Contrast Ultrasound Of Microvascular Imaging For Evaluating Tumor Angiogenic Activity Of Breast Carcinoma

Objective1.To study the effects and mechanisms of angiogenesis in the tumorgenesis andprogression of breast precancerous lesion and carcinoma. To find a simple andobjective method to screen breast carcinoma in high risk population.2.To compare the differences in histological morphology, hemodynamics andrelated molecules between benign and malignant tumor, different regional bloodperfusion in the same type tumor. To offer reliable indicators for breast cancer earlydiagnosis and identification, and therefore to lay experimental basis for molecularimaging of neovascularization of breast carcinom.Materials and methods1.Objects95 patients were included in our study which were divided into 5 groups: normalcontrols, 10 cases,simple hyperplasia, 20 cases; atypical hyperplasia, 20 cases;intraductal carcinoma in situ, 15 cases;invasive ductal carcinoma, 30 cases.30 malignant tumor patients had 33 foci, the histological typing were as follows:infiltrating ductal carcinoma, 23 foci; infiltrating lobular carcinoma, 7 foci;intraductal carcinoma in situ, 3 foci. 15 benign tumor patients had 17 foci that wereall breast fibroadenoma.All patients were accepted histological examination.The recruitment criteria for hyperplasia patients was refered to Rosen's Breast Pathology. The histological typing and diagnosis of breast tumor were refered to"histological typing of breast tumor" made by WHO in 2003.2. Instruments and methods2.1 InstrumentsThe ultrasound imaging was performed with iU22 ultrasound system (PhilipsTechnologies) connected to a L9-3 wide frequency linear array probe. The settings ofpulse inversion harmonic(PIH) contrast imaging were as follows: the frame rate was16 HZ, mechanical index(MI) was 0.07.The perfluoropropane-containing human albumin microphere was prepared bypharmacology experiment base of Nanfang hospital. The contrast medium (0.02ml/kg) was administered with intravenous bolus injection.2.2. Image collection and analysisImage collection:Routine procedure for breast checking was used. Color Dopplerflow imaging and power Doppler imaging (PDI) to observe the blood flow rich area andimages were frozen and stored. After contrast was injected, the continuously dynamicimaging was stored synchronously.Image analysis:The regions of interest(ROI) with PDI can be divided into 4grades according to the blood flow rich degree.Turn on QLAB software and MVI procedure to observe the imaging track ofmicrosphere in microvessels in real time. To sample twice at blood flow rich area toobtain time-intensity curve of regions of interest (ROI). The related parameters suchas peak intensity, area under the curve, time to peak enhancement and wash-out timewere obtained accordingly.To sample at the outer area of foci and blood flow rich area to obtain time-intensity curve of ROI and the perfusion parameters of different region was obtainedaccordingly. (mean perfusion parameter=marginal perfusion parameters+interiorperfusion parameter/2)3. pathology examination3.1 HE staining: The tissues were fixed in 10% formalin, embedded in paraffin,serially sectioned at 4μm, and stained with hematoxylin and eosin. 3.2 Immunohistochemistry:Immunohistochemistry was performed according to the protocol of the kit. Thepositive cell was determined by the appearances of light brown granules in cytoplasmor cell membrane.Microvessel density (MVD) was determined by Weidner's method. Theexpression of VEGF and Flk-1/KDR was determined by semi-quantitative integralmethod: positive cells≤5%, score 0; 6%～25%, score 1; 26%～50, score 2; 51%～75%,score 3;＞75%, score 4. The score of stain: no specific staining, score 0; yellowstaining, score 1; light brown, score 2; brown, score 3. To multiply the score marks,the scores were definited as flows: 0～1, negative (-), 2～4, weakly positive (+); 5～8,middle posivive (++); and 9～12, strong positive (+++).3.3 Electron microscopy observationThe samples of breast cancers (6 cases), breast fibroadenoma (4 cases) andnormal breast tissues (4cases) was analysed routinely with transmission electronmicroscopy (CM10, PHILIPS).4 Statisticalal analysisThe measurement data of the five group was presented with mean±standarddeviation. The Kolmogorov-Smimof test was used to examine the normality of thevalues' distribution. One-way ANOVA was used when test for homogeneity ofvariance and Games Howell test was used when test for heterogeneity of variance andenumeration data analysis.For the difference between benign and malignant tumor, measurement data wastested with independent sample t test, enumeration data was tested with x~2, twoindependent sample was performed using the non-parametric