than of children ( 23%); no
other correlation was found . In nodular desmoplastic medulloblastomas, tumor cells in peripheral regions of
nodules and in internodular regions were bcl-2-positive and MIB-1-positive, while the cells within nodules were
p53-positive. The same co-expression of bcl-2 and MIB-1 was observed in perivascular cuffs of tumor cells
infiltrating the cerebellar cortex and subpial region. This finding is consistent with the role of bcl -2 in helping
cells to escape death promoting cell proliferation; it contradicts the hypothesis that bc l-2 expression in
medulloblastoma is related to differentiation. The semiquantitative evaluation of bcl -2 expression is not a
prognostic factor of survival, however the role apparently played by bcl -2 in medulloblastoma indicates that
treatments based on bcl-2-modulating approaches might be successful.
Efficient in vivo gene transfer and sustained gene expression by a SIV -based lentiviral vector in ovarian
cancer cells
Walter Habeler, Stefano Indraccolo, Laura Stievano, Sonia Minuzzo, Veronica Tisato , Luigi Chieco-Bianchi,
and Alberto Amadori.
Dipartimento di Scienze Oncologiche e Chirurgiche,Universit a degli Studi di Padova.Italy
Aim of the study: Local gene therapy could be a therapeutic option for ovarian carcinoma, a leading cause of
mortality among gynecological malignancies, because the large majority of women presents with disease
contained within the peritoneal cavity. In this study, we compared gene transfer efficiency by retroviral and
lentiviral vectors in ovarian cancer cells.
Methods: Mo-MLV-based and SIV-based retroviral vectors expressing the enhanced green fluorescent protein
(EGFP) gene were generated and used to transduce ovarian cancer cells both in vitro and in vivo. Efficiency of
gene delivery was determined by confocal scanning microscopy of tumor sections, cytofluorimetric analysis of
EGFP expression and by a quantitative real-time PCR assay.
Results: We found that the ovarian cancer cell line Igrov-1 could be infected with comparable efficiency in vitro
by either retroviral vectors or SIV-based lentiviral vectors carrying the vesicular stomatitis virus G protein
(VSV-G) as the envelope. However, the lentiviral vector was much more efficient than the Mo -MLV-based
vector in transducing aphidylcoline-treated non-proliferating cancer cells. Gene transfer was env-mediated, as it
was neutralized by an anti-VSV-G antibody, and it strictly required the presence of viral particles. In vivo gene
transfer experiments in a xenograft model indicated that the lentiviral vector was much more effic ient than the
retroviral vector to deliver the reporter gene to tumor cells, as evaluated by confocal scanning microscopy of the
tumors and quantitative analysis by flow cytometry. Proviral DNA analysis by a quantitative real -time PCR
assay confirmed these findings at the genomic level. Finally, gene expression by the SIV-based vector was also
considerably more resistant to silencing in vivo, compared to the Mo -MLV-based vector.
Conclusions: These findings indicate that lentiviral vectors deserve attention in the design of future gene therapy
approaches to ovarian cancer aimed at achieving long -term expression of therapeutic genes.
A laboratory and clinical study of the synergy between radiation and the Herpes Simplex Virus
mutant,HSV 1716 as a treatment for brain tumours.
Harrow S, Quigg M, Boyd M, Mairs R, Brown S M, Rampling R.
Glioblastomas are the most common adult brain tumour with a median survival of less than one year. Standard
treatment comprises surgery followed by radiotherapy, however following this regime local recurrence occurs in
about 80% of cases. This treatment failure is due to tumour infiltration into normal brain and the high rate of
tumour cell proliferation following incomplete surgical resection. New strategies are therefore sought to improve
the prognosis for brain tumour patients.
One such strategy is the utilisation of a genetically modified oncolytic Herpes Simplex Virus, HSV 1716, which
is deleted in both copies of the RL1 gene that encodes the virulence factor ICP34.5. This virus is avirulent but
can replicate and lyse actively dividing cells. We have performed phase 1 clinical studies with HSV 1716 to
demonstrate its safety following direct injection in patients with primary and relapsed high grade glioma. No
toxicity has been demonstrated. Phase 2 trails are in progress.
