Objective:To explore the capability of vascular endothelial growth factor receptor 2 (VEGFR2)/integrinα vβ 3 dual-targeted microbubbles in assessing the expression level of pro-angiogenic factors during renal cell carcinoma (RCC) growth. Methods:VEGFR2/integrinα vβ 3 dual-targeted microbubbles were prepared by using biotin-avidin linkage method. Twenty subcutaneous RCC xenografts in nude mice were established by subcutaneously injecting 786-O cells and then divided into 2 groups randomly. The targeted contrast-enhanced ultrasound (t-CEUS) examination was performed for all 10 mice in the first group when xenograft tumors were metered from 5 to 10 mm and >10 to 20 mm respectively. And the quantitative parameters of RCC on t-CEUS were longitudinally evaluated during tumor growth. The second group were divided into two subgroups according to xenograft tumors′ diameter, which was 5 to 10 mm and >10 to 20 mm respectively, and underwent t-CEUS examination. Quantitative analysis was performed for all t-CEUS images to obtain the targeted quantitative parameters, which including peak intensity (PI), area under the time-intensity curve (AUC), the differential tissue enhancement (dTE, presenting the difference in PI before (P 1) and after (P 2) the process of Flash). All xenograft tumors in the second group were harvested for immunohistochemical staining to observe the expression of VEGFR2, integrinα vβ 3 and CD31, and their differences in RCC with different tumor sizes. And the correlations between quantitative parameters and VEGFR2, integrinα vβ 3 and CD31 were analyzed. Results:The longitudinal comparison showed that there were statistically significant differences between AUC and dTE of RCC with different tumor sizes (all P<0.001). The larger the tumor size, the smaller the parameters were. According to the horizontal comparison, the expression levels of VEGFR2 and integrinα vβ 3 in larger RCCs were higher than those of RCCs with smaller size (both P<0.05), but there was no significant difference in CD31 expression between the two subgroups ( P=0.754). Both the targeted quantitative parameters (AUC anddTE ) and pro-angiogenic factors (VEGFR2 and integrinα vβ 3) were negatively correlated with tumor size ( rs=-0.83, -0.81, -0.70, -0.88; all P<0.05). Further more, there were good positive correlations between AUC and VEGFR2, integrinαvβ ( rs=0.76, 0.72; all P<0.05). There were good positive correlations between dTE and VEGFR2, integrinα vβ 3 ( rs=0.81, 0.70; all P<0.05). Additionally, the parameter PI was positively correlated with the expression of CD31 ( rs=0.70, P=0.025). Conclusions:The t-CEUS, mediated by VEGFR/integrinα vβ 3 dual-targeted microbubbles, allows noninvasive assessment of the expression levels of VEGFR2 and integrinα vβ 3 in RCCs, which decrease gradually with the increase of tumor size.

