Initial evaluation of (4S)-4-(3-[¹⁸F]fluoropropyl)-l-glutamate (FSPG) PET/CT imaging in patients with head and neck cancer, colorectal cancer, or non-Hodgkin lymphoma

Tumor cells require an adequate supply of nutrients to meet anabolic and energetic needs while maintaining appropriate redox balance for growth, proliferation, and survival. Recent research revealed several interesting insights into tumor metabolism, including how tumor cells adopt metabolic pathways to cope with such demands in challenging environments [1, 2]. Whereas the upregulation of the glycolytic pathways has long been described and [¹⁸F]FDG PET is widely used in clinical routine for tumor imaging, other dominant metabolic pathways in tumors such as the glutaminolytic pathway or glutathione biosynthesis/redox-balancing pathway are now being explored in more detail. The glutaminolytic pathway provides both energy and building blocks for tumor growth [3] and can be investigated by PET probes targeting the alanine serine cysteine-preferring transporter 2 (ASCT2 or SLC1A5) [4]. The glutathione biosynthesis/redox-balancing pathway can be investigated by targeting the system x_C⁻ transporter [5]. This antiporter consist of two subunits, SLC7A11 (also known as xCT subunit), which is the catalytic subunit that mediates transport function, and SLC3A2 (also known as 4F2hc or CD98hc subunit), which functions as a chaperone and recruits the SLC7A11 subunit to the plasma membrane. Extracellular cystine gets imported in exchange for efflux of intracellular glutamate. Cystine is then reduced intracellularly to two molecules of cysteine. The sulfur-containing cysteine molecules can be incorporated into proteins or used in the biosynthesis of the antioxidant agent glutathione. Improved access to glutathione provides advantages for tumor cell survival by allowing better detoxification of chemotherapeutics and reactive oxygen species while inhibition of system x_C⁻ has been shown to induce tumor-selective ferroptosis and suppresses tumor growth in various tumor models [6, 7].

[¹⁸F]FDG remains the primary radiotracer used clinically in the evaluation of various cancers [8‐10]. However, [¹⁸F]FDG also has its known limitations. Prominent background uptake in the brain, kidneys, and often the gastrointestinal tract can reduce the sensitivity of [¹⁸F]FDG in those regions. Another challenge is the prominent [¹⁸F]FDG uptake seen in benign, inflammatory lesions such as infections, granulomatous processes, and sarcoidosis [11‐13]. This can reduce the specificity of [¹⁸F]FDG for malignancy and make the interpretation of certain scans difficult, demanding the development of other tumor imaging probes. [¹⁸F]FDG PET uptake shows correlation with many parameters such as tumor aggressiveness, proliferative activity, and prognosis [14, 15] but does not necessarily inform about other tumor characteristics. These limitations stimulated the development of new PET tracers to study other important aspects of tumor metabolism.

For example, (4S)-4-(3-[¹⁸F]fluoropropyl)-l-glutamate (previously BAY 94-9392 and herein referred to as [¹⁸F]FSPG) is an investigational ¹⁸F-labeled glutamate derivative for PET imaging of system x_C⁻ transporter activity. Specific transport of [¹⁸F]FSPG via the x_C⁻ transporter was demonstrated in cell competition assays and xCT knock-down cells, and excellent tumor visualization was achieved in animal tumor models [16]. Biodistribution analysis in rodents showed rapid blood clearance via the kidneys and low background activity from healthy tissue, providing high contrast for tumor imaging. In cancer models, [¹⁸F]FSPG demonstrated the ability to identify drug-resistance by detecting upregulated antioxidant pathways and provides an early redox indicator of tumor response to treatment, preceding other markers such as tumor shrinkage and decreased glucose utilization [17, 18]. [¹⁸F]FSPG did not accumulate in the inflammatory model tested in animals [16], although subsequent clinical studies reported uptake in sarcoidosis [19]. Pilot clinical studies examining dosimetry and biodistribution in healthy volunteers [20, 21] and tumor detection in patients with non-small cell lung cancer, hepatocellular carcinoma, and brain tumors showed promising results and confirmed preclinical data [22‐25]. In particular, low background uptake in the brain, lung, and bowel was observed. Other PET agents targeting the system x_C⁻ transporter, [¹⁸F]hGTS13 and [¹⁸F]FASu, have also been recently described in preclinical models [26‐28].

