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382
Table 3: Methods of gene transfer
Method Examples Advantages Disadvantages Comments
1. Viral vectors Adenoviruses Efficient Technically Adenoviruses transfer genetic
Lentiviruses Long-lasting difficult material epigenetically (does not
Retroviruses Potential for integrate with host genome)
Adeno-associated toxic reactions Retroviral, lentiviral and adenoviruses associated viral genes integrate into
Herpes simplex host genome.
virus-1
PET Imaging: Potential Applications in Gene and Stem Cell Therapy
2. Non-viral Liposomes Relatively Inefficient Less widely used than viral vectors
vectors Electrophoresis simple
Particle-mediated Non-toxic
uptake Larger DNA
Direct uptake of capacity
naked DNA
Examples of Gene Therapy
Hemophilia B is a hereditary condition involving the X- chromosome and a genetic defect
leading to a deficiency of factor IX, a vital clotting factor. In an initial human trial, Adenoassociated viruses carrying the gene for factor IX were injected at multiple sites in a skeletal
muscle that could be excised in the event of an unanticipated adverse event. Genetic expression
of factor IX and low levels of factor IX were detected in the circulation. Patients’ need for
factor IX replacement injections (protein therapy) was reduced. However, no significant
reduction in clotting time was observed (2).
In cancer gene therapy, a commonly studied paradigm is of suicide genes. A suicide
gene is a gene whose expression in tumor cells leads to production of enzymes which can
convert a pro-drug (which is inactive on its own) into a cytotoxic active drug and hence,
makes the tumor cells more susceptible to chemotherapy. In a clinical trial of incurable
hepatocellular carcinoma (3), tumor cells were transfected with adenoviral vector carrying
HSV1-tk (Herpes Simplex1-thymidine kinase) gene, by direct injection into lesions. A prodrug gancyclovir was administered which was expected to be activated by phosphorylation
by the expressed enzyme and produce cytotoxic effects. The conventional methods of
monitoring gene expression in such an experiment would involve traditional biopsies followed
by conventional molecular biology techniques in tissue samples. However, the role of PET
imaging in such a paradigm lies in its ability to non-invasively monitor the gene expression
in target tissue. In this particular example, the gene expression can be monitored by
administering a radio labeled substrate (e.g. 18F-FHBG, 9-(4-[18F]-Fluoro-3hydroxymethylbutyl)-guanine, a pencyclovir derivative) of the expressed enzyme (HSV1TK) and detecting its intracellular trapping by PET scanner. This substrate thus, acts as a
reporter probe which “reports” on the presence, location, magnitude, and persistence of
PET Imaging: Potential Applications in Gene and Stem Cell Therapy
383
gene expression. The gene HSV1-tk acts as the reporter gene whose protein product (in this
case, HSV1-TK enzyme) can interact with a reporter probe and “report” about its own
expression. Thus, in this particular example, HSV1-tk gene acts as both the reporter gene as
well as the suicide gene. However, this may not always be the case. A therapeutic gene
(which may or may not be a suicide gene) may not have an available radiolabeled probe or
the development of such a probe maybe deemed difficult, expensive or technically unfeasible.
Thus, to image the expression of such genes, a reporter gene has to be coupled along with
the therapeutic gene using one of the several established methodologies. The aim, in such an
approach will be to ensure the simultaneous expression of both the genes so that imaging of
the expression of the reporter gene provides indirect information about the expression of the
therapeutic gene.
The various approaches to such coupling are summarized in table 4.
Table 4: Methods of coupling therapeutic and reporter genes
Method Aim Advantages Pitfalls
1. Fusion-gene
technology
2. Bicistronic
transcription units
3. Bidirectional vectors
4. Double vector
approach
A unique polypeptide
that retains activities of
two different proteins:
therapeutic gene product
and reporter gene
product
cis-linked genes joined by
an internal ribosomal
entry site (IRES)
Expression of two genes
from a common promoter
Two different gene delivery vectors; one carrying
therapeutic gene, the
other reporter gene
Reporter gene-therapeutic
gene coupling
Synthesis of two
different proteins from a
single mRNA chain;
single reporter gene can
be used with ideally any
therapeutic gene
Encouraging results in
initial studies
Single reporter gene
vector can be used to
track the expression of
many therapeutic genes
Loss of function of
either peptide sequence
due to interactions with
the other sequence or
altered localization in
intracellular compartments
Both proteins may form
at different rates
complicating
quantification of
therapeutic gene
expression.
