An Experimental Study On MR Perfusion And Diffusion Imaging Of The Liver Before And After TACE For Hepatocellular Carcinoma

BackgroundTranscatheter arterial chemoembolization(TACE) is considered to be an effective treatment for hepatocellular carcinoma.However,angiogenesis,the process leading to the formation of new blood vessels from preexisting vessels,plays a central role in the survival of tumor cells,in local tumor growth,and in the development of distant metastasis and reduces therapeutic effectiveness.Early detection of residual tumor and vascularity after interventional treatments is critical before TACE and during follow up.Generally,computed tomographic(CT) images could be considered as a routine modality to judge the efficacy of TACE.The pattern and distribution of iodized oil in the tumor are useful for assessing the therapeutic effects of TACE.A greater amount of accumulation of iodized oil within tumor indicates a greater area of necrosis.During follow-up,the focal defect or washout of iodized oil in the mass with the contrast-enhanced area suggests the presence of a viable tumor and that additional treatment is needed.The contrast-enhanced CT is a reliable method for the assessment of therapeutic efficacy.On contrast-enhanced CT,necrotic tissues were unenhanced and viable tumors were enhanced.But it was difficult to judge the viability and necrosis of the tumors correctly due to the inhomogeneous deposition,because lipiodol negative areas could not actually represent the viability of the tumors.It can also be difficult to evaluate contrast enhancement in a tumor with partial retention of iodized oil on contrast-enhanced CT because of the beam-hardening artifacts produced by the high attenuation of iodized oil.MR imaging was valuable in the evaluation of therapeutic efficiency of TACE, especially on T₂WI,most of viable tumors were hyperintense and the coagulative necrosis within the tumors considered as a positive response to TACE was hypointense. But a signal intensity of the tumors after TACE was variable on SE T₁WI and T₂WI,all of viable tumors,hemorrhage,liquefied necrosis and inflammatory infiltration could also result in hyperintensity on the T₂WI.Therefore,it was difficult to assess the viable tumors of HCC after TACE by conventional SE imaging.Dynamic contrast scanning also has a great value in the evaluation of therapeutic efficacy of TACE.The residual viable tumors were shown as rapidly enhanced portions within the lesions,when necrotic portions had no enhancement at the contrast early phase scanning.At the late phase scanning,the enhancement of the most lesions became hypointense,and just a few lesions showed a persistent enhancement.Both viable tumors and inflammatory infiltration could present such changes,so the contrast early phase scanning was not more reliable in the evaluation of viable tumors.Existing diagnostic imaging techniques provide limited evaluation of tissue characteristics beyond morphology.Perfusion imaging ultimately may have a role in the earlier identification of tumor recurrence if subtle differences in enhancement of benign versus malignant tissue can be captured earlier.CT perfusion imaging has the potential to provide both high-temporal-resolution and high-spatial-resolution imaging of the entire liver for the detection of HCC,however,the radiation doses must be considered.A whole-liver CT perfusion study would result in radiation doses that are approximately three to six times greater than those of a routine diagnostic CT examination.Perfusion MR imaging represents a promising technique for HCC surveillance.T1 weighted perfusion MRI has been used to assess tumor angiogenesis of uterine cervical carcinoma, and has a good correlation with microvessel density(MVD).Diffusion-weighted imaging can provide an insight about water composition within a tumor and the degree of tumor viability.The apparent diffusion coefficients of malignant masses were significantly lower than those of benign masses.A study of an animal model of hepatocellular carcinoma showed that:diffusion-weighted imaging clearly distinguished the regions of tumor necrosis from viable tumor.So,DWI may offer significant utility in the longitudinal assessment of novel liver tumor treatments.The purpose of this study was to observe angiogenesis and necrosis of HCC before and after TACE by MR perfusion Imaging and diffusion imaging,compared with microvessel density and histological evaluation,and to find out their feasibility and value in assessing therapeutic effectiveness of HCC during follow