RESEARCH ARTICLE
Evaluation of Positron Emission Tomographic Tracers for Imaging of Papillomavirus-Induced Tumors in Rabbits Sonja Probst,* Stefan Wiehr,* Frederic Mantlik, Holger Schmidt, Armin Kolb, Peter Mu¨nch, Maria Delcuratolo, Frank Stubenrauch, Bernd J. Pichler, and Thomas Iftner
Abstract In this study, simultaneous positron emission tomography (PET)/magnetic resonance (MR) imaging was employed to evaluate the feasibility of the PET tracers 2-deoxy-2-18F-fluoro-D-glucose (18F-FDG),
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C-choline, and
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F-fluorothymidine (18F-FLT) to detect
papillomavirus-induced tumors in an established rabbit model system. The combined PET/MR allowed the analysis of tracer uptake of the tumors using the morphologic information acquired by MR. New Zealand White rabbits were infected with cottontail rabbit papillomavirus genomes and were imaged for up to 10 months with a simultaneous PET/MR system during the course of infection. The uptake characteristics of the PET tracers
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C-choline and
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F-FLT of tumors and reference tissues were
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examined relative to the clinical standard, F-FDG. Tracer biodistribution of various organs was measured by gamma-counting after the last PET scan and compared to the in vivo PET/MR 18F-FDG uptake. Increased tracer uptake was found 2 months postinfection in primary tumors with
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F-FDG and
points. Our data show that the PET tracer 11
C-choline and
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F-FLT. However,
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18
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C-choline, whereas
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F-FDG.
UMAN PAPILLOMAVIRUSES (HPVs) are the causative agents for cervical cancer and have been implicated in other anogenital cancers and a subset of head and neck carcinomas.1–4 Cervical cancer is the third most frequent cancer in women worldwide. It is estimated that approximately 530,000 women developed cervical cancer and 275,000 died by reason of the disease in 2008.1 Twelve high-risk HPV types recognized as class I carcinogens by the World Health Organization are necessary risk factors for the development of cervical cancer.2 HPV has also been
H
*Authors who contributed equally to this work. From the Division of Experimental Virology, Institute of Medical Virology, University Hospital Tu¨bingen; Department of Preclinical Imaging and Radiopharmacy, Werner Siemens Imaging Center, Eberhard Karls University; Department of Empirical Inference, MaxPlanck Institute for Intelligent Systems; and Department of Diagnostics and Interventional Radiology, Eberhard Karls University Tu¨bingen, Tu¨bingen, Germany. Address reprint requests to: Thomas Iftner, PhD, Medical Virology, Section Experimental Virology, University Hospital Tu¨bingen, ElfriedeAulhorn-Str. 6, 72076 Tu¨bingen, Germany; e-mail: thomas.iftner@ med.uni-tuebingen.de. #
2014 Decker Publishing
F-FLT failed to detect the tumors at all measured time
F-FDG is superior for imaging papillomavirus-induced tumors in rabbits compared to
C-choline imaging, which has previously been applied to detect various tumor entities in
patients, appears to be an alternative to
DOI 10.2310/7290.2013.00070
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linked to the development of nonmelanoma skin cancer, which represents the most common malignancy in whites worldwide.5 An excellent animal model to study papillomavirus-induced cancer formation in vivo is the New Zealand White (NZW) rabbit, where infections with cottontail rabbit papillomavirus (CRPV) cause the development of skin tumors within 4 to 6 weeks postinfection, which then progress to invasive cancer without additional cofactors within 8 to 14 months.6,7 Although skin tumor progression may be directly evaluated by visual inspection, the depth and progression of subcutaneous invasion and its temporal molecular changes are difficult to assess. In this study, we used a simultaneous positron emission tomography (PET)/magnetic resonance (MR) system8 to combine anatomic and molecular information for more precise analyses of skin tumor growth or regression9 to develop a noninvasive method of monitoring papillomavirus-induced skin tumor growth in CRPV-infected NZW rabbits. This study aimed at identifying suitable tracers for future studies monitoring various therapy regimens, which include the use of potential tumor-inhibiting substances. Imaging methods, such as computed tomography (CT) or MR imaging, provide excellent morphologic information.9,10 One potential problem with these techniques is the lack of metabolic information, which is essential for
Molecular Imaging, Vol 13, 2014.
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the reliable prediction of the physiologic response of the cancer tissue to the respective treatment. Functional changes in tumor biology, such as apoptosis on treatment, occur much earlier than morphologic changes affecting the size of the tumor.11 With this in mind, PET reveals the metabolic and functional parameters of living cells and is capable of distinguishing between normal and malignant tissues through the use of various PET tracers. PET has previously been performed on mice with chemically induced skin tumors12,13 and in patients with sinonasal inverted papilloma,14,15 of which more than 70% are HPV negative,16 using 2-deoxy-2-18F-fluoro-D-glucose (18F-FDG) as a PET tracer. Here we evaluate the uptake of 18 F-FDG, 39-deoxy-39-18F-fluorothymidine (18F-FLT) and 11 C-choline into papillomavirus-induced tumors to determine the most suitable PET tracer for examining physiologic skin tumor growth and progression causally induced by papillomaviruses in rabbits. In addition to the widely used PET tracer 18F-FDG for glucose metabolism, the 18F-radiolabeled thymidine derivative 18F-FLT was used. 18F-FLT is a PET marker for cell proliferation and is consumed by proliferating cells via the salvage pathway, which incorporates nucleotides into replicating deoxyribonucleic acid (DNA) during the S phase of the cell cycle.17 18F-FLT is, however, rarely incorporated into the DNA18 and has been used in preclinical and clinical studies to monitor treatment efficacy in cancers.19 11CCholine is another promising PET tracer that can be used to detect membrane synthesis. It is initially phosphorylated within the tumor cell. The resulting phosphatidylcholine is then trapped in the lecithin of the cell membrane.20 Dividing tumor cells are characterized by increased cell membrane synthesis, leading to an enhanced uptake of 11C-choline. Thus, although not directly correlated with the cellular proliferation rate, 11Ccholine is an interesting candidate for cancer imaging where 18F-FDG and 18F-FLT fail. 11C-Choline was first used for imaging brain tumors21 and is currently employed primarily for detection and staging of prostate cancer,22–25 because of its low bladder activity. Thus, for detection of gynecologic malignancies 11C-choline might be superior to 18F-FDG and 18F-FLT, which are excreted via the urinary tract, because of reduced masking of the pelvic region.25,26 In this study, the clinical gold standard in PET oncology, 18F-FDG, showed a markedly increased uptake into the tumor tissue of rabbits, whereas 18F-FLT failed. However, 11C-choline revealed an uptake comparable to 18 F-FDG, thus representing an alternative PET tracer to 18 F-FDG for imaging papillomavirus-induced tumors.
