Published OnlineFirst September 30, 2013; DOI: 10.1158/0008-5472.CAN-13-1199
`
`Integrated Systems and Technologies
`
`Cancer
`Research
`
`Free Somatostatin Receptor Fraction Predicts the
`Antiproliferative Effect of Octreotide in a Neuroendocrine
`Tumor Model: Implications for Dose Optimization
`
`Pedram Heidari1, Eric Wehrenberg-Klee1, Peiman Habibollahi1, Daniel Yokell1,
`Matthew Kulke2, and Umar Mahmood1
`
`Abstract
`
`Somatostatin receptors (SSTR) are highly expressed in well-differentiated neuroendocrine tumors (NET).
`Octreotide, an SSTR agonist, has been used to suppress the production of vasoactive hormones and relieve
`symptoms of hormone hypersecretion with functional NETs. In a clinical trial, an empiric dose of octreotide
`treatment prolonged time to tumor progression in patients with small bowel neuroendocrine (carcinoid) tumors,
`irrespective of symptom status. However, there has yet to be a dose optimization study across the patient
`population, and methods are currently lacking to optimize dosing of octreotide therapy on an individual basis.
`Multiple factors such as total tumor burden, receptor expression levels, and nontarget organ metabolism/
`excretion may contribute to a variation in SSTR octreotide occupancy with a given dose among different patients.
`In this study, we report the development of an imaging method to measure surface SSTR expression and
`occupancy level using the PET radiotracer 68Ga-DOTATOC. In an animal model, SSTR occupancy by octreotide
`was assessed quantitatively with 68Ga-DOTATOC PET, with the finding that increased occupancy resulted in
`decreased tumor proliferation rate. The results suggested that quantitative SSTR imaging during octreotide
`therapy has the potential to determine the fractional receptor occupancy in NETs, thereby allowing octreotide
`dosing to be optimized readily in individual patients. Clinical trials validating this approach are warranted. Cancer
`Res; 73(23); 6865–73. Ó2013 AACR.
`
`Introduction
`Neuroendocrine tumors (NET) are a heterogeneous group of
`malignancies that are thought to originate from endocrine
`progenitor cells located in various organ systems including the
`lung, pancreas, and gastrointestinal tract. Very commonly,
`these NETs secrete a variety of biologically active peptides
`and amines that can lead to symptoms of wheezing, nausea,
`abdominal pain, flushing, and diarrhea, among others (1).
`Somatostatin receptor (SSTR) agonists have been employed
`with great success for controlling these symptoms (1, 2). With
`80% to 100% of well-differentiated NETs expressing high levels
`of SSTR (3), somatostatin analogs, such as octreotide, have
`become the treatment of choice for symptomatic relief through
`the reduction of NET hormone production.
`
`Authors' Affiliations: 1Division of Nuclear Medicine and Molecular Imag-
`ing, Department of Radiology, Massachusetts General Hospital, Harvard
`Medical School and 2Department of Medical Oncology, Dana-Farber
`Cancer Institute, Harvard Medical School, Boston, Massachusetts
`
`P. Heidari and E. Wehrenberg-Klee contributed equally to this work.
`
`Corresponding Author: Umar Mahmood, Division of Nuclear Medicine
`and Molecular Imaging, Department of Radiology, Massachusetts General
`Hospital, Boston, MA 02114. Phone: 617-726-6477; Fax: 617-726-6165;
`E-mail: umahmood@mgh.harvard.edu
`
`doi: 10.1158/0008-5472.CAN-13-1199
`Ó2013 American Association for Cancer Research.