Preclinical research has recently shown that increased system x_C⁻ activity enhances cancer cell dependency on glucose and a previously unappreciated role of system x_C⁻ was uncovered [29, 30]. This demonstrates that both the glycolytic and the glutathione pathways are connected. Limiting glucose supply with inhibitors of glucose transporters can selectively kill cancer cells with high levels of system x_C⁻ or suppress tumor growth. This may further assist with future therapeutic strategies to target the metabolic vulnerability in tumors with high system x_C⁻ expression.

The work presented here is the initial evaluation of [¹⁸F]FSPG in patients with head and neck cancer (HNC), colorectal cancer (CRC), or non-Hodgkin lymphoma (NHL). The selection of these malignancies was based upon the tracer’s favorable performance in preclinical studies. For example, strong tumor uptake was demonstrated in subcutaneous human NCI-HT29 colon tumor models [16]. Moreover, xCT-targeted therapy has shown potential use for arresting tumor growth and/or sensitizing these cancer cells, reemphasizing the transporter’s role in disease pathogenesis [31‐34]. The physiologic biodistribution of [¹⁸F]FDG was also considered for this selection of malignancies, as high [¹⁸F]FDG accumulation in the brain and bowel inevitably limits the contrast for tumor imaging in these regions. Due to the pilot nature of this investigation with [¹⁸F]FSPG, imaging with [¹⁸F]FDG was an inclusion criterion and was used for general comparison.

The purpose of this study was twofold: (1) to provide preliminary data regarding the pattern of [¹⁸F]FSPG PET uptake in patients with HNC, CRC, or NHL and (2) to compare these profiles with [¹⁸F]FDG PET, thus exploring possible clinical applications for this radiotracer in these disease entities.

The protocol for this study was reviewed and approved by the U.S. Food and Drug Administration (eIND 108509), the Institutional Review Board at Stanford University, and the Scientific Review Committee at the Stanford Cancer Institute. Fifteen subjects with histologically confirmed, newly diagnosed, or recurrent head and neck cancer (HNC, n = 5), colorectal cancer (CRC, n = 5), or non-Hodgkin lymphoma (NHL, n = 5) were recruited (ClinicalTrials.​gov Identifier: NCT01186601). Detailed inclusion and exclusion criteria are listed in the Supplementary data section. To be eligible, each subject was required to have a positive whole-body [¹⁸F]FDG scan beforehand. The average time between the [¹⁸F]FDG scan and the [¹⁸F]FSPG scan was 10 ± 6 days. [¹⁸F]FDG PET/CT studies were done as a standard-of-care clinical scan, approximately 60 (± 15) min after a standard radioactive dose of 555 MBq ± 20% (15 mCi ± 20%) was administered. A low-dose, non-contrast CT was performed for attenuation correction and anatomic localization for all scans [35, 36].

Prior to the [¹⁸F]FSPG PET/CT scan, a brief physical exam was performed, and vital signs, blood, and urine samples were collected. [¹⁸F]FSPG was administered as a slow intravenous bolus injection over 60 s. The mean ± standard deviation of the radioactive dose given was 301.4 ± 28.1 MBq (8.1 ± 0.8 mCi), with a range of 270.1–336.7 MBq (7.3–9.1 mCi). After each respective tracer injection, the cannula and injection system were flushed with 10 mL normal saline (0.9% NaCl).

