Purpose: This study sought to evaluate the reproducibility of [11C]choline-positron emission tomography and the effect of trastuzumab in breast cancer.

Experimental Design: Twenty-one patients with newly diagnosed and recurrent breast cancer stage II-IV had a baseline dynamic [11C]choline-PET scan, 10 patients had a second [11C]choline-PET scan to examine reproducibility, and 6 patients had a second scan within a month after trastuzumab. Analysis of [11C]choline uptake was measured as the semiquantitative standardized uptake value at 30 and 60 minutes (SUV30 and SUV60), and quantitatively as the net irreversible retention of the radiotracer at steady-state (Ki) and plasma to tissue exchange at 60 minutes (IRF60min).

Results: Breast tumor lesions in all patients were visualized by [11C]choline PET. The difference in tumor versus normal tissue uptake was significant for SUV30, SUV60, Ki, and IRF60 minutes (Wilcoxon P < 0.0001). At 60 minutes postinjection, 15.1 ± 2.16% of plasma radioactivity was due to unmetabolized [11C]choline radioactivity. [11C]Choline uptake was reproducible in breast tumor lesions (r2 = 0.9 for SUV, 0.9 for Ki, and 0.8 for IRF60). Early responses to trastuzumab measured by [11C]choline-PET were significant in three lesions occurring in two patients who responded clinically.

Conclusions: [11C]Choline-PET uptake variables can be reproducibly assessed. Initial studies show that trastuzumab decreases [11C]choline uptake. Clin Cancer Res; 16(16); 4236–45. ©2010 AACR.

Translational Relevance

[11C]Choline-positron emission tomography (PET) is a marker of choline kinase activity, which is upregulated in tumorigenesis, and as such is a promising imaging technique in cancer. In breast cancer, we have recently shown that tumor [11C]choline uptake by PET imaging correlates with grade in estrogen receptor (ER)–positive tumors. This current study evaluates repeatability of [11C]choline-PET in both ER-positive and ER-negative tumors; we also assessed the effect of trastuzumab on choline uptake. We show that [11C]choline tumor standardized uptake at 60 minutes is reproducible to within 9% (% coefficient of variation). We further provide initial evidence that [11C]choline tumor uptake decreases after treatment with trastuzumab in patients who subsequently had a clinical response, a finding that warrants further evaluation of [11C]choline as a response/resistance imaging biomarker.

Radiolabeled choline has been explored as a potential radiotracer for use in positron emission tomography (PET) oncology studies (14). Increased choline uptake reflects intracellular choline kinase and activity, which is higher in malignantly transformed cells than in normal human mammary epithelial cells (5, 6). Our group has recently explored the use of [11C]choline in estrogen receptor (ER)–positive breast cancer and found a good correlation with tumor grade, a measure of tumor aggressiveness (r = 0.7; ref. 7). This study aimed to explore the utility of [11C]choline in both ER-positive and ER-negative breast tumors, and to determine the test-retest variability of [11C]choline pharmacokinetics in the absence of treatment in the same population.

Choline is an essential component of cell membranes and is thus necessary for cell division; it acts as a source of lipid second messengers through the Kennedy pathway (8). Following transport into the cell, the initial step results in the formation of phosphocholine from the phosphorylation of choline by the enzyme choline kinase (CK; in a reaction dependent on ATP and Mg2+); phosphocholine is then effectively trapped intracellularly (9). Human mammary epithelial cells oncogenically transformed by Her2 (5), Ras (10), Src (11), and Mos (11) exhibit increased levels of phosphocholine compared with untransformed cells due to increased constitutive activity of CKα. Alterations in membrane choline phospholipid metabolism are evident in breast cancer tumorigenesis (5). In mitogenic signaling, CKα is thought to play a critical role in the regulation of cell cycle progression through G1→S transition and has been previously identified as a therapeutic target (1215).

[11C]Choline-PET has mainly been studied in prostate cancer diagnosis (2, 1618), and to a lesser extent in other tumors such as glioma (19), lung cancer (20), and hepatocellular carcinoma (21). When compared with [18F]fluorodeoxyglucose (FDG), [11C]choline shows higher sensitivity for the detection of lung tumors, prostate tumors, esophageal tumors in the mediastinum, and brain tumors measuring ≥5 mm (19). Other anatomic regions (liver, pancreas, small bowel) showed high physiologic uptake of choline, potentially limiting the role of [11C]choline-PET for tumor detection/monitoring in these sites (19). It is recognized that [11C]choline uptake is not absolutely specific to cancer; other processes such as inflammation or infection may lead to increased uptake, although previous studies have shown that [11C]choline is more sensitive in detecting lymph node metastases in the mediastinum from esophageal and lung tumors than FDG (1, 4, 22).

Before being routinely incorporated into clinical practice, a new radiotracer requires thorough validation to determine the difference between tumor and background uptake and the extent of the effect of metabolism on tumor uptake, and to quantify the intrinsic variability in the absence of treatment. The intrinsic variability of each pharmacokinetic parameter describing uptake and retention should be quantified to provide a robust objective criteria that can be used to determine responses similar to those for Response Evaluation Criteria in Solid Tumors and the more recent guidelines for FDG-PET (known as the Positron Emission Tomography Response Criteria in Solid Tumors; refs. 23, 24).

