BR55 Ultrasound Molecular Imaging of Clear Cell Renal Cell Carcinoma Reflects Tumor Vascular Expression of VEGFR-2 in a Patient-Derived Xenograft Model.

Standard imaging cannot reliably predict the nature of renal tumors. Among malignant renal tumors, clear cell renal cell carcinoma (ccRCC) is the most common histological subtype, in which the vascular endothelial growth factor 2 (VEGFR-2) is highly expressed in the vascular endothelium. BR55, a contrast agent for ultrasound imaging, consists of gas-core lipid microbubbles that specifically target and bind to the extracellular portion of the VEGFR-2. The specific information provided by ultrasound molecular imaging (USMI) using BR55 was compared with the vascular tumor expression of the VEGFR-2 by immunohistochemical (IHC) staining in a preclinical model of ccRCC. Patients' ccRCCs were orthotopically grafted onto Nod-Scid-Gamma (NSG) mice to generate patient-derived xenografts (PdX). Mice were divided into four groups to receive either vehicle or axitinib an amount of 2, 7.5 or 15 mg/kg twice daily. Perfusion parameters and the BR55 ultrasound contrast signal on PdX renal tumors were analyzed at D0, D1, D3, D7 and D11, and compared with IHC staining for the VEGFR-2 and CD34. Significant Pearson correlation coefficients were observed between the area under the curve (AUC) and the CD34 (0.84, p < 10-4), and between the VEGFR-2-specific signal obtained by USMI and IHC (0.72, p < 10-4). USMI with BR55 could provide instant, quantitative information on tumor VEGFR-2 expression to characterize renal masses non-invasively.

Carbonic Anhydrase IX in Renal Cell Carcinoma, Implications for Disease Management.

Carbonic Anhydrase IX (CAIX) is a well-described enzyme in renal cell carcinoma, with its expression being regulated by the hypoxia-inducible factor 1 alpha, it is known for interfering with hypoxia processes. Renal carcinoma encompasses a broad spectrum of histological entities and is also described as a heterogeneous malignant tumor. Recently, various combinations of checkpoint inhibitors and targeted therapies have been validated to manage this disease. Reliable markers to confirm the diagnosis, estimate the prognosis, predict or monitor the treatment response are required. Molecular imaging developments allow a comprehensive analysis of the tumor, overcoming the spatial heterogeneity issue. CAIX, being highly expressed at the tumor cell surfaces of clear cell renal carcinoma, also represents a potential treatment target. In this manuscript we reviewed the current knowledge from the literature on the pathophysiological interactions between renal cell carcinoma and CAIX, the role of CAIX as a marker for diagnosis, prognosis, treatment monitoring and molecular imaging, and the potential target for therapeutic strategies.

A Novel Microflow Phantom Dedicated to Ultrasound Microvascular Measurements.

Tumor microvascularization is a biomarker of response to antiangiogenic treatments and is accurately assessed by ultrasound imaging. Imaging modes used to visualize slow flows include Power Doppler imaging, dynamic contrast-enhanced ultrasonography, and more recently, microvascular Doppler. Flow phantoms are used to evaluate the performance of Doppler imaging techniques, but they do not have a steady flow and sufficiently small channels. We report a novel device for robust and stable microflow measurements and the study of the microvascularization. Based on microfluidics technology, the prototype features wall-less cylindrical channels of diameters ranging from as small as 147 up to 436 µm, cast in a soft silicone polymer and perfused via a microfluidic flow pressure controller. The device was assessed using flow rates from 49 to 146 µL/min, with less than 1% coefficient of variation over three minutes, corresponding to velocities of 6 to 142 mm/s. This enabled us to evaluate and confirm the reliability of the Superb Microvascular Imaging Doppler mode compared with the Power Doppler mode at these flow rates in the presence of vibrations mimicking physiological motion.

New calibration setup for quantitative DCE-US imaging protocol: Toward standardization.

