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Clinical Investigations |
Cotzias Neuro-Oncology Laboratory [R. G. B., B. B.] and Radiochemistry/Cyclotron Core Facility [R. D. F.], Memorial Sloan-Kettering Cancer Center, New York, New York 10021; PET Program [U. R., I. G., P. V., J. M., R. P. M., K. L. L.] and Institute of Medical Radiobiology [N. E. A. C., R. W., J. K.], Paul Scherrer Institute, Villigen CH-5232, Switzerland; Department of Neurosurgery, University Hospital, Zürich CH-8091, Switzerland [K. v. A., Y. Y.]; Department of Neurosurgery, Cantonal Hospital, Aarau CH-5001, Switzerland [H. L.]; and Department of Nuclear Medicine, University Hospital, Essen, D-45122, Germany [E. J. K.]
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ABSTRACT |
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The plasma half-life of [124I]IUdR was short (2–3 min), and the arterial plasma input function was similar between patients (48 ± 12 SUV*min). Plasma clearance of the major radiolabeled metabolite ([124I]iodide) varied somewhat between patients and was markedly prolonged in one patient with renal insufficiency. It was apparent from our analysis that a sizable fraction (15–93%) of residual nonincorporated radioactivity (largely [124I]iodide) remained in the tumors after the 24-h washout period, and this fraction varied between the different tumor groups. Because the SUV and Tm:Br ratio values reflect both IUdR-DNA incorporated and exchangeable nonincorporated radioactivity, any residual nonincorporated radioactivity will amplify their values and distort their significance and interpretation. This was particularly apparent in the meningioma and glioblastoma multiforme groups of tumors.
Mean tumor Ki values ranged between 0.5 ± 0.9 (meningiomas) and 3.9 ± 2.3 µl/min/g
(peak value for glioblastoma multiforme, GBM). Comparable SUV and Tm:Br
values at 24 h ranged from 0.13 ± 0.03 to
0.29 ± 0.19 and from 2.0 ± 0.6 to
6.1 ± 1.5 for meningiomas and peak GBMs, respectively.
Thus, the range of values was much greater for Ki
(
8-fold) compared with that for SUV (2.2-fold) and Tm:Br
(3-fold). The expected relationships between Ki, SUV,
and Tm:Br and other measures of tumor proliferation (tumor type and
grade, labeling index, and patient survival) were observed. However,
greater image specificity and significance of the SUV and Tm:Br values
would be obtained by achieving greater washout and clearance of the
exchangeable fraction of residual (background) radioactivity in the
tumors, i.e., by increased hydration and urinary
clearance and possibly by imaging later than 24 h after
[124I]IUdR administration.
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSAppendix 1REFERENCES |
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The potential for obtaining functional images of DNA synthesis using PET and SPECT has been recognized for some time (4, 5, 6) . PET and SPECT provide the opportunity to perform noninvasive measurements of uptake, distribution, and clearance of radiolabeled precursors of DNA in both tumor and normal tissues of patients with cancer. Our proposed use of radiolabeled IUdR ([124I]IUdR) is substantially different from other investigations that use [11C]TdR as a radiolabeled precursor of DNA synthesis (7 , 8) . The application of a time-dependent strategy for "wash-out" of radiolabeled metabolites (mainly iodide) to reduce background activity and improve image specificity is feasible with [124I]IUdR and PET imaging because of the 4.2-day physical half-life of 124I. A "wash-out strategy" is not possible with compounds labeled with 11C or 18F because of the short physical half-lives of these radionuclides (20 and 110 min, respectively). Alternatively, kinetic modeling based on knowing the time course of parent compound and radiolabeled metabolites in blood can be used to correct measured tissue activity for non-DNA incorporated radioactivity.
This work represents an extension of our previous studies using [131I]IUdR (9) and [123I]IUdR (10) and SPECT imaging. These earlier studies demonstrated that improved tomographic sensitivity would be beneficial, if not necessary. Because of the low count rates in the SPECT images, it was apparent that IUdR-SPECT could only identify the most active regions of tumor proliferation. We decided to initiate the studies reported here using [124I]IUdR to achieve better tomographic sensitivity and counting statistics than are possible with PET (in comparison with SPECT). In 20 patients with primary brain tumors, we demonstrate the first clearly defined relationships between [124I]IUdR uptake and retention and several independent measures (indices) of tumor proliferative activity, i.e., we compare the image-derived values of the IUdR-DNA incorporation constant (Ki), the SUV, and the Tm:Br radioactivity concentration ratio to other independent indices of tumor proliferation (tumor type and grade, labeling index, and survival).
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSAppendix 1REFERENCES |
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Fourteen of the 20 patients underwent surgical resection of their tumor within 3 days of completing their PET study, and BrdUrd LIs were determined. In those patients who underwent partial, subtotal, or total tumor resection, multiple tissue specimens (up to six) were obtained from different tumor sites. The individual LI values were averaged to result in a "global" LI for each tumor. A "peak" LI (highest individual) value was also used in patients with GBM because the individual LI values frequently varied in specimens obtained from this tumor. Peak LIs in patients with GBM were also correlated with peak IUdR uptake measures in the same tumor, although there was no attempt to confirm spatial correspondence between the measures. In the single patient who underwent tumor biopsy (patient 12), only one tissue sample was available.