`
`Over the past several years, it has further been demon-
`strated that SSTR agonists may also have an antiproliferative
`effect on NETs (4, 5) and may have a role as antineoplastic
`therapy for NETs. It was shown in a double-blind randomized
`controlled trial that patients with metastatic well-differen-
`tiated mid-gut NETs who received monthly intramuscular
`injections of a standard dose (30 mg) of long-acting octreo-
`tide (octreotide LAR) had a significantly increased progres-
`sion-free survival (PFS) compared to those who received
`placebo (15.6 vs. 5.9 months, respectively; ref. 6). The patients
`in this study benefited from octreotide LAR therapy regard-
`less of tumor functional status. The results of this trial
`suggest that octreotide may possess antiproliferative prop-
`erties on NETs that are enacted through SSTR-mediated
`signaling, irrespective of activation of pathways involved in
`bioactive peptide and amine production. This paradigm shift
`in the use of SSTR agonists for their antitumor effects has
`been empiric in nature, without an understanding of what
`fraction of the somatostatin receptors were bound during
`agonist therapy, how this changed over the course of a
`monthly treatment cycle, and the association with prolifer-
`ation. The development of a noninvasive approach to quan-
`tify the concentration of SSTR in tumors, the change in free
`receptor fraction with therapy, and the resultant downstream
`effects on proliferation would provide a method to optimize
`this treatment, both at the individual level and across treat-
`ment populations.
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`Published OnlineFirst September 30, 2013; DOI: 10.1158/0008-5472.CAN-13-1199
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`Heidari et al.
`
`Radionuclide imaging of SSTR through 111In-octreotide or
`68Ga–DOTATOC has largely been qualitative in nature, using the
`high contrast gained from the images to locate foci of disease not
`apparent on other imaging modalities or to monitor disease
`progression by the assessment of tumor volume changes over
`time (7–9). In fact, tumor imaging with these agents is typically
`performed either for primary staging before initiation of octreo-
`tide LAR therapy or monitoring of disease progression during
`the expected nadir blood levels of octreotide LAR to aid in tumor
`visualization (10, 11). The objective in this study was to use the
`quantitative nature of 68Ga–DOTATOC PET imaging to develop
`a technique that allows one to compute the receptor density in a
`tumor volume and monitor the fraction that is occupied with
`agonist treatment at a given time, based on changes in the
`available (unbound) receptor density. In combination with
`0
`0
`another positron-emitting radiotracer, 18F-fluoro-3
`-deoxy-3
`-
`L-fluorothymidine (18F-FLT), a proliferation marker (12), we
`were able to correlate the changes in unoccupied SSTR with
`proliferation status in an animal model. The imaging techniques
`and quantitation methods thus developed have the potential to
`be readily translated to patients with NETs to more effectively
`monitor treatment and improve dosing regimens.
`
`Materials and Methods
`Cell culture
`AR42J [American Type Culture Collection (ATCC)], an STTR
`expressing rat pancreatic carcinoma, was cultured in F-12K
`medium (ATCC), supplemented by 20% (v/v) FBS, 100 U/mL
`penicillin, and 100 mg/mL streptomycin. A549 (ATCC), an
`SSTR-negative human alveolar basal epithelial carcinoma cell
`line, was cultured in F-12K medium (ATCC), supplemented by
`10% (v/v) FBS, 100 U/mL penicillin, and 100 mg/mL strepto-
`mycin. Cultures were maintained in a humidified incubator at
`
`37
`C, 5% CO2. Subculturing was performed employing a 0.25%
`trypsin/0.1% EDTA solution. The cell lines were obtained from
`ATCC and were used in this study for less than 6 months after
`resuscitation. Cell lines undergo comprehensive quality con-
`trol and authentication procedures by ATCC before shipment.
`These include testing for mycoplasma by culture isolation,
`Hoechst DNA staining, and PCR, together with culture testing
`for contaminant bacteria, yeast, and fungi. Authentication
`procedures used include species verification by DNA barcoding
`and identity verification by DNA profiling.
`
`68Ga labeling of DOTATOC
`A 68Ge/68Ga generator (iThemba Labs) was eluted with 6 mL
`of 0.6 N HCl. The eluant was added to a buffer system of 2 mol/L
`HEPES at pH 3.5 to 4.0 with 5 mg of DOTATOC. The reaction
`
`solution was heated at 100
`C for 20 minutes. The reaction
`product was loaded on a reverse-phase C18 Sep-Pak mini
`cartridge and eluted with 200 mL of 200-proof ethanol. The final
`formulation was adjusted to 10% ethanol in saline. The chemical
`and radiochemical purity of 68Ga-DOTATOC was measured
`through radio thin-layer chromatography (TLC; refs. 13, 14).
