[CANCER RESEARCH 61, 110 –117, January 1, 2000]
Synthesis and Evaluation of 18F-labeled Choline as an Oncologic Tracer for
Positron Emission Tomography: Initial Findings in Prostate Cancer
Timothy R. DeGrado,1 R. Edward Coleman, Shuyan Wang, Steven W. Baldwin, Matthew D. Orr, Cary N. Robertson,
Thomas J. Polascik, and David T. Price
Departments of Radiology [T. R. D., R. E. C., S. W.] and Surgery [C. N. R., T. J. P., D. T. P.], Duke University Medical Center, Durham, North Carolina 27710, and Department
of Chemistry, Duke University, Durham, North Carolina 27708 [S. W. B., M. D. O.]
ABSTRACT
The up-regulation of rates of choline uptake and phosphorylation in
certain malignancies has motivated the development of positron-labeled
choline analogues for noninvasive detection of cancer using positron
emission tomography (PET). The choline analogue, no-carrier-added
[18F]fluoromethyl-dimethyl-2-hydroxyethyl-ammonium (FCH), was synthesized through the intermediate [18F]fluorobromomethane. FCH was
evaluated in relationship to 2-[18F]fluoro-2-deoxyglucose (FDG) as an
oncological probe in cultured PC-3 human prostate cancer cells, a murine
PC-3 human prostate cancer xenograft model, and in PET imaging studies
of patients with prostate cancer. FCH was synthesized in 20 – 40% radiochemical yield and >98% radiochemical purity. Accumulation of FCH
and FDG were comparable in cultured prostate cancer cells, whereas only
FCH was inhibited (90%) by hemicholinium-3, a specific inhibitor of
choline transport and phosphorylation. FCH showed similar biodistribution to [14C]choline in the tumor-bearing mouse, with prominent renal
and hepatic uptake. Tumor uptake of FCH was similar to choline and
FDG in the mouse model, although tumor:blood ratios were moderately
higher for FCH. Initial PET imaging studies in prostate cancer patients
showed high uptake of FCH in advanced prostate carcinoma and detection of osseous and soft tissue metastases. FCH uptake by tumors was
markedly reduced in patients rescanned during androgen deprivation
therapy. It is concluded that FCH closely mimics choline uptake by
normal tissues and prostate cancer neoplasms. FCH is potentially useful as
a PET tracer for detection and localization of prostate cancer and monitoring effects of therapy.
lived radioisotope fluorine-18 (T1/2 ⫽ 110 min) led Hara et al. (11) to
synthesize and preliminarily evaluate the choline analogue FEC. The
more rapid accumulation of radioactivity in the urinary bladder with
the F-18-labeled analogue rendered it less preferable to CH for imaging of primary prostate carcinoma and metastatic prostate carcinoma in the pelvic lymph nodes (11).
We recently reported the synthesis and preliminary evaluation of
no-carrier-added FCH as an analogue of choline for oncological
imaging with PET (12). It was anticipated for reasons of structural
similarity that the [18F]fluoromethylated FCH would mimic CH transport and metabolism more closely than the [18F]fluoroethylated FEC,
resulting in a biodistribution similar to choline with lower urinary
excretion of 18F radioactivity. FCH is presently evaluated in cultured
PC-3 human prostate cancer cells, a murine PC-3 human prostate
cancer xenograft model, and PET imaging studies of patients with
prostate cancer.
MATERIALS AND METHODS
Equipment. Nuclear magnetic resonance spectra were recorded on a Varian INOVA 400 MHz spectrometer. High-resolution fast atom bombardment
mass measurements were made using a JEOL JMS-SX102A mass spectrometer operating at 10k resolution.
N,N-Dimethyl-N-Fluoromethylethanolamine (Fluorocholine, [19F]FCH).
To a 50-ml pressure tube containing 20 ml of dry tetrahydrofuran at ⫺78°C
was added 5 ml of (0.0498 mole) N,N-dimethylethanolamine (Aldrich Chemical Co.). Chlorofluoromethane (Synquest Labs, Alachua, FL) was bubbled
INTRODUCTION
through the solution for 15 min upon which the tube was sealed with a teflon
PET2 is uniquely suited to evaluate metabolic activity in human screw cap. The mixture was allowed to warm to room temperature over 18 h,
neoplasms for diagnostic imaging purposes. The glucose analogue during which time a white solid precipitated. The solid was isolated by
FDG has proven successful as a PET imaging agent for detection and filtration, washed several times with cold tetrahydrofuran, and dried under
localization of many forms of cancer. The elevated rate of glycolysis vacuum. N,N-dimethyl-N-fluoromethylethanolamine was isolated as a hygro1
in many types of tumor cells enhances the uptake of FDG in neo- scopic, amorphous white solid (1.386 g, 17.7%); mp 184 –185°C (dec.); H
plasms relative to normal tissues (1–3). However, FDG-PET has been NMR (400 MHz, D2O), ␦ 3.08 (d, J⫽2.1 Hz,13 6 H), 3.45–3.48 (m, 2 H),
3.90 –3.93 (m, 2 H), 5.28 (d, J⫽44.9 Hz, 2 H); C NMR (100 MHz, D2O), ␦
found to have less sensitivity and/or specificity for assessment of
47.18, 55.28, 63.09, 95.77, 97.97; 19F NMR (376.5 MHz, D O) ␦ 106.45 (mt,
some types of cancer, motivating efforts to develop new oncological J⫽45.2 Hz); HRMS (FAB) Calcd for (M-H)⫹ C H ONF: 2122.0981, Found
5 13
tracers for PET. Carbon-11 (T1/2 ⫽ 20 min)-labeled choline (CH) has 122.0984.
