Eur J Nucl Med Mol Imaging (2010) 37:1861–1868 DOI 10.1007/s00259-010-1493-2

ORIGINAL ARTICLE

[11C]Choline as pharmacodynamic marker for therapy response assessment in a prostate cancer xenograft model Bernd J. Krause & Michael Souvatzoglou & Ken Herrmann & Axel W. Weber & Tibor Schuster & Andreas K. Buck & Roman Nawroth & Gregor Weirich & Uwe Treiber & Hans-Jürgen Wester & Sibylle I. Ziegler & Reingard Senekowitsch-Schmidtke & Markus Schwaiger

Received: 16 December 2009 / Accepted: 1 May 2010 / Published online: 30 May 2010 # Springer-Verlag 2010

G. Weirich Institute of Pathology, Technische Universität München, Munich, Germany

muscle (T/M) ratios. Every week the size of the implanted tumour was determined with a sliding calliper. Results The PC-3 tumours could be visualized by [11C] choline PET. Before treatment the T/Mmean ratio was 1.6±0.5 in the control group and 1.8±0.4 in the docetaxel-treated group (p=0.65). There was a reduction in the mean [11C] choline uptake after docetaxel treatment as early as 1 week after initiation of therapy (T/M ratio 1.8±0.4 before treatment, 0.9±0.3 after 1 week, 1.1±0.3 after 2 weeks and 0.8±0.2 after 3 weeks). There were no decrease in [ 11 C]choline uptake in the control group following treatment (T/M ratio 1.6±0.5 before treatment, 1.7±0.4 after 1 week, 1.8±0.7 after 2 weeks and 1.7±0.4 after 3 weeks). For analysis of the dynamic data, a generalized estimation equation model revealed a significant decrease in the T/Mdyn ratios 1 week after docetaxel treatment, and the ratio remained at that level through week 3 (mean change −0.93±0.24, p<0.001, after 1 week; −0.78±0.21, p<0.001, after 2 weeks; −1.08±0.26, p<0.001, after 3 weeks). In the control group there was no significant decrease in the T/Mdyn ratios (mean change 0.085±0.39, p=0.83, after 1 week; 0.31±0.48, p=0.52, after 2 weeks; 0.11±0.30, p=0.72, after 3 weeks). Metabolic changes occurred 1 week after therapy and preceded morphological changes of tumour size during therapy. Conclusion Our results demonstrate that [11C]choline has the potential for use in the early monitoring of the therapeutic effect of docetaxel in a prostate cancer xenograft animal model. The results also indicate that PET with radioactively labelled choline derivatives might be a useful tool for monitoring responses to taxane-based chemotherapy in patients with advanced prostate cancer.

R. Nawroth : U. Treiber Department of Urology, Technische Universität München, Munich, Germany

Keywords Prostate cancer . Xenograft . MicroPET/CT . Therapy . Choline

Abstract Purpose [11C]Choline has been established as a PET tracer for imaging prostate cancer. The aim of this study was to determine whether [11C]choline can be used for monitoring the effects of therapy in a prostate cancer mouse xenograft model. Methods The androgen-independent human prostate cancer cell line PC-3 was implanted subcutaneously into the flanks of 13 NMRI (nu/nu) mice. All mice were injected 4–6 weeks after xenograft implantation with 37 MBq [11C] choline via a tail vein. Dynamic imaging was performed for 60 min with a small-animal PET/CT scanner (Siemens Medical Solutions). Six mice were subsequently injected intravenously with docetaxel twice (days 1 and 5) at a dose of 3 mg/kg body weight. Seven mice were treated with PBS as a control. [11C]Choline imaging was performed prior to and 1, 2 and 3 weeks after treatment. To determine choline uptake the images were analysed in terms of tumour-toB. J. Krause (*) : M. Souvatzoglou : K. Herrmann : A. W. Weber : A. K. Buck : H.-J. Wester : S. I. Ziegler : R. Senekowitsch-Schmidtke : M. Schwaiger Department of Nuclear Medicine, Klinikum rechts der Isar, Technische Universität München, Ismaninger Str. 22, 81675 München, Munich, Germany e-mail: [email protected] T. Schuster Department of Statistics, Technische Universität München, Munich, Germany

