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Object. Due to their surgical inaccessibility or aggressive behavior, some meningiomas cannot be cured with cur-
rent treatment strategies. Gene therapy is an emerging strategy for the treatment of brain tumors, which the authors in-
vestigated to determine whether adenoviruses could be used for gene transfer in meningioma cells.
Methods. The presence of the high-affinity Coxsackievirus and adenovirus receptor (CAR) for adenovirus type 5,
as well as endothelial growth factor receptor (EGFR) and alphav integrins (ITGAVs), were analyzed in primary tumors
by using immunohistochemical studies and in primary meningioma cell cultures by using fluorescence-activated cell
sorting. Targeting of adenoviruses to EGFR was achieved using bispecific antibodies, whereas targeting of adenovi-
ruses to the ITGAVs was accomplished by insertion of an RGD (arginine-glycine-aspartic acid) motif in the adenovi-
rus fiber HI loop. Gene transfer efficiency of untargeted and targeted vectors was compared in primary cell cultures
and in spheroids derived from patients’ resected tumor material.
The presence of CARs was observed in all tumors and in all but one of the derived primary meningioma cells. The
higher expression of EGFRs and ITGAVs indicated that these receptors could be used as alternative targets to redirect
the adenoviruses. Redirection of adenoviruses to the EGFRs or integrins enhanced gene transfer threefold (range two–
sevenfold) for EGFRs in primary meningioma cells and ninefold (range three–23-fold) for integrins (p = 0.002, analy-
sis of variance). The effect of adenovirus targeting was confirmed in spheroids composed of primary meningioma cells.
Conclusions. Gene transfer with adenoviruses targeted to tumor-specific receptors is very effective in primary me-
ningioma cells and spheroids. These vectors are promising agents for gene therapy of meningiomas.
account for approximately 20% of all es.2,28 Therefore, an alternative treatment is required for pa-
M
ENINGIOMAS
primary intracranial tumors.6 They are mostly be- tients with either a malignant or a recurrent benign menin-
nign lesions that can be cured by total surgical re- gioma that is surgically inaccessible or carries too high a
section, although 10% exhibit an aggressive course even surgical risk.
after macroscopically complete removal. In patients older Radiation therapy is effective for both malignant and be-
than 60 years, surgical treatment has been shown to result nign meningiomas.15,50 In more than half of patients, howev-
in significant mortality and morbidity rates in 15% of cas- er, the tumor will recur within 5 years, and almost half of
those with atypical and malignant tumors will die within 10
Abbreviations used in this paper: ANOVA = analysis of variance; years after diagnosis.10,22 In patients with residual or recur-
CAR = Coxsackievirus and adenovirus receptor; CHO = Chinese ring benign tumors, there is increasing concern about radi-
hamster ovary; CMV = cytomegalovirus; DMEM = Dulbecco mod- ation-related side effects that may occur even with highly
ified Eagle medium; EGFR = endothelial growth factor receptor;
FACS = fluorescence-activated cell sorting; FCS = fetal calf serum;
accurate therapies such as radiosurgery.33 Chemotherapy
IgG = immunoglobulin G; ITGAV = alpha v integrin; ITGAVB3 = and hormonal therapy have not acquired a place in the stan-
alpha v beta 3 integrin; ITGAVB5 = alpha v beta 5 integrin; mAb = dard treatment of recurring meningiomas, despite inciden-
monoclonal antibody; PBS = phosphate-buffered saline; SD = stan- tal reports of tumor responses and stable disease after treat-
dard deviation; vps = viral particles. ment with these modalities.9,12,27,34,35,43,44,46
Gene therapy, especially with adenoviral vectors, has de- Cell Lines
veloped as a promising option for treating tumors. Infection The U373MG (anaplastic astrocytoma) and the human glioma-
of a large number of tumor cells is crucial for the success derived cell line U118MG (glioblastoma multiforme) were grown in
of this type of therapy. Clinical trials in malignant gliomas DMEM supplemented with 10% FCS and antibiotic agents.