Materials and Methods Animal Model and Infection Procedure Specific pathogen-free (SPF) female NZW rabbits were obtained from Charles River (Kisslegg, Germany) and were maintained in an SPF facility under standard environmental conditions (18 6 2uC room temperature, 50 6 10 % relative humidity, 12-hour light/dark cycle). The rabbits were anesthetized using 35 mg of ketamine hydrochloride and 0.25 mg of medetomidine hydrochloride per kilogram body weight. The infection sites at the flanks of the rabbits were marked, and viral DNA was injected into the skin using a Helios Gene Gun (BioRad, Munich, Germany) as described previously.27,28 Animal experiments were approved by the review board for animal experiments (permission no. H1/08). Simultaneous PET/MR Imaging Simultaneous PET/MR imaging was performed using the human BrainPET system (Siemens Healthcare, Knoxville, TN), which allows nearly whole-body scans of the rabbits. The BrainPET PET/MR system consists of an MRcompatible PET system inserted into a modified 3 T MR scanner (Magnetom Tim Trio, Siemens Healthcare). The technical specifications of the PET system have been described previously.29 The PET scanner has an axial field of view (FOV) of approximately 19.1 cm and a transversal FOV of 32.0 cm. The image volume consisted of 153 transaxial images, with a matrix size of 256 3 256 and an isotropic voxel size of 1.25 mm3. All image data were acquired in list mode for retrospective data rebinning and reconstruction. CRPV-infected NZW rabbits were imaged at 2, 4, and 10 months postinjection under anesthesia using the PET tracers 18F-FDG (50–60 MBq), 18F-FLT (50–60 MBq), and 11 C-choline (100–110 MBq), which were injected via an ear vein catheter. The imaging schedule contained the consecutive measurement of one cohort of rabbits during 1 week (day 1: 18F-FLT; day 3: 11C-choline; day 5: 18FFDG), which allowed us to significantly reduce the number of animals used in this study according to the guidelines of animal care. For all tracers, 30- or 60-minute dynamic PET scans were performed. The 30-minute scans started after an uptake time of 30 minutes and the 60-minute scan directly during tracer injection. Simultaneously to the PET acquisition, the MR examination protocol was performed, which included gradient-echo localizers, transverse T1-weighted three-dimensional spoiled gradient echo
PET/MR Imaging of Papillomavirus Tumors in Rabbits
sequence (FLASH) for PET attenuation correction (repetition time [TR]/echo time [TE] 4.6 ms/2.46 ms, excitation angle 10u, matrix size 256 3 256 3 160, isotropic voxel size of 1.25 mm3, bandwidth 590 Hz/px, parallel imaging acceleration factor 2, acquisition time [TA] 3 minutes 13 seconds). For the morphologic analysis, a sagittal T1weighted MPRAGE (TR/TE 1,900 ms/2.23 ms, excitation angle 9u, matrix size 246 3 256 3 192, isotropic voxel size of 1.25 mm3, bandwidth 190 Hz/px, TA 10 minutes 44 seconds) was used. Additionally, a two-dimensional transversal T2-weighted spoiled gradient echo sequence (TR/TE 250 ms/2.46 ms, excitation angle 70u, matrix size 160 3 256 3 40, voxel size 0.35 3 0.35 3 3 mm, bandwith 320 Hz/px, TA 1 minute 20 seconds) was acquired. PET attenuation maps for scatter and attenuation correction were computed from the FLASH image. To remove artifacts in the MR image originating from the living animal, for example, breathing and heart motion, a combination of thresholding, morphologic operations, and connected component analysis was employed. After thresholding the MR image and shrinking connected regions to remove small artifacts, only the largest connected region was retained, discarding larger MR motion artifacts outside the rabbit. The MR image was then segmented into four classes by intensity thresholding, assigning the corresponding CT values (air, 21,000 HU; lung, 2650 HU; fat, 270 HU; soft tissue, 50 HU). Image smoothing was performed using a gaussian filter (5.625 mm full width at half maximum), and the assigned CT values were converted to linear attenuation coefficients at 511 keV (yielding air 0 cm–1, lung 0.0336 cm–1, fat 0.08928 cm–1, and soft tissue 0.10065 cm–1). PET listmode files were rebinned into multiple frames depending on the tracer. PET data were reconstructed using a three-dimensional ordered subset expectation maximization (OSEM) algorithm (6 iterations, 16 subsets) and resolution modeling using the software provided by the manufacturer. Corrections for randoms, attenuation, and scatter were applied. PET images were analyzed using Inveon Research Workplace software (Siemens Preclinical Solutions, Knoxville, TN). Data sets from PET/MR were coregistered, and regions of interest (ROI) were drawn around the viable papilloma tissue, based on the anatomic information from the MR images and the molecular information of the PET. Volumetry of the viable tumor tissue was assessed from the PET/MR data obtained by the 18F-FDG imaging using these ROI. Absolute quantification as a percentage of the injected dose or standard uptake values (SUVs) of the PET data sets was not possible in this study
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because the BrainPET was not cross-calibrated. Therefore, the uptake of the tracers used is expressed as a ratio of tumor to muscle or tumor to liver uptake. PET/CT Imaging To enable quantification and report clinically relevant SUVs, 11C-choline-PET/CT was performed for two animals using a clinical scanner. PET data acquisition (Hi-Rez Biograph 16, Siemens Healthcare) was initiated 50 minutes after injection of 11C-choline (50–60 MBq) and lasted for 4 minutes (one bed position). For attenuation correction, a non–contrast-enhanced low-dose CT scan was acquired, and CT for the anatomic information was sequentially performed. Attenuation-corrected trans-axial slices (matrix size 256 3 256, corresponding to a FOV of 45.5 cm 3 45.5 cm) were reconstructed using the standard software of the scanner (Fourier rebinning, two-dimensional OSEM with 8 iterations, 16 subsets, 5 mm gaussian filter). PET/CT images were analyzed and compared to PET/MR images. Ex Vivo Biodistribution Following the final 18F-FDG PET scan, the animals were euthanized by injection of T61 (Intervet, Unterschleissheim, Germany) under deep anesthesia and were subsequently dissected. Tumors and organs were removed and measured together with an aliquot of the injected radiotracer solution by gamma-counting (Wizard singledetector gamma-counter; PerkinElmer, Waltham, MA) using an energy window of 350 to 650 keV. The results are expressed as percent injected dose per gram (% ID/g) of tissue. Statistical Analysis Statistical analysis was performed using analysis of variance (ANOVA) with a post hoc Bonferroni test carried out with Origin 8 software (OriginLab Corporation, Northampton, MA). Data were considered statistically significant for p , .05. All quantitative results are shown as the mean 6 1 standard deviation (SD).
