S.M. Rodigheri; C.R. Daleck; R.L. Amorim; S.G. Calazans; J.L. Sequeira; M.M.P. Rodrigues; P. Pinczowski; A.B. De Nardi; S.C. Fernandes; J.R.F. Cesar
Lymphomas are usually diagnosed in dogs (Vail & Young 2007). Thus, the identification of biologically prognostic factors in lymphomas must be investigated (Korkolopoulou et al. 2005). The vascular endothelial growth factor (VEGF) is a 46 kD dimeric glycoprotein secreted by neoplastic cells, macrophages, plasma cells and lymphocytes, which triggers endothelial cell proliferation by interacting with specific receptors in a paracrine or autocrine fashion (Ferrara 2004). VEGF is one of most important mediators of neoplastic angiogenesis and lymphangiogenesis (Hirakawa et al. 2005). Angiogenesis is an integral component of the viability of all tumor types, reflecting the inviolable demand of a growing tumor for sufficient levels of nutrients and oxygen exchange (Korkolopoulou et al. 2005). Lymphangiogenesis is also important for tumors expansion, especially the for lymph node metastasis (Kadowaki et al. 2005). The aim of this study was to investigate the expression of VEGF in dogs with lymphoma by means of immunohistochemistry and its relationship to biological behavior of canine lymphomas.
Materials and Methods
Fifteen dogs with lymphoma referred to Oncology Unit in the Veterinary Hospital of São Paulo State University (UNESP/Jaboticabal) were included in this study. Control group consisted of five healthy dogs. Clinical staging was accomplished, (stage I--involvement limited to a single lymph node, or lymphoid tissue in a single organ; stage II--involvement of many lymph nodes in regional area; stage III--generalized lymph node involvement; stage IV--liver and/or spleen involvement or stage V--manifestations in the blood and involvement of bone marrow and/or other organ systems), as well as anatomical classification (multicentric, alimentary, cutaneous, mediastinal or extranodal). Tumor samples were collected by means of incisional or excisional biopsy. All tissue samples were fixed in 10% buffered formalin solution and embedded in paraffin. Histopathological diagnosis was performed on the slides stained with hematoxylin and eosin, according to Kiel classification. Immunohistochemistry was performed to identify T or B cell origin, using anti-CD3 polyclonal antibody (Dako) and anti-CD79a monoclonal antibody (Dako), respectively. VEGF immunohistochemistry was performed as following: consecutive 3μm sections were cut from each block, deparaffinized and rehydrated. Endogenous peroxidase activity was blocked with hydrogen peroxide for 20 minutes and washed in distillated water. Before incubation with the primary antibody, sections were heated in steamer (95°C), for 30 minutes. The antibody mouse anti-VEGF monoclonal (Dako) was used at a 1:25 dilution. Sections were incubated with primary antibody overnight at 4°C, following the two-step horseradish peroxidase technique and using Envision Dual-link Kit (Dako). Negative controls were performed using non-specific IgG as the primary antibody. Five fields in each section were examined at x400 magnification for the VEGF analysis. The percentage of VEGF-positive tumor cells was scored at 0 to 4 levels (0: less than de 5% of labeled cells to 4: more than 75% of labeled cells). Staining was scored as weak (1), moderate (2) or strong (3). Final scoring was defined at 0 to 12, based on two scoring criteria described. Statistical comparisons between groups were made with the Mann-Whitney test and p<0.05 was considered significant. Spearman correlation test was used to correlate immunolabeling, histologic grade, cellular morphology and immunophenotype.
Histological classification showed high-grade lymphomas prevalence (86,7%), corresponding to morphological subtypes: centroblastic (5), immunoblastic (4), lymphoblastic (2) and anaplastic (2). Two low-grade lymphomas were identified: a centrocytic and a centroblastic-centrocytic lymphoma. The majority of lymphomas were of B-cell origin (67%). VEGF showed a diffuse cytoplasmatic staining pattern, detected in neoplastic and normal lymph nodes. VEGF mean score was higher (p < 0,0001) in dogs with lymphoma (lymphoma: 9.07±2.34; control: 2.70±2.35). Ninety per cent of control lymph nodes showed VEGF immunostaining, with average score 0 to 6. All lymphomas presented VEGF immunostaining, with average score 6 to 12. No correlation was verified among VEGF immunostaining and histological classification or immunophenotype (p > 0.05) in dogs with lymphoma.
Discussion and Conclusions
More than 35 years ago, Judah Folkman suggested that cancer arising depends on vascular support within the tumor. After that, several studies have been focused on angiogenesis, trying to find biomarkers which could predict prognosis, detect cancer in early stages and provide a model for new anticancer agents (Lamontagne 2005). VEGF immunostaining is a prognostic factor for different solid tumor in humans (Ferrara 2004). Some studies have been shown that VEGF expression is higher in dogs with malignant mammary tumors, squamous cell carcinomas, seminomas and mastocytomas (Maiolino et al. 2000, Restucci et al. 2002, Restucci et al. 2003, Rebuzzi et al. 2007). Although it is known that solid tumor development requires vigorous vascularization, the influence of angiogenesis in hematopoietic tumors was just recognized (Koster & Raemaekers 2005). Jorgensen (2005) evaluated the VEGF immunostaining in different histological types of human non-Hodgkin lymphomas, and it was stronger in more aggressive tumors. Previous studies reported the correlation between VEGF expression and some prognostic factors in human lymphomas (Hazar et al. 2003, Korkolopoulou et al. 2005). In this present study, from 15 samples evaluated, 12 (80%) showed high scores (8, 9 or 12). Wolfesberger et al. (2007) detected 60% of high VEGF expression by evaluating percentage of positive cells. Hazar et al. (2003) reported VEGF expression in 33.8% of human non-Hodgkin lymphomas. Ten per cent of control lymph nodes presented no VEGF immunostaining, and 60% presented low scores (1 and 2). Wolfesberger et al. (2007) also verified VEGF expression in normal lymph nodes, suggesting the need for VEGF in physiological maintenance of lymph nodes. Immunohistochemical technique used monoclonal anti-VEGF-A antibody. VGF-A is an important angiogenesis and lymphangiogenesis inducer (Hirakawa et al. 2005, Kadowaki et al. 2005). Our results showed that VEGF mean score was higher (p < 0.0001) in canine lymphomas, suggesting the influence of VEGF in biological behavior of this neoplasm. Although we cannot conclude that VEGF superexpression detected in canine lymphomas results in lymphatic and/or sanguineous vascularization, lymphangiogenesis seems to be essential to dissemination of neoplastic cells to lymph nodes and to other tissues.
Authors gratefully acknowledge the financial support of the Fundação de Amparo à Pesquisa do Estado de São Paulo (Fapesp).
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