Acta Neuropathol (1994) 88 : 334-348
9 Springer-Verlag 1994
A. Jouvet 9 M. F6vre-Montange 9 R. Besanfon E. Derrington 9 G. Saint-Pierre 9 M.E Belin 9 J. Pialat C. Lapras
Structural and ultrastructural characteristics of human pineal gland, and pineal parenchymal tumors
Received: 8 February 1994/ Revised, accepted: 20 May 1994
We have studied 20 pineal parenchymal tumors (PPT) and 4 normal or cystic pineal glands both by light and electron microscopy and immunohistochemistry with antibodies against glial markers [glial fibrillary acidic protein (GFAP) and protein S-100] or neural/neuroendocrine markers [neurofilaments (NF), synaptophysin and chromogranin A]. Light microscopy revealed the cellular organization of pinealocytes in the normal gland and in different morphological types of pineal tumors (typical pineocytomas, PPT with intermediate differentiation, mixed PPT exhibiting elements of both pineocytoma and pineoblastoma and pineoblastomas). Immunohistochemistry showed the presence of GFAP and protein S-100 in interstitial cells in nonneoplastic pineal gland. Cell processes were labeled with anti-synaptophysin and anti,NF antibodies. No immunoreactivity was found for chromogranin A in non-neoplastic pineal gland. In pineocytomas, GFAP and protein S-100 were observed in interstitial cells. Synaptophysin and NF were present in the large rosettes of pineocytomas. Synaptophysin, NF and chromogranin A were present in pineocytomas with a lobular arrangement of cells. Anti-chromogranin A immunoreactivity was also seen in lobular areas of some PPT with intermediate differentiation. Analysis of normal human pineal gland by electron microscopy showed the presence of vesicle-crowned rodlets (VCR or" synaptic ribbons), fibrous filaments (F), paired twisted filaments but few dense-core vesicles (DCV) in normal pinealocytes. Tumoral pineal cells appeared to differentiate Abstract
Supported by grants from the R6gion Rh6ne Alpes and from INSERM (CJF 90-10)
either towards a neurosensory pathway characterized by the presence of sensory cells elements (VCR and F), or towards a neuroendocrine pathway, with the occurrence of many DCV. Immunogold labeling demonstrated the presence of chromogranin A in neurosecretory granules. Human pineal gland 9 Pineal parenchymal tumors 9 Ultrastructure 9 Chromogranin A Key words
Introduction The ultrastructural morphology of the human pineal gland in both normal and pathological circumstances is quite poorly documented. Only a few studies describe the ultrastructure of fetal [27, 34, 35] and adult human pineal gland [1, 13, 26]. Parenchymal pineal tumors (PPT: pineocytomas, pineoblastomas) are very rare. Their ultrastructure has been described in 12 cases [9, 14, 15, 17, 23, 31, 36] or partially reported [2, 10, 28, 48]. Only a few immunohistochemical studies have been reported; these describe the presence of glial fibrillary acid protein (GFAP), S-antigen, rhodopsin, neuron-specific proteins (NSE), neurofilaments (NF) and recoverin in normal human pineal gland [18, 25, 29, 38, 39] and in PPT [4, 6, 9, 17, 19, 24, 37, 39]. Here, we report findings using electron microscopy and immunohistochemical techniques in an analysis of a series of 4 normal or cystic pineal glands and 20 PPT.
Materials and methods Case material
A. Jouvet (I8]) 9E. Derrington - J. Pialat - C. Lapras H6pital Neurologiqne, BP Lyon Montchat, F-69394 Lyon Cedex 03, France M. F6vre-Montange 9R. Besangon 9M.E Belin Laboratoire d'Anatomie Pathologique, Facult6 de M6decine Alexis Carrel, INSERM CJF 90-10, F-69372 Lyon Cedex 08, France
The main clinical features are summarized in Table 1. Normal tissue was obtained from two normal pineal glands, one in the vicinity of an angioma (patient A) and another one from a patient suffering from aqueduct stenosis (patient B). Normal tissue was also collected from the residual parenchyma of two cysticpineal glands (patients C and D).
335 T u m o r s o f t h e p i n e a l r e g i o n (n = 157) w e r e surgically e x c i s e d in t h e N e u r o l o g i c a l H o s p i t a l o f L y o n (France) f r o m 1975 to 1992. O f t h e s e t u m o r s 49 w e r e PPT; o f t h e s e p i n e o c y t o m a s or p i n e a l o b l a s t o m a s 20 h a v e b e e n e x a m i n e d by e l e c t r o n m i c r o s c o p y ( E M ) a n d 18 w e r e a n a l y z e d b y b o t h E M a n d i m m u n o h i s t o c h e m i s t r y (see Table 1).