BACKGROUND: The DCE-US (Dynamic Contrast-Enhanced Ultrasonography) imaging protocol predicts the vascular modifications compared with Response Evaluation Criteria in Solid Tumors (RECIST) based mainly on morphological changes. A quantitative biomarker has been validated through the DCE-US multi-centric study for early monitoring of the efficiency of anti-angiogenic cancer treatments. In this context, the question of transposing the use of this biomarker to other types of ultrasound scanners, probes and settings has arisen to maintain the follow-up of patients under anti-angiogenic treatments. As a consequence, radiologists encounter standardization issues between the different generations of ultrasound scanners to perform quantitative imaging protocols. PURPOSE: The aim of this study was to develop a new calibration setup to transpose the DCE-US imaging protocol to the new generation of ultrasound scanners using both abdominal and linear probes. METHODS: This calibration method has been designed to be easily reproducible and optimized, reducing the time required and cost incurred. It is based on an original set-up that includes using a concentration splitter to measure the variation of the harmonic signal intensity, obtained from the Area Under the time-intensity Curve (AUC) as a function of various contrast-agent concentrations. The splitter provided four different concentrations simultaneously ranging from 12.5% to 100% of the initial concentration of the SonoVue contrast agent (Bracco Imaging S.p.A., Milan, Italy), therefore, measuring four AUCs in a single injection. The plot of the AUC as a function of the four contrast agent concentrations represents the intensity variation of the harmonic signal: the slope being the calibration parameter. The standardization through this method implied that both generations of ultrasound scanners had to have the same slopes to be considered as calibrated. This method was tested on two ultrasound scanners from the same manufacturer (Aplio500, Aplioi900, Canon Medical Systems, Tokyo, Japan). The Aplio500 used the settings defined by the initial multicenter DCE-US study. The Mechanical Index (MI) and the Color Gain (CG) of the Aplioi900 have been adjusted to match those of the Aplio500. The reliability of the new setup was evaluated in terms of measurement repeatability, and reproducibility with the agreement between the measurements obtained once the two ultrasound scanners were calibrated. RESULTS: The new setup provided excellent repeatability measurements with a value of 96.8%. Once the two ultrasound scanners have been calibrated for both types of probes, the reproducibility was excellent with the agreement between their respective quantitative measurement was at the lowest 95.4% and at the best 98.8%. The settings of the Aplioi900 (Canon Medical Systems) were adjusted to match those of the Aplio500 (Canon Medical Systems) and these validated settings were for the abdominal probe: MI = 0.13 and CG = 34 dB; and for the linear probe: MI = 0.10 and CG = 38 dB. CONCLUSION: This new calibration setup provided reliable measurements and enabled the rapid transfer and the use of the DCE-US imaging protocol on new ultrasound scanners, thus permitting a continuation of the therapeutic evaluation of patients through quantitative imaging.

Combining functional imaging and interstitial pressure measurements to evaluate two anti-angiogenic treatments.

BACKGROUND: Interstitial hypertension is responsible for poor capillary blood flow and hampered drug delivery. The efficacy of combined sorafenib/bevacizumab treatment given according to different administration schedules has been evaluated by measuring both interstitial pressure (IP) and quantitative dynamic contrast-enhanced ultrasonography (DCE-US) parameters in melanoma-bearing mice. MATERIAL AND METHODS: [corrected] Sixty mice were xenografted with B16F10 melanoma. Animals received a daily administration over 4 days (D0 to D3) of either sorafenib at 30 mg/kg, bevacizumab at 2.5 mg/kg alone, or different schedules of combined treatments. Perfusion parameters determined using an Aplio® sonograph (Toshiba) with SonoVue® contrast agent (Bracco) were compared to IP measurements using fiberoptic probes (Samba®) at D0, D2, D4, D8. RESULTS: The mean baseline IP values ranged between 6.55 and 31.29 mmHg in all the groups. A transient IP decrease occurred at D2 in all treated groups, and especially in the concomitant group which exhibited a significant IP reduction compared to D0. A significant decrease in both the peak intensity and the area under the curve was observed at D4 in the group with concomitant administration of both molecules which yielded maximal inhibition of the tumor volume and the number of vessels. No correlation was found between IP values and volume or perfusion parameters, indicating complex relationships between IP and vascularization. No IP gradients were found between the center and the periphery but IP values in these two regions were significantly correlated (R = 0.93). CONCLUSION: The results suggest that IP variations could be predictive of vascular changes and that one single IP measurement is sufficient to fully characterize the whole tumor.

