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BACKGROUND: Individuals with chronic kidney disease (CKD) have decreased kidney cortical microvascular perfusion, which may lead to worsening kidney function over time, but methods to quantify kidney cortical microvascular perfusion are not feasible to incorporate into clinical practice. Contrast-enhanced ultrasound (CEUS) may quantify kidney cortical microvascular perfusion, which requires further investigation in individuals across the spectrum of kidney function. METHODS: We performed CEUS on a native kidney of 83 individuals across the spectrum of kidney function and calculated quantitative CEUS-derived kidney cortical microvascular perfusion biomarkers. Participants had a continuous infusion of the microbubble contrast agent (Definity) with a flash-replenishment sequence during their CEUS scan. Lower values of the microbubble velocity (β) and perfusion index (β×A) may represent lower kidney cortical microvascular perfusion. Multivariable linear regression models tested the associations of the microbubble velocity (β) and perfusion index (β×A) with estimated glomerular filtration rate (eGFR). RESULTS: Thirty-eight individuals with CKD (mean age±SD 65.2±12.6 years, median [IQR] eGFR 31.5 [18.9–41.5] ml/min per 1.73 m(2)), 37 individuals with end stage kidney disease (ESKD; age 54.8±12.3 years), and eight healthy volunteers (age 44.1±15.0 years, eGFR 117 [106–120] ml/min per 1.73 m(2)) underwent CEUS without side effects. Individuals with ESKD had the lowest microbubble velocity (β) and perfusion index (β×A) compared with individuals with CKD and healthy volunteers. The microbubble velocity (β) and perfusion index (β×A) had moderate positive correlations with eGFR (β: r(s)=0.44, P

The ability to provide prompt, real-time, easily accessible and radiation-free diagnostic assessments makes ultrasound (US) one of the most versatile imaging modalities. The introduction and development of stable microbubble-based ultrasound contrast agents (UCAs) in the early 1990s improved visualization of complex vascular structures, overcoming some of the limitations of B-mode and Doppler imaging. UCAs have been used extensively in the adult population to visualize vasculature and to evaluate perfusion and blood flow dynamics in organs and lesions. Since the first observations that air bubbles within a liquid can generate a strong echogenic effect, to the early makeshift approaches with agitated saline, and later to the development of industrially produced and federally approved UCAs, these agents have evolved to become both clinically and commercially viable. Perhaps the most exciting potential of UCAs is being uncovered by current research that explores the use of these agents for molecular imaging and therapeutic applications. As contrast-enhanced ultrasound (CEUS) becomes more widely available, it is important for pediatric radiologists to understand the physics of the interaction between the US signal and the microbubbles in order to properly utilize them for the highest level of diagnostic imaging and interventions. In this article we introduce the composition of UCAs and the physics of their behavior in US, and we offer a brief history of their development over the last decades.

The role of contrast-enhanced ultrasound (CEUS) imaging is being widely explored by various groups for its use in the pediatric population. Clinical implementation of new diagnostic or therapeutic techniques requires extensive and meticulous preclinical testing and evaluation. The impact of CEUS will be determined in part by the extent to which studies are oriented specifically toward a pediatric population. Rather than simply applying principles and techniques used in the adult population, these studies are expected to advance and augment preexisting knowledge with pediatric-specific information. To further develop this imaging modality for use in children, pediatric-focused preclinical research is essential. In this paper we describe the development and implementation of the pediatric-specific preclinical animal and phantom models that are being used to evaluate CEUS with the goal of clinical translation to children.

Ultrasound contrast agent (UCA) use in radiology is expanding beyond traditional applications such as evaluation of liver lesions, vesicoureteral reflux and echocardiography. Among emerging techniques, 3-D and 4-D contrast-enhanced ultrasound (CEUS) imaging have demonstrated potential in enhancing the accuracy of voiding urosonography and are ready for wider clinical adoption. US contrast-based lymphatic imaging has been implemented for guiding needle placement in MR lymphangiography in children. In adults, intraoperative CEUS imaging has improved diagnosis and assisted surgical management in tumor resection, and its translation to pediatric brain tumor surgery is imminent. Because of growing interest in precision medicine, targeted US molecular imaging is a topic of active preclinical research and early stage clinical translation. Finally, an exciting new development in the application of UCA is in the field of localized drug delivery and release, with a particular emphasis on treating aggressive brain tumors. Under the appropriate acoustic settings, UCA can reversibly open the blood-brain barrier, allowing drug delivery into the brain. The aim of this article is to review the emerging CEUS applications and provide evidence regarding the feasibility of these applications for clinical implementation.

Background Fetal surgery is increasingly performed to correct congenital defects. Currently, fetal brain perfusion cannot be assessed intra-operatively. The purpose of this study was to determine if contrast-enhanced ultrasound (CEUS) could be used to monitor fetal cerebral perfusion during fetal surgery and if parameters correlate with fetal hemodynamics or acid/base status. Methods Cannulated fetal sheep were insufflated with carbon dioxide gas in an extra-uterine support device and in utero to mimic fetal surgery. Fetal heart rate, mean arterial pressure, and arterial blood gases were serially measured. CEUS examinations of the brain were performed and time-dependent metrics were quantified to evaluate perfusion. The relationships between measured parameters were determined with mixed linear effects models or two-way repeated measures analysis of variance. Results 6 fetal sheep (113 ± 5 days) insufflated at multiple time-points (n = 20 experiments) in an extra-uterine support device demonstrated significant correlations between time-dependent perfusion parameters and fetal pH and carbon dioxide levels. In utero, 4 insufflated fetuses (105 ± 1 days) developed hypercarbic acidosis and had reductions in cerebral perfusion parameters compared to age-matched controls (n = 3). There was no significant relationship between cerebral perfusion parameters and fetal hemodynamics. Conclusions CEUS-derived cerebral perfusion parameters can be measured during simulated fetal surgery and strongly correlate with fetal acid/base status.

Brain contrast-enhanced ultrasound (CEUS) is an emerging application that can complement gray-scale US and yield additional insights into cerebral flow dynamics. CEUS uses intravenous injection of ultrasound contrast agents (UCAs) to highlight tissue perfusion and thus more clearly delineate cerebral pathologies including stroke, hypoxic-ischemic injury and focal lesions such as tumors and vascular malformations. It can be applied not only in infants with open fontanelles but also in older children and adults via a transtemporal window or surgically created acoustic window. Advancements in CEUS technology and post-processing methods for quantitative analysis of UCA kinetics further elucidate cerebral microcirculation. In this review article we discuss the CEUS examination protocol for brain imaging in children, current clinical applications and future directions for research and clinical uses of brain CEUS.