`
`Competitive binding study
`To evaluate the specific binding of 68Ga-DOTATOC, a com-
`petitive binding assay using a fixed concentration of radio-
`
`tracer and increasing concentration of octreotide acetate was
`performed. AR42J (SSTR2-expressing) and A549 (SSTR-nega-
`tive) cells were seeded in 24-well plates (2.5  105/well) and
`allowed to grow to 80% confluence. Wells were incubated for 1
`hour with 0.01 to 1,000 mmol/L concentration of octreotide
`acetate (Abbiotec). Then, 25 mCi 68Ga-DOTATOC (9.5 nmol/L
`DOTATOC peptide) was added to each well and plates were
`
`incubated at 37
`C for 1 hour. The medium was removed and
`
`C PBS. Cells were collected
`wells were washed 3 times using 4
`after trypsin treatment, and the number of cells in each well
`was counted using an automated cell counter (Countess,
`Invitrogen). 68Ga activity in the cells in each well was assayed
`using an automated gamma counter (Wizard 2480, Perkin
`Elmer) and decay corrected for the beginning of incubation
`with 68Ga-DOTATOC.
`
`In vitro cell-cycle assay
`To assess the effect of octreotide on cell-cycle progression,
`AR42J and A549 were seeded in 6-well plates and incubated at
`
`C for 24 hours (A549) or 2 days (AR42J) in cell culture
`37
`medium. The medium was then removed and fresh medium
`was added to each well. Wells were randomized to receive
`octreotide acetate at a concentration of 1 mmol/L or no
`
`octreotide acetate and all wells were incubated at 37
`C for
`0
`-deoxyuridine;
`24 hours before the addition of EdU (5-ethynyl-2
`Click-it EDU kit, Invitrogen), a fluorescent DNA analog that is
`incorporated during DNA synthesis. Treated cells were then
`sorted for cell-cycle phase using fluorescence-activated cell
`sorting (FACS) and the percentage of the cells in S-phase
`determined in octreotide treated versus nontreated groups
`for each cell line. Studies were performed in triplicate.
`
`Western blotting for SSTR2 expression
`Nude (nu/nu) mice were injected subcutaneously with 106
`AR42J cells suspended in Matrigel (BD Biosciences) in the left
`upper flank. After PET imaging, AR42J tumors were removed
`and extracted and whole protein extract purification per-
`formed. Protein samples (30 mg) were loaded onto SDS-PAGE
`and run at 120 V and 14 mA for 1.5 hours. Gels were blotted on
`polyvinylidene difluoride (PVDF) membrane and the blots
`
`incubated overnight at 4
`C with SSTR2 monoclonal antibody
`(Abcam) at 1:500 dilution. b-Actin monoclonal antibody (Santa
`Cruz) at 1:1,000 dilution was used as an internal control.
`Detection was performed using the BM Chemiluminescence
`Western Blotting Kit (Mouse/Rabbit; Roche) and imaged on
`the Carestream In-Vivo Multispectral FX Imaging System.
`Quantitation of SSTR2 and b-actin expression was performed
`by drawing a region of interest around the protein bands on
`chemiluminescence images acquired with the Carestream
`Molecular Imaging Software. The acquired data were normal-
`ized using b-actin expression and corrected for tumor weight.
`
`In vivo imaging studies
`AR42J-bearing mice were divided randomly in 4 groups
`(n ¼ 3 in each group) that received vehicle, 1.25, 2.5, or 10
`mg/kg octreotide acetate, delivered via intraperitoneal injec-
`tion every 6 hours for a total of 5 injections to reach steady-
`state blood levels. Five hours following injection of the fifth
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`Published OnlineFirst September 30, 2013; DOI: 10.1158/0008-5472.CAN-13-1199
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`Free Somatostatin Receptor Predicts Proliferation
`
`In vitro studies. A, 68Ga-DOTATOC demonstrated significantly higher binding to AR42J compared to A549 cells (P < 0.0001). B, in vitro competitive
`Figure 1.
