Molecular Imaging
Molecular imaging transports diagnosis to
the next level
Novel imaging protocols are paired with
contrast enhancing probes to report gene expression
By James Brice
In October 1927, delegates to the 5th Solvay Conference in Brussels, Belgium,
assembled for a extraordinary group photograph [Fig. 1]. Twenty-nine scientists were portrayed; 17 of them,
including Arthur Compton, Marie Curie, Albert Einstein, Hendrik Lorenz, and Max
Planck, would win Nobel prizes. More important, these individuals defined how
space, time, and the structure of atoms would be understood in the 20th
century.
In September 1999, another group of scientists gathered for a photograph,
with the Grand Teton mountain range providing a backdrop [Fig. 2]. Doctors all, among them were Ronald Blasberg,
Thomas Meade, Michael Phelps, and Ralph Weissleder. That group is defining how
radiology will be practiced in the 21st century.
Their work is based on the understanding that disease is a genetic process
and that a new model for radiology, called molecular imaging, is required for
molecular therapies. The methods employed are so sensitive that diseases
measured on the molecular level can potentially be detected and corrected before
the patient is aware of their symptoms. While conventional medicine of the 20th
century treated the effects of disease, molecular medicine in the 21st century,
with its complementary molecular imaging techniques, will treat its causes.
According to Weissleder, director of the Center for Molecular Imaging
Research (CMIR) at Massachusetts General Hospital, molecular imaging will allow
earlier detection and characterization of disease and an earlier assessment of
treatment efficacy. Instead of waiting weeks to observe the results of
chemotherapy, the oncologist will know its effects within a few days and can
initiate a substitute therapy, if necessary, while it can do the most good.
Molecular imaging answers key clinical questions associated with gene
therapies. Through this in vivo medium, clinicians can determine if
gene-altering therapies have reached their cellular targets. It reveals the
anatomic region where the introduced genes are expressed as well as the onset,
magnitude, and duration of expression.
Many new molecular therapies are cytostatic, rather than cytotoxic. This
means the therapies inhibit cell growth, but they don't kill cells outright, so
radiology's mainstay measures of tumor location, diameter, and volume may no
longer provide an accurate reading of a patient's condition. The traditional
standards for drug dosing are rendered obsolete because molecularly targeted
therapies may be largely free of side effects. Instead of monitoring physical
symptoms, clinicians will use molecular imaging to determine whether a larger or
smaller dose of a drug should be administered.
Molecular imaging would be no more than a fanciful concept without the Human
Genome Project. The $4 billion enterprise, completed last year, catalogued the
sequence of 30,000 genes that make up the human genome. In addition to supplying
researchers with the basic ingredients for molecular medicine and imaging, the
project created methods and instruments that have accelerated the pace of
discovery. Twenty enabling technologies are contributing to molecular imaging
research, according to Weissleder. Microdevice engineering, high-throughput drug
testing, and improved data processing have all made a mark.
The National Cancer Institute has been the main force behind molecular
imaging research. NCI director Dr. Richard D. Klausner has funneled research
money into molecular imaging since 1997, approving $44 million for molecular
imaging research in fiscal year 2000. About $54 million will be allocated for
molecular imaging in fiscal year 2001, which began in October 2000.
Massachusetts General Hospital, Memorial Sloan-Kettering Cancer Institute,
and the University of California, Los Angeles, designated as the NCI's in vivo
cellular and molecular imaging centers, are among the focal points of
NCI-sponsored research. Each ICMIC receives about $2 million in grant funding
annually, along with invaluable connections to National Institutes of Health
laboratories and scientists around the country. Sixteen academic departments
have been awarded "pre-ICMIC" grants and the NCI plans to award two ICMIC grants
in each of the next three years.
"Klausner's vision and his genius are behind all this," Weissleder said. "He
provided the money and the stimulus for folks like myself to interact with
molecular biology colleagues to translate the extraordinary things they have
done with ex vivo reagents into strategies that will work in an in vivo imaging
environment."
Although molecular imaging may seem exotic, the principles guiding it are
familiar to anyone who has practiced nuclear medicine, said Dr. Sam Gambhir,
director of the Crump Institute for Molecular Imaging at UCLA. Many molecular
imaging protocols use a radioisotope as a tracer, but even techniques that
employ optical or MR imaging modalities rely on some pharmacological means to
track the pharmacokinetic properties of the molecular therapies with which they
are paired.
