Participants
The study was approved by the Institutional Ethics Committee of the First Hospital of Jilin University, Changchun, China. Written informed consent was obtained from the patients by email or letter. We retrospectively reviewed the Picture Archiving and Communication System for renal cell carcinoma patients who had undergone renal CT plain scan plus three-phase enhancement before surgery (January 2017 to December 2019). The inclusion criteria were as follows: 1) patients aged between 18 and 80 years; 2) renal CT plain scan plus three-phase enhancement before surgery; 3) patients who had undergone CT performed according to our standard MM protocol; 4) patients who had received standard nephrectomy; 5) There is an interval between CT scan and surgery and the interval was no more than 2 weeks.
The exclusion criteria were as follows: 1) patients with renal cell carcinoma complicated with hemorrhage were excluded; 2) Imaging data with poor quality due to body movements or other artifacts.
Energy Spectrum CT Imaging
Renal three-phase enhancement CT were performed on a Revolution GSI CT scanner (GE Healthcare). Scanning range was conducted from “diaphragmatic apex to lower pole of kidney” with supine position. The CT scan was conducted with the following parameters: tube voltage, automatically selected by the machine according to the patient's weight; tube current, 485 mA; pitch, 0.984; field of view (FOV), medium; image matrix,512×512; rotation speed, 0.6; slice thickness/gap, 5/5mm. Renal three-phase enhancement scan: high pressure syringe was used to inject non-ionic contrast agent iohexol or iopatol through elbow vein, with the dosage of 1.0-1.5ml/kg and the speed of 3.5ml/s. The cortical phase scanning was performed at 25s after injection, renal parenchymal phase scanning was performed at (60 ± 5)s, and excretion phase scanning was performed at (180 ± 30)s after injection. During the scanning, the patient was asked to hold his breath after deep inspiration to avoid motion artifacts affecting the image quality.
Acquisition of iodine and water concentration
The iodine and water concentration of renal three-phase enhancement CT were measured in Picture Archiving and Communication Systems (Neusoft, Version 5.5, China). The IC of each tumor needs to take the average of six values on the decomposition images for iodine (120kev). These six values include four values in the axial maximum plane and two values in the coronal maximum plane. The four measurement values of the maximum axial plane include the measurement values of the front, back, left and right directions, and the region of interest (ROI) of these four directions should not cross each other as far as possible, and avoid obvious necrosis. And keep the area of ROI consistent. The WC of each tumor was obtained on the water equivalent images in the same way as above. The iodine concentration in the aorta (ICao) at the same slice of the lesion was obtained. Normalized iodine concentration (NIC) was normalized to the ICao using the formula NIC = IC/ICao. In the same way, the normalized WC (NWC) was obtained using the formula NWC = WC/WCao. WCD1 = The difference of WC between renal cortical phase and parenchymal phase. NWCD1 = The difference of NWC between renal cortical phase and parenchymal phase. WCD2 = The difference of WC between renal parenchymal phase and excretory phase. NWCD2 = The difference of NWC between renal parenchymal phase and excretory phase. WCD3 = The difference of WC between renal cortical phase and excretory phase. NWCD3 = The difference of NWC between renal cortical phase and excretory phase. ICD1 = The difference of IC between renal cortical phase and parenchymal phase. NICD1 = The difference of NIC between renal cortical phase and parenchymal phase. ICD2 = The difference of IC between renal parenchymal phase and excretory phase. NICD2 = The difference of NIC between renal parenchymal phase and excretory phase. ICD3 = The difference of IC between renal cortical phase and excretory phase. NICD3 = The difference of NIC between renal cortical phase and excretory phase.
Interpretation of clinicopathological results
According to the electronic medical records, the pathologic findings such as tumor types, tumor classifications, and stage of tumor were assessed. Distant metastasis was defined by imaging examinations and clinical follow-up[12].
Immunohistochemical analysis
Immunohistochemistry was used to stain tumor microvessels. The first antibody was CD34. The thickness of paraffin fixed sections was 5 µ m, which were dewaxed and hydrated. Microwave antigen repair, 2% citrate buffer, PH 6.0, 15min. After the first antibody was dripped, the slices were incubated in a wet box at 4 ℃ overnight. After the universal second antibody was dripped, the slices were placed in a wet box and incubated at 37 ℃ for 30 min. The slices were washed with TBS (0.01 mol / L, pH7.4) for 3 times. After DAB staining, the nuclei were stained with hematoxylin. Finally, seal the slide.
Analysis of tumor microvessels
Five sections per patient, and the average value of microvessel density, area and grading was taken from the five sections. When no less than one slice has characteristic blood vessels, the characteristic blood vessels can be defined as positive.
1) Microvessel density (MVD): After staining, the high vascular density area was found under low power microscope. The top five regions with the higher vascular density in each slice were selected. The number of microvessels in each field of vision was counted. The average value of the five regions is calculated as MVD. When counting, only a single endothelial cell or cell cluster is counted, otherwise the vascular lumen is not counted as microvessel.
2) Microvascular area (MVA): Percentage of microvessel area in tumor tissue area.
3) Microvascular classification: Punctiform microvessels showed that the microvessels were divided by tumor cells and distributed in punctiform shape, and there was no communication between the microvessels. The linear microvessel showed that there were punctate microvessels on both sides of the microvessel, and the middle was long and narrow, which was linear. The annular microvessels are characterized by thick microvessels, which encircle the tumor cells in an island shape and communicate with each other. Strip type microvessels showed thick microvessels, parallel to tumor trabeculae, communicating with each other, showing a thick network. The tumor microvascular classification was semi quantified: the punctiform type was grade I, the linear type was grade II, and the annular or strip type was grade III. The microvascular grade of the tumor was based on the majority of the grading results in any 10 high-power fields.
4) Analysis of special vascular structure: Record whether there are vascular lakes, vascular trees (One main vessel accompanied by branch vessels), thick muscular vessels and vessels with diameter more than 50 microns appearing.
Statistical analysis
Pearson’s or Spearman’s correlation test was used to test the correlation between WC and IC values (mean WC, mean IC, random WC, random IC, mean NWC and mean NIC in renal cortical phase, parenchymal phase and excretory phase; WCD, NWCD, DIC and NICD parameters) with microvessel parameters (MVD, MVA, specific microvascular, microvascular grading, vessel diameter and tumor diameter). All statistical analyses were performed using SPSS 21.0 (SPSS). P < 0.05 was considered statistically significant.