Feasibility of [¹⁸F]fluoropivalate hybrid PET/MRI for imaging lower and higher grade glioma: a prospective first-in-patient pilot study

Glioma is the most common primary malignant brain tumour in adults, and accurate malignancy characterisation is important to optimise treatment pathways, as survival can range from 10 to 20 years in the case of IDH mutated, 1p/19q co-deleted lower-grade glioma (LGG) to 15 months for IDH wild-type glioblastoma, the most aggressive higher-grade glioma (HGG) [1‐4]. Conventional contrast-enhanced MR imaging (CE-MRI) is routinely used in the initial evaluation of brain tumours but has a limited role in discriminating LGG from HGG; tissue confirmation of the diagnosisis, therefore, important in patients suspected of harbouring a primary glial neoplasm. Other MRI methods [5] that quantify perfusion, blood volume, diffusion and tumour metabolite levels provide improved discrimination of malignancy [4, 6] as well as determining recurrence and therapy response [7, 8]. Several PET radiotracers have also been tested in the setting of brain tumour detection [9]. The most widely used of these are amino acid tracers like [¹⁸F]fluoroethyl-L-tyrosine, yet for these, conventional static imaging has limited application [10, 11]; dynamic imaging improves lesion characterisation [12‐15]. Newer amino acid tracers such as [¹⁸F]fluciclovine offer static imaging capabilities [16]. Hybrid PET/MRI, having overcome numerous technical challenges, including the efficient performance of PET within a magnetic field and use of MR-based attenuation correction [17], now provides the opportunity to combine novel PET and multi-parametric MRI for single-visit pre-surgical brain tumour characterisation [18]. The benefit of MRI in PET/MRI investigations has been incremental and has rarely made full use of either multi-modal MRI or kinetic co-analysis, and a saltatory approach would require simultaneous investigation of detailed multi-modal multi-parametric PET/MRI data.

The critical role of short-chain fatty acids (SCFAs) in brain tumour biology has come to the forefront. In particular, the report by Machimo and co-workers [19] that brain tumours have the capacity to oxidise acetate more so than glucose (but not glutamine) indicates a unique adaptation within the brain niche. The authors further assert that tumour grade-correlated ACSS2 is a key enzyme required for this metabolic vulnerability and potential therapeutic target of tumours. The ability of acetate to characterise brain tumours has been exploited in PET with [¹¹C]acetate [20], a radiotracer that lacks widespread clinical application. We recently reported the discovery of a non-metabolised fluorine-18 radiolabelled compound for imaging SCFA transcellular flux [21‐23]. The radiotracer, [¹⁸F]fluoropivalate (FPIA), shows low background activity in most organs except eliminating organs – liver and bladder – and has appropriate dosimetry for routine clinical use [24]. In the present pilot study, we assess multi-parametric multi-modal dynamic FPIA PET/MRI to determine if this approach provides discrimination of glioma grade.

We report the first study of FPIA PET in human tumours. In this exploratory PET/MRI phenotyping study, we show that FPIA PET tumour uptake (SUV_{60_max}) increases in a glioma grade-related manner and that DCE-MRI-derived extravascular-and-extracellular volume fraction (v_e) was higher in GBM (grade IV) compared to grades II–III tumours. FPIA is a novel tracer under evaluation for brain tumours. Several lines of evidence indicate that this tracer is a candidate for imaging glioma: (a) The tracer shows high contrast and growth-related uptake in orthotopic models of glioma in mice, with contrast surpassing FDG PET [22]; (b) biodistribution of the tracer in healthy human volunteers shows low background uptake, except in eliminating organs [24]; and (c) this present study shows that the tracer has high contrast in a grade-dependent manner, with associated survival duration in our cohort: 25–64 months, 29–61 months, and 1.6 to 13.7 months, respectively, for WHO grades II, III, and IV, respectively.

The evolving understanding of the differential use of SCFAs by human brain tumours has come to the fore [19], and tracers for imaging this phenotype beyond [¹¹C]acetate – which is difficult to distribute to other sites without on-site cyclotron – will enable this biology to be appreciated and form the basis of clinical diagnostic programmes for brain tumours. Ki (net solute transfer at steady state) and K1 (product of blood flow and the unidirectional first-pass extraction fraction) were higher in HGG compared to LGG, with K1 increasing in the order WHO grade II < WHO grade III < WHO grade IV. FPIA being a SCFA transports across cell membranes supported by the ubiquitous monocarboxylate transporters (MCTs) and sodium-coupled monocarboxylate transporters with several members, whose expression and functions in tissue are complex [37]. We show that mRNA expression of the prototypical MCT, SLC16A1 (MCT1), for instance, is higher in gliomas compared with corresponding normal brain tissue but otherwise non-discriminatory, while that of SLC25A20 is more emphasised in HGG (Supplementary Fig. S6). As FPIA is positively correlated with SLC25A20 protein expression (together with ACSS2, SLC22A5) [22], and our current study shows higher SLC25A20 protein expression in human HGG (grades III and IV), we suggest that SLC25A20, a mitochondrial enzyme that transports acylcarnitines from inside the cell into mitochondria, is worthy of further elaboration as an enzyme that contributes to the higher FPIA uptake in HGG. SLC25A20 studies were conducted in a separate, larger cohort to understand variations of enzymes that regulate SCFA metabolism. Future studies should examine, particularly SLC25A20, in relation to FPIA uptake. Of note, the mechanism of uptake of FPIA is distinct from that of amino acid tracers including [¹⁸F]fluoroethyl-L-tyrosine and [¹⁸F]fluciclovine [10, 11, 16]. Dynamic scans were conducted to verify the best imaging time point within 66.5 min. Based on the temporal changes in FPIA uptake in most tumours, we suggest an uptake time of 60 min (mid-time frame) for future static FPIA PET protocol. Indeed, this suggested protocol has been implemented in a new FPIA study (clinicaltrials.gov/ct2/show/NCT05801159).