Three [¹⁸F]FSPG PET/CTs were then acquired sequentially to capture different time points after tracer injection for evaluation of temporal change in biodistribution. The images were obtained using a GE Discovery PET/CT scanner (either model D600 or D690). The first image acquisition, with a total duration of 45 min, was performed immediately after the injection of tracer. It was performed as five sequential whole-body (vertex to mid-thigh) PET scans after obtaining one CT (140 kV, range 10–85 mAs) for attenuation correction and anatomic localization. Each of the 5 PET scans gradually increased in the number of minutes per bed position as follows: 30 s/bed, 30 s/bed, 1 min/bed, 2 min/bed, and 2 min/bed. The second and third whole-body PET/CT scans, each with a duration of approximately 30 min (3 min/bed position), were started at 60 and 105 min post-tracer injection, respectively. The patients were asked to void before the second and third scan session to reduce their radiation exposure and to improve visualization of the pelvic structures.

After the scans were completed, and again the next day, additional sets of ECG, vital signs, and blood and urine samples were obtained. Any adverse events either noted by the participant or the research team were recorded. Seven days later, the patient was contacted by phone to determine if there were any interim adverse events or medication changes.

[¹⁸F]FSPG and [¹⁸F]FDG PET images were analyzed as described previously [23].

Twelve men and three women, with an average age of 66 years (range 45–87), were recruited. All five subjects with HNC had squamous cell carcinoma (SCC), and all CRCs were adenocarcinomas. The subjects with NHL had a variety of subtypes including diffuse large B cell (n = 1), follicular (n = 1), cutaneous T cell (n = 1), and mantle cell (n = 2). The full patient demographics are given in Table 1. Whole-body maximum intensity projection (MIP) images are shown in Fig. 1 of a representative participant with HNC, CRC, or NHL.

No adverse events were observed, either in terms of self-described symptoms or clinically relevant deviations in their vital signs or laboratory values. As observed before in prior studies [21], visual analysis of the PET images showed consistent physiologic biodistribution of [¹⁸F]FSPG across the subjects, with prominent diffuse activity throughout the pancreas (average SUV at 60 min was 7.2 ± 1.5). There was additional low-grade and variable activity in the oral cavity and oropharynx, salivary glands, thyroid gland, mediastinal blood pool, and liver. Some subjects also showed mild diffuse activity in the stomach. The tracer showed predominant clearance through the kidneys and excretion into the bladder. Beyond this, there was very low background activity in the brain, chest, abdomen, and pelvis. Time-activity-curve analysis for all seven time points per subject showed rapid clearance of the radiotracer from the blood pool (Supplementary Figure 1). More specific results separated by cancer type are presented next.

A consistent physiologic biodistribution pattern for [¹⁸F]FSPG was found in patients with HNC, CRC, or NHL. The subjects with HNC all showed uptake in tumor lesions with both [¹⁸F]FSPG and [¹⁸F]FDG PET, although there were some key differences between these scans. Most notably, the lack of any [¹⁸F]FSPG background uptake in the normal brain allows for easier interpretation of [¹⁸F]FSPG PET scans, especially for skull base lesions close to the brain (Fig. 2). This includes one subject with a large nasal mass and another with a nasopharyngeal mass extending to the clivus. Evaluation of regional nodal and distant pulmonary metastases was visually indistinguishable between the two radiopharmaceuticals, although several of these lesions were quite small (subcentimeter in size). SUV analysis showed that the primary tumors had a higher SUV value on the [¹⁸F]FSPG PET scan (by approximately 1.0 SUV more) in comparison to the metastases. The absolute [¹⁸F]FSPG SUV values for these lesions were also lower than for the comparative [¹⁸F]FDG PET scan, although the tumor-to-background ratios were higher. The only exception was a patient with recurrent SCC of the left tongue base. In that particular case, the [¹⁸F]FDG PET scan had a notably higher uptake than the [¹⁸F]FSPG PET scan. Subsequent biopsy confirmed SCC recurrence. Incidentally, this subject was given antibiotic therapy for a presumed oral infection for 1 week between his [¹⁸F]FDG and [¹⁸F]FSPG PET scans. As such, the [¹⁸F]FDG PET signal presumably reflects both the tumor and the associated infection and may also have been lower if done after the antibiotic therapy. [¹⁸F]FSPG uptake has been associated with an active inflammatory disease state by measuring xCT activity in activated M1 macrophages [19], but its role in inflammatory or infectious lesions remains to be explored in more detail.