Radiation appears to act synergistically with ICP34.5 null HSV with respect to tumour cell killing. However the
mechanism and optimum scheduling of this synergy are unclear. We are investigating the relationship between
HSV 1716 replication in a human glioma cell line that has been subjected to external beam irradiation. By
maintaining virus replication we hope to define the optimum scheduling of virus plus radiation. In addition we
are using MTT assays to assess the cytotoxicity of HSV1716 with external beam radiation. The activity of
HSV1716 in response to external beam radiation has dir ect clinical relevence as the current treatment of brain
tumours involves external beam irradiation.
COX-2 promoter drives tumor specific transgene expression in primary glioma and meningioma
Idema S(1,2), Lamfers MLM(1,2), Grill J(1,3), Yamamoto M(4), Curiel DT(1,4), van Beusechem VW(1),
Vandertop WP(2), Dirven CMF(2), Gerritsen WR(1).
1)Dept. of Medical Oncology, Division of Gene Therapy, VUMC, Amsterdam,
The Netherlands
2)Dept. of Neurosurgery, VUMC, Amsterdam, The Netherlands
3)Dept. of Pediatrics, Gustave Roussy Institute, Villejuif, France.
4)Gene Therapy Center, University of Alabama at Birmingham, Birmingham, Alabama.
For gene therapeutic strategies for the treatment of gliomas to be successful, high levels of transgene expression
in tumor and low levels of transgene expression in normal brain are desired. To achieve such tumor specificity,
tissue-specific promoters may offer benefit.
Cyclooxygenase-2 (COX-2), involved in the synthesis of inflammatory prostaglandins, is strongly exp ressed in
glioma samples and associated with increased malignancy. In addition, expression is low or non -present in
normal brain tissue.
To utilize the differential expression of COX-2 for achieving tumor specificity, adenoviral vectors were applied
which encode the marker gene luciferase, driven by the COX -2 promoter (Ad.COX.Luc). Promoter activity of
this vector was compared to the control adenoviral vector Ad.CMV.Luc in a panel of glioma cells. Expression of
luciferase under the COX-2 promoter was 1.26 fold (range 0.33 &#8211; 3.32) of CMV -induced luciferase
expression in glioma cell lines and 1.27 fold (range 0.66 &#8211; 1.8) of CMV in primary glioma cells,
indicating the strong activity of the promoter in glioma. In KATO -3 cells, negative for COX-2, a 34% decrease
in luciferase expression relative to CMV was noted.
More importantly, the effects of Ad.COX.Luc were studied on primary organotypic spheroids of glioma and
meningioma tumors and compared to spheroids of normal brain tissue. Spheroids retain th e complex architecture
present in tumors thereby offering a clinically relevant 3 -dimensional in vitro model for studying gene
therapeutic strategies. Infection of spheroids with Ad.COX.Luc strongly decreased luciferase expression in
normal brain (mean 0.1 6 fold of CMV), but gave only a slight decrease in glioma and meningioma spheroids
(mean 0.74 fold of CMV). Whether the results with the marker gene can be translated to a therapeutic effect was
demonstrated using COX-2-driven Thymidine Kinase expression in combination with ganciclovir. Ad.COX-
2.Tk infection on glioma cell lines was found to give a cytocidal effect comparable to that of Ad.CMV.Tk.
These results suggest that COX-2 promoter-based vectors can induce selectivity in transgene expression in
tumors compared to normal brain and offer potential for application in gene therapy approaches using
therapeutic genes.
Coexpression of Shc-A and Shc-C in human glioma.
