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Primary germinoma of the brain
Primary germinoma of the brain. Immunocytochemical demonstration of tumour cells in the cerebrospinal fluid.
E. S t a r k * , A . K n e h a n s * , U. W u r s t e r * , a n d U. P a t z o l d *
Introduction Summary Germinomas are one of the most common midline neoplasms of the brain, although their absolute occurrence is rare. Most frequently they appear in the pinealis region. The diagnosis of these tumours is not easy as surgery is difficult, especially if they are growing in atypical sites. As these turnouts are sensitive to radiation and chemotherapy, diagnosis without operation is desirable. Even if the turnout disseminates to the meninges, cytologic diagnosis by investigation of the CSF is often missed, particularly as reactive inflammation with appearance of plasma cells and oligoclonal bands is not uncommon and the tumour cells often resemble lymphocytes and histiocytes. Immunocytochemistry might distinguish even a few tumour cells from the predominant imflammatory cells. We describe a young man, suffering from a dysgerminoma of the third ventricle. In this case, immunocytochemically staining for human chorionic gonadotropin (hCG) and alpha-fetoprotein (AFP) have been demonstrated in CSF cells.
Case report A.K., a 17-year-old male was taken ill in summer 1984 with headache, nausea, photophobia and occasional vomiting. About half a year later
A patient is presented, who developed a suprasellar tumour. Stereotactical biopsy of the tumour revealed the diagnosis of a dysgerminoma. Immunocytochemical examination of the CSF showed neoplastic cells staining for human chorionic gonadotropin and for alphafetoprotein. The autors stress the possibility to diagnose primary intracranial germ cell tumours without operation. Key words: Brain tumour, germinoma, immunocytochemistry, cerebrospinal fluid.
his parents noticed a progredient mental change, he got more and more depressive and apathic and developed an infantile behaviour.In April 1985 he complained of painful legs, general weakness and visual disturbance. He was hospitalized. Some weeks later extreme electrolyte disturbance arised, so he was referred to our hospital in the end of June. He was apathic, had pupils non reacting to light and an incomplete vertical gaze palsy. A CCT showed a suprasellar tumour in the hypothalamic region covering the walls of the third ventricle. The investigation of the CSF gained by lumbar puncture in conventional staining only revealed inflammatory disturbance with some atypical cells, probably originating from the tumour.
* Division of Neurology and Clinical Neurophysiology Hannover Medical School, FRG Address for correspondence and reprint requests': E. Stark, Neurol. Klinit rnit klin. Neurophysiol. Medizinische Hochschule Hannover, Konstanty-Gutschow-Str. 8, 3000 Hannover 61. Accepted 10. 7.86 Clin Neurol Neurosurg 1986. Vol. 88-4
263
CSF from an Omaya reservoir, implanted August 1985, revealed some more cells suspicious for malignancy. A stereotactic biopsy of the tumour was performed, histologically it showed the typically two-cell pattern germinoma without trophoblastic cells. A combined therapy with radiation and intrathecal application of Methotrexate was started. The next month he improved consecutively. On September 24 he died six hours after the onset of a fulminant meningitis caused by Acinetobacter anitratum.
