Cytogenetic and ultrastructural study of a pineocytoma case report

Journal of Neuro-Oncology 48: 131–134, 2000. © 2000 Kluwer Academic Publishers. Printed in the Netherlands.

Clinical Study

Cytogenetic and ultrastructural study of a pineocytoma case report Alessandro Dario1 , Michele Cerati2 , Monica Taborelli2 , Giovanna Finzi2 , Marco Pozzi1 and Alberto Dorizzi1 Neurosurgical Department, 2 Service of Histology and Pathological Anatomy, Varese Regional Hospital and Macchi Foundation, Italy

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Key words: chromosome, pineocytoma, ultrastructure Summary The authors report a case of pineocytoma in a 44-year-old woman suffering from headache, vomiting and Parinaud syndrome. At histopathological examination the neoplasm showed a ill-defined lobulate pattern with some small pineocytomatous rosettes. The electron-microscopy revealed cells of moderate size and oval nuclei with smooth nuclear envelopes; well-developed organelles were found in the, abundant cytoplasm. The chromosome analysis revealed this kariotype: 58–59, XXX, −4, −5, −13, −14, −15, +19. This is the first report of a pineocytoma with ultrastructural and cytogenetic study; it confirms the literature findings of the electron-microscopy, whereas there is partial accordance with the previous cytogenetic studies.

Introduction The pineocytoma is a rare tumor which develops in the parenchyma of the pineal body. This neoplasm constitutes only 0.1% of all brain tumors [1] and represents approximately 45% of the pineal parenchymal origin neoplasms. In literature only two cytogenetic studies of pineocytomas have been reported [2,3]; we present the first ultrastructural and cytogenetic study of a pineocytoma. Case report A 44-year-old woman was admitted to our hospital suffering from headache, vomiting and Parinaud syndrome. The CT-scans showed a pineal mass with calcification in the caudal part of the neoplasm and heterogeneous enhancement after contrast; there was also supratentorial hydrocephalus present. The T1-weighted MR images confirmed the heterogeneous enhancement after contrast of the slightly lobulated pineal neoplasm (Figure 1). The alpha-fetoprotein (AFP), Beta-human chorionic gonadotropin (b-HGC) and carcino embrionic antigen (CEA) markers of the serum and cerebrospinal fluid (CSF) were negative. After an external shunt a stereotactic biopsy was carried out to elucidate the histopathological diagnosis.

Figure 1. T1-weigthed MRI with contrast showing heterogeneous enhancement of the irregular pineal neoplasm with hydrocephalus.

The specimens revealed a pineocytoma. Because of this diagnosis the neoplasm was removed one week later by a supracerebellar–subtentorial approach and the tumoral tissue was submitted to histopathological and cytogenetic study. In consequence of persistent ventricular dilation an internal shunt was implanted. After discharge the

132 patient did not receive radiation therapy. At the present time, 34 months after surgery, she enjoys good health.

Histopathological study Tissue samples of the tumor collected during surgery were fixed in formalin and embedded in paraffin. The samples for routine examination were stained with hematoxylin and eosin (HE). The samples for immunohistochemical examination were stained for neuronspecific enolase (NSE) and synaptophysin (SYN) using the avidin–biotin–peroxidase (ABC) method. At histopathological examination (Figure 2) the tumor showed an ill-defined lobulate pattern with some ‘pineocytomatous rosettes’. The tumor cells were uniform and small in size with round nuclei and homogeneous eosinophilic cytoplasm. The tumor cells revealed considerable immunoreactivity for NSE and SYN (Figure 3). For ultrastructural studies tissues were fixed in aldehydes and post-fixed in osmic acid. Sections stained with uranyl acid and lead showed round cells with long processes which terminated as bulbous endings. Nuclei varied from oval to round, showing peripheral granules of heterochromatin. Prophiles or rough and smooth endoplasmic reticulum were sparse in the cytoplasm as well as Golgi apparatus, mitochondria, lysosomes and numerous small dense-core granules and clear vesicles of about 250 nm diameter (Figure 4). Cytoplasmic borders showed some small cell junctions. Cytoplasmic processes showed numerous parallel microtubules and clear vesicles, the latter accumulating

Figure 3. Immunohistochemical study (SYN immunostaining ×200). Tumor cells show immunoreactivity for synaptophysin.