Mann-CWhitney U-test.A value of P＜0.05 was considered statisticalally significant.Results:1.Results of ChaptⅠ1.1 pathology results1.1.1 Breast carcinoma combined with atypical breast hyperplasia: in all 45Breast carcinoma patients, 64.44% patients had Atypical breast hyperplasia in peri-cancer tissue in which the incidence rate of Atypical breast hyperplasia inIntraductal carcinoma patients was 73.33% (11/15) while the rate in invasive ductalcarcinoma was 60%(18/30).1.1.2 CD34,VEGF,Flk-1/KDR expressionCD34 was expressed positively in each group. CD34 was expressed in thecytoplasm or on the cell membrane that its distribution show significant heterogeneitythat expressed mainly in the cancer nest margin or hyperplasia active tissues.The MVD increased with the progression of disease. The MVD was minimal innormal breast group (17.10±4.44) and was maximal in invasive ductal carcinoma(51.89±3.05). The MVD had significant difference among groups(P＜0.05) and hadsiginificant difference between each group except normal group and hyperplasiagroup, atypical breast hyperplasia group and intraductal carcinoma group.VEGF expressed mainly in the cytoplasm and on the cell membrane of breastcancer cells and new vessel endothelial cells, and expressed on the duct epithelial cellof atypical hyperplasia and vascular endothelial cells of interstitial tissues. VEGFhardly expressed in normal breast and simple epithelium hyperplasia while expressedmaximally in the invasive ductal carcinoma that had significant different amonggroups. Compare means between groups, VEGF expression had significant differencebetween groups except normal and simple hyperplasia group.Flk-1/KDR expressed mainly in the cytoplasm and on the cell membrane ofbreast cancer interstitial vascular endothelial cells, and expressed on the vascularendothelial cells of atypical hyperplasia. Flk-1/KDR hardly expressed in vascularendothelial cells of normal breast and simple epithelium hyperplasia group, andexpressed increaseingly with the progression of disease while expressed maximally inthe invasive ductal carcinoma.1.2 Ultrasound results1.2.1 PDI resultsWith the progression of breast disease, the blood flow detected by PDI becomericher that the normal breast group was in grade 0 or 1 while the invasive ductalcarcinoma group was the richest that most was in grade 3. 1.2.2 Contrast enhanced ultrasound imaging1.2.2.1 MVI resultsThe MVI of blood flow improved significantly after use of contrast agents. Innormal breast tissues, the arrangements of the blood vessels were regularly, the sizeswere homogeneous, the distributions were natural. The number of blood vesselincreased around the focus in simple epithelium hyperplasia tissues. We could seedilated and distorted vessels extended from the margin to the interior in atypicalhyperplasia except the increased vessel number. In intraductal carcinoma in situ, theblood vessel distortion and anastomosis was obvious. In invasive ductal carcinomagroup, we could see either the blood vessel distention, distortion, cystiform dilationand the disorderly distribution and agglomerated new born blood vessel network.1.2.2.2 Time-intensity curve analysisThe peak intensity(PI) among 5 groups had significant diffierence that thelowest was normal breast group(38.26±8.52) and the highest was invasive ductalcarcinoma group (120.06±11.00). Compare means between each group, there weresignificant difference between groups except normal breast group and hyperplasiagroup, atypical hyperplasia group and intraductal carcinoma in situ group.The time to peak enhancement(TTP) among 5 groups had significant differencethat the longest was simple hyperplasia group (22.80±1.94s) and the shortest wasinvasive ductal carcinoma group (14.59±1.02s). Compare the difference betweenevery 2 groups, there weren't significant difference between normal breast group andhyperplasia group, normal breast group and atypical hyperplasia group, atypicalhyperplasia group and intraductal carcinoma in situ group while the others hadsignificant differences.The area under the curve(AUC) among 5 groups had significant difference thatthe lowest was normal breast group(363.09±28.14) and the highest was invasiveductal carcinoma group (1300.63±124.86). Compare means of AUC between eachgroup, there were significant difference between groups except normal breast groupand hyperplasia group, atypical hyperplasia group and intraductal carcinoma in situgroup. The wash-out time (WOT) among 5 groups had significant difference that thelowest was normal breast group(31.39±1.14) and the highest was invasive ductalcarcinoma group (74.93±3.19). The WOT showed an increasing trend with theprogression of breast disease. Compare means between each