PET imaging is a promising tool for clinical management of cancer patients. It is useful for diagnosis of the primary lesions, status of lymph node and distant metastasis, and monitor of treatment effect.1,2 In 1924, Otto Warburg first described a high rate of glucose metabolism leading to production of lactic acid under aerobic condition in cancer cells.3,4 The “Warburg effect” is the biochemical basis for using 18F-FDG in conjunction with PET (now commonly refer to as 18F-FDG PET) for the detection and monitoring of malignant tumors in cancer patients.5,6 However, due to its high accumulation in the normal brain, 18F-FDG PET imaging displayed a poor sensitivity for detecting primary brain tumors.7,8 The 2 most essential nutrients for cancer cells are glucose and glutamine, and metabolism of these 2 nutrients (glycolysis and glutaminolysis) is vital for survival and maintaining growth of cancer cells.4,9 Noninvasive imaging of glutamine metabolism may represent a useful biomarker for cancer detection and evaluation of therapy response.10 Therefore, molecular imaging of cancer-associated markers is urgently needed to improve and enhance the diagnosis and to potentially improve the survival rate of brain metastasis patients. Additional tracers or probes for PET imaging that can measure tumor metabolism are worth exploring, especially because the metastatic brain lesion is a common prognostic marker for solid extracranial tumors.
Recently, 18F-(2S,4R)-4-fluoroglutamine (18F-FGln) has been developed and characterized as metabolic indicator for glutamine. It was demonstrated to have a relatively high tumor cell uptake and retention in animal models.11–13 Therefore, it might serve as a suitable probe for monitoring the glutamine addiction/metabolism in cancer cell. The first clinical trial of 18F-FGln PET imaging in gliomas has showed that it is capable of differentiating the clinically progressing disease from the stable gliomas.14 Further studies in gliomas and other 9 kinds of human cancers showed a high diagnostic rate (17/25), where avidity of 18F-FGln PET was significantly correlated with 18F-FDG PET in all tested patients.10 However, to our best knowledge, there is no systematic evaluation on 18F-FGln PET imaging for brain metastasis patients. The goal of the present study was to explore the characteristic of 18F-FGln PET for brain metastasis patients and to compare with that of 18F-FDG PET and/or contrast-enhanced MRI in the same patient lesion-by-lesion.
MATERIALS AND METHODS
Radiosynthesis and Quality Control of 18F-(2S,4R)-4-Fluoroglutamine
18F-(2S,4R)-4-Fluoroglutamine was synthesized using a 2-step chemical reaction as described before with minor modifications.13 The chemical structure (Supplemental Fig. S1, Supplemental Digital Content 1, http://links.lww.com/CNM/A130) and radio-HPLC analysis (Supplemental Fig. S2, Supplemental Digital Content 2, http://links.lww.com/CNM/A131) of 18F-FGln were provided in the supplemental information (Supplemental Digital Content 3, http://links.lww.com/CNM/A132).
Patients (7 men and 7 women; age range, 25–67 years) with specific primary malignant tumor and suspected cerebral metastasis were enrolled in this study. Local institutional review board approved this protocol (no. 2017KT38), and all patients signed a consent form before enrollment to the study. Pathologic analyses of the primary lesions in all patients were performed before this clinical trial. Tumor genomic profiling was obtained in 6 patients. Table 1 provides the details of patient information. One patient received brain radiotherapy, preceding to 18F-FGln PET and 18F-FDG PET scan (Table 1).
For all patients, no specific subject preparation was required on the day of 18F-FGln PET scanning. After an IV injection of 18F-FGln (3.7 MBq/kg), 3 patients underwent dynamic whole-body PET/CT scans by using a Biograph mCT Flow 64 scanner (Siemens, Erlangen, Germany) for 60 minutes. Another 11 patients underwent a static whole-body PET/CT scan at 30 ± 10 minutes after injection. All patients underwent a follow-up 18F-FDG PET scan on the same imaging system within a week after the 18F-FGln PET imaging. For 18F-FDG PET imaging, all patients were asked to fast at least 6 hours before dose administration; the glucose concentrations in the blood of all subjects were below 10 mmol/L. After injection of approximately 3.7 MBq of 18F-FDG per kilogram, the patients were asked to rest quietly in a waiting room for about 1 hour until scanning.
