Rosa Patruno

Vascular Endothelial Growth Factor (VEGF) as a Target of Bevacizumab in Cancer: From the Biology to the Clinic

Angiogenesis is important in the growth and progression of solid tumours. The main pro-angiogenic factor, namely vascular endothelial growth factor (VEGF), also known as vascular permeability factor, is a potent angiogenic cytokine that induces mitosis and also regulates the permeability of endothelial cells. The soluble isoform of VEGF is a dimeric glycoprotein of 36-46 kDa, induced by hypoxia and oncogenic mutation and it binds to two specific tyrosine-kinase receptors: VEGF-1 (flt-1) and VEGF-2 (KDR/flk1). An increase in VEGF expression in tumour tissue or some blood compartments (i.e. serum or plasma) has been found in solid and haematological malignancies of various origins and is associated with metastasis formation and poor prognosis. Bevacizumab, a recombinant humanised monoclonal antibody developed against VEGF, binds to soluble VEGF, preventing receptor binding and inhibiting endothelial cell proliferation and vessel formation. Pre-clinical and clinical studies have shown that bevacizumab alone or in combination with a cytotoxic agent decreases tumour growth and increases median survival time and time to tumour progression. Bevacizumab is the first anti-angiogenetic treatment approved by the American Food and Drug Administration in the first-line treatment of metastatic colorectal cancer. It has shown preliminary evidence of efficacy for breast, non-small-cell lung, pancreatic, prostate, head and neck and renal cancer as well as haematological malignancies. Common toxicities associated with bevacizumab include hypertension, proteinuria, bleeding episodes and thrombotic events. This review summarises the critical role of VEGF and discusses the data available on bevacizumab, from the humanisation of its parent murine monoclonal antibody (mAb) A.4.6.1 to its use in cancer clinical trials.

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.

Mast Cell Positivity to Tryptase Correlates with Metastatic Lymph Nodes in Gastrointestinal Cancer Patients Treated Surgically

Background: Angiogenesis has been found to be a reliable prognostic indicator for several types of malignancies. Tryptase is a serine protease stored in mast cell (MC) granules, which plays a role in tumor angiogenesis. MCs can release tryptase following c-Kit receptor activation. Method: In this study, immunohistochemistry, image analysis methods and clinical aspects were employed in a series of 41 gastrointestinal cancer patients with stage T3-4N2a-bM₀ (by the American Joint Committee on Cancer, AJCC, for colorectal cancer, 7th edition) and T3N2-3M₀ (by AJCC for gastric cancer, 7th edition) to evaluate the possible correlation between MCs positive to tryptase (MCPT) in tumor tissue and the number of metastatic lymph nodes harvested. Results: Data demonstrated a positive correlation between MCPT in tumor tissue and the number of metastatic lymph nodes; the validity of these data needs confirmation in larger patient cohorts. Conclusion: This is the first report considering MCPT in tumor tissue as a potential tool for a valid indication of the type of surgical treatment and its radicality, and it might be considered for the prognosis of patients before radical surgical treatment. Our pilot data need confirmation in a larger patient cohort.

Sorafenib (BAY 43-9006) in Hepatocellular Carcinoma Patients: From Discovery to Clinical Development

Angiogenesis and signaling through the RAS/RAF/mitogen-activated protein/extracellular signal-regulated kinase (ERK) kinase (MEK)/ERK cascade have been reported to play important roles in the development of hepatocellular carcinoma (HCC). Sorafenib (Nexavar), a novel bi-aryl urea BAY 43-9006, is an orally administered multikinase inhibitor with activity against RAS/RAF kinases multikinase inhibitor with activity against RAF kinases and several receptor tyrosine kinases, including vascular endothelial growth factor receptor (VEGFR), platelet-derived growth factor receptor (PDGFR), FLT3, Ret, and c-Kit. It is involved in angiogenic pathway and cell proliferation. Sorafenib has demonstrated potent anti-tumor activity in in vitro studies, preclinical xenograft models of different tumor types and human clinical trials. This review summarizes the history of sorafenib from its discovery by the medicinal chemistry approach through to clinical development and ongoing trials on the combination between sorafenib and trans-arterial chemoembolization (TACE) in HCC patients.

