Published OnlineFirst January 10, 2012; DOI: 10.1158/1078-0432.CCR-11-2462 Clinical Cancer Research Imaging, Diagnosis, Prognosis Evaluation of Deuterated 18F- and 11C-Labeled Choline Analogs for Cancer Detection by Positron Emission Tomography Timothy H. Witney1, Israt S. Alam1, David R. Turton2, Graham Smith1, Laurence Carroll1, Diana Brickute2,  Nguyen1, Giampaolo Tomasi1, Ramla O. Awais2, and Eric O. Aboagye1 Frazer J. Twyman1, Quang-De Abstract Purpose: 11C-Choline–positron emission tomography (PET) has been exploited to detect the aberrant choline metabolism in tumors. Radiolabeled choline uptake within the imaging time is primarily a function of transport, phosphorylation, and oxidation. Rapid choline oxidation, however, complicates interpretation of PET data. In this study, we investigated the biologic basis of the oxidation of deuterated choline analogs and assessed their specificity in human tumor xenografts. Experimental Design: 11C-Choline, 11C-methyl-[1,2-2H4]-choline (11C-D4-choline), and 18F-D4-choline were synthesized to permit comparison. Biodistribution, metabolism, small-animal PET studies, and kinetic analysis of tracer uptake were carried out in human colon HCT116 xenograft–bearing mice. Results: Oxidation of choline analogs to betaine was highest with 11C-choline, with reduced oxidation observed with 11C-D4-choline and substantially reduced with 18F-D4-choline, suggesting that both fluorination and deuteration were important for tracer metabolism. Although all tracers were converted intracellularly to labeled phosphocholine (specific signal), the higher rate constants for intracellular retention (Ki and k3) of 11C-choline and 11C-D4-choline, compared with 18F-D4-choline, were explained by the rapid conversion of the nonfluorinated tracers to betaine within HCT116 tumors. Imaging studies showed that the uptake of 18F-D4-choline in three tumors with similar radiotracer delivery (K1) and choline kinase a expression—HCT116, A375, and PC3-M—were the same, suggesting that 18F-D4-choline has utility for cancer detection irrespective of histologic type. Conclusion: We have shown here that both deuteration and fluorination combine to provide protection against choline oxidation in vivo. 18F-D4-choline showed the highest selectivity for phosphorylation and warrants clinical evaluation. Clin Cancer Res; 18(4); 1063–72. 2012 AACR. Introduction Choline is required for the biosynthesis of phosphatidylcholine, a key component of the plasma membrane. Following transport into the cell, choline is phosphorylated by choline kinase to phosphocholine and then is further metabolized to phosphatidylcholine via CDP-choline, known as the Kennedy pathway. Once phosphorylated, phosphocholine is trapped within the cell. Diacylglycerol, Authors' Affiliations: 1Comprehensive Cancer Imaging Centre at Imperial College, Faculty of Medicine, Imperial College London; and 2Hammersmith Imanet Ltd., Hammersmith Hospital, London, United Kingdom Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/). Corresponding Author: Eric O. Aboagye, Comprehensive Cancer Imaging Centre, Faculty of Medicine, Imperial College London, Room 240 MRC Cyclotron Building, Hammersmith Hospital, Du Cane Road, London, W12 0NN, United Kingdom. Phone: 020-83-83-3759; Fax: 020-83-83-17-83; E-mail: eric.aboagye@imperial.ac.uk doi: 10.1158/1078-0432.CCR-11-2462 2012 American Association for Cancer Research. a product of phosphatidylcholine degradation, is mitogenic, playing a role in the regulation of cell-cycle progression from G1 to S via increased cyclin D1 and cyclin D3 expression (1). Furthermore, aberrant activation and expression of several oncogenes results in elevated choline kinase activity and intracellular levels of phosphocholine (2–4). Choline kinase overexpression is a common feature of several human cancers (5) and in early stage non–small cell lung cancer, choline kinase has been shown to have prognostic significance (6). The expression of choline transporters, including CTL1 and OCT3, is also increased following malignant transformation and may contribute to radiotracer uptake (7, 8), with choline transport closely associated with cell growth (9). 11C-choline has become a viable alternative to 18F-2-fluoro-2-deoxyglucose for positron emission tomography (PET) imaging of the prostate (10– 12), in which the increased choline kinase activity in tumors provides the basis for tumor-specific contrast in comparison with surrounding nonneoplastic tissues. A fluorinated analog, 18F-fluoromethylcholine, has also been developed for PET imaging of choline metabolism (13), with the longer half-life of fluorine-18 (109.8 vs. 20.4 minutes for www.aacrjournals.org Downloaded from clincancerres.aacrjournals.org on March 3, 2014. © 2012 American Association for Cancer Research. 