Light microscopy P i n e a l t u m o r s p e c i m e n s w e r e fixed in 4 % p a r a f o r m a l d e h y d e cont a i n i n g 15 % picric acid a n d e m b e d d e d in paraffin. R o u t i n e staining w a s p e r f o r m e d with h e m a l i n - p h l o x i n e - s a f r o n , D e G i r o l a m i silv e r c a r b o n a t e i m p r e g n a t i o n [8], M a l l o r y ' s p h o s p h o t u n g s t i c acid h e m a t o x y l i n ( P T A H ) a n d B o d i a n ' s silver i m p r e g n a t i o n .
Immunohistochemistry N o r m a l cystic p i n e a l g l a n d s a n d 18 cases of P P T w e r e s t u d i e d by i m m u n o h i s t o c h e m i s t r y . A f t e r fixation as d e s c r i b e d a b o v e , i m m u n o h i s t o c h e m i s t r y w a s p e r f o r m e d by t h e i m m u n o p e r o x i d a s e ( p e r o x i d a s e - a n t i p e r o x i d a s e , P A P ) t e c h n i q u e u s i n g polyclonal antisera to G F A P ( D a k o , dilution 1/200), p r o t e i n S-100 (PS100, D a k o , dilution 1/300) a n d m o n o c l o n a l a n t i b o d i e s to N F (70 a n d 2 0 0 - k D a f o r m s ; D a k o 2 F l l , dilution 1/50), s y n a p t o p h y s i n ( B o e h r i n g e r , c l o n e 5438, 10 ~tg/ml) a n d c h r o m o g r a n i n A ( H y b r i t e c h ; L K 2 H 1 0 , dilution 1/1000). A f t e r i n c u b a t i o n with p r i m a r y a n t i b o d i e s ( i n c u b a t i o n t i m e s f r o m 2 h to o v e r n i g h t ) , b o u n d a n t i b o d i e s w e r e r e v e a l e d with seco n d a r y a n t i b o d i e s a n d P A P c o m p l e x . T h e color r e a c t i o n was d e v e l o p e d with 3 , 3 ' - d i a m i n o b e n z i d i n e . C o n t r o l sections were t r e a t e d with n o r m a l s e r u m in place of specific a n t i s e r u m . T h e specificity of t h e a n t i b o d i e s w a s t e s t e d o n c o n t r o l tissue s p e c i m e n s k n o w n to c o n t a i n t h e a n t i g e n s .
T a b l e 1 Clinical a n d light m i c r o s c o p y findings of n o r m a l a n d cystic p i n e a l gland, p i n e o c y t o m a s a n d p i n e o b l a s t o m a s
(Z1 l o b u l a t e d z o n e ,
Z2 typical p i n e o c y t o m a with r o s e t t e s , PG p i n e a l g l a n d , / n o t t e s t e d , - n o t s e e n , + rarely s e e n ) Age Tumor or pineoClub shape (years) normal size cytomatous process rosette (De Girolami)
Neurite process (Bodian)
Interstitial cell
/
/
0,5 cm 1,5 cm
/ +
/ +
15
1 cm
/
F
22
2 cm
2.
M
14
2 cm
3.
F
15
2,5 cm
4.
F
64
5.
F
6.
No.
Sex
A
M
8
0,5 cm
B
M
C
F
6 8
D
M
1.
Homer Wright rosette
Subtype
Operation
+
Normal
• •
Normal Normal (Cyst)
_+
Normal (Cyst)
total removal biopsy total removal total removal total removal total removal total removal total removal total removal total removal total removal total removal total removal total removal total removal incomplete removal total removal incomplete removal
/
/
•
/
/
3 cm
++
/
/
50
3 cm
++
+
F
35
2,5 cm
+
/
+
7.
M
49
3 cm
•
/
+
8.
M
13
3 cm
/
+
9.
F
24
4 cm
+
+
10.
F
63
'4 cm
+
+
11.
M
19
2 cm
/
+
12.
F
59
4 cm
/
/
13.
F
56
>3 cm
/
+
14.
F
27
> 2 cm
/
+
15.
M
25
> 3 cm
+
pineocytoma like PG pineocytoma like PG pineocytoma like PG typical pineocytoma typical pineocytoma intermediate differentiation intermediate differentiation intermediate differentiation intermediate differentiation intermediate differentiation intermediate differentiation intermediate differentiation intermediate differentiation mixed pineocytoma/ pineoblastoma pineoblastoma
16.
M
1
> 4 cm
§
pineoblastoma
17.
F
24
> 4 cm
§
pineoblastoma pineoblastoma
•
Z l Z2 --IZ1 Z2 -§
+
•
•
•
Z2+ Z2 +
18.
M
11
> 4 cm
§
19.
F
23
> 4 cm
§
pineoblastoma
20.