Results PET/MR Imaging and Biodistribution Tumors induced by CRPV are depicted at different time points in Figure 1. At 2 months postinfection, small
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papillomas are visible in the inoculated skin on the back of the rabbits (see Figure 1A), which successively progress to larger tumors (see Figure 1, B–D). To monitor tumor growth in the rabbits noninvasively, the animals were imaged using the PET tracers 18F-FDG, 11C-choline, and 18 F-FLT at 1, 2, 4, and 10 months after infection by simultaneous PET/MR imaging. No tumor formation was detected in PET images from all three tracers in the first month postinjection. Tumor volumetry on the basis of the 18 F-FDG PET/MR data revealed 0.0 6 0.0 cc 2 weeks postinjection; 4.1 6 3.5 cc 2 months postinjection; 7.6 6 5.2 cc 4 months postinjection, and 17.4 6 1.1 cc 10 months postinjection. Figure 2 shows a representative rabbit at 10 months postinjection. The imaging results are presented as CRPV-induced tumor to organ ratios in Figure 3. The glucose analogue 18F-FDG showed the highest uptake values compared to the other PET tracers tested (see Figure 3A). At 2, 4, and 10 months postinjection, we found an 18F-FDG tumor to muscle ratio of 11.22 6 3.53 (n 5 3), 13.57 6 0.68 (n 5 5), and 16.2 6 0.91 (n 5 3), respectively. Tumor to liver ratios showed lower values compared to the tumor to muscle ratios but revealed the same trend at 2, 4, and 10 months postinjection (0.76 6 0.14, 1.11 6 0.12, and 1.31 6 0.35, respectively). 11CCholine also showed high tumor to muscle ratios after 4 (6.69 6 1.47; n 5 4) and 10 months postinjection (6.34 6 1.04; n 5 3; see Figure 3B), whereas, after 2 months postinjection, the 11C-choline uptake was lower (2.73 6 1.5, n 5 2) than the uptake measured for 18F-FDG. Due to the high 11C-choline uptake in the liver of the animals, the tumor to liver ratios at 2 months postinjection (0.27 6 0.18), 4 months postinjection (0.35 6 0.12), and 10 months postinjection (0.34 6 0.03) were quite low compared to the results obtained with 18F-FDG. Interestingly, no uptake of the proliferation marker 18F-FLT was observed at any time point after infection (see Figure 3C). The tumor to muscle
ratios obtained using image analyses showed no differences between the three imaged infection time points (0.8 6 0.06 [n 5 2], 2 months postinjection; 0.75 6 0.25 [n 5 3], 4 months postinjection; 1.0 [n 5 1], 10 months postinjection). Similar results were obtained when tumor to liver ratios were calculated (0.95 6 0.15, 2 months postinjection; 0.71 6 0.09, 4 months postinjection; 0.57, 10 months postinjection). After injection of 18F-FDG, the time-activity curves (TACs) of the tumor uptake increased during the dynamic acquisition time of 60 minutes (see Figure 3A, right panel). The 18F-FDG TACs of the muscle tissues remained low during the entire acquisition time, which is in agreement with previous findings, for example, in mice.20 In contrast, the 18F-FDG TACs of the liver tissue peaked immediately after the injection due to the high blood-pool concentration of the tracer at this early time point and declined rapidly before it reached a stable plateau after about 25 minutes acquisition time. The level (arbitrary values) was similar to the one reached in the tumor tissue, thus explaining the low tumor to liver ratios at 50 to 60 minutes after 18F-FDG injection (see Figure 3A). 11C-Choline TACs showed a very low uptake into the muscle tissue, whereas values steadily increased for tumor and liver tissue. However, no plateau was reached in the TACs of the tumor or liver tissue (see Figure 3B). 18F-FLT uptake remained low for tumors and muscle for the entire 60 minutes of acquisition time. The liver 8F-FLT TAC revealed a perfusion peak in the first 5 minutes after tracer injection (see Figure 3C). The results of the 18F-FDG biodistribution are shown in Figure 4. After 6 months postinjection, the organ to muscle ratios showed a relative 18F-FDG uptake of 10.8 6 2.05 in tumor tissue, 3.55 6 0.27 in uninfected skin, 2.88 6 0.36 in blood, 7.78 6 0.17 in the liver, 9.72 6 0.64 in the spleen, 6.03 6 1.33 in the kidneys, 2.7 6 0.45 in heart muscle tissue, 6.22 6 0.92 in lung tissue, and 1.69 6 1.66 Figure 1. Overview photograph of a cottontail rabbit papillomavirus (CRPV)-infected New Zealand White rabbit illustrating the site of infection (A). Close-up photographs of infected sites at various time points postinjection are shown in B to D. CRPV induces tumor development in the skin of the rabbits, which becomes visible at 2 months postinjection (B) and which progresses to larger tumors at 4 months postinjection (C). Successively, highly keratinized tumors are visible (D).
PET/MR Imaging of Papillomavirus Tumors in Rabbits
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Figure 2. Representative maximum intensity projections (MIP; left) and coronal MR (center left), fused PET/MR (center right), and correlating PET (right) images of a cottontail rabbit papillomavirus–infected rabbit imaged with the PET tracers 18F-FDG (top), 11C-choline (center), and 18FFLT (bottom). The PET data are shown as sagittal (upper left) and coronal (lower left) MIP. 18F-FDG showed the highest uptake, but 11C-choline uptake was also increased in tumor tissue. No uptake was observed in tumors using the proliferation marker 18F-FLT. The figure depicts the precise coregistration of the molecular and anatomic information due to the simultaneous acquisition of PET and MR.
in fat (see Figure 4). After 10 months postinjection, the organ to muscle ratios revealed an even higher relative uptake of 18F-FDG of 15.69 6 1.54 in tumors and of 12.93 6 2.21 in liver tissue. Since the muscle tissue did not show an increased uptake of 18F-FDG at the tested time points (0.011
6 0.0036 percentage of injected dose per gram in muscle tissue at 6 months postinjection and 0.018 6 0.015 %ID/g at the late infection stage), the increase in relative 18F-FDG uptake in the tested organs is due to an increased 18FFDG consumption, most probably because of the
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Figure 3. Analysis of the PET/MR images and PET quantification. Imaging with 18F-FDG revealed an increased relative tumor uptake of the PET tracer in cottontail rabbit papillomavirus–infected rabbits over time (A). Choosing the muscle (A) or the liver (B) as the reference tissue, the overall highest uptake was observed for 18F-FDG. Using 11C-choline as PET tracer, the tumor to muscle ratios were also highly increased at 4 and 10 months postinjection (p.i.) but less pronounced after 2 months postinjection. No increased uptake of 18F-FLT was observed in tumors of the infected rabbits at any imaging time point. The time-activity curves (TACs) depict the biodistribution of the tracers during acquisition time (C). The results are shown as tumor to muscle ratios (A), tumor to liver ratios (B), or arbitary units (C). Error bars represent standard deviation; the asterisk indicates significant differences (p , .05).
activated immune system at this stage of infection. 18FFDG organ to muscle ratios at 10 months postinjection were as follows: 3.73 6 2.23 in uninfected skin, 4.26 6 2.5 in blood, 12.93 6 0.74 in spleen, 8.4 6 4.54 in kidneys, 4.17 6 0.26 in heart muscle tissue, 5.84 6 3.59 in lung tissue, and 0.79 6 0.26 in fat. Similar results were obtained at 4 months postinjection. PET/CT Imaging For quantification of the 11C-choline uptake into tumors of CRPV-injected rabbits, PET/CT scans were performed at 10 months postinjection and were compared to PET/ MR imaging. Two animals underwent PET/CT measurements with 11C-choline. SUVs from the 11C-choline-PET/ CT were 1.46 6 0.02 for tumor, 0.69 6 0.02 for muscle, and 7.74 6 0.73 for liver tissue (Figure 5).
Discussion The early assessment of applied therapeutics in cancer patients is most critical to manage efficient chemotherapy and an optimal quality of life.30 Infection of NZW rabbits with CRPV serves as an excellent animal model system to study the role of the viral oncoproteins in tumor development. In this study, we employed the animal model system for CRPV-induced tumors to evaluate the PET tracers 18F-FDG, 18F-FLT, and 11C-choline for tumor detection using simultaneous PET/MR imaging. All primary tumors were detectable after 2 months postinjection in 18F-FDG and 11C-choline PET images, whereas 18 F-FLT failed to image CRPV-induced tumors in the rabbits. Functional changes in tumor biology, such as apoptosis or senescence on treatment, occur much earlier than morphologic changes affecting the size of the tumor.11
PET/MR Imaging of Papillomavirus Tumors in Rabbits
Figure 4. Ex vivo 18F-FDG biodistribution of organs of infected rabbits after the final PET/MR scan at 6 or 10 months postinjection (p.i.) measured in a gamma-counter. An increased uptake of 18F-FDG was observed in tumors of infected rabbits 6 and 10 months postinjection, which confirmed the results obtained by PET/MR imaging.