`binding studies demonstrate binding affinity of 68Ga-DOTATOC to SSTR. Octreotide concentration  10 mmol/L completely binds all sites, whereas doses of
`1 mmol/L allow for 68Ga-DOTATOC accumulation in graded manner in AR42J cells. A549 serve as a negative control with minimal expression of
`SSTR. C, gated FACS EDU binding data demonstrates that treatment with octreotide significantly decreased (P < 0.001) the number of AR42J cells in S-phase,
`whereas it had no effect on the number of A549 cells in S-phase. D, gated FACS EDU graph shows decreased proliferation in octreotide-treated versus
`control cultures for AR42J (top row), compared to A549 cells (second row), which showed no difference. Arrows, the cells in the S-phase.
`
`dose (trough blood level of octreotide acetate), the mice
`underwent dynamic PET imaging with 68Ga-DOTATOC.
`Immediately following, the mice were imaged in static mode
`and then a sixth dose of octreotide was administered. Five
`hours after receiving the last dose of treatment solution the
`mice were imaged using 18F-FLT PET in static mode.
`
`Static PET imaging protocol
`Approximately 400 mCi of 68Ga-DOTATOC prepared as
`described above and diluted into a final volume of 150 to
`200 mL that was injected intravenously via tail vein, and 1 hour
`later, static PET images were acquired for 15 minutes in 2 bed
`positions using the Sedecal Argus microPET. Static PET imag-
`ing with 18F-FLT was performed 2 hours after intravenous
`injection of 400 mCi 18F-FLT. Images were reconstructed using
`2D-OSEM (4 iterations, 16 subsets) and were corrected for
`scatter and randoms. The mean standard uptake value (SUV-
`mean) for each tumor was calculated in a 3-dimensional (3D)
`region of interest autodrawn around the tumor using a 30%
`isocontour threshold.
`
`Dynamic PET imaging protocol and compartmental
`modeling
`Mice were placed under anesthesia with 2% isoflurane in O2
`and positioned on the scanner such that heart and tumor were
`both in the field of view. Dynamic PET data were acquired in
`list mode for 60 minutes beginning immediately before injec-
`tion of 400 mCi 68Ga-DOTATOC in 150 to 200 mL of volume via
`tail vein. The list mode data were then reframed in 40 fifteen-
`second, 20 thirty-second, 16 sixty-second, and 16 ninety-sec-
`ond frames. Scans were reconstructed and a 3D region of
`interest was set around the tumor, as described above. The
`input function was measured from a spherical region of
`interest with a 3-mm diameter over the center of the mouse
`heart. Time activity curves were plotted for tumor and blood
`pool. To determine the best compartmental model fit for 68Ga-
`DOTATOC binding, an octreotide challenge study was per-
`formed during a dynamic 68Ga-DOTATOC PET study in AR42J-
`bearing mice. The octreotide dose (150 mg) was injected via tail
`vein 10 minutes after the scan start and the administration of
`68Ga-DOTATOC.
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`Published OnlineFirst September 30, 2013; DOI: 10.1158/0008-5472.CAN-13-1199
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`Heidari et al.
`
`Ki-67 staining
`To determine tumor cell proliferation changes in response
`to octreotide therapy in vivo, Ki-67 staining was performed.
`Tumor samples from AR42J tumor–bearing mice from the
`different treatment groups described above were excised and
`kept in frozen tissue-embedding fixative (Fisher Scientific) at
`80
`
`C for further immunofluorescent staining. Briefly, slide-
`mounted 5-mm-thick sections were prepared and fixed using
`ice-cold acetone for 10 minutes followed by 3 wash with PBS
`(5 minutes each). Tissue sections were blocked for 30 minutes
`with 1% bovine serum albumin in PBS with Tween, washed
`with PBS, and incubated overnight at þ4
`
`C with Ki-67 anti-
`body (Abcam). The slides were washed again with PBS,
`mounted with mounting medium for fluorescence-containing
`DAPI (Vector Laboratories), and visualized by confocal fluo-
`rescent microscopy (Nikon). Ki-67–stained and -unstained
`cells in the resulting images were segmented using ImageJ,
`software and the percentage of cells stained for Ki-67 was
`determined.