CSF investigations Methods Immediately after lumbar resp. puncture of the Omaya reservoir the CSF was centrifugated at 4 ~ and 200 g for 10 minutes in a conic tube. The sediment was resuspended in a mixture of medium 199 with fetal calf serum. For each slide 200 ~tl of this suspension were cytocentrifugated at 80 g for 10 minutes 1. One slide was Pappenheim stained, the other used for immunocytochemistry. As not all antibodies were immediately available, these slides were fixed in acetone and stored at -70 ~ until further processing. After thawing they were incubated successively with the primary antibody, rabbit antimouse and goat antirabbit antibody. As primary antibodies monoclonal antibodies directed against the following antigens were used: Leu 2a, Leu 3a (Becton-Dickinson), kappa and lambda immunoglobulin light chain (Dianova),
Fig 1. ImmunocytochemicalstainingforAFP. Erythrocytes, lymphocytes and one tumour cell with nonhomogeneous positive stainingcytoplasm. 264
hCG, AFP (Serotec), glial fibrillary acid protein (GFAP), vimentin and cytokeratin. All antibodies were used in an appropriate dilution in phosphat buffered saline (PBS). The second and the third antibody were peroxidase labelled. After each incubation step the slides were washed in PBS and finally developed in tris buffered saline (TBS) containing 0.06 mg/ml diaminobenzidine and 0.01% H202 at pH 7.6 for 10 minutes. Then they were fixed in PBS with 0.6% glutardialdehyd, counterstained in hemalaun and mounted in aquamountL Intrathecal IgG synthesis rate was calculated according to Tourtellotte's formula3, olicoclonal bands were investigated by isoelectric focusing using silver stain4. [3-hCG and AFP were serologically detected by radioimmunoassay.
Results The CSF showed a mild pleocytosis (lumbar puncture at 28.6.: 13/~tl, ventricular puncture at 9.8.: 104/~tl) with predominantly lymphocytes and a few bigger, presumptively neoplastic cells. These cells had a high nuclearcytoplasmic ratio, vesicular nuclei and large, irregular nucleoli. The protein level was increased (0.55 resp. 2.87 g/l) with a pattern of mixed barrier dysfunction and intrathecal IgG synthesis (Tourtellotte-Index 25.3 resp. 134.2 mg/dat). Oligoclonal bands were positive. CSF levels of I3-hCG and AFP were increaed to 437 mU/ml resp. 218 ng/ml (Serum standards < 5 mU/ml resp. < 15 ng/ml, CSF standards not known). Immunocytological
Fig 2. Immunocytochemicalstainingfor hCG:Erythrocytes and a tumourcell withgranularpositivityof the cytoplasm.
stain for l y m p h o c y t e m a r k e r s r e v e a l e d a inflamm a t o r y p a t t e r n with 18% s u p p r e s s o r , 46% helper T-cells and 3% polyclonal B - l y m p h o c y t e s . T h e t u m o r cells did not stain for v i m e n t i n , c y t o k e r a t i n and G F A P . Staining for A F P a n d h C G s h o w e d positivity in s o m e of the n e o p l a s t i c cells (Fig. 1, 2).
Discussion T h e t h e r a p y of m i d l i n e t u m o u r s even i f - t h e y a p p e a r in t h e i r typical site, the p i n e a l r e g i o n , is still u n d e r discussion. In case of n o n - m a l i g n a n t t u m o u r s , e s p e c i a l l y p i n e a l o m a s , surgical t r e a t m e n t m a y be n e c e s s a r y , as t h e s e t u m o u r s are r a d i o r e s i s t e n t 5. C o n t r a r y , m a l i g n a n t m i d l i n e t u r n o u t s as g e r m i n o m a s and t e r a t o m a s a r e highly r a d i o s e n s i t i v e a n d s h o u l d be t r e a t e d w i t h o u t surgery. L o n g p e r i o d s o f r e m i s s i o n can be a c h i e v e d by r a d i a t i o n 6. In view of t h e s e facts it is d e s i r a b l e to d i a g n o s e t h e s e t u m o u r s without o p e r a t i o n . In a b o u