Hence, treatment efficacy may be monitored by PET imaging combining morphologic and molecular information without applying additional radiation doses arising from CT measurements. 18 F-FDG, which is the current gold standard for oncologic PET imaging, has been shown to accumulate in normal tissue, such as the liver, due to physiologic glucose uptake and in sites of inflammation due to increased glucose metabolism.31 We used the glucose analogue 18F-FDG to determine whether an increased glucose uptake into the papillomavirus-induced tumors can be detected by PET imaging of CRPV-infected NZW rabbits and were able to detect tumors ever since the 2 months postinjection examined time points. However, a major drawback for the clinical application of 18F-FDG PET is the hampered detection of HPV-associated tumors in the pelvic region due to the excretion of radioactivity into the bladder complicating the interpretation of acquired images in this area.32,33 Use of the tracer 11C-choline has the advantage of not complicating imaging of pelvic tumors as it is not excreted via the bladder. Choline is incorporated into cells via phosphorylcholine synthesis and integrates into the phospholipids of cell membranes.34 Various cancers have elevated levels of phosphorylcholine compared to the low or even undetectable levels in normal tissues.35,36 Indeed 11 C-choline has been used successfully in PET imaging of tumors in the pelvic region, including cervical cancer.22–24,26,37,38 Therefore, the simultaneous use of noninvasive, high–tissue contrast MR imaging in combination with 11C-choline PET has the potential to improve diagnosis and support future clinical staging of cancers. In addition, we confirmed reports from previous studies
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Figure 5. Coronal PET/CT images of a cottontail rabbit papillomavirus– infected rabbit with the PET tracer 11C-choline.
demonstrating that the uptake of 11C-choline into tumors was lower than the uptake of 18F-FDG.37,39 Interestingly, the proliferation marker 18F-FLT failed to detect the CRPV-induced tumors in rabbits. However, we cannot rule out that this might be due to the slow proliferation rate of the tumors, which is implicated by the low relative uptake values in the TACs over the acquisition time of 60 minutes (see Figure 3). To our knowledge, no commercial Ki-67 marker is available for staining rabbit tissues preventing us to proof this hypothesis by histology. Fuchs and colleagues showed recently that ketamine/ xylazine reduces 18F-FLT uptake in carcinomas in mice.40 Since imaging was here performed under ketamine/ medetomidine anesthesia, this could have an impact on 18 F-FLT uptake. However, the TAC indicates a rapid washout of FLT and nearly no specific binding, whereas Fuchs and colleagues found a decent uptake under ketamine/xylazine anesthesia, which was reduced (4.3 6 0.9 %ID/cc) compared to animals scanned under isoflurane anesthesia (6.4 6 1.5 %ID/cc). Therefore, the use of ketamine anesthesia might influence 18F-FLT uptake. In future studies, we also plan to compare the functional parameter obtained from extended MR measurements such as diffusion-weighted magnetic resonance imaging (DW-MRI) while acquiring the PET data simultaneously. This approach will allow multiparametric data analysis comparing the DW-MRI quantification via the apparent diffusion coefficient with the PET data. In this study, the aim was to evaluate clinically used tracers in the animal model of papillomavirus-induced tumors. However, future PET/MR experiments using a papillomavirusspecific antibody against a putative tumor antigen would be advantageous to distinguish between secondary inflammation reactions and tumor tissue.31,41 At present, we cannot ensure that the enhanced 18F-FDG uptake in tumor
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tissue is solely based on the increased glycolysis in papillomavirus-induced malignant cells. In summary, our data show that papillomavirusinduced tumors in rabbits can be visualized by both 18FFDG and 11C-choline.
Conclusion This study shows the feasibility of 18F-FDG but also of 11Ccholine PET imaging to detect papillomavirus-induced tumors in the rabbit animal model system. It therefore provides a basis for future studies to investigate the applicability of simultaneous PET/MR imaging using the tracers 18F-FDG or 11C-choline in preclinical settings for tumor characterization, staging, and treatment monitoring of papillomavirus-induced cancers.
Acknowledgments We are grateful to Denis Lamparter and Walter Ehrlichmann for producing the PET tracers, Henriette Heners for expert technical assistance with the PET/CT scans, and Juliane Haedicke for critical revision of the manuscript. Financial disclosure of authors: This research was supported by the Deutsche Forschungsgemeinschaft SFB 773. Financial support from the Deutsche Forschungsgemeinschaft was also provided through grants PI771/1-1 and PI771/5-1. Financial disclosure of reviewers: None reported.
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8. Kolb A, Wehrl HF, Hofmann M, et al. Technical performance evaluation of a human brain PET/MRI system. Eur Radiol 2012;22: 1776–88, doi:10.1007/s00330-012-2415-4. 9. Hricak H, Yu KK. Radiology in invasive cervical cancer. AJR Am J Roentgenol 1996;167:1101–8, doi:10.2214/ajr.167.5.8911159. 10. Subak LL, Hricak H, Powell CB, et al. Cervical carcinoma: computed tomography and magnetic resonance imaging for preoperative staging. Obstet Gynecol 1995;86:43–50, doi:10.1016/ 0029-7844(95)00109-5. 11. Brindle K. New approaches for imaging tumour responses to treatment. Nat Rev Cancer 2008;8:94–107, doi:10.1038/nrc2289. 12. Ishikawa TO, Kumar IP, Machado HB, et al. Positron emission tomography imaging of DMBA/TPA mouse skin multi-step tumorigenesis. Mol Oncol 2010;4:119–25, doi:10.1016/j.molonc. 2010.01.005. 13. Beltrami E, Valtorta S, Moresco R, et al. The p53-p66Shc apoptotic pathway is dispensable for tumor suppression whereas the p66shcgenerated oxidative stress initiates tumorigenesis. Curr Pharm Des 2013;19:2708–14, doi:10.2174/1381612811319150005. 14. Jeon TY, Kim HJ, Choi JY, et al. 18F-FDG PET/CT findings of sinonasal inverted papilloma with or without coexistent malignancy: comparison with MR imaging findings in eight patients. Neuroradiology 2009;51:265–71, doi:10.1007/s00234-009-0510-2. 15. Shojaku H, Fujisaka M, Yasumura S, et al. Positron emission tomography for predicting malignancy of sinonasal inverted papilloma. Clin Nucl Med 2007;32:275–8, doi:10.1097/01.rlu. 0000257334.65253.cc. 16. Lawson W, Schlecht NF, Brandwein-Gensler M. The role of the human papillomavirus in the pathogenesis of Schneiderian inverted papillomas: an analytic overview of the evidence. Head Neck Pathol 2008;2:49–59, doi:10.1007/s12105-008-0048-3. 17. Shields AF, Grierson JR, Dohmen BM, et al. Imaging proliferation in vivo with [F-18]FLT and positron emission tomography. Nat Med 1998;4:1334–6, doi:10.1038/3337. 18. Rasey JS, Grierson JR, Wiens LW, et al. Validation of FLT uptake as a measure of thymidine kinase-1 activity in A549 carcinoma cells. J Nucl Med 2002;43:1210–7. 19. Been LB, Suurmeijer AJ, Cobben DC, et al. [18F]FLT-PET in oncology: current status and opportunities. Eur J Nucl Med Mol Imaging 2004;31:1659–72, doi:10.1007/s00259-004-1687-6. 20. Kukuk D, Reischl G, Raguin O, et al. Assessment of PET tracer uptake in hormone-independent and hormone-dependent xenograft prostate cancer mouse models. J Nucl Med 2011;52:1654–63, doi:10.2967/jnumed.110.086702. 21. Hara T, Kosaka N, Shinoura N, Kondo T. PET imaging of brain tumor with [methyl-11C]choline. J Nucl Med 1997;38:842–7. 22. Kotzerke J, Prang J, Neumaier B, et al. Experience with carbon-11 choline positron emission tomography in prostate carcinoma. Eur J Nucl Med 2000;27:1415–9, doi:10.1007/s002590000309. 23. de Jong IJ, Pruim J, Elsinga PH, et al. Preoperative staging of pelvic lymph nodes in prostate cancer by 11C-choline PET. J Nucl Med 2003;44:331–5. 24. Scher B, Seitz M, Albinger W, et al. Value of 11C-choline PET and PET/CT in patients with suspected prostate cancer. Eur J Nucl Med Mol Imaging 2007;34:45–53, doi:10.1007/s00259-006-0190-7. 25. de Jong IJ, Pruim J, Elsinga PH, et al. Visualization of prostate cancer with 11C-choline positron emission tomography. Eur Urol 2002;42:18–23, doi:10.1016/S0302-2838(02)00129-X.
