Journal of Neuro-Oncology 15: $1-$30, 1993. S 1

MEDULLOBLASTOMA RECURS AS MALIGNANT GLIOMA WITHIN THREE YEARS AFTER CESSATION OF THERAPY: REPORT OF TWO PATIENTS.

Tore. G. Abrahamsen, Finn Wesenberg and Sverre MCrk. Dept. of Pediatrics, Rikshospitalet National Hospital, Oslo and Depts. of Pediatrics and Pathology, Haukeland Hospital, Bergen, Norway.

Medulloblastoma is composed of primitive neuroectodermal ceils. However, areas with more differentiated tumorcells of unknown prognostic significance, may be found.

We describe two patients, a 6 1/2 years old boy and a 7 years old girl, both with a primary medulloblastoma of regular histology. They received surgery (gross total removal), radiation (SLOP 83) and chemotherapy (SIOP 83, sandwich regimen). Unfortunately, within 3 years off therapy they both died from a recurrent, cerebellar tumor with the histology of malignant glioma with mainly astrocytic or oligodendroglial differentiation respectively. The boy had been treated with growth hormon for 1 year prior to the appearance of the recurrent tumor.

This rapid development of a second tumor of different histology in these two patients has no obvious explanation. However, a possible therapy induced differentiation of residual tumor cells or of a new malignancy, the occurence of residual cells unresponsive to treatment or a sampling error can not be excluded.

IL-2/LAK IMMUNOTHERAPY FOR LEPTO- M E N I N G E A L METASTASES (LM) F R O M PEDIATRIC BRAIN TUMOR. Jeffrey Allen, Roberta Hayes, Robert DaRosso & Anita Nirenberg. NYU Medical Center, New York, USA.

Intracavitary administration of interleukin-2 and lympholdne activated killer cells (IL-2/LAK) has induced- objective responses and prolonged survival in adults with high ~rade gliomas. Little data exists regarding its activaty in LM following intrathecal or intraventricular administration. This phase I/II study was designed to determine a biologically effective, maximum tolerable dose of IL-2 when administered with LAK via an Ommaya reservoir into the lateral ventricle of children with LM from a recurrent primary brain tumor. Autologous LAK cells were produced ex- vivo following leukapheresis 3-4 days prior to administration. Eight pts (mean age-15 yrs) received IL-2 on Mo/We/Fri for 2 weeks, constituting 1 cycle. Their diagnoses were PNET (3), high grade astrocytoma (4) and germinoma (1). LAK cells were only administered with the first and ~econd IL-2 doses (mean LAK cells per cycle-5.5xl0 ). For 3 shunt dependent pts, IL-2/LAK was administered via a lumbar catheter. A second cycle was repeated in 2 weeks, constituting 1 course, and the study permitted a total of 3 courses over 1 year. The first two IL-2 dose levels, 0.3 & 0.6 MIU (Cetus)/dose, have been completed without significant toxicity. One 14 y.o. boy with a thalamic glioblastoma and diffuse LM had a Prolonged partial response for 14 months. The other

evaluable pts had progressive disease. A CSF eosinophilia was noted in 5 of 7 pts. In conclusion, the optimum biologic and maximally tolerable dose of IL-2 has not been reached and one pt experienced a significant clinical and radiographic response. (Supported by FDA-R-00690).

ISOLATION AND MOLECULAR CHARACTERIZATION OF NOVEL GLUTATHIONE S-TRANSFERASE-I1 GENE VARIANTS IN HUMAN MALIGNANT GLIOMA CELLS. Francis Ali-Osman and Nike Akande. Section of Molecular Therapeutics, Dept of Experimental Pediatrics. and The Brain Tumor Center, M.D. Anderson Cancer Center, Houston TX. 77030 USA.

Recent evidence suggest a critical role for glutathione S- transferase-~ in the progression, clinical behavior and therapeutic response of many human tumors. Data from our laboratory have shown that the level of GST-r~ expression in human glioma cells correlates with their resistance to 1,3-bis[2-chloroethyl]-l-nitrosourea. To date only one human GST-n gene, clustered with other tumor-associated genes (including, BCL1 and INT2) on chromosome 1 lq13, has been described. We report here the cloning, sequencing and structural characterization of three novel variants of the GST-n geue isolated from both primary and cultured human malignant astrocytomas. For comparison, we also cloned and sequenced the previously described human placenta GST-n gene. The gene variants were characterized by two nucleotide substitutions in the ORF of the GST-~ gene, (A to T) at nt position 313 and (A to G) at nt position 341, resulting in changes at codon 105 [Ile to Val] and at codon 114 [Ala to Val]. The substitutions also gave rise to several new resn'iction enzyme sites in exons V and VI of the variant GST-~ genes. PCR-RFLP analysis was used to confirm the gene alterations. We will present data on the significance of these gene variants in malignant gliomas and their effects Oll the cellular phenotype and drug sensitivity. Supported by grant CA55835 fi'om the N1C, NIH and a grant from the Kleberg Foundation.

CHARACTERIZATION OF A NEW METALLO-PROTEASE EXPRESSED BY C6 GLIOBLASTOMA CELLS.

V. Amberger, H. Seniberger, P~A. Paganetti and M.E. Schwab.

Glioblastornas are known as highly invasive toumors, which infiltrate the brain in a very diffuse manner. The C6 gblioblastoma cells are a good model to study invasive behaviour. As shown in several in vitro experiments the C6 cells are able to overcome the inhibitory substrate properties for cell migration expressed by oligedendrocytes and CNS white matter (Schwab and Caroni, 1988). This cellular behaviour is correlated with the expression of a proteolytic activity by the C6 ceils which might play a role in the inactivation of the myelin associated inhibitors. Using various known protease blockers we identified dais activity as a metalloprotease (Paganetti et al., 1988). Its blocker profile is distinct from that of known rnetaUoproteases. Newly developed oligopeptides can specifically inhibit the spreading of the C6 ceils on CNS. By using a specific peptide degradation assay we characterized the protease as tightly bound to the membrane, which can be sohibilized only with detergents. The rnetalloprotease is insensitive to blockers of the serin-, aspartyl- and cystein-proteases, but the activity can be destroyed completely by chelating agents and restored with cobalt and partly with zinc. The C6 protease can be stabilized with dithiothreitol and is highly sensitive to phosphoramidon, but not to thiorphan, wifich distinghuishes it clearly from enkephalinase and endothelin converting enzyme. The pH-optirnum has a peak at 6.0. An active enzyme fragment can be cleaved from the membrane with a short trypsin-treatment. Further purification of the metalloprotease is currently in progress using an affinity column and a size exclusion column in series.

V. Amberger +, H. Seulberger #, P.A. Paganetti* and M.E. Schwab+~ + Brain Research Institut, University of Zurich, CH-8029 Zurich # BASF, section ZHB, DW-6700 Ludwigshafen * Sandoz Research Institute Berne Ltd, CH-3001 Berne

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TENASCIN EXPRESSION IN HUMAN GLIOMA CELL LINES AND TISSUE. Arita N 1, Ohnishi T 2 , Hiraga S 2, Yamamoto H 2 , Taki T 2 , Izumoto S 2 , Higuchi M 2 , Hayakawa T 2, Kusakabe M 3, Sakakura T 3. Depts of Neurosurg, Kinki Universi- ty t and Osaka Universi~ 2, Lab of Cell Biol, Tsukuba Life Sci Cente#

Tenascin f iN) is an extracellular matrix glycopro- tein and has been shown to have critical roles in cancer cell proliferation, adhesion and metastasis. We examined TN expression in five human glioma cell lines and 56 surgical specimens of gliomas. All the cell lines expressed TN as examined by immunocytochemistry and fiowcytometry. By ELISA, levels of TN in the conditioned medium varied in each cell. In cells with higher level of TN, adhesion to fibronectin(FN) was revealed to be enhanced when incubated with an antibody to TN in cell adhesion assay. By immunohistochemistry, TN was positive in the basement membrane of the tumor vessels in anaplastic astrocytomas (53%) and glioblastomas (85%), but negative in astrocy- tomas. FN was localized in the basement mem- brane of the vessels in all astrocytomas, 80% of anaplastic astrocytomas, and 67% of glioblasto- mas. In the serial sections, it was revealed that TN and FN were not co-localized in the same vessels. In meningeal giiomatosis, disseminated glioma was chiefly composed of TN-positive and GFAP-nega- tive tissue as compared to the primary site. TN was negative in the infiltrated tissue of the primary site. These results indicate human glioma cells produce various degrees of TN. TN may have specific roles in the process of tumor invasion and angiogenesis.

A D O P T I V E L Y TRANSFERRED G L I O M A - S E N S I T I Z E D LYMPHOCYTES TRAFFIC TO AND ALTER THE HISTOLOGY OF GLIOMAS IN A RAT MODEL. NG Baldwin, CD Rice, RE Merchant, MCV-VCU, Richmond, VA, 23298 USA

We previously reported that tumor-sensitized lymphocyte populations could be successfully expanded in number in vitro using Bryostatin-1 (Br-1, a protein kinase C activator) and the Ca ++ ionophore, Ionomycin (Io); two agents which enhance intracellular signal transduction. In this phase of our investigation, we have focused on the potential therapeutic effects these cells may have for an intraeranial glioma. Viable RT-2 glioma ceils (10 ~) were injected into each hind foot pad of syngeneic Fischer 344 rats and the draining lymph nodes (DLNs) of each limb were harvested after 9-10 days. Lymphocytes were isolated and pulsed 18 h in complete medium (CM; RPMI-1640, 10% FCS, 20 U/ml human recombinant IL-2) with 5 nM Br-1 and 1 #M Io at 37 ~ C and then maintained in CM for one week. In the meantime, another set of rats was inoculated intracerebrally with 104 RT-2 gliorna. After 7 days, animals received an IV infusion of 106 ~ttIndium-labelled or unlabelled lymphocytes/g rat. Control glioma models were treated with only IV saline. Gamma camera images made 18 h following IV injection of H~In-labelled DLN cells indicated trafficking of cells into the side of brain with tumor. Actual tissue counts from tumor averaged 5.2 times higher than that in the contralateral hemisphere. Under these experimental conditions, there was no significant difference in survival between controls and treated models although postmortem examinations revealed that the size and histopathology of gliomas in treated animals differed from those of controls. Gliomas of treated rats showed no necrosis and appeared to be less infiltrative than those of controls. Furthermore, there were more areas of hemorrhage and, compared to controls, pronounced infiltration of lymphocytes and greater peritumoral edema. Gliomas of controls showed more necrosis and greater numbers of mitotic figures. Our results suggest that specifically-sensitized DLN lymphocytes whose numbers have been expanded in vitro can traffic to an intracerebral glioma and effectively lower its apparent histologic grade. Edema and hemorrhage seen in these animals most likely reflect cytokine effects.

COMPARISON OF THE EXPRESSION OF P-GLYCOPROTEIN AND BOTH WILD TYPE AND MUTANT FORMS OF P53 IN ADULT HUMAN MAUGNANT GUOMA CULTURES. Sally M Ashmore and JL Darling. Department of Neurological Surgery, Institute of Neurology, Queen Square, London WC1N 3BG.

P-glycoprotein, the product of the MDR1 gene, is expressed in many human tumours including malignant gliomas. The p53 tumour suppressor gene product may regulate expression of P-glycoprotein. Mutant p53 has been shown to specifically stimulate the MDR1 gene promoter and wild type p53 showed specific repression. This study compares the expression of p53 and P- glycoprotein in t3 short term malignant glioma cell lines. Fluorescence immunocytochemistry was used to detect the p53 and mdrl proteins. The antibodies used were JSB-1, against P-glycoprotein, and three antibodies against p53. Antibodies DO1 and 421, recognizing both wild type and mutant forms, and 240 which is specific for the mutant form. All cell lines showed some staining with JSB-1, the brightest, positively staining cells ranged from 6-77% of the total cell populations. Four of the lines showed more than 50% of cells expressing the mutant form of p53 indicated by nuclear staining with 240. It might be expected that the most P-glycoprotein would be found in those cell lines expressing the most mutant p53, but in this study there was no obvious correlation, other factors must be involved.

INTERNATIONAL SOCIETY OF PAEDIATRIC ONCOLOGY (SIOP) MEDULLOBLASTOMA STUDIES II & III DR. C.C. BAILEY ON BEHALF OF THE SIOP STUDY GROUP SIOP II • trial assigned patients to high risk (residual post surgical disease, brain stem involvement, intracranial or spinal spread) or low risk groups. All patients were randomised to receive or not a pre-radiotherapy chemo- therapy with Vincristine/Procarbazine/ Methotrexate. High risk patients received craniospinal radiotherapy (55GY to tumour site, 35Gy to "prophylactic" fields) followed by maintenance chemotherapy with Vincristine and OCNU. Low risk patients were further random- ised to standard radiotherapy as above) or to reduced dose (25Gy to "prophylactic fields + 55Gy to tumour site). Low risk patients did not receive any maintenance therapy. 364 patients were entered, 135 were high risk 229 were low risk. With 76 months median follow up (48-106 months) overall survival is 58.9%. No group derived advantage from the chemotherapy (high risk 5yr E.F.S. 56.3% vs 52.8%, low risk 63.3% vs 59.5%, non significant differences). Low risk patients randomised for reduced dose radiotherapy did less well than those receiving standard dose (55.3% vs 6?.6% 5yr E.F.S. P = 0.073). Low risk patients who received chemotherapy followed by reduced dose radiotherapy faired worst of all (5yr E.F.S. 41.7%). Analysis os high risk patients confirmed that the presence off metastases is a prognostic indicator but failed to show any significance for residual disease after surgery or the presence of brain stem involvement.

SIOP III fnls is a study in which patients with P.N.E.T. are randomised to radiotherapy alone or to pre- radiotherapy chemotherapy with Carboplatin/ Cyclophosphamide/Etoposide/Vincristine. 21 patients have been randomised thus far. A further 25 patients have been notified but not randomised. Details of the study will be displayed.

CHEMOTHERAPY ONLY STRATEGY IN PRIMARY CENTRAL ,NERVOUS SYSTEM GERM CELL TUMORS (CNS GCT): RESULTS OF AN INTERNATIONAL STUDY.

Balmaeeda C, Diez B, Villablanca J, Walker R, Finlay J . Memorial Sloan-Ketterlng Cancer Center, New York and participating centers.

CNS GeT are traditionally treated with irradiation (XRT). With such therapy alone, patients (pts) with Germinomas (G) enjoy a 60-100% 5-yr survival, while those with non germinomatous GCT (NGGCT), a 20-50% 5-yr survival. Concern for the deleterious effects of XRT in pts with G, the relative ineffectiveness of XRT for NGGCT, and the known sensitivity of GCT to platinum- containing regimens led to the design of a new therapeutic strategy. Our study represents an international effort: 26 institutions, spanning 20 cities in 5 countries. Pts receive 4 cycles (cyc) of induction chemotherapy (IC) consisting of Carboplatinttrn (500mg/M2/day(d)x2d), Etopnside (150mg/M2/dx3d) and Bleomycin(15mg/M2/d). Pts with residual tumor after 4 cye proceed to XRT and then intensified chemo with Cydophosphamide. Pts with complete response (CR) receive 2 more eye of IC, without XRT. 59 pts [36 males,19 females, median] age 13 yrs (range 3-35)]are enrolled, 52 evaluable for response. Median follow-up is 13 months (range 1-43).There are 29 G and 23 NGGCT. Of 23 G with negative tumor markers (TM) 21 achieved CR and 2 partial response (PR). There were no relapses (R). Of 29 pts with G or NGGCT with elevated TM, 16 achieved CR and 13 PR. 6 had R and 3 had progressive disease. 3 pts had radiological abnormalities after IC: second look surgery revealed no tumor (2) and mature teratoma (1). There were 6 toxic deaths. There was no impact of tumor location or AFP on outcome. Its with elevated BHCG had a 50% R rate, while those without such an devation had no R. Future recommendations include mandatory tumor marker studies at diagnosis and 2nd-surgery in cases of questionable response.Use of granulocyte colony stimulating factors may decrease mortality, while identification of certain pts with an unfavorable prognosis may allow those to be treated with more intensive chemotherapy.

E X P R E S S I O N OF ESTRAMUSTINE- l l INDING P R O T E I N IN E P E N D Y M O M A TUMOURS AND IN D E V E L O P I N G E P E N D Y M A L CELLS. A.Tommy Bergenheim 1,2 Magdalena Hamnan 3, Jonas Bergh 4, Per- ~3,:eRidderheim2andRoger Heilriksson I. DepartmentsofOncology 1 and Neumsmgery 2, UmeS, and Departmeots of Netu'opathology 3 and Oncology 4, Uppsala, Sweden,

Backgroun0: The mainstones in the treatment of ependymoma are aggressive surgery followed by radiotherapy. Chemotherapy is still not established aud no ultimate drug have so far beeo found. Estramustiue (EM), with a demonstrated effect oil astrocytoma ill vitro, has beou showu to penetrate the blood-turnout banier and to accumulate ill human brain lumoar tissue including ependymoma. The cytotoxic efl?ct of EM is proposed to party depend on the presence of estramustiue- binding protein (EMBP). To investigale the expression of EMBP in ependymoma turnouts, in normal developing rat ependymal cells, and normal human epeodyma au immuno-histochemical study was performed. Me~hod$; Semi-thin sections fiom 10 ependymoma tumours, sections from fetal, neonatal, and adult rat brains and human autopsy material were collected. The Avidin-Biotin-Peroxidase technique was used to delrlollstrale the presence of EMBP usiug a primary mouse monoclonal antibody raised against purified rat EMBP. Results: An EMBP-Iike protein was demonstrated in all ependymoma lumours at varying degree. Mosl turnout cells were uniformly staiuedi However, in some lumours very highly slained cells were found scattered in the tissue. An increase in staining intensity with increasing malignancy was found. In fetal neonatal, and the adult brain a high staining was found ill all ependynral cells. Two human specimens did also show a high immuno-rcactivity ill the ependymal cells. The immunoreactivity was within Ihc cyloplasm of the tumour cells as well as the normal cells. Conclusion: An EMBP-Iike protein has been dclected ill early appearing ependymal cells ill fctak UeUllNal, and adult ral. The same immunoreaclivily was fouud ill huln[lll elx3i/dymolna [muours and normal human ependyma.

CYTOSKELETAL REMODELING AND MIGRATION BEHAV- IOR OF HUMAN GLIOMA CELLS ON DIFFERENT EXTRA- CELLULAR MATRIX PROTEINS. Michael E. Berens, Moulque D. Rief and Alf Giese, Neuro-Oncelogy Laboratory, Barrow Nanrologieal Institute, Phoenix, AZ, USA, 85013-4496.

The hypothesis of this investigation is that specifie ligands in the extraeellular matrix of the brain are used as locemotive attachment sites for invasive glioma cells. The mechanical drive for cell movement is largely derived from polymerization of actin linked to adhesion plaques on the advancing cell membrane. We have studied the migration patterns and ectin eytoskeletal organization of human glioma cells SF763 and SF767, and culnued normal human glial cells on different extrasellular matrix proteins. A migration assay was developed in which the distribution of a population of cells on specific proteins plated within a cloning ring was followed after the ring is removed. Collagen type IV, fibronectin, laminin and vitronectin (each human- derived) were studied for their influence on eel1 growth and movement. Growth curve and BrdU-labeling index studies demonstrated that none of these proteins altered the proliferative rates of the glinma cens from that seen on albumin-blocked tissue culture plastic. For cells plated on ftbronectin, cellagen, and vitronectin, there was minimal migration away from the initial monolayer boundary after 4 days of culture. In marked contrast, the distribution area of glioma cells on lsmlnin was found to increase significantly after 4 days. The aetin eytuskeleton of normal glia was eonfined to the eytoplesm as defined by a smooth membrane boundary, and changed in response to different ECM proteins. Actin fibers in glia on laminin were more elongated but maintained a well defined, linear architecture. Astroeytoma cells had, in general, a profoundly disorganized eytoskeleton showing numerous small projections perpendicular to the membrane. In contrast to the eytoskeletal structure on other surfaces, astroeytoma cells on laminin showed a polarization. Amidst a very large number of small aetin projections, these cells contained solitary regions of 'leading edge' morphology, characterized by a smooth membrane with polymarizad aetin parallel to the membrane. The adhesion of glioma cells to ECM shows protein specificity, and alters the assembly of the eytoskeleton. These changes influence the migration rate of the cells.

S Y N E R G I S M B E T W E E N E ST RA M U ST I N E AND R A D I O T H E R A P Y IN A RAT GLIOMA MODEL. A. Tommy Bergenheim 1 2 BjOrn Zacta'isson 2, J~rgeu Elfversson 1, Roger Henriksson 2. Departments of 1Neurosargery and 2Oncology. University Hospital, Ume~, Sweden.

Back-.mund: Estramustine (EM), a cytotoxic drug used th advauced prostatic carcinoma, has been shown to excert antiproliferative effects on several humarl glioma celMines in vitro. EM is demonstrated to arrest the tumour cells in the G2/M phase of the cell cycle, to induce free oxygen radicals, aud to enhance blood flow which may increase the radioseusibility. An uptake and uccumulu0on of the cytoloxic metabolites has also been delnoustraled iu aslrocyloma lissue ill patients treated with EM, To iuves0gate whether EM could polenfiate the effect of i~xadiation in vivo a rat glioma model was set up. Methods: The nitrosourea induced BT4C turnout was stereotactically inoculated in the fight caudate nucleus of the rats. To eliminate differences ill gastrointestinal absorption the rats were given EM intraperitoneally daily. One group got EM in the dosage of 5 mg/kg and oue got 20 mg/kg. In addition to EM radiotherapy was given to two groups with 4 Gy for 5 days. One group recieved only radiotherapy and one group served as a non-treated control.The rats were sacrificed oll day 24 after turnout implantation and the turnout area was approximately calculated as the product of width and height at the largest coronar see[toll, Results:

t r e a t m e n t no tulnnur area (ram 2) p-value control 7 51.5 RT 6 47.3 ns EM 5 mg/kg l0 43.9 ns EM 20 mg/kg 8 33.7 <0.05 EM 5 mg/kg+RT l0 26.2 <0.001 EM 20 mo~o~o~o~o~o~o~o~o~o~kg+RT 10 26.3 <0.001 Conciusiou: Estrumusline seeins In polentiale the effect of radiotherapy in rat gliomu in vivo.

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ACUTE IODINE-125 BRACHYTHERAPY INJURY IN RAT BRAIN: EDEMA IS EQUIVALENT TO BREAKDOWN OF BLOOD- BRAIN-BARRIER AND IS NOT REDUCED BY TIRILIZAD Mark Bernstein MD, FRCSC, Alberto Cabantog MD, Jennifer Glen, David Mikulis MD, Phil Leung PhD; Brain Tumour Research Laboratory, Playfair Neuroscience Unit, The Toronto Hospital, University of Toronto, Toronto, Canada

F-344 rats were administered a minimum brachytherapy dose of 80 Gy to a 5.5 mm radius volume of the right frontal lobe with a stereotacticatly-placed removable high-activity iodine-125 seed. Tirilizad (5 mg/.kg) or vehicle was administered intravenously via an indwelling jugular line every 6 hours during the implant and for 24 hours thereafter. Rats were then studied with T2 and gadolinium-enhanced T1 MRI. Experiments were done on normal rats and on rats with 5 mm diameter 9L/SF gliosarcomas. In both normal and tumor- bearing animals, tirilizad did not reduce the volume of brain injury as measured on MRI. Tirilizad had no effect on tumor growth. In both experiments the volume of brain injury measured by T2 MRI (ie. edema) was equivalent to the volume of brain injury measured by gadolinium-enhanced T1 MRI (ie. blood-brain-barrier breakdown). This study was supported by the Upjohn Company.

G l i a l c e l l m i g r a t i o n i n v i v o and i n v i t r o . Rolf Bjerkvig 12, Paa l -Henning Pede r sen 2, Berit M a t h i s e n I, Rupava t hana Mahespa ran 1 and Hans Kristian Haugland2.1Department of Anatomy and Cell Biology and The Gade Institute, 2Departmente of Pathology, Haukeland Hospital, Bergen Norway.

Mal ignan t b r a in t u m o r s are cha rac t e r i s ed b y extens ive invas ive growth into the s u r r o u n d i n g b ra in t issue. Whe the r this diss iminat ion of t umor cells is e n h a n c h e d by specific proper t ies of these cells or by h o s t f ac to r s is unc l ea r . In the d e v e l o p i n g n e r v o u s s y s t e m t h e i m m a t u r e neuroec todermal cells show a high mitotic activity and have, like glioma ceils, the capacity to migrate over c o n s i d e r a b l e d i s tances . Fetal ne ra l cells homogra f t ed into the adul t bra in can also migrate over considerable distances in the CNS.

The p re sen t s tudy describes the the migra tory b e h a v i o u r o f b o t h fe ta l a n d g l ioma cells t r a n s p l a n t e d above the corpus cal losum of adul t rats. The ceils were s ta ined with the c a b o c y a n i n e dye(DiI) before implanta t ion. In cryosections, t he i m p l a n t e d cells were v i sua l ized showing ce l l migra t ion in the per ivascular space along blood vessels and along fiber tracts. These findings were also compared to the migratory pa t te rn of h u m a n lac-z t ransfec ted glioma cells t ransp lan ted into the adul t rat brain.

Several extracellular matr ix (ECM) prote ins a re k n o w n to i n f l u e n c e cell p r o l i f e r a t i o n , cel l migra t ion and cell a t tachement . The influence of these componen t s on cell migrat ion and invasion will be discussed.