Molecular imaging differs from conventional techniques because it identifies
specific gene products and intracellular processes. Its designers must overcome
biocompatibility, vascular, interstitial, and cell membrane barriers to deliver
the imaging probes to their molecular targets. Minimizing the size and
concentration of probe molecules is essential. Typical target concentrations are
on pico- or millimolar levels. Because so few molecules are involved, novel
strategies must be created to amplify the probe's signal to the point that it
can be detected.
PET Marker Genes
Imaging reporter genes fall into two classes. One class encodes intracellular
enzymes, requiring the imaging probe to cross cell membranes to access the
enzyme target. This approach is relatively uncomplicated and doesn't risk
stimulation of the immune system. The second class of reporter genes works by
encoding cell surface proteins or receptors. The advantage of surface expressed
receptors and acceptors is favorable kinetics, Weissleder said. In some cases,
the tracer need not penetrate the cell. Synthetic receptors can also be
engineered to recognize already approved imaging drugs such as
pertechnetate.
Reporter gene imaging exemplifies the intracellular approach, according to
Dr. Michael Phelps, chair of molecular and medical pharmacology at UCLA.
Reporter genes describe the genetic or enzymatic events initiated by the
molecular therapies to which they are assigned. They are capable of covering
many types of molecular therapies, thereby simplifying the design of paired
therapeutic/reporter agents.
"Approaches developed with one imaging technology are being used to develop
approaches in another modality or to bring data from each technology together to
provide a more informative result," Phelps said. "This illustrates how the
individual imaging technologies are coming together to build a new school of
thought of molecular imaging."
The reporter gene model at UCLA actually has several components, according to
Gambhir. A PET reporter gene is composed of a herpes simplex virus gene for the
thymidine kinase enzyme (HSV1-tk) that has been inserted into an adenovirus or
retrovirus, depending on the clinical application [Fig. 3]. The DNA molecules of the reporter gene and the
therapeutic gene are linked through an internal ribosomal entry site. When the
therapeutic gene is expressed, the reporter gene is also expressed and begins
producing HSV1-tk enzyme. A reporter probe (fluorine-18 penciclovir, for
example) is then administered intravenously in picomole amounts to avoid drug
reaction. It penetrates and then is washed out of cells everywhere in the body,
except where it encounters the thymidine kinase enzyme. The enzyme
phosphorylates the radioactive probe, changing its electrical charge and
trapping the probe inside the cell, where its presence indicates gene expression
at that site. The signal from the cell is amplified as the phosphorylated
reporter probe accumulates. The anatomic distribution pattern of F-18 is then
revealed with PET imaging.
The Sloan-Kettering molecular imaging laboratory, led by Dr. Ronald Blasberg,
is working with the same reporter gene concept but has combined it with green
fluorescence protein and ex vivo techniques to validate uptake. Researchers have
various options when they examine the tissue culture in a petri dish.
Fluorescence indicating gene expression ex vivo can be performed before
attempting the more costly and technically demanding in vivo imaging techniques.
Blasberg's group developed a radiolabeled probe that is selectively
phosphorylated in much the same way that fluorodeoxyglucose is phosphorylated by
hexokinase. From that starting point, they devised strategies to examine
endogenous molecular events. The most straightforward of these involves
endogenous gene transcription, in which a reagent places hexokinase or other
endogenous enhancer/promoter elements upstream from the viral-tk or gene
fluorescent reporter probe.
Some of the institute's most exciting projects have involved an imaging
technique designed to monitor the ubiquitous tumor suppressor gene, p53 [Fig. 4]. This work has shown that endogenous p53 expression
can be imaged in tissue and that p53 and downstream pathways are upregulated in
tumor cells after chemotherapy and radiotherapy. Following the administration of
BCNU chemotherapy to tumor-bearing animals, Sloan-Kettering researchers imaged
upregulation of endogenous p53, signaling activation of the enzymatic cascade
that leads to apoptosis in the tumors.
Molecular Imaging With MRI
A reporter gene protocol developed at MGH could potentially match the
effectiveness of the PET HSV1-tk technique while capitalizing on the spatial
resolution possible with MRI.
Monocrystalline iron oxide nanoparticles, or MIONs, an MR contrast agent that
Weissleder and colleagues developed 10 years ago, is a leading candidate for
this role. Each particle comprises about 2000 atoms of iron in a crystalline
form wrapped with dextrin. When the dextrin is cross-linked, the particles are
called CLIONS (cross-linked iron oxide nanoparticles).
