Applied Radiology

Dr. MacKenzie is currently a fourth-year Radiology Resident at Brigham and Women's Hospital, Boston, MA. He earned his BS double major in Computer Science and Biological Science and his MS in Biological Science from Stanford University, Palo Alto, CA, and his MD from Albert Einstein College of Medicine, New York, NY. Dr. MacKenzie is an American Board of Radiology B. Leonard Holman Research Pathway Resident with a focus on Molecular Imaging applications for arthritis. Dr. MacKenzie will start a Fellowship in Musculoskeletal Radiology at the University of Pennsylvania, Philadelphia, PA, in July 2005.

Molecular imaging may be defined as the imaging of specific biological processes at the molecular and cellular level in living organ-isms. ^1,2 The goal is to reveal the early underlying biochemical and genetic events responsible for disease rather than indirect and late changes (eg, altered blood flow or tumor size) as seen with most current clinical diagnostic imaging modalities. Direct imaging of events fundamental to disease processes with molecular imaging should ultimately translate into better patient care through earlier and more specific detection and intervention.

Magnetic resonance imaging (MRI) is uniquely suited to play a large role in molecular imaging. When compared with other imaging modalities, the excellent anatomical resolution ³ and multiplanar capabilities make MRI particularly worthy to pinpoint molecular events (Table 1). ⁴ With molecular MRI, there is no radiation exposure or need for coregistration of molecular activity with anatomic structures as there is with positron emission tomography coupled with computed tomography (PET/CT). The expense and the relatively large and possibly toxic concentrations of contrast probe required to detect molecular events are some of the challenges facing molecular MRI. ⁵

In recent years, there has been intense interest in molecular imaging with MR techniques. This growing research discipline has emerged, in a large part, due to rapid advances in our understanding of specific molecular pathways from contributions in fields such as biochemistry, molecular biology, cellular biology, and genetics. Numerous examples illustrate the recent advances that have been critical in transitioning the concept of molecular MRI into a working reality. The human genome provides a map of the fundamental building blocks for the biomarkers that may be detected with molecular imaging; molecular cloning allows for the rapid production of novel DNA and proteins that may be suitable imaging targets or probes; X-ray crystallography creates a 3-dimensional (3D) structure of biomolecules that may serve as imaging probes or targets. Chip arrays, bioinformatics, gene therapy, and proteomics are other important advances in the mainstream or near horizon of basic science investigation. The list of tools available to aid in the development of molecular imaging techniques continues to grow.

Advances in our understanding of the molecular and genetic basis for disease have led to the need for noninvasive imaging techniques that can reveal molecular events in vivo. In general, there are 3 criteria that must be met for successful molecular MRI applications: 1) sufficient production of MR contrast to depict the molecular event; 2) favorable pharmacokinetics for the molecular probe; and 3) proven usefulness of the probe- that is, validated for basic science or clinical applications.

Production of contrast on MRI

Three different classes of contrast agents may be tailored for molecular applications to produce visible signal changes on MR images: paramagnetic contrast agents, superparamagnetic particles, and metabolite detection with MR spectroscopy. Each class has unique properties that must be considered for the contrast agent to be useful for molecular applications.

The majority of MR images are based upon the nuclear MR signal from water protons. The signal intensity produced in any given voxel (3D volume) is a function of the imaging sequence (eg, gradient echo, spin echo, fast spin echo, etc.) and the selected sequence parameters, such as the repetition time (TR), and echo time (TE), as well as of the intrinsic tissue properties. These are primarily the water proton spin density, and water relaxation times T1, T2, and T2*. Local variations in these intrinsic tissue parameters provide the image contrast offered by MR. The paramagnetic and superparamagnetic contrast agents primarily affect the local microenvironment to produce image contrast by altering the tissue relaxations times, in particular T2*, which dramatically decreases the signal intensity in typical gradient-echo acquisitions. ^4-7

A third, and substantially different, means of imaging molecular events is with MR spectroscopy. In MR spectroscopy, instead of using image contrast, a metabolite that is produced by or heralds the molecular event is detected by the metabolite's spectroscopic peak at a precise anatomic location. Although MR spectroscopy may not be considered molecular imaging when the molecular event is rigidly defined as a ligand-receptor interaction, ⁸ systems have been designed with MR spectroscopy to detect precisely controlled genetic events such as genetically engineered conversion of a prodrug into its active chemotherapeutic agent. ⁹

Molecular probe design and development

Most current molecular probes for MRI combine either a paramagnetic or superparamagnetic contrast agent with a high-affinity ligand that is specific for a particular molecular target or receptor. The ligand on the molecular probe is specific for a molecular target, an imaging biomarker, used to help establish the presence or severity of disease. ¹⁰ Targets may be any molecular process and range from 2 copies of DNA per cell to hundreds of thousands of intra- or extracellular proteins or metabolites (Figure 1). ¹ High-affinity imaging probes are fundamentally different from nonspecific contrast agents, such as the widely used intravenous gadolinium diethylenetriamine penta-acetic acid (Gd-DTPA). Molecular contrast agents are generally distributed throughout the body based on a dynamic interplay between the physiochemical properties of the probe and the physiology of the body. While physiologic parameters primarily regulate the distribution of Gd-DTPA (eg, blood flow, ability to diffuse into the extracellular space), the ligand on the molecular probe helps the contrast agent to accumulate at the site of interest. ⁷