Lorenzo MAGRASSI (1), Luciano CONTI (2), Andrea LANTERNA (2, 3) Giorgio BUTTI (1), Ele na CATTANEO
(2). 1 Neurosurgery Dept. Of Surgery, University of Pavia - IRCCS Policlinico S. Matteo, Pavia, Italy. 2
Institute of Pharmacological Sciences - University of Milan, Milano, Italy. 3 Neurosurgery University of Milano
Bicocca - Ospedale S. Gerardo, Monza, Italy.
Shc adapter proteins are important modulators of development from undifferentiated neuroepithelial precursors
to mature neurons. Shc like protein are adapters with multiple phosphotyrosine binding domains and play an
important role in receptor tyrosine kinase pathways and other signal transduction pathways where tyrosine
phosphorilation is involved. During normal CNS development maturation from proliferating neuroepithelial
precursors to neurons is characterized by a switch from Sch -A to Shc-C expression. Switching expression from
Shc-A to Shc-C is functionally important for appropriate maturation and survival of neurons. We investigated by
immunocytochemistry and western blot analysis the presence of Shc-A and Shc-C protein isoforms in human
high grade and low grade gliomas. We found that Shc -A proteins are expressed in both low and high grade
gliomas while Shc-C expression is restricted to high grade gliomas. Coexpression of the two genes is never
found during normal development and may re present an important peculiarity of high grade glioma tumors.
Analysis of Italian glioblastoma patiens treated by gene therapy based on HSV -TK and GCV
administration (GTI-0115 protocol)
Milanesi I, Benedetti S, Boiardi A, Silvani A, Bruzzone MG, Far ina L, Fariselli L, Savoiardo M, Giombini S,
Solero CL, Broggi G, Finocchiaro G.
Istituto Nazionale Neurologico Besta, Milano, Italy
The aim of the study was to evaluate the efficacy and feasibility of combining gene therapy based on retroviral -
mediated gene of the HSV-TK gene and GCV administration and radiotherapy in primary glioblastoma patients
(GBM). We enrolled six patients in two randomized arms of treatment. For the whole group of patients the
median age and KPS were 56 yr. (range 38 - 73) and 80, respectively. Two patients were submitted to a total
resection, 4 to a sub-total surgery. After surgery 3 patients were treated by injection of HSV -TK retroviral
producer cells and GCV iv administration, and with radiotherapy (conventional irradiation with 2 Gy/ fraction up
to a total dose of 57- 60 Gy). The control patients received only radiotherapy (same dose and schedule). In the
gene therapy group we observed one CR, one PR and one PD, respectively. Two months after surgery one of the
gene therapy patients was re-operated for an abscess (growing in the surgical cavity). This patient died for a
multifocal evolution of the disease 14 months later. The second patient died one month after surgery because of a
pulmonar thromboembolism. The third died 18 mo nths after surgery for a local recurrence.
The mean survival time was 16 쳌 16 months for the control group and 16.7 months 쳌 13.6 for the gene therapy
group. Thus, although based on very small number of patients, these results are in agreement with the gener al
outcome of a larger study based one more than 250 patients.
This modality of treatment altought feasible and non -toxic does not give any result in term of survival.
Improvements are necessary in term of gene delivery and targeting of the therapy.

G. Paganelli M. Bartolomei, M. Ferrari, M. Cremonesi, G. Broggi*, G. Maira**, C. Sturiale***, C. Grana and
M. Chinol
Division of Nuclear Medicine, European Institute of Oncology, Milan, Italy
*Istituto Neurologico ”C. Bestaö, Milan, Italy
**Division of Neurosurgery, Policlinico ”A. Gemelliö, Rome, Italy
***Department of Neurosurgery, Ospedale ”Bellariaö, Bologna, Italy
The aim of this study was to determine the maximum -tolerated dose, of a pre-targeting three-step (3-S) method
employing 90Y-biotin in the locoregional radioimmunotherapy (RIT) of recurrent high grade glioma, and to
investigate the antitumor efficacy of this new treatment.
Twenty-four patients with recurrent glioma underwent second surgical debulking and implantation of a catheter
into the surgical resection cavity (SRC), in order to introduce the radioimmunotherapeutic agents [biotinylated
monoclonal antibody (MoAb), avidin and 90Y-biotin].