Patients with tuberous sclerosis complex (TSC) can develop solid kidney masses from childhood. Imaging surveillance is done to detect renal cell carcinoma (RCC) and angiomyolipomas (AML), including AMLs at risk for hemorrhage. Intravenous contrast-enhanced ultrasound (CEUS) may be useful for screening as ultrasound is well tolerated by children and ultrasound contrast agents (UCA) are not nephrotoxic. Retrospective review of kidney CEUS exams of pediatric TSC patients. Qualitative CEUS analysis by consensus of 3 radiologists assessed rate, intensity, and pattern of lesion enhancement. Quantitative CEUS analysis was performed using Vuebox®. Where available, abdominal MRI was analyzed qualitatively for the same features and quantitatively by in-house-developed software. Time-intensity curves were generated from both CEUS and MRI where possible. Appearance of lesions were compared between CEUS and MRI and histology where available. Nine masses in 5 patients included one histologically proven RCC and 8 AMLs diagnosed by imaging. Quantitative CEUS of RCC showed malignant features including increased peak enhancement 162%, rapid wash-in rate 162%, and elevated washout rate 156% compared to normal kidney tissue; versus AML which was 68%, 105%, and 125%, respectively. All masses were hypoenhancing on MRI compared to normal kidney tissue; MR dynamic contrast study offered no distinction between RCC and AML. The only MRI feature differentiating RCC from AML was absence of fat. Temporal resolution afforded by CEUS was useful to distinguish malignant from benign kidney masses. CEUS may prove useful for screening, characterizing, and follow-up of kidney lesions in pediatric TSC patients.

Objectives Contrast-enhanced ultrasound (CEUS) can provide quantitative perfusion metrics and may be useful to detect cerebral pathology in neonates and premature infants, particularly in extrauterine environments. The effect of hemodynamics on cerebral perfusion metrics is unknown, which limits the clinical application of this technology. We aimed to determine associations between systemic hemodynamics and concurrently measured brain perfusion parameters in an animal model of extrauterine support. Methods Nine fetal lambs were transferred to an extrauterine support device. Lumason® ultrasound contrast (0.1-0.3 ml) was administered via the umbilical vein and 90-second cine clips were obtained. Time-intensity-curves (TICs) were generated and time-dependent and area-under-curve (AUC) parameters were derived. Associations between brain perfusion metrics and hemodynamics including heart rate (HR) and mean arterial pressure (MAP) were evaluated by multilevel linear mixed-effects models. Results Eighty-six ultrasound examinations were performed and 72 examinations were quantifiable. Time-dependent measurements were independent of all hemodynamic parameters (all p ≥.05). Oxygen delivery and mean blood flow were correlated with AUC measurements (all p ≤.01). Physiologic HR and MAP were not correlated with any measurements (all p ≥.05). Conclusion Detected aberrations in time-dependent CEUS measurements are not correlated with hemodynamic parameters and are thought to reflect the changes in cerebral blood flow, thus providing a promising tool for evaluation of brain perfusion. CEUS brain perfusion parameters are not correlated with physiologic HR and MAP, but AUC-dependent measurements are correlated with oxygen delivery and blood flow, suggesting that CEUS offers additional value over standard monitoring. Overall, these findings enhance the applicability of this technology.

Brain injury remains a leading cause of morbidity and mortality in children. We evaluated the feasibility of using a pediatric swine model to develop contrast-enhanced ultrasound (CEUS)-based measures of brain perfusion for clinical application in various types of brain injury monitoring. Six-week-old, 10-kg swine (N = 10) were anesthetized, and an acoustic window was created in the right frontal cranium to provide visualization of an oblique coronal plane and bilateral thalami. Ultrasound contrast agent was administered via a femoral venous catheter as a weight-based (0.03 mL/kg) bolus. After localization of the imaging plane, CEUS cine clips were acquired for 90 seconds. Bolus injection of contrast agent provided global visualization of cerebral perfusion and highlighted microvasculature in the brain. Preliminary evaluation of bolus kinetics in piglets showed a central gray nuclei-to-cortex ratio similar to human infants with a steep wash-in that crossed and remained above the 1.0 threshold for most of the enhancement period. We demonstrated the similarity in brain perfusion between piglets and human infants, specifically central gray nuclei-to-cortex ratio, showing preliminary feasibility of its use as a pediatric model of brain perfusion. Contrast-enhanced ultrasound can be performed at the bedside as a minimally invasive procedure, and quantitative CEUS may provide critical information regarding changes in brain perfusion as a result of injury or as a response to therapy.

When performing contrast-enhanced ultrasound (CEUS), ultrasound (US) scanner settings, examination technique, and contrast agent dose and administration must be optimized to ensure that high-quality, diagnostic and reproducible images are acquired for qualitative and quantitative interpretations. When carrying out CEUS in children, examination settings should be tailored to their body size and specific indications, similar to B-mode US. This review article details the basic background knowledge that is needed to perform CEUS optimally in children, including considerations related to US scanner settings and US contrast agent dose selection and administration techniques.

With the development of an artificial environment to support the extremely premature infant, advanced imaging techniques tested in this extrauterine system might be beneficial to evaluate the fetal brain. We evaluated the feasibility of (a) performing contrast-enhanced ultrasound (CEUS) and (b) quantifying normal and decreased brain perfusion in fetal lambs maintained on the extrauterine environment for neonatal development (EXTEND) system. Twin premature fetal lambs (102 days of gestational age) were transferred to the EXTEND system. Twin B was subjected to sub-physiological flows (152 mL/kg/min) and oxygen delivery (15.9 mL/kg/min), while Twin A was maintained at physiological levels. We administered Lumason contrast agent into the oxygenator circuit and performed serial CEUS examinations. We quantified perfusion parameters and generated parametric maps. We also recorded hemodynamic parameters, serum blood analysis, and measurements across the oxygenator. Postmortem MRIs were performed. No significant changes in hemodynamic variables were attributable to CEUS examinations. On gray-scale images, Twin B demonstrated ventriculomegaly and progressive parenchymal volume loss culminating in hydranencephaly. By CEUS, Twin B demonstrated decreased peak enhancement and decreased overall parenchymal perfusion when compared to Twin A by perfusion parameters and parametric maps. Changes in perfusion parameters were detected immediately following blood transfusion. Postmortem MRI confirmed ultrasonographic findings in Twin B. In this preliminary experience, we show that CEUS of the brain is feasible in fetal lambs maintained on the EXTEND system and that changes in perfusion can be quantified, which is promising for the application of CEUS in this extrauterine system supporting the premature infant.

