Journal of Neuro-Oncology 24: 125-140, 1995. 9 1995 Kluwer Academic Publishers. Printed in the Netherlands. PILOCYTIC ASTROCYTOHA PRESENTING AS HULTIFOCAL HENINGEAL GLIOHATOSIS. CASE REPORT. S. Rosemberg, M.J. Teixeira, V.A. F.' Alves Dept. of Pathology (Neuropathology Division), University of Sao Paulo School of Medicine. A 3l-year-o]d previous healthy white man presented 40 and 30 days before hospitalization two episodes of dysphasia, paresis and parestesisof the right limbs, followed by progressive general weakness, and signs of intracranial hypertension. At admission, the neurological exam showed brisk tendon reflexes in the lower limbs, a right ankle c)onus, and mild hypoaIgesia below the level of C4. The MRI showed a meningeal focal thickning (lOmm) beneath both frontal poles and at the ventral aspect of the bra;n-stem. A similar lesion was present at the anterior aspects of the 3rd and 4th cervical segments of the spinal cord,whereas the whole dorsal cord was cuffed by the abnormal tissue. The was no intra-cerebra] or in intra-spinal masses. A biopsy of the frontal lesion revealed a pilocytic astrocytoma. A few days after the procedure, the patient became paraplegic and died from respiratory insufficiency. Autopsy was not performed. As far as we know, pilocytic astrocytoma with a such presentation, which can be considered as a m u l t i f o c a I meningeal gliomatosis, has not been reported in the

literature.

Clinlenpathologic Features of Primary and Post-Radiatlon Cerebra] Gliosarenma. Perry JR, Ang LC, Bilbao IM, Muller PJ. Divisions of Neurology and Pathology Sunnybrook Health Sciences Centre, and Pathology and Neurosurgery St. Michael's Hospital, Toronto, Ontario, Canada. 32 patients (pts) with histologically confirmed cerebral gliosatooma (GS) were reviewed. 25 were found at the dlne of first iee~cfion while in 7 resection of recurrent turnout originally diagnosed as glioblastoma multiforme (GBM) and treated with radiotherapy (XRT) revealed GS. Histological features included a dimorphic malignant cell population with mixed gfiomatuns (GFAP +re) and sarc~natons (spindle cell GFAP -re with reticulin and vimentin +ve) elements in all cases. Mean age of the primary and post-XRT GS pts was 65.6 (43-85) and 49.6 (3736) years respectively. Mean Karnofsk'y performance status (KPS) (76.8, 81.4), presenting features (language deficit > seizur~ > hemiparesis), sex distribution, and tumour location was similar for both groups without evidence of site predilection. For prinmy tumours initial treatment was gross total resection in 20 %, paxtial in 76 %, and biopsy with cyst aspiration in 4 ~. 12/25 had XRT (5,000 CGy/25) while 12% received chemotherapy. Overall median survival was 25 0-66) weeks with pts receiving XRT (46 weeks, mean KPS 78.3) surviving longer than those t~eated conservatively (13 weeks, mean KPS 75.3). Pts with GS diagnosedat recurrence (postXP,T) originally bad a diagnosis of GBM in 6, and grade 3 astrocytoma in one. Mean time to local recurrence was 38 weeks. One pt had a firm turnout with dural attachment whereas all others had soft intra-axial lesions. Therapy at recurrence included oral CCNU in 3 pts and intra-cavitary BCNU and PDT in one each. Median survival was 53 weeks and similar to primary GS pts treated with XRT. Younger mean age may have also conferred a sm'vival advantage for the GS group. Smooth muscle acdn immunoreactivity was seen in all tumour vessels and extended tu the sarcomatous cells in 60% of the primary turnouts while others revealed features of malignant fibrous histiocytoma, fibrosarcoma, and osteesarr Most postXRT cases revealed the laRer sarcomatons features. We confirm, in a large series, that clinical behavinur end survival nharacterisdcs of GS are similar to GBM. Pts with GS may benefit from post-opsrative X'RT although selection bias favouring XRT may have been present in our series. GS appears not to be a single entity and may arise from at least two pathways including vascular proliferation within GBM and from a muldpotential progenitor cell. These mechanisms may be influeeced by XRT, although we cannot rule out sampling error as the cause of the original diagnosis of GBM in some of our cases.

Low Grade Glioma of Childhood with Ostanblastic Metastases to Pelvic Bones: Report of Two Kyung-Whan Min, M.D., Department of Pathology University of Oklahoma Health Seiences Center, Oklahoma City, OK Extraeranial metastases of giiomns are relatively rare and occur most frequently in high grade gliomas with or without surgical intervention. Childhood astrosytomas are commonly of low grade and manifest benign growth characteristics and clinical behavior in contrast to adult examples. In spite of their benign growth characteristics a small percentage of childhood low grade giiomas may present with disseminated metastasis. We report two children with low grade astrocytoma presenting with biopsy proven disseminated osteoblastic metastases to lower vertebrae and pelvic bones. The first ease was a 9 year old girl diagnosed with a low grade giioma of bralnst~m which was irradiated following a partial resection. One year later, she developed multiple osteoblastic metastasis to the lumbar vermbrae and pelvic bones. The second ca~ was a 9 year old girl who was diagnosed as a low grade giioma of the bralnstem end almost simultanenuc multiple osteoblastic lesinns in lower vertebrae and pelvic bones identical to the first case. In both, biopsies of the lilac crest revealed that the marrow spaces are filled with spindle coils arranged in bundles. There was a slight variation of the size and shape of nuclei. The microscopic appearance simulated that of a neurilemmoma. "me tumor cells in cases were strongly positive for gtial fibrillasy acidic protein and S-100 promin and an electron microscopy done in the first case revealed intracytoplesmic bundles of intermediate filamemts consistent with gila1 fibrils. The immunohistuebemical and ultmstmctural features of the bone tumors were consistent with metastatic astrooytomes. The cases reported herein document metastatic potential of low grade gliomes end indicate an existence of a subset of low grade gliomas which possess a unique invesive property end metastatic capability. The paRem end distribution of matastases in both cases support the claim that shedding of tumor cells into the cerebrospinal fluid, resulting in seeding of tumor cells in the lower spinal cohinm With subsequent transvenous dissemination, may be one of the mechanisms of metastasis in low grade giiomas.

Glinneurocytomns: A Composite Tmaor of the Brain. Kyung-When Min, M.D., Robert Cashman, M.D., Roger A. Brumback, M.D. Department of Pathology, University of Oklahoma Health Sciences Center, Oklahoma City, OK We present two cases of low grade giinma in which multiple cellular components including cells with dense core granules consistent with "ucurocytes" were identified on electron microscopic studies. The first case was a 13 year old bey with a left paraeaggital parieto-uccipital mass which presented with a four month hism~ of seizures end declining school performance. The tumor was composed of sheets of estrocytes with dark, hypeschromatie, ploomorphie nuclei in a fibrillary end microcystic background. The tumor contained the pleomosphismseen in the edult varient of pilocytic astrs~ytoma as well as the microcystic component seen in the juvenile variety. The second case was an apparently normal boy until the onset of seizures at age 1O months. Initially, seizaav.~improved on phenobarbital treatment, but good seizure control was never achieved. CT scan at age 23 months showed a calcified non-enhancing 5 x 4 cm left parietal mass. This tumor was composed of sheets of cells with clear cytoplasm end round to oval nuclei. Mucinous intercellular material stained positively with PAS, mucicarmine, end alcien blue. Foci of calcification were evident. Ultsnstrocturally in both cases, there were occasional tumor cells having round to oval nuclei with moderate amounts of cytoplasm containing 150 250 nm diameter dense core grenules. These cells were admixed with the majority of tumor cells which in case 1 had the ultrastructurni features of protoplasmic or piincytic astrocytes and in case 2 had features of astrocytes end oligodendrucytes. The tumors presented herein bear superficial resemblance to dysembryoplastic neureepithelial tumors (DNT). However, dysplastic ganglion cells in DNT are an essential component for the diagnosis, and there was no evidence of dysplastic ganglion cells in either of our cases. Cells containing degse core granules (nearocytes) were found unexpectedly in both cases by electron microscopy end appear to be a part of neoplastic process. The significanceof neurocytes in low grade gliomas is not known. Cerebral tumors consisting of neurohasts/nenrocytes in tote or in part are being increasingly recognized in recent years and our cases add to the spectrum of such neoplasms.

126 Cerebral glioneurocytoma : a variant of central neurocytoma ? M. M. Ruehoux, K. Medard, J. Hassoun, Lille and Marseille, France ; Abidjan, CSte-d'Ivoire. The patient, a 23 year old man, was hospitalized for one month history, of headaches, vomiting, blurred vision and right hemiparesia. CT scan revea!ed a huge, heterogenous and calcified tumor of the left frontal lobe, slightly enhanced by contrast. At surgery, the tumor, 6 cm in diameter, ill-circumscribed, lied in the brain parenchyma and was totally removed. Histologically, it was composed of densely packed small round cells with regular, non mitotic nuclei. The tumor cells were dispersed within a fibrillary stroma. No typical or large rosettes, no more than honeycomb (oligodentrogliaMike) pattern were found. Calcifications with occasional bone metaplasia were numerous. Immunohistochemical investigations showed synaptophysin positivity of the fibrillary stroma and less often of cytoplasmas and GFAP positivity of tumor cells and processes. Neurofilaments were scanty within the tumor. A few cells were Leu 7 (HNK) and-NSE positive. No tumor recurrence has been noted eight months after surgery. Several clinical (the age of the patient), histologicai (non mitotic small round cell tumor with a fibrillary stroma) and immunohistochemical (synaptophysin immunoreactivity) features linked this tumor to central neurocytoma (Hassoun et al., Acta Neuropathol 1982, 56 : 151 ; Brain Pathol 1993, 3 : 297). However the extraventricular location and the GFAP immunoreactivity of the tumor constituted atypical features. This tumor could be compared to some rare cases previously reported of frontal (Harada et al. No Shinkei Geka 1991, 19 : 89 ; Nishio et al., Cancer 1992, 70 : 529) or spinal (Louis et ai., J Neuroncol 1990,9 : 231 ; Cocca et al., Acta Neuropathol 1994, 87 : 537) neurocytomas and of related tumors coexpressing neuronal and glial markers ( Von Deimling et al., Acta Neuropathol 1990,79 : 473 ; Lab Invest 1991, 64 : 585). The concept of benign small neuronal cell tumor should be probably widened and include ubiquitary central nervous system tumors involved into a preferential but not exclusive neuronal differentiation process.

Cooocurrence of Ganglioglioma and Pleomorphie Xanthoastroeytoma in the Temporal Lobe. C. Rao, A. Abdu, D, Deloso, M. Wrzolek and R. Swanson. Dr. Joanna Sher Neuropathology Laboratory, Departments of Pathology and Neurosurgery KCHC-SUNY-HSC at Brooklyn, NY. Gangliogliomas and pleomorphic xanthoastroeytomas (PXA) share several common features, notably rarity, age, site of occurrence and good prognosis. Temporal lobe is the most common location for both the tumors. Cooecurrence of ganglioglloma and PXA is a rare event and only two cases have been reported, one in the temporal lobe (Furuta et al '92) and one in the cerebellum (Lindboe, Cappelen, Kepes '92). We report here a second case of cooceurrence of ganglioglloma and PXA in the temporal lobe of a 19 yea r old black male who presented with a new onset seizure. CT scan of the brain revealed a 2x2 em left temporo-parietal cystic mass with mural nodule and minimal surrounding edema and~hyperdense areas suggestive of calcifications. Submitted surgical specimen revealed distinct areas of both types of neoplasms. Ganglioglioma component revealed many neuronal cells, neoplastic astrocytes, inflammatory cells and calcifications. A few binucleated neurons were seen. Neurons and astrocytes were respectively positive for synaptophysin and glial fibrillary acidic protein (GFAP). PXA component which was contiguous but distinct revealed pleomorphic cells, vacuolated cells with foamy cytoplasm which were GFAP positive. Reticulin was abundant in P X A p o r t i o n of the tumor.

