Published OnlineFirst March 31, 2012; DOI: 10.1158/2159-8290.CD-11-0316
Article Name BRIEF
research
Ulmert et al.
Research BRIEF
Imaging Androgen
Receptor Signaling
with a Radiotracer
Targeting Free
Prostate-Specific
Antigen
David Ulmert1,7, Michael J. Evans2, Jason P. Holland3,4, Samuel L. Rice3,4, John Wongvipat2, Kim Pettersson9,
Per-Anders Abrahamsson7, Peter T. Scardino1,3, Steven M. Larson3,4, Hans Lilja1,5,8, Jason S. Lewis3,4, and
Charles L. Sawyers2,6
Despite intense efforts to develop radiotracers to detect cancers or monitor
treatment response, few are widely used as a result of challenges with demonstrating clear clinical use. We reasoned that a radiotracer targeting a validated clinical biomarker
could more clearly assess the advantages of imaging cancer. The virtues and shortcomings of measuring secreted prostate-specific antigen (PSA), an androgen receptor (AR) target gene, in patients with prostate cancer are well documented, making it a logical candidate for assessing
whether a radiotracer can reveal new (and useful) information beyond that conferred by serum
PSA. Therefore, we developed 89Zr-labeled 5A10, a novel radiotracer that targets “free” PSA.
89
Zr-5A10 localizes in an AR-dependent manner in vivo to models of castration-resistant prostate
cancer, a disease state in which serum PSA may not reflect clinical outcomes. Finally, we demonstrate that 89Zr-5A10 can detect osseous prostate cancer lesions, a context where bone scans fail
to discriminate malignant and nonmalignant signals.
Abstract
SIGNIFICANCE: This report establishes that AR-dependent changes in PSA expression levels can be
quantitatively measured at tumor lesions using a radiotracer that can be rapidly translated for human
application and advances a new paradigm for radiotracer development that may more clearly highlight
the unique virtues of an imaging biomarker. Cancer Discov; 2(4); 320–7. ©2012 AACR.
Authors' Affiliations: 1Urology Service of the Department of Surgery,
Human Oncology and Pathogenesis Program, 3Program in Molecular
Pharmacology and Chemistry, Departments of 4Radiology and
5
Laboratory Medicine and Medicine (Genitourinary Oncology Service),
6
Howard Hughes Medical Institute, Memorial Sloan-Kettering Cancer
Center, New York, New York; Departments of 7Urology and 8Laboratory
Medicine, Lund University, Skåne University Hospital, Sweden;
9
Department of Biotechnology, University of Turku, Turku, Finland
2
Corresponding Authors: Hans Lilja, Department of Laboratory Medicine,
Memorial Sloan-Kettering Cancer Center, 1250 York Avenue, New York, NY
10065. Phone: 212-639-6982; Fax: 646-422-2379; E-mail: [email protected]
Jason S. Lewis, Radiochemistry Service, Department of Radiology, Memorial
Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10065.
Phone: 646-888-3038; Fax: 646-422-0408; E-mail: [email protected]
Note: Supplementary data for this article are available at Cancer
Discovery Online (http://www.cancerdiscovery.aacrjournals.org). Charles L. Sawyers, Human Oncology and Pathogenesis Program,
Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York,
NY 10065. Phone: 646-888-2594; Fax: 646-888-2595
E-mail: [email protected]
D. Ulmert and M.J. Evans contributed equally to this work.
doi: 10.1158/2159-8290.CD-11-0316
J.S. Lewis and C.L. Sawyers are senior cocorresponding authors.
320 | CANCER DISCOVERY april 2012 ©2012 American Association for Cancer Research.
www.aacrjournals.org
Downloaded from cancerdiscovery.aacrjournals.org on June 16, 2017. © 2012 American Association for
Cancer Research.
Published OnlineFirst March 31, 2012; DOI: 10.1158/2159-8290.CD-11-0316
PET Imaging of Androgen Receptor Signaling
Introduction
18
The serendipitous discovery that F-fluorodeoxyglucose
uptake reflects aberrant c-KIT signaling (and predicts response to imatinib therapy) in gastrointestinal stromal tumors has greatly simplified patient management (1) and,
more generally, underscores the promise of molecular imaging in oncology. However, deliberately engineering radiotracers to achieve similar success in other tumors has proven
challenging, resulting in the high attrition rate of novel radiotracers in the clinic.
The target of a radiotracer necessarily frames its potential
context of use (i.e., detection, response indicator), and candidates are often selected on the basis of preclinical evidence
pointing to an upregulation in cancer. In this regard, it can
be challenging to appropriately evaluate novel radiotracers
in patients without a thorough appreciation of the pathobiologic mechanism of target upregulation. Therefore, we
reasoned that a radiotracer targeting a well-studied, tumorspecific clinical biomarker reflective of oncogenic pathway
activation could more rapidly document the unique advantages of studying patient response with a cognate noninvasive imaging tool.
To establish this concept, we selected prostate-specific
antigen (PSA) based on the large body of research highlighting the virtues and shortcomings of measuring the secreted
forms of this protein in prostate cancer (2–4). Originally, it
was hoped that measuring total concentrations of PSA in
serum might revolutionize prostate cancer management by
allowing early detection of subclinical disease and precise
monitoring of residual disease after therapy. This hope was
based on the fact that PSA is expressed almost exclusively by
prostate epithelia and, as an androgen receptor (AR) target
gene, its expression reflects AR pathway activity. Years of
work to clinically validate this biomarker have revealed certain limitations. For example, despite strong associations
between metastasis or death from prostate cancer and PSA
levels in blood (5), the inability to distinguish PSA produced
by normal versus malignant prostate tissue limits its general use in primary screening. Moreover, apart from a few
contexts in which a dramatic reduction confirms successful
therapeutic intervention (e.g., postradical prostatectomy),
interpreting changes in serum PSA levels in response to
therapy has been problematic. At first glance, this may seem
surprising because PSA expression is tightly coupled to AR
signaling. However, the ability to detect PSA in serum not
only requires expression, but also secretion and leakage into
the circulation—two processes that are very poorly understood (2). Also, it is well documented that only a very small
percentage of intratumoral PSA is secreted into perivascular
space (6), and the rate-determining step to serum circulation is undefined. These considerations raise the possibility
that a noninvasive tool measuring tumor-associated PSA
expression could more clearly reflect AR-driven changes in
PSA expression.
PSA is initially produced as a catalytically active serine protease [“free” PSA (fPSA)], and subsequent to its release into the
perivascular space, it is rapidly and irreversibly converted to noncatalytic forms [“complexed” PSA (7, 8)]. We therefore reasoned
that 5A10, a monoclonal antibody that specifically recognizes
research BRIEF
an epitope adjacent to the catalytic cleft of PSA (9–11), and
therefore selectively binds fPSA, could in principle target tumorassociated PSA. In considering radiolabeling strategies, we noted
that our recent studies with 89Zr-labeled monoclonal antibodies
(mAb) yielded high-contrast images of tumors with low radiotracer uptake in normal tissues (12–14). Based on these observations, we prepared 89Zr-labeled 5A10.