Assessment of quantitative perfusion parameters by dynamic contrast-enhanced sonography using a deconvolution method: an in vitro and in vivo study.

OBJECTIVES: The purpose of this study was to investigate the impact of the arterial input on perfusion parameters measured using dynamic contrast-enhanced sonography combined with a deconvolution method after bolus injections of a contrast agent. METHODS: The in vitro experiments were conducted using a custom-made setup consisting of pumping a fluid through a phantom made of 3 intertwined silicone pipes, mimicking a complex structure akin to that of vessels in a tumor, combined with their feeding pipe, mimicking the arterial input. In the in vivo experiments, B16F10 melanoma cells were xenografted to 5 nude mice. An ultrasound scanner combined with a linear transducer was used to perform pulse inversion imaging based on linear raw data throughout the experiments. A mathematical model developed by the Gustave Roussy Institute (patent WO/2008/053268) and based on the dye dilution theory was used to evaluate 7 semiquantitative perfusion parameters directly from time-intensity curves and 3 quantitative perfusion parameters from the residue function obtained after a deconvolution process developed in our laboratory based on the Tikhonov regularization method. We evaluated and compared the intraoperator variability values of perfusion parameters determined after these two signal-processing methods. RESULTS: In vitro, semiquantitative perfusion parameters exhibited intraoperator variability values ranging from 3.39% to 13.60%. Quantitative parameters derived after the deconvolution process ranged from 4.46% to 11.82%. In vivo, tumors exhibited perfusion parameter intraoperator variability values ranging from 3.74% to 29.34%, whereas quantitative ones varied from 5.00% to 12.43%. CONCLUSIONS: Taking into account the arterial input in evaluating perfusion parameters improves the intraoperator variability and may improve the dynamic contrast-enhanced sonographic technique.

Ultrasound Molecular Imaging of Renal Cell Carcinoma: VEGFR targeted therapy monitored with VEGFR1 and FSHR targeted microbubbles.

Recent treatment developments for metastatic renal cell carcinoma offer combinations of immunotherapies or immunotherapy associated with tyrosine kinase inhibitors (TKI). There is currently no argument to choose one solution or another. Easy-to-use markers to assess longitudinal responses to TKI are necessary to determine when to switch to immunotherapies. These new markers will enable an earlier adaptation of therapeutic strategy in order to prevent tumor development, unnecessary toxicity and financial costs. This study evaluates the potential of ultrasound molecular imaging to track the response to sunitinib in a clear cell renal carcinoma model (ccRCC). We used a patient-derived xenograft model for this imaging study. Mice harboring human ccRCC were randomized for sunitinib treatment vs. control. The tumors were imaged at days 0, 7, 14 and 28 with ultrasound molecular imaging. Signal enhancement was quantified and compared between the two groups after injections of non-targeted microbubbles and microbubbles targeting VEGFR1 and FSHR. The tumor growth of the sunitinib group was significantly slower. There was a significantly lower expression of both VEGFR-1 and FSHR molecular ultrasound imaging signals in the sunitinib group at all times of treatment (Days 7, 14 and 28). These results confirm the study hypothesis. There was no significant difference between the 2 groups for the non-targeted microbubble ultrasound signal. This study demonstrated for the first time the potential of VEGFR1 and FSHR, by ultrasound-based molecular imaging, to follow-up the longitudinal response to sunitinib in ccRCC. These results should trigger developments for clinical applications.

Multimodal imaging for tumour characterization from micro- to macroscopic level using a newly developed dorsal chamber designed for long-term follow-up.

Optical imaging of living animals is a unique method of studying the dynamics of physiological and pathological processes at a subcellular level. One-shot acquisitions at high resolution can be achieved on exteriorized organs before animal euthanasia. For longitudinal follow-up, intravital imaging can be used and involves imaging windows implanted in cranial, thoracic or dorsal regions. Several imaging window models exist, but none have proven to be applicable for long-term monitoring and most biological processes take place over several weeks. Moreover, none are compatible with multiple imaging modalities, meaning that different biological parameters cannot be assessed in an individual animal. We developed a new dorsal chamber that was well tolerated by mice (over several months) and allowed individual and collective cell tracking and behaviour analysis by optical imaging, ultrasound and magnetic resonance tomography. This new model broadens potential applications to areas requiring study of long-term biological processes, as in cancer research.