Dr. James P. Basilion, an assistant professor of molecular pharmacology, has
conjugated these particles to ligands for a cell surface receptor, such as the
transferrin receptor. The key to effective conjugation, Basilion said, is to
ensure that chemical modifications to the ligand are made without destroying its
affinity for the receptor because of the mass of the material used to generate a
signal for detection with MRI. With this in mind, the MGH lab devised
conjugation methods to create a maximally activated transferrin and developed
ways to conjugate CLION or MION to transferrin, directing the contrast agent to
attach itself to the receptors of cells that overexpress the transferrin
receptor. The presence of a contrast agent changes the magnetic susceptibility
of those cells.
Although this contrast-enhancing behavior can be used to improve the efficacy
of MRI for cancer diagnosis, the MGH team plans to report gene expression with
the aid of the agent. It has developed vectors that position a therapeutic gene
and a transferrin gene side by side in a vector. The expression of the
transferrin receptor gene product therefore is a surrogate measure for the
expression of the therapeutic gene product.
While the MION protocol is an example of a surface receptor encoding,
researchers at the California Institute of Technology are developing MRI
contrast agents that become activated at an intracellular level. EgadMe is the
most advanced agent thus far developed in this class of selectively activated
agents, said Dr. Thomas J. Meade, senior research associate in biology. It
consists of a chelator that occupies eight of the nine coordination sites on a
gadolinium contrast ion. A galactopyranose residue caps off the remaining
coordination site on a gadolinium ion. In this water-inaccessible configuration,
the contrast agent is "inactive," meaning it does not affect the T1 times of MRI
images.
The agent is turned on, however, when b-galactosidase in the cells
enzymatically cleaves the galactopyranose from the coordination site [Fig. 5]. A water molecule almost immediately occupies the
site, thereby changing its magnetic susceptibility and T1 relaxivity and
charging the particle so it is trapped inside the cells. b-galactosidase is an
enzyme manufactured by the gene lac-Z plasmid, a marker gene often used by
developmental biologists. The presence of b-galactosidase means the lac-Z gene
in those cells is turned on. While the "turned-off" EgadMe washes out of the
cells, the "turned-on" agent lingers to light up the cells where gene therapy is
expressed.
The Cal Tech team created an agent, similar to EgadMe, to study autoimmune
function triggered by the absence or presence of caspase enzymes that contribute
to the onset of apoptosis. A peptide that is a substrate for one of the caspase
enzymes is used instead of galactopyranose as the cap. Other versions of the
agents target caspase-3 and caspase-9. Both are crucial to the apoptotic
cascade, Meade said.
Another chemical configuration may reveal the presence or absence of matrix
metalloprotease peptides (MMPs). Researchers believe cancer cells manufacture
the peptides to eliminate the interstitial space around the cancer cell in order
to start angiogenesis. By further altering the EgadMe model to selectively turn
on the MR agent when it encounters MMPs, one can potentially gain an early
measure of a cell cluster's transformation into cancer.
Functional MRI Approaches
Dr. Robert J. Gillies and his group at the Tumor Physiology and Imaging Lab
of the Arizona Health Science Center are finding ways to apply functional MR
techniques to molecular imaging. They have tested a protocol on animals and
humans to examine the value of apparent diffusion coefficient (ADC) in tumors
for measuring therapeutic response. The data suggest a strong relationship
between an early rise in ADC and the ultimate therapeutic response, Gillies
said. Dr. Brian Ross at the University of Michigan, who pioneered this
technique, has applied it with favorable results in children with brain tumors
and in animal models.
Gillies believes that MR perfusion measures the growth of interstitial volume
caused by cellular necrosis or apoptosis. His lab is further refining its
techniques to differentiate between the two types of cell death. An early
response of apoptosis is cell shrinkage, he said. An export of cations,
particularly sodium and potassium, and a concomitant loss of cell water occur.
The early response in necrosis is cell swelling, which produces an effect
opposite to early apoptosis. It is followed by a loss in the integrity of the
plasma membrane, which then makes the intra- and extracellular volume
coincide.
The ability to distinguish apoptosis from necrosis would allow clinicians to
differentiate the two mechanisms of gene expression treatments. Low doses
generally can lead to apoptosis and higher doses induce necrosis. An oncologist
could use this information to determine how well therapy is working soon after
its administration, and dosing could be modified to optimize its effectiveness,
Gillies said.
Optical Imaging
Optical imaging will also become an important molecular imaging modality, in
part because of new activatable fluorescent contrast agents. Optical imaging
uses light waves in a manner similar to x-rays at much higher frequencies.
