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@ 1990 The Japanese Society of Pathology

Ganglioglial Differentiation in Medulloblastoma

Motoshige Kudo', Masakuni Shimizu2s3, Yasuhiko Akutsu2~*, Hisatoshi lrnaya2s5, Mau Nan Chen2, and Myota Miura2

A case of cerebellar medulloblastoma with clusters of mature ganglion cells and glial cells is described. The patient, a 15 -year -old girl, underwent three operations followed each time by radiation and chemotherapy during the four-year clinical course. Histologically, the ganglion cells were clearly identifiable by their abundant eosino- philic cytoplasm, round nuclei with prominent nucleoli, tigroid granules, and argyrophilic fibrils and axons. Im- munohistochemically, the cells were NSE- and NF-positive, and ultrastructurally they contained abundant tubules and filaments, neurosecretory granules and well developed rough endoplasmic reticulum. There were many cells transitional in appearance between primitive cells and mature ganglion cells. The tumor also had many mature yet atypical astrocytes and oligodendrocytes. The exact mechanism of the extensive neuronal and glial maturation of medulloblastoma cells is unclear, but the repetitive surgical interventions, radiation and chemotherapy might have had certain cytostatic effects on rapidly dividing medulloblastoma cells, giving them a chance to mature into postmitotic cells with potential for neuronal and glial differentiation. Acta Pathol Jpn 40 : 50-56, 1990.

Key words : Cerebellar medulloblastoma, Light microscopy, Ganglioglial maturation, lmmunohistochemistry

INTRODUCTION

Cerebellar medulloblastoma has been recognized as a distinct clinicopathologic entity. However, the question of whether the medulloblastoma possesses the potential to differentiate into neurons and/or glia has long been

Received July 4, 1989. Accepted for publication September 22, 1989. 'Department of Pathology, Toho University School of Medi- cine, Tokyo. 2Departments of Neurosurgery and Pathology Kosei General Hospital, Tokyo. Present address : 3Dept. of Plastic and Reconstructive Sur- gery, Toho University, 'Dept. of Neurosurgery, 5Dept. of Pathology, Nippon Medical School, Tokyo. Mailing address: Motoshige Kudo, M.D. ( I ~ ~ ~ ) , Depart- ment of Pathology, Toho University School of Medicine, Omori, Ota-ku, Tokyo 143, Japan.

debated, together with the histogenesis. Using conven- tional histological methods, there is no obvious evidence of neuronal and/or glial differentiation in most cases of medulloblastoma. Therefore, in recent years, immuno- histochemistry has been extensively performed for the identification of neuronal and/or glial differentiation in medulloblastomas, and for estimating its frequency and extent (1 -10).

We recently encountered a cerebellar medulloblastoma showing extensive maturation to adult ganglion cells and glial cells following repetitive surgical interventions, radi- ation and chemotherapy during the four-year clinical course. This report demonstrates unequivocal ganglion cells and glial cells in the medulloblastoma using conven- tional light microscopy and immunohistochemistry. Although the appearance of ganglion cells has been rarely described in medulloblastomas (9-1 3), the present case seems exceptional in the degree of maturation, number and distribution of the ganglion cells.

CASE HISTORY

The patient, a 15-year-old girl, was first admitted to the Kosei General Hospital, Tokyo, on September 6, 1983, with complaints of tumbling, general malaise, headache, nausea and vomiting. Physical examination disclosed that she also suffered from hypotonus, truncal ataxia, a positive finger to nose test, and a choked optic disc. Consciousness, eye movement, and pupil size were normal. CT examination showed a tumor in the cerebel- lar vermis with enhancement effects and mild hydroceph- alus (Fig. 1). Angiography showed the tumor to be avascular. On September 16, 1983, a subtotal (about two/thirds) tumorectomy was performed. The patho- logic diagnosis was medulloblastoma. Radiation ther- apy and chemotherapy were then carried out. Total amounts of radiation were 50 Gy (1.6 Gy/day) to the whole brain, and 30 Gy (1.6 Gy/day) to the spinal canal. Chemotherapy was performed twice with vincristine (0.3 mg and 0.2 mg iv) and twice with ACNU (30 mg and 20