Patient follow-up has extended over a 17–30-month period, and survival data are available on 19 of the 20 patients. Living patients were documented as of January 1999 and include 10 of the 19 evaluable patients. Note that patient 19 died of a pulmonary embolus.
Production of [124I]Iodide.
124I was routinely produced by The Cyclotron
Corporation CV 28 compact cyclotron in Essen, Germany, via the nuclear
reaction 124Te(d,2n) 124I
by irradiation of enriched
124TeO2 (89.6%) with 14
MeV deuterons. After thermodistillation (6 min at 740°C), the
124I product was adjusted to a specific activity
of 12 Ci/µmol by iodide carrier (11)
. The
solution [0.02 M NaOH (12)
] was obtained in
a volume not higher than 100 µl. It was transported by car to the
Paul Scherrer Institute. The product had the following contaminants
[their average relative yield at the time of patient administration
(45 h after target irradiation) is given in
parentheses]: 13.2-h 123I (0.15%), 60-d
125I (1.89%), 13.0-d 126I
(1.43%), 12.4-h 130I (0.87%), and 8.02-d
131I (0.35%; Ref. 13
).
Synthesis and Quality Assurance of
[124I]Iododeoxyuridine.
[124I]IUdR was routinely synthesized by
direct electrophilic labeling of 2'-deoxyuridine with
124I (12)
. Six hundred µg of
2'-deoxyuridine were dissolved in 250 µl of 0.2 M
phosphate buffer (pH 7.4). In an IodoGen-coated ReactiVial, the
solution was allowed to react for 15 min at 65°C under stirring with
185 MBq (5 mCi) 124I in 100–200 µl of solution
(including the rinsing solution). The product was separated by SEP-PAK
C-18 cartridges and was washed with 30 ml of water that eluted
deoxyuridine, unbound iodide, and the phosphates. The labeled compound
was then eluted with 2 ml ethanol, and the solvent was slowly
evaporated at 65°C under nitrogen atmosphere. The residue was taken
up with 8 ml of physiological saline solution and filtered through a
0.22-µm Millex GS sterile filter. The radiochemical yield was
60%.
Quality control was performed by TLC (Silicagel 60, F254, 5 x 20, acetone) directly after synthesis and by radio-HPLC (Ultrasphere RP 18, 5 µm; eluent of water:methanol, 80:20 v/v) shortly before administration. On the basis of >30 syntheses, the average contamination grade was determined to be 2.9% by TLC (mainly iodate) and 4.3% by HPLC. A purity grade of 95% [124I]IUdR could be kept routinely prior to injection into patients. Analysis of [124I]IUdR metabolites showed that the in vitro stability of the solution allowed a time window of 2 days following the end of synthesis for patient administration (13) .
[124I]IUdR PET Imaging.
On the day of study, a radial artery and a peripheral hand or arm vein
were catheterized for arterial blood sampling and radiopharmaceutical
administration, respectively. Patients were positioned in parallel to
the orbitomeatal line. Head movements were restricted by an
individually formed plastic head support. Before tracer injection, a
10-min transmission scan was acquired in each scanning position (see
below) using a
[68Ga]/[68Ge] ring
source. Patients received a 10-ml i.v. infusion of
[124I]IUdR (28.0–64.4 MBq) over 3 min. A
dynamic series of arterial blood samples and image acquisitions were
obtained over 48 min (time frames: 3 x 1 min,
10 x 3 min, and 3 x 5 min) using an
ECAT 933/04-16 (Siemens-CTI PET Systems, Knoxville, TN). Given the
limited axial sampling of the ECAT 933/04-16 (seven slices over 5.6 cm
with an in-plane resolution of 8–9 mm FWHM), the couch was
moved, and the head was re-imaged (50 to 65 min) to obtain a 14-slice
image set of the whole head. The initial dynamic sequence was always
obtained with the bulk of the tumor within the axial field of view. At
completion of the first day of study, contour marks of the patient’s
head were administered to the plastic head support to allow
repositioning of the head in the scanner with the same support at the
time of the washout study. Twenty-four h after
[124I]IUdR administration, a repeat imaging
session (60 min acquisition time, 30 min per 7-slice image set) was
performed. Two venous blood samples were also taken at that time and
were averaged. Reconstructed images were obtained from the PET data.
Corrections were made for randoms, scatter, dead-time, detector
inhomogeneity, and attenuation. Image data were calibrated to Bq/ml
based on a series of [124I] phantom studies and
decay corrected to the time of injection.
[124I]Iodide PET Imaging.
The same data acquisition protocol (PET, arterial blood) and data
analysis was used in these experiments as described for the
[124I]IUdR studies. Similar doses of
[124I]iodide were injected (50–62.4 MBq), as
an i.v. infusion over 3 min.
Image Registration.
In selected patients, both the 24-h and 0–65-min PET images were
registered to the MR image. For this purpose, PET image data sets of
both studies were processed to form a contiguous volume of 14 adjacent
planes. Scalp contours were defined on each of the 14 image planes of
this volume, and similar contours of the scalp were derived from the MR
images. The PET to MR registration was conducted using the Pelizzari
(head and hat) algorithm (14)
. The registered PET images
were resliced using trilinear interpolation along the planes of the MR
scan.
Measurement of [124I]IUdR and Radiolabeled
Metabolites in Plasma.