shown potential use in two applications: brain tumors (4, 5), where
Synthesis of [18F]FCH. FCH was synthesized via the intermediate FBM
FDG has suboptimal specificity due to uptake by normal brain and (Fig. 1). The synthesis of FBM was essentially that of Eskola et al. (13), which
some posttherapy responses (6), and prostate carcinoma (7), where was modified from Coenen et al. (14). The alkylation with FBM of dimethFDG shows inadequate sensitivity (3, 8). CH was initially synthesized ylethanolamine, isolation of the resultant FCH, and performance of quality
and evaluated as a physiological probe for choline uptake by normal control HPLC were modified from the techniques used by Hara et al. (4) for
11
11
tissues (9, 10). The practical advantages of working with the longer synthesis and quality control of [ C]CH from [ C]methyliodide. FBM was
produced by reaction of dibromomethane (0.05 ml) with no-carrier-added
[18F]fluoride assisted by (Kryptofix 2.2.2/K)2CO3 (10 ␮mol) in dry acetonitrile
Received 4/6/00; accepted 11/1/00.
(0.7 ml). FBM was isolated by gas chromatography (Poropak Q, 80/100 mesh,
The costs of publication of this article were defrayed in part by the payment of page
7.8 ⫻ 700 mm, 100°C, helium flow ⫽ 75 ml/min, retention time ⫽ 6 min) and
charges. This article must therefore be hereby marked advertisement in accordance with
trapped in a solution of 0.1 ml of dimethylethanolamine in acetone (1.5 ml)
18 U.S.C. Section 1734 solely to indicate this fact.
1
To whom requests for reprints should be addressed, at Department of Radiology,
within a 2.5-ml conical glass vial kept at ⫺5°C using a Peltier cooling/heating
Duke University Medical Center, Box 3949, Durham, NC 27710. Phone: (919) 684-7727;
device (15). The vial was sealed and heated to 100°C for 10 min. The solvent
Fax: (919) 684-7130; E-mail: [email protected].
2
was evaporated under a stream of helium, and the residue taken up in sterile
The abbreviations used are: PET, positron emission tomography; FDG, 2-([18F]fluorowater (5 ml) and transferred to a cation exchange SEP-PAK cartridge (Accell
2-deoxyglucose; CH, trimethyl-2-hydroxyethyl-ammonium); FEC, 2-[18F]fluoroethyl-dimeth18
yl-2-hydroxyethyl-ammonium; FBM, [ F]fluorobromomethane; HPLC, high-performance
Plus CM Light; Walters). After further washing of the cartridge with sterile
liquid chromatography; HC-3, hemicholinium-3; EGF, epidermal growth factor; FCH,
water (10 ml), the product was eluted with sterile isotonic saline (⬎2 ml) and
[18F]fluoromethyl-dimethyl-2-hydroxyethyl-ammonium; PSA, prostate-specific antigen; CT,
passed through a 0.22-␮m sterile filter (Millex GS; Millipore). Radiochemical
computed tomography; MRI, magnetic resonance imaging; SUV, standardized uptake value;
purity of FCH was measured by analytical HPLC (C-18 250 ⫻ 4.6 mm, 0.05
PC, phosphocholine; CK, choline kinase; PI-3 kinase, phosphatidylinositol 3-kinase.
110
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F-LABELED CHOLINE FOR IMAGING PROSTATE CANCER
Fig. 1. Reaction sequence for synthesis of FCH.
M phosphoric acid and 1 mM 2-naphthalenesulfonic acid in 80% water/20%
methanol, 0.5 ml/min, retention time ⫽ 4.4 min) using nonradioactive fluorocholine as a reference standard. The sample was doped with 0.1 mg of choline
chloride before administration on the HPLC to avoid variable retention of the
high specific activity [18F]FCH on the column.
Accumulation of Radiotracers by PC-3 Human Prostate Cancer Cells.
Cells (2.5 ⫻ 105/well) were seeded on 6-well plates and incubated for 2 days
at which time ⬎90% confluency was reached. The incubation medium was
DMEM supplemented with 10% calf serum. The medium contained 15 mM
glucose. On the day of the study, the medium was refreshed using a volume of
1 ml in each well. Cells were incubated in control conditions or with the
addition of metabolic and growth factor receptor inhibitors to test the sensitivity of uptake of the radiotracers to specific inhibitions. The inhibitor of
choline uptake and phosphorylation, HC-3 (Research Biochemicals, Natick,
MA), was added to give a concentration of 5 mM. The PI-3 kinase inhibitor
LY294002 (Calbiochem, San Diego, CA) was used at a concentration of 15
␮M. The EGF receptor kinase inhibitor AG1478 (Calbiochem) was added at a
concentration of 50 nM. The concentrations of the inhibitors were ⱖ10 times
their respective literature in vivo IC50 values for choline phosphorylation
(HC-3; Ref. 16), PI-3 kinase inhibition (LY294002; Ref. 17), and EGF receptor kinase inhibition (AG1478; Ref. 18). Following a 30-min incubation
period, the radiotracers, FDG or FCH, were added (⬃2 ␮Ci/well) and the cells
were incubated for 2 h. The cells were washed three times with PBS solution,
released from the plates by briefly incubating with 0.05% trypsin in DMEM,
transferred to test tubes, and counted for F-18 radioactivity in a gamma
counter. The amount of radioactivity in the cells was normalized by the dose
administered to each well.
