MI community builds framework for broad-based clinical translation

Molecular imaging is maturing as a scientific force in preclinical research and as a mechanism to guide imaging discoveries toward clinical practice.

The first wave of MI appeared with SPECT and PET imaging targeted to specific cellular surface receptors, the expression of glycolysis in cells, and the metabolic and pathophysiological features of Alzheimer's disease. MR spectroscopy is used to report on the relative concentrations of metabolites to guide biopsies. Diffusion and perfusion imaging guide the initial therapeutic response to acute stroke and help assess the prognosis for patients with primary brain tumors.

In addition to emerging clinical roles, MI has prompted new methods for testing drug safety and efficacy. Preclinical small-animal imaging with microPET/CT, high-field MRI, and near-infrared fluorescence optical imaging is helping pharmaceutical companies decide if they should invest in preclinical trials for investigational agents. MI could save them hundreds of millions of dollars from FDA drug trial costs and potentially could translate into faster regulatory approvals, lower prescription drug costs, and better clinical outcomes.

Molecular imaging is no longer the exclusive province of futurists and bench scientists, according to Dr. Martin Pomper, an associate professor of radiology at Johns Hopkins Medical Center.

"The future is now," he said in the opening lecture of a two-day symposium on imaging in molecular medicine that preceded the 2006 Society for Molecular Imaging annual meeting in September.

The symposium, sponsored by the SMI, the RSNA, and the Society of Nuclear Medicine, defined state-of-the-art molecular imaging. The SMI meeting also featured research that is expanding those horizons.

Few words were wasted on the use of fluorine-18 FDG-PET for staging and recurrence because these applications have already entered the clinical mainstream. The National Oncologic PET Registry, established in May, has broadened the reach of this molecular technique to all cancers. As of mid-October, 11,112 exams had been compiled in the NOPR database from 1294 facilities in the U.S.

FDG AS BIOMARKER

Much was said, however, about the potential use of FDG-PET as a biomarker for therapeutic response to cancer therapy. Such use would constitute an important step because it would allow drug developers to use FDG uptake measurements as surrogate markers for response of disease to investigational drugs in FDA clinical trials. Researchers agreed on standard guidelines for these issues during a workshop sponsored by the National Cancer Institute in January. The guidelines were published in June (JNM 2006; 47(6):1059-1066).

The prognostic capabilities of FDG standardized uptake values have been established for lung cancer, esophageal cancer, and thyroid cancer. It can also assist in differentiating between indolent and aggressive lymphomas, said Dr. Steven Larson, director of nuclear medicine at Memorial Sloan-Kettering Cancer Center.

Larson described how Ga-68 F(ab)2-Herceptin can measure HER2/neu inhibition from Herceptin, an FDA-approved drug for breast cancer and a promising treatment for prostate cancer (Figure 1).

"This is molecular imaging," he said. "This is looking at molecular events and key biomolecules that are responsible for cancer change."

CANCER STAGING

Ultrasmall superparamagnetic iron oxide nanoparticles use a different molecular mechanism to detect lymph node metastasis. The USPIO ferumoxtran-10 (Combidex; Advanced Magnetics), for example, is taken up by macrophages in functioning lymph nodes, making them appear dark on T2-weighted MRI. Malignant nodes do not contain macrophages to capture the agent. They appear as light structures on T2-weighted MRI.

"Independent of size, you are able to elegantly show nodes that are infiltrated with cancer," said Dr. Mukesh G. Harisinghani, director of abdominal imaging at Massachusetts General Hospital in Boston.

Nanoparticles and nanotechnologies were a major theme during SMI scientific sessions, said Dr. Ralph Weissleder, director of the Center for Molecular Imaging Research at MGH.

"Nanoparticles can be engineered to dial in or dial out to optimize pharmacokinetics," Weissleder said. "They can be constructed for multimodality use or for either diagnostic or therapeutic applications. They often have new optical properties or ultrasound properties. This multivalency concept allows high affinity targeting."