Choline has been proposed as a pathway marker of the extracellular receptor kinase/mitogen-activated protein kinase pathway (MAPK; ref. 10). Based on its regulation, we proposed that [11C]choline pharmacokinetics would be modified by an agent such as trastuzumab. Breast tumor–derived cells expressing Her2 have high levels of phosphocholine (5). The human epidermal growth factor receptor (Her2/neu) is overexpressed in 20% to 30% of invasive breast cancers. Her2 promotes tumor cell proliferation through the RAS/MAPK pathway and inhibits apoptosis through phosphatidylinositol 3′-kinase–AKT–mammalian target of rapamycin (25). Trastuzumab (Herceptin, Genentech) is a humanized monoclonal antibody that targets the extracellular domain of Her2, leading to reduced proliferation and survival of Her2 dependent tumors (26).

A reliable biomarker that can be used in vivo such as the one proposed here ([11C]choline-PET) would have an obvious advantage in being able to detect de novo resistance in vivo to agents such as trastuzumab so that alternative, more effective treatments can be offered to patients. Given that Her2 and other effectors of the Ras-MAPK pathway increase choline phosphorylation (5, 10, 27), we hypothesized that the retention of [11C]choline will be attenuated through inhibition of Her2 and/or the MAPK pathway.

Patients

Our previous study assessed choline uptake only in ER-positive tumors (7). We included ER-negative tumors in this study to extend our knowledge of choline metabolism in breast cancer. Ethical approval for the study was granted by the Hammersmith Hospitals NHS Trust Research Ethics Committee. All patients gave fully informed consent to participate in the study, which was done under the Declaration of Helsinki guidelines. The administration of radioactivity for the PET scans was approved by the Administration of Radioactive Substances Advisory Committee, United Kingdom. Patient recruitment for the study was divided into two arms: (A) To determine the reproducibility of [11C]choline-PET, patients had a baseline scan followed by a second scan within 1 week in the absence of treatment. (B) To assess response to trastuzumab, patients who were eligible for single-agent trastuzumab therapy had a baseline scan followed by a posttreatment scan within 1 month of initiating treatment (mean 9 days, range 2–28 days). Trastuzumab was given intravenously as a loading dose of 8 mg/kg followed by 6 mg/kg every 3 weeks (see Table 3). Patients in group B had a clinical response assessment scheduled for three or more cycles of trastuzumab.

Patients were recruited from the medical oncology clinics at Charing Cross Hospital, London. Inclusion criteria are as follows: ages ≥18 and ≤80 years, with histologic or cytologic evidence of breast cancer (American Joint Committee on Cancer stage II–IV) including at least one site of measurable disease ≥2 cm, a treatment-free interval of ≥3 weeks (for cytotoxic chemotherapy, hormonal therapy, or cytostatic agents), 4 weeks applied for radiotherapy to the imaging site, a life expectancy of ≥3 months, hemoglobin of ≥10 g/dL, granulocyte count of ≥1.5 × 109/L, platelet count of ≥100 × 109/L, adequate creatinine clearance (calculated ≥ 50 mL/min), adequate hepatic function (bilirubin ≤1.5 upper limit of normal, serum transaminases ≤2.5 upper limit of normal), Eastern Cooperative Oncology Group performance status 0-2 inclusive, and able to give fully informed consent and comply with the protocol. In addition, those patients in the drug treatment arm had to have breast tumors with Her2 receptor positivity of grade 3+ by immunohistochemistry, or positive by a fluorescent in situ hybidization test.

Radiotracer synthesis and scanning

[11C]Choline was synthesized through the method previously described by Pascali et al. (28). The target dose for radioactivity was 370 MBq for each scan (equivalent to 1.036 mSv). A short transmission scan lasting 2 minutes for subject positioning, and a second longer transmission scan for attenuation correction were done before radiotracer injection. All patients were scanned on a CTI/Siemens HR+ ECAT962 tomograph (Siemens). The field of view was centered on the target tumor lesion(s) that had been identified previously by clinical/radiological examination.

[11C]Choline was administered through bolus intravenous injection over 10 to 30 seconds followed by a 6 mL normal saline flush. Patients were scanned for 65 minutes in two-dimensional mode. Data were reconstructed by filtered back projection and binned into time frames as follows: 1 × 30 (background), 5 × 10, 2 × 20, 2 × 30, 5 × 60, 3 × 120, 2 × 180, 3 × 300, and 3 × 600 seconds.

Blood sampling and [11C]choline metabolite analysis

The concentration of [11C]choline in arterial plasma was measured using continuous arterial blood sampling over time during the scan to quantify the arterial plasma input function. Blood was sampled through a 22G cannula inserted in the radial artery at a rate of 300 mL/h for the first 10 minutes after radiotracer injection. Total blood radioactivity was measured using an online radioactivity counter. This was compared with that in discrete samples taken at baseline and at 2.5, 3.5, 5, 7.5, 10, 15, 20, 30, 45, 50, and 60 minutes. The relative contributions of the [11C]choline parent fraction (and the metabolite, [11C]betaine) were determined using reverse-phase high-performance liquid chromatography as previously described (7).

Image and data analysis

Image analysis of tumor and normal tissues was done using Analyze software. The perimeter of each suitable lesion was manually defined by visual inspection of 20 to 60 minutes summed image derived from the dynamic scan. The time course of radioactivity from each region of interest was extracted and displayed as time versus radioactivity curves, decay corrected and normalized to body surface area.