The plasma input function of [124I]IUdR
was determined from 21 arterial blood samples drawn from a catheterized
radial artery during the initial scan. Samples were immediately placed
on ice and centrifuged at 4°C to obtain cell-free plasma. Plasma and
whole-blood radioactivity was measured in a well counter that was
cross-calibrated with the PET camera and expressed as Bq/ml and
percentage of injected dose/ml of plasma. Up to 13 plasma samples,
collected during the first 15 min after
[124I]IUdR administration, were further
analyzed for parent compound and radioactive metabolites using an
HPLC-radiation detection system as described previously
(15)
. Seven hundred µl of cell-free plasma was mixed
with an equal volume of ice-cold perchloric acid (0.8 M
perchloric acid, 1%
Na2S2O5,
and 0.1% EDTA) for deproteinization. After centrifugation, 1 ml of the
supernatant was directly injected into the HPLC system. Metabolite
separation was accomplished with a reverse-phase column (Beckman
Ultrasphere RP18, 5 µm, 250 x 4.6 mm) using
methanol:water (20:80 v/v) with a flow rate of 1 ml/min as mobile
phase. The retention time of the unchanged tracer (7 min) and the
metabolites (unbound [124I]iodide at 2.5 min
and [124I]iodouracil at 5 min) were verified
using unlabeled standards and UV absorption at 254 nm. The integrated
peaks of tracer and metabolites were expressed as percentage of total
plasma activity at the sampling time.
Data Analysis.
ROIs were determined using PET image data acquired from summed 33–48
min postinjection image frames. Elliptical whole tumor ROIs were then
drawn on two or more adjacent planes of the summed images; their
positioning was guided by visual comparison with the T1- and
T2-weighted MRI or contrast-enhanced CT scans. Particular attention was
paid to avoid inclusion of necrotic or cystic areas into the ROIs
(glioblastomas). Peak tumor ROIs were obtained from the GBM tumor
regions with highest tracer retention at 24 h, because GBMs
frequently demonstrated intratumoral heterogeneity of radioactivity
concentration. Elliptical ROIs for normal brain were obtained
contralateral to the side of the tumor ROIs. The ROIs were then applied
to the full dynamic series and to the 24-h images, and decay corrected
tissue time-activity curves were generated.
Three tumor proliferation parameters were calculated from the ROI data: (a) the IUdR-DNA incorporation clearance constant, Ki; (b) the SUVs in the 24-h image; and (c) the tumor:normal brain radioactivity ratio in the 24 h image, Tm/Br.
The calculation of the IUdR-DNA incorporation clearance constant,
Ki, involves a two-step process. The first step involves an
estimation of the IUdR-iodide tissue plasma volume (Vp) and
tissue distribution volume (Ve) from a kinetic analysis of
the 0–48-min dynamic uptake data (see the "Appendix"). The second
step involves the calculation of Ki:
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CpIUdRdt (Bq*min/ml) is the plasma
IUdR concentration-time integral (input function).
Cp24h largely reflects the
concentration of radiolabeled iodide. Eq. A assumes that the IUdR-DNA
incorporated radioactivity is irreversibly trapped during the course of
the study (16
, 17)
.
The SUV is a commonly used parameter in the clinical evaluation and
comparison of nuclear medicine studies; it is unitless and was
calculated in the standard manner (18
, 19)
:
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where the injected dose (Bq) and body weight (g) are known. SUV corrects for differences in dose of radiopharmaceutical administered and body weight between individual patient studies; it provides a better index for a comparisons between different patients and for the comparison of serial studies in a single patient.
The tumor:normal brain radioactivity concentration ratio (Tm:Br) was also calculated. An advantage of SUV and Tm:Br for routine clinical studies is that the arterial input function does not have to be determined. The disadvantage in expressing results in terms of SUV and Tm:Br is that the measured tissue radioactivity is not corrected for residual, non-DNA incorporated radioactivity in the tissue [i.e., Cp24h * (Ve + Vp)] or for differences in the [124I]IUdR input function between different subjects (20) .
The calculation of the fraction of total tissue radioactivity (Am24h) in the vascular (Vp), extracellular (Ve), and DNA-incorporated compartments at 24 h is based on the estimates of Vp and Ve, and the concentration of radioactivity in plasma at 24 h (Cp24h); where the percentage of activity in Vp = 100*(Vp* Cp24h)/Am24h, the percentage activity in Ve = 100*(Ve* Cp24h)/Am24h, and the percentage of activity in DNA = 100 - (% in Vp + % in Ve).
Labeling Index.
The LI was determined based on a modified assay first used to
determine tumor potential doubling times (21)
. The method
has been described in detail elsewhere (22)
. Multiple
biopsy samples were derived from resected tumors from patients
administered BrdUrd 20 min previously. The biopsies were immediately
fixed in 50% ethanol at 4°C. A single-cell suspension for flow
cytometric evaluation was obtained from each biopsy as described
previously (22)
. The cell density of the final suspension
was monitored using an electronic counter (Coulter counter). The pellet
was then resuspended in 100 ml HBSS and 20 ml of anti-BrdUrd FITC
antibody (Becton Dickinson, Basel, Switzerland), incubated at room
temperature for 25 min with occasional manual agitation, washed, and
stained with 20 ml of propidium iodide (1 mg/ml) prior to analysis
using a FACScan flow cytometer (Becton Dickinson).
