Abstract
We explored system 
transporter activity and the detection of inflammatory or infectious lesions using (4S)-4-(3-18F-fluoropropyl)-l-glutamate (18F-FSPG) PET. Methods: In 10 patients with various inflammatory or infectious diseases, as many as 5 of the largest lesions were selected as reference lesions. 18F-FSPG images were assessed visually and quantitatively. Expression levels of xCT, CD44, and surface markers of inflammatory cells were evaluated by immunohistochemistry. Results: 18F-FSPG PET detected all reference lesions. 18F-FSPG uptake in sarcoidosis was significantly higher than that in nonsarcoidosis. The lesion–to–blood-pool SUV ratio for 18F-FSPG was comparable to that for 18F-FDG in sarcoidosis. In nonsarcoidosis, however, it was significantly lower. In 5 patients with available tissue samples, the SUVmax for 18F-FSPG and CD163 were negatively correlated (ρ = –0.872, P = 0.054). Conclusion: 18F-FSPG PET may detect inflammatory lesions when activated macrophages or monocytes are present, such as in sarcoidosis.
System 
is composed of xCT and 4F2hc, mediating cellular cystine uptake for glutathione synthesis to protect cells from oxidative stress (1). Several lines of evidence suggest that system 
may play a role in the innate and adaptive immune system. Upregulation of system 
expression was observed in activated macrophages and granulocytes (2,3). Induction of system 
may be an autoprotective mechanism from the high levels of released reactive oxygen species (4). Activation of T-lymphocytes has been reported to involve expression of system 
in antigen-presenting cells (5,6). Upregulation of xCT occurs during activation and differentiation of B lymphocyte (7). On the other hand, very low or no expression of xCT was detected in peripheral leukocytes, thymus, spleen, and lymph nodes in humans (8). These results suggest that system 
is a key player in the active phase of inflammation.
(4S)-4-(3-18F-fluoropropyl)-l-glutamate (18F-FSPG) is a new l-glutamate derivative that is specifically taken up by system 
as previously demonstrated in tumor models and cancer patients (9,10). Our primary objective was to explore 18F-FSPG PET detection of inflammatory lesions of infectious or noninfectious cause. Secondary objectives were to perform quantitative analyses of 18F-FSPG uptake and compare the results with those for 18F-FDG. In addition, we evaluated the expression of xCT, CD44, and surface markers of inflammatory cells by immunohistochemistry to explore their correlation with 18F-FSPG uptake.
MATERIALS AND METHODS
Study Design
The protocol of this open-label, nonrandomized, single-dose explorative study was approved by both the Institutional Review Board and the Korea Food and Drug Administration. All patients provided written informed consent before participating.
Radiopharmaceutical Preparation
18F-FSPG was radiolabeled as described previously (9). The specific activity of 18F-FSPG formulated for intravenous administration was 74.4 ± 59.6 GBq/μmol (range, 35.0–235.6 GBq/μmol), and the radiochemical purity was 91.6% ± 1.5% (range, 90.1%–94.4%).
Patients
Patients were enrolled if they had inflammatory or infectious diseases with strong clinical or laboratory evidence of an inflammatory or infectious focus or foci in defined anatomic regions preferably visible on 18F-FDG PET/CT scans. Full eligibility criteria are shown in Supplemental Appendix A (supplemental materials are available at http://jnm.snmjournals.org).
PET/CT Procedure
18F-FDG and 18F-FSPG PET/CT (10) was performed as previously described using the same PET/CT scanner (Biograph True Point 40; Siemens). 18F-FSPG PET/CT was conducted during 3 time windows (0–45, 60–75, and 105–120 min) after injection of 300 ± 10 MBq of 18F-FSPG.
Image Analysis
The PET/CT images were assessed visually and quantitatively by 2 board-certified nuclear medicine physicians as previously reported (10). SUVmean was obtained to generate a time–activity profile of 18F-FSPG uptake. The PET/CT images at 60 min after injection were used for visual and quantitative analysis. The largest inflammatory or infectious lesion was chosen as the representative lesion for patient-based analysis. As many as 5 of the largest lesions were selected as reference lesions. The SUV ratio was calculated by dividing the SUVmax of the reference lesion by the SUVmean of blood-pool activity.
Immunohistochemical Staining of xCT, CD44, and Surface Markers of Inflammatory Cells
The immunohistochemical staining methods are shown in Supplemental Appendix A.