IODINE-125 BRACHYTHERAPY FOR MALIGNANT BRAIN TUMORS Mark Bernstein MD, FRCSC, Normand Laperriere MD, FRCPC, Cindy Thomason PhD, Phil Leung PhD; Division of Neurosurgery, The Toronto Hospital, and Departments of Radiation Oncology and Physics, Princess Margaret Hospital, University of Toronto, Toronto, Canada

We have treated 108 patients with malignant brain tumors with high-activity iodine-125 stereotactic brachytherapy. Indications were: "up-front" glioblastoma on a randomized study in 52, recurrent glioblastoma in 46, and recurrent solitary metastatic tumor in 10. Minimum tumor dose was 60 Gy for "up-front" glioblastoma, and 70 Gy for recurrent glioblastoma and metastasis. Median survival for patients treated for recurrent glioblastoma is about 50 weeks post implant. On the randomized study (which has accrued 120 of 161 total patients required) the median survival of the implanted patients is about 64 weeks post diagnosis. Reoperation for a combination of recurrent tumor and radiation necrosis has been performed in 30% of the patients. Complications have occurred in 16% of patients; the most important is temporary increase in intracranial pressure requiring heroic medical and/or surgical therapy, seen in 6% of patients. 15% of recurrences of tumor following brachytherapy have occurred at a distance from the treatment volume. Brachytherapy has definite efficacy for highly selected patients with malignant brain tumors but complications are significant, reoperation frequent, and recurrence outside the treatment volume common. The true role (and benefit) of brachytherapy for patients with malignant brain tumors has yet to be fully defined.

Role of O6-alkylguanine-DNA alkyltransferase in the resistance of human brain tumor cell lines to alkylating agents. Bobola MS, Berger MS, and Silbcr JR. Dept. of Neurological Surgery, Univ. of Washington, Seattle WA 98195. O6-alkylguanine-DNA alkyltransferase (O6-AGT) has been implicated as a mechanism of resistance of diverse tumors to chemotherapeutic alkylating agents. We have assessed the role of O'~6--AGT in determining the resistance of human brain neoplasms to alkylating agents by evaluating its role in limiting the sensitivity of 12 human brain tumor-derived cell lines to 1,3-bis(2-chloroethyl)-l- nitrosourea (BCNU), ethylnitrosourea (ENU) and N- methyl-N'-nitrosoguanidine (MNNG). We found the 0 6- AGT content, which ranged from 0 to 120 fmol/106 cells in the cell lines, did not correlate with sensitivity to the three alkylating agents. Furthermore, ablation of O 6 - A G T activity with the potent inhibitor O6-benzylguanine had limited or no effect on sensitivity to BCNU or ENU. Elimination of O6-AGT, however, did have a marked effect on MNNG sensitivity in the majority of the cell lines. Our results show that O~6--AGT can be a primary determinant of sensitivity of brain tumor cell lines to MNNG but not to BCNU and ENU. Moreover, our findings demonstrate that O6-AGT content is not indicative of sensitivity of these brain tumor cell lines to the three alkylating agents.

INDUCIBLE EXPRESSION OF HUMAN GFAP cDNA IN GLIOMA CELLS UNDER THE CONTROL OF A TETRACYCLINE-RESPONSIVE PROMOTER

Erik Bongcam-Rudloff, Jia-Lun Wang , Monica Nist6r and Bengt Westermark. Department of Pathology, University Hospital, S-751 85, Sweden.

Intermediate filaments, together with microtubules and actin microfilaments, constitute the cytoskeleton of most higher eukaryotic cells.iThe glial fibrillary acidic protein (GFAP) is a member of the intermediate filament protein family, and a constituent of the cytoskeleton of cells of the astrocyte lineage. GFAP is well conserved between species and shows a high sequence homology with two qther intermediate filament proteins, viz. vimentin (expressed in cells of mesenchymal origin) and desmin (expressed in muscle cells). GFAP is generally present in primary brain tumor of asrrocytic origin. However, GFAP expression is often lost in subpopulations of glioblastoma cells, and most glioblastoma cell lines lack GFAP. The biological functions of GFAP in normal and neoplastic glia ceils remain elusive.

In the present study, we have constructed an expression plasmid that allows for ~an inducible GFAP expression. A full length GFAP cDNA was .inserted into the multiple cloning site of pUHD 10-3, downstrem~ of a hCMV minimal promoter with heptamerized tet- operator's (constructed by Dr. M. Gossen, Heidelberg). The plasmid was then cotransfeeted with pUHD 15-1, coding for the tTA transcription factor, and a neomycin resistance gene, into the the GFAP negative cell line U-1242 MG. This experimental system creates an " on/off" situation where GFAP expression is suppressed by tetracyclin. Immunofluorescence analysis using confocal microscopy proved the validity of the system; GFAP was absent from control cells and transfected cells in the presence of tetracyclin. Upon removal of tetracyclin, GFAP was expressed, initially in a punctate pattern, and later as typical GFAP fibrils. Double fluorescence analysis showed that the GFAP fibrils were generated on the framework of vimentin fibrils. The present experimental system is currently being used in studies on the biological functions of GFAP.

C A P T O P R I L I N H I B I T S C6 G L I O M A A N D E N D O T H E L I A L CELL G R O W T H IN VITRO. Steven Brem, Gary Breslow, Jason Ho, Stephen Gately, Shingo Takano , and Wil l iam Ward. Divis ion of Neurological Surgery, Northwestern Memorial Hospital and School of Medicine, Chicago, Illinois, 60611-2906.

Captopril inhibits the conversion of angiotensin I to angio tens in II (Ang II), a pept ide reported to be an angiogenie factor. Ang II receptors have been found associated with rat brain microvessels, and complete renin- angintensin systems have been reported in gl ioma cells. We have inves t iga ted the potent ia l ang iosuppress ive and ant ineoplast ic properties of captopri l on C6 g l ioma and bovine pulmonary artery endothelial cell (BEC) growth in vitro. For growth inhibition studies, C6 gl ioma and BEC in s ix -wel l plates were a l lowed to attach overnight , then exposed for five days at 0.1 mM to 10 mM of captopril and the number of adherent cells counted. Captopril, 1.0 mM, inhibited C6 gl ioma growth, 240 + 20 cells compared to the control (non-treated) 814 + 387 cells, P < 0.0001. Captopril t reatment of endothel ia l cel ls showed a trend towards inhibit ion with the control, 1225 + 332 cells, greater than captopri l- treated cells, 707 + 247, P < 0.08. Captopri l inhibited the C6 gl ioma bromodeoxyuridine labeling index with the control cel ls showing a label ing index of 47.2 4.89% and captopri l 5.0 mM, 26.5 + 3.89%, P< 0.001. BUdR labeling of BEC was also inhibited, but at a higher concentration; the control BEC labeling was 34 + 1.96% and captopril 10 mM, 25.2 + 3.56%, P < 0.002. In conclusion, e ap top r i l me r i t s fu r ther i n v e s t i g a t i o n as a nove l angiosuppressive and antineoplastic agent.

MANAGEMENT OF RECURRENT GLIOMA$ WITH FRACTIONATED STEREOTACTIC RADIOTHERAPY (SRT); ROOM FOR IMPROVEMENT.

M. Brada, R Laing, J Warrington Neuro-oncology Unit & Academic Unit of Radiotherapy and Oncology, Institute of Cancer Research and Royal Marsden Hospital, Downs Road, Sutton, Surrey SM251-if, UK.

Twenty-eight patients with recurrent high grade glioma were treated with fcactionated SlIT on a dose escalation protocol with doses ranging from 20-50 Gy. Median survival from SRT was 15 months which is comparable to interstitial RT. Eight patients developed presumed late radiation damage with steroid responsive neurological deterioration, but this did not compromise survival. The risk increased with dose to the target volume.

The risk of radiation damage to normal brain depends on a number of parameters including the volume of normal tissue/brain treated to high radiation doses. Theoretical calculations using technique of dose volume histograms suggests that for non-spherical lesions stereotactically guided conformal radiotherapy (SGCR) using multiple fixed beams with shaped shielding blocks achieves superior sparing compared to conventional multiple arc SRT.

As the majority of cranial lesions are non-spherical and often larger than 4.5cm diameter the future development of SRT should involve SGCR. As with other techniques it will be important to demonstrate reduced toxicity as well as improved efficacy for this high precision technique of radiation.

D - P E N I C I L L A M I N E , A NOVEL ANTIGLIOMA AGENT, SELECTIVELY SUPPRESSES ENDOTHELIAL CELL GROWTH AND M I G R A T I O N . Steven Brem, Herbert Engelhard, Shingo Takano, and Stephen Gately. Division of Neurological Surgery, Northwestern Memorial Hospital and School of Medicine, Chicago, Illinois, 60611-2906.

Copper deple t ion and D-penic i l l amine (D-Pen) therapy inhibits angiogenesis, tumor growth (Am J Pathol, 137: 1121, 1990) and neoplastic invasiveness in the brain (Neurosurgery, 26: 391, 1990), but the mechanisms of action are unclear. To test the cell-specific growth inhibitory properties of D-pen in vitro, established human gl ioma cell l i n e s - - U-87, U-373, U-138, A-172, bovine pulmonary artery (BEC) and fetal aortic endothelial cells, GM7373, AGO7680, were exposed to varying concentrations of D- Pen in DMEM + 10% fetal calf serum, or control ( media + serum without D-Pen), for five days and the number of adherent cel ls counted to determine the ICs0 values. The endothelial cells were sensitive to D-Pen; the BEC IC5o was 133 ~tg/ml, GM 7373 was 55 gg/ml, and AGO7680, 111 gg/ml. In contrast, human glloma lines showed significantly higher inhibitory ICso values: U-87, 1050 gg/ml; U-373, 844 gg/ml ; U-138, 1033 gg/ml ; and A-172, 844 gg/ml . DNA flow cytometry of D-pen treated cells was indicative of a b lock in ear ly S-phase. Endothel ia l cel ls were more sensi t ive to D-Pen treatment, wi th S-phase changes at concentra t ions as low as 10 Bg/ml. D-Pen inhib i ted endothelial cell migration to 67% of control at 200 gg/ml, 48% at 500 Bg/ml, and 35% at 1000 Bg/ml, P < 0.002. These findings support the concept that the antineoplastic properties of penici l lamine may be l inked to the selective suppression of endothelial cell growth and migration.

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UROKINASE-TYPE PLASMINOGEN ACTIVATOR AND PLASMINOGEN ACTIVATOR INHIBITORS IN HUMAN BRAIN TUMORS. Steven Brem, Barry Landau., Hau Kwaan*, and Elaine Verrusio*, Division of Neurological Surgery and Department of Hematology*, Northwestern University School of Medicine, Chicago, IL. 60611

The mechanisms that regulate angiogenesis and tumor invasion in malignant central nervous system (CNS) tumors are unknown. Because urokinase-type plasminogen activator (uPA) has been implicated in the malignant phenotype outside the CNS, we quantified uPA and its inhibitors in 33 human brain tumors. The malignant glioblastoma multiforme (n=12) and metastatic tumors (n=2) demonstrated the largest amount of uPA activity by zymography. In contrast, anaplastic astrocytoma (n=6) and acoustic shwannoma (n=4) showed intermediate activity; low grade glioma (n=3), meningioma (n--4) and normal brain (n=2) had only trace uPA activity. The uPA antigen as detected by ELISA was significantly elevated in glioblastoma, 3.53 + 3.50 ng/mg total protein, as compared to anaplastic astrocytoma and low grade glioma, 0.22 + 0.33 rig/mg (p<0.01) and was not detected in normal brain at the limit of sensitivity of 0.04 rig/rag. PAI-1 was also elevated in glioblastoma, 7.89 + 6.04 ng/mg bat was not detected in lower grade gliomas nor normal brain at a sensitivity of 0.05 ng/mg. PAI-2 was not detected in any of the tumors except for one meningioma. These data suggest that increased uPA and PAI-1 could mediate the invasive spread and angiogenesis characteristic of human glioblastoma.

PERIPHERAL BENZODIAZEPINE RECEPTOR GENE EXPRESSION IN MALIGNANT GLIOMAS

William C. Broaddus, Kathryn Hager-Loudon, Randall E. Merchant and William Loudon

Division of Neurosurgery, Medical College of Virginia Richmond, Virginia USA

The peripheral benzodiazepine receptor (PBR) is thought to be a mitochondrial membrane component which is distinct from the central-type receptor associated with the GABA receptor complex. We have demonstrated the presence of PBR in human malignant glial tumors and cultured cells, but not in normal human brain samples, making the PBR a potential target for labeling or directing therapies at tumor cells. We have also shown the presence of a 17 kilodalton component which can be photoaffinity labeled with a compound specific for the PBR (~H-PK14105). The availability of cDNA probes for human and rat PBR 17kD components led us to test the expression of PBR mRNA by human malignant gliomas and cultured cells. We wished to further test whether manipulations of the malignant phenotype of the ceils would lead to alterations in expression of PBR and its mRNA.

We report here the results of Northern blot analysis of a series of malignant glial tumors and cell lines, demonstrating expression of PBR mRNA. The effects of addition of cyclic-AMP analogues and corticosteroids to induce cellular differentiation and arrest of proliferation on PBR expression will also be presented.

TOXICITY OF INTERFERON-ALPHA (IFN-a), BCNU, AND RADIATION (RT) IN HIGH-GRADE GLIOMA PATIENTS. J. C. Buckner, T. L. Cascino, P. S. Schomberg, J. R. O'Fallon, R. P. Dinapoli, P. A. Burch, and E. G. Shaw

Preclinical evidence in a vadety of human tumor cell lines suggests that interferons may act as biomodulators of radiation and chemotherapy. In order to assess toxicity and efficacy in patients (pts) with high grade gUoma, we treated 11 pts in a pilot tdal with radiation (RT : 6,480 cGy in 36 fractions) plus BCNU (200 mg/m 2 on day 1 RT) followed by interferon alpha (IFNa : 12 x 10 s u/m 2 subcutaneously days 1-3 each week) and BCNU 150 mg/m 2 intravenously every 7 weeks upon completion of RT. Four additional pts enrolled but did not receive IFNa (2 pts: refusal; 2 pts: intervening medical complications precluded further treatment). Excessive fatigue occurred with weekly iFNa but treatment was tolerable with IFNa given at weeks 1,3, and 5 of each 7-week cycle. In an ongoing trial, 289 pts have been registered to receive BCNU and RT (as in pilot), followed by randomization (192 pts) either to BCNU alone (200 mg/m 2 ) or BCNU + IFN-a weeks 1,3 & 5 (as in pilot). Myelosuppression remains the dose limiting toxicity despite a 25% dose reduction in BCNU when combined with IFN-a. Median nadirs are:

!.,eukocvtes Platetets BCNU (n=83) 2700/mcL 67,000/mcL BCNU + IFN-a (n=77) 2400/mcL 70,000/mcL. Other toxicities on the IFN-a arm include flu-like illness: 70%; vomiting: 49%, reversible neuro-cortical toxicity: 32%; seizures: 12%. In the pilot trial, 5 pts remain alive at 2.8-3.8 years. Optic atrophy and hemorrhagic infarct occurred in 1 patient, but permanent cognitive impairment has not occurred. In summary, myelosuppression, vomiting, flu-like illness, reversible neuro- cortical toxicity, and seizures remain the major side effects of therapy with IFN-a. Significant long-term neuro-cortical toxicity has not occurred. Survival data in the pilot study remain encouraging, warranting continuation of the phase III trial.

Protein Kinase C Isozyme Prof'de of Malignant Low- Passage and Established Glioma Lines

William T. Couldwell, M.D., Ph.D., Jack B. Jiang, Ph.D. and David Burns, Ph.D.

Malignant gliomas express high Protein Kinase C (PKC) activity when compared to non-transformed glia, and this high activity correlates strongly with their proliferation rates in vitro. In an attempt to elucidate the mechanism for this increased activity, a Western blot analysis of both established glioma lines and low-passage glioma specimens was performed. All established lines tested (A172, U87, U138, U373) and low-passage tumors from surgical specimens (92-604, 92-625,542-6677, 567-1472) displayed identical PKC isozyme profiles. All specimens exhibited no evidence of the/3 (either I or II) or the 3' isozymes. All displayed strong signal for a, ~, and g" isozymes. The administration of a potent inhibitor of the c~ isozyme (compound #270; Sphinx Pharmaceuticals, Durham, NC) demonstrated marked inhibition of growth of all tumor cell lines (eg., in A172 an IC50 of I~M), suggesting that activity of this isozyme may have particular functional significance in regulating proliferation in vitro. The authors postulate that the loss of expression of the /3 isozyme and dysregulation of the system may have implications for altered growth regulation manifest in these cells.

Treatment of Recurrent Malignant Gliomas with High Dose Tamoxifen. William T. Couldwell, M.D., Ph.D., Martin H. Weiss, M.D., Michael L.J. Apuzzo, M.D.

Previous work has demonstrated the importance of the Protein Kinase C (PKC) signal transduction system in regulating the growth rate of malignant gliomas in vitro. Tamoxifen was administered orally in very high dosages (to achieve PKC-inhibitory tissue concentrations) to 14 patients (10 males:4 females, age range 26-74, mean 47 years) with malignant gliomas (anaplastic astrocytoma or glioblastoma multiforme) who had failed treatment with external beam radiation therapy (and additional chemotherapy in 3). The dosage administered was approximately 5-6 times that used for standard antiestrogen therapy. Significant tumor reduction on radiographic images (>50% on volumetric MRI with decrease in metabolic activity on PET scans) with clinical improvement occurred in 4 patients, with one patient (glioblastoma) attaining complete remission after 22 months of therapy. An additional patient has stabilized with no change in clinical or radiographic examinations. Of the remaining nine non-responders, four patients had marked and rapid progression of their disease despite treatment. Complications of treatment included one deep venous thrombosis requiting anticoagulation, nausea in one patient, "hot-flashes" in two young female patients, and an increase in proliferative retinopathy in a diabetic patient. Mean follow-up of the 5 patients that have either stabilized or improved is 14.8 months, with median survival of the entire cohort 11 months. These encouraging preliminary results in a minority of these patients suggests potential for this type of therapy, and prompts clinical trials using existing PKC inhibitors with increased potency.

MONOCYTE CHEMOATTRACTANT PROTEIN-I (MCP-I) IS SECRETED BY HUMAN ASTROCYTIC TUMORS IN VITRO AND IN VIVO. Desbaillets I., Tada M., de Tribolet N., Diserens A-C, Hamou M-F and E. Van Meir. Departement of Neurosurgery, CHUV, I011 Lausanne, Switzerland.

Expression of monocyte ehemoattractant protein- 1 (MCP-I) was examined in human central nervous system tumors (glioblastomas and astrocytomas) and normal human brain. Northern blot analysis on twelve human glioblastoma cell lines demonstrated constitutive expression of MCP-I mRNA in 6 of 12 cell lines. Stimulation with cytokines interleukin-l~ and tumor necrosis factor-~ increased or induced MCP-I mRNA expression in all cell lines tested. Immunoprecipitation demonstrated the secretion of both isoforms (15 kDa; MCP-I~, 13 kDa; MCP- I~) of the MCP-I protein. Reverse-transcription polymerase chain reaction and Northern blot analysis on tissues demonstrated MCP-I mRNA expression in 15 out of 15 glioblastomas, 2 out of 4 anaplaatic astrocytomas and 5 out of 5 low grade astrocytomas, as well as in fetal brain, but not in normal adult brain. In situ hybridization on 2 glioblastomas and 1 low grade astrocytoma indicates that neoplastic astrocytes and endothelial cells express MCP-I mRNA in vivo. Bioaasay using a Boyden chamber method with antibody blocking showed considerable monocyte chemoattractant bioactivity of MCP-I in tumor cyst fluids of patients with glioblastomas and/or astrocytomas. The present results suggest that in vivo production of MCP-I by tumor cells may play an important role in the infiltration of monocytes/macrophagea into astroglial tumor tissue.

LOW GRADE INFILTRATING ASTROCYTOMAS WITH ELEVATED BUDR LABELLING INDICES: COMPARISON WITH MIB- 1 LABELLING INDEX AND WITH OUTCOME. RL DAVIS, K. ONDA, MD PRADOS, BRAIN TUMOR RESEARCH CENTER, UNIVERSITY OF CALIFORNIA SAN FRANCISCO, SAN FRANCISCO, CA, USA 94143- 0506

A previously reported subset of low-grade infiltrating astrocytomas with elevated BUDR LI and with rapid recurrence or death has been studied using MIB-1 to see if this antibody also identified this group of cases. Thus far 12 cases have been studied, with BUDR LI 's of 1.5- 6.2%. These cases show MIB-1 LI 's of 2.4-10.4%. In the KI-67 literature the range of LI 's for these low grade tumors is < 1% to 4.8%; MIB-1 ranges have not yet been published. Of the 12 patients, only 2 are known to be alive without evidence of tumor progression. These data suggest that it may be possible to prospectively identify patients with low grade infiltrating astrocytomas with poorer prognosis and treat them more aggressively anticipating the tumor recurrence.

Supported in part by NCI grants 13525 and 50210.

BIODISTRIBUTION AND EFFECTIVENESS OF 06- BENZYLGUANINE ADMINISTERED IN POLYETHYLENE GLYCOL OR CREMOPHOR IN D456 BRAIN TUMOR XENOGRAFTS. M. Eileen Dolan, Matthew o r. Flaig and Henry S. Friedman, The University of Chicago, Chicago, IL and Duke University, Durham, NC

O~-Benzylguanine effectively inactivates the DNA repair protein, O~-alkylguanine-DNA alkyltransferase and has been shown to increase the therapeutic efficacy of BCNU in brain tumor xenografts. This study was undertaken to ascertain the influence of vehicle (polyethylene glycol-400 (PEG400) or cremophor) on the biodistribution and effectiveness of Ot-benzylguanine as an adjuvant therapy with BCNU. Nude mice bearing subcutaneous D456MG glioblastoma xenogmfts were administered i.p. 10 mg/kg 06- ber~ylguanine dissolved in 40% PEG400/saline or the same dose of drug in 10%cremophor/saline. Ot-Benzylguanine administered in PEG vehicle was more rapidly distributed to the tumor than when administered in eremophor. There was more than three-fold the amount of O6-benzylguanine remaining in the small intestine of mice 1 h after an ip injection of the drug in cremophor compared to PEG. In contrast, at 1 and 6 h following administration in PEG400, the tumor had almost twice as much 06-benzylguanine than when cremophor was used as a vehicle. The number of tumor regressions after treatment with 10 mg/kg Ot-benzylguanine followed by 38 mg/m 2 BCNU was 8/9 for the drug administered in PEG and 1/10 for the drug in cremophor. Using the same treatment regimen but increasing the dose of Otbenzylguanine to 30 mg/kg led to a growth delay of 45.2 and 11.5 days using PEG and eremophor, respectively. These studies demonstrate that 06- benzylguanine is a much more effective enhancer of the anfitumor activity of BCNU when administered in PEG400 compared to cremophor which may be due to a more rapid distribution of the drug to the tumor.

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FLqqCTIONAL ANALYSIS OF AN EPIDERMAL (~ROT#TH FACTOR RECEPTOR T~ITH A TRUNCATION IN THE EXTRACELLULAR DOMAIN THAT IS FREQS)ENTLu PRODUCED IN [4LIOBLASTOHAS WITH E@FR @ENE AMPLIFICATION A. Jonas EAst,^arid i Nicola Longo 2. V .Pete_r.

Collins 3 arid C.David James i. iLaboratory of Molecul~r ~e~xco~<3ncol~gy, Dept Netlro stir gery,

2Dept. P e d i a t r i c s , Emory U n i v e r s i t y , A t l a n t a , USA and 2Lud~ig Institute for Cancer Reseat'oh, Kat'olit]ska Instituter, Stock'holm, S~.zeden.

Recently }Te presented a cl'mracter'isation of mutant epidermal 9~o~,th factor receptor (E@FR) genes and transcripts 6ssociated ~{ith E@FR gene amplification in glioblastorms (Ekstz'and et al., PNA$ 89;~S09-13, 1992), The most co[mon of the alterations result in expression of s receptor vhose extracel l~Jlar domain is partially truncated, ~e ~m,ve cloned the cDNA encoding this mutant receptor and tr~nsfected it into Chinese hamster ovary (CHO) cells, a cell line that is devoid of endogenous E@FR expression Anti E@F- r e c e p t o r a n t i b o d y - m e d i a t e d i~un , : )p re c i p i t a t i on of cell lysates from CH0-transfiectants reveal a 140-145 kD protein; a size shift from that os the ]Tild type i70 kD E@F-t~.ceptor, consistent ~{ith the deletion of the 267 ~Lino acid residues caused l>y the corresponding w,',.',t~tion. Cells transfected with the mutant E,$F-reseptot ~ eDNA does no~ bind 125I-E(~F and ~re ll.ot g~'o~,:th stimulated by the addition of E@F to serur~t-free media. Analysis off E~$F-receptor tyrosine kinsse activity reveals~ floweret, th~.t the mutant receptor is constitutively active Consequently, these results suggests tt~mt E,3FR gene alterations ~hich result in production of th!s mutant receptor activate a signal transduction p~.th~.~ay associated ~,ith cell gro~.,, th/prol i - ferafion.

RADIATION-INDUCED BRAIN IN.IURY IS REDUCED BY THE POLYAMINE INHIBITOR DFMO. JR Fike, GT Gobbel, D Chou, B Wijnboven, M Bellinzona, M Nakagawa. Brain Tumor Research Center, University of California, San Francisco, California, 94143, USA.