Statistics.
The Pearson product moment correlation coefficient was determined for
combinations of LI with each of the three PET-derived measures
(IUdR-DNA Ki, SUV, and Tm:Br). In addition, each of the
PET-derived parameters plus LI was correlated with survival. Plots were
generated for each of the above-described relationships.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSAppendix 1REFERENCES |
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. Fourteen of the 20 patients had a surgical resection of their
tumor within 3 days of the IUdR PET study, and LIs were determined in
multiple tumor fragments. Three additional patients were injected with
[124I]iodide, and the pharmacokinetics of
iodide uptake and clearance from tumor and brain tissue was studied in
the same manner as that described above for
[124I]IUdR; these results are presented in the
"Appendix."
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. The total plasma activity peaked at
the end of the infusion (3 min). HPLC analysis revealed very fast
metabolism of [124I]IUdR and revealed two
radiolabeled metabolites, iodouracil and iodide. The fraction of
iodouracil, the product of the initial degradation step catalyzed by
nucleoside phosphorylases, remained small because of rapid
dehalogenation to uracil and iodide. At 5 min after injection,
[124I]iodide was the major radiolabeled
metabolite, and the concentrations of
[124I]IUdR and
[124I]iodide were approximately equal. After 10
min, [124I]iodide accounted for >85% of total
plasma radioactivity. The mean plasma half-time of
[124I]IUdR was calculated from exponential
curve fits of the individual patient data (Fig. 1, C and D)
; the mean clearance half-time was estimated as
2.5 ± 0.5 min. These results are similar to those that
we reported previously for [131I]IUdR
(9)
. The input function (plasma concentration x time) of IUdR was calculated from the HPLC determined time
course of [124I]IUdR in plasma and was similar
for all tumor groups (Table 2)
.
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for low- and high-grade tumors,
respectively. The uptake profiles differed considerably in low-grade
astrocytomas and glioblastomas and between normal brain and the higher
grade tumors. The tissue and plasma radioactivity profiles were fitted
to several possible pharmacokinetic models to account for IUdR and
iodide transport as well as IUdR incorporation into tissue (tumor) DNA
and yielded multiple solutions or highly unlikely rate constants with
large error estimates. The inclusion of reasonable constraints was also
applied to the model to reduce the number of parameter estimates, but a
substantial improvement could not be obtained.
Therefore, we used a simpler, two-compartment model (blood and
tissue) to estimate tissue plasma volume (Vp), the initial
plasma clearance (influx) constant (K1), and the tissue
distribution volume (Ve) of
[124I]IUdR-derived radioactivity (see the
"Appendix"). Estimates of Vp and Ve were
necessary to calculate the IUdR-DNA incorporation constant,
Ki (Eq. A), from the images that were obtained at 24 h.
Mean Vp, Ve, and K1 values for the different
tumor groups and contralateral brain tissue are shown in the
"Appendix"
and can be compared with the values obtained
in an additional three patients after
[124I]iodide injection (Appendix;
).
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compared with that at 1 h (Fig. 2, B and E)
. The level of radioactivity in the tumor
at 24 h was also substantially less than that at 1 h,
indicating that considerable "washout" of radioactivity had
occurred (radioactivity that was not incorporated into DNA).
Radioactivity retained in tumor tissue at 24 h after IUdR
administration was shown previously to predominantly reflect IUdR-DNA
incorporation in rodent tumors (17)
.
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, and individual patient
data points are shown graphically in Fig. 3A
. Only the Ki value for meningiomas was not
significantly different from zero. A comparison between low-grade
gliomas (WHO grade II), anaplastic gliomas (WHO grade III), and GBM
(WHO grade IV) revealed no significant differences in Ki,
whereas the Ki of anaplastic gliomas and glioblastomas was
significantly higher than the Ki of meningiomas.
|
; Fig. 3, B and C
). The SUV for each of the different tumor groups was
significantly higher than that of contralateral brain tissue, although
these values include background radioactivity that did not completely
clear during the 0–24-h postinjection period. The Tm:Br ratio was
significantly greater than unity for all tumor groups, and significant
differences between the tumor groups was observed, except between
meningiomas and low-grade gliomas (Table 2)
. The expected progression
in mean Ki, SUV, and Tm:Br values with tumor grade was
observed (Fig. 3)
.
Comparisons.
An independent measure of tumor proliferation, the BrdUrd LI, was
determined on multiple tumor samples obtained at surgery in 14 patients
(Table 2
and Fig. 3D
). The expected relationship between
Ki, SUV, Tm:Br ratio, and the BrdUrd LI was observed in
those patients who underwent surgery after the IUdR PET study (Fig. 4)
.
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. The Tm:Br ratio demonstrated the highest correlation to the BrdUrd LI
(Fig. 4C)
and to survival (Fig. 5C)
.
