A case of a 18-year-old male student having suffered from headache and visual impairments for about two months is reported. Neurological examination was negative. CT-scan performed in urgency revealed the presence of triventricular hydrocephalus caused by stenosis of the cerebral aqueduct. The most urgent therapeutic goal was mainly the management of the hydrocephalus, therefore a ventricular-peritoneal shunt placement (Medos Hakin-programmable valve) was used to provide an alternative flow pathway for the cerebrospinal fluid. The critical location of the lesion discouraged the STB procedure to obtain histopathological specimens available for the diagnosis. In addition, biochemical analysis of cerebrospinal liquor resulted normal with regard to cell count and protein content. Flow chart adopted in the diagnostic phase is reported in  Figure 1 .
Post-shunting (20 days) MRI showed a significant decrease of the ventricular size. Moreover, the MRI revealed a nodular lesion within the mesencephalic tectum with hypo-intense areas on the T1-weighted images ( Figure 2A ) and hyper intense signals on the T2-weighted sequence ( Figure 2B ). Gadolinium- T1-weighted image showed a low focal enhancement of the lesion in the periaqueductal area which suggested a reduced damage of the blood-brain barrier ( Figure 2C ). Proton magnetic resonance spectroscopy (H-MRS) revealed elevated choline peaks (choline/creatinine ratio at 1,9) in addition to reduced NAA (N-acetylaspartate) ( Figure 2D ). The perfusion-weighted imaging (PWI) of the lesion showed a low value of relative cerebral blood volume (CBV) ( Figure 2E ). The MR signal alteration was non-specific and could suggest, as alternative diagnostic hypothesis to a neoplasm, inflammatory lesions of various types, demyelinating and vascular.
The impossibility, given the mesencephalic location of the lesion, to perform a STB confirms the importance of the information which could be obtained with the cytological observation of peripheral blood CTCs shortly cultured in vitro and obtained applying a protocol previously described.
Biomolecular pathways and cell culture plays a pivotal role in cancer research. However, culture-induced changes in biological properties of tumor cells might profoundly affect research reproducibility and translational potential. For this reason, the cytological preparations we used in this diagnostic procedure were prepared with a protocol of short-time in vitro expansion, which we have widely shown to maintain phenotypic and genotypic features of CTCs. The volume of the starting blood sample was 5 ml. Briefly, adensity gradient was applied to the blood sample. The cellular suspension isolated in correspondence of the working density phase ( Figures 3a, b ) was seeded on chamber slides ( Figure 3d ) for a short-time expansion of 14 days. The procedure of expansion had two objectives: i) to unmask rare non-haematological cells with atypical proliferation ability in vitro and ii) to highlight rare cells numerically sufficient and viable for further characterization. After 14 days, the adherent cells were fixed and stained for cytological examination. The culture density of CTCs in the total cultivated cells was of 1:50. The lower rate of atypical cells in the blood-derived culture suggested finding these elements directly in peripheral blood difficult. In fact, the analysis for the presence of negative CD45 cells (CD45 is a marker of the haematological population) was negative in the patient's whole blood sample.
In this case, as shown in  Figure 3 , the Hematoxylin and Eosin (H&E) stained cytological preparations revealed the presence of bulky, polinucleated or spindled cell atypical elements ( Figures 3A, B ). In addition, these atypical cells found in the blood resulted, at the immunohistochemical analysis performed through methods previously described, positive for the expression of the astrocytes markers S-100 ( Figures 3C, D ) and glial fibrillary acidic protein (GFAP) ( Figures 3E, F ). A section of brain human tissue was used as internal positive control. Moreover, as negative control, demonstrating that the reaction visualized is due to the interaction of the epitope of the target molecule and the paratope of the antibody/affinity reagent, parallel assays with the primary antibodies omitted was performed. Moreover, as positive control for the presence of CTCs from cerebral lesions in the peripheral blood we used cytological preparations obtained with the same method from peripheral blood collected from one case of glioblastoma and from one case of brain metastasis from melanoma (cerebral metastasis) ( Figures 3G, H ). As negative control was used the cytological pattern isolated by peripheral blood collected from a case of post-stroke frontal lobe scar ( Figure 3I ). The final cytological diagnosis was of pilocytic astrocytoma because the protean features of the proliferating glial elements ( Figures 3A-F ).
The control MRI (6 months after shunt placement) documented that the lesion was stable. Moreover, the area of altered signal at the quadrigeminal plate, hyperintense on T2/Fluid Attenuation Inversion Recovery (FLAIR), hypointense on T1-weighted images, placed at the entrance of the aqueduct of Sylvius, decreased in width. MRI was repeated evert 6 months in the first 2 years; afterwards, an annual MRI was performed, without changes in the radiological picture. Clinical outcome (68 months) supported by follow up corroborates the pathological diagnosis of pilocytic astrocytoma/low grade glioma.
The patient provided written consent in accordance with ethical principles, relevant guidelines and regulations of the Declaration of Helsinki in accordance with experimental protocols included in the study approved by the local ethical committee, with study number 2013.34. The consent includes also authorization to publish the collected data for medical/scientific purposes in accordance with local laws.