Acta Pathologica Japonica 40 (1): 1990 51

Figure 1. CT scan on admission showing tumor in cerebellar vermis with hydrocephalus.

mg iv). She was discharged with no residual tumor evident by CT on December 16, 1983. A follow-up CT examination in November, 1985, showed tumor recur- rence in the vermis, despite intermittent chemotherapy. A second operation was performed on January 17, 1986. The pathologic diagnosis was recurrent medulloblas- toma. Postoperatively, radiation therapy with a total dose of 49.6 Gy was carried out. Vincristine (0.75 mg iv) and ACNU (100 mg iv) were also administered once each. She was discharged with residual tumor (about 2 cm in size) as determined by CT on March 31, 1986. She was followed up in the outpatient clinic on medica- tions which included vincristine (0.75 mg iv) and ACNU (100 mg iv) five times until January 11, 1987, when she experienced headache, vomiting and tonic seizures. Because of the tumor regrowth in the cerebellar vermis areas, a third operation was carried out. The pathologic diagnosis was cerebellar medulloblastoma with neuronal and glial differentiation. She received postoperative radiation (total dose, 44.8Gy) and ACNU (100 mg, iv, once) chemotherapy until April 9, 1987, when she was dischargsd. Thereafter, however, her clinical course continued to worsen, and CT examination indicated tumor extension to the third ventricular wall and a moderate degree of hydrocephalus. She died on July 30, 1987. Permission for autopsy was not granted.

MATERIALS AND METHODS

Tissues were fixed in 10% formalin, embedded in paraffin, and stained with hematoxylin-eosin (HE), Bodian stain, phosphotungstic acid-hematoxylin (PTAH), cresyl violet (Nissl stain), Masson’s trichrome stain, and silver stain for reticulin fibers. Peroxidase- anti peroxidase (PAP) immuno histoc hemist ry was a Is0

carried out. The primary antibodies and their dilutions used were anti-neurofilament (NF), 1 : 4 (purchased from Ortho, Tokyo), anti-neuron-specific enolase (NSE), 1 : 200, anti-$100 protein, 1 : 400, anti-glial fibrillary acidic protein (GFAP), 1 : 300, anti-myoglobin, 1 : 400 (all from Dakopatts, Glostrup, Denmark), and anti- myelin basic protein (MBP), 1 : 1,200, a gift from Dr. T. Noguchi, Department of Physiology, Toho University School of Medicine, Tokyo. As a negative control, nor- mal serum was used instead of these primary antibodies. Various tissues previously known to be positive for each antibody were used as positive controls. Furthermore, an ultrastructural study was attempted using tissue blocks embedded in paraffin. The blocks were deparaffinized and again fixed in 1% osmium tetroxide, dehydrated in a graded ethanol series and propylene oxide, and then embedded in Epon 812. Semithin sec- tions were made and stained with toluidine blue. Ultrathin sections were made, stained with uranyl ace- tate and lead citrate, and observed with a Hitachi H-600 electron microscope.

PATHOLOGIC FINDINGS

Light microscopic study

The first and second surgical specimens were very similar and the highly cellular tumors were composed entirely of primitive undifferentiated cells. Both speci- mens contained numerous Homer-Wright rosettes and mitotic figures (Fig. 2). The stroma was very scanty and fibrovascular except for multifocal necrosis and scars. No desmoplasia was present, and no reticulin fibers were found between tumor cells.

The specimens obtained from the third operation also showed a highly cellular tumor but apparently differed from the previous two operative specimens due to the presence of well mature, often gigantic, ganglion cells. The ganglion cells were composed of abundant eosino- philic cytoplasm with tigroid granules and large round nuclei with prominent nucleoli, and they were usually aggregated in large clusters and often also mingled, singly or in small groups, with the primitive cells (Figs. 3a, b). Tigroid granules were usually lost from the central part of each cell. Ganglion cells with two or more nuclei were frequent. Mitotic figures, mostly atypical, were scattered. Bodian stain clearly demon- strated prominent neurofibrils and axons in the cells (Fig. 3c). In addition, they showed various degrees of im- munohistochemical positivity for NSE (Fig. 3d) and NF (Fig. 3e), but were negative for GFAP, S-100 protein and myoglobin. Many cells transitional in appearance between primitive cells and mature ganglion cells were

52 Ganglioglial Maturation in Medulloblastoma (Kudo et a/.)

Figure 2. Two previous surgical specimens are very similar and entirely composed of undifferentiated primitive cells with Homer- Wright rosettes. First surgical specimens. (HE).