PET/MR Imaging of Papillomavirus Tumors in Rabbits
26. Sofue K, Tateishi U, Sawada M, et al. Role of carbon-11 choline PET/CT in the management of uterine carcinoma: initial experience. Ann Nucl Med 2009;23:235–43, doi:10.1007/s12149009-0230-z. 27. Xiao W, Brandsma JL. High efficiency, long-term clinical expression of cottontail rabbit papillomavirus (CRPV) DNA in rabbit skin following particle-mediated DNA transfer. Nucleic Acids Res 1996;24:2620–2, doi:10.1093/nar/24.13.2620. 28. Jeckel S, Loetzsch E, Huber E, et al. Identification of the E9/E2C cDNA and functional characterization of the gene product reveal a new repressor of transcription and replication in cottontail rabbit papillomavirus. J Virol 2003;77:8736–44, doi:10.1128/JVI.77.16. 8736-8744.2003. 29. Schlemmer HP, Pichler BJ, Schmand M, et al. Simultaneous MR/ PET imaging of the human brain: feasibility study. Radiology 2008; 248:1028–35, doi:10.1148/radiol.2483071927. 30. Therasse P, Arbuck SG, Eisenhauer EA, et al. New guidelines to evaluate the response to treatment in solid tumors. European Organization for Research and Treatment of Cancer, National Cancer Institute of the United States, National Cancer Institute of Canada. J Natl Cancer Inst 2000;92:205–16, doi:10.1093/jnci/ 92.3.205. 31. Rosenbaum SJ, Lind T, Antoch G, Bockisch A. False-positive FDG PET uptake--the role of PET/CT. Eur Radiol 2006;16:1054–65, doi:10.1007/s00330-005-0088-y. 32. Sugawara Y, Eisbruch A, Kosuda S, et al. Evaluation of FDG PET in patients with cervical cancer. J Nucl Med 1999;40:1125–31. 33. Williams AD, Cousins C, Soutter WP, et al. Detection of pelvic lymph node metastases in gynecologic malignancy: a comparison
34.
35.
36.
37.
38.
39.
40.
41.
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of CT, MR imaging, and positron emission tomography. AJR Am J Roentgenol 2001;177:343–8, doi:10.2214/ajr.177.2.1770343. Roivainen A, Forsback S, Gronroos T, et al. Blood metabolism of [methyl-11C]choline; implications for in vivo imaging with positron emission tomography. Eur J Nucl Med 2000;27:25–32, doi:10.1007/PL00006658. Miller BL, Chang L, Booth R, et al. In vivo 1H MRS choline: correlation with in vitro chemistry/histology. Life Sci 1996;58: 1929–35, doi:10.1016/0024-3205(96)00182-8. Tedeschi G, Lundbom N, Raman R, et al. Increased choline signal coinciding with malignant degeneration of cerebral gliomas: a serial proton magnetic resonance spectroscopy imaging study. J Neurosurg 1997;87:516–24, doi:10.3171/jns.1997.87.4.0516. Torizuka T, Kanno T, Futatsubashi M, et al. Imaging of gynecologic tumors: comparison of (11)C-choline PET with (18)F-FDG PET. J Nucl Med 2003;44:1051–6. Picchio M, Treiber U, Beer AJ, et al. Value of 11C-choline PET and contrast-enhanced CT for staging of bladder cancer: correlation with histopathologic findings. J Nucl Med 2006;47:938–44. Tian M, Zhang H, Oriuchi N, et al. Comparison of 11C-choline PET and FDG PET for the differential diagnosis of malignant tumors. Eur J Nucl Med Mol Imaging 2004;31:1064–72. Fuchs K, Kukuk D, Mahling M, et al. Impact of anesthetics on 39[18F]fluoro-39-deoxythymidine ([18F]FLT) uptake in animal models of cancer and inflammation. Mol Imaging 2013;12:277–87. Ponomareva ON, Rose JA, Lasarev M, et al. Tissue-specific deletion and discontinuous loss of heterozygosity are signatures for the mutagenic effects of ionizing radiation in solid tissues. Cancer Res 2002;62:1518–23.
Evaluation of Positron Emission Tomographic Tracers for Imaging of Papillomavirus-Induced Tumors in Rabbits Sonja Probst,* Stefan Wiehr,* Frederic Mantlik, Holger Schmidt, Armin Kolb, Peter Mu¨nch, Maria Delcuratolo, Frank Stubenrauch, Bernd J. Pichler, and Thomas Iftner
Abstract In this study, simultaneous positron emission tomography (PET)/magnetic resonance (MR) imaging was employed to evaluate the feasibility of the PET tracers 2-deoxy-2-18F-fluoro-D-glucose (18F-FDG),
11
C-choline, and
18
F-fluorothymidine (18F-FLT) to detect
papillomavirus-induced tumors in an established rabbit model system. The combined PET/MR allowed the analysis of tracer uptake of the tumors using the morphologic information acquired by MR. New Zealand White rabbits were infected with cottontail rabbit papillomavirus genomes and were imaged for up to 10 months with a simultaneous PET/MR system during the course of infection. The uptake characteristics of the PET tracers
11
C-choline and
18
F-FLT of tumors and reference tissues were
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examined relative to the clinical standard, F-FDG. Tracer biodistribution of various organs was measured by gamma-counting after the last PET scan and compared to the in vivo PET/MR 18F-FDG uptake. Increased tracer uptake was found 2 months postinfection in primary tumors with
18
F-FDG and
points. Our data show that the PET tracer 11
C-choline and
18
F-FLT. However,
11
18
11
C-choline, whereas
18
F-FDG.
UMAN PAPILLOMAVIRUSES (HPVs) are the causative agents for cervical cancer and have been implicated in other anogenital cancers and a subset of head and neck carcinomas.1–4 Cervical cancer is the third most frequent cancer in women worldwide. It is estimated that approximately 530,000 women developed cervical cancer and 275,000 died by reason of the disease in 2008.1 Twelve high-risk HPV types recognized as class I carcinogens by the World Health Organization are necessary risk factors for the development of cervical cancer.2 HPV has also been
H
*Authors who contributed equally to this work. From the Division of Experimental Virology, Institute of Medical Virology, University Hospital Tu¨bingen; Department of Preclinical Imaging and Radiopharmacy, Werner Siemens Imaging Center, Eberhard Karls University; Department of Empirical Inference, MaxPlanck Institute for Intelligent Systems; and Department of Diagnostics and Interventional Radiology, Eberhard Karls University Tu¨bingen, Tu¨bingen, Germany. Address reprint requests to: Thomas Iftner, PhD, Medical Virology, Section Experimental Virology, University Hospital Tu¨bingen, ElfriedeAulhorn-Str. 6, 72076 Tu¨bingen, Germany; e-mail: thomas.iftner@ med.uni-tuebingen.de. #
2014 Decker Publishing
F-FLT failed to detect the tumors at all measured time
F-FDG is superior for imaging papillomavirus-induced tumors in rabbits compared to
C-choline imaging, which has previously been applied to detect various tumor entities in
patients, appears to be an alternative to
DOI 10.2310/7290.2013.00070
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linked to the development of nonmelanoma skin cancer, which represents the most common malignancy in whites worldwide.5 An excellent animal model to study papillomavirus-induced cancer formation in vivo is the New Zealand White (NZW) rabbit, where infections with cottontail rabbit papillomavirus (CRPV) cause the development of skin tumors within 4 to 6 weeks postinfection, which then progress to invasive cancer without additional cofactors within 8 to 14 months.6,7 Although skin tumor progression may be directly evaluated by visual inspection, the depth and progression of subcutaneous invasion and its temporal molecular changes are difficult to assess. In this study, we used a simultaneous positron emission tomography (PET)/magnetic resonance (MR) system8 to combine anatomic and molecular information for more precise analyses of skin tumor growth or regression9 to develop a noninvasive method of monitoring papillomavirus-induced skin tumor growth in CRPV-infected NZW rabbits. This study aimed at identifying suitable tracers for future studies monitoring various therapy regimens, which include the use of potential tumor-inhibiting substances. Imaging methods, such as computed tomography (CT) or MR imaging, provide excellent morphologic information.9,10 One potential problem with these techniques is the lack of metabolic information, which is essential for
Molecular Imaging, Vol 13, 2014.