Objective:To evaluate the ability of vascular endothelial growth factor receptor 2(VEGFR2)/integrin α vβ 3 dual-targeted microubble (MBD) to target angiogenesis of renal cell carcinoma (RCC) in vivo. Methods:Non-targeted microbubble (MBN) USphere LA was employed as a template to prepare single- and dual-targeted microbubbles which could bind VEGFR2 and/or integrin α vβ 3 (MBV and MBI) by the biotin-avidin bridging method. A total of 40 RCC nude mice models were established by subcutaneously injecting 786-O cells.Twenty of the models were all injected with MBN, MBV, MBI and MBD in a random order, and the other 20 models were registered for antibody blocking assays. The results of ultrasound images were used for quantitative analyses, and the following quantitative parameters were obtained: intensity increment (a 1), peak halving speed (a 2), curve rising slope (a 3), perfusion time (t 0), time to peak (TTP), peak intensity (PI), mean transit time (MTT) and area under the curve (AUC) for the first three minutes, peak intensity at 10 s before (P 1) and after (P 2) ultrasound destruction, and the differences of tissue enhancement (dTE) between P 1 and P 2 (dTE=P 1-P 2). All the quantitative parameters of four contrast agents and the antibody blocking assays were compared.Besides, the immunohistochemical assays were performed to evaluate the expression of CD31, VEGFR2 and integrin α vβ 3 in tumor tissues. Results:The differences of parameters of a 1, a 3, t 0, TTP, PI and P 2 among four different microbubbles had no statistical significances (all P>0.05), and all parameters between the two single-targeted contrast agents were not statistically different (all P>0.05). The parameters of AUC, MTT, P 1 and dTE all showed a trend that dual-targeted bubbles > single-targeted bubbles > non-targeted bubbles (all P<0.05). On the contrary, the trend of dual-targeted bubbles < single-targeted bubbles < non-targeted bubbles (all P<0.05) was observed for a 2. In the antibody blocking experiment, a 2 was faster after the antibody injection ( P<0.001), while AUC, MTT, P 1 and dTE were all lower than those before the antibody injection ( P<0.001), and the other parameters were not statistically different before and after the antibody injection (all P>0.05). Immunohistochemical analyses confirmed the high expression of CD31, VEGFR2 and integrin β 3 in tumor tissues. Conclusions:The VEGFR2 and integrin α vβ 3 dual-targeted microbubble has a good potential to target the angiogenesis of human RCC in vivo.

Objective:To investigate the contrast-enhanced ultrasound (CEUS) features of inflammatory hepatocellular adenoma (I-HCA).Methods:The contrast-enhanced ultrasound features I-HCA of 28 cases from April 2009 to November 2019 in Zhongshan Hospital, Fudan University were retrospectively analyzed, including arterial phase enhancement pattern, the homogeneity of enhancement, subcapsular enhancement, and the internal perfusion defect. All I-HCA lesions were divided into >5 cm group( n=9) and ≤5 cm group( n=19), the CEUS features between the two groups were compared. Results:All I-HCA lesions were hyper-enhanced in the arterial phase, among which 39.3% (11/28) showed diffuse filling, 39.3%(11/28) showed centripetal filling, and 21.4%(6/28) showed centrifugal filling pattern. Twenty-five percent (7/28) of I-HCAs showed heterogeneous enhancement, 10.7% (3/28) revealed unenhanced areas within the lesions. Subcapsular vessels were observed in 67.7 (21/31) of I-HCA lesions. Heterogeneous enhancement and unenhanced areas were more frequently detected in lesions >5 cm ( P=0.020, 0.026, respectively), while there was no difference in the enhancement pattern and subcapsular vessels between the two groups ( P>0.05). Inportal venous phase, 42.9%(12/28) of I-HCAs showed hypo-enhancement, and 57.1%(16/28) of lesions showed washout in late phase. According to "hyper-enhancement in arterial phase, sustained hyper- or iso-enhancement in portal venous and late phase" by CEUS, the diagnostic accuracy of benign lesion was 42.9%(12/28). According to any of hyper-enhancement pattern in arterial phase, subcapsular vascular enhancement, and sustained hyper- or iso-enhancement in portal venous and late phase, the diagnostic accuracy of I-HCA was 71.4% (20/28). Conclusions:CEUS is valuable in the diagnosis of inflammatory hepatocellular adenoma.

