General information
Patients who underwent surgical resection of renal tumors between January and December 2018 at the First Affiliated Hospital of Suzhou University with a pathology diagnosis of RCCC were enrolled. Exclusion criteria included a history of autoimmune disease or infectious disease. A total of 66 patients were enrolled, including 40 males and 26 females, age 35–82 years, with an average age of 61.62 ±10.87. There were 40 patients in which the tumor was less than 4 cm in diameter, 17 in which the diameters were 4 cm to 7 cm, and nine patients in which diameters were greater than 7 cm. There were 27 patients with R. E. N. A. L. scores of less than 6 and 37 patients with scores of more than 6. According to Fuhrman’s histological classification, 24, 28, 11 and 3 tumors were classified as grade I, II, III and IV respectively. According to TNM staging of American Cancer Federation in 2010, there were 46 tumors in stage I and 20 in stage II.
Immunohistochemistry
Specimen collection
We collected specimens from renal tumors and corresponding adjacent normal tissues (more than two centimeters away from the edge of the tumor and no invasion of tumor cells confirmed on post-surgical pathology). The specimens were fixed in formalin (10% formaldehyde solution) and then embedded in paraffin and sectioned for archiving.
Immunohistochemistry (SP method)
Preserved paraffin slices were sequentially treated by dewaxing, hydration, phosphate buffer saline (PBS) washing and rinsing with citric acid buffer. To inactivate endogenous peroxidase, 3% hydrogen peroxide was dropped onto the slices, maintained at room temperature for 10 minutes and washed again in PBS. Subsequently, 5% bovine serum albumin (BSA) 50μl were added to the slides and incubated at room temperature for 10 minutes. After adding primary antibody against IL–9 (diluted at 1:200), the slices were incubated overnight at 4 °C, and washed with PBS thereafter. Next, second antibody was added, incubated at room temperature for 60 minutes and then washed with PBS again. Then sections were reacted with DAB color reagent for 5 minutes, re-stained with hematoxylin, dehydrated with a gradient concentration of ethanol, and finally sealed with neutral resin for microscopic observation.
Criteria for determining results
Because IL–9 is mainly expressed in the cytoplasm, the appearance of pale yellow, brown yellow or brown granules in the cytoplasm at 200-fold magnification was considered positive. For a section judged positive, ten microscopic views with 400-fold magnification were randomly observed and scored according to the proportion of positive cells and staining intensity. Specifically, when the proportion of positive staining cells was less than 5%, 5%–30%, 31%–60% and more than 60%, the scores were zero, one, two and three, respectively. The scoring criteria for staining intensity were as follows: colorless scored zero, light yellow scored one, brown yellow scored two and brown scored three. Finally, the score of positively stained cell proportion was multiplied by the staining intensity score, and the product of less than two points were classified as negative (-); otherwise it was considered positive (+) [10].
Follow-up
Patients were followed up for six to seven months with the deadline of June 1, 2019. Progression-free survival (PFS) was defined as the time from the day of surgery to the deadline or the time point when progression was observed. Patients with stage T1-T2 were followed up every six weeks after surgery, then every six months for up to three years, and annually thereafter. Patients with stage T3-T4 were followed up every six weeks after surgery, every three months up to two years, then every six months from the third year and annually thereafter. Follow-up was conducted by telephone and outpatient consultation.
Statistical analysis
The quantitative data were expressed as mean ± standard deviation (SD). Qualitative data were expressed as ratios; comparisons between two groups were determined using the 2 test. Statistical analyses were performed using SPSS 22.0. GraphPad Prism 8.0 was used to draw survival curves. Kaplan-Meier and Log-rank tests were used for survival analysis. All p-values were considered statistically significant if P <0.05.