F
2
4 cm
§
pineoblastoma
total removal total removal incomplete removal total removal total removal total removal
Radio therapy
Survival
> 2 years > 6 months > 9 years > 3 mouths > 11 years > 13 years > 7 years > 3 months > 11 years §
> 9 years > 4 years
§
> 6 years > 14 years
§
8 m after radioth. > 4 years
+
> 4years
+
> 4 years
+
7 years
+
> 8 years
+
2 months
+
9 months
+
6 months 3 days
+
not known
336 Electron microscopy Biopsies of normal pineal glands or tumor fragments were fixed at the time of operation by immersion for 1 day in 4% glutaraldehyde or in 4% paraformaldehyde and 1% glutaraldehyde as described by MacDowell and Trump [30], post-fixed in 2% osmium tetroxide, dehydrated in a graded series of ethanols and embedded in epoxy resin. Sections were cut with an ultramicrotome, stained with uranyl acetate and lead citrate and then examined on a Jeol electron microscope (100 cx II). Immunoelectron microscopy To perform chromogranin immunolabeling, a two-step protein Agold technique was used, as described by Hearn [16]. Briefly, thin sections were collected on nickel grids and floated for 1 min on drops of 5 % hydrogen peroxide. The grids were rinsed in distilled water and placed in drops of 1% bovine serum albumin in TRIS buffer (pH8.2) for lh, then immersed in drops of antibody (LK2H10) used at a 1/2000 dilution in the same buffer, and incubated 18h at 4~ After rinsing, the grids were placed in drops of rabbit anti-mouse IgG (Jackson Immunoresearch Laboratories), diluted 1/500 in buffer for 1 h at room temperature. After washing, the grids were incubated in drops of protein A-gold (20 rim) for 1 h, rinsed in buffer, then distilled water and counterstained in uranyl acetate and lead citrate prior to observation. Histological typing and tumor grading Tumor grading was first established by analysis using light microscopy according to the World Health Organization classification [21, 45] and the recent review by Schild et al. [46]. This classification was revised on the basis of the ultrastructural and immunohistochemical results. The differentiation of PPT varied from well-differentiated pineocytomas (n=5) with favorable biological behavior to highly malignant undifferentiated pineoblastoma (n=6). Tumors with intermediate histological characteristics [PPT with intermediate differentiation (n=8) and mixed pineocytoma/pineoblastoma (n=1)] with uncertain prognosis, were also observed.
Results Table 1 summarizes the main clinical and light microscopy findings. The age of the patients with p i n e o c y t o m a ranged f r o m 13 to 64 years and those with pineoblast o m a ranged f r o m 1 to 25 years. T h e sex ratio was 13 females to 7 males. P i n e o c y t o m a tumors consisted of well-defined masses that ranged in diameter f r o m 2 - 4 cm. Pineoblastomas were larger and m o r e heterogeneous.
Light microscopy In normal pineal gland, a lobular organization of the p a r e n c h y m a was observed. In cystic pineal gland this organization was sometimes disrupted. It was easy to distinguish pinealocytes f r o m the interstitial cells because of the larger size of their nuclei. The silver technique of D e Girolami and Zvaigzne [8] showed club-shaped pinealocytic processes and Bodian's staining showed argyrophilic processes.
Fig. la-f Photomicrographs of representative sections of the dif- 9 ferent types of parenchymal pineal tumors, a Pineocytoma showing a morphological aspect similar to normal pineal gland and partial formation of perivascular pseudorosettes, b Pineocytoma showing a typical appearance with large fibrillary pineocytomatous rosettes (arrow). c Pineocytoma with intermediate differentiation showing transitional morphological features, lobulated regions and typical areas with pineocytomatous rosettes, d Pineocytoma with intermediate differentiation showing a lobular organization of cells and an endocrine-like vascularization (arrow). e Pineocytoma with intermediate differentiation showing diffuse cell proliferation, f Pineoblastoma composed of small round cells. a-f Hemalin-phloxine safron stain; a, b, fx370; e-ex237