A Siemens workstation (MultiModality Workplace) was used for postprocessing. Two experienced nuclear medicine physicians blinded to the prior findings independently reviewed all studies. Any discordant results were resolved by consensus. Compared with the uptake in the adjacent normal tissue, the focal uptake of 18F-FGln PET in the tumor lesion was considered as positive. A single experienced radiologist blinded to the results of both PET/CT scans retrospectively reviewed all MRI studies. Because histologic proof was not feasible for all of the metastatic lesions, the composite of all performed anatomic and functional imaging studies (ie, PET imaging and/or CT, and/or MRI) was considered the imaging comparator. A “positive” finding on any of the imaging was counted as true-positive for the presence of the disease.15 The region of peritumoral edema was estimated on the base of the maximum distance from the tumor margin to the outer edge of edema and was graded as follows: minor (≤1 cm) and major (>1 cm).16
For the in vivo distribution of 18F-FGln PET in patients, 3-dimensional volumes of interest (VOIs) of major normal organs/tissue were drawn from the first set of the serial images, then the VOI was copied to other sets of images. The VOIs were constructed on the PET images and their locations were verified and confirmed using the corresponding CT images as described previously.17 For the uptakes in the tumor lesions, an ellipsoidal VOI was placed over each metabolically active lesion that was suspected for malignancy. The quantitative analyses of healthy organs/tissue and all cancer lesions were performed using the average/maximal standard uptake values (SUVmean/SUVmax). To compute tumor-to-normal brain ratios (SURs), SUV values of normal brain were obtained from disease free areas within the contralateral brain tissue. All SURs of the corresponding 18F-FDG PET and 18F-FGln PET scans were obtained from the same normal brain area on both types of scans.
All statistical tests were conducted using the IBM SPSS Statistics Version 19.0 for Windows (SPSS, Inc, IBM Company). Categorical variables are expressed as number (%) and quantitative variables as mean ± SD. The independent samples nonparametric test was used to compare the difference of 2 quantitative groups. Pearson product-moment correlation coefficient (r) was used for correlation analysis between continuous variables. A P value of less than 0.05 was considered statistically significant.
Biodistribution of 18F-FGln in Patients With Brain Metastasis
After administration of 18F-FGln (3.7 MBq/kg), there was no adverse effect being noticed or reported in all tested patients, which indicated that 18F-FGln was well tolerated in all patients. The 1-hour dynamic imaging in 3 brain metastasis patients (containing 6 brain metastatic lesions and 3 primary tumors) showed that 18F-FGln accumulated in the tumor rapidly, and all tumor lesions were visualized within the first 30 minutes. As to primary tumors, there was a rapid increase in the early phase, and a limited or slow decrease in the late phase. However, the radioactivity remained in metastatic brain lesions consistently throughout the 1-hour scanning period (Fig. 1). This is consistent with results from a previous study in glioma animal models.14 It was reasonable to selectively perform static imaging in all of the other 11 patients at a period of 30 ± 10 minutes after injection. The biodistribution of 18F-FGln in healthy organs was listed in Supplemental Figure S3 (Supplemental Digital Content 4, http://links.lww.com/CNM/A133), which was also obtained from dynamic PET/CT scans of the 3 patients. A predominant uptake and excretion of radioactivity was observed in kidneys (SUVmean ≥ 5; SUVmax ≥ 7), and moderate uptakes were also seen in pancreas, liver, heart, and spleen (3 ≤ SUVmean < 5; 4 ≤ SUVmax < 7). There was no significant accumulation in the normal brain, lung, and muscle (all SUVmean ≤ 1; all SUVmax ≤ 2).
Brain Metastasis Visual Assessment
This study evaluated the concordance between 18F-FDG PET/CT, 18F-FGln PET/CT, and contrast-enhanced MRI in image detection of presumed metastatic brain lesions. All patients were positive for brain metastasis on the imaging studies that demonstrated 38 lesions in 6 anatomic regions (frontal, parietal, temporal and occipital lobe, deep brain structures, and cerebellum) on the imaging comparator. Thirty-one lesions have been identified as positive in the 18F-FGln PET scan, and only 14 lesions in 18F-FDG PET were identified (Fig. 2). The per-lesion detection rates for 18F-FGln PET and 18F-FDG PET imaging were 81.6% and 36.8%, respectively (Supplemental Table S1, Supplemental Digital Content 5, http://links.lww.com/CNM/A134). Finally, the detection rates of per-segment (frontal, parietal, temporal and occipital lobe, deep brain structures, and cerebellum) was 75.0%, 75.0%, 100%, 100%, 100%, and 100% for 18F-FGln PET and 25.0%, 24.0%, 75.0%, 0%, 50%, and 25.0% for 18F-FDG PET imaging, respectively. The distribution of brain lesions in 6 anatomical areas by 18F-FGln, 18F-FDG, and contrast-enhanced MRI was shown in Supplemental Figure S4 (Supplemental Digital Content 6, http://links.lww.com/CNM/A135).