t 80% o f i n t r a c r a n i a l germ i n o m a s t u m o u r cells a r e f o u n d in the C S F 7, b u t t h e s e cells are o f t e n r a r e a n d not easy to distinguish f r o m the a b u n d a n t i n f l a m m a t o r y r e a c t i o n . T u r n o u t cells have b e e n d e s c r i b e d as synthesizers of local i m m u n o g l o b u l i n s 8. Morphologically these tumours are ind i s t i n g u i s h a b l e f r o m g e r m i n o m a s o f the o v a r or s e m i n o m a s o f the testis. A s histological diagnosis of t h e s e t u r n o u t s e s p e c i a l l y in m e t a s t a s e s is n o t always easy, i m m u n o c y t o c h e m i c a l m e t h o d s have b e e n e s t a b l i s h e d . If d i f f e r e n t i a t i o n to y o l k sac t u m o u r s o c c u r the cells will c o n t a i n A F P , in case of d i f f e r e n t i a t i o n to a c h o r i o c a r c i n o m a h C G . D i f f e r e n t i a t i o n to an e m b r y o n a l carc i n o m a will p r o d u c e b o t h m a r k e r s 9'~~ This diff e r e n t i a t i o n can i m m u n o h i s t o c h e m i c a l l y m o r e often be d e m o n s t r a t e d t h a n m o r p h o l o g i c a l l y 11. T h e case p r e s e n t e d h e r e shows, t h a t imm u n o c y t o c h e m i c a l diagnosis of p r i m a r y int r a c r a n i a l g e r m cell t u m o u r s is p o s s i b l e b a s e d on
C S F cells. W e s u p p o s e , that in all cases o f m i d l i n e t u m o u r s d i a g n o s e d by C C T , e s p e c i a l l y when there are no metastases, immunocytochemistry of CSF should be done. As in a high p e r c e n t a g e of p r i m a r y i n t r a c r a n i a l g e r m cell t u m o u r s n e o p l a s t i c cells can be f o u n d in C S F by r e p e a t e d e x a m i n a t i o n s , m a n y o f t h e s e cases can b e e x c l u d e d f r o m s u r g e r y a n d i m m e d i ately t r e a t e d by safer r a d i o t h e r a p y .
References 1
WURSTER U, STARK E, ENGELHARDT P. L i q u o r z y t o l o g i e nach kombinierter Zentrifugation und Zytozentrifuga-
tion im Vergleich zu Sedimentation und Membranfiltration. Arztl Lab 1984; 30:184-8. 2 STARKE. Differenzierung von Oberflaechenantigenen auf Liquorzellen mit monoctonalen Antik6rpern. Akt Neurol 1985; 12:180-2. 3
TOURTELLO'ITE WW, POTVIN RA, FLEMING ID, MURTHY KN. LEVY J,
SYNDULKO K, POTVIN J.
Multiple
Sclerosis:
Measurement and validation of central nervous system IgG synthesis rate. Neurology 1981; 30:240-4, 4 WURSTERU. Demonstration of oligoclonal IgG in the unconcentrated CSF by silverstain. In: STATHAKOSP. ed, Electrophoresis 1982. Berlin-New York: W. de Gruyter 1983: 250-9. 5
JOOMA R, KENDALL BE. D i a g n o s i s
and management
of
pineal tumors. J Neurosurg 1983; 58:654-65. 6
ALLEN JC, N1SSELBAUM J, EPSTEIN F, ROSEN G, SCHWARTZ
MK. Alphafetoprotein and human chorionic gonadotropin determination in cerebrospinal fluid. An aid to the diagnosis and management of intracranial germ-cell tumors. J Neurosurg 1979; 51:368-74. 7
UEKI K, TANAKA R. T r e a t m e n t s
and prognoses of pineal
tumors - experience of 110 cases. Neurol Med Chir 1980; 20:1-26. 8 STATZ A, FELGENHAUER K. Liquorbefunde bei intrakraniellen Germinomen. In: Verhandl Dtsch Ges Neurol 1983; 2:933-5. 9
MORINAGA S, OJIMA M, SASANO N. H u m a n
chorionic
gonadotropin and alphafetoprotein in testicular germ cell tumors. Cancer 1983; 52:1281-9. 10
BJORNSSON J, SCHEITHAUER BW, OKAZAKI H, LEECH RW.
Intracranial germ cell tumors: Pathobiological and immunohistochemical aspects of 70 cases. J Neuropathol Exp. Neurol 1985; 44:32-46. 11
BUTCHER DN, GREGORY WM, GUNTHER PA~ MASTERS JRW,
PARKINSONMC. The biological and clinical significance of HCG containing cells in seminoma. Br J Cancer 1985; 51:473-8.