Pineocytomas without mitosis and necrosis (cases 1 - 5 ) , occurred as two types. Some a p p e a r e d similar to the n o r m a l pineal gland, but lacked the lobular organization of cells and presented perivascular pseudorosettes (Fig. la). Others were typical pineocytomas, with proliferation of uniform cells characterized by scant amphophilic cytoplasm and round or indented nuclei. In these typical pineocytomas, the cells f o r m e d large but irregular fibrillary p i n e o c y t o m a t o u s rosettes (Fig. lb). In pineocytomas with intermediate differentiation (cases 6-13), a dense lobular a r r a n g e m e n t of cells (Fig. ld) with an endocrine vascularity was observed in one case (case 11). Transitional forms presenting lobulated regions and typical areas with p i n e o c y t o m a t o u s rosettes (Fig. lc) were also found (cases 9, 10). In one transitional f o r m (case 9), a few giant cells and some large gangliocytes were observed. Papillary figures were found in case 13. Cases 6 and 7 presented a m o r e diffuse proliferation (Fig. le). In case 14 different regions of the t u m o r presented morphological features that were in some cases typical of p i n e o c y t o m a and others of pineoblastoma. Pineoblastomas (cases 15-20) (Fig. 10 were highly cellular with small round cells resembling those of cerebellar medulloblastomas. Cell processes were not seen, but occasionnal Homer-Wright rosettes were observed. Immunohistochemistry Results of the immunohistochemical analysis of the cystic pineal gland and tumors f r o m patients with pineocytomas or pineoblastomas are s u m m a r i z e d in Table 2. In the normal p a r e n c h y m a of cystic pineal gland a few GFAP-positive cells with proximally thick, darkly stained processes were found. T h e interstitial cells were also labeled by an antibody to anti-PSi00. A n intense gliosis was o b s e r v e d around the cystic cavity. A dense infiltration of the residual pineal p a r e n c h y m a by antiGFAP-labeled processes was observed. Synaptophysin and N F were distributed in a m o r e widespread m a n n e r with an intense i m m u n o r e a c t i o n confined to the pineal cell processes. C h r o m o g r a n i n A immunolabeling was not detected. In pineocytomas, only interstitial cells were i m m u nolabeled with anti-GFAP (Fig. 2a) and anti-PSI00
337
338
339 Table 2 Immunohistochemical findings in cystic pineal gland and in tumors from patients with pineocytomas or pineoblastomas (/ not tested, Z1 endocrinoid area, Z2 pineocytomatous rosettes, IC interstitial cells) Patients GFAP PS100 Synapthophysin A
l
/
B
/
/
C D 1. 2. 3. 4.
+ IC + IC • IC / / • IC
+IC +IC + IC / / + IC
NF
/ / + +
/ / + +
/ / + + rosettes + rosettes
/ / +
ChromograninA
normal pineal gland and in the majority of pineocytomas, the junctions between vascular endothelial cells appeared tight but, in two cases fenestrae were present in the endothelial cells (see Fig. 6d). The ultrastructural characteristics of normal pinealocytes observed in norreal or cystic pineal gland and in those of pineal tumor cells are summarized in Table 3.
Ultrastructural features in the normal pinealocyte
Juxtaposition of clear and dark cells was observed in all cases of normal or cystic pineal glands with the pres5. ence of long zonulae adherentes (Fig. 3c). Clear cells 6. were more abundant in cystic pineal glands than in nor7. • • IC mal pineal glands. T h e pinealocytes consisted of round 8. / or elongated cell bodies with a process that terminated ZI+Z2/ ZI+ Z1 + Z 2 9. ZI+ Z2Z2 / in a club-shaped end. The pinealocyte nuclei were oval ZI+Z2Z1 + ZI+Z210. Z1 / or spherical, sometimes invaginated, located eccentriZ2Z2cally in the cell and were less dense than glial cell nu+ + + 11. / clei. Nucleolar material was not very prominent. / / + 12. +- IC / + + + Numerous organelles were scattered throughout the 13, • IC / / / 14. cytoplasm, especially in dark cells. Parallel microtubu15, les of 20-25 n m were found in cell perikarya but were / / 16. preferentially concentrated in cell processes. A moder/ / 17. / / ate a m o u n t of cilia ( 9 + 0 configuration), microtubular 18. / / 19. sheaves (MTS) and clusters of centrioles were seen. A / / 20. granular endoplasmic reticulum and little smooth endoplasmic reticulum were randomly distributed in the cytoplasm and many free ribosomes were present. The .41 Fig. 2a-f Photomicrographs of the immunohistochemical staining of paraffin-embedded sections, a GFAP immunostaining in typical mitochondria were large and numerous in the cytopineocytoma: few labeled astrocytes (arrow). b Protein S-100 plasm and sometimes packed in clusters in cell pro(PS100) immunolabeling of interstitial ceils in typical pineocy- cesses. Multivesicular bodies, clear vesicles, rare dense toma. e Neurofilament immunolabeling in pineocytomatous ro- core vesicles (DCV) of 8 0 - 1 2 0 n m , surrounded by a settes, d Synaptophysin immunolabeling in pineocytomatous rosettes, e Neurofilament immunolabeling in lobulated pineocy- clear space separating the granule from the vesicle toma (arrow). f Chromogranin A immunolabeling in lobulated m e m b r a n e , and dark lysosomes (Fig. 3a, d) were seen pineocytoma (arrow). a-f Counterstained with hemalin; ax370; scattered throughout the cytoplasm, and were more b-dx237; e-f• abundant in cell processes. Cytoplasmic melanin granules were rarely observed. O t h e r distinguishing features