The standardized uptake value at 30 and 60 minutes (SUV30 and SUV60) was calculated by normalizing tumor [11C]choline activity data at 30 and 60 minutes postinjection time points to body surface area and injected dose. SUV = measured tissue activity/([injected activity/body surface area]); AUC was the integral of the SUV over the imaging period. Modified Patlak analysis, which takes account of tissue and plasma metabolites of [11C]choline, was used to derive Ki, a measure of irreversible retention within a region of interest. For flux constant measurements, the presumption of the graphical method used is that no metabolite is retained in the tumor (7, 2932). Spectral analysis was used to calculate the tumor impulse response function (plasma to tissue exchange) at 60 minutes (IRF60) using the metabolite-corrected plasma input function and the tumor time-activity curve (33). K1 was calculated using a two-tissue compartmental model as previously described (29).

Statistical analysis

The difference between tumor and normal tissue uptake was evaluated on a lesion-by-lesion basis using the Wilcoxon matched pairs test. Analysis of reproducibility of PET pharmacokinetic parameters was done on a lesion-by-lesion basis. For reproducibility measurements, individual parameter values for the two pretreatment scans were plotted against each other; high reproducibility was represented by all data distributed along a line of unity. Pearson's correlation coefficient and Wilcoxon signed rank test were used to assess reproducibility.

We also assessed the normality of the data as a probability plot of the ordered relative differences versus the normal probability (cumulative frequency of ranks) for each lesion. Relative differences in parameter estimates were defined as follows: scan A − scan B)/(mean value for scans A and B). Intraclass correlation coefficient (ICC), a measure of reliability of the two measurements, was assessed; if the measurements were identical, the ICC will be 1. A two-way random mixed model with absolute agreement was used. The variance of the relative difference for the different parameters was tested using the F test, which compares the SDs of two normally distributed data sets.

The 95% reference ranges for spontaneous fluctuations in the parameters was calculated as 1.96 × SD of the relative difference in the two pretreatment scans. Data from reproducibility analyses were then used to determine if treatment with trastuzumab resulted in a significant difference in [11C]choline pharmacokinetics. [11C]Choline response to treatment was the relative difference between posttreatment and the pretreatment measurement; that is, (scanpre − scanpost)/(scanpre). Where two pretreatment scans were available, their average was used. The Mann-Whitney test was used to assess the significance of the change in [11C]choline uptake between (a) clinical responders (complete or partial response, stable disease) and (b) progressive disease. A P value of ≤0.05 was considered significant. Statistical analyses were done using SPSS for Windows version 12 (SPSS) and GraphPad Prism version 3.0 (Graph Pad Software).

Patient characteristics

In total, 21 patients between 39 and 71 years old entered the study, all of whom had a baseline [11C]choline scan; 10 patients had a second scan for repeatability analysis, and 6 patients had scans pre- and post-trastuzumab. The groups were well balanced with regard to stage (eight stage II, six stage III, and seven stage IV). Eleven of the 21 patients had tumor samples that were ER positive, whereas the remainder were ER negative. The scans were well tolerated. The mean radioactivity administered per scan was 347.43 ± 6.29 MBq (specific activity 361–1,316 MBq/μmol). The characteristics of the study patients are shown in Table 1.

Table 1.

Patient characteristics

PatientTumor typeAJCC clinical stage*Histologic gradeEstrogen receptor staining
Lobular II ER++ 
Inflammatory IV Negative 
Ductal II Negative 
Ductal II Negative 
Ductal IV ER+ 
Ductal IV ER++ 
Mucinous II ER+++ 
Ductal III ER+++ 
Ductal IV ER++ 
10 Ductal IV Negative 
11 Ductal III Negative 
12 Ductal II ER+++ 
13 Ductal II Negative 
14 Ductal II ER+ 
15 Ductal II Negative 
16 Ductal III Negative 
17 Ductal III Negative 
18 Ductal III Negative 
19 Ductal III ER+++ 
20 Ductal IV Negative 
21 Ductal IV ER++ 
PatientTumor typeAJCC clinical stage*Histologic gradeEstrogen receptor staining
Lobular II ER++ 
Inflammatory IV Negative 
Ductal II Negative 
Ductal II Negative 
Ductal IV ER+ 
Ductal IV ER++ 
Mucinous II ER+++ 
Ductal III ER+++ 
Ductal IV ER++ 
10 Ductal IV Negative 
11 Ductal III Negative 
12 Ductal II ER+++ 
13 Ductal II Negative 
14 Ductal II ER+ 
15 Ductal II Negative 
16 Ductal III Negative 
17 Ductal III Negative 
18 Ductal III Negative 
19 Ductal III ER+++ 
20 Ductal IV Negative 
21 Ductal IV ER++ 

Comparison of tumor and normal tissue uptake of [11C]choline

All 21 patients were included in this part of the analysis, which sought to evaluate the differences in pharmacokinetic parameters describing [11C]choline uptake and retention between breast tumors/metastases and normal tissues on a lesion-by-lesion basis (total number of tumor lesions = 25). Tracer uptake was visible in all tumor lesions; no new lesions were identified by [11C]choline-PET, which had not been previously identified by other clinical/radiological methods. Data for the various parameters in breast tumor/metastases and corresponding normal tissues are shown in Supplementary Table S1 and are depicted graphically in Supplementary Fig. S1A to D. Comparisons of SUV and Ki in ER-positive and ER-negative tumors are shown in Supplementary Fig. S1E and F. No arterial samples were available from patient 20; therefore, only semiquantitative analysis (SUV) was done in this particular patient.