Results
We conjugated 89Zr to 5A10 with the chelator desferrioxamine B through a previously established synthetic route (see
Methods and Supplementary Fig. S1) (12, 14). To determine
the affinity of 89Zr-5A10 for fPSA, competition binding assays
were conducted, and the bioconjugation of 5A10 resulted in
virtually no loss of affinity for purified fPSA (Supplementary
Fig. S2).
We began in vivo studies by administering 89Zr-5A10 to
intact male mice inoculated with subcutaneous xenografts
of LNCaP-AR [a PSA-positive prostate cancer model derived
from parental LNCaP overexpressing wild-type AR (15)]. This
model was chosen because the magnitude of PSA production
and circulation in mouse serum closely approximates that
observed in patients with advanced disease (Supplementary
Table S1). Tissues were harvested at multiple time points
postinjection to determine the kinetics of radiotracer biodistribution (Fig. 1A). Peak tumor-associated activity was
observed 24 hours postinjection (19.59% ± 4.9% ID/g, tumor-to-muscle ratio: 22.80 ± 18.6), and with few exceptions,
little 89Zr-5A10 accumulation was observed in host tissues
(Fig. 1A, Supplementary Fig. S3, and Supplementary Tables
S2 and S3). Positron emission tomography (PET) studies
showed a region of contrast at the tumor, supportive of the
biodistribution data (Fig. 1B). Using 100-fold excess of unlabeled 5A10 to compete 89Zr-5A10 uptake, we confirmed
the specificity of the biologic interaction between 89Zr-5A10
and LNCaP-AR (Fig. 1C, Supplementary Fig. S4, and
Supplementary Tables S4 and S5). Moreover, there was little incorporation of the nonspecific radiotracer 89Zr-labeled
mouse IgG1 in LNCaP-AR at 24 hours postinjection (Fig. 1C,
Supplementary Fig. S5, and Supplementary Tables S6 and
S7). As expected, PC3 xenografts, an AR- and PSA-negative
model of human prostate cancer, showed little avidity for
89
Zr-5A10 at 24 hours postinjection (Fig. 1C, Supplementary
Fig. S6, and Supplementary Table S8). Finally, subcutaneous CWR22Rv1 xenografts were avid for 89Zr-5A10, another AR- and PSA-positive prostate cancer model (Fig. 1C,
Supplementary Fig. S6, and Supplementary Table S9).
We next asked whether 89Zr-5A10 could detect androgen-regulated elevations in fPSA expression. Castrated
male mice were inoculated with LNCaP-AR, and after tumor formation, animals received no manipulation or a
surgically implanted subcutaneous testosterone pellet.
89
Zr-5A10 was administered 7 days postmanipulation,
and biodistribution studies were conducted 24 hours
postinjection. 89Zr-5A10 localization was significantly
higher in LNCaP-AR xenografts exposed to testosterone
compared with control (Fig. 1D, Supplementary Fig. S7,
and Supplementary Table S10). Intratumoral PSA levels
april 2012 CANCER DISCOVERY | 321
Downloaded from cancerdiscovery.aacrjournals.org on June 16, 2017. © 2012 American Association for
Cancer Research.
Published OnlineFirst March 31, 2012; DOI: 10.1158/2159-8290.CD-11-0316
Ulmert et al.
research BRIEF
4 hours
12 hours
24 hours
48 hours
72 hours
aP-AR
22Rv1
% ID/g
Tumor
uptake
Coronal
(% ID/g)Trans.
C
*
7520
10.0% ID/g
LNCaP-AR
No treatment
PC3
Testosterone
CWR22Rv1
15
50
**
**
10
25 5
0
% ID/g
**
0.0% ID/g
89 Time 89
Zr-5A10 Zr-5A10
(hours)
Tumor
Muscle
75
Liver
D
CaP-AR
3
R22Rv1
+ 5A10
Lungs
Zr-5A10
89
Heart
Muscle Tumor
Zr-5A10
89
489Zr-5A10 24
Zr-5A10 8972
Zr-IgG
Blood
0
Tumor
25
0
Tumor
Zr-5A10
25
89
Muscle
(hours)
Muscle Tumor
Muscle
0.0%
ID/g
0Zr-5A10 89Zr-5A10 89Zr-IgG 89Zr-5A10
89
Zr-5A10
Time
4Blood + 5A10
24
72 Muscle
Heart
Liver
Tumor
89
Liver
0
5
Blood
5
% ID/g
Tumor uptake
(% ID/g)
**
1010
% ID/g
*
Coronal
% ID/g Trans.
B
Heart Liver Muscle Tumor
4 hours
12
hours
89 25
Zr-5A10
24 hours
10.0% ID/g
25
LNCaP-AR
48 hours
20
PC3
20
72 hours
CWR22Rv1
1515
Liver
Blood
Lungs
0
Lungs
5
Heart
10
Heart
15
Blood
% ID/g
20
Figure 1. 89Zr-5A10 specifically localizes to multiple preclinical
10.0%
models of ARandID/g
fPSA-positive prostate cancer. A, biodistribution
data of selected tissues from intact male mice bearing LNCaP-AR
xenografts at multiple time points show that peak intratumoral
uptake of 89Zr-5A10 is observed at 24 hours. Over time, activity
depleted from the blood pool, represented by the blood and heart,
and like many mAbs, persistently high uptake was observed in the
liver. B, representative transverse (Trans.) and coronal PET slices of
0.0%bearing
ID/g
intact male mice
LNCaP-AR xenografts shows localization of
Time
4
24 89Zr-5A10
72 to the
tumor (T) and uptake in the murine liver (L). The
(hours)
tissues from these
animals were incorporated into the biodistribution
profile at the 120-hour time point. C, biodistribution data showing
89
89
Zr-5A10
tumor-associated
in multiple subcutaneous prostate
No treatment
10.0%Zr-5A10
ID/g
75
cancer Testosterone
models and several treatment conditions in intact male mice.
89
The localization of Zr-5A10 to LNCaP-AR was entirely competed
50
by coinjection with excess unlabeled 5A10 (1 mg unlabeled mAb).
The nonspecific radiotracer 89Zr-IgG did not localize to LNCaP-AR,
25
and 89Zr-5A10 did not localize to PC3, an AR- and fPSA-null model
of prostate cancer. Intermediate localization of 89Zr-5A10 to
CWR22Rv1 xenografts
0.0% ID/gwas observed, consistent with the lower
0
of fPSA in this model compared with LNCaP-AR.
Time
4
24basal expression
72
(hours)
*P , 0.01 compared
with all conditions. **P , 0.01 compared with
PC3. D, surgical implantation of a subcutaneous testosterone
pellet in castrated mice bearing LNCaP-AR tumors resulted in
No treatment
75
increased tumor-associated 89Zr-5A10, whereas uptake in other
Testosterone
organs was
unchanged. Biodistribution data were acquired at
24
hours
postinjection.
