Preoperative Staging Of Rectal Cancer Ultrasound Imaging And Its Expression On TrkB And D2-40

IntroductionColon cancer is the third most common cancer worldwide, and the incidence of colon cancer is increasing. Numerous improvements in the surgical, radiologic, and oncologic treatment have been made over the past two ecades. Its prognosis mainly depends on choosing the most effective treatment. Prerequisites for this treatment option include accurate preoperative tumor staging with regard to tumor detection, mesorectal fat infiltration, mesorectal fascia status, nodal involvement and distal metastatic disease. A variety of imaging studies have played an important role.Staging accuracy for CT ranges from 33%to 77%for T-stage accuracy and 53% for N-stage accuracy. MRI has shown improved T-stage accuracy, with rates of 86% and 67%on N-stage. TRUS has shown T-stage accuracy 65%and N-stage accuracy ranging from 57%to71%. Therefore,we can't find any dominance on ultrasonography in rectal cancer.Angiogenesis has gained much attention in recent years. It has been shown to be an essential event for tumor growth and metastases. Several studies have demonstrated that tumoral angiogenesis is an independent prognostic factor in rectal cancer. Therefore, the assessment of this factor would seem to be important when evaluating patients with cancer of rectal.Color Doppler Flow Imaging(CDFI) and Color Doppler Energy(CDE) ultrasonography may reflect dynamics of bloodstream, "Vascular index (VI) is under the CDE calculating the ratio of the number of color pixel to the total number of pixels within the tumor section. Three-dimensional power Doppler ultrasound (3D-CPA) is a non-invasive evaluation of visceral perfusion of the method, which can clearly show low tumor blood flow and blood vessels within the construction and distribution of changes, and can do quantitative analysis. In addition, ultrasound is the research focus,it can show blood flow in capillaries and other microvessels, has been applied to diagnosis of focal lesions in the liver, and in the thyroid, breast, uterus and other parts of the qualitative lesions there are also diagnostic study, the research in recal cancer has not been reported. This study is designed to investigate the value of vascular and stage in rectal cancer by CDFI,CDE,3D-CPA and contrast-enhance ultrasonography.The prognosis of patients with colon cancer principally correlates with the proliferative, apoptotic and invasive potentials of tumor cells, which is regulated by some critical genes. Tropomysin-related kinase B (TrkB) is a member of Trk family, functions as a receptor tyrosine kinase, which is necessary for the normal evelopment of nervous system. Recent studies have been initiated to show the suppression of anoikis and induction of metastasis by TrkB. TrkB is up-regulated in various primary human tumors. Therefore, the overexpression of TrkB might play an important role in the progression of malignant tumors. However despite that point mutation was observed in colorectal cancer, whether TrkB positively participates in primary colon cancer has not yet been determined. This study is designed to investigate the expression and clinical significance of TrkB in surgically resected colon cancer with different clinicopathological features.MethodsPart one1. Two-dimensional model and CDFI, CDE modeRectum and the tumor's location, shape, size, internal echo and around the infiltration area was observed. CDFI was started to observe the tumor blood flow. Detection of tumor blood flow within the spectrum, recorded peak systolic velocity (PSV) and resistance index (RI). Start CDE mode, the frozen section in five different images, select the most blood flow signal aspect display, storage image. CDE image will be input into the computer, and computer image analysis software to obtain the number of quantitative parameters of the blood vessel vascular index (VI).2.Three-dimensional imagingCPA mode for 3D-CPA rectal tumor imaging, shows three-dimensional tumor blood vessels course and branches, semi-quantitative method to tumor blood flow grading, Qlab software GI3DQ tool measured the volume of cancer, calculated vascular strips, calculating vascular index VI.3. Contrast-enhance ultrasound.In dual-rate contrast-enhance imaging mode ultrasound imaging. Real-time observation of colorectal cancer cases and contrast-enhanced dynamic process, store the image data imaging process. Finally, image analysis, review the process of image contrast, increased observation time of rectal lesions, and enhance the level and changes in enhanced mode. Open QLAB analysis software, start the region of interest (ROI) quantitative procedures, access to regions of interest perfusion time-intensity curves. Start microvascular imaging (MVI) process, real-time observation of contrast agent microbubbles in the capillaries in the imaging path.4. All patients with International Union Against Cancer