Rosette forming glioneuronal tumor of the fourth ventricle in squash cytology smear.

Rosette forming glioneuronal tumor (RGNT) is a recently recognized and extremely rare glioneuronal tumor occurring in the fourth ventricle. It is crucial for the cytopathologist to be aware of this entity as it can be easily mistaken for more common neoplasms occurring at this site. We present here the cytology of such a rare case of RGNT that was misdiagnosed as ependymoma. The varying cytological features of this entity, as well as the common diagnostic difficulties encountered in cytology, are highlighted in this report.

Introduction

Rosette forming glioneuronal tumor (RGNT) a rare slowly growing tumor affecting mainly the young adults which has been included as one of the glioneuronal tumors in the 2007 version of the World Health Organization (WHO) classification of central nervous system tumors.[1] The neoplasm is composed of distinct neurocytic rosettes, perivascular pseudorosettes and an astrocytic component mostly pilocytic astrocytoma. The tumor most often occurs in the fourth ventricle.[12] The cytomorphology of this rare entity has been described only in a few isolated case reports. Here, we are addressing the potential role of cytology for the diagnosis of RGNT as well as the close differential diagnoses which can cause difficulties during squash smear cytological examination.

Case Report

A 15-year-old male presented with complaints of headache and vomiting of 6 months duration. He also had one episode of seizure. He was diagnosed to have a fourth ventricular lesion by imaging and empirically treated with antituberculous drugs. The patient had no relief of complaints and hence was referred to our institute. On clinical examination, he had no neurological deficits. A magnetic resonance imaging brain was done, which showed a solid cystic lesion near the fourth ventricle. The tumor was hyperintense on T2-weighted images [Figure 1a] and showed minimal enhancement with Gadolinium contrast [Figure 1b]. The imaging findings were interpreted as pilocytic astrocytoma. The patient underwent midline suboccipital craniectomy and C1 arch excision with subtotal decompression of the tumor.

Figure 1

(a) Magnetic resonance imaging T2-weighted axial image showing a welldefined hyper intense solid cystic lesion in the region of 4th ventricle, with focal peripheral iso intense areas (b) Sagitial image showing irregular peripheral enhancement of the lesion with central nonenhancing area. (c) Smear cytology of a neoplasm with pilocytic astrocytes, rosenthal fibres,eosinophilic granular bodies (H and E, ×200) (d) Cytology with neurocytic cells around central eosinophilic neuropil (H and E, ×200) (inset a rosetie in smear – H and E, ×400)

Cytopathologic findings

The intra-operative squash smears were fixed in 95% ethanol and stained with hematoxylin and eosin as well as toluidine blue stains. Cytology showed a neoplasm composed of glial cells with oval dark nuclei, long hairlike processes and rosenthal fibers [Figure 1c]. Also seen are small cells with scanty cytoplasm and round nuclei with fine chromatin. These cells were seen in groups, forming rosettes [Figure 1d] with central eosinophilic fibrillary material and some arranged around vessels forming pseudo rosettes [Figure 2a]. Microvascular proliferation was also seen. A cytologic diagnosis of ependymoma was considered based on the age and the site of the lesion.

Figure 2

(a, b) Cytology and histology showing perivascular pseudorosetie patiern of small cells with myxoid stoma. (H and E, ×200) Histology of the neoplasm with pilocytic astrocytes, rosenthal fibres and eosinophilic granular bodies. (H and E, ×200) Histology of neurocytic cells around central eosinophilic neuropil (H and E, ×200) (inset showing roseties with neuropil showing S-100 positivity (Avidin biotin, ×200)

Histopathologic findings

The paraffin sections showed features of a typical RGNT with small neurocytes forming rosettes and perivascular pseudorosettes [Figure 2b and d]. This was admixed with pilocytic form of glioma having bipolar astrocytes, rosenthal fibers and eosinophilic granular bodies [Figure 2c]. The neurocytic cells were distributed in an abundant myxoid and neuropil background [Figure 2b and d]. The neurocytic rosettes and pseudorosettes with neuropil material showed strong S-100 (monoclonal, Novacastra, 1:100) immunopositivity [Figure 2d inset]. These areas were glial fibrillary acidic protein (GFAP), synaptophysin and Neuron specific enolase (monoclonal, Novacastra, 1:100) negative on immunostaining. The neurocytic cells were positive for Neurofilament protein (monoclonal, Novacastra, 1:100). The pilocytic component was strongly positive for GFAP. The postoperative period of the patient was unremarkable, and he is now under regular follow-up.

