Pharmacokinetic Modeling of Targeted Ultrasound Contrast Agents for Quantitative Assessment of Anti-Angiogenic Therapy: a Longitudinal Case-Control Study in Colon Cancer.

PURPOSE: To evaluate quantitative and semi-quantitative ultrasound molecular imaging (USMI) for antiangiogenic therapy monitoring in human colon cancer xenografts in mice. PROCEDURES: Colon cancer was established in 17 mice by injection of LS174T (Nr = 9) or CT26 (Nn = 8) cancer cells to simulate clinical responders and non-responders, respectively. Antiangiogenic treatment (bevacizumab; Nrt = Nnt = 5) or control treatment (saline; Nrc = 4, Nnc = 3) was administered at days 0, 3, and 7. Three-dimensional USMI was performed by injection at days 0, 1, 3, 7, and 10 of microbubbles targeted to the vascular endothelial growth factor receptor 2 (VEGFR2). Microbubble binding rate (kb), estimated by first-pass binding model fitting, and semi-quantitative parameters late enhancement (LE) and differential targeted enhancement (dTE) were compared at each day to evaluate their ability to assess and predict the response to therapy. Correlation analysis with the ex-vivo immunohistological quantification of VEGFR2 expression and the percentage blood vessel area was also performed. RESULTS: Significant changes in the USMI parameters during treatment were observed only in the responders treated with bevacizumab (p-value < 0.05). Prediction of the response to therapy as early as 1 day after treatment was achieved by the quantitative parameter kb (p-value < 0.01), earlier than possible by tumor volume quantification. USMI parameters could significantly distinguish between clinical responders and non-responders (p-value << 0.01) and correlated well with the ex-vivo quantification of VEGFR2 expression and the percentage blood vessels area (p-value << 0.01). CONCLUSION: USMI (semi)quantitative parameters provide earlier assessment of the response to therapy compared to tumor volume, permit early prediction of non-responders, and correlate well with ex-vivo angiogenesis biomarkers.

Intra-Animal Comparison between Three-dimensional Molecularly Targeted US and Three-dimensional Dynamic Contrast-enhanced US for Early Antiangiogenic Treatment Assessment in Colon Cancer.

Purpose To perform an intra-animal comparison between (a) three-dimensional (3D) molecularly targeted ultrasonography (US) by using clinical-grade vascular endothelial growth factor receptor 2 (VEGFR2)-targeted microbubbles and (b) 3D dynamic contrast material-enhanced (DCE) US by using nontargeted microbubbles for assessment of antiangiogenic treatment effects in a murine model of human colon cancer. Materials and Methods Twenty-three mice with human colon cancer xenografts were randomized to receive either single-dose antiangiogenic treatment (bevacizumab, n = 14) or control treatment (saline, n = 9). At baseline and 24 hours after treatment, animals were imaged with a clinical US system equipped with a clinical matrix array transducer by using the following techniques: (a) molecularly targeted US with VEGFR2-targeted microbubbles, (b) bolus DCE US with nontargeted microbubbles, and (c) destruction-replenishment DCE US with nontargeted microbubbles. VEGFR2-targeted US signal, peak enhancement, area under the time-intensity curve, time to peak, relative blood volume (rBV), relative blood flow, and blood flow velocity were quantified. VEGFR2 expression and percentage area of blood vessels were assessed ex vivo with quantitative immunofluorescence and correlated with corresponding in vivo US parameters. Statistical analysis was performed with Wilcoxon signed rank tests and rank sum tests, as well as Pearson correlation analysis. Results Molecularly targeted US signal with VEGFR2-targeted microbubbles, peak enhancement, and rBV significantly decreased (P ≤ .03) after a single antiangiogenic treatment compared with those in the control group; similarly, ex vivo VEGFR2 expression (P = .03) and percentage area of blood vessels (P = .03) significantly decreased after antiangiogenic treatment. Three-dimensional molecularly targeted US signal correlated well with VEGFR2 expression (r = 0.86, P = .001), and rBV (r = 0.71, P = .01) and relative blood flow (r = 0.78, P = .005) correlated well with percentage area of blood vessels, while other US perfusion parameters did not. Conclusion Three-dimensional molecularly targeted US and destruction-replenishment 3D DCE US provide complementary molecular and functional in vivo imaging information on antiangiogenic treatment effects in human colon cancer xenografts compared with ex vivo reference standards. © RSNA, 2016 Online supplemental material is available for this article.