Objective:To explore the diagnostic performance of ultrasound attenuation imaging (ATI) in grading the degree of hepatic steatosis in metabolic dysfunction-associated fatty liver disease (MAFLD).Methods:The liver gray-scale ultrasound and ATI examinations were performed on 212 subjects who were treated in Zhongshan Hospital Affiliated to Fudan University from August 2020 to March 2021. The attenuation coefficient(AC) values among different degrees of hepatic steatosis were analyzed and the diagnostic performance of ATI was evaluated. Relationships between AC values and clinical characteristics were assessed by Pearson′s correlation analysis.Results:The AC values for normal liver, mild, moderate and severe fatty liver were (0.56±0.05)dB·cm -1·MHz -1, (0.68±0.09)dB·cm -1·MHz -1, (0.82±0.09)dB·cm -1·MHz -1, (0.94±0.09)dB·cm -1·MHz -1, respectively. There were significant differences in AC values among different hepatic steatosis divisions( P<0.008). There was highly significant correlation between AC values and the degree of hepatic steatosis( r=0.860, P<0.01), moderate correlation between AC values and BMI( r=0.425, P<0.01), weak correlation between AC values and HDL-C( r=-0.237, P=0.029), no correlations between AC values and age, TC, TG, LDL-C ( r=0.083, 0.055, 0.133, -0.039, all P>0.05) .The areas under the receiver operating characteristics curve of ATI for mild fatty liver and above, moderate fatty liver and above, severe fatty liver and above were 0.958, 0.962, 0.918; the sensitivity were 90.1%, 95.8%, 94.9%, the specificity were 96.1%, 87.1%, 73.9%, and the cut-off values were 0.666 dB·cm -1·MHz -1, 0.719 dB·cm -1·MHz -1, 0.803 dB·cm -1·MHz -1, respectively. Conclusions:ATI is a reliable and convenient method for evaluating the degree of hepatic steatosis in MAFLD.

Objective:To investigate the value of contrast-enhanced ultrasonography (CEUS) in detecting minute renal cell carcinoma (MRCC) smaller than 15 mm (by ultrasonic measurement) and the strategy to improve its detection rate.Methods:Fifty-three pathologically confirmed MRCCs by surgery from November 2007 to October 2019 at Zhongshan Hospital of Fudan University were enrolled in this retrospectively study. All of them underwent both conventional ultrasound and CEUS examinations. The clinical and imaging data were collected and analyzed. Common features, such as tumor size, location, echogenicity, morphology, border, and blood flow signals were observed on conventional ultrasound. On CEUS, the presence of enhancement, wash in and wash out pattern, perfusion uniformity within the lesions were observed.Results:Post-operative pathology confirmed 48 clear cell carcinomas, 4 papillary carcinomas, and 1 chromophobe cell carcinoma. On conventional ultrasound, 12/53 lesions showed no protrusion out of the kidney, and 41/53 cases slightly protruded out of the kidney. There was considerable difficulty in the detection of ten lesions, which was achieved with the guidance of CT/MRI, due to their dorsal location of the kidney. On conventional ultrasound, solid, hyper-echoic, color flow signal with varying degrees were the main features of MRCC.The boundary could be well- or ill-defined, and cystic changes existed in part of cases. On CEUS, most MRCCs showed simultaneous enhancement in cortical phase, iso- to hyper-enhancement at peak, and rapid washout in parenchymal phase. The comparisons of imaging features demonstrated that the characteristics were significantly different between conventional ultrasound and CEUS with regard to boundaries, blood supply, and perfusion uniformity (χ 2=12.425, 20.247, 7.185; all P<0.01). Conclusions:CEUS can significantly improve the detection rate of MRCC, which is superior to conventional ultrasound.

Objective:To explore the value of contrast-enhanced ultrasound(CEUS) in distinguishing of renal oncocytoma(RO) and chromophobe renal cell carcinoma(chRCC).Methods:The ultrasonic image features of 49 ROs and 72 chRCCs between October 2007 and January 2020 were retrospectively analyzed, all lesions underwent ultrasonic examination (including 19 ROs and 70 chRCCs with CEUS imaging) and were pathologically approved in our institution. The statistically significant parameters from univariate analyses were then entered for further multivariable Logistic regression. The value of each ultrasonic imaging feature in differentiating RO and chRCC was evaluated.Results:According to the univariate analyses, all imaging features on conventional ultrasound were not statistically different between RO and chRCC (all P>0.05), while the characteristics of tumor wash-in/out pattern, enhancement degree and homogeneity at peak time and pseudocapsule around tumor were significantly different (all P<0.05). After multivariable analyses, tumor wash-in and wash-out pattern were excluded for tumor differentiation ( P>0.05), and the parameters of enhancement degree or homogeneity at peak time and pseudocapsule around tumor were still significantly different between tumor types (all P<0.05, odd ratio was 8.683, 6.667 and 18.774 respectively). The overall sensitivity, specificity and accuracy of these three parameters in diagnosing RO was 68.4%, 91.4% and 86.5%, respectively. Conclusions:CEUS can provide some useful information for the differentiation of RO and chRCC.