have shown that the vectors used so far were unable to pro-
duce efficient transgene delivery into tumor cells, which ex- Recombinant Adenoviruses
plains the lack of significant therapeutic effect.39,40 Our pres- A recombinant E1-deleted adenovirus expressing the luciferase
ent research is focused on the development of new vectors reporter gene under the CMV promoter, AdCMVLuc, was used. The
that are targeted toward tumor cells in such a way that gene vector Ad5LucRGD, which expresses luciferase under the same
CMV promoter and contains an integrin-targeting peptide (arginine-
transfer is enhanced in tumor cells and diminished in nor- glycine-aspartic acid, the RGD-4C peptide) in the HI loop of the
mal ones. To this end, an advantage is realized from knowl- fiber knob, was generated as described previously.14 The presence of
edge of the basic mechanisms for adenovirus infection. the RGD binding motif in the HI loop was confirmed by polymerase
Human adenovirus serotype 5 entry is a two-step process chain reaction and restriction enzyme analysis. The functionality of
involving high-affinity binding of the fiber of the virus to the RGD binding motif was verified by the ability of the vector to en-
the CAR, followed by interactions of the penton-base pro- hance gene transfer in a CAR-negative but ITGAV-positive cell line
(U118MG). Recombinant adenoviruses were propagated on the per-
tein with ITGAVs for internalization.4,26 Targeting strate- missive 293 cell line and purified using CsCl gradient banding. Sub-
gies based on immunological principles use bispecific mol- sequently, they were titered in parallel: in vps by using the OD260
ecules that bind to the adenoviral capsid on one side and to method and in plaque-forming units by using end-point titration on
a cellular receptor on the other.13,20,32,51 Such molecules re- 293 cells. The titers were 3.3 1012 vps/ml and 4.1 1012 vps/ml
direct the infection to a pathway independent of the cell for AdCMVLuc and Ad5LucRGD, respectively. The ratio of infec-
membrane receptor, CAR, by making a molecular bridge tious vps/total vps was 1:4 and 1:10 for AdCMVLuc and Ad5Luc-
RGD, respectively. When the two viruses were compared, the same
between the virus and the tumor cell. Genetic targeting is number of vps was used.
achieved by direct modifications of the adenoviral genome,
that is, insertion of the DNA sequence encoding for a pep- Immunohistochemical Reagents
tide with specific binding affinity. Only a few attempts with The mouse anti-CAR mAb (RmcB4) was prepared as ascites fluid.
this second approach have been successful, generally by in- The anti-ITGAVB5 mAb clone P1F6 and the anti-ITGAVB3 mAb
sertion of the sequence of small peptides into the fiber knob clone LM609 were used. A supernatant of the 425 hybridoma47 cul-
sequence.26,49 ture was used as a source for anti-EGFR mAb.
Because low CAR expression has been shown to limit A new mouse mAb against firefly luciferase (LUC-Y) was used to
gene transfer with adenoviruses in many tumor types,1,29 we detect luciferase expression in infected cells (details of this mAb will
be published elsewhere).
undertook this study to define the efficacy of adenovirus for
transferring genes in primary meningioma cells and sphe- Flow Cytometry
roids and to evaluate which targeting strategies can improve Cultured cells were trypsinized, washed with PBS, and centri-
gene transfer in these settings. fuged. Half a million cells were incubated with the first antibody for
1 hour on ice; the antibody had been diluted in PBS containing 0.1%
bovine serum albumin. Concentrations of the primary antibody were
Materials and Methods 10 g/ml, 2.5 g/ml, and 10 g/ml for RmcB, P1F6, and LM609,
Histological Studies respectively. For the 425 mAb, undiluted supernatant of the hybrido-
ma was used at a concentration of 1 g/ml of IgG-1, according to en-
The histological features of the tumors were reviewed by one of zyme-linked immunosorbent assay. The irrelevant primary antibody
the authors (P.v.d.V.) and classified according to the revised World used, at a concentration of 10 g/ml, was mouse mAb IgG-1 323A3
Health Organization system.25,38 The number of mitoses per 10 hpfs directed against an epithelial marker not expressed on glioma cells.