Biodistribution Studies in a Murine PC-3 Human Prostate Cancer
Xenograft Model. All animal experiments were conducted under a protocol
approved by the Duke University Institutional Animal Care and Use Committee. Androgen independent prostate cancer cells (PC-3) suspended in matrigel
(Collaborative Research, Bedford, MA) at a concentration of 1 ⫻ 106 cells/100
␮l were injected s.c. into the flank of male athymic mice (BALB/c nu/nu), 4 – 6
weeks of age. The mice were maintained in pathogen-free conditions as
described previously (19). Body weight and tumor volume were measured
weekly, and tumor volume (mm3) was calculated using the formula S2 ⫻ L/2,
where S and L represent the large and small diameters of the tumor, respectively.
After the tumor volume had surpassed 0.5 cm3, the mice were anesthetized with pentobarbital (75 mg/kg) before injection of the radiotracer and
remained anesthetized throughout the study. [18F]FCH (20 – 40 ␮Ci) and
[14C]choline (4 ␮Ci) were simultaneously injected into a tail vein. A
prescribed duration of time was allowed before procurement of heart, liver,
lung, blood, kidney, bone (femur), brain (whole), prostate gland, tumor,
bladder, and skeletal muscle. The tissues were weighed, and counted for
18
F in a gamma counter, then dissolved in Solvable (DuPont, Boston, MA)
and counted for 14C in a liquid scintillation counter. For the bladder, the
percentage of the injected dose in the urine was determined. For all other
tissues, radiotracer uptake was calculated as:
of FCH will be published elsewhere. The subjects were informed of all risks
associated with the study, and written informed consent was obtained. Table 1
shows clinical data on the patients evaluated with FCH-PET. Patients 1 and 4,
having hormone naı̈ve prostate cancer, underwent FCH-PET scanning before
and after initiation of androgen deprivation therapy. Imaging was performed
using the Advance PET scanner (GE Medical Systems, Milwaukee, WI),
having an intrinsic spatial resolution of ⬃5 mm in all directions (20). A
transmission scan of the pelvic region was obtained before administration of
radiotracer. [18F]FCH (2.5–5 mCi) was administered i.v. In the first three
patients, dynamic imaging of the lower pelvis region was commenced for 20
min (frame sequence ⫽ 12 ⫻ 10 s, 2 ⫻ 30 s, 1 ⫻ 2 min, 3 ⫻ 5 min). After
the dynamic scan, a whole-body emission scan (4 min per bed position) was
performed without attenuation correction. During image reconstruction, the
emission data in the pelvic region were corrected for photon attenuation using
the transmission scan. In the fourth patient, both transmission and emission
data were collected over the entire thorax to provide an attenuation-corrected
whole-body scan. The emission imaging was commenced over the lower pelvis
at 5 min after injection to obtain an image of this region before the arrival of
radioactive urine at the bladder. The images were reconstructed using an
Ordered Subset Expectation Maximum algorithm. Regions of interest were
drawn manually on the attenuation-corrected images for evaluation of FCH
kinetics in tissues. Standardized uptake values of FCH uptake in tissues were
calculated using the attenuation-corrected images according to the equation:
SUV ⫽
Body Wt. (g) CFCH(nCi/ml)
(2)
Dose(nCi)
where CFCH is the concentration of FCH in the tumor region of interest. Patient
3 also underwent an attenuation-corrected whole-body FDG-PET study within
4 days of the FCH-PET study for comparison of image quality and SUV
values. Emission imaging was commenced 45 min after i.v. administration of
[18F]FDG (9.7 mCi).
RESULTS
Synthesis of [18F]FCH. [18F]FCH was synthesized in 20 – 40%
radiochemical yield (not decay corrected) in a synthesis time of under
40 min. The radiochemical yield was determined primarily by the
yield of the intermediate synthon, [18F]FBM, because the yield of the
alkylation reaction of FBM with dimethylethanolamine was ⬎90%. In
a subset of samples, the final preparation was subjected to mass
spectrometry for detection of impurities. No organic material was
detected in the samples, consistent with the high specific activity of
the no-carrier-added F-18-labeled tracer and adequate isolation of
FCH from dimethylethanolamine by the cation exchange SEP-PAK
method. Radiochemical purity of ⬎98% of FCH was verified by
analytical HPLC (Fig. 2).