FISH IMMUNOLOGY. I. BINDING AND ENGULFMENT OF CANDIDA ALBICANS BY ERYTHROCYTES OF RAINBOW TROUT (SALMO GAIRDNERI RICHARDSON)

ABSTRACT The role of fish erythrocytes (FE) as phagocytic cells has poorly been investigated, until now. Here, we have focussed our attention on the interplay between rainbow trout (Salmo gairdneri Richardson) erythrocytes and Candida albicans (CA). At the same time, the intervention of autologous head kidney macrophages (MØ) in the CA processing by FE has been studied. Data show that CA particles bind to FE, which, in turn, are able to engulf but not kill them. In the presence of MØ, a decrease of FE with bound CA occurs and, in some microscopic images, FE form rosettes with MØ. Phagocytosis of CA is higher in rosetting MØ than in non-rosetting ones. According to our findings, it appears that FE represent a reservoir of engulfed CA and rosetting is an efficacious phenomenon of presentation of pathogens to MØ, where an effective clearance of them can take place.

Correlation between Serum Tryptase, Mast Cells Positive to Tryptase and Microvascular Density in Colo-Rectal Cancer Patients: Possible Biological-Clinical Significance

Background Tryptase is a serin protease stored and released from mast cells (MCs) that plays a role in tumour angiogenesis. In this study we aimed to evaluate serum tryptase levels in colo-rectal cancer (CRC) patients before (STLBS) and after (STLAS) radical surgical resection. We also evaluated mast cell density positive to tryptase (MCDPT) and microvascular density (MVD) in primary tumour tissue. Methods A series of 61 patients with stage B and C CRC (according to the Astler and Coller staging system) were selected. Serum blood samples were collected from patients one day before and one day after surgery. Tryptase levels were measured using the UniCAP Tryptase Fluoroenzymeimmunoassay (Pharmacia, Uppsala, Sweden). Tumour sections were immunostained with a primary anti-tryptase antibody (clone AA1; Dako, Glostrup, Denmark) and an anti CD-34 antibody (QB-END 10; Bio-Optica Milan, Italy) by means of immunohistochemistry and then evaluated by image analysis methods. Results The mean ± s.d. STLBS and STLAS was 5.63±2.61 µg/L, and 3.39±1.47 µg/L respectively and a significant difference between mean levels was found: p = 0.000 by t-test. The mean ± s.d. of MCDPT and MVD was 8.13±3.28 and 29.16±7.39 respectively. A strong correlation between STLBS and MVD (r = 0.83, p = 0.000); STLBS and MCDPT (r = 0.60, p = 0.003); and MCDPT and MVD (r = 0.73; p = 0.001) was found. Conclusion Results demonstrated higher STLBS in CRC patients, indicating an involvement of MC tryptase in CRC angiogenesis. Data also indicated lower STLAS, suggesting the release of tryptase from tumour-infiltrating MCs. Serum tryptase levels may therefore play a role as a novel bio-marker predictive of response to radical surgery. In this context tryptase inhibitors such as Gabexate and Nafamostat Mesilate might be evaluated in adjuvant clinical trials as a new anti-angiogenic approach.

A possible role of thymidine phosphorylase expression and5-fluorouracil increased sensitivity in oropharyngeal cancer patients

Thymidine Pi deoxyribosyltransferase (TP) is an enzyme involved in DNA synthesis up‐regulated in tumours and it is also a pro‐angiogenic factor. TP cannot activate capecitabine, because capecitabine first needs conversion by carboxylesterase and cytidine deaminase into 5‐deoxy‐fluorouridine. This compound can be activated by TP to 5‐fluorouracil (5‐FU). Although TP is not necessary for 5‐FU toxicity, experimental data suggest that high levels of TP correlate with an enhanced response to 5‐FU therapy. In this study, we have analysed by immunohistochemistry CD34, CD68 and TP positive cells in bioptic samples from 53 patients with T1–3 N0–1 M0 oropharyngeal squamous cell carcinoma (OSC) and from 24 patients with non‐dysplastic oropharyngeal leukoplakia (NDOLP). Results showed that the mean of TP‐positive cells, CD68 positive macrophages and CD34 positive endothelial cells eval‐uated as microvessel density (MVD) was significantly higher in OSC than in NDOLP. Moreover, at a median follow‐up of 19 months, patients with TP expression and higher MVD showed a better survival rate as compared to those with low MVD, probably as a consequence of 5‐FU‐based therapy.We hypothesized a role for TP in oropharyngeal tumourigenesis and 5‐FU activation in the adjuvant setting of OSC patients.

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.