THERAPY RESPONSE

University of Michigan researchers have demonstrated that functional MRI diffusion can be a robust biomarker for cancer treatment response. The intensity of diffusion can be calculated and plotted voxel by voxel on an apparent diffusion coefficient (ADC) map. The color-coded renderings are well-suited for quick visual assessments of changes in tissue density.

ADC mapping has been used to predict the prognosis of patients with grade III/IV gliomas and to track the response of germ cell cancers to therapy, according to radiology department co-chair Dr. Brian Ross.

A case study presented by Ross showed a 15% increase in ADC compared with a baseline study seven days after initiating therapy. ADC measures rose 54% after three weeks and reached 63% after six weeks, indicating positive treatment response (Figure 2).

"So in a responsive tumor, you can easily, and in one week, detect a tremendous change in diffusion. This bodes well for translating this outside of the brain to other tumor sites," he said.

MR spectroscopy is already applied clinically in oncology and neurology. MRS protocols developed at the University of California, San Francisco aid prostate cancer staging. Radiologists elsewhere have adopted MR spectroscopy to differentiate between tumor recurrence and radiation necrosis, said Dr. John W. Chen, an assistant radiologist in the neuroradiology department at MGH. It helps differentiate between acute stroke and patients with mitochondrial myopathy, encephalopathy, lactic acidosis, and strokelike episodes. It is also used to monitor treatment for the condition.

CV BIOMARKERS

Descriptions of preclinical MI applications for atherosclerosis by Dr. Farouc A. Jaffer, director of the cardiovascular imaging program at the CMIR, reveal how specific molecular targets create opportunities for earlier diagnosis and effective treatment. Jaffer and CMIR scientists are developing investigational MI techniques that identify when the risk of vulnerable plaque failure becomes ominous enough to require angioplasty or stenting.

Inflammation is the cardinal biological signature of high-risk plaque, Jaffer said. Molecules, cells, and mechanisms relevant to its noninvasive measurement include macrophages, endothelial cell activation, proteases, apoptosis, and angiogenesis. Each is a pathway for diagnosis and treatment.

USPIOs were a natural choice for isolating macrophages. CMIR researchers bonded dextran-coated iron oxide particles to near-infrared fluorochromes to amplify the signal of the particles. This allowed direct observation of macrophage cell activity in the aortic roots of atherosclerotic mice with laser-scanning fluorescence microscopy. Inflammatory carotid plaque identified with hot-spot FDG-PET imaging correlated with patients who had previously suffered a transient ischemic attack.

As one of the earliest markers of inflammation, the adhesion molecule VCAM-1 was another logical candidate for vulnerable plaque imaging, Jaffer said. Its ability to internalize ligands suggested that many VCAM-1 molecules could be imprinted on magnetofluorescent nanoparticles. The resulting MRI probe has shown a strong affinity for murine endothelial cells. This characteristic has helped researchers examine VCAM-1 expression in atheroma in vivo, including the anti-inflammatory effects of statin therapy in mice placed on high-cholesterol diets.

The CMRI group has also established the feasibility of near-infrared fluorescent (NIRF) probes that can be activated to detect the enzymatic action of matrix metalloproteinases (MMPs) in inflamed atherosclerotic plaques. This is important because MMPs appear to predispose the fibrous caps of atheroma to rupture, Jaffer said.

Angiogenesis is an early step in atheroma development. Dr. Greg Lanza, an associate professor of medicine at Washington University in St. Louis, has developed a versatile perfluorocarbon nanoparticle that can be loaded with gadolinium for MRI enhancement and with various ligands for surface receptor targeting. For this application, Lanza studded the nanoparticle with gadolinium, avb3-integrin, and fumagillin, an anti-angiogenic therapy. In a separate experiment involving rabbits, Dr. Patrick Winter, a senior scientist at Washington University, demonstrated that the probe can image early angiogenic activity and inhibit it as well.

