Ultrasound emerges as versatile molecular option

Modality plays dual roles in diagnosis and treatment

By: Charles Bankhead

Although much of the interest in molecular imaging has centered on optical, MR, and nuclear imaging, ultrasound also has attracted a large and enthusiastic following that encompasses a diversity of clinical and scientific fields.

Ultrasound has several characteristics that distinguish it from other molecular imaging modalities and that some investigators tout as advantages over other modalities:

  • real-time imaging;

  • relatively short and efficient imaging protocols, compared with MRI or nuclear imaging;

  • noninvasiveness (in the absence of contrast) to minimize patient discomfort;

  • low operating costs;

  • large installed equipment base; and

  • complementary therapeutic and imaging applications.

    Ultrasound has many potential advantages that other imaging modalities can't offer,

    said Flemming Forsberg, Ph.D., head of the Jefferson Ultrasound Research and Education Institute at Thomas Jefferson University.

    "With some of the ultrasound biomicroscopy systems, we can get resolution down to 30 to 60 microns with commercially available machines. That's not to the cellular level, but it sure is a lot better than we get with MRI and PET," he said. "We can get that kind of resolution in real-time, so we can perform manipulations and see what happens."

    The diagnostic potential of ultrasound is being evaluated in a growing list of clinical conditions. Some of the major areas of investigation include angiogenesis (both endogenous and therapeutic), thrombus detection, identification of atherosclerotic plaque at risk for rupture, and detection and quantification of markers of inflammation.

    Much of the work has involved use of contrast-enhanced ultrasound. In many instances, the contrast is in the form of microbubbles, originally developed as blood-pool contrast agents.

    "The contrast agents are really gas-filled microbubbles about the same size as red blood cells," Forsberg said. "To survive in the circulation, the gas bubbles have to be encapsulated with surfactant or proteins or polymers or some other material. Once we have created the bubbles, we don't have to fill them with gas; we can fill them with something else, such as a drug, creating a very appealing potential for targeted drug delivery."

    The other approach to contrast-enhanced ultrasound imaging involves use of nanoparticles, which were developed specifically for molecular imaging. Nanoparticles are very small and essentially impossible to see with ultrasound when they circulate in the bloodstream, which is why liquid nanoparticles were not effective as blood-pool contrast agents, said

    Dr. Gregory Lanza, an assistant professor of medicine and of biomedical engineering at Washington University in St. Louis.

    "When the particles bind to a surface, it's like silvering a mirror. You can't see one nanoparticle, just like you can't see one grain of silver," he said. "However, as the particles accumulate, the surface becomes more reflective. The coating on a surface changes the acoustic impedance difference between blood and the surface, making the surface acoustically more reflective. Then we can detect it with ultrasound."

    The ultrasound contrast agents remain within the vasculature, so leakage is not an issue as it is with MR, Forsberg said.


    Considerable interest surrounds the potential of ultrasound to detect tumor-related angiogenesis. Investigators at the University of Virginia, for example, have reported use of contrast-enhanced ultrasound with microbubbles targeted to the av▀3 integrin expressed on the neovascular endothelium in a rat model of malignant glioma (Circulation 2003;108:336-341).

    "In this tumor model, we were able to diagnose the tumor in a very early stage," said Dr. Jonathan Lindner, a professor of medicine at UVA. "We used perfusion imaging with contrast ultrasound and found a nice correlation between the amount of angiogenesis

    by perfusion or blood volume and the amount of signal generated by the targeted microbubbles."

    The Virginia researchers are particularly excited by the fact that they can also detect angiogenesis in normal tissue, just outside the margin of very small tumors.

    "The normal tissue adjacent to a tumor that is just starting to grow will demonstrate an angiogenic phenotype to supply the feeding vessels for the tumor, and we can detect that," Lindner said.

    Forsberg and his colleagues recently reported the use of contrast-enhanced ultrasound to identify molecular markers of angiogenesis in a model of malignant melanoma (Ultrasound Med Biol 2002;28:445-451). The group has used ultrasound to evaluate angiogenic activity in several other tumor models (Figure 1).

    Extensive drug development activity in the oncology field involves strategies to inhibit tumor-related angiogenesis. Development of effective antiangiogenic therapies could lead to a potentially large role for contrast-enhanced ultrasound in monitoring response to therapy, Forsberg said.

    In cardiovascular medicine, the ability to detect angiogenesis at a very early stage has potential for identifying early response to molecular therapies intended to stimulate blood vessel expansion or growth in ischemic tissue (Figure 2). Strategies to stimulate endogenous angiogenesis will proliferate in the future, Lindner said, as the population of patients with coronary disease not amenable to bypass surgery or angioplasty continues to increase.

    "We have been able to create a rat model of peripheral vascular disease and then monitor endogenous angiogenesis," he said. "We could follow the development of natural collateral circulation and angiogenesis in the ischemic hindlimb. We also stimulated therapeutic angiogenesis by delivering (a growth factor) to the muscle. We saw an intense signal from the microbubbles, and it actually preceded the increase in blood flow. We were able to do phenotypic imaging and predict angiogenic response, either with endogenous or therapeutic angiogenesis."