*P , 0.01 compared with no treatment.
50
Error bars represent the standard deviation from mean.
Zr-5A10
89
Coronal Trans.
25
Coronal Trans.
A
No treatment
Testosterone
50
25
Tumor
Muscle
Liver
Lungs
Heart
Zr-5A10
89
Blood
0
Zr-5A10
89
also increased, as expected (Supplementary Table S11).
Similar results were observed with CWR22Rv1 xenografts
(Supplementary Fig. S8 and Supplementary Tables S12
and S13). Collectively, these results show that 89Zr-5A10
can faithfully reflect intratumoral AR signaling.
We next tested if pharmacologic inhibition of AR can
be quantified in vivo with 89Zr-5A10 PET using the antiandrogen MDV3100, whose clinical activity is correlated
with responses in the LNCaP-AR model (16, 17). Castrated
male mice were inoculated with subcutaneous LNCaP-AR
xenografts, and tumor-bearing mice were randomized into
groups receiving a daily oral gavage of vehicle or MDV3100
at 10, 40, or 80 mg/kg. Seven days postinitiation of treatment, 89Zr-5A10 was administered, and biodistribution
studies were conducted 24 hours postinjection (Fig. 2A,
Supplementary Fig. S9, and Supplementary Table S14). As
expected, this course of therapy inhibited tumor growth
and fPSA protein in the tumor (Supplementary Fig. S10
and Supplementary Table S15). Accordingly, tumor-associated 89Zr-5A10 was significantly decreased by a 40- and
322 | CANCER DISCOVERY april 2012 80-mg/kg dose of MDV3100 (Fig. 2A). Statistically significant changes in tumor-associated 89Zr-5A10 were observed
by PET between groups of animals receiving a daily oral
gavage of vehicle or 80 mg/kg MDV3100 (Fig. 2B and C
and Supplementary Table S16). In addition, a significant
increase in tumor-associated 89Zr-5A10 was observed in a
separate treatment arm of mice receiving subcutaneous
testosterone pellets. Notably, the fold change in tumorassociated 89Zr-5A10 closely mirrored the fold change in
expression of intratumoral fPSA. For example, the testosterone pellet resulted in an approximate 2.5-fold increase
in tumor-associated 89Zr-5A10 (Supplementary Table S10),
very close to the approximate 2.6-fold increase in fPSA expression in the LNCaP-AR tumors (Supplementary Table
S11). Collectively, these results highlight the ability of
89
Zr-5A10 to measure pharmacologically triggered changes
in intratumoral AR signaling.
One of the challenges in evaluating experimental therapies like MDV3100 is assessing therapeutic effects on individual metastatic lesions in patients with diffuse disease (18).
www.aacrjournals.org
Downloaded from cancerdiscovery.aacrjournals.org on June 16, 2017. © 2012 American Association for
Cancer Research.
Published OnlineFirst March 31, 2012; DOI: 10.1158/2159-8290.CD-11-0316
Time
0
4
5
(weeks)
Time
PET Imaging
of Androgen
Receptor
Signaling
0
4
5
Tumor
*
MDV3100
MDV3100
C
25% ID/g
25% ID/g
Test.L
Veh.
25% ID/g
0.0% ID/g
LNCaP-AR
L
0.0% ID/g
LNCaP-AR
0.0% ID/g
T
*
20
15
20
10 15
MDV3100
L
T
T
*
Veh.
Veh.
Tumor-associated
89
Tumor-associated
Zr-5A10 (% ID/g)
Tumor-associated
Zr-5A10 (%
89 ID/g)
Zr-5A10 (% ID/g)
Test.
Test.
10
520
0155
*
*
Test.
**
**
Vehicle MDV3100
100
5
Test.
Vehicle MDV3100
**
89
B
Coronal
*
Tumor
Lungs
Muscle
Heart
Muscle
Muscle
0
Lungs
5
Lungs
10
Blood
15
Heart
0
020
Blood
5
Vehicle
MDV3100 (10 mg/kg)
MDV3100 (40 mg/kg)
MDV3100 (80 mg/kg)
525
10
Tumor
10
Blood
15
% ID/g
% ID/g
20
Vehicle
MDV3100 (40
mg/kg) Harvest
Inoculate
Initiate
20 MDV3100
mg/kg)
MDV3100
(80 mg/kg) Inject
mice (10treatments
15 MDV3100 (40 mg/kg) 89Zr-5A10
MDV3100 (80 mg/kg)
Heart
% ID/g
25
5
Trans.
89
Time Vehicle
0
4
25
(weeks)
MDV3100 (10 mg/kg)
Trans.
Inject
Zr-5A10
A
research BRIEF
Coronal
Trans.
Inoculate Initiate
Harvest
mice Initiate
treatments Harvest
Inject
Inoculate
89
Zr-5A10
mice treatments
Coronal
(weeks)
0
LNCaP-AR
Test.
Vehicle MDV3100
Figure 2. 89Zr-5A10 detects pharmacologic inhibition of AR in vivo. A, biodistribution data from castrated male mice bearing LNCaP-AR
xenografts show that MDV3100 inhibits localization of 89Zr-5A10 to tumor. Animals were treated with vehicle, or the indicated dose of MDV3100 for
7 days, at which time 89Zr-5A10 was injected, and animals were harvested for biodistribution studies 24 hours postinjection *P , 0.01 for the 40-mg/
kg and 80-mg/kg dose of MDV3100 compared with vehicle or 10 mg/kg MDV3100. B, representative transverse (Trans.) and coronal PET slices of
intact male mice bearing LNCaP-AR xenografts on the right flank and imaged with 89Zr-5A10 24 hours postinjection after manipulation with a
subcutaneous testosterone (Test.) pellet or a daily oral gavage of vehicle (Veh.) or MDV3100 (80 mg/kg) for 7 days. Clear visual differences in tumorassociated 89Zr-5A10 can be seen between the groups. Arrows indicate the position of the tumor (T) and the murine liver (L). C, region-of-interest
analysis of the tumors from the PET study shows statistically significant changes in tumor-associated 89Zr-5A10. *P , 0.01 compared with vehicle.
**P , 0.05 compared with vehicle. Error bars represent the standard deviation from mean.
Although bone is the most common site of metastatic spread
in prostate cancer (19), metastases remain particularly challenging to characterize, because the most widely used nuclear medicine technologies (e.g., 99mTc-MDP, 18F-NaF) do not
directly image the tumor but, rather, target nearby normal
bone repair (20). Therefore, these scans cannot distinguish
between malignant and nonmalignant disease (e.g., injury or
degenerative joint disease). Furthermore, response to antiandrogen therapy cannot be efficiently assessed because resolution of tumor-induced bone repair can lag clinical response
by months or years.
