Development of a flow cytometric panel to assess prognostic biomarkers in fine needle aspirates of canine cutaneous or subcutaneous mast cell tumors

Mast cell tumor (MCT) is a common skin cancer in dogs that has a wide range of clinical behaviors. The purpose of this study was to develop a novel multicolor flow cytometry (FC) panel that will enable the quantification of candidate prognostic markers (Ki-67 and pKIT) in fine needle aspirate (FNA) samples prior to surgical removal of the tumors. FNA of canine MCTs and the NI-1 cell line were utilized to develop a FC panel that includes a viability dye (FVS620, BD Biosciences; 7-AAD, Invitrogen) and the following primary conjugated antibodies: CD117-PE (ACK45, BD Biosciences), pKIT-A647 (polyclonal bs-3242R, BIOSS) and Ki-67-FITC (20Raj1, eBioscience; MIB-1, DAKO). A total of nine FNA samples of canine MCTs were collected, seven out which produced sufficient cells for FC analysis. The Ki-67 antibody clone 20Raj1 produced a positive signal when applied to blood leukocytes but failed to provide robust labeling of neoplastic mast cells. The Ki-67 antibody clone MIB-1 delivered a superior staining quality in both the NI-1 cells and primary MCT cells. CD117-PE signal was adequate post fixation and permeabilization and in the combination of 7-AAD. pKIT produced non-specific staining and was not suitable for this multicolor FC panel. In conclusion, FNA samples of canine MCTs can often yield adequate cell numbers for FC analysis, and a multicolor FC panel was developed that can detect Ki-67 in canine mast cells. This would permit further studies into the potential use of this panel for canine cutaneous and subcutaneous MCT prognostication purposes

Altered Erythropoiesis in Mouse Models of Type 3 Hemochromatosis

Type 3 haemochromatosis (HFE3) is a rare genetic iron overload disease which ultimately lead to compromised organs functioning. HFE3 is caused by mutations in transferrin receptor 2 (TFR2) gene that codes for two main isoforms (Tfr2α and Tfr2β). Tfr2α is one of the hepatic regulators of iron inhibitor hepcidin. Tfr2β is an intracellular isoform of the protein involved in the regulation of iron levels in reticuloendothelial cells. It has been recently demonstrated that Tfr2 is also involved in erythropoiesis. This study aims to further investigate Tfr2 erythropoietic role by evaluating the erythropoiesis of two Tfr2 murine models wherein either one or both of Tfr2 isoforms have been selectively silenced (Tfr2 KI and Tfr2 KO). The evaluations were performed in bone marrow and spleen, in 14 days’ and 10 weeks’ old mice, to assess erythropoiesis in young versus adult animals. The lack of Tfr2α leads to macrocytosis with low reticulocyte number and increased hemoglobin values, together with an anticipation of adult BM erythropoiesis and an increased splenic erythropoiesis. On the other hand, lack of Tfr2β (Tfr2 KI mice) causes an increased and immature splenic erythropoiesis. Taken together, these data confirm the role of Tfr2α in modulation of erythropoiesis and of Tfr2β in favoring iron availability for erythropoiesis

The role of vascular endothelial growth factor and matrix metalloproteinases in canine lymphoma: in vivo and in vitro study

Background: Canine lymphoma represents the most frequent haematopoietic cancer and it shares some similarities with human non-Hodgkin lymphoma. Matrix metalloproteinases (MMPs) and vascular endothelial growth factor (VEGF) play a coordinated role during invasion and proliferation of malignant cells; however, little is known about their role in canine haematologic malignancies. The aim of this study was to investigate the mRNA and protein expression of VEGF and the most relevant MMPs in canine lymphoma. Lymph node aspirates from 26 B-cell and 21 T-cell lymphomas were collected. The protein expression levels of MMP-9, MMP-2 and VEGF-A were evaluated by immunocytochemistry, and the mRNA levels of MMP-2, MMP-9, MT1-MMP, TIMP-1, TIMP-2, RECK, VEGF-A and VEGF-164 were measured using quantitative RT-PCR.Results: MT1-MMP, TIMP-1 and RECK mRNA levels were significantly higher in T-cell lymphomas than in B-cell lymphomas. Higher mRNA and protein levels of MMP-9 and VEGF-A were observed in T-cell lymphomas than in B-cell lymphomas and healthy control lymph nodes. A positive correlation was found between MMP-9 and VEGF-A in T-cell lymphomas. Moreover, MMP-9, MT1-MMP, TIMP-1 and VEGF-A were expressed at the highest levels in high-grade T-cell lymphomas.Conclusions: This study provides new information on the expression of different MMPs and VEGF in canine lymphoma, suggesting a possible correlation between different MMPs and VEGF, immunophenotype and prognosis

New molecular and therapeutic insights into canine diffuse large B cell lymphoma elucidates the role of the dog as a model for human disease

open21siopenAresu, Luca; Ferraresso, Serena; Marconato, Laura; Cascione, Luciano; Napoli, Sara; Gaudio, Eugenio; Kwee, Ivo; Tarantelli, Chiara; Testa, Andrea; Maniaci, Chiara; Ciulli, Alessio; Hillmann, Petra; Bohnacker, Thomas; Wymann, Matthias P; Comazzi, Stefano; Milan, Massimo; Riondato, Fulvio; Dalla Rovere, Giulia; Giantin, Mery; Giannuzzi, Diana; Bertoni, FrancescoAresu, Luca; Ferraresso, Serena; Marconato, Laura; Cascione, Luciano; Napoli, Sara; Gaudio, Eugenio; Kwee, Ivo; Tarantelli, Chiara; Testa, Andrea; Maniaci, Chiara; Ciulli, Alessio; Hillmann, Petra; Bohnacker, Thomas; Wymann, Matthias P; Comazzi, Stefano; Milan, Massimo; Riondato, Fulvio; Dalla Rovere, Giulia; Giantin, Mery; Giannuzzi, Diana; Bertoni, Francesc