Metabolism of [11C]choline

Arterial plasma samples were available for metabolite analysis in 20 patients and showed that the fraction of radioactivity in plasma due to [11C]choline decreased rapidly postinjection such that at 60 minutes, the mean (±SE) radioactivity fraction in arterial plasma due to [11C]choline was 15.15 ± 2.17% (range 7.98–41.99%; see Fig. 1A). The 11C-radioactivity profile in plasma showed [11C]choline and one other component more polar than [11C]choline, [11C]betaine; evidence that this is the main metabolite of choline in humans has been published elsewhere (32, 34). In five patients who had arterial sampling pre- and post-trastuzumab, there was no significant difference in metabolism of choline (Wilcoxon P = 0.44), indicating that the drug had not affected radiotracer metabolism.

Fig. 1.

A, summary of [11C]choline metabolism in plasma. The plot represents the proportion of unmetabolized [11C]choline in arterial plasma expressed as percentage of total plasma radioactivity measured in 20 patients. At 60 min, the mean activity was 15.2 ± 2.2%, with the remainder due to a single metabolite, [11C]betaine. B, time versus radioactivity curve for tumor and normal tissues in patient 4. C, comparison of a modified Patlak model in tumor and normal tissue. D, range of distinct kinetic components within the tumor lesion determined by spectral analysis—log β values to the right of the graph represent reversible components including blood volume (rightmost) and trapped components (leftmost).

Fig. 1.

A, summary of [11C]choline metabolism in plasma. The plot represents the proportion of unmetabolized [11C]choline in arterial plasma expressed as percentage of total plasma radioactivity measured in 20 patients. At 60 min, the mean activity was 15.2 ± 2.2%, with the remainder due to a single metabolite, [11C]betaine. B, time versus radioactivity curve for tumor and normal tissues in patient 4. C, comparison of a modified Patlak model in tumor and normal tissue. D, range of distinct kinetic components within the tumor lesion determined by spectral analysis—log β values to the right of the graph represent reversible components including blood volume (rightmost) and trapped components (leftmost).

Close modal

Tumor [11C]choline pharmacokinetics

The uptake of [11C]choline was significantly higher in the tumor compared with the corresponding normal tissue (i.e., normal breast tissue or lung tissue) for SUV30, SUV60, IRF60, and Ki (Wilcoxon P < 0.0001). Tumor uptake was lowest in a skin metastasis from a ductal tumor (Ki = 4.08 × 10−4 mL plasma/s/mL tissue, SUV60 2.86 × 10−5 m2/mL) and highest in a lung metastasis (Ki = 18.21 × 10−4 mL plasma/s/mL tissue, SUV60 × 10−5 12.89 m2/mL). Examples of typical time versus radioactivity curves of [11C]choline uptake in tumor and corresponding normal tissues are shown in Fig. 1B and Supplementary Fig. S2. There was high uptake in the liver and spleen thought to be due to choline metabolism and blood pooling, respectively.

There was a good fit of the Patlak and spectral models to the raw tumor data (see Fig. 1C). Tumor Ki calculated using plasma corrected for choline metabolism but non–metabolite-corrected tissue data as an input function was 17.9% higher than Ki calculated using modified Patlak analysis; the linear part of the Patlak plots suggests that a net irreversible retention component of the tracer is present within our 60-minute data, which was also evident in the spectrum of kinetic components (Fig. 1D).

Reproducibility of [11C]choline PET

Ten patients were able to complete two [11C]choline scans to determine reproducibility of radiotracer uptake and retention. In total, 13 lesions were assessed on an individual basis. The mean number of days (±SD) between the first and second scans was 3.75 ± 1.75 days, during which none of the patients received any treatment. Most (70%) of the patients had relapsed disease; the remainder were newly diagnosed. [11C]Choline uptake was found to be reproducible for all the parameters under investigation. The Pearson correlation coefficient (r) for Ki (modified Patlak) was 0.89, P < 0.0001, and for SUV was r = 0.945, P < 0.0001 (see Table 2). Results of the correlation analysis are also depicted graphically in Fig. 2A to D for the different imaging parameters. The pharmacokinetic parameters studied were all reproducible, with ICC values approaching 1 in nearly all cases. The semiquantitative parameters seemed only minimally (nonsignificant) superior compared with the modeled parameters Ki and IRF60.

Table 2.

Reproducibility of [11C]choline-PET in breast tumor lesions

COVPearson correlation coefficient rr2Wilcoxon PSD of mean relative difference (%)F testICC
SUV30 15.4 0.9 0.8 0.1 20.3 1.0 0.9 
SUV60 8.4 0.9 0.9 0.5 12.4 0.7 0.9 
SUVMAX60 16.9 0.8 0.7 0.9 24.8 0.8 0.8 
AUC 9.4 0.9 0.9 0.7 14.8 0.6 0.9 
Ki 14.7 0.9 0.8 0.8 21.2 0.8 0.9 
IRF60 13.4 0.9 0.8 0.5 19.5 1.0 0.9 
K1 15.3 0.8 0.6 1.0 22.4 0.5 0.8 
COVPearson correlation coefficient rr2Wilcoxon PSD of mean relative difference (%)F testICC
SUV30 15.4 0.9 0.8 0.1 20.3 1.0 0.9 
SUV60 8.4 0.9 0.9 0.5 12.4 0.7 0.9 
SUVMAX60 16.9 0.8 0.7 0.9 24.8 0.8 0.8 
AUC 9.4 0.9 0.9 0.7 14.8 0.6 0.9 
Ki 14.7 0.9 0.8 0.8 21.2 0.8 0.9 
IRF60 13.4 0.9 0.8 0.5 19.5 1.0 0.9 
K1 15.3 0.8 0.6 1.0 22.4 0.5 0.8 