Quantitative Assessment of Inflammation in a Porcine Acute Terminal Ileitis Model: US with a Molecularly Targeted Contrast Agent.

PURPOSE: To evaluate the feasibility and reproducibility of ultrasonography (US) performed with dual-selectin-targeted contrast agent microbubbles (MBs) for assessment of inflammation in a porcine acute terminal ileitis model, with histologic findings as a reference standard. MATERIALS AND METHODS: The study had institutional Animal Care and Use Committee approval. Acute terminal ileitis was established in 19 pigs; four pigs served as control pigs. The ileum was imaged with clinical-grade dual P- and E-selectin-targeted MBs (MBSelectin) at increasing doses (0.5, 1.0, 2.5, 5.0, 10, and 20 × 10(8) MB per kilogram of body weight) and with control nontargeted MBs (MBControl). For reproducibility testing, examinations were repeated twice after the MBSelectin and MBControl injections. After imaging, scanned ileal segments were analyzed ex vivo both for inflammation grade (by using hematoxylin-eosin staining) and for expression of selectins (by using quantitative immunofluorescence analysis). Statistical analysis was performed by using the t test, intraclass correlation coefficients (ICCs), and Spearman correlation analysis. RESULTS: Imaging signal increased linearly (P < .001) between a dose of 0.5 and a dose of 5.0 × 10(8) MB/kg and plateaued between a dose of 10 and a dose of 20 × 10(8) MB/kg. Imaging signals were reproducible (ICC = 0.70), and administration of MBSelectin in acute ileitis resulted in a significantly higher (P < .001) imaging signal compared with that in control ileum and MBControl. Ex vivo histologic grades of inflammation correlated well with in vivo US signal (ρ = 0.79), and expression levels of both P-selectin (37.4% ± 14.7 [standard deviation] of vessels positive; P < .001) and E-selectin (31.2% ± 25.7) in vessels in the bowel wall of segments with ileitis were higher than in control ileum (5.1% ± 3.7 for P-selectin and 4.8% ± 2.3 for E-selectin). CONCLUSION: Quantitative measurements of inflammation obtained by using dual-selectin-targeted US are reproducible and correlate well with the extent of inflammation at histologic examination in a porcine acute ileitis model as a next step toward clinical translation.

MR angiography with parallel acquisition for assessment of the visceral arteries: comparison with conventional MR angiography and 64-detector-row computed tomography.

The purpose of the study was to retrospectively compare three-dimensional gadolinium-enhanced magnetic resonance angiography (conventional MRA) with MRA accelerated by a parallel acquisition technique (fast MRA) for the assessment of visceral arteries, using 64-detector-row computed tomography angiography (MDCTA) as the reference standard. Eighteen patients underwent fast MRA (imaging time 17 s), conventional MRA (29 s) and MDCTA of the abdomen and pelvis. Two independent readers assessed subjective image quality and the presence of arterial stenosis. Data were analysed on per-patient and per-segment bases. Fast MRA yielded better subjective image quality in all segments compared with conventional MRA (P = 0.012 for reader 1, P = 0.055 for reader 2) because of fewer motion-induced artefacts. Sensitivity and specificity of fast MRA for the detection of arterial stenosis were 100% for both readers. Sensitivity of conventional MRA was 89% for both readers, and specificity was 100% (reader 1) and 99% (reader 2). Differences in sensitivity between the two types of MRA were not significant for either reader. Interobserver agreement for the detection of arterial stenosis was excellent for fast (kappa = 1.00) and good for conventional MRA (kappa = 0.76). Thus, subjective image quality of visceral arteries remains good on fast MRA compared with conventional MRA, and the two techniques do not differ substantially in the grading of arterial stenosis, despite the markedly reduced acquisition time of fast MRA.