An understanding of the pharmaco-kinetics of the molecular probe is essential for successful implementation. The ability of a probe to detect the target molecule is governed by classic pharmacology: the route of administration/absorption, distribution/delivery to the target, and elimination through metabolism or excretion. An ideal molecular probe is one with favorable pharmacokinetics such that the probe can be administered easily, distributes efficiently to the biomarker, and is cleared from the patient with minimal side effects. ^5-7

Clearly, the ligand-receptor interaction is a dynamic interaction and will affect the MRI parameters. The timing of imaging after the probe administration is paramount. For example, many contrast agents require a 24-hour delay after administration before sufficient quantities of the probe have accumulated at the target, necessitating careful registration of pre- and postcontrast images. Resolution and speed of image acquisition required to detect signal changes from the molecular probe are also equally important considerations. Many examples illustrate the various factors that must be considered when developing the imaging parameters and can be found in applications of oncologic and arthritis imaging, thrombosis detection, and genetic and cell-based therapies.

Clinical applications

Tumor imaging

Many aspects of tumor biology are governed by molecular events, and it is likely that molecular MRI will enhance tumor detection, provide accurate pretreatment staging, monitor response to therapy, and survey for reoccurrence after remission. The molecular MRI application that has been best described and has the potential for widespread clinical practice is the use of lymphotropic superparamagnetic nanoparticles ¹¹ in the nodal staging of prostate cancer (Figure 2). Harisinghani and others ¹² found that the majority (71%) of malignant nodes detected with the superparamagnetic nanoparticles were smaller than the threshold size (10 mm) used to identify nodal disease on conventional CT and MRI. MRI with super-paramagnetic nanoparticles had a high overall sensitivity, specificity, and accuracy on both a per-patient and a node-by-node basis, and the negative predictive value of the test was very high (100%). The implications of detecting metastasis are considerable, because patients with positive lymph nodes receive androgen-deprivation therapy with radiation and are spared a radical prostatectomy. ^13-17

A goal of molecular MRI contrast agents is to improve the detection of malignant cells both at the primary site of development and at locations of metastasis. For example, the formation of de novo blood vessels is a common characteristic of many tumors. MRI probes specific to molecules responsible for angiogenesis have been used to assess tumor growth and malignant potential. ¹⁸

Another means of improving the early detection of malignancy could be with gadolinium (Gd)-encapsulated liposomes that preferentially target tumor cells. Similar to 2-18F-fluoro-2-deoxyglucose positron emission tomography (FDG-PET) that measures increased glucose metabolism to mark areas of tumor, an MRI contrast agent was developed that presents ligands bearing glucose conjugates at the liposome surface. ¹⁹ Active targeting of tumor cells with liposomes is attractive not only because high concentrations of MRI contrast material can be delivered in the liposome, but also because liposomes can encapsulate drugs. ^20-22 Thus, a liposome approach could simultaneously show tumor burden and deliver chemo-therapeutic agents. Although potential pitfalls include immunogenicity and a relatively large size that may prevent liposome access into the extracellular compartment, ²³ methods have been devised to decrease immunogenicity ^24,25 or increase delivery of bulky molecular probes into the extracellular compartment or across the blood-brain barrier. ^26-29

A novel MRI contrast agent has also been developed to monitor tumor progression and response to treatment. Zhao and coworkers ³⁰ developed a superparamagnetic probe specific to cells expressing synaptotagmin I, a molecule that binds to cell membranes of apoptotic cells. The degree of programmed cell death after chemotherapy and radiotherapy has been shown to correlate with tumor growth delay and cure ^31,32 and the superparamagnetic probe conjugated to synaptotagmin I showed good correlation with apoptosis both in vitro and in vivo.

Arthritis imaging

The spectrum of diseases that comprises inflammatory arthritis is largely mediated by immune mechanisms, some of which are well characterized on the molecular level and are ripe for molecular MRI probe development. Activated macrophages in areas in which inflammation can be labeled with superparamagnetic agents, presumably through macrophage phagocytosis, can be detected with MRI. ³³ For example, the use of superparamagnetic particles in a rat model of antigen-induced arthritis showed potential in detecting the degree of macrophage infiltration. ³⁴ The superparamagnetic particles indicated areas of early inflammation and correlated with treatment response, suggesting that molecular MRI might provide advanced detection for early intervention before the development of irreversible bone erosions.

Detection of thrombosis

Molecular imaging approaches for the detection of arterial or venous thrombosis would benefit patients by providing a specific, noninvasive test. Current MRI methods of clot detection have limitations. Blood on traditional MRI sequences shows variable signal characteristics depending on the age of the clot, which makes interpretation of MR images for thrombosis challenging. ^35-38 Furthermore, many clinically significant thrombotic events occur in small, distal coronary or pulmonary arteries that are prone to motion artifact and are below the resolution of current fast MR sequences. ³⁹ In these scenarios, a molecular probe would provide a specific marker to improve the detection of small thrombi.