—
Greater viral load: greater
chance of toxicity;
interaction between genes
altering correlation
between gene expressions
The foregoing examples have shown HSV1-tk as the prototypic example of reporter
genes. However, there are three broad categories of PET reporter genes:
a. Enzymes (of which HSV1-tk is an example)
b. Receptors
c. Transporters
384
PET Imaging: Potential Applications in Gene and Stem Cell Therapy
In the receptor category of PET Reporter genes, a gene coding for a receptor such as
human dopamine D2 receptor (D2R) or the human somatostatin receptor subtype2 (SSTr2)
is introduced in the target cells. The advantage of using these receptor systems is that they
have a limited expression in the body (D2Rs in nigro-striatal system of the brain and SSTr2
mainly in the pituitary gland, many carcinoids, and some other types of neuroendocrine
tumors). The radiolabeled ligands for these receptors can be used as their corresponding
reporter probes. Similarly, in the transporter category of PET reporter genes, a gene encoding
a transporter protein is introduced in the target cells. Specific examples of the transporter
approach include the sodium-iodide symporter (NIS) and the norepinephrine transporter.
The various radioisotopes of iodine can be used as the corresponding reporter probe for NIS
reporter gene. The examples of various PET reporter genes and their corresponding reporter
probes are summarized in table 5.
Table 5: Examples of PET reporter genes and their corresponding probes
PET Reporter Gene Class PET Reporter Probe
1. HSV-1tk Enzyme 5-[
2. D2R Receptor 3-(2-[18F]fluoroethyl)spiperone ([18F]FESP),
3. SSTr2 Receptor
4. NIS Transporter
124
I]iodo-2’-fluro-2’-deoxy-1--D-arabinofuranosyl-5iodouracil ([
9-(4-[18F]-Fluoro-3-hydroxymethylbutyl)-guanine
([18F]FHBG)
O-[11C]raclopride
64
Cu-TETA-octreotide,
68
Ga-DOTA-Phe1-Tyr3-octreotide
124
I
124
I]FIAU),
Stem cell Therapy and PET
Stem cells are the undifferentiated progenitor cells that have self-renewing capabilities and
can develop into highly specialized cells that form the various organs. Stem cell therapy
comes under the broad category of cell therapy. Human stem cells can be derived from the
embryo, or the adults. A broad classification of stem cells is as follows:
1. Totipotent stem cells can form a placenta and can develop into a complete embryo.
These cells are derived from the first few divisions of the fertilized oocyte.
2. Pluripotent stem cells can form tissues derived from all three major germline
layers-endoderm, mesoderm, and ectoderm. These cells are derived from the inner
cell mass of the blastocyst. The common reference of embryonic stem cells refers to
these cells.
3. Multipotent stem cells are the tissue specific progenitor cells found in adults.
Tissues such as bone marrow, skin, intestinal tract, and liver have tremendous
regenerative potential due to the presence of such cells. Recent data suggest that
PET Imaging: Potential Applications in Gene and Stem Cell Therapy
adult stem cells can generate differentiated cells beyond their own tissue boundaries,
a process termed “developmental plasticity” (4). Hematopoietic stem cells are the
best studied adult stem cells. Adherent mesenchymal cells in the bone marrow
contain a second class of stem cell, called a stromal stem cell or a mesenchymal
stem cell, which have the apparent potential to form bone, cartilage, tendon, adipose
tissue, muscle, marrow stroma, and neural cells.
The potential applications of stem cell therapy are summarized in table 6.
Table 6: Potential applications of Stem Cell Therapy
Disease Stem Cell Therapy Goal
Cardiovascular diseases Replace ischemic cells/ Generate cardiomyocytes
Myocardial ischemia/ infarction
Heart failure
Neurodegenerative diseases Replace dopaminergic neurons in PD
Parkinson’s disease (PD) Replace motor neurons in ALS
Amyotrophic Lateral Sclerosis Replace oligodendrocytes in MS
Multiple Sclerosis
Musculoskeletal disorders Replace myoblasts
Duchenne muscular dystrophy
385
Hepatic disease Replace or regenerate hepatocytes
Hepatic failure
Hepatitis B or C
Metabolic Disorders Regenerate pancreatic islets or induce beta cell differentiation
Diabetes Regenerate bone
Osteoporosis
Adapted from (2)
Role of non-invasive imaging in stem cell therapy is broadly directed towards:
(a) monitoring the success of cell transplantation and engraftment in vivo,
(b) serving as a surrogate endpoint in clinical trials, and
(c) determining individual clinical benefit and prognostic implications.