With these considerations in mind, we asked if 89Zr-5A10
can more clearly distinguish a skeletal prostate cancer lesion. Osseous tumors were established in intact male
mice through injection of LNCaP-AR in the tibia of the left hindlimb, and tumor development was confirmed
by MRI after 7 weeks (Supplementary Figs. S11 and S12).
PET/computed tomography (CT) studies showed high
contrast in the tumor-bearing hindlimb compared with
the contralateral limb (Fig. 3A). Consistent with this observation, coregistered PET/MRI images showed an
alignment of the PET and MRI contrast in the tibia (Fig.
3B and Supplementary Fig. S13). Postmortem autoradiography of a surgically excised tibia showed that positron emissions from 89Zr-5A10 coaligned with the topography of
the LNCaP-AR lesion defined by histology (Supplementary
Fig. S14). To further confirm that 89Zr-5A10 does not crossreact with bone remodeling, bone fractures were induced surgically in a separate cohort of mice by puncturing the tibia
in the right hindlimb. Both 18F-NaF and 99mTc-MDP readily
localized to the site of repair, whereas 89Zr-5A10 did not (Fig.
3C). Collectively, these results highlight the unique specificity of 89Zr-5A10 for prostate cancer tumors in the bone.
Discussion
In this report, we show that changes in the expression of
PSA, an AR-regulated, prostate-specific gene, can be measured
noninvasively with the novel radiotracer 89Zr-5A10. In support
of its suitability for quantifying AR signaling in castration- resistant prostate cancer, 89Zr-5A10 readily localized to multiple
AR- and PSA-positive prostate cancer models and quantitatively
measured declines in fPSA synthesis induced by antiandrogen
therapy in a clinically validated xenograft model of castrationresistant prostate cancer. Because 89Zr-5A10 specifically targets
prostate cancer cells rather than the nonmalignant skeletal pathologies that phenocopy the changes induced by cancers on
bone scans, this radiotracer offers the opportunity for more
accurate staging and better treatment selection. Owing to the
abundant expression of PSA also in benign pathologies of the
prostate (2), unambiguously detecting prostate cancer lesions
within the prostate gland with 89Zr-5A10 may be challenging.
In this respect, the most exciting immediate clinical application for 89Zr-5A10 is likely the opportunity to study AR-driven
tumor activity in individual lesions in a heterogeneous disease
to enhance the clinical assessment of advanced disease.
We previously reported that changes in prostate-specific
membrane antigen (PSMA), a cell surface protein whose expression is suppressed by AR, can also serve as a noninvasive marker
for imaging of AR signaling (21). Although PSMA imaging with
PET is attractive because several human-ready targeting agents
already exist (22, 23), PSMA expression is not prostate specific and the clinical impact of AR-directed therapy on PSMA
expression is not known (24). Also, a formal comparison in
LNCaP-AR xenografts showed that 89Zr-5A10 PET resulted in
a more compelling change in tumor localization posttherapy
than 89Zr-J591, a radiotracer derived from a humanized monoclonal antibody to PSMA (Supplementary Fig. S15).
april 2012 CANCER DISCOVERY | 323
Downloaded from cancerdiscovery.aacrjournals.org on June 16, 2017. © 2012 American Association for
Cancer Research.
Published OnlineFirst March 31, 2012; DOI: 10.1158/2159-8290.CD-11-0316
Ulmert et al.
research BRIEF
Time
C
(days)
Time 0
TimeTime(days) 0
(days)
0
(days) 0
Wound10
A
10
10
12
10
12
12
Image 12 Image with
18
with
Wound with [Image
tibia
F]-NaF 89Image
Zr-5A10
and
89 99m with
Image
with [18F]-NaF
Zr-5A10
and
[
Tc]-MDP
Image
with
WoundWound tibia
ImageImage
18
89
[99mTc]-MDP
tibia
with
[
F]-NaF
Zr-5A10
and
18
89
tibia
with [ F]-NaF
Zr-5A10
and
[99mTc]-MDP
[99mTc]-MDP
89
18
[99mTc]-MDP
F]-NaF
[ [18
89 Zr-5A10
[99mTc]-MDP
Zr-5A10
F]-NaF
[99mTc]-MDP
[99mTc]-MDP
0.0% ID/g
0.0% ID/g
0.0% ID/g
0.0% ID/g
Trans.
89
89
Zr-5A10
Zr-5A10
10%
ID/g
10%
ID/g
10% ID/g
10% ID/g
Coronal
PET/MRI
3.9% ID/g
PET/MRI
3.9% ID/g
Trans.
Trans.
MRI
Coronal
Coronal
MRI
PET/MRI
3.9% 3.9%
ID/g ID/g
PET/MRI
Trans.
B
MRI
Coronal
MRI
[18F]-NaF
[18F]-NaF
0.0% ID/g
0.0% ID/g
0.0% ID/g
0.0% ID/g
Figure 3. 89Zr-5A10 specifically targets prostate cancer in the bone microenvironment in vivo. A, a PET/CT image shows that 89Zr-5A10
localizes to an osseous LNCaP-AR graft located in the left tibia of an intact male mouse. The image is a 3-dimensional volume rendering of PET data
overlaid onto surface-rendered CT data. To generate this image, a weighted average of the intensities along projections through the PET is
computed and this in turn is blended with the CT-rendered image. The PET data are represented with a semiquantitative blue (low)–green (high)
color scale and show a region of contrast in the animal’s left (tumor-bearing) tibia compared with the right (normal) tibia. B, a coregistered PET/MRI
image shows the colocalization of positron emissions from 89Zr-5A10 with the tumor-associated contrast detected by MRI. C, intact male mice
received a fracture in the tibia, and 10 days postsurgery, bone remodeling was evaluated with 18F-NaF. A clear region of contrast was identified in
the fractured tibia by PET. Two days after the first image, animals received a coinjection of 99mTc-MDP and 89Zr-5A10. Single‑photon emission
computed tomography imaging showed that 99mTc-MDP also localized to the region of healing bone, as expected. In contrast, PET imaging showed no
detectable 89Zr-5A10 at the wound site, pointing to the high specificity of this reagent for prostate cancer compared with contemporary clinical
radiotracers. The white line represents the location of the fracture.
The precise mechanism by which 89Zr-5A10 allows visualization of AR- and fPSA-positive prostate cancer models is
unclear. Our data and published work suggest the following
model. Before secretion, fPSA exists as a proteolytically active
protein (25) and then is transiently present in the pericellular
space before sequestration by extracellular-binding proteins
that preclude recognition by 5A10. We propose that visualization of prostate cancer cells by 89Zr-5A10 is dependent on
this ability of 5A10 to recognize fPSA in this unique context.
Future studies are required to refine our understanding of
the biologic basis of localization of 89Zr-5A10 to tumor in
situ, which may also further enhance our understanding of
the pathobiologic mechanism(s) of fPSA secretion and processing. Regardless of the mechanism, the molecular imaging tool presented here could have near-term clinical impact,
particularly because the dosimetry of other 89Zr-labeled mAbs
has been determined to be favorable for humans and will
soon be examined in clinical trials (26).