Abbreviations: COV, coefficient of variation; SUV30 and SUV60, average SUV value at 30 and 60 minutes, respectively; SUVMAX60, maximum SUV value at 60 minutes; AUC, AUC at 60 minutes, Ki, Patlak Ki corrected for radioactive metabolites of [11C]choline present in plasma and tissue; IRF60, IRF value at 60 minutes; K1, delivery of [11C]choline determined by compartmental analysis.

Fig. 2.

Repeatability for the PET pharmacokinetic parameters: A, SUV30; B, SUV60; C, Ki; D, IRF60, with the corresponding Pearson correlation coefficient value (r).

Fig. 2.

Repeatability for the PET pharmacokinetic parameters: A, SUV30; B, SUV60; C, Ki; D, IRF60, with the corresponding Pearson correlation coefficient value (r).

Close modal

Relationship of [11C]choline-PET parameters with ER status

There was a significant difference in [11C]choline uptake measured by Ki between ER-positive and ER-negative tumors (Mann-Whitney = 0.032) with significant overlap. The difference was not significant for SUV60 or SUV30 (Mann-Whitney P = 0.11 and 0.14, respectively).

Measurement of response to trastuzumab with [11C]choline

Six patients with eight lesions were imaged before and after treatment with trastuzumab. Posttreatment scans were done a median of 3.5 days (range 2–28 days) after single-agent trastuzumab. Typical changes in [11C]choline-PET images in a patient with a partial clinical response (patient 17) are shown in Fig. 3. The percentage changes in parameter values from baseline and the corresponding clinical responses (complete response, partial response, stable disease, and progressive disease) are shown in Table 3. As we proposed to evaluate the most suitable pharmacokinetic parameter using dynamic imaging, the field of view was centered on one region of interest; whole body scanning was not done in this study. Hence, the utility of [11C]choline response measurements in metastases outside the field of view (e.g., brain lesions as in patient 20) was not evaluable, making comparison between PET response and overall clinical response difficult.

Fig. 3.

[11C]choline-PET images in a patient who responded to trastuzumab. The posttreatment scan was conducted 3 d after initiating single-agent trastuzumab (8 mg/kg).

Fig. 3.

[11C]choline-PET images in a patient who responded to trastuzumab. The posttreatment scan was conducted 3 d after initiating single-agent trastuzumab (8 mg/kg).

Close modal
Table 3.

Percentage change in [11C]choline parameters post-trastuzumab

graphic
 
graphic
 

Only one patient had a complete response clinically; this occurred in a palpable axillary node, which became impalpable. One other patient had a partial response in two lesions—all of these regions had significant decreases in [11C]choline uptake. One lesion with progressive disease (patient 21) showed an increase in [11C]choline uptake, which was more significant when measured using the modeled parameters Ki and IRF60 than the semiquantitative parameters SUV60 and AUC. The small number of lesions in this study prohibits any definitive statistical analysis to assess if the [11C]choline-PET parameter changes post-trastuzumab are different between responders and nonresponders.

The 95% confidence limits of the reproducibility data were used to determine if parameter changes for individual lesions post-trastuzumab were truly statistically significant when compared with baseline measurements. As seen in Table 3, there was no absolute agreement for the significant responses among pharmacokinetic parameters for each lesion. In summary, for SUV60, there were five lesions that showed a significant decrease; these corresponded with three clinical responses in two patients. Comparison with clinical response was not possible in the two other lesions due to progression outside the imaging field; the results for AUC were similar. Many of the changes in the kinetic parameters were nonsignificant post-trastuzumab. The modeled parameter Ki detected CR and PD, but not PR, whereas IRF60 only detected PR.

We have shown in this study that serial measurements of choline metabolism by [11C]choline-PET can be done reliably and used for monitoring response to drug therapy in patients with breast cancer. [11C]Choline-PET is gaining prominence as an alternative radiotracer to [18F]FDG-PET, particularly in prostate cancer staging and measurement of residual disease postsurgery (2, 18). We recently showed, for the first time, that [11C]choline-PET also has utility in ER-positive breast cancer. The present study extends that knowledge to ER-negative breast cancer with a more detailed analysis of radiotracer kinetics, and importantly verifies the reliability of performing serial [11C]choline-PET studies.