Figure 3. a. Third surgical specimens contain mature ganglion cells often mingled with primitive cells. (HE). b. Mature ganglion cells are usually aggregated in large clusters and possess abundant cytoplasm, tigroid granules and round nuclei with prominent nucleoli. (HE). c. lntracytoplasmic neurofibrils and axons are evident in ganglion cells. (Bodian stain). d. Ganglion cells are positive for NSE. (NSE immunostain).

Acta Pathologica Japonica 40 (1): 1990 53

observed, containing varying a mounts of cytoplasm, spindle- to oval-shaped nuclei with less prominent nu- cleoli, and axons (Figs. 3f, g).

Another type of tumor cell was found in both ganglion cell clusters and primitive cell areas. These cells were strongly positive for GFAP and considered to be astro- cytic. The population and appearance of astrocytes varied considerably from area to area. Some clusters of ganglion cells or primitive cells contained few astrocytes, whereas other clusters possessed numerous astrocytes both singly and in groups (Figs. 4a, b). The astrocytes often possessed large, hyperchromatic atypical nuclei and pleomorphic, often abundant plump cytoplasm. Others were indistinguishable in appearance from the surrounding unstained primitive cells except for the presence of varying amounts of GFAP-positive cyto- plasm. Multinucleated astrocytes were found occasion- ally. These astrocytes were considered to represent astrocytic differentiation of medulloblastoma cells. In addition, the astrocytes in and near the peripheral tumor- infiltrating areas had oval vesicular nuclei and relatively

Figure3. e. Ganglion cells are positive for NF. (NF immuno- stain). f. Cells transitional to mature ganglion cells are evident. (HE). g. Transitional cells possess argyrophilic fibrils and axons. (Bodian stain).

well developed cytoplasm and processes. They were considered to be preexisting ordinary reactive astrocytes entrapped in the tumor.

The presence of oligodendroglia was also suggested in conventional histologic sections. In some areas there were groups of relatively small cells with round nuclei and some cytoplasm, often with perinuclear haloes. lmmunohistochemically, many of these cells were MBP- positive, and were also scattered singly or in groups throughout (Fig. 4c). Some of the MBP-positive cells possessed nuclei similar to those of the surrounding MBP-negative cells. No apparent features suggestive of ependymal differentiation were observed in the speci- mens.

There were widespread areas of multifocal necrosis, hemorrhage, and fibrohyalinous scars with calcium deposits. Traversing, thin to thick strands of reticulin and/or collagen fibers were noted along with fine capil- lary networks in the clusters of ganglion cells. Vascular walls were generally hyalinized. No desmoplasia was present.

54 Ganglioglial Maturation in Medulloblastoma (Kudo et a/.)

Electron microscopic study

Preservation of the specimens was poor, but fortunate- ly certain organelles were preserved well enough to permit recog nit ion. Ganglion cells contained numerous tubular structures (about 18-24 nm in diameter), filaments (about 8-10 nm in diameter), and well devel- oped rough endoplasmic reticulum. Dense cored gran- ules were also found, and with some reservations they were considered to be neurosecretory granules, partly because of their poor preservation. The neuropil was well formed in the surrounding areas. Astrocytes pos- sessed abundant glial filaments (about 8-10 nm in diam- eter).

DISCUSSION The present case appears exceptional among medullo-

blastomas in terms of the degree of maturation, number and distribution pattern of ganglion cells. The unique distribution pattern of ganglion cells with transitional

Figure 4. a. Astrocytes are observed in ganglion cell clusters, often being uni- and bipolar between ganglion cells. (GFAP immunostain). b. Astrocytes are often indistinguishable from surrounding GFAP-negative primitive cells except for their GFAP-positive cytoplasm. (GFAP immunostain). c. Oligoden- droglia-like cells are positive for MBP. (MBP immunostain).

cells and mitotic activity precludes the interpretation that the neurons previously existed in the host tissue.