`
`Statistical analysis
`The statistical analysis was performed using GraphPad
`Prism 5. Unpaired t test was used to compare the number of
`cells in S-phase in control and treatment groups. One-way
`ANOVA was employed to discern the differences in SUVmean
`and Ki among different treatment groups in mice. Tukey
`multiple comparison test was used to compare the significance
`between groups. The 68Ga-DOTATOC influx rate and SUV-
`mean were plotted against 18F-FLT SUVmean, and correlation
`between the measurements was determined using linear
`regression. P < 0.05 was considered statistically significant.
`Mean values are reported  SEM.
`Results
`Competitive binding and in vitro cell-cycle assays
`68Ga-DOTATOC demonstrated 8.6-fold greater binding to
`AR42J compared to A549 cells (P < 0.0001; Fig. 1A). This is
`consistent with the high expression of SSTR type 2 in AR42J
`cells and undetectable expression of SSTR2 in A549 cells. In
`the competition receptor-binding assay, nonlabeled octreotide
`competed specifically with the 68Ga-DOTATOC for binding to
`the AR42J cells. As shown in Fig. 1B, treatment of AR42J cells
`with increasing doses of octreotide acetate led to decreased
`68Ga-DOTATOC uptake. 68Ga-DOTATOC influx was complete-
`ly inhibited at an octreotide concentration of 10 mmol/L or
`higher. A549 did not show considerable 68Ga-DOTATOC
`uptake or displacement with octreotide treatment. This find-
`ing shows that 68Ga-DOTATOC can be used to monitor the
`SSTR octreotide occupancy in SSTR expressing cells. Cell-cycle
`assays were performed to demonstrate the effect of octreotide
`on cell proliferation. As seen in Fig. 1C and D, for SSTR-
`expressing AR42J cells, treatment with octreotide decreased
`the percentage of cells in S-phase by 53% compared to control
`(P < 0.001). In SSTR nonexpressing A549 cells, treatment with
`octreotide did not lead to a significant difference in S-phase
`compared to control (P > 0.5). These data suggest that octreo-
`tide exerts a downstream inhibitory effect on cell proliferation
`through somatostatin receptors.
`
`Correlation of imaging findings with SSTR2 protein
`levels
`There was a consistent ratio between SSTR2 expression
`level and 68Ga-DOTATOC quantitative imaging measures
`irrespective of tumor size (tumors with diameter 3.9–8.2
`mm) in AR42J tumors. The total 68Ga-DOTATOC uptake of
`the tumors as measured by the molecular tumor burden
`(MTB; ref. 15) on PET studies, which were acquired in the
`static mode, strongly correlates with the total SSTR2 content
`in tumors (R2 ¼ 0.99, P < 0.0001), as seen in Fig. 2A. MTB is
`the product of SUVmean and molecular tumor volume (total
`volume of the voxels in the region of interest with 68Ga-
`DOTATOC uptake above the defined threshold; ref. 16).
`Moreover, the mean 68Ga-DOTATOC uptake in tumors
`(SUVmean) also strongly correlates (Fig. 2B) with the expres-
`sion of SSTR2 normalized for b-actin expression (R2 ¼ 0.85,
`P < 0.0004). These findings demonstrate that the noninva-
`sively measured 68Ga-DOTATOC uptake is a true reflection
`of the SSTR2 levels in the tumors.
`
`Dynamic 68Ga-DOTATOC PET imaging of AR42J tumors
`Octreotide challenge studies showed that 68Ga-DOTATOC
`is partially displaced by octreotide but a large fraction was not
`
`Figure 2. Western blotting results demonstrate a consistent ratio between
`SSTR2 expression level and 68Ga-DOTATOC quantitative measures
`irrespective of tumor size (tumors with diameter 3.9–8.2 mm) in AR42J
`tumors. A, total 68Ga-DOTATOC uptake of the tumors (MTB) strongly
`correlates with the total SSTR2 content in tumors. B, the mean 68Ga-
`DOTATOC uptake in tumors (SUVmean) also strongly correlates with the
`SSTR2/b-actin expression ratio.
