TSPO PET signal using [¹⁸F]GE180 is associated with survival in recurrent gliomas

Diffuse gliomas are the most frequent adult primary brain tumors [1], and almost all relapse after initial treatment. Efforts to understand the molecular mechanisms and prognostic factors of these heterogeneous tumors have led to the discovery of molecular markers which shape the most recent 2021 World Health Organization (WHO) classification of brain tumors to distinguish different glioma subgroups [2]. This molecular classification is mirrored in the current treatment guidelines [3]. All guidelines emphasize magnetic resonance imaging (MRI) as the gold standard for diagnostic imaging to gain information about the presumable histology and composition of the lesion as well its extent and, as a consequence, its amenability for treatment and its potential prognosis [4]. Beyond MRI, amino acid PET such as [¹⁸F]Fluoroethyltyrosine (FET) positron emission tomography (PET) has proven valuable to delineate tumor extent, identify intratumoral heterogeneity, and distinguish recurrent disease from pseudoprogression [5‐7]. Subsequently, amino acid PET has entered current diagnostic guidelines for primary and recurrent glioma [8, 9]. However, it is becoming increasingly clear that the interplay between tumor cells and the tumor microenvironment plays an important role in disease progression and treatment response or resistance. In this context, tumor-associated macrophages and microglia gain considerable attention, also in recurrent glioma [10‐12]. Thus, PET imaging of the respective cellular elements is of interest to provide insight into the tumor microenvironment and tumor-host interaction. As such, PET imaging of the 18 kDa translocator protein (TSPO) as a marker of activated microglia and neuroinflammation [13] has shown increased uptake in glioma patients, as well [14‐17].

TSPO is a mitochondrial membrane protein with a variety of functions in health and disease. Beyond classical mitochondrial functions such as respiration and oxidative stress regulation, more diverse functions such as cell proliferation and apoptosis have recently been implied [18]. TSPO is expressed ubiquitously and upregulated in steroid-synthesizing cells and microglial and malignant cells [18].

Preliminary data show an upregulation of TSPO expression in high-grade glioma and hint at a correlation between histologically increased TSPO expression and shorter survival, yet this was before description of molecularly defined glioma subgroups [19, 20]. To visualize TSPO expression and its spatial distribution in vivo, different radiolabeled TSPO ligands such as [¹¹C]-(R)PK11195 were used and shown to correlate with histological TSPO expression [21], but usability was limited by a low binding affinity or a short half-life of [¹¹C]. In contrast, the third-generation TSPO radioligand [¹⁸F]GE180 shows a high binding affinity [22] and convenient half-life for the clinical use due to the labelling with [¹⁸F]. In glioma patients, tracer uptake volumes were reported to exceed areas of contrast enhancement on MRI [23]. Several clinical case series showed a trend of higher TSPO tracer uptake in histologically or molecular biologically more aggressive tumors, such as isocitrate dehydrogenase (IDH) wild-type tumors [24, 25]. We here aim to describe the relationship between TSPO tracer uptake and clinical outcome in molecularly defined groups of recurrent glioma patients.

Glioma grading according to molecular features has improved prognostication in recent years, currently resulting in the 2021 revised edition of the WHO classification of CNS tumors [2]. Yet, prognosis for glioma patients remains poor, especially in the almost inevitable case of tumor recurrence. As no standard therapy for recurrent glioma is defined, treatment has to be tailored to the individual patient. For optimally fitting treatments, further markers of tumor aggressiveness are essential.

To optimally tailor treatments, [¹⁸F]FET PET has been established as a valuable imaging method to delineate tumor extent in vivo [8]. As a biomarker for prognostication uptake intensity on FET PET does not consistently predict survival [29], particularly not in glioblastoma [32]. In search of novel diagnostic and therapeutic tools, TSPO has gained interest recently, and earlier works could indeed show an association of tracer uptake on TSPO PET with IDH mutation status as a marker of glioma aggressiveness [24, 25]. To our knowledge, this is the first study analyzing the prognostic value of TSPO PET using [¹⁸F]GE180 in a larger cohort of recurrent glioma patients.