MI-GUIDED INTERVENTIONS

The next phase of MI clinical adoption may aid interventional procedures, said Dr. Umar Mahmood, an associate professor of radiology at Harvard Medical School. Benefits from intraoperative NIRF optical scanners and targeted probes include:

- guiding biopsies by improving the target-to-background ratios that distinguish enhanced regions of interest from the surrounding tissues;

- aiding cancer staging by noninvasively directing the radiologist to the most aggressive area of a tumor for histological sampling;

- identifying sentinel nodes to diagnose metastatic spread;

- uncovering small, hidden structures, such as ureters, during surgery;

- avoiding nerves and vessels during surgical excision; and

- defining tumor margins to assure complete excision of cancerous tissue.

Large field-of-view intraoperative systems, handheld devices, endoscopes, microscopes, and confocal endoscopes have been developed for interventional applications. Some devices have secured FDA clearance, and at least two nontargeted NIRF dyes are available for use.

The SMI trade exhibit featured a marked increase in new fluorescence tomography equipment vendors, said Dr. Sam Gambhir, director of molecular imaging at Stanford University. Improved time domain and frequency domain performances are raising signal-to-background ratios by reducing autofluorescence. True cross-section bioluminescence imaging is now possible.

Plastic surgeons are also implementing clinical applications, according to Dr. John V. Frangioni, an assistant professor of radiology at Beth Israel Deaconess Medical Center in New York City. Using itacyanine green or methylene blue dyes under optical guidance, plastic surgeons are identifying perforated arteries and veins that could compromise the health of skin selected for transplantation.

Mahmood is optimistic about applying NIRF imaging to endoscopy and bronchoscopy. Preclinical applications performed on mice at CMIR are actually more technically challenging than those that will likely be used in humans, he said. Imaging catheters for these small-animal studies are just 0.8 mm in diameter. Full-spectrum white light illuminates the region of interest, while near-infrared signal produces fluorescence from the targeted lesions. Minimal disease is uncovered because of the 9 to 1 target-to-background ratio.

With the knowledge that half of the 1.5 million new cancer cases diagnosed in the U.S. this year will be cured with excision surgery, Frangioni has focused on using NIRF imaging as a tool for real-time surgical guidance. He and his colleagues at Beth Israel Deaconess have reengineered their intraoperative imaging system for multiwavelength operation at 15 frames per second. They constructed a high-powered, multiwavelength wide field light source and implemented techniques for developing targeted and nontargeted NIRF probes.

Their fluorescence-assisted resection and exploration imaging system uses invisible near-infrared light to enable surgeons to noninvasively visualize critical structures intraoperatively and in real-time. The technology has been applied to detecting the margins of a tumor in a swine's distal foot and sentinel lymph node mapping of the gastrointestinal tract (Figure 3). Potentially, it can guide the excision of tumors containing as few as 50,000 malignant cells, he said.

CELL TRACKING

MI will play an indispensable role in cell tracking, although the techniques are still being refined in large-animal models and clinical trials, said Jeff W.M. Bulte, Ph.D., a professor of radiology at Johns Hopkins School of Medicine. Cells can be labeled with iron oxide nanoparticles, such as Feridex, providing a tracking mechanism to confirm cell delivery and monitor their short-term migration into surrounding tissues. The value of MR-enhancing iron oxide diminishes over time, however, as its concentration is diluted because of cell division.

Because the PET reporter genes alter the genetic makeup of the stem cells they penetrate, the progeny of those cells inherit that modified genetic signature over many generations. The actual imaging process involves injecting the patient with a reporter probe bonded to a radioisotope. It quickly washes out of irrelevant cells but is locked into cells that express the reporter gene. As a result, reporter gene strategies are well suited for monitoring stem cell survival, division, and differentiation.

