Preliminary results using PET/MR in glioblastoma patients treated with regorafenib: an 18F-FET and DWI-ADC comparison

Introduction: The use of regorafenib in recurrent glioblastoma patients has been recently approved by the Italian Medicines Agency (AIFA) and added to the National Comprehensive Cancer Network (NCCN) 2020 guidelines as a preferred regimen. Given its complex effects at the molecular level, the most appropriate imaging tools to assess early response to treatment is still a matter of debate. DWI and 18 F–FET PET are promising methodologies providing additional information to the currently used RANO criteria. The aim of this study was to evaluate the variations in DWI/ADC- and 18 F-FET PET-derived parameters in patients who underwent PET/MR at both baseline and soon after starting regorafenib. Method: We retrospectively selected 16 consecutive GBM patients who underwent 18 F–FET PET/MR before and after two cycles of regorafenib. Patients were sorted into stable (SD) or progressive disease (PD) categories in accordance with RANO criteria. We were also able to analyze 4 SD patients who underwent a third PET/MR after another 4 cycles of regorafenib. 18 F–FET uptake greater than 1.6 times the mean background activity was used to dene an area to be superimposed on an ADC map at baseline and soon after treatment. A number of metrics were then derived and compared. Result: The average increases in FET and ADC pathological volumes were higher in PD than in SD patients, although in neither case did the difference reach signicance. However, when the percentage difference in FET volumes was plotted against the corresponding percentage difference in ADC, a correlation was observed (R = 0.54). Patients with a twofold increase in FET after regorafenib showed a signicantly higher increase in ADC pathological volume than the remaining subjects (p = 0.0023). Conclusion: In recurrent glioblastoma patients treated with regorafenib, 18 F-FET and ADC metrics, being obtained from completely different measures, could serve as semi-quantitative independent biomarkers of response to treatment. These promising parameters should be tested in a larger cohort of glioblastoma patients treated with regorafenib. A reconstruction of single frame PET images obtained at to minutes after tracer injection used for the present study as by EANM analysis of dynamic pattern beyond scope of present Standard corrections for dead time performed. A clinical Germany) included in AC clinical and PET. quality of UTE map was visually assessed in all patients. The PET data were reconstructed using a 3D ordered subset expectation maximization algorithm with 8 iterations, 21 subsets and a 3-mm Gaussian lter, from which PET images with a 256 x 256 matrix size (voxel size = 2.32 × 2.32 × 2.03 mm) were derived.

access to their data for research purposes.

Patient selection
We retrospectively selected sixteen consecutive recurrent GBM patients who underwent 18 F-FET PET/MR from May 2019 to October 2020 at the Nuclear Medicine Unit of Padua University Hospital before and after two cycles of regorafenib; 4/16 patients were followed up with a third PET/MR; all of the patients were treated at the Veneto Institute of Oncology-IRCCS in Padua. Excluded Patients were those who underwent 18 F-FET PET/MR but lacked one of the following inclusion criteria: 1. Histologically con rmed glioblastoma; 2. Radiologically and/or histologically con rmed disease relapse after conventional treatment according to RANO criteria (maximal safe resection followed by chemoradiotherapy); 3. Acquisition of baseline 18 F-FET PET/MR no sooner than one week before starting regorafenib; 4. Acquisition of a second 18 F-FET PET/MR no later than two weeks after two cycles of regorafenib; 5. No treatment changes between baseline and post-regorafenib 18 F-FET PET/MR.