Here, we could confirm an association of [¹⁸F]GE180 uptake with known markers of malignancy such as histological tumor grade and, in the subgroup of IDH-wild-type glioblastoma, with the number of recurrences. Interestingly, as opposed to the primary situation [25], both recurrent IDH-mutant and IDH-wild-type glioblastoma show a highly increased maximum uptake value. This discrepancy between tumors in the primary and recurrent setting might have to do with an increased aggressiveness of recurrent IDH-mutant tumors as opposed to the primary situation (only 8 of 46 recurrent IDH-mutant tumors in our cohort did not show histological features of malignization).Whether hypermutation [33] or immune modulation induced by previous therapies drives a change in either TSPO expression or activation remains to be described in detail.

We found a strong negative correlation with survival time: Survival was more than three times longer in patients with sub-median SUV_max compared with those with supra-median SUV_max. TTF was also significantly longer in cases with sub-median SUV_max. Notably, this association between [¹⁸F]GE180 PET signal intensity and poor outcome was also found within the subgroups of IDH-wild-type and IDH-mutant tumor patients. A significant difference in TTF and PRS could even be seen in the largest homogenous patient subgroup of IDH-mutant astrocytoma CNS WHO 2021 grade 3, possibly due to a more nuanced substratification of malignancy than through the cut-off values set by histological grading. This clear association with survival even within molecularly homogenous subgroups suggests an added value of [¹⁸F]GE180 PET imaging to the clinically established molecular tumor stratification. This is notable because although molecular stratification greatly improved prognostication, diverging clinical courses are seen, especially after tumor recurrence, often but not always showing more malignant courses. If [¹⁸F]GE180 PET allowed further sub-stratification, therapy regimens and control intervals could be optimized.

Comparing these prognostically different groups of patients with low SUV_max versus high SUV_max, significant differences were particularly found for the tumor size measured by contrast-enhanced MRI, and, among IDH-mutant glioma patients, also measured by [¹⁸F]FET PET-based tumor volume. Patients with high SUV_max had significantly larger contrast-enhancing tumor volumes, and it is tempting to speculate about a causal relationship between these parameters (e.g., high TSPO expression leads to fast tumor growth). Preclinical studies implicating a role of the TSPO protein in cellular functions such as reduced apoptosis [34], increase of proliferation [35], and cell migration [36] provide possible mechanistic explanations. However, only a low to moderate association could be found between [¹⁸F]GE180 SUV_max values and volume of contrast enhancement or [¹⁸F]FET PET-based tumor volume. Another conspicuity was the higher uptake intensity on [¹⁸F]FET PET in the group of patients with high [¹⁸F]GE180 SUV_max, which can be explained by a moderate degree of correlation between both parameters. However, the strong association with survival outcomes was restricted to the [¹⁸F]GE180 PET signal and not found for uptake intensity on [¹⁸F]FET PET, which is in line with previous data demonstrating that TBR_max on [¹⁸F]FET does not serve as reliable prognostic biomarker [37, 38].

