Contrast-Enhanced Sonographic Appearance Of Hepatic Malignant Tumors And The Correlation With MVD And VEGF

IntroductionPrimary hepatocellular carcinomas are typical carcinomas which have massive blood supplies. The neoangiogenesis plays crucial effect during the survival, development and metastasis of tumors. Vascular endothelial growth factor (VEGF) is known as one of the strongest factors which can promote the generation of blood vessels. It can promote cell proliferation and division, migration and then massive angiogenesis formation. It has been presented and proved that stain vascular endothelial cells with immunohistochemical technique and microvessel density (MVD) obtained by measuring "hot spot" in the tumor was an index of angiogenesis. Most researchers have pointed out that MVD represents the degree of angiogenesis and it is a reliability index of the prognosis of tumor. The relationship between the parameters of contrast enhanced sonography time-intensity curve and MVD and the expression of VEGF was discussed in our study.MethodsPart one35 patients confirmed by surgery pathololgy were reviewed in this study.we analyzed the enhancement appearance according to the type and degrees of pathologic differentiation.group A:14 moderately to poorly differentiated hepatocellular carcinomas(HCCs); group B:10 well- differentiated HCCs; group C:6 intrahepatic cholangiocarcinamas; group D: 5 hepatic metastatic carcinomas (of which 3 colon cancer,2 ovarian cancer).Toshiba Aplio color sonographic system,the contrast agent (Sonovue,Bracco company)consists of sulfur hexafluoride(SF6)surrounded by phospholipids.The liver was scanned with a conventional sonogram to select an appropriate section,the specific mode of contrast-enhanced sonogram began,frame frequency(FR) was 15,dynamic range (DR) was 40,the acoustic power was lowered to a mechanical index of less than 0.1,Sonovue was injected IV as a bolus of 2.4ml through the antecubital vern ,the entire scanning process was recorded for 5-8 minutes.The enhancement and washout patterns were analyzed by three physicians,the time-intensity curve was made by ImageLab sofeware.The regions of interests was in the lesions and the liver appoximately the same depth,avoiding necrosis and vessels . The parameters were calculated: enhancement time (peak time of—starting time ), ascending slope,relative peak intensity(peak intensity of lesion—intensity of the same depth normal liver ); peak enhancement rate((peak intensity of lesion—intensity of the same depth normal liver)/ intensity of the same depth normal liver);falling slope .Part twoOf the 25 patients suspected for Primary hepatocellular carcinomas (HCCs), 24 patients diagnosed finally as HCCs by surgery pathology were reviewed in this study. 14 moderately to poorly differentiated HCCs and 10 well-differentiated HCCs. The CEUS methods and parameters were same to part one.The histological samples were fixed with Formalin and embedded with Paraffin routinely.The tumor slices were stained by CD34 with immunohistochemical technique (s-p technique). The positive cells with CD34 dyeing were vascular endothelial cells and the color was brown. Those cells were single layer under microscope, there were no vascular cavity formation in most of them, and even only scatter or cluster vascular endothelial cells. MVD count: firstly the slice was observed with 100 times light microscopy to find the 5 regions with the highest vascular density which were the "hot-spot". Secondly MVD count was performed under 400 times light microscope and any brown-stained cell or cell cluster even without tubular structure was recognized as a MVD number. This course was completed by two senior pathologic doctors who did not know the pathologic results and the final results were the average of the two .Analyze the correlation between the parameters of the contrast-enhanced time-intensity curve and the MVD.Part threeOf the 25 patients suspected for Primary hepatocellular carcinomas(HCCs), 24 patients diagnosed as HCCs by surgery pathology were reviewed in this study. Among them there were 14 moderately to poorly differentiated HCCs and 10 well-differentiated HCCs. The CEUS methods and parameters were same to part one.Of the tumor tissues cut in the operation,fresh hepatic carcinoma tissue (avoiding necrosis area)and the adjacent tissue (about 2cm beyond the tumor)were reserved and frozen in -80℃. The histological samples of primary hepatic cellular carcinomas were fixed with Formalin and