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`Published OnlineFirst September 30, 2013; DOI: 10.1158/0008-5472.CAN-13-1199
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`Free Somatostatin Receptor Predicts Proliferation
`
`Figure 3. Dynamic 68Ga-DOTATOC scans of 2 AR42J-bearing mice. A, in this dataset, octreotide IV challenge at 10 minutes resulted in partial displacement of
`tracer from the receptor, but a component of the uptake could not be displaced even though the Patlak plots (B) show essentially no tracer influx after
`octreotide challenge in steady-state condition (orange dots). Please note that the slope of the best-fit line (Ki) in the steady state is approximately zero
`(R2 ¼ 0.98). C, without an octreotide drug challenge, the tracer accumulates in the tumor with a constant rate, as is evident in the Patlak plot (D); in the steady-
`state condition, the orange dots the slope of the best-fit line (Ki) is 0.69 (R2 ¼ 0.99). E, this pattern of tracer uptake is compatible with an irreversible two-tissue
`Rt
`CPðtÞdt
`in the x-axis is plotted against CTissueðtÞ
`compartment model. F, the x- and y-axes of the Patlak plots (such as B and D) are calculated using formula 1;
`CPðtÞ
`CPðtÞ
`the y-axis to draw the Patlak plot and K is measured using linear regression. The net tracer influx rate (Ki) is measured using formula 2. t, time after
`tracer injection; CTissue, the amount of tracer in the region of interest; CP(t), the concentration of tracer in plasma; K, the rate of entry into the
`irreversible compartment; V0, the distribution volume of the tracer in the central compartment.
`
`in
`
`0
`
`displaceable following competitive challenge. The displace-
`able fraction was the 68Ga-DOTATOC bound to SSTR but not
`yet internalized, whereas the remaining component was
`already internalized and non-displaceable (Fig. 3A and C).
`These findings were compatible with an irreversible 2-com-
`partment tissue model (Fig. 3E; ref. 17). Thus, on the basis of
`the equations in Fig. 3F, we employed a Patlak graphical plot
`and calculated the tumor influx constant (Ki; refs. 18, 19). We
`noted that for all studies, steady state was achieved in less
`than 25 minutes, and thus a 25-minute cutoff was used for
`fitting the Ki. The net 68Ga-DOTATOC influx rate, measured
`using a Patlak plot, following IV challenge of octreotide
`1/min
`1 (Fig.
`decreased to 0.0 (mL plasma)/(mL tissue)
`
`3B), whereas the influx rate was approximately 0.7 (mL
`1/min
`1 without an octreotide chal-
`plasma)/(mL tissue)
`lenge (Fig. 3D).
`The parameter Ki, which is the net rate of 68Ga-DTOATOC
`influx, is independent of tumor perfusion and reflects the
`number of available receptors as well as the rate of receptor
`trafficking (17–19). Measurement of the Ki in AR42J tumors
`demonstrated
`that
`octreotide
`treatment
`significantly
`decreased the rate of tracer influx in all treatment groups
`compared to control and that the mean Ki monotonically
`decreased with higher doses. The mean Ki was 0.67  0.02,
`0.61  0.03, 0.23  0.02, and 0.17  0.03 (mL plasma)/(mL
`
`1/min1 in vehicle, 1.25, 2.5, and 10 mg/kg treatment
`tissue)
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`Heidari et al.
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`Figure 4. Dynamic PET imaging of AR42J tumors in vehicle (A), 1.25 mg/kg (B), 2.5 mg/kg (C), and 10 mg/kg (D) octreotide groups with accompanying Patlak
`plot on top right corner of each panel (orange). There is a graded decrease in the slope of the Patlak plot (Ki) after reaching steady state with increasing doses
`of octreotide treatment (blue, blood pool; red, tumor uptake; purple, tumor/blood pool ratio; green, data points in Patlak plot before reaching steady-state
`blood levels; orange, data points in Patlak plot after reaching steady-state blood levels).
`
`groups, respectively, as shown in Fig. 4. There was a significant
`decrease in the Ki of all treatment groups compared to the
`vehicle group. The decreasing Ki reflects the number of recep-
`tors occupied by octreotide and that are unavailable to bind
`68Ga-DOTATOC.