was assessed and meningiomas were classified as malignant if this Subsequently, the cells were washed and incubated with fluorescein
number was at least 20 or atypical if it was between four and 20. Ad- isothiocyanate–conjugated rabbit anti–mouse antibody for 30 min-
ditional immunohistochemical studies (most often epithelial mem- utes on ice and in the dark. After washing in PBS, the cells were re-
brane antigen) were performed if required. suspended in 500 l of PBS containing 1% formaldehyde. Analysis
was performed using an FACS apparatus. The human glioma–de-
Primary Meningioma Cell Cultures and Spheroids rived cell line U373MG served as a positive control for CAR, EGFR,
We used fresh material collected during brain tumor surgery in ITGAVB3, and ITGAVB5 because high positivity has been shown
adult patients, after informed consent had been obtained. previously for these four antigens, and U118MG served as a nega-
tive control for CAR.32 The fluorescence intensity of each sample
Primary meningioma cell cultures were obtained after mechanical was quantified as the ratio between the median fluorescence inten-
dissociation, according to the method described by Darling.11 The sities of the stained sample and of the irrelevant antibody control.
cells were cultured in DMEM or the Ham F-12 medium supplement- These ratios were compared with those of the positive control cell
ed with 10% FCS and antibiotic agents. Nonglial origin was con- line, U373MG, and expressed as a percentage of the latter value.
firmed by morphological findings and by the absence of staining
with the anti–glial fibrillary acidic protein mAb clone 6F2. To pre- Immunohistochemical Studies
vent endothelial cell contamination, large blood vessels were sep-
arated from the tumor material. All the experiments on these cells Immunohistochemical studies were performed on 4-m frozen
were completed before the fifth passage, usually done between the sections fixed with acetone. We used a frozen pellet of 293 cells and
second and third passage. normal human liver as positive controls for CAR. Normal human
Organotypic multicellular meningioma spheroids were grown skin was used as a positive control for EGFR and normal human ton-
from small explants of fresh human tumor in 48-well plates coated sil as a positive control for ITGAVB3 and ITGAVB5. Nonspecific
with agarose (one spheroid per well), according to the method origi- antibody binding was blocked by incubation with 2% normal rabbit
nally described by Bjerkvig, et al.,5 for glioma. After checking them serum. Tumor slides and positive controls were incubated for 1 hour
for viability by morphological appearance and trypan blue exclusion, at room temperature in blocking buffer (1% bovine serum albumin
we used spheroids of similar diameters (400–500 m) for gene trans- in PBS) with the same mouse mAbs that were used for cytometry.
fer experiments. Concentrations were 1 g/ml for 425 and 10 g/ml and for RmcB,
LM609, and P1F6, respectively. Two negative controls were used TABLE 1
for each staining; these controls were treated either with blocking Clinical and pathological characteristics
buffer only or with normal mouse IgG-1. Specimens were then of the meningiomas cultured*
washed in PBS and incubated with biotinylated rabbit anti–mouse
IgG antibody diluted 1:500 in PBS for 30 minutes. The second anti- Case Age Histological Mitoses/
body binding was visualized with a peroxidase-conjugated streptavi- No. (yrs) Tumor Location Finding 10 hpf
din avidin–biotin complex kit diluted 1:200 in PBS for 1 hour.
VU-16 68 convexity (rt frontal) benign 1
Immunocytochemical Studies VU-18 63 tentorial (posterior fossa) benign 0
For immunocytochemical studies, infected cells were fixed direct- VU-22 42 skull base (parasellar) benign 0
ly in the plates with methanol/acetone (1:1). After washing in PBS, VU-29 36 skull base (petrosal) benign 0
the fixed cells were incubated with 5 g/ml purified LUC-Y anti- VU-36 72 skull base (sphenoidal) malignant 20
body diluted in diluent for 1 hour at room temperature. After wash- VU-38 28 cervical (patient w/ NF2) benign 0
ing in PBS, the cells were then incubated with the second antibody, VU-41 44 convexity (lt frontal) atypical 5
10 g/ml goat anti–mouse Ig antibodies conjugated to peroxidase, VU-44 62 skull base (temporal fossa) atypical 8
for 30 minutes at room temperature. After a subsequent wash, the red * NF2 = neurofibromatosis Type 2.
conversion of chromogen -amino-9-ethylcarbazole was used to de-
tect the presence of LUC-Y bound to the luciferase protein in the
cells. Cells were counterstained with hematoxylin and eosin.