TNM staging system for staging, pathological staging data collection. The parameters derived from ultrasound compared with TNM staging.5. SPSS13.0 software used for statistical analysis.PSV, RI, VI compared with the TNM stage grouping data using the ordered linear trend test.3D-CPA vessel count and pathological staging rectal cancer comparison, Pearson Chi-Squareχ2 test. Quantitative information on 3D-CPA vascular index compared with pathological staging using two independent samples t test.Results were considered statistically significant when the p-value was less than 0.05.Part two1. Tissue samples and patients:90 cases of rectal tissue from the paraffin blocks were the First Affiliated Hospital of China Medical University, of which 50 cases were from the pathology investigation,40 cases of surgical specimens of tumor preparation on their own.Formalin-fixed paraffin-embedded tumor tissue sections of conventional HE staining. All of the included patients underwent surgical resection without having chemotherapy or radiation therapy. Formalin-fixed paraffin-embedded sections of tumor were stained with hematoxylin and eosin (H&E) routinely,2. Western blot.:Frozen tissues (including tumor and non-tumorous portion) or cells were washed twice with ice-cold PBS, homogenized on ice in lysis buffer containing 20mM Tris-HCl, 1mM EDTA,50mM NaCl,50mM NaF, 1mM Na3VO4, 1%Triton-X100 and 1mM PMSF. The homogenate was centrifuged at 15000 rpm for 30 min at 4℃The supernatant was extracted and protein content was determined by the BCA assay (Pierce).80μg of total protein was separated by 6%SDS-PAGE and then transferred to PVDF membranes. After blocking with 5%BSA, primary antibodies were incubated on the membranes for TrkB (sc-8316,1:200) and p-actin (1:200) (both from Santa Cruz) overnight at 4℃. The membranes were then incubated for 2 h at 37℃with goat anti-rabbit IgG(1:2000) (ZhongShan, China). Immunoreactive straps were identified using the ECL system (KaiJi,China), as directed by the manufacturer. The DNR Imaging System was used to catch up the specific bands, and the optical density of each band was measured using the Image J software. The ratio between the optical density of TrkB andβ-actin of the same sample was calculated as relative content and expressed graphically.3.Immunohistochemistry:90 paraffin sections of colon tumor were deparaffinized and rehydrated routinely. The antigen recovery was performed by heating slides in an autoclave sterilizer for 2 min in 0.1 mol/L Tris-HCl buffer at pH10. The sections were incubated overnight with primary rabbit polyclonal antibody detecting TrkB (sc-8316,1:100) and mouse monoclonal antibody for D2-40 (ZhongShan, China), following 3%H2O2 and 5%rabbit serum treatment at 37℃for 1h. After which they were incubated with second antibody and SP complex for 30 min (SP kit), and visualized with DAB (DAB kit)(both from MaiXin, China). Negative controls were non-immune rabbit IgG at the same dilution as for the primary antibody.4.MTT assay: The MTT assay was applied to evaluate the proliferation of cells transfected. Cells were detached and seeded in 5 96-well plates (1×103/200μl) in parallel, and transfected for 24h before detection. During the following 5d, the optical density in one indicated plate was examined and cells in other plates were cultured continuously. Everyday,20μl MTT (5mg/ml) was added in each well,4h later the liquids were removed and 150μl DMSO was added. After 10min in vortex, the optical density was measured. The cell growth curves were drawn according to time (X-axis) and average optical density (Y-axis). Data presented are representative of three individual wells.5.Cell apoptosis assay:The cell apoptosis was examined by flow cytometry using an Annexin V-FITC apoptosis detection kit (BD), following the manufacturer's protocol. Cells were washed twice in ice-cold PBS and resuspended in 1×binding buffer (1×106/ml). Cells of 100μl (1×105) were gently mixed with 5μl Annexin V-FITC and 5μl PI, and then incubated for 15min at room temperature away from light. After supplemented another 400μl 1×binding buffer, cell apoptosis was detected in flow cytometer. Results are representative of three individual experiments.6.Cell invasion assay:The cell invasion assay was performed using a 24-well Transwell chamber (Costar). At 24h following transfection, cells (1×104) were detached and seeded in the upper chamber of a 8μm pore size insert precoated with Matrigel (BD) and cultured for another 24h. Cells were allowed to migrate towards medium containing 15% FBS in the bottom chamber. The non-migratory cells on the upper membrane surface were removed with a cotton tip, and the migratory cells attached to the lower membrane surface were fixed with 4% paraformaldehyde and stained with hematoxylin. The number of migrated cells was counted in 5 randomly selected 400×power fields under microscope. Data presented are representative of three individual wells. 