Exploratory Clinical Trial of (4S)-4-(3-[¹⁸F]fluoropropyl)-l-glutamate for Imaging x_C⁻ Transporter Using Positron Emission Tomography in Patients with Non–Small Cell Lung or Breast Cancer

This article is featured in Highlights of This Issue, p. 5155

One of the hallmarks of cancer is reprogramming energy metabolism (1). Cancer cells use both glucose and glutamine as a substrate to generate energy and to provide building blocks, such as amino acids, nucleosides, and fatty acids (2–4). Currently, increased glucose uptake and enhanced glycolytic metabolism of tumors is used for tumor positron emission tomography (PET) imaging with 2-[¹⁸F]fluoro-2-deoxyglucose ([¹⁸F]FDG). However, FDG has limitations in several tumor entities and settings. Therefore, imaging of other fundamental processes in tumors would be of great interest for early cancer detection, therapy monitoring, or prediction of resistance to chemotherapy (5).

The high rate of glucose and glutamine flux to increase cell mass results in an increase of oxidative intermediates, an altered redox potential, and excessive reactive oxygen species (3). Glutathione is the major thiol-containing endogenous antioxidant and serves as a redox buffer against various sources of oxidative stress (4, 6). The system x_C⁻ is a heterodimeric transporter, composed of a light chain, xCT (SLC7A11), and heavy chain, 4F2hc (SLC3A2), which mediates the sodium-independent cellular uptake of cystine in exchange for intracellular glutamate at the plasma membrane (7). Intracellularly, cystine can then be converted to 2 molecules of cysteine, which is used for glutathione synthesis. Another main function of system x_C⁻ is maintenance of a cysteine–cystine redox cycle in the extracellular compartments (8). In patients with cancer, it has become evident that the x_C⁻ transporter plays an important role in growth (9) and progression of cancer and glutathione-based drug resistance (10). CD44 is an adhesion molecule important for tumor cell invasion and metastasis and is a marker for cancer stem cells (11, 12). Quite recently, a CD44 splice variant has been reported to regulate redox status in cancer cells by stabilizing the xCT subunit of system x_C⁻ (13). (4S)-4-(3-[¹⁸F]fluoropropyl)-l-glutamate (BAY 94-9392, and herein referred to [¹⁸F]FSPG) is a novel ¹⁸F-labeled glutamate derivative for PET imaging. Specific transport of [¹⁸F]FSPG via the x_C⁻ transporter was shown in cell competition assays and xCT knockdown cells, and an excellent tumor visualization was achieved in animal tumor models (14). Tumor-specific adaptations of the intermediary metabolism and the function of system x_C⁻ and CD44 in glutathione biosynthesis and the cysteine–cystine redox cycle are schematically illustrated in Supplementary Fig. S1.

[¹⁸F]FSPG PET that examines x_C⁻ activity in vivo would provide information on oxidative stress of tumors and have a potential for the better understanding of tumor biology and chemoresistance mechanisms. The purpose of this study was to assess the tumor detection rate of [¹⁸F]FSPG PET in patients with lung or breast cancer relative to [¹⁸F]FDG. Furthermore, the dynamic biodistribution of [¹⁸F]FSPG in normal organs and tumors and the correlation of the uptake of [¹⁸F]FSPG with immunohistochemical staining intensity of xCT and CD44 were assessed.

This is an open-label, nonrandomized, single-dose explorative study conducted to evaluate the safety, tolerability, and diagnostic performance of [¹⁸F]FSPG positron emission tomography/computed tomography (PET/CT) in patients with non–small cell lung cancer (NSCLC) or breast cancer that showed lesions in previously conducted [¹⁸F]FDG PET/CT. The trial was registered at http://www.clinicaltrial.gov as NCT01103310. Primary outcome measure was visual assessment of the tumor detection rate with [¹⁸F]FSPG compared with that of [¹⁸F]FDG. The secondary outcome measures were quantitative analysis of [¹⁸F]FSPG uptake in tumors and assessment of safety variables including vital signs and laboratory findings. Effective radiation doses for [¹⁸F]FSPG as extrapolated from biodistribution studies in mice to humans were 5.1 mSv for a male and 6.5 mSv for a female subject (unpublished data). Our study protocol was approved by both the Institutional Review Board of the Asan Medical Center (University of Ulsan College of Medicine, Seoul, Republic of Korea) and the Korea Food and Drug Administration. This study was conducted in accordance with the Helsinki Declaration. All patients provided written informed consent before participation in the study.

