Child's Nerv Syst (1992) 8:326-331 mllN$ �9 Springer-Verlag 1992

Age-related immunoreactivity pattern in medulloblastoma Stephan Patt and Claus Zimmer

Institut fiir Neuropathologie, Freie Universit/it Berlin, Klinikum Steglitz, Hindenburgdamm 30, W-1000 Berlin 45, Federal Republic of Germany

Received December 13, 1991/Revised March 20, 1992

Abstract. Thirty-five paraffin-embedded medulloblastomas (19 from children and 16 from adults; 24 classic medul- loblastomas, 10 desmoplastic medulloblastomas, 1 tumor with neuronal differentiation) were examined for reac- tions with antibodies against glial fibrillary acidic protein (GFAP), cytokeratins KL1 and M N F l l 6 , desmin, and vimentin. Only the tumor from the youngest patient, a 152-day-old boy, showed a positive immunoreact ion for cytokeratins. Because of this age-related expression of cytokeratins in medulloblastomas primarily in very young children, cytokeratin positivity was interpreted as a sign of tumor immaturity. Five medulloblastomas showed scattered GFAP-posi t ive reactive astrocytes and/or other positive, probably neoplastic, cells. Only two tumors showed GFAP immunoreact ivi ty in unequivocally neo- plastic cells. Of six tumors that reacted with vimentin, three showed strong reactivity throughout, one being the tumor from the 152-day-old boy. The remaining three demonstrated nests of vimentin-positive cells with weak or intense somatic immunoreact ivi ty for vimentin. None of the 35 cases showed positivity for desmin, indicating that mesenchymal differentiation is restricted to the rare so-called medul lomyoblastomas.

Key words: Medul loblastomas - Immunohis tochemis t ry - Cytokerat ins - Electron microscopy

It has long been recognized that medulloblastomas, al- though usually appearing as undifferentiated round-cell tumors, may have the potential not only for neuronal and astrocytic [27], but occasionally also for oligodendroglial and ependymal differentiation [20]. Mesenchymal differ- entiation with muscle fibers can also be observed in rare cases. The origin of the myogenic components in these so-called medul lomyoblas tomas is a subject of contro- versy [25].

Correspondence to: S. Patt

Expression of cytokeratins has not been described in any large series of medulloblastomas [7, 9, 33]. It was found by Schwechheimer [31] in just one melanotic tumor comprised of typical medul loblastoma and epithelial ar- eas, but only in the latter areas.

We have examined 35 medulloblastomas by light mi- croscopy and immunohistochemistry, concentrating par- ticularly on desmin and keratin immunoreactivity, in order to elucidate "unusual" differentiation potentials of a relatively large series of medulloblastomas. Expression of neuronal markers was not investigated, since it has already been examined by many other authors [5, 16, 28, 32, 35]. Electron microscopy was performed in 23 cases.

Material and methods

Formalin-fixed, paraffin-embedded, 7-gm-thick sections from biop- sies of 35 medulloblastomas taken from patients between 152 days and 32 years old (Table 1) were studied using routine histological stains (hematoxylin and eosin, Nissl, periodic acid-Schiff, Goldner). Nineteen were children (below 15 years) with a mean age of 6.7 years, and 16 were adults with a mean age of 24.5 years. There were 12 female and 23 male patients.

For immunohistochemical examination, 5-gm-thick paraffin sections were incubated with polyclonal and monoclonal antibodies using the avidin-biotin-peroxidase reaction (ABC) according to Hsu et al. [19] for the polyclonal antibodies and the alkaline-phos- phatase-antialkaline-phosphatase (APAAP) method of Cordell et al. [8] for the monoclonal antibodies. Endogenous peroxidase activity was blocked by 0.3% H202 in phosphate-buffered saline (PBS) for 30 min. For reduction of unspecific staining, the sections were preincubated in PBS containing 5% bovine serum albumin and 10% normal horse serum for I h, then incubated with the primary antibody for 12 h. This was followed by incubation with biotinylated horse anti-mouse-IgG antibodies (Vector, Wiesbaden, FRG) for 30 min and ABC (Vector, Wiesbaden, FRG) for 45 min. Peroxidase activity was visualized using 3,3-diaminobenzidine. The APAAP method involved the use of a monoclonal mouse APAAP complex (Dianova, Hamburg, FRG) and BCIP/NBT (Sigma, Dei- senhofen, FRG) as substrate. The sections were counterstained with hematoxylin. Negative controls were performed by omitting the primary antibody and replacing it with nonimmune serum. The following antibodies were used: glial fibfillary acidic protein (GFAP; rat, polyclonal, Dako, Hamburg, FRG), cytokeratin KL1 (mouse,