Eight patients with anaplastic astrocytoma (AA) and 16 patients with glioblastoma (GBM)
were injected first with biotinylated anti-tenascin MoAb (2 mg), then with avidin (10 mg; 24
h later) and finally 90Y-biotin (18 h later). Each patient received two of these treatments 8-10
weeks apart. The injected activity ranged from 0.555 to 1.110 GBq (15-30 mCi). Dosage was
escalated by 0.185 GBq (5 mCi) in four consecutive groups. The treatment was well tolerated
without acute side effects up to 0.740 GBq (20 mCi). The maximum tolerated activity was
1.110 GBq (30 mCi) limited by neurological toxicity. None of the patients developed
hematologic toxicity. In three patients infection occurred around the catheter. The average
absorbed dose to the normal brain was minimal compared with that received at the SRC
At first control (after 2 months), partial (PR) and minor (MR) responses were observed in
three GBM (1 PR; 2 MR) and three AA patients (1 PR; 2 MR) with an overall objective
response rate of 25%. Stable disease (SD) was achieved in seven GBM and five AA patients
(50%). There was disease progression in six GBM patients (25%), but in none of the AA
At the dosage of 0.7-0.9 GBq per cycle, locoregional 3 -S-RIT was safe and produced an objective response in
25% of patients. Based on these encouraging results, phase II studies employing 3 -S-RIT soon after first
debulking are justified.
Keywords: locoregional radioimmunotherapy; glioma; monoclonal antibody.
Detection and molecular characterization of JC virus in human brain tumors
Pagani E., Mancuso R., Delbue S., Tarantini L., Borghi E., Omodeo -Zorini E., Monga G., Car G.B., Boldorini
R. and Ferrante P.
Several recent reports have suggested the possible involvement of JC virus (JCV) in the pathogenesis of human
brain tumors. JCV, like BK virus, the other human polyomavirus, and SV40, is capable of inducing different
types of brain neoplasm in animals after intracerebral inoculation, and to induce transform ation in human cell
cultures. JCV is a very common virus that establishes a latent infection in the kidney of up to 80% of the adult
normal population worldwide and induces progressive multifocal leukoencephalopathy (PML) in
immunodeficient individuals. The discovery of various JCV genotypes and the observation of rearrangements of
the transcriptional control region (TCR) have suggested that a particular genomic organization could be
associated with a more pronounced neurotropic capability.
To verify the possible involvement of JCV in human brain tumors, we collected brain tumor tissue, peripheral
blood cells and cerebrospinal fluid (CSF) from 30 histologically diagnosed cases of neural derived tumors. In all
the collected samples JCV DNA was searched using a nested PCR designed to amplify the highly conserved
large T antigen (LT) coding region. LT DNA was amplified in eight of fourteen glioblastomas (57.1%), in two of
seven meningiomas (28.6%), in one of three astrocytomas (33.3%) and in none of the other ty pes of tumor.
JCV genotype distribution has been studied by nucleotide sequencing of VP1 region. Only JCV genotype 1 has
been detected, and in particular the subtype a was found in four tumor tissues and one CSF, and the subtype b in
two tumor tissues and one CSF. TCR nucleotide sequencing revealed the presence of one archetypal derived
(type II) and four Mad-4 TCR rearrangements.
On the whole the data obtained give a further support to the possible involvement of JCV in human brain tumors,
and suggest that JCV genotype 1, Mad-4 strain, could be of particular relevance in their pathogenesis.
Ceramide levels in human glial tumors
Riboni L, Campanella R, Bassi R, Villani R, Gaini SM, Viani P, Tettamanti G.