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: creatinine 108 µmol (normal range 53–94), urea 10.8
mmol/l (1.8–8.2)] had substantially higher levels of plasma
radioactivity at 24 h (predominantly
[124I]iodide). The plasma concentration was
1.08 SUV/ml, compared with a mean value of 0.31 ± 0.16
SUV/ml for the other 19 patients at 24 h. Not surprisingly, the
tumor:plasma activity ratio was comparatively low (0.58), compared with
a mean value (0.89 ± 0.19) for the other six GBM
patients at 24 h. The Ki value calculated by Eq. A (2.9
µl/min/ml; which corrects for plasma and non-DNA incorporated
radioactivity) was more consistent with that of other patients with GBM
(2.0 ± 0.3 µl/min/ml). Without this correction, the
calculated value of Ki would be 8-fold higher and would
reflect a substantial error by including non-DNA incorporated
radioactivity. Of note, the "peak" Ki for this
patient’s GBM (patient 1) was considerably higher (3-fold) than the
"peak" values for other GBMs, suggesting that part of this tumor
was growing very rapidly (which is consistent with a very short
poststudy survival, 1.6 months; Table 1
). The tumor and "peak"
tumor SUVs, which are not corrected for non-DNA incorporated
radioactivity or the arterial input function of IUdR, were very high
(0.63 and 0.69 SUV, respectively) in this patient compared with that of
the other six grade IV tumors (0.16 ± 0.06 and
0.23 ± 0.08 SUVs, respectively; Table 2
). On the basis
of our kinetic analysis of the data (see the "Appendix"), only 12
and 34% of measured radioactivity (SUV) in the "viable-appearing"
and "peak" regions, respectively, of the tumor at 24 h
reflects IUdR-DNA incorporated activity. This is because of the
combined effects of a comparatively high blood volume
(Vp = 0.075 ml/cc) and extracellular
space (Ve = 0.435 ml/cc) and high blood
radioactivity level (Cp = 1.08 SUV/ml) at the
time of imaging. In contrast, the Tm:Br ratio for patient 1 was not
substantially different from that of the other GBMs.
FDG and IUdR Imaging.
In one patient (no. 1), both IUdR and FDG imaging was performed;
[18F]FDG preparation and the clinical imaging
protocol have been described (23)
. The 24-h IUdR image is
somewhat compromised by high levels of residual plasma radioactivity,
as described above. Nevertheless, an interesting relationship is seen
by a direct comparison of the IUdR and FDG images (Fig. 6, A and B)
. This comparison shows foci of both
high IUdR and high FDG activity, but they are spatially
separated in the tumor. A pixel-by-pixel scattergram of the
registered IUdR and FDG images also demonstrates the differences in the
two images (Fig. 6C)
.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSAppendix 1REFERENCES |
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40-fold, with an improvement of 8.5-fold in the relative number of
detected events. A 2–14-fold further improvement in tomograph
sensitivity and relative number of detected events is obtained by
current-generation PET tomographs compared with the ECAT 933/04-16.
Comparisons.
The critical issue addressed in this study is whether the
[124I]IUdR PET images (and the derived values
of Ki, SUV, and the Tm:Br activity ratio) correlate with
other independent measures of tumor malignancy, including tumor grade,
BrdUrd labeling index, and patient survival. We present the first such
comparisons in 20 patients with primary brain tumors (see Figs. 3
4
5
),
and the results are both encouraging and discouraging. All three
measures of tumor proliferation (Ki, SUV, and Tm:Br) yielded
similar results. The expected correlation of these three measures with
an independent assessment of tumor proliferation (LI) was particularly
strong, and this was encouraging (see Fig. 4
). Similarly, the
correlation of these three measures with patient survival was good,
although there was some scatter in the data and one patient death
(pulmonary embolus) that was not related to his tumor. It should be
noted that histological features (e.g., tumor type, grade,
and presence of necrosis) as well as patient age are among the
strongest predictors of survival in patients with primary brain tumors
of glial origin (26
, 27)
.
A discouraging aspect of our analysis was the finding that the Tm:Br
ratio demonstrated the best (or nearly the best) correlation with the
three independent measures of tumor malignancy. Our expectation was
that Ki would provide the most accurate assessment of tumor
proliferation. Reviewing the data provides some insight into this issue
(see the "Appendix," particularly the assessment of Ve
and Vp). The calculated percentage of measured (imaged)
radioactivity at 24 h in the tissue compartments was different in
the different tumor groups (Table 3)
, and this difference impacts on the calculation of Ki (Eq.
A) as well as SUV and the Tm:Br ratio. The calculated fraction of
reversible (non-DNA incorporated) radioactivity in the 20 patients
reported in this study was substantially greater than that obtained in
our previous studies in animals, where <10% of measured tumor
radioactivity at 24 h was not incorporated in DNA
(17)
.
|
and Fig. 3
. This is because of the
substantial differences in the percentage of measured radioactivity in
Vp, Ve, and DNA (Table 3)
. The presence of poor renal
function and reduced renal clearance of radiolabeled iodide further
amplifies the magnitude and fraction of exchangeable radioactivity in
the tissue, even after a 24-h period of washout (patient no. 1, see
above). A large residual fraction of exchangeable radioactivity in the
tumor results in higher tissue background activity and a loss of
IUdR-DNA image specificity, and it will amplify the SUV and the Tm:Br
values.
The calculation of Ki attempts to correct for the
exchangeable fraction of intravascular and extracellular radioactivity.
A question could be raised whether this calculation is accurate,
i.e., whether the estimates of Ve and
Vp from a kinetic analysis of IUdR-derived radioactivity
data are accurate. We suggest that our numbers for the exchangeable
fraction of radioactivity in brain and low-grade and anaplastic tumors
with low vascular permeability (low K1; see
in the "Appendix") may be an underestimate, whereas in high grade
gliomas with comparatively high vascular permeability (high
K1) the estimate of Ve and the exchangeable
fraction of measured radioactivity may be somewhat overestimated. The
basis for this assessment is discussed in the "Appendix."