Accumulation of FCH and FDG by Cultured PC-3 Human
Prostate Cancer Cells. Under control conditions, cultured PC-3 human prostate cells accumulated FCH at a slightly lower rate in
comparison with FDG (Fig. 3). Specific inhibition of choline transport
and phosphorylation by HC-3 resulted in a 90% (P ⬍ 0.001) decrease
in FCH uptake, whereas FDG uptake was unchanged, demonstrating
the specificity of uptake of FCH to the choline processing pathway.
Table 1 Clinical data on patients
Uptake (%dose kg/g) ⫽
CPM(tissue) ⫻ Body Wt. (kg) ⫻ 100
Tissue Wt. (g) ⫻ CPM (dose)
Primary disease
(1)
where cpm ⫽ counts per min.
In a separate experiment, the biodistribution of [18F] FDG was determined
in the same animal model with a time of sacrifice of 45 min after injection.
PET Imaging Studies. The biodistribution of FCH was investigated in
four patients with prostate cancer. The FCH-PET studies were approved by the
Duke University Medical Center Investigational Review Board and Cancer
Research Committee. The supportive data on toxicity and radiation dosimetry
Patient
Age
Clinical stage
Gleason grade
Serum PSA (ng/ml)
1
2
3
4
59
79
80
65
T3NXM1b
T2NXM0
TXNXM1b
TXNXM1b
4.4
4.4
22.1
7.6
4172
25.4c
a
b
a
Patient status after radical prostatectomy and bilateral scrotal orchiectomy now with
hormone refractory disease.
b
Patient status after radical prostatectomy with hormone naı̈ve disease.
c
Measured 8 months before initial PET study.
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F-LABELED CHOLINE FOR IMAGING PROSTATE CANCER
Fig. 2. Reverse phase HPLC radiochromatogram of FCH. The sample was doped with
0.1 mg of choline chloride.
Fig. 3. Effect on metabolic and growth factor inhibitors on FDG and FCH uptake by
cultured PC-3 human prostate cancer cells. Cells were preincubated in control conditions
(䡺) or with 5 mM HC-3 (high-affinity choline transporter and CK inhibitor; f), 15 ␮M
LY294002 (PI-3 kinase inhibitor; o), or 50 nM AG1478 (EGF receptor kinase inhibitor;
s). Cells were incubated with radiotracers for 2 h. Values represent mean and SD of three
wells in each condition. The asterisk denotes statistical significance (P ⬍ 0.01) relative to
control conditions. A 90% decrease in accumulation of FCH was observed in HC-3treated cells. Inhibition of PI-3 kinase by LY294002 reduced uptake of FDG and FCH by
26% and 15%, respectively.
Inhibition of PI-3 kinase by LY294002 reduced uptake of FDG and
FCH by 26% and 15%, respectively. The EGF receptor kinase inhibitor AG1478 had no affect on either FCH or FDG uptake.
Biodistribution of FCH in a Murine PC-3 Xenograft Model.
Table 2 and Fig. 4 show the biodistribution of [18F]FCH, [methyl14
C]CH (DuPont NEN Research Products, Boston, MA), and
[18F]FDG in mice. The kidneys and liver were found to be the primary
sites of uptake for both FCH and CH, similar to previous findings with
radiolabeled choline (4, 21). Tumor uptake of the choline analogues
and FDG were comparable at 45– 60 min after injection. However, the
tumor:blood ratio, a diagnostically important parameter, was higher
(P ⬍ 0.05) at 60 min for FCH (5.3 ⫾ 2.4) than for the other two
tracers. Uptake of FCH by normal brain was one-tenth that for FDG
(P ⬍ 0.0001) and one-half that of CH (P ⬍ 0.05). At 30 min, there
was ⬃1% and ⬃10% of the injected dose in the urinary bladder for
CH and FCH, respectively. Together with the observed slower renal
clearance of radioactivity from the kidneys and lower blood radioactivity concentrations for FCH relative to CH, these findings are
consistent with less reabsorption and excretion of radioactivity from
the renal proximal tubular filtrate into the circulation for FCH. Liver
uptake was lower (P ⬍ 0.05) for FCH than for CH.
PET Imaging in Patients. Patient 1: FCH-PET imaging of a
59-year-old male with untreated locally advanced clinical stage T3
prostate cancer (serum PSA, 22 ng/ml; Gleason grade, 4.4) demonstrated accumulation of FCH in the primary prostate carcinoma and an
osseous metastasis in the left ischium (Fig. 5A). The latter finding was
correlated with Tc-99m MDP bone scan, CT, and MRI findings of a
single focus of metastatic prostate cancer. PET images acquired at 3–5
min demonstrated accumulation of FCH in the prostate gland before
the arrival of activity at the bladder. Fig. 5B shows the kinetics of
FCH in the prostate, metastasis, and a region of interest drawn within
the urinary bladder. Radioactivity concentration rose rapidly in the
prostate and metastasis and reached a plateau by 5 min after injection.
Radioactivity began to arrive in the bladder at about 8 min after
injection, and the concentration increased rapidly over the next 20
min. The SUV of both the primary tumor and the metastasis was 7.7
after 5 min. Radioactivity concentration in the brain of the patient was
measured to be ⬍2% that measured in the prostate gland or metastasis. However, the pituitary gland and choroid plexus, which do not
have a blood-brain barrier, showed relatively high uptake as previously noted in [11C]CH scans (4). Also in agreement with CH distribution (4), kidneys, liver, scalp tissue and salivary glands showed
notable uptake of tracer. Uptake in these normal tissues were observed
in all patients and considered normal sites of FCH localization in
Fig. 4. Kinetics of FCH and [14C]choline in PC-3 human prostate cancer tumor-bearing
mice.