Synthesis of the precursor and subsequent ¹⁸F labeling using ¹⁸F-fluoride were conducted as described recently (14). Each batch of [¹⁸F]FSPG that was produced met the criteria listed in the specification for appearance, identity, radiochemical and chemical purity, radioactivity concentration, specific activity, pH, bacterial endotoxin level, and sterility before being released. The final product was formulated as a sterile solution for intravenous injection. The amount of drug substance per injected unit was 300 ± 30 MBq and 100 μg or less mass dose. The specific activity was 18.2 GBq/μmol or more. The decay-corrected radiochemical yield was 29.1% ± 5.0% (range, 20.5%–40.5%), and the radiochemical purity was 90.8% ± 0.5% (range, 90.2%–91.6%).

The inclusion criteria consisted of patients who underwent [¹⁸F]FDG PET/CT and showed tumor mass with high certainty by visual analysis, histologically confirmed NSCLC or adenocarcinoma of the breast (female patients), age ≥35 and ≤75 years, an interval between [¹⁸F]FDG and [¹⁸F]FSPG PET/CT of 4 weeks or more, adequate recovery (excluding alopecia) from previous anticancer treatment, an Eastern Cooperative Oncology Group performance status of 0 to 2, and adequate function of major organs. No chemotherapy, radiotherapy or immune/biologic therapy, or biopsy were allowed between the [¹⁸F]FDG and the [¹⁸F]FSPG PET/CT. Patients were excluded from the study if any of the following applied to them: pregnancy, lactation, a concurrent, severe and/or uncontrolled, and/or unstable medical disease other than cancer, a lifetime history of alcohol or drug abuse, if they were a relative of any of the investigator, student of an investigator or a dependent, or if they were participating in or had participated in another clinical study involving the administration of an investigational drug during the preceding 4 weeks. Patients were recruited by referral from investigators.

[¹⁸F]FDG PET/CT imaging was conducted as described previously (15). [¹⁸F]FSPG PET/CT images were obtained using a PET/CT scanner (Biograph True Point 40 or Biograph 16; Siemens) on which [¹⁸F]FDG PET/CT scans were acquired for clinical reasons. Oral hydration with water was encouraged, and no food restriction was required before [¹⁸F]FSPG PET/CT studies. [¹⁸F]FSPG PET/CT acquisition was conducted during 3 time intervals. The first interval ranged from 0 (immediately after tracer injection) to 45 minutes, the second interval from 60 to 75 minutes, and the third interval from 105 to 120 minutes. For attenuation correction of the PET scan, a low-dose CT (80 kV CARE Dose 4D, 31 mAs) without contrast medium administration was acquired for each imaging window. The total radiation exposure from the 3 CT examination did not exceed 3 mSv. Five, consecutive skull base-to-mid thigh or whole-body tumor imaging (top of the skull to the mid-thigh or feet) were acquired for the first imaging interval of 0 to 45 minutes along with the injection of 300 ± 10 MBq of [¹⁸F]FSPG. Each PET scan was acquired for 0.5, 0.5, 1, 2, and 2 minutes per bed position, respectively. During the second and the third imaging intervals, 1 skull base-to-mid thigh or whole-body tumor image per each imaging window was acquired for 2 minutes per bed using the same acquisition parameter applied for the previous scan during the first imaging interval. Patients were asked to void their urinary bladder immediately after the first scan. Urinary bladder voiding was also encouraged before and after the second and third imaging session. Scans were corrected for random and scattered using the models implemented by the software supplied by the scanner manufacturer; they were also corrected for attenuation as estimated by the CT image. Data were reconstructed using the manufacturer-provided ordered-subset expectation maximization algorithm. No correction for partial volume effects was conducted.

