[CANCER RESEARCH 51. 1930-1933. April I. 1991]
A Fluorine-18 Labeled Progestin as an Imaging Agent for Progestin Receptor
Positive Tumors with Positron Emission Tomography1
Aalt Verhagen,2 Philip H. Elsinga, Tjibbe J. de Groot, Anne M. J. Paans, Cornelis C. J. de Goeij, Mels Sluyser,
and Willem Vaalburg
Department of Nuclear Medicine, University Hospital, Groningen [A. V., P. H. E., T. J. d. G., A. M. J. P., W. V.¡,and Division of Tumor Biology, The Netherlands
Cancer Institute, Amsterdam fC. C. J. d. G., M. S.J, The Netherlands
ABSTRACT
The potential of the fluorine-18 labeled progestin 21-|'8F|fluoro-16aethyl-19-norpregn-4-ene-3,20-dione (|'*F]FENP) as an imaging agent for
the in vint assessment of progestin receptor (PR) positive neoplasms
with positron emission tomography has been investigated. Tissue distri
bution studies in immature estrogen primed female rats revealed high
uptake of radioactivity, expressed as the differential absorption ratio, by
uterine tissue. After simultaneous administration with unlabeled FENP,
a significant decrease (83%) in uterine uptake was observed 60 min after
injection. Uterine uptake was highly selective. The ratio of uptake of
radioactivity by uterine tissue to that by blood was 39 at 180 min. In
mice bearing transplanted Grunder strain mammary carcinomas tissue,
distribution studies demonstrated a selective uptake of |"T |l I M' by PR
positive tumors. Pretreatment with unlabeled FENP caused a significant
decrease (66%) in tumor uptake. Uptake by other tissues was not affected
by the presence of unlabeled progestin. The ratio of uptake of radioactiv
ity by tumor tissue to that by blood was 4.7 at 180 min. For FENP
pretreated mice and mice bearing PR negative tumors, this ratio was 1.7
and 1.1, respectively. It is concluded that the uptake of |'*F]FENP by
uterine and by PR positive mammary tumor tissue in vivo is primarily
receptor related, presumably to the PR. Furthermore, |'*F|FENP appears
to be suitable for imaging of PR positive human neoplasms with positron
emission tomography.
INTRODUCTION
Receptor imaging with PET' has focused primarily on recep
tor sites for specific neurotransmitters
in the brain and the
possible disturbance or dysfunction of these sites in neurological
and psychiatric disorders (1). In addition, the principles of the
i/i vivo measurement of neuroreceptors have been extended to
oncology (2-4).
The presence and measurement of ER and PR are important
for prognosis and therapy of breast cancer. It has been demon
strated that the concomitant presence of these receptors in
breast cancer tissue indicates a high probability of responding
to endocrine treatment (5, 6). Thus, in vivo imaging and quan
tification of ER and PR in human breast cancer with PET may
be useful in the assessment of the potential hormone respon
siveness of a primary tumor, and particularly of metastatic foci,
and in monitoring the effect of various hormonal therapeutic
regimens in individual patients. The ability of PET to evaluate
ER in human breast cancer was demonstrated with the fluorine18 labeled estrogen 16a-[l8F]fluoro-estradiol (3).
Received 9/19/90; accepted 1/15/91.
The costs of publication of this article were defrayed in part by the payment
of page charges. This article must therefore be hereby marked advertisement in
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
' This study was supported by a grant from the Jan Kornelis de Cock Stichting.
2 To whom requests for reprints should be addressed, at Department of Nuclear
Medicine, University Hospital. Oostcrsingel 59. 9713 EZ Groningen. The
Netherlands.
1The abbreviations used are: PET. positron emission tomography; ER, estro
gen receptor(s); PR. progestin rcceptor(s); FENP. 2l-fluoro-16u-ethyl-19-norpregn-4-ene-3,20-dione; ORO 2058, 21-hydroxy-l6ir-ethyl-19-norpregn-4-enc3,20-dione; ("F|FENP. 21-('8F]iluoro-16(i-ethyl-I9-norpregn-4-ene-3,20-dione;
GR. Grunder strain; DAR. differential absorption ratio; HD. hormone dependent;
HI. hormone independent.
Since steroid receptors occur at minute concentrations in
tumor tissue, a very small quantity of radioactive steroid must
be administered to achieve a ratio between receptor and nonreceptor binding. This can be accomplished by using radiopharmaceuticals with high pharmacological selectivity and high
affinity for the particular steroid receptor and labeled with high
specific radioactivity (GBq/^mol). For imaging PR with PET,
the progestin FENP appears to be a very suitable ligand. FENP
is an analogue of the synthetic progestin ORG 2058 in which
the 21-hydroxy group has been replaced by a fluorine atom.
Both ORG 2058 and FENP are high affinity progestins (7).