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the reliable prediction of the physiologic response of the cancer tissue to the respective treatment. Functional changes in tumor biology, such as apoptosis on treatment, occur much earlier than morphologic changes affecting the size of the tumor.11 With this in mind, PET reveals the metabolic and functional parameters of living cells and is capable of distinguishing between normal and malignant tissues through the use of various PET tracers. PET has previously been performed on mice with chemically induced skin tumors12,13 and in patients with sinonasal inverted papilloma,14,15 of which more than 70% are HPV negative,16 using 2-deoxy-2-18F-fluoro-D-glucose (18F-FDG) as a PET tracer. Here we evaluate the uptake of 18 F-FDG, 39-deoxy-39-18F-fluorothymidine (18F-FLT) and 11 C-choline into papillomavirus-induced tumors to determine the most suitable PET tracer for examining physiologic skin tumor growth and progression causally induced by papillomaviruses in rabbits. In addition to the widely used PET tracer 18F-FDG for glucose metabolism, the 18F-radiolabeled thymidine derivative 18F-FLT was used. 18F-FLT is a PET marker for cell proliferation and is consumed by proliferating cells via the salvage pathway, which incorporates nucleotides into replicating deoxyribonucleic acid (DNA) during the S phase of the cell cycle.17 18F-FLT is, however, rarely incorporated into the DNA18 and has been used in preclinical and clinical studies to monitor treatment efficacy in cancers.19 11CCholine is another promising PET tracer that can be used to detect membrane synthesis. It is initially phosphorylated within the tumor cell. The resulting phosphatidylcholine is then trapped in the lecithin of the cell membrane.20 Dividing tumor cells are characterized by increased cell membrane synthesis, leading to an enhanced uptake of 11C-choline. Thus, although not directly correlated with the cellular proliferation rate, 11Ccholine is an interesting candidate for cancer imaging where 18F-FDG and 18F-FLT fail. 11C-Choline was first used for imaging brain tumors21 and is currently employed primarily for detection and staging of prostate cancer,22–25 because of its low bladder activity. Thus, for detection of gynecologic malignancies 11C-choline might be superior to 18F-FDG and 18F-FLT, which are excreted via the urinary tract, because of reduced masking of the pelvic region.25,26 In this study, the clinical gold standard in PET oncology, 18F-FDG, showed a markedly increased uptake into the tumor tissue of rabbits, whereas 18F-FLT failed. However, 11C-choline revealed an uptake comparable to 18 F-FDG, thus representing an alternative PET tracer to 18 F-FDG for imaging papillomavirus-induced tumors.
Materials and Methods Animal Model and Infection Procedure Specific pathogen-free (SPF) female NZW rabbits were obtained from Charles River (Kisslegg, Germany) and were maintained in an SPF facility under standard environmental conditions (18 6 2uC room temperature, 50 6 10 % relative humidity, 12-hour light/dark cycle). The rabbits were anesthetized using 35 mg of ketamine hydrochloride and 0.25 mg of medetomidine hydrochloride per kilogram body weight. The infection sites at the flanks of the rabbits were marked, and viral DNA was injected into the skin using a Helios Gene Gun (BioRad, Munich, Germany) as described previously.27,28 Animal experiments were approved by the review board for animal experiments (permission no. H1/08). Simultaneous PET/MR Imaging Simultaneous PET/MR imaging was performed using the human BrainPET system (Siemens Healthcare, Knoxville, TN), which allows nearly whole-body scans of the rabbits. The BrainPET PET/MR system consists of an MRcompatible PET system inserted into a modified 3 T MR scanner (Magnetom Tim Trio, Siemens Healthcare). The technical specifications of the PET system have been described previously.29 The PET scanner has an axial field of view (FOV) of approximately 19.1 cm and a transversal FOV of 32.0 cm. The image volume consisted of 153 transaxial images, with a matrix size of 256 3 256 and an isotropic voxel size of 1.25 mm3. All image data were acquired in list mode for retrospective data rebinning and reconstruction. CRPV-infected NZW rabbits were imaged at 2, 4, and 10 months postinjection under anesthesia using the PET tracers 18F-FDG (50–60 MBq), 18F-FLT (50–60 MBq), and 11 C-choline (100–110 MBq), which were injected via an ear vein catheter. The imaging schedule contained the consecutive measurement of one cohort of rabbits during 1 week (day 1: 18F-FLT; day 3: 11C-choline; day 5: 18FFDG), which allowed us to significantly reduce the number of animals used in this study according to the guidelines of animal care. For all tracers, 30- or 60-minute dynamic PET scans were performed. The 30-minute scans started after an uptake time of 30 minutes and the 60-minute scan directly during tracer injection. Simultaneously to the PET acquisition, the MR examination protocol was performed, which included gradient-echo localizers, transverse T1-weighted three-dimensional spoiled gradient echo
PET/MR Imaging of Papillomavirus Tumors in Rabbits
sequence (FLASH) for PET attenuation correction (repetition time [TR]/echo time [TE] 4.6 ms/2.46 ms, excitation angle 10u, matrix size 256 3 256 3 160, isotropic voxel size of 1.25 mm3, bandwidth 590 Hz/px, parallel imaging acceleration factor 2, acquisition time [TA] 3 minutes 13 seconds). For the morphologic analysis, a sagittal T1weighted MPRAGE (TR/TE 1,900 ms/2.23 ms, excitation angle 9u, matrix size 246 3 256 3 192, isotropic voxel size of 1.25 mm3, bandwidth 190 Hz/px, TA 10 minutes 44 seconds) was used. Additionally, a two-dimensional transversal T2-weighted spoiled gradient echo sequence (TR/TE 250 ms/2.46 ms, excitation angle 70u, matrix size 160 3 256 3 40, voxel size 0.35 3 0.35 3 3 mm, bandwith 320 Hz/px, TA 1 minute 20 seconds) was acquired. PET attenuation maps for scatter and attenuation correction were computed from the FLASH image. To remove artifacts in the MR image originating from the living animal, for example, breathing and heart motion, a combination of thresholding, morphologic operations, and connected component analysis was employed. After thresholding the MR image and shrinking connected regions to remove small artifacts, only the largest connected region was retained, discarding larger MR motion artifacts outside the rabbit. The MR image was then segmented into four classes by intensity thresholding, assigning the corresponding CT values (air, 21,000 HU; lung, 2650 HU; fat, 270 HU; soft tissue, 50 HU). Image smoothing was performed using a gaussian filter (5.625 mm full width at half maximum), and the assigned CT values were converted to linear attenuation coefficients at 511 keV (yielding air 0 cm–1, lung 0.0336 cm–1, fat 0.08928 cm–1, and soft tissue 0.10065 cm–1). PET listmode files were rebinned into multiple frames depending on the tracer. PET data were reconstructed using a three-dimensional ordered subset expectation maximization (OSEM) algorithm (6 iterations, 16 subsets) and resolution modeling using the software provided by the manufacturer. Corrections for randoms, attenuation, and scatter were applied. PET images were analyzed using Inveon Research Workplace software (Siemens Preclinical Solutions, Knoxville, TN). Data sets from PET/MR were coregistered, and regions of interest (ROI) were drawn around the viable papilloma tissue, based on the anatomic information from the MR images and the molecular information of the PET. Volumetry of the viable tumor tissue was assessed from the PET/MR data obtained by the 18F-FDG imaging using these ROI. Absolute quantification as a percentage of the injected dose or standard uptake values (SUVs) of the PET data sets was not possible in this study
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because the BrainPET was not cross-calibrated. Therefore, the uptake of the tracers used is expressed as a ratio of tumor to muscle or tumor to liver uptake. PET/CT Imaging To enable quantification and report clinically relevant SUVs, 11C-choline-PET/CT was performed for two animals using a clinical scanner. PET data acquisition (Hi-Rez Biograph 16, Siemens Healthcare) was initiated 50 minutes after injection of 11C-choline (50–60 MBq) and lasted for 4 minutes (one bed position). For attenuation correction, a non–contrast-enhanced low-dose CT scan was acquired, and CT for the anatomic information was sequentially performed. Attenuation-corrected trans-axial slices (matrix size 256 3 256, corresponding to a FOV of 45.5 cm 3 45.5 cm) were reconstructed using the standard software of the scanner (Fourier rebinning, two-dimensional OSEM with 8 iterations, 16 subsets, 5 mm gaussian filter). PET/CT images were analyzed and compared to PET/MR images. Ex Vivo Biodistribution Following the final 18F-FDG PET scan, the animals were euthanized by injection of T61 (Intervet, Unterschleissheim, Germany) under deep anesthesia and were subsequently dissected. Tumors and organs were removed and measured together with an aliquot of the injected radiotracer solution by gamma-counting (Wizard singledetector gamma-counter; PerkinElmer, Waltham, MA) using an energy window of 350 to 650 keV. The results are expressed as percent injected dose per gram (% ID/g) of tissue. Statistical Analysis Statistical analysis was performed using analysis of variance (ANOVA) with a post hoc Bonferroni test carried out with Origin 8 software (OriginLab Corporation, Northampton, MA). Data were considered statistically significant for p , .05. All quantitative results are shown as the mean 6 1 standard deviation (SD).
