In response to: Anatomy of ¹⁸F-GE180, a failed radioligand for the TSPO protein

Dear Sir,

We read with interest the recently published letter by Zanotti-Fregonara et al. [1] that very critically discusses the characteristics of ¹⁸F-GE180 as a PET tracer for the 18-kDa mitochondrial translocator protein (TSPO) in human brain. This follows a previously published and rather similar letter and thus offers little information that is substantially new [2].

The main hypotheses of Zanotti-Fregonara et al. [1] are still that

The evidence that Zanotti-Fregonara et al. present in support of these hypotheses is not valid. From our point of view, the complete discreditation of a tracer is a harsh assessment and one that must be firmly based on scientifically conclusive evidence. Taking into consideration all published data as well as our own experience with the tracer, the arguments of the authors, although partially understandable, are not sufficient to support their general dismissal of ¹⁸F-GE180.

In our studies of glioma patients [3‐5], we found that ¹⁸F-GE180 PET tracer uptake could be clearly visualized on the PET image in areas outside of the gadolinium enhancement area on MRI and even in gliomas without any visible contrast enhancement on MRI; Zanotti-Fregonara et al. argue that the BBB in these areas might be disrupted in terms of a microdisruption which is simply not depicted on MRI. We fully agree that the presence of an “MRI-invisible microdisruption” of the BBB in these cases cannot be ruled out. However, even if there was a microdisruption of the BBB, which can only be passed by the PET imaging probe and not by gadolinium, the hypothesis that the accumulation of ¹⁸F-GE180 beyond the BBB is merely nonspecific signal and is a speculation that is not firmly based on scientifically conclusive evidence. We claim that TSPO-expressing tissue beyond the BBB is required for the highly increased tracer signal found in our glioma studies. Our conclusion is based on several findings:

Zanotti-Fregonara et al. insist on the importance of a correlation of in vivo ¹⁸F-GE180 uptake with histopathological analysis using stereotactic brain biopsies. We have an ongoing publicly funded study correlating ¹⁸F-GE180 uptake and histopathology in brain tumours exactly addressing this issue (German Research Foundation, FOR 2858; project A1 and A2). We evaluate here in-depth the correlation between TSPO expression and ¹⁸F-GE180 uptake in humans and hope to reproduce the promising results obtained in preclinical studies; e.g., Parhizkar et al. were able to demonstrate a strong correlation between the phagocytosis marker CD68 and the ¹⁸F-GE180 μPET signal in a transgenic amyloid mouse model [7]. Significant associations between ¹⁸F-GE180 PET and microglia immunohistochemistry were also proven for the more general microglia activation marker Iba1 in different amyloid mouse models [8, 9]. Considering this variety of evidence together with the minor impact of BBB leakage in amyloid mouse models, it seems very unlikely that a parallel BBB-related phenomenon confounds all observed associations of PET with gold standard assessments.

Zanotti-Fregonara et al. are of course correct in stating that the disruption of the BBB has an influence on the ¹⁸F-GE180 uptake, but this is a phenomenon that is more or less shared among all other TSPO tracers. To our knowledge, no publication has proven that the uptake of any other TSPO tracers e.g. is not influenced by the BBB disruption. Zanotti-Fregonara et al. themselves provide an example for the TSPO tracer ¹¹C-ER176, which shows moderate uptake within the area of increased ⁸²Rb-PET signal and contrast enhancement on MRI, but not beyond [1]. This tracer together with several other TSPO tracers has, compared with ¹⁸F-GE180, the disadvantage of exhibiting a very high fraction of metabolised tracer potentially contributing to tracer signal in regions with disrupted BBB. In contrast, ¹⁸F-GE180 has a favourably high parent fraction even at late time points [10, 11].

Furthermore, the statement by Zanotti-Fregonara et al. attributing the reported positive correlation of ¹⁸F-GE180 signal and the WHO grade in glioma [3] to increased levels of BBB disruption only is not correct. It may be true that the level of BBB disruption is—in many cases—higher in high-grade than in low-grade gliomas; however, up to 40% of gliomas without contrast enhancement on MRI are high-grade gliomas [12, 13]. TSPO expression itself is, independent of potential BBB disruption, also highly correlated with the WHO grade as shown by neuropathological studies [14]. Nonetheless, we agree with Zanotti-Fregonara et al. that the data on normal volunteers indicate that a certain basic microdisruption might be necessary for ¹⁸F-GE180 accumulation and therefore, TSPO-expression in an area completely without BBB-disruption might be underestimated by ¹⁸F-GE180 PET. However, as mentioned above, there is barely any clinical situation, in which brain pathologies (e.g. neuroinflammatory processes, gliomas, or their local co-existence) are not accompanied by at least a minor disruption of the BBB. Thus, this phenomenon cannot be ruled and is not of major practical relevance in investigating the diseased brain. It is of course highly desirable to understand in-depth the influence of BBB breakdown on ¹⁸F-GE180 uptake and similar tracers, e. g. by using adequate methodology including multi-modal PET and elaborate MRI analyses.