embedded with Paraffin.The expression of vascular endothelial growth factor (VEGF) was detected with S-P method. VEGF protein was mainly distributed in cytoplasm and the positive cells were stained as brownish-yellow or brown-black granules. The stained intensity was judged through coloring strength and positive cell number synthetically. A: count scores according to the cells stained strength, cells without stained color (-) was 0, light yellow (+) was 1 and dark yellow (++) was 2. B: count scores according to the proportion of stained cells, below 1/3 is 1, between 1/3 and 2/3 is 2 and above 2/3 is 3. Rank the scores obtained by multiplying the scores of A and B from high to low, 0 is "-", 1 -3 is "+", between 4-6 is "++".The VEGF expression of HCC and adjacent tissue was qualitatively analyzed with Western-blot method. Analyze the relationship between the parameters of enhancement time-intensity curve and VEGF expression.ResultsPart 11. 14 moderately to poorly differentiated HCCs enhanced in the arterial phase homogeneously or heterogeneously, 92% (13 lesions) of them quickly washed out in the portal venous phase. 10 well-differentiated HCCs enhanced in the arterial phase homogeneously or heterogeneously, 50% (5 lesions) of them slowly washed out in the portal venous or the late phase.2. 6 intrahepatic cholangiocarcinomas enhanced in the peripheral area of the tumors in the arterial phase, and cord-like light band extending from the peripheral areas to the center. The enhanced range was expanded as time prolonged. And washed out in the portal venous phase.3. The falling slope of moderately to poorly differentiated HCCs group was more than that of well differentiated HCCs group,the relative peak intensity of HCCs was more than that of intrahepatic cholangiocarcinomas . The difference between them was significant.Part 21. The MVD number of moderately to poorly differentiated HCCs group was more than that of well differentiated HCCs group, The difference between them was significant..2. The relative peak intensity and falling slope of HCCs of time-intensity curve were correlate with MVD number. The correlation coefficients were 0.44 and 0.50 respectively and the P value was 0.03 and 0.01 respectvely. There was no relationship between the enhanced time, ascending slope , peak enhancement rate of time-intensity curve and MVD number (p>0.05).Part 31. Immunohistochemistry manifestations: VEGF of HCCs mainly distribute in cell plasma. The VEGF expression grade of moderately to poorly differentiated HCCs group was higher than that of well differentiated HCCs group. But not significantly different statistically (p>0.05).2. protein qualitative analysis: the VEGF areas between moderately to poorly differentiated HCCs group, well differentiated HCCs and contrast group was significantly different(p<0.05). the VEGF areas between moderately to poorly differentiated HCCs group and well differentiated HCCs was significantly different(p<0.05).The VEGF density between moderately to poorly HCCs and well differentiated HCCs,contrast group was significantly different(p<0.05).3. The relative peak intensity of HCCs was correlate with VEGF area and density, the correlation coefficient were 0.50 and 0.44 respectively, the P value was 0.02 and 0.04 respectvely. There was no relationship between the VEGF expression and enhanced time, ascending slope, falling slope and peak enhancement rate (p>0.05).conclusions1. the enhancement appearance of different differentiation HCC is different , the enhancement appearance of different pathologic type is different, the enhancement manifestations of hepatic malignancies are related to pathologic types and grades. 2. the falling slope rate of moderately to poorly differenteated HCC is more than the well -differentiatd ones,the relative peak intensity of HCC is more than intrahepatic cholangiocarcinimas,the parameters of contrast-enhanced time-intensity curve can reflect indirectly the blood supply of tumors and differentiation.3. the relative peak intensity of HCC is correlate to MVD, the parameters of contrast-enhanced time-intensity curve obtained before operation can evaluate haemodynamic circulation, reflect indirectly the generation of neoangiogenesis and evaluate prognosis.4. the relative peak intensity of HCC is correlate to VEGF expression area and density, the parameters of contrast-enhanced time-intensity curve obtained before operation can evaluate haemodynamic circulation, reflect indirectly the expression of VEGF.