Methods
Preparation of Zirconium-89
Zirconium-89 was produced through the 89Y(p,n)89Zr transmutation reaction on an EBCO TR19/9 variable-beam energy cyclotron
(Ebco Industries, Inc.) in accordance with previously reported methods (27). 89Zr-oxalate was isolated in high radionuclidic and radiochemical purity > 99.9% with an effective specific activity of 195 to
497 MBq/μg (5.27–13.31 mCi/μg).
Preparation of Radiolabeled Constructs
Desferrioxamine B (DFO) was conjugated to antibodies using
the following protocol. Antibody (in phosphate-buffered saline) was
324 | CANCER DISCOVERY april 2012 added to a centrifuge vial and the pH adjusted to 9.5 to 10.0 with
Na2CO3(aq.). Four equivalents of [Fe(N-succDFO-TFP)] were added,
and the reaction was conducted at room temperature for 1 hour. The
pH of the reaction was then adjusted to 3.9 to 4.2 by the slow addition of 0.25 M H2SO4(aq.), and a 10-fold excess of ethylenediaminetetraacetic acid disodium salt was added. The reaction was incubated
in a water bath at 38°C for 1 hour. The DFO-conjugated antibody
was purified by size-exclusion chromatography (Sephadex G-25 M,
PD-10 column; GE Healthcare).
Typical radiolabeling reactions were conducted according to previously reported methods (12) used for labeling monoclonal antibodies with 89Zr. Briefly, 89Zr-oxalate [429 MBq (11.6 mCi)] in
1.0 M oxalic acid (250 μL) was adjusted to pH 7.1 to 7.7 with
1.0 M Na2CO3(aq.). DFO-conjugated 5A10 [500 μL, 2.0 mg/mL
(1.0 mg of protein), in sterile saline] was added and the reaction
was mixed gently. The reaction was incubated at room temperature for between 1 and 2 hours and progress was monitored with
instant thin-layer chromatography [ITLC (diethylene triamine
pentaacetic acid, 50 mM, pH 7)]. After 2 hours, crude radiolabeling yields and RCP were typically > 80% to 90%. 89Zr-5A10 was
purified by using spin-column centrifugation [4 mL total volume,
> 30 kDa particle retention (Amicon Ultra-4; Millipore), washed
with 4×3 mL sterile saline]. The radiochemical purity of the final 89Zr-5A10 (formulation: pH 5.5–6.0; < 500 μL; sterile saline)
was measured by ITLC and size-exclusion chromatography. In the
ITLC experiment, the 89Zr-5A10, 89Zr-IgG, and 89Zr-DFO remain
at the baseline (Rf = 0.0), whereas 89Zr4+(aq.) ions and the complex
89
Zr-diethylenetriaminepentaacetic acid (DTPA) elute with the solvent front (Rf = 1.0). The final radiochemical yield of the purified
89
Zr-5A10 was typically > 70% and the product was formulated
in sterile saline with a radiochemical purity > 99% (n = 5) and a
specific activity of 195.0 ± 8.0 MBq/mg (5.27 ± 0.2 mCi/mg) of
protein. Supplementary Figure S1 shows a typical radio-ITLC chromatogram of the crude and purified (formulated) 89Zr-5A10.
www.aacrjournals.org
Downloaded from cancerdiscovery.aacrjournals.org on June 16, 2017. © 2012 American Association for
Cancer Research.
Published OnlineFirst March 31, 2012; DOI: 10.1158/2159-8290.CD-11-0316
PET Imaging of Androgen Receptor Signaling
Cell Lines
PC3 and CWR22Rv1 were purchased from American Type Culture
Collection, and AR and PSA expression was annotated by immunoblot and ELISA. The cell lines were cultured according to the manufacturer’s instructions. LNCaP-AR was previously developed and
reported by the Sawyers laboratory (15). The cell line was authenticated for AR overexpression and PSA expression by immunoblot.
Animal Studies
All animal experiments were conducted in compliance with institutional guidelines at Memorial Sloan-Kettering Cancer Center. Male
CB-17 severe combined immunodeficient (SCID) mice (6–8 weeks
old) were obtained from Taconic Farms, Inc.; LNCaP-AR, 22Rv1, and
PC3 tumors were inoculated in the right flank by subcutaneous injection of 1.0 × 106 cells in a 200-μL cell suspension of a 1:1 v/v
mixture of media with Matrigel (Collaborative Biomedical Products,
Inc.). Tumors developed after 3 to 7 weeks. MDV3100 was dissolved
in dimethyl sulfoxide (DMSO) so that the final DMSO concentration when administered to animals would be 5%. The formulation of
the vehicle is 1% carboxymethyl cellulose, 0.1% polysorbate 80, and
5% DMSO. MDV3100 or vehicle was administered daily by gavage.
Tumor volume (V/mm3) was estimated with caliper measurements.
Preparation of Osseous Tumor Grafts and Bone Fracture Model
Before surgery, castrated male SCID mice were anesthetized
with ketamine, and an incision was made in the left hindlimb. The
tibia was punctured using a bone drill, and 1 × 105 cells (22Rv1 or
LNCaP-AR) were injected into the cavity. The puncture was closed
with bone wax, the incision sutured, and animals received a palliative
dose of carprofen (5 mg/kg) once daily for 3 days postsurgery. Tumor
development was followed with bioluminescence imaging and confirmed with MRI. The bone fracture model was prepared similar to
the osseous tumor model, excluding injection of cells and application of bone wax.
Biodistribution Studies
Biodistribution studies were conducted to evaluate the uptake of
Zr-5A10 in human prostate cancer xenograft models. Mice received
89
Zr-5A10 [1.11–1.85 MBq (30–50 μCi), 5.7–9.5 μg of protein, in 200
μL sterile saline for injection] through intravenous tail-vein injection
(t = 0 hour). Animals (n = 4–5 per group) were euthanized by CO2 asphyxiation at 1, 4, 12, 24, 48, 72, 96, and 120 hours postinjection and
blood was immediately harvested by cardiac puncture. Sixteen tissues (including the tumor) were removed, rinsed in water, dried in air
for 5 minutes, weighed, and counted on a gamma-counter for accumulation of 89Zr radioactivity. The tumor tissues were partitioned for
biodistribution or PSA ELISA. The mass of 89Zr-5A10 formulation
injected into each animal was measured and used to determine the
total number of counts per minute by comparison to a standard syringe of known activity and mass. Count data were background- and
decay-corrected and the tissue uptake [measured in units of percentage injected dose per gram (% ID/g)] for each sample was calculated
by normalization to the total amount of activity injected.