Table 2 Uptake (% dose kg/100 g) of radiotracers in tissues of mice with PC-3 xenografts
Tissue
FCH (n ⫽ 5)
10 min
FCH (n ⫽ 3)
30 min
FCH (n ⫽ 5)
60 min
CH (n ⫽ 5)
10 min
CH (n ⫽ 3)
30 min
CH (n ⫽ 5)
60 min
FDG (n ⫽ 2)
45 min
Tumor
Blood
Heart
Brain
Lung
Liver
Kidney
Skeletal muscle
Prostate
3.6 ⫾ 0.6
2.7 ⫾ 0.9
15.5 ⫾ 5.9
0.8 ⫾ 0.3
18.0 ⫾ 5.3
50.7 ⫾ 15.3
127.7 ⫾ 27.6
4.4 ⫾ 1.8
6.6 ⫾ 2.2
7.1 ⫾ 2.1
3.3 ⫾ 0.2
13.2 ⫾ 2.6
1.0 ⫾ 0.2
17.1 ⫾ 1.4
56.7 ⫾ 13.2
116 ⫾ 17
1.1 ⫾ 1.0
7.1 ⫾ 2.1
7.9 ⫾ 5.0
1.5 ⫾ 0.6
12.7 ⫾ 3.2
0.8 ⫾ 0.1
21 ⫾ 4.4
58.4 ⫾ 40.6
94.3 ⫾ 31.0
4.1 ⫾ 0.6
7.1 ⫾ 3.0
3.2 ⫾ 1.8
2.1 ⫾ 0.3
20.3 ⫾ 7.2
1.4 ⫾ 0.7
26.0 ⫾ 9.6
52.3 ⫾ 11.9
99.0 ⫾ 12.9
4.7 ⫾ 1.7
9.0 ⫾ 6.8
4.8 ⫾ 1.6
1.5 ⫾ 0.9
9.7 ⫾ 1.9
1.0 ⫾ 0.8
9.6 ⫾ 6.6
65.2 ⫾ 19.4
53.4 ⫾ 7.3
2.5 ⫾ 1.5
5.9 ⫾ 2.5
6.7 ⫾ 2.5
2.2 ⫾ 1.0
9.1 ⫾ 2.5
1.7 ⫾ 0.6
16.7 ⫾ 3.7
67.1 ⫾ 49.7
41.5 ⫾ 15.0
2.8 ⫾ 1.7
5.5 ⫾ 2.6
8.9 ⫾ 0.7
2.8 ⫾ 0.009
48.2 ⫾ 17.9
8.0 ⫾ 0.7
7.4 ⫾ 3.1
1.5 ⫾ 0.4
5.3 ⫾ 1.9
8.3 ⫾ 0.4
2.3 ⫾ 0.4
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F-LABELED CHOLINE FOR IMAGING PROSTATE CANCER
Fig. 5. A, attenuation-corrected [18F]FCH-PET
image (coronal projections, 3–5 min after injection)
of the pelvis region of patient 1 having an untreated
primary prostate carcinoma (P) and an osseous
metastasis in the left ischium (M). The slice thickness is 12.9 mm. In these early images, radioactivity had not yet arrived at the urinary bladder, allowing excellent delineation of the prostate gland.
The metastasis was also apparent on bone scan, CT,
and MRI. B, time-activity curves for FCH in the
same patient, showing the rapid clearance of radioactivity from the arterial blood (iliac artery region
of interest), rapid uptake by prostate cancer in
prostate and ischial metastasis, and arrival of radioactivity in the urine at about 8 min after injection.
correspondence with choline uptake by tissues. The patient was
rescanned with FCH at 2 months after initiating androgen deprivation
therapy, at which time his PSA level had decreased to 0.9 ng/ml. Both
the primary tumor and osseous metastasis were visualized in the
follow-up study; however, the SUVs for FCH uptake by the tumors
were substantially lower than in the initial study [SUV, 3.0 in primary
tumor (61% decrease); SUV, 2.4 in ischial metastasis (68% decrease)].
Patient 2: Transmission and FCH emission scans of a 79-year-old
male with hormone naı̈ve clinical stage T2 prostate cancer were
commenced over the lower pelvis, but the prostate was not in the
field-of-view as revealed by the later whole-body scan. Radioactivity
uptake was demonstrated in the prostate gland in the whole-body scan,
but it was not quantifiable due to lack of attenuation correction in this
region. No metastatic lesions were detected in the PET images. A
recent radionuclide bone scan of the patient also showed no evidence
of osseous metastases. Positioning of the prostate gland within the
field-of-view of the PET scanner in the initial attenuation-corrected
scans was made difficult by the obesity of the patient (height, 1.8 m;
weight, 147 kg).