1063 Published OnlineFirst January 10, 2012; DOI: 10.1158/1078-0432.CCR-11-2462 Witney et al. Translational Relevance 11 C-Choline–positron emission tomography (PET) is a marker of choline kinase expression and activity, which is upregulated during carcinogenesis. To date, 11C-choline–PET has been used for the detection of a range of human cancers and has emerged as a viable alternative to 18 F-2-fluoro-2-deoxyglucose for the imaging of prostate adenocarcinoma. 11C-choline, however, is rapidly oxidized to betaine in an unwanted side reaction, complicating data interpretation. Here, we designed novel choline analogs and tested their metabolic profiles and sensitivity for cancer detection. The doubly fluorinated and deuterated analog 18F-D4-choline showed lowest betaine oxidation. This radiotracer could be used for cancer detection, irrespective of histologic type. Therefore, the development of new choline radiotracers with an improved metabolic profile should provide a means to simplify interpretation of clinical PET data, while increasing selectivity for phosphorylation. carbon-11), potentially enabling more widespread adoption of choline imaging in the clinic and the ability to image at later time points posttracer injection. Within the imaging time window (60 minutes), tumor radiolabeled choline uptake is a function of perfusion, transport of the radiotracer from the extracellular space into cells, where it is either converted into phosphocholine by the action of choline kinase or oxidized by choline oxidase to betaine. Further incorporation of phosphocholine to membrane phosphatidylcholine is negligible within this time window (7, 8, 14, 15). Chromatographic analysis also indicates that further betaine metabolism or conversion to acetylcholine is negligible (16, 17). Hence, radiotracer uptake broadly represents transport and phosphorylation on the one hand, and transport and oxidation on the other. One key limitation of choline–PET is the rapid oxidation to radiolabeled betaine, making it difficult to assess choline kinase–specific trapping of activity (as phosphocholine) within tumors without plasma metabolite evaluation using complex kinetic analysis. We have recently developed a novel tracer, 18F-fluoromethyl-[1,2-2H4]-choline (18F-D4-choline), with reduced in vivo oxidation to betaine and improved sensitivity, for the detection of choline metabolism in comparison with the nondeuterated 18F-fluoromethylcholine (16). This improved metabolic profile was shown (16, 17) to be based on the deuterium isotope effect (18–21). Here, we sought to further evaluate the structural determinants of deuteration on substrate metabolism, as well as the effect of deuteration on tumor-specific uptake. To this end, we developed a novel choline tracer, 11C-[1,2-2H4]-choline (11C-D4-choline) and compared its in vivo tumor uptake, kinetics, and metabolic profile to 11C-choline and 18F-D4choline PET tracers. 1064 Clin Cancer Res; 18(4) February 15, 2012 Materials and Methods Cell lines HCT116 colorectal carcinoma (LGC Standards) and PC3M prostate adenocarcinoma cells (kind donation from Dr Matthew Caley, Prostate Cancer Metastasis Team, Imperial College London, United Kingdom) were grown in RPMI1640 media, supplemented with 10% fetal calf serum (FCS), 2 mmol/L L-glutamine, 100 U/mL penicillin, and 100 mg/mL streptomycin (Invitrogen). A375 malignant melanoma cells were a kind donation from Professor Eyal Gottlieb, Beatson Institute for Cancer Research, Glasgow, United Kingdom, and were grown in high glucose (4.5 g/L) Dulbecco’s modified Eagle’s medium media, supplemented with 10% FCS, 2 mmol/L L-glutamine, 100 U/mL penicillin, and 100 mg/mL streptomycin (Invitrogen). All cells were maintained at 37 C in a humidified atmosphere containing 5% CO2. Western blots Western blotting was done using standard techniques (22, 23). For detailed methodology, see Supplementary Materials. In vivo tumor models All animal experiments were conducted by licensed investigators in accordance with the United Kingdom Home Office Guidance on the Operation of the Animal (Scientific Procedures) Act 1986 and within the newly published guidelines for the welfare and use of animals in cancer research (24). Male BALB/c nude mice (aged 6–8 weeks; Charles River) were used. Tumor cells (2  106) were injected subcutaneously on the back of mice and animals were used when the xenografts reached approximately 100 mm3. Tumor dimensions were measured continuously using a caliper and tumor volumes were calculated by the equation: volume ¼ (p/6)  a  b  c, in which a, b, and c represent 3 orthogonal axes of the tumor. In vivo tracer metabolism Radiolabeled metabolites from plasma and tissues were quantified using a method adapted from Smith and colleagues (17). Briefly, tumor-bearing mice under general anesthesia (2.5% isofluorane; nonrecovery anesthesia) were administered a bolus intravenous injection of one of the following radiotracers: 11C-choline, 11C-D4-choline (18.5 MBq), or 18F-D4-choline (3.7 MBq), and sacrificed by exsanguination via cardiac puncture at 2, 15, 