We and others have labeled FENP isotopically with fluorine18 (8, 9).
In this paper, we describe our investigations concerning [18F]
FENP as a potential imaging agent for the in vivo assessment
of PR positive neoplasms with PET. The uptake of [I8F]FENP
by uterine tissue is investigated in immature estrogen primed
Wistar rats. GR mouse mammary carcinomas are used to
examine [18F]FENP for tumor uptake. Mammary carcinomas
can be induced in GR mice by treatment with estrone and
progesterone (10, 11). Growth of these tumors is hormone
(estrogen and progesterone) dependent. However, when the
tumors are serially transplanted, a progressive decrease in hor
mone dependency is observed which eventually leads to com
plete hormone independency. This is accompanied by a decrease
in tumor ER and PR densities (10, 11). Tumor uptake of [I8F]
FENP is investigated
carcinomas.
MATERIALS
in
these
transplanted
mammary
AND METHODS
Synthesis of I'TIFENP. |"F]FENP was synthesized by nucleophilic
(Inori 11:11
¡onof the corresponding 21-tosylate (8). Briefly, the 21-tosylate was subjected to a substitution reaction with an aminopolyether/
K*-complex containing ['"Fjfluoride. The ['"Flfluoride was prepared
from '"O enriched water by the '*O(p.n)l8F nuclear reaction. Purifica
tion and measurement of specific radioactivity were performed accord
ing to the method described previously (8). A typical batch had a
specific radioactivity of at least 185 GBq/Mmol and a radiochemical
purity of >99%. For animal studies [I8F]FENP was dissolved in
ethanol/propylene glycol/saline (1:2:2, v/v) and passed through a 0.2fim membrane filter.
Tissue Distribution Studies in Normal Rats and Tumor Bearing Mice.
Three groups of immature female Wistar rats (28-32 days) were used
for tissue distribution studies. Each group consisted of 5 rats. Prior to
the experiments all rats received 5 Mgestradici i.p. in 0.1 ml of 5%
ethanol/sunflower oil for 3 successive days. Rats were used within 24
h after the last injection. The average animal body weight at the time
of the experiment was 88 ±10 (SD) g. Two groups were treated with
with 3.7 MBq ['"FJFENP i.v. At 60 and 180 min after injection the
animals were killed by cervical dislocation. To ascertain receptor related
uptake by uterine tissue, the third group of rats was given [iaF)FENP
together with 20 ¿jgunlabeled FENP. These rats were killed 60 min
after injection. Blood and tissue samples were weighed and assayed for
radioactivity in a calibrated well counter. Tissue uptake was expressed
as the DAR and calculated from the formula:
1930
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1991 American Association for Cancer Research.
ASSESSMENT OF PR POSITIVE TUMORS WITH PET
DAR = (radioactivity/g
radioactivity)
tissue) x (g body weight/total
Table 1 Tissue distribution of'T-radioactivity after i.v. injection of["F]FENP
into immature estrogen primed female rats
DAR"
injected
For each animal, the selectivity of uterine uptake was estimated by
calculating the ratio of the uptake of radioactivity by uterine tissue to
that by blood or muscle tissue.
HD mammary carcinomas were induced in ovariectomized GR mice
by treatment with estrone and progesterone. Serial transplantation and
evaluation of the hormone dependency of the tumors were carried out
as described previously (10, 11). HD and HI mammary tumor trans
plants from 4 different tumor lines were used. Experiments were carried
out within 3 weeks after discontinuation of progesterone treatment.
Two groups of mice bearing HD tumors and one group of mice bearing
HI tumors were used for tissue distribution studies. Each group con
sisted of 4 mice. The average mouse body weight and tumor weight at
the time of the experiment were 33 ±4 and 3.8 ±1.5 g, respectively.
All mice received 3.7 MBq [I8F]FENP i.p. and were killed by cervical
min*0.36
TissueUterusOvariesBloodMuscleLiverKidneyLungBrainBoneFat60
min2.08
min1.69
0.38f1.17
±
0.05*0.70
±
+0.65
±0.270.15
0.200.15
±
±0.04
+0.11
±0.030.29
±0.020.27
0.051.59
±
0.021.63
±
±0.76
0.280.55
+
±0.200.52
±0.23
0.060.32
±
0.080.36
±
±0.09
0.080.36
±
±0.100.36
±0.09
+1.48±1.57
0.061.14
±
0.051.24
±
0.211.28
+
±0.211.37
±0.3060
±0.440.180.010.020.170.070.020.020.200.33RatioUterus/bloodUterus/muscle13.78±
±0.33180
2.567.34
0.451.35
±
±15.89±10.875.50
±1.452.49
±0.1939.42
* In order to block receptor related uterine uptake. [18F]FENP was coinjected
with 20 MgFENP.