Patient 3: FCH-PET imaging of an 80-year-old male status after
radical retropubic prostatectomy and bilateral scrotal orchiectomy
with progressive hormone refractory prostate cancer (PSA, 4172)
demonstrated extensive uptake of tracer in both bones and soft tissue
lesions (Fig. 6). SUV values in osseous lesions of the pelvis region
ranged between 3.8 and 8.0. SUV values for soft tissue lesions could
not be quantified due to the lack of attenuation correction in the
corresponding regions, but their signal intensities were similar to
those of nearby osseous metastases. The same patient was scanned
with FDG within the same week (Fig. 6). The FDG-PET images
demonstrated fewer lesions and less pronounced uptake in the detected lesions. SUV values for FDG were approximately one-half
those observed for FCH in the same lesions. No radioactivity was
observed in the urinary bladder on the FCH whole-body scan obtained
at ⬃25–29 min after injection. Following the PET scan (at ⬃1 h after
injection), the patient produced 70 ml of urine that was measured to
contain 1.3% of the injected dose of radioactivity. The experience in
the first three patients led us to modify the scanning protocol to allow
for imaging of the prostate gland with minimal chance of confounding
activity in the urinary bladder: a single whole-body scan would be
acquired, commencing over the pelvis 4 –5 min after injection of FCH.
Patient 4: A 65-year-old man with a history of clinical stage T3b
prostate cancer treated by radical retropubic prostatectomy was diagnosed with metastatic disease by bone scan. FCH-PET imaging was
performed before and after initiating androgen deprivation therapy. In
the first study, whole-body emission scanning was started 5 min after
injection, beginning at the pelvic region to image the pelvis and
prostatic bed before arrival of urinary radioactivity. The images (Fig.
7) demonstrated FCH uptake (SUVs, ⬎8) in several locations presumed to be local recurrence in the prostatic bed and metastatic
prostate cancer in the pelvic lymph nodes and bone. The osseous
metastases seen on FCH-PET images were corroborated by recent
bone scan results. The patient was rescanned 2 weeks after initiating
androgen deprivation therapy, at which time his serum PSA was 121
ng/ml. The repeat scan showed 35– 40% decreases of SUVs in the
various tumors (Fig. 7). Of particular note, a positive single lesion in
the left pelvis presumed to be prostate carcinoma within a pelvic
lymph node was not visualized on the repeat scan.
DISCUSSION
PET is used to detect and stage cancer because of its unique
strength in providing noninvasive assessment of metabolic and physiological rates through tracer techniques. The present results with
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F-LABELED CHOLINE FOR IMAGING PROSTATE CANCER
Fig. 6. Comparison of FCH (A) and FDG (B) PET images in patient 3 having advanced metastatic prostate carcinoma. The images are not attenuation corrected. Images are coronal
projections having a slice thickness of 12.9 mm. The patient had undergone radical retropubic prostatectomy and limited pelvic lymphadenectomy 12 years before. The serum PSA
level was 4172. Bone scans showed extensive osseous metastases. Both scans reveal soft tissue and osseous metastases, but FCH allowed detection of more lesions, and showed 2-fold
higher tracer uptake in osseous lesions as estimated by the SUV index (FCH, 8.0; FDG, 4.1).
FCH confirm the potential use of positron-labeled choline analogues
(7) for detection of prostate cancer. Differentiation of malignant
cancer tissue from neighboring nonmalignant tissues may be accomplished by exploiting changes in choline processing that may occur in
response to metabolic, genetic, or microstructural changes in malignant cells.
Choline is transported into cells and used for synthesis of phospholipids and sphingomyelin. Intracellular choline is rapidly metabolized
to PC or oxidized by choline dehydrogenase and betaine-aldehyde
dehydrogenase to betaine (mainly in liver and kidneys). Phosphoryl-
ation of choline, catalyzed by CK, is an obligatory step for incorporation of choline into phosphatidylcholine. Once phosphorylated, the
polar PC molecule is trapped within the cell. Extensive studies using
magnetic resonance spectroscopy (22) and biochemical analyses (23–
26) have revealed elevated levels of choline, PC, and phosphoethanolamine in many types of cancer cells. The activity of CK has been
found to be up-regulated in malignant cells (24 –26), providing a
potential mechanism for the enhanced accumulation of radiolabeled
choline analogues by neoplasms. These findings have motivated the
development of magnetic resonance spectroscopic imaging techniques
Fig. 7. Coronal projections of attenuationcorrected FCH-PET images in patient 4 having
hormone naı̈ve prostate cancer after radical prostatectomy (A) and in same patient 2 weeks after
initiating androgen deprivation therapy (B). The
slice thickness is 12.9 mm. Emission imaging was
commenced over the pelvic region at 5 min after
injection, preceding the arrival of radioactivity at
the urinary bladder. Before hormonal therapy, FCH
uptake is high (SUVs exceeding 8) in several osseous and soft tissue metastases in the pelvic region
and vertebrae. The accumulation of FCH in metastases was less pronounced in the follow-up study,
showing SUVs that were 35– 40% decreased. The
small focus of uptake (presumably in a lymph
node), demonstrated by the arrow, was not visualized in the follow-up study.