30, or 60 minutes postradiotracer injection. For automated radiosynthesis methodology see Supplementary Materials. Tumor, kidney, and liver samples were immediately snap-frozen in liquid nitrogen. Aliquots of heparinized blood were rapidly centrifuged (14,000  g, 5 minutes, 4 C) to obtain plasma. Plasma samples were subsequently snap-frozen in liquid nitrogen and kept on dry ice prior to analysis. For analysis, samples were thawed and kept at 4 C immediately before use. To ice-cold plasma (200 mL) was Clinical Cancer Research Downloaded from clincancerres.aacrjournals.org on March 3, 2014. © 2012 American Association for Cancer Research. Published OnlineFirst January 10, 2012; DOI: 10.1158/1078-0432.CCR-11-2462 Deuterated Choline–PET Radiotracers for Cancer Detection added ice-cold methanol (1.5 mL) and the resulting suspension centrifuged (14,000  g; 4 C; 3 minutes). The supernatant was then decanted and evaporated to dryness on a rotary evaporator (bath temperature, 40 C), then resuspended in high-performance liquid chromatography (HPLC) mobile phase [solvent A: acetonitrile/water/ethanol/acetic acid/1.0 mol/L ammonium acetate/0.1 mol/L sodium phosphate (800/127/68/2/3/10); 1.1 mL]. Samples were filtered through a hydrophilic syringe filter (0.2-mm filter, Millex PTFE filter, Millipore) and the sample (1 mL) then injected via a 1-mL sample loop onto the HPLC for analysis. Tissues were homogenized in ice-cold methanol (1.5 mL) using an Ultra-Turrax T-25 homogenizer (IKA Werke GmbH and Co. KG) and subsequently treated as per plasma samples. Samples were analyzed on an Agilent 1100 series HPLC system (Agilent Technologies), configured as described above, using the method of Leyton and colleagues (16). A mBondapak C18 HPLC column (7.8  3,000 mm; Waters), stationary phase and a mobile phase comprising of solvent A (vide supra) and solvent B [acetonitrile/water/ethanol/ acetic acid/1.0 mol/L ammonium acetate/0.1 mol/L sodium phosphate (400/400/68/44/88/10)], delivered at a flow rate of 3 mL/min were used for analyte separation. The gradient was set as follows: 0% B for 5 minutes; 0% to 100% B in 10 minutes; 100% B for 0.5 minutes; 100% to 0% B in 2 minutes; 0% B for 2.5 minutes. PET imaging studies Dynamic 11C-choline, 11C-D4-choline, and 18F-D4choline imaging scans were carried out on a dedicated small animal PET scanner (Siemens Inveon PET module, Siemens Medical Solutions USA, Inc.) following a bolus intravenous injection in tumor-bearing mice of either approximately 3.7 MBq for 18F studies or approximately 18.5 MBq for 11C, accommodating for substantially shorter half-life of 11C (20.38 minutes for 11C vs. 109.77 minutes for 18F). Dynamic scans were acquired in list mode format over 60 minutes. The acquired data were then sorted into 0.5-mm sinogram bins and 19 time frames for image reconstruction (4  15, 4  60, and 11  300 seconds), which was done by filtered back projection. For input function analysis, data were sorted into 25 time frames for image reconstruction (8  5, 1  20, 4  40, 1  80, and 11  300 seconds). The Siemens Inveon Research Workplace software was used for visualization of radiotracer uptake in the tumor; 30- to 60-minute cumulative images of the dynamic data were employed to define 3-dimensional (3D) regions of interest (ROI). Arterial input function was estimated as follows: a single voxel 3D ROI was manually drawn in the center of the heart cavity using 2 to 5 minutes of cumulative images. Care was taken to minimize ROI overlap with the myocardium. The count densities were averaged for all ROIs at each time point to obtain a time versus radioactivity curve (TAC). Tumor TACs were normalized to injected dose, measured by a VDC-304 dose calibrator (Veenstra Instruments) and expressed as percentage injected dose per mL www.aacrjournals.org tissue. The area under the TAC, calculated as the integral of %ID/mL from 0 to 60 minutes, and the normalized uptake of radiotracer at 60 minutes (%ID/mL60) were also used for comparisons. Kinetic analysis in HCT116 tumors A 2-tissue irreversible compartmental model was employed to fit the TACs, as has been previously established for 11C-choline (25, 26), described extensively in Supplementary Data. Here, both a Single Input 3k model (irreversible binding of the parent) and Double Input [3þ2]k model (irreversible binding of the parent, reversible binding of the metabolite) were used to describe radiotracer kinetics. K1 (radiotracer delivery; mL/mL/min) and k2 (1/ min) are the rate constants of transfer from plasma to tissue and from tissue to plasma, respectively. k3 (1/min) represents the rate at which the parent tracer is phosphorylated. In this model the irreversible uptake rate constant Ki (mL/ mL/min) can be expressed as a function of the microparameters as K1k3/(k2 þ k3). K10 (mL/mL/min) and k20 (1/min) are the rate constants of transfer from plasma to tissue and from tissue to plasma of labeled betaine. A schematic