Table 1. Clinicopathological data of 35 patients with medulloblas- tomas. H, Cerebellar hemisphere; V, vermis; C, classic medulloblas- toma; D, desmoplastic medulloblastoma; N, neuronal medulloblas- toma

Age Sex Location Histological type (years) (case no.)

152 days M H C (1065/85) 4 F V D (567/76) 5 M H C (61/71) 5 M H C (471/72) 5 M H D (1029/83) 6 M H C (465/79) 6.5 M H C (564/73) 8 F V C (205/79) 8 M H D (1183/86) 8 M V + H C (294/89) 9 F V C (419/74) 9 M V C (547/77)

10 F H D (607/74) 11 M V C (374/77) 11 M V C (823/87) 12 F V D (495/74) 13 M V D (270/76) 14 M V N (442/75) 15 M V C (288/76)

17 M H C (596/74) 19 F U D (473/79) 19 M V C (840/77) 19 M H D (1535/84) 20 F H D (213/70) 22 F H C (622/78) 22 M H C (703/89) 23 M H C (24/78) 25 F H C (349/77) 26 M H C (1591/87) 28 F V + H C (1346/83) 29 M H D (619/75) 30 F H C (658/82) 30 M H C (1002/86) 31 F H C (459/80) 32 M H C (618/75)

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Fig. 1. a - c Vimentin expression in medulloblastoma, a Tumor with scattered weakly positive cells (arrows), b tumor with neuronal differentiation and vimentin-positive Homer-Wright rosettes, e tu- mor of the youngest patient (152-day-old boy) with strong positivity in all tumor cells ( x 300). d Cytokeratin expression. A few strongly cytokeratin-positive medulloblastoma cells in the tumor from 152- day-old boy (arrows; x 300)

monoclonal, Immunotech, Marseille, France), cytokeratin MNF116 (mouse, monoclonal, Dako, Hamburg, FRG), desmin (mouse, monoclonal, Dako, Hamburg, FRG), and vimentin (mouse, mono- clonal, Dako, Hamburg, FRG).

For electron microscopy, several intraoperative biopsy speci- mens from 23 tumors were fixed in 2.5% buffered glutaraldehyde solution and washed in cacodylate buffer. Postfixation was per- formed in 1% osmium tetroxide. Following dehydration with ace- tone, the samples were embedded in araldite. Semithin sections (0.5 gm) were stained according to Richardson et al. [26]. Ultrathin sections were cut on a Reichert ultramicrotome, stained with uranyl acetate and lead citrate, and examined with a Zeiss EM10 transmis- sion electron microscope.

Results

Microscopic findings

Twenty- four t u m o r s (12 f rom 19 ch i ldren a n d 12 f rom 16 adul ts) were classic m e d u l l o b l a s t o m a s . The t u m o r s showed the typica l fea tures o f round-ce l l t u m o r s wi th a h igh cell densi ty. The neop l a sms were c o m p o s e d o f small ,

p o o r l y d i f fe ren t ia ted cells wi th h y p e r c h r o m a t i c nuclei and scanty cy top lasm. Mi toses were f requent . Necroses were p resen t in some cases. The cells were somet imes a r r a n g e d in roset tes . The t u m o r s h a d a sparse, m o d e r a t e - ly vascu la r ized connect ive t issue s t roma.

Ten t u m o r s (6 f rom 19 ch i ldren and 4 f rom 16 adul ts ) were desmop la s t i c var iants , cha rac te r i zed by " pa l e , " less cel lular a reas s u r r o u n d e d by c row de d d a r k e r cells. One m e d u l l o b l a s t o m a ( t umor in a 14-year -o ld boy) showed neu rona l d i f fe ren t ia t ion wi th a b u n d a n t H o m e r - W r i g h t roset tes.