Conventional cell labeling techniques using
111
In-oxine and
99m
Tc-HMPAO have been
employed for tracking stem cells in vivo. 18F-FDG has also been employed for labeling bone
marrow mesenchymal stem cells. However, this technique was limited owing to the short
half life of 18F. All these techniques are also limited by their inability to determine the
viability of the labeled cells.
To overcome these limitations, the paradigm of genetically modifying stem cells with
reporter genes ex vivo before introducing them in the body has been developed. Stem cells
386
PET Imaging: Potential Applications in Gene and Stem Cell Therapy
can also be genetically modified with the therapeutic genes of interest. The latter has the
advantage that such genetically modified stem cells serve as a continuous source of gene
products as compared to somatic gene therapy which needs repeated gene transfers as the
target cells undergo turnover (except in cancer gene therapy where the gene expression is
required for a short duration). It is in this sense that stem cell therapy and gene therapy are
converging, as mentioned in the beginning of this chapter. Lentiviral vector is preferred for
stable integration of reporter genes into stem cells because it can transfect both dividing and
nondividing cells and lead to high levels of gene expression. It has been demonstrated in
animal studies that genetic modification of stem cells with reporter genes does not interfere
with their function. However, this possibility cannot be ruled out completely (5,6).
Functional evaluation of the target tissue following stem cell therapy can be performed
using conventional nuclear medicine techniques, e.g.,
201
Tl,
99m
Tc-sestamibi, 82Rb for
myocardial viability, function and blood flow; 11C-Raclopride imaging following fetal brain
tissue grafting in patients with Parkinson’s disease demonstrating the functional status of
the grafted cells by the displacement of 11C-Raclopride from the post-synaptic D2 receptors
following synthesis of dopamine by the grafted cells (2).
Other imaging modalities in gene therapy and stem cell therapy
Optical imaging and Magnetic Resonance Imaging are the other imaging modalities found
useful in imaging of stem cell therapy and gene therapy (3,5,7). A detailed outline of these
modalities is beyond the scope of this chapter. However, a few pertinent points are as
follows:
(1) Optical imaging is primarily a tool in animal models for imaging genes expressed
on the surface of body. Also, unlike PET/SPECT/MRI, optical imaging does not
involve tomographic imaging and hence, is limited in its resolution. The most
commonly used gene product targeted in optical imaging is the luciferase enzyme.
Green fluorescent protein (GFP) is another example of a target for optical imaging.
Combined optical and PET reporter probe imaging (dual reporter imaging) has been
attempted in gene therapy models (5).
(2) Superparamagnetic iron oxide (SPIO) particles are well known for their ability to
generate MR contrast. SPIOs and Ultra small superparamagnetic Iron oxide (USPIO,
<30nm) particles are endocytosed by cells and have been used for stem cell tracking
using MRI. Superparamagnetic derivatives of transferrin have been used to monitor
the expression of transferring receptor as a reporter gene (3,5). However, mass
limitations make this a less sensitive technique for gene expression and stem cell
imaging.
Conclusion
Positron Emission Tomography, with the use of a large number of positron labeled chemical
PET Imaging: Potential Applications in Gene and Stem Cell Therapy
387
compounds, provides high spatial resolution, generates tomographic images, and gives precise
quantitative data in imaging of gene therapy and stem cell therapy. Moreover, excellent
improvements in PET resolution in small animal imaging using micro-PET can serve to
bridge the gap between pre-clinical and clinical studies using PET. In future, Positron
Emission Tomography imaging is expected to play a vital role in the ever expanding and
evolving fields of gene therapy and stem cell therapy.
References
1. Phelps ME. Positron emission tomography provides molecular imaging of biological processes. PNAS
2000; 97(16): 9226-9233.
2. Bodine D, Jameson JL, McKay R. Stem Cell and Gene Transfer in Clinical Medicine. In Kasper et al.
(Eds.) Harrison’s Principles of Internal Medicine 16th Edition. McGrawHill Companies, Inc. pp.39297, 2004.