The PET/CT studies were assessed visually and quantitatively by the consensus of 2 experienced nuclear medicine physicians who were informed of all available [¹⁸F]FDG PET imaging, clinical, and laboratory findings. The readers reviewed all images to determine whether the image quality was adequate for interpretation. The number, location, size, extent, and intensity of all abnormal uptakes in relation to the background uptake in normal comparable tissues were described. Intensity features were classified as major, minor accumulation, or absent. Lesions with minor or major accumulation were regarded as positive. For dynamic assessment of [¹⁸F]FSPG uptake in tumor and normal organs, spherical volumes of interest with a diameter of 1.5 or 1.2 cm were placed on normal organs and tumor, respectively. The selected tumor lesion was histologically proven to be cancer. A volume of interests on liver and descending thoracic aorta as the blood pool was drawn as previously recommended (16). For quantitative analysis, the volume of interest was drawn semiautomatically using the vendor's software (TrueD, Siemens). Mean standardized uptake values (SUV_mean) of the volume of interest were obtained on each time frame to generate a time activity profile of the [¹⁸F]FSPG uptake. All SUVs were normalized to the injected dose and the patients' body weight and were defined as follows:

The [¹⁸F]FSPG PET/CT image acquired 60 minutes after injection was used for visual and quantitative analysis. As many as 5 of the largest, malignant-looking lesions seen on [¹⁸F]FDG PET/CT were selected and defined as reference lesions for lesion-based analysis and positive percentage agreement with [¹⁸F]FSPG. Selected lesions were visually assessed with regard to their [¹⁸F]FSPG uptake, which was then compared with that of [¹⁸F]FDG. The maximal standardized uptake value (SUV_max) of each selected tumor lesion was measured using the single maximum pixel count within the lesion. The SUV ratio (SUVR) was obtained by calculating the ratio of the SUV_max of the tumor lesion and the SUV_mean of blood pool activity. Finally, the lesion detection rates of [¹⁸F]FDG and [¹⁸F]FSPG PET/CT were determined by measuring the total number of cancerous lesions using both [¹⁸F]FDG and [¹⁸F]FSPG PET/CT and based on subjective visual interpretation and available radiologic information.

The efficacy of [¹⁸F]FSPG PET/CT was assessed in terms of patient-based as well as lesion-based detection, and histology served as the gold standard for patient-based sensitivity analysis. The positive percentage of agreement was calculated using positive [¹⁸F]FDG uptake as a comparator. Additional lesion detection using [¹⁸F]FSPG PET/CT was also analyzed.

For subjects enrolled, the safety of [¹⁸F]FSPG was assessed on the basis of laboratory parameters (Supplementary Table S1), vital functions (blood pressure, heart beat, body temperature, etc.), electrocardiograms, and physical examinations at baseline, 2 hours after intravenous administration of [¹⁸F]FSPG, and again at about 24 hours. Adverse events were continuously recorded, beginning with patient enrollment until the last patient contact between 3 to 8 days after [¹⁸F]FSPG administration.

Tumor tissues from core-needle biopsies for routine diagnostic pathologic examinations were obtained before or after [¹⁸F]FSPG PET/CT and used for immunohistochemistry (IHC) studies of x_C⁻ transporter and CD44. If patients underwent surgery after [¹⁸F]FSPG PET/CT, this tumor specimen was used for further pathologic examination. A protocol for automatic immunohistochemical staining device (Benchmark XT, Ventana Medical Systems) using formalin fixed, paraffin-embedded tissue sections was used. Briefly, 4-μm thick whole tissue sections were transferred onto poly-l-lysine–coated adhesive slides and dried at 74°C for 30 minutes. After standard heat epitope retrieval for 1 hour in EDTA, pH 8.0, in the autostainer, the samples were incubated with antibodies to both of the x_C⁻ transporter (1:250 dilution; NB300-318, polyclonal anti-xCT antibody, Novus Biologicals; ref. 17), and CD44 (1:100 dilution; Clone DF 1485, DakoCytomation). The sections were subsequently incubated with ultraView Universal DAB kit (Ventana Medical Systems). Slides were counterstained with Harris hematoxylin. Pancreatic tissue and tissue microarrays of variable cancer tissues including breast and lung were used as positive controls for xCT and tonsil for positive CD44 controls. Incubation with the primary antibody was omitted in the negative controls. The level of expression of x_C⁻ transporter in both membrane and cytoplasm and of CD44 in the membrane of malignant tumor cells was examined by an experienced pathologist who was completely blinded to any patient and imaging information. The results of the immunoassay for x_C⁻ transporter and CD44 were semiqualitatively assessed using a scale of 0, 1+ (weak), 2+ (medium), and 3+ (strong) with a sample being reported as positive if greater than 10% of the cells in the sample were positively stained. The correlation between the intensity of immunohistochemical staining and SUV_max of the corresponding lesion on the PET/CT was then assessed.