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F-LABELED CHOLINE FOR IMAGING PROSTATE CANCER
for mapping of choline levels in prostate cancer (27, 28) and brain
tumors (29, 30). For localization of prostate cancer in a sextant of the
prostate (27) and diagnosis of extracapsular extension of prostate
cancer (28) using proton MR imaging, the addition of magnetic
resonance spectroscopy imaging data on the ratio of choline plus
creatine to citrate was found to improve diagnostic sensitivity and
accuracy, particularly for less experienced readers (28).
The present results indicate that FCH closely mimics choline in its
uptake and sequestration by prostate cancer cells. Pretreatment of
cultured PC-3 human prostate cancer cells with 5 mM HC-3, a specific
inhibitor of choline uptake and phosphorylation (16, 31), resulted in a
90% decrease in FCH accumulation. However, because the biochemical form of F-18 radioactivity was not determined in our studies and
HC-3 acts as an inhibitor of both choline transport and CK-mediated
phosphorylation (16, 31), we cannot strictly conclude that FCH uptake
by the cells was dependent on CK activity. The inhibitory effects of
PI-3 kinase and EGF receptor kinase inhibitions on FCH accumulation were less pronounced than for choline transport/phosphorylation
inhibition by HC-3, suggesting the existence of other signaling pathways that are able to activate choline transport and phosphorylation
within malignant cells.
FCH showed accumulation in PC-3 prostate cancer xenografts in
mouse similar to radiolabeled choline. By 1 h after injection the
tumor:blood ratio had reached about 5:1, suggesting a trapping of
tracer in the tumor cells consistent with metabolic trapping via phosphorylation by CK. However, the accumulation of tracer in tumor
tissue was low relative to the normal uptake by kidneys, liver, heart,
and lung. The human imaging data also showed high normal uptake of
FCH in kidneys and liver but relatively low uptake in lung and heart
in comparison with the mouse data. The prostate cancer metastases in
the ribs were well differentiated from normal lung in the images.
These data suggest a species difference in the lung and heart uptake
of FCH.
Consistent with previous data with [11C]CH (4, 7), blood clearance
for FCH is very rapid in the human, and excellent tumor to background contrast is obtained in PET images by 3 min after injection.
The rapidity of the uptake process reveals the avidity of the choline
transport system in prostate cancer as well as certain normal tissues.
Tissue perfusion, transporter density, and CK activity are presumably
important determinants of tracer uptake and sequestration by tissues.
The short residence time of FCH in the blood may limit the diffusion
of tracer into areas that are poorly perfused. The potential effects of
perfusion, particularly during interventions that may affect tumor
vascularity and vasomuscular tension, should be carefully considered
when interpreting the uptake data. Also, there is potential for poor
sensitivity of this technique to detect malignant tissue that is poorly
perfused. On the other hand, the rapid and extensive clearance of
tracer from the bloodstream minimizes the effects of any potential
radiolabeled metabolites on the tissue kinetics. It also makes the SUV
parameter more useful than for a tracer that has a slow (and, therefore,
more variable) blood clearance at the time of measurement of tissue
uptake. Finally, the rapid blood clearance allows PET imaging to be
commenced as early as 4 –5 min after injection.
FCH differs from a previously reported (11) analogue, FEC, by a
single methylene group. It is not clear from the limited data provided
in abstract form on FEC (11) what differences may exist between the
tracer kinetic properties of FCH and FEC. Our finding that FCH and
CH have similar biodistributions in mice suggest that electronegativity effects of the ␣-fluorine atom are well tolerated by the processes
(presumably CK catalyzed phosphorylation) that are responsible for
the trapping of choline analogues in tissues. The notably higher
urinary clearance of FCH relative to choline was also seen with FEC
(11). Choline is efficiently reabsorbed by renal proximal tubular cells
under normal conditions. The primary route of clearance of choline
from the body depends on oxidation via choline dehydrogenase and
betaine-aldehyde dehydrogenase (primarily in the kidney) to betaine.
Betaine is then excreted in the urine. The more pronounced early
urinary clearance of the radiofluorinated choline analogues could be
explained by incomplete tubular reabsorption of intact tracer or enhanced excretion of oxidative metabolites.
Of high interest for future investigations is determining the metabolic fate of FCH in the cancer cell in relationship to choline processing in normal and transformed (malignant) cells. The finding of
avid uptake of FCH in prostate cancer with prolonged retention
strongly suggests metabolic sequestration of the tracer consistent with
trapping in the cell subsequent to phosphorylation in analogy to the
known processing of choline. However, detailed biochemical studies
are required to confirm this indication. It is known that transformation
is accompanied by increases in both choline and PC levels in a variety
of cancer types (22–26). The activation of the choline uptake and
phosphorylation seems to be a later event involved in a cascade of
intracellular signal transduction events that result in transformation,
including activation of ras-GTPase-activating protein (32), PI-3 kinase (33), and various protein and tyrosine kinases (33–35). Cuadrado
et al. (36) showed that PC triggered DNA synthesis in quiescent
NIH3T3 fibroblasts, whereas choline, phosphorylserine, and phosphoethanolamine had no effect. The CK inhibitor, HC-3, was found to
block proliferation induced by growth factors, but the blockade was
bypassed by PC addition. Thus, PC seemed to act as a prerequisite
second messenger for mitogenic activity, implicating CK activity as a
critical step during regulation of cell proliferation by growth factors
(36). If, as suspected, CK-mediated phosphorylation of radiolabeled
choline analogues determines their retention in tissues, these radiotracers may not only be useful as cancer detection probes, but may
also be used in experimental studies as unique tools to allow noninvasive monitoring of the regulation of an important signal transduction pathway.