3. Penuelas I, Boan JF, Marti-Clement JM et al. Positron emission tomography and gene therapy: basic
concepts and experimental approaches for in vivo gene expression imaging. Mol Imaging Biol 2004;
6(4): 225-238.
4. Körbling M, Estrov Z. Adult Stem Cells for Tissue Repair -A New Therapeutic Concept? N Engl J
Med 2003; 349:570-82.
5. Zhou R, Acton PD, Ferrari VA. Imaging stem cells implanted in infracted myocardium. J Am Coll
Cardiol 2006;48:2094 –106
6. Bengel FM. Nuclear imaging in cardiac cell therapy. Heart Fail Rev (2006) 11:325–332
7. Wunderbaldinger P, Bogdanov A, Weissleder R. New approaches for imaging in gene therapy. European
Journal of Radiology 2000; 34:156-165.
Computed Tomography in PET/CT
Mahadevappa Mahesh
Imaging modalities are commonly differentiated as functional or anatomical in nature.
However, both categories of information are complimentary for integration of data for
diagnosis, for the planning performance, and the evaluation of therapy.
The advantage of using a Computed Tomography (CT) scanner with a Positron Emission
Tomography (PET) scanner as in a PET/CT scanner are that a PET scan is followed by the
CT scan over the same area. Both the scans are fully registered, which allows online fusion
of two sets of images. PET provides functional image of radiopharmaceutical distribution,
while CT provides anatomical images that are used for localizing radiopharmaceutical update
and alternation correction from PET reconstruction.
The advantages of using a CT scanner with PET scanner in the so-called hybrid imagers
are that the CT data improves anatomical landmarks for localization of imaging uptakes.
This is due to high photon flux, which improves accuracy and reduced noise levels of
attenuation measurements. CT also provides high special resolution images that can generate
highly accurate localization maps and also attenuation correction maps. Since CT energy is
in the range of less than 100 KeV and the PET energy is in the range of 511 KeV, there are
no cross talks between the transmission and emission images. PET/CT is designed such that
it is neither only PET nor only CT; it is truly a new imaging tool. It combines two x-ray
medical imaging technologies. X-ray computed tomography for anatomical imaging and
attenuation corrections, while PET for functional imaging. The PET/CT scanner can operate
as a single system, which can be used to acquire only CT scans, only PET scans, or PET/CT
scans. Figure 1 shows a schematic drawing of the PET/CT scanner. The CT gantry is
positioned prior to PET gantry. Both the CT gantry and the PET gantry are assembled such
that; they are positioned on an assembly line; so that it can easily separated to performing
repairs or maintenance services on individual scanners.
38 8
Computed Tomography in PET/CT
389
CT Gantry
Table
CT Scan
Figure 1: Schematic Diagram of CT & PET Gantry in a PET CT scanner. The diagram shows the
CT gantry followed by the PET gantry.
PET Scan
PET
An initial challenge in the development of PET/CT scanner was the stability of the
patient table. When the table is extended such that the head of the table is positioned all the
way inside the PET gantry. Even with a minimal flex or bending of the table could result in
a significant error in the image fusion process. Hence the stability of the patient table when
positioned with maximum extension was a challenge. A typical PET/CT protocol includes,
moving the patient first under CT gantry to perform CT scan of the region of interest and
then moving the patient further to the PET gantry to acquire PET scans. For example, during
a whole body PET/CT scan, the patients are scanned with CT scanner from mid-ear to midthigh, and then the patients positioned at a different location/fields under PET gantry to
acquire PET images.
In this chapter, we discuss the fundamentals of CT as applied to PET/CT scanners. We
also discuss the radiation doses for typical PET/CT scans, particularly from the CT scan
portion.
Fundamentals of CT
Computed tomography is a method of acquiring and reconstructing an image of thin
cross-section on the basis of measurements of attenuation. In comparison with conventional
radiographs, CT images are free of superimposing tissues and are capable of much higher
contrasts due to elimination of scatter. Fundamentally, a CT scanner makes many
390
x
m w
Computed Tomography in PET/CT
measurements of attenuation through the plane of a finite thickness of a cross-section of the
body. The system uses this data to reconstruct a digital image of the cross-section with each
pixel in the image representing a measurement of the mean attenuation of a box-like element
also called ‘voxel’ (three-dimensional pixel element) that extends through the thickness of
the section. As CT image is based on the measurement of the x-ray attenuation through the
section plane using many different positions. This is achieved by rotating both the x-ray
tube and the detectors around the patient. An attenuation measurement quantifies the fraction
of radiation removed in passing through a given amount of specific material of thickness.