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Introduction Prostate cancer is one of the most commonly diagnosed cancers and one of the leading causes of death in men. Most prostate cancers show an androgen dependency in their growth. Therefore antiandrogen therapy is an important therapeutic approach. However, some patients develop hormone resistance under antiandrogen therapy, while others continue to respond. Clinical trials have shown that chemotherapy can play an important role in palliative therapy in these patients [1]. Docetaxel has become a standard chemotherapeutic agent for treating metastatic hormone-resistant prostate cancer [2]. However, response to therapy is observed in only 20–50% of patients [2–4]. To select patients who will possibly benefit from single-agent or combination therapies, assessment of response to therapy is usually performed using biological markers (i.e. PSA level, PSA doubling time, PSA velocity, PSA density) [5] and imaging techniques [6]. However, it can often take weeks to months before a response to a given therapy can be assessed in terms of morphological changes. Therefore, surrogate markers of early therapy response are needed. Assessment of response with such a surrogate marker should be possible early in the course of treatment and would help to individualize therapy. Evaluation of tumour metabolism by PET is a promising tool for the assessment of the early effects of cancer therapy as has been shown in combination with [18F]FDG as metabolic marker in a variety of tumours. FDG has been evaluated for its usefulness in monitoring the effectiveness of therapy in prostate cancer in animal models [7] and in humans [8]. Due to the limitations of [18F]FDG, alternative tracers for molecular imaging of prostate cancer have been introduced [9–12], and among these [11C]- and [18F]labelled choline derivatives have shown promising results for restaging prostate cancer in patients with biochemical recurrence [13–15] and advanced prostate cancer [16]. This study was designed to determine if [11C]choline can be used as a marker to monitor response to docetaxel in prostate cancer. Therefore we performed [11C]choline small-animal PET/CT studies in a PC-3 prostate cancer xenograft model.

Material and methods Synthesis of [11C]choline [11C]Choline was synthesized according to the method of Pascali [17] with minor modifications. [11C]CO2 was converted to [11C]CH3I by a catalytic gas-phase iodination reaction via [11C]CH4 (GE MeI MicroLab). [11C]CH3I, in a flow of He at 50 ml/min, was passed through a Light-CM

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cartridge loaded with N,N-dimethylethanolamine (25 μl). The column was washed with 10 ml EtOH followed by 10 ml water before the product was eluted with isotonic saline (2–5 ml of 0.9% NaCl) through a Millipore filter (Millex GS, 0.22 μm) into a sterile vial. The pH of the formulated solution was about 7. Quality control was performed by HPLC (LiChrosorb RP18, 250×4.6 mm; 1 mM sodium naphthalene sulphonic acid, 50 mM M H3PO4, 1.5 ml/min; k=3.7). Prostate cancer tumour model The prostate cancer tumour model used in this study was a human prostate cancer/athymic mouse xenograft model implanted with cells of the human prostate cancer cell line PC-3. PC-3 is an androgen-independent androgen receptornegative PSA-negative human prostate cancer cell line [18]. Animal experiments were performed according to a protocol approved by the Technische Universität München. The subcutaneous prostate tumour xenografts were generated from 16 male athymic nu/nu mice (NMRI nu/nu Naval Medical Research Institute, Charles River Laboratories) at 6–8 weeks of age. To generate PC-3 tumours, the animals were injected into the left flank with 1x107 cells/injection suspended in 0.9% NaCl in a volume of approximately 100 µl per animal without Matrigel. Palpable tumours developed within 4 to 6 weeks after implantation. Animal PET/CT imaging Approximately 4–6 weeks after tumour implantation, PC-3 tumour-bearing mice were imaged in the prone position in a microPET FOCUS 120 scanner (Siemens Preclinical Solutions, Knoxville, TN, USA; for the performance characteristics of the microPET FOCUS 120 scanner see Kim et al. [19]). The resolution of the microPET FOCUS 120 scanner is between 1.1 and 2.4 mm full-width at half-maximum in each of the three dimensions within a 2-cm imaging field of view around the central axis of the tomograph. To define the tumour contour, data from a small-animal CT scanner (Inveon, Siemens Preclinical Solutions) were used. The CT exposure settings were 80 kV x-ray tube voltage, 300 µA xray tube current, and 400 ms exposure time for each of the 270 x-ray projections during one 360° rotation. CT images were reconstructed with a modified Feldkamp algorithm. microPET FOCUS 120 data were not corrected for attenuation. Tumour/muscle (T/M) ratios were used for data analysis since SUVs could not be calculated. The mice were anaesthetized by inhalation anaesthesia using isoflurane. Temperature, respiration and reflexes were monitored during imaging. List-mode PET data were acquired for 60 min beginning at the time of injection of 37 MBq [11C]choline via a microcatheter placed in a tail