One approach by Botnar and colleagues ⁴⁰ shows the potential for a Gd-based probe to detect acute and subacute thrombosis. Four atoms of Gd-DTPA were attached to a peptide specific for fibrin, and this molecular probe showed high contrast among thrombus, thrombus-free vessel wall, and blood (Figure 3). This probe combines high molecular specificity for thrombus formation by binding the product of an activated coagulation system, with an MRI contrast agent with excellent signal due to the increased T1 relaxivity conferred by multiple atoms of Gd per probe mole-cule. ⁵ Long-circulating ultrasmall superparamagnetic contrast agents have also been used to image macrophage and monocyte activity in atherosclerotic plaques. ^41,42

Gene therapy

As clinical applications for gene therapy are developed, molecular MRI is expected to play a role in multiple areas. MRI may monitor the progression and quantify the amount of gene delivered to the site of interest as well as report on the efficiency and duration of transgene expression. One example is EGadMe, a "smart" molecular imaging probe that irreversibly transitions from a weak to a strong relaxivity state in the presence of a common reporter gene product, β-galactosidase (β-gal). ⁴³ Cells that express the therapeutic gene generally also express the reporter gene β-gal and produce MRI contrast enhancement as a result of β-gal cleavage of EGadMe to the strong relaxivity state (Figure 4).

EGadMe is important because before inducing the therapeutic transgene, the efficacy of most viral vectors is first tested with a reporter gene such as β-gal. In addition, noninvasive imaging with a probe specific for transgene activity may evaluate which tissues are preferentially transduced and quantify gene expression over time without having to sacrifice test organisms.

Molecular MRI may also quantify and localize gene activity by detecting the metabolites that are produced by a transgene. For instance, the transgene for tyrosinase has been incorporated into cells and its activity measured by its production of melanin. Tyrosinase catalyzes the production of melanins, which have a remarkably high metal-binding capacity (up to 35% by weight). During tyrosinase expression, investigators have shown a resulting bright signal of iron containing melanin on T1-weighted images. ^44,45 MR spectroscopy has also been used to measure cytosine deaminase transgene expression in vivo by quantifying the transgene's enzymatic production of 5-fluorouracil (Figure 5). ⁹

Cell-based therapies

Similar to gene therapy, cell-based therapies are becoming increasingly more common options, and molecular MRI may speed development and augment treatment monitoring. Bone marrow transplant is in wide clinical practice and may benefit from in vivo tracking of transplanted hematopoietic cells ⁴⁶ as therapies are improved and new treatment regimens are tested. Currently, the amount of MR contrast agent delivered to cells can be increased with transfection techniques, ⁴⁷ and single cells may be imaged. ⁴⁸ As more cell-based therapies are envisioned and tested (eg, transplanting cardiac myocytes to rescue cardiac function ⁴⁹ ), the increasing momentum for in vivo and noninvasive monitoring will further develop the field of molecular MRI.

Validation

The success or failure of molecular MRI lies in the validation of the particular MR-based probe as a clinically useful tool. A molecular probe generally detects a surrogate end point, a marker of the natural history and factors associated with disease progression rather than clinical outcome measurements such as morbidity and mortality (Table 2). ^10,50 There should be a strong link of the biomarker with the true end point sought-usually decreased morbidity and/or mortality. ⁵¹

Generally, validation through extensive patient experience is required for a surrogate to be accepted for clinical use to minimize the uncertainty of unexpected and inaccurate information that may result from limited experience with a relatively untested biomarker. ¹⁰ The biomarker should reflect the effect of therapy, and the detection of the biomarker must be accurate, reproducible, and feasible over time. ¹⁰ Furthermore, the side effect profile or toxicity should be balanced with the benefit gained from the information provided by the molecular imaging study. It follows that the molecular MRI probes most likely to be successful will be those that target surrogate end points that have been well studied and characterized in large groups of patients.

Conclusion

The enormous potential for molecular MRI is the reality that the fundamental basis for most medical disease is the alteration of molecular parameters that may be readily visualized with sophisticated imaging techniques. Unlike most current clinical diagnostic imaging modalities, the aim of molecular MRI is to reveal the biochemical and genetic basis of disease in addition to demonstrating altered anatomy and physiology. The success of molecular MRI will rely on the synergy of sophisticated MRI techniques and the development of novel contrast agents that not only take advantage of our expanding knowledge of molecular process/disease, but also show well-validated clinical or basic science applications.

Acknowledgment

The author wishes to thank Drs. Frank Rybicki and Philipp Lang for their thorough review and insightful comments regarding the contents of the manuscript and Drs. Philipp Lang, Frank Rybicki, David Lee, and Prof. Ashfaq Mahmood for their mentorship and support (Brigham and Women's Hospital, Harvard Medical School). Many thanks are due to Prof. René M. Botnar (Beth Israel Deaconess Medical Center, Harvard Medical School), who kindly contributed a sample of her work for reproduction here.