89
Small-Animal Positron Emission Tomography Imaging
PET imaging experiments were conducted on a micro-PET Focus
120 scanner (Concorde Microsystems). In repeated studies (n = 4)
mice were administered formulations of 89Zr-5A10 (10.4–12.6 MBq
[280–340 μCi], 53.1–64.5 μg of protein, in 200 μL sterile saline for
injection) through intravenous tail-vein injection. Approximately 5
minutes before recording PET images, mice were anesthetized by
inhalation of 1% to 2% isoflurane (Baxter Healthcare)/oxygen gas
mixture and placed on the scanner bed. PET images were recorded
at various time points between 1 and 120 hours postinjection.
research BRIEF
List-mode data were acquired using a γ-ray energy window of 350
to 750 keV and a coincidence timing window of 6 nanoseconds. For
all static images, scan time was adjusted to ensure a minimum of 20
million coincident events were recorded. Data were sorted into 2-dimensional histograms by Fourier rebinning, and transverse images
were reconstructed by filtered back-projection into a 128 × 128 × 63
(0.72 × 0.72 × 1.3 mm) matrix. The reconstructed spatial resolution
for 89Zr was 1.9 mm full-width half-maximum at the center of the
field of view. The image data were normalized to correct for nonuniformity of response of the PET, dead-time count losses, positron
branching ratio, and physical decay to the time of injection, but no
attenuation, scatter, or partial-volume averaging correction was applied. An empirically determined system calibration factor [in units
of (mCi/mL)/(cps/voxel)] for mice was used to convert voxel count
rates to activity concentrations. The resulting image data were then
normalized to the administered activity to parameterize images in
terms of percentage injected dose per gram. Manually drawn 2-dimensional regions of interest were used to determined the maximum
and mean percentage injected dose per gram (decay corrected to the
time of injection) in various tissues. Images were analyzed by using
ASIPro VMTM software (Concorde Microsystems).
Coregistered Positron Emission Tomography/Computed
Tomography
CT images were acquired on a small-animal Siemens/CTI microCAT II (Siemens Medical Solutions) scanner with an 8.5-cm axial
by 5.0-cm transaxial field of view. Coregistered PET/CT images were
recorded and mapped to a matrix in accordance with previously reported methods (28).
Magnetic Resonance Imaging
Mouse prostate MR images were acquired on a Bruker 4.7-T
Biospec scanner operating at 200 MHz and equipped with a 400
mT/m inner diameter 12-cm gradient coil (Bruker Biospin MRI
GmbH). A custom-built quadrature birdcage resonator with inner diameter of 32 mm was used for radiofrequency excitation and acquisition (Stark Contrast MRI Coils Research, Inc.). Mice were
anesthetized with oxygen and 1% isoflurane gas. Animal breathing was monitored by using a small-animal physiologic monitoring
system (SA Instruments, Inc.). T2-weighted scout images along 3
orthogonal orientations were first acquired for animal positioning.
The T2-weighted fast spin-echo rapid acquisition with relaxation enhancement (RARE) sequence was used to acquire axial mouse pelvic
images with a slice thickness of 0.8 mm and field of view 30 mm ×
34 mm with a spatial resolution of 117 × 133 μm. The following
acquisition parameters—TR = 4.5 s, TE = 40 ms, RARE factor 8, and
an acquisition time of 20 minutes—were used.
PSA Detection in Serum and Tumor Tissues
Free PSA and total PSA were measured with a dual-label immunofluorometric assay (DELFIA ProstatusTM PSA Free/Total PSA;
Perkin-Elmer Life Sciences) according to the manufacturer’s recommendations. This assay measures free PSA and complexed PSA in
an equimolar fashion (29), and the crossreactivity of PSA-ACT for
free PSA is less than 0.2% (10). The lower limits of detection are 0.05
μg/L for total PSA [coefficient of variation (CV) = 5.0% at 2.32 μg/L]
and 0.04 μg/L for free PSA (CV = 5.9% at 0.25 μg/L). For detection,
the 1235 automatic immunoassay system from Perkin-Elmer Life
Sciences was used. Complexed PSA concentrations were calculated by
subtracting free PSA from total PSA.
Affinity Tests of 89Zr-Labeled 5A10
The capture mAb H117 was immobilized onto microtiter plates
through physical adsorption by using low-fluorescence Maxisorp
april 2012 CANCER DISCOVERY | 325
Downloaded from cancerdiscovery.aacrjournals.org on June 16, 2017. © 2012 American Association for
Cancer Research.
Published OnlineFirst March 31, 2012; DOI: 10.1158/2159-8290.CD-11-0316
Ulmert et al.
research BRIEF
strips (Nunc). The wells were coated with 1 μg of the mAb H117 in
100 L of buffer containing 0.2 mol/L NaH2PO4 buffer overnight at
35°C. Coated wells were washed twice with DELFIA wash solution
and were then saturated for 3 hours at room temperature with 300
μL of a solution containing DTPA-treated bovine serum albumin
(1 g/L), sorbitol (60 g/L), diazolidinyl urea (1 g/L), and 50 mmol
of NaH2PO4. After saturation, the wells were aspirated, dried, and
stored at 4°C in sealed plastic bags with desiccant until use.
Using a fresh sample of 89Zr-5A10, affinity assays were conducted
by adding a solution of fPSA (25 μL, 47.7 ng/mL) in 100 μL of
DELFIA assay buffer to each well from the previously prepared
plates. After incubation for 1 hour at room temperature, the solution was aspirated, and the wells were washed twice with assay buffer. The binding assay was initiated with the addition of 200-μL
aliquots of assay buffer containing 20 μg of 89Zr-5A10 and 0.0, 0.002,
0.02, 0.2, 0.5, 1, 2, 4, 6, 8, or 10 μg of unlabeled 5A10. All reactions
were conducted in duplicate. The reactions were incubated, with slow
shaking, for 2 hours, at which time the wells were aspirated and
rinsed 4 times with DELFIA wash solution. The bound activity was
determined in a NaI (Tl) well counter (Perkin Elmer 2480 Automatic
Gamma Counter).
Autoradiography and Tissue Histology
After mice were euthanized, the tibia, including the tumor, was surgically excised and embedded in optimal cutting temperature compound (Miles, Inc.) and snap-frozen on dry ice in a cryomold. Sets of
10 contiguous 5-μm-thick tissue sections were cut using a Microm
HM500 cryostat microtome (Microm International) and arrayed onto
poly-L-lysine–coated glass microscope slides. Tissue sections were fixed
in 10% phosphate-buffered formalin for 5 minutes, washed twice, airdried, and stained with hematoxylin and eosin (H&E). Stained tissue
sections were placed in a film cassette against a Fuji film BAS-MS2325
imaging plate (Fuji Photo Film Co.) to acquire digital autoradiograms.
The slides were exposed for 48 hours, approximately 168 hours after
injection of 89ZrCl or 89Zr-5A10. Exposed phosphor plates were read by
a Fujifilm BAS-1800II bio-imaging analyzer (Fuji Photo Film Co.) generating digital images with 50-μm pixel dimensions. Digital images
were obtained with an Olympus BX60 System Microscope (Olympus
America, Inc.) equipped with a motorized stage (Prior Scientific, Inc.).