Attenuation is expressed as shown in equation- 1, where It is the x-ray intensity measured
with the material of thickness ‘“x’ in the x-ray beam, Io is the x-ray intensity measured
without the material in the x-ray beam and the µ is the linear attenuation co-efficient of the
specific material.
I I e
t o
(1)
Characteristics of an unknown material can be determined, and given its physical thickness
“x, then one can measure Io and It and derive the characteristic of the material by using the
above equation. The image reconstruction process is basically a process of determining the
attenuation values for each voxel by using many x-ray projections obtained by scanning the
object from different angles. The CT signal is basically acquired because of the tissue
discrimination, which is due to attenuation of radiation between voxel, which depends on
differences in tissue density, atomic number of elements presented in the voxel and the
detected mean photon energy.
The other fundamental of CT imaging is that the attenuation values are scaled to a more
convenient scale and is normalized to voxel containing water and are expressed as CT
numbers in Hounsfield units. The CT number of a material is defined as in equation-2,
where µw is the attenuation coefficient of water, µm is the attenuation coefficient of material
of interest and ‘k’ is a scaling factor.
#
CT k
w
(2)
With the scaling factor of 1000, the CT number of water is always ‘0’, and the CT
number of air is -1000. Tissues denser than water have positive CT numbers and tissues less
dense than water have negative CT numbers. The CT numbers are represented or mapped
onto a grayscale that is visible as a CT image.
The other fundamental in CT is regarding the CT gantry. Even though the CT gantry has
a physical opening of close to 60-70 cm in diameter, however the actual sampling area
where the attenuation measurements are made is less than the physical opening, which is
Computed Tomography in PET/CT
391
usually 50-55 cm in diameter. Because of the sampling area being shorter than the physical
gantry opening, CT image often results in eliminating part of patient’s shoulder or hip that
lies outside the sampling area. The plane of the gantry opening is called the transaxial plane
(x-y plane) and the longitudinal direction that is in and out of the gantry is noted as zdirection.
PET/CT
PET-CT became practical with helical CT and more recently with multiple-row detector
CT (MDCT). The principle behind helical CT or spiral CT is that the patient is transported
continuously through the gantry while data is acquired continuously during the several 360
degrees rotations. Helical CT was made possible because of three major technological
advances. The development of slip-ring gantry, allowed the x-ray tube to rotate around the
patient without having to stop to unwind the wires. The development of interpolation
algorithms allowed interpolation of data from adjacent helix during helical scan and
development of high-powered x-ray tubes that allowed continuous data acquisition.
In the 1990’s, the introduction of helical CT revolutionized the field of diagnosis because
it allowed for a continuous volumetric data acquisition. In the late 1990’s the introduction of
multiple-row detectors CT (MDCT) revolutionized the field even further. With the
introduction of multiple-row detector CT (MDCT), the single-row of detectors was replaced
with multiple-rows of detectors. This automatically increased scan volume per rotation.
Also, it enabled to acquire multiple thin sections providing higher z-axis resolution.
The key differences between a single-row detector CT (SDCT) and a multiple-row detector
CT are the number of multiple-row detectors in the longitudinal direction. Also, the x-ray
beam width is larger with the MDCT than in SDCT. For the same scan time of the gantry
rotation, one could acquire four times the scan volume with the MDCT compared to the
SDCT. The evolution of MDCT introduced many new clinical applications and is increasing
at a rapid pace. The early commercial PET/CT scanners installed in 2001 consisted of an
MDCT scanner assembled next to a PET gantry. It is important to examine the multiple-row
detector array design in order to understand the various scan acquisition during PET/CT.
Multiple-row detector design arrays
PET/CT became practical with helical CT and more recently with multiple-row Detector
CT. By late 1998 all major CT manufacturers launched Multiple-row Detector CT also
known as MDCT scanners capable of yielding at least four-transaxial slices per x-ray tube
rotation. The key difference between the MDCT and single-row detector CT is in the number
of row detectors in the longitudinal or z direction which enabled for increased scan volume
per tube rotation and improved z-axis resolution, key to improving 3D reconstructed image.
The number of transaxial slices that can be obtained per gantry rotation is determined by the
data acquisition system (DAS) channels and not by the available number of physical detectors
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