Despite promising survival data hinting at a role of TSPO in glioma tumorigenesis and progression [20, 39], the histological and molecular equivalent of a high TSPO tracer uptake remains to be evaluated in detail. Histologically and on mRNA level, tumor cells express high levels of TSPO, especially glioblastoma as opposed to low-grade glioma [19, 39]. This difference in TSPO expression even occurs among glioblastoma and other homogeneous molecular groups and correlates with higher tumor aggressiveness [20]. Although the mechanisms leading to this phenomenon are as yet unclear, an association with regulation of proliferation, apoptosis, migration, and/or mitochondrial functions such as respiration and oxidative stress regulation can be speculated [18]. While tracer uptake on [¹⁸F]FET PET is considered as surrogate marker of tumor cells due to overexpression of l-amino acid transporters particularly on tumor cells, upregulated TSPO expression in glioma is not only found in tumor cells but likewise in tumor-associated macrophages, endothelial cells, pericytes, and especially microglia [39]. As a microglia activation marker, TSPO PET visualizes neuroinflammation and has been established as a tool for imaging inflammatory CNS processes in neurodegenerative diseases [40, 41] or in multiple sclerosis [13, 42]. In the tumor microenvironment, inflammatory processes are increasingly recognized to play a role in gliomagenesis [43], treatment resistance [44], and tumor recurrence [12, 45]. As effectors of these processes, immunosuppressive myeloid-derived suppressor cells and regulatory T-cells outweigh activating immune effector cells such as T-cells and natural killer cells [46‐49]. This mainly immunosuppressive glioma microenvironment is maintained and reinforced by expression and secretion of immune-suppressing molecules by glioma cells and tumor-associated astrocytes [10]. Nevertheless, the tumor microenvironment and immune status is highly heterogeneous across tumor entities: For example, immunosuppression more strongly prevails in IDH-wild-type glioblastoma, whereas IDH-mutant astrocytomas secrete granulocyte colony-stimulating factor which increases the ratio of non-suppressive neutrophils [11]. Yet, heterogeneity is not only seen between different molecular tumor entities, but also both spatially and temporally within the same tumor [50]. This heterogeneity adds to the difficulty of therapeutically stimulating an anti-glioma immune response [44]. Therefore, illustrating the tumor immune environment in vivo and monitoring changes over time is promising for prognostication and especially in light of recent advances in immunomodulating therapies [51].

A possible perspective would be to use [¹⁸F]GE180 PET imaging to augment standard FET PET imaging to guide treatment aggressiveness: Undertreatment is fatal in glioma recurrence, yet overtreatment is also to be avoided to minimize side effects and maintain quality of life in a situation where no standard treatment exists and treatment is mainly guided by clinical experience and individual, patient- and tumor-specific factors. In vivo information about tumor aggressiveness is therefore highly valuable for an informed treatment decision. Furthermore, non-invasive characterization of tumor heterogeneity could prompt multimodal treatment decisions such as resection or high-dose re-radiotherapy of especially aggressive but locally treatable areas. Scanning intervals could also be optimized, especially in histologically low-grade tumors showing signs of increased aggressiveness during initial or follow-up imaging.

Some limitations of this study must be noted. As it contains an unselected population of glioma patients undergoing molecular imaging, statistical power is limited by low numbers in individual subgroups. However, evaluating these homogeneous subgroups is extremely important to assess the added value of [¹⁸F]GE180 PET to routine neuropathological and molecular assessment. While further improvements in prognostication might be attained by extraction of radiomic or pharmacokinetic features, this study chose a straightforward method for image analysis, which is easily applicable in the clinical routine. As a purely observational study, the evaluated associations are not statistically influenced by therapy. As a prognostic value of [¹⁸F]GE180 PET could be shown in recurrent glioma patients, a longitudinal analysis of individual patients would be of high interest to address changes of TSPO expression and their prognostic value: An ideal study should obtain serial [¹⁸F]GE180 PET imaging of a cohort of, e.g., IDH wild-type glioblastoma patients prior to initial resection, before and after radiochemotherapy, during and after adjuvant chemotherapy, and at the time of (suspected) recurrence. It would be highly valuable to monitor changes in TSPO expression during disease progression and its association with individual survival to better understand tumor- as well as treatment- or patient-specific influences. These data would be boosted greatly by tissue samples at the individual timepoints to disentangle the cellular sources of the TSPO signal over time, particularly the proportions of tumor cells vs. inflammatory cells. Unlike in a human study, where tissue sampling is ethically warranted in case of newly diagnosed tumors and suspected recurrence only, an analogous animal study could include tissue sampling during and after therapy. This would be especially useful to determine how TSPO expression and its cellular distribution changes during treatment as well as early and later after treatment, and if an increase at different timepoints has to be interpreted differently, such as in the context of an inflammatory reaction to treatment [52, 53]. These aspects will be covered in upcoming studies.

TSPO PET with [¹⁸F]GE180 is a promising imaging tool for prognostication in patients with recurrent IDH-mutant and IDH-wild-type glioma. The biological background and the usefulness of TSPO PET as a personalized read-out need to be elucidated in mechanistic and prospective studies.