HYPOTHALAMIC GANGLIOGLIOMAWITH MULTIPLE CONGENITAL ANOMALIES SUGGESTIVE OF PALLISTER-HALL SYNDROME. C. Rao, D. Deloso, V. Anderson, A. Seymour and M. Wrzolek. Dr. Joanna Sher Neuropathology Laboratory, Department of Pathology,Kings County Hospital Center and State University of New York Health Science Center at Brooklyn, New York. Pallister-Hall Syndrome (congenital hypothalamie hamartoblastoma syndrome) is a rare malformation characterized by polydactyly, nail dysplasia, skeletal dysplasia, flat nasal bridge, low set ears, renal, cardiac and pulmonary anomalies, imperforate anus, undescended or hypoplastic testes, micropenis and a hypothalamic hamartoblastoma. It has been suggested that inclusion of diencephalic lesions other than hamartoblastomas associated with some of the above described non central nervous system malformations would be an appropriate extension of the definition of the syndrome. We report here an autopsy study of such a case of a 5 month old baby boy with flat nasal bridge, low set ears, bilateral inguinal hernias, undescended testes, multiple rib and vertebral anomalies, cardiac and renal anomalies in association with a large suprasellar hypothalamic ganglioglioma. Histologically, inspite of predominance of a hamartomatous appearance, the diagnosis of ganglioglioma was favored because of definite neoplastic astrocytic component and the growth pattern showing aneasement of the brainstem and encirclement of the vessels of Circle of Willis and posterior pituitary.

Immunocytochemicaland cell proliferation studies in dysembryoplastie neuroepithelial tumours M. Honavar, K.B. Waters and S.M. Wise Department of Neuropathology, Institute of Psychiatry, London SE5 8AF, UK Dysembryoplastic neuroepithelial tumour (DNT) is a recently recognised morphologically heterogeneous intrinsic neoplasm seen in young patients with chronic epilepsy. Their indolent behaviour and certain morphological features have often placed these into the category of hamartoma.. To characterise DNT further and to investigate its neoplastic nature, immunocytochemical and cell proliferation studies were carried out. Glial fibfillary acidic protein (GFAP), was seen in the astrocytic component. Synaptophysin was seen in the oligodendroglia-like cells in some of the cases and in the pefikarya of mature neurones within some of the tumours. Rare carbonic anhydrase II staining was seen. Neurone specific enolase and neurofilament marker RT97 were not expressed in any of the tumours. In many, much of the tumour failed to express any markers other than GFAP. Proliferating cell nuclear antigen (PCNA) expression was studied and the labelling index (LI) was calculated .21 DNT had no PCNA expression. In 11 the LI was less than 1%, in another 6 between 1-4.9%, in 13 between 5-20% and in 6 the LI was in the range of that of high-grade astrocytomas, 20.545.5%. The LI did not correlate with any clinical or morphological parameters. The focal high labellit~g supports the concept that DNT are neoplasms.

127 C l i n i o o p a t h o l o g i e a l Study of O l i g o d e n d r o g l i o m a - l i k e Tumors in the C e r e b r u n T. Kubota, K. Sato, M. Kabuto, T. Nakagawa, R. Kitai, H. Nitta*, J. Yamashita* Department of Neurosurgery, F u k u i M e d i c a l School, Fukui, Japan *Department of Neurosurgeryl K a n a z a w a U n i v e r s i t y School of Medicine, Kanazawa, Japan. The identification of oligodendroglioma has not generally been established. Ten adult cerebral tumors largely exhibiting a honeycomb appearance were i m m u n o h i s t o c h e m i c a l l y and u l t r a s t r u c t u r a l l y studied. Their clinical features were also evaluated. Tumors diagnosed as o l i g o a s t r o c y t o m a w e r e excluded. The distributions of glial fibrillary acidic protein (GFAP), p h o s p h o r y l a t i o n - d e p e n d e n t (P-D) n e u r o f i l a ~ e n t protein (NFP, 200K), p h o s p h o r y ! a t i o n - i n d e p e n d e n t (P-I) NFPs (68 K, 160 K, 200 K), ~ - t u b u l i n (B-T), m y e l i n basic protein (MBP) and s y n a p t o p h y s i n (SNY) were examined. Numerous G F A P p o s i t i v e t u m o r cells w e r e seen in six. Their u l t r a s t r u c t u r a l features e x h i b i t e d astrocytic nature w i t h abundant i n t e r m e d i a t e filaments on which GFAP were usually stained immunoelectronmicroscopically. A f t e r the tumor removal and chemoradiotherapy, all tumors r e c u r r e d and five cases died following one to eight years. O t h e r four cases had abundant NFPs and o c c a s i o n a l GFAP p o s i t i v e tumor cells. P-I 68K and 200K NFPs w e r e u s u a l l y stained. SNY and ~-T were focally stained. GFAP positive cells were seen at times. U l t r a ~ t r u c t u r a l l y , some tumor cells revealed neuronal differentiation, others showed glial nature. After the tumor extirpation and chemoradiotherapy, three recurred following two, five and ten years, respectively. One could not be followed. In conclusion, ten cases largely showing a honeycomb a p p e a r a n c e are d i a g n o s e d that six are clear cell a s t r o c y t o m a and four are m i x e d neuronal - glial tumor.

Four cases of psam~omatous melanotic schwannoma (PMS) and the Carney syndrome. C. Vital*, J. Rivel*, F. Sangalli*, B. Benjelloun*, A. Vital*, F. Leger*, V. Riemens*, B. Epardeau**, J. Guerin*, J.M. Coindre*. *Bordeaux II University, **H6pital Foch Suresnes (France). There were 3 males, 39, 45 and 36 years old, and a 30-year-old female. Two tumors were located in a spinal nerve root, and 2 in the region of the trigeminal ganglion. Each tumor was blue to black at gross examination, and exhibited mainly sheets of spindle cells and sometimes epithelioid cells. Large nuclei with prominent nucleoli were present in the last case. Each specime~ contained brown pigment and cytoplasms were always marked by SlO0 protein and HMB45, and were negative for GFAP and cytokeratin. In addition, each tumor contained several psammomatous bodies, and adipose tissue was present in one specimen. Ultrastructural examination was performed in one case and evidenced premelanosomes and melanosomes in various stages of maturation. The last patient had peri-umbilical spotty pigmentation, which was also present in 6 siblings and their mother. All four cases fulfilled the criteria for PMS, and 3 of them had no other patent component of the Carney syndrome. Two PMS were located in the region of the trigeminal ganglion which is a rather rare location for such a tumor.

Xanthoependym0ma, a new entity ! MM.Ruchoux ~ P.DheUemmes*, M.Hamon#, M.Lecomte ~ J.Hassoun+ ~ Neuropathology Department LILLE, FRANCE * Neurosurgery Department, LILLE, FRANCE # Neuroradiology Department, LILLE, FRANCE + Pathology Department, MARSEILLE, FRANCE The patient, a 13 years old boy, was admitted to our hospital with generalized seizure, without noticeable history. Computed tomography disclosed a left parieto-temporal space occuping lesion. MRI showed an heterogeneous mass with hyperintensity on T1 weighted scan, and hypointensity on T2 weighted scan evocating lipid contents. Pathological examination found a pleomorphic ependymoma with compact, cystic and papillary areas and confirmed the presence of scattered or gathered large cells with heavily lipidized cytoplasm. Often, the lipid droplet was sole and occupied much of the cell-s body. Immunoperoxidase stain for glial fibrillary protein showed strong ring shaped staining for the remaining cytoplasm of tumour cells around the area occupied by lipid droplet. The ultrastructural study objectived large lipid droplets and cells filled with small lipid droplets. As far as now, no description of lipid contents in ependymoma have been reported. The evidence of lipid in cells with ependymoma phenotype support the concept that ependymocytes are also capable of lipid storage.

A comparative immunohistochemical and ultrastructural study of neuroblastoma and primitive neuroectodermal tumor Saburo Yagishita, Nobuyuki Kawano, Tom Kameya Department of Pathology Kanagawa Rehabilitation Center and Department of Neurosurgery and Pathology, Kitazato University, Kanagawa, 243-01, Japan "Primitive neuroectodermal tumor (PNET) " including neuroblastoma (NB) of the brain has been recognized as a distinct ciinico-pathological entity. Because of the primitive nature of the tumor, however, its differentiating capacity are still of dispute. We studied to 2 PNET, 1 cerebral (primary and recurrent) and 4 cerebellar NB, 2 neurocytomas, two immature teratomas and a set of developing brains of rat (gestational days of 10 to t8), both by electron microscopy and immunohistochemistry. Many glial tumors, benign and malignant, meningiomas, and schwannomas were sewed as controls. The antibodies applied are beta tubulin 1+ II, Ill and TuJ1, PGP 9.5, vimentin, cytokeratin, synaptophysin (SYN 38), neurofilaments (70 kd, 160 Kd, and 200 Kd), gila] fibrous acidic protein (GFAP) and chromogranin A. All neurocytomas, primary cerebral NB and two mature NB were positive for SYN 38 and TuJ1. While, recurrent cerebral NB was positive for vimentin and TuJ1, and negative for SYN 38. Two PNET showed vimentin-positivity and TuJl-negativity. Some ceils comprised in primitive neural tube were vimentin-positive and outer ceil layer (neuroblastic differentiation) TuJl-pesitive. Electron microscopically, PNET were composed of immature cells with only few cytoplasmic processes between the tumor cells and intercellular spaces were very scanty. Recurrent NB had many slender processes reminiscent of developing axons. As a result, immature neuronal ceils first became positive to vimentin and later TuJ1- positive when the primitive neuronal ceils start to form slender processses from the cytoplasm. Tubulin III, which is considered to be specific to neuronal cells, was immunoreactive to various tumor cells as did some other antibodies. The reason of one TuJl-positive glioblastoma remains to be settled.

128 Static Image Analysis of Central Nervous System (CNS) and Peripheral Nervous System (PNS) Primitive Neuroectodermal tumors (PNET) R Bowman, BH Liwnicz, N Peckham, Loma Linch University Medical Center, Loma Linch, California U.S.A. Twenty-one cases of 5am cut paraffm-embedded PNET's were stained by immuno-peroxidase methods for GFAP, neurofdament, synaptophysin, Ki-67, p53 and S100 with appropriate controls for each. Cases were analyzed for antibody content using static image cytometry on the CAS200. Results were compared to the location of the tumor, either CNS (n=10) or PNS (n=l 1). The strongest correlation, regardless of site, was a negative linear correlation between Ki-67 and GFAP (-.45, p=.04) indicating that as these tumors increase in proliferation their glial differentiation diminishes. Group means by site indicate that tumors located in the CNS had an increased amount of GFAP, p53, and S 100 while containing decreased amounts of synaptophysin. All tumors, regardless of site, tended to have high proliferation (mean 27%, range 8.7-54%) and were essentially negative for neurofilament. Using multiple regression, two significant variables p53, and GFAP (p<.05), there was a moderately-strong linear correlation in determining the site, CNS (r2=.43, p<.005). Using discriminant analysis and two significant variables, GFAP and p53 (p<.05) the site, either PNS or CNS, could be determined in 17/21 (81%)of the cases. This limited study suggests that the use of immunohistochemical staining analyzed by static image cytometry can separate tumors into CNS and PNS.