Epstein-Barr virus (EBV) status was available for three patients. EBV-positive lesions showed lower SUV values on both scans in comparison to those that were EBV-negative. [¹⁸F]FDG PET functional parameters have previously been shown to be significantly associated with plasma EBV DNA load [37], and the roles of both have been demonstrated for prognostication in HNC [38]. As a core biomarker in the setting of tumorigenesis, the possibility of [¹⁸F]FSPG also correlating with EBV would be noteworthy.

All subjects with CRC showed uptake on both [¹⁸F]FSPG and [¹⁸F]FDG PET scans. In these subjects, all of whom had recurrent, metastatic adenocarcinoma, both radiopharmaceuticals visually performed quite similarly with no major discrepancies noted in terms of lesion detection. SUV analysis shows a very similar pattern to the HNC cases where the absolute SUV values for the primary and metastatic lesions were lower for the [¹⁸F]FSPG PET scan than in comparison to the [¹⁸F]FDG PET scan, but with higher tumor-to-background levels. Overall, these findings indicate that both radiopharmaceuticals were similarly effective for detecting CRC in this small patient cohort. K-ras mutation status was also available for three patients with thoracic metastases. Mutated k-ras has been shown to increase [¹⁸F]FDG uptake, possibly by upregulation of GLUT1 [39, 40]. The pulmonary nodule positive for k-ras mutation showed higher uptake with [¹⁸F]FDG than the wildtype (3.5 versus 1.9), whereas the [¹⁸F]FSPG SUV values were comparable (1.2 versus 1.0), which may allude to the different pathways targeted by the two tracers.

The subjects with NHL showed the greatest amount of variability between the [¹⁸F]FSPG and [¹⁸F]FDG PET scans. In fact, not all the subjects with NHL showed significant uptake above background with [¹⁸F]FSPG PET. One subject with diffuse large B cell lymphoma and another with follicular lymphoma both had uptake levels essentially equivalent to the liver background. Both of these subjects showed mild-to-moderate uptake with [¹⁸F]FDG. Another subject with cutaneous T cell lymphoma showed very similar mildly increased uptake in comparison to [¹⁸F]FDG. The most interesting subtype of NHL was mantle cell lymphoma, of which there were two subjects. The first showed the lowest SUV values of all 5 subjects with NHL, while the other showed the highest. In fact, the latter subject’s [¹⁸F]FSPG SUV values were nearly double that of the comparative [¹⁸F]FDG scan although both scans were visually intensely active. As a marker of the proliferative index, the Ki-67 staining for the first subject with mantle cell lymphoma was 6% while for the second subject was 20%. The SUV of [¹⁸F]FDG rises with increased proliferative activity and biological aggressiveness of the tumor tissue. SUV was shown to have a statistically significant positive correlation with the proliferative index Ki-67 across a variety of subtypes of non-Hodgkin’s lymphoma [41]. This may explain some of the discrepancy between their [¹⁸F]FSPG and [¹⁸F]FDG scans. Moreover, the lesions from the indolent follicular and cutaneous T cell lymphomas had lower [¹⁸F]FSPG SUV and uptake ratios than the more aggressive types (mean SUV of 3.4 versus 5.4; mean uptake ratio of 1.7 versus 3.0). Counterintuitive to the consensus in the literature which supports lower [¹⁸F]FDG uptake in indolent lymphomas as well [42], the corresponding [¹⁸F]FDG values were comparable between the two groups (mean SUV of 5.2 versus 5.0; mean uptake ratio of 1.9 versus 1.9).

As in other [¹⁸F]FSPG PET studies, the scalp showed incidental prominent physiologic uptake, possibly corresponding to the x_C⁻ transporter’s role in hair pigmentation [43]. Intense diffuse activity was also noted throughout the pancreas on [¹⁸F]FSPG PET, which would limit evaluation of primary pancreatic cancers. However, [¹⁸F]FSPG PET has been recently studied in patients with metastasized pancreatic ductal adenocarcinoma with promising results [44]. Due to prominent radioactive clearance through the kidneys and urinary bladder, evaluation of these regions is additionally difficult, although concomitant diuretic administration was not utilized.