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vein. List-mode data were sorted according to the following framing scheme: 20 s × 15 frames, 60 s × 10 frames, 300 s × 9 frames (34 frames). Three-dimensional data were Fourier-rebinned into two dimensions and reconstructed with an ordered subset expectation maximization algorithm with 16 subsets and four iterations. The data were not corrected for attenuation or scatter. The animals were divided into two groups. Six mice were treated with 3 mg/kg body weight docetaxel on days 1 and 5 via a microcatheter placed in the tail vein. Seven (control) mice had no treatment (phosphate-buffered saline). On days 7, 14 and 21 after the baseline PET/CT scan, PET/CT imaging was repeated using the same protocol as described for the baseline study. Before each PET/CT scan, the maximum diameters of the implanted tumours were determined with a sliding calliper. Two mice (one control group, one treatment group) had died by 1 week after the baseline PET/CT imaging. One mouse in the control group was killed 2 weeks after PET/ CT imaging following institutional regulations because of a heavy tumour burden. Therefore only the results from 13 animals were included in the data analysis. All remaining mice were killed 3 weeks after the baseline PET/CT scan following institutional regulations. [11C]Choline small-animal PET/CT data analysis Images summing all frames were calculated and coregistered with the CT data. Three square regions of interest (ROI) measuring 5×5 mm were placed in three transaxial slices within the tumour (the ROIs were not placed over the whole tumours due to a variable amount of necrosis so they covered only parts of the tumour; ROIT) and on the opposing muscle tissue (thigh, ROIM) on the CT image and the ROIs were transferred to the coregistered PET data. For statistical calculation the maximum count rate was used for these ROIs. To eliminate the dependency on the injected activity and the body weight, tumour/muscle (T/M) ratios were used. In each mouse three T/M ratios were calculated, which were defined as the counts in ROIT divided by the counts in ROIM in three slices (T/Mmean ratio). Time–activity curves for ROIT and ROIM were generated from the 34 acquired PET frames. For this purpose the ROIs drawn on the CT images were transferred to every single frame of the dynamic PET data. Time–T/M ratio curves were calculated by dividing the time–activity curves of ROIT and ROIM (T/Mdyn ratio). T/Mdyn ratios reflect the relative radiotracer clearance from the tumour. [11C]Choline T/Mmean ratio and T/Mdyn ratio at baseline were statistically compared with the T/Mmean and T/Mdyn ratios 1, 2 and 3 weeks after treatment (in the same animal). New ROIs were created after 1, 2, and 3 weeks because of potential changes in tumour configuration.