Ceramide represents an important sphingoid mediator involved in the signaling pathways that control cell
proliferation, differentiation and death. To determine whether ceramide levels correlate with the malignant
progression of human astrocytomas, we investigated these levels in surgical specimens of glial tumors of low -
and high-grade malignancy. Tumor samples, obtained from 52 patients who underwent therapeutic removal of
primary brain tumors were used. The tumors were classified according to standard mor phologic criteria and they
were grouped into tumors with low -grade and high-grade of malignancy. Sections of normal brain tissue
adjacent to the tumor were also analyzed in 11 of the 52 patients. After extraction and partial purification,
ceramide was measured by quantitative derivatization to ceramide -1-phosphate using diacylglycerol kinase.
Ceramide levels were significantly lower in the combined high-grade tumors compared to low-grade ones and in
both tumor grou!
ps compared to peritumoral tissue. The re sults indicate an inverse correlation between the amount of ceramide
and tumor malignancy as assessed by both the histological grading and ganglioside pattern. Moreover, the
highest ceramide levels were found in patients with a longer survival time. The pr esent findings indicate that
ceramide is inversely associated with malignant progression of human astrocytomas and poor prognosis. The
down regulation of ceramide levels in human astrocytomas emerges as a novel alteration that may contribute to
glial neoplastic transformation. The low ceramide levels in high -grade tumors may underlie their rapid growth
and apoptotic resistant features. This study supports the rationale for the potential benefits of a ceramide -based
Thalidomide in primary brain tumours
Riva M, Landonio G, Ferrante E, Arena O, Collice M, Siena S, Defanti CA
Proof has recently been accumulated that angiogenesis is essential in HGG formation and growth. Thalidomide,
developed as a hypnotic/sedative agent in the late 1950s, shows, at higher doses, antiangiogenetic properties due
to TNF antagonism tehrefore is a promising agent for long-term adjuvant therapy in patients with malignant
9 female and 12 male supratentorial HGG Pts [28 - 74 years, median 56; 16 gliobl astomas 1 gliosarcoma and 4
mixed anaplastic gliomas; 3 were newly diagnosed HGG and 18 residual and/or relapsing tumor, after surgery
and Rt] were treated with Thalidomide in a Phase II open trial. The starting dose was 100 mg o.i.d and increased
until a final dose of 800-1200 mg was achieved. The drug was taken as a single dose at night. Our aim was to
obtain two therapeutic regimens: the former at a maximum dose of 800 -1200 mg if well-tolerated and the latter
at 300-400 mg, in presence of side effects, sedation and costipation. 16 relapsing pts took concomitant Ct with
Temozolomide (in 4 of them as a 2nd line Ct).
The minimum follow-up required was 2 months from the final dose achievement (range in present series 2 - 10
months). 5 Pts are in dose titration.
6 Pts reached the highest dose of drug, 4 took the lowest. 6 Pts dropped out either for sedation or inadequate
follow-up. 2 Pts showed intratumoural bleeding,1 pt a slight sensory peripheral neuropathy and another Pt skin
Thalidomide is the only antiangiogenetic drug easily found and generally well tolerated .
We suggest using this drug :
1] in Pts with HGG during Rt in the hope of a synergistic potentiation of microvascular density inhibition and
with concomitant Ct to increase its clinical efficacy;
2] in Pts with a Low-Grade Glioma to prevent progression towards a more malignant tumour.
The combined therapeutic effect of cytokine producing autologous cancer cell vaccines and local tumor
irradiation in a murine glioma model
Safrany G1, Lumniczky K,1 Desaknai S,1 Szende B,2 Hidvegi E,1 and Hamada H,3
1 - Department of Molecular and Tumor Radiobiology, National Research Institute for Radiobiology and
Radiohygiene, Budapest, Hungary; 2 - 1st Institute of Pathology and Experimental Cancer Research,
Semmelweis University, Budapest, Hungary; 3 - Department of Molecular Medicine, Biomedical Research
Center, Sapporo Medical University, Sapporo, Japan
The combined therapeutic effect of cytokine producing cancer cell vaccines and local radiother apy was studied
in a mouse glioma 261 (Gl261) brain tumor model. Brain tumor bearing mice were treated with cytokine (IL -2,
IL-4, IL-6, IL-7, IL-12, GM-CSF, TNFa, LIF, LT) producing vaccines made by in vitro transduction of Gl261
cells with corresponding adenoviral vectors. Vaccines producing either IL-2, IL-4, IL-12 or GM-CSF cured
about 20-40% of mice. The anti-tumor effect strongly depended on the secreted cytokine level. Vaccination
therapy induced specific activation of cytotoxic T lymphocytes measured by cytotoxicity assay. Brain tumors
were heavily infiltrated by CD4+ lymphocytes after treatment with IL -2, IL-4, IL-12 or GM-CSF secreting cells.