Comparison of tumor to normal tissue localization of [11C]choline-derived radioactivity by PET indicated high tumor to normal tissue contrast for both ER-positive and ER-negative breast cancer. In general, the technique shows promise for imaging supradiaphragmatic disease; the high physiologic uptake in the liver and spleen will present a challenge for imaging in those regions. To define an optimal time for assessment of breast tumors by [11C]choline-PET, we examined both metabolism of the radiotracer in arterial blood and the time course of tumor radiotracer uptake. In agreement with the pilot studies of Roivainen and coworkers, [11C]choline was metabolized such that only 15.1% of radioactivity was present at 60 minutes postinjection; the only radioactive metabolite observed was the oxidation product of choline oxidase, [11C]betaine. Objectively, these data would indicate that time points ≤30 minutes may be more appropriate to avoid significant contribution of [11C]betaine to the tumor signal as has been observed in animal tumor models (27). Notably, however, the ability of [11C]choline-PET to discriminate breast tumors from normal tissues was found to be similar whether data were assessed at 30 minutes (SUV30) or 60 minutes (SUV60). This latter observation is possibly due to very rapid and efficient phosphorylation and trapping of [11C]choline in tumors, supported by observation of high net irreversible uptake (Ki) and the presence of irreversible components in the spectrum of kinetic components (Fig. 1D). The time versus radioactivity curves remain at steady state after ∼10 minutes, making it unlikely that [11C]betaine contributes significantly to the intracellular tumor signal, for example, through donation of the radiolabeled methyl group to produce labeled methionine from homocysteine (35). Thus, although present at significant levels in blood, [11C]betaine does not seem to alter the outcome of the data acquisition at 30 or 60 minutes.

The retention of choline within tumor cells, in PET studies, is largely due to the activity of CK, which is overexpressed in breast cancer (9). Estrogen receptor status had no effect on [11C]choline tumor uptake (SUV60P = 0.11) but had an effect with respect to retention (Ki P = 0.03), in contrast to previous work by Ramirez de Molina et al. (9) where higher CKα expression was seen in ER-negative tumors. However, this occurred with a large overlap and would have to be confirmed in larger studies. A recent magnetic resonance spectroscopy study by Chen et al. (36) suggested that ER activity had negligible effect on membrane choline phospholipid metabolism. The mechanism for increased choline uptake and retention in ER-negative and ER-positive tumors in this study is thought to be due to elevated CK expression (5, 6), although upregulation of the choline transmembrane transporter may also be important at early time points (37). There was no significant difference between Her2-negative and Her2-positive patients with respect to [11C]choline uptake (data not shown).

For all imaging methods, knowledge of the precision of repeated quantitative end points is a prerequisite for serial measurements, and by extension, reliability for detecting response to drug therapy. In this first test-retest study of [11C]choline-PET in any cancer, we show that serial studies can be done with high reproducibility. ICC values for SUV30, SUV60, SUV60MAX, AUC, Ki, and IRF60 were ∼0.9 or greater (Table 2); as expected, macropharmacokinetic parameters (K1, K3) were more variable. We showed that [11C]choline SUV60 was reproducible to within 9% (% coefficient of variation). [11C]Choline-PET was less reproducible than 3′-deoxy-3′-[18F]Fluorothymidine (FLT) and FDG (38, 39). This could be attributed to reduced counts due to the use of 11C compared with 18F, or to more rapid metabolism of [11C]choline; unfortunately, there are limited data on the reproducibility of 11C-labeled PET radiotracers in human tumors for comparison. These studies again justify the use of SUV30 and SUV60 in the assessment of choline metabolism by PET. The [11C]choline-PET objective response was defined in this study as the reduction in imaging parameters below the 95% confidence limits in the test-retest study. Thus, with the current protocol (without correction for respiratory motion), a decrease of 39.8 for SUV30, 24.2% for SUV60, 41.6% for Ki, 59.3% for IRF60, and 27% for AUC will be classified statistically as response. As with other oncology radiotracers, the precision of the modeled pharmacokinetic parameters (Ki, IRF60) were not necessarily superior to semiquantitative parameters (SUV). Whether the accuracy of the two approaches differs in the assessment of drug therapy remains to be seen.

The final part of this study sought to determine whether [11C]choline could be used to measure response to treatment, in this case treatment of Her2-positive patients with trastuzumab. We selected this patient group because Her2 overexpression increases the levels of phosphocholine in immortalized mammary epithelial cells (5). Using the objective response criteria defined above, significant reductions in [11C]choline parameters were observed in some patients within 1 month of initiating trastuzumab therapy (Table 3). That response to trastuzumab was detectable; the technique allows detection of changes outside the 95% reference range of spontaneous fluctuations. The small number of patients included in this part of the study (partly attributed to the switch of clinical treatment protocols within the hospital from single-agent trastuzumab to combination regimen during the trial period) precludes definitive inferences to be made regarding the optimal parameter for assessing response, and whether both increases (seen for Ki in a patient with progressive disease) and decreases (seen in responding patients) are definitive for categorizing clinical response. To date, no previous PET studies using [11C]choline have evaluated tumor response to a therapeutic agent. Most studies have analyzed choline metabolism in tumors using magnetic resonance spectroscopy. Meisamy et al. (40) studied a group of breast cancer patients undergoing neoadjuvant treatment with doxorubicin-based chemotherapy with quantitative 1H-magnetic resonance spectroscopy. A positive correlation was found between percentage change in total choline (%Δtcho) and change in longest diameter. Interestingly, %Δtcho at 24 hours posttreatment was able to distinguish between responders and nonresponders. Another recent study by Baek et al. (41) evaluated the relationship between 1H-magnetic resonance spectroscopy measurements of total choline compounds (phosphocholine, glycerophosphocholine, and choline) and pathologic complete response in patients receiving neoadjuvant treatment for breast cancer, and found that, in responders, the change in choline metabolism was greater than change in tumor size. However, the reduction in total choline between responders and nonresponders only became significant at 69 days. Whether the PET technique allows earlier assessment of response will become apparent in future studies.