7. The SPSS 13.0 software was applied to complete data processing. "Two-independent samples of nonparametric test" was used to assess the levels of TrkB in colon tumors and non-tumorous counterparts. "Binary logistic regression" analysis was applied to analyze the correlations between TrkB expression and clinicopathological characteristics, and "Independent samples t-test" was used to compare LVD between groups of higher and lower TrkB for the results of immunohistochemistry. One-way ANOVA was used to compare the differences between cells with various treatments. All data were represented as mean±SD and results were considered statistically significant when the p-value was less than 0.05.ResultsPart one1. There is significant relationship between VI and TNM stage in rectal cancer. (P=0.001, r=0.476)2.Stage I and II, the rich blood of cancer, tumor blood vessels could be found around most of the internal probe and may be blood vessels, blood flow to the main grade II, III period, IV of rectal cancer may be peripheral and internal Exploration and blood vessels, mainly to grade III, showing the rich vascular tree, branch complex, tortuous irregular vessels Traveling. I, II, and III, IV grade of difference between the blood vessels, were significantly different (P<0.05)3. After the use of contrast agents, blood signal enhanced significantly around and inside the rectal tumour quickly. We can see the borden of the tumor clearly.The inner part exhibited earlier washout than that of the consecutive colon in the later phase,while the peripheral area still remaind hyperenhancement.The extension and border line could be determined clearly.Shape of the curve in stageⅢand IVrectal cancer was characterized as ascend rapidly and drop slowly than that of stage I andⅡ. Part two1. Western blot analysis was used to evaluate TrkB expression in 30 colon tumors and non-tumorous tissues distant from the primary tumor of the same case. The overexpression of TrkB was found in 26 tumorous samples in comparison with the non-tumorous counterparts (p=0.000).2.TrkB expression in 90 cases of colon cancer by immunohistochemistry. TrkB immunoreactivity was detected in 74 (82.2%) colon tumors. We considered that 65 tumors(72.2%) were higher expression (scores of 4 or 6) and 25 cases (27.8%) were lower expression (scores of 0,1,2, or 3), as described in Materials and methods. Clinicopathological correlations of TrkB expression revealed by immunohistochemistry were then analyzed statistically. No statistical differences were found between the higher TrkB expression and the characteristics of tumor size(T≥5cm versus T<5cm, p=0.407), as well as differentiation (well versus poor-moderate, p=0.867).However, immunostaining showed a statistically significant correlation between higher TrkB expression and lymph node metastasis at the time of resection (p=0.035).LVD in positive and negative node group were 169.0±29.1 and 125.7±37.5, respectively. More lymphatic vessels were present in tumors with higher TrkB expression (p=0.000).3.5 days after TrkB-siRNA transfection, the O.D. of LoVo cells in TrkB, non-silencing and control group were 1.41±0.09,1.58±0.02 and 1.55±0.06, respectively (p=0.035). From the proliferative curves we observed that cells of TrkB-siRNA group exhibited less tendency to proliferation during the 5 days examined compared with non-silencing and control group.4.The apoptotic rates of LoVo cells in TrkB-siRNA, non-silencing and control group were 26.5±1.9%,8.1±0.5%and 5.4±1.2%, respectively (p=0.000,).5.TrkB-siRNA transfected cells exhibited extremely diminished TrkB in comparison to those non-silencing and control cells. The numbers of invasive cells in TrkB-siRNA, non-silencing and control group were 16.2±3.9,26.4±3.2 and 25.9±3.3, respectively (p=0.020).Conclusions1. The number of vessels detected on CDE within the unit area is linear correlation with pathological stage in rectal cancer, more number of vessels per unit area with increased staging. Transrectal 3D-CPA classification and detection of colorectal tumor blood vessels per unit volume within the article number in stage III and IV were significantly higher than those of stage I and II, that can be used as preoperative staging of rectal ultrasound for the diagnosis. Ultrasound imaging can clearly show the level of rectal capillary blood perfusion and microcirculation lesion directly reflect the situation. Rectal ultrasound sonography typical for the preoperative staging of rectal cancer to provide more information.2. Overexpression of TrkB was common in rectal cancer. The increased expression of TrkB was correlated with higher metastatic facility, possibly by lymphoangiogenesis. The augmented apoptosis, attenuated proliferation and invasion of LoVo cells were also observed by the interruption of TrkB expression using specific siRNA. Taken together, TrkB may provide a helpful target for inhibitory therapies of progression in colon cancer.