Objective: To investigate the value of contrast-enhanced ultrasound(CEUS) in differential diagnosis of complex renal cysts and clear renal cell carcinoma with cystic change(CRCCC). Methods: The ultrasonographic datas of 82 lesions in 82 patients with complicated renal cysts or CRCCC confirmed by pathology were analyzed. The characteristics of conventional ultrasound and CEUS were observed and evaluated. The lesions were graded according to Bosniak classification criteria. Results: Pathological examination showed that 36 cases were complicated renal cysts and 46 cases were CRCCC. Routine ultrasound showed there were 9 cases (25.0%) with cystic masses and 27 cases (75.0%) with solid and cystic masses in complex renal cysts, of which 14 cases (38.9%) could detect color flow signals. In CRCCC, 2 cases (4.3%) were with cystic masses and 44 cases (95.7%) were with solid and cystic masses, of which 33 cases (75.0%) could detect color flow signals. CEUS showed that only 18 cases (50.0%) of the complex renal cysts showed enhancement of cystic wall or septum, with equal or low enhancement at the peak, 9 cases (50.0%) accompanied by decrease of renal cortex, 35 cases (97.2%) had thin and regular cystic wall, no enhancement of cystic wall in all lesions, and 33 cases (91.7%) had septal thickness less than 1 mm. Forty-five cases (97.8%) of CRCCC showed enhancement of cystic wall or septum, 40 cases (88.9%) showed equal or high enhancement at peak, 30 cases (66.7%) were faster than the decrease of renal cortex, 37 cases (80.4%) showed uneven thickness of cystic wall, 24 cases (52.2%) showed enhancement of cystic wall nodules, and 28 cases (60.9%) showed uneven thickness of septum. After CEUS, 33 cases (91.7%) of complex renal cysts were classified as grade Ⅰ and Ⅱ, while 42 cases (91.3%) of CRCCC were classified as grade Ⅲ and Ⅳ. Conclusions The CEUS manifestations of complex renal cysts are different from those of CRCCC. The application of Bosniak criteria in CEUS is helpful for the differential diagnosis of complex renal cysts and CRCCC.

Objective To explore the value of contrast-enhanced ultrasound (CEUS) in improving the diagnosis ability for xanthogranulomatous cholecystitis ( XGC ) and wall-thickening gallbladder cancer ( GBC) . Methods Forth-three patients with XGCs and 31 patients with wall-thickening GBCs proved by pathology were enrolled in this study ,the features on conventional ultrasound and CEUS were recorded ,and the preliminary diagnosis before and after CEUS were given by doctors . Results Significant differences were found in continuous gallbladder inner wall and arterial blood supplement on conventional ultrasound , 58 .1％ (25/43) had continuous inner wall and 34 .9％ (15/43) had arterial blood flow in XGCs compared to 19 .4％ (6/31) and 100％ in GBCs . On CEUS ,72 .1％ (31/43) demonstrated continuous gallbladder inner wall and 48 .8％ (21/43) had hypoechoic nodules in the wall in XGCs compared to 16 .1％ (5/31) and 19 .4％ (6/31) in GBCs ,respectively ( P <0 .05) . No significant difference was found in intra-calcification , infiltration to adjacent organs ,gallbladder stones and fast-in and fast-out enhanced pattern( P >0 .05) . The area under ROC curve was improved from 0 .701 to 0 .899 after combining with CEUS ( P < 0 .05 ) . Conclusions Conventional ultrasound combining with CEUS could help acquiring more effective ultrasonic information and may improve the differential diagnosis ability of XGCs and GBCs .