Data were reported as mean ± SD unless otherwise specified. A P value of <0.05 was considered to be statistically significant. Comparison of quantitative parameters was conducted using the paired t test. The correlation of [¹⁸F]FSPG uptake (SUV_max) with the intensity of x_C⁻ transporter and CD44 IHC was assessed using Spearman rank correlation coefficients (ρ). The significance level was calculated assuming that t [t = rs□[(n − 2)/(1 − rs²)] in which rs is the sample Spearman rank correlation coefficient] is distributed approximately as Student t distribution with n − 2 degrees of freedom under the null hypothesis. All statistical tests were conducted using the IBM SPSS Statistics Version 19 for Windows (SPSS, Inc., IBM Company).

Among patients assessed for eligibility, 1 patient with NSCLC declined to participate before receiving [¹⁸F]FSPG injection and was replaced by an additional patient. Ten patients with NSCLC and 5 with breast cancer were included in this study and were examined at Asan Medical Center between April 2010 and December 2010 (8 men, 7 women; ages 35–70 years). All but 1 patient (patient 10 in Table 1) was examined on a Biograph True Point 40 scanner. [¹⁸F]FSPG PET/CT studies were completed without any scanner- or patient-related problems. The mean time interval between [¹⁸F]FDG and [¹⁸F]FSPG PET/CT was 2.7 ± 3.7 days (range, 1–12 days). The patient and lesion characteristics are listed in Table 1. All but 2 patients were newly diagnosed as NSCLC or breast cancer. Patient 14 had right modified radical mastectomy followed by adjuvant chemotherapy 3 years ago. Local recurrent lesions were treated with mass excision and radiotherapy 4 months before the enrollment. Patient 15 underwent skin-sparing mastectomy for primary breast cancer 2 years ago. Both patients were on hormonal treatment. All patients void their urinary bladder immediately after the first imaging session. All [¹⁸F]FSPG PET/CT procedures were conducted as planned.

Administration of [¹⁸F]FSPG and the PET/CT procedures were well tolerated by all 15 patients. No clinically relevant changes in safety parameters were observed. There were no study drug–related adverse events.

The overall image quality was adequate for diagnosis in all patients, and all patients showed initially high uptake in the kidneys and pancreas. The kidneys showed a rapid, intense uptake, which gradually decreased (11.1 ± 1.5 SUV_mean at 60 minutes postinjection), whereas the pancreas and scalp activity continuously increased, reaching a plateau at approximately 15 to 60 minutes postinjection (SUV_mean value determined at 60 minutes for the pancreas was 8.6 ± 4.5 and the scalp 2.0 ± 0.5, respectively). This uptake and excretion pattern resulted in prominent signals from the kidney, pancreas, and bladder, as seen on delayed images (Fig. 1). Liver, breast, and bone marrow showed prolonged uptake, which resulted in normal visualization on delayed images. [¹⁸F]FSPG cleared rapidly from the blood pool with 0.8 ± 0.2 SUV_mean of blood pool activity 60 minutes following injection. However, vascular activity was visible on the most of the PET acquisitions but was then barely distinguishable from the low-level background activity 105 minutes following injection (SUV_mean, 0.4 ± 0.1). No focal or elevated uptake was observed in the brain, muscle, small or large intestinal track, or on the cortical or trabecular bone surfaces. In some patients, delayed activity accumulation in the stomach was observed.