Cerebral biopsies of multiple sclerosis (MS) lesions m m i c k i n g a brain tumor: a report of 3 cases. A. Vital*, C. Vital*, H. Loiseau*, L. Mouton*, M.B. Delisle**, C. Rumlens*. *Bordeaux II University, **Toulouse University (France). Three patients underwent a cerebral biopsy because of clinical and radiological features initially suggesting a brain tumor. In all cases, the lesion contained large pleomorphic reactive astrocytes often with bizarre nuclei, but most of these astrooytes were well separated from each other and presented well-developed processes. Mitotic figures, endothelial hyperplasia and necrosis were absent. In one case, the cellularity was high, but atypical astrocytes were intermingled with foamy macrophages. Aggregated perivascular foamy macrophages were observed in the 3 cases, and mild perivascular lymphocytic infiltrates were found in one. L oyez and Bodian stains showed, respectively, myelin loss and relative preservation of axons in all 3 cases. The main difficulty in these patients was to eliminate a diagnosis of grade II gemistocytic astrocytoma. The most helpful features were the well separated astrocytes more suggestive of a reactive gliosis, the presence of foamy macrophages and the relative preservation of axons. The diagnosis of MS was confirmed by the subsequent development of remitting and relapsing symptoms in all cases.

(This is an abstract of up-to-date results. The poster will present a larger number of samples, which can change the statistical values).

Haemangiopericytic type of Meningeoma: does it exist? L.M. Barbosa-Coutinho, L.M. and A. Hilbig Fundacao Faculchde Federal de Ciencias Medicas de Porto Alegre, Department of Pathology, Porto Alegre, Brazil The haemanginpericytic type of meningeoma was recently excluded of the WHO classification (1990), because their similar behaviour to haemangio- pericytoma originating in other parts of the body. But Rubinstein (1988) already had inquestionable examples of mixed or transitional forms which link both haemangioblastic to the haemangiopericytic variants and each of these to the meningotheliomatous and fibroblastic types of meningeoma. The authors report a ease of a 47 year old white man with a right parietal mass, that had an heterogeneous hyperdensity at the computed tomography. The tumor was excised and the histopathological examination showed a highly cellular tumor, with compact arrangement of polygonal ceils, which separate the thin-walled vascular spaces, showing frequent mitotic figures. There are several necrotic areas in the tumor. Some of the polygonal cells are arranged in sheets and form whorls, where the transition area between the meningotheliomatous and haemangioporicytic type of meningeema is seen. This report, in addition to the others found in the literature, confirmed the existence of such transitional and mixed forms of meningeoma, linking both haemangiopericytic to other types of meningeema. This confirms the theory of the polyblastic potential of the arachnoid cell, with divergent differentiation into pericytes.

Coreprotein of the psammoma bodies in meningioma. F. Akai, M. Taneda, H. lwasaki and Y. Suzuki, Kinki University School of Medicine, Osaka, Japan Osteopontin(Opn) is a phosphorylated glycopmtein, which promotes bone formation and mediates bone destruction. We demonstrated complete identity between urinary stone protein and human Opn. The fine structure of psammoma bodies in meningiomas has been described. The initial loci of the calcification is the matrix vesicles and remnants of degenerated cells. The organic matrix of the calcification is poorly understood, especially in its chemical nature. The gene of human osteopontin was cloned into pMAL-c2 vector and expressed in E. coli and finally human ostcopontin was purified. The antibody against Opn was raised in the rabbit. We examined psammoma bodies in 71 cases of meningioma at microscopic and ultrastructual levels. Immunohistochemical staining was performed at 1,000 dilution. Female predominance was significant in the frequency ofpsammoma bodies( 26% of female cases and 8.7% of male ones). Opn immunoreactivity was found in the psammoma bodies without exception. Concentric circular arrangement was common. The reaction products were also found in the cytoplasm, which contribute the menigocytic whorl. Under the electron microscope, Opn bearing cells had the scarce microorganella, though they showed the conventional features of meningothelial cells with interdigitations and junctional complexes. DAB reaction was strong on the small vesicles and weak reaction was found in ER and mitoehondria. Collagen fibers were free from the reaction products. Tumor cells, which showed degeneration Sprocess, e.g.. formy appearance, were demonstrated the reaction products in their cytoplasm and inflammatory cell infiltration was usual. These findings indicate that tumor cells comprising the meningocytic whorls express Opn during their degenerative process. And it may serve as the coreprotein of carcification, so the initial site of calcification in the psammoma bodies. Upregulation of the Opn by hormones or their receptors was responsible for the female predominance of the psammoma bodies.

129 EXPRESSION OF HORMONAL RECEPTORS AND P53 PROTEIN IN BENIGN AND MALIGNANT MENINGIOMAS A.M.C. Tsanaclis, P.H.P. Aguiar, A.F. Logullo, M.R. Matamoros and A. Yacubian University of Sao Paulo School of Medicine, Sao Panlo, Brazil The aim of this study is to assess the possible relation between the malignant transformation of the meningiomas and the expression of p53 protein, and oestrogen and progesterone receptors. Criteria for the diagnosis of malignant meningioma were: invasion of the cerebral cortex and elevated labelling index (> 1%), evaluated by the MIB-1 monoclonal antibody. Immunocytochemical methods using MIB-1 and monoclonal antibody against the p53 protein (Immunotech, Marseilles) and monoclonal antibodies against the oestrogen (Dako, California) and progesterone receptors were performed with the Dako PAP kit (mouse, universal) in 19 meningiomas surgically excised. Expression of oestrogen receptors was restricted to two examples and progesterone receptors to one, p53 protein was detected in very few nuclei in all malignant tumors and in two tumors with elevated proliferation index ( > 1.5%). The paucity of expression of the p53 protein, detected by immunocytochemistry, suggests that this gene either does not play a major role in malignant transformation of meningiomas or it may be altered but the alteration is not detected immunocytochemieally by the current available antibodies. High proliferative rates of meningiomas seem to encompass malignant transformation.

"Hemangioblastoma" in a limited ~nse: Immunohistochemical and Electron Microscopic Study N. Kawano, H. Komatsu, S. Yagishita, H. Oka, T. Suwa Department of Neurnsurgery, Kitasato University School of Medicine, Sagamihara, Kanagawa, and Department of Pathology, Kanagawa Sougou Rehabilitation Center, Kanagawa, Japan In order to determine the histogenesis of the stromal cell of hemangioblastoma (HB), we studied 18 HBs from our file. The original pathologic diagnoses were made by well-experienced neuropathologists. Sixteen HBs were located in the cerebellum and two HBs in the spinal cord. All 18 HBs were studied immunohisto-chemieally using GFAP, vimentin, actin, LN3, factor-VHI, UEA-1, EMA, NSE, neurofilament and S-100. Electron microscopic study was performed in all tumors. The results revealed that 4 tumors were clear cell ependymoma as the tumor cells were diffusely GFAP positive and showed welldeveloped intercellular junctions, microvilli and occasional cilia. Features suggesting an astrocytie origin were seen in 3 tumors. Vascular endothelial cells were often fenestrated in these 7 cases (4+3). In the remaining 11 tumors, immunohistochemical common features were positive for vimentin, NSE and S-100. Stromal ceils of this group did not show any specific ultrastructures that could identify their origin. Endothelial cells were not fenestrated in this group. We conclude that clear cell ependymoma is apt to be diagnosed as HB when it occurs in the cerebellum. For differentiation, H&E staining is of little help, and GFAP stain and electron microscopy are needed. As tumors which show classic histologic features of HB seem to contain various tumors, a rule-out study is necessary. After this procedure, remaining tumors are correctly called HB in a ilarrower sense.

Meningeomas after Embolization with Micropartioles G. Stoltenburg-Didinger, C. Gotzia and G. n e n n d o r f Institute of Neuropathology and Department of Radiology, Free University, Berlin, Germany. Purpose: Evaluation of the p a t h o h i s t o l o g i c a l effects and distribution of embolic m a t e r i a l in m e n i n g e o m a s following preoperative embolization with microparticles. Material and Methods: In 13 patients with m e n i n g e o m a s a preoperative embolization was performed. Microparticles of Tantalumpowder (I-2~) and PVA (150350p) were used. After e m b o l i z a t i o n and surgical removal, size and number of vessels c o n t a i n i n g embolic material were evaluated. Results: In 7 of the 13 tumors embolic m a t e r i a l was present in the lumina of vessels of d i f f e r e n t sizes. The smallest vessel diameter containing T a n t a l u m p o w d e r was llp. In small vessels no embolic m a t e r i a l was found in any of the tumors investigated. In vessels larger than 91~ Tantalumpowder was e q u a l l y missing. The vessels containing T a n t a l u m p o w d e r ranged between ii@ and 30~. The lumina were p a r t i a l l y or c o m p l e t e l y occluded by Tantalumpowder alone or t o g e t h e r w i t h fibrin and platelets indicating intravascular coagualation. PVA reached and almost c o m p l e t e l y occluded the larger vessels with diameters of 121~ and more. The walls of vessels c o n t a i n i n g embolic material or the surrounding tissue never e x h i b i t e d concomitant exudation or inflammatory changes. The necroses resulting from e m b o l i z a t i o n w e r e anemic. Conclusion: The smallest vessels of m e n i n g e o m a s are not reached by Tantalumpowder despite the m u c h smaller size of the particles. The effect of the e m b o l i z a t i o n seems to be due to intravascular clotting of the embolic material in vessels proximal to the capillary bed. The resulting disturbances in m i c r o c i r c u l a t i o n finally cause intravascular coagulation. As the vascular wall of vessels in m e n i n g e o m a s is similar to capillaries or small venules all over the tumor, an anatomical distinction between arteries and veins is impossible. The anemic nature of the n e c r o s e s and the missing occlusion of the capillaries indicate the arterial nature of the vessels c o n t a i n i n g embolic material.

Primary cerebral T cell lymphoma in an 11 year old boy Mirakhur, M, Cameron, S. and Allen, I.V. Drs.

This eleven year old boy presented with a three month history of headache, dizziness, vomiting and blurred vision. On clinical examination he was alert and orientated but had papilloedema. A C T scan showed an irregular hyperdense lesion in the low right frontal area with marked oedema. A right frontal craniotomy was performed with debulking of tumour. At operation there was dural infiltration. Pathological examination using H & E, immunocytochemistry and electron microscopy revealed a T cell lymphoma. Haematological parameters eg. peripheral smear and bone marrow showed no abnormality. There was no evidence of immune deficiency or extracranial involvement. The child is HIV negative. Post-operatively he continues to do well T cell neoplasia in the cranium and at this age is rare. Its association with HTLV-1 infection will be discussed.