Rationale and Objectives Breast cancer is the leading type of cancer among women. Visualization and characterization of breast lesions based on vascularity kinetics was evaluated using three-dimensional (3D) contrast-enhanced ultrasound imaging in a clinical study. Materials and Methods Breast lesions (n = 219) were imaged using power Doppler imaging (PDI), 3D contrast-enhanced harmonic imaging (HI), and 3D contrast-enhanced subharmonic imaging (SHI) with a modified Logiq 9 ultrasound scanner using a 4D10L transducer. Quantitative metrics of vascularity derived from 3D parametric volumes (based on contrast perfusion; PER and area under the curve; AUC) were generated by off-line processing of contrast wash-in and wash-out. Diagnostic accuracy of these quantitative vascular parameters was assessed with biopsy results as the reference standard. Results Vascularity was observed with PDI in 93 lesions (69 benign and 24 malignant), 3D HI in 8 lesions (5 benign and 3 malignant), and 3D SHI in 83 lesions (58 benign and 25 malignant). Diagnostic accuracy for vascular heterogeneity, PER, and AUC ranged from 0.52 to 0.75, while the best logistical regression model (vascular heterogeneity ratio, central PER, and central AUC) reached 0.90. Conclusion 3D SHI successfully detects contrast agent flow in breast lesions and characterization of these lesions based on quantitative measures of vascular heterogeneity and 3D parametric volumes is promising.

To evaluate the use of transtemporal brain contrast-enhanced ultrasound (CEUS) to assess cerebral blood perfusion in a cohort of children without neurological disorders.We included pediatric patients who were undergoing a clinically-indicated CEUS study. Brain scans were performed with a Siemens Sequoia scanner and a 4V1 transducer, that was placed on the left transtemporal bone. Brain scans were performed simultaneously with the images of the clinically-indicated organ of interest. Qualitative and quantitative analysis was performed to evaluate the hemispherical blood flow at the level of the midbrain during the wash-in and wash-out phases of the time-intensity curve. Clinical charts were reviewed to evaluate post-CEUS adverse events.Five patients were evaluated (mean age 5.8 ± 5.1 years). Qualitatively, more avid enhancement in the midbrain than the cortex was observed. Structures depicted ranged between the centrum semiovale at the level of the lateral ventricles and the midbrain. A quantitative analysis conducted on four patients demonstrated less avid perfusion on the contralateral (i.e. right) side, with a mean left/right ratio ranging between 1.51 and 4.07. In general, there was a steep positive wash-in slope starting at approximately 10 s after contrast injection, reaching a peak intensity around 15-26 s on the left side, and 17-29 s on the right side. No adverse events were reported.Transtemporal brain CEUS is feasible and safe in the pediatric population and allows qualitative and quantitative assessment of cerebral perfusion.

Neurodevelopmental impairment is common in survivors of congenital diaphragmatic hernia (CDH). Altered cerebral perfusion in utero may contribute to abnormal brain development in CDH patients.5 fetal lambs with surgical left-CDH and 5 controls underwent transuterine cranial Doppler and contrast enhanced ultrasound (CEUS). Global and regional perfusion metrics were obtained. Biometric and perfusion data were compared between groups via nonparametric Mann Whitney U test and Spearman's rank order correlation.No significant differences in cerebral Doppler measurements were identified between groups. By CEUS, CDH animals demonstrated significantly decreased global brain perfusion and increased transit time. With focal regions-of-interest (ROIs), there was a tendency towards decreased perfusion in the central/thalamic region in CDH but not in the peripheral brain parenchyma. Transit time was significantly increased in both ROIs in CDH, whereas flux rate was decreased in the central/thalamic region but not the peripheral brain parenchyma. Biometric CDH severity was correlated to perfusion deficit. There was no difference in cardiomyocyte histology.The fetal lamb model of CDH shows altered cerebral perfusion as measured by CEUS, correlating to disease severity. This suggests a physiological abnormality in fetal cerebrovascular perfusion that may contribute to abnormal brain development and neurodevelopmental impairment in survivors.

Grayscale ultrasound (US) is decisive in stratifying which thyroid nodules benefit from fine-needle aspiration to evaluate for malignancy. Unfortunately, a significant percentage of nodules remain indeterminate.Herein, we review the clinical considerations and diagnostic accuracy of advanced US, Doppler US, contrast-enhanced US, and US elastography techniques in the evaluation of indeterminate nodules.We conclude that these techniques may be used in combination with grayscale US to improve the assessment of lesion vascularity and tissue property.

OBJECTIVE: Breast cancer is the most frequent type of cancer among women. This multi-center study assessed the ability of 3D contrast-enhanced ultrasound to characterize suspicious breast lesions using clinical assessments and quantitative parameters. METHODS: Women with suspicious breast lesions scheduled for biopsy were enrolled in this prospective, study. Following 2D grayscale ultrasound and power Doppler imaging (PDI), a contrast agent (Definity; Lantheus) was administrated. Contrast-enhanced 3D harmonic imaging (HI; transmitting/receiving at 5.0/10.0 MHz), as well as 3D subharmonic imaging (SHI; transmitting/receiving at 5.8/2.9 MHz), were performed using a modified Logiq 9 scanner (GE Healthcare). Five radiologists independently scored the imaging modes (including standard-of-care imaging) using a 7-point BIRADS scale as well as lesion vascularity and diagnostic confidence. Parametric volumes were constructed from time-intensity curves for vascular heterogeneity, perfusion, and area under the curve. Diagnostic accuracy was determined relative to pathology using receiver operating characteristic (ROC) and reverse, step-wise logistical regression analyses. The κ-statistic was calculated for inter-reader agreement. RESULTS: Data were successfully acquired in 219 cases and biopsies indicated 164 (75%) benign and 55 (25%) malignant lesions. SHI depicted more anastomoses and vascularity than HI (P < .021), but there were no differences by pathology (P > .27). Ultrasound achieved accuracies of 82 to 85%, which was significantly better than standard-of-care imaging (72%; P < .03). SHI increased diagnostic confidence by 3 to 6% (P < .05), but inter-reader agreements were medium to low (κ < 0.52). The best regression model achieved 97% accuracy by combining clinical reads and parametric SHI. CONCLUSIONS: Combining quantitative 3D SHI parameters and clinical assessments improves the characterization of suspicious breast lesions.

Objectives Developmental dysplasia of the hip (DDH) is one of the most common developmental deformities of the lower extremity. Although many children are successfully treated with a brace or harness, some require intraoperative closed or open reduction and spica casting. Surgical reduction is largely successful to relocate the hip; however, iatrogenic avascular necrosis is a major source of morbidity. Recent research showed that postoperative gadolinium-enhanced magnetic resonance imaging (MRI) can depict hip perfusion, which may predict a future incidence of avascular necrosis. As contrast-enhanced ultrasound (CEUS) assesses blood flow in real time, it may be an effective intraoperative alternative to evaluate femoral head perfusion. Here we describe our initial experience regarding the feasibility of intraoperative CEUS of the hip for the assessment of femoral head perfusion before and after DDH reduction. Methods This single-institution retrospective Institutional Review Board-approved study with a waiver of informed consent evaluated intraoperative hip CEUS in children with DDH compared to postoperative contrast-enhanced MRI. Pediatric radiologists, blinded to prior imaging findings and outcomes, reviewed both CEUS and MRI examinations separately and some time from the initial examination both independently and in consensus. Results Seventeen patients had 20 intraoperative CEUS examinations. Twelve of 17 (70.6%) had prereduction hip CEUS, postreduction hip CEUS, and postreduction gadolinium-enhanced MRI. Seven of 12 (58.3%) were evaluable retrospectively. All CEUS studies showed blood flow in the femoral epiphysis before and after reduction, and all MRI studies showed femoral head enhancement after reduction. The CEUS and MRI for all 7 patients also showed physeal blood flow. Conclusions Contrast-enhanced ultrasound is a feasible intraoperative tool for assessing adequate blood flow after hip reduction surgery in DDH.