Study of the reliability of quantification methods of dynamic contrast-enhanced ultrasonography: numerical modeling of blood flow in tumor microvascularization.

Dynamic contrast-enhanced ultrasonography is a recent functional dynamic imaging technique that allows evaluation of the efficacy of anti-angiogenic treatments by quantifying changes in specific parameters of the tumor vasculature. Preclinical and clinical experimental studies now reveal the existence of sources of variability in the quantitative methods. In order to study the reliability of quantification methods (both semi-quantitative and quantitative), we have developed the first numerical model of blood flow and contrast agents in vascular networks with computational fluid dynamics Fluent software version 15.0 (ANSYS, France). We studied four vascular networks (1.84 × 10-3, 2.28 × 10-3, 2.4 × 10-3 and 2.54 × 10-3 ml) and four blood velocities (0.01, 0.02, 0.03 and 0.05 m s-1). For variations in tumor vascular volume the quantitative method is more sensitive, with variations of parameter perfusion of 25.7%, in contrast to variations of the semi-quantitative parameters between 14.9 and 19.5%. For changes in blood velocity the semi-quantitative method is more sensitive, with variation of the area under the enhancement curve (64%), the maximum of the enhancement curve (60%), and the slope of the enhancement curve (73%). The transit time parameters from the two quantitative methods were weakly sensitive to both blood volume and blood flow variations. This study is hopeful and may be extended to the treatment of more complex vascular networks, to approach clinical conditions, and to the evaluation of quantification methods in contrast imaging.

Variations of microstructure, mineral density and tissue elasticity in B6/C3H mice.

200-MHz scanning acoustic microscopy (SAM) and synchrotron radiation microCT (SR-microCT) were used to assess microstructural parameters, acoustic impedance Z and tissue degree of mineralization of bone (DMB) in site-matched regions of interest in femoral bone of two inbred strains. Transverse femoral sections taken from 5 C57BL/6J@Ico (B6) and 5 C3H/HeJ@Ico (C3H) mice (5.5 months old) were explored. Mass density rho, elastic coefficient c(11) and Young's modulus E(1) were locally derived in the distal epiphysis, distal metaphysis for trabecular bone and mid-diaphysis for cortical bone using a rule-of-mixture model. Structural parameter estimations obtained from X-ray tomographic and acoustic images were almost identical. Both strains had the same bone diameter, but the C3H mice had greater cortical thickness and smaller cancellous diameter than did B6 mice. The average DMB and impedance values were in the range between 1.13 and 1.33 g cm(-3) and 5.8 and 7.8 Mrayl, respectively. All tissue parameters were lower in B6 mice than in C3H mice. However, interstrain differences of DMB were much less (up to 3.8%) than differences of Z (up to 13.2%). SAM and SR-microCT fulfill the requirement for a simultaneous evaluation of cortical bone microstructure and material properties at the tissue level. However, SAM provides a quantitative estimate of elastic properties at the tissue level that cannot be captured by SR-microCT. The strong differences in the measured acoustic impedances among the two inbred strains indicate that the impedance is a good parameter to detect genetic variations of the skeletal phenotype in small animal models.

Toward a Standardization of Ultrasound Scanners for Dynamic Contrast-Enhanced Ultrasonography: Methodology and Phantoms.

The standardization of ultrasound scanners for dynamic contrast-enhanced ultrasonography (DCE-US) is mandatory for evaluation of clinical multicenter studies. We propose a robust method using a phantom for measuring the variation of the harmonic signal intensity obtained from the area under the time-intensity curve versus various contrast-agent concentrations. The slope of this measured curve is the calibration parameter. We tested our method on two devices from the same manufacturer (AplioXV and Aplio500, Toshiba, Tokyo, Japan) using the same settings as defined for a French multicenter study. The Aplio500's settings were adjusted to match the slopes of the AplioXV, resulting in the following settings on the Aplio500: at 3.5 MHz: MI = 0.15; CG = 35 dB and at 8 MHz: MI = 0.10; CG = 32 dB. This calibration method is very important for future DCE-US multicenter studies.