Extremely high spatial resolution is possible with optical imaging, but the
clinical value of unenhanced coherent tomography has been limited to transparent
objects or opaque tissue thinner than 150 mm.
Optical imaging has improved considerably, however, especially through the
work of Dr. Britton Chance at the University of Pennsylvania. His efforts have
led to proposed applications in diagnosing breast cancer and melanoma and
interrogating atherosclerotic disease. Catheter-based microtransducer
advancements are extending the modality's reach intraluminally into the female
reproductive system, stomach, and bowel.
A new generation of fluorescent contrast agents, developed at CMIR, is
extending its reach into molecular imaging, said Dr. Umar Mahmood, a radiologist
and staff researcher. In certain chemical configurations, these agents have no
effect on the viewed fluorescent image while they are moving through the body
because the fluorochromes are bound together and emit no signal. The
fluorochromes are unbound after specific enzymatic interaction, however, and
these encounters cause them to glow. In some cases, the emission of photons
boosts the contrast 1000-fold. This enables target detection with near-infrared
fluorescence imaging down to the 10-8 molar concentration level.
The Future
What does this all mean for radiology in 2010? New diagnostic procedures and
agents will help identify either the genotype or molecular phenotype of
abnormalities in vivo, making cancer diagnosis possible, according to Dr. Daniel
C. Sullivan, associate director of the NCI's biomedical imaging program. Breast
adenocarcinoma, for example, is thought to be at least two different diseases.
It is a safe bet that by 2010, radiologists will be determining optimal therapy
by using in vivo molecular imaging to identify these unique genetic
profiles.
Today's mainstream diagnostic modalities will all play a role. Ultrasound,
CT, and digital fluoroscopy will, at a minimum, guide microcatheters to anatomic
targets to deliver angiogenic inhibitors and paired therapeutic/imaging agents.
Microbubbles may be used as a delivery medium; they will burst and release their
therapeutic load when they encounter a focused ultrasonic beam, so gene therapy
may avoid the numerous molecular hazards otherwise encountered during transit.
Angiogenesis inhibitors will probably be used in 10 years, and molecular imaging
will monitor their efficacy, according to Weissleder.
Memorial Sloan-Kettering's Blasberg envisions gene and protein expression
arrays that redefine the rules of pathology and histological confirmation.
Evaluations that seek out molecular abnormalities are already being added to
some pathological and histological evaluations. Gene and protein expression
arrays will be considered routine by the end of this decade. Imaging's
traditional role in diagnosis may actually be diminished, while its importance
in monitoring therapies following their administration will increase, he
said.
Functional MR techniques such as perfusion and spectroscopy are not so exotic
as to rule out their use in 10 years. Activatable MR agents may play a role if
researchers can discover how to get these large molecules to penetrate cell
membranes and to deal with the high mass levels of probes required to produce
sufficient signal for imaging. Although MR will continue to be renowned for
exquisite spatial resolution, it may not always be as popular as it is today,
because it can't match the tracer ability of PET, Gillies said. But MR will be
valued for providing high-contrast registered images of morphology and perfusion
and identifying spectroscopic signatures for cancer diagnosis and therapy
monitoring.
Optical imaging will become prominent because it provides benefits beyond the
capabilities of other imaging technologies, Weissleder said. It will identify
molecular abnormalities, such as p53 absence, overexpression of a protease
enzyme, or the presence of an oncogene.
"This will be the classic case of early diagnosis with molecular imaging,
perhaps before morphologic or clinical phenotypic signs of disease can be seen,"
he said.
Mahmood predicts that some of today's mainstream applications will appear
quaint 10 years from now. At that time, no one will recommend serial scans
separated by three-month intervals to monitor the efficacy of chemotherapy based
on the size of a tumor. Instead, Mahmood foresees that a genetic profile of the
tumor or precancerous lesion will be generated when it is first detected.
Individualized therapeutic plans will be formulated based on genetic profiles,
and therapeutic efficacy will be monitored during the first cycle of therapy.
Serendipity makes accurate prediction difficult, and random events interfere
with well-intentioned forecasting, Phelps said.
"It is clear, however, that the first gene imaging to obtain FDA approval for
clinical use will be with PET, because the imaging probes are used in such low
mass amounts that they will not produce pharmacologic or physiologic effects,"
he said.
Also uncertain is how much value students of scientific history will attach
to the photo of the molecular imaging researchers grouped in front of the Grand
Tetons. Only time will tell if these researchers will be accorded the same
esteem that Madame Curie and her colleagues earned after pausing for their
photograph nearly 100 years ago.