Gliomas can be very heterogeneous and may include areas of necrosis and exhibit a wide range of intratumoral cellular density (particularly high-grade gliomas), which will impact on the measurements. To address these issues in part, the grade IV gliomas were also analyzed with respect to the "peak" area of radioactivity observed in the 24-h images. The rationale for "peak" tumor assessments has been discussed previously (28 , 29) and was performed with the goal of identifying the most proliferative region of the tumor and presumably reflecting a more homogenous component. The 24-h images of the anaplastic and low-grade gliomas were considerably more uniform, and no attempt was made to perform "peak" activity analysis of these tumors.
Limitations and Advantages.
Some limitations of current methods to image tumor proliferation are
related to the rapid metabolic degradation of the nucleoside
(e.g., radiolabeled IUdR and TdR) in blood, because of
hydrolysis of the glycosidic bond by TdR phosphorylase. TdR
phosphorylase is homologous with platelet-derived growth factor and
limits the magnitude of the input function. The arterial input function
of IUdR (48 ± 12 SUV*min; Table 2
) is low compared with
the input functions of FDG [170 ± 30 SUV*min;
calculated from previous FDG experiments (23)
]. In
addition, the fraction of tumor cells that are undergoing division and
are in S-phase at the time of IUdR injection is low in brain tumors,
which further limits the amount of IUdR incorporated into DNA. Thus,
the level of radioactivity in the tumors reflecting DNA incorporation
is relatively low (0.13–0.29 SUV; Table 1
) compared with other
tumor-imaging agents, such as FDG, where brain cortical values are
substantially higher (3.8 ± 1.3 SUV; Ref.
23
).
A variable common to all cell metabolism, transport, or cell epitope imaging studies is cell density within the field of view. Glioblastomas can have substantial differences in regional morphology (ranging from densely packed viable cells with high proliferative indices to areas of low cell density, microcystic changes, and areas of frank necrosis or cyst formation). Low-grade gliomas can also vary with respect to cell density and microcystic changes, whereas anaplastic gliomas tend to be more homogeneous and more densely packed. Thus, the level of radioactivity in the images is influenced by several factors and should be considered as a reflection of the magnitude of proliferating cells within the field of view. For this reason, we elected to express our data for GBM as a "peak" ROI value as well as a whole tumor value (excluding frankly cystic and necrotic components identified on the MR image).
A morphologically heterogeneous tumor is a potential source of error, particularly when cystic or necrotic regions of a tumor cannot be clearly identified and excluded on the basis of the MR image. Fluid-containing cysts or necrotic tumor regions can act as "reservoirs" of residual (exchangeable) radioactivity that clear more slowly compared with other parts of the tumor. Some of these areas were initially mistaken for active proliferative regions on the 24-h PET images, prior to the registration and comparison with the MR images. Thus, frankly necrotic and cystic tumor regions must be identified and excluded from the analysis to avoid potential confusion in the interpretation of the PET images.
Nevertheless, there are specific advantages for [124I]IUdR proliferation imaging. The long (4-day) physical half-life of 124I, as well as other isotopes of iodine (123I and 131I) that can be used to label IUdR, provides the opportunity to use a "washout" strategy and "late" imaging. We have described this imaging strategy previously (9 , 17) . A 24-h washout period was used in this study; iodide is cleared by the kidney and gastrointestinal tract, and >70% of radiolabeled iodide is excreted in the urine during the first 24 h (16 , 30 , 31) . Thus, background radioactivity will be substantially lower 24 h after IUdR administration compared with that at 1 or 2 h. As described above, the 24-h washout period did not achieve the level of tissue clearance nor image specificity that was achieved in our previous animal experiments (17) . We estimate that only 53–85% of glioma radioactivity measured at 24 h reflected IUdR-DNA incorporation; in meningiomas, it was only 7%.
A similar washout strategy is not possible with
[11C]TdR imaging of tumor proliferation.
Measurements of 11C radioactivity in tumor
and normal tissues within 1 h of
methyl-[11C]TdR administration
include a large fraction of radiolabeled metabolites (6
, 32)
. The 20-min physical half-life of 11C
is too short to allow for sufficient tissue and body clearance of these
metabolites. Recently, it has been suggested that
11C labeling of TdR on the 2' position of the
pyrimidine ring results in fewer radiolabeled metabolites and
that an increased percentage of total tissue radioactivity is measured
in the acid insoluble or DNA fraction at 1 h after
2'-[11C]TdR (80%) compared with
methyl-[11C]TdR (40%) injection
(33
, 34)
. However, substantially greater amounts of
11C-labeled carbon dioxide and bicarbonate are
measured after administration of 2'-[11C]TdR.
It has been shown in the dog that the predominant fraction of blood
radioactivity is
[11C]CO2/[11C]CO3-
3 min after 2'-[11C]TdR administration;
[11C]CO2/[11C]CO3-
accounts for 70% of total blood activity between 5 and 60 min, and
only 47% of total administered radioactivity is exhaled over 10 min
(32)
. At least 25% of blood radioactivity will be
reflected in background tissue radioactivity attributable to
bicarbonate (33)
. To address these issues, various groups
have resorted to more "sophisticated" analytic methods and complex
modeling to obtain reliable measures and parametric images of tissue
(tumor) proliferation (7
, 8
, 36) .