Normal brain injury limits the dose of radiation used in the treatment of malignant brain tumors. The pathophysiologic characteristics of radiation-induced normal brain injury include necrosis, edema, changes in vascular permeability (breakdown of the blood-brain barrier, BBB), and aiterations in blood flow. We quantified the extent to which the polyamine inhibitor DFMO affected 1-125 radiation damage in normal dog brain. DFMO doses were 37.5, 75 and 150 mg/kg/day and total drug treatment time ranged from 4-18 days; radiation dose was 20 Gy at a point 0.75 cm from the 1-125 source. Pathophysiologic parameters were m e a s u r e d us ing quan t i t a t i ve CT; quan t i t a t ive immnnohistochemical methods were used to determine cellular response. Intravenous infusion of DFMO resulted in significant reductions in edema volume in all treatment schedules. Significant reductions in the volume of necrosis and BBB breakdown were observed at 75 and 150 mg/kg/day given for 18 days. The results suggest that DFMO may affect edema and necrosis through different mechanisms. Measurements of vascularity and permeability (transfer constant) suggest that DFMO's effect on edema might be mediated by its effect on BBB breakdown. DFMO may reduce necrosis by affecting the general cell response after irradiation, facilitating completion of cell recovery processes by delaying DNA synthesis/mitosis. Astrocyte and endothelial cell densities (labeled cells/mm2) increased as a function of time after irradiation, but DFMO delayed the onset of that response. BUdR labeling showed that cell proliferation was inhibited by DFMO treatment. Although the precise mechanism(s) responsible for the effects of DFMO on normal brain radiation response is unknown, it appears to involve altered cell kinetics and/or altered tissue physiology. Supported by CA 13525

BE-4-4-4-4 1S EFFECTIVE IN TREATING HUMAN BRAIN TUMOR CELL LINES IN TISSUE{ CULTURE AND NUDE MICE Feuerstein BG 1,2, Basu HS 1 , Dolan ME 3, Bergeron C 4, Pellariu M 1 , Deen DF 1, Marton LJ 5, Brain Tumor Res Ctr 1 , and Dept Lab Med 2 Univ California San Francisco; Dept Medicine 3, Univ of Chicago; 4Dept Pediatrics, University of Reanes, France; 5Depts Path/Lab Med, Oncol, and Human Oncol, Univ Wisconsin, Madison

Our laboratory is developing analogs of polyamines as antitumor agents. One new agent is 1, 19-bis-[ethylamino]-5, 10, 15 triazanonadeeane (BE-4-4-4-4). We tested this agent in human brain tumor cell lines in tissue culture and implanted in the flanks of nude mice. For tissue culture experiments, we grew the lines in minimal essential medium supplemented with enriched calf serum, and added BE- 4-4-4-4 directly into the medium. We monitored cell number by particle counting and survival by colony forming assay. Alkylaimg agents were added 1 hour before we performed the survival assay. 5 I.LM BE-4-4-4-4 given to the cell lines in tissue culture for more than 96 hr inhibited growth, decreased levels of polyamines, and decreased colony forming ability. The cytotoxicity varied among the lines: SF763 and SF-767 were most sensitive and most resistant, respectively. Cis platinum and BCNU were both synergistic with BE-4-4-4-4. In nude mice, one cycle of BE-4-4-4-4 treatment consisted of 5-6 mgfKg bid for 4 days, 3 days off the drug, followed by another 4 days of drug treatment. Tumors of U251-MG and U87-MG cells grown in nude mice were delayed in growth after one cycle of BE-4-4-4-4. Tumors of SF-767 cells were also delayed in growth after one cycle of BE-4~-~--4; and after a second course, 5/8 tumors regressed and 3/8 showed complete regression. We are presently planning large animal toxicity studies. BE-4-4-4-4, is a new agent extremely effeclive in treating human brain tumor cell lines in tissue culture and in nude mice, and may provide effective therapy in glioma patients. Supported by CA13525, 49409, 47228 and the National Brain Tumor Foundation.

HIGH-DOSE CHEMOTHERAPY WITH AUTOLOGOUS MARROW RESCUE (HDCx+ABMR) IMPROVES SURVIVAL OF PATIENTS WITH RECURRENT MALIGNANT GLIOMA (MG) COMPARED WITH CONVENTIONAL OR NO CHEMOTHERAPY. Jonathan Finlay, The Departments of Pediatr ics and Neurol- ogy, Memorial Sloan-Ketter ing Cancer Center, New York, New York, USA.

Since 1986, 43 pat ients (p ts ) , median age 13 yrs (range 0.7-45 yrs) with recurrent MG (30 GBM, 9 AA and 4 mixed MG) have received HDCx+ABMR with I o f 3 regimens: th iotepa/etoposide (T/E), n=15 pts; T/E+BCNU, n=l l pts; T/E+carbeplat in, n=17 pts. 28 pts were t reated a f t e r Is t re lapse/pre~ ression (R/PD), 12 a f t e r 2nd R/PD and 1 each a f t e r 3rd, 4th and 5th R/PD. The median time to death (MTTD) post-ABMR is 6 mos, r e f l ec t i ng a 16% tox ic mor ta l i t y wi th in 2 mos of ABMR. 28% of pts are a l i ve , of whom 8 (3 GBM, 5 AA) are f ree of disease from 12+ to 53+ mos post-ABMR. This outcome post-HDCx+ABMR may be compared with the post-relapse surv iva l o f pts aged 3-21 yrs t reated i n i t i a l l y with an XRT+Cx regimen, CCG-945 in the USA between 1985 and 1990. Of 101 with R/PD (59 AA, 42 GBM) 51 received no fu r the r Cx, with a MTTD pos t - l s t R/PD of 2 mos, and only l p t - a f t e r an equivocal PD-alive beyond 12 mos. Of 42 pts who received conventional Cx pos t - l s t R/PD, the MTTD was 7 mos, but only 2 pts (5%) remain a l i ve beyond 12 mos, both with fu r the r PD. Age, sex, extent o f resect ion, residual tumor volume or tumor locat ion did not impact upon outcome. The T/E/BCNU regimen produced an unacceptable tox ic mor ta l i t y (27%).

The. gutcome:~fo]lowing=HDCx*ABMR fo r pts w i t h relapsed MG~indicates adurab ]e progression- f ree sumv~:a}:can:belachievedt~n almost 20% of such pts, representing a s i gn i f i can t improvement.

CONTINUOUS LOW-DOSE ORAL VP-16 FOR PATIENTS WITH RECURRENT MALIGNANT GLIOMA Fulton DS, Urtasun RC, Cross Cancer Institute, Edmonton, Canada VP-16 is effective for treatment of patients with malignant glioma when given intravenously in high doses. However, because the percentage of dividing ceils in malignant glioma may be small, cell cycle specific drugs, such as VP-16 may be more effective if given continuously over a prolonged period. When prolonged oral VP-16 is given at a dose of 50 mg/m2/day, temporary interruptions of therapy are required because of myelosuppresion, in this study, the dose of 50 rag/day was chosen in the hope that interruptions of therapy would not be required. VP-16, g0 mg/day was given orally until the neutrophil count dropped to <1.0 or the platelets fell to <75,000 and resumed when the counts rose to normal levels. Fourteen patients with anaplastic astrocytoma (aa), 12 with glioblastoma multiforme (gbm), and 2 with anaplastic oIigodendroglioma were treated at the time of tumor progression. All had KPS>_7O at study entry. All patients had prior RT, 8 with adjuvant nitrosourea. Eighteen had prior nitrosourea chemotherapy for initial tumor recurrence, 2 had no prior chemotherapy. Ten patients were treated with VP- 16 at first recurrence, 18 at 2rid and 2 at 3rd recurrence. All patients had CT or MR scans and clinical evaluation every 8 weeks. Only one patient required temporary discontinuation of the drug for myelotoxicity. No patients had nausea or vomiting. A partial response (R) was noted in 2 patients (both aa) lasting 29.7 and 36.3+ weeks. Ten patients (7 aa, 2 gbm) had stable disease (S) for at least 8 weeks. The rate of partial response plus stabilization was 43%. The response rate (R+S) was 2/10 patients treated at first recurrence and 10/18 treated at 2nd or 3rd recurrence. The median time to tumor progression was 9.3 weeks for all patients and 14.4 weeks for R+S patients. The median survival for all patients was 23.1 weeks and 35.7 weeks for R+S patients. The study suggests that prolonged low-dose oral VP-16 is effective for patients with malignant glioma previously treated with nitrosourea and is more effective for aa than gbm.

UNTEGRIN ~6fl4 MEDIATES SPECIFIC ATTACHMENT AND MORPHOLOGICAL CHANGES IN HUMAN GLIOMA CELLS ON LAMININ. Alf Giese, Mohique D. Rief, Adrienne C. Scheek and Michael E. Bemns, Nanro-Oncology Laboratory, Barrow Neurological Institute, Phoenix, AZ, USA, 85013-4496.

lntegfins are dimefized transmembrane proteins comprised of a and B subunits which mediate attachment, migration, proliferation and differentiation responses of cells to Iigands in the extraeellular matrix. The unique pattern of glioma cell invasion within the brain and the rarity of extra-CNS dissemination of glioma cells suggest that hitegrius may play a key role in the behavior of this tumor. We investigated the attachment of human glioma cells (SF763 and SF767) to a panel of ECM proteins and the sensitivity of this attachment to inhibition by anti-integrinantibodies. Gliomacells attached to collagan, fibronectin, vitronectin and laminin to varying degrees and in a dose-dependent fashion. Human larninln elicited the most rapid attachment of the cells and also induced a segregated cell distribution in marked contrast to the other proteins, where the ceils formed contiguous clusters. Furthermore, in contrast to attachment to the other proteins, attach- mezt to laminin was not blocked by anti-61 antibodies, lntegrin e~6B4 has been proposed as a putative laminln receptor. Although anti-B4 antibodies did not inhibit attachment to laminin, RT-PCR analysis indicated the presence of mRNA encoding 64 integrin. Immnnopreci- pitation using AI/B2 antibodies against 61 integrin confirmed the presence of this subtmlt. Our results show that human glioma cells are able to discriminate among tigands in basement membrane proteins. The attachment and morphology of glioma cells shows ligand- specificity, especially to laminim. We speculate that changes in the expression or distribution of integrins on glioma ceils may lead to specific invasion patterns in the brain, and may, in part, account for the low ineidance of extra-CNS metastasis.

Supported by NS27030 (MEB and MDR) and the Deutsche For- schangsgemeinschaft (AG).

EXPRESSION OF THE TRANSMEMBRANE DRUG EFFLUX PUMP, P-GLYCOPROTEIN IN NORMAL EPENDYMA AND EPENDYMOMA

J. Geddes, GM Vowles, SM Ashmore and ]L Darling Royal London Hospital and the Institute of Neurology, London, United Kingdom

There is limited data which suggests that ependymal cells express P-glyceprotein and this may be related to putative role of these cells in CSF-braln transport. Samples of normal foetal and adult ependenyma and formalin fixed, paraffin wax embedded material from 29 cases of spinal or cerebral ependymoma were stained using a standard avidin-biotin complex peroxidase labelling method with two antibodies, JSB-1 and C-219, which reco~dse separate epitopes of the P-glycoprotein molecule. Normal ependyma were diffusely positive using either antibody. 87% of tumour samples had P- glycoprotein positive cells as evidenced by one or other antibody. Only four cases, two cellular, one myxopapillary and one papillary ependymoma, failed to stain Cellular ependymomas had fewer positive cells than either myxopaplllary or subcutaneous ependym- omas. 3/7 myxopapillary ependymomas and all subcut- aneous ependymomas had virtually all cells stained positive with either antibody. Irrespective of tumour type or site, P-giycoprotein levels tended to be higher in recurrent tumours than in equivalent tumours at diagnosis. It is clear that normal ependyrnal cells and a majority of ependymomas express P-glycoprotein. In the light of the observation it would seem reasonable to concentrate efforts in developing combination chemo- therapy regimens which did not rely on cytotoxic agents associated with the MDR phenotype.

MULTIPLE GROWTH FACTOR PRODUCTION BY MALIGNANT GLIOMAS: A UNIFYING PARADIGM OF TUMOR ANGIOGENESIS AND GROWTH. G.Y. Gillespie, C.K. Goldman, M.T. Tucker, E. Lyon and J.-C. Tsai. Brain Tumor Research Laboratories, Division of Neurosurgery, University of Alabama, Birmingham, AL 35294-0006

Malignant gliomas can produce and respond to a broad variety of growth factors that can result in progressive tumor growth and extensive neovascularization. Glioma cells in culture express EGF receptors and can be stimulated by EGF at physiological levels (f-10ng/mL) to proliferate and generate Transforming Growth Factor-alpha and Vascular Endothelial Growth Factor. TGFe can auto- induce its own gene expression, providing further evidence for a candidate autocrine stimulation mechanism in gliomas with intact EGFr . Expression of VEGF has been documented in every glioma tissue and glioma cell line that we have examined. VEGF is endothelial celt-specific and elicits endothelial cell growth as well as sprout/tube formation, both of which are essential initial elements in neovascularization. VEGF also markedly elevates [Ca]i f lux resulting in increased vascular permeability, the clinical result of which is brain edema and increased intracranial pressure. Enhanced activation of thromboplastin and increased expression of Factor-VIII-related antigen by endothelial cells are also important effects of VEGF stimulation. These endothelial cell products may contribute to hypercoagulability in glioma patients. Together with Tumor Necrosis Factor-e which we have shown malignant glioma cells can produce, VEGF can induce endothelial cell death, perhaps causing focal necrosis frequently seen within malignant gliomas. Production of a bioactive high molecular weight (MW 186kD) Transforming Growth Factor-6 by malignant gliomas may provide selective growth and survival advantages for glioma cells. TGF6 biofluid levels appear to correlate inverselywith I~a~ienLsurvivat,

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RESPONSE OF NORMAL RAT ASTROCYTES AND CEREBRAL ENDOTHELIAL CELLS TO RADIATION. G.T. Gobbel and P.H. Chan, Brain Tumor Research Center, Dept. Neurosurg., and CNS Injury & Brain Edema Center, Dept. Neurol., University of California, San Francisco, CA 94143

Radiation damage to normal tissue including cerebrovascular alterations limits the dose that can be delivered to malignant brain tumors. If the basis of the sensitivity of the cerebral vasculature to radiation could be determined, melhods of ameliorating radiation brain injury might be developed. We have isolated primary cultures of cerebrovascular cells (endothelial cells and astrocytes) and are examining the response of these cell types to radiation. Cerebral endothelial cells were isolated from 14 day old Sprague-Dawley rats, plated onto collagen- coated plastic, and irradiated the following day with doses of 0-6 Gy. Multiplicity (endothelial cells/irradiated colony) was determined by staining irnmunohistochemically for the expression of an endothelial cell specific marker, Factor VIII- related antigen. At 12 days after isolation, the cells were stained with crystal violet and the surviving fraction (SF) of colonies (>50 cells) at each dose determined. SFs were 0.73, 0.37, and 0.13 at 2, 4, and 6 Gy, respectively prior to correction for multiplicity and 0.26, 0.029, and 0.007 after correction. In addition, 16 or 32 Gy increased the basal level of lipid peroxidation (measured by malondialdehyde level) by 12.4% and 73.4%, respectively. For comparison, astrocytes were isolated from newborn rat pups, plated on plastic, and immediately irradiated. At 14 days after isolation, the cells were stained for colony formation or expression of an astrocyte specific marker, glial fibrillary acid protein (GFAP). Over 94% of the colonies were positive lot GFAP, and SFs (0.38, 0.088, and 0.029 at 2, 4, and 6 Gy, respectively) were higher than for the endothelial cells. These initial results suggest that cerebral endothelial cells may be the most sensitive cerebrovasoular cell. The high levels ot the antioxidant glutathione in astrocytes compared to the high levels of the pro-oxidant xanthine oxidase and production of nitric oxide radicals in endothelial cells may be responsible for the apparent differences in radiation sensitivity. Supported by NIH grants NS 14543, AG 08938, NS 25732, and the American Brain Tumor Association.

Radiosensitization with Carotid Arterial Infusion (IA) of Bromodeoxyuridine (BUdR) + 5- Fluorouracil (5-FU) Biomodulation with Focal External Beam Radiation (FEBT) for Malignant Astrocytomas (MAs): Final Report

Harry S. Greenberg, W.F. Chandler, W.D. Ensminger, L. Junck, H. Sandier, J. Bromberg, and P. McKeever, Ann Arbor, MI

The objective was to investigate continuous IA halopyrimidine radiosensitization in the treatment of MA. A permanently implantable pump system was developed for continuous IA carotid BUdR + 5-FU infusion because of BUdR regional advantage. Sixty- two patients (48 grade IV, 14 grade III) were entered into one of two studies with IA BUdR + 5-FU infused continuously for 8 1/2 weeks with FEBT to 5,940 cGy the last 6 1/2 weeks. Twenty-three patients received BUdR alone and 39 patients received BUdR + 5-FU, wi th a median fo l low-up of 53 months. The max imum tolerated dose (MTD) of BUdR was 400 mg/m2/day for 8 1/2 weeks. The Kaplan Meier median survival (KMS) was 20 months. In the second trial, the MTD was 400 mg/m2/day of BUdR and 5 mg/m2/day of 5-FU, with a KMS of 17 months. The KMS of patients in both trials 1 and 2 was 19 months. The KMS of the 48 grade IV patients was 13.5 months. Two-year survival of grade III patients was 71%. The dose-limiting toxicity was a reversible unilateral focal forehead dermatitis, blepharitis and conjunc t iv i t i s . Cont inuous IA ha lopy r imid ine infusion may enhance the effectiveness of FEBT in the treatment of MA. Study supported by National Cancer Institute RO1, Program Project, and Shiley Infusaid Co.

EFFECT OF 1,19-BIS-(ETHYLAMINO)-5,10,15- TRIAZANONADECANE (BE-4-4-4-4) AND 1,15-BIS- (ETHYLAMINO),4,12-DIAZAPENTADECANE (BE-3-7-3) ON RADIATION SENSITIVITY AND PLDR IN SF-767 AND DAOY HUMAN BRAIN TUMOR CELLS. Gonzalez, G.G., Sarkar, A., Basu, H., Feuerstein, B.G. and Deen, D.F. Brain Tumor Research Center, University of Califomia, San Francisco, CA 94143-0520.

Polyamine analogs mimic some regulatory functions of the natural polyamines. Unlike the natural polyamines, however, analogs can both decrease biosynthesis and increase catabolism of polyamines. Previous studies with c~-difluoromethylomithine (DFMO), which inhibits polyamine biosynthesis, showed inhibition of potentially lethal damage recovery (PLDR) after irradiation of some human brain tumor cells. DFMO combined with radiation is currently in clinical trial. Several polyamine analogs are presently being evaluated for clinical use, including BE-4-4-4-4. We are interested in whether there is a rationale for combining these analogs with radiation in the clinic. We investigated the effects of the spermine analogs BE-4-4-4-4 and BE-3-7-3 on radiation survival and PLDR in a human glioblastoma (SF-767) and a medulloblastuma (Daoy) cell line. Cells were preincubated with 0.5 ~tM BE-4-4-4-4 or BE-3-7-3 for 96 h, irradiated with graded doses of X rays, and assayed for colony forming efficiency. PLDR after irradiation was also estimated by holding cells at 37~ in EBSS for various times before assay. BE-4- 4-4-4 and BE 3-7-3 decreased putrescine, spermidine and spermine and inhibited cell growth in both cell lines. Neither analog affected radiation killing in SF-767 cells, but BE-3-7-3 potentiated X-ray killing slightly in Daoy cells. In SF-767 cells, BE-4-4-4~1 inhibited PLDR markedly, while BE-3-7-3 inhibited PLDR only slightly. Similar studies on Daoy cells are in progress. Our data suggest that these analogs have different effects on X-ray cell killing and PLDR, and these effects are cell-line dependent. Supported by NIH grants CA-13525 and CA-49409 and The National Brain Tumor Foundation.

GROWTH AND DIFFERENTIATION OF HUMAN GLIOMA CELL LINES IN VARIOUS SERUM-FREE MEDIA. Hans Kr. Haugland and Ole-BjCrn Tysnes. The Gade Institute, Department of Pathology, University of Bergen, N-5021 Bergen, Norway.

Serum-supplemented growth media are widely used to study mammalian cell growth in culture. These media stimulate and maintain a wide range of cells in vitro. However, due to the complex composition of serum, there is a need for serum-free growth cond i t ions when ef fec ts of spec i f i c ly added components are to be evaluated. The present study compare the properties of four different defined media with a serum containing medium on three gl ioma cell lines. Monolayer and spheroid growth, and migratory capacity was studied in the human glioma cell lines GaMg, D37Mg and D54Mg. The studies were done on polylysine coated plasticware. Growth was seen in the cell lines with all media, a l though the growth curves var ied considerably. The most potent of the serum-free media (SF-X, Costar) , compr i sed 30-40% of the growth in m o n o l a y e r when compared wi th the se rum containing medium. The spheroid growth comprised 65-75% when the same comparison was made. For the cells to be able to migrate, the tumor spheroids had to attach to the surface. This abil i ty varied considerably between the media. The MCDB 105 medium (Sigma), was uniformly the best serum-free medium in this aspect. Further, the same medium proved to comprise the greatest migratory capacity. These results will be exploited in further studies on the role of ECM-components in glioma malignancy.

N-ETHYL-N-NITROSOUREA (ENU) INDUCED MALIGNANT TRANSFORMATION OF RAT TYPE-1 ASTROCYTES IN VITRO AND THE ALTERATIONS OF p53 TUMOR SUPPRESSOR GENE: Dept. of Neurosurgery, Osaka Univ. Med. School: Shoju Hiraga, Norio Arita, Takanori Ohnishi, Takuyu Taki, Hiroshi Yamamoto, Masahide Higuchi and Toru Hayakawa

Abstract: In ENU induced rat glioma model, the location of tumor is unpredictable and serial observation of neoplastic changes in a single lesion is not possible. We induced neoplastic transformation of cultured type-I astrocyte (AS-l) by exposure to ENU and observed the morpho- logical changes of them. Since the importance of p53 alteration is stressed in human glial carcinogenesis, we also focused on the role of p53 alteration in this model. [Materials and Methods] Rat AS-I prepared from fetal brains (day 20 of gestation) was treated with a single dose of ENU (10 to 200 ug/ml). The ceils were passaged biweekly and morphological changes were serially observed under a phase contrast microscope. Cell characteristics were assessed by monoclonal antibodies to GFAP, galactocerebroside(GC) and A2B5. When transformed cells appeared, they were isolated and cell biological parameters of them were examined. The expression of mutant p53 was determined using Northern blot analysis and immunocytochemistry with a mutant specific anti- body, PAb240. p53 mutation was confirmed by RT-PCR and DNA sequencing using a dye primer method. [Results and Conclusions] Among the cells exposed to a high dose of ENU (150 to 200 ng/ml), transformed loci composed of piled-up cells appeared 42 to 57 days after treatment. Morphological transformation of the cells was closely asso- ciated with sudden emergence of immunocytochemically p53 positive cells. Six clonal cell lines isolated from these loci maintained immano- cytochemical characters specific to AS-l; they were positive for GFAP, but negative for A2B5 and GC. They exhibited increased proliferative capacity as judged by shorter doubling time, increased saturation density and tumorigenicity in nude mouse. Northern blot analysis demonstrated a marked increase of p53 mRNA level in all the transformed cell lines and DNA sequence revealed point mutations and alteration of amino acids in exon 5-8 regions. These results indicated that rat AS-1 can be transformed by single exposure to a high dose of ENU and mutations of p53 gone were suggested to be closely involved in this process. This transforming system of AS-1 in vitro has a great advantage for further investigation of astrocytie tumorigenesis.

AN ISOTOPE-AGAROSE ASSAY FOR RADIOSENSITIVtTY TESTING OF HUMAN MALIGNANT GLIOMAS IN PRIMARY CULTURE.

Erik ]sern, Geirmund Unsgaard, Anne Beate Langeland Marthinsen, Trond Strickert, Eirik Helseth. Institute of Cancer Research, University of Trondheim, Department of Neurosurgery, Department of Oncology, Trondheim Regional Hospital, Trendheim, Norway.

Several recently published studies use in vitro methods for measuring the inherent cellular sensitivity to radiation of malignant gliomas and investigate the possibility of using this information to predict respons to radiation therapy. There is considerable overlap between the radiosensitivity (as measured with 'surviving fraction at 2 Gy' = SF2) of malignant gliomas in these studies and SF2 of other malignant tumors known to exhibit better clinical responses to radiotherapy than gliomas. Here we present an assay for quantifing in vitro radiosensitivity and show that with this method malignant gliomas come out with SF2 values that are significantly higher than SF2 for endometrial carcinoma. Primary cultures from 27 tumor biopsies (13 glioblastomas, 3 anaplastic astrocytomas, 6 intracerebral metastases of adenocarcinomas, 5 endometrial carcinomas from the primary tumor) were studied in a colony forming assay with soft agar medium. The cells were radiated in increasing doses from 0,5-10 Gy. Proliferation was assessed by 3H-thymidine incorporation 7 days later.The surviving fraction at 2 Gy (SF2) + standard deviation and range was 84+ 13% (58-104) for the gliomas, 68+ 30% (8-84) for the metastases and 42+ 15% (32-67) for carcinomas of the endometrium. The difference in SF2 between the gtiomas and the endometrial carcinomas is significant with p<0,01 (Wilcoxons rank sum test). The results suggest that inherent cellular radiosensitivity as measured in our assay may be indicative of the clinical response to radiotherapy.

CEREBRAL INTERSTITIAL RADIOSURGERY WITH A MINIATURE X-RAY PROBE Hochberg F "l" , Cosgrove R "I', Valenzuela R 4" , Pardo F ~ , Zervas N +. +Neurology, § and ~Radiation Oncology, Massachusetts General Hospital and Harvard Medical School.

OBJECTIVE: To perform a safety and feasibility study of an interstitial radiosurgical probe as therapy for selected brain tumors. METHOD: The probe is 10 cm. in length and 3.2 nun. in diameter. It is powered by a 9 Volt battery, the peak current from the thermionic emitter is 40 incA. It generates a spherical beam of Xray photons at 40 KVp. Normal tissue receives minimal radiation as dose faUoffis near 1/r 3, One hundred thousand rads can be delivered at the tip surface of the probe. Therefore 1500 rods can be given to the perimeter ofa 3 cm. diameter spherical lesion with a scalp dose of <100 rods and near background exposure to personnel. The probe is placed in the lesion through the needle tract used to confirm the nature of the tumor. Three hours are required for the combination of biopsy and treatment. RESULTS: Ten patients, with clinico-mdiologic diagnosis of tumor (8 metastatic and 2 primary), were treated. In one patient there was a small asymptomatic hemorrhage during the biopsy procedure prior to treatment. Patients, between 37 and 80 years, received 1000 to 1500 fads in a single dose of durations from 6.5 to 28 minutes. The lesions treated were all below 30 mm diameter. With follow-up between 2 and 5 months, all patients exhibit improved or unchanged functional status. Local failures have not been observed to date. One patient has exhibited failure beyond the treatment edge. CONCLUSION: The procedure is pmmising for treatment of critically located tumors with minimal morbidity. A Phase III study will compare this device to surgical resection OR external beam irradiation. It can potentially be applied to other organ systems.