' Mean ±SD.
a The uptake by the uterus and the ovaries was significantly decreased after
coinjection of 20 //g FENP (two sided Õ
test, P< 0.001 and P< 0.02. respectively).
dislocation after 180 min. One group of mice bearing HD tumors
received 30 /ig unlabeled FENP 30 min before the administration of
the labeled progestin. Measurement of tissue uptake and tumor uptake
selectivity was identical to that described for normal rats.
Positron Emission Tomography. A radiopharmaceutical labeled with
a positron emitting radioisotope can be measured as a result of the
annihilation of the positron in tissue. At the point of each annihilation,
two photons are emitted in opposite directions. These coincidence
photons, rather than the positron itself, are detected. We used a
longitudinal positron camera system to image the distribution of [18F]
able uptake of the lipophilic progestin by fat tissue was noticed.
Furthermore, radioactivity appeared to accumulate markedly
in the bones. Most likely, this is attributable to metabolic
defluorination of the labeled progestin and subsequent uptake
of [18F]fluoride by bone tissue. Our results concerning the tissue
distribution of [18F]FENP in normal rats are comparable with
FENP (12). The system consists of two uncollimated large field of view
gamma cameras operating in a coincidence mode. The cameras are
placed in a fixed colinear position. A computer system is used for data
acquisition and reconstruction of the images.
The PET study was carried out with two tumor bearing mice. We
used an HD and HI mammary tumor transplant. Both of the mice were
given an i.p. injection of 1.9 MBq [18F]FENP. At 102 min after
those reported previously (9). However, when we consider the
relatively high PR density in uterine tissue, the potential of
[18F]FENP is perhaps overestimated. This prompted us to
investigate whether selective PR related mammary tumor up
take is measurable. By using different transplant generations of
GR mouse mammary carcinomas, we can compare tumor up
take of [18F]FENP by HD tumors with that of HI tumors. HD
administration, data acquisition was started at a frame rate of 1 frame/
3 min for 150 min. After 2 days the mouse bearing the HD tumor was
imaged again according to the same procedure. However, in this experi
ment the animal was pretreated with 30 ^g unlabeled FENP to dem
onstrate the blocking of tumor uptake. During data acquisition mice
were under mild anesthesia with sodium pentobarbitone. Tumor size
was estimated by measuring length, width, and height of the tumor and
calculating the volume assuming an ellipsoid shape. PET data were
corrected for nonuniform sensitivity and physical decay (12). At 105,
165, and 225 min after injection the data were summed into 15-min
images. The PET images were displayed and regions of interest were
defined, representing the tumor and the total body. The amount of
radioactivity within the assigned regions was retrieved. Subsequently,
DAR values for tumor uptake were calculated. Because minced tumor
tissue was transplanted by s.c. inoculation into the flank of the animals,
the tumors were imaged free from surrounding tissue.
RESULTS
mammary tumors are ER and PR positive, whereas HI mam
mary tumors are practically devoid of these receptors (11, 12).
Tissue distribution data in mice bearing GR mammary tumor
transplants showed that the uptake of [18F]FENP by HD tumor
tissue was selective, as was evident by the uptake of this tissue
and by the tumor to blood ratio (Table 2). Tumor uptake was
considerably less than the observed uterine uptake in normal
rats. Receptor related tumor uptake was indicated by a statis
tically significant decrease in uptake when mice were pretreated
with an excess of unlabeled FENP. Uptake in other tissues was
not affected by the presence of unlabeled progestin. In contrast,
Table 2 Tissue distribution of'*F-radioactivity at 180 min after i.p. injection of
[ISFJFENP into mice bearing GR mammary tumor transplants
AND DISCUSSION
DAR"
In immature estrogen primed female rats the uterus demon
strated a pronounced uptake of [18F]FENP (Table 1). In addi
Tissue
tion, uterine uptake remained relatively high even after 180
min. A statistically significant decrease in uptake by the uterus
and the ovaries was observed when rats were cotreated with an
excess of unlabeled FENP. Moreover, the decrease in uptake
of radioactivity by the uterus, and to some extent the ovaries,
appeared to be selective, whereas all the other tissues showed
equivalent uptake in the absence and presence of the blocking
dose of unlabeled progestin. The selectivity of uterine uptake
was measured by the ratios of radioactivity localized in uterine
tissue to that localized in blood or in muscle tissue. Although
uterine uptake decreased during the specified period of time,
the ratios continued to increase, as [18F]FENP was cleared more
rapidly from tissues with known undetectable PR. A consider-
HD tumor
HD tumor"
BloodMuscleLiverKidneyLungBrainBoneTumor1*0.06
T0.05±0.0
0.020.05
0.010.62
0.010.66
0.090.26
0.160.27
0.090.10
0.090.09
0.010.03
0.020.04
0.012.02
0.012.00
0.480.29
0.600.10
0.02r0.06
0.130.06
HI tumor
0.010.04
±
0.010.59
±
0.060.21
±
±0.050.10
±0.020.04
±0.011.67
±0.320.06
±0.02
Ratio
Tumor/blood
4.66 ±1.30
1.71 ±0.61
1.09 ±0.34
* In order to block receptor related tumor uptake mice were pretreated with
30 f<gFENP.