265
E. S t a r k * , A . K n e h a n s * , U. W u r s t e r * , a n d U. P a t z o l d *
Introduction Summary Germinomas are one of the most common midline neoplasms of the brain, although their absolute occurrence is rare. Most frequently they appear in the pinealis region. The diagnosis of these tumours is not easy as surgery is difficult, especially if they are growing in atypical sites. As these turnouts are sensitive to radiation and chemotherapy, diagnosis without operation is desirable. Even if the turnout disseminates to the meninges, cytologic diagnosis by investigation of the CSF is often missed, particularly as reactive inflammation with appearance of plasma cells and oligoclonal bands is not uncommon and the tumour cells often resemble lymphocytes and histiocytes. Immunocytochemistry might distinguish even a few tumour cells from the predominant imflammatory cells. We describe a young man, suffering from a dysgerminoma of the third ventricle. In this case, immunocytochemically staining for human chorionic gonadotropin (hCG) and alpha-fetoprotein (AFP) have been demonstrated in CSF cells.
Case report A.K., a 17-year-old male was taken ill in summer 1984 with headache, nausea, photophobia and occasional vomiting. About half a year later
A patient is presented, who developed a suprasellar tumour. Stereotactical biopsy of the tumour revealed the diagnosis of a dysgerminoma. Immunocytochemical examination of the CSF showed neoplastic cells staining for human chorionic gonadotropin and for alphafetoprotein. The autors stress the possibility to diagnose primary intracranial germ cell tumours without operation. Key words: Brain tumour, germinoma, immunocytochemistry, cerebrospinal fluid.
his parents noticed a progredient mental change, he got more and more depressive and apathic and developed an infantile behaviour.In April 1985 he complained of painful legs, general weakness and visual disturbance. He was hospitalized. Some weeks later extreme electrolyte disturbance arised, so he was referred to our hospital in the end of June. He was apathic, had pupils non reacting to light and an incomplete vertical gaze palsy. A CCT showed a suprasellar tumour in the hypothalamic region covering the walls of the third ventricle. The investigation of the CSF gained by lumbar puncture in conventional staining only revealed inflammatory disturbance with some atypical cells, probably originating from the tumour.
* Division of Neurology and Clinical Neurophysiology Hannover Medical School, FRG Address for correspondence and reprint requests': E. Stark, Neurol. Klinit rnit klin. Neurophysiol. Medizinische Hochschule Hannover, Konstanty-Gutschow-Str. 8, 3000 Hannover 61. Accepted 10. 7.86 Clin Neurol Neurosurg 1986. Vol. 88-4
263
CSF from an Omaya reservoir, implanted August 1985, revealed some more cells suspicious for malignancy. A stereotactic biopsy of the tumour was performed, histologically it showed the typically two-cell pattern germinoma without trophoblastic cells. A combined therapy with radiation and intrathecal application of Methotrexate was started. The next month he improved consecutively. On September 24 he died six hours after the onset of a fulminant meningitis caused by Acinetobacter anitratum.
CSF investigations Methods Immediately after lumbar resp. puncture of the Omaya reservoir the CSF was centrifugated at 4 ~ and 200 g for 10 minutes in a conic tube. The sediment was resuspended in a mixture of medium 199 with fetal calf serum. For each slide 200 ~tl of this suspension were cytocentrifugated at 80 g for 10 minutes 1. One slide was Pappenheim stained, the other used for immunocytochemistry. As not all antibodies were immediately available, these slides were fixed in acetone and stored at -70 ~ until further processing. After thawing they were incubated successively with the primary antibody, rabbit antimouse and goat antirabbit antibody. As primary antibodies monoclonal antibodies directed against the following antigens were used: Leu 2a, Leu 3a (Becton-Dickinson), kappa and lambda immunoglobulin light chain (Dianova),
Fig 1. ImmunocytochemicalstainingforAFP. Erythrocytes, lymphocytes and one tumour cell with nonhomogeneous positive stainingcytoplasm. 264
hCG, AFP (Serotec), glial fibrillary acid protein (GFAP), vimentin and cytokeratin. All antibodies were used in an appropriate dilution in phosphat buffered saline (PBS). The second and the third antibody were peroxidase labelled. After each incubation step the slides were washed in PBS and finally developed in tris buffered saline (TBS) containing 0.06 mg/ml diaminobenzidine and 0.01% H202 at pH 7.6 for 10 minutes. Then they were fixed in PBS with 0.6% glutardialdehyd, counterstained in hemalaun and mounted in aquamountL Intrathecal IgG synthesis rate was calculated according to Tourtellotte's formula3, olicoclonal bands were investigated by isoelectric focusing using silver stain4. [3-hCG and AFP were serologically detected by radioimmunoassay.