Results PET/MR Imaging and Biodistribution Tumors induced by CRPV are depicted at different time points in Figure 1. At 2 months postinfection, small
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papillomas are visible in the inoculated skin on the back of the rabbits (see Figure 1A), which successively progress to larger tumors (see Figure 1, B–D). To monitor tumor growth in the rabbits noninvasively, the animals were imaged using the PET tracers 18F-FDG, 11C-choline, and 18 F-FLT at 1, 2, 4, and 10 months after infection by simultaneous PET/MR imaging. No tumor formation was detected in PET images from all three tracers in the first month postinjection. Tumor volumetry on the basis of the 18 F-FDG PET/MR data revealed 0.0 6 0.0 cc 2 weeks postinjection; 4.1 6 3.5 cc 2 months postinjection; 7.6 6 5.2 cc 4 months postinjection, and 17.4 6 1.1 cc 10 months postinjection. Figure 2 shows a representative rabbit at 10 months postinjection. The imaging results are presented as CRPV-induced tumor to organ ratios in Figure 3. The glucose analogue 18F-FDG showed the highest uptake values compared to the other PET tracers tested (see Figure 3A). At 2, 4, and 10 months postinjection, we found an 18F-FDG tumor to muscle ratio of 11.22 6 3.53 (n 5 3), 13.57 6 0.68 (n 5 5), and 16.2 6 0.91 (n 5 3), respectively. Tumor to liver ratios showed lower values compared to the tumor to muscle ratios but revealed the same trend at 2, 4, and 10 months postinjection (0.76 6 0.14, 1.11 6 0.12, and 1.31 6 0.35, respectively). 11CCholine also showed high tumor to muscle ratios after 4 (6.69 6 1.47; n 5 4) and 10 months postinjection (6.34 6 1.04; n 5 3; see Figure 3B), whereas, after 2 months postinjection, the 11C-choline uptake was lower (2.73 6 1.5, n 5 2) than the uptake measured for 18F-FDG. Due to the high 11C-choline uptake in the liver of the animals, the tumor to liver ratios at 2 months postinjection (0.27 6 0.18), 4 months postinjection (0.35 6 0.12), and 10 months postinjection (0.34 6 0.03) were quite low compared to the results obtained with 18F-FDG. Interestingly, no uptake of the proliferation marker 18F-FLT was observed at any time point after infection (see Figure 3C). The tumor to muscle
ratios obtained using image analyses showed no differences between the three imaged infection time points (0.8 6 0.06 [n 5 2], 2 months postinjection; 0.75 6 0.25 [n 5 3], 4 months postinjection; 1.0 [n 5 1], 10 months postinjection). Similar results were obtained when tumor to liver ratios were calculated (0.95 6 0.15, 2 months postinjection; 0.71 6 0.09, 4 months postinjection; 0.57, 10 months postinjection). After injection of 18F-FDG, the time-activity curves (TACs) of the tumor uptake increased during the dynamic acquisition time of 60 minutes (see Figure 3A, right panel). The 18F-FDG TACs of the muscle tissues remained low during the entire acquisition time, which is in agreement with previous findings, for example, in mice.20 In contrast, the 18F-FDG TACs of the liver tissue peaked immediately after the injection due to the high blood-pool concentration of the tracer at this early time point and declined rapidly before it reached a stable plateau after about 25 minutes acquisition time. The level (arbitrary values) was similar to the one reached in the tumor tissue, thus explaining the low tumor to liver ratios at 50 to 60 minutes after 18F-FDG injection (see Figure 3A). 11C-Choline TACs showed a very low uptake into the muscle tissue, whereas values steadily increased for tumor and liver tissue. However, no plateau was reached in the TACs of the tumor or liver tissue (see Figure 3B). 18F-FLT uptake remained low for tumors and muscle for the entire 60 minutes of acquisition time. The liver 8F-FLT TAC revealed a perfusion peak in the first 5 minutes after tracer injection (see Figure 3C). The results of the 18F-FDG biodistribution are shown in Figure 4. After 6 months postinjection, the organ to muscle ratios showed a relative 18F-FDG uptake of 10.8 6 2.05 in tumor tissue, 3.55 6 0.27 in uninfected skin, 2.88 6 0.36 in blood, 7.78 6 0.17 in the liver, 9.72 6 0.64 in the spleen, 6.03 6 1.33 in the kidneys, 2.7 6 0.45 in heart muscle tissue, 6.22 6 0.92 in lung tissue, and 1.69 6 1.66 Figure 1. Overview photograph of a cottontail rabbit papillomavirus (CRPV)-infected New Zealand White rabbit illustrating the site of infection (A). Close-up photographs of infected sites at various time points postinjection are shown in B to D. CRPV induces tumor development in the skin of the rabbits, which becomes visible at 2 months postinjection (B) and which progresses to larger tumors at 4 months postinjection (C). Successively, highly keratinized tumors are visible (D).