Molecular Imaging to Predict Response to Targeted Therapies in Renal Cell Carcinoma.

Molecular magnetic resonance imaging targeted to an endothelial integrin involved in neoangiogenesis was compared to DCE-US and immunochemistry to assess the early response of three different therapeutic agents in renal cell carcinoma. Human A498 renal cells carcinoma was subcutaneously inoculated into 24 nude mice. Mice received either phosphate-buffered saline solution, sunitinib, everolimus, or bevacizumab during 4 days. DCE-US and molecular MRI targeting αvß3 were performed at baseline and 4 days after treatment initiation. PI, AUC, relaxation rate variations ΔR2⁎, and percentage of vessels area quantified on CD31-stained microvessels were compared. Significant decreases were observed for PI and AUC parameters measured by DCE-US for bevacizumab group as early as 4 days, whereas molecular αvß3-targeted MRI was able to detect significant changes in both bevacizumab and everolimus groups. Percentage of CD31-stained microvessels was significantly correlated with DCE-US parameters, PI (R = 0.87, p = 0.0003) and AUC (R = 0.81, p = 0.0013). The percentage of vessel tissue area was significantly reduced (p < 0.01) in both sunitinib and bevacizumab groups. We report an early detection of neoangiogenesis modification after induction of targeted therapies, using DCE-US or αvß3-targeted MRI. We consider these outcomes should encourage clinical trial developments to further evaluate the potential of this molecular MRI technique.

Bone microstructure and elastic tissue properties are reflected in QUS axial transmission measurements.

Accurate clinical interpretation of the sound velocity derived from axial transmission devices requires a detailed understanding of the propagation phenomena involved and of the bone factors that have an impact on measurements. In the low megahertz range, ultrasonic propagation in cortical bone depends on anisotropic elastic tissue properties, porosity and the cortical geometry (e.g., thickness). We investigated 10 human radius samples from a previous biaxial transmission study using a 50-MHz scanning acoustic microscope (SAM) and synchrotron radiation microcomputed tomography. The relationships between low-frequency axial transmission sound speed at 1 and 2 MHz, structural properties (cortical width Ct.Wi, porosity, Haversian canal density and material properties (acoustic impedance, mineral density) on site-matched cross-sections were investigated. Significant linear multivariate regression models (1 MHz: R(2) = 0.84, p < 10(-4), root-mean-square error (RMSE) = 38 m/s, 2 MHz: R(2) = 0.65, p < 10(-4), RMSE = 48 m/s) were found for the combination of Ct.Wi with porosity and impedance. A new model was derived that accounts for the nonlinear dispersion relation with Ct.Wi and predicts axial transmission velocities measured at different ultrasonic frequencies (R(2) = 0.69, p < 10(-4), RMSE = 52 m/s).

Molecular ultrasound imaging using contrast agents targeting endoglin, vascular endothelial growth factor receptor 2 and integrin.

Expression levels of endoglin, αv integrin and vascular endothelial growth factor receptor 2 (VEGFR2) were investigated using targeted, contrast-enhanced ultrasonography in murine melanoma tumor models. Microvasculature and expression levels of biomarkers were investigated using specific contrast agents conjugated with biotinylated monoclonal antibodies. Ultrasound signal intensity from bound contrast agents was evaluated in two groups of mice: control mice and mice treated with sorafenib. Expression levels were analyzed by immunohistochemistry. Endoglin biomarkers were more highly expressed than αv integrin and VEGFR2. Endoglin decreased in the sorafenib group, whereas it tended to increase with time in the control group. Targeted ultrasound contrast agents may be used for non-invasive longitudinal evaluation of tumor angiogenesis during tumor growth or therapeutic treatment in preclinical studies. Endoglin protein, which plays an important role in angiogenesis, seems to be a target of interest for detection of cancer and for prediction of therapeutic efficacy.

Dynamic contrast-enhanced ultrasound parametric maps to evaluate intratumoral vascularization.