were the presence of antibody (Fig. 2b). Synaptophysin and sometimes N F filaments and vesicle-crowned rodlets ( V C R or "synlabeling was seen in the large pineocytomatous rosettes aptic ribbons"). Isolated filaments were rarely found, of typical pineocytomas (Fig. 2 c - d ) . but bundles of 5- to 8-nm packed filaments (Fig. 3a), Labeling of synaptophysin and N F (Fig. 2e) was also more dense than those of fibrous astrocytes, were found in pineocytomas with a lobular cell arrangement. observed. These were elongated or ovoid and were conMoreover, these contained chromogranin A labeled centrated in the processes. In the proximity of these cells. A positive chromogranin reaction was also seen in filaments, paired twisted filaments (PTF; filament lobular areas of transitional pineocytomas (Fig. 2f). In diameter: 10nm; Fig. 3a), arranged in parallel short case 13, which had papillary features, immunolabeling bundles, were also found (2-7 ~tm in length and 2 - 3 btm of NF, synaptophysin and chromogranin A was in width). T h e periodicity of their constrictions was, on observed. average, 26 n m and the maximal width of the helix was In pineoblastoma cells labeling for GFAP, PS100, 16 nm. Parallel bundles of 20-30 P T F could be associsynaptophysin, N F and chromogranin were not ated and f o r m e d crystalline-like inclusions in cross secobserved. tion. A high frequency of V C R was found in all h u m a n pineal glands. T h e y consisted of a central osmiophilic Electron microscopy area of variable length (about 700 nm), 50 n m in width, and surrounded by 60-nm diameter clear vesicles. T h e y Glial cells, blood vessels and nerve fibers were seen in were linear or archiform and arranged parallel or perthe parenchyma of normal and cystic pineal glands. In pendicular to the plasma m e m b r a n e (Fig. 3b, e, f).
340
VCR were observed in both perikarya and cell processes, most of them lying close to the plasma membrane.
Fig. 3a-e Photomicrographs illustrating the ultrastructural a p p e a r a n c e i n n o r m a l o r c y s t i c p i n e a l g l a n d , a P a i r e d t w i s t e d filam e n t s ( P T F ) a r r a n g e d i n p a r a l l e l s h o r t b u n d l e s (one arrow), e l o n g a t e d b u n d l e s o f p a c k e d f i l a m e n t s (double arrow) a n d p r o g r a n u l e s (small arrow), b V e s i c l e c r o w n e d r o d l e t s ( V C R ) (short arrow) a n d f i l a m e n t s (long arrow), c J u x t a p o s i t i o n o f c l e a r a n d d a r k c e l l s , p r e s e n c e o f l o n g z o n u l a e a d h e r e n t e s , d O v o i d fibro u s f i l a m e n t s (double arrow), i m m a t u r e g r a n u l e (one arrow), e, f V e s i c l e c r o w n e d r o d l e t s (one arrow), a, b , d, f x 4 9 , 0 0 0 ; c x 14,000; e x 3 5 , 0 0 0
Ultrastructural features in pineal parenchymaI tumors Certain characteristics of the normal pineal gland were observed in all pineocytomas studied, such as alternation of clear and dark cells (Fig. 4a) which were joined with extensive zonulae adherentes (Fig. 4e), pleiomorphic intermingled processes, rare microvilli (Fig. 4 0 and endocytosis profiles (Fig. 4d-g). Nuclei were indented. However, they were rounder in PPT with intermediate differentiation and pineoblastomas. Annulate lamellae (Fig. 4a) were sometimes observed in the perikaryon near the nucleus. As in the normal pineal gland, the organelles found included synapticlike vesicles (Fig. 4b), cilia (Fig. 4c), DCV (Fig. 4d), numerous mitochondria (Fig. 4g) and multivesicular bodies (Fig. 4h). The qualitative and quantitative distribution of organelles varied as a function of the pineocytoma subtype.
In pineocytoma with morphological features akin to those of normal pineal gland under light microscopy, the ultrastructural aspect was similar to those of normal glands (Fig. 5d-f). However, the PTF were organized in shorter bundles (Fig. 5g). Mitochondrial clusters were more frequently observed (Fig. 5c). VCR were always observed but were less numerous (Fig. 5a, b). Smooth reticulum was found, but peculiar arrangements resembling membranous whorls (Fig. 5e) were observed in almost all these tumors. D C V were not frequently observed. However, dusters of centrioles, sparse cilia and MTS were present in all pineocytomas.
T a b l e 3 U l t r a s t r u c t u r a l f i n d i n g s o f n o r m a l a n d c y s t i c p i n e a l g l a n d , p i n e o c y t o m a s a n d p i n e o b l a s t o m a s (DCVdense c o r e v e s i c l e s , VCR v e s i c l e c r o w n e d r o d l e t s , PTF p a i r e d t w i s t e d f i l a m e n t s , CE c e l l e n d i n g , PG p i n e a l g l a n d , + + n u m e r o u s o r g a n e l l e s o b s e r v e d , + s o m e organelles observed, + few organelles observed, 0 no organelles) No.
