Correlation Study Between CT Perfusion Imaging And Angiogenesis, Invasion And Metastasis Of Colorectal Cancer

PrefaceHuman colorectal cancer is one of the commonest malignant tumors,ranking second only to lung cancer as the leading cause of mortality in malignant tumors.There has been a marked increase in the incidence in recent years.Conventional CT scanning has a good spatial resolution and density resolution,in particular the application of MSCT multiplanar reformation(MPR) image is flexible which can better show the tumor form of colorectal cancer and determine violations of the surrounding circumstances and metastasis,but can not evaluate the changes in organ function.CT perfusion imaging is different from the conventional CT scan.It can evaluate organs, tissues and the blood supply of the hemodynamic status,and make a quantitative and semi-quantitative analysis of perfusion process to reflect the malignancy,the prognosis and recurrence of the tumor.It has brought about the change from morphological observation to the micro-analysis of metabolic and functional status and played an important role in diagnosis,the determination of clinical stage and the prediction and evaluation of curative effects.With the application of multi-slice spiral CT,perfusion imaging allows a more efficient,higher temporal resolution,and it has been widely used in abdominal organs.Invasion and metastasis are the most important and most essential biological characteristics of malignant tumors,and remain the main causes of death in patients with malignant tumors,and colorectal cancer has no exception.The growth,metastasis and dissemination of malignant tumors can't do without its own blood supply,and the process of setting up blood supply is related to the increasing release of angiogenic factors(such as vascular endothelial growth factor,vascular endothelial growth factor, VEGF) and the subsequent increase in the density of capiliaries.Matrix metalloproteinase-2(MMP-2) plays a crucial role during the course of tumor invasion and metastasis by promoting cancer cells to invade the surrounding tissues and the occurrence of metastasis by way of degrading the extracellular matrix.This experiment aims to provide a basis for the prediction of invasion and metastatic ability in colorectal cancer with CT perfusion imaging by studying the correlation between CT perfusion imaging parameters,MMP-2 and VEGF expression,and MVD count of colorectal cancer.Materials and Methods1.Clinical DataFrom June 2008 to December 2008,among treatments at the First Affiliated Hospital of China Medical University,61 cases were clinically diagnosed with colorectal cancer by colorectal multi-slice spiral CT perfusion imaging.Forty-one cases which were confirmed by surgery within 2 weeks after diagnosis and had general access to the tumor specimens,as well as complete reports were involved in this study.There are 30 male cases and 11 female cases.Age ranges from 36 to 81 years old,with an average age of 62.05±12.13. Twenty cases of tumors were located in the rectum,4 cases at the junction of rectum and sigmoid colon,4 cases of sigmoid colon,3 cases in descending colon,3 cases of transverse colon,and 7 cases of cecal-ascending colon.All 41 cases were adenocarcinoma,including 4 well-differentiated, 34 differentiated,and 3 poorly differentiated cases.Clinicopathological stage was estimated by the Modified Duke Staging System.The standard is as follows:Modified Duke A,the tumor penetrates into the mucosa of the bowel wall but no further.Modified Duke B,B1:tumor penetrates into,but not through the muscularis propria(the muscular layer) of the bowel wall.B2:tumor penetrates into and through the muscularis propria of the bowel wall.Modified Duke C,C1:tumor penetrates into, but not through the muscularis propria of the bowel wall;there is pathologic evidence of colon cancer in the lymph nodes.C2:tumor penetrates into and through the muscularis propria of the bowel wall;there is pathologic evidence of colon cancer in the lymph nodes.Modified Duke D,the tumor,which has spread beyond the confines of the lymph nodes(to organs such as the liver,lung or bone).Among the 41 cases,there are 7 Duke A,19 Duke B,10 Duke C,and 5 Duke D cases.2.CT perfusion imaging,image post-processing and analysis(1)CT perfusion scanningMake sure of no digestive tract imaging a week before CT scan.Fast for six hours and perform respiratory training in patients with fixed supporter before scanning.Fill in the patient's digestive tract with one percent of oral diatrizoate 500ml,ask to expand bladder and rectum with rectal inflation of 50～100ml gas.Make patients lie on back to perfom abdominal pelvic CT scan.After that,choose the maximum level of disease as the target level of perfusion imaging.18G intravenous catheter system was used to inject contrast agent Omnipaque(dosage 50ml,injection rate is 6ml/s),then start perfusion scan mode after 10 seconds delayed period,with 50s duration,120kv tube voltage,50mAs tube Current,7～10mm slice thickness,and 200 images.Inject 50ml omnipaque with the high-pressure syringe(the speed is 3ml/s) the instant the perfusion imaging is done,and perform conventional abdominal CT again with 30s and 70s delayed periods as a diagnostic image.(2)CT perfusion image post-processing and analysisCT perfusion images will be sent to the Siemens image post-processing workstations,and processed by pre-installed software for data processing Body Perfusion.Siemens pose perfusion CT software is non-deconvolution algorithm.CT perfusion image post-processing can shed light on lesions,the target artery time density curve(time-density curve,TDC),the flow through the TDC calculation hemorrhage (blood flow,BF),blood volume(blood volume,BV),the initial reperfusion period (time to start,TTS),time to peak perfusion(time to peak,TTP),the surface of vascular permeability(permeability surface,PS),Patlak volume(Patlak blood volume,PBV) and other parameters,as well as BF,BV,TTS,TTP,PS,PBV pseudo-color at the same time.ROI region needs to select specific solid part with multi-point averaging acquisition.According to different lesion levels of colorectal cancer,ROI is attribute to the abdominal aortic artery check,external iliac artery(or femoral artery) axial images of the central region.2.Pathology specimens processing method and immunohistochemical analysis.Preparation of histopathological sections,staining were done under the guidance of teachers from Department of Pathology.