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Statistical evaluation Statistical analysis was performed using SPSS software (version 15.0; SPSS, Chicago, IL). Quantitative values are expressed as means±standard deviation. All statistical tests were performed two-sided and a p values less than 0.05 were considered statistically significant. T/Mmean ratios To present the results in a descriptive way, T/Mmean ratios at baseline and for each week in the both groups were calculated. The T/Mmean ratios were calculated based on the area under the curve instead of using the flawed simple sample mean, because the scan lengths of the PET frames were not equal. For the tumour size the mean tumour diameters in both groups (±standard deviation) were calculated. Analysis of dynamic data A generalized estimation equation (GEE) model approach was used to evaluate the measured T/Mdyn ratios and tumour sizes. The GEE approach takes into account the fact that multiple measurements in the same animal are used for statistical analysis [20]. Regression coefficients (±standard errors) from the GEE models are reported to show mean group differences and time effects. The changes in the T/Mdyn ratios were assessed in a linear regression framework by the GEE and the estimated mean changes in T/M ratio with time are presented for 10-min increments. Two interaction analyses were also carried out: 1. Week × therapy: This analysis was carried out to test if there was a statistically significant change in the effect of therapy with respect to the T/Mdyn ratio over 3 weeks. 2. Week × dynamics: This analysis was carried out for each treatment group to test if there was a statistically significant change in the radiotracer clearance over 3 weeks (different slopes of T/Mdyn ratio over the course of 3 weeks). Tumour growth was compared between groups by evaluating the mean change in tumour diameter per time unit. For this purpose the slope of the regression line of the mean tumour diameter in the treated group was compared to the slope of the regression line n the control group.

Results [11C]Choline small-animal PET visualized PC-3 tumours implanted in the left flank of athymic nu/nu mice (Fig. 1).

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The PET images also showed a high [11C]choline accumulation in the liver and the kidneys. There was a variable amount of necrosis in all tumours (personal communication, data not shown; tumour necrosis assessed by G. Weirich); however, this did not affect tumour delineation or ROI placement (Fig. 1). T/Mmean ratios The T/Mmean ratios in relation to week and therapy group are presented in Table 1. Before treatment the mean T/Mmean ratio was 1.6±0.5 in the control group and 1.8±0.4 in the treatment group (not significantly different, p=0.65). As early as 1 week after docetaxel treatment initiation there was a decrease in [11C]choline uptake in the tumours and the level remained low during the study period (T/Mmean ratio 1.8±0.4 before treatment, 0.9±0.3 after 1 week, 1.1±0.3 after 2 weeks and 0.8±0.2 after 3 weeks). There were no differences in [11C]choline uptake in the control group during the study period (T/Mmean ratio 1.6±0.5 before treatment, 1.7±0.4 after 1 week, 1.8±0.7 after 2 weeks and 1.7±0.4 after 3 weeks).

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Analysis of time dependency of the effect of treatment (week × therapy) revealed a significant increase in the difference between groups in T/Mdyn ratios after 1, 2 and 3 weeks compared to baseline (−1.04±0.46, p=0.025, after 1 week 1; −1.10±0.53, p=0.037, after 2 weeks; −1.22±0.41, p=0.003, after 3 weeks), while there were no significant differences in the T/Mdyn ratios in control and treated animals for the baseline PET scan (0.14±0.30, p=0.65, at baseline). The difference between groups increased over the weeks as a result of a substantial decrease in the T/Mdyn ratios in treated animals, whereas no significant changes were seen the control animals. Analysis of changes in dynamics over the course of 3 weeks (week × dynamics) in the groups revealed a significant reduction in the slope of the T/Mdyn ratios 1, 2 and 3 weeks after docetaxel treatment (mean increment of descent per minute compared to baseline: 0.008±0.004, p=0.032, after 1 week; 0.007±0.003, p=0.003, after 2 weeks; 0.012±0.004, p=0.002, after 3 weeks). There was no significant change in the slope of the T/Mdyn ratios at baseline and after 1, 2 and 3 weeks in the control animals (−0.002±0.008, p=0.816, after 1 week; 0.005±0.007, p=0.496, after 2 weeks; 0±0.005, p=0.963, after 3 weeks).