Noninvasive measurements of the regional microvascular perfusion might lead to sensitive biomarkers for the changes in intracranial hemodynamics that could guide timely surgical interventions for neonatal brain injuries. The current work utilizes a clinically available contrast enhanced ultrasound (CEUS) system and particle tracking velocimetry to perform ultrasound localization microscopy for measuring the microcirculation in piglets. A new deep learning method based on U-net is proposed for enhancing noisy raw CEUS images and detecting the microbubbles. Subsequently, the bubbles are tracked using a Kalman filter based method, which incorporates conditions of spatio-temporal consistency in flow direction and globally optimizes the assignment of bubbles to trajectories. Based on analysis of synthetic data, the U-net results demonstrate significant improvement in the processing speed and localization accuracy over a conventional blind deconvolution method. Visualization of the microvasculature is performed by superposing the bubble trajectories, enabling depiction of a complex micro-vessel network, where neighboring vessels separated by 40 µm can be distinguished. The corresponding perfusion map shows the velocity distribution in these vessels. Based on the current frame rate (44 fps), speeds in the 0.1 to 12 cm/s range can be well captured. These methods show promise as potential clinical tools for bedside measurement of cerebral microcirculation.

Objectives We explore the correlation of contrast-enhanced ultrasound (CEUS) parameters to intracranial pressure (ICP) in a porcine experimental model of pediatric cardiac arrest. Methods Eleven pediatric pigs underwent electrically induced cardiac arrest followed by cardiopulmonary resuscitation. ICP was measured using intracranial bolt monitor and CEUS was monitored through a cranial window. Various CEUS parameters were monitored at baseline, immediately post return of spontaneous circulation (ROSC), 1 hour-post ROSC, and 3 hours post-ROSC. Results There was significant ICP correlation with wash-out slope assessed by CEUS time intensity curve analysis at immediate post-ROSC. At 3 hours post-ROSC there was also significant negative correlation between ICP and peak enhancement which may be due to the evolution of anoxic injury. Conclusion The use of CEUS in assessing disruption of cerebral hemodynamics and ICP post cardiac arrest will need future validation and comparison to other imaging modalities. The correlation between CEUS parameters and ICP may be due to the alterations in cerebral autoregulation that result from anoxic brain injury.

We evaluated the use of quantitative contrast-enhanced ultrasound (CEUS) to study wash-out behavior of ultrasound contrast agents in the pediatric brain in cases of hypoxic ischemic injury (HII). Six neonates and young infants were imaged using CEUS for suspected HII in the Neonatal Intensive Care Unit/Pediatric Intensive Care Unit. After receiving a bolus of ultrasound contrast agent Lumason (Bracco Diagnostics Inc.), analysis was performed in the whole brain, cortex, cortical/subcortical gray and white matter and central gray nuclei to quantify wash-out metrics and ratios. On magnetic resonance imaging clinical imaging findings, 3 children were classified as unaffected and 3 with classical imaging findings consistent with HII. A lower wash-out rate was found in the case of HII compared with the unaffected cases. Here, we present initial work exploring the wash-out behavior for differentiation between unaffected and HII in the brain. These preliminary findings are indicative of altered hemodynamics in HII and are promising for the potential use of CEUS to quantitatively differentiate between the unaffected and HII brain. Little is known about the CEUS wash-out dynamics, especially in the setting of the pediatric brain injury. Our preliminary findings are encouraging and warrant further investigation into the mechanisms behind delayed clearance of the ultrasound contrast agent in the setting of HII.

Purpose: Children born with congenital diaphragmatic hernia (CDH) have intrinsic lower lung compliance. Shear wave elastography (SWE) is an emerging, noninvasive tool to evaluate lung stiffness with the potential to predict postnatal lung function. SWE of the lung in CDH has never been performed in utero. We aimed to evaluate lung elastography in a fetal lamb model of CDH. Methods: Following IACUC-approved protocols, 3 fetal …

The role of contrast-enhanced ultrasound (CEUS) imaging is being widely explored by various groups for its use in the pediatric population. Clinical implementation of new diagnostic or therapeutic techniques requires extensive and meticulous preclinical testing and evaluation. The impact of CEUS will be determined in part by the extent to which studies are oriented specifically toward a pediatric population. Rather than simply applying principles and techniques used in the adult population, these studies are expected to advance and augment preexisting knowledge with pediatric-specific information. To further develop this imaging modality for use in children, pediatric-focused preclinical research is essential. In this paper we describe the development and implementation of the pediatric-specific preclinical animal and phantom models that are being used to evaluate CEUS with the goal of clinical translation to children.

Ultrasound contrast agent (UCA) use in radiology is expanding beyond traditional applications such as evaluation of liver lesions, vesicoureteral reflux and echocardiography. Among emerging techniques, 3-D and 4-D contrast-enhanced ultrasound (CEUS) imaging have demonstrated potential in enhancing the accuracy of voiding urosonography and are ready for wider clinical adoption. US contrast-based lymphatic imaging has been implemented for guiding needle placement in MR lymphangiography in children. In adults, intraoperative CEUS imaging has improved diagnosis and assisted surgical management in tumor resection, and its translation to pediatric brain tumor surgery is imminent. Because of growing interest in precision medicine, targeted US molecular imaging is a topic of active preclinical research and early stage clinical translation. Finally, an exciting new development in the application of UCA is in the field of localized drug delivery and release, with a particular emphasis on treating aggressive brain tumors. Under the appropriate acoustic settings, UCA can reversibly open the blood-brain barrier, allowing drug delivery into the brain. The aim of this article is to review the emerging CEUS applications and provide evidence regarding the feasibility of these applications for clinical implementation.

Brain contrast-enhanced ultrasound (CEUS) is an emerging application that can complement gray-scale US and yield additional insights into cerebral flow dynamics. CEUS uses intravenous injection of ultrasound contrast agents (UCAs) to highlight tissue perfusion and thus more clearly delineate cerebral pathologies including stroke, hypoxic-ischemic injury and focal lesions such as tumors and vascular malformations. It can be applied not only in infants with open fontanelles but also in older children and adults via a transtemporal window or surgically created acoustic window. Advancements in CEUS technology and post-processing methods for quantitative analysis of UCA kinetics further elucidate cerebral microcirculation. In this review article we discuss the CEUS examination protocol for brain imaging in children, current clinical applications and future directions for research and clinical uses of brain CEUS.

