Nicola Zizzo

A model of study for human cancer: Spontaneous occurring tumors in dogs. Biological features and translation for new anticancer therapies

Murine cancer models have been extremely useful for analyzing the biology of pathways involved in cancer initiation, promotion, and progression. Interestingly, several murine cancer models also exhibit heterogeneity, genomic instability and an intact immune system. However, they do not adequately represent several features that define cancer in humans, including long periods of latency, the complex biology of cancer recurrence and metastasis and outcomes to novel therapies. Therefore, additional models that better investigate the human disease are needed. In the pet population, with special references to the dog, cancer is a spontaneous disease and dogs naturally develop cancers that share many characteristics with human malignancies. More than 40 years ago, optimization of bone marrow transplantation protocols was undertaken in dogs and recently novel targeted therapies such as liposomal muramyl tripeptide phosphatidylethanolamine and several tyrosine kinase inhibitors, namely masitinib (AB1010) and toceranib phosphate (SU11654), have been developed to treat dog tumors which have then been translated to human clinical trials. In this review article, we will analyze biological data from dog tumors and comparative features with human tumors, and new therapeutic approaches translated from dog to human cancer.

VEGF concentration from plasma-activated platelets rich correlates with microvascular density and grading in canine mast cell tumour spontaneous model.

Canine cutaneous mast cell tumour (CMCT) is a common cutaneous tumour in dog, with a higher incidence than in human. CMCT is classified in three subgroups, well and intermediately differentiated (G1 and G2), corresponding to a benign disease, and poorly differentiated (G3), corresponding to a malignant disease, which metastasize to lymph nodes, liver, spleen and bone marrow. In this study, we have evaluated serum (S), platelet‐poor plasma (P‐PP), plasma‐activated platelet rich (P‐APR) and cytosol vascular endothelial growth factor (VEGF) concentrations, microvascular density (MVD) and mast cell density (MCD) in a series of 86 CMCTs and we have correlated these parameters with each other, by means of ELISA detection of VEGF and immunohistochemistry. Results show that VEGF level from cytosol P‐APR and MVD were significantly higher in G3 CMCTs as compared to G1 or G2 subgroups. Moreover, a significantly strong correlation among VEGF levels from P‐PAR and cytosol, MVD and MCD was found in G3 subgroup. Because VEGF levels from P‐APR well correlated with MVD and malignancy grade in CMCT, we suggest that VEGF might be secreted from MCs and it may be a suitable surrogate inter‐species angiogenetic markers of tumour progression in CMCT. Finally, CMCT seems to be a useful model to study the role of MCs in tumour angiogenesis and inhibition of MCs degranulation or activation might be a new anti‐angiogenic strategy worthy to further investigations.

Masitinib (AB1010), from canine tumor model to human clinical development: Where we are?

Masitinib mesylate (AB1010) is a novel potent and selective tyrosine kinase inhibitor, targeting mainly wild-type and mutated c-Kit receptor (c-KitR), Platelet Derived Growth Factor Receptor-alfa/beta (PDGFRa/ß), Lymphocyte-specific kinase (Lck), Lck/Yes-related protein (LYn), Fibroblast Growth Factor Receptor 3 (FGFR3) and Focal Adhesion Kinase (FAK). It is the first anticancer therapy approved in veterinary medicine for the treatment of unresectable canine mast cell tumors (CMCTs), harboring activating c-KitR mutations, at dose of 12.5mg/kg once daily. Considering its anti-proliferative action, principally given by inhibiting the MCs c-KitR anti-angiogenic pathway that leads cancer progression, and its role as chemosensitizer, masitinib is under clinical investigation in several human malignancies (Gastro-Intestinal Stromal Tumors, acute myeloid leukemia, systemic mastocytosis, pancreatic cancer, multiple myeloma, non-small cell lung cancer, melanoma, ovarian and prostate cancer), which are characterized by similar canine c-KIT proto-oncogene mutations. Here, we analyze masitinib structure activity, its pharmacokinetics compared to imatinib, the c-KitR pathway referring to the most frequent c-KIT mutations sensitive or resistant to this novel drug compared to imatinib, and masitinib safety profile. We, also, explore preclinical and clinical (completed and ongoing) trials with the aim to emphasize as this recent anti-angiogenic therapy, at first approved in CMCTs and, currently in development for the treatment of several human neoplasms, could be represent a milestone in translational oncology, in which the murine experimental model of cancer research could be integrated by canine spontaneous tumor model.