130 T-cell rich B-cell lymphomas of the brain. A report of 3 cases. J. J. Kepas, Department of Pathology, Kansas University Medical Center, Kansas City, KS, USA and R. Baba-Ahmed, Department of Pathology, Hopital Parnet, Hussein-Dey, Algiers The concept of T-cell rich B-cell lymphoma was introduced into general pathology by Ramsay et al (I). These authors felt that large numbers of reactive T-cells may accompany a specific subtype of B-cell lymphomas. A broader review of such cases revealed that such tumors comprise a hererogenous group, i.e. different types of B-cell lymphomas m a y b e accompanied by massive T-cell reaction (Krishnan et al, 2.) We report three cases of T-cell rich B-cell lymphomas in the brain. In each instance the B-cells represented the neoplastic elements and the T-cells were entirely benign. The reactive response by T-cells supported the original concept of "a lymphocyte civil war" by daVita. (3). Within the brain lymphomas the reactive Tcells formed an inner ring around blood vessels and they were in turn surrounded by neoplastic B-cells. At the margins of the tumor, the T-cells formed what appeared like a defensive layer between the B-cell tumor and the surrounding uninvolved brain. In one of the cases the original biopsy showed a "pure" B-cell lymphoma, a recurrent tumor at some distance of the original mass contained numerous reactive T-cells in addition, indicating that even within one and the same tumor the reactive T-cell component may not be present at all times. References: i. Ramsay AD, Smith WJ, Isaacson PH: T-cel~ rich B-cell lymphoma. Am. J. Surg. Pathol. 1988; 12: 43343 2. Krishnan J, Wallberg K, Frizzera; T-cell rich B-cell lymphoma. A study of 30 cases, supporting its histologic heterogeneity and lack of clinical distinctiveness. Am. J. Surg. Pathol. 1994; 18: 455-65, 3. 3. deVita AD: Lymphocyte reactivity in Hodgkin's disease: A lymphocyte civil war. NEJM 1973; 289:801-2

Pituitary Adenoma: An Unsuspected Diagnosis For A Ciival Mass K. Wong*, J. Raisanen*, S.L .Taylor**, M.W. McDermott**, P. Gutin** *Department of Pathology and **Department of Neurosurgery, Univeristy of California, San Francisco, California, USA Pituitary adenomas are common tumors accounting for approximately 10% of intracranial neoplasms. Most arise from the adenohypophysis and are confined to the region of the sella turcica. Other sites may be involved as a result of extension, infiltration or ectopic location. However, posterior extention or ectopic involvement of the clivus of the sphenoid and occipital bones is rare. Six patients with destructive clival masses were referred to our institution with presumptive diagnoses of chordomas. In all cases histologic work up revealed pituitary adenomas. Because they represent a subset of adenomas, the clinical histories of the patients and the histologic features of the tumors were reviewed. Five of the patients were male and ages ranged from 31 to 67. Four had extremely high plasma concentrations of prolactin (8,132 to 22,424 ng/ml, nl
Primary Ki-1 (Anaplastic Large Cell) Lymphoma of the Brain and Spinal Cord. A Case Report. Necat Havlioglu, Anantha Manepalli, Lorenzo Galindo, Cirilo SoteloAvila, and Leonard Grosso. Department of Pathology, Saint Louis University School of Medicine. A case of primary Ki-1 lymphoma of the brain is presented. The patient was a 4'h year old African-American girl who presented with a 4-5 day history of headaches, nausea, vomiting, neck stiffness, and difficulty in walking. Cerebrospinal fluid on examination showed a mild lymphocytosis. CT scan of the brain showed two discrete densities in the left occipital lobe and in the brain stem. An MRI showed multiple densities scattered over the brain surface and brain stem. The mother had had a recent skin PPD conversion without apparent active disease. The clinical impression, based on the presentation and the radiologic features, was an infection involving the central nervous system. An extensive workpp to rule out an infectious process was negative. Antituberculous medication was started with little or no clinical improvement in 2 weeks. A brain biopsy was performed. Histological examination showed an anaplastic neoplasm diffusely infiltrating brain parenchyma. The neoplastic cells were large with amphophilic cytoplasm, large nuclei with irregular nuclear contours and prominent nucleoli. A high mitotic rate including atypical mitotic figures was noted. There was a lymphoplasmacytic infiltrate in the background. Immanohistocbemical stains showed diffuse strong positivity for CD30 and moderate staining for epithelial membrane antigen. Leukocyte common antigen, cytokeratin, neuron specific enolase and B and T marker stains were negative. The histology was characteristic for Ki-1 large cell lymphoma. This is one of the first reports of this variant in the pediatric population.

Squamous Metaplasia at the Periphery of a Necrotic Pituitary Adenoma C. Rao, S. Kavavattathayyil, and R. Swanson.

A. Abdu, D. Deloso, P. Chen

Dr. Joanna Sher Neuropathology Laboratory, Departments of Pathology and Neurosurgery, KCHC-SUNY-HSC at Brooklyn, NY. Squamous metaplasia of glandular epithelium in the infundibular region is seen in up to 25-30% of pituitary glands studied at autopsy. However squamous metaplasia as a tissue response to ischemic necrosis of a non neoplastic pituitary gland or to hemorrhagic necrosis of an adenoma has been documented only once (Kepes, Sayler, Hiszezynskyj '82). We report here on another case of squamous metaplasia at the periphery of a necrotic pituitary adenoma in a 52 year old black male patient who presented with a progressive decline of visual function with precipitous loss of vision on the day of admission. He had no history suggestive of any endocrinological abnormalities. MRI revealed a large sellar-suprasellar mass encroaching on to the optic chiasm. Transsphenoidal specimen revealed necrotic fragments strongly suggestive of a necrotic pituitary adenoma. A craniotomy was done fifteen days later, specimen from which revealed a viable and extensively necrotic pituitary adenoma with recent and old hemorrhages, granulation tissue and hyalinization. At the periphery of the necrotic adenoma, squamous metaplasia was noted, which appeared to be arising from the remnants of the surrounding non neoplastic adenohypophyseal cells. The metaplastic squamous cells were positive for Keratin and negative for the battery of pituitary harmones. The pituitary adenoma revealed focal positivity for thyroid stimulating harmone (TSH) and follicular stimulating harmone (FSH).

131 Central pontine myelinolysis (CPM) in a patient with surgically-treated craniopharyngioma complicated by diabetes insipidus (DI). M.A. Wrzolek, C. Rao, State University of New York, Health Science Center at Brooklyn. CPM [s a recognized complication of rapid overcorrection of hyponatremia. We present a case of a 23year-old Hispanic woman who developed DI after surgical resection of craniopharyngioma. One year later, she underwent cholecystectomy which was complicated by Klebsielia sepsis and respiratory arrest. Control of DI has been difficult in postoperative period, with several episodes of hyponatremia and hypernatremia, one of which was associated with severe hyperglycemia. She died of massive pulmonary embolism two months after surgery. At autopsy, there was a symmetrical demyelinated lesion in the base of pons, typical of CPM. Other neuropathological findings included residual craniopharyngioma in the hypothalamic region and an old frontal lobe abscess. This case underscores the labile electrolyte balance in patients treated for craniopharyngioma who, in addition to DI, may have impaired thirst control mechanisms. The electrolyte balance may prove to be particularly difficult to control in patients with superimposed infection, setting ground for development of CPM. Even though DI is the most frequent endocrinological complication in craniopharyngioma survivors, the association of craniopharyngioma and CPM has been previously described in only four cases.

Resection of 61iomas and Life Expectancy. J. Cook, D.E. Woodward, P. Tracqui, G.C. Cruywagen, J.D. Murray, G.T. Bartoo and E.C. Alvord, Jr., University of Washington, Seattle, WA 98195, USA. Our mathematical model illustrates why an advancing f i r e is d i f f i c u l t to control from behind. When a tumor is invasive, not only is i t rarely possible to remove i t a l l , but i t is also impossible, using current techniques, to accurately estimate the amount of tumor remaining. We model an expanding brain tumor using a linear reaction-diffusion equation to represent cell proliferation and i n f i l t r a t i o n . Death occurs when the cell density reaches a critical level at a critical radius or when the total cell count reaches a critical level. An exact expression can be derived for l i f e expectancy in the absence of surgical intervention. The same expression can be used to estimate the earliest time at which a tumor could theoretically be identified with current clinical technology. We model surgical resection as the removal of a central area of the tumor, and derive approximations for the time taken for the remaining tumor to reach a critical size. We show that only i f resection is on a sufficiently large scale, and is sufficiently early, can i t postpone death by a period greater than the cell doubling time. We graph l i f e expectancy as a function of time and extent of surgery. Tumors with highly diffusive cells are shown to be more d i f f i c u l t to treat than rapidly proliferating tumors. The former are detectable for a shorter period and require resection of significantly more than the visibly affected tissue. We conclude that gliomas can be classified not only according to the speed at which they grow but also according to their density profile.

Janusz Szymas, Jacek Jelonek, Roman Slowinski

Krzysztof

Krawiec,

Application of artificial neural network to computer-aided diagnosis of neuroepithe!ial tumours of the central nervous system. Department of Pathology, University of Medical Science and Institute of Informatics, Technical University, Poznan, Poland We have developed a computer system ONCOBASE that can support the pathologist's diagnosis concerning the classification of neuroepithelial tumours of the central nervous system. The system employs a multi-layer feed-forward neural network with a backpropagation algorithm. The network has 159 input units corresponding to histological attributes, 32 and 12 units in two hidden layers, and 12 output units corresponding to different classes of neuroepithelial tumours. It was taught using 267 training examples acquired from the expert. Several parameters of the network and the learning algorithm have been tuned: the number of hidden units, stop condition, convergence threshold, randomization range of the weights and the learning speed. Additionally, rough sets have been applied as an intelligent front-end for the neural network. In particular, they are used to reduce the input information to the smallest set of relevant attributes. It gives an important gain in the learning time and improves the prediction ratio. The verification of ONCOBASE system on the set of 66 new cases gives very satisfactory results (prediction ratio of 94.7%).

132 The significance of the identification of mitoses in gliomas $W Coons and PC Johnson Barrow Neurological Institute, Phoenix, Arizona The histological identification of mitotic activity is a cornerstone of several highly predictive grading systems for gliomas. In the St. Anne/Mayo and WHO classifications, the identification of even a single mitotic figure is a sufficient criterion for classifying an astrocytoma as high grade. However, the amount of tumor available for microscopic review ranges from only a few mm2 in small needle biopsies, up to several cm2 in open resections. The question arises as tO whether the presence of a singte mitosis in the smaller specimen has a different significance than in the larger. 473 primary gliomas (370 astrocytomas and 103 oligcx:lendrogliomas/oligo-astrocytcmas (O-A)) were reviewed until the first mitosis was identified, or until 100 400x fields had been reviewed without identifying a mitosis. Among the 401 cases in which mitoses were identified, they were found in the first 10 fields in 80%; 20 fields in 90%, and 30 fields in 95%. Long-term survival data was available on 286 diffuse astrocytomas (20 grade 2, 34 grade 3 and 232 grade 4) and 56 oligodendrogliomas/O-A. The median survival of the grade 2 astrocytomas (963 days) was significantly longer than that of the grade 3 tumors (194 days), confirming the prognostic significance of the presence of mitoses (p<0.0001). Among the grade 3 tumors, survival was correlated with the field in which the first mitosis was found. When a mitosis was seen in the first 10 fields, median patient survival was 120 days, compared to 384 days when the first mitosis was found later (p<0.002). A similar relationship was seen in the oligodendrogliomas/O-A. Median survival in patients with tumors with no mitoses in the first 10 fields was 1197 days, compared to 451 days when found in the first 10 fields (p<0.002). Although the presence of mitoses is by itself a powerful prognostic marker, the evaluation of mitotic activity offers more prognostic information than can be obtained by the desirably simple approach of noting just their presence or absence.

lmmunohistochemical investigation of relationships between monoclonal and polyclonal Ki-67 antibodies on frozen and paraffin sections from human glioblastomas. S.H. Torp, E. Johannesen, and C.F. Lindboe, Deptartment of Pathology, University Hospital of Trondheim, Trondheim, Norway.