describing the kinetic models used here is described in Supplementary Fig. S1. Statistics Data were expressed as mean  SEM, unless otherwise shown. The significance of comparison between 2 data sets was determined using Student t test. ANOVA was used for multiparametric analysis (Prism v5.0 software for windows, GraphPad Software). Differences between groups were considered significant if P  0.05. Results Deuteration leads to enhanced renal radiotracer uptake Time course biodistribution was done in nontumor– bearing male nude mice with 11C-choline, 11C-D4-choline, and 18F-D4-choline tracers. Supplementary Fig. S2 shows tissue distribution at 2, 15, 30, and 60 minutes. There were minimal differences in tissue uptake between the 3 tracers over 60 minutes, with uptake values in broad agreement with data previously published for 18Fcholine and 18F-D4-choline (13, 17). In all tracers there was rapid extraction from blood, with the majority of radioactivity retained within the kidneys, evident as early as 2 minutes postinjection. Deuteration of 11Ccholine led to a significant 1.8-fold increase in kidney retention over 60 minutes (P < 0.05; Supplementary Fig. S2A and B), with a 3.3-fold increase in kidney retention observed for 18F-D4-choline when compared with 11Ccholine at this time point (P < 0.01; Supplementary Fig. S2A and C, respectively). There was a trend toward increased urinary excretion for 11C-D4-choline and 18 F-D4-choline, in comparison with the nature identical tracer, 11C-choline, although this increase did not reach statistical significance. Clin Cancer Res; 18(4) February 15, 2012 Downloaded from clincancerres.aacrjournals.org on March 3, 2014. © 2012 American Association for Cancer Research. 1065 Published OnlineFirst January 10, 2012; DOI: 10.1158/1078-0432.CCR-11-2462 Witney et al. Figure 1. Metabolic profile of 11 C-choline (A and D), 11C-D4choline (B and E), and 18F-D4choline (C and F) in the liver (A–C) and kidney (D–F) of BALB/c nude mice. Radiolabeled metabolite profile was assessed at 2, 15, 30, and 60 minutes after intravenous injection of parent radiotracers using radio-HPLC. Mean values (n ¼ 3) and SEM are shown. a, P < 0.05 when 11C-D4-choline is compared with 11C-choline; b, P < 0.05 when 18F-D4-choline is compared with 11C-choline; c, P < 0.05 when 18F-D4-choline is compared with 11C-D4-choline. Bet-ald, betaine aldehyde; p-Choline, phosphocholine. Deuteration of 11C-choline results in modest resistance to oxidation in vivo Tracer metabolism in tissues and plasma was done by radio-HPLC (Fig. 1). Peaks were assigned as choline, betaine, betaine aldehyde, and phosphocholine, using enzymatic (alkaline phosphatase and choline oxidase) methods to determine their identity (Supplementary Figs. S3 and S4, respectively; ref. 16). In the liver, both 11C-choline and 11C-D4-choline were rapidly oxidized to betaine (Fig. 1A and B), with 49.2  7.7% of 11C-choline radioactivity already oxidized to betaine by 2 minutes. Deuteration of 11C-choline provided significant protection against oxidation in the liver at 2 minutes postinjection, with 24.5  2.1% radioactivity as betaine (51.2% decrease in betaine levels; P ¼ 0.037), although this protection was lost by 15 minutes. Notably, a high proportion of liver radioactivity (80%) was present as 18F-D4-phosphocholine by 15 minutes with 18F-D4choline (Fig. 1C). This corresponded to a much reduced liver-specific oxidation when compared with the 2 carbon- 1066 Clin Cancer Res; 18(4) February 15, 2012 11 tracers (15.0  3.6% of radioactivity as betaine at 60 minutes; P ¼ 0.002). In contrast to the liver, deuteration of 11C-choline resulted in protection against oxidation in the kidney over the entirety of the 60-minute time course (Fig. 1D and E). With 11C-D4-choline there was a 20% to 40% decrease in betaine levels over 60 minutes when compared with 11Ccholine (P < 0.05), corresponding to a proportional increase in labeled phosphocholine (P < 0.05). As shown in Fig. 1F, 18 F-D4-choline was more resistant to oxidation in the kidney than both carbon-11–labeled choline tracers. There was a relationship between levels of radiolabeled phosphocholine and kidney retention when data from all 3 tracers were compared (R2 ¼ 0.504; Supplementary Fig. S5). In the plasma, the temporal levels of betaine for both 11C-choline and 11C-D4-choline were almost identical; it should be noted that total radioactivity levels were low for all radiotracers. At 2 minutes, 12.1  2.6% and 8.8  3.8% of radioactivity was in the form of betaine for 11C-choline and 11C-D4-choline, respectively, rising to 78.6  4.4% and Clinical Cancer Research Downloaded from clincancerres.aacrjournals.org on March 3, 2014. © 2012 American Association for Cancer Research. Published OnlineFirst January 10, 2012; DOI: 10.1158/1078-0432.CCR-11-2462 Deuterated Choline–PET Radiotracers for Cancer Detection 79.5  2.9% at 15 minutes. Betaine levels were significantly reduced