Possible biological and translational significance of mast cells density in colorectal cancer

Mast cells (MCs), located ubiquitously near blood vessels, are descended from CD34(+) hematopoietic stem cells. Initially, although their role has been well defined in hypersensitivity reactions, the discovery of their sharing in both innate and adaptive immunity has allowed to redefine their crucial interplay on the regulatory function between inflammatory and tumor cells through the release of mediators granule-associated (mainly tryptase and vascular endothelial growth factor). In particular, in several animal and human malignancies it has been well demonstrated that activated c-Kit receptor (c-KitR) and tryptase (an agonist of the proteinase-activated receptor-2) take pivotal part in tumor angiogenesis after the MCs activation, contributing to tumor cells invasion and metastasis. In this review, we focused on crucial MCs density (MCD) role in colorectal cancer (CRC) development and progression angiogenesis-mediated; then, we will analyze the principal studies that have focused on MCD as possible prognostic factor. Finally, we will consider a possible role of MCD as novel therapeutic target mainly by c-KitR tyrosine kinase inhibitors (imatinib, masitinib) and tryptase inhibitors (gabexate and nafamostat mesylate) with the aim to prevent CRC progression.

Mast cells density positive to tryptase correlates with angiogenesis in pancreatic ductal adenocarcinoma patients having undergone surgery

Background. Literature data suggest that cells such as mast cells (MCs), are involved in angiogenesis. MCs can stimulate angiogenesis by releasing of several proangiogenic cytokines stored in their cytoplasm. In particular MCs can release tryptase, a potent in vivo and in vitro proangiogenic factor. Nevertheless few data are available concerning the role of MCs positive to tryptase in primary pancreatic cancer angiogenesis. This study analyzed MCs and angiogenesis in primary tumour tissue from patients affected by pancreatic ductal adenocarcinoma (PDAC). Method. A series of 31 PDAC patients with stage T2-3N0-1M0 (by AJCC for Pancreas Cancer Staging 7th Edition) was selected and then underwent surgery. Tumour tissue samples were evaluated by means of immunohistochemistry and image analysis methods in terms of number of MCs positive to tryptase (MCDPT), area occupied by MCs positive to tryptase (MCAPT), microvascular density (MVD), and endothelial area (EA). The above parameters were related to each other and to the main clinicopathological features. Results. A significant correlation between MCDPT, MCAPT, MVD, and EA group was found by Pearsons t-test analysis (r ranged from 0.69 to 0.81; P value ranged from 0.001 to 0.003). No other significant correlation was found. Conclusion. Our pilot data suggest that MCs positive to tryptase may play a role in PDAC angiogenesis and they could be further evaluated as a novel tumour biomarker and as a target of antiangiogenic therapy.

C-Kit Expression, Angiogenesis, and Grading in Canine Mast Cell Tumour: A Unique Model to Study C-Kit Driven Human Malignancies

Canine cutaneous mast cell tumour (CMCT) is a c-Kit driven tumour sharing similar c-Kit aberrations found in human gastrointestinal stromal tumour. CMCT is classified into three forms: well- (G1), intermediately (G2) (more benign diseases), and poorly (G3) differentiated (malignant) forms. We assess a correlation between c-Kit status, grading, and angiogenesis in CMCTs to explore their potential significance in humans. C-Kit receptor (c-KitR) expression, microvascular density (MVD), and mast cell granulated and degranulated status density (MCGD and MCDD, resp.) were analyzed in 97 CMCTs, by means of histochemistry, immunohistochemistry double staining, and image analysis system. Data showed that predominantly diffuse cytoplasmic- and predominantly focal paranuclear- (Golgi-like) c-Kit protein (PDC-c-Kit and PFP-c-Kit, resp.) expression correlate with high MVD, G3 histopathological grade, and MCDD. Moreover, predominant cell membrane-c-KitR (PCM-c-KitR) expression status correlates with low MVD, G1-G2 histopathological grade, and MCGD. These findings underline the key role of c-Kit in the biopathology of canine MCTs, indicating a link between aberrant c-Kit expression, increased angiogenesis, and higher histopathological grade. CMCT seems to be a model to study contributions of c-Kit activated MCs in tumour angiogenesis and to evaluate the inhibition of MCs activation by means of c-Kit tyrosine kinase inhibitors, currently translated in humans.