Objective To prepare the vascular endothelial grow th factor receptor 2 ( VEGFR2 )/Integrinαv β3 dual‐targeted contrast ultrasound agent ,and further evaluating the physical properties ,imaging characteristics and targeted ability in v itro . Methods VEGFR2 targeted microbubble ( MBV ) ,Integrinαv β3 targeted microbubble ( MBI) and VEGFR2/Integrinαv β3 dual targeted microbubble ( MBD) were prepared by attaching VEGFR2 antibody ,Integrinαv β3 antibody and both of VEGFR2/Integrinαv β3 antibody with no targeted USphere LA respectively , using biotin‐avidin linkage method . USphere LA with no antibody attached were used as non‐targeted microbubble( MBN ) . M icrobubble′s physical properties were observed , and its stability was detected by caculating bubble′s concentration at different time points . MBD′s ultrasound imaging characteristics were evaluated by comparing its grey level of ultrasound imaging with SonoVue at the time of preparation and 3 days after preparation . To detect the targeted ability of microbubble ,different types of microbubble were added into Hepa 1‐6 and C3H10 cells ,respectively . T he above procedure was repeated using the pre‐antibody blocking test in Hepa 1‐6 cell . Results MBD had a mean size of 1 256 nm .T he concentration of microbubble in the first duration of three days was declining slowly ,and its speed was accelerated after five days of preparation . T he grey level of new prepared MBD was similar to that of SonoVue in the same concentration ( P ＝0 .113) ,while the level was higher than that of SonoVue within 3 days after preparation( P ＜0 .001) . T he number of microbubble binding to Hepea1‐6 led a tendency of MBD＞ single targeted microbubble ＞ MBN ( all P ＜0 .05 ) . T he number of C3H10 and pre‐blocked Hepa1‐6 cell attached to each group of microbubble had no statistical difference ( all P ＞0 .05) . In addition ,following the pre‐blocked precedure ,the number of Hepa 1‐6 cell attached to each group of microbubble had no statistical difference either ( all P ＞ 0 .05 ) . Conclusions VEGFR2/Integrinαv β3 dual‐targeted contrast ultrasound agent is a stable microbubble and it has excellent ultrasound imaging and targeting ability in v itro .

Objective: To analyze and summarize the ultrasonographic features of fibrolamellar hepatocellular carcinoma (FLHCC). Methods: The ultrasound images were retrospectively analyzed in 23 FLHCC cases which were confirmed by pathology, including the size, echogenicity, boundary and other features on gray scale ultrasound, color Doppler flow imaging and enhancement patterns on contrast-enhanced ultrasound (CEUS). Results: Twenty-three patients had 23 lesions of FLHCC. The average age was (40.0±17.1) (15-77) years old and among them 3 cases had liver cirrhosis(13.0%). The maximum diameter of FLHCC lesions was 2.5-16.7 cm and the average was (7.2±4.3)cm. On gray scale ultrasound, 82.6% (19/23) lesions were hypoechoic, 78.3% (18/23) lesions showed cord-like or sheet-like hyperechoic area and 47.8% (11/23) lesions displayed strip-like echogenic attenuation in the center, with calcification in 17.4% (4/23) lesions. There were 91.3% (21/23) lesions showed peripheral and internal rich color flow signals on color Doppler flow imaging and 60.9% (14/23) with subcapsular thick blood vessels. All 8 FLHCC lesions displayed early and hyper-enhancement in the arterial phase and wash-out in the portal and delayed phases on CEUS. Internal nonenhanced scar-like area was appeared in all lesions on CEUS. Conclusions Patients with FLHCC are mostly young and few with liver cirrhosis. Ultrasonographic features are characterized by relatively large-sized mass with internal acoustic attenuation or calcification on gray scale ultrasound and hypervascularity with central scar on CEUS.