Statistical Analysis
Comparison was conducted using the Mann–Whitney U test, Fisher exact test, and Spearman rank correlation coefficients (ρ) on SPSS Statistics, version 21 (IBM), for Windows (Microsoft).
RESULTS
Patients and18F-FSPG PET/CT Procedure
Five men and 5 women were enrolled (age range, 42–66 y). All but 2 patients underwent 18F-FDG PET/CT. The mean interval between 18F-FDG and 18F-FSPG PET/CT was 2.0 ± 1.8 d (range, 1–6 d). The baseline characteristics are summarized in Supplemental Table 1. Twenty-four reference lesions in 10 patients were selected (lung, 17; lymph node, 7).
Biodistribution of 18F-FSPG
SUVmean data for normal tissues and inflammatory or infectious lesions are summarized in Supplemental Figure 1. The kidney and pancreas showed high uptake, whereas the spleen exhibited low uptake. There was no or negligible uptake in the brain, myocardium, muscle, intestinal track, and bone.
18F-FSPG Uptake in Inflammatory or Infectious Lesions
The patient-based (10/10, 7 major and 3 minor accumulations) and lesion-based (24/24, 15 major and 9 minor accumulations) detection rates by visual assessment were 100%.
All sarcoid reference lesions (10/10) showed major uptake (Fig. 1), whereas only 36% (5/14) of the nonsarcoid lesions had the same uptake (Supplemental Fig. 2, P = 0.002). SUVmax was significantly higher in sarcoidosis than in nonsarcoidosis, as was SUV ratio (P < 0.001, Fig. 2). SUVmax and SUV ratio for 18F-FDG were also significantly higher in sarcoidosis than in nonsarcoidosis (P = 0.001 and 0.006, respectively, Fig. 2).
A 53-y-old man with sarcoidosis (patient 5). 18F-FDG (A) and 18F-FSPG (B) present similar major uptake involving pleura, supraclavicular lymph nodes, and thoracic lymph nodes. The only differences are normal physiologic uptake in brain, pancreas, and kidney. On immunohistochemistry evaluation (×400), proportion of inflammatory cells positive for xCT (C), CD44 (D), CD68 (E), and CD163 (F) was 80%, 80%, 80%, and 1%, respectively.
18F-FSPG and 18F-FDG uptake in reference lesions at 60 min after injection. (A) SUV ratio (SUVR) did not significantly differ between 18F-FSPG and 18F-FDG in sarcoidosis but was significantly lower in nonsarcoidosis. (B) SUVmax for 18F-FSPG was significantly lower than that for 18F-FDG in both sarcoidosis and nonsarcoidosis.
The visually assessed intensity of 18F-FSPG accumulation in reference lesions was the same as that of 18F-FDG (100%, 10/10) in sarcoidosis, whereas 92% (11/12) of the lesions in nonsarcoidosis had lower 18F-FSPG accumulation (Supplemental Fig. 3). Quantitative analysis showed that SUV ratio for 18F-FSPG in sarcoid lesions at 60 min after injection was comparable to that for 18F-FDG (P = 0.481, Fig. 2A) although SUVmax was significantly lower for 18F-FSPG than for 18F-FDG (P < 0.001, Fig. 2B). In nonsarcoidosis, SUVmax and SUV ratio were significantly lower for 18F-FSPG than for 18F-FDG (P < 0.05, Fig. 2).
18F-FSPG Uptake and Correlation with Immunohistochemical Staining
Eight patients underwent core-needle biopsy, but the tissue sample was sufficient from only 5 patients. The results of immunohistochemical analysis are shown in Supplemental Table 2. All 5 patients showed positive xCT expression in at least 50% of inflammatory cells. Histiocytes and plasma cells showed positive xCT staining (Fig. 1, Supplemental Figs. 2 and 4). In 2 of 3 patients with a major accumulation, the proportion of CD44 expression was at least 80% (Fig. 1 and Supplemental Fig. 4), whereas 2 patients with minor uptake showed no more than 30% positive cells (Supplemental Fig. 2). The proportion of immunohistochemical staining of xCT correlated significantly with that of CD68 (ρ = 0.9, P = 0.037). The SUVmax for 18F-FSPG correlated negatively with CD163-positive cells, with borderline significance (ρ = –0,872, P = 0.054). However, no correlation was found with other surface markers of inflammatory cells.