Furthermore, all brain metastasis detected by 18F-FGln PET, including small lesions (diameter ranging from 6–25 mm) could clearly be separated from the surrounding brain tissue. Boundaries delineated by 18F-FGln PET scan showed a good correspondence with the matched CT and MRI scans (Fig. 3; Supplemental Fig. S5, Supplemental Digital Content 7, http://links.lww.com/CNM/A136). As part of this study, we compared 18F-FGln uptake (SUVmax) in metastatic brain lesions with the characteristic of enhanced MRI. The avidity of 18F-FGln PET (SUVmax ≥ 3) was noted in 21 lesions with nodular enhancement in MRI. In contrast, tumors with rim enhancement (n = 10) showed a minimal 18F-FGln avidity (SUVmax < 3) on PET imaging (Fig. 3, Table 2). In addition, the SUVmax of 18F-FGln PET in certain smaller lesions (the maximum diameter ≤ 1) were higher than large lesions (the maximum diameter > 1), although this was not statistically significant. The activity of 18F-FGln uptake in the brain metastasis was also independent of the extent of peritumoral edema (Fig. 4, Table 3).
Quantitative Analysis of Brain Metastasis
As depicted in Figures 2, 3, and 4, 18F-FGln PET images for all of the 14 patients showed that there was a low-level uptake of 18F-FGln in normal brain tissues (white matter and cortical gray matter), showing an SUVmax of 0.48 ± 0.19 (ranging from 0.08 to 0.78). In contrast, high glucose consumption in normal brain tissues resulted in a high 18F-FDG accumulation, where the SUVmax of 18F-FDG PET reached 9.24 ± 2.78 (ranging from 4.1 to 13.56). In all brain metastasis lesions, an elevated accumulation of 18F-FGln was observed with the SUVmax of 3.46 ± 1.52 (ranging from 1.04 to 6.02) at 30 ± 10 minutes after injection. The SUR of 18F-FGln PET reached 4.97 ± 2.23 (ranging from 1.26 to 8.70) (Supplemental Table S1, Supplemental Digital Content 5, http://links.lww.com/CNM/A134). Compared with 18F-FGln PET imaging, 18F-FDG PET showed a significantly higher accumulation SUVmax of 11.85 ± 6.08 (ranging from 2.3 to 23.2) in the detected tumor lesions (P < 0.001), but the SUR was 1.22 ± 0.69 ranging from 0.56 to 3.12 (Fig. 5A). Furthermore, in 14 brain metastatic lesions visualized by both 18F-FDG PET and 18F-FGln PET imaging, a positive correlation of SUVmax was observed (r = 0.780, P < 0.01) (Fig. 5B).
Evaluation of Extracranial Lesions
In addition of the brain metastasis, multiple extracranial lesions existed in all 14 patients were also visualized by both whole-body 18F-FDG and 18F-FGln PET/CT. Although 18F-FGln did show a relatively low SUVmax in the primary lesions than that of 18F-FDG (the SUVmax were 4.47 ± 1.08 vs 12.53 ± 7.87), all primary tumor sites in 10 patients (no surgery resection before imaging) were clearly delineated by whole-body 18F-FGln or 18F-FDG PET/CT imaging, whereas 4 patients who received primary tumor resection surgery before did not show any radiotracer uptake. The representative images were included in Supplemental Figure S6 (Supplemental Digital Content 8, http://links.lww.com/CNM/A137). As to the extracranial metastasis, both tracers showed a concordant increased radioactive uptake except in liver and bone (Figs. 6, 7; Table 4). Further, the SUVmax of 2 tracer agent were similar in bone metastases (18F-FDG: SUVmax = 7.92 ± 3.56; 18F-FGln: SUVmax = 7.16 ± 2.85). However, the SUVmax of 18F-FDG in most other metastatic lesions were 3 to 4 times greater than that of 18F-FGln (Table 4).
Brain metastasis originated from primary tumor of peripheral organs or tissues, which is one of the most important prognostic indicators for staging and monitoring of cancer patients. Although contrast-enhanced MRI is excellent sensitivity in diagnosis of brain metastasis, this modality in measuring anatomical abnormality is insufficient for a specific evaluation of this type of neoplasm.18,19 Therefore, molecular imaging of cancer-associated markers is urgently needed to enhance the diagnosis rate and to improve the survival rate of brain metastasis patients.20 In this study, we do not propose that 18F-FGln is superior to 18F-FDG and many of other neuroimaging method, but that 18F-FGln may provide complementary metabolic information specifically about glutamine uptake relevant to brain metastasis pathology. Herein, we report on the characteristic and feasibility of in vivo tumor detection with noninvasive 18F-FGln PET for brain metastasis patients. Our results indeed suggest that 18F-FGln PET is a promising investigational radiologic probe for patients with brain metastasis.