Image acquisition and reconstruction
All 18 F-FET PET/MR images were acquired with a 3-T Biograph integrated PET/MR scanner (Siemens Healthcare, Germany) at the Nuclear Medicine Unit of Padua University Hospital, Italy. Following the most recent recommendations by the European Association of Nuclear Medicine, all study patients were required to fast for a minimum of 4 hours before the intravenous administration of approximately 250 MBq of 18 F-FET. Dynamic PET data were acquired from the time of tracer administration to 50 minutes post-injection [23], while at the same time a standardized MR protocol was performed. The latter included: 1-mm isotropic 3D T1-weighted magnetization-prepared rapid acquisition gradient echo (MPRAGE) (TR 2400 ms, TE 3.24 ms, slice thickness 1 mm, matrix size 256 x 256, FOV 256 x 256 mm) before and after contrast enhancement, 3D isovolumetric uid-attenuated inversion-recovery (FLAIR) (TR 5000 ms, TE 394 ms, TI 1800 ms, slice thickness 1 mm, matrix size 256 x 256, FOV 250 x 250 mm) and RESOLVE® sequence (Siemens Healthcare, Germany) (TR 5,000 ms, TE1 72 ms, TE2 122 ms, voxel size 1.56 x 1.56 x 3.12 mm), a high-resolution DWI sequence based on a readout-segmented echoplanar imaging (EPI) strategy [24]. Apparent diffusion coe cient (ADC) images were calculated from acquired DWI images with a b-value of 1000 s/mm 2 and 0 s/mm 2 . The contrast medium used with all patients was gadobutrol 0.1 mmol/Kg (Gadovist®, Bayer Inc., Mississauga, Ontario).
A reconstruction of single frame PET images obtained at 20 to 40 minutes after tracer injection was used for the present study as suggested by EANM guidelines. The analysis of the 50 min dynamic curve pattern is beyond the scope of the present paper. Standard corrections for decay, scatter and dead time were performed. A clinical UTE sequence (Siemens Healthcare, Germany) was included in the MR protocol (because more advanced AC methods are limited to a research settings and not directly applicable to a standard clinical setting) and used for attenuation correction of PET. The quality of the derived UTE map was visually assessed in all patients. The PET data were reconstructed using a 3D ordered subset expectation maximization algorithm with 8 iterations, 21 subsets and a 3-mm Gaussian lter, from which PET images with a 256 x 256 matrix size (voxel size = 2.32 × 2.32 × 2.03 mm) were derived.

Qualitative image analysis
One neuroradiologist and one nuclear medicine physician (with 7 and more than 10 years' experience in the eld of neuro-oncology, respectively), blind to the patients clinical outcomes and the follow-up imaging, jointly reviewed all 18 F-FET PET/MR images at both baseline and post-regorafenib. In accordance with the latest Response Assessment Criteria for High-Grade Gliomas by the Response Assessment in Neuro-Oncology (RANO) Working Group [11,25], study patients were divided into the following response-assessment categories: Complete Response (CR), Partial Response (PR), Stable Disease (SD), Progressive Disease (PD).
In cases of assumed CR or PR at the post-regorafenib time point, a follow-up MR scan was performed at least 4 weeks later and reviewed for con rmation. Progressive Disease was de ned (according to RANO) as the ful llment of one or more of the following conditions: 1. ≥ 25% increase in the sum of the products of the perpendicular diameters of the enhancing lesions compared with the smallest tumor measurement at baseline; 2. Appearance of any new contrast-enhancing lesion; 3. Signi cant increase in T2/FLAIR non-enhancing lesion.
Patients fell into the Stable Disease category if they did not meet the conditions for complete response, partial response, or progressive disease, and were administered the same or a lower dose of corticosteroids.
The mean SUV of a crescent-shaped VOI (BG FET ), manually drawn in the hemisphere contralateral to the tumor, was used as the 18 F-FET background [26].
The pathological FET volume (FET vol/pat ) was segmented through a 3D semiautomatic contouring process, excluding areas with an 18 F-FET uptake less than 1.6 times the background mean activity. This threshold was based on an 18 F-FET biopsy-controlled study, where it was proven to accurately differentiate between tumoral and non-tumoral tissue [27]. The derived segmented volume was visually re ned to exclude areas of non-speci c 18 F-FET spillover (major blood vessels, cranial bones, meninges etc.) using the aligned MDC images as the morphological reference.
FET vol/pat was then superimposed onto the ADC images ( Figure 6) to obtain the corresponding ADC volume (ADC vol ). The details are as follows: 1. FET vol/pat was imported into the aligned ADC image; 2. Areas with non-speci c high ADC values were automatically subtracted (with the aim also to correct for anatomical distortions induced by metal implants and air lled cavities) from the original volume, pinpointing the ADC values in the cerebrospinal uid of the lateral ventricles; 3. Areas of the original volume located outside the brain parenchyma were analogously subtracted, pinpointing a region external to the head.
A standard spherical volume (radius = 5 mm) was then placed on the ADC images in the hemisphere contralateral to the tumor, carefully avoiding lateral ventricles and major vessels, in order to derive the mean ADC value of the normal brain parenchyma (BG ADC ). This method was chosen in view of the stability of the ADC values in the "healthy" brain parenchyma during treatment with antiangiogenetic agents [13]. A qualitative assessment of the high resolution DWI and ADC derived maps was performed in every patient and revealed no signi cant distortions or misregistrations affecting the selected tumor area or background area.
The quality of alignment and segmentation was nally checked by an experienced nuclear medicine physician (with more than 10 years' experience in the eld of neuro-oncology).