AN E V A L U A T I O N OF MICROSATELLITE REPEAT EXPANSION IN H U M A N GLIOMAS. Robert B. Jenkins, Steven R. Ritland, Kevin C. Hailing and Stephen N. Thibodeau, Mayo Clinic and Foundation, Rochester, Minnesota 55905 USA

Recently, somatic genomic expansion of microsatellite DNA markers has been observed in sporadic colon carcinoma (Thibodeau et at. Science 260:816-819, 1993). This instability is expressed as a small (1 repeat unit) or a large ( > 1 repeat) expansion in the number of microsatellite repeat units at one or more genetic locus. In colon carcinoma, microsatellite instability usually occurs simultaneously at multiple non-linked loci, generally in the absence of loss of heterozygosity, and associated with a good prognosis. We were interested in learning if the phenomenon of microsatellite expansion occurs in gliomas, and if it occurs, what are the characteristics and clinical correlations of this expansion. We examined 16 microsatellite loci mapped to chromosomes 9, 10 13, 17, 19, 22, and X in 48 gliomas. These chromosomes were selected because loss of alleles mapped to these chromosomes are associated with glioma pathogenesis. Among this group of tumors we observed no cases with expansion at these microsatellite loci. Conversely, the incidence of loss of alleles at loci mapped to chromosomes 9, 10, and 17 in this group of tumors was 38 %, 73 %, and 25 %, respectively. Our results suggest that genomic instability, as demonstrated by microsatellite repeat unit expansion, is an uncommon event in gliomas and is unlikely to be an important genetic event in glioma pathogenesis. (Supported in part by NIH grant CA50905.)

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PRESENCE OF INCREASED AMINOPEPTIDASE A ACTIVITY IN BLOOD VESSELS OF BRAIN TUMORS Juillerat,L.; Darekar,P.; Janzer,R.C.; Hamou, M.F.; Division of Neuropathology and Division of Neurosurgery, Lausanne, Switzerland

In normal conditions, cerebral micro- vasculature expresses low levels of various aminopeptidases. We have observed that in blood vessels invading brain tumors of various origin the activity of aminopeptidase A (APA) was specifically increased. The known function of this enzyme is to hydrelyse N-terminal aspartic acid from angiotensin. The overexpression of this enzyme was not strictly linked to tumor malignity. This APA activity could not be induced in vitro in endothelial cells which did not express this enzyme, either by addition of glioblastoma-conditioned culture medium or by coculture of gioblastoma cells with endothelial cells. In cerebral endothelial cell lines expres sing constitutively a high level of APA activity, the same culture conditions did not modify that expression. In these cells, inhibition of APA activity by amastatin neither modified their growth nor their response to added angiotensin. Then APA activity did not seem linked to vascular growth. Addition of dexame- thasone, a substance able to diminish brain edema, to these cerebral endothelial cells in culture decreased APA activity. It is hypothetized that the production of des-Asp l- angiotensin by APA in cerebral vessels of tumors might be linked to a modulation of vascular permeability.

INDUCTION OF DIFFERENTIATION AND ANTIPROLIFERATIVE EFFECT OF TNF-a ON HUMAN GLIOBLASTOMA CELLS

Tsutomu Kato, Yutaka Sawamura, Mitsuhiro Tada, Shirou Sakuma, Masako Sudo, Hiroshi Abe

Although TNF has been applied to early clinical trials for patients with malignant glioma in Japan, majority of human glioma cells has been reported to be resistant to TNF cytocydal effect. We studied antiproliferative effect associated with induction of differentiation by TNF on glioblastoma cell lines. Fourteen human glioblastoma cell lines were treated with low- dose TNF, up to 250 units/ml and for 7 days. All of the cell lines expressed p55-TNF receptor assessed by PCR and flow cytometory, but not p75 type receptor. MTT, colony forming and thymidine incorporation assays revealed antiproliferative effects of TNF with inhibition of DNA synthesis. Flow cytometory with BrdU and PI dual staining technique demonstrated that this antiproliferative effect of TNF, even at I unit/ml, was attributed to accumulation of glioma cells in G0/GI phase. Furthermore the TNF stimulation enhanced productions of bioactive molecules including IL--6, IL-8, GM-CSF, pGE2, and manganous superoxide dismutase. Most of the glioma cells lines expressing p55-TNF receptor well responded to the low-dose TNF stimulation, but not simultaneously showed sensitivity to its cytocydal effect. In conclusion, iow~ose TNF arrests human gliohlastoma cells in G0/GI phase and inhabits DNA synthesis resulting in suppress proliferation of the cells, furthermore increases bioactive molecules. We would llke suggest that TNF may be utilized as an agent for a differentiation therapy of human glioblastomas.

UPTAKE OF 99mTC-LABELED LOW DENSITY LIPOPROTEIN

(LDL) BY GLIOMAS

Kallio M(1), Leppal~ J(2), Nikula T(5), Nikkinen P(3), Gylling H(4), F~rkkil~t M(1), Hiltunen J(5), Jgt,~skel~inen J(2), and Liewendahl K(3). Departments of Neurology(l), Neurosurgery(2), Clinical Chemistry(3), II Department of Medicine(4), Helsinki University, 00290 Helsinki, Finland and MAP Medical Technologies Inc.(5), Finland.

As a part of the Finnish BNCT project we studied whether LDL, a potential carrier of boron in the neutron capture therapy, accumulates in cerebral gliomas. The uptake of LDL by these tumors in vivo is poorly known. Nine patients participated in the study. Four had a newly diagnosed and untreated high-grade glioma; four had a recurrent high-grade glioma and had previously been operated on and radiated, and one had a previously treated recurring low-grade glioma. We la- beled autologous LDL with 99m-technetium and injected it intra- venously. A scintigraphy of the brain was taken 2 hours and 15 to 20 hours after the injection. As a control we used albumin (HSA). The activity of labeled LDL injected varied from 19.7 to 34.7 mCi and that of HSA from 14.4 to 28.7 mCi. In the 2 hour scan the tumor area was clearly visualized in seven patients with activity ratios tumor vs nor- mal brain varying from 0.9 to 1.7 for LDL and 1.4 to 1.8 for HSA. In the 20 hour scan the tumor area could be seen in all patients with ac- tivity ratios tumor vs normal brain varying from 1.4 to 3.0 for LDL and 1.7 to 2.8 for HSA. The activity ratios between tumor and normal brain increased from the first to the second scan, more obviously with HSA than with LDL. In the recurrent tumors the tumor to brain ratio tended to be larger than with untreated tumors. The biological elimina- tion of 99m-technetium-LDL from blood resembled that of 1-125-LDL which is used commonly as a standard in studies on LDL-metabolism. In conclusion it seems that LDL accumulates in the tumor area. On the basis of the study cannot be distinguished whether LDL is taken up into the tumor cells or remains in the extracellular space in the tumor area. Further studies are needed to elucidate the behavior of LDL in gliomas and warranted on the way to developing a specific boron carrier for neutron capture therapy.

THE EFFECT OF EXTENT OF RESECTION ON RECURRENCE PATTERNS IN PATIENTS WITH IOW GRADE GLIOMAS

G. Evren Keles, Mitchel S. Berger, Anna Deliga- nis, George 0jemann.Univ of Washington, Seattle, WA, USA To evaluate the role of radical resection for low grade gliomas, we analyzed the pre and post- operative radiographic tumor volumes (CT hypo- density, MR T2 hyperintensity) in 53 patients, using a previously described (J Neurosurg 77:151 1992) method of computerized image analysis, to determine if percent of resection (POR) and vo- lume of residual disease (VRD) postoperatively, influences the incidence of recurrence (IOR), time to tumor progression (TTP) and histology of the recurrent tumor. No recurrence was detected, regardless of POR and VRD, with preoperative tumor volumes less than I0 cm3. Tumors measuring 10-30 cm3 had an I0R and TIP of 13.6% and 58 months, respectively, compared with tumors mea- suring more than 30 cm3 which had an IOR and TIP/ of 41.2% and 30 months, respectively (p=0.016)./ All patients who underwent 100% resection had a/ recurrence-free follow-up period. In the remain- ing patients, as the POR decreased, the IOR in- creased along with a shorter TTP (p=0.03). Pa- tients with a VRD of greater than i0 cm3 had both a higher ION (46.2%) and a shorter TIP (30 months) when compared to patients with a VRD of less than I0 em3 (IOR, 14.8% end TTP, 50 months) (p=0.002). Forty-six percent of patients with a VRD of more than i0 cm3 developed a higher grade recurrence and this was significantly more fre- quent then patients with a VRD less than i0 cm3 (3.7%) (p=0.0009). Our data suggests that a greater POR and smaller VRD convey a significant advantage to patients with low grade gllomas.

CYCLOPHOSPHAMIDE AND CONTINUOUS INFUSION VINCRISTINE IN PAEDIATRIC NEUROONCOLOGY: A CLINICAL AND PHARMACOKINETIC STUDY KELLIE S.J., DE GRAAF S.S.N., BLOEMHOF H., JOHNSTON I., UGES D.D.R., BESSER M., CHASELING R.W., OUVRIER R.A. The Children's Hospital, Sydney, Australia, and University Hospital, Groningen, the Netherlands.

Vincristine (VCR) is active as a single agent against a range paediatric CNS tumours and is frequently included in maltiagent chemotherapy protocols. VCR neurotoxicity was previously found to be dose-limiting using conventional IV bolus doses during early clinical trials, however continuous infusion studies in adults suggest an improved therapeutic index. We studied the relationship between the plasma and CSF pharmacokinetics of VCR administered by 96- hour continuous infusion and nenrotoxicity. Treatment comprised two cycles of cyclophosphamide 65mg/m 2 IV on day 1 followed by VCR 1.5mg/m 2 IV bolus on day 2 followed by VCR 0.5mg/n~/24 hours on days 2-5. Nineteen cycles were administered to 10 patients ages 1-15 years with astrocytoma (n=4), ependymoma (n=3), medalloblastoma (n=2), and ganglioglinma (n=l). Five patients were newly diagnosed and 5 were treated at recurrence, including 4 after prior radiation therapy. Our investigations also included a feasibility study of VCR pharmacokinetics in plasma and CSF. To achieve this aim, we developed a new specific and highly sensitive HPLC assay for the quantitation of VCR in plasma and CSF with a detection limit of 0.5 ng/ml. Jaw pain, constipation, mild abdominal pain and depressed muscle stretch reflexes were common however, no Grade 3 toxicity (motor weakness, gait impairment, wrist/foot pain, severe abdominal pain or sensory loss), nor Grade 4 toxicity (cranial nerve palsies, motor paralysis, ileus, SIADH, seizures, or hallucinations) occurred. Haematologic toxicity was mild. Using our HPLC methodology, measurable concentrations of VCR could be demonstrated in plasma throughout the 96 hour infusion. Our investigation demonstrates that the dose of VCR by continuous infusion can be safely escalated in children with CNS tumours, and studies correlating drug exposure with VCR efficacy and toxicity are now possible.

INTERSTITIAL BRACHYTHERAPY FOR RECURRENT MALIGNANT GUOMA N.D.Kitchen I , S.Hughes 2, R.Beaney 2 and D.G.T.Thomas 1 Department of Neurological Surgery 1 , Institute of Neurology and St.Thomas' Hospital 2, London, UK.

24 adult patients with recurrent malignant gliomas have been treated with high dose iodine-125 brachytherapy. Dosimetry is performed using 3D computer graphics and is based on contrast enhanced CT to deliver 5OGy to the tumour edge. Recently FDG-PET has been used to plan treatment and assess response. The GilI-Thomas-Cosman repeat stereotactic Iocaliser has been used in all cases and an afterioading technique has been employed. Selection criteria have been strict and only one quarter of referred patients have been treated. Median survival time from tumour recurrence and implantation has been 36 and 25 weeks respectively. Patient age (range 23-65 years), prior chemotherapy (13 patients), time to recurrence (mean 70.3 weeks), treatment volume size (range 4-66cc) and initial histology were not significantly correlated with survival (Cox proportional hazards regression and the log rank test), although the pre-operative Kamofsky Performance Status (KPS; range 50-90, mean 69) was for both implantation (p=0.O137) and recurrence (p=O.016) to death. Compared with controls (9 patients who declined brachytherapy for non-medical reasons) brachytherapy appears to confer some survival benefit though we have found this to be less dramatic than those obtained by others. Reasons for this are discussed, particularly timing of treatment and patient selection. However, we are encouraged to continue brachytherapy in the small number of patients with discrete recurrent tumours with high KPS.

MOLECULAR CYTOGENETICS OF MALIGNANT GLIOMAS Kim DH 1,2 Maeda T 1, Mohapatra G 3, Park S 1, Waldman FW 3, Gray JW 3, Feuerstein BG3; Brain Tumor Res Ctr 1, Dept.s Neurosurg 2, Lab Med 3, and Div Molec Cytometry 3, University of California San Francisco

Our laboratory is working to map and quantify regions of deletion and amplification in human malignant gliomas. We have studied 30 primary glioblastoma multiforme (GBM) using comparative genomic hybridization (CGH) and fluorescence in situ hybridization (FISH)~ CGH compares how well tumor DNA and normal DNA bind to normal metaphase chromosomes. In amplified regions, tumor DNA binds more than normal DNA; in deleted regions, tumor DNA binds less than normal DNA. Using CGH, we have found the following abnormalities in 40% or more GBM: -lp, -16p, -19, -9p, -10, -22, -Y, and +7. The former 3 lesions are present at rates far above those previously reported in GBM. We have also found, at lower frequencies, deletions and amplifications not previously described in GBM. We are presently extending our analysis to lower grade tumors. FISH uses probes for centromeres and chromosomal loci to determine whether single interphase tumor cells show amplifications or deletions at those specific sites. We have used FISH to characterize Epidermal Growth Factor Receptor (EGFR), p53, and centromeres of chromosomes 7, 10, and 17. These studies show that EGFR amplification is present in 18/30 GBM, but also document a surprising amount of heterogeneity. Seven of the amplified cases had <1- 5% of cells amplified; and amplification level varied 20 fold between cells in the same case. This heterogeneity was also seen using pericentromefic probes, where minor subpopulations of aneusomic cells were found at varying frequencies. We found that p53 is not physically deleted in GBM, implicating mitotic recombination as a probable mechanism of loss at this locus. We are presently studying probes at 9p, lp, and 19 by FISH. We are also correlating FISH and CGH results with clinical data to determine whether these parameters have applicability to the patient. Supported by CA13525 and the National Brain Tumor Foundation.

O6-alkylguanine-DNA alkyltransferase activity in a human meduiloblastoma cell line treated with 0 6- benzylguanine.

D. Koala , J. Si lber , M. Berger , Dept . o f Neurologica l . S u r g e r y , Un iv . o f W a s h i n g t o n , Sea t t l e W A 9 8 1 9 5 . Dep le t ion o f O 6 - a t k y l g u a n i n e - D N A a l k y l t r a n s f e r a s e (AGT) , a D N A r e p a i r p ro t e in i m p l i c a t e d in t u m o r res is tance to ch lo roe thy ln i t rosoureas (CENUs) , wi th the substrate a n a l o g u e inh ib i to r O 6 - b e n z y l g u a n i n e (BG) increases the sensit ivity o f a number o f human tumor cell lines to CENUs. To fur ther evaluate the cl inical potential o f B G in the C E N U - b a s e d ad juvan t c h e m o t h e r a p y o f h u m a n b ra in t umor s , the c o u r s e o f d e p l e t i o n a n d r e a p p e a r a n c e o f A G T ac t iv i ty w a s d e t e r m i n e d in the medul loblas toma cell line UW228-1 . Treatment o f UW228- 1 with 20 ~ M BG reduced act ivi ty to non-detectable levels (<300 molecules per cell) wi thin 1 hour. E l imina t ion o f act ivi ty was main ta ined for at least 24 hours when cells: were g rown in the presence o f the inhibitor. The course o f r eappea rance o f A G T act iv i ty was de te rmined af ter a 2: hoa r exposure to 20 g M BG. A G T levels increased to 39% of that o f untreated cells (24 versus 61 fmol /106 cells) by 24 hours af ter the r emova l o f the inhibitor. Subsequent to this A G T ac t iv i ty s l o w l y d e c r e a s e d and w a s a g a i n undetectable by 120 hours af ter removal o f BG. Act ivi ty o f unt rea ted ceils p roduced addi t ive A G T activity indicat ing that i n c o m p l e t e r e s to r a t i on o f A G T was no t due to persistent i n t r a c e l l u l a r p o o l s o f B G . O u r r e su l t s demons t r a t e that a s ingle , t rans ient exposure to B G is capable o f p roduc ing a large, protracted depression of A G T activity.

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PNET IN ADULTS

KRAUSENECK P, MOLLER B, STRIK H, WARMUTH-METZ M

Since 1979 26 adult pts. with PNET were seen in our clinic: age range: 17 - 63; 7 females, 19 males; 21 infratentorial, 5 supratentorial.

Mean follow-up and median survival t ime is in excess of 3 years. Different treatment strategies regarding to radiation port and application of chemotherapy were fol lowed over the years. The more recent exper ience with aggressive polychemotherapy showed high responsiveness of relapsing tumours with several long lasting complete remissions. Therefore and since CSF dissemination even in pts. with neuraxis irradiation is common, an adjuvant i.v. and i.th. chemotherapy is advisable.

MONOCYTE CHEMOATTRACTANT PROTEIN(MCP-1 )

DERIVED FROM GLIOMA

Jun-ichi Kuratsu, Hideo Takeshima, Yukitaka Ushio Kumamoto University Medical School, Kumamoto, Japan

The presence of macrophages within malignant gliomas has been well documented. We purified, sequenced, and cloned Monocyte chemoattractant protein-1 (MCP-I) from one of the cell lines (UIOSMG). Expression of MCP-I in human glioma cell lines and surgical specimens was studied by Northern blot analysis and in situ hybridyzation and immunohistochemistry. Both mRNA and MCP-I protein were located inside glioma cel ls by in si tu h y b r i d i z a t i o n and immunohistochemistry. The degree of macrophage infiltration appeared to correlate with the level of the MCP-1 expression. MCP-1 concentration in CSF's from patients with malignant glioma was significantly higher than that from patients with benign glioma or patients without tumor. CSF's from patients with subarachnoid dissemination of malignant glioma contained significantly higher amounts of MCP-1 than that from patients without dissemination. These results indicate that MCP-1 is produced by malignant glioma and that measuring for MCP-1 level in CSF may be useful to differentiate malignant glioma from benign glioma and to detect the presence of subarachnoid dissemination of tumor cells.

INTENSIVE ALTERNATING CHEMOTHERAPY IN GHILDREN WITH HIGH-RISK CNS TUMORS. C. Kretschmar 1, H. Grodman 1, R. Linggood2; Boston Floating 1 and Mass General Hospitals 2, Boston, Mass.

Since May, 1990, 9 children with newly diagnosed high- risk (7 pts) or refractory (2 pts) primary CNS tumors have been treated with a 5-drug pre-radiation (RT) regimen: vincristine 1.5 mg/m2/wk x 6, cytoxan 2.25 g/m2, VP-16 450 rag/m2, alternating q 4 wks with cis- platinum 120 mg/m2, BCNU 120 mg/m2. Response and progression-free survivals are below:

age- p r io r RT f /u vrs tumor therapy response (Gy) -mos 10 ol igo- P D l m o PR ( p r i o r ) 25+

astro, posbRT 15 parietal PD 5 yrs CR ( p r i o r ) 3+

PNET post-RT 6 pontine biopsy SO 6.6 35+

glioma 15 pontine none SO 7.2 30

gioma 8 pineo- part ial CR 5.5 10+

blastoma 18 GBM-2nd VAC+RT PR (prior at 4+

malig, for rhabdo age 5) 5 pontine none-off PR too early 2 +

glioma steroids 6 pontine none-off PR too early 2 +

glioma steroids 13 anaplas, part ial neuro improv, too early 2 +

astro, resect. (scan pending)

In summary, all 9 children have shown clincial or radiologic (2 CR, 4 PR) response to this regimen, although it is too early to evaluate effects on survival.

GENETIC ALTERATIONS IN MULTIFOCAL GLIOMAS. A. P. KYRITSIS, M. BONDY, J. CUNNINGHAM, M. XIAO, V. LEVIN, N. LEEDS, J. BRUNER, W. K. A. YUNG, AND H. SAYA. DEPARTMENT OF NEURO-ONCOLOGY, THE U. T. M. D ANDERSON CANCER CENTER, HOUSTON, TX 77030.

Multiple cerebral masses usually represent metastatic disease, even if the primary site of malignancy cannot be identified. However, malignant glioma can also present as multifocal lesions. We examined the incidence of multifocal gliomas in 596 consecutive cases of gliomas seen in our institution from August 1988 to May 1992. We found 65 patients with multifocal gliomas (10.9%), 36 at the initial presentation of the disease (early multifocal gliomas), and 29 at a later change during recurrence or progression (late multifocal gliomas). There was high incidence of secondary malignancies in the early multifocal gl ioma g roup (27.8%), compar ing wi th late multifocal glioma group (6.8%), or the non-multifocal glioma group (4.9%). The secondary malignancies included four cases of melanoma, three of colon carcinoma (one in the late rnultifocal glioma group), one of thyroid carcinoma, one of osteosarcoma, one cervical carcinoma, one breast carcinoma (late group) and one of bladder carcinoma. Multifocal gliomas are more frequent than generally believed and, therefore, multiple cerebral masses should be thoroughly evaluated and not always presumed to be of metastatic origin. We found high incidence of germ-line p53 gene mutations (27.8%) in patients with multifocal glioma, patients with unifocal glioma and another primary malignancy (20%) and patients with unifocal gl ioma and a family history of cancer but no second malignancies (20%). Our results indicate that heritable germ- line p53 mutations are frequent in patients with multifocal glioma, glioma as another pr imary malignancy, or glioma associated with a family history of cancer and that they may mediate increased susceptibility to cancer in pat ients ' relatives.

AN 1N~RNA~P-ROTEIN AS A TUMOR ANTIGEN L.A. Lampson, M.R. Nichols, M.A. Lampson, and A.D. Dunne Dept. of Neurology, Brigham and Women's Hospital & Harvard Medical School, Boston MA 02115

Actively migrating leukocytes may be particularly useful against disseminated tumor in the brain. Our goal is to define the nature and distribution of molecules that can serve as target antigens for cell-mediated immunotherapy. It has long been assumed that only cell surface molecules can serve as target antigens. Yet, current understanding of basic immunology suggests that internal molecules can also serve as targets of T ceil- mediated immunity. Here, we extend our studies of this in the 9L/lacZ brain tumor model (Cancer Res. 52:1018, 1992; 53: 176, 1993). Methods. 9L/lacZ cells stably express E coli-derived b-galactosidase (b-gal) as a cytoplasmic protein. Rats were immunized with b-gal protein or unrelated proteins, each in complete Freund's adjuvant, or unimmunized. After 3 weeks, b-gal+ 9L/lacZ tumor was implanted into the brain. After 3 more weeks, brains were examined histologically for b- gal+ tumor, or for tumor that had lost the b-gal marker (antigenic modulation). Computer-assisted image analysis was used to measure tumor growth. A survival study was also performed. Results. Pre-immunization with b-gal protected against the growth of implanted b- gal+ tumor. Rats immunized with b-gal showed reduced tumor size, and increased survival, as compared to controls. Abnormal b-gal- areas were shown to contain responding cells, not tumor. Conclusions. Tnmor target antigens need not he limited to cell surface proteins. This widens the spectrum of novel gene products that can be considered as potential tumor antigens.

COMPARISON OF CD44 EXPRESSION PATTERNS BETWEEN PRIMARY BRAIN TUMORS AND BRAIN METASTASES

Hong Li, Marie-France Humou, Rehana Janfeerally, Annie- Claire Diserens, Eiwin Van Meir and Nicolas de Tribolet Department ofNeurosurgery, CHUV, Lausanne, Switzerland

CD44, as lymphocyte homing receptor and adhesion molecule, is expressed in several forms which have identical end sequences but differ in the middle, a highly variable extracellular region. The CD44 molecule without these variations is called standard CD44 (CD44s), otherwise, variant CD44s (CD44v). Recent data reveal that CD44v can confer a metastatic potential to low- or non-metastatic rat tumor cells and play certain role(s) in multistep carcinogenesis and metastasis of some human cancers. In order to investigate a possible correlation between CD44v expression and metastatie ability, as well as invasiveness, 37 glioblastoma tumors/cell lines, 10 meinngiomas, 13 neurinomas, 9 medulleblastomas and 4 ependymomas and 23 brain metastases were studied by RT/PCR, Northem blotting and immunocytochemistry. Although most of primary CNS tumors expressed CD44s, none of them produced CD44v. In contrast, a high incidence of CD44v expression (13/23, 56.5%) was found in brain metastases of various origins. CD44v transcripts in BMs were then characterised. Heterogeneity of CD44v expression could be noticed, but Domains II and III of CD44v always existed, indicating the importance of these two domains in metastatic processes. Our data thus support a good correlation between CD44v expression and tumor dissemination and suggest that lack of CD44v expression may be of biological significance in the very weak metastatic potential of primary CNS tumors. Since CD44s was highly expressed in less invasive neurinomas, this molecule might not be a key factor in tumor invasion.

DM88-I13, A PHASE II STUDY OF ACCELERATED FRACTIONATED RADIOTHERAPY WITH CARBOPLATIN FOLLOWED BY PCV CHEMOTHERAPY FOR GLIOBLASTOMA PATIENTS V.A. Levin, M. Maor, W.K.A. Yung, R. Sawaya, M. Leavens, J. Bruner, S. Woo, P. Thall, M.J. Gleason. Brain Tumor Center, University of Texas M.D. Anderson Cancer Center, Houston, Texas.

Between 1988 and 1992, 91 patients received 190-200 cGy three times/day with 2 hr infusions of 34 mg/m 2 of carboplatin for two 5-day cycles separated by two weeks (wks); following radiotherapy, patients received CCNU, procarbazine, and vincristine (PCV) until tumor progression or one year. An increase in radiation toxicity was seen with 200 cGy fractions; toxicity to 190 cGy fractions was not observed. 84 patients, with a median age of 55 years were evaluable for response. The median survival was 56 wks for the entire group. Analysis showed a best median survival of 104 wks for certain age, surgical, and carboplatin dose subsets. Total resection was performed in 19% and subtotal resection in 71%; reoperation was performed once in 33 and twice in 6 patients. Survival was worst for those ~61 years (35 wks). The study shows the feasability and relative safety of accelerated fractionated radiotherapy with chemotherapy.