The results of this pilot study are encouraging but have limitations. Foremost is the small sample size for each of the cancer indications associated with the preliminary nature of this project. However, the goal was not to definitively characterize the behavior of [¹⁸F]FSPG for each of these cancer indications, but rather to understand whether there is any uptake or role at all for this radiopharmaceutical in each cancer type. Indeed, the cancer types chosen were based on known increased activity and the proven role by [¹⁸F]FDG. It is noteworthy that this also poses a strong inherent potential bias against [¹⁸F]FSPG due to an underestimation of the added clinical impact in non-FDG-avid disease. The goal was not to outperform [¹⁸F]FDG in these indications, but to learn about the potential utility of [¹⁸F]FSPG in these indications while addressing a different metabolic pathway for imaging. In this light, the results of this study show that [¹⁸F]FSPG indeed performed well for all these cancer types except for certain subtypes of lymphoma. However, since only 1–2 subjects per type were imaged, results should be cautiously evaluated. Overall, the SUV for [¹⁸F]FSPG was generally similar but slightly lower than [¹⁸F]FDG across all 15 subjects, and when evaluated as a ratio relative to background uptake, it was slightly higher than [¹⁸F]FDG. Statistically significant differences were observed in some instances (absolute uptake in HNC and the tumor-to-muscle ratios in CRC and NHL) but should be interpreted with caution since, again, the small sample sizes limit the power for each of these evaluations.

It is generally well accepted that system x_C⁻ mediated uptake of cystine and glutathione biosynthesis have pro-survival functions under stress conditions. The unexpected and unique observation made by Koppula et al. [29] on the pro-cell death function of system x_C⁻ in the context of glucose starvation is of special interest. It was reported from preclinical investigations that tumor cells with high system x_C⁻ activity would have a more limited metabolic flexibility and more reliance on glucose for survival than those with low system x_C⁻ activity. High intracellular levels of cystine from increased system x_C⁻ activity can be potentially toxic, if the constitutive reduction to the more soluble cysteine is limited. Constant replenishing of the cellular NADPH pool is required and renders such cells dependent on the pentose phosphate pathway and high glycolytic activity [30]. This may also explain the high concordance between [¹⁸F]FDG and [¹⁸F]FSPG in this study. Another publication reports about similar observations on reduced nutrient flexibility upon increased system x_C⁻ expression [45]. Accordingly, tumors with high [¹⁸F]FSPG uptake could represent those that would be more vulnerable than those with low [¹⁸F]FSPG uptake when glucose is limited. More research is needed to verify the model for the proposed role of system x_C⁻ on glucose dependence to better understand the possible implications of these observations.

These initial promising results with [¹⁸F]FSPG warrant further evaluation in a larger cohort of cancer patients to confirm these preliminary findings. Moreover, it would be beneficial to investigate the possible role of [¹⁸F]FSPG PET in imaging [¹⁸F]FDG non-avid disease and assessing therapy response. Additional information on the metabolic phenotype and adaptations of tumors against oxidative stress may provide a better understanding of the underlying tumor biology and chemoresistance mechanisms that can potentially be useful for therapy selection and monitoring with [¹⁸F]FSPG PET.

[¹⁸F]FSPG is a promising PET radiopharmaceutical that specifically targets the x_C⁻ transporter. Avid uptake of [¹⁸F]FSPG was observed in subjects with HNC and CRC, and variable uptake was seen in subjects with NHL. Overall, the results are encouraging and show concordant visualization with [¹⁸F]FDG PET across 15 subjects with 3 different cancer types. Future studies based on larger numbers of subjects and those with a wider array of primary and recurrent or metastatic tumors are planned to further evaluate the utility of this novel radiopharmaceutical.