Data analysis
A pixel dump of FET vol/pat , BG FET , ADC vol , and BG ADC was imported into the R software [28] for further analyses. The mean ADC value of the BG ADC was used as a threshold for ADC vol . Only those pixels below the threshold were considered pathologic (ADC vol/pat ). The percentage differences in ADC vol/pat and FET vol/pat before and after regorafenib were calculated and compared (ΔADC vol/pat = ADC vol/pat (T1-T0)/T0 and ΔFET vol/pat = FET vol/pat (T1-T0)/T0).

Statistical analysis
All statistical analyses were performed using the R Software. The Shapiro-Wilk normality test was performed on the distribution of all the parameters. Where normal distributions could not be assumed, non-parametric tests were performed. The percentage changes in ADC vol/pat and FET vol/pat before and after regorafenib were plotted and the Pearson correlation coe cient (PCC) calculated, assuming a linear correlation between the two variables. The differences in the percentage changes in FET vol/pat and ADC vol/pat between the response groups determined according to RANO criteria [11] were compared using the Wilcoxon signed-rank test for repeated measures. The signi cance level (α) was set at 0.05.

Patients
Our study population consisted of 15 IDH-wt and 1 glioblastoma NOS patients (6 females, 10 males, median age: 54.4 years, age range: 31 -73 years). All the study patients had undergone maximal safe resection, adjuvant chemoradiotherapy with temozolomide and subsequent maintenance temozolomide (from 1 to 12 cycles) before relapsing. The median time elapsed between radiotherapy and baseline 18 F-FET PET/MR was 319 days. In one subject, reirradiation was given in a single fraction about 4 weeks before starting regorafenib. Eight of the 16 patients had been surgically retreated before being scheduled for regorafenib, and at least 19 days passed before the rst 18 F-FET PET/MR was performed. All the study patients received 2 cycles of regorafenib (160 mg per day; 3 weeks on, 1 week off) without treatment interruption. The characteristics of the population are summarized in Table 1.

18 F-FET PET/MR image analysis
After 2 cycles of regorafenib, 7/16 (44%) patients were observed to have stable disease (SD), and the remaining 9/16 (56%) to have disease progression (PD) according to the RANO criteria ( Table 2). The values of the 18 F-FET PET/MR-derived parameters before and after treatment with regorafenib (FET vol/pat , TBR mean , TBR max [23], ADC vol/pat , and mean ADC vol/pat ) are listed in Table 2 and summarized in Table 3. Their absolute and percentage variations after treatment are presented in Table 4. Although the absolute and percentage increases in FET vol/pat were on average higher in PD than SD patients (21605 mm 3 and 168% versus -1160 mm 3 and 70%), the differences between the two groups were not statistically signi cant (p = 0.17). Similarly, the average absolute and percentage increases in ADC pathological volume were also higher in PD than SD subjects (501 mm 3 and 554% versus 33 mm 3 and 297%), and also failed to reach statistical signi cance (p = 0.53). The percentage variations in mean ADC, TBR max and TBR mean did not differ signi cantly between SD and PD patients (Tables 3 and 4, Figure 1). When the percentage difference in FET pathological volumes was plotted against the corresponding percentage difference in ADC pathological volumes, a linear regression model revealed a correlation between the two variables (R = 0.54) (Figure 2). We found no evident correlation between the percentage variation in mean ADC values and the corresponding percentage variation in FET vol/pat (R = 0.04).
Patients with at least a twofold increase in FET pathological volume (Figure 3) after regorafenib showed a signi cantly higher increase in ADC pathological volume than the remaining subjects (p = 0.0023). In 2/9 subjects classi ed as progressive after two cycles of regorafenib, the FET pathological volume decreased by 76% and 31%, respectively. Consistent with this, a decrease in the ADC pathological volume was observed in the former (-93%), while no residual pathological ADC areas could be detected in the latter. In contrast, in 3/7 patients classi ed as stable after treatment, an increase in FET pathological volume (5895 mm 3 , 2057 mm 3 , and 1009 mm 3 , respectively) was observed; in the same patients, the ADC pathological volume increased at similar rates (273 mm 3 , 88 mm 3 , and 425 mm 3 ).