Evidence for Allelic Imbalance (AI) of Chromosome 6 in Human Astrocytomas

Bertrand C. Liang, D.A. Ross, P.S. Meltzer, J.M. Trent, and H.S. Greenberg, Ann Arbor, MI

The objective was to identify the contribution of a putative tumor suppressor gene on chromosome 6 in the development of gliomas. Recent work from our laboratory has demonstrated that transfer of human chromosome 6 can suppress the malignant phenotype of melanoma (Science 1990;247:568). Because of the neural crest origin of melanoma and since cytogenetic analysis has revealed that up to 30% of gliomas have abnormalities involving chromosome 6, we have performed restriction fragment polymorphism analysis to determine the importance of allelic loss on chromosome 6 in gliomas. DNA samples from tumor and WBC were obtained from patients with pathologically verified gliomas. Of the 15 paired samples, there were 1 grade I, 4 grade II, 2 grade III, 7 grade IV, and 1 ganglioglioma. DNA was hybridized with polymorphic probes D6S29 (6p21), C-MYB (6@3.3-24), SOD2 (6q25), D6S37 (6q26), and ESR (6q27). All grades of tumor revealed areas of genetic loss. AI was noted in 9/35 (26%) of informative loci on 6q, and 5/6 (83%) on 6p. Genetic loss from chromosome 6 is a frequent event in glial neoplasms. Further analysis is underway to define the importance of this observation in the genesis and/or progression of gliomas. Study supported by American Brain Tumor Association.

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Local Sustained Release Of Cisplatin From Biodegradable Polymer For The Treatment Of Intracranial Glioma In The Rat Lillehei KO, Kong Q, DeMasters BK, Withrow SJ

We investigated the effects of a biodegradable polymer, open polylactic acid (OPt.A), containing 8.2% platinum (OPLA-Pt), on normal rats and rats with one week established 9L gliosarcoma tumor. Permanent cannulas were installed in the right frontal lobe of male Fischer 344 rats (200-250 gm). Seven days later (day 0) 5 x 103 tumor cells were innoculated intracerebrany and the tumor was allowed to establish for 7 days. OPLA-Pt was then instilled at doses of polymer equivalent to 17.5 ug, 175 ug, 875 ug cisplatin. All surviving animals were sacrificed on day 30. Tumor control animals died between days 20-25. Normal rats receiving OPLA-Pt showed varying degrees of toxicity. High dose OPLA-Pt created extensive white matter necrosis with death within 24 hrs. Low dose OPLA-Pt demonstrated only focal microscopic changes with ne deaths. OPLA-Pt completely eradicated tumor in all tumor bearing animals at each of the three dosage levels. White matter toxicity remained extensive in the high dose group with early deaths. No deaths were seen in the low dose group, with microscopic white matter damage limited to the margin of the tumor cavity. These results are encouraging and support the potential use of local, low dose OPLA-Pt in the treatment of brain tumors.

NCIC CTG PHASE II STUDY OF CHEMOTHERAPY FOR ANAPLASTIC OLIGODENDROGLIOMA. Macdonald DR, Cairncross JG, Ludwin S, Lee D, Cascino T, Buckner J, Dropcho E, Fulton D, Stewart D, Schold C, Wainman N, Eisenhauer E. NCIC CTG, Queen's University, Kingston, Canada.

The National Cancer Institute of Canada Clinical Trials Group (NCIC CTG) conducted a phase It study of "intensive" PCV (procarbazine 75 mg/m 2 po days 8-21, CCNU (Iomustine) 130 mg/m 2 po day 1, vincristine 1.4 mg/m 2 days 8 & 29, q6 weeks x 6 cycles) for new or recurrent (prior RT) contrast enhancing anaplastic oligodendroglioma. Central pathology and radiology review were required. The minimum criteria for partial response were ->50% decrease in maximum X-sectional area of contrast enhancing tumor on consecutive CT/MR scans 6 weeks apart, steroid dose stable or reduced, clinically stable or improved (J Clin Onco11990; 8: 1277-1280). Of 33 patients entered onto study, 9 were excluded, 7 for inel ig ib le p a t h o l o g y ( < 9 0 % "pure" anaplast ic oligodendroglioma). Of 24 patients (13M, 11 F; ages 18-73 median 45) eligible and evaluable for response, 15 had recurrent tumor; 18 responded (75%), 9 completely (38%), 9 partly (38%); 4 had stable disease and 2 progressed during cycle 1. The median time to progression was 20 months for patients with complete response, 10 months for partial response and 6 months for stable disease. Toxicity included: mild-to-moderate nausea or vomiting in 70-80% of cycles; grade 3 or 4 neutrepenia in 40% of cycles without nadir sepsis; grade 3 or 4 thrombocytopenia in 15% of cycles; rash from procarbazine allergy in 30% of patients; and mild-to-moderate sensory neuropathy in 70% of patients. This study confirms that AO is a chemosensitive tumor and responds predictably to PCV. Central pathology review of anaplastic oligodendrogtioma is important.

NCIC CTG PHASE II STUDY OF TOPOTECAN FOR MALIGNANT GLIOMA. Macdonald DR, Cairncross JG, Eisenhauer E. London and Kingston, Ontario, Canada.

The National Cancer Institute of Canada Clinical Trials Group (NCIC CTG) is conducting a phase II study of topotecan (1.5 mg/m 2 iv daily x 5 days, each 3 weeks) in adult patients with biopsy proven malignant glioma and recurrent or progressive contrast enhancing measurable (->2 x 2 cm) disease. Topotecan, a semi-synthetic analogue of camptothecin, is a topoisomerase I inhibitor and produces single strand DNA breaks in vitro. To date, 10 patients have been treated: 3 remain on study: 6 are off-study, 2 for toxicity and 4 for disease progression. Of 8 patients evaluable for response, 1 had a complete radiologic response, 1 has a minor response (33% reduction in enhancing tumor), 2 are stable, and 4 had disease progression; 2 are too early to evaluate. Toxicity was primarily hematologic; of 19 cycles in 8 patients evaluable for toxicity 15 had grade 4 neutropenia (<0.5 x 109/L), 5 had grade 4 thrombocytopenia (<25 x 109/L), 2 had nadir sepsis (1 fatal), and 2 required RBC and platelet transfusions. There has been no significant non- hematologic toxicity so far. This study is continuing. Further data will be presented.

PRE-RADIATION CHEMOTHERAPY FOR MALIGNANT GLIOMA. Macdonald DR, Kirby S, Fisher B J, Caimcross JG. London, Canada.

We investigated the efficacy of chemotherapy (CTX) prior to radiation (RT) for malignant gliomas. Twenty-nine patients (10 females, 19 males; Karnofsky score ~ 60) were treated with at least one cycle of PCV (procarbazine, CCNU and vincfistine) or CCNU before RT (6000 cGy). This allowed response to CTX to be assessed independently of RT and ineffective CTX discontinued. Responses were assessed using rigorous criteria (J Clin Oncol 8: 1277- 1280, 1990). Nineteen patients with glioblastoma (66%) received PCV (15 patients) or CCNU (4). Three had partial responses, 7 were stable, 7 progressed, and 2 with complete resections did not progress. Five progressive cases stabilized with RT and 2 continued to progress. Seven with anaplastic astrocytomas (24%) received PCV (6 patients) or CCNU (1). Four were stable, 1 progressed (this patient did not stabilize with RT), and 2 with complete resections did not progress. Three with anaplastic oligodendrogliomas or anaplastic mixed gliomas (10%) received PCV. Two had partial responses and 1 was stable after one cycle - pre-RT CTX continues. Overall, 5 had partial responses (17%), 12 were stable (41%), 8 progressed (28%), and 4 with complete resections did not progress (14%). Five of 8 patients who progressed on CTX stabilized with RT. Toxicity was mainly hematologic and tolerable. We conclude that one cycle of CTX prior to RT is well tolerated by most patients and allows an objective assessment of response to CTX in many. Follow-up and further experience are needed.

EFFECTS OF VITAMIN D AND RETINOIC ACID ON HUMAN GLIOBLASTOMA CELL LINES

L. Magrass i , G. Butti , S. Pezzotta, G. Milanes i ~ Dep. of Surgery-Section of Neurosurgery, University of

Pavia, ~ CNR Pavia, ITALY

Previous works from our and other laboratories demonstrated the presence of vitamin D receptor (VDR) and retinoic acid receptors (RARs) in human brain tumors. We have shown that VDR mRNA is often expressed at high levels in human glioblastoma. However data on Ihe biological effects of vitamin D active metabolites and their interaction with retinoic acid on human GBM are laking. Furthermore the antineoplasfic activity of the vitamin D in experimental models of solid tumors and leukemia suggests potential clinical applications of these compounds. We tested the effects of cholecalciferol (1OHD3), 1,25-dihydroxyvitamin D 3 (2OHD3) and retinoic acid (RA) at concentrations ranging from 80nM to 100gM on two stable human glioblastoma cell lines HU70 and HU197. The expression of VDR and RAR (RAR a , RAR ]3, and RXR) mRNAs was previously demonstrated by reverse-lranscription amplification and sequencing. The cell were exposed to the tested compounds since plating. The medium with fresh substance was replaced every two days. After six days from plating the MTT assay for cell proliferation was performed. At concentration of 65p.M RA completely inhibited cell adhesion and proliferation. 1OHD 3 and 2OHD 3 induced the same effects at 25gM. When the effects of RA and vitamin D were tested at the above concentrations on non confluent cultures in logarithmic growth cell death was induced. Under these conditions no cell was still viable after 48 hours. When cells were tested with combinations of vitamin D and RA cell death was induced at shorter times of exposure and with lower concentration of the substances. This last result may suggest a direct interaction between RXR and VDR, as previously described in other systems.

TREATMENT RESULTS OF I01 PATIENTS

WITH CEREBRAL GLIOBLASTOMA

Masao Matsutani, M.D.

Department of Neurosurgery

Univ. of Tokyo, Tokyo, Japan

In 71 patients treated by conventional

radiation therapy combined with radio-

sensitizing agents, the combination of

ACNU and vineristine resulted in sig-

nificantly longer survival consisting

of the median survival of 101.1 weeks

and the two-year survival rate of 46~

than other combinations. Factors cor-

related to longer survival were a pa-

tient age, extensive resection, a good

performance status, a radiation dose

of 88-72Gy, no visible tumor after ir-

radiation, etc.. Intraoperative radiat-

ion therapy (IORT) with a high energy

electron beam was applied for 30 pa-

tients. A median survival and the 2-

year survival rate were 118.9 weeks and

@l.Z ~. Our results lead to the conclu-

sion that maximum resection of the

tumor follwed by localized high dose

irradiation and 70Gy conventional ir-

radiation with ACNU and VCR would be an

effective treatment for glioblastoma.

INTERACTIONS BETWEEN FETAL RAT BRAIN CELLS AND MATURE BRAIN TISSUE IN VIVO AND IN VITRO. K i r s t e n M a r i e n h a g e n 1, Paa l -Henn ing P e d e r s e n l , S v e r r e M o r k 1, Ole D i d r i k L a e r u m 1 a n d Rolf B j e r k v i g l , 2. iThe Gade Institute, Department o f Pathology and 2Department of Anatomy and Cell Biology, Haukeland Hospital, Bergen, Norway.

Feta l a n d m a t u r e n e u r a l cells in t h e f o r m o f d i f f e r e n t i a t e d b r a i n ce l l a g g r e g a t e s w e r e h o m o g r a f t e d in to the cal losal b o d y of adu l t ra ts . Th e g r a f t e d cells w e r e p r e s t a i n e d wi th t h e v i ta ! c a r b o c y a n i n e d y e (DiI) to d i s c r i m i n a t e b e t w e e n g r a f t e d cells a n d hos t b r a i n t issue. Both fe ta l a n d d i f f e r e n t i a t e d cells s h o w e d little i n f i l t r a t i o n in to t h e s u r r o u n d i n g b r a i n t i ssue a t t h e i m p l a n t a t i o n site. The fetal cells showed o p p o sed to d i f fe ren t i a ted cel ls e x t e n s i v e cell m i g r a t i o n a l o n g t h e w h i t e m a t t e r of t h e co rpus ca l losum. Fetal cells were also ex t en s iv e ly d i s t r i b u t e d in the s u b a r a c h n o i d space of b o t h c e r e b r a l h e m i s p h e r e s a n d showed a dis t inct a s s o c i a t i o n wi th l a r g e r b lo o d vessels . Cells f r o m m a t u r e b r a i n cell agg rega t e s d id no t m i g r a t e as f a r as t h e i m m a t u r e cells a n d s h o w e d no assos ia t ion wi th b lood vessels.

To d i s t ingu i sh b e t w e e n pass ive cell d i s p l a c e m e n t a n d a c t i v e cell m i g r a t i o n , i n e r t f l u o r e s c e n t m i c r o s p h e r e s as well as fixed fetal b r a i n cells were i m p l a n t e d , e i t h e r a l o n e o r in c o m b i n a t i o n wi th v i ta l cells. It is c o n c l u d e d t h a t t h e m i g r a t o r y b e h a v i o u r a lso has a p a s s i v e c o m p o n e n t s i n ce i n e r t m i c r o s p h e r e s m a y s p r e a d into ce r t a in a r e a s of the CNS.

SUCCESSFUL IMMUNOTHERAPY OF A GLIOMA MODEL WITH ADOPTIVELY TRANSFERRED LYMPHOCYTES. RE Merchant, NG Baldwin, CD Rice, MCV-VCU, Richmond, VA, 23298 USA

Preclinical testing of a new adoptive immunotherapy protocol employing tumor-sensitized lymphocyte populations was performed in rats with an intradermat (ID) or intracerebral (IC) glioma. Effectur cells were isolated from popliteal lymph nodes of Fischer 344 rats whose hind foot pads had been injected 9-10 days earlier with 106 syngeneic RT-2 glioma ceils. Draining lymph node (DLN) cells were incubated overnight in a complete medium (CM; RPMI-1640 + 10% fetal calf serum, and 20 U/ml human recombinant interleukin-2) with 5 nM Bryostatin-1 and 1 gM Ionomycin. The lymphocytes were then maintained in CM. Up to a 20-fold expansion in their numbers was achieved after 7 days in vitro. Tumor models were produced in rats by ID inoculation with 2 X 106 RT-2 ceils or IC inoculation with 104 RT-2 cells. After 3 days, these animals received a tail vein infusion of DLN cells or saline (controls). ID gliomas of controls (n=18) had tumors in excess of 1 cm diameter after 3 weeks while no tumors grew in 17/18 (94%) of the ID RT-2 models treated with 1~ sensitized DLN cells/g. In the IC glioma models, all controls (n=6) died of their tumors by day 17 while 5/6 IC models treated with 4 x 104 DLN cells/g survived. The one treated model which died of tumor, did so on day 27. Histopathological examination of the brains of survivors indicated that at the site of glioma inoculation, there were no tumor cells but rather only lymphocytes and maerophages. Other parts of the brain showed no apparent pathology. Lethally irradiating the RT-2 ceils prior to foot pad inoculation did not hamper their capacity to "sensitize" the DLN lymphocytes as these cells were equally efficacious in the glioma models. Finally, immunologic memory was achieved. Twelve ID RT-2 models who had responded to immnnotherapy were re-inoculated ID with 2 X l0 s RT-2 3 wks after their first tumor challenge. None of these rats developed tumors. Immunotherapy had no apparent toxicity; treated animals gained weight and displayed normal behavior. These results indicate that a reproducible and sustained eradication of a malignant glioma can be achieved by the adoptive transfer of tumor-sensitized DLN lymphocytes.

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DIFFERENTIAL EFFECT OF GROWTH FACTORS ON HUMAN GLIOMA CELL MIGRATION AND INVASION.

Abderrahim MERZAK, Shahriar KOOCHECKPOUR, Yannis ROXANIS, Geoffrey J. PILKINGTON.

Department of Neuropathology, Institute of Psychiatry, De Crespigny Park, Denmark Hill, London SE5 8AF.

Gliomas represent the major type of human brain turnout. Among the most important characteristics of these turnouts is their extensive diffuse spread into the normal structures of the brain. The factors involved in the control of this phenomenon are not well understood. In this work, we studied the involvement of epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), platelet derived growth factor (PDGF), and transforming growth factor betal (TGF-gl) on migration and invasiveness of two human glioma cell lines derived from a grade 3 astrocytoma (IPSB-18) and a glioblastoma multiforme (IPRM-5) respectively. This was performed using 8 #m polycarbonate filters in Boyden chambers for migration and the same filters coated with a thin layer of the reconsituted basement menbrane protein cemposite, Matrigel. We found that migration of the two cell lines was stimulated to a considerable extent by EGF and to a lower extent by bFGF and PDGF. Interestingly, TGF-BI was found to inhibit the stimulatory effect of all the other factors in the grade 3 astrocytoma derived cell line while it was found to potentiate their effect in the glioblastoma multiforme derived cell line. TGF-B1 alone was found to be migration stimulatory in the two cell lines studied. In addition, there was no effect of TGF-II1 on invasiveness of the anaplastic astrocytoma while the other factors elicited an increase of this potential. Invasion potential of the glioblastoma multiforme cell line was stimulated by all the growth factors used including TGF-f/1. In conclusion, migration and invasion of these two cell lines were stimulated by EGF, bFGF, and PDGF at different extents and this effect is differentially modulated by TGF-I~I in the two cell lines.

CLINICAL USE OF HUMAN MONOCLONAL

ANTIBODY CLN-IgG AND INDUCTION OF ANTI-

ANTI-IDIOTYPIC ANTIBODY IN .THE SERUM OM GLIOMA PATIENTS. Masakatsu Nagal 1, Kunihiko

Watanabe, Jun-ichi Narita, Hideaki Hagiwara 2. 1Dept of

Neurosurg. Dokkyo Univ School of Med., Tochigi, Japan, 2Hagiwara Institute of Health, Kasai, Hyogo,

Japan

CLN-IgG is monoclonal antibody (Ab) produced from

human-human hybridoma, recognizing tumor common

antigen (Ag) JA226 (60k/53k). Phase II clinical trial of

CLN-IgG was conducted in 10 cases of malignant glioma.

1 mg of the Ab was administered to patient twice a week

for more than 8 weeks. Concentrated induction of tumor Ag unspecific anfi-anti-idiotypic Ab (Ab3) in the serum of the patient was analysed using anti-CLN-IgG idiotype Ab

(idio33) which was obtained from hybridoma of Balb/c

mouse immunized by CLN-IgG. In 2 cases of partial

remission treated with the Ab, high levels (50-100 folds)

of Ab3 were measured. Induction of Ab3 would be

expected as anti-growth action on glioma and it corresponded to the clinical course of the patents.

SHORTENED POLYCHEMOTHERAPY IN MALIGNANT GLIOMA WITH ACNU, ARA-C AND VM 26 MOLLER B, KRAUSENECK P

Several studies including the German-Austrian-Glioma study showed, that at least in patients with better risk polychemethe- rapy is more effective than treatment with a nitrosourea com- pound alone, but there are no data regarding to the minimum duration of chemotherapy [n malignant glioma. Commonly che- motherapy is applied for 1-2 years. To examine the effects of a shortened (6 months) and intensified chemotherapy we con- ducted a phase-II-study in newly diagnosed malignant glioma patients after surgery and radiotherapy with KPS _> 70.

Results 40 patients, 22:18 m:f, age 47-+12 y, KPS 17:13:10 70:80:90%, WHO Grade 4: 32, residual tumour: 28 A marked (WHO 3-4), but short nadir of leuko- or throm- bocytes was seen in over 1/3 of cycles, but complications (bleeding, serious infections) were rare ( < 10%). No other main adverse effects occured, especially no relevant pulmon- ary dysfunction. 3 pts. are n.e., 8 progressed, 9 remained sta- ble and 1 showed a PR, in 19 patients CR continued or could be achieved (in 11 with residual tumour) and chemotherapy was stopped after a median of 7 months. 9/19 pts. are still in remission 6m - > 2y after discontinuing CT. Only 3 early re- lapses were seen in these 19 patients. Relapses responded to reinduction of therapy. Median survival was slightly better (16 m. n.s.) than in the corresponding subgroup of the German- Austrian-Glioma-Study. We conclude, that for pts. in good condition intensifying and shortening CT may be a chance for longer survival with less cumulative toxicity, better "quality of life" at lower cost.

F r o m g l i a l p r e c u r s o r s t o g l i a l t u m o u r s

Mark Noble, Ludwig Institute for Cancer Research, 91 Riding House Street, London W1P 8BT, U.K.

O u r s t u d i e s o n the b i o l o g y o f

o l i g o d e n d r o c y t e - t y p e - 2 a s t r o c y t e ( O - 2 A )

p rogen i to r cells of the central nervous sys tem

will be discussed as a pa rad igm for studies on

precursor cell b io logy and gl ioma cell biology.

Specific topics addressed will be the control of

p recursor cell division and d i f ferent ia t ion , the

use of p r ecu r so r t ransplanta t ion in the repair

o f d e m y e l i n a t i n g d a m a g e , the b i o l o g i c a l

d i f f e r e n c e s b e t w e e n p e r i n a t a l and adul t

p r e c u r s o r cel ls , the ident i f ica t ion o f g r o w t h

factors w h i c h m a y be involved in p r o m o t i n g

p recu r so r cell se l f - renewal and remyel ina t ion

in v i v o , a n d t h e d e r i v a t i o n a n d

cha rac te r i za t ion o f a h u m a n O - 2 A l ineage

glioma.

STEREOTACTiC MULTIPLE ARC RADIOTHERAPY FOR BRAIN TUMORS K. Nomura, H. Oyama, M Motoo. S. Shibui, K. Tokuue*, Y. Akine*. Dept. of Neurosurgery, * Dept. of Radiotherapy. National Cancer Center, Tokyo, Japan.

A stereotactic radicsurgery which can perform fractionated irradiation has been developed using a linear accelerator. This method requires a linac, a CT, a CT simulator and a treatment planning system for common radiotherapy. The special point of this method is a invention of the new device to immobilize a patient during irradiation by using a custom made device made of synthetic material which become flexible with heating. The target in the brain can be located at the isocenter of the tinac through the CT data and the CT simulator. The x-ray source can circled around the isocenter on multiple planes by rotating a treatment couch. Dose distribution by this method is confirmed to be comparable to that of the gamma unit.

Twenty patients with brain tumor (6 metastatic tumors, 6 gliomas, 3 pituitary adenomas, 2 sarcomas, 1 medullc- blastoma, 1 germinoma and 1 meningioma) were treated with this method during 1991-1993. Dose of radiation was varied from 3Gy to 6Gy/day and 18-120Gy in total, determined case bay case basis. Of these, 10 patients have had the full dose irradiation on the same region in the past. Four patients showed increase of the tumor size after irradiation, of which two cases were operated and turned out to be radiation necrosis. The others showed the good local control.

This stereotactic multifractionated radiotherapy would be very useful not only to be able to get the same sharpness of dose distribution as gamma unit but also to treat the patients observing the influence of irradiation,

ROLE OF GLIOMA-DERIVED MOTILITY FACTOR (GMF) IN TUMOR INVASION

-I-. Ohnishi, N. Arita, S. Hiraga, 3-. Hayakawa Dept. of Neurosurg., Osaka Univ Med Sch, Osaka

We have prev ious ly demonst ra ted that human mal ignant gl ioma cells, T98G, secrete a protein fac tor that s t imulates the moti l ty of the producer cel ls (1). T h e g l i o m a - d e r i v e d moti l i ty fac tor (GMF) has been puri f ied to homogene i ty from the s e r u m - f r e e condi t ioned medium of T98G by gelat in af f in i ty ch romatography and hepar in a f f in i t y - , DEAE- , hyd roxyapa t i t e - , gel permiat ion- and sulfopropyl HPLC. GMF showed a s ingle protein band on non - reduc ing S D S - po lyacry lamide gel e lec t rophores is with a molecular mass of 145 kDa. GMF stimulated the migrat ion of T98G cel ls in a d o s e - d e p e n d e n t manner and was act ive at above around 300 pM. Checkerboard analys is demonst ra ted that GMF had not only a chemotactic effect but also a chemokinet ic effect on T98G ceils. C. gl ioma cells and T98G cells, both of which ~ high invas iveness in an in vitro invasion assay with reconst i tu ted basement membrane, Ma t r i - gel, migrated to the GMF with great intensity, while A172 and 9L gl ioma cells and normal gila] cells, all of which week ly inf i l t rated the Mat r i - gel barr ier, migrated to the GMF with much less intensity. On the other hand, act iv i ty of co l lagenases in g l ioma cel ls was not re lated to the degree of invas ion into the basement membrane. These resul ts indicate that cell migrat ion st imulated by GMF may play an important role(s) in the process of g l ioma cell invasion. (1) J Neurosurg 73:881-888, 1990

INHIBITORY EFFECTS OF BRADYKININ AND CALCIUM

IONOPHORE ON GLIOMA CELLS" MIGRATION AND GROWTH IN A SERUM-FREE MEDIUM. Svein J. Tjoflaat Nygaard and Ole Bj~m Tysnes. The Gade Institute, Department of Pathology, University of Bergen,N-5021 Bergen, Norway.

Bradykinin (BK) and calcium-ionophores have been shown to mobilize intracellular calcium in several glioma cell lines. The present study evaluate the effects of of BK and the calcium ionophore A 23187

on growth and directional migration in the glioma cell lines GaMg, D-54 Mg and D-37 Mg. Monolayer

cells and spheroids were grown in medium

containing serum. Subsequently, the medium was changed to a BK- or A23187-containing serum-free defined medium. BK in increasing concentration induced a decrease in GaMg cell growth and migration. At 300 nM BK the growth rate and

migration was 64% and 75% of the control, respectively. This effect was less abundant in D-54 Mg and D-37 Mg. A23187 considerably reduced the glioma migration in all cell lines. A concentration of

3 ~M A23187 reduced the migration to 70% for GaMg and D-54 Mg and 20% for D-37 Mg as compared to control cells. It is concluded that the calcium homeostasis play a significant roIe in glioma growth and migration.