up.Chapter One:The establishment and improvement of VX2 Carcinoma modelObjective To establish suitable metastatic rabbit vx2 hepatic carcinoma model,to compare the success rate of different tumor implantation methods.Methods Thirty New Zealand white rabbits were randomly divided into three groups,10 rabbits in each group.Group 1:Solution of the VX2 carcinoma cells were injected into left liver by syringe needle.Group 2:Solution of the tumor tissue masses was transplanted into liver.Group 3(improvement group):VX2 carcinoma masses were injected into the liver using percutaneous puncture inoculation method.The operation was guided by CT scanner in each group.CT scanning was performed in all experimental animals 2 week after implantation.Results The success rates of transplanted carcinoma were 30%,50%and 90%in group 1,group2 and group3 respectively,and the statistical difference was found.The respective ectopic implantation incidence at group 1,group2 and group3 were 1(3),1(5) and 0(8),no statistical difference was observed.The success rates of single nodule implanted tumor were the highest in group 3(80%) than in group 1(10%) and group 2 (20%).The difference was statistically different(p=0.003).Conclusion It is successful to improve the rabbit modes bearing VX2 metastatic hepatic carcinoma.And the method of percutaneous puncture carcinoma masses implanation guided by CT was a good method to establish the hepatic carcinoma models with a high success rates,especial rates of single nodule implanted tumor,low ectopic implantation rates and little damage of animals.Chapter Two:Observation of the value of MR Diffusion Weight Imaging in diagnosis of hepatocellular carcinomaObjective To investigate the value of MR DWI in diagnosing hepatic carcinoma by studying the appearances of rabbit vx2 hepatic carcinoma in MR T1 WI,T2WI and DWI series at different times after the tumors were transplanted.Methods The metastatic rabbit vx2 hepatic carcinoma models were presented in 30 New Zealand white rabbits.VX2 carcinoma masses were injected into the liver using percutaneous puncture inoculation method in guiding by CT scanner in each group.MR T1WI,T2WI,T2WI SiP and DWI were performed in all experimental animals at 4day,1, 2 and 3 weeks after implantation with a Sense-Flex-M.T2W_TSE_SENSE and T2W TSE_SPIR parameters:TR 3544ms,TE 81ms,FOV 150mm,slice thickness 4mm, intersection gap of-1 mm,matrix 256×256,restruction matrix 512×512,scan time 74s, NSA 2.The MR image parameters with T1WI SENSE were as follows:TR 407ms,TE 17ms,FOV 150mm,slice thickness 3mm,intersection gap of- 1 mm,matrix 256×256,restruction matrix 512×512,scan time 94s,NSA 2,EL=3.Diffusion-weighted images of the liver were acquired using an SE-EPI sequence with the following imaging parameters:b-values of 0,300/mm²,TR 6000ms,TE 49ms, FOV 150mm,slice thickness 3mm,intersection gap of 0mm,matrix 112×112,restruction matrix 256×256,flip angle 90°,scan time 156s,NSA 6.The diagnosis rates of different methods in different time were observed.The ADC values in cores and peri-areas were analyzed.Results Of 30 rabbits,25 were transplanted vx2 hepatic carcinoma.The success rate of carcinoma transplanted was 81.25%.The tumor lesion could be found in every examination series at 4 days after the implantation.The diagnosis rates of DWI,T1WI, T2WI and T2WI SPIR were 80%,60%,72%and 72%respectively.Statistical difference was found(p=0.007).The diagnosis rates were 100%in all MR series at 1,2 and 3 week after implantation.In comparison of diagnosis rates between 4days and 1 to 3 week after the tumor implantation in different series,no significant difference was only observed on DWI series(p＞0.05) and significant difference on other series(p＜0.05).The ADC values of peri-areas were 1862.58±150.97,1636.70±152.96, 1273.43±173.59,1005.68±192.00 respectively,at 4day,1,2 and 3 weeks after implantation.The significant difference was observed(F= 153.997,p=0.000).The values decreased with the times developing from 4 days to 3 weeks after the implantation.The ADC values of cores were 1498.81±264.41,1078.92±153.58,662.751±174.27, 1553.19±179.01 respectively at 4day,1,2 and 3 weeks.There were statistical difference in each group ADC values(F=149.442,p=0.000),which decreased with the times developing before 2 weeks,but increased at 3 weeks.Conclusion MR DWI is a sensitive series in examination of liver tumor,,and will be used in diagnosis of liver tumor more and more.The values of ADC may help discriminate between the necrosis areas and normal areas,will play a important role in surgical operation and