The size of histologically proven NSCLC or breast cancer for dynamic assessment of [¹⁸F]FSPG uptake was 3.9 ± 2.4 cm. Tumor activity continuously increased, reaching a plateau at about 60 minutes. There were large variations in the SUV_mean among individual patients and in different tumor types (NSCLC: 5.4 ± 4.8; breast: 1.8 ± 1.2 at 60 minutes postinjection). Because of the clearance of background activity over time, the tumor-to-background ratios of 1 representative lesion increased over time up to 105 minutes (tumor/blood ratio of SUV_mean: 5.7 ± 5.4 at 60 minutes postinjection and 9.2 ± 9.0 at 105 minutes postinjection). For more detailed information, see Supplementary Table S2 and Supplementary Fig. S2.

The results of the visual and quantitative analysis of [¹⁸F]FSPG PET are summarized in Table 2. All 10 NSCLC and 3 of the 5 breast cancer lesions that were confirmed by pathology examination could also be visually identified by [¹⁸F]FSPG PET. Two missed breast cancer lesions were sized more than 7 cm and were identified by clinical examination and conventional imaging studies. Thirty lesions with NSCLC and 19 with breast cancer were selected as reference. The size of these reference lesions of patients with NSCLC or breast cancer for lesion-based analysis was 2.6 ± 1.6 cm and 2.8 ± 2.2 cm, respectively. We included 1 brain lesion that was scanned 10 hours after FDG injection under the principle of intention-to-diagnose (patient 8). The visually assessed signal intensity activity of [¹⁸F]FSPG accumulation was the same as that of [¹⁸F]FDG in 70% (21/30) of the reference lesions in patients with NSCLC (Fig. 2 and Supplementary Movie S1). However, with breast cancer, only 11% (2/19) of the lesions showed the same uptake visually (Fig. 3), whereas 89% (17/19) of the lesions had lower [¹⁸F]FSPG accumulation (Table 1). In the NSCLC patient group, only 1 lesion was better visualized on [¹⁸F]FSPG than on [¹⁸F]FDG PET (brain metastasis). When the total number of cancerous lesions using both [¹⁸F]FDG and [¹⁸F]FSPG PET/CT were compared, [¹⁸F]FSPG detected 59 of 67 (88%) positive [¹⁸F]FDG lesions in NSCLC, and 30 of 73 (41%) in breast cancer. Seven lesions (brain 1, pleura 3, lung 2, and lymph node 1) were additionally detected only on [¹⁸F]FSPG in 2 patients with NSCLC (Fig. 2). Pathologic diagnosis of the additional lesions on [¹⁸F]FSPG PET was not made, but MRI and chest CT revealed 3 metastatic lesions in the brain, lung, and supraclavicular lymph node station.

Quantitative analysis of [¹⁸F]FSPG also showed similar findings. The SUV_max of tumor lesions was significantly higher for [¹⁸F]FDG PET than that for [¹⁸F]FSPG PET in NSCLC and breast cancer (P < 0.001, Table 2). There was a modest correlation between SUV_max of [¹⁸F]FDG and [¹⁸F]FSPG (NSCLC, ρ = 0.69, P < 0.001; breast cancer, ρ = 0.61, P < 0.005; Supplementary Fig. S3). The SUVR of NSCLC were comparable (P = 0.12), although in breast cancer, the SUVR of [¹⁸F]FDG differed significantly from that of [¹⁸F]FSPG (P < 0.05, Table 2). For more information on a patient with low [¹⁸F]FSPG uptake, see the Supplementary Fig. S4 and Supplementary Movie S2.