CE-T1-CE MRI is the gold standard for high radiation dose boost and surgical resection, with T2/FLAIR regions considered less relevant due to oedema confounding tumour infiltration. With re-irradiation and aggressive resection becoming routine, the ability to accurately define the correct tumour target is urgent. A major consideration in evaluating new brain radiotracers is their ability to transit the blood–brain barrier (BBB). The criteria often used for asserting BBB transit are ‘visible PET uptake in the absence of MRI contrast and discordance between MRI and PET data’. These criteria depend, however, on how many such lesions exist in the cohort studied; on the basis of such criteria, [¹¹C]methionine and 6-[¹⁸F]-fluoro-L-DOPA (FDOPA) are considered tracers that cross the BBB [38]. Another approach is to determine the rate constant for the net irreversible transit of solute from blood to tissue at a steady state (non-zero Ki) described by Patlak et al. [39, 40]. The non-zero Ki seen in CWM and all grades of brain tumours (Fig. 3C) suggests that FPIA crosses the intact BBB. This inference is supported by the high and variable partitioning of FPIA between blood and healthy brain tissue (V_T), despite low K1, as well as the presence, in the spectral analysis output, kinetic components that are slower than simply delivery (Fig. 3B). SCFAs, charged at physiologic pH, can be transported into brain tissue by transporters including the monocarboxylate transporters, which are abundant in healthy brain tissue, blood–brain barrier and brain tumours; thus, the non-zero Ki and high V_T of FPIA are unsurprising [37, 41]. Expectedly, other properties of the variably compromised BBB in tumours, e.g. perfusion, may also contribute to overall SUV. The discordance between SUV and CE-MRI (Fig. 2A,B) further supports the notion that FPIA tumour uptake does not simply reflect compromised BBB. It should be noted that while the present report shows that FPIA tumour uptake correlates with tumour grade, albeit in a small cohort of patients, a consensus has not been reached for either [¹¹C]methionine or FDOPA regarding correlations between PET uptake and WHO grades II, III and IV, with high FDOPA and [¹¹C]methionine uptake seen in WHO grade II oligodendroglioma compared to IDH-mutant astrocytoma in some reports [42, 43].

A limitation of this study is its exploratory nature in 10 patients. Thus, any potential implied use of the technology can only form the basis of future studies. These potential applications include (a) the ability to use FPIA as a virtual biopsy of glioma malignancy to detect LGG transformation, target surgical biopsy and plan surgical resections, and (b) to exploit the tracer for target volume delineation to plan stereotactic radiosurgery. The period between imaging and histological diagnosis was variable (> 100 days in P3 and P7); furthermore, for P3, the biopsy was clinically indicated, which could have missed the most aggressive part of the tumour. These variations, which were beyond our control, are limitations of the study. The grading of glioma has been recently modified [2]. In our study, there were five IDH mutant tumours from two grade II diffuse glioma and three mixed/transforming WHO grade III HGG patients, and five WHO grade IV patients (GBM, IDHwt). Thus, the magnitude of FPIA uptake is not due to mutation status per se but likely the combination of factors that determine glioma grade, with the transformation of grade II to grade III in this small cohort implied for SUVmax ≥ 1.2 or TBRmax ≥ 2.4. Against the backdrop of a radiotracer with non-zero Ki, we speculate that TBRmean signifies TBR across the entire lesion, and for LGG, this could include many regions that are not transforming admixed with transforming regions and healthy brain tissue, with TBRmax representing maximum transformation within the region of interest. A counterargument is that noisy voxels could contribute to the TBRmean, given the low absolute value, and this fact should be examined in future studies. Further studies show that FPIA uptake is also high in brain metastasis (Islam et al., unpublished), which supports the notion of malignancy-related uptake. Other MRI methods that examine perfusion, blood volume, diffusion and tumour spectra have been investigated for malignancy prediction [44]. For example, v_e (DCE-MRI-derived extravascular-and-extracellular volume fraction; proposed cellularity marker) was higher in GBM compared to grades II and III tumours, consistent with v_e reported to be predictive of progression-free and overall survival in HGG patients [6], and DCE-MRI enhancing variables, in general, have been shown to been shown to be different in low- and high-grade gliomas [45]. We did not have sufficient patient numbers to comment on DCE variables and CBV in grade III patients with and without 1p/19q del, and this will be a future objective. ADC in HGG and IDHwt gliomas were lower than in LGG and IDHmu gliomas. For hybrid PET/MRI, the combination of FPIA and v_e was not selected by the penalised regression method, which instead suggested that combinations of FPIA PET and perfusion variables may be worthy of investigation. Further investigation of these variables and methodology in a larger patient population is warranted. Another area that needs further work is the use of FPIA PET to support volume delineation, as seen in discordance PET/MRI data in Fig. 2A and Supplementary Figs. S3 and S4. Validation of tumour extent by FPIA will transform the use of imaging to guide radiotherapy volume delineation as well as efficient surgical planning.

In conclusion, FPIA PET shows uptake in glioma, increasing in the order LGG (WHO grade II) < (WHO grade III) < HGG (WHO grade IV). The combined use of FPIA PET and v_e or perfusion variables in a PET/MRI context appear worthy of investigation in future studies.