Results The CSF showed a mild pleocytosis (lumbar puncture at 28.6.: 13/~tl, ventricular puncture at 9.8.: 104/~tl) with predominantly lymphocytes and a few bigger, presumptively neoplastic cells. These cells had a high nuclearcytoplasmic ratio, vesicular nuclei and large, irregular nucleoli. The protein level was increased (0.55 resp. 2.87 g/l) with a pattern of mixed barrier dysfunction and intrathecal IgG synthesis (Tourtellotte-Index 25.3 resp. 134.2 mg/dat). Oligoclonal bands were positive. CSF levels of I3-hCG and AFP were increaed to 437 mU/ml resp. 218 ng/ml (Serum standards < 5 mU/ml resp. < 15 ng/ml, CSF standards not known). Immunocytological
Fig 2. Immunocytochemicalstainingfor hCG:Erythrocytes and a tumourcell withgranularpositivityof the cytoplasm.
stain for l y m p h o c y t e m a r k e r s r e v e a l e d a inflamm a t o r y p a t t e r n with 18% s u p p r e s s o r , 46% helper T-cells and 3% polyclonal B - l y m p h o c y t e s . T h e t u m o r cells did not stain for v i m e n t i n , c y t o k e r a t i n and G F A P . Staining for A F P a n d h C G s h o w e d positivity in s o m e of the n e o p l a s t i c cells (Fig. 1, 2).
Discussion T h e t h e r a p y of m i d l i n e t u m o u r s even i f - t h e y a p p e a r in t h e i r typical site, the p i n e a l r e g i o n , is still u n d e r discussion. In case of n o n - m a l i g n a n t t u m o u r s , e s p e c i a l l y p i n e a l o m a s , surgical t r e a t m e n t m a y be n e c e s s a r y , as t h e s e t u m o u r s are r a d i o r e s i s t e n t 5. C o n t r a r y , m a l i g n a n t m i d l i n e t u r n o u t s as g e r m i n o m a s and t e r a t o m a s a r e highly r a d i o s e n s i t i v e a n d s h o u l d be t r e a t e d w i t h o u t surgery. L o n g p e r i o d s o f r e m i s s i o n can be a c h i e v e d by r a d i a t i o n 6. In view of t h e s e facts it is d e s i r a b l e to d i a g n o s e t h e s e t u m o u r s without o p e r a t i o n . In a b o u t 80% o f i n t r a c r a n i a l germ i n o m a s t u m o u r cells a r e f o u n d in the C S F 7, b u t t h e s e cells are o f t e n r a r e a n d not easy to distinguish f r o m the a b u n d a n t i n f l a m m a t o r y r e a c t i o n . T u r n o u t cells have b e e n d e s c r i b e d as synthesizers of local i m m u n o g l o b u l i n s 8. Morphologically these tumours are ind i s t i n g u i s h a b l e f r o m g e r m i n o m a s o f the o v a r or s e m i n o m a s o f the testis. A s histological diagnosis of t h e s e t u r n o u t s e s p e c i a l l y in m e t a s t a s e s is n o t always easy, i m m u n o c y t o c h e m i c a l m e t h o d s have b e e n e s t a b l i s h e d . If d i f f e r e n t i a t i o n to y o l k sac t u m o u r s o c c u r the cells will c o n t a i n A F P , in case of d i f f e r e n t i a t i o n to a c h o r