Targeted microbubbles for imaging tumor angiogenesis: assessment of whole-body biodistribution with dynamic micro-PET in mice.

PURPOSE: To evaluate in vivo whole-body biodistribution of microbubbles (MBs) targeted to tumor angiogenesis-related vascular endothelial growth factor (VEGF) receptor 2 (VEGFR2) by using dynamic micro-positron emission tomography (PET) in living mice. MATERIALS AND METHODS: Animal protocols were approved by the Institutional Administrative Panel on Laboratory Animal Care. Lipid-shell perfluorocarbon-filled MBs, targeted to VEGFR2 via anti-VEGFR2 antibodies, were radiolabeled by conjugating the radiofluorination agent N-succinimidyl-4-[(18)F]fluorobenzoate (SFB) to the anti-VEGFR2 antibodies. These MBs were then injected intravenously into nude mice (n = 4) bearing angiosarcomas, and the whole-body biodistribution of these probes was assessed for 60 minutes by using dynamic micro-PET. Results were compared with ex vivo gamma counting (n = 6) and immunofluorescence staining (n = 6). Control studies in angiosarcoma-bearing mice were performed with injection of the radiolabeled antibodies alone (n = 3) or free SFB (n = 3). A mixed-effects regression of MB accumulation on fixed effects of time and tissue type (tumor or muscle) and random effect of animal was performed. RESULTS: VEGFR2-targeted MBs rapidly cleared from the blood circulation (50% blood clearance after approximately 3.5 minutes) and accumulated in the liver (mean, 33.4% injected dose [ID]/g +/- 13.7 [standard deviation] at 60 minutes) and spleen (mean, 9.3% ID/g +/- 6.5 at 60 minutes) on the basis of micro-PET imaging. These findings were confirmed with ex vivo gamma counting. Uptake of targeted MBs was significantly higher (P < .0001) in tumor than in adjacent skeletal muscle tissue. Immunofluorescence staining demonstrated accumulation of the targeted MBs within hepatic Kupffer cells and splenic macrophages. Biodistribution of the radiolabeled antibodies and free SFB differed from the distribution of the targeted MBs. CONCLUSION: Dynamic micro-PET allows assessment of in vivo biodistribution of VEGFR2-targeted MBs.

Dual-targeted contrast agent for US assessment of tumor angiogenesis in vivo.