Discussion

Rosette forming glioneuronal tumor was first described in 2002 as a distinct variant of mixed glioneuronal tumor by Komori et al.[2] which was later confirmed by Preusser.[3] Until date, approximately 43 cases of RGNT have been reported.[4] This tumor is considered as Grade I according to the WHO classification.[1] The pathologic features of RGNT can be easily diagnosed in excision biopsies with neurocytic-like cells, forming neurocytic and perivascular rosettes dispersed in a microcystic myxoid matrix admixed with pilocytic astrocytes.[12]

Approximately, there are eight case reports describing the intra-operative squash smear features of this tumor. Initially, Komori et al.[2] and Jacques et al.[5] had described the cytology of this entity as smears showing cells with round nuclei having granular chromatin, inconspicuous nucleoli, and scant cytoplasm admixed with piloid astrocytes. They did not stress the typical appearance of neurocytic rosettes on smears Kinno et al.[6] and Ghosal et al.[7] had described the combination of neurocytic rosettes as well as pilocytic astrocytes in RGNT in their case reports. However, due to sampling error only part of the tumor may be represented in the smear preparation. Hence in squash smears, we can easily miss the classical neurocytic rosettes, especially with other elements dominating the cytology.

On cytology the main differential diagnosis of RGNT include pilocytic astrocytoma, dysembryoplastic neuroepithelial tumor (DNT), oligodendroglioma, neurocytoma, pineocytoma and ependymoma.[67] Pilocytic astrocytoma have an almost similar clinical setting and in smears show long hair such as cytoplasmic processes, rosenthal fibers, eosinophilic granular bodies and occasionally an abundant mucinous background.[8] But they lack the neurocytic rosettes seen in RGNT. Before the introduction of the terminology of RGNT many such cases were misdiagnosed as cerebellar DNTs.[9] DNT in smears may also show sheets of small round oligo like cells and fibrillary cells in an abundant mucinous background. However, DNTs are extremely rare in the fourth ventricle. They occur usually as epilepsy associated tumors in the temporal lobe. A few scattered floating neurons, ganglioid cells as well as calcospherites can be seen on cytology of DNT.[8] It is to be emphasized here that any tumor in the fourth ventricle with features of DNT on cytology, the possibility of RGNT should be considered.

The fourth ventricle is an uncommon site for oligodendroglioma. It is composed of monomorphous round cells with a finely branching capillary network as in RGNT cytology.[8] However, in oligodendrogliomas the smears will be more cellular and crowded, with calcified spherules and have no rossetes or gliofibrillary matrix. The oligodendroglial cells in cytology will be larger with coarse nuclear chromatin. The neurocytic cells of RGNT are small and round with more fine and uniform nuclear chromatin.[8] Neurocytoma and pineocytoma, composed of monotonous population of small cells forming neuropil rosettes, are differentiated from RGNT by the absence of pilocytic astrocytic component, mucoid background and by the specific site of the tumor. The dominant perivascular pseudorosettes and gliofibrillary processes in RGNT can be mistaken for ependymoma of the fourth ventricle in smears. The nuclei of ependymoma when compared with RGNT are larger and round with pale stippled chromatin and have one or more prominent nucleoli.[8] The presence of pilocytic features, mucoid background, neurocytic rosettes with central neuropil and absence of distinct tubular ependymal canals can help in distinguishing RGNT from ependymoma.

Despite its rarity, RGNT should always be considered in the differential diagnosis of tumors occurring in the fourth ventricle. A review of literature on RGNT cytology highlights that on adequate sampling as well as correlation with the relevant clinical picture including the site it is fairly easy to diagnose this entity on squash smears.