The aim of this study was to compare Ki-67 labelling index (LI) in a prospective group of ten uniformly handled frozen and formalin fixed, paraffin embedded glioblastomas using monoclonal (moab) and polyclonal (polyab) Ki-67 antibodies. The LIs (%) were found to be (median and range):

Ki-67 moab (Dako) Ki-67 polyab (Dako)

Frozen sections

Paraffin sections

5.9 (2.6-11.4) 13.7 (6.7-21,5)

0.004 (0.0-4.0) 12.0 (2.2-22.7)

On frozen sections the moab Ki-67 gave lower indices than the polyab Ki-67 which might be due to reactivity against different epitopes. The moab Ki-67 revealed only occasionai positive staining on paraffin sections and only after microwave treatment. The polyab Ki-67 worked we}l on both frozen and paraffin sections and the indices were comparable (r=0.72). As paraffin sections provide morphology superior to frozen sections, we recommend the use of Ki-67 polyab on paraffin sections to assess the proliferative activity in glioma tissues.

Comparison of Ki 67 and MIB 1 monoclonal antibodies in gUal tumors E. Uro, P.H. Bousquet, M.B. Delisle C.H.U. Rangueil, Toulouse, France

Janusz Szymas, Michael Beil

The aim of this study was to compare the labelling index (LI) obtained when using two monoclonai antibodies directed against the antigen Ki 67, in gliomas. Both immunohistochemicai methods were applied on surgical specimens from 33 brain tumors, including 5 astrocytomas, 5 anaplastic astrocytomas, 15 glioblastomas, and 8 oligodendrogliomas. Ki 67 LI was determined on cryosections, using the pcroxidase-antiperoxidase method. MIB 1 technique was performed on sections obtained from adjacent paraffin-embedded material. Following microwave processing, a streptavidin-biotin method was used. The highest LI was, respectively, 19,8 for Ki 67, 22 for MIB 1. In most cases, there was a mild difference between both values ( < 25%). MIB 1 LI was not always superior to KI 67 LI, as previously determined in tumors outside the brain. The MIB 1 expression was, like that of Ki 67, heterogenous, inside each tumor. This confirms that the immunostaining for If/67 can be done on both paraffin and frozen sections, allowing retrospective studies to evaluate proliferating activity of brain tumors.

Department of Pathology, University of Medical science, Poznan. Poland. IBSB. Berlin. Federal Republic of Germany.

Proliferating cell nuclear antigen (PCNA) and nucleolar organizer region-associated proteins (AgNORs): Expression in human astrocytic gliomas.

In this retrospective study, 96 astrocytic tumours were stained with (i) monoclonal antibody PC10 which recognizes the proliferating cell nuclear antigen (PCNA) and (2) modified silver technique visualized nucleolar organizer regionassociated proteins (AgNORS) in formalin fixed, paraffin-wax embedded sections. All the cases were typed and graded using the WHO Classification System. The PCNA labelling indices (LIs) and the mean number and area of AgNORs per nucleus of tumour cells of glioblastoma (n=25), anaplastic astrocytoma (n=22) and fibrillary (n=14) protoplasmic (n=13) gemistocytic (n=22) astrocytoma was correlated with the histological diagnosis and grade. For evaluation computer-aided image analysis system was applied. In general PCNA LI increased with tumour grade, but there was considerable variation of LIs, especially in grade III and IV tumours. We found significant differences in the mean number and area of AgNORs per nucleus between iscmorphous astrocytomasf anaplastic astrocytomas and glioblastomas. Both of these methods were reproducible but showed no correlation. The combination of PCNA immunohistochemistry and AgNORs morphometry seems to give various important prognostic information about astrocytomas independent of the appraised arbitrary grade of biological malignancy.

133

Expression of various PCNA clones in glioblastomas. C.F. Lindboe, E. Johannesen and S.H. Torp, Department of Pathology, Trondheim University Hospital, Trondheim, Norway. Ten glioblastomas were stained with commercial monoclonal antibodies against the PCNA clones 19A2 (BioGenex, dilution i:80), 19F4 (Boehringer, dil. 1:160) and PC 10 (Boehringer, dil. 1:40 on frozen sections and 1:200 on paraffin sections). Immunohistochemical staining was performed on frozen sections and paraffin sections of the same specimens (formalin fixation for 6 hrs) with and without microwave pre~eatment for 2 x 5 min. in citrate buffer. None of the three tested antibodies revealed positive staining of frozen sections from the controls (human tonsils) and glioblastomas. In paraffin sections without microwave pretreatment no positive staining could be demonstrated for the clones 19A2 and 19F4 whereas the controls and two cases showed staining for PC 10 (labelling index (L1) < 1%). In microwave heated paraffin sections positive staining for PC10 could be demonstrated in all cases with a LI in the range of < 1 - 17.2%. However, in seven of these cases there was a disturbing unspecific background staining. Staining for 19F4 was positive in eight cases (LI < 1 - 3.5%), but for 19A2 in only three cases (LI < 1%). Thus, in spite of the disturbing background staining, PC 10 staining on microwave pretreated paraffin sections gave the best results with the highest number of positive cases as well as the highest labelling indices.

Immunohistochemical study of proliferative indexes in childhood medulloblastomas L. Albuquerque*, J. Pimentel*, L. Tbvora**, N.L. Antunes*** *Laborat6rio Neuropatologia, **Servic,,o Neurocirurgia, ***Servi;o Pediatria, Hospital de Santa Maria, 1600 Lisboa, Portugal Proliferative capacity has been evoked as an independant prognostic factor in childhood medulloblastomas. We studied in retrospect 18 medulloblastomas diagnosed between 1983 and 1993. The aims of the study were: a) to evaluate some histological features i.e. necrosis and desmoplasia, to define cell differentiation by NSE and GFAP, and to calculate proliferative indexes (L.I), by using antibodies against PCNA or Ki-67 / MIB1; b) to correlate those parameters with outcome. There were 11 male and 7 female with a median age of 6,7 years. Ten patients were poor-risk (Packer, 1991 ). Radiotherapy of the neuraxis was performed in 4 children, chemotherapy was concurrently added to another twelve. LIs were calculated counting 750-1000 cells, in the areas of higher staining. Necrosis and desmoplasia were present in 6116 cases, each one; 6 cases disclosed NSE staining. PCNA LI was 12+_14% (range 0-50%), Ki-67 [_1 17+_8% (range 5-36%), no correlation being found between both values. In 6 cases Ki-67/I was <15%. For a follow-up time (16 patients) of 35,2 months, 3 patients died, 13 remained alive, 10 of them disease-free. Kaplan-Meyer estimates of disease4ree probability and Iogrank Z2 comparisons disclosed: a) no evidence of clinical significant risk-factors; b) absence of correlation either with the histological parameters or with the PCNA El; c) MIB1 1_1was the only prognostic significant factor: the group with I_l _> 15% harboured a better outcome, possibly due to the effectiveness of adjuvant therapy on proliferating cells.

Comparative immunohistochemical study on the expression of five stress-response proteins [srpg0, srp72, srp27, a Bcrystallin ( a B C ) and ubiquitin (Lib)], p53 protein and

SIGNIFICANCE OF PROLIFERATION MARKERS IN RF~ CURRENT MENINGIOMAS

proliferating cell nuclear antigen (PCNA) labeling index (LI) in brain tumors.

S. Weis, D. Protopapa, U./Vlfierz*, P.A. Winkler*, H.J. Reulen* and, P. Mehraein Institute of Neuropathology and * Department of Neurosurgery, Ludwig-Maximilians-University, D-80337 Munich, Germany

S. Kato~, T. Motita ~, M. Kato~, F. Herz3, A. Hirano4, and E. Obama~, 'Div. of Neuropathol. & 2Dep. of Pathol. ,Tottori Univ., Yonago, Japan. ~ I ~ . of Pathol. & 'Div. of Neuropathol., Montefiore Medical Center, Bronx, NY 10467, USA. This retrospective study was carried out on 123 primary brain tumors and 21 carcinoma metastasis to the central nervous system. Serial sections of formalin-fixed, paraffin-embedded tissues were used. Our results show that except for oligodendrogtionm, proportions of all the other t u ~ exanfined expressed four or five srp's, that a certain percenlage of all tumors had p53-posifive cells and lhat the mean PCNALI varied frem 0.1% among pituitary adeaomas to 30% in glioblastomas (GBM) and 31% in breast ~ metastasis (Br-Met). While the highest proportion of srp90-positive tumors was seen among astrocytomas (46%), GBM (50%), meningiomas (46%) and Br-Met (50%), that of srp72-positive tumors was observed in GBM (50%), meningiomas (58%) and Br-Met (80%). On the other hand, 86% of the schwannomas (Schw), 80% of Br-Met and 50% of GBM were positive for srp27. The highest frequency of ~ BC-positive tumors was seen among Sehw (76%), GBM (67%) and ependymomas (60%). By contrast, llb-positive tumors were detected less often: 30% of Br-Met, 27% of lung carcinoma metastasis and 25% of GBM. By comparison, most GBM (75%) and Br-Met (55%) and many medulloblastomas (42%) were stained for p53 protein. These tumors also had the highest PCNA-LI values. We suggest that slp expression in brain tumors could be related to the immunohistoehemieally-detectable p53 protein, the expression of which may depend, at least in part, on the growth potential of a given tumor.

The significance of proliferation markers in predicting recurrenties of meningiomas was investigated by means of immunhistochemistry and morphometry. Antibodies against PCNA (P10) and MIB-1 were used. The immunopositive cells were counted following the "random systematic sampling"; the numerical density as well as the cell index were determined. One group consisted of 10 meningotheliomatous meningiomas which recurred and became malignant meningiomas (M1). In the second group (M2), the recurring tumors were meningotheliomatous meningiomas. The third group (M3) consisted of meningotheliomatous meningiomas without recurrency during the last three years. The majority of the cases with recurrencies showed a higher numerical density as compared to the primary tumor. In some cases with more then one recurrency, the number of positive cells decreased to increase in the subsequent recurrent tumor. There was no significant difference between the primary tumors of group M1 and M2 and control meningiomas (M3) with no recurrencies. Thus, proliferation markers do not give significant information for predicting the potential of the tumor to recur.

134 Immunohistochemical Analysis of Meningioma Recurrence using MIB-I combined with eimpson's Grade. Dr. Xiao Di, M.D., Department of Meurosurgery, Affiliated Hospital, Hainan Medical College Haikou, nainan, People's Republic of China. We investigated a relationship b e t w e e n the r e c u r r e n c e and the proliferation rate of various subtypes using the monoclonal antibody MIB-I c o m b i n e d w i t h Simpson's grade in 36 surgically treated patients with intracranial meningiomas. The initial surgical procedure was graded according to Simpson's classification from grade I to V. The conventional formalin or alcohol-fixed, p a r a f f i n - e m b e d d e d sections of archival meningioma materials were incubated with MIB-I monoclonal antibody using a m o d i f i e d avidinbiotin peroxidase complex (ABC) technique. A new technique "boiling method" for antigens r e t r i e v a l from paraffin-embedded tissues was e s t a b l i s h e d in the procedure of heating tissue section instead microwave. The percentages of MIB-I labelling index (LI) were observed in the meningothelial (syncytial), transitional, fibrous and angiomatous types from less than 1% to 14.7%. There were increases of proliferating cells ranging from 3.2% to 19.3 for atypical and from 4.9% to 20.4% for anaplastic meningiomas. Eight recurrent tumors including two of Simpson's grade I show r e m a r k a b l y higher M I B - I LIs than 9% except two, whose Simpson's grade were III and IV respectively on initial surgery. It was noted that tumors with a higher MIB-I LI may have a higher proliferative potential and greater biological malignancy than those with a lower MIB-I LI. Immunohistological labelling of proliferating prediction of the recurrence using MIB-I can help the traditional Simpson's grades to elucidate the biological behaviour of meningioma and clinical prognosis of the patients.