The quality of contrast-enhanced ultrasound (CEUS) imaging performed with high-frequency convex and linear transducers is often suboptimal. A common solution to improving the microbubble signal is by increasing the volume of the ultrasound contrast agent being administered. An alternative technique to improve the signal from the contrast agent is to adjust the mechanical index (MI). This study aimed to compare the manufacturer's default MI to an optimal MI (as determined by the best contrast-to-tissue ratio) for improving the CEUS image quality using linear and convex transducers. This study found that in most cases, the default CEUS MI setting by the manufacturer is often suboptimal, and increasing the MI is necessary to improve the contrast-to-tissue ratio and image quality. The MI can be modified by the clinician during the study to improve the quality of the clinical CEUS examination.

Background Minimally invasive fetal surgery, or fetoscopy, is an alternative to open fetal surgery to repair common birth defects like myelomeningocele. Although this hysterotomy-sparing approach reduces maternal morbidity, the effects of in utero insufflation on the fetus are poorly understood. Our purpose was to determine the optimal fetal insufflation conditions. Methods Fetal sheep at gestational age 104 to 107 days were studied under insufflation conditions in utero and ex utero. The ex utero fetuses were cannulated via their umbilical vessels into a support device, the EXTra-uterine Environment for Neonatal Development (EXTEND). EXTEND fetuses were exposed to four different insufflation conditions for four hours: untreated carbon dioxide (CO2) (n = 5), warm humidified (whCO2) (n = 4), whCO2 with the umbilical cord exposed (n = 3), and whCO2 without amniotic fluid (skin and cord exposed) (n = 3). Results In utero insufflation led to significant increases in fetal CO2 and reductions in fetal pH. Ex utero insufflation with whCO2 did not lead to changes in fetal blood gas measurements or cerebral perfusion parameters. Insufflation with whCO2 with an exposed umbilical cord led to reduced umbilical blood flow. Conclusions Insufflation with warm humidified CO2 with an amniotic fluid covered umbilical cord is well tolerated by the fetus without significant changes in hemodynamics or cerebral perfusion parameters. Type of study Basic science Level of evidence N/A

With the development of an artificial environment to support the extremely premature infant, advanced imaging techniques tested in this extrauterine system might be beneficial to evaluate the fetal brain.We evaluated the feasibility of (a) performing contrast-enhanced ultrasound (CEUS) and (b) quantifying normal and decreased brain perfusion in fetal lambs maintained on the extrauterine environment for neonatal development (EXTEND) system.Twin premature fetal lambs (102 days of gestational age) were transferred to the EXTEND system. Twin B was subjected to sub-physiological flows (152 mL/kg/min) and oxygen delivery (15.9 mL/kg/min), while Twin A was maintained at physiological levels. We administered Lumason contrast agent into the oxygenator circuit and performed serial CEUS examinations. We quantified perfusion parameters and generated parametric maps. We also recorded hemodynamic parameters, serum blood analysis, and measurements across the oxygenator. Postmortem MRIs were performed.No significant changes in hemodynamic variables were attributable to CEUS examinations. On gray-scale images, Twin B demonstrated ventriculomegaly and progressive parenchymal volume loss culminating in hydranencephaly. By CEUS, Twin B demonstrated decreased peak enhancement and decreased overall parenchymal perfusion when compared to Twin A by perfusion parameters and parametric maps. Changes in perfusion parameters were detected immediately following blood transfusion. Postmortem MRI confirmed ultrasonographic findings in Twin B.In this preliminary experience, we show that CEUS of the brain is feasible in fetal lambs maintained on the EXTEND system and that changes in perfusion can be quantified, which is promising for the application of CEUS in this extrauterine system supporting the premature infant.

Objectives To evaluate the effects of ultrasound contrast agent (UCA) administration on hemodynamic parameters and support equipment in in vitro and in vivo models of extracorporeal support. Methods In vitro, incrementally increasing bolus doses of a UCA were administered proximal to a membrane oxygenator, and ultrasound cine clips were obtained. The rates of microbubble destruction across the oxygenator and over time were calculated from time-intensity-curves. Measurements across the membrane oxygenator were recorded and compared by a repeated-measures analysis of variance. In vivo, 7 premature fetal lambs were transferred from placental support to the extrauterine environment for neonatal development. Contrast agent boluses were administered for contrast-enhanced ultrasound (CEUS) examinations. Hemodynamic parameters and serum laboratory values were evaluated before and after the examinations by paired t tests. For oxygenator staining, oxygenator membranes from the in vitro circuit, study animals (n = 4), and control animals (n = 4) were stained for the adherent UCA. Results In vitro, with all doses (0.1-4 mL), there was no difference in measured parameters across the oxygenator (P ≥ .09). Contrast agent destruction (3%-14%) across the oxygenator was observed at the first pass with a progressive decline in contrast intensity over time. In vivo, there was no difference in hemodynamic parameters or serum laboratory values (P ≥ .08) with any CEUS examination (n = 17). For oxygenator staining, all oxygenator membranes were negative for UCA with lipid staining. Conclusions The UCA had no detectable effect on the oxygenator or measured parameters in in vitro and in vivo studies, thus providing additional safety data to support the use of CEUS in the setting of extracorporeal support.

Purpose To determine whether three-dimensional subharmonic aided pressure estimation (SHAPE) and subharmonic imaging can help predict the response of breast cancer to neoadjuvant chemotherapy. Materials and Methods In this HIPAA-compliant prospective study, 17 women (age range, 45-70 years) scheduled to undergo neoadjuvant therapy for breast cancer underwent ultrasonography (US) immediately before therapy and at completion of 10%, 60%, and 100% of chemotherapy. All patients provided written informed consent. At each examination, radiofrequency data were collected from SHAPE and subharmonic imaging during infusion of a US contrast agent. Maximum-frequency magnitude and mean intensity were calculated for SHAPE and subharmonic imaging. The signal differences in the tumor relative to the surrounding area were compared with the final treatment response by using the Student t test. Results Four patients left the study, and data from two patients were discarded because of technical problems. Eight patients completed the entire imaging protocol, and an additional three patients dropped out after the imaging session at completion of 10% of chemotherapy as a result of disease progression (these patients were counted as nonresponders). Patients' imaging outcomes consisted of six responders (tumor volume reduction90%) and five partial responders or nonresponders. The results at completion of 10% of therapy showed that the subharmonic signal increased more in the tumor than in the surrounding area for responders than in partial responders or nonresponders (mean ± standard deviation, 3.23 dB ± 1.41 vs -0.88 dB ± 1.46 [P = .001], respectively, for SHAPE and 1.32 dB ± 0.73 vs -0.82 dB ± 0.88 [P = .002], respectively, for subharmonic imaging). Moreover, three patients whose tumor measurements initially increased were correctly predicted to be responders with SHAPE and subharmonic imaging after completion of 10% of therapy. Conclusion SHAPE and subharmonic imaging have the potential to help predict response to neoadjuvant chemotherapy for breast cancer as early as completion of 10% of therapy, albeit on the basis of a small sample size.