Analysis of dynamic data Tumour volume The radiotracer clearance (T/Mdyn ratio) is shown for control animals in Fig. 2 and for docetaxel-treated animals in Fig. 3. After initiation of treatment, the T/Mdyn ratios showed significantly decreased values. The decrease in the T/Mdyn ratios was evident as early as 1 week after treatment initiation and remained at the decreased level throughout the observation period of 3 weeks (Fig. 3). In the control animals there was almost no change in the T/Mdyn ratios (Fig. 2). Radiotracer clearance showed a slight decrease (negative slope) over time in control animals and in the treated animals before treatment. After treatment the T/Mdyn ratios were more or less constant at a significantly lower level over the whole time period. The GEE model revealed a significant decrease in the T/Mdyn ratios as early as 1 week after docetaxel treatment, and the ratios remained at that level through week 3 (mean change −0.93±0.24, p<0.001, after 1 week; −0.78±0.21, p<0.001, after 2 weeks; −1.08±0.26, p<0.001, after 3 weeks). There were no statistically significant changes in the T/Mdyn ratios after 2 weeks and 3 weeks compared to 1 week. In the control animals there was no significant decrease in the T/Mdyn ratios (mean change 0.085±0.39, p=0.83, after 1 week; 0.31±0.48, p=0.52, after 2 weeks; 0.11±0.30, p=0.72, after 3 weeks). In both groups there were slight but significant decreases in the T/Mdyn-ratios over the whole time period of 60 min at baseline with a negative slope (mean change per minute: −0.007±0.0012, p<0.001, and −0.016±0.0017, p<0.001).

Figure 4 shows the differential tumour growth rates between the treatment and the control group. The maximum tumour diameter increased in the control animals after the baseline measurement, whereas the growth of tumours in the treated animals was inhibited. Comparing tumour growth between the groups over the whole period of 3 weeks, revealed a significant difference in differential tumour growth rates between the treatment and the control group (p=0.025).

Discussion In this study small-animal [11C]choline PET was able to visualize all PC-3 tumours in athymic mice. This is partly in contrast to the findings of Zheng et al. [21] who evaluated [11C]choline as PET biomarker in different prostate cancer tumour animal models. In that study tracer retention in some PC-3 tumours was unclear in micro-PET images. In contrast to the study by Zheng et al. [21], we additionally used CT that helped to delineate the tumours. The [11C]choline T/Mmean ratio in our study was 1.6 in the control group and 1.8 in the treatment group before docetaxel treatment. which is close to the T/M-ratio of 2.1 reported by Zheng et al. [21]. The analysis of the dynamic data in our study revealed a slight but significant decrease in the T/Mdyn ratio over the

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A Control 100%

Before

1 wk

2 wk

3 wk

B Docetaxel therapy

0% Before

1 wk

2 wk

3 wk

Fig. 1 Transverse small-animal [11C]choline PET images in example animals from each group (a control group, b docetaxel-treated group; arrows tumour). The [11C]choline uptake remains high in the control group animal with tumour growth during the 4-week period. The [11C]

choline uptake in the tumour of the treated animal has decreased after only 1 week and tumour growth remains inhibited during the entire observation period

whole imaging period in the control animals. In the study by Zheng et al. [21], the T/M ratios in the mouse bearing PC-3 tumour was more or less constant over the whole study period. This difference might be explained by the fact that, for the PC-3-tumour, Zheng et al. present [11C]choline small-animal PET imaging for only one mouse with three implanted tumours. The T/M ratios versus time were plotted for each of the tumours separately. There was considerable variability among the three time curves. The authors did not present mean values and did not carry out a statistical analysis. In our dynamic data analysis we included two groups of PC-3-tumour bearing mice (six in the treatment group, seven in the control group) for the baseline scan. For the baseline scan in both groups the

mean T/M(dyn) ratios decreased significantly during the experiment. Distinct differences were shown in T/M ratios over time between different tumour xenograft models (PC-3 vs. LNCaP and CWR22rv [21]). This might be indicative of differences in cell biology between the various cell lines. Lower [11C]choline uptake was also shown in androgenindependent and less differentiated PC-3 tumours than in androgen-dependent tumours (LNCaP, C4-2). This is complemented by in vitro and in vivo xenograft studies on prostate cancer that have shown that FDG uptake in prostate cancer also depends on the prostate tumour characteristics and the growing conditions [22, 23]. Price et al. [24] showed that FDG uptake is equal to or greater