The ganglion cells were clearly shown by classical HE and silver impregnation stainings, showing abundant eosino philic cytoplasm co nta ining uneq uivoca I t ig roid granules, round nuclei with prominent nucleoli, and argyr- ophilic fibrils and axons. Their neuronal nature was further confirmed by immunohistochemical and electron microscopic studies. They were positive for NSE and NF, and possessed organelles such as neurotubules and filaments, neurosecretory granules, and well developed rough endoplasmic reticulum. Even though many of the ganglion cells appeared well mature, the results of Bodian staining suggested that some were abnormal, because it is known that Bodian stain does not usually reveal intracytoplasmic fibrils in ganglion cells unless the neurofibrils are physically and/or chemically altered.

On the other hand, modern immunohistochemistry was required for more conclusive assessment of glial differentiation, as was the case, in the present tumor, although there was evidence suggestive of glial

Acta Pathologica Japonica 40 (1): 1990 55

differentiation b y HE sta in ing. The present immunohistochemical findings are similar to those previ- ously reported (1-8, 10). Some of the GFAP-positive cells could be considered to be preexisting non-neoplastic astrocytes entrapped in the tumor because of their morphological similarity to the reactive astrocytes in gliotic areas of the same specimens. However, many other GFAP-positive cells exist, which are often mor- phologically indistinguishable f rom the surrounding un- stained primitive cells except for GFAP positivity. These atypical GFAP-positive cells with abundant cytoplasm and irregular, bizarre, hyperchromatic nuclei with occa- sional multinucleation, appear to fulfill the stringent criteria suggested earlier for astrocytic differentiation in medulloblastomas (1, 5, 8, 10). I n addition, the astrocytes in ganglion cell clusters could be considered to be the result of simultaneous bidirectional differentiation of medulloblastoma cells toward glia and neurons.

MBP is known to be a marker of myelin-forming glia, namely oligodendroglia, in the developing brain (14, 15), and is a possible marker of oligodendroglioma cells (16). The purity and specificity of the anti-MBP serum used in the present study has already been confirmed (17, 18). Therefore, together with the oligodendroglia observed by HE staining, the presence of MBP-positive cells strongly suggests that the present tumor had oligodendroglial differentiation as well, which has been documented in medulloblastomas (1 0, 12, 13).

The exact mechanism of the unusual extensive matura- tion to adult ganglion cells and glial cells in the present case is not clear. Nevertheless, since surgical interven- tions, radiation and chemotherapy had been carried out repetitively over the four-year clinical course, one may speculate that such therapeutic procedures must have produced cytostatic effects, particularly on the cell kinetics of rapidly dividing medulloblastoma cells (1 9, 20), thus giving a maturing growth advantage to the postmitotic cell population with potential for neuronal and glial differentiation. Incidentally, the presence of fibrous tissue that had undergone almost complete hyalinization with focal calcification, and tumor cells apparently undergoing multinucleation and giant cell formation may, to some extent, suggest the existence of radiation effects in the present case.

In brief, the present case strongly suggests that medulloblastoma is a primitive neuroectodermal tumor possessing the potential to differentiate to both neurons and glia.

Acknowledgements : The authors thank Dr. Tetsuya No- guchi for supplying anti-MBP serum, Ms. Machiko Mizutani, Mrs. Yoko Maeda and Mrs. Toshie Shimozeki for technical

assistance, Mr. Tadahiko Naoe and Mr. Jikoku Kuroda for photographic assistance, and Mr. Steven McKay and Dr. Sumie Shimizu for help in manuscript preparation.

REFERENCES 1.

2.

3.

4.

5.

6.

7.

8.

9.

10.

11.

12.

13.

14.