with 18F-D4-choline, with 43.7  12.4% of activity present as betaine at 15 minutes. A further increase in plasma betaine was not observed with 18F-D4-choline over the remainder of the time course. Fluorination protects against choline oxidation in tumor 11 C-choline, 11C-D4-choline, and 18F-D4-choline metabolism were measured in HCT116 tumors (Fig. 2). With all tracers, choline oxidation was greatly reduced in the tumor in comparison with levels in the kidney and liver. At 15 minutes, both 11C-D4-choline and 18F-D4-choline had significantly more radioactivity corresponding to labeled phosphocholine than 11C-choline; 43.8  1.5% and 45.1  3.2% for 11C-D4-choline and 18F-D4-choline, respectively, in comparison with 30.5  4.0% for 11C-choline (P ¼ 0.035 and P ¼ 0.046, respectively). By 60 minutes, the majority of radioactivity was phosphocholine for all 3 tracers, with 11 11 18 Figure 2. Metabolic profile of C-choline, C-D4-choline, and F-D4choline in HCT116 tumors. Radiolabeled metabolite profile in HCT116 tumor xenografts was assessed at 15 (A) and 60 minutes (B) after intravenous injection of parent radiotracers using radio-HPLC. Mean values (n ¼ 3) and SEM are shown.  , P < 0.05;   , P < 0.01;    , P < 0.001. p-Choline, phosphocholine. www.aacrjournals.org labeled phosphocholine levels increasing in the order of 11 C-choline < 11C-D4-choline < 18F-D4-choline. There was no difference in the tumor metabolic profile for 11C-choline and 11C-D4-choline at 60 minutes, although reduced choline oxidation was observed for 18F-D4-choline; 14.0  3.0% betaine radioactivity with 18F-D4-choline compared with 28.1  2.9% for 11C-choline (P ¼ 0.026). Choline tracers have similar sensitivity for imaging tumors by PET Despite the low systemic oxidation of 18F-D4-choline, tumor radiotracer uptake in mice by PET was no higher than with 11C-choline or 11C-D4-choline (Fig. 3). Figure 3A–C shows typical (0.5 mm) transverse PET image slices showing accumulation of all 3 tracers in HCT116 tumors. For all 3 tracers, there was heterogeneous tumor uptake, but tumor signal-to-background levels were identical, confirmed by normalized uptake values at 60 minutes and values for the tumor area under the time versus radioactivity curve (data not shown). It should be noted that the PET data represent total radioactivity. In the case of 11C-choline or 11C-D4choline, a significant proportion of this radioactivity is betaine (Fig. 2). Tumor tracer kinetics Despite there being no difference in overall tracer retention in the tumor, the kinetic profiles of tracer uptake varied between the 3 choline tracers, detected by PET (Fig. 3D). The kinetics for the 3 tracers were characterized by rapid tumor influx over the initial 5 minutes, followed by stabilization of tumor retention. Initial delivery of 18F-D4-choline over the first 14 minutes of imaging was higher than for both 11Ccholine and 11C-D4-choline (expanded TAC for initial 14 minutes shown in Supplementary Fig. S6). Slow wash-out of activity was observed with both 18F-D4-choline and 11CD4-choline between 30 and 60 minutes, in contrast to the gradual accumulation detected with 11C-choline. Parameters for the irreversible trapping of radioactivity in the tumor, Ki and k3, were calculated from a 2-tissue irreversible model, using metabolite-corrected TAC from the heart cavity as input function (Fig. 4A and B). A double input model, accounting for the contribution of metabolites to the tissue TAC, was used for kinetic analysis, described in Supplementary Data. There was no significant difference in flux constant measurements between deuterated and undeuterated 11C-choline. Addition of methylfluoride, however, resulted in 49.2% (n ¼ 3; P ¼ 0.022) and 75.2% (n ¼ 3; P ¼ 0.005) decreases in Ki and k3, respectively; that is, when 18F-D4-choline was compared with 11C-D40 choline. K1 values were similar between all 3 tracers: 0.106  0.026, 0.114  0.019, and 0.142  0.027 for 11C-choline, 11 C-D4-choline, and 18F-D4-choline, respectively. It is possible that intracellular betaine formation (not just presence of betaine in the extracellular space) led to a higher than expected irreversible uptake; there was a significant 388% and 230% increase in the ratio of betaine:phophocholine at 15 and 60 minutes, respectively (P ¼ 0.045 and 0.036) with 11 C-choline in comparison with 18F-D4-choline (Fig. 4C). Clin Cancer Res; 18(4) February 15, 2012 Downloaded from clincancerres.aacrjournals.org on March 3, 2014. © 2012 American Association for Cancer Research. 1067 Published OnlineFirst January 10, 2012; DOI: 10.1158/1078-0432.CCR-11-2462 Witney et al. 11 11 18 Figure 3. C-choline (*), C-D4-choline (~), and F-D4-choline (&) PET image analysis. HCT116 tumor uptake profiles were examined following 60-minute dynamic PET imaging. A–C, representative axial PET-CT images of HCT116 tumor–bearing mice (30–60 minutes of summed activity) for 11C-choline (A), 11 C-D4-choline (B), and 18F-D4-choline (C). Tumor margins, indicated from CT image, are outlined in red. D, the tumor TAC. Mean  SEM (n ¼ 4 mice per group). 