Bergh at the Department of Radiobiology, Vrije Universiteit Med-
Bispecific Single-Chain Antibody Constructs ical Center, and Dr. J. T. Douglas at the Gene Therapy Center,
University of Alabama. The AdCMVLuc was provided by Dr. R.
Bispecific single-chain antibody constructs were made as de- D. Gerard at the University of Texas Southwestern Medical Cen-
scribed previously.20 The 425-S11 fusion protein recognizes the ter, Dallas, TX. The mouse anti-CAR mAb (RmcB) was obtained
EGFR on one side and the fiber knot on the other; it also contains the from Dr. R. L. Crowell at Hahnemann University, Philadelphia,
secretion signal from IgG-1. PA. The FCS was purchased from Life Technologies, Grand Island,
An undiluted supernatant of CHO cells, stably transfected with the NY. The Ham F-12 medium was supplied by Invitrogen, Carlsbad,
plasmid encoding for the bispecific single-chain antibody construct CA. The mAb clone P1F6 was acquired from Life Technologies,
425-S11, was used for the targeting experiments. The cells were Breda, The Netherlands, and the mAb clone LM609 was purchased
cultured in a cultivation system. Concentrations of bispecific sin- from Chemicon, Temecula, CA. A supernatant of the 425 hybrido-
gle-chain antibody constructs were determined using enzyme-linked ma culture was supplied by the American Type Culture Collection,
immunosorbent assays. The optimal ratio between the CHO cell Rockville, MD.
supernatant and vps was determined by targeting experiments on The fluorescein isothiocyanate–conjugated rabbit anti–mouse an-
U118MG. A single batch of supernatant from transfected CHO cells tibody was acquired from Dako A/S, Glostrup, Denmark, as were
was used for all experiments. the biotinylated rabbit anti–mouse IgG antibody, the avidin–bio-
tin complex kit, the diluent, the goat anti–mouse Ig antibodies, and
Gene Transfer Assays the chromogen 3-amino-9-ethylcarbazole. The FACScan device
To assess adenoviral infection in cells, 105 cells/well were plated was purchased from Becton-Dickinson, Erembodegem-Aalst, Bel-
in a 24-well plate in triplicate and incubated overnight in 1 ml of cul- gium. The cell cultivation system (model CL350) was obtained
ture medium to allow adherence. Before infection, 108 vps of adeno- from INTEGRA Biosciences AG, Wallisellen, Switzerland. The
virus were incubated with 25 l of the 425-S11 containing CHO su- luciferase chemiluminescence assay system was purchased from
pernatant for 30 minutes at room temperature. Next, the mixture was Promega, Madison, WI. The luminometer (model LB 9507) was
diluted in DMEM containing 2.5% FCS to a concentration of 5 acquired from EG&G Berthold, Bad Wildbad, Germany.
107 vps/ml, and 200 l was added to each well, that is, 100 vps/cell.
The cells were incubated at 37˚C in 5% CO2 for 1 hour, washed with
PBS, and then supplemented with 1 ml of DMEM containing 10%
FCS. Twenty-four hours after infection, the cells were assayed for Results
luciferase expression.
To assess adenoviral infection in spheroids, each one was plated
in a separate well of a 96-well plate coated with agarose, in 150 l Clinicopathological Correlation
of DMEM with 10% FCS, then 107 vps of the conjugated viruses or Primary meningiomas resected between May 2000 and
of the control viruses, diluted in 50 l of DMEM containing 2.5% October 2000 in the neurosurgery department of the Vrije
FCS, were added to the wells. The luciferase assay was performed
after 24 hours of incubation at 37˚C in 5% CO2. Universiteit Medical Center were used for this study. One
tumor was malignant, two were atypical, and five were
Luciferase Assay benign according to the new World Health Organization
To measure luciferase activity in the cells, we used the luciferase criteria (Table 1). Half of the lesions were located on the
chemiluminescent assay system. After removing the culture medi- skull base.