OBJECTIVES: The purposes of this study were to assess the reliability of parametric maps from dynamic contrast-enhanced ultrasound (DCE-US) to reflect the heterogeneous distribution of intratumoral vascularization and to predict the tissue features linked to vasculature. This study was designed to compare DCE-US parametric maps with histologic vascularity measurements. MATERIALS AND METHODS: Dynamic contrast-enhanced ultrasound was performed on 17 melanoma-bearing nude mice after a 0.1-mL bolus injection of SonoVue (Bracco SPA, Milan, Italy). The parametric maps were developed from raw linear data to extract pixelwise 2 semiquantitative parameters related to perfusion and blood volume, namely, area under the curve (AUC) and peak intensity (PI). The mathematical method to fit the time-intensity curve for each pixel was a polynomial model used in clinical routine and patented by the team. Regions of interest (ROIs) were drawn on DCE-US parametric maps for whole tumors and for several local areas of 15 mm within each tumor (iROI), the latter reflecting the heterogeneity of intratumoral blood volume. As the criterion standard correlation, microvessel densities (MVDs) were determined for both ROI categories. In detail, for all iROI of 15 mm, MVD and maturity were divided separately for vessels of 0 to 10 µm, 10 to 40 µm, and greater than 40 µm in diameter, and the results were correlated with the ultrasound findings. RESULTS: Among the 17 studied mice, a total of 64 iROIs were analyzed. For the whole-tumor ROI set, AUC and PI values significantly correlated with MVD (rAUC = 0.52 [P = 0.0408] and rPI = 0.70 [P = 0.0026]). In the case of multiple iROI, a strong linear correlation was observed between the DCE-US parameters and the density of vessels ranging in their diameter from 0 to 10 µm (rAUC = 0.68 [P < 0.0001]; rPI = 0.63 [P < 0.0001]), 10 to 40 µm (rAUC = 0.98 [P = 0.0003]; rPI = 0.98 [P = 0.0004]), and greater than 40 µm (rAUC = 0.86 [P = 0.0120]; rPI = 0.92 [P = 0.0034]), respectively. However, the DCE-US parameter values of perfusion and blood volume were not significantly different according to the diameters (AUC: P = 0.1731; PI: P = 0.2918) and maturity of blood vessels. CONCLUSIONS: Parametric maps of DCE-US can be reliably established from raw linear data and reflect the heterogeneous histological measures of vascularization within tumors. In contrast, the values of DCE-US parametric maps (AUC, PI) do not allow deduction of heterogeneous tissue features such as the diameters and maturity of vascular networks.

Establishment and characterization of new orthotopic and metastatic neuroblastoma models.

BACKGROUND/AIM: Treatment of metastatic neuroblastoma remains a challenge in pediatric oncology. Relevant preclinical models may improve exploration of oncogenesis and new therapies. We developed new orthotopic and metastatic models derived from stage 4 neuroblastoma. MATERIAL AND METHODS: Orthotopic and systemic models were established in BalbC Rag2(-/-)gammaC(-/-) mice following adrenal and intravenous injection of luciferase-transfected IMR-32 and IGR-N91 cells, respectively. RESULTS: All four models exhibited 100% tumor take rate. Metastatic spread of orthotopic IMR-32-Luc cells was observed mainly to the lung, liver and bone; that of IGR-N91-Luc cells to liver, spleen and adrenals. Interestingly, systemic IMR-32-Luc cells metastasized rather to the lung, liver and bone, and IGR-N91-Luc to liver, lung, spleen and adrenals. Feasibility of non-invasive, real-time antitumor response evaluation was validated in the systemic models. CONCLUSION: These neuroblastoma models with distinct patterns of metastatic spread represent relevant tools for exploring local and metastatic tumor cell tropism, mechanisms of spread and evaluating new cancer therapeutics.

Impact of the arterial input function on microvascularization parameter measurements using dynamic contrast-enhanced ultrasonography.