In conclusion, [11C]choline-PET can be used in serial measurements of breast tumors with high precision; there seems to be a decline in choline uptake in response to trastuzumab in some patients; and further study is warranted for the use of choline as a response marker.

No potential conflicts of interest were disclosed.

Grant Support: Medical Research Council UK grant U1200.02.005.00001.01, Clinician Scientist Grant, National Institute of Health Research (NIHR) CS009/009 (L.M. Kenny), Cancer Research UK grant C37/A5610, Experimental Cancer Medicine Centre grant C37/A7283, and Biomedical Research Centre grant.

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. Section 1734 solely to indicate this fact.

1
Hara
T
,
Inagaki
K
,
Kosaka
N
,
Morita
T
. 
Sensitive detection of mediastinal lymph node metastasis of lung cancer with 11C-choline PET
.
J Nucl Med
2000
;
41
:
1507
13
.
2
Hara
T
,
Kosaka
N
,
Kishi
H
. 
PET imaging of prostate cancer using carbon-11-choline
.
J Nucl Med
1998
;
39
:
990
5
.
3
Hara
T
,
Kosaka
N
,
Shinoura
N
,
Kondo
T
. 
PET imaging of brain tumor with [methyl-11C]choline
.
J Nucl Med
1997
;
38
:
842
7
.
4
Kobori
O
,
Kirihara
Y
,
Kosaka
N
,
Hara
T
. 
Positron emission tomography of esophageal carcinoma using (11)C-choline and (18)F-fluorodeoxyglucose: a novel method of preoperative lymph node staging
.
Cancer
1999
;
86
:
1638
48
.
5
Aboagye
EO
,
Bhujwalla
ZM
. 
Malignant transformation alters membrane choline phospholipid metabolism of human mammary epithelial cells
.
Cancer Res
1999
;
59
:
80
4
.
6
Glunde
K
,
Jie
C
,
Bhujwalla
ZM
. 
Molecular causes of the aberrant choline phospholipid metabolism in breast cancer
.
Cancer Res
2004
;
64
:
4270
6
.
7
Contractor
KB
,
Kenny
LM
,
Stebbing
J
, et al
. 
[11C]choline positron emission tomography in estrogen receptor-positive breast cancer
.
Clin Cancer Res
2009
;
15
:
5503
10
.
8
Marty
M
,
Cognetti
F
,
Maraninchi
D
, et al
. 
Randomized phase II trial of the efficacy and safety of trastuzumab combined with docetaxel in patients with human epidermal growth factor receptor 2-positive metastatic breast cancer administered as first-line treatment: the M77001 study group
.
J Clin Oncol
2005
;
23
:
4265
74
.
9
Ramirez de Molina
A
,
Gutierrez
R
,
Ramos
MA
, et al
. 
Increased choline kinase activity in human breast carcinomas: clinical evidence for a potential novel antitumor strategy
.
Oncogene
2002
;
21
:
4317
22
.
10
Liu
D
,
Hutchinson
OC
,
Osman
S
,
Price
P
,
Workman
P
,
Aboagye
EO
. 
Use of radiolabelled choline as a pharmacodynamic marker for the signal transduction inhibitor geldanamycin
.
Br J Cancer
2002
;
87
:
783
9
.
11
Hernandez-Alcoceba
R
,
Saniger
L
,
Campos
J
, et al
. 
Choline kinase inhibitors as a novel approach for antiproliferative drug design
.
Oncogene
1997
;
15
:
2289
301
.
12
Ramirez de Molina
A
,
Gallego-Ortega
D
,
Sarmentero-Estrada
J
, et al
. 
Choline kinase as a link connecting phospholipid metabolism and cell cycle regulation: implications in cancer therapy
.
Int J Biochem Cell Biol
2008
;
40
:
1753
63
.
13
Banez-Coronel
M
,
de Molina
AR
,
Rodriguez-Gonzalez
A
, et al
. 
Choline kinase α depletion selectively kills tumoral cells
.
Curr Cancer Drug Targets
2008
;
8
:
709
19
.
14
Gallego-Ortega
D
,
Ramirez De Molina
A
,
Gutierrez
R
, et al
. 
Generation and characterization of monoclonal antibodies against choline kinase α and their potential use as diagnostic tools in cancer
.
Int J Oncol
2006
;
29
:
335
40
.
15
Lacal
JC
. 
Choline kinase: a novel target for antitumor drugs
.
IDrugs
2001
;
4
:
419
26
.
16
Coleman
R
,
DeGrado
T
,
Wang
S
, et al
. 
9:30-9:45. Preliminary evaluation of F-18 fluorocholine (FCH) as a PET tumor imaging agent
.
Clin Positron Imaging
2000
;
3
:
147
.
17
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
.
18
Richter
JA
,
Rodriguez
M
,
Rioja
J
, et al
. 
Dual tracer (11)C-Choline and FDG-PET in the diagnosis of biochemical prostate cancer relapse after radical treatment
.
Mol Imaging Biol
2010
;
12
:
210
7
.
19
Hara
T
. 
11C-choline and 2-deoxy-2-[18F]fluoro-d-glucose in tumor imaging with positron emission tomography