18 F-D4-choline shows good sensitivity for the PET imaging of prostate adenocarcinoma and malignant melanoma Having confirmed that 18F-D4-choline has the most desirable metabolic profile for in vivo studies, with good sensitivity for the imaging of colon adenocarcinoma, we wanted to evaluate its suitability for cancer detection in other models of human cancer, including malignant melanoma A375 and prostate adenocarcinoma PC3-M. In vitro uptake of 18F-D4-choline was similar in the 3 cell lines over 30 minutes (Supplementary Fig. S7), relating to near-identical levels of choline kinase expression (Supplementary Fig. S7 insert). Retention of radioactivity was shown to be dependent on both choline transport and choline kinase activity, as treatment of cells with the dual choline transport and choline kinase inhibitor, hemicholinium-3, resulted in more than 90% decrease in intracellular tracer radioactivity in all 3 cell lines. Similar intracellular trapping of 18F-D4choline in these cancer models were translated to their uptake in vivo (Fig. 5A), showing similar values for flux constant measurements, including rates of delivery (K1; Supplementary Table S1) and other and PET imaging variables. There was a trend toward increased tumor retention of 18F-D4-choline in the order of A375 < HCT116 < PC3-M, reflected by the expression of choline kinase in these lines 1068 Clin Cancer Res; 18(4) February 15, 2012 (Fig. 5C). There was no discernible difference in tumor metabolite profiles between the 3 cell cancer models at either 15 or 60 minutes of postinjection (Fig. 5B). Discussion Aberrant phospholipid metabolism is a hallmark of many cancers (5), resulting in upregulated mitotic signaling and an increase in plasma membrane biosynthesis. One such mediator of phospholipid metabolism, choline kinase, has been shown to be a biomarker of malignant transformation (2). Proton and phosphorous magnetic resonance spectroscopic (MRS) techniques have provided a means to measure the product of choline kinase activity, phosphocholine, from tumor tissue biopsies ex vivo and from noninvasive spectroscopic imaging measurements in vivo (27). MRS, however, is hampered by inherently poor in vivo sensitivity, making it difficult to resolve individual choline metabolite resonances, complicating data interpretation, whereas ex vivo measurements requires invasive sampling from a small, possibly unrepresentative, region of interest. Given the current drawbacks of choline metabolite analysis by MRS, a more viable alternative has been the use of radiolabeled choline for noninvasive tumor imaging. PET-labeled choline tracers provide vastly improved Clinical Cancer Research Downloaded from clincancerres.aacrjournals.org on March 3, 2014. © 2012 American Association for Cancer Research. Published OnlineFirst January 10, 2012; DOI: 10.1158/1078-0432.CCR-11-2462 Deuterated Choline–PET Radiotracers for Cancer Detection Figure 4. Pharmacokinetics of 11 C-choline, 11C-D4-choline, and 18 F-D4-choline in HCT116 tumors. A, modified compartmental modeling analysis, taking into account plasma metabolites and their flux into the exchangeable space in tumor, was used to derive Ki, a measure of irreversible retention within the tumor. B, the kinetic parameter, k3, an indirect measure of choline kinase activity, was calculated using a 2-site compartmental model as previously described (36, 37). C, ratio of betaine to phosphocholine in tumors. Metabolites were quantified by radio-HPLC at 15 and 60 minutes of postinjection of tracer. Mean values (n ¼ 4) and SEM are shown.  , P < 0.05;   , P < 0.001. p-choline, phosphocholine. sensitivity, when compared with MRS, and enable dynamic measurements of choline metabolism, but without the chemical resolution of MRS. To date, 11C-choline has successfully been used for the clinical imaging of prostate, brain, breast, and esophageal carcinomas (10, 25, 28–30). Despite its relative success, 11C-choline-PET imaging has not been widely adopted in the clinic; the short half-life of carbon-11 requires an on-site cyclotron and rapid metabolism of the choline tracer presents complications for data interpretation and limits the imaging time frame to early time frames (25). We have recently developed a fluorinated choline analog, 18F-D4-choline, labeled with a longer lived isotope and with improved metabolic profile, required for late tumor imaging (16). The substitution of deuterium for Figure 5. Dynamic uptake and 18 metabolite analysis with F-D4choline in tumors of different histologic origin. A, the tumor TAC obtained from 60-minute dynamic PET imaging. Mean  SEM (n ¼ 3–5 mice per group). B, metabolic profile of 18F-D4-choline in tumors. Radiolabeled metabolite profile in HCT116 tumor xenografts was assessed post-PET imaging using radio-HPLC. Mean values (n ¼ 3) and SEM are shown. C, choline kinase expression in malignant melanoma, prostate adenocarcinoma, and colon carcinoma tumors. Representative Western blot from tumor lysates (n ¼ 3 xenografts per tumor cell line). Actin was used as a loading control. CKa, choline kinase alpha; %ID, percentage injected dose. www.aacrjournals.org Clin Cancer Res; 18(4) February 15, 2012 Downloaded from clincancerres.aacrjournals.org on March 3, 2014. © 2012 American Association for Cancer Research. 