i o c a r c i n o m a h C G . D i f f e r e n t i a t i o n to an e m b r y o n a l carc i n o m a will p r o d u c e b o t h m a r k e r s 9'~~ This diff e r e n t i a t i o n can i m m u n o h i s t o c h e m i c a l l y m o r e often be d e m o n s t r a t e d t h a n m o r p h o l o g i c a l l y 11. T h e case p r e s e n t e d h e r e shows, t h a t imm u n o c y t o c h e m i c a l diagnosis of p r i m a r y int r a c r a n i a l g e r m cell t u m o u r s is p o s s i b l e b a s e d on
C S F cells. W e s u p p o s e , that in all cases o f m i d l i n e t u m o u r s d i a g n o s e d by C C T , e s p e c i a l l y when there are no metastases, immunocytochemistry of CSF should be done. As in a high p e r c e n t a g e of p r i m a r y i n t r a c r a n i a l g e r m cell t u m o u r s n e o p l a s t i c cells can be f o u n d in C S F by r e p e a t e d e x a m i n a t i o n s , m a n y o f t h e s e cases can b e e x c l u d e d f r o m s u r g e r y a n d i m m e d i ately t r e a t e d by safer r a d i o t h e r a p y .
References 1
WURSTER U, STARK E, ENGELHARDT P. L i q u o r z y t o l o g i e nach kombinierter Zentrifugation und Zytozentrifuga-
tion im Vergleich zu Sedimentation und Membranfiltration. Arztl Lab 1984; 30:184-8. 2 STARKE. Differenzierung von Oberflaechenantigenen auf Liquorzellen mit monoctonalen Antik6rpern. Akt Neurol 1985; 12:180-2. 3
TOURTELLO'ITE WW, POTVIN RA, FLEMING ID, MURTHY KN. LEVY J,
SYNDULKO K, POTVIN J.
Multiple
Sclerosis:
Measurement and validation of central nervous system IgG synthesis rate. Neurology 1981; 30:240-4, 4 WURSTERU. Demonstration of oligoclonal IgG in the unconcentrated CSF by silverstain. In: STATHAKOSP. ed, Electrophoresis 1982. Berlin-New York: W. de Gruyter 1983: 250-9. 5
JOOMA R, KENDALL BE. D i a g n o s i s
and management
of
pineal tumors. J Neurosurg 1983; 58:654-65. 6
ALLEN JC, N1SSELBAUM J, EPSTEIN F, ROSEN G, SCHWARTZ
MK. Alphafetoprotein and human chorionic gonadotropin determination in cerebrospinal fluid. An aid to the diagnosis and management of intracranial germ-cell tumors. J Neurosurg 1979; 51:368-74. 7
UEKI K, TANAKA R. T r e a t m e n t s
and prognoses of pineal
tumors - experience of 110 cases. Neurol Med Chir 1980; 20:1-26. 8 STATZ A, FELGENHAUER K. Liquorbefunde bei intrakraniellen Germinomen. In: Verhandl Dtsch Ges Neurol 1983; 2:933-5. 9
MORINAGA S, OJIMA M, SASANO N. H u m a n
chorionic
gonadotropin and alphafetoprotein in testicular germ cell tumors. Cancer 1983; 52:1281-9. 10
BJORNSSON J, SCHEITHAUER BW, OKAZAKI H, LEECH RW.
Intracranial germ cell tumors: Pathobiological and immunohistochemical aspects of 70 cases. J Neuropathol Exp. Neurol 1985; 44:32-46. 11
BUTCHER DN, GREGORY WM, GUNTHER PA~ MASTERS JRW,
PARKINSONMC. The biological and clinical significance of HCG containing cells in seminoma. Br J Cancer 1985; 51:473-8.
265