puncture biopsy.Chapter Three:MR diffusion and perfusion imaging of the liver before and after transcatheter hapatic chemoembolization for hepatocellular carcinoma Objective To observe angiogenesis and necrosis of HCC before and after TACE by MR perfusion imaging and diffusion imaging,compared with microvessei density and histological evaluation,and to find out their feasibility and value in assessing therapeutic effectiveness of HCC during follow up.Methods1.Animals and tumor models30 New Zealand white rabbits(weight:2.5-3.0kg) with VX2 liver tumors were randomly divided into three groups,10 rabbits in each group(group 1,pre-TACE;group2, 3 day post- TACE;group 3,1 week post- TACE).The MR scan was performed before chemoembolization,at 3 day and 1 week after chemoembolization,respectively at group 1,2 and 3.The technique for tumor implantation was basically similar to that described in Group 3 of Chapter One.All three groups received interventional therapy after three weeks of the VX2 tumor was implanted.The laboratory animal center of Guangdong province provided all animals.2.ChemoembolizationEach rabbit was successfully catheterized with DSA.Conventional DSA was performed by using a C-arm unit guidance.Rabbits were initially anesthetized by using sumianxin(0.1-.02mg/kg).Vascular access was achieved in the femoral artery through transfixion using a transfixion pin with plastic sleeve.A 4-F catheter was advanced over a guidewire into the celiac trunk.DSA of arteries was performed by using 2-3 ml manual injections of contrast agent.The 3-F catheter was advanced into the left hepatic artery.1-3 ml lipiodol emulsion(lipiodol mixed with 1mg MMC and 1mg pharmorubicin) were superselectively delivered into tumor vessel by manual injection.Follow-up angiography was performed and confirmed the complete stasis of antegrade blood flow to the liver tumors.After chemoembolization,the catheter was removed and the femoral artery was ligated.Then skin closure was finished.3.MR imagingMR perfusion and diffusion imaging examination were performed in the supine position by using a clinical Sense-Flex-M coil.The scans were done before chemoembolization,at 3 day and 1 week after chemoembolization,at group 1,2 and 3 respectively.MR diffusion imaging parameters has been described in Chapter Two. The MR perfusion imaging protocol consisted of T1-weighted fast field echo(FFE) sequences with a field of view of 355 mm×75 ram,matrix 89×256,TR/TE of 4.3/1.4 milliseconds,slice thickness of 5 mm,intersection gap of 1 mm,NSA of 1.This sequence was repeated 65 times,in4 slice(in total,80 seconds),a dose of 0.2 mmool/kg of Gadoteric Acid contrast medium was injected after the start of the scan.Injection of Gadodiamide was performed through a 22-gauge needle via ear vein at a flow rate of 2 ml/sec followed by injection of 3ml bolus of saline(NaCl 0.9%).4.Histological evaluationEach animal was then sacrificed with overdose penthiobarbital sodium intravenous injection for pathology observation after MR examination.The livers were removed and fixed in 10%acetic formalin.A slice was cut at the same level as the slice imaged in MR and the general pathology was observed.Each VX2 tumor was divided into the periphery area and center area.The specimens of both areas were embedded in paraffin,and thinner sections of 3mm were cut and stained with hematoxylin-eosin-safran.These slices were examined with an optical microscope at 20 magnifications.Immumohistochemical staining was performed using the SP method.The specimens were observed under a low power microscope(×40),then the sections with most dense area of microvessels were selected and observed under a high power(×400).Microvessels were evaluated under a microscope,microvessel density in the center and rim of the lesion were calculated.5.Statistical analysisThe values of ADC,MSI and MVD in the center and rim of the lesion were calculated.All data were expressed as mean±SD,the statistical differences between different groups were analyzed by ANOVA,and the correlation between MSI and MVD, ADC and MVD was assessed by Spearman correlation analysis.Significance was accepted when p＜0.05. Results1.Lesion signal intensity characteristics in DWI images before and after TACEThe lesions assessed before TACE were hypo- intense relative to the surrounding liver parenchyma on T1-weighted images,and the most portion of tumors was hyperintense relative to the surrounding liver parenchyma on T2-weighted images and DWI images,and the heterogeneous signal intensity was observed when liquefied necrosis or fat degeneration