In their most recent letter, Zanotti-Fregonara et al. state: “The substantial role of BBB disruption in the uptake of ¹⁸F-GE180 is clearly shown in a recent pilot study in patients with ischemic lesions […]. The authors found that a significant part of the high uptake in the lesions is nonspecific and driven by BBB damage. Furthermore, up to 50% of the signal in nonischemic brain is due to vascular noise.” [1]. Keeping in mind that the signal fraction caused by vasculature has been reported to be about 20% for ¹⁸F-GE180 [11] (and not 50%) of the signal in healthy tissue at late time points (best representing distribution volumes), we do not support the above-drawn conclusions of Zanotti-Fregonara et al. A high vascular proportion of tissue signal of up to 50% is only true for the very early time frames (15–30 min post injection) used by Visi et al., which do not represent distribution volume [11]. Their hypothesis that high ¹⁸F-GE180 uptake in lesions is mainly non-specific is only based on the observation that a high uptake in one low-affinity binder (LAB) lesion was found in early static images, see [15]. Visi et al. observed otherwise a high correlation of lesion-to-reference ratios of the early ¹⁸F-GE180 signal with distribution volume ratios obtained using ¹¹C-(R)-PK11195, indicating very similar in vivo behaviour [15]. This included the uptake in both observed LAB lesions. Due to the unclear susceptibility to TSPO polymorphism in vivo [10, 11, 16, 17], we believe that the high uptake in one LAB lesion does not prove non-specificity of the ¹⁸F-GE180 signal, but rather likely represents disease severity and TSPO expression level similar to ¹¹C-(R)-PK11195.

With respect to the authors’ second claim that “the quality of the images is so poor that even a difference this large cannot be consistently detected” [1], we primarily refer to our previous reply letter [6]. The controversial results reported for ¹⁸F-GE180 uptake in healthy volunteers with different binding affinity status could be attributed to the low target expression in healthy tissue itself and the technical difficulties associated with ¹⁸F-GE180 quantification, see e. g. [17]. Moreover, one has to discuss to what extent the diagnostic quality of a tracer is limited by a very low signal of the tracer in healthy brain. A high target-to-background ratio allowing sensitive detection of TSPO expressing diseased tissue is the primary goal in a clinical setting. We believe that ¹⁸F-GE180 has suitable characteristics for that. It is quite obvious that specific tracer uptake is (besides the extensively discussed influence of BBB disruption) heavily influenced by the pathology itself, in such a way that potential differences induced by the binding affinity status in pathological lesions are very hard to detect; e.g., in patients with brain tumours, tumoural aggressiveness and inflammatory activity have a major effect on TSPO expression and will mask differences between the diverging binding affinity status at least for high- and medium-affinity binders, which represent the majority of the patients.

In summary, we reject the claim of Zanotti-Fregonara et al. that ¹⁸F-GE180 is a “failed radioligand”. Vice versa, we would like to clarify that ¹⁸F-GE180 cannot legitimately be called “superior” or an ideal radioligand in the light of the given limitations highlighted in the joint discussion [1, 2, 6]. We believe that a “perfect” radioligand targeting the TSPO does not (yet) exist—neither ¹⁸F-GE180 nor any other ligand so far—as every existing TSPO radioligand suffers drawbacks [18], which have to be weighed and thoughtfully considered during data interpretation. However, for beneficial routine clinical use, one has to compromise when dealing with the limitations of TSPO ligands. We believe—despite the discussed drawbacks of ¹⁸F-GE180—that imaging with ¹⁸F-GE180 can add comprehensive clinical information: First, we think that ¹⁸F-GE180-uptake in patients with multiple sclerosis and clinical worsening despite unchanged and non-enhancing lesions on MRI can indicate remaining disease activity [19, 20]—independent of the very exact contribution of the BBB on ¹⁸F-GE180 uptake. In these patients, standard imaging with contrast-enhanced MRI is less sensitive in assessing the change of the clinical situation. Second—also independent of the very exact contribution of the BBB on ¹⁸F-GE180 uptake—we believe that imaging with ¹⁸F-GE180 can significantly add clinical information in brain tumour patients by delineating the tumour extent beyond contrast enhancement on MRI [3‐5, 21] and, in future, probably by depicting in more detail, compared with labelled amino acids, the tumour characteristics. Therefore, in summary, we believe that despite the repeated articulation of ¹⁸F-GE180 PET drawbacks, its use can significantly contribute to clinical issues that thus far cannot be resolved with standard imaging alone.

Finally, we wanted to strongly emphasize our appreciation of Dr. Owen’s previous and current work. We do not doubt Dr. Owen’s theses in personal communications at all, as probably inadvertently indicated by Zanotti-Fregonara et al. [1]. By contrast, we rather wanted to encourage the authors to further obtain and publish data on in vitro affinity to TSPO polymorphism in order to foster fruitful exchange and help to solve the common controversial and important issues.