Subtype
A B C
Normal Normal Normal (cyst) Normal (cyst) Pineocytoma like P G Pineocytoma like P G Pineocytoma like P G typical pineocytoma typical pineocytoma intermediate intermediate intermediate intermediate intermediate intermediate intermediate intermediate mixed Pineoblastoma Pineoblastoma Pineoblastoma Pineoblastoma Pineoblastoma Pineoblastoma
D 1. 2. 3. 4. 5. 6. 7. 8. 9. I0. 11. 12. 13. 14. 15. 16. 17. I8. 19. 20.
Light or Zonulae dark cells adherentes
Cilia 9+0
Microtubules
CE Membramitonous chondria whorl
VCR
Ovoid and fibrous bodies
PTF
DCV
Clear synaptic like vesicles
Tumor differentiation
+ + +
++ ++ ++
+ + +
++ ++ ++
0 0 +
0 0 0
++ ++ ++
++ ++ ++
+++ +++ ++
+ + +
++ ++ ++
+
++
+
++
+
0
++
++
++
•
++
+
++
_+
++
+
+
++
++
++
_+
++
neurosensory
+
+
+
++
0
0
+
++
+
•
+
neurosensory
+
+
0
+
+
0
++
++
++
+
++
neurosensory
+
+
+
++
++
+
++
++
++
+_
++
neurosensory
+
+
++
++
+
++
+
++
+
+
++
neurosensory
+ + + + + + + + • •
+ + ++ + + ++ + + + +
+ ++ + ++ + + + 0 0 +
+ + + ++ ++ ++ ++ ++ + •
++ + + ++ ++ 0 + + + •
+ + + + + 0 0 0 0 0
0 + + 0 0 0 0 0 0 0
+++ ++ ++ • • 0 • + 0 0
+ 0 0 0 0 0 0 0 0 0
+ + + +++ +++ +++ ++ ++ + +
+ + + ++ +++ +++ ++ + + +
neurosensory neurosensory neurosensory transitional transitional neurosecretory neurosecretory neurosecretory undifferentiated undifferentiated
0
0
0
0
+
0
+
0
0
+
++
undifferentiated
+
+
0
0
+
+
0
0
0
+
0
undifferentiated
•
+
0
+
+
+
0
0
0
+
+_
undifferentiated
0
+
+
•
0
0
0
0
0
0
0
undifferentiated
0
0
0
+
0
0
0
0
0
•
•
undifferentiated
341
342
Fig. 4a-h
Photomicrographs showing the common ultrastructural features of pineocytoma, a Alternation of clear and dark cells (long and short arrows), cell processes rich in organelles, annulate lamellae in the perikaryon (small arrow), b Synaptic-like vesicles (long arrow), c Cilia (9+0) (arrow), centriole (double arrow), d
Dense-core vesicles and vesicular endocytosis (arrow). e Extensive cell junctions (arrow). f Microvilli (arrows). g Numerous mitochondria and vesicular endocytosis (arrows). h Multivesicular bodies (one arrow) and filaments (double arrow), a, f• b• e• d, h• e• g•
343
In the cases of typical pineocytoma, generally similar ultrastructural features were found. Moreover, the cell processes, which form the pineocytomatous rosettes, were rich in organelles, especially VCR, PTF, oriented microtubules and clear vesicles. In intermediate differentiated PPT with lobular organization, PTF and VCR were absent. Mitochondria were abundant but were not organized in clusters (Fig. 6c). Oriented microtubules as well as clear synaptic like vesicles (Fig. 6a) and numerous DCV (Fig. 6b, c) were found in the cell processes. Chromogranin Apositive neurosecretory granules (Fig. 6b) were detected by immunogold labeling. In other PPT with intermediate differentiation and in mixed pineocytoma! pineoblastoma the number of organelles was lower. In pineoblastomas, the tumor cells were smaller with a round nucleus. Cell processes were absent and cell junctions were rare and short. Only a few organelles were observed.