Moleclar Genetics Analysis of Oligodendroglial Tumors Shows Preferential Allelie Losses on Chromosome Arms 19q and lp Julia Reifenberger, Guido Reifenberger, Lu Liu, C.David James, Wolfgang Wechsler, and V. Peter Collins Department of Pathology, Sahlgrenska Hospital, Gothenburg, Sweden, and Department of Neuropathology, Heinrich-Heine-University of DDsseldorf, Germany The molecular genetic alterations of oligodendroglial tumors and mixed gliomas of the central nervous system were studied in a series of 37 cases. All chromosomal arms were examined for losses of genetic information by performing restriction fragment length polymorphism analysis for 180 loci. Loss of heterozygosity was most frequently observed for loci on the long arm of chromosome 19 with a commonly deleted region at igq13.2-q13.4 distal to the CYp2a gene and proximal to the DIGS22 locus. The incidence of a l l e l i c loss on Igq was particularly high (8}%) in oligodendroglial tumors and equal to 31% in mixed~. gliomas. More than 75% of the tumors with alleliC deletions on 19q also showed loss of heterozygosi~y for loci on ip with a commonly deleted region distal to the NGFBgene (1p13-pter). Seven tumors (Ig%) had lost alleles from 17p with the deleted region including the TP53 tumor suppressor gene in all cases. In addition to losses of genetic information from chromosomes Igq and ip, anaplastic tumors from this series showed a high incidence of a l l e l i c losses on gp and 10.

C-MYC Amplification in Medunoblestoma Biopsies (Bxs) end Xenografts (Xgs). R.E. MeLendon, S.K. Batra, H.S. Friedman, B.K.A. Rasheed, D.D. Bigner, S.K. Bigner, Duke University Medical Center, Durham NC, USA. The t r e a t m e n t of medulloblastomas has been hindered by inabilities to discern patients with aggressive neoplasms from those with indolent neoplasms. Recent studies have suggested a relationship between hyperexpression of myc family oncogenes and clinical aggressiveness in a variety of neoplasms. Eleven medulloblastoma Xgs derived from neoplasms of 11 patients ranging in age from 1 to 12 years were studied to determine the relationship between e-myc amplification (AMP) and hyperexpression and the relationship between (AMP) of c-myc DNA in the original Bxs and these derived Xgs. Medulloblastoma Xgs grown in athymic mice were snap frozen either in liquid nitrogen or in mounting media and analyzed by Southern blot, Northern blots and immunohistochemical techniques. Southern blot analysis revealed c-myc AMP in 8 (D283 MED-X, D341 MED-X, D382 MED-X, D384 MED-X, D425 MED-X, D487 MED-X, D511 MED-X, D556 MED-X) of 11 Xgs with probable DNA rearrangement in 2 (D283 MED-X and D384 MED-X). Northern blot analysis and immunohistochemistry performed in the 8 Xgs with c-myc AMP demonstrated hyperexpression of c-myc in all 8. Previous studies have shown t h a t e-myc hyperexpression was not identified in one Xg (DAOY) also not exhibiting DNA AMP. Two Xgs (D306 MED-X and D386 MED-X) which had been found not to exhibit DNA AMP did not survive beyond early passages and were not studied for hyperexpression. Analysis of 6 Bxs by slot blot (D283 MED-Bx, D306 MED-Bx, D382 MED-Bx, D384 MED-Bx, D386 MED-Bx, D425 MED-Bx) revealed grossly detectable e-myc AMP in 3 (D341 MED-Bx, D382 MED-Bx, D384 MED-Bx). In a further study of 43 medulloblastoma Bxs, including the 6 from whom Xgs were derived, no additional Bxs were found to exhibit c-mye AMP. None of the Bxs or Xgs exhibited N-myc AMP. These studies support the hypothesis that c-myc is biologically active in some medulloblastomas and that AMP of c-myc DNA is the primary mechanism mediating hyperexpression in medulloblastoma.

Subtypes of human glioblastomas def'med by cytogenetic

analysis S. Part and G. Thiel Institute of Neuropathology, Free University of Berlin, and Institute of Medical Genetics, Humboldt-University, Berlin, Germany Using classical cytogenetic methods, we studied 22 human g|ioblastomas. Seventeen cases could be subdivided into two types: Type 1 contained two tumors with a complete loss of chromosome 17, but without loss of chromosome 10 and without trisomy 7. These tumors occurred in somewhat younger patients (mean age 48,51 years). Type 2 consisted of 15 glioblastomas, which showed trisomy 7, loss of chromosome 10 but no alterations concerning chromosome 17. The mean age was higher in this group (59,1 years). A similar subclassification of glioblastomas was described by van Deimling et al. g r a i n Pathology 3:19-26, 1993], who investigated a larger series of tumors using molecular genetic methods. Their type 1 subset was characterized by the loss of the short arm of chromosome 17 and the absence of amplification of the epidermal growth factor receptor (EGFr) gene which is located on chromosome 7. This type ocurred primarily in younger patients and corresponded to "secondary" glioblastomas of classical terms. Type 2 subset was characterized by EGFr amplification without loss of chromosome 17 and occurred in older patients with "primary" glioblastomas. Loss of chromosome 10 was found in both subsets. Both studies, which used different methods, revealed very similar results. Our cytogenetic study is, to our knowledge, the first investigation that confirmed the analysis based on molecular genetics. This suggests that glioblastoma subtypes really exist. However, further studies are needed to elucidate the value of such a subclassification, particularly regarding prognosis.

135 Glinl Fibrillary

Acidic

Protein and Vimentin Isoforms in Oliomas.

F. Labrousse (1,3), B. de N~chaud (2), D. Gom~s (3), C. Daumas-Duport (4), C. Allarmarget (I), P. Dupouey (3). Departments of : (I) Pathology, H6pital Universitaire Dupuytren, Limoges. (2) Cellular Biochemistry, Coll~ge de France, Paris. (3) Antigens Biochemistry, Instltut Pasteur, Paris. (4) Neuropathology, H6pital Saints-Anne, Paris, France. The isoform patterns of glial fibrillary acidic protein (GFAP) and of vimentin were studied in a series of 22 gliomas classified according to the system used at the H6pital Sainte-Anne, Paris. There were 12 gliobastomas or grade 4 astrocytomas, 2 grade 3 astrocytomas, 2 juvenile pilocytic astrocfyomas (JPA) and 6 oligodendrogliomas. T w o samples of normal brain and 2 samples of reactive gliosis were comparatively studied. T w o dimensional gel electrophoresis of Triton-insoluble cytoskeleton preparations revealed the protein isoforms, defined by their isoelectric point (Ip) and their molecular

weight (respectively 5.8 and 50 Kd for GFAP and 5.6. and 56 Kd for vimentin in normal brain). A protein has been considered as heterogeneous when more than 3 isoforms were found. In glioblastomas and in most cases of astrocytomas, GFAP and vlmentin were predominant and often heterogeneous. In JPA numerous vimentin isoforms were observed while GFAP was not modified. In these cases, isoforms of G F A P and of vimentin could correspond to phosphorylated forms. In 3 astrocytomas, the pattern was comparable to the one of normal brain. A basic isoform of G F A P (Ip > 5.8.), without alteration of vimentin was selectively expressed in reactive gliosis, in all cases of ollgodendrogliomas and in 2 (grade 4 and 3) gemistocytic astrocytomas. This isoform, which is not expressed in other tumors, might be secondary to a modification in G F A P D N A transcription.

Enhancing the efficiency of interaction between microscopic brain tumor and therapeutic ceils L.A. Lampson, A. Chen, and A.O. Vortmeyer Dept. of Neurology, Brigham and Women's Hospital & Harvard Medical School, Boston MA, USA Even for new biological therapies, Specific delivery to microscopic CNS tumor remains a challenge. One approach is to use inflammatory cells, either as effectors themselves, or as vehicles for BRMs, drugs, vectors, or other agents. To fully exploit therapeutic leukocytes, enhanced CNS entry and dissemination are needed. Here, we use 2- and 3-D analysis to demonstrate one way in which T cell entry and dissemination can be enhanced in a controlled way. Methods. Intracerebra[ injection of proinflammatory cytokine (IFN-g or TNF-a) was used to activate cerebral vessels. Peripheral immunization with a neural antigen was used to activate T cells. Immunocytochemistry double-labeling was used to identify T cells and vessel walls on frozen brain sections. Quantitative results were obtained using computer-assisted image analysis, in 2 and 3 dimensions (Can. Res. 53: 176, 1993; Brain Pathol. 4: 125, 1994). T cell extravasation, and passage from the perivascular space (PVS) into the parenchyma proper, were both measured. Findinas. Each treatment alone enhanced T cell entry. Optimal T cell dissemination through the brain was obtained when both activation of endothelial cells and activation of T cells were combined: Total T cells, parenchymal T cells, and the volume of infiltrating T cells were each enhanced. Although widespread, the inflammation was controlled. The numbers of T cells per vessel, or per high power field, remained moderate. _Conclusions. Complementary activation of endothelial cells and of T cells can enhance T ceil entry and dissemination in the brain, without overwhelming inflammation. Using the same system, quantitative analysis of the effects on microscopic CNS tumor can now be performed.

Tumor cells expressing the herpes simplex virus-thymidine kinase gene in the treatment of meningeal neoplasia in rats.

ABLATION OF THE RETINOBLASTOMAPROTEIN FAMILY LEADS TO A P O P T O S I S IN PI9 E M B R Y O N A L C A R C I N O M A C E I ~ S U N D E R G O I N G NEUROECTODERMAL DIFFERENTIATION

F. Vrinnis, P. Qi, V. Chvrington, G. Cano and J. Wu. TuftsNew England Medical Center, Boston, USA.

R.S.Slack, I.s.skerjanc, B.Lac~, J.Craigt K.Jardine, M.W.McBurney Department of Medicine and *Department of Laboratory Medicine, University of Ottawa and Ottawa Civic Hospital, Ottawa, ON Canada KIH 8M5

A promising strategy in treating neoplastic meningitis involves the use of Herpes Simplex Virus-thymidine kinase (HSV-tk+) modified cells. In our experiments we used HSV-tk+ ceils to treat meningeal carcinomatosis in the Walker 256 model. Intrathecal administration of 2 x l0 s Walker cells results in median survival of 15 days. 50% of animals implanted with Walker-tk+ cells and treated with ganciclovir (GCV 30 mg/kg i.p. for 14 days) showed long term survival, whereas the remaining 50% died from tumor growth 50 days after implantation. Tumor cells from the last group of animals were cultured in vitro and were shown to be still sensitive to GCV, suggesting that multiple cycles of GCV may be beneficial. Histopathology studies confirmed the leptomeningeal infiltration in the untreated Walker or Walker tk+ animals. Walker cells were premixed with Walker-tk+ cells in 1:1, 1:10 or 1:50 ratio, respectively, implanted intrathecally and the animals were treated with GCV. All groups treated with GCV showed statistically significant increased survival with maximal effect being observed in the 1:10 and 1:50 groups. Interestingly, irradiated Walkertk+ cells are not tumorigenic and exert a bystander cytocidal effect on Walker cells in vitro similar to the one seen by nonirradiated Walker-tk+ cells. However, irradiated Walker-tk+ ceils, in contrast to nonirradiated Walker-tk+ cells, had no effect on survival in vivo. We conclude that Walker-tic+ cells in the presence of GCV could be used in the treatment of experimental meningeal neoplasia in rats.