This multi-center study evaluated the ability of contrast-enhanced, nonlinear 3D ultrasound imaging to characterize indeterminate breast lesions using quantitative parametric maps and clinical assessments. In total 236 women with biopsy-proven breast lesions were enrolled in this study. Following B-mode and power Doppler imaging (PDI), an ultrasound contrast agent (Definity®, Lantheus Medical Imaging, N Billerica, MA, USA) was administrated. Contrast-enhanced 3D harmonic imaging (HI; transmitting/receiving at 5.0/10.0 MHz) as well as 3D subharmonic imaging (SHI; transmitting/receiving at 5.8/2.9 MHz) were performed using a modified Logiq 9 scanner (GE Healthcare, Waukesha, WI, USA) with a 4D10L probe. Five radiologists blinded to the reference independently scored the imaging modes using a 7-point BIRADS scale. Parametric volumes were constructed from time-intensity curves for vascular heterogeneity, perfusion (PER as the slope of the curve) and area under the curve (AUC) based on individual voxel values in the lesion. ROC analysis were applied to assess diagnostic accuracy with biopsy results as the reference. Out of the 236 cases, 219 were successfully scanned and biopsies resulted in 164 (75%) benign and 55 (25%) malignant lesions. 3D HI showed flow in 8 lesions (5 benign and 3 malignant), whereas 3D SHI visualized flow in 83 lesions (58 benign and 25 malignant). Hence, extracting quantitative parameters was restricted to the SHI volumes that demonstrated sufficient flow for processing. Diagnostic accuracy for the quantitative SHI parameters ranged from 0.52 to 0.75. Diagnostic accuracies from the clinical assessments ranged from 0.55-0.94 for baseline ultrasound, 0.52-0.93 for PDI, 0.59-0.85 for HI and 0.55-0.91 for SHI. The best logistical regression model achieved an accuracy of 0.91. In conclusion, combining quantitative SHI perfusion estimates and conventional B-mode imaging may increase the accuracy for characterizing indeterminate breast masses.

Background Two-dimensional ultrasounds (2D US) have low reproducibility of given images in later sessions and often depend on the operator’s experience. Purpose Due to difficulties in reproducing images in 2D US separated in time, a device to track the position of the ultrasound probe was created. This device aims to measure the probe position angle in two axes for 2D US to provide accurate repositioning of the 2D US probe for repeatability of the desired imaging plane. Materials and methods The device uses an inclinometer with an Arduino microprocessor to determine the angle of the ultrasound probe to subsequently establish the specific position coordinates of the probe. To test the effectiveness of this device, a 2D US probe was placed on a frontal burr hole of a neonatal swine to image the brain. Results When fixing the point of contact, using the probe with the angle sensing device helped the operator reproduce images that were both visually and positionally similar. This attachment allows the probe to return to the same image with at least 95% similarity in between different sessions, based on evaluations using an image analysis Matlab program. The sensor can return images of the same location with landmarks within the tolerance of 1.400°. Conclusion This device could potentially improve 2D US image reproducibility such that accuracy of images between sessions is increased, thereby increasing the reproducibility of 2D US in diagnostic and interventional procedures.

Here we report an ultra-long-acting tunable, biodegradable, and removable polymer-based delivery system that offers sustained drug delivery for up to one year for HIV treatment or prophylaxis. This robust formulation offers the ability to integrate multiple drugs in a single injection, which is particularly important to address the potential for drug resistance with monotherapy. Six antiretroviral drugs were selected based on their solubility in N-methyl-2-pyrrolidone and relevance as a combination therapy for HIV treatment or prevention. All drugs released with concentrations above their protein-adjusted inhibitory concentration and retained their physical and chemical properties within the formulation and upon release. The versatility of this formulation to integrate multiple drugs and provide sustained plasma concentrations from several weeks to up to one year, combined with its ability to be removed to terminate the treatment if necessary, makes it attractive as a drug delivery platform technology for a wide range of applications., Patient drug regime compliance is a major issue; sustained release implants could address this. Here, the authors report on a phase inverted in situ forming implant of PLGA for the sustained release of antiretroviral drugs and optimize and demonstrate the release of 6 different drugs over a period of up to a year.

Objectives Subharmonic imaging (SHI) is a technique that uses the nonlinear oscillations of microbubbles when exposed to ultrasound at high pressures transmitting at the fundamental frequency ie, fo and receiving at half the transmit frequency (ie, fo/2). Subharmonic aided pressure estimation (SHAPE) is based on the inverse relationship between the subharmonic amplitude of the microbubbles and the ambient pressure change. Methods Eight waveforms with different envelopes were optimized with respect to acoustic power at which the SHAPE study is most sensitive. The study was run with four input transmit cycles, first in vitro and then in vivo in three canines to select the waveform that achieved the best sensitivity for detecting changes in portal pressures using SHAPE. A Logiq 9 scanner with a 4C curvi-linear array was used to acquire 2.5 MHz radio-frequency data. Scanning was performed in dual imaging mode with B-mode imaging at 4 MHz and a SHI contrast mode transmitting at 2.5 MHz and receiving at 1.25 MHz. Sonazoid, which is a lipid stabilized gas filled bubble of perfluorobutane, was used as the contrast agent in this study. Results A linear decrease in subharmonic amplitude with increased pressure was observed for all waveforms (r from −0.77 to −0.93; P

OBJECTIVE. Breast cancer is the most frequent type of cancer among women (25% of all cancers). The angiogenic process that fuels the growth of tumors is a potential early indicator for differentiating between malignant and benign tumors. Recently, the use of microbubble-based contrast agents combined with ultrasound has allowed the development of contrast agent–specific imaging modes that provide visualization of tumor neovascularity. CONCLUSION. Contrast-enhanced Doppler, harmonic, and subharmonic imaging are some of the imaging modes that have been investigated for visualizing and quantifying the vascularity in breast tumors.