Fig. 5a-g Photomicrographs showing ultrastructural features of 9 neurosensory differentiation, a Cluster of VCR (arrow). b VCR parrallel to the plasma membrane, e Clusters of mitochondria (arrow). d Representative field from cells with neurosensory differentiation: numerous microtubules and mitochondria, bundles of packed filaments (double arrow) in the vicinity of VCR (arrow). e Membranous whorl (arrow). f Ovoid (shortarrows) or elongated (long arrow) bundles of packed filaments, g Paired twisted filaments (arrow). a• b• c• dx17,500; e• 12,500; f• g•
glioma rather than pineocytoma, whereas pineocytoma would be confirmed by the presence of NF or synaptophysin. Our results with EM confirm and extend those of previous studies of the normal pineal gland of human fetus [34, 35], a young girl [26] and middle aged and elderly humans [13], as well as of normal [1, 20, 42, 52] and tumoral animal pineal gland [51] and human pineal tumors [14,15, 23, 31, 36]. In normal pineal gland, we observed 9+0 type cilia, typical of retina and ear hair cells, and VCR, which are Discussion thought to be vestigial structures corresponding to synaptic ribbons present in true neurosensory cells. This In this study we report the characteristics of the human clearly supports the phylogenic origin of the vertebrate pineal gland and pineal tumors as seen by light and and human pinealocyte as a modified neurosensory cell electron microscopy. In this large series of cases, [5, 33, 53]. VCR have been described in the pinealoobservations made by light microscopy are in agree- cytes of most mammalian species [1, 20, 26, 52] but ment with previous descriptions of normal pineal gland were not mentioned in the study of fetal human pineal or PPT [2, 3, 7, 14, 15, 17, 46, 47, 49, 50]. There is, how- gland [34]. ever, a dearth of published studies concerning ultraNumerous large fibrous filaments have been structural features that characterize the normal differ- described in human fetal pineal gland [34]. It has been entiation of human pinealocytes. We have sought to suggested previously that these correspond to glial filaidentify these characteristics using electron microscopy ments [2, 17], if so they might be restricted to interstitial and evaluate whether their appearance in the cells of astrocytes. In our study, they were also observed in cells PPT may aid in the diagnosis and evaluation of the that contained VCR. This confirms that they are also prognosis of these tumors. characteristic of normal pinealocytes. In our study, PTF were present in all four normal or cystic pineal glands. They were particularly abundant in the vicinity of VCR Normal and cystic pineal gland and near fibrous filaments. PTF were suggested to correspond to the paired helical filaments in Alzheimer's At the light microscopy level, the characteristic lobular brain [13] but their presence in the pineal of young subarrangement of normal pinealocytes was not always jects in this study and that of Kurumado and Mori [26] conserved in cystic pineal glands. This makes it more suggest that this resemblance is incidental rather than difficult to distinguish between cystic pineal gland and pathological. The large number of filaments observed pineocytoma. The greater cellularity associated with the in pineal cells revealed by EM resemble those of photolatter, combined with the presence of radiating perivas- receptors cells of lower vertebrates [40]. cular structures and of slight cytonuclear atypias should The presence of rare mitochondria clusters, similar be distinctive. However, these features are also shared to those found in the inner segment of photoreceptors, by some low-grade-appearance ependymomas. In such concludes the list of ultrastructural features of normal cases De Girolami staining, which reveals the "club- pinealocytes that have been conserved during evolution shaped" cell endings, allows these cells to be distin- in a rudimentary form and persist as evidence of the guished from those of ependymomas. Furthermore, original pineal gland photoreceptor function. though GFAP staining has previously been described to In contrast the observation of rare dense-core vesicindicate an astrocytic differentiation of certain pineocy- les in normal pinealocytes is consistent with their tomas [2, 17], in our study, in all cases, GFAP and adaptation to a neurosecretory function, whereas the PTAH labeling was always restricted to interstitial numerous small, clear synaptic-like vesicles are consistastrocytes. Thus, GFAP labeling of tumor cells in such ent with both neurosecretory and neurosensory roles. cases would tend to confirm low-grade ependymoma/
344
Fig. 5a-g
345
Fig. 6a-d Photomicrographs showing the ultrastructural features of neurosecretory differentiation, a Clear and dense-core vesicles (DCV) (short and double arrow), microtubules in cell process (long arrow), h DCV, chromogranin A immunogold labeling in
DCV (arrows). c Numerous mitochondria and DCV (arrow). d Fenestra in the vascular endothelial cell (arrow). ax49,000; hx70,000; cx28,000; dx35,000
346 Pineocytomas The typical pineocytoma, with large pineocytomatous rosettes, is easily identified by light microscopy. Neuronal differentiation has been identified by the presence of NF [6], NSE [4, 6, 9, 50] and synaptophysin [4, 6]. Pineocytomas with rosettes were also labeled with antibodies to NF and synaptophysin in this study. Other PPT apparently demonstrated either intermediate or mixed levels of differentiation. These are much more difficult to distinguish from pineoblastomas on the basis of only light microscopy. Thus, some pineocytomas have a pleiomorphic appearance (PPT with intermediate differentiation). Their cellular density is sometimes very high and smaller pineocytomatous rosettes that can resemble Homer-Wright rosettes can be observed. Tumors presenting these morphological features could also show evidence of high vascularity and giant cells. Although these are normally characteristic of highly malignant PPT, in the absence of mitotic figures and necrosis and associated with positive immunolabeling of NF and synaptophysin, the above-mentioned histological features need not indicate high malignancy. EM revealed that all pineocytomas, including the intermediate and mixed tumors described above, exhibited many of the ultrastructural features characteristic of normal pinealocytes, although to varying degrees. These were observed less often and some of them not at all in pineoblastomas. We have identified three subtypes of pineocytoma as a function of the type of organelles they contained. A primarily neurosensory/photoreceptor differentiation was associated with the presentation of mitochondria clusters, fibrous bodies and PTF, the last of which has previously been described in pineocytoma by Hassoun et al. [15]. Membranous whorls with an ultrastructure that resembles that of the outer segment of photoreceptors were also observed in this type of tumor. The second population, which was characterized by high numbers of DCV, was designated as exhibiting neurosecretory/neuroendocrine differentiation. The third class of pineocytoma was designated "transitional" and characterized by the presence of different pineocytoma cells that preferentially expressed either neurosecretory or neurosensory phenotypic characteristics. The number of "neurosensory" structures, in particular mitochondrial clusters, was higher in pineocytomas with "neurosensory" differentiation than in normal pinealocytes. Membranous whorls were characteristics 9 of this type of tumor but not found in pinealocytes. Furthermore, in the neurosecretory type o f i u m o r , there was a dramatic augmentation in t h ~ n u m b e r of DCV observed compared with the normal pinealocyte. This rendered all the five tumors in the neurosecretory/transitional group susceptible to immunolabeling with the antibody to chromogranin A. None of the other tumors or normal pinealocytes was labeled by this marker. This neurosecretory type of PPT presents the characteristics of neuroendocrine neoplasms or neuroendocrine cells [4, 11, 12, 43].