DISCUSSION
We found that 18F-FSPG uptake was significantly higher in patients with sarcoidosis than in patients with nonsarcoidosis. Inflammatory mechanisms of sarcoidosis include activation of macrophages and dendritic cells (11). Of note, 2 patients with sarcoidosis had elevated serum angiotensin-converting enzyme and systemic symptoms. On the other hand, patients with nonsarcoidosis presented with radiologic abnormalities without systemic symptoms. The inclusion of nonsarcoidosis patients was influenced by other diagnostic imaging studies that may have selected patients with a less active disease process and may have led to spectrum bias. This explanation is supported by higher 18F-FDG uptake in patients with sarcoidosis than in patients with nonsarcoidosis.
The proportion of xCT-positive cells correlated significantly with that of CD68-positive cells. However, SUVmax for 18F-FSPG did not correlate with xCT, CD44, or CD68 expression. The small number of patients and the limited amount of tissue available for immunohistochemical staining may have prevented us from obtaining a positive correlation. By contrast, previous examinations in tissue samples from tumor patients showed a correlation, suggesting that different or additional components contribute to uptake and retention in tumors and inflammatory tissue. The lack of correlation between 18F-FSPG and xCT or CD44 may also result from the possibility that xCT expression has an additional role to play in remitting the chronic inflammatory response (4). In a mouse model of chronic inflammation induced by 3-methylcholanthrene, xCT messenger RNA was significantly upregulated, whereas xCT expression contributed to the termination of inflammation (3,4). In lipopolysaccharide-induced inflammation, a high initial rate of cystine uptake in macrophages was followed by a decrease in uptake at a later phase of inflammation (2). Finally, the regulation of CD44 on system 
in inflammation may be different from that in cancer. There is a need for more studies on the regulation of system 
activity in inflammation.
The SUVmax for 18F-FSPG correlated negatively with CD163-positive staining, which is predominantly associated with M2 macrophages (12). Only 1% of cells showed positive CD163 staining in patient 5, who had sarcoidosis (Fig. 1), suggesting that most macrophages in this patient had the features of M1 macrophages. Our results may indicate that high 18F-FSPG uptake represents an active disease state that is detectable by measuring xCT transporter activity in activated M1 macrophages. M1 and M2 macrophages appear to be oppositive, such as proinflammatory versus antiinflammatory (13). Our results suggest a potential usefulness of 18F-FSPG PET in differentiating macrophage polarization. Our immunohistochemical results may also explain why 18F-FSPG uptake was negligible 3 d after turpentine oil injection into the calf muscle (9).
The biodistribution of 18F-FSPG here is consistent with previously reported data from cancer patients (10). The low background uptake would be especially advantageous in detecting inflammatory or infectious lesions in the brain, heart, and intestine, where the application of 18F-FDG is limited because of normal physiologic uptake.
This study had several limitations. The detection rate must be considered with caution because of the small number of patients. Furthermore, most patients had pulmonary diseases. Immunohistochemical analysis was limited by the small amount of tissue available. Therefore, the current data are too limited to draw any firm conclusions from the immunohistochemical results.
CONCLUSION
18F-FSPG PET may detect inflammatory lesions when activated macrophages or monocytes are present, such as in sarcoidosis. Further studies on a larger number of patients should be performed to validate whether 18F-FSPG PET can detect foci in a greater variety of inflammatory or infectious diseases and from different body locations.
DISCLOSURE
The costs of publication of this article were defrayed in part by the payment of page charges. Therefore, and solely to indicate this fact, this article is hereby marked “advertisement” in accordance with 18 USC section 1734. This study was sponsored and financially supported by Bayer Pharma AG/Piramal Imaging GmbH, Berlin (Germany), and a Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health and Welfare, Republic of Korea (HI06C0868). Drs. Oh and Moon received a research grant from Piramal Imaging GmbH. Drs. Koglin, Stephens, and Dinkelborg were employed by Piramal Imaging GmbH. Dr. Koglin has ownership interests in patents covering the applications of 18F-FSPG. No other potential conflict of interest relevant to this article was reported.
Footnotes
Published online Oct. 15, 2015.
- © 2016 by the Society of Nuclear Medicine and Molecular Imaging, Inc.
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- Received for publication August 5, 2015.
- Accepted for publication October 5, 2015.