OBJECTIVES To investigate whether quantitative contrast-enhanced ultrasound (CEUS) can accurately identify neonates and infants with hypoxic ischemic brain injury. METHODS In this prospective cohort study, 8 neonates and infants with a suspicion of hypoxic ischemic injury were evaluated with CEUS. RESULTS An interesting trend was observed in the central gray nuclei-to-cortex perfusion ratios. The ratios at the peak enhancement, wash-in area under the curve, perfusion index, and maximum wash-in slopes were lower in all of the affected cases compared to the normal group but not statistically significant given the small sample size (P = .0571). Additionally, when the central gray nuclei-to-cortex perfusion ratio was plotted for all time points along the time-intensity curve, it was observed that the affected cases showed a trend that was qualitatively different from that of the normal cases. In the affected cases, the ratio time-intensity curves either stayed below 1.0 for the entire enhancement period or reached 1.0 close to peak wash-in before falling just below 1.0 for the remaining period of enhancement. However, in the unaffected patients, there was a steep wash-in that crossed the 1.0 threshold and remained above 1.0 for most of the enhancement period. CONCLUSIONS Bedside CEUS is an easily obtainable brain-imaging modality that has the potential to effectively identify infants and neonates with evolving brain injury. A larger prospective study evaluating the correlation between CEUS findings and the reference standard of diffusion- and perfusion-weighted magnetic resonance imaging is needed to establish it as a diagnostic tool.

The ability to visualize breast lesion vascularity and quantify the vascular heterogeneity using contrast-enhanced 3-D nonlinear ultrasound imaging was investigated in a clinical population. Patients (n = 236) identified with breast lesions on mammography were scanned using power Doppler imaging, contrast-enhanced 3D HI, and 3D SHI on a modified Logiq 9 scanner (GE Healthcare). Time-intensity curve volumes were developed corresponding to ultrasound contrast agent flow in the lesions after being identified in 4DView (GE Medical Systems). Time-points corresponding to baseline, peak intensity and complete washout of contrast were identified to generate vascular heterogeneity plots of the lesion volume (in the center and periphery as well as the ratio of the two). Vascularity was observed with power Doppler imaging in 93 lesions (69 benign and 24 malignant). The 3D HI showed flow in 8 lesions (5 benign and 3 malignant), whereas 3D SHI visualized flow in 83 lesions (58 benign and 25 malignant). Parametric volumes, that contained a single parametric value for every voxel within the 3D volume, were generated based on perfusion (PER) and area under the curve (AUC). ROC analysis and reverse, step-wise logistical regression were used to assess diagnostic accuracy with biopsy results as the reference. Analysis of vascular heterogeneity in the 3D SHI volumes found benign lesions having a significant difference in vascularity between central and peripheral sections (1.8 ± 0.16 vs. 1.2 ± 0.09 dB, p = 0.0003, respectively), whereas malignant lesions showed no difference (1.7 ± 0.33 vs. 1.3 ± 0.21 dB, p = 0.23), indicative of more vascular coverage. Diagnostic accuracy (i.e., area under the ROC curve) for heterogeneity, PER and AUC ranged from 0.52 to 0.75. The best logistical regression model (heterogeneity ratio, PER central and AUC central) achieved an area of 0.88. In conclusion, 3D SHI is able to detect UCA flow in vascular breast masses. Evaluation of vascular heterogeneity and parametric maps suggests such quantitative parameters might aid in the characterization of breast lesions.

Point shear wave elastography (pSWE) provides noninvasive measures of mechanical stiffness of biological tissue. Over the past decade, research has demonstrated that pSWE is effective at diagnosing a multitude of pathologies such as liver fibrosis and cancer. Life science researchers stand to benefit tremendously from access to pSWE but a number of challenges have limited its use in small animals. First, high frequency pSWE imaging suffers from transducer heating challenges and bandwidth requirements. Second, pSWE measurements are highly susceptible to variations in the environment and operator (e.g. precompression of tissue, transducer placement, etc.) which bias measurements and introduce error. Therefore, the objective of this work is to develop a robotically controlled, non-contact pSWE system capable of measuring liver stiffness in rodents.

Microbubble based contrast-enhanced ultrasound (CEUS) enables the visualization of vascularity given the tendency of microbubbles to function as a blood pooling agent. Using contrast specific ultrasound (US) imaging, it is possible to quantify the kinetics of these agents and derive various perfusion metrics. In this ongoing clinical study, we evaluate the feasibility of using these microbubble based contrast agents (CA) to develop qualitative and quantitative measures of perfusion in patients with chronic kidney disease (CKD). CEUS imaging may provide information about kidney perfusion that will aid in early diagnosis, disease progression and response to therapy. Patients (n = 63) with CKD and 3 healthy controls were imaged using CEUS. Imaging was performed on a Sequoia 512 (Siemens Healthcare) by trained sonographers using a 4C1 transducer (1–4 MHz). Definity (Lantheus Medical Imaging) was administered to the patient as a continuous IV infusion based on recommended dosage guidelines. Multiple sequences of microbubble flash-replenishment were used to generate signal intensity versus time (TIC) curves for specific regions of interest (ROIs). Maximum intensity projections (MIPs) and perfusion maps were also generated based on flash-replenishment cine sequences. Flow rate (β) based on the slope of the TIC was compared with CKD staging to study the correlation between the metrics. Preliminary evaluation of the perfusion maps showed a marked difference between the healthy controls and CKD patients. Significant correlations (r2=0.20, p=0.03) and (r2=0.16, p

OBJECTIVES The ability to estimate tissue perfusion (in milliliter per minute per gram) in vivo using contrast-enhanced 3-dimensional (3D) harmonic and subharmonic ultrasound imaging was investigated. MATERIALS AND METHODS A LOGIQ™ 9 scanner (GE Healthcare, Milwaukee, WI) equipped with a 4D10L probe was modified to perform 3D harmonic imaging (HI; f(transmit), 5 MHz and f(receive), 10 MHz) and subharmonic imaging (SHI; f(transmit), 5.8 MHz and f(receive), 2.9 MHz). In vivo imaging was performed in the lower pole of both kidneys in 5 open-abdomen canines after injection of the ultrasound contrast agent (UCA) Definity (Lantheus Medical Imaging, N Billerica, MA). The canines received a 5-μL/kg bolus injection of Definity for HI and a 20-μL/kg bolus for SHI in triplicate for each kidney. Ultrasound data acquisition was started just before the injection of UCA (to capture the wash-in) and continued until washout. A microvascular staining technique based on stable (nonradioactive) isotope-labeled microspheres (Biophysics Assay Laboratory, Inc, Worcester, MA) was used to quantify the degree of perfusion in each kidney (the reference standard). Ligating a surgically exposed branch of the renal arteries induced lower perfusion rates. This was followed by additional contrast-enhanced imaging and microsphere injections to measure post-ligation perfusion. Slice data were extracted from the 3D ultrasound volumes and used to generate time-intensity curves offline in the regions corresponding to the tissue samples used for microvascular staining. The midline plane was also selected from the 3D volume (as a quasi-2-dimensional [2D] image) and compared with the 3D imaging modes. Perfusion was estimated from the initial slope of the fractional blood volume uptake (for both HI and SHI) and compared with the reference standard using linear regression analysis. RESULTS Both 3D HI and SHI were able to provide visualization of flow and, thus, perfusion in the kidneys. However, SHI provided near-complete tissue suppression and improved visualization of the UCA flow. Microsphere perfusion data were available for 4 canines (1 was excluded because of an error with the reference blood sample) and showed a mean (SD) perfusion of 9.30 (6.60) and 5.15 (3.42) mL/min per gram before and after the ligation, respectively. The reference standard showed significant correlation with the overall 3D HI perfusion estimates (r = 0.38; P = 0.007), but it correlated more strongly with 3D SHI (r = 0.62; P < 0.001). In addition, these results showed an improvement over the quasi-2D HI and SHI perfusion estimates (r = -0.05 and r = 0.14) and 2D SHI perfusion estimates previously reported by our group (r = 0.57). CONCLUSIONS In this preliminary study, 3D contrast-enhanced nonlinear ultrasound was able to quantify perfusion in vivo. Three-dimensional SHI resulted in better overall agreement with the reference standard than 3D HI did and was superior to previously reported 2D SHI results. Three-dimensional SHI outperforms the other methods for estimating blood perfusion because of the improved visualization of the complete perfused vascular networks.