AIM: To evaluate the sources of variation influencing the microvascularization parameters measured by dynamic contrast-enhanced ultrasonography (DCE-US). METHODS: Firstly, we evaluated, in vitro, the impact of the manual repositioning of the ultrasound probe and the variations in flow rates. Experiments were conducted using a custom-made phantom setup simulating a tumor and its associated arterial input. Secondly, we evaluated, in vivo, the impact of multiple contrast agent injections and of examination day, as well as the influence of the size of region of interest (ROI) associated with the arterial input function (AIF). Experiments were conducted on xenografted B16F10 female nude mice. For all of the experiments, an ultrasound scanner along with a linear transducer was used to perform pulse inversion imaging based on linear raw data throughout the experiments. Semi-quantitative and quantitative analyses were performed using two signal-processing methods. RESULTS: In vitro, no microvascularization parameters, whether semi-quantitative or quantitative, were significantly correlated (P values from 0.059 to 0.860) with the repositioning of the probe. In addition, all semi-quantitative microvascularization parameters were correlated with the flow variation while only one quantitative parameter, the tumor blood flow, exhibited P value lower than 0.05 (P = 0.004). In vivo, multiple contrast agent injections had no significant impact (P values from 0.060 to 0.885) on microvascularization parameters. In addition, it was demonstrated that semi-quantitative microvascularization parameters were correlated with the tumor growth while among the quantitative parameters, only the tissue blood flow exhibited P value lower than 0.05 (P = 0.015). Based on these results, it was demonstrated that the ROI size of the AIF had significant influence on microvascularization parameters: in the context of larger arterial ROI (from 1.17 ± 0.6 mm(3) to 3.65 ± 0.3 mm(3)), tumor blood flow and tumor blood volume were correlated with the tumor growth, exhibiting P values lower than 0.001. CONCLUSION: AIF selection is an essential aspect of the deconvolution process to validate the quantitative DCE-US method.

Estimation of intra-operator variability in perfusion parameter measurements using DCE-US.

AIM: To investigate intra-operator variability of semi-quantitative perfusion parameters using dynamic contrast-enhanced ultrasonography (DCE-US), following bolus injections of SonoVue(®). METHODS: The in vitro experiments were conducted using three in-house sets up based on pumping a fluid through a phantom placed in a water tank. In the in vivo experiments, B16F10 melanoma cells were xenografted to five nude mice. Both in vitro and in vivo, images were acquired following bolus injections of the ultrasound contrast agent SonoVue(®) (Bracco, Milan, Italy) and using a Toshiba Aplio(®) ultrasound scanner connected to a 2.9-5.8 MHz linear transducer (PZT, PLT 604AT probe) (Toshiba, Japan) allowing harmonic imaging ("Vascular Recognition Imaging") involving linear raw data. A mathematical model based on the dye-dilution theory was developed by the Gustave Roussy Institute, Villejuif, France and used to evaluate seven perfusion parameters from time-intensity curves. Intra-operator variability analyses were based on determining perfusion parameter coefficients of variation (CV). RESULTS: In vitro, different volumes of SonoVue(®) were tested with the three phantoms: intra-operator variability was found to range from 2.33% to 23.72%. In vivo, experiments were performed on tumor tissues and perfusion parameters exhibited values ranging from 1.48% to 29.97%. In addition, the area under the curve (AUC) and the area under the wash-out (AUWO) were two of the parameters of great interest since throughout in vitro and in vivo experiments their variability was lower than 15.79%. CONCLUSION: AUC and AUWO appear to be the most reliable parameters for assessing tumor perfusion using DCE-US as they exhibited the lowest CV values.

Assessment of bone structure and acoustic impedance in C3H and BL6 mice using high resolution scanning acoustic microscopy.

Two hundred-MHz time-resolved scanning acoustic microscopy was applied for the investigation of acoustic and structural bone properties of mice from two inbred strains. Transverse sections of femur taken from 5 C57BL/6J@Ico and 5 C3H/HeJ@Ico mice were explored. Both strains had the same bone diameter, but the C3H/HeJ@Ico mice had greater cortical thickness, smaller cancellous diameter, and greater acoustic impedance values than C57BL/6J@Ico mice. The strong differences in the measured acoustic impedances among the two inbred strains indicate that the impedance is a good parameter to detect genetic variations of the skeletal phenotype in small animal models.