PURPOSE: To develop and validate a dual-targeted ultrasonographic (US) imaging agent with microbubbles (MBs) that attaches to both vascular endothelial growth factor (VEGF) receptor 2 (VEGFR2) and alpha(v)beta(3) integrin and to compare the US imaging signal obtained from dual-targeted MBs (MB(D)) with that from single-targeted MBs (MB(S)) in a murine model of tumor angiogenesis. MATERIALS AND METHODS: Animal protocols were approved by the institutional Administrative Panel on Laboratory Animal Care. Single- and dual-targeted US imaging agents were prepared by attaching anti-VEGFR2, anti-alpha(v)beta(3) integrin, or both antibodies to the shell of perfluorocarbon-filled MBs. Binding specificities of targeted MBs compared with isotype-matched immunoglobulin G-labeled control MBs (MB(C)) and nontargeted nonlabeled MBs (MB(N)) were tested with VEGFR2-positive and alpha(v)beta(3) integrin-positive cells (mouse SVR cells) and control cells (mouse 4T1 cells). In vivo imaging signals of contrast material-enhanced US by using anti-VEGFR2-targeted MBs (MB(V)), anti-alpha(v)beta(3) integrin-targeted MBs (MB(I)), MB(D), and MB(C) were quantified in 49 mice bearing SK-OV-3 tumors (human ovarian cancer). Tumor tissue was stained for VEGFR2, alpha(v)beta(3) integrin, and CD31. RESULTS: Attachment of MB(D) to SVR cells (mean, 0.74 MBs per cell +/- 0.05 [standard deviation]) was significantly higher than attachment to 4T1 cells (mean, 0.04 +/- 0.03), and attachment to SVR cells was higher for MB(D) than for MB(V) (mean, 0.58 +/- 0.09), MB(I) (mean, 0.42 +/- 0.21), MB(C) (mean, 0.11 +/- 0.13), and MB(N) (mean, 0.01 +/- 0.01) (P < .05). Imaging signal in the murine tumor angiogenesis model was significantly higher (P < .001) for MB(D) (mean, 16.7 +/- 7.2) than for MB(V) (mean, 11.3 +/- 5.7), MB(I) (mean, 7.8 +/- 5.3), MB(C) (mean, 2.8 +/- 0.9), and MB(N) (mean, 1.1 +/- 0.4). Immunofluorescence confirmed expression of VEGFR2 and alpha(v)beta(3) integrin on tumor vasculature. CONCLUSION: Dual-targeted contrast-enhanced US directed at both VEGFR2 and alpha(v)beta(3) integrin improves in vivo visualization of tumor angiogenesis in a human ovarian cancer xenograft tumor model in mice. SUPPLEMENTAL MATERIAL: http://radiology.rsnajnls.org/cgi/content/full/248/3/936/DC1.

Assessment of aortoiliac and renal arteries: MR angiography with parallel acquisition versus conventional MR angiography and digital subtraction angiography.

PURPOSE: To prospectively compare the image quality, sensitivity, and specificity of three-dimensional gadolinium-enhanced magnetic resonance (MR) angiography accelerated by parallel acquisition (ie, fast MR angiography) with MR angiography not accelerated by parallel acquisition (ie, conventional MR angiography) for assessment of aortoiliac and renal arteries, with digital subtraction angiography (DSA) as the reference standard. MATERIALS AND METHODS: The study was approved by the institutional review board; informed consent was obtained from all patients. Forty consecutive patients (33 men, seven women; mean age, 63 years) suspected of having aortoiliac and renal arterial stenoses and thus examined with DSA underwent both fast (mean imaging time, 17 seconds) and conventional (mean imaging time, 29 seconds) MR angiography. The arterial tree was divided into segments for image analysis. Two readers independently evaluated all MR angiograms for image quality, presence of arterial stenosis, and renal arterial variants. Image quality, sensitivity, and specificity were analyzed on per-patient and per-segment bases for multiple comparisons (with Bonferroni correction) and for dependencies between segments (with patient as the primary sample unit). Interobserver agreement was evaluated by using kappa statistics. RESULTS: Overall, the image quality with fast MR angiography was significantly better (P=.001) than that with conventional MR angiography. At per-segment analysis, the image quality of fast MR angiograms of the distal renal artery tended to be better than that of conventional MR angiograms of these vessels. Differences in sensitivity for the detection of arterial stenosis between the two MR angiography techniques were not significant for either reader. Interobserver agreement in the detection of variant renal artery anatomy was excellent with both conventional and fast MR angiography (kappa=1.00). CONCLUSION: Fast MR angiography and conventional MR angiography do not differ significantly in terms of arterial stenosis grading or renal arterial variant detection.

Prospective intraindividual comparison between respiratory-triggered balanced steady-state free precession and breath-hold gradient-echo and time-of-flight magnetic resonance imaging for assessment of portal and hepatic veins.