The t u m o r in the ce rebe l lum o f the 152-day-o ld b o y was a classic m e d u l l o b l a s t o m a con ta in ing n u m e r o u s ne- croses.

Immunohistochernical findings

Reactivity for GFAP. Seven t u m o r s con ta ined some G F A P - p o s i t i v e cells. F ive t u m o r s showed a few cells wi th G F A P - p o s i t i v e c y t o p l a s m and G F A P express ion in

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Fig. 2. Electron micrograph ( x 2500) of a filament-containing tu- mor cell in the medulloblastoma from the 152-day-old boy. The 6 to ll-nm-thick filaments form irregular whorls and bundles and show circumscribed dense accumulations. (Inset x 8200)

longer processes, which could be either GFAP-pos i t ive t umor cells or scattered reactive astroeytes. Two tumors exhibi ted GFAP-pos i t ive cells central ly and had a few short s tubby immunopos i t i ve processes. These were p robab ly GFAP-pos i t ive neoplast ic cells. Twenty-eight t umors were completely GFAP-nega t ive .

Reactivity f o r desmin. Sta in ing for desmin was absent in the neoplast ic cells of all tumors , whereas the walls of some vessels showed desmin positivity.

Reactivity f o r vimentin and coexpression o f vimentin and GFAP. Twenty-n ine tumors were v iment in-negat ive . Two had scattered weakly posit ive cells (Fig. 1 a). The neuro- blastic var iant showed vimentin-posit ive rosettes (Fig. 1 b). The t u m o r of the 152-day-old boy and two other medul - lob las tomas were compIetely v iment in-pos i t ive (Fig. 1 c). Blood vessel walls and connect ive tissue showed s t rong v imen t in posi t ivi ty in all t u m o r s . D o u b l e s ta in ing for G F A P a n d v imen t in revealed a coexpression of bo th ant i- gens only in some reactive: astrocytes scattered th rough- out the t u m o r tissue. No s imul taneous s ta in ing was ob- served in cells which were unequivoca l ly neoplast ic.

Reactivity fo r eytokeratins. The medul loblas tomas were all cytokerat in-negative except in the 152-day-old boy. This t u m o r showed s t rong posi t ivi ty for K L I (Fig. 1 d) a nd

Table 2. Immunohistochemical findings in medulloblastoma (35 cases: 19 children and 16 adults), e, Few or probably reactive astro- cytes; +, some positive tumor cells; + +, many positive tumor cells; + + +, all tumor cells positive; -, no immunoreaction; GFAP, glial fibrillary acidic protein; KL1, Pan cytokeratin; MNF116, Pan cy- tokeratin

Age Sex GFAP KL1 MNF Des- Vimen- (years) 116 min tin

152 days M �9 + + - + + + 4 F . . . . . 5 M . . . . . 5 M - - - + 5 M . . . . 6 M �9 . . . . 6.5 M . . . . . 8 F . . . . . 8 M . . . . . 8 M . . . . . 9 F . . . . . 9 M . . . . .

10 F . . . . + + + 11 M . . . . . 11 M . . . . . 12 F . . . . . 13 M �9 . . . . 14 M . . . . + + 15 M . . . . .

17 M . . . . + + + 19 F . . . . + 19 M . . . . . 19 M �9 . . . . 20 F . . . . . 22 F . . . . . 22 M . . . . . 23 M . . . . . 25 F . . . . . 26 M �9 . . . . 28 F �9 . . . . 29 M + + . . . . 30 F + . . . . 30 M . . . . . 31 F �9 . . . . 32 M . . . . .

Total : 2(7)/35 1/35 1/35 0/35 6/35

M N F I I 6 in cell clusters located near bu t unconnec t ed with vessels.

The results are summar ized in Table 2.

Electron microscopical f indings

Most tumors were comprised of undi f ferent ia ted cells with a r o u n d or ovoid, i r regularly con toured nucleus which con ta ined dist inct nucleoli a nd marg ina t ed chro- mat in . The cytoplasm was scant in re la t ion to the nuclear vo lume and occasional ly formed short processes. The cytoplasm con ta ined r ibosomes as well as a few mito- chondr ia and some m e m b r a n e s of rough endoplasmic ret iculum. The intercel lular spaces were nar row. The tu- m o r cells were separated f rom the b lood vessel walls by basement me mbr a ne .