    Another potentially large area of ultrasound application in cardiovascular medicine relates to identification of vulnerable plaque-atherosclerotic lesions that are prone to rupture. Vulnerable plaques are associated with inflammation and increased concentrations of proinflammatory markers. Contrast-enhanced ultrasound has demonstrated the ability to identify these markers and the vulnerable plaques associated with them, said Dr. Evan Unger, a professor of radiology at the University of Arizona and chair, president, and CEO of ImaRx Therapeutics.

    Unger has also been a leading investigator of contrast ultrasound with targeted microbubbles to identify clots such as left atrial thrombi, which can break off, embolize, and lead to stroke in patients with atrial fibrillation. The ability to identify thrombin, fibrin, and platelets could have widespread application in conditions such as unstable atherosclerotic plaque, pulmonary embolism, and deep vein thrombosis, Lanza said.


    Lindner and colleagues have applied contrast-enhanced ultrasound to evaluation of several inflammatory conditions, including inflammatory bowel disease. To identify and monitor inflammation, they use microbubbles targeted to activated leukocytes or to cell adhesion molecules such as P-selectin, VCAM, or ICAM.

    Inflammatory bowel disease is a particularly exciting area of investigation because the condition is difficult to diagnose and assess, he said. It can be treated with steroids and immunosuppressive agents, but many patients are young and cannot stay on chronic steroids and immunosuppressives for the rest of their lives. By the time they have an exacerbation or relapse, the disease has progressed to the point of causing serious damage. Bowel scarring and dilation become progressively worse over time.

    "Contrast-enhanced ultrasound imaging might offer a way to screen for recurrences at a very early stage, before symptoms occur. Ultrasound also might be a way to follow response to therapy in patients with IBD," Lindner said.


    Investigation into therapeutic applications of ultrasound is at least as active as research into the diagnostic potential of the imaging modality. Some researchers see the therapeutic potential of contrast-enhanced ultrasound as holding the brightest future for the technology.

    "I really think that therapeutic applications of ultrasound offer the most promise," Unger said. "That is the direction in which my own work is going. We will see more and more investigation into use of ultrasound for therapeutic purposes."

    The therapeutic potential of ultrasound comprises several strategies. The energy generated by ultrasonic waveforms has been used to create therapeutic cavitation injuries. Unger has developed a microbubble technology to enhance the cavitation effect. In one application using the ultrasound agent SonoLysis (Figure 3), specially coated bubbles adhere to the surface of a clot before being popped. The strategy could have applications in a range of clot-related conditions, including myocardial infarction, stroke, deep vein thrombosis, and thrombi in renal dialysis grafts. A similar strategy could be used to create enhanced cavitation injury to tumors.

    Lanza's group is using ultrasound and nanoparticle technology to deliver therapeutic agents to tumors.

    "Our current focus is on incorporating antiangiogenic therapies into the particles and then using ultrasound to push the nanoparticles against the surface we want the drug delivered to," he said.


    Other groups are using a similar approach with microbubbles. Investigations include the use of contrast-enhanced ultrasound to deliver gene therapy. One strategy is to burst microbubbles at the surface of interest and release a gene (or drug) into the microcirculation (J Cardiovasc Pharmacol Ther 2002;7:171-180).

    At the Society for Molecular Imaging meeting in August, Dr. Chrit Moonen, a professor of

    radiology at Universite Victor Segalen in Bordeaux, France, described the use of MR-guided focused ultrasound to generate therapeutic hyperthermia. Temperature is a key factor in control of gene expression, local drug delivery, enhancement of drug transport, ablation, and other therapeutic strategies, he said.

    Focused ultrasound offers a noninvasive means of achieving targeted hyperthermia. However, efficient and effective temperature conduction is dependent on factors that include tissue composition and perfusion. MR is useful for providing guidance because of its ability to map temperature and to identify and characterize anatomy.

    Moonen and his colleagues have used MR-guided focused ultrasound to achieve local control of a temperature-sensitive gene promoter in a modified glioma tumor cell line. Their work has demonstrated the potential of MR-guided focused ultrasound to control expression of therapeutic proteins in gene therapy.

    "This coupling of MR thermometry and focused ultrasound has the advantage of real-time targeting and temperature monitoring," said Moonen. "MR-guided focused ultrasound allows automatic and precise control over temperature regulation and provides for image-guided molecular therapies by guaranteeing the desired temperature trajectory in the region of interest."


    Moonen's work illustrates that ultrasound should not necessarily be viewed as competing with other molecular imaging modalities. Ultrasound can be used as a stand-alone imaging modality or as complementary to other modalities, according to Lanza.

    "For instance, you wouldn't use ultrasound to look at lung cancer or at rupture of plaque in coronary arteries," he said. "However, you can use ultrasound in the carotid arteries, the prostate, and the breast."

    In those circumstances, ultrasound might be useful by itself. In other cases, MRI might be used, for example, to see nanoparticles, which are both acoustically reflective and paramagnetic. In the operating room during resection, ultrasound might be used to find the margins of the cancer or to evaluate lymph nodes for dissection. Then the use of ultrasound would be complementary to another imaging modality, Lanza said.

    Ultrasound can be done rapidly and easily at the patient's bedside, Lindner said. And almost everyone has an ultrasound machine, even in their office.

    "There are real advantages in tracers for things such as markers of inflammation and in the fact that the contrast agent remains intravascular and doesn't leak out," he said. "However, the real advantage of ultrasound over other modalities is its potential therapeutic application and possibly the ability to diagnose and treat with the same modality."