The mean time interval between [¹⁸F]FSPG PET/CT and surgery or biopsy for routine diagnostic pathologic study was 2.5 ± 11.0 days. Immunohistochemical staining of tumors revealed that 12 patients had x_C⁻ transporter-positive tumors (8 NSCLC and 4 breast cancer, 7 with 2+ immunostaining, and 5 with 3+ immunostaining). In 1 patient with breast cancer, no x_C⁻ transporter expression was identified (Table 1). [¹⁸F]FSPG SUV_max values at 60 minutes after injection showed high variability and overlapped between tumors with 2+ score (range, 1.3–11.4) and 3+ (range, 3.9–22.5, Fig. 4A). Samples from 4 patients with breast cancer showed lowest [¹⁸F]FSPG uptake (SUV_max < 5) despite a xCT 2+ immunostaining score. Among 12 patients, 8 had CD44-positive tumors (7 NSCLC and 1 breast cancer; 2 with 1+, 2 with 2+, and 4 with 3+ immunostaining) and 4 patients with breast cancer had CD44-negative tumors. Tumors with CD44 3+ immunostaining showed highest [¹⁸F]FSPG SUV_max values at 60 minutes, whereas CD44 0 to 2+ immunostaining resulted in lower SUV_max values (Fig. 4B). Statistical analyses revealed a significant correlation of [¹⁸F]FSPG SUV_max 60 minutes after injection with both x_C⁻ transporter (ρ = 0.68, P < 0.01) and CD44 expression (ρ = 0.77, P < 0.01, Fig. 4). SUVR also correlated with immunohistochemical staining results in the same manner (data not shown). A significant correlation was also observed between immunohistochemical staining of xCT and CD44 as shown in Supplementary Fig. S5 (ρ = 0.65, P < 0.05). Two patients with NSCLC and 1 patient with breast cancer (liver metastasis) with both strong positive xCT and CD44 expression showed a very high [¹⁸F]FSPG SUV_max, as seen in Figs. 2 and 3, whereas 1 with both negative immunohistochemical staining had a low SUV_max (patient 15). All patients with negative CD44 showed a low [¹⁸F]FSPG SUV_max of ≤ 4.0 (patient 11, 12, 13, and 15). No significant relationship was observed between [¹⁸F]FDG and immunohistochemical staining scores of xCT and CD44.

The data reported in this manuscript are the first human data on [¹⁸F]FSPG in patients with NSCLC or breast cancer. [¹⁸F]FSPG showed a favorable biodistribution and clearance pattern allowing its use as a potential PET tracer in patients with cancer. [¹⁸F]FSPG showed a relatively high tumor detection rate and high tumor to background ratios that were comparable with that of [¹⁸F]FDG in NSCLC but not in breast cancer. Particularly, additional lesions not seen on [¹⁸F]FDG were detected by [¹⁸F]FSPG in patients with NSCLC. We found that [¹⁸F]FSPG was well tolerated, safe, and without adverse events in all 15 study patients. Correlation of [¹⁸F]FSPG uptake with the staining intensity of 2 immunohistochemical markers (xCT and CD44) suggests the potential ability of [¹⁸F]FSPG PET to assess the system x_C⁻ activity in patients with cancer.

The biodistribution data showed increasing uptake up to 60 minutes after injection in pancreas and scalp, whereas predominant renal excretion resulted in high initial uptake in the kidneys, which decreased slowly over time. In human studies of a wide variety of tissues and cells examined, x_C⁻ transporter is predominantly expressed in the brain, pancreas (18), stromal, and immune cells. In monkeys, xCT protein distribution was shown in the kidney and duodenum (19). A recent study suggests that the Slc7a11 gene is a major genetic regulator of pheomelanin in hair and melanocytes (20). The critical role of x_C⁻ transporter in the control of pigmentation may correspond with the scalp uptake of [¹⁸F]FSPG in this study. Our biodistribution data are therefore in accordance with previous studies with xCT and [¹⁸F]FSPG biodistribution in animals (14). No uptake in healthy brain tissue is likely due to inability of the tracer to cross the intact blood brain barrier. High normal uptake in the pancreas and kidneys and excretion in bladder likely precludes the use of [¹⁸F]FSPG in assessing tumors in these organs, as shown in Fig. 2. However, low background in other organs is very advantageous in detecting lesions, especially in the abdomen and brain in which [¹⁸F]FDG PET has limitations in differentiating tumor from normal uptake. In addition, low bone uptake of [¹⁸F]FSPG was observed, which might be useful for the assessment of metastatic bone lesions. An accompanying study to assess biodistribution, stability, and radiation dosimetry of [¹⁸F]FSPG in healthy volunteers showed that the critical organs for the radiation dose were the kidney and the urinary bladder wall followed by the pancreas (Smolarz et al., manuscript in preparation). The effective dose of [¹⁸F]FSPG was slightly lower than that of [¹⁸F]FDG. Only the parent compound was detected by analysis of blood samples from healthy volunteers for up to 4 hours postinjection indicating high stability of the compounds in humans (unpublished data; Smolarz et al., manuscript in preparation).