occurred.At 3d after TACE,on DWI images,the heterogeneous hypo-intense was observed,which was coagulative necrosis confirmed by morphological observation.At 1w after TACE,the heterogeneous hypo-intense areas became larger.The coagulative necrosis progressed with the development of times.2.Changes of ADC valuesThe ADC values of peri-areas were 1089.31±107.24,1865.38±157.28 and 1860.97±194.46 respectively,at pre-TACE group,3 days post-TACE group and 1week post-TACE group.The significant difference was observed(F=80.875,p=0.000).The ADC values of cores in each group were 1595.92±197.51,1633.38±193.82, 1848.14±183.01 respectively.Significant difference was found between pre-TACE and after TACE(F=5.049,p = 0.014)3.Characteristics on PWI images before and after TACEAll tumors lesions were depicted on HBF,HBV images.Time intensity curves derived from ROIs drawn in the peri-areas and cores of the tumor showed a rapid initial increase in signal intensity,representing hypervascularity before and after TACE.The peak values in peri-areas were higher than cores.The intensity curves in post-TACE groups showed less steep than pre-TACE group.4.Changes of MSI valuesThe MSI values of peri-areas were 7.35±1.20,8.70±0.96and 2.85±0.96 respectively at pre-TACE group,3 days post-TACE group and lweek post-TACE group.The significant difference was observed(F=85.986,p= 0.000).The MSI values of cores in each group were 2.41±0.59,1.50±0.38 and 1.33±0.46 respectively.Significant difference was found between pre-TACE and after TACE(F= 14.414,p= 0.000)5.Results of morphological observationUnder low magnification,HCC was seen as invasive carcinoma in the liver,there was no clear margin between neoplasm and the hepatic parenchyma,neoplastic cells distributed straggly,there was separating fibre with abundant capillary of newly developed;under high magnification,the volume of neoplasm was large with irregular morphology,cells distributed irregularly with abundant cytoplasm.Nucleus was hypertrophic with different size and shape,staining was non-uniform,phase of nucleous mitosis showed in many cells,a great number of lymphocytes and plasma cells could be seen infiltrating in the mesenchyma.After TACE lesions underwent un-complete necrosis,karyopycnosis and nuclear fragmentation was seen at the center of the lesions.6.Results of immunohistochemistryCD34 was expressed in HCC and various degree of yellow stain could be seen in the epithelial cells of hepatic sinusoid in the HCC section.But MVD of HCC tissues were lower significantly after TACE than before TACE.The MVD values of peri-areas were 51.14±19.71,26.88±12.76 and 11.07±6.52 respectively at pre-TACE group,3 days post-TACE group and lweek post- TACE group.The significant difference was observed(F=20.582,p=0.000).The MVD values of cores in every group were 13.49±5.17,8.52±3.64,8.32±5.11 respectively.Significant difference was found between pre-TACE and after TACE(F=3.899,p=0.033)7.Correlate analysisThe values were analyzed by Pearson's correlation coefficient.The results showed that there was negative correlation between ADC and MVD(r=-0.534,p=0.000),positive correlation between MSI and MVD(r=0.694,p=0.000). Conclusion1.Time intensity curves derived from ROIs drawn in the peri-areas and cores of the tumor showed a rapid initial increase in signal intensity.The peak values in peri-areas were higher than cores.The intensity curves in post-TACE groups showed less steep than pre-TACE group.2.Significant difference in MVD values of peri-areas and cores was found at pre-TACE group,3 days post-TACE group and 1 week post-TACE.That indicated capillary vessels decrease obviously after TACE.3.MVD values of peri-areas and cores decreased along the times of TACE developing. The statistical significant was found.There was a positive correlation between MSI and MVD.The hepatic blood flow decreased along with the MVD,which was the pathological basis of MSI values decreasing.4.MR perfusion imaging has a potential to evaluate the angiogenesis of liver tumors in vivo,and could demonstrate the haemodynamics variation of new vessels.It provides a new,valuable technology for hemodynamics study of HCC and evaluation of perfusion level and therapeutic effect after TACE.5.Significant difference of ADC values in peri-areas and cores was found between pre-TACE and after TACE,which demonstrated that the diffusion of tumor tissues increased after TACE.There was negative correlation between MVD and ADC values. Values of ADC could reflect the changes of tumor after TACE,was a reliable index to monitor the tumor necrosis.