' Mean ±SD.
''Of each tumor 2 samples »ereweighed and assayed for radioactivity.
'The uptake by tumor tissue was significantly decreased after pretreatment
with 30/ig FENP (two sided t test, P < 0.001).
1931
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1991 American Association for Cancer Research.
ASSESSMENT OF PR POSITIVE TUMORS WITH PET
HI mammary tumors hardly showed any uptake of radioactiv
ity. Moreover, the low uptake by HI mammary tumors empha
sized the observed selective tumor uptake of ['"FJFENP by HD
0.60
mammary tumors. Again a considerable accumulation of radio
activity in the bones was noticed.
The most commonly affected distant sites of metastatic dis
ease in breast cancer are lung, bone, and liver. With regard to
imaging of métastases,especially the nonspecific uptake by
bone and liver tissue appears to reduce selective uptake of [I8F]
r
Q
a."
g
0.40
FENP by PR positive tumors. However, to arrive at definite
conclusions clinical investigation is required.
PET images revealed a receptor related uptake of ['"FjFENP
by an HD mammary tumor transplant (Fig. 1). These images
were used to calculate the differences in uptake of radioactivity
before and after pretreatment with an excess of unlabeled FENP
and were compared with similar images of a mouse bearing an
HI mammary tumor transplant (Fig. 2). Initially, the uptake by
the HD mammary tumor declined slowly and remained con
stant thereafter. The difference with tumor uptake after pre
treatment with unlabeled FENP increased during the specified
period of time. The ratios of tumor uptake before and after
pretreatment were 1.3 and 2.1 at the second and third time
interval after injection, respectively. In addition, during the
specified time intervals the tumor region of the mouse bearing
the HI mammary tumor hardly showed any visible uptake of
radioactivity (images not shown). At 165 to 180 min after
injection the DAR values for HD and HI tumor uptake, result
ing in a ratio of about 7.4, correlated well with the results
obtained with the tissue distribution studies (Table 2). However,
in the PET images the HD tumor uptake after pretreatment
with unlabeled FENP appeared to be higher.
Whether [I8F]FENP and PET can be used clinically for the
in vivo assessment of PR in neoplasms is currently under
investigation. We are studying patients with breast cancer and
patients with meningiomas. The latter neoplasms have also
been shown to contain high PR densities (13).
In summary, our present results obtained with two animal
models indicate a selective uptake of [I8F]FENP by uterine
0.20
105-120
165-180
225-240
IMAGE TIME AFTER INJECTION(min)
Fig. 2. Tumor uptake of |"F]FENP, expressed as the DAR. at three time
intervals after i.p. injection. In the PET images regions of interest were selected
for the tumor and the total body. The amount of radioactivity within these regions
of interest »asused to calculate the DAR values for the tumors. A. mouse with
HD mammary tumor treated with |"F]FENP; •mouse with HD mammary
tumor after pretreatment with 30 fig FENP; •,mouse with HI mammary tumor
treated with ['"FJFENP.
FENP suppresses the uptake of radioactivity to the level ob
served in tissues with known undetectable PR. Therefore, we
conclude that uterine and HD mammary tumor uptake in vivo
is primarily receptor related, presumably to the PR. This sug
gests the potential applicability of [ISF]FENP and PET for
imaging PR positive neoplasms.
ACKNOWLEDGMENTS
The authors are grateful to Dr. M. B. Groen of Organon Interna
tional BV, The Netherlands, for providing samples of ORG 2058 and
ORG OH-06 (FENP), and to the operating team of the cyclotron of
the Kernfysisch Versneller Instituut, Groningen, The Netherlands, for
expert technical assistance.
tissue and by PR positive mammary tumor tissue. Unlabeled
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Fig. 1. Anterior whole body PET ¡magesof a mouse bearing an H D mammary
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was visible after administration of |"F]FENP with high specific radioactivity (A).
After pretreatment with excess unlabeled FENP. hardly any tumor uptake was
observed (B). Organs involved in steroid metabolism and excretion showed high
uptake in the images. Arrows, location of the tumor.
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A Fluorine-18 Labeled Progestin as an Imaging Agent for
Progestin Receptor Positive Tumors with Positron Emission
Tomography
Aalt Verhagen, Philip H. Elsinga, Tjibbe J. de Groot, et al.
Cancer Res 1991;51:1930-1933.
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