Hemangiopericytoma (HPT) is a rare mesenchymal tumor of fibroblastic type and for its rarity is poorly studied. The most common sites of metastatic disease in patients with intracranial HPT are the bone, liver, and lung, suggestive for an hematogenous dissemination; for this reason, we investigated, for the first time, the presence of circulating tumor cells (CTCs) in hemangiopericytoma patient by CellSearch® and SceenCell® devices. Peripheral blood samples were drawn and processed by CellSearch, an EpCAM-dependent device, and ScreenCell®, a device size based. We found nontypical CTCs by CellSearch system and the immunofluorescence analysis performed on CTCs isolate by ScreenCell demonstrated the presence of single CTCs and CTC clusters. The molecular characterization of single CTCs and CTC clusters, using antibodies directed against EpCAM, CD34, cytokeratins (8, 18, and 19), and CD45, showed a great heterogeneity in CTC clusters. We believe that the present study may open a new scenario in the rare tumors: the introduction of the liquid biopsy and the molecular characterization of circulating tumor cells could lead to personalized targeted treatments and also for rare tumors.
Hemangiopericytoma (HPT) is a rare mesenchymal tumor of fibroblastic type [
Circulating tumor cells were detected through two methods, one EpCAM-dependent (CellSearch) and the other size based (ScreenCell). Peripheral blood samples were drawn after informed consent into two tubes, after discarding the first milliliters to avoid contamination with cutaneous epithelial cells taken by the needle during the sampling. 7.5 mL of blood was collected in CellSave preservative tube (Menarini Silicon Biosystems, Castel Maggiore, Bo, Italy) and processed by CellSearch (Menarini Silicon Biosystems), employing CellSearch CTC kit, according to manufacturer’s instructions. Six milliliters of blood was collected in tube containing K2EDTA and processed by ScreenCell Cyto kit (ScreenCell, Sarcelles, France) to isolate fixed cells for cytological studies. Briefly, in order to fix the cells and lyse red blood cells, 3 mL of blood was diluted in 4 mL of FC2 filtration buffer (ScreenCell). After 8 min of incubation at room temperature, 7 mL of diluted sample was added into device tank and filtered under a pressure gradient using a vacutainer tube. After washing with PBS to remove red blood cells debris, the filter was left on absorbing paper to dry at room temperature and then stored at −20°C. The filtration was carried out in duplicate and completed within 3 min.
For immunofluorescence, nonspecific protein binding was blocked by 5% normal goat serum. Specimens were permeabilized in PBS-Tween 20 (PBS-T) for 20 min.
Protocol 1: the filter was first incubated overnight at 4°C with primary mouse monoclonal anti-EpCAM (VU1D9; 1 : 50; Invitrogen Ltd., Paisley, UK) antibody and then, after washing twice with PBS-T, probed for 1 h with antimouse AlexaFluor-488 secondary antibody (1 : 50, Invitrogen). Next, section was incubated overnight at 4°C with staining solution of phycoerythrin- (PE-) conjugated cytokeratins (CK) 8, 18, and 19 and allophycocyanin- (APC-) conjugated CD45 antibodies (CellSearch CTC kit). Nuclei were counterstained with 4
Protocol 2: the filter was first incubated overnight at 4°C with primary mouse monoclonal anti-EpCAM and rabbit polyclonal anti-CD45 (1 : 50; Cell Signaling Technology, Danvers, MA, USA) antibodies and then, after washing twice with PBS-T, probed for 1 h with antimouse AlexaFluor-350 and antirabbit AlexaFluor-594 (1 : 50; Invitrogen) secondary antibodies. Next, section was incubated overnight at 4°C with CD34 class III/FITC (Dako North America Inc., Carpinteria, CA, USA). Finally, specimen was counterstained with DRAQ5 (Cell Signaling Technology), a cell permeable far-red fluorescent DNA dye, for visualization of cell nuclei.
All primary antibodies were diluted in 1% bovine serum albumin in PBS-T.
The filters were scanned by a digital scanner (Aperio ScanScope FL System, Aperio Technologies, Inc., Oxford, UK) and were also analyzed by confocal microscopy (Leica TCS-SP2).
CTC clusters were defined as groups of CTCs containing ≥3 distinct nuclei according to previous publication [
According to the mesenchymal nature of hemangiopericytoma, we failed to detect typical epithelial-like CTCs, but we were able to identify 5 DAPI+/EpCAM+/CK-/CD45- events, which might be referable to cells with nonepithelial features. According to the CellSearch guidelines, all objects with no clear cytokeratin staining are defined as “suspicious objects.” Similar events have been previously described by our group to have prognostic significance in other cancer types, such as renal cell carcinoma, frequently lacking cytokeratin expression [
Phenotype of circulating tumor cells isolated using ScreenCell Cyto kit. In representative images, nuclei are displayed in blue. Pseudocolors were changed for each of the four channels to optimize the visualization of the different signals. (a) Immunofluorescence for CD34, EpCAM, and CD45. Clusters of CD45- cells coexpressing EpCAM and CD34 are displayed. (b) and (c) Immunofluorescence for EpCAM, cytokeratins (CK) 8, 18, and 19, and CD45. Single CD45-/CK- cells expressing EpCAM (b). Cluster of CD45- cells coexpressing EpCAM and CK (c).
The authors have no conflicts of interest to declare.
Paola Gazzaniga and Walter Gianni contributed equally to this work.
This work was supported by A.R.Ger.On. Onlus.