(1)slice productionMake 4 serial slices of the selected cases of paraffin-embedded tissues with 4μm each slice.The first slice was proceed with the HE staining,and the remaining three were for immunohistochemical staining.(2)immunohistochemical analysisPeroxidase labeled streptomycin-avidin method(SP) was used.CD34,VEGF, MMP-2 monoclonal antibody and SP immunohistochemical kit were purchased from the Jinqiao Biotechnology Ltd,Beijing.Make known colorectal cancer-positive biopsy as positive control,PBS buffer as negative control.(3)Judgement of immunohistochemical resultsMVD(microvascular density,MVD) count:MVD unit area is defined as tissue stained with CD34-positive microvascular cells.Cancer isolated brown with vascular endothelial cells or cell clusters on behalf of an individual microvessel.First view the whole film at low magnification(100×),larger microvessel density to identify"hot spots"(hot spot) with high-power microscope(200×),each slice records five"hot spots".Choose the average number of cases as the MVD.criterion for VEGF:cytoplasmic membrane with homogeneous or brown coloring was taken positive,non-staining cell was negative.Park,such as criterion-referenced to determine[19]method:First observe a comprehensive cross-section with the low-fold mirror(40×) to select the strongest VEGF staining,then count 100 cells at 200 times magnification.Positive staining cells＜5%is for the negative(--);5～15%for weakly positive(+);16 ~ 50%of(++);＞50%positive for the strong positive(+++)。criterion for MMP-2:Mainly positive for microscopic tumor cells,vascular endothelial,mesenchymal appear brown,yellow or particle mass.Criteria:First observe a comprehensive cross-section with the low-fold mirror(40×) to select the strongest MMP-2 staining,then count 100 cells at 200 times magnification.Positive staining cells＜5%is for the negative(--);5～15%for weakly positive(+);16～50% of(++);＞50%) positive for the strong positive(+++)。4.Statistical treatment of experimental dataAll data were analysed with SPSS 13.0 statistical software.Measurement data to both the standard deviation of the number of people said that for the normal distribution, homogeneity of variance of the measurement data,to compare the two groups using t test or paired t test,comparison between groups using one-way ANOVA analysis,then the use of SNK-q test for multiple comparisons are a few.Skewed distribution of the information or Level measurement data was analysed by non-parametric test, comparison between two groups was analysed by Wilcoxon test,and multi-group comparison was analysed by Kruskal Wallis test.If the p-value＜0.05,the difference has statistical significance.Results 1.In this study,lesions of colorectal cancer was the target arterial time density curve,and a complete set of MIP maps and color-coded map,including BF,BV,TTS, TTP,PS,PBV map information and CT perfusion parameter values.MIP map shows the enhancement of tumor,which is similar to the conventional CT;The red and yellow regions on the perfusion map stand for rich blood flow,green for medium blood flow, and blue for poor blood flow in the low level of regional perfusion.Such color-coded map shows the tumor blood perfusion status directly.2.the average of CT perfusion parameters in colorectal cancerAmong forty-one cases of colorectal cancer,BF values(61.9 persons 31.2 ml/100ml/min),BV values[83.9 disabilities 39.2(a ratio of 1/1000)],TTS values [30.0 disabilities 25.2(1/10s)]),TTP values[145.7 disabilities 38.9(1/10s)],PS values [56.1 disabilities 34.4(0.5ml/100ml/min)],and PBV values[84.7 disabilities 59.6(a ratio of 1/1000)].3.the correlation analysis between CT perfusion parameter values,the MVD counts,VEGF and MMP-2 expressionPearson correlation analysis and Spearman rank correlation analysis showed that: tumor parenchyma CT perfusion parameters of BF,BV,PS and VEGF expression were positively correlated(P＜0.05);tumor parenchyma CT perfusion parameters of BF,BV, PS and MVD count were positively correlated(P＜0.05);MVD count and VEGF expression were also positively correlated(P＜0.05);and tumor parenchyma PS values and MMP-2 expression were positively correlated(P＜0.05).Conclusion1.BF,BV values of serosal invasion group were higher than those of without serosal invasion group,and liver metastasis group were higher than those of without liver metastasis group(P value＜0.05).PS values of serosal invasion,liver metastasis, and lymph node metastasis groups were higher than those of without serosal invasion, no liver metastasis,no lymph node metastasis groups(P value＜0.05).2.some parameters of CT Perfusion imaging can reflect the characteristics of blood perfusion,which further lays the foundation for studying tumor microvascular in order to identify the correlation between tumor perfusion and immunohistochemical parameters of VEGF,MMP-2 as well as evaluating metastasis.3.CT perfusion parameters of BF,BV and PS values were positively correlated to VEGF expression in Parenchyma tumor.CT perfusion parameters of BF,BV and PS values were positively correlated to MVD expression in Parenchyma tumor,MVD expression and VEGF expression were also positively correlated.CT perfusion imaging parameters of PS and MMP-2 expression were significantly correlated.4.CT perfusion parameters can reflect not only the angiogenesis of colorectal cancer of the MVD,but also the VEGF and MMP-2 expression,indicating that CT perfusion parameters of BF,BV and PS can be used as an in vivo assessment of angiogenesis,tumor invasion and metastatic ability.