We generated stable clones of Plg embryonal carcinoma lEe) cells in which the activity of the retinoblastoma (Rb) family of proteins has been ablated. We used EIA p3OO-binding deletion mutants that could be expressed at high levels in undifferentiated EC cells without modifying their embryonic stem cell character. Following induction of neuroectodermal differentiation with retincic acid, cells lacking functional Rb exhibited massive cell death relative to parental cells expressing control plasmid only. DNA laddering, indicative of internucleosomal cleavage of DNA, characteristic of apoptotic cell death, was clearly visible in all clones possessing EIA mutants capable of inactivating pRb, p107 and p130. Electron microscopy confirmed wide-spread apoptosis in the mutant cell line cultures. With the use of cell type specific markers, we found that cells most likely committed to the neural lineage underwent apoptosis. Stable clones expressing EIA mutants defective in the binding of pRb, pl07 and pl3O, differentiated normally and did not exhibit apoptosis. We examined the regulation of a series of genes believed to be targets for regulation by the Rb family. The most striking correlation was seen in the regulation of c-fos, a harbinger of cell death. While c-los levels remain low in parental PI9 cells regardless of differentiation status, clones lacking functional Rb exhibit a dramatic upregulation of c-los coinciding with the time at which apoptosis is initiated. Our results suggest that the Rb family may serve as a molecular link to the cascade of events leading to apoptosis of certain populations of terminally differentiated neurons.

136 HMGCoA Reductase is Highly Expressed in Medulloblastomas and Medulloblastoma Cell Lines; Inhibition In Vitro Induces Apoptosis. R.J.B. Macaulay, I. DimiU'oulakos, L.E. Becker and H. Yeger. Department of Pathology, the Hospital for Sick Children and the University of Toronto, Toronto, Canada. Manipulation of medulloblastoma cell lines in vitro may yield potential avenues of therapeutic intervention. Recently we used differential display-polymerase chain reaction to isolate genes involved in the neuronal differentiation of neuroblastoma, the peripheral nervous system counterpart to medulloblastoma. One highly expressed gene was found to encode 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCoA reductase). In medulloblastomas, high levels of HMGCoA reductase expression were observed by northern analysis in 6/7 tumours as well as in 3/3 permanent cell lines (DAOY, D283Med and UW228). Lovastatin, an irreversible inhibitor of HMGCoA reductase, enables synchronization of mitotically active cells at concentrations of 20-60 ~tM. In medulloblastoma cell lines, as little as 1.25 I.tM lovastadn was able to inhibit growth of DAOY and UW228. At concentrations of 5 ~tM the majority of cells detached, and above 10 ~tM, only rare ceils remained attached. Attached UW228 cells showed marked elongation of cytoplasmic processes with no ultrastructural or immunocytochemical evidence of differentiation. Detached DAOY and UW228 cells were shown to be non-viable usifig a live/dead assay, and showed ultrastrnctural evidence of apoptosis. D283Med cells, which grow in suspension, were more resistant to lovastatin, but at 40 IIM the majority of cells showed apoptotic changes. Northern analysis showed markedly increased expression of HMGCoA reductase in D283Med but not DAOY or UW228 cells exposed to 10 ~tM lovastatin for 24 hours. Since lovastatin induced cell death in each of the cell lines, the potential therapeutic benefit of inhibition of HMGCoA reductase in medulloblastomas should be further explored. (Supported by NCI Canada. Lovastatin generously supplied by Merck, USA.)

The Use of Oncogene Amplification or p53 Gene Mutations as Markers for TumorDerived DNA in Cerebrospinal Fluid C. Harker Rhodes, Charles Honsinger, George D. Sorenson Department of Pathology, Dartmouth-Hitchcock Medical Center, Lebanon, NH 03756 The sensitivity of PCR-based methods for the detection of DNA offers opportunities for tumor diagnosis from the small amounts of tumorderived DNA released into body fluids. We report the detection of tumor DNA in the cerebrospinal fluid (CSF) of three patients with intracranial neoplasms. One patient had a metastatic breast carcinoma which contained amplified HER-2/neu genes and amplified HER-2/neu gene sequences were present in her CSF. The second patient had a glioblastoma which contained amplified epidermal growth factor receptor (EGFR) genes and amplified EGFR gene sequences were present in her CSF. The third patient had a glioblastoma with a mutation in codon 273 of the p53 gene and DNA with the mutant p53 sequence was demonstrated in his CSF by allele-specific PCR. This report demonstrates that CSF sometimes contains tumor-derived DNA and suggests that PCR examination of CSF DNA m a y be diagnostically useful.

Coordinate Expression of Neurotrophin-3 and trk C Linked to a More Favorable Prognosis in M e d u l l o b l a s t o m a . Goumnerova LC, Segal RA, Kwon YK, Stiles CD, Pomeroy SL, Depts. of Neurology and Neurosurgery, Children's Hospital, Dept. of C e l l u l a r and M o l e c u l a r Biology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA, 02115. We have examined the expression of neurotrophins in m e d u l l o b l a s t o m a , a m a l i g n a n t brain tumor believed to be d e r i v e d from the e x t e r n a l germinal layer of the cerebellum. Northern and western analyses were performed on tumor samples (n=ll) which were snap frozen in the operating room to preserve RNA and protein integrity. All of the tumors were found to express mRNA encoding neurotrophin-3 (NT-3) and its cognate r e c e p t o ~ trk C. Due to limited tissue, 3 tumor samples were t e s t e d and found to have NT-3 protein and trk immunoreactivity. Patients with tumors expressing high levels of trk C mRNA had significantly longer intervals without disease progression than those with low levels (KaplanMeier, P=.02). Expression of trk A, trk B, or p75 was not associated with favorable disease outcome. C o o r d i n a t e expression of ligand and receptor implies the constitutive a c t i v a t i o n of trk C receptors in patients w i t h a more f a v o r a b l e outcome. Conceivably, this activation promotes the differentiation of medulloblastoma and may be relevant to the development of new therapeutic strategies for the tumor. At minimum, our findings i n d i c a t e that trk C expression is a marker of prognostic value.

Ras mediated signal transduction pathway in human estrocytoma cell lines: A Guha1'2, N Lau2, A Pawson2:1-Div. of Neurosurgery & Surgical Oncology, Univ. of Toronto; 2Lunenfeld Research Inst., Univ. of Toronto, Toronto, CANADA Platelet-Derived Growth Factor (PDGF) and Epidermal Growth Factor (EGF) may stimulate the growth of human astrocytomas, by activation of the ras signal transduction pathway. Malignant human astrocytoma cell Ilines were examined for expression and activation status of PDGF and EGF receptors. Complexes of the receptors with Shc and Grb2 (upstream activators of ras) were evaluated with immunoprecipitations and western blotting. Two of the four cell lines expressed constitutively activated PDGF-a receptors, but only one of the four lines had constitutively activated PDGF-b receptors, though it was expressed by all lines. EGF receptors were expressed by all four but constitutively active in three of the cell lines. With PDGF or EGF stimulation, the receptors complexed with Shc, which became phosphoryllated and bound Grb2. Blocking ras signalling using the Ash17 ras dominant-negative mutant, induced with a metallothionine promoter, decreased growth of astrocytoma cells as seen on colony formation assay. Stable clones are being characterized for both in-vitro and in-vivo growth. If our results show that activation of ras is vital to the proliferative signal in astrocytomas, reported pharmacological methods of blocking ras may be useful in decreasing the growth of astrocytomas in the future.

137 Epidermal growth factor is mitogenic for a human glioblastoma cell line (Ru-O-2A/Gbl) with properties of the oligodendrocyta-type 2. astronyte (O-2A) lineage Ute Engel, Nick J. Gutowski , Karen Bevan and Mark Noble, Ludwig Institute for Cancer Research, London, U.K. We have studied the cell biology of a human glioblastoma derived cell line (Hu-O-2A/Gbl) and have applied immunohistochemical and cell culture techniques which have been established for rodent glial precursor cells I. The Hu-O-2A/Gbl derived cell population has been grown for up to 19 passages and its behaviou~ has been investigated in different growth factor conditions - in astroctye conditioned medium, in chemically defined medium with additional platelet derived growth factor and epidermal growth factor (EGF). The antigenic profile of the cells in each growth condition was examined using rabbit anti-GFAP and the mouse monoclonal antibodies A2B5, O1, 04, anti GalC and anti-EGF-receptor. We have found that these cells resemble rodent O-2A cells An some respects, including antigenic phenotype and morphology and their response to different defined growth factors: We found that EGF enhances the proliferation of Nu-O-2A/Gbl cells and that EGF either promotes more cells to bind the A2B5 mAb or preferentially stimulates the proliferation A2B5+ cells. In chemically defined medium with EGF the cells showed the highest rate of BrdU-incorporation, responding accordingly to various concentrations of EGF and double their division rate after 3 days in culture at a distinct cell density. Using the confocal microscopy we show that all Hu-O-2A/Gbl cells bind an anti-EGF receptor antibody. These experiments have provided new insights in the biology of human glioma-derived cell lines. 1 Raff, Miller and Nobel (1983) Nature, 303:390-396.

Uroklnase Gene Expression Marks the Malignant Astrocyte and Localizes to the Invading Tumor Margin In Situ. C.L. Gladson, V. Pijuan, M.A. Olman, G.Y. Gillespie and I. Yacoub, University of Alabama at Birmingham, Birmingham, Alabama, U.S.A. Tumor cell protease secretion is known to be involved in tumor cell invasion. In order to understand the role urokinase (U-PA) and the urokinase receptor (U-PAR) play in malignant astrocytoma cell invasion of normal brain, expression of U-PA and U-PAR mRNAs were investigated in astrocytoma and non-tumorous brain biopsies, by riboprobe in situ hybridization. We found U-PA and U-PAR mRNA expression in 8 of 8 glioblastoma multiforme tumors; while in contrast, U-PA mRNA was not expressed in 8 non-tumorous brain biopsies nor in 4 low grade astrocytorna brain biopsies. U-PAR mRNA was expressed by astrocytes in 1 of 8 non-tumorons brain biopsies and by astrocytoma cells in 1 of 4 low grade astrocytomas (Table 1). Consistent with the gene expression, U-PA and U-PAR proteins were expressed by malignant astrocytas in 5 of 5 glioblastoma multiforme tumors, analyzed by immunohistochemistry on cryostat sections. In vitro, adherent human U-251MG malignant astrocytoma cells expressed U-PA and U-PAR proteins at sites of cell-matrix contact (focal contacts), when plated in serum free media. To study the tumor margin, U-251MG cells were propagated intraeerebra]ly in a scid mouse xenograff (28 days), and U-PA mRNA and protein were found to localize to the invading tumor margin, while U-PAR mRNA was expressed throughout the tumor. These data indicate that U-PA expression marks the malignant astrocyte phenotype, and that U-PA may play a role in malignant astrocytoma invasion of normal brain. TABLE l. U-PA AND U-PAR EXPRESSION IN ASTROCYTOMAS U-PA mRNA U-PAR mRNA Non-tumorous Brain (0/8) (1/8) Astrocytoma (0/4) (1/4) Anaplastic Astrocytoma (2/4) (4/4) Glioblastoma Multiforme (8/8) (8/8)

Role of calcium signal t r a n s d u c t i o n in glioma progression T. Yamasaki, K. Enomoto, K. Moritake, Y. Akiyama, M. Kawahara, a n d T . Maeno, Departments of Neurosurgery and Physiology, Sh}mane Medical University, Izumo 693, Japan Role of calcium signal transduction in glial tumor progression was investigated in vitro by using a methylcholanthrene-induced mouse malignant glioma cell line. A fura-2 fluorescence image of the cell line showed a 4-to 8-fold increase in intracellular calcium level with tumor proliferation. The calcium oscillation spread to adjacent ceils with a speed and distance of 20 Fzm/sec and up to 200/zm, respectively. The calcium transmission among surrounding cells was noted even in a cell that was separate from the initially stimulated cell, provided the distance was within 200# m. Microinjection of Lucifer yellow indicated no gap junction between cells. Depletion of extraceLlular calcium ion inhibited intrace[}ular calcium rise and its transmission to neighboring cells. Microinjection of inositol trisphosphate onto the cytoplasm accelerated calcium mobilization from internal stores. An intracellular calcium blocker, TMB-8, eliminated the cytosolic calcium oscillation in initially stimulated cells, while it had no effect on calcium diffusion to surrounding cells. Antagonists of voltage-dependent calcium channels, nifedipine and verapam[1, exhibited no action on the calcium response. This suggested that calcium transmission is triggered by some releasing factor, which may be ejected from initially stimulated cells, and mediated via a membrane receptor but not through a gap junction. Taken together, calcium signal transduction in glioma progression may be related to both an influx of extracellular calcium and a redistribution of intracellular calcium from internal stores, while calcium propagation to neighboring cells may involve calcium influx alone.