Table 1 T/Mmean ratios by week and therapy group

Baseline Week 1 Week 2 Week 3 a

Initial T/M ratio (means±SD)a

Change of T/M-ratio per 10min intervals (means±SE)b

Area under the curve (means ±SD)c

Overall mean (means±SD)d

Control group

Treatment group

Control group

Treatment group

Control group

Treatment group

Control group

Treatment group

2.1±1.0 2.3±2.0 2.7±1.1 2.5±1.3

2.2±1.2 1.1±0.5 1.3±0.7 0.8±0.6

−0.03±0.04 −0.16±0.04 −0.18±0.04 −0.14±0.03

−0.14±0.04 −0.05±0.01 −0.06±0.01 −0.02±0.01

90±26 93±24 102±40 95±20

100±21 53±15 63±15 47±12

1.6±0.5 1.7±0.4 1.8±0.7 1.7±0.4

1.8±0.4 0.9±0.3 1.1±0.3 0.8±0.2

T/M ratio of the first PET frame.

b

Change over time assessed via the slope of the linear regression fit line; the slope represents the estimated mean change in the T/M ratio over a 10-min period. c

Area under the curve of the T/M ratio over time taking into account non-equidistance between the measurement time points.

d

Represents an approximation of the T/Mmean ratio over all time points.

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3.00

Baseline

2.50

d7 after PBS d21 after PBS

d14 after PBS

2.00 1.50 1.00 0.50 0.00 1

6

11

15

20

45

60

Time (min)

Fig. 2 Mean T/M ratio versus time in PC-3 tumours in control PBSuntreated animals. The data presented are the averages for all seven animals

than [18F]fluorocholine uptake in nude mice with PC-3 and LNCaP xenografts. In contrast to FDG, there is evidence that [11C]choline is taken up in differentiated as well as in undifferentiated prostate cancers, although the uptake is dependent on the differentiation of the tumour. Therefore [11C]choline could represent an imaging biomarker that might be more broadly used for therapy monitoring in prostate cancer. In prostate cancer, increased choline uptake is associated with an increased content of phosphorylcholine and an increased phosphatidylcholine turnover [25, 26]: Key enzymes of choline metabolism are upregulated [27, 28], and prostate cancer cells show an increased expression of choline transporters and an upregulated transport rate [29–31]. This was an initial study of the use of [11C]choline as pharmacodynamic marker for assessing response to therapy in a prostate cancer xenograft model using small-animal PET/CT. As an important result, docetaxel treatment caused a significant and early decrease in [11C]choline uptake in vivo in the PC-3 tumours as early as 1 week after initiation of treatment. The early changes in [11C]choline uptake during the course of treatment preceded morphological changes.

T/M ratio

3.50 3.00

Baseline

d14 after DTx

2.50

d7 after DTx

d21 after DTx

The question arises as to why there was a decrease in choline-uptake as shown by [11C]choline PET imaging in our study following docetaxel treatment. Docetaxel exerts multiple mechanisms of antineoplastic activity: First, docetaxel inhibits microtubular depolymerization; Second, docetaxel is able to induce apoptosis. Third, docetaxel also causes alterations in a large number of genes, many of which contribute to the molecular mechanism by which it affects prostate cancer cells [32–35]. Tumour growth inhibition in the PC-3 mouse xenograft model is most likely related to different therapy-induced processes [32, 36–38]. Davoodpour et al. [37] found a significant increase in apoptotic nuclei in PC-3 prostate cancer cell aggregates following therapy. In vitro docetaxel has been shown to induce cell lysis in PC-3 prostate cancer cells [36] and it resulted in approximately 50% cell kill in a PC-3 prostate cancer cell line [38]. Furthermore, docetaxel has been shown to result in a dose- and time-dependent inhibition of proliferation of PC-3 prostate cancer cells in vitro [32]. There are at least two possible explanations for the early decrease in [11C]choline uptake after docetaxel treatment in our study. First, docetaxel treatment induces apoptosis with subsequent cell death paralleled by a decrease in [11C] choline uptake. Second, docetaxel treatment could lead to a change in choline metabolism, for example through modulation of choline transport and/or choline kinase activity. The possibility of early treatment response assessment using [11C]choline represents a major advantage compared to simple size measurements, as morphological differences in tumour growth were most pronounced late in the course of therapy (i.e. weeks after therapy onset). Early assessment 16