um, lysis buffer was added to the wells and the whole plate was snap-
frozen on dry ice. After thawing, the luciferase activity was mea- Expression of CAR and the ITGAVs
sured during 10 seconds immediately after initiation of the light
reaction in a luminometer. Values were normalized per number of The expression of CAR, ITGAVB3, and ITGAVB5 was
viable cells by using trypan blue exclusion. analyzed using immunohistochemical studies performed in
To measure luciferase activity in the spheroids, they were re- the frozen tumor specimens and by FACS. We were able to
moved from the wells, rinsed with PBS, and resuspended individu- obtain matched samples for immunohistochemical studies
ally in 100 l of lysis buffer. After three cycles of freezing and thaw-
ing, and vortexing to ensure complete lysis of the spheroids, the and FACS in six of the eight tumors.
luciferase measurement was performed using 25 l of the sample. The CAR was present in all meningiomas, although its
Values were expressed per spheroid. level of expression was quite variable. In the tumors that
had a high level of CAR expression, staining was found in
Sources of Supplies and Equipment the cytoplasm, whereas in ones with lower levels, only peri-
The U373MG and U118MG cell lines were supplied by J. Van der nuclear staining could be detected (Fig. 1a and e). In all
TABLE 2
Expression of target antigens in primary meningioma cell cultures (FACS) and in the corresponding tumors (IHC)*
ITGAVB3 ITGAVB5
CAR (RmcB) EGFR (425) (1976) (P1F6)
Case
No. IHC† FACS‡ IHC FACS IHC FACS IHC FACS
adenoviral gene therapy for these tumors will face the same
problems as are encountered in gliomas, that is, low selec-
tivity because of high CAR expression in surrounding nor-
mal brain cells.16 Therefore, new strategies that target the
virus to meningioma cells are needed to increase the se-
lectivity and eventually the efficacy of gene transfer. We
have shown that targeting strategies can, on the one hand,
increase gene transfer to tumor cells, and on the other, make
adenoviral infection more selective, thereby sparing nor-
mal cells.18,21 Integrins play a central role in cell adhesion to
the extracellular matrix and also in cell migration, signal
transduction, and induction of angiogenesis by tumors. As
shown in our study and in the literature, the cellular recep-
tors ITGAVB3 and ITGAVB5 are highly expressed in me-
ningioma cells and in meningioma vasculature, but not in
microvessels surrounding normal dura or in normal arach- FIG. 6. Bar graph showing gene transfer efficiency with target-
noidal cells.3,17 In addition, the ligand for ITGAVB3s, fibro- ed adenoviruses, in meningioma organotypic spheroids. Organo-
nectin, is also highly expressed in the extracellullar matrix typic multicellular spheroids derived from the meningioma of the
of meningiomas.37 This differentiated integrin expression, patient in Case VU-18 were grown on agarose-coated wells in a
which was high in the tumor cells and vessels and low in the 48-well plate. After 2 weeks in culture, eight spheroids per group
were infected with 107 vps of each of the four types of viruses:
normal surrounding cells, led us to examine the role of in- AdCMVLuc (untargeted control vector); AdCMVLuc 425-S11
tegrins as possible targets for improved adenoviral trans- (vector targeted to the EGFR); Ad5LucRGD (vector targeted to
gene delivery. Using the integrin-targeted Ad5LucRGD the ITGAVs); and Ad5LucRGD 425-S11 (vector targeted to
vector, a significant increase in transgene delivery to me- the EGFR and the ITGAVs simultaneously). Infections were per-
ningioma cells was obtained compared with the untarget- formed as described in Fig. 4. The mean amount of gene trans-
ed vector (three–23-fold improvement, median ninefold). fer SD is indicated by the numbers over the bars.
We found that, the lower the CAR expression, the higher
the gain observed with RGD-targeted vectors. S11 bispecific single-chain antibody constructs, a further
Alternatively, adenoviruses can be redirected toward a increase in gene transfer was obtained, resulting in a 30-fold
tumor-specific receptor by bispecific antibodies. Because increase of transgene delivery into meningioma spheroids.