1069 Published OnlineFirst January 10, 2012; DOI: 10.1158/1078-0432.CCR-11-2462 Witney et al. hydrogen on the ethyl alcohol portion of choline resulted in a large observed isotope effect in the oxidation of choline to betaine by choline oxidase. Further studies showed that the magnitude of the 1H/2D isotope effect was more profound when all protons were substituted for deuterium, in comparison with partial deuteration of the fluorocholine (17). Urinary radioactivity, however, is higher with fluorinated choline analogs relative to 11C-choline (16, 31), potentially limiting their use for the detection of pelvic cancers, such as prostate adenocarcinoma. Here, we developed a novel choline tracer, 11C-D4-choline, which, based on previous work with fluorinated and deuterated choline tracers, was predicted to have reduced oxidation to betaine and a favorable urinary excretion profile. The kidney has high choline kinase activity along the nephron (32), shown to exhibit the greatest tissue retention for choline-PET and, therefore, is the radiation-dose-critical organ (13, 17). Kidney retention increased in the order of 11 C-choline < 11C-D4-choline < 18F-D4-choline over the 60minute time course (Supplementary Fig. S2), with total kidney radioactivity shown to be proportional to the % radioactivity retained as labeled phosphocholine (Supplementary Fig. S5; R2 ¼ 0.504). The increased conversion of choline to phosphocholine with 11C-D4-choline relative to 11 C-choline corresponded with a significant decrease in choline oxidation to betaine and could be ascribed to increased substrate availability for phosphorylation. Further attenuation of choline oxidation was observed with 18 F-D4-choline, indicating that the magnitude of the 1H/2D isotope effect is influenced by fluorination. Protection against choline oxidation by deuteration of 11C-choline was shown to be tissue specific, with a decrease in betaine radioactivity measured in the liver at just 2 minutes postinjection when compared with 11C-choline (Fig. 1). This effect is presumably due to the lower capacity of choline oxidase in rodent kidney compared with liver. 18F-D4choline provided substantially reduced betaine oxidation in the liver over 60 minutes postinjection, again suggesting that fluorination, in part, drives this reduced capacity to oxidize choline pseudosubstrates to betaine. Despite systemic protection against choline oxidation with 18F-D4-choline, the reduction in the rate of choline oxidation was much more subtle in implanted HCT116 tumors (Fig. 2). At 15 minutes postinjection, there were 43.6% and 47.9% higher levels of radiolabeled phosphocholine when 11C-D4-choline and 18F-D4-choline, respectively, were injected relative to 11C-choline. By 60 minutes there was no difference in labeled phosphocholine levels between the 3 tracers, although there was a significant decrease in betaine-specific radioactivity with 18F-D4-choline. This equilibration of phosphocholine-specific activity can be explained by a saturation effect, with parent tracer levels reduced to a minimum by 60 minutes, severely limiting substrate levels available for choline kinase activity. Lower betaine levels were observed in the tumor with all 3 tracers over the entire time course when compared with liver and kidney, likely resulting from a lower capacity for choline oxidation or increased washout of betaine. It should be 1070 Clin Cancer Res; 18(4) February 15, 2012 noted that the capacity of rodents to metabolize choline is substantially higher than that of humans (14, 33). The slower metabolic rate in humans may, therefore, provide a better differential between these choline tracers. Despite this, deuteration of 11C-choline per se provided less than expected protection against choline oxidation in the liver, tumor, and kidney, especially in the context of improved metabolic profile shown with deuterated fluorocholine versus nondeuterated fluorocholine (16, 17) Comparison of the 3 choline radiotracers by PET showed no significant differences in overall tumor radiotracer uptake and hence sensitivity (Fig. 3), despite large changes observed in other organs. Initial tumor kinetics (at time points when metabolism was lower), however, varied between tracers, with 18F-D4-choline characterized by rapid delivery over approximately 5 minutes, followed by slow wash-out of activity from the tumor. This compared with the slower uptake, but continuous tumor retention of 11Ccholine. At 60 