Subsequently, H&E images were acquired to the same resolution as the
DAR data. DAR images were manually aligned to the H&E images using rigid planar transforms.
Statistical Analyses
Data were analyzed by using the unpaired, 2-tailed Student t test.
Differences at the 95% confidence level (P , 0.05) were considered to
be statistically significant.
Disclosure of Potential Conflicts of Interest
C.L. Sawyers is a coinventor of MDV3100 and owns stock in the
company (Medivation) that is developing the drug for prostate cancer treatment. H. Lilja is a coinventor of fPSA assays for in vitro diagnostics in blood (Arctic Partners). This article does not make any
claims about the efficacy of MDV3100 or the diagnostic value of
fPSA measurements in the blood; it merely uses MDV3100 and fPSA
measurements in serum as tools to evaluate the 5A10 antibody-based
radiotracer described herein.
Author Contributions
Conception and design: D. Ulmert, M.J. Evans, J.P. Holland,
S.M. Larson, H. Lilja, J.S. Lewis, C.L. Sawyers
Development of methodology: D. Ulmert, M.J. Evans, J.P. Holland,
K. Pettersson
Acquisition of data: D. Ulmert, M.J. Evans, J.P. Holland, S.L. Rice,
J. Wongvipat, H. Lilja, J.S. Lewis
326 | CANCER DISCOVERY april 2012 Analysis and interpretation of data: D. Ulmert, M.J. Evans,
J.P. Holland, S.L. Rice, P-A. Abrahamsson, S.M. Larson, H. Lilja, J.S. Lewis
Writing, review, and/or revision of the manuscript: D. Ulmert, M.J.
Evans, J.P. Holland, S.L. Rice, K. Pettersson, P-A. Abrahamsson, S.M.
Larson, H. Lilja, J.S. Lewis, C.L. Sawyers
Administrative, technical, or material support: D. Ulmert,
J.P. Holland, J. Wongvipat, P-A. Abrahamsson, H. Lilja
Study supervision: D. Ulmert, P-A. Abrahamsson, P.T. Scardino,
S.M. Larson, H. Lilja, J.S. Lewis, C.L. Sawyers
Providing the critical reagent (antibody) for the study: K. Pettersson
Acknowledgments
We thank Drs. Howard Scher, Naga Vara Kishore Pillarsetty,
Brett Carver, and Pat Zanzonico for comments; and Valerie Longo,
Thomas Ku, Michael Doran, Dr. Sara Cheal, Dr. Katharina Braun,
Dr. Sean Carlin, Dr. Carl Le, Dov Winkleman, William Golden, and
Brad Beattie for technical assistance.
Grant Support
D. Ulmert, M.J. Evans, C.L. Sawyers, and J.S. Lewis were supported in
part by the Geoffrey Beene Cancer Research Center of Memorial SloanKettering Cancer Center (MSKCC). D. Ulmert was supported in part
by the Tegger Foundation and the David H. Koch Young Investigator
Award from the Prostate Cancer Foundation. M.J. Evans was supported in part by the Brain Tumor Center at MSKCC. J.S. Lewis, S.L.
Rice, and J.P. Holland were supported by the Office of Science (BER),
U.S. Department of Energy (DE-SC0002456). M.J. Evans and S.L. Rice
were supported in part by a training grant in molecular imaging from
the NIH (R25-CA096945). S.M. Larson was supported by the Ludwig
Center for Cancer Immunotherapy at MSKCC and the National Cancer
Institute (P50-CA86438). C.L. Sawyers was supported in part by the
Howard Hughes Medical Institute. H. Lilja was supported in part by
the National Cancer Institute (R33 CA127768-02), the Swedish Cancer
Society (3455), the Swedish Research Council (Medicine-20095), and the
Sidney Kimmel Center for Prostate and Urologic Cancers. P.T. Scardino
and H. Lilja were supported in part by the Prostate Cancer SPORE
through the National Cancer Institute (P50-CA92629) and the David H.
Koch Fund through the Prostate Cancer Foundation. Technical services
provided by the MSKCC Small-Animal Imaging Core Facility were supported in part by the NIH (R24-CA83084, P30-CA08748).
Received November 30, 2011; revised January 20, 2012; accepted
February 6, 2012; published OnlineFirst March 31, 2012.
References
1. Gayed I, Vu T, Iyer R, Johnson M, Macapinlac H, Swanston N, et al.
The role of 18F-FDG PET in staging and early prediction of response
to therapy of recurrent gastrointestinal stromal tumors. J Nucl Med
2004;45:17–21.
2. Lilja H, Ulmert D, Vickers AJ. Prostate-specific antigen and prostate cancer: prediction, detection and monitoring. Nat Rev Cancer
2008;8:268–78.
3. Ulmert D, Serio AM, O’Brien MF, Becker C, Eastham JA, Scardino
PT, et al. Long-term prediction of prostate cancer: prostate-specific
antigen (PSA) velocity is predictive but does not improve the predictive accuracy of a single PSA measurement 15 years or more before
cancer diagnosis in a large, representative, unscreened population. J
Clin Oncol 2008;26:835–41.
4. Ulmert D, Cronin AM, Bjork T, O’Brien MF, Scardino PT, Eastham JA, et al. Prostate-specific antigen at or before age 50 as a predictor of advanced prostate cancer diagnosed up to 25 years later: a case–control
study. BMC Med 2008;6:6.
5. Vickers AJ, Cronin AM, Bjork T, Manjer J, Nilsson PM, Dahlin A, et al. Prostate specific antigen concentration at age 60 and death or metastasis from prostate cancer: case-control study. BMJ 2010;341:c4521.
www.aacrjournals.org
Downloaded from cancerdiscovery.aacrjournals.org on June 16, 2017. © 2012 American Association for
Cancer Research.
Published OnlineFirst March 31, 2012; DOI: 10.1158/2159-8290.CD-11-0316
PET Imaging of Androgen Receptor Signaling
6. Stege RH, Tribukait B, Carlstrom KA, Grande M, Pousette AH. Tissue
PSA from fine-needle biopsies of prostatic carcinoma as related to serum PSA, clinical stage, cytological grade, and DNA ploidy. Prostate
1999;38:183–8.
7. Lilja H. A kallikrein-like serine protease in prostatic fluid cleaves the
predominant seminal vesicle protein. J Clin Invest 1985;76:1899–903.
8. Christensson A, Laurell CB, Lilja H. Enzymatic activity of prostatespecific antigen and its reactions with extracellular serine proteinase
inhibitors. Eur J Biochem 1990;194:755–63.
9. Lilja H, Christensson A, Dahlen U, Matikainen MT, Nilsson O,
Pettersson K, et al. Prostate-specific antigen in serum occurs predominantly in complex with alpha 1-antichymotrypsin. Clin Chem
1991;37:1618–25.