`
`Static 68Ga-DOTATOC PET imaging of AR42J tumors
`Static scan results confirmed and paralleled the results
`of dynamic 68Ga-DOTATOC PET imaging. Treatment with
`increasing doses of octreotide acetate led to progressively
`significant decreases in tumor SUVmean compared to con-
`trol. Representative images are shown in Fig. 5. The mean
`SUVmean was 0.96  0.05, 0.88  0.08, 0.42  0.03, and 0.21 
`0.04 in vehicle, 1.25, 2.5, and 10 mg/kg treatment groups,
`respectively (Fig. 6B). As shown in the Fig. 6D, there was a
`very strong correlation between tumor SUVmean and Ki
`measured by static and dynamic 68Ga-DOTATOC PET imaging
`(R2 ¼ 0.95, P < 0.0001). The high agreement of quantitative
`parameters between static and dynamic 68Ga-DOTATOC PET
`in our study suggests that SUVmean of the tumors in static
`PET, although not as accurate as Ki measured by dynamic
`PET for free receptor density, can be effectively employed to
`monitor SSTR occupancy with octreotide treatment.
`
`Static 18F-FLT PET imaging and its correlation with
`68Ga-DOTATOC PET imaging
`Treatment with octreotide acetate decreased the SUVmean
`of 18F-FLT tumor uptake in all treatment groups compared to
`the control group. As with dynamic and static 68Ga-DOTATOC
`
`PET imaging, higher doses of octreotide led to monotonically
`decreasing 18F-FLT PET SUVmean. The mean SUVmean was
`1.40  0.10, 1.30  0.08, 0.34  0.06, and 0.18  0.03 in vehicle,
`1.25, 2.5, and 10 mg/kg treatment groups, respectively, as seen
`in Fig. 6. There was a strong correlation between the SUVmean
`of the tumors in 18F-FLT PET scans and both SUVmean and
`Ki of tumors in 68Ga-DOTATOC PET scans (R2 ¼ 0.95, P <
`0.0001 and R2 ¼ 0.97, P < 0.0001, respectively; Fig. 6E and F).
`This demonstrates that increased occupancy of SSTR with
`octreotide results in a reduced rate of tumor proliferation in
`vivo, assessed by static18F-FLT PET; the magnitude of reduc-
`tion in tumor proliferation rate is directly correlated with
`and dependent on the level of SSTR octreotide occupancy
`and thus can be potentially monitored using 68Ga-DOTA-
`TOC PET imaging.
`
`Ki-67 staining results
`Treatment of AR42J tumors with increasing doses of octreo-
`tide led to a reduction in the percentage of cells staining for Ki-
`67. The mean percentage of cells stained for Ki-67 was 25  1.2,
`23  0.8, 11  0.9, and 5  1.1 in the vehicle, 1.25, 2.5, and 10
`mg/kg treatment groups, respectively. Increase in the SSTR
`octreotide occupancy results in a corresponding decrease in
`the rate of tumor proliferation shown by the decrease in
`relative number of cells stained for Ki-67 (Fig. 5). These results
`are consistent with the results obtained by noninvasive
`18F-FLT PET imaging and further confirm the enhanced anti-
`proliferative effects of octreotide on tumor cells with increas-
`ing level of SSTR octreotide occupancy.
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`Free Somatostatin Receptor Predicts Proliferation
`
`Figure 5.
`Imaging of AR42J tumors
`using static 68Ga-DOTATOC (A-D),
`18F-FLT (E-H), and staining for Ki-
`67 in tumors samples (I-L) in 4
`groups of mice treated with vehicle,
`1.25, 2.5, or 10 mg/kg groups.
`There is high 68Ga-DOTATOC and
`18F-FLT uptake as well as 25%
`cells stained for Ki-67 in tumors in
`the control group; there is a graded
`decrease across all the biomarkers
`with increased dose of octreotide
`treatment. The 10 mg/kg
`octreotide dose shows near
`background levels of 68Ga-
`DOTATOC and 18F-FLT uptake
`and 5% staining for Ki-67.