EGFR expression has been regularly shown in meningio- The question arises, whether our approach would also
mas and is absent from normal brain, in our series of tumors be of value in vivo, meaning that the targeting strategies
we analyzed whether EGFR could serve to redirect the ade- we have described will enhance transgene delivery to the
noviruses.7,8,30 tumor, diminish liver toxicity, and spare the normal brain
All the meningioma specimens analyzed, fresh tumor cells. Using a similar strategy with adapter molecules, in vi-
material as well as primary cell cultures, revealed EGFR vo targeted gene transfer has been shown in an ovarian can-
expression. Expression of EGFR in normal brain cells was cer model and in the lung endothelium.41,42 Furthermore,
almost absent, except in a few microglial cells. Infection of it has also been shown previously that fibroblast growth
primary meningioma cell cultures with the EGFR-target- factor receptor–targeted adenovirus demonstrates efficient
ed adenovirus resulted in an increase of transgene delivery liver detargeting after intravenous administration, thereby
by a factor of three (range two–seven) compared with un- mitigating hepatic toxicity of the treatment.19 Concerning
targeted adenovirus. This increase might be explained by the risk of unwanted gene delivery to normal brain cells,
a larger number of EGFRs present on the cell membrane this will be dependent on the presence of the targeted recep-
compared with CAR, as is indicated by our FACS data, tor in these cells. Because the receptors targeted by our
which shows a higher labeling ratio for EGFR than for strategies are not highly expressed in the brain, whereas the
CAR in each tumor. The single-chain antibody construct CAR is, one would expect to invert the unfavorable thera-
that binds to the fiber knob blocks the knob’s interaction peutic ratio with normal adenoviruses toward a more favor-
with CAR. In theory, incubating the adenovirus with 425- able one with targeted adenoviruses. To explore adequate
S11 conjugates should ablate native tropism of the virus. In tumor targeting and brain detargeting, one would need to
practice, however, it is very difficult to saturate completely develop an orthotopic human xenograft meningioma mod-
all the CAR-binding sites of the adenovirus with bispecific el. Such a model did not exist until recently, when Mc-
antibodies. For this reason, although native tropism can be Cutcheon, et al.,31 described their xenograft model in nude
tremendously reduced in this setting, some residual CAR mice. For intraarterial administration in the clinic, however,
binding can be observed. A further improvement may be one could take advantage of the distinct vascularization of
possible if mutant adenoviruses that do not bind to CAR meningiomas that arise in the external carotid artery circu-
anymore could be targeted with a similar approach. lation. Theoretically, the targeting strategies we used would
From our transfection experiments it can be concluded then be of benefit, because we showed that meningioma en-
that RGD targeting yields higher levels of transgene deliv- dothelial cells express EGFR and ITGAVs. It is very unlike-
ery than EGFR targeting. Theoretically the combination of ly that the use of bispecific antibodies in combination with
these two targeting strategies would redirect viral infec- the adenovirus, or the insertion of the small RGD motif in
tion toward the EGFR and the integrins, thereby increasing the fiber, will alter the capability of the virus to enter the tu-
the chances for the adenovirus to find a receptor for infec- mor after intravenous injection, because the size of the anti-
tion. Indeed, when Ad5LucRGD was incubated with 425- body is only a fraction of the viral size.
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mented therapeutic benefit in a murine model of ovarian cancer. tion de France/Federation Nationale des Centres de Lutte Contre le
Clin Cancer Res 4:2455–2461, 1998 Cancer, the 1997 Spinoza Award to Dr. Pinedo from the Nether-
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nary endothelium in vivo. Mol Ther 2:562–578, 2000 and P50 CA83591 from the United States Department of Defense to
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44. Sharif S, Brennan P, Rawluk D: Non-surgical treatment of menin- Department of Neurosurgery, Vrije Universiteit Medical Center,
gioma: a case report and review. Br J Neurosurg 12:369–372, De Boelelaan 1117, 1007 MB Amsterdam, The Netherlands. email:
1998 c.dirven@vumc.nl.