10. Pettersson K, Piironen T, Seppala M, Liukkonen L, Christensson A,
Matikainen MT, et al. Free and complexed prostate-specific antigen
(PSA): in vitro stability, epitope map, and development of immunofluorometric assays for specific and sensitive detection of free PSA and
PSA-alpha 1-antichymotrypsin complex. Clin Chem 1995;41:1480–8.
11. Piironen T, Villoutreix BO, Becker C, Hollingsworth K, Vihinen M,
Bridon D, et al. Determination and analysis of antigenic epitopes
of prostate specific antigen (PSA) and human glandular kallikrein 2
(hK2) using synthetic peptides and computer modeling. Protein Sci
1998;7:259–69.
12. Holland JP, Divilov V, Bander NH, Smith-Jones PM, Larson SM,
Lewis JS 89Zr-DFO-J591 for immunoPET of prostate-specific membrane antigen expression in vivo. J Nucl Med 2010;51:1293–300.
13. Holland JP, Sheh Y, Lewis JS. Standardized methods for the production of high specific-activity zirconium-89. Nucl Med Biol
2009;36:729–39.
14. Holland JP, Caldas-Lopes E, Divilov V, Longo VA, Taldone T,
Zatorska D, et al. Measuring the pharmacodynamic effects of a novel
Hsp90 inhibitor on HER2/neu expression in mice using Zr-DFOtrastuzumab. PLoS One 2010;5:e8859.
15. Chen CD, Welsbie DS, Tran C, Baek SH, Chen R, Vessella R, et al.
Molecular determinants of resistance to antiandrogen therapy. Nat
Med 2004;10:33–9.
16. Tran C, Ouk S, Clegg NJ, Chen Y, Watson PA, Arora V, et al.
Development of a second-generation antiandrogen for treatment of
advanced prostate cancer. Science 2009;324:787–90.
17. Scher HI, Beer TM, Higano CS, Anand A, Taplin ME, Efstathiou E, et al. Antitumour activity of MDV3100 in castration-resistant prostate cancer: a phase 1–2 study. Lancet 2010;375:1437–46.
research BRIEF
18. Mehra R, Kumar-Sinha C, Shankar S, Lonigro RJ, Jing X, Philips
NE, et al. Characterization of bone metastases from rapid autopsies of prostate cancer patients. Clin Cancer Res 2011;17:3924–32.
19. Cooper CR, Chay CH, Gendernalik JD, Lee HL, Bhatia J, Taichman RS, et al. Stromal factors involved in prostate carcinoma metastasis to
bone. Cancer 2003;97:739–47.
20. Even-Sapir E, Metser U, Mishani E, Lievshitz G, Lerman H,
Leibovitch I. The detection of bone metastases in patients with highrisk prostate cancer: 99mTc-MDP Planar bone scintigraphy, singleand multi-field-of-view SPECT, 18F-fluoride PET, and 18F-fluoride
PET/CT. J Nucl Med 2006;47:287–97.
21. Evans MJ, Smith-Jones PM, Wongvipat J, Navarro V, Kim S, Bander NH,
et al. Noninvasive measurement of androgen receptor signaling with
a positron-emitting radiopharmaceutical that targets prostate-specific
membrane antigen. Proc Natl Acad Sci U S A 2011;108:9578–82.
22. Milowsky MI, Nanus DM, Kostakoglu L, Vallabhajosula S, Goldsmith
SJ, Bander NH. Phase I trial of yttrium-90-labeled anti-prostate-specific membrane antigen monoclonal antibody J591 for androgenindependent prostate cancer. J Clin Oncol 2004;22:2522–31.
23. Chang SS, Reuter VE, Heston WD, Bander NH, Grauer LS, Gaudin
PB. Five different anti-prostate-specific membrane antigen (PSMA)
antibodies confirm PSMA expression in tumor-associated neovasculature. Cancer Res 1999;59:3192–8.
24. Noss KR, Wolfe SA, Grimes SR. Upregulation of prostate specific
membrane antigen/folate hydrolase transcription by an enhancer.
Gene 2002;285:247–56.
25. Kessenbrock K, Plaks V, Werb Z. Matrix metalloproteinases: regulators of the tumor microenvironment. Cell 2010;141:52–67.
26. Borjesson PK, Jauw YW, de Bree R, Roos JC, Castelijns JA, Leemans
CR, et al. Radiation dosimetry of 89Zr-labeled chimeric monoclonal
antibody U36 as used for immuno-PET in head and neck cancer patients. J Nucl Med 2009;50:1828–36.
27. Holland JP, Sheh Y, Lewis JS. Standardized methods for the production of high specific-activity zirconium-89 Nucl Med Biol
2009;36:729–39.
28. Beattie BJ, Forster GJ, Govantes R, Le CH, Longo VA, Zanzonico PB, et al. Multimodality registration without a dedicated multimodality
scanner. Mol Imaging 2007;6:108–20.
29. Mitrunen K, Pettersson K, Piironen T, Bjork T, Lilja H, Lovgren T.
Dual-label one-step immunoassay for simultaneous measurement of
free and total prostate-specific antigen concentrations and ratios in
serum. Clin Chem 1995;41:1115–20.
april 2012 CANCER DISCOVERY | 327
Downloaded from cancerdiscovery.aacrjournals.org on June 16, 2017. © 2012 American Association for
Cancer Research.
Published OnlineFirst March 31, 2012; DOI: 10.1158/2159-8290.CD-11-0316
Imaging Androgen Receptor Signaling with a Radiotracer
Targeting Free Prostate-Specific Antigen
David Ulmert, Michael J. Evans, Jason P. Holland, et al.
Cancer Discovery 2012;2:320-327. Published OnlineFirst March 31, 2012.
Updated version
Supplementary
Material
Cited articles
Citing articles
E-mail alerts
Reprints and
Subscriptions
Permissions
Access the most recent version of this article at:
doi:10.1158/2159-8290.CD-11-0316
Access the most recent supplemental material at:
http://cancerdiscovery.aacrjournals.org/content/suppl/2012/04/06/2159-8290.CD-11-0316.DC
2
http://cancerdiscovery.aacrjournals.org/content/suppl/2012/02/08/2159-8290.CD-11-0316.DC
1
This article cites 29 articles, 14 of which you can access for free at:
http://cancerdiscovery.aacrjournals.org/content/2/4/320.full#ref-list-1
This article has been cited by 12 HighWire-hosted articles. Access the articles at:
http://cancerdiscovery.aacrjournals.org/content/2/4/320.full#related-urls
Sign up to receive free email-alerts related to this article or journal.
To order reprints of this article or to subscribe to the journal, contact the AACR Publications
Department at [email protected].
To request permission to re-use all or part of this article, contact the AACR Publications
Department at [email protected].
Downloaded from cancerdiscovery.aacrjournals.org on June 16, 2017. © 2012 American Association for
Cancer Research.