HIGH P53 EXPRESSION IS PROGNOSTIC OF OVERALL SURVIVAL IN ADULT SUPRATENTORIAL ASTROCYTOMA PATIENTS

F.S. Pardo, D.W. Hsu, E.T. Hedley-Whytr L Efird and E.V. Schmidt

Dcpts. of Radiation Oncology and Neuropathology, Massachusetts General Hospital and Harvard Med. School, Boston MA

Sixty-six patients with adult supratentorial astrocytoma evaluated at NIGH bctwr 1970 and 1990 were studied. The clinical characteristics of the patients were entered into multivariate analyses and representative tumor tissue was assessed for p53 expression using a standard immunoperoxidase technique employing both primary and lot- matched prc-absorbed p53 antibodies,, Patients with high levels of p53 expression were more likely to fail therapy and demonstrated significantly decreased overall survival times (p<.05) (Figure). Grade (p<.001), age (p<.05) and p53 expression status (p<.05) were found to bc independent prognostic factors for overall survival.

Event-Free~ Survival ~ ~ l ~ ~ '~ :

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THE MIGRATORY PATTERN OF FETAL BRAIN CELLS AND GLIOMA CEUS IN THE ADULT BRAIN

P a a l - H e n n i n g P e d e r s e n l K i r s t e n M a r i e n h a g e n 1, S v e r r e M o r k I a n d Rolf B je rkv ig l , 2.

1 The Gade Institute, Department of Pathology and 2Department of Anatomy and Cell Biology,

Haukeland Hospital, Bergen, Norway.

W h e t h e r t h e i n v a s i v e b e h a v i o u r t h a t c h a r a c t e r i s e p r i m a r y b r a i n t u m o r s is e n h a n c e d b y i n t r i n s i c p r o p e r t i e s o f t h e g l i o m a cel ls o r c a u s e d b y h o s t f a c t o r s t h a t is u n k n o w n . In t h e d e v e l o p i n g n e r v o u s s y s t e m t h e n e u r a l cells s h o w h i g h m i t o t i c a c t i v i t y a n d h a v e , l ike g l i o m a cells, t he c a p a c i t y to m i g r a t e o v e r c o n s i d e r a b l e d i s t a n c e s . The p r e s e n t s t u d y d e s c r i b e s t h e m i g r a t o r y b e h a v i o u r o f f e t a l a n d h u m a n g l i o m a cells t r a n s p l a n t e d i n to the s a m e a r e a o f t h e r a t b r a i n . The c a r b o c y a n i n e d y e (DiD w a s u s e d to s t a i n t h e i m p l a n t e d cells. F l u o r e s c e n c e m i c r o s c o p y o f c r y o s e c t i o n s s h o w e d t h a t b o t h cel l t y p e s f o l l o w e d t h e s a m e m i g r a t o r y p a t t e r n s a l o n g b l o o d v e s s e l s a n d f i b e r t r a c t s . H u m a n l a c - z t r a n s f e c t e d g l i o m a cells t r a n s p l a n t e d i n to a d u l t r a t b r a i n a l so c o n f i r m e d t h e m i g r a t o r y p a t t e r n o f g l i o m a cel ls . S e v e r a l e x t r a c e l l u l a r m a t r i x (ECM) p r o t e i n s a r e k n o w n t o i n f l u e n c e cel l b e h a v i o u r a n d m a y p l a y a n i m p o r t a n t r o l e in t h e i n v a s i v e p r o c e s s . S e c t i o n s f r o m i m p l a n t e d ce l l s w e r e t h e r e f o r e f u r t h e r c h a r a c t e r i s e d b y i m m u n o - c y t o c h e m i s t r y f o r l a m i n i n , f i b r o n e c t i n , c o l l a g e n t y p e 1V a n d c h o n d r o i t i n s u l p h a t e . It is c o n c l u d e d m a l i g n a n t g l i o m a s fo l low the s a m e m i g r a t o r y p a t t e r n as t h a t o b s e r v e d f o r t he fe ta l ce l ls o f n e u r o e c t o d e r m a l o r i g i n a n d t h a t s e v e r a l ECM c o m p o n e n t s a r e e x p r e s s e d in a s s o s i a t i o n wi th t h e g r a f t e d s p e c i m e n s ,

HYALURONIC ACID AND CD44 EXPRESSION IN HUMAN BRAIN TUMOUR-DERIVED CELL LINES: SIGNIFICANCE TO INVASIVE BEHAVIOUR.

Geoffrey J.PILKINGTON 1 , Tracey M.CLARKE 1, Hui Tian YU 1, l Joan P.ROGERS and Robert STERN.

1Department of Neuropathology, Institute of Psychiatry, De Crespigny Park, Deranark Hill, London SE5 8AF and 2Department of Pathology, University of California, San ~'h'andsco, USA.

Hyaluronic acid (HA) is a glycosaminoglycan which has been implicated in the metastasis of non-neural turnouts. Host fibroblast secretion of HA occurs as a result of growth factor-mediated stimulation from neoplastic ceils. In the brain, however, HA is produced by astrocytes; neoplastic glia showing higher levels than their normal counterparts. It is our contention, therefore, that HA stimulation may take place via an autocrine loop or by paraerine interaction between sub-populations of neoplastic astrocytes in brain tumours. The mechanisms thought to be involved in HA mediation of invasion include: taking on water of hydration, thereby increasing the extracellular space, facilitation of motility by HA receptor/actin microfilament Interaction and angioganesis as a censequance of HA degradation by hyahironidase. As little is known about HA in the context of local invasiveness of brain turnouts, we examined a large series of human brain turnout-derived cell lines for expression of HA, both intracelhilarly and on the cell surface by immunocytochemistry. In addition, the CD44 monoclonal antibody, which recoginses the HA receptor, was studied in the same cell lines (Pilkington et al NeuroReport 4: 259-262). Quantitative data on secreted HA in scrota- free culture supematants has been obtained using a modified enzyme- linked immunosorbant assay (ELISA) which utilises HA binding protein. Differential expression of CD44 and surface-bound HA may indicate the invasive potential of tumours of different histological type and grade of malignancy. Preliminary results also suggest a relationship between tumour grade and HA secretion in vitro.

SUCCESSFUL TREATMENT OF MALIGNANT GLIOMA~ LONG-TERM REMISSION OVER FIVE YEARS. CT/MRI/PET STUDY. Surasak Phuphanich, M.D., Harvey Greenberg, M.D., R. Murtagh~ M.D., Jesus Viloria, M.D., and Joseph Ransohoff, M.D., Neuro-Oncology Program, H. Lee Moffitt Cancer Center, University of South Florida (USF), Tampa, Florida, U.S.A.

The mean survival of patients (pts) with malignant glioma is six months and two- year survival is only 7.5%. Between 198~ and 1992, 402 pts with brain tumors were seen at USF. We identified six pts who are in long-term remission over five years (range of 5-11.5 years) with excellent quality of life, mean Karnofsk~ score of 90% and are actively working. There were 3 glioblastoma multiforme and 3 anaplastic astrocytoma pts with age at the time of operation ranging from 8-34 years. Four of six pts received adjuvant chemotherapy initially in addition to surgery and radiation therapy, and two a t the time of tumor recurrence. Serial brain CT/MRI scans showed complete dis- appearance of tumor and correlated with area of focal hypometabolism by positron emission tomography-fluorodeoxyglucose (PET-FDG) studies. All pts presented initially with seizures, but only one still requires anticonvulsant therapy. Our data suggests that long-term remission is possible in malignant gliomaptswho receive multimodality therapy despite poor prognosis.

GANGLIOSIDES AND GLIOMA CELL MOTILITY.

Geoffrey J.PILKINGTON, Kimberly MARTIN, Abderrahim MERZAK, V.Erika HATVA, Joan P.Rogers, Yannis ROXANIS and Shahriar KOOCHECKPOUR.

Deparhnent of Neuropathology, Institute of Psychiatry, De Crespigny Park, Denmark Hill, London SE5 8AF.

Since ganglioside expressing cells in the developing mammalian nervous system have been shown to be highly migratory in nature (Small et al Nature 328: 155-157, 1987), a similar relationship between cell surface ganglioside expression and motility in neoplastic gila may have important implications to the local invasive potential of brain turnouts in man. We have shown recently that expression of the ganglioside-recognising monoclonal antibody, A2B5, may be modulated on neoplastic astrocytes by basic fibroblast growth factor and epidermal growth factor (Pilkington et al Neuroscience Letters 149: 1-5, 1993). There is also considerable evidence that ganglioside exression is related to cell cycle phase. In order to determine whether A2B5 expression, cell cycle phase and motility are related, cells from both human and experimental gliomas were seeded onto 8/xm porosity polycarbooate filters in "transwell" migration chambers under a variety of growth factor-sopplemanted conditions. Cells which had migrated through the filter were fluorescence immono-stained using A2B5 and proliferating cell nuclear antigen (PCNA) antibodies and nuclear counterstained with propidium iodide, then migration counts per unit time were made. In all turnout lines examined the majority of migratory cells were A2B5- positive while PCNA staining was restricted to approximately 5 % of migratory cells. PCNA and A2B5 expression appeared to be mutually exclusive. Moreover, incubation of glioma cell lines in medium with the exogenous gangliosides, GM1, GM3, GD3, GDla, GDlb and GTlb prior to migration assays, inhibited proliferation and stimulated motility.

This work was supported by the Humane Research Trust and the Leverhulme Trust.

ROLE OF UROKINASE TYPE PLASMINOGEN ACTIVATOR AND UROKINASE RECEPTORS AND OF 92 KDa TYPE IV COLLAGENASE IN GLIOBLASTOMA INVASION USING AN IN VITRO MATRIGEL MODEL. Jasti S. Rat, S. Mohanam and R. Sawaya, Department of Nearosurgery, The University of Texas M.D. Anderson Cancer Center, Houston, Texas.

The invasive nature of human gliomas represents a major factor in preventing their total resection. Clues to the underlying mechanisms of the tumor cell invasion are lacking. In this study, we have quantitatively assayed a glioblastoma cell lines for its ability to invade a polycarbonate filter coated with matrigel which contains a complex basement membrane components. At 48 hours, the glioblastoma cell lines (UWRI, U251, LGll) showed a rate of invasiveness of 64%, 42% and 41% respectively. The invasiveness rate of the astrncytes was 12%. The antibodies for urokinase, urokinase receptors and 92 KDa type Iy collagenase inhibited the iuvasion from 42% to 12%, 14% and 21% respectively. Similarly e- amino caproic acid, and tissue inhibitors of metalloprotease, inhibited the invasion rate of U251 from 42% to 14% and 10% respectively. Fibrin/gelatin zymography showed the presence of uPA and 92 KDa type IV collagenase in serum free conditioned media. U251 cell line was found to bind rpro-uPA saturable and reversibly indicated the presence of urokinase receptors which was further shown by northern analysis. Invasion assay also further confh'med that the cell lines with more receptors had more % invasion rate. It was also shown that U251 cell invasion was dependent upon plasminogen and matrigel concentration. Finally, the addition of hyaluronic acid, a constituent of brain extracelhilar maaix, enhanced the rate of invasion. In conclusion, these findings provide evidence for the role of proteases, receptors and their inhibitors in the invasion of glioblastoma cell line and suggest a therapeutic role for protease inhibitors in attempting to minimize the invasive propensity of gliomas.

LOSS OF HETEROZYGOSITY FOR LOCI ON CHROMOSOME 10 IS ASSOCIATED WITH MORPHOLOGICALLY MALIGNANT MENINGIOMA PROGRESSION. Sandra A. Rempel, Karl Schwechheimer, Richard L. Davis, Webster K. Cavenee, and Mark L, Rosenblum.

Meningioma is a common tumor of the central nervous system which displays morphological heterogeneity. Genotypes at several loci on chromosomes 22 and 10 were examined by CA dinucleotide repeat polymorphism analysis to determine whether Ihis phenotypic wtriability is associated with distinct or overlapping genetic lesions. We concentrated on loci on chromosomes 22 and 10 because these genomic regions have previously been shown to be altered in the former in sporadic and familial meningiomas and in the latter as a late stage event in progression of another common brain tumor, astrocytoma. A total of 38 tumors classified as benign, atypical, or malignant by morphological criteria, invasive characteristics, or both were examined. We found that loss of heterozygosity (LOH) for loci on chromosome 22 occurred in 5/15 benign, 2/2 atypical, and 5/10 malignant meningiomas. Alterations on chromosome 10 were found in 0/20 benign, 1/2 atypical, and 4/13 malignant meningiomas. Among the malignant meningiomas, LOH for loci on chromosome 10 occurred in 2/4 morphologically malignant tumors, in 2/4 morphologically and invasively malignant tumors, and in 0/5 invasively malignant tumors. LOH for loci on chromosome 22 accompanied but was not restricted to allelic loss of loci on chromosome 10. These data suggest that progression of meningiomas from arachnoidal cells to the morphologically malignant phenotype may, in part, entail the loss of a tumor suppressor gene(s) on chromosome 22 early in the process and that this may be compounded by alterations of chromosome 10, the LOH of which is associated with morphological signs of malignancy,

Amplification and Overexpression of the MDM2 Gene in a Subset of Hmnan Malignant Gliomas Without

p53 Mutations

Guido Reifenherger ~, LuLiu~3,Koiehi Iehimura ~, Esther E.SchmidP, and V. Peter Collins L'2

~Division of Neuropathology,Department of Pathology I, Sahigrenska Hospital, S-41345 Gothenburg, Sweden 2Ludwig Institute for Cancer Research,Stockholm Branch, S-10401 Stockholm, Sweden

The MDM2 (murine double minute 2) gene has recently been shown to code for a cellular protein that can complex the p53 tumor suppressor gene product and inhibit its function. We studied a series of 157 primary human brain tumors by using three different RT-PCR generated probes for MDM2 on southern and northern blots. We found the MDM2 gene amplified and highly overexpressed in 8-10 % of gliobastomas and anaplastic astrocytomas. Thus, MDM2 represents the second most frequently amplified gene after the epidermal growth factor receptor gene in these tumor types. Sequencing of the p53 transcripts in the eases with MDM2 amplification revealed no mutations and restriction fragment length polymorphism analysis showed, with one exception, no losses of alleles on chromosome 17. Our results indicate that amplification and overexpression of MDM2 may be an alternative molecular mechanism by which a subset of human malignant gliomas escapes from p53-regulated growth control.

R E L I A B I L I T Y O F H I S T O L O G I C A L G R A D I N G O F A S T R O C Y T I C G L I O M A S B I O P S I E D BY

S T E R E O T A C T I C T E C H N I Q U E

Revesz T, Scaravilli F, Cockburn H, Thomas D G T

Since it had been suggested that needle biopsies tend to underestimate the grade of malignancy in gliomas, tumour grade was correlated with survival in 160 supratentorial diffuse astrocytic turnouts.

The grading systems applied were those of Kernohan and Daumas-Duport . Using the former there was a generally good correlation between histological grade and survival (p < 0.0001), although no distinction could be made between grade 3 (anaplastic astrocytoma) and grade 4 (glioblastoma) groups. On the other hand tumour grades obtained by using the Daumas-Duport grading system not only correlated with survival (p<0 .0001) , but also enabled us to identify a group of patients with intermediate grade malignancy (grade 3).

One of the advantages the Daumas-Duport grading system can offer over other grading systems, especially when small tissue samples are used, is its sensitivity in identifying glioblastoma as necrosis is not a prerequisite for the diagnosis.

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EFFICACY OF CTL ADOPTIVE IMMUNOTHERAPY FOR GLIOMA CORRELATES WITH CYTOKINE PRODUCTION RATHER THAN IN VITRO CYTOTOXICITY. CD Rice, RT Biron, RE Merchant, MCV-VCU, Richmond, VA, 23298, USA

Our laboratory has recently been involved in studies involving the in vitro generation of glioma-specific cytotoxic T lymphocytes (CTL) and their adoptive transfer into rat glioma models. We have investigated responses using two different glioma cell lines; the nitrosourea-induced line, D74, and the rolls sarcoma-induced line, RT- 2. Rats were sensitized to D74 or RT-2 by inoculation of one of the irradiated cell lines into each hind foot pad. After 10 days, tumor draining lymph nodes (DLNs) were removed and made into a single- cell suspension. These tumor-DLN lympbocytes were next activated overnight in medium containing IL-2 (20 U/ml), Bryostatin-1 (5nM), and Ionomycin (1 #M). Only medium and IL-2 were necessary for in vitro expansion of CTL number. A 100-fold increase in lymphocytes was usually observed after 7 days and, at this time, greater than 95% of the cells expressed the CTL phenotype (i.e. CD8 +). In vitro 51Cr- release assays for cytotoxieity indicated that RT-2 sensitized CTL were more toxic for RT-2 targets than D74 and also that D74 effectors were more cytotoxic for RT-2 targets as well. These data suggested that RT- 2 CTL would be more effective than D74 CTL in controlling RT-2 or D74 tumors in vivo. To test this hypothesis, intradermal glioma models were established using the D74 and RT-2 lines. On day three post- tumor inoculation, each animal received 106 RT-2 or D74 effector cells/g body weight via their tail vein. Tumor diameters were measured daily. Contrary to the in vitro cytotoxicity results, D74 CTL were more effective in slowing glioma growth in both D74 and RT-2 models. Subsequent studies were conducted to determine if tumor necrosis factor-e~ (TNF-a) and ~'-interferon (~'IFN) were produced when D74 and RT-2 CTL were co-cultured with the glioms targets. Both D74 and RT-2 effectors produced comparable amounts of TNF-a in the presence of either target while D74 effectors released significantly more ~-IFN under the same conditions. Our results indicate that CTL specificity in vitro as well as in vivo immunotherapeutic efficacy correlates with ~-IFN-release rather than in vitro eytoxieity.

CYSTEINE PROTEASE INHIBITORS ARREST INVASION OF BRAIN TUMOR CELLS IN VITRO. Mark L. Rosenblum, MD, James MeKerrow, MD, Bonnie Sloane, Phi), Tom Mikkelsen, MD, Norbert Roosen, MD, Peter Coopersmith, BS, Robert Smith, PhD (Midwest Neuro-Oneology Center, Henry Ford Hospital, Detroit, MI)

We analyzed the presence of extracellular matrix (ECM) degrading enzymes in biopsy samples of normal brain (NB), benign meningiomas (Mug), highly proliferative but noninvasive aggressive meaingiomes (AMng), invasive malignant meningiomas (MMng), and glioblastoma mnitiforme (GM). Analysis showed evidence for elevated levels of cysteine proteases (CPs). MnGs and AMngs expressed slightly higher levels of CP than did NB. MMngs frequently expressed the highest levels of CP. Values for cathepsin B were 116 4- 44 (SD) for 4 NB specimens, 252 4- 92 for 11 Mugs, 66 4- 40 for 2 AMngs, 844 + 592 for 11 MMngs, and 359 4- 146 for 7 GM. In 1 patient a Mng and a MMng were removed in 1 operation and showed eathepsin B levels of 317 and 850, respectively. The significant increase (P=0.0033) in CP levels for MMngs over Mugs suggests that this ECM degrading enzyme has a role in its invasive phenotype. The level of eathepsin B in GMs was significantly higher than for NB, suggesting that invasion in GM could be in part influenced by that enzyme. Based on these observations, we carried out a pilot sercen of the effect of a new irreversible CP inhibitor, fluoromethylketone (CPI#9), on the in vitro invasion of ceils freshly dissociated from biopsies of MMngs, Mugs, GMs, and NB. A dose-responsive decrease in tumor cell invasion through Matrigel was noted using 10-1000 #M of CPI#9. Ceils from all 4 MMng biopsies showed decreased invasion to 20-30% levels with 100 #M, whereas the small amount of invasion for cells from 2 Mugs was not affected. The degree of invasion remaining after CPI treatment of MMngs was about the same as observed for Mngs. Furthermore, treatment of 3 of 4 freshly dissociated samples of GM cells showed partial inhibitionof invasion through Matrigel by CPI#9, where.as the minimal invasion of NB cells was not inhibited by CPI#9. These studies show the potential for developing novel antiinvasive ~ e n t s through studies of the invasive process.

SECRETION OF METALLOPROTEINASES BY HUMAN BRAIN TUMOURS IN VITRO.

Harcharan Kaur ROOPRAI 1, Steven MAIDMENT 1, Garry Rucklidge 2, Geoffrey J. PILKINGTON 1.

1Deparlment of Neuropathology, Institute of Psychiatryz De Crespigny Park, Denmark Hill, London SE5 8AF and "The Rowett Research Institute, Buckburn, Aberdeen AB2 9SB.

Metalloproteinases (collagenase, gelatinase and stromelysin) are a family of Zn 2+ dependent endopeptidases with a broad spectrum of proteolytic activity for several components of the extracellular matrix (ECM). These enzymes have been implicated in turnout invasion of the normal brain by virtue of their ability to degrade proteins of the ECM. A number of control mechanisms exist to regulate the enzyme activities. These mechanisms operate at different levels, and include control of the synthesis and secretion of the enzymes and the presence of tissue inhibitors of metalloproteinases (TIMP). This work reports the expression of metalloproteinases from serum-free medium of twelve cell lines derived from brain tumours of different histological type and grade of malignancy by zymogram analysis. The enzymes were inhibited by the metal chelators, EDTA (10 mM) and 1:10 phenanthroline (2 raM) thus confirming that the enzymes are metalloproteinases. Results show that no degradation was found in the gels containing casein (substrate for stromelysin) for all culture media tested while gelatinase and collagenase were differentially expressed in these cell lines.

This work was supported by the Association for Internatinal Cancer Research.

Post-treatment bioenergetic changes in murine glioma. Jack M. Rozental, MD, PhD and Anton Volovsek. Department of Neurology, VA Lakeside Medical Center and Northwestern University, Chicago, IL 60611.

Using PET-FDG, we showed that the peak CMRGlu in gliomas of patients treated with either BCNU or "eight-drugs-in-one-day" chemotherapy increased acutely 24 hours after treatment. Patient survival was inversely proportional to the degree of increase. This suggested that tumors re-energized after treatment, a property which determined their ability to repair sublethal treatment-induced damage. To further study this phenomenon in vitro, cultured murine glioma was exposed to 50 pM or 100 pM BCNU for 2 hours. The resultant changes in phosphocreatine, AMR, ADP, and ATP, as well as in the ATP/ADP ratio, were measured with HPLC at 24, 48, 72, 96 and 120 hours after treatment. Phosphocreatine levels peaked 24 hours after treatment. AMP and ADP decreased after BCNU exposure, reaching a nadir after 72 hours. ATP levels increased after 24 hours and remained high through 120 hours. The ATPIADP ratio (the measure of "energy charge") eeaked after 72 hours. The change in energy charge correlated with resultant in vitro clonogenicity. These results support our hypothesis that tumors re-energize after treatment, a property which impacts on their resistance to the treatment.

EXPRESSION OF TIMP-1, TIMP-2 AND TYPE IV COLLAGENASE TRANSCRIPTS IN PEDIATRIC BRAIN TUMORS: Correlation with histopathological tumor grade

Rutka JT, Smith SL, Fukuyama K, Matsuzawa K, Brain Tumor Research Laboratory, Hospital for Sick Children, University of Toronto

H u m a n m a l i g n a n t as t rocytomas are h ighly invasive neoplasms which infiltrate diffusely into regions of normal brain. In general, tumor cell invasion is a complex cellular process mediated in part by the elaboration of tumor- associated metal loproteases (MPs) and modula ted by various protease inhibitors (TIMPs). We have recently shown that among astrocytoma cell lines, TIMP-1 and TIMP-2 transcript levels correlate inversely with in vitro tumor cell invasion (Proc AACR 34: 70,1993). In the present study, we sought to determine if a relationship exists between t ranscr ip t levels of MPs and TIMPs and histopathological tumor grade in a series of pediatric brain tumors. Northern and in situ hybridization analysis was performed on 20 pediatric brain tumors (5 low grade astrocytomas, 5 high grade astrocytomas, 5 PNET's, 2 ependymomas, 1 rhabdoid tumor, 1 meningioma, and 1 gliosarcoma) using cDNA probes for TIMP-1, TIMP-2, and type W collagenase. While there was some variability of transcript expression within this g roup of tumors, in general, t ranscript levels for type IV collagenase were much higher in the high grade or anaplastic tumors than in the low grade tumors; by way of contrast, transcript levels for TIMPs 1 & 2 were higher in the low grade, well- differentiated tumors than in the high grade tumors. These data support the notion that high grade pediatric brain tumors are invasive neoplasms in part because of higher expression of MPs than of TIMPs.

HIGH RESOLUTION 1 H-MAGNETIC RESONANCE SPECTROSCOPY STUDIES ON CULTURES DERIVED FROM MALIGNANT GLIOMAS WITH DIFFERENTIAL SENSITIVITY TO CCNU AND TEMOZOLAMIDE IN VITRO. A.A. SankarL J.L. DarlingL S.R. Williams 2. D.G.T. Thomas 1 . 1Dept. of Neurological Surgery, Institute of Neurology. U.K. zRCS Unit of Biophysics, Institute of Child Health. U.K.

Cell cultures derived from human malignant gliomas display a wide range of sensitivity to chemotherapeutic drugs. High resolution magnetic resonance spectroscopy (MRS) can be used to produce a metabolic profile of extracted cells, the analysis of which may reflect in situ chemosensitivity. Fourteen cell cultures, derived from adult human gliomas, have been investigated, with respect to their intrinsic chemosensitivity as determined by measuring IDs0 with the MTT reduction method. For CCNU, sensitivity ranged from 0.6/Ig.ml- i to 8.0/ig.ml- i For temozolamide sensitivity ranged from 3.7/Jg.ml - I to 105pg.ml - I . A degree of cross-resistance was observed, i H-M RS analysis showed that spectra from all cultures shared some common peaks. Variation was observed in peaks at three positions - 1.46, 3.55 and 3.7 parts per million, which we assign to alanine, glycine (tentatively) and a mixture of other amino-acids. Cultures sensitive to CCNU, which were resistant .to temozolamide tended to have higher levels of these metabolites than cultures resistant to CCNU, but sensitive to temozolamide. These

, results may aid in the interpretation of the relationship between chemosensitivity and 1H-M R S.