We have directly compared the radioactivity uptake of 18F-FGln PET and 18F-FDG PET in brain metastasis. Our study demonstrated the discordant positive lesions in brain by both 18F-FDG PET/CT and 18F-FGln PET/CT. In addition, the SUVmax of 18F-FGln PET in certain smaller lesions were usually higher, although this was not statistically significant. The reason for more prominent relative uptake in smaller lesions should be related to metabolic variation in individual tumor types or with varying degree of substrate availability. It is generally recognized that brain metastasis is usually accompanied by peripheral edema. In previous studies, it is reported that pathophysiology of peripheral edema in brain metastasis is different from primary brain tumors.21 The causes were likely due to the increased permeability of capillary endothelium in metastatic tumors (vasogenic edema), and these abnormal capillaries are similar to capillaries from the tissue of origin.22 Interestingly, our results suggest that 18F-FGln uptake (SUVmax) in brain metastasis appeared to be independent of peripheral edema. This may support results reported previously by Venneti et al14 that the increased blood-brain barrier permeability do not significantly contribute to 18F-FGln uptake. Furthermore, when compared 18F-FGln uptake (SUVmax) in all positive lesions with the characteristic of enhanced MRI, 18F-FGln uptake was positive in metastatic lesions with solid nodular contrast enhancement on MRI. Tumors with rim-enhancement and central necrosis showed low uptake of 18F-FGln.
In a normal physiologic condition, the metabolism in the brain requires dynamic cross-talk between various cell types to establish a cohesive metabolic signaling network.23,24 Although glioblastoma and brain metastases represent a broad range of cancer subtypes with distinct cellular origins and diverse genetic programs, they exhibit common metabolic characteristics that may be the result of reprogramming to enable rapid growth in the brain.25 18F-FGln uptake is mainly mediated by the amino acid transporter SLC1A5,11,12 which was minimally expressed in the normal brain but markedly increased in gliomas.14 Interestingly, previous studies on immunohistochemical staining analysis of a tissue microarray containing 77 cases of metastatic carcinoma by Kim et al26 showed SLCIA5 also prominently expressed on metastatic carcinoma. Consequently, the up-regulation of SLCIA5 could be one of the primary reasons for higher 18F-FGln uptake (by using this glutamine transporter) observed in glioma and brain metastases.
Tumor cells will increase anaplerotic glutamine utilization when relying on increased aerobic glycolysis to obtain anabolic precursors for macromolecular synthesis toward cancer cell multiplication and tumor growth.27 Our results showed the positive correlation between 18F-FGln and 18F-FDG in 14 brain metastases support this metabolic paradigm.
As PET/CT is a kind of commonly used whole-body diagnostic tool for cancer patients, we further compared 18F-FGln imaging with 18F-FDG imaging in extracranial lesions patient-by-patient. We found that 18F-FGln uptake in the liver was more intense than tumor, so that the tumor sites appeared as cold spots. Further, this study showed that certain bone metastasis lesions could be missed for the slight radioactive uptake within metastases or be confused by the intense normal uptake of 18F-FGln within the bone morrow. Hence, further clinical trials are needed for conclusion for a purpose of sufficient statistical power.
Other radiotracers have been used to assess brain tumors (including brain metastasis). Radiolabeled choline was reported as a tracer superior to 18F-FDG in depicting brain lesions or relapse.28 Nevertheless, Mertens et al1,29 has reported a relative difficulty in recognizing the existence and/or the border of brain tumors around choroid plexus and pineal gland. Recent studies emphasized the possible role of several small molecule tracers, 11C-methionine (11C-MET),30,31 18F-fluorethyltyrosine (18F-FET),32 18F-thymidine (18F-FLT),33 and 18F-dihydroxy-L-phenylalanine (18F-DOPA).34,35 A common feature of these tracers is the very low rate of physiological distribution in the brain, which allows better diagnostic accuracies in detecting primary brain tumors.1,34 None of the reported studies have compared these tracers with 18F-FGln PET in the present clinical context. Future prospective comparisons of 18F-FGln PET with other amino acid tracers in larger populations would better define the role of these tracers for evaluation of brain tumors.
18F-FGln PET/CT imaging takes advantage of glutamine addiction in brain metastasis and may serve as a valuable tool to assess metabolic glutamine uptake in patient with brain metastasis. Low uptake in the normal brain may also help in evaluating more central nervous system metastasis, and its utility in the liver and bone metastatic lesions may require more caution due to uptake in normal structures.
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18F-(2S,4R)-4-fluoroglutamine; glutamine metabolism; biodistribution; brain metastasis; FDG
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