In the cases with light-microscopically detected GFAP- positive tumor cells, the cytoplasm of some cellS demon- strated bundles of filaments which measured approxi- mately 8-10 nm across but were variable in length: The nuclei looked similar to those in cells without filaments. Scattered astrocytes were identifiable by their longer pro- cesses and darker cytoplasm.

No myogenic elements were found in the tumor cells. In the medulloblastoma of the 152-day-old boy, which

showed anticytokeratin positivity, cells with bundles of 6- to ll-nm-thick intracytoplasmic filaments were found (Fig. 2). These intermediate filaments, which formed irreg- ular whorls and bundles and had characteristic circum- scribed dense accumulations, may represent cytokeratin.

Discussion

The multidirectional potential of medulloblastomas for astrocytic, neuronal, oligodendroglial, ependymal, and mesenchymal differentiation has repeatedly been dis- cussed on the basis of light-microscopic, ultrastructural, and immunohistochemical findings mainly of antibodies against GFAP, neurofilaments, synaptophysin, neuron- specific enolase, S 100 protein, and vimentin [5, 7, 9, 16, 28, 29, 32, 33, 35]. Expression of desmin and cytokeratins has seldom been examined. Giordana et al. [12] examined laminin expression in five medulloblastomas.

Cytokeratins are relatively specific for normal and neoplastic epithelial cells [23] but have also been found in nonepithelial tumors [4, 6, 11, 17, 24, 33, 34]. Cruz- Sanchez et al. [9], Sime et al. [33], and Coffin et al. [7] did not find cytokeratin expression in their large series of medulloblastomas. In a melanotic infratentorial tumor in a 8-year-old boy, Schwechheimer [31] described cytoker- atin-positive cells only in the epithelioid areas and not those comprised of typical medulloblastoma cells.

It is noteworthy that, in our series, cytokeratin expres- sion was found only in the tumor of the boy who was nearly 5 months old (our series, however, lacked any other cases of very young children, i.e., under 5 years). Similarly, cytokeratin expression was described by Zim- mer et al. [36] in a primitive neuroectodermal tumor ("supratentorial medulloblastoma") of a 5-month-old boy. Cruz-Sanchez et al. [9], however, did not observe cytokeratin positivity in their youngest patient, who was 2 months old.

Rare cytokeratin expression in medulloblastomas can be interpreted in various ways. (1) It could be seen as a real epithelial differentiation, though this seems unlikely in view of its presence in different nonepithelial tumors as well as in a variety of CNS neoplasms. (2) It could be a sign of anaplasia, i.e., evidence of abnormal protein syn- thesis due to an altered antigene pattern during neoplas- tic transformation [13]. (3) Cytokeratin expression, how- ever, most probably indicates immaturity of the medul- loblastomas. "More mature" medulloblastomas of older individuals would, according to this interpretation, have lost their potential to express cytokeratins, comparable to the situation in ependymomas, where cytokeratin positiv- ity is no longer found at later stages [13, 21]. This is

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compatible with the transient cytokeratin expression during the development of neuroectodermal cells [2]. However, it is not clear whether immunohistochemistry sufficiently enables us to differentiate between mature (differentiated) and immature (undifferentiated) tumors. In his critical report on the value of immunohistochemi- cal investigations in childhood brain tumors, Gullotta [16] emphasized that a clear-cut positivity of antibodies is usually seen in differentiated tumors, but in undifferenti- ated tumors, interpretation of the immunohistochemical findings is very difficult and problematic and is fairly subjective. We are aware of the subjectivity of our inter- pretion of the keratin expression observed in the youngest of our cases. Nevertheless, this finding is not a cross-reac- tion artifact, as Western blotting analysis in a similar study demonstrated [14]. The cytokeratin expression therefore, in fact, may have the above cited meaning.