It will be interesting to establish whether pineocytomas expressing a primarly neurosensory/photoreceptor differentiation correspond to those reported to express S-antigen or rhodopsin [19, 24, 28]. These observations suggest that in normal human pinealocytes, although the primary function is neurosecretory, a delicate balance is nevertheless maintained between the expression of neurosecretory and perhaps neurosensory phenotypic characteristics. In pineocytomas, this balance may be deregulated, leading to the increased expression of a single facet of this duality. It has also been proposed that two populations of pinealocytes exist in normal mammalian pineal gland that differ with respect to their expression of neurosensory! photoreceptor-associated antigens [32, 41]. It is unknown whether they originate from one or more different precursors. The two subclasses of pineocytoma described above may indeed correspond to tumors originating from cells of these different subpopulations. However, the third class of pineocytoma that we designated "transitional" may be in favor of the single pluripotent precursor hypothesis. Pineoblastomas Pineoblastomas are similar to the tumors of the medulloblastoma-neuroblastoma group classified by Rorke et al. [44] as primitive neuroectodermal tumors. Indeed, their similarities extend to the presence of occasional Homer-Wright rosettes. In our study the immunohistochemical stainings perfomed were negative. However, labeling of NF and NSE in one case of pineoblastoma [37] and immunoreactivity against chromogranin A [22] have been reported previously. EM revealed that pineoblastoma cells exhibited relatively fewer of the ultrastructural characteristics found in pineocytoma, normal or cystic pineal gland and in much smaller numbers. This, not surprisingly, is in agreement with their identity as very poorly differentiated, highly malignant tumor cells. Conclusions Our study shows a close correlation between observations made at the level of light and electron microscopy in pineocytomas and pineoblastomas. In general morphological staining appears insufficient to discriminate between cystic pineal gland and pineocytoma or lowgrade glioma, and certain pineocytomas and pineoblastoma with confidence. Immunohistochemistry increases the confidence with which they can be discriminated and EM results allow confirmation of immunohistochemical staining. We show that pineocytomas can be subdivided into three classes that differ in their expression of either neurosensory, neuroendocrine or both neurosensory and neuroendocrine phenotypic characteristics on the basis of EM.
347 It w o u l d be interesting to d e t e r m i n e w h e t h e r t h e y differ in prognosis; however, at p r e s e n t we lack sufficient n u m b e r s o f cases a n d lapsed time to analyze this adequately. Nevertheless, it a p p e a r s t h a t increased differentiation in either p a t h w a y m i g h t suggest a favorable prognosis. C h r o m o g r a n i n A a p p e a r s to be an a d e q u a t e label for n e u r o s e c r e t o r y differentiation w h e n E M is n o t available. A l t h o u g h the ultrastructural studies p r o v i d e p o w e r f u l m e a n s to characterize differentiation, t h e y suffer f r o m the small a m o u n t o f tissue t h a t can be analyzed. This is a p r o b l e m given the disparate n a t u r e of different regions o f i n t e r m e d i a t e a n d m i x e d p i n e o c y t o mas. F o r this r e a s o n , it is best used in c o n j u n c t i o n with i m m u n o h i s t o c h e m i s t r y which allows the rapid e x a m i n a tion o f large areas of the t u m o r biopsy.
Acknowledgements We are grateful to Paul P6vet for his helpful suggestions. We thank Gdrard Berger for his help with gold immunolabeling and Nicole Dubois for skillful secretarial assistance.
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