minutes, a 2.7-fold and 4.0-fold higher unmetabolized parent tracer was seen with 18F-D4-choline in tumor compared with 11C-choline and 11C-D4-choline, respectively, (Fig. 2). Deuteration did not, however, alter total tumor radioactivity levels and the modeling approach used did not distinguish between different intracellular species. Although all tracers were converted intracellularly to phosphocholine, the higher rate constants for intracellular retention (Ki and k3; Fig. 3A and B) of 11C-choline and 11 C-D4-choline, compared with 18F-D4-choline, were explained by the rapid conversion of the nonfluorinated tracers to betaine within HCT116 tumors, indicating greater specificity with 18F-D4-choline. Compared with 18F-D4choline, the tumor betaine-to-phosphocholine metabolite ratio increased by 388% (P ¼ 0.045) and 259% (P ¼ 0.061, nonsignificant) for 11C-choline and 11C-D4-choline, respectively (Fig. 4C). It is also important to note that the compartmental modeling is subject to some minor experimental limitations. In humans and larger animals, a more accurate input function can be obtained by continuous blood sampling following radiotracer injection. Individual plasma metabolite data can also be easily obtained and fitted instead of the averaged data taken from a separate cohort of animals used here. It has been reported elsewhere that fluorination increases urinary excretion in comparison with 11C-choline (13, 16). However, in this study, we did not observe these undesirable urinary excretion properties. This may be due to use of anesthesia for immobilizing mice during imaging. There was a trend toward increased urinary excretion in the two deuterated tracers (suggesting a trade-off between reduced oxidation and renal excretion) when compared with 11Ccholine, although these did not reach significance. Low radioactivity levels in the urine prevented accurate metabolite analysis (data not shown). The low radioactivity levels in the urine should enable accurate prostate imaging with 18 F-D4-choline, especially if patients void to reduce bladder radioactivity prior to late time point imaging. Given the favorable urinary excretion properties and greatly superior systemic metabolic profile of 18F-D4-choline, PET imaging Clinical Cancer Research Downloaded from clincancerres.aacrjournals.org on March 3, 2014. © 2012 American Association for Cancer Research. Published OnlineFirst January 10, 2012; DOI: 10.1158/1078-0432.CCR-11-2462 Deuterated Choline–PET Radiotracers for Cancer Detection was carried out in 2 further models of human cancer to assess generic utility in tumors of different origins: A375 malignant melanoma and PC3-M prostate adenocarcinoma. PC3-M cells were chosen as a clinically relevant model for choline imaging, whereas A375 have constitutively active mitogen-activated protein kinase (MAPK) due to a BRAFV600E mutation (34); the MAPK pathway is known to regulate choline kinase activity (35). 18F-D4-choline uptake in vitro (where betaine formation is negligible) was similar in all 3 cell lines, reflecting near-identical levels of choline kinase a expression. The delivery of 18F-D4-choline (K1) was similar between the different tumor types in vivo, suggesting that choline transporter expression was probably not deficient in any of the tumors. These in vitro findings translated well in vivo, with comparable tumor uptake, kinetics, choline kinase a expression, and metabolism for all the tumor types, suggesting that 18F-D4-choline may have utility for tumor detection, irrespective of histologic type. In conclusion, we have shown here that deuteration of 11 C-choline provides a smaller than expected protection against choline oxidation. Despite a significant increase in labeled phosphocholine at early time points, this did not increase the overall sensitivity for the detection of choline metabolism in vivo. More promising is the substantial decrease in betaine oxidation illustrated here with 18FD4-choline, which may permit the clinical imaging of choline without invasive blood sampling. Fluorine-18 labeling may also lead to wider clinical adoption and permit imaging at late time points. We have further validated 18F-D4-choline using 3 models of human cancer, including a clinically relevant model of human prostate adenocarcinoma. Disclosure of Potential Conflicts of Interest A patent on novel choline imaging agents has been filed. Acknowledgments The authors thank Dr. Magdy Khalil for his help with the PET imaging studies and for advice about kinetic analysis, and Dr. Matthew Caley and Professor Eyal Gottlieb for provision of cell lines. Grant Support This work was funded by Cancer Research UK–Engineering and Physical Sciences Research Council grant C2536/A10337. E.O. Aboagye’s laboratory receives core funding from the UK Medical Research Council (MC US A652 0030). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. 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