Mannoji H, Takeshita I, Fukui M, Ohta M, and Kitamura K. Glial fibrillary acidic protein in medulloblastoma. Acta Neuropathol (Berl) 55: 63-69, 1981. Palmer JO, Kasselberg AG, and Netsky MG. Differentiation of medulloblastoma. Studies including immuno histochemica I localization of glia I fibrillary acidic protein. J Neurosurg 55: 161-169, 1981. Rorke LB. The cerebellar medulloblastoma and its relationship to primitive neuroectodermal tumors. J Neuropathol Exp Neurol 42: 1-15, 1983. Roessmann U, Velasco ME, Gambetti P, and Autilio- Gambetti L. Neuronal and astrocytic differentiation in human neuroepithelial neoplasms. An immunohisto- chemical study. J Neuropathol Exp Neurol 42 : 113- 121, 1983. Schindler E and Gullotta F. Glial fibrillary acidic pro- tein in medulloblastomas and other embryonic CNS tumours of children. Virchows Arch [Pathol Anat]

Dickson DW, Hart MN, Menezes A, and Cancilla PA. Medulloblastoma with glial and rhabdomyoblastic differentiation. A myoglobin and glial fibrillary acidic protein immunohistochemical and ultrastructural study. J Neuropathol Exp Neurol 42: 639-647, 1983. Herpers MJHM and Budka H. Primitive neuroectoder- ma1 tumors including the medulloblastoma : Glial differentiation signaled by immunoreactive for GFAP is restricted to the pure desmo pla st ic medullo blastoma ("arachnoidal sarcoma of the cerebellum"). Clin Neu- ropathol 4 : 12-18, 1985. Kumanishi T, Washiyama K, Watabe K, and Sekiguchi K. Glial fibrillary acidic protein in medulloblastomas. Acta Neuropathol (Berl) 67 : 1-5, 1985. Velasco ME, Ghobrial MW, and Ross ER. Neuron- specific enolase and neurofilament protein as markers of differentiation in medulloblastoma. Surg Neurol

Burger PC, Grahmann FC, Bliestle A, and Kleihues P. Differentiation in the medulloblastoma. A histological and immunohistochemical study. Acta Neuropathol (Berl) 73: 115-123, 1987. Kane W and Aronson SM. Gangliogliomatous matura- tion in cerebellar medulloblastoma. Acta Neuropathol (Berl) 9: 273-279, 1967. Rubinstein LJ. Tumors of the central nervous system : Atlas of tumor pathology, 2nd Series, Fascicle 6. Armed Forces Institute of Pathology, Washington, D. C., 1972. Rubinstein LJ. The cerebellar medulloblastoma : Its origin, differentiation, morphological variants, and bio- logical behavior. In Vinken PJ and Bruyn GW, eds. Handbook of clinical neurology, Vol. 18. American Elsevier, New York, 167-1 93, 1975. Hartman BK, Agrawal HC, Kalmbach S, and Shearer WT. A comparative study of the immunhistochemical localization of basic protein to myelin and oligoden-

398: 263-275, 1983.

23: 177-182, 1985.

56 Ganglioglial Maturation in Medulloblastorna (Kudo et a/.)

drocytes in rat and chicken brain. J Comp Neurol

15. ltoyama Y, Sternberger NH, Kies MW, ef a/. Im- munocytochemical method to identify myelin basic protein in oligodendroglia and myelin sheaths of human nervous system. Ann Neurol 7 : 157-166, 1980.

16. Tanaka J, Hokama Y, and Nakamura H. Myelin basic protein as a possible marker for oligodendroglioma. Acta Pathol Jpn 38: 1297-1303, 1988. Noguchi T, Sekiguchi M, Sugisaki T, Tsukada Y, and Shimai K. Faulty development of cortical neurons in the Snell dwarf cerebrum. Dev Brain Res 10: 125-

188: 273-290, 1979.

17.

138, 1983. 18. Kudo M and Noguchi T. lmmunoreactive myelin basic

protein in tumor cells associated with carcinomatous neuropathy. Am J Clin Pathol 84: 741-743, 1985.

19. Venes JL, Mclntosh S, OBrien RT, and Schwartz AD. Chemotherapy as an adjunct in the initial management of cerebellar medulloblastomas. A preliminary report. J Neurosurg 50: 721-724, 1979. Hoshino T, Kobayashi S, Townsend JJ, and Wilson CB. A cell kinetic study on medulloblastomas. Cancer 55 :

20.

1 71 1-1 7 1 3, 1985.