Applications for Tumor Proliferation Imaging.
It must be emphasized that tumor proliferation imaging is not
limited to brain tumors and can be applied to most systemic solid
tumors as well (7
, 8
, 36, 37, 38)
. It is also important to
note that the spatial location of high
[124I]IUdR uptake does not necessarily
correspond to the location of high metabolic activity measured with
[18F]FDG in the same tumor (see Fig. 6
). A
comparison of the registered images presented in Fig. 6
shows that
there are foci of high IUdR and high FDG activity, but they are
spatially separated in the tumor. It should also be noted that regions
of high FDG accumulation within experimental tumors have been shown to
be associated with morphological changes of hypoxia-ischemia, impending
necrosis, and the infiltration of macrophages (39, 40, 41)
.
Tumor proliferation imaging provides significantly different
information from that provided by FDG or other commonly used imaging
agents. This was demonstrated in our initial studies comparing
[131I]IUdR SPECT with
[201Tl]thallium SPECT and
[18F]FDG PET imaging (9)
. Images
obtained with IUdR, FDG, methionine, or thallium reflect different
biochemical and biological processes that contribute to the
accumulation and retention of these radiopharmaceuticals and
provide substantially different information about tumor phenotype.
An important issue for future applications of tumor proliferation imaging is whether the values, or more specifically a change in the value of Ki, SUV, and Tm:Br, are predictive of treatment response, i.e., can tumor proliferation imaging with [124I]IUdR, [11C]TdR, or any other radiopharmaceutical predict treatment response "early" in the course of therapy, prior to changes in tumor volume or energy (glucose) metabolism? Early assessment is particularly relevant to the newer biological treatments involving cytostatic (in contrast to cytolytic) drugs, where the evaluation of treatment response can be protracted over months. An extended period of observation is often required before significant changes in the patient’s clinical status or in the radiographic evaluations are observed, and these criteria are currently used to define treatment response. Can tumor proliferation imaging accurately predict treatment response before the standard assessments of "time to progression of disease" and "survival" can be completed (38 , 42) ?
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Appendix 1 |
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. For the [124I]iodide experiments, the
0–48-min and 0–24-h data sets for each of the three patients were
fitted using the same two-compartment model; the results from the
0–24-h data set are shown in
. A comparison of the 0–24-h fitted values
and the 0–48-min
fitted values (not shown) suggests that the values from the 0–48-min
fit approximate the values from the 0–24-h fit when the permeability
of the vessels (reflected in K1 and k2) is high,
whereas in contralateral brain and in the anaplastic astrocytoma, where
vascular permeability is comparably low, the value of
Veiodide (Ki/k2) obtained
from the fit of the 0–48-min data underestimates the value obtained
from the fit of the 0–24-h data. The value of
Veiodide for brain tissue estimated
from the 0–24-h data set
approximates the reported values
of brain extracellular space, whereas brain
Veiodide estimated from the 0–48-min
data set was substantially lower (0.030 ± 0.003 ml/g).
These differences are most likely attributable to the low count rate in
many of the images and resultant noise in the images. For the [124I]IUdR studies, we fitted the tissue and plasma radioactivity profiles to several possible pharmacokinetic models to account for IUdR and iodide transport as well as IUdR incorporation into tissue (tumor) DNA. These fits yielded multiple solutions or highly unlikely rate constants with large error estimates. The inclusion of reasonable constraints were also applied to the model to reduce the number of parameter estimates, but a substantial improvement could not be obtained. Our inability to obtain reliable parameter estimates for IUdR-DNA incorporation in tumor tissue from pharmacokinetic modeling and fitting the data were primarily related to: (a) the low IUdR-DNA signal relative to the total signal measured during the initial 48-min period of imaging (when the images primarily reflected [124I]iodide); and (b) the relatively low count rate, resulting in image frames with a low signal:noise ratio. A substantial improvement in scanner count rate (>5-fold) is expected using current-generation tomographs and 3-dimensional (septa out) image acquisitions.
The two-compartment model that we used to fit the 0–48-min
[124I]IUdR data set assumed similar
transcapillary exchange parameters and distribution volumes (plasma and
extracellular) for IUdR and iodide, and it also assumes that the
IUdR-DNA component of measured tissue radioactivity during the
0–48-min period is small (see Fig. 1, A and B
).
The time course of total tissue and plasma radioactivity was used in
the fits, and no attempt was made to account for different radiolabeled
species (e.g., [124I]IUdR,
[124I]IU, or
[124I]iodide) or irreversibly bound
radioactivity (IUdR-DNA). The two-compartment model has the advantage
of simplicity, and reasonably good and consistent fits of the data were
obtained (see Fig. 1, E and F
).
The model simplifications described above are reasonable given:
(a) the octanol-water partition coefficients of IUdR and
iodide;4(b) the fraction of IUdR binding to serum proteins
[e.g., non-bound fraction = 56.0 ± 11.9%
(n = 6)];5(c) the low fraction of measured radioactivity that
represents IUdR-DNA during the 0–30-min period (see Fig. 1, A and B
); and (d) the fact that beyond
5 min the dominant radioactive species in blood is
[124I]iodide (see Fig. 1, C and D
). For high-grade gliomas, only 19 ± 18%
of the radioactivity imaged at 1 h was estimated to be
attributable to IUdR-DNA incorporation; for meningiomas, the value was
<4%.