GM-CSF vaccination induced moderate CD8+ infiltration, as well. Depleting either CD4+ or CD8+ lymphocyte
subsets abolished the anticancer effect of GM-CSF expressing cells. Strong synergism was observed by
combining cytokine vaccination with local tumor irradiation: about 80 -100% of glioma bearing mice was cured.
The high efficiency of combined treatment was maintained even under sub-optimal conditions when neither of
the modalities alone cured any of the mice. This suggests that vaccination therapy might open a new potential on
the clinical treatment of high-grade gliomas when applied as adjuvant to existing tre atment modalities.
Clinical immunisation with autologous glioma cells transduced with Interferon -gamma by Adenovirus.
Salford LG1, Siesj¨ P1, Skagerberg G1, Persson BRR2, Kjellman C3, Lindvall M3, Visse E3
and Widegren B3.
Dept. of Neurosurgery1, Medical Radiation Physics2 and Tumour Immunology3, Lund University, Sweden

In collaboration with the BRIGTT group, Rausing Laboratory, Lund University
Based upon earlier experimental work by our group, we have started a human immuno -gene therapy study. The
goal for this translational study is to investigate the effects of immunisation with autologous tumour cells
expressing gene-sequences for human interferon-g. Since more than two decades we have sought for efficient
treatment against malignant gliomas. Our most successful treatment in the animal models is immuno -gene
therapy where murine genes for the cytokines IFN -g, IL-7 and B7-1 were chosen for their ability to stimulate
different stages of the pathway for cytotoxic T lymphocyte (CTL) activation. Rats of the syngeneic inbred strain
Fischer 344 had rat glioma cells of the N32 line inoculated in the right caudate nucleus and 1 or 3 days later, N32
cells transfected with either IFN-g, IL-7 or B7-1 genes were injected subcutaneously (and in some studies
intraperitoneally). This treatment was repeated three to four times with seven to fourteen days interval and
resulted in significantly improved survival compared with treatment with wild type rat glioma cells (eg not
transfected with the cytokine genes). The continued work concentrated on treatment with IFN-g secreting tumour
cells of both the N32 line and also a newly developed ENU i!
nduced rat glioma cell line called N29. This work, proved the effectiveness of the technique. Cure was achieved
in 72% of the animals treated with the IFN-g cells. Tumour infiltrating leukocytes from N32 -IFN-g immunised
animals showed a significantly stronger infiltration by CD8+ T -cells; significantly more NK cells; a significantly
stronger infiltration by CD4+ T-cells; an increased number of CD25 expressing T-cells. These results confirmed
the possible usefulness of IFN-g transfected tumour cells in the immune -therapy of rat brain tumours.
The animal experiments have motivated us to start a human immuno -gene therapy study including 20 patients
with glioblastoma multiforme, where >80% of the tumour can be surgically removed. The goal is to ascertain
whether immunisation with autologous tumour cells expressing gene -sequences for human interferon-g : is safe
for the patients; gives rise to an immunological response and adds any beneficial effect to conventional therapy
(tumour growth, prolonged survival). The study started in June 2000 and hitherto, eleven patients are included
out of which 2 have received 6 and 9 immunisations respect ively (May 2001).
The immunisation takes place in the dermis of the upper arm. Seven days after each immunisation, a skin biopsy
is taken from the centre of one of the injection sites.