Tumor diameter (mm)

T/M ratio

3.50

14 12 10 8

2.00

p = 0.025

1.50

6

1.00

Baseline

0.50 0.00 1

6

11

15

20

45

60

Time (min)

Fig. 3 Mean T/M ratio versus time in PC-3 tumours in docetaxeltreated animals. The data presented are the averages for all six animals. DTx docetaxel

Week 1

Week 2

Week 3

No therapy 7.8 ± 3.3

9.7 ± 2.9

12.6 ± 3.4

12.9 ± 2.5

7.8 ± 1.5

9.6 ± 2.8

10.7 ± 3.6

9.6 ± 1.6

Therapy

Fig. 4 Course of the mean tumour diameter (±SD) in the two groups. There was a significant difference in differential tumour growth rates between the treatment and the control group (p=0.025)

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of response to treatment could have clinical consequences for the management of prostate cancer patients. First, in patients not responding to treatment, therapy could be discontinued and therapy-associated side effects and the cost of an ineffective therapy could be avoided. Second, an alternative treatment regimen could be considered early in patients not responding to therapy. At present, in the event of failure of chemotherapy with docetaxel in CRPC (castration-resistant prostate cancer) there are no standard second-line therapy options available with proven survival benefit [2]. Various therapeutic approaches have been evaluated in clinical studies, including new antiandrogens, new chemotherapeutic agents, targeted drugs (tyrosine kinase inhibitors, monoclonal antibodies), gene therapies and oncolytic viruses [1, 39, 40]. Early assessment of response to docetaxel treatment was achieved in our study using T/Mmean and T/Mdyn ratios. Furthermore, analysis of the time-dependent changes in radiotracer clearance added important information. This is of interest first because there is evidence that timedependent changes in radiotracer clearance differ between different tumour xenograft models [21], and second the radiotracer clearance changed after treatment: after treatment there was a significant reduction in the slope compared to untreated tumours which could have been related to the effects of the docetaxel treatment. Limitations of the study Given the performance characteristics of the microPET FOCUS 120 scanner used in this study, partial volume effects might have influenced the ROI values from the small-animal [11C]choline PET images of the tumour xenografts for several reasons. First, the tumours were initially small and near the limit of resolution of this system. Second, there was a variable amount of necrosis in all tumours during the longitudinal imaging. Third, the tumour configuration might have changed after treatment. We tried to correct for this effect using the maximum count rates in ROIs for data analysis. This approach seems reasonable as for small lesions underestimation of mean count rates is a well-known phenomenon, and thus the maximum count rates in ROIs should be used. Nevertheless, a potential effect of the limited spatial resolution of the scanner cannot be excluded.

Conclusion [11C]Choline was shown to be a useful pharmacodynamic marker for early assessment of treatment response after docetaxel therapy in an animal xenograft prostate cancer model. The investigation of choline metabolism may prove

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useful in predicting response to therapy in advanced androgen-independent prostate cancer early in the course of treatment, providing important clinical information with respect to disease management. Further preclinical studies are necessary to further assess the suitability of radioactively labelled choline derivatives for early assessment of therapy response also including histopathological and immunohistochemical analyses. Acknowledgments The authors thank Sybille Reder, Elisabeth Aiwanger, Annette Frank and Birgit Pfost for their excellent and extensive technical support. Conflicts of interest None.

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