The 18A2/mtsl metastasis-associated gene and the TIMP2 gene are expressed and down-regulated, respectively, in invasive human glioma cell lines ~. A. Merzak 1, C. Parker2, S. Kooeheckpour1, G.V. Sherbet2and G.J. IKlkington1. Department of Neuropathology, Institute of Psychiatry, De Crespigny Park, Denmark Hill, London SE5 8AF, UK. z Cancer Research Unit, The Medical School, University of Newcastle upon Tyne, Newcastle upon Tyne, NE2 4HH, UK. 8 ~tm-porosity polyearbonate filters in modified Boyden chambers, coated with the reconstituted extracellular matrix composite, Matrigel, were used to measure the invasiveness of eight human glioma-derived cell lines and human fetal brain cells. With the exception of one low grade glioma derived cell line, all lines studied proved to be invasive, while normal fetal brain cells failed to invade. This invasive potential was independent of the histological grade of the turnout from which the cell lines originated. In addition, the expression of the metastasisassociated gene 18A2/mtsl as well as the tissue inhibitor of metalloproteinases-2 (TIMP-2) was analysed in each of the gllomaderived cell lines. The 18A2/mtsl was expressed in all the cell lines studied with the exception of fetal brain cells and the low grade non invasive glioma-derived IPRK-7 cell line. The 18A2/mtsl related genes coding for the S100 subfamily of calcium binding proteins were found to be both differentially and over-expressed in invasive cell lines. TIMP-2 was expressed only in non invasive cell lines. These results suggest that the 18A2/ratsl and TIMP-2 genes could play an important role in the invasive behaviour of human glioma ceils in vitro. " Supported by a travel bursary from the Journal: Neuropathology and Applied Neurobiology. This work was supported by the Leverhulme Trust.

138 Non-expression

of

CD15

precludes

in vitro a d h e s i o n

of

neoplastic gila to cultured endothelial cells: a possible barrier to metastasis. K. Martin, J. Akinwunmi, H.K. Rooprai, A. Kennedy, A. Linke, N. Ognjenovic and G.J. Pilkington Intrinsic brain turnouts are charactcrised by diffuse local invasion and an apparent inability to metastasise. We believe that the absence of the adhesive carbohydrate epitope CD15 on human glioma cells may help to explain the general failure of primary brain tumours to metastasise by precluding adhesion of circulating neoplastic glia to 'targef organ endothelia. We also postulate that the presence of CD 15 on cultured experimental rat glioma cell lines contributes to their documented metastatic potential and casts doubt on the utility of these cell lines as a model for human primary brain tumours. CDI5 functions as a ligand for the seleetin family of cytoldne-inducible glycoproteins on endothelial cells. Selectincarbohydrate interactions have been implicated in the metastatic spread of cancer ceils. We immunostained a series of cultured intrinsic human brain tumours, three cell lines derived from experimental rat gliomas, two metastatic carcinoma cell lines and human umbilical vein endothelial cells (HUVEC) using MAbs which reeognise CD15. Expression of the seleetin CD62 by HUVEC was confirmed by immunocytocbemistry. The animal glioma cells, HUVEC and carcinoma cells were all strongly positive for CD15, whereas the human gliomas showed limited positivity. Chromium radiolabelling binding assays of CD15positive and -negative cell lines, using HUVEC as an attachment substrate, were carded out in the presence and absence of CD15 antibody. The level of adhesion of neoplastic cells to HUVEC corresponded to CD15 expression and CD15 antibodies reduced adhesion. This w o r k was s u p p o r t e d by the Association for International Cancer Research and the Leverhulme Trust.

Q u a n t i t a t i v e analysis of DNA topoisomerase I activity in camptothecin-ll -seisitive and -resistant glioma cells T.Fujiwara, Y.Matsumoto, K.Miyake, M.Shin, S.Nagao, Department of Neurological Surgery, Kagawa Medical School, Kagawa, Japan. Camptothecin-1 I(CPT-11) is a new derivative of camptothecin, a plant alkaloid antitumor agent. Antitumor activity of CPT-11 is considered to be mediated through interaction of the drugs with its target enzyme, DNA topoisomerase I(topo I). In this study, we studied the relation between sensitivityto CPT-11 and topo I activity of glioma cells. Farthermore, we established CPT-I 1 resistant cell lines in order to elucidate the potential mechanisms of drug resistance. We used t 1 glioma cells grown from specimens obtained at surgery and 2 glioma cell lines(C6 and T98G). CPT-11 resistant cells (C6/CPT11, T98G/CPT-11) were selected by stepwise exposure to the drug. Topo 1 activity was determined by the relaxation of supercoiled Escherichal coli DNA(pBR32). The sensitivity of these cells was examined by the MTT assay. Among the glioma ceils from biopsy specimens, activity of topo I in the CPT-l 1-sensitive group tended to be higher than that in the CPT-11resistant group. Topo[ activity may serve as a novel marker to predict the sensitivityto topo I inhibitors. CPT- 11-resistant cell lines C6/CPT- 11 and T98G/CPT-11,respectively, exhibit 5.4 and 7.3 fold increase in resistance to CPT-11. No differences in topo I activity of CPT-11 ,howevere, were observed between the parent and CPT-II resistant lines. On the other hand, topo I from resistant lines was 2 to 4 fold resistant to the inhibitory effect of the CPT-11 on the relaxation activity of topo I, in comparison with their parent lines. This enzymological difference may be responsible for the resistance to CPT-11.

ANALYSIS OF LEUCINE AMINOPEPTIDASE AND CELLULAR NDP KINASE LEVELS AS PROGNOSTIC MARKERS OF BRAIN TUMOUR INVASIVENESS AND METASTASIS. G. Pulido-Cejudo3, K. Jamison3, B. Lach2, H. Hugenhaltz1 and J. Campione-Pieeardo 3. 1Department of Neurosurgery, Ottawa General Hospital. 2 Department of Pathology, Ottawa Civic Hospital. 3National Laboratory for Viral Oncology,LCDC Canada, K1A 0L2. The quantitative evaluation of interdependent cellular markers is imperative to assist

patient prognosis and appraisal of tamour

malignancy. In cultures obtained from primary (astrocytomas/gliomas) and metastatic (breast /lung) tumours, cellular NDP-Kinase (NDPK) activities together with serum levels of Lencine Aminopeptidase (LAP) were determined to asses tumour malignancy. Based on both the net increase in seric LAP exopeptidase activity and tumanr histopathology, three grades of tumanr malignancywere tentatively defined: TUMOUR GRADE LAP ACTIVITY (U/lag) 1. Highly malignant/metastatic 1 X 10-5 to 2 X 10-4 2. Malignant/metastatic

2 X 10-6 to 1 X 10-5

3. Poorly malignant/metastatic 1 X 10"7 to 2 X 10-6 NDPK activities were determined in gemistocytic astrecytomas, glioblastoma multifarme, metastatic breast and lung carcinomas ranked as grades 1-2. A greater proportion of stromal NDPK was found in comparison with their corresponding cytosolic fractions when reacted with anti-NDPK antibodies. Interestingly, NDPK activity staining after native PAGE as well as total enzyme activity revealed that only cytosolic fractions contained NDPK activity. In contrast, negligible NDPK enzyme activity levels were found in the stroma. Furthermore, cytasolic NDPK activity staining showed three distinct species in comparison to a single cytasolic form detected in benign tumours. Hence, based on seric LAP levels as well as on NDPK activity, distribution and oligomeric composition of cytosolic NDPK, a quantitative appraisement of tumanr malignancy can be achieved. In conclusion, malignant and metastatic brain tumours possess a greater proportion of stromal inactive NDPK in comparison to a less abundant but active cytosolic enzyme.

Secretion of metalloproteinases by h u m a n brain t u m o u r s in vitro: modulation by e x o g e n o u s gangliosides. S . L M a i d m e n t , A . M e r z a k , S. K o o e h e c k p o u r , H.K. R o o p r a i and G.J. Pilkington

Matrix metalloproteinases (MMPs) are a large family of zincdependent endopeptidases with broad affinities for ECM proteins. The family includes type I eollagena~e, the type IV collagenases (gelatinases) and the stromelysins; secretion of metalloproteinases by neoplastic ceils is thought to facilitate invasion and metastasis. Gangliosides are a family of sialic acidcontaining glycosphingolipids found in the membranes of all eukaryotie cells, being particularly abundant in the CNS. The ganglioside profiles of CNS neoplasms have been found to differ from normal CNS tissue in that a higher proportion of the structurally simpler gangliosides are expressed. In this study we have employed two SDS-PAGE techniques to assess the effect that incubation of cells with a range of different gangliosides has upon the secretion of four different MMP enzymes. The cell-conditioned media f~om eight different cell lines derived from inttinsie brain turnouts of varying histological type and grade of malignancy served as the source of MMPs for this study. We conclude that all six gangliosides tested (GM1, GM3, GD3, GDla, G D l b and GTlb) substantially increase the secretion of all four MMPs investigated in all eight of the cell lines used. This w o r k was supported by the Association for International C a n c e r Research and the L e v e r h u l m e Trust.

139 Thyroid hormone induced morphological changes in a human ependymal tumor established as a cell line C. Lins, C.M. Takyia, J. Garcia-Abreu, F.F. Rodrigues, F. Duarte, C. Chagas, H. Chneiweiss, V. Moura Neto Insituto de Biofisica Carlos Chagas Filho, UFRJ, Brasil Hospital Universitario Clementino Fraga Filho, UFRI, Brasil INSERM Ul14, College de France, Paris, France

Ependymal ce!ls exhibit a variety of distinct morphological features: cilia, central pore on the apical surface, microvilli and secretory structures. There are three major morphological types of ependymoeytes: ciliated cuboidal or columnar cells, tanycytes and small nonciliated secretory calls. Ependymal tumors can show these different subtypes, which are positively immmunostained for the cytoskeletal protein vimentin. We have analyzed a human ependymal tumor maintained in culture, using either explants or dissociated tumor cells. Although cilia and gland-like vesicles were observed in these cells by electronic microscopy, more than 90% of plated dissociated cells showed a flat or ovoid morphoIogy and the tanycyte-like morphology was infrequent. Cells cultivated in the presence of the Thyroid hormone (T3) exhibited a progressive morphological transformation from flat shaped to process-bearing cells. The same transformation was obtained in normal rat glial cells and related to an indirect effect of T3, most likely going through the release of cytokines, themselves responsible for the morphological change. This suggests that like astroglia, tumoral ependymocytes are able to release T3-induced factors. These cytokine-like molecules released by the cells can play an important role in the morphology, proliferation and differentiation of ependymal tumor cells.