Although, time–activity curve of tumor SUV showed variable patterns, the average tumor uptake of [¹⁸F]FSPG increased until 60 minutes and remained constant over the following study period. Tumor to background ratio increased over the time up to 105 minutes, as background SUV decreased more rapidly than that of tumors during the whole imaging period. As tumor detection rates of [¹⁸F]FSPG images obtained after 60 and 105 minutes were the same (data not shown), and [¹⁸F]FDG PET 60 minutes after injection was used as a comparator, we selected the 60-minute images of [¹⁸F]FSPG for further analysis. Our data showed that tumor uptake and tumor detection rate of [¹⁸F]FSPG was remarkably high in NSCLC but lower in breast cancer. A positive [¹⁸F]FDG PET scan was an inclusion criteria for patients in this study. Thus, no conclusions in terms of superiority/inferiority can be drawn from this study, and further studies with [¹⁸F]FSPG in an unselected patient cohort are needed. As [¹⁸F]FSPG targets a completely different metabolic pathway than glycolysis, tumors with a high rate of glycolysis do not have to be necessarily avid for [¹⁸F]FSPG in parallel. Nevertheless, in NSCLC, a high degree of overlap between tumors with a high glycolytic phenotype and [¹⁸F]FSPG-positive phenotype was observed, whereas [¹⁸F]FDG-positive tumors in breast cancer showed lower detection rate and accumulation of [¹⁸F]FSPG. More studies in NSCLC, breast cancer subtypes, and other cancer indications are needed to further elucidate and define the future role of [¹⁸F]FSPG.

Our results showed higher [¹⁸F]FSPG uptake in patients with NSCLC but substantially lower in patients with breast cancer. Cancer cells may possibly use alternative pathways to ensure cysteine availability in the absence of x_C⁻ transporter. For example, cysteine can be synthesized from l-methionine via the transsulfuration pathway as shown in C6 glioma cells (21). NCI60 panel studies revealed that the expression level of cystathionine β-synthase, the key enzyme of the transsulfuration pathway, is the highest in breast cancer cell lines but lower in lung cancer cell lines (22). Another pathway for cysteine availability is the γ-glutamyl cycle, which allows the efficient use of glutathione for cysteine storage (23). Altered expression of γ-glutamyl transpeptidase has been found in human tumors of the liver, lung, and breast (24, 25). Fibroblasts, activated macrophages, or dendritic cells may also supply cancer cells with cysteine as shown in lymphoid cells, which are unable to express the system x_C⁻ (26, 27). These cells may take up cystine from the extracellular space by using the system x_C⁻ transporter, convert cystine to cysteine intracellularly, and release cysteine into the extracellular space, where it is available to cancer cells via transporters, such as those from the alanine-serine-cysteine family. All these mechanisms may partially explain the observed differences in [¹⁸F]FSPG uptake between NSCLC and patients with breast cancer. The relative contribution of different mechanisms for cysteine availability needs to be further explored.

Our data showed that even a 2+ immunostaining score for xCT does not necessarily result in a high [¹⁸F]FSPG uptake. Meanwhile, all patients with negative xCT or CD44 had a low level of [¹⁸F]FSPG uptake. Interestingly, 3 patients with both strong positive xCT and CD44 expression showed a very high [¹⁸F]FSPG SUV_max. All these findings suggest a relationship of [¹⁸F]FSPG uptake with the staining intensity of IHC if both the xCT subunits of system x_C⁻ and CD44 are considered. A recent study reported a role of a CD44 splice variant in regulating the redox status in cancer cells by stabilizing the xCT subunit at the cell membrane (13). Although the CD44 antibody used in this study is not able to distinguish between normal and the CD44 splice variant, the CD44 splice variant can be assumed to be the dominant CD44 form in tumors (28). Our results may confirm an important role of CD44 for the proper functioning of the system x_C⁻. However, IHC data show only protein expression and does not provide any information on the functional activity, which is of great importance when studying transporter molecules. Staining of the xCT subunit was observed in the membrane and/or cytosol. No relationship was found between [¹⁸F]FSPG uptake and localization of xCT staining. In addition, we may not explain a low level of [¹⁸F]FSPG uptake even though with positive xCT 2+ and CD44 2+ immunostaining in patient 5. We need more studies to clarify these issues. IHC using a specific antibody to the CD44 splice variant may help to better understand this situation.