TRANSFECTION OF HUMAN ASTROCYTOMA CELLS WITH ANTI-SENSE GFAP cDNA: Analysis of astrocytoma morphology, proliferation and tumorigenicity

Rutka Jr , Smith SL, Fukuyama K, Matsuzawa K; Brain Tumor Research Laboratory, Hospital for Sick Children, University of Toronto

Glial fibrillary acidic protein (GFAP) is a 50-kDa intracytoplasmic f i lamentous protein that constitutes a port ion of, and is specific for the cytoskeleton of the astrocyte. A critical observation from histopathological studies is that there is progressive loss of GFAP expression with increasing astrocytic anaplasia. We have previously shown that transfection of GFAP-negative astrocytoma cells with an expression vector carrying a full-length GFAP cDNA results in a restoration of GFAP expression, altered tumor cell morphology, and reduced proliferation and growth of astrocytoma cells in monolayer and soft agar (Proc AACR 33: 50, 1992). In the present study, we have transfected GFAP-positive U 251 MG astrocytoma cells with an anti-sense GFAP cDNA construct. Post-transfection, U 251 MG a s t r o c y t o m a cel ls we re GFAP-nega t i ve by immunofluorescence analysis. GFAP-negative U 251 MG cells demonstrated marked morphological alterations in monolayer culture. Cytoplasmic processes were no longer present, and the cells assumed an epithelioid configuration. By analysis of the incorporat ion of tritiated thymidine, GFAP-negative U 251 MG cells demonstrated a two-fold increase in proEferation over controls. Finally, anti-sense GFAP-transfected U 251 MG cells grew into colonies which were larger and twice as numerous as controls. These data demonstrate that the elimination of GFAP expression by human astrocytoma cells has affected astrocytoma growth rate and further support the role of GFAP per se as an impor tan t as t rocyt ic cytoskeletal prote in capable of modulating the growth of astrocytoma cells.

ENHANCEMENT OF NITROSOUREA INDUCED CYTOTOXICITY IN NITROSOUREA-RESISTANT AND SENSITIVE CELLS UNDER OXIC AND HYPOXIC CONDITIONS. Sarkar, A., Marton, L.J. and Dean, D.F. Brain Tumor Research Center, University of California, San Francisco, CA 94143-0520.

Chlomethylnitrosoureas (CENUs) are used as chemotherapeutic agents for a variety of human tumors. CENU resistant cells cart be sensitized to 1,3-bis(2- chloroethyl)-l-nitmsonrea (BCNU) by pretreatment with c~- difluoromethylomithine (DFMO), an inhibitor of omithine decarboxylase (ODC). O6-alkylguanine-DNA alkyltransferase (AGT) is a major determinant of cellul% resistance to BCNU. Recently, it has been shown that O - benzylguanine (BG), an irreversible inhibitor of AGT sensitizes CENU-resistant cells to CENU. We have investigated the effects of 5 different CENUs on several rat and human brain tumor cell lines with varying degrees of resistance to CENUs under oxic and hypoxic conditions. Ceils were treated with graded concentrations of CENU for 1 h without breaking the hypoxia. Cell survival was assayed with a colony forming efficiency assay and in some cases DNA crosslinking was measured using the alkaline elutinn assay. Both CENU-resistant and sensitive cell lines show more cell killing under hypoxic than under oxic conditions. Our results indicate that enhanced CENU cytotoxicity may be partially related to reduced polyamine levels or ODC activity. We found that BG pretreatment sensitized BCNU- resistant cells more under hypoxic than oxic conditions. Moreover, combination treatment with DFMO and BG increased BCNU-induced cell killing more than did treatment with DFMO or BG plus BCNU. Our results suggest that a combination of DFMO and BG can be used to overcome a significant amount of CENU resistance in ceils. Supported by N1H grant CA-42779.

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A ~EW CHEMOTHERPY-BASED PROTOCOL FOR MANAGEMENT OF INTRACRANIAL GERM CELL TUMORS

Yutaka Sawamura,Jun Ikeda,Tsutomu Kato,Hiroshi Abe Dept. Neurosurgery, Hokkaido University, Sapporo, Japan

Seventy seven patients with intracranial germ cell tumors including 54 germinomas, 13 teratomas, 8 embryonal carcinomas and 2 endodermal sinus tumors have been treated in our institute. Our previous protocol mainly comprised 40 to 45 Gy irradiation to the whole ventricle field, and for recurrent cases chemotherapy using either PVB (CDDP/Vinblastin/Bleomycin) or EP (VP-16/CDDP) regimen. The overall survival rate was approximately 80%, however the radiation therapy induced remarkable mental and neuroendoerinological deficits, especially all patients with suprasellar germinomas had pituitary disfuction. Outcome of the patients with embryonal carcinoma or endodermal sinus tumor was extremely poor. PVB and EP therapy regimens were also insufficient to control recurrent lesions. Therefore, we have recently conducted a new chemotherapy-based treatment regimen as the followings. When a germ cell tumor is suspected, a radical surgery firstly is performed to obtain precise histological diagnosis and to reduce tumor bulk. The classification into good, intermediate and poor prognosis group is carried out owing to histological findings and tumor markers such as AFP and HCG. The good prognosis group includes solitary pure germinoma with negative tumor markers and mature teratoma. In this good prognosis group, EP therapy regimen is given 3 courses after surgery and 24 Gy local irradiation is added only for the pure germinoma. In the poor prognosis group including embryonal carcinoma, endodermal sinus tumor, immature teratoma, and AFP highly positive tumors, patient receives 4 to 6 courses PEI (CDDP/VP-16/Ifosfamide) therapy followed with 30 to 50 Gy of focused irradiation. This paper reviews 77 cases of germ cell tumors and presents preliminary results of our new protocol.

KINETICS OF EXTRACRANIAL METASTASIS OF THE GLIOBLASTOMA: R. G. Selker, F. T. Vertosick, M. T. Goldenberg West Penn Center for Neuro-Oncology, Pittsburgh, PA 15224

In a survey of over 1200 glioblastoma patients evaluated in the past 23 years, we have recognized 8 instances of extracranial metastasis to bone, skin lung, li.ver and spinal e• space (,75%). The literature: documents an occurrence rate of 3-40%. Craniotomy was not a prerequisite. Using the stand~ doubling theory of Aivord and others (1:0 x 10 x 10~ Ceil), and assuming clinical i,nt, racranial symptomatology occurs at 30 doublings (2.5 cm diamete:r-8 gms)and death at 37-39 doublings, extracranial metastasis must have occurred ~- Operatively~order to achieve clinical recognition (27,~30 doublings = 125 mg or i 6 cm ~) during the patient's lifetime f~50 Weeks), In most instances, therefore, three L(3): cyto- reductive,intracranial efforts wou]d have been required to avoid an intracranial death, allowing time ~or the metastasis to achieve clinical relevance; a most unusual occurrence for most~atients. As with other isolid tumors (colon, breast, lung) ceil dissemination occurred before surgical intervention and may become clinically relevant as survival times increase.

SURGICAL TREATMENT OF MULTIPLE BRAIN METASTASIS

R. Sawaya, M.D., R. Bindal, B.A., M. Leavens, M.D.

We conducted a retrospective chart review of 56 patients with multiple brain metastases who underwent craniotomy, of whom 30 bad one or more lesions left unremoved (Group A) and 26 had all lesions removed in a single operation (Group B). Additionally, 26 patients with a single metastasis who underwent craniotomy (Group C) were selected to match Group B with regard to the type of primary tumor, time from first diagnosis of cancer to diagnosis of brain metastases, and presence or absence of systemic cancer at the time of surgery. Median survival was 6 months for Group A, 15 months for Group B, and 14 months for Group C. There was a statistically significant difference in survival times between Groups A and B (p =0.003) and Groups A and C (p = 0.012) but not between Groups B and C (p>0.5).

Lesions recurred in the brain in 31% of patients in Group B and 36% of patients in Group C. There was no significant difference in the incidence of relapse (p>0.5). Immediate improvement of symptoms occurred in 65 % of patients in Group A, 75 % of patients in Group B, and 84% of Group C. Worsening of symptoms occurred in 13% of patients in Group A, 10% of Group B, and 4% of Group C. Groups A, B, and C had peri-craniotomy complication rates of 8%, 9%, and 8%, respectively and 30-day mortality rates of 3%, 4%, and 0%, respectively. We conclude that patients with multiple metastases undergoing surgical removal of all lesions have a prognosis similar to that of patients undergoing surgery for a single metastasis.

THE CLINICO-PATHOLOGICAL STAGING OF MEDULLOBLASTOMA Sevlever G.; Diez B.: Taratuto A.; Schultz M.; Picco P.; Monges J. Children's Hospital "Rica~lo Gutierrez" and FLENI- Buenos Aires, Argentina.

From 1975 to 1988, 123 cases with clinical follow-up, were chosen in order to identify prognostic factors. Features analyzed were histological subtype (desmoplasic or classical), necrosis, nuclear pleomorphism, mitosis, rosettes, calcium and immunoreactive cells for GFAP, NSE and NF. PCNA immunostaining was performed in formol-fixed paraffin embedded material (96 cases); results were expressed as percentage of positive cells. Clinico-therapeutic characteristics were: age, sex, radio and chemotherapy, tumoral size and dissemination according to T and M- stage of Chang system and extent of surgical resection. Overall 5-year survival was 46%. Sex, age and histologic features (except necrosis) did not affect prognosis. Better outcome was observed in patients with smaller tumors. Main prognostic factors were T-stage of Chang system and low values of PCNA-Li (labelling index). Patients with T2 stage and PCNA-Li less than 8.5% had a 60% survival rate at 60 months compared with 23% in cases with PCNA-Li higher than 8.5% and T3b Stage (p < 0.005). A "low dsk" group was defined combining PCNA- Li < than 8.5%, T2 stage and total resection with 5-year survival of 71% opposed to a "high risk" cohort with PCNA-Li higher than 8.5%, partial resection and T3b stage (p < 0.001). Nevertheless, clear-cut grouping is possible only in a minority of the whole series. A multidisciplinary approach to prognostic factors is needed to allow a better classification and perhaps a "biological" staging of these tumors.

COMPARATIVE ASSESSMENT OF PROLIFERATION RATE, DNA CONTENT AND HISTOPATHOLOGY IN MEDULLOBLASTOMA Sevlever G.; Martinez M; Pacheco G.; Gamboni M.; Schultz M; Taratuto A. Instituto de Investigaciones Neuroi6gicas "Ra01 Carrea"- Sanator[o Mater Dei. Buenos Aires, Argentina.

DNA analysis and cellular proliferation rate are becoming a part of the diagnostic and prognostic approach in tumor evaluation. Several reports point-out these techniques as predictors of biologic behavior with potential for guiding therapy. This ability is critical in medulloblastomas where patients are at high risk for neurologic and endocrinologic sequelae. DNA content, pioidy pattern and proliferative activity were studied in 54 medulioblastoma patients by means of a CAS-200 image analysis system and [mmunodetection of PCNA in formol-fixed paraffin-embedded material. PCNA labelling index (PCNA-Li) showed high values (mean !9.12%; S/D 10.2%; range 4.5-66%). Image analysis with Feulguen stain yielded 37 cases (68.51%) with a diploid pattern and 17 (31.48%) with aneuploid content. Proliferative fraction (S+G2+M) in diploid cases showed a good correlation with PCNA-Li. Aneuploid tumors demonstrated higher PCNA-Li (mean 23.17%) than diploid cases (mean 16.84%). Desmoplasic MB with "pale islands" showed lower PCNA-Li (mean 14.8%) than diffuse lesions (mean 21.7%). When analyzing jointly with tumor size (T-stage of Chang system), diploid tumors disclosed a trend toward smaller size lesions while aneuploid cases were more uniformly distributed. Patients with disseminated disease at diagnosis (M-stage of Chang classification) tend to have a diploid DNA content. We conclude that diploid tumors, as defined by image analysis and Feulgen stain, are more frequent than aneuploid, tend to be of low T- stage, have a high incidence of desmoplasic "pale islands" histologic subtype, disclose low PCNA-Li but show early dissemination (this shoud be confirmed in larger series). Alternatively, aneuploid lesions are a minority, are generally of diffuse histologic subtype, exhibit higher PCNA-Li and homogeneous tumoral size distribution. Multivariate statistical analysis is mandatory in order to assess independent prognostic value of these features, for the purpose of defining biological behavior.

06-alkylguanine-DNA alkyltransferase activity in brain tumors and adjacent normal brain. Silber JR, *Mueller BA, *Ewers TG, and Berger MS,

University of Washington Depts. of Neurological Surgery and *Epidemiology, Seattle WA 98195. We have assayed the activity of the DNA repair protein O6-alkylguanine DNA aikyltransferase (AGT) in 60 human brain tumors to assess the effects of tumorigenesis in brain on DNA repair capability. No detectable activity (<0.5 fmol/106cells, i.e. <300 molecules/106ceUs) was found in 25% of the tumors. Measurable AGT vaired by more than two orders of magnitude, from 0.5 to 104 fmol/106 cells. We also assayed AGT activity in overlying, histologically disease- free brain resected with 25 tumors. Of these, 52% had no detectable AGT activity and the remainder had activity ranging from 0.7 to 22 fmol/106 cells. For 15/25 paired samples, tumor activity was about 2 to 17 times greater than that of normal brain; for 4 pairs tumor activity was at: least 40% or less than that of normal brain; the remaining 6; had nondetectable activity for both tumor and normal tissue. These differences in the magnitudes and distributions of activities for tumor vs. normal brain tissue were significant (p=.02), demonstrating that tumorigenesis fin the brain is often accompanied by marked elevation of AGT.

APOPTOSIS AND BCL-2 EXPRESSION IN GLIOMA CELLS T. Shiraishi, K. Tabuchi, S. Nakagawa and S. Kihara Dept. of Neurosurgery, Saga Med. Sch. Japan

The cause of eukaryotic cell death can be divided into two categories, such as incidental cell death and programmed cell death (apoptosis). Since apoptotic cell dies without inflammatory reaction, induction of apoptosis seems to be useful for clinical treatment. Recently, bcl-2 protooncogene has been reported to suppress apoptosis. The purposes of this paper is (1) to evaluate the expression of Bcl-2 protein and (2) to induce apoptosis in glioma cells (T98G). Mitochondria and cytoplasm in perinuclear region were positively stained with anti-human Bcl-2 protein (DAKO) by cenfocal laser microscopy. Approximately 60% of T9 8G cells expressed Bcl-2 protein by flowcytometry. DNA of T98G cells treated with either anti-Fas antibody, ethanol or ACNU for 48 hrs showed the bands with multiple of 180-200 base pairs. Electron micrograph showed typical apoptotic changes, such as condensation of chmmatin, shrinkage of cell volume and apoptotic bodies. Our results showed that apoptosis could be induced in glioma cells and Bcl-2 might contribute to survival of glioma cells. We will discuss the significance of apoptosis in relation to the therapy of glioma.

MULTIPLE RESISTANCE MODULATORS COMBINED WITH CISPLATIN, MITOMYCIN-C, AND RADIATION FOR GLIOMAS. DJ Stewart, L Eapen, O Agboola, Ottawa Regional Cancer Centre, Ottawa, Canada, K1Y 4K7.

We are assessing the feasibility of adding 7 concurrent resistance modulators (Mods) to therapy of high grade gtiomas. We combined Mods with differing mechanisms of action or non-overlapping toxicity since: a) most cancers are resistant due to >1 mechanism; b) clinically achievable doses of many Mods are < those needed in vitro to $ resistance. Patients get 60 Gy radiation with chemotherapy courses 1 and 2, cisplatin 20-25 mg/m 2 iv days 0-4 q3wk (5 courses) & mitomycin 10 mg/m 2 iv day 0 (courses 1 and 3). Cohort (Coh) 1 (6 patients/Coh) got metronidazole (M) (to $ cell glutathione & 1" hypoxic cell kill) 1.5 glm 2 po q8h days -1 to 5 each course, M 0.5 g/m 2 pr q8h days -1 to 5, & pentoxifylline (to 1" drug/O 2 delivery & to $ repair of sublethal damage) 400 mg po tid continuously from day -3, course 1. M dose was $ by 50% due to excess CNS toxicity, & cisplatin was $ to 20 mg/m2/day. Coh 2 got revised Coh 1 drugs + dipyridamole (to 1" cell drug uptake) 100 mg/m ~ po q6h days -1 to 5. Coh 3 (current level) gets Coh 2 drugs + beta carotene 300 mg/day po from day -1, course 1 until tumor progression. Coh 4 will get Coh 3 drugs + novobiocin (inhibits topoisomerase II) 600 mg/m 2 po q12h days -1 to 7. Cohort 5 will get Coh 4 drugs + tamoxifen (inhibits protein kinase C) 100 mg/m 2 po daily days -1 to 5. Coh 6 will get Coh 5 drugs + coumadin (to $ DNA synthesis & inhibit DT-diaphorase) 5 mg po daily from day -1, course 1 to 4 weeks after last chemotherapy. To date, 13 patients have been entered. CNS toxicity was excessive early in Coh 1, but was tolerable once cisplatin & M doses were $. Nausea is moderately severe. It is too early to comment on efficacy.

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CELL MEMBRANES IN CISPLATIN-SENSITIVE VS RESISTANT GLIOMA CELLS. DJ Stewart, P Popovic, R Goel, P Raaphorst, PTT Wong. Ottawa Regional Cancer Centre & National Research Council, Ottawa, Canada.

Pressure tuning infrared spectroscopy (PTIS) (in which pressure distorts chemical bonds and --> 1" IS data) was done in cisplatin-sensitive U373MG human glioblastoma cells and a 2-fold resistant variant. In our prior studies, OV2008 human ovarian cells and EMT-6 subcutaneous murine mammary tumors differed most from their variants (resistant due to ,!. cisplatin uptake) in CH 2 symmetric stretching bands of methylene chains of membrane lipids: in sensitive cells, the disorder-order transition pressure increased by 1.5-2 kbars (ie, methylene chain conformational structure of membrane lipids is more disordered or fluid than in resistant cells). In model membranes, eisplatin diffused through phosphatidyl choline but not through more rigid phosphatidyl serine and phosphatidyl ethanolarnine bilayers. In U373MG glioblastoma cells, the resistant variant had a J, disorder- order transition pressure (by 0.25 kbars) in the CH 2 symmetric stretching bands (ie, more rigid cell membranes), and had a new peak at 1378 cm 1 (suggesting 1" cell membrane methyl branches and end methyl groups of acyl chains). The 1" membrane rigidity and 1 ̀membrane fatty acid branching could ---> $ drug diffusion through membrane lipid bilayers. Sensitive and resistant cell spectra were similar in regions of vibration modes from functional groups in nucleic acids and proteins. Conclusions: Cell membrane lipid changes could

$ transmembrane cisplatin diffusion, $ uptake, and 1" resistance.

VASCULAR ENDOTHELIAL GROWTH FACTOR(VEGF) EXPRESSED IN BRAIN TUMORS

K. Tabuchi, S. Shimokawa, M. 0h-uchida and K. Hori

Departments of Neurosurgery and Biochem- i s t r y , Saga Medical School, Saga, Japan

The growth of bra in tumor is c l o s e l y re- la ted to the tumor angiogenes is tha t is gene ra l l y thought to be induced by f ac to r s derived from the tumor c e l l s themselves. The VEGF(vascular endothe l ia l grwoth fac- tor) is 46kD pro te in and a l igand of the fms- l ike ty ros ine k i n a s e ( f l t ) . The VEGF is known as the spec i f i c mitogen for endo- t h e l i a l c e l l s , having a s t rong e f f e c t for tumor angiogenesJs . In order to know as to whether bra in tumor c e l l s produce VEGF, we examined the mRNA of human VEGF in 54 bra in tumor samples(49 su rg ica l specimens and 5 glioma ce l l l ines) by Northern blot ana ly s i s . Twelve out of 54 samples(22~) expressed VEGF mRNA. There observed 2 smal ler bands of mRNA(1.2 and 1.0kb) in 8 samples in add i t ion to the expected 3 l a r - ger bands (5 .5 ,4 .2 and 3. Tkb). These unique smal le r mRNAs have not been reported p rev i - ously. Our p resen t f ind ings sugges t tha t the smal ler mRNAs may be t r ansc r ibed by a d i f f e r e n t t r a n s c r i p t i o n a l system of the tumor c e l l s .

RELEVANCE OF CONTINUOUS CSF MONITORING IN PCL AND PNETS IN ADULTS

STRIK H, MOLLER B, MARKERT C, PFLUGHAUPT K-W, KRAUSENECK P

We report on 35 pts with primary cerebral lymphoma (PCL n=20) or PNET (infra- and supratentorial, n=15) with continuous CSF sampling during routine follow-up after surgery and radiotherapy. Mean follow-up was more than 2 years. In 10 PCL and 9 PNET pts. a CSF relapse with no or minimal symptoms could be diagnosed by this routine CSF examination and treatment could be resumed early. In 15 of these pts. again a complete remission was achieved mainly by combined i.th. and i.v. chemotherapy. 3 PCL pts. and 3 PNET pts. live in CR after a CSF relapse.

In contrast to other series in the literature a high rate of CSF dissemination was disclosed in these entities and could be effectively treated. This is demonstrated by unusual long survival time AFTER meningeal relapse (median 2 years) and many CRs after early treatment. In conclusion, a continuous CSF monitoring has to be recommended for these tumours.

PRODUCTION OF IL-1 RECEPTOR ANTAGONIST BY GLIOBLASTOMA CELLS IN VITRO AND IN V1VO-ITS REGULATION AND ANTAGONISM TO 111-1 AUTOCRINE LOOP Tada M, Diserens A-C, 1auferrally R, Desbaillets I, Hamou M-F, de Tribolet N. Department of Neurosurgery, CHUV, Lausanne, Switzerland

We investigated the expression and production of interleukin 1 receptor antagonist (IL-1RA) in 16 glioblastoma cell lines. RT-PCR and Northern blot analyses demonstrated strong constitutive expression of IL-1RA mRNA in 2 cell lines (LN443, LN444) and weak expression in other 14 cell lines. ELISA on culture supernatants and immunocytochemical stain showed IL-1RA production in the 2 cell lines.

Stimulation with IL-1, TNF and PMA, but not with LPS or GM-CSF, increased the mRNA expression and protein production of IL-1RA. Of the 16 cell lines assessed by RT-PCR, all the cell lines expressed type I IL-1 receptor. Among them, 11 cell lines presented IL- l(x, suggesting an autocrine loop through IL-lct and type I receptor. IL-1 type II receptor was expressed by 13 out of 16 cell lines. Among them, 7 cell lines expressed both IL-113 and IL-1[3 convertase, suggesting a possible but less predominant autocrine loop through IL-113 and type II receptor. In vivo study with RT-PCR and Northern blot analyses demonstrated IL-1RA mP, NA in 8 out of 19 glioblastoma or anaplastic astrocytoma tissues. These results, taken together, suggest an important role of IL-11LA in the regulation of the 1L-1 autocrine loop and modification of cytokine network in glioblastoma tissues.

Dominan t negative effect of novel form of the beta retinoic acid receptor generated by alternative sp l i c ing

Takeshima, H., Mochizuki, H., Clifford, J. L., Nishi T., Levin, V. A., Lotan, R. and Saya, H. Deparmaent of Neuro-Oncology and Department of Tumor Biology, The University of Texas, M. D. Anderson Cancer Center, Houston Texas 77030, USA

Nuclear retinoic acid (RA) receptors, which belong to the steroid hormone receptor superfamily, Include two subfamilies: the RARs (-o~, -13, and -y) and the RXRs (-cq -

13, and -y). These two types of retinoid-activated receptors form heterodimers that bind to DNA and modulate transcription for a large number of genes associated with the differentiated phenotype in a wide variety of cell types. In the present study, we identified a novel form of human RAR-13 transcript, which appears to be generated by

alternative splicing. This novel shorter transcript (RAR-~S) is missing one exon (205 base) in the retinoid-binding domain. The translation of the RAR-13S transcript is expected to be terminated prematurely leading to the synthesis of a protein that has lost its 200 C-terminal amino acids, thus truncating the ligand binding domain. The RAR- 13S transcript is expressed in a wide variety of cell lines including neuroblastomas and retinoblastomas. Transfection experiments revealed that the RAR-13S dominantly suppressed RA-induced transcriptional activation regulated by the normal form of RAR-13. We propose that the relative expression of endogenous RAR-bS and the full length normal form of RAR-13 may be an important factor in the regulation of RA-induced cellular responses.

P R O L I F E R A T I O N , M I G R A T I O N A N D I N V A S I O N OF H U M A N G L I O M A C E L L S EXPOSED TO ANTIFOLATE DRUGS A.J.A. Terzis 1, H. Arnold t, O.D. Laerum 2, and R.Bjerkvig 3- 1Department of Neurosurgery, University of Liibeck, Germany, 2The Gade Institute Dept. of Pathology and 3 Department of Anatomy, University of Bergen, Norway.

The present study describes the effects of two folate

antagonists, methotrexate (MTX) and the lipophilic

antifolate trimetrexate (TMX) on two permanent human

glioma cell lines (GaMg and D-54Mg) grown as

multicellular tumor spheroids. In addition, the effects of

drug exposure on tumor cell invasion was studied using a

three-dimensional organ co-culture system. When tumor

spheroids were exposed to MTX or TMX at concentrations

that caused 65-70% inhibition of ceU migration, there was a

latent period of 4 to 5 days before Inhibition of spheroid

growth ensued. Tumor cell invasion was studied in a co-

culture system, where tumor spheroids were confronted

with fetal rat brain cell aggregates. It is shown that both

drugs had little effect on tumor cell Invasion.

MEDULLOBLASTOMA -MORPHOLOGICAL VARIANTS AND CORRELATION WITH IMMUNOPHENOTYPE, KINETIC DATA AND DNA CONTENT IN A PEDIATRIC SERIES. Taratuto A, L,; Sevlever G.; Schultz M.; Diaz D.; Di Tella M.; Cuccia V,; Pomata H,; Monges J.; Gallo G., Hospital Nac de Pediatria "Juan P. Garrahan"-Inst de Invest Neurol "Raul Carrea"FLENI Buenos Aires - Argentina.