The purpose of this study was to compare respiratory-triggered balanced steady-state free precession (bSSFP) with breath-hold contrast-enhanced dynamic two-dimensional (2D) gradient-echo (GRE) and time-of-flight (TOF) magnetic resonance imaging (MRI) for portal and hepatic vein visualization and assessment of portal and hepatic venous variants. Sixty patients with liver disease underwent nonenhanced bSSFP and contrast-enhanced GRE, bSSFP, and TOF imaging. Contrast-to-noise ratios (CNRs) for portal and hepatic veins were measured. Two readers rated the quality of portal and hepatic vein visualization on a 5-point Likert scale. The diagnostic performance of each MRI series in the detection of portal and hepatic venous variants was assessed in 40/60 patients who also underwent contrast-enhanced multidetector-row computed tomography (MDCT). CNRs for portal and hepatic veins were highest on contrast-enhanced bSSFP images. Image quality of portal and hepatic veins was rated higher for nonenhanced bSSFP than for contrast-enhanced GRE (p<0.03) and TOF (p<0.003) and higher for contrast-enhanced than for nonenhanced bSSFP (p<0.003). Compared with MDCT, portal and hepatic venous variants were identified with an accuracy of 99% on bSSFP images, with an excellent interobserver agreement (kappa=0.97). Compared with MDCT, presence of surgically important portal and hepatic venous anatomical variants can be predicted with high accuracy on bSSFP images.

Assessment of the abdominal aorta and its visceral branches by contrast-enhanced dynamic volumetric hepatic parallel magnetic resonance imaging: feasibility, reliability and accuracy.

The purpose of this study was to evaluate a new three-dimensional gradient-echo (GRE) MR sequence performed with a parallel acquisition technique to shorten breath-hold times (parallel GRE MRI) in the detection of arterial variants and stenosis of the abdominal aorta and its visceral branches. A total of 102 patients underwent dynamic parallel GRE MRI, timed to the arterial phase by a test bolus (mean breath-hold time, 17 s). For both quantitative and qualitative analysis, the abdominal aorta and its visceral branches were divided into 13 arterial segments. In a subanalysis of 55/102 patients, the accuracy of parallel GRE MRI compared to MDCT in the detection arterial variants and stenosis was calculated for two independent readers. Mean SNRs and CNRs were 47.2 and 35.6, respectively. Image quality was rated good or excellent in 1,234/1,326 segments (93%). Hepatic and renal arterial variants were identified with an accuracy of 93 and 95%, respectively (reader 1) and 98 and 100%, respectively (reader 2). Both readers detected arterial stenosis with an accuracy of 98%. Interobserver agreement was good to excellent for the detection of hepatic (kappa=0.69) and renal (kappa=0.92) variants and for the diagnosis of stenosis (kappa=0.96). Dynamic three-dimensional parallel GRE MRI is feasible and allows a reliable and accurate diagnosis of arterial variants and stenosis of the abdominal aorta and its visceral branches in a short breath-hold-time.

Assessment of skeletal muscle perfusion by contrast medium first-pass magnetic resonance imaging: technical feasibility and preliminary experience in healthy volunteers.

PURPOSE: To probe the potential and pitfalls of contrast medium first-pass skeletal muscle perfusion imaging under reproducible stress conditions. MATERIALS AND METHODS: Magnetic resonance (MR) signal dynamics in calf muscle and lower-leg arteries of 20 healthy volunteers were analyzed under postarterial occlusion reactive hyperemia and concurrent contrast medium first pass, using a saturation recovery spoiled gradient-echo type sequence without heartbeat synchronization. The signal vs. time curves were analyzed descriptively and by two-compartment deconvolution analysis. RESULTS: Highly significant changes in calf muscle signal dynamics in the hyperemic leg vs. those in the contralateral leg at rest were found in phenomenological and deconvolution analysis. Although a distortion of the arterial signal derived input function by inflow effects was found to cause large variations of the deconvolution results, the magnitude of the observed effects suggested a potential for immediate visual detection of areas with reduced tissue perfusion. CONCLUSION: The first-pass approach appeared promising for visual evaluation. However, a disentanglement of inflow and contrast medium-induced effects on arterial signal intensity was deemed a prerequisite for input function-based numerical assessment.