The ability to delineate atherosclerotic plaque from the surrounding tissue using custom-developed subharmonic imaging (SHI) digital filtering techniques was investigated in vivo using a commercially available system. Atherosclerosis was induced in the aorta of two Watanabe Heritable Hyperlipidemic rabbits following which injections of an ultrasound contrast agent (UCA) Definity (Lantheus Medical Imaging, N Billerica, Massachusetts) were administered. Imaging was performed using a Galaxy intravascular ultrasound (IVUS) scanner (Boston Scientific, Natick, Massachusetts) equipped with an Atlantis® SR Pro Imaging Catheter (Boston Scientific). Four preliminary band-pass filters were designed to isolate the subharmonic signal (from surrounding tissue) and applied to the radio-frequency (RF) data. Preliminary filter performances were compared in terms of vessel-tissue contrast-to-tissue ratio (CTR) and visual examination. Based on preliminary results, a subharmonic adaptive filter and a stopband (SB) filter were designed and applied to the RF data. Images were classified as fundamental, SHI, and SB. Four readers performed qualitative analysis of 168 randomly selected images (across all three imaging modes). The images were scored for overall image quality, image noise, plaque visualization, and vessel lumen visualization. A Wilcoxon signed-rank test was used to compare the scores followed by intraclass correlation (ICC) evaluation. Quantitative analysis was performed by calculating the CTRs for the vessel-to-plaque and vessel-to-tissue (compared using a paired student’s t test). Qualitative analysis showed SHI and SB to have significantly less image noise relative to the fundamental mode ( p < 0.001). Fundamental mode scored significantly higher than SHI and SB for the remaining three categories. ICC showed mixed results among reader evaluation for delineation of plaque. However, quantitatively, SHI produced the best vessel-plaque CTR.

The ability to visualize breast lesion vascularity and quantify the vascular heterogeneity using contrast-enhanced 3-D nonlinear ultrasound imaging was investigated in a clinical population. Patients (n = 236) identified with breast lesions on mammography were scanned using power Doppler imaging, contrast-enhanced 3D HI, and 3D SHI on a modified Logiq 9 scanner (GE Healthcare). Time-intensity curve volumes were developed corresponding to ultrasound contrast agent flow in the lesions after being identified in 4D View (GE Medical Systems). Time points corresponding to, wash-in, baseline, peak intensity, and washout of ultrasound contrast agent were identified and used to generate and compare vascular heterogeneity plots for malignant and benign lesions. Vascularity was observed with power Doppler imaging in 93 lesions (69 benign and 24 malignant). The 3D HI showed flow in 8 lesions (5 benign and 3 malignant), whereas 3D SHI visualized flow in 83 lesions (58 benign and 25 malignant). Analysis of vascular heterogeneity in the 3D SHI volumes found benign lesions having a significant difference in vascularity between central and peripheral sections (1.8 ± 0.16 vs. 1.2 ± 0.09 dB, p = 0.0003, respectively), whereas malignant lesions showed no difference (1.7 ± 0.33 vs. 1.3 ± 0.21 dB, p = 0.23), indicative of more vascular coverage. Parametric volumes, that contained a single parametric value for every voxel within the 3D volume in order to visualize localized variations, were generated based on perfusion (PER) and area under the curve (AUC). These maps highlighted the variations in the vascularity for individual voxels in the lesion volume. Finally, a preliminary measure for lesion characterization, based on vascular heterogeneity, achieved an area under the ROC of 0.72. These preliminary results suggest quantitative evaluation of vascular heterogeneity in breast lesions using contrast-enhanced 3D SHI is feasible and able to detect variations in vascularity between central and peripheral sections for benign and malignant lesions to aid in characterization.

Neoadjuvant chemotherapy is standard of care for localized breast. The patient response to neoadjuvant chemotherapy correlates with survival and early response assessment is therefore beneficial. The purpose of this study was to determine if 4D subharmonic aided pressure estimation (SHAPE) can predict the response of breast cancer to neoadjuvant chemotherapy based on changes in interstitial fluid pressure and breast tumor vascularity. Seventeen patients scheduled for neoadjuvant therapy of a breast cancer underwent 4 ultrasound exams: prior to therapy, at 10%, 60%, and 100% completion of chemotherapy. Ultrasound exams were performed using a modified Logiq 9 scanner with a 4D10L probe (GE Healthcare, Milwaukee, WI, USA). Modified software enabled the collection of radiofrequency data from 4D pulse inversion subharmonic imaging (transmitting at 5.8 MHz and receiving at 2.9 MHz) before and during a continuous infusion of Definity (Lantheus Medical Imaging, N Billerica, MA, USA) at acoustic settings optimized for SHAPE. The ratios of maximum subharmonic magnitude acquired during infusion relative to unenhanced signals were calculated offline in the tumor and surrounding areas for all 4 exams. The difference between ratios in the tumor and the surrounding area were then compared to the final tumor treatment response. Four patients left the study and 2 patients' data were discarded. Patients' clinical outcomes consisted of 6 responders (tumor volume reduction > 90%) and 5 partial/non responders. The results from 10% completion of therapy showed the subharmonic signal increased more in the tumor than in the surrounding area for responders compared to partial/non responders (3.23 ± 1.41 dB vs. −0.88 ± 1.46 dB; p = 0.001). Moreover, 3 patients whose tumor size initially increased during therapy were correctly predicted by SHAPE to be responders. In conclusion, SHAPE has the potential to predict the neoadjuvant chemotherapy response of breast cancer as early as at 10% completion of the therapy; albeit based on a small sample size.