Figure 4. Tumor cells often show a narrow process which terminate in a bulbous ending; prophiles of rough and smooth endoplasmic reticulum, mitochondria, lysosomes, dense core granules, microtubules and clear vesicles are evident in cytoplasms (uranyllead ×22400).

in bulbous endings (Figure 5) where symmetric membrane thickening suggestive of synaptic junctions were also evident (Figure 6). Cytogenetic study

Figure 2. Histopathology of the tumor (HE ×200). The tumor show a lobulated pattern and focally tumor cells from the ‘pineocytomatous rosette’.

Chromosome analysis was performed on direct preparation. Suspension of tumor cells were obtained by mincing small pieces of the tumor in petri dish and incubating for 72 h at 37◦ C with 5% of CO2 . The medium used was DMEM-F12 enriched by high

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Figure 5. Bulbous endings of tumor cells show numerous clear vesicles (uranyl-lead ×44800).

Figure 7. Metaphase plate Q-banded of pineocytoma showing an hypotriploid chromosome complement.

G banding techniques on ten metaphases. Structural abnormalities were identified as clonal if found in two or more cells. Numerical changes (two or more cells for gain, three or more cells for loss) were described relatively to the ploidy of the abnormal modal population, as recommended by ISCN 1995. The tumoral karyotype was: 58–59, XXX, −4, −5, −13, −14, −15, +19 (Figure 7). The pathological ultrastructural, and cytogenetic features of this tumor are summarized in Table 1. Discussion

Figure 6. Bulbous endings. Asymmetric membrane thickening suggestive of a synaptic junction (uranyl-lead ×88200).

glucose. The tumor cells were exposed to colcemid overnight and harvested by hypotonic treatment in 1% sodium citrate and repeated fixations in methanol– acetic acid (3 : 1). The cell suspension was obtained using a solution of 70% acetic acid and the metaphase spread was performed on a warm plate at 40◦ C. Karyotype analysis was performed using QFQ and

In our patient the considerable immunoreactivity for NSE and SYN confirms the histological findings of the tissue typical in pinecytoma; this tumor can be also classified as grade 2 in the histological classification of malignancy in pinecytoma by Tsunoda et al. [1]. The ultrastructural studies of the pineal parenchimal tumors (PPT) have refined the diagnostic criteria of differentiation by a more accurate balance of the presence of dendrites, synapse-like junctions, bulbous endings and dense core granules/clear vesicles [4]. Between the three subtypes of pineocytoma differentiation (neurosensory/photoreceptor, neurosecretory/ neuroendocrine, transitional differentation) diagnosed by electron-microscopy from Jouvet et al. [5] our patient had the characters of the neurosecretory/neuroendocrine differentation.

134 Table 1. Pathological ultrastructural, and cytogenetic feature of the pineocytoma From literature Ultrastructural characteristics [4,5] Neurosensory/photoreceptor differentiation: Fibrous bodies, paired twisted filaments, membranous whorls, microtubules Neurosecretory/neuroendocrine differentiation: Granules and dense-core vesicles, microtubules Transitional differentiation: Cells with neurosecretory or neurosensory phenotypic characteristics Chromosome loss [2,3] −X, −Y, −3, −4, −6, −8, −9, −10, −11, −12, −13, −14, −15, −16, −17, −18, −19, −20, −21, −22 Chromosome gain +5 Chromosome alterations 1, 3, 11, 12, 21, 22