A small number of patients and the exploratory nature of this study limit its statistical power. Validation of [¹⁸F]FSPG in a different patient population with further refinements in quantitative PET measurement (29) and IHC (30) is therefore needed. In addition, patients were not fasted before the [¹⁸F]FSPG PET. In view of similar affinities of cystine and glutamate for x_C⁻ transporter, plasma cystine or glutamate may potentially inhibit [¹⁸F]FSPG uptake (14, 31). However, overall postprandial changes were reported to be small, and peak levels are expected to be less than 100 μmol/L (32–34). We believe that the nonfasting status did not affect the [¹⁸F]FSPG uptake significantly in most patients. Finally, tumors sometimes show high heterogeneity, and immunohistochemical staining of tumor biopsy samples will show different results compared with PET imaging in which the entire tumor lesion is measured. Further studies will further examine the heterogeneity of system x_C⁻ expression across the tumor bed and might better explain sometime discordant imaging results.

In conclusion, [¹⁸F]FSPG is safe and seems to be a promising novel tumor imaging agent with a favorable biodistribution and high cancer detection rate in patients with NSCLC. [¹⁸F]FSPG PET imaging may assess x_C⁻ transporter activity in tumor tissue noninvasively. A significant correlation between [¹⁸F]FSPG and xCT as well as CD44 using IHC suggests a role in the assessment of oxidative stress–induced signaling, which may lead to a better understanding of chemoresistance mechanisms. Given that CD44 has broad functions in cellular signaling cascades (12), additional roles of [¹⁸F]FSPG PET are expected. More studies are needed to elucidate a correlation with tumor progression, metastasis, and chemoresistance, which may provide more insights into potential clinical applications of this new tumor PET tracer.

S.J. Oh and D.H. Moon have a commercial research grant from Bayer. N. Koglin and C. Hultsch have ownership interest (including patents) in Patents covering BAY 94-9392. L.M. Dinkelborg is employed in Piramal Imaging GmbH as a managing director. L.M. Dinkelborg also has ownership interest (including patents) in Piramal Imaging GmbH. No potential conflicts of interest were disclosed by the other authors.

Conception and design: L. Fels, L.M. Dinkelborg, S.S. Gambhir, D.H. Moon

Development of methodology: S. Baek, S.J. Oh, L. Fels, N. Koglin, C. Hultsch, C.A. Schatz

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): S. Baek, G. Gong, J.-S. Ryu, L. Fels, L.M. Dinkelborg, D.H. Moon

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): S. Baek, C.-M. Choi, S.H. Ahn, J.W. Lee, L. Fels, N. Koglin, L.M. Dinkelborg, S.S. Gambhir, D.H. Moon

Writing, review, and/or revision of the manuscript: S. Baek, C.-M. Choi, J.-S. Ryu, L. Fels, N. Koglin, L.M. Dinkelborg, E.S. Mittra, S.S. Gambhir, D.H. Moon

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): L. Fels, D.H. Moon

Study supervision: L. Fels, D.H. Moon

Bayer sponsored clinical research trial: C. Bacher-Stier

The authors thank all of the investigators who participated in this trial in the Nuclear Medicine Department, especially Ji Young Kim, Seol Hoon Park, Kwang Ho Shin, Seon Hee Yoo, Sin Ae Kim, and Jung Eun Kim, for their support of the trial. The authors also thank Sabine Jabusch, Woo Young Chung, K.S. Kim, and Seung Yong Park for their excellent technical assistance; and the cyclotron team, particularly Sung Jae Lim, Woo Yeon Moon, Soo Jeong Lim, Dong Ryeol Lee, and Sang Ju Lee, for conducting radiotracer synthesis. The authors also thank all the patients who participated in this study.

The trial was sponsored and financially supported by Bayer HealthCare.

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.