Some authors have reported on vimentin expression in medulloblastomas. Schwechheimer [31] found vimentin- positive tumor cells in four of six cases. Cruz-Sanchez et al. [9] observed vimentin expression in 28% of 50 cases, Sime et al. [33] in 13% of 30 cases, and Coffin et al. [7] in 38% of 53 cases. We have found vimentin expression in neoplastic cells of six patients. The strongest vimentin reaction was observed in the medulloblastoma of the 152-day-old child and in two other medulloblastomas. Vimentin is expressed in early stages of neuroectodermal stem cell maturation [3, 10, 30]. In astrocytes, vimentin is expressed first during ontogenesis, is later coexpressed with GFAP, and is then lost. Vimentin expression in our medulloblastomas was not age-related and is thus probably not typical of an early tumor stage (immaturi- ty), in contrast to cytokeratin expression (see above). In gliomas, vimentin positivity of neoplastic astrocytes is well known, and gliomas are often both vimentin- and GFAP-positive. In medulloblastomas, however, our dou- ble staining examinations yielded results that were in agreement with those reported by Cruz-Sanchez et al. [9], inasmuch as cases with unequivocally GFAP-positive tumor cells exhibited double staining in reactive astro- cytes but no coexpression of vimentin and GFAP in neo- plastic cells.

Regarding GFAP expression in medulloblastomas, we found GFAP positivity in neoplastic cells of only two tumors, while GFAP positivity in the other five tumors was not clearly neoplastic. Others have reported a higher percentage of GFAP positivity in neoplastic cells (10- 20%) [1, 5, 18]. While these authors considered that the GFAP-positive cells were derived from medulloblastoma cells, Coffin et al. [7], Mannoji and Becker [21], Schindler and Gullotta [29], and those who advocate a mesenchy- mal hypothesis of medulloblastoma origin [15, 22] inter- preted them as preexistent normal or reactive astrocytes intermingled with neoplastic cells. However, Schindler and Gullotta [29] and Gullotta [16] also used the term "few GFAP-positive tumor cells" for cells found in 10% of their cases, but they stressed that GFAP-positivity in neoplastic cells does not exclude the possibility that one is dealing with "pseudopositivity", i.e., that some neo- plastic cells or even cells other than glial have phagocy- tized GFAP from degnerating astrocytes. To date, no

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total agreement regarding G F A P positivity in medullo- b las tomas has been reached [28].

Desmin positivity, characterist ically found in muscle cells, was absent in our series using immunohis tochemi- cal methods and electron microscopy. Cruz-Sanchez et al. [9] and Schwechheimer [31] likewise repor ted no desmin-posit ive cases, whereas Coffin et al. [7] described desmin positivity in 5 o f 53 medul loblas tomas . These observat ions demons t ra te tha t mesenchymal differentia- t ion is lacking in mos t medul lob las tomas and is obvious- ly restricted to the rare medu l lomyob las tomas with myo- genic elements [25]. Desmin negativity, however, is com- patible with the various hypotheses o f medul lob las toma origin, and agrees par t icular ly with the hypothesis tha t medu l lob las toma is a primitive neuroepithelial t umor with or wi thout a capaci ty for differentiation. Disagree- ment with a neuroepithelial origin is expressed by those who believe that a medul lob las toma is an embryona l mesenchymal neop lasma [15, 22]. G i o r d a n a et al. [12] interestingly demons t ra ted a ne twork o f the specific base- ment m e m b r a n e glycoprote in laminin in desmoplast ic medul lob las tomas separat ing small and large lobules o f t u m o r cells. The authors explained the occurrence o f laminin by the hypothesis that basement membranes are p roduced by t u m o r cells when facing mesenchymal struc- tures. This might be suppor ted by the interpreta t ion o f Gul lo t ta [15] and Ma t a ka s et al. [22] on the mesoecto- dermic origin o f medul loblas toma.

In summary , cy tokera t in expression was found only in one single case: it was interestingly restricted to the younges t pat ient in our series. Thus, cytokera t in expres- sion could p r o b a b l y be interpreted as a sign o f immatur i - ty, a l though it also m a y be purely accidental. G F A P , vimentin, and desmin immunoreac t iv i ty were largely comparab le with those in o ther repor ted series.

Acknowledgements. The authors wish to thank Ms. Gabriele Kluge and Ms. Rita Benz for their excellent technical work.

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