The composition of the cellular infiltration in the skin is studied by markers for T lymphocytes (CD3); Helper
cells, subset of T cells (CD4); Cytolytic T-cells, subset of T cells (CD8); Natural killer cells (CD16) and B
lymphocytes, B cells (CD20). Also the expre ssion of cytokines for functional T cell subsets are studied: IL-2, IL-
4, IL-10, IL-12, IL-18, TNF-a, TNF-b, IFN-g and TGF-b 1,2 and 3.
Peripheral blood is sampled both before and after operation and also after each immunisation event. Co -culture
of this blood with tumour cells from the patient allows for a selection of T -cells that can recognise tumour-
specific antigens.
Results from the first human treatments will be described.

The combination of the protein kinase Ca dna enzyme and endostatin ha s a synergistic effect on
intracranial gliosarcoma growth
Sorensen, D.R., Leirdal, M., and Sioud, M.
Malignant brain tumors have a poor prognosis, in spite of aggressive multimodality therapy. Thus, the search for
novel therapeutic strategies should facilitate the design of effective therapeutic agents. Here, we have
investigated the combination of endostatin, an anti-angiogenic protein and a nuclease resistant protein Ca DNA
enzyme on gliosarcoma BT4C in vivo. Endostatin was delivered via a mini osmotic pu mp for a period of up to
28 days, whereas the PKCa DNA enzyme was given as a single intracranial injection on day 3 following tumor
cell inoculation. The DNA enzyme inhibited glioma cell proliferation in vitro and PKCa gene expression in a
dose dependent manner as compared with its inactive form. Rats treated with both the endostatin and the PKCa
DNA enzyme survived longer (P<0.0002) than those treated with either endostatin (P< 0.025) or PKCa (P<
0.0467) alone. This data validate the PKCa signaling pathway and angiogenesis an attractive combined

Patrizia Tunici*, Antonia Follenziò, Luigi Naldiniò and Gaetano Finocchiaro*
*Istituto Neurologico Besta, Milano; òIRCC Candiolo (Torino); Italy

The high grade of recurrence of glioblastoma multiforme (GBM) is correlated to the capacity of GBM cells to
infiltrate the normal brain parenchima. An effective treatment of these cancers will require the develo pment of
new ways to kill cells that have dispersed significant distances from their site of origin. Since immortalized
neural progenitor cells have been reported to possess an extensive tropism for intracranial gliomas, we have
derived neural progenitor cells (npc, growing in vitro as neurospheres) from the subventricular zone of newborn
mice brain to transduce in these cells the antitumor cytokine interleukin 4 (IL -4). Previously, we transduced IL-4
cDNA in npc by MMLV-derived retroviral vectors. To impro ve transduction efficacy we have cloned the cDNA
of mouse IL-4 into the lentivirus transfer vector pRRLsin.PPT.hCMV.pre. The plasmid was successively co -
transfected with the packaging and envelope vectors (CMVR8.74 and VSV -G, respectively) in the 293 T cells.
The medium was collected 36 and 48 hours after transfection and concentrated by ultracentrifugation to a value
of 500 ng/microl of p24. The concentrated virus was used to transduce npc leading to high production of IL -4, as
assayed by ELISA: 1 microl of virus solution used to transduce 1x 10 6 npc led to the production of 0.9 microg
of IL-4/106 cells/48 hours. Previously MMLV trasduction of npc, followed by G418 selection led to the
production of 0.18 microg of IL-4/106 cells/48 hours. In vivo experiments are in progress, based on injection of
105 IL-4 npc or control npc transduced by GFP into established gliomas derived from injection of 5 x 10 4 or 2 x
104 GL261 cells. In parallel we are testing the migration capacity of these primary progenitor cells by injecting
in parallel npc in the right hemisphere and GL261 cells in the left hemisphere.
Combined low dose irradiation and Ad.d24 infection enhances oncolytic activity on glioma cell lines
van Warmerdam, J, Lamfers M, Grill, J, Lafleur M, van de Berg J, Curiel D, Fueyo J, Vandertop W, Gerritsen
W, Dirven C.