Gambhir's group and oncologists at City of Hope Medical Center and UCLA have successfully collaborated on the first application of reporter gene imaging in humans. The procedure met FDA good manufacturing practice standards for production of the reporter gene and probes and used direct infusion instead of viral transporter to overcome regulatory obstacles to the protocol. The technique was initially applied by Dr. Michael C. Jensen, director of pediatric oncology at City of Hope Medical Center, on T cells that were genetically reengineered to target recurrent glioma.

"Reporter genes not only show the delivery of cells to their targets, they also show what happens to the T cells as they home in on recurrent gliomas in living patients," Gambhir said.

At Johns Hopkins, Bulte is working with Dr. Aravind Arepally, Brad Barnett, and Dr. Dara Kraitchman, to address the problem of allogeneic rejection of transplanted human pancreatic islet cells by housing them in synthetic microcapsules before infusion into the liver in swine. The capsules were fabricated from poly-L-lysine-alginate labeled with Feridex to permit MRI tracking. Individual capsules can be identified with 3T MRI, and a characteristic imaging pattern can be seen when the capsules rupture. In a preclinical trial, the three- and six-week islet viability and insulin secretion rates of 94% and 82%, respectively, did not differ markedly from unlabeled microcapsules.

ALZHEIMER'S DISEASE

Molecular imaging researchers are examining FDG-PET as well as more targeted probes that potentially can diagnose the precursors of Alzheimer's disease early enough for effective therapy and recovery, said Dr. Dima A. Hammoud, an assistant professor of neuroradiology at Johns Hopkins.

FDG should not be counted out as a possible mechanism for early disease diagnosis. According to one published study, cognitively normal adults can exhibit the same patterns of reduced FDG uptake in the brain as Alzheimer's patients.

"This is very important because one can use this population of patients to follow up on disease progression and possibly evaluate treatment," Hammoud said.

Rather than assessing neuronal death and atrophy, C-11 Pittsburgh Compound B (PIB) measures the presence of b-amyloid plaques, and F-18 FDNNP and C-11 SB-13 identify amyloid and neurofibrillary tangles, the two leading suspects for the cause of Alzheimer's.

C-11 PIB's value in the search for effective Alzheimer's disease treatments was demonstrated this year in a collaborative study between Washington University and the University of Pittsburgh. It found an inverse relationship between positive PIB binding, indicative of brain amyloid plaque load, and amyloid-b 42 protein levels in cerebrospinal fluid, Hammoud said. However, three of the seven subjects with increased PIB uptake and low amyloid-b levels were cognitively normal. More research is needed to learn if elevated PIB precedes the onset of AD symptoms, enabling physicians to use it to predict the development of Alzheimer's disease.

Dr. Jorge Barrio, a professor of molecular and medical pharmacology at the University of California, Los Angeles, has established a correlation between FDDP uptake rates in the cerebellum and neocortex and the worsening of memory and other cognitive functions among Alzheimer's patients. Preliminary studies of C-11 SB-13 show that it exhibits the same binding characteristics as C-11 PIB.

Evidence suggesting that soluble amyloid-b protease may be the most neurotoxic manifestation of AD has led the skeptics of amyloid plaques' neurofibrillary tangles to question their value as surrogate markers, Hammoud said. The debate has led researchers to search for probes to target suspected proteases.

Microglial activation, associated with neuronal inflammation, may also cause AD. Preliminary research presented at the SMI meeting suggests that C-11 PK-00095, a ligand that selectively binds to peripheral vascular endothelial growth factor receptors that are known to be overexpressed in activated microglia, may play a diagnostic role.

From drug discovery to stem cell therapy to NIRF imaging, molecular imaging is setting the course for diagnostic imaging's future. Mature applications are already influencing diagnosis, treatment, and clinical outcomes. A powerful engine for scientific discovery of new MI methods is in motion, helping promising concepts make the transition from preclinical testing to clinical implementation.