.
Mol Imaging Biol
2002
;
4
:
267
73
.
20
Pieterman
RM
,
Que
TH
,
Elsinga
PH
, et al
. 
Comparison of (11)C-choline and (18)F-FDG PET in primary diagnosis and staging of patients with thoracic cancer
.
J Nucl Med
2002
;
43
:
167
72
.
21
Yamamoto
Y
,
Nishiyama
Y
,
Kameyama
R
, et al
. 
Detection of hepatocellular carcinoma using 11C-choline PET: comparison with 18F-FDG PET
.
J Nucl Med
2008
;
49
:
1245
8
.
22
Roivainen
A
,
Yli-Kerttula
T
. 
Whole-body distribution of (11)C-choline and uptake in knee synovitis
.
Eur J Nucl Med Mol Imaging
2006
;
33
:
1372
3
.
23
Eisenhauer
EA
,
Therasse
P
,
Bogaerts
J
, et al
. 
New response evaluation criteria in solid tumours: revised RECIST guideline (version 1.1)
.
Eur J Cancer
2009
;
45
:
228
47
.
24
Wahl
R
,
Jacene
H
,
Kasamon
Y
,
Lodge
MA
. 
From RECIST to PERCIST: evolving criteria for PET response criteria in solid tumours
.
J Nucl Med
2009
;
50
Suppl 1
:
122
50S
.
25
Pegram
M
,
Slamon
D
. 
Biological rationale for HER2/neu (c-erbB2) as a target for monoclonal antibody therapy
.
Semin Oncol
2000
;
27
:
13
9
.
26
Slamon
DJ
,
Leyland-Jones
B
,
Shak
S
, et al
. 
Use of chemotherapy plus a monoclonal antibody against HER2 for metastatic breast cancer that overexpresses HER2
.
N Engl J Med
2001
;
344
:
783
92
.
27
Leyton
J
,
Smith
G
,
Zhao
Y
, et al
. 
[18F]fluoromethyl-[1,2-2H4]-choline: a novel radiotracer for imaging choline metabolism in tumors by positron emission tomography
.
Cancer Res
2009
;
69
:
7721
8
.
28
Pascali
C
,
Bogni
A
,
Iwata
R
,
Cambiè
M
,
Bombardieri
E
. 
[11C]Methylation on a C18 Sep-Pak cartridge: a convenient way to produce [N-methyl-11C]choline
.
J Labelled Comp Radiopharm
2000
;
43
:
195
203
.
29
Kenny
LM
,
Vigushin
DM
,
Al-Nahhas
A
, et al
. 
Quantification of cellular proliferation in tumor and normal tissues of patients with breast cancer by [18F]fluorothymidine-positron emission tomography imaging: evaluation of analytical methods
.
Cancer Res
2005
;
65
:
10104
12
.
30
Mankoff
DA
,
Shields
AF
,
Graham
MM
,
Link
JM
,
Krohn
KA
. 
A graphical analysis method to estimate blood-to-tissue transfer constants for tracers with labeled metabolites
.
J Nucl Med
1996
;
37
:
2049
57
.
31
Patlak
CS
,
Blasberg
RG
. 
Graphical evaluation of blood-to-brain transfer constants from multiple-time uptake data. Generalizations
.
J Cereb Blood Flow Metab
1985
;
5
:
584
90
.
32
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
.
33
Cunningham
VJ
,
Jones
T
. 
Spectral analysis of dynamic PET studies
.
J Cereb Blood Flow Metab
1993
;
13
:
15
23
.
34
Barak
AJ
,
Tuma
DJ
. 
Betaine, metabolic by-product or vital methylating agent?
Life Sci
1983
;
32
:
771
4
.
35
Zeisel
SH
. 
Dietary choline: biochemistry, physiology, and pharmacology
.
Annu Rev Nutr
1981
;
1
:
95
121
.
36
Chen
JH
,
Baek
HM
,
Nalcioglu
O
,
Su
MY
. 
Estrogen receptor and breast MR imaging features: a correlation study
.
J Magn Reson Imaging
2008
;
27
:
825
3
.
37
Henriksen
G
,
Herz
M
,
Hauser
A
,
Schwaiger
M
,
Wester
HJ
. 
Synthesis and preclinical evaluation of the choline transport tracer deshydroxy-[18F]fluorocholine ([18F]dOC)
.
Nucl Med Biol
2004
;
31
:
851
8
.
38
Kenny
L
,
Coombes
RC
,
Vigushin
DM
,
Al-Nahhas
A
,
Shousha
S
,
Aboagye
EO
. 
Imaging early changes in proliferation at 1 week post chemotherapy: a pilot study in breast cancer patients with 3′-deoxy-3′-[(18)F]fluorothymidine positron emission tomography
.
Eur J Nucl Med Mol Imaging
2007
;
34
:
1339
47
.
39
Weber
WA
,
Ziegler
SI
,
Thodtmann
R
,
Hanauske
AR
,
Schwaiger
M
. 
Reproducibility of metabolic measurements in malignant tumors using FDG PET
.
J Nucl Med
1999
;
40
:
1771
7
.
40
Meisamy
S
,
Bolan
PJ
,
Baker
EH
, et al
. 
Neoadjuvant chemotherapy of locally advanced breast cancer: predicting response with in vivo (1)H MR spectroscopy—a pilot study at 4 T
.
Radiology
2004
;
233
:
424
31
.
41
Baek
HM
,
Chen
JH
,
Nie
K
, et al
. 
Predicting pathologic response to neoadjuvant chemotherapy in breast cancer by using MR imaging and quantitative 1H-MR spectroscopy
.
Radiology
2009
;
251
:
653
62
.