Department of Neurosurgery, VU University Medical Center, Amsterdam, The Netherlands
Division of Gene Therapy, Department of Medical Oncolgy, VU University Medical Ce nter, Amsterdam, The
Department of Radiotherapy, VU University Medical Center, Amsterdam, The Netherlands
Laboratoire de Pharmacotoxicologie et de Pharmacogenetique, Institut Gustav Roussy,Villejuif,France
Gene Therapy Center, University of Alabama, Birmingham, USA
Department of Neuro-Oncology, University of Texas, M.D. Anderson Cancer Center, Houston, USA
Malignant glioma is known to be almost refractory to any currently available treatment methods and is
associated with a poor prognosis. The use of oncolytic viruses in the treatment of braintumors has shown to be
effective in vitro and in animal studies. Recent studies report a synergistic effect of ONYX-015 with irradiation
in a mouse model for glioma (Georger, ASCO 2001). A new Conditional Replicating Adenovirus (CRAd),
which carries a 24-bp deletion in the E1A region (Fueyo, 2001), proved to be a m ore efficacious oncolytic virus
than ONYX-015 in the same mouse model. We have analyzed on a panel of glioma cell lines the effects of
dosing and scheduling of this new CRAd in combination with irradiation. Since E1A has been suggested to play
a role in adenoviral-induced radiosensitivity (Martin-Duque, 1999), we also compared the effects of Ad.d24, to
wild-type adenovirus in combination with irradiation. The WST -assay was used to measure viability at different
ts after infection.
Oncolytic activity of the replication-competent adenoviruses tested was most enhanced when cells were
irradiated 48 hours prior to infection. A subtherapeutic irradiation dose of 2 Gy resulted in the same killing effect
with 10-fold less virus particles per tumor cell. N o significant differences were found in the results of irradiated
glioma cells infected with the E1A-mutated Ad.d24 compared to wild -type adenovirus.
Thus, our data show that subtherapeutic iradiation 48 hours prior to infection with CRAd&#8217;s results in an
enhanced oncolytic effect on glioma cells. In addition, the 24 -bp deletion in the E1A viral protein of Ad.d24
does not interfere with the reported role of E1A induced radiosensitivity.

Prevention of metastasis formation after transfection with interleukin -4 or haemagglutinin antigen
Weilemann F, Kirsch M, Steinmetz A, Kunze S, Schackert HK, Schackert G
The purpose of our study was to examine the effects of HA or IL -4 expression on brain metastases formation.
Previous data have shown that expression of haemagglutinin antigen of influenza virus (HA) by the murine
colon carcinoma cell line (CT-26) produces systemic immunization against tumor challenges in the cecum, liver
and lungs but not in the brain of BALB/c-mice. Immunization with IL-4 expressing CT-26 cells inhibits lung
metastases formation.
We have used a cerebral metastasis model employing selective internal carotid artery injections of tumor cells.
Brain metastases formation of HA or IL-4 expressing CT-26 cells with and without immunization was evaluated
in Balb/c mice.
Systemic preimmunization with HA or IL-4 expressing tumour cells cannot protect against brain metastases,
while the local, intracerebral expression of HA or IL-4 inhibits the growth of haematogenous brain metastases.
Therefore, local intracerebral expression of cytokines is necessary to prevent tumor formation in a murine model
of cerebral metastases.


Microsoft word - j&j complaint -- 1-15-10.doc

Case 1:07-cv-10288-RGS Document 81 Filed 01/15/10 Page 1 of 34UNITED STATES OF AMERICA, ex rel. COMPLAINT OF THE UNITED STATES This is an action against defendant pharmaceutical manufacturer Johnson & Johnson and two of its subsidiaries (collectively, “J&J”) to recover treble damages, restitution, and civil penalties under the False Claims Act, 31 U.S.C. §§ 3729-33, and the

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