`
`Discussion
`Octreotide continues to play a key role in the treatment of
`patients with metastatic NET, both for the control of symp-
`toms of hypersecretion and, more recently, for control of tumor
`growth (1, 20–22). In an efficacy clinical trial called the PRO-
`MID trial (6), it was shown that a fixed monthly injection of
`octreotide LAR significantly increased the time to tumor
`progression in patients with metastatic mid-gut carcinoids by
`8.3 months compared to placebo, demonstrating the antipro-
`liferative effect of octreotide LAR on well-differentiated mid-
`gut NETs. However, the potential for optimized individual
`dosing of octreotide to lead to further significant improve-
`ments in PFS has never been explored. Quantitative noninva-
`sive assessment of somatostatin receptor occupancy and
`downstream pharmacodynamic assessment of proliferation,
`as demonstrated in this study, could directly guide such
`personalized dosing optimization.
`This paradigm shift from the use of imaging for disease
`detection to disease characterization, including the direct and
`downstream molecular effects of therapy on specific tumors,
`provides an opportunity to use such assessment to prospec-
`tively guide tailored therapy rather than retrospective report-
`ing on treatment effectiveness measured by tumor volume
`changes. Such paired imaging of upstream intracellular sig-
`naling from cell surface receptors and downstream effects such
`as proliferation changes or alterations in apoptosis rates could
`be applied to a broad range of targeted therapies for individual
`patient drug dosing optimization. This includes therapies
`
`targeted at receptor tyrosine kinases, estrogen receptors, and
`androgen receptors, among other targets.
`The quantitative nature of the 68Ga-DOTATOC PET measure-
`ments allows an indirect assessment of fractional SSTR occu-
`pancy. Our kinetic model is similar to that developed by Henze
`and colleagues (17) for characterization of the kinetics of 68Ga-
`DOTATOC uptake in brain meningiomas. Using dynamic PET
`imaging, we established that 68Ga-DOTATOC and octreotide
`directly compete for binding to SSTR and that binding of
`somatostatin analogs results in irreversible internalization
`(23) of the ligand receptor complex. Using this tracer kinetic
`model, we could then reliably calculate SSTR free fraction with
`increasing doses of octreotide. We demonstrated a highly sig-
`nificant correlation between the net tracer influx rate (Ki) and
`the SUV measurement. The net tracer influx rate (Ki) is mea-
`sured by Patlak graphical analysis of dynamic PET data based on
`an irreversible 2-compartment kinetic model, which removes
`the effects of perfusion from the calculated receptor-mediated
`uptake values, whereas SUV, which is calculated from static PET
`data, is more routinely employed in the clinic and does not
`separate perfusion effects from the receptor mediated uptake.
`For clinical translation, both the net tracer influx rate (Ki) and
`SUV measurements could be determined in patients. Given the
`typical enhancement pattern seen on CT scanning of carcinoid
`tumors (24, 25) suggestive of high tumoral perfusion, these may
`correlate clinically as well as in preclinical assessment per-
`formed in this study, providing a means to more simply translate
`this approach for patient assessment.
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`Heidari et al.
`
`Figure 6. A–C, results of PET imaging in 4 treatment groups of mice bearing AR42J tumors (n¼ 3 in each treatment group). A, net tracer influx rate (Ki) in dynamic
`68Ga-DOTATOC shows a graded and significant decrease in uptake with increased dose of octreotide. B, the same pattern of decrease is observed in
`SUVmean of tumors in static 68Ga-DOTATOC PET (B) and 18F-FLT PET (C). D, there is strong correlation between SUVmean of tumor in static 68Ga-DOTATOC
`PET scans and Ki in dynamic 68Ga-DOTATOC PET analysis. E and F, correlation between SUVmean of tumor in 18F-FLT PET scans and Ki in dynamic
`68Ga-DOTATOC PET (D) as well as SUVmean in static 68Ga-DOTATOC PET (E). There is very strong correlation between 18F-FLT uptake in the tumor and both
`Ki and SUVmean of tumor in 68Ga-DOTATOC PET scans. , P < 0.05.
`
`One possible limitation to measuring receptor occupancy
`level using PET imaging is that it is most useful when the
`receptor-targeted therapy such as octreotide is in the subsatur-
`ating range. If the administered dose of octreotide is high enough
`to completely saturate receptors, then there will be no tracer
`uptake in the intracellular compartment;
`in these circum-
`stances, receptor quantitation using PET imaging shows com-
`plete occupancy and the degree of excess octreotide dose cannot
`be assessed. In practice, the majority of carcinoid patients is
`given subsat