Vascular endothelial growth factor concentrations from platelets correlate with tumor angiogenesis and grading in a spontaneous canine non-Hodgkin lymphoma model

Published data strongly suggest that tumor progression and malignancy are associated with increased angiogenesis. However, no data have been published concerning the relationship between microvascular density (MVD), tumor cytosol, and blood vascular endothelial growth factor (VEGF) concentrations in canine non-Hodgkin lymphoma (C-NHL), a neoplasm that shares biological and clinical characteristics with human NHL. We have evaluated MVD and tumor cytosol, serum (S), platelet-poor plasma (P-PP), plasma-activated platelet rich (P-APR) VEGF concentration in a series of 63 B-cells C-NHL by means of immunohistochemistry and enzyme-linked immuno-sorbent assay (ELISA) detection of VEGF. We found that MVD, VEGF from cytosol, and VEGF from P-APR are significantly correlated (p ranging from 0.001 to 0.003) and that these parameters paralleled with the malignancy degree of NHL. Accordingly, spontaneous C-NHL seems to be an interesting model to study the role of angiogenesis as interspecies pathway of tumor malignancy and we suggest that VEGF from P-APR might be a novel useful circulating bio-marker of tumor angiogenesis.

Endothelial area and microvascular density in a canine non-Hodgkin's lymphoma: an interspecies model of tumor angiogenesis

Experimental and clinical data indicate that tumor progression is associated with angiogenesis and that an increase in microvascular density (MVD) is associated with a poor prognosis, in both solid and hematological malignancies. No data have been published concerning the relationship between angiogenesis and malignancy grade in canine non-Hodgkins lymphoma (NHL), which is a neoplasm that shares several biological and clinical characteristics with human NHL. In the present study, we evaluated this relationship in a series of 43 cases of canine NHL. The results demonstrate that both MVD and endothelial area (EA) were significantly higher in high-grade compared to low-grade lymphoma and a good statistical correlation was found between MVD and EA. These data indicate that increased angiogenesis paralleled with increased malignancy grade in canine NHL, which represents an interesting tumor model for studying the role of angiogenesis as an interspecies pathway of tumoral malignancy and biological aggressiveness.

Microvascular density and endothelial area correlate with Ki-67 proliferative rate in the canine non-Hodgkin's lymphoma spontaneous model.

Experimental and clinical data indicate that tumor progression and malignancy are associated with increased angiogenesis and higher Ki-67 proliferation rate. Furthermore, increased angiogenesis and higher Ki-67 proliferation rate are associated with a poor prognosis, in both solid and hematological malignancies. However, no data have been published concerning the relationship between angiogenesis and Ki-67 proliferation rate in canine non-Hodgkins lymphoma (NHL), a neoplasm that shares several biological and clinical characteristics with human NHL. This study has evaluated the relationship between angiogenesis and Ki-67 proliferation rate in a series of 58 canine NHL. Results showed that microvascular density (MVD), endothelial area (EA) and Ki-67 (MIB-1) are significantly correlated and that all the above indexes paralleled with the malignancy degree of NHL. These data suggest a biological link between angiogenesis and Ki-67 proliferation rate in canine NHL, which represents an interesting model to study the role of angiogenesis and proliferative activity as inter-species pathways of tumoral malignancy and biological aggressiveness.

Infiltrating mast cells correlate with angiogenesis in bone metastases from gastric cancer patients.

While gastric cancer is a well established angiogenesis driven tumor, no data has been published regarding angiogenesis stimulated by mast cells (MCs) positive for tryptase in bone metastases from gastric cancer patients (BMGCP). It is well established that MCs play a role in immune responses and more recently it was demonstrated that MCs have been involved in tumor angiogenesis. We analyzed infiltrating MCs and neovascularization in BMGCP diagnosed by histology. A series of 15 stage T3-4N2-3M1 (by AJCC for Gastric Cancer Staging 7th Edition) BMGCP from bone biopsies were selected. Tumour tissue samples were evaluated by mean of immunohistochemistry and image analysis methods in terms of MCs density positive to tryptase (MCDPT), MCs area positive to tryptase (MCAPT), microvascular density (MVD) and endothelial area (EA). A significant correlation between MCDPT, MCAPT, MVD and EA groups to each other was found by Pearson and t-test analysis (r ranged from 0.68 to 0.82; p-value ranged from 0.00 to 0.02). Our very preliminary data suggest that infiltrating MCs positive for tryptase may play a role in BMGCP angiogenesis, and could be further evaluated as a novel target of anti-angiogenic therapy.