group, there weresignificant difference between groups except normal breast group and hyperplasiagroup, atypical hyperplasia group and intraductal carcinoma in situ group.From the data above, we found that the PI, AUC and WOT in each groupincreased with the progression of breast cancerous disease while the TTP showed asharp increase in simple hyperplasia group,then decreased gradually.2. Results of chaptⅡ2.1 Pathology results2.1.1 CD34,VEGF,Flk-1/KDR expressionExpression of CD34: The distribution of CD34 in breast carcinoma washeterogeneous. The micro-vessel rich regions distributed diffuse around the margin ofcancer nest. The MVD of malignant group(34.48±8.34) was significantly higherthan that of benign group(18.65±4.69).Expression of VEGF: the high VEGF expression distributed diffuse or focally atthe margin of cancer nest and necrotic tissue. The VEGF expression betweenmalignant and benign group had significant difference.Expression of CD34: Flk-1/KDR expressed diffuse or focally in breastcarcinoma, especially high at the margin of cancer nest and necrotic tissue. TheFlk-1/KDR expression between malignant and benign group had significantdifference.2.1.2 Transmission electron microscope resultsThe newborn blood vessels of breast cancer were composed of monolayerendothelial cells, the gap between endothelial cells became wide. The basementmembrane not continuously had obvious interstitial edema. The shapes of endothelialcells were abnormal, the cell body became bigger and cytoplasm became richer andabnormal cell nucleus appeared and pinocytotic vesicle increased. The endothelial cell of fibroadenoma and normal breast had tight gap, thecytoplasm was smooth, the basement membrane was integral. The cell was long-flatthat had normal cytoplasm, nuclei and the pinocytotic vesicles.2.2 Contrast enhanced ultrasound imaging2.2.1 Blood signal of malignant breast carcinoma with or without contrast: thepositive rate for blood flow had no significant difference before the use of contrastagents. After the use of contrast agents, the positive rate enhanced significantlyaround and inside the focal.2.2.2 MVI: MVI inside of the focus was increased heterogeneously that hadthe characteristic of malignant tumor such as filling defect and vessel distortion.2.2.3 Time-intensity curve analysisShape of the curve: the curve of malignant tumor group was characterized asascend rapidly and drop slowly while the benign group presented as ascend slowlyand drop rapidly.The AUC and WOT of malignant tumor group were significantly higher thanthat of benign group while the PI and time to peak had no statistical difference.In malignant tumor group, the PI, AUC and WOT of the margin of focus weresignificantly higher that of inside region of focus, while time to peak was the opposite.However, the perfusion parameter between inside and outside of focus in benigngroup had no statistical differences.Conclusions1. The tumorgenesis of breast carcinoma is complicated process that affected bymulti-factors. Angiogenesis played pivotal role in the transformation process fromprecancerous lesion to malignant breast carcinoma. The ultrasound findings such asPDI, MVI and main parameters of time-intensity curve have good accordance withthe immunohistochemical results. With the progression of breast cancer, theexpression VEGF and its receptor, Flk-1/KDR, increased; at the same time, the MVDincreased, the framework of the vessel become disorder and the perfusion parameterssuch as PI, AUC, WOT increased either. However, in the very early stages, theparameters mentioned above have no obvious changing while they changed significantly from the atypical hyperplasia stage. PDI, contrast MVI and the mainperfusion parameters could reflect the rule of angiogenesis activities changing indifferent precancerous lesions that it could be a useful, non-invasive method to screenhigh risk population.2. The perfusion pattern, shape of time-intensity curve, mean perfusionparameter and variance of regional perfusion parameters are valuable diagnostic basisin discriminating benign and malignant breast tumor. The expression of endothelialmolecules of neovasculation and the heterogeneity of mierovasculature are the basisfor the ultrasound evaluation of angiogenesis in breast carcinoma.3. The VEGF and Flk-1/KDR expression are continuously high in intraductalcarcinoma in situ and invasive ductal carcinoma patients while they were hardlyexpressed in benign breast lesions. The ultrastructures of newly mierovessel in breastcarcinoma are different from that of normal vascular endothelium. The results give usa hint that the molecular imaging that targeted to VEGF and Flk-1/KD may providean new idea to explore the early diagnosis of breast carcinoma.