Study design
This was analytical cross-section study among patients undergoing surgery at TMH in a period October 2015 to September 2017. This was conducted in oncology and pathology department at Tongji medical hospital; the said hospital has capacity of 4000-6000 beds.
Specimen Collection
Tissue samples analyzed in this study were collected from Chinese patients (n=161) who had undergone either a palliative surgical procedure or a therapeutic surgical procedure between October 2015 and September 2017 at Tongji medical hospital (Hubei, Wuhan, China). Patient data retrieved were age, sex, date of birth, history of smoking and alcohol consumption, TNM stage, anatomical location of tumor, histopathological pattern of the cancer, tumor grade, Bormann’s classification, vascular and lymphatic involvement by tumor, tumor grading (well differentiated, moderate differentiated, and poorly differentiated), and positive VEGF status. TMN staging criteria were used according to the American Joint Committee on Cancer (AJCC) [17].
Immunohistochemical analysis of VEGF -C
Immunohistochemical staining was done using 4-μm thick paraffin-embedded sections and treated with 0.3% hydrogen peroxide at room temperature for ten minutes. These sections were heated in a solution (pH 6.0) of 1% mmol/L of trisodium citrate in a microwave for the extraction of antigen. Incubation of sections was done at a humidity condition of 4°C of primary antibodies (mouse monoclonal VEGF-C antibody) [1:100, DAKO]. Chosen slides were then washed three times in 0.1 mmol/L PBS for about 2 minutes and then incubated at standard room temperature with horseradish peroxidase (Envision, DAKO) conjugated mouse secondary antibody for 30 minutes. Negative control for VEGF-C detection was done using normal rabbit antibody IgG after development was done with 3,3'-Diaminobenzidine.
VEGF -C scoring according to immunohistochemistry
Two pathologists who were unaware of the clinical outcome of the patients did a pathological analysis of immunohistochemistry. The analysis of the staining was exclusively restricted to tumor cell reactions. VEGF-C stained results were classified, according to the intensity of color and percentage of epithelial cells that showed specific immunoreactivity, into 0, 1, 2, and 3 designations. If the summation of intensity and percentage was in the 0-2 ranges, it was considered a weak expression of VEGF-C, while a range of 3-6 was regarded as a strong expression of VEGF-C [18, 19, 20].
Statistical data analysis
Data were entered into Excel, imported for analysis into Stat software version 13.v, and analyzed according to the objectives. Continuous variables were analyzed using mean and standard deviation. The significance of the associations between the predictor and outcome variables was calculated using a Chi-square test on the categorical variables. The odds ratio was used to test the strength of the association between predictor and outcome variables. A p-value of less than 0.05 was seen as significant. Predictor variables, which were found to be significant on univariate analysis, were subjected to multivariate logistic regression analysis to test the significance of the association between these variables and the outcome.