Implications for Clinical PET Studies. There is keen interest in
the development of a sensitive and accurate noninvasive imaging
technique for prostate cancer. The existing imaging technologies,
including CT (37, 38), MRI (38, 39), ultrasound (39), nuclear medicine scanning with 111In-capromab pendetide (40), and PET imaging
with FDG (3, 8, 41– 44) are not sufficiently sensitive to obviate the
need for surgical staging pelvic lymphadenectomy. Radioisotope bone
scans are highly sensitive in detection of osteoblastic bone metastases,
but false positive readings may occur due to tracer uptake associated
with degenerative joint disease and related benign bone diseases and
abnormalities. Furthermore, bone scans cannot detect metastases in
lymph nodes and other soft tissue. Hara et al. (7) reported superior
sensitivity of [11C]CH-PET relative to FDG for the detection of
metastatic prostate cancer. Initial findings reported herein with FCH
in prostate cancer patients seems to agree with the data on [11C]CH.
The longer half-life of fluorine-18 (110 min) is better suited for the
demands of clinical PET and may allow off-site production and
distribution of the radiotracer. The rapid uptake kinetics of the
positron-labeled choline analogues allow transmission scanning before emission scanning while maintaining efficient use of the PET
scanner. The trapping of FCH in neoplasms makes PET scanning a
straightforward process uncomplicated by the effects of redistribution
of tracer over time. Furthermore, the simple kinetics of FCH may
support the usefulness of the ratio of tumor to reference tissue (e.g.,
lung) concentrations as a quantitative index, rather than the SUV
parameter. However, the presence of radioactivity in the urine encourages early imaging of the pelvic region before arrival of radioactivity
at the urinary bladder.
Preliminary data obtained in four patients with prostate cancer
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18
F-LABELED CHOLINE FOR IMAGING PROSTATE CANCER
suggest that further clinical research is warranted to determine the
diagnostic accuracy and sensitivity of 18F-labeled choline analogues
in prostate cancer detection and staging. FCH-PET imaging of two
patients with advanced disease demonstrated increased uptake in soft
tissue lesions in the pelvis that may correlate with pelvic lymph nodes.
Thus, FCH-PET imaging may represent a potential new imaging
technique for identifying nodal metastases and/or local recurrences in
patients with advanced disease; however, the validity of the technique
must be correlated with histological specimens in future prospective
studies before a definitive conclusion can be reached. Nevertheless,
preliminary data demonstrating increased uptake of FCH in known
metastatic lesions identified by bone scan in patients with advanced
prostate cancer does provide a rationale for future prospective clinical
investigations to evaluate the usefulness of this technique in prostate
cancer detection and staging.
Fluorine-18-labeled choline analogues may be useful in other malignancies where elevated choline and PC levels have been demonstrated, such as tumors in the breast, lung, colon, and brain (22, 23, 26,
29, 30, 45, 46). FCH-PET imaging may also be useful for monitoring
the therapeutic efficacy of traditional and novel chemotherapeutic
agents. The SUV index should be a fairly robust parameter to indicate
tracer trapping in neoplasms, but the potential effects of therapy on
tumor blood flow needs to be carefully considered since the uptake
kinetics are likely to be highly dependent on tissue perfusion.
Conclusions. The findings of up-regulation of choline uptake
and phosphorylation in neoplasms have motivated the development
of positron-labeled choline analogues for PET imaging of cancers.
In this work, a practical synthesis of the [18F]fluoromethylated
analogue of choline, FCH, was developed. FCH demonstrated
HC-3-sensitive accumulation in cultured PC-3 human prostate
cancer cells. FCH showed similar biodistribution to [14C]choline in
a murine PC-3 xenograft model with accumulation of tracers in
tumor tissue, but with more pronounced urinary excretion of radioactivity. Preliminary investigations using FCH-PET in patients
with prostate cancer demonstrated uptake in primary and metastatic lesions. These data demonstrate that FCH closely mimics
choline uptake and sequestration in prostate cancer cells and support the need for future investigations to determine the sensitivity
and accuracy of FCH-PET imaging for detection, staging, and
evaluation of recurrence of prostate carcinoma. Furthermore, investigations on FCH processing within the malignant cell in relationship to choline processing and mitogenic signal transduction
pathways are also of high interest.
ACKNOWLEDGMENTS
We thank Sharon Hamblen, Mary Traynor, and DeAndre Starnes for excellent technologist assistance and Collin McKinney for assistance with the
radiosynthesis apparatus. We recognize Ray Liao and Dawn Geverd for
assistance with biological experiments. Dr. Olof Solin of the Turku PET
Center provided helpful information on the synthesis of [18F]fluorobromomethane.
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Synthesis and Evaluation of 18F-labeled Choline as an
Oncologic Tracer for Positron Emission Tomography: Initial
Findings in Prostate Cancer
Timothy R. DeGrado, R. Edward Coleman, Shuyan Wang, et al.
Cancer Res 2001;61:110-117.
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