Seventy-two surgical specimens of medulloblastoma in infancy and childhood diagnosed between 1988 and 1993 in a pediatric hospital were studied in an attempt to correlate morphological variants with immunophenotype mainly expression of Synaptophysin (SYN) and Glial Fibdllary Acidic Protein (GFAP), kinetic data (PCNA immunostaining in alcohol -fixed, paraffin embedded) as well as DNA analysis with static cytometry (CAS-2O0) in some desmoplastic cases with "pale islands", Thirteen were considered morphologically mainly undifferentiated, 13 with neuroblasfic differentiation (Homer Wright rosettes), 12 desmoplastic diffuse with tumor cells aligned in Indian file and/or nodular with "dark nodes", and 34 desmoplastic with '*pale islands"; although larger samples showed some coexistence of morphological variants. SYN was expressed in 47 out of 55 cases immunostained; 4 out of 6 undifferentiated, 11 out of 13 with Homer Wright rosettes, 5 out of 9 desmoplastic diffuse and/or with "dark nodes" (within the nodes), and aU the 27 desmoplastic with "pale islands" cases immunostained (within the "pale islands"). Neuronal specific enolase as well as class [11 Beta tubuUn (TU J I) were co-expressed with SYN within "pale islands", Homer Wright rosettes, and neuroblastic areas (cytoplasm and short processes ) in 5 cases studied, further supporting the strong tendency towards neuronal differentiation. GFAP was expressed mainly in reactive astrocytes in 53 out of 60 cases immunostained; 8 out of 9 undifferentiated, 8 out of 12 with Homer Wright rosettes, 9 out of 10 desmoplastic diffuse and/or with "dark nodes", and 28 out of 29 with "pale islands". Glial "differentiation" was difficult to assess, although in 12 cases (1 with H W rosettes, 3 desmoplasfio diffuse with "dark nodes", 8 desmoplastic with "pale islands*') a few neoplastic cells showed GFAP positive cytoplasmic rim. Thus its incidence in prognosis is questionable. Mean PCNA-Li was 23.26 for 49 cases studied (range 4.1-51.3; S/D 11.31); undifferentiated tumors ( 5 cases) 24,76% (StD 8.14); with Homer Wright rosettes(10 cases) 25.48% (S/D 13.85), diffuse desmoplastic or with "dark nodes" (8 cases) 33.23% (S/D 5.4) and with "pale islands" (26 cases) 19.05% (S/D 9.74), the last group suggesting less active proliferation. SYN positivity in desmoplastic tumors within "dark nodes" as well as in "pale islands" supports that most of them have a tendency to neuroblastic differentiation. PCNA labelling in these tumors within as well as outside the "dark nodes" but mainly outside of the "pale islands" suggests the latter further differentiated from the "dark nodes", DNA analysis with static cytometry performed in 8 cases, disclosed 4 diploid and 4 aneuploid pattern. Proliferative fraction (S+G2+M); was always lower within "pale islands" than outside. Further correlation with longer follow-up, which at this stage is not possible, would disclosethe prognostic impact of each feature.

EFFECTS OF PHOTODYNAMIC THERAPY ON P R O L I F E R A T I O N , M I G R A T I O N AND INVASION OF G L I O M A SPHEROIDS A.J.A. Terzis 1, A. Dietze 1, U. Knopp 1, H. Arnold t, O.D. Laerum 2, and R.Bjerkvig 3. 1Department of Neurosurgery, University of L~ibeck, Germany, 2The Gade Institute Dept. of Pathology and 3 Department of Anatomy, University of Bergen, Norway.

The effects of PHOTOSAN/ARGON PUMPED DYE laser

on cell growth and directional migration in spheroid

cultures were investigated on 2 human glioblastoma cell

lines (GaMG, U-251MG). In addition, the effects on tumor

cell invasion in vitro was studied. Tumor spheroids from

both cell lines, when treated, showed a dose dependent

reduction of migration and proliferation. Histological

examination revealed a severe cell damage. Invasion was

investigated in a co-culture system where treated tumor

spheroids were confronted with fetal rat brain cell

aggregates. The toxic effects caused by the photodynamic

therapy did not seem to affect the invasiveness of the

tumors.

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DIFFUSION CHARACTERISTICS OF INTRALESIONAL ADMINISTERED 1 131 MONOCLONAL ANTIBODY IN PATIENTS WITH HIGH GRADE GLIOMAS.

R Thomas, M Brada, P Carnochan, G Flux, N Kitchen, D Thomas, M Zalutsky, D Bigner, Neurn-oncology Unit, Institute of Cancer Research & Royal Marsden Hospital. The National Hospital, Queen Square, London. Duke University, Durham.

A phase one study is currently underway in patients with recurrent high grade gliomas infusing 1 131 labelled antitenascin monoclonal antibody (mab) into the solid component of their tumour. An important early aim, is to establish the distribution of mab following injection. We report accurate post infusion diffusion results in a patients evaluated by two separate methods:-

1) Three-dimensional image integration of the post infusion Single Photon Emission Computerised Tomography ISPECT) with diagnostic computed tomography scans using a stereotactic head frame and Analyse computer programme.

2) At autopsy 9 weeks after infusion the whole brain was cut into I cm coronal slices and catheter track recorded in relation to tumour and normal tissue. The radioactivity in 1 cm cubes was measured by a scintillation counter and results transposed onto the coronal slices.

Both methods produced similar results with diffusion distances of 2-2.5cm in the three orthogonal planes giving a total volume of 8-15.6 cm 3. Furthermore, the good correlation between SPECT and autopsy adds confidence to the ability of SPECT to assess mab -131 I volume in future patients, where pathological mapping may not be available.

Radia t ion- induced expression of bFGF in rat astrocyte cul tures . Phi l ip Tof i lon and Paul Borchard t . U.T. M.D. Anderson Cancer Center, Houston, TX, U S A

Although the histological changes accompanying r a d i a t i o n - i n d u c e d CNS d a m a g e have been wel l documented , the under ly ing mechan i sms are poor ly understood. The prol i ferat ion and differentiat ion of glial and endothelial cells, suggested radiat ion targets wi th in the CNS, are dependent on g rowth fac tors p roduced by astrocytes. In other tissue, radiat ion has been shown to modulate the expression of genes coding for a var ie ty o f g rowth factors and cytokines . To determine whether changes in gene express ion may p lay a role in CNS injury after radiotherapy, we have begun to invest igate the effects o f X rays on gene expression in type I astrocyte cultures derived f rom the per inatal rat cerebral cortex. The expression o f the early response gene c-fos was evaluated as a function of dose and time after radiation. A significant increase in the levels o f c- fos m R N A was de tec ted af ter irradiation of astrocyte cuttures with doses as low as 1 Gy. c-fos expression reached a m a x i m u m at 1 h and returned to control levels by 2-4 h postirradiation. We then evaluated the expression o f bFGF, a putative AP-1 responsive gene. The expression o f bFGF was barely detectable in unirradiated cultures. However , after 1 G y b F G F m R N A levels i nc r ea sed s i g n i f i c a n t l y reaching a m a x i m u m at 8-24 h postirradiat ion. After i r radia t ion with 20 Gy, there was a s imi la r b F G F induction, but it reached a maximum at 4 h and returned to essent ial ly control levels by 24 h post i r radiat ion. B e c a u s e O - 2 A p r o g e n i t o r p r o l i f e r a t i o n a n d ang iogenes i s are in f luenced by bFGF, these da ta suggest that the induct ion o f bFGF in astrocytes m a y play a role in CNS radioresponse.

EPIDERMAL GROWTH FACTOR RECEPTOR EXPRESSION IN HUMAN GLIOMAS Sverre H. Torp I, Eirik Helseth 2, Are Dalen 3, and Geirmund Unsgaerd 2 1The Institute of Cancer Research, 2 Department of Neurosurgery, 3 Department of Microbiology, University Hospital of Trondheim, Norway

Expression of epidermal growth factor receptor (EGFR) was determined in frozen sections of 42 human gliomas using biotinylated epidermal growth factor (B-EGF) and anti-EGFR monoclonal antibodies (moabs). All gliomas were found to express EGFR assessed by positive B-EGF binding; the highest expression levels were found in glioblastomas. About 80% ofthe gliomas were shown to express EGFR with the moabs. Reactive astrocytes and diverse neurons were also EGFR positive while resting gliaI cells did not express detectable levels of EGFR. In conclusion, B-EGF is a more sensitive reagent than anti-EGFR moabs to detect EGFR in tissue sections. Further, EGFR is more frequently expressed in human gliomas of all grades than shown by other investigators. Also neurons and reactive astrocytes were EGFR positive, which may be taken into account in EGFR directed clinical trials of human gliomas.

CO-EXPRESSION OF LOW MOLECULAR WEIGHT NEUROFILAMENT PROTEIN AND GLIAL FIBRILLARY ACIDIC PROTEIN IN ESTABLISHED HUMAN GLIOMA CELL LINES

rakashi Tohyama 1,2, Osami Kubo 1 , Kintomo Takakura 1 , Virginia M.- Y. Lee 2 and John Q. Trojanowski 2 IDept. of Neurosurgery, Tokyo Women's Medical College, Tokyo, Japan 2Dept. of Pathol & Lab. Med. U. of Pennsylvania, Philadelphia, U.S.A.

This report describes the expression of glial and neuronal cytoskeletal proteins and their mRNAs in established cell lines derived from human primitive neuroectodermal tumors (PNETs) and malignant gliomas. Northern blot analyses revealed neurofilamant (NF) protein mRNAs in 6 of 7 PNET cell lines, but no glial fibrillary acidic protein (GFAP) mRNA. Six of these cell lines contained mRNA for the microtubnle- associated proteins (MAPs) known as MAPlb, while MAP2 mRNAs were detectexl only in 1 of the PNET cell lines. These findings closely paralleled previously published data on the expression of these cyloskeletal proteins in the same group of PNET cell lines. Although GFAP mRNA was detected only in 2 of 5 glioma cell lines, 4 of these cell lines contained mRNAs for the low molecular weight (Mr) NF protein (NF-L). Western blot analysis confn'med the expression of both GFAP and NF-L protein in 2 of the gtioma cell lines( i.e. U251 MG and U373 MG) that contained GFAP and NF-L mRNAs. Further, double immanofluoreseence studies showed that GFAP and NF-L co- localized in the same glioma cells. In contrast, neither the middle (NF- M) or high (NF-I-I) M r NF pmteins or their mRNAs were detected in any of these glioma cell lines. Finally, MAPlb mRNA was expressed in all 5 glioma cell lines while MAP2 mRNAs were detected only in 3 of the cell lines. This is the ftrst documentation of the expression of both glial specific and neuron specific cytoskeletal proteins in human malignant glioma-derived cell lines. These data may reflect the aberrant induction of neuron specific gene products in some neoplastic glial cell lines. Alternatively, our findings may indicate that some glioma cell lines correspond to transformed bipotential human CNS precursors of cells res~eted to a nettronal or glial lineage.

DIFFERENTIAL EFFECTS OF 12-O-TETRADECANOYL- 13-PHORBOL ACETATE {TPA) ON GLIOMA GROWTH, MIGRATION AND INVASION. Ole-Bjorn Tysnes, Svein Nygaard and Ole Didrik Laerum. The G a d e Ins t i tu te , D e p a r t m e n t of Pathology, Un ive r s i ty of Bergen, N-5021 Bergen, Norway.

To e v a l u a t e t h e e f fec t s of p r o t e i n k i n a s e C (PKC} a c t i v a t i o n on a g l i o m a cell l ine (GaMG), th i s s t u d y e x a m i n e d t h e e f fec t s of t h e p h o r b o l e s t e r 1 2 - 0 - t e t r a d e c a n o y l - 1 3 - p h o r b o l a c e t a t e (TPA} on t he t u m o r p rope r t i e s prol i fera t ion, m i g r a t i o n a n d invas ion . G a M G m o n o l a y e r g r o w t h w a s r e d u c e d b y 30% (p<0.05) in t he p r e s e n c e of TPA a n d c o n c o r n m i t a n t l y cells a c c u m u l a t e d in the G2M p h a s e of the cell cycle a s d e t e r m i n e d b y flow- cy tome t r i c ana lyses . In s t u d i e s of d i rec t iona l m ig ra t i on of cells f r o m G a M G sphe ro ids low c o n c e n t r a t i o n s of TPA i n c r e a s e d t h e a r e a of m ig ra t i on b y 60% (p<0.005). The effects a t h ighe r concen t r a t i ons of t he phorbo l e s t e r we re still p r e s e n t b u t less p r o n o u n c e d . G l ioma invas ion w a s a s s e s s e d w i t h a g l i oma s p h e r o i d / b r a i n a g g r e g a t e co- cu l tu re t ecn ique a n d r evea led TPA- induced e n h a n c e m e n t of the invas ive p r o c e s s (p<0.05} a t low c o n c e n t r a t i o n s of the phorbo l es ter . Down- regu la t i on of PKC b y p a s s a g i n g t h e cel ls in a TPA c o n t a i n i n g m e d i u m s ign i f i can t ly d e c r e a s e d t he m i g r a t o r y r e s p o n s e to TPA. Morever , the PKC i n a c t i v e p h o r b o l e s t e r 4 - a - p h o r b o l - 1 2 , 1 3 - d i d e c a n o a t e h a d no effect on t he m i g r a t o r y r e s p o n s e of GaMG cells. It is c o n c l u d e d t h a t metabo l ic p a t h w a y s u n d e r cont ro l of PKC m a y p l a y a n i m p o r t a n t role in t he cont ro l of g l ioma g rowth , m i g r a t i o n a n d i nvas ion . T h e ef fec ts o f PKC a c t i v a t i o n a r e p r o b a b l y d e p e n d e n t o n t h e g r o w t h c o n d i t i o n s . S t u d i e s of c u l t u r e c o n d i t i o n s in t h r e e - d i m e n s i o n a l s y s t e m s a s wel l a s in vivo m o d e l s a r e r e q u i r e d to f u r t h e r e v a l u a t e t he role of PKC in g l ioma bio logy.

NECROSIS AND POOR BLOOD PERFUSION BUT NO BINDING OF 123I-IAZA IN HUMAN GLIOBLASTOMAS. R. C. Urtasun, M. B. Parliament, A. J. McEwan, R. H. Mannan, L. I. Weibe, Cross Cancer Institute and University of Alberta, Edmonton, Canada.

Eight previously untreated patients with the diagnosis of glioblastoma were studied non-invasively prior to a surgery or radiation in order to assess: a) The tumor hypoxic fraction using 123I-iodoazomycin arabinoside (123I-IAZA). b) The tumor blood peffusion using 99mTe-hexamethylpropyleneamine oxime (99mTc-HMPAO). The microscopic specimens prior to the study revealed the presence of necrosis in all tumors. The tumors were left intact except for the diagnostic burr-hole needle biopsy. 99mTc-HMPAO and 123I-IAZA scans were performed within two days of each other. All imaging procedures were performed at 1, 16 and 24 hrs post injection using a gamma camera and SPECT images, coronal, axial and sagittal reconstructions. Although, there was evidence of necrosis in the biopsy specimens of all the tumors arid blood perfusion defects were uniformly seen in all the patients who had 99mTc- HMPAO, only 1/8 patients showed a marginal 123I-IAZA hypoxic fraction, which re-oxyganated after radiation treatment, suggesting that chronic biological hypoxia might not be the primary cause of radio resistance in these tumors.

1HNMR SPECTROSCOPY STUDY OF HUMAN BRAIN AND GLIOBLASTOMA EXTRACTS COMPARED TO PRIMARY MURINE BRAIN CELL CULTURES, UDP-N:ACETYLSUGARS AND HYPO TAURINE AS TUMOR MARKERS G . U N S G ~ -1 , U.SONNEWALD 2, I. GRIBBESTAD 2, E. ISERN 1 , S. B. PETERSEN 2, 1. UNIVERSITY OF TRONDHEIM, DEP. OF NEUROSURGERY; 2. MR-CENTER SINTEF-UN1MED, TRONDHEIM, NORWAY.

Using 1HNMR spectroscopy on tissue or cell extracts, detailed information can be obtained about small watersoluable molecules such as amino acids, sugars, glycolysis and Krebs cycle products. The special advantage of NMR spectroscopy lies in the in vivo applicability of this technique. Proton spectra of brain and neuronal cultures reveal the presence of N-acetyl aspartate and GABA exclusively in neurons whereas glutamine is produced in astrocytes. Recently interest has increased in the area of uridine diphospho (UDP) sugars, since elevated levels have been found in a number of tumors, including prostatic adenocarcinoma and breast tumor. IHNMR spectra revealed the presence of elevated levels of UDP-N- acetylglucoseamine and N-acetylgaiactoseamine in glioblastoma extracts and the glioblastoma cell line U87, in comparison with normal brain tissue and primary cell cultures of neurons and astrecytes. Hypo taurine has been identified in oligodendrocyte cultures and in cerebellar granule cells. In normal brain it was not observed by NMR spectroscopy, but was easily detectable in glioblastoma tissue extracts. NMR spectroscopy is a valuable technique to identify metabolites not known to be present in the tissue or organ of interest.

FACTORS INFLUENCING MEASUREMENT OF DNA DOUBLE STRAND BREAKS IN HUMAN BRAIN TUMOR SPECIMENS USING PULSED FIELD GEL ELECTROPHORESIS (PFGE). Wang, J, Delgado D.A., and Deen, D.F. Brain Tumor Research Center, University of California, San Francisco, CA 94143-0520.

The importance of DNA double strand breaks (DSB) in cell killing by ionizing radiation has long been recognized, and we are interested in measuring DSB as a possible predictor of the clinical outcome of radiation therapy. We are proposing a protocol that uses densitometry to quantitate ediidinm bromide stained DNA in gels following PFGE. "This is a very sensitive method and avoids prelabeling the cells with radioactive isotopes. Using human brain tumor cell lines and frozen human brain tumor tissues, we optimized variables that affect the measurement of DNA DSB, such as cell lysis conditions, pulse time, running buffer, film exposure time and DNA concentration in agarose plugs. We found that the measured DSB value depends on the DNA concentration in plugs. Therefore, we established a quick assay to determine DNA concentration in tumor samples using TO-PRO-l, a DNA-specific dye. Using optimal conditions, we measured radiation- induced DNA DSB in human brain tumor specimens (frozen) obtained from the BTRC Tissue Bank. The dose-response curves varied from tumor to minor, but we did not find a good correlation with the clinical response to radiation in these patients. This is probably because freezing destroys most if not all the repair ability in these cells, and it is the residual damage remaining after repair that should correlate with clinical outcome. Therefore, we need to measure this residual damage in fresh human brain tumor tissue. Experimental conditions will be optimized in xenografts and then be used to assay fresh human brain tumor tissues from patients. Supported by NIH grant CA-13525.

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ANALYSIS OF FIVE EPENDYMOMA CELL LINES USING FLUORESCENT IN SITU HYBRIDISATION Tracy J Warr 1 , Denise Sheer 2, Pat Gorman 2, and JL Darling 1 . Department of Neurological Surgery, Institute of Neurological Surgery1, Queen Square, London WC1N 3BG. ICRF 2, Lincoln's Inn Fields, London WC2A 3PX.

Although ependymomas constitute 5-10% of primary childhood brain tumours, karyotypic studies on these neoplasms are scarce, often due to the poor quality of metaphase preparations. Approximately 40% of the few tumours analysed have been found to be monosomic for chromosome 22 and structural aberrations of the long arm of 22, particularly in band ql 3, have been observed. We have used fluorescent in situ hybridisation (FISH) to analyse numerical and structural aberrations of chromosome 22 in low passage cultures of 5 ependymoma cell lines. Hybridisation of biotinylated centromeric probes to metaphase spreads and interphase nuclei revealed that all 5 celt lines had retained both copies of chromosome 22. Using a panel of cosmids from along the length of chromosome 22 we analysed the cell lines for deletions and translocations. Simultaneous hybridisation of the biotinylated centromere probe and a digoxygenin- labelled cosmid probe allowed differential two-colour detection of the probes with FITC and Texas Red. Using this technique we have detected a translocation at 22ql 3.3 in one cell line and characterised it within the band. This study illustrates the advantages of FISH in the identification of chromosome aberrations in brain tumours which may be undetectable using traditional cytogenetic methods.

DIFFERENTIAL EXPRESSION OF TWO FGF RECEPTOR G E N E S IS ASSOCIATED WITH MALIGNANT PROGRESSION IN HUMAN GLIOMAS. F. Yamagnehi, H. Saya, I. Brunet, M. Berger and R.S. Morrison. Dept. of Neurosurgery, M.D. Anderson Cancer Center, Houston, TX 77030.

Structurally and functionally diverse forms of the fibroblast growth factor receptor (FGFR) are generated by alternative splicing. Cell and tissue-specific expression of alternatively spliced FGF receptors may provide selective responses to different FGF family members during development and in some pathological processes. In the present study we evaluated FGFR expression in normal and neoplastic human brain. FGFR1 mRNA expression was abundant in normal cortex (grey matter), but was expressed at low to non- detectable levels in normal white matter. Conversely, white matter expressed abundant levels of FGFR2. Consistent with its expression in normal white matter, benign astrocyte tumors also expressed abundant levels of FGFR2. However, FGFR2 expression diminished in astrocytic tumors in relation to their loss of differentiation. In contrast, FGFR1 mRNA levels were significantly elevated in malignant glial tumors relative to normal white matter. Alternate use of an exon coding for an 89 residue amino terminal immunoglobulin-like (lg) disulfide loop produces isoforms of FGFR-1 containing three (FGFR-lc0 and two (FGFR- 11~) lg-like loops in the extracellular domain. Normal cortex expressed an alternatively spliced form of FGFR1 mRNA containing 3 lg domains whereas, malignant astrocytes primarily expressed a form of FGFR1 containing 2 lg domains. Intermediate grades of glial tumors shifted their expression from FGFR-la to FGFR-11~ as they progressed from a benign to a malignant phenotype. The results of the present study suggest that 1) glial cells may preferentially express FGFR2 while, FGFR1 expression is principally restricted to neurons. 2) FGFR2 expression may be required for astrocyte differentiation and 3) transformed astrocytes express an alternatively spliced form of FGFR1 that may provide them with an increased growth advantage.

SUPPRESSOR GENE STATUS IN HUMAN GLIOMAS IN ESTA- BLISHED CELL LINES AND IN PRIMARY SPECIMENS. M Westphal, L Anker, W Hamel, M LHcke, M She- pard(*), P Kleihues (+), HD Herrmann Laboratory for Brain Tumor Biology, Univ. Hospi- tal Eppendorf, Universit~ of Hamburg, Germany, (*) Canji, San Diego, (+) Neuropathology Z~rich

A panel of human cell lines derived from gliomas was investigated for abnormal expression and possible mutations in the p53 and the retinobla- stoma (Rb) genes. The highly conserved regions of the p53 gene were screened for mutations by SSCP and sequencing. Mutations were found in 6 of 17 cell lines. Additional evaluation of the original tumors from which the cell lines were derived revealed identical mutations in tumor specimens and corresponding cell lines in all but one case. Elevated p53 protein expression was not always associated with p53 gene muta- tions. Using the Rb specific antibody MAb-1, loss of Rb expression was found in immunoprecipitates of 8 of 24 glioma cell lines. Using direct detection with Western blot and a much more sensitive antibody, 3C8, Rb was undetectable in only 3 of these 8 lines and weakly expressed in 4 of the others. Rb loss was rare in primary tumors, but it is possible that not only loss of expression occurs but also reduction of expression which adds a quantitative perspective to the function of Rb. By Southern blotting no alterations of the Rb gene were found in the cell lines, p53 and Rb genes may both be altered in gliomas with p53 being affected more frequently. Supported by the Deutsche Krebshilfe W41/90/He-2

M O D U L A T I O N O F E G F R / T G F - a A U T O C R I N E G R O W T H R E G U L A T I O N IN HUMAN G L I O M A BY A N T I S E N S E A P P R O A C H E S - P O T E N T I A L A G E N T S F O R M O L E C U L A R T H E R A P Y . W.K. ALFRED YUNG, SCOTT TAYLOR, AND PETER A. STECK

Epidermal Growth Factor Receptor (EGFR) has been shown to be amplified and overexpressed in over 40% of glioblastoma. Altered EGFR protein has also been reported. TGF-a, a ligand to EGFR, has been shown to be over-expressed in malignant gliomas. Several antisense approaches have been utilized in our laboratory to modulate the growth regulatory activity of the EGFR/TGF-a autocrine loop. I) An antisense oligonucleotide (TGF-AC) was synthesized against the translation initiation site of TGF-a mRNA. Growth studies demonstrated a dose dependent growth inhibition of U251 cells by TGF-AC. More importantly, RT-PCR studies showed specific depletion of TGF-a mRNA in TGF-AC treated ceils, and most interestingly, EGFR mRNA was also noted to be decreased, clearly indicate the ligand/reccptor relationship of TGF-a/EGFR in U251 cells. II) An antisense expression vector (pTGF-AS) was construced to include the TGF-a coding sequence in an antisense orientation under the control of a inducible MMTV promoter. Several clones, in preliminary screening, showed inhibition of growth when induced with dexamethasone and failed to form tumors in nude mice. III) A specific triple-helix forming oligonucleotides (Triplex) was designed against the promotor of the EGF-R gene (EG4-ap). Growth studies with U251 and LG glioma ceils showed dose dependent growth inhibition with the ED50 at 5 mM concentration. Colony forming assay also demonstrated decrease in number and size of colonies by treated ceils. RT-PCR analysis for EGFR mRNA showed specific inhibition of EGFR transcription by EG4-ap. Most excitingly, receptor binding analysis clearly showed depletion of receptor expression on the ceil surface, suggesting that EG4-ap triplex was capable of inhibiting EGFR mRNA transcription, which in tom lead to decrease in receptor protein expression and growth inhibition. These studies illustrate several potentially exciting molecular agents/approaches that can be applied in the clinic for the treatment of m a n a n a n t l ~ r ~ n t n m ~ r ~ . . . . . o . . . . . . . . . . . . . . . . . . . .