Present case

Dense-core vesicles, microtubules

−4, −5, −13, −14, −15

+19 None

Although the neuronal differentation has been considered as a ‘benign hallmark’, further investigations are necessary to assess the utility of this prognostic parameter in the pineocytomas [6]. In particular the potential for neuronal differentation confirmed by cell culture and mRNA expression suggests a good prognosis for this pineocytoma [7]. Cytogenetic investigation using direct preparation to avoid the culturing selection revealed an hypotriploid chromosome complement. Clonal aberrations identified were losses of chromosomes 4, 5, 13, 14 and 15 and the gain of chromosome 19. Noteworthy losses of chromosomes 4, 5, 14 and 15 are frequent events in low and high grade astrocytomas and neuroblastomas. Loss of chromosomes 4 and 5 are also characteristic of meningiomas whereas gain of chromosome 19 is a typical feature of gliomas, neuroblastomas and retinoblastomas [8,9]. No structural aberrations were observed in this case. Tumoral karyotypes were previously described in only two pineocytomas showing near diploid chromosome complements with losses of different chromosomes, the gain of chromosomes 5 and the rearrangement of chromosomes 1, 3, 11, 12, 21 and 22 [2,3]. The common abnormalities in these three cases are losses of chromosomes 4, 5, 14

and 15 (Table 1) and interestingly, these chromosomes are reported as losses in more other solid tumors suggesting the presence of tumor suppressor genes in these regions. Our case along with the other two reported by literature, indicate that chromosome instability is a consistent feature of pineocytomas and that genes responsible for the correct segregation of chromosomes at mitosis may be involved in these types of neoplasms. This is the first case of polyploid pineocytoma reported but more karyotyped cases are needed to ascertain a possible correlation between morphological ultrastructural features and chromosome alterations in order to obtain a more precise identification of chromosomal regions non-randomly involved in the development of this neoplasm.

References 1. Tsunoda S, Sakaki T, Tsujimoto M, Yabuno T, Tsuzuki T, Nakamura M, Hiramatsu K, Morimoto T, Boku E, Iwanaga H, Asada H: Clinicopathological study on pineocytoma. Brain Tumor Pathol 12: 31–37, 1995 2. Rainho CA, Rogatto SR, Correa de Moraes L, BarbieriNeto J: Cytogenetic study of a pineocytoma. Cancer Genet Cytogenet 64: 127–132, 1992 3. Bello MJ, Rey JA, De Campos JM, Kusak ME: Chromosomal abnormalities in a pineocytoma. Cancer Genet Cytogenet 71: 185–186, 1993 4. Min KW, Scheithauer BW, Bauserman SC: Pineal parenchymal tumors: an ultrastructural study with prognostic implications. Ultrastruct Pathol 18: 69–85, 1994 5. Jouvet A, Fevre-Montange M, Besancon R, Derrington E, Saint-Pierre G, Belin MF, Pialat J, Lapras C: Structural and ultrastructural characteristics of human pineal gland and pineal parenchymal tumors. Acta Neuropathol 88: 334–348, 1994 6. Numoto RT: Pineal parenchymal tumors: cell differentiation and prognosis. J Cancer Res Clin Oncol 120: 683–690, 1994 7. Fevre-Montagne M, Jouvet A, Privat K, Korf HW, Champier J, Reboul A, Aguera M, Mottolese C: Immunohistochemical, ultrastructural, biochemical and in vitro studies of a pineocytoma. Acta Neuropathol 95: 532–539, 1998 8. Poulsgard L, Ronne M: Cytogenetic analysis of common nervous system tumors in adults. In: Molecular Genetics of Nervous System Tumors. Levine AJ, Schmidek HH (eds) Wiley-Liss, New York, 1993, p 225 9. Mitelman F: Catalog of chromosome aberrations in cancer. 5th edn., Johansson B, Mertens F (eds), Wiley-Liss, New York

Address for offprints: Alessandro Dario, Macchi Foundation Via Torriani, 32 1-22100, Como, Italy; Fax: + 39 332 261045