PET/MR Imaging of Papillomavirus Tumors in Rabbits
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Figure 2. Representative maximum intensity projections (MIP; left) and coronal MR (center left), fused PET/MR (center right), and correlating PET (right) images of a cottontail rabbit papillomavirus–infected rabbit imaged with the PET tracers 18F-FDG (top), 11C-choline (center), and 18FFLT (bottom). The PET data are shown as sagittal (upper left) and coronal (lower left) MIP. 18F-FDG showed the highest uptake, but 11C-choline uptake was also increased in tumor tissue. No uptake was observed in tumors using the proliferation marker 18F-FLT. The figure depicts the precise coregistration of the molecular and anatomic information due to the simultaneous acquisition of PET and MR.
in fat (see Figure 4). After 10 months postinjection, the organ to muscle ratios revealed an even higher relative uptake of 18F-FDG of 15.69 6 1.54 in tumors and of 12.93 6 2.21 in liver tissue. Since the muscle tissue did not show an increased uptake of 18F-FDG at the tested time points (0.011
6 0.0036 percentage of injected dose per gram in muscle tissue at 6 months postinjection and 0.018 6 0.015 %ID/g at the late infection stage), the increase in relative 18F-FDG uptake in the tested organs is due to an increased 18FFDG consumption, most probably because of the
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Figure 3. Analysis of the PET/MR images and PET quantification. Imaging with 18F-FDG revealed an increased relative tumor uptake of the PET tracer in cottontail rabbit papillomavirus–infected rabbits over time (A). Choosing the muscle (A) or the liver (B) as the reference tissue, the overall highest uptake was observed for 18F-FDG. Using 11C-choline as PET tracer, the tumor to muscle ratios were also highly increased at 4 and 10 months postinjection (p.i.) but less pronounced after 2 months postinjection. No increased uptake of 18F-FLT was observed in tumors of the infected rabbits at any imaging time point. The time-activity curves (TACs) depict the biodistribution of the tracers during acquisition time (C). The results are shown as tumor to muscle ratios (A), tumor to liver ratios (B), or arbitary units (C). Error bars represent standard deviation; the asterisk indicates significant differences (p , .05).
activated immune system at this stage of infection. 18FFDG organ to muscle ratios at 10 months postinjection were as follows: 3.73 6 2.23 in uninfected skin, 4.26 6 2.5 in blood, 12.93 6 0.74 in spleen, 8.4 6 4.54 in kidneys, 4.17 6 0.26 in heart muscle tissue, 5.84 6 3.59 in lung tissue, and 0.79 6 0.26 in fat. Similar results were obtained at 4 months postinjection. PET/CT Imaging For quantification of the 11C-choline uptake into tumors of CRPV-injected rabbits, PET/CT scans were performed at 10 months postinjection and were compared to PET/ MR imaging. Two animals underwent PET/CT measurements with 11C-choline. SUVs from the 11C-choline-PET/ CT were 1.46 6 0.02 for tumor, 0.69 6 0.02 for muscle, and 7.74 6 0.73 for liver tissue (Figure 5).
Discussion The early assessment of applied therapeutics in cancer patients is most critical to manage efficient chemotherapy and an optimal quality of life.30 Infection of NZW rabbits with CRPV serves as an excellent animal model system to study the role of the viral oncoproteins in tumor development. In this study, we employed the animal model system for CRPV-induced tumors to evaluate the PET tracers 18F-FDG, 18F-FLT, and 11C-choline for tumor detection using simultaneous PET/MR imaging. All primary tumors were detectable after 2 months postinjection in 18F-FDG and 11C-choline PET images, whereas 18 F-FLT failed to image CRPV-induced tumors in the rabbits. Functional changes in tumor biology, such as apoptosis or senescence on treatment, occur much earlier than morphologic changes affecting the size of the tumor.11
PET/MR Imaging of Papillomavirus Tumors in Rabbits
Figure 4. Ex vivo 18F-FDG biodistribution of organs of infected rabbits after the final PET/MR scan at 6 or 10 months postinjection (p.i.) measured in a gamma-counter. An increased uptake of 18F-FDG was observed in tumors of infected rabbits 6 and 10 months postinjection, which confirmed the results obtained by PET/MR imaging.
Hence, treatment efficacy may be monitored by PET imaging combining morphologic and molecular information without applying additional radiation doses arising from CT measurements. 18 F-FDG, which is the current gold standard for oncologic PET imaging, has been shown to accumulate in normal tissue, such as the liver, due to physiologic glucose uptake and in sites of inflammation due to increased glucose metabolism.31 We used the glucose analogue 18F-FDG to determine whether an increased glucose uptake into the papillomavirus-induced tumors can be detected by PET imaging of CRPV-infected NZW rabbits and were able to detect tumors ever since the 2 months postinjection examined time points. However, a major drawback for the clinical application of 18F-FDG PET is the hampered detection of HPV-associated tumors in the pelvic region due to the excretion of radioactivity into the bladder complicating the interpretation of acquired images in this area.32,33 Use of the tracer 11C-choline has the advantage of not complicating imaging of pelvic tumors as it is not excreted via the bladder. Choline is incorporated into cells via phosphorylcholine synthesis and integrates into the phospholipids of cell membranes.34 Various cancers have elevated levels of phosphorylcholine compared to the low or even undetectable levels in normal tissues.35,36 Indeed 11 C-choline has been used successfully in PET imaging of tumors in the pelvic region, including cervical cancer.22–24,26,37,38 Therefore, the simultaneous use of noninvasive, high–tissue contrast MR imaging in combination with 11C-choline PET has the potential to improve diagnosis and support future clinical staging of cancers. In addition, we confirmed reports from previous studies
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Figure 5. Coronal PET/CT images of a cottontail rabbit papillomavirus– infected rabbit with the PET tracer 11C-choline.
demonstrating that the uptake of 11C-choline into tumors was lower than the uptake of 18F-FDG.37,39 Interestingly, the proliferation marker 18F-FLT failed to detect the CRPV-induced tumors in rabbits. However, we cannot rule out that this might be due to the slow proliferation rate of the tumors, which is implicated by the low relative uptake values in the TACs over the acquisition time of 60 minutes (see Figure 3). To our knowledge, no commercial Ki-67 marker is available for staining rabbit tissues preventing us to proof this hypothesis by histology. Fuchs and colleagues showed recently that ketamine/ xylazine reduces 18F-FLT uptake in carcinomas in mice.40 Since imaging was here performed under ketamine/ medetomidine anesthesia, this could have an impact on 18 F-FLT uptake. However, the TAC indicates a rapid washout of FLT and nearly no specific binding, whereas Fuchs and colleagues found a decent uptake under ketamine/xylazine anesthesia, which was reduced (4.3 6 0.9 %ID/cc) compared to animals scanned under isoflurane anesthesia (6.4 6 1.5 %ID/cc). Therefore, the use of ketamine anesthesia might influence 18F-FLT uptake. In future studies, we also plan to compare the functional parameter obtained from extended MR measurements such as diffusion-weighted magnetic resonance imaging (DW-MRI) while acquiring the PET data simultaneously. This approach will allow multiparametric data analysis comparing the DW-MRI quantification via the apparent diffusion coefficient with the PET data. In this study, the aim was to evaluate clinically used tracers in the animal model of papillomavirus-induced tumors. However, future PET/MR experiments using a papillomavirusspecific antibody against a putative tumor antigen would be advantageous to distinguish between secondary inflammation reactions and tumor tissue.31,41 At present, we cannot ensure that the enhanced 18F-FDG uptake in tumor
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tissue is solely based on the increased glycolysis in papillomavirus-induced malignant cells. In summary, our data show that papillomavirusinduced tumors in rabbits can be visualized by both 18FFDG and 11C-choline.
Conclusion This study shows the feasibility of 18F-FDG but also of 11Ccholine PET imaging to detect papillomavirus-induced tumors in the rabbit animal model system. It therefore provides a basis for future studies to investigate the applicability of simultaneous PET/MR imaging using the tracers 18F-FDG or 11C-choline in preclinical settings for tumor characterization, staging, and treatment monitoring of papillomavirus-induced cancers.
Acknowledgments We are grateful to Denis Lamparter and Walter Ehrlichmann for producing the PET tracers, Henriette Heners for expert technical assistance with the PET/CT scans, and Juliane Haedicke for critical revision of the manuscript. Financial disclosure of authors: This research was supported by the Deutsche Forschungsgemeinschaft SFB 773. Financial support from the Deutsche Forschungsgemeinschaft was also provided through grants PI771/1-1 and PI771/5-1. Financial disclosure of reviewers: None reported.
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