Implications of K1, Ve, and Vp
for Imaging Tumor Proliferation with IUdR.
The tumor estimates of Ve and Vp obtained
from the two-compartment fit of the 0–48-min
[124I]IUdR data are reasonable
,
although they are not perfect. Nevertheless, they were used in the
calculation of Ki (Eq. A). The 5-fold higher K1
for brain in the [124I]IUdR studies (compared
with the [124I]iodide studies) probably
reflects the higher lipid solubility of IUdR (compared with
iodide4), although IUdR is partially bound to
plasma proteins (see above). Also of note is the 40% lower estimate of
brain Ve in the IUdR study (Table
) compared with that in
the iodide study
and to other published estimates of brain
extracellular space (43
, 44) . This is partially explained
by the noisy data and shorter duration (0–48 min) of the IUdR data set
used in the fit
, compared with the longer duration (0–24 h)
of the iodide data set
as discussed above. Only 50% of
brain radioactivity measured at 24 h is accounted for by using the
values of Ve and Vp shown in
. In
contrast, 81 and 105% of measured brain radioactivity can be
accounted for if the published values of brain extracellular space
(Ve) for white matter (0.11 ml/g brain) and gray matter
(0.15 ml/g brain), respectively, are used in these calculations. This
comparison is appropriate and consistent with many studies showing
little or no proliferation (DNA synthesis) of adult brain tissue
(45
46)
and very low rates of cell proliferation in brain
vascular structures (49)
. This suggests that our
Ve estimates in tumors with low vascular permeability (low
K1 values) may also be low, and that the exchangeable
(nonincorporated) radioactivity correction used in Eq. A is likely to
be underestimated under these conditions. Because the residual
(exchangeable) radioactivity in low-grade gliomas at 24 h may
account for a sizable fraction of the measured radioactivity (Table 3)
,
an underestimate in Ve would result in higher than expected
Ki values. This was seen in a comparison of the values of
Ki, Ve, and K1 for some low-grade (grade II) and
anaplastic (grade III) gliomas in Tables 2
. This
observation illustrates the importance of using a "washout"
strategy with [124I]IUdR and suggests that a
longer washout period would be beneficial with respect to increasing
image specificity (e.g., lowering the fraction of
exchangeable background radioactivity). Not surprisingly, the estimate
of brain Vp in both studies
was nearly
identical.
Another issue to consider is the basis for the difference in the
pattern of radioactivity distribution in the 1- and 24-h images (see
Fig. 2
). The pattern of radioactivity observed at 1 h generally
resembled the pattern of blood-brain barrier disruption observed in the
contrast-enhanced MR or CT images. This similarity is attributable to
the fact that the distribution of IUdR-derived radioactivity is largely
governed by the vascular volume (Vp), vascular permeability
(e.g., K1), the size of the extracellular space
(Ve) of the tumor, and the level of IUdR-derived
radioactivity in blood ([124I]iodide). As noted
above, IUdR-DNA incorporation accounts for only a small fraction of the
radioactivity in the 0–60-min images, and these images largely reflect
[124I]iodide (see Fig. 1, A and B
).
The values of K1 varied substantially among the
different tumor groups and contralateral brain tissue
. Not
surprisingly, meningiomas had the highest values, followed by GBM
(grade IV). These K1 values are consistent with previous
studies of brain tumor vascular permeability in patients using
[68Ga]-labeled EDTA (50)
,
[82Rb]rubidium (51
, 52)
, and PET
imaging. Similarly, the values of Ve and Vp for
the different tumor groups are consistent with published values
(50
51
52)
.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported in part by a Fogarty Senior
International Fellowship Award F06 TW02120-01 (to R. G. B.) and
Department of Energy Grant DOE 86ER60407 (to R. D. F.). 
2 To whom requests for reprints should be
addressed, at Memorial Sloan-Kettering Cancer Center, Department of
Neurology, Room C799, 1275 York Avenue, New York, NY 10021. Phone:
(212) 639-7337; Fax: (212) 717-3063.
3 The abbreviations used are: CT, computed
tomography; MR, magnetic resonance; PET, positron emission tomography;
SPECT, single photon emission computed tomography; IUdR,
iododeoxyuridine; TdR, thymidine; SUV, standard uptake value; Tm:Br,
tumor:brain; BrdUrd, bromodeoxyuridine; LI, labeling index; GBM,
glioblastoma multiforme; HPLC, high-performance liquid chromatography;
ROIs, regions of interest; FDG, fluorodeoxyglucose; FWHM, full width
half maximum.
4 The octanol:water partition coefficient of IUdR
and iodide at 20°C and pH 7.4 was measured [the log octanol:water
partition coefficients were -0.631 ± 0.026
(n = 3) and -1.76 ± 0.03
(n = 3), respectively].
5 R. G. Blasberg, unpublished results.
Received 6/22/99. Accepted 12/ 2/99.
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, and living patients were
documented as of January 1999. Note that patient 19 (*) died of a
pulmonary embolus. The correlation coefficient for the plots are 0.595
(A), 0.452 (B), 0.818 (C),
and 0.720 (D), respectively.