Discussion
The main nding to emerge from the present study was the correlation between the percentage changes in the pathological FET and ADC volumes in recurrent GBM patients treated with regorafenib at their rst disease relapse. To our knowledge, this is the rst work assessing the variation in the ADC signal in the FET-positive volume in patients undergoing 18 F-FET PET/MR both at baseline and soon after beginning this new bene cial second-line therapy.
The value of DWI-derived parameters in treatment monitoring of GBM patients has already been extensively investigated [29][30][31][32], and many authors have suggested that the DWI methodology could play an important role in guiding response assessment, particularly when conventional contrast-enhanced and T2-weighted/FLAIR sequences are less reliable. Although DWI sequences are routinely acquired as part of the standard MR protocol for brain tumor imaging, the most recent recommendations [33] only describe how diffusion-weighted images should be acquired and provide no guidance for clinically interpreting and quantifying the extent of the tumor for the purpose of response evaluation. Two major issues consequently arise, the rst regarding the strategy to identify the region on the DWI-ADC images to be analyzed, the second regarding the threshold for pathological ADC values. In most of the published studies, the tumor volume was outlined and the VOI constructed on contrast-enhanced T1-weighted images, which were subsequently transferred to the corresponding DWI-ADC images. Buemi et al. [34], for example, manually drew the VOIs encompassing the areas of tumor-related contrast enhancement, and T2-weighted/FLAIR abnormalities were mapped onto the corresponding ADC images, thus deriving the CE-ADC and T2/FLAIR-ADC volumes, respectively. Histogram analysis and curve tting using a two-mixture normal distribution model were carried out to calculate the mean ADC of the lowest ADC values in these areas (CE-ADC-L and T2/FLAIR-ADC-L) [35]. Interestingly, only the mean ADC in CE-ADC-L turned out to be signi cantly predictive of progression-free survival and overall survival in GBM patients treated with bevacizumab and fotemustine. The predictive value of the low-ADC areas is con rmed by other published papers [36,37]. Zeiner et al. [38] calculated the ADC-ratio by measuring the minimum ADC values in the tumor and normalizing them by the ADC values of the contralateral, normal appearing brain tissue. In our study, instead, a standard VOI contralateral to the lesion was used to determine the appropriate threshold for the selection of pathological ADC values. This approach allowed for a more direct identi cation of the low ADC values in the de ned VOIs, avoiding the need for complex mathematical models. However, the post-regorafenib variation in the mean ADC thus calculated did not signi cantly correlate with the corresponding change in FET-positive volume, nor with the RANO response categories. A possible explanation for this discrepancy may lie in the different methodological approaches used here and in the previously published studies [13,34,[43][44][45][35][36][37][38][39][40][41][42]. This highlights the importance of future efforts towards standardizing the analysis of ADC maps before considering the inclusion of this methodology in the response assessment criteria.
It is important to note that the changes in neither the pathological FET volume nor the pathological ADC volume were signi cantly different in the stable and progressive patients as assessed by RANO criteria. These criteria are based mostly on changes in T2/FLAIR and contrast-enhanced areas, which are known to be affected by edema, in ammation, gliosis, and disruption of the blood-brain barrier. DWI, instead, is sensitive to microscopic water motion, resulting in relatively restricted diffusion in areas of tightly packed tumor cells. However, diffusion may be altered by causes other than increased cellularity in neoplastic tissue, and the diagnostic performance of this methodology is in uenced by the choice of the appropriate DWI parameter to analyze [17,46,47]. Moreover, the heterogeneity of the ADC signal may have translated into the wide variability we observed in the changes in the pathological ADC volume after regorafenib. This, in turn, may explain why a relatively high threshold of increase in FET pathological volume was needed to subdivide our population into groups with signi cantly different pathological ADC volumes.
In three cases which were classi ed according to the RANO criteria as stable after treatment with regorafenib, both the ADC and FET pathological volumes increased compared with the baseline examinations (patients #5, #8, and #13; Figure 5). Information from subsequent follow-ups was available for two of these patients: a) patient #5 (interestingly, classi ed as SD by the RANO criteria) showed a slight increase in FET vol/pat (and to a lesser extent also in ADC vol/pat ) at a PET/MR examination performed two months later (TP3 in Fig 4), and presented disease progression at an MR scan performed 4 months later; b) patient #8 showed a signi cant increase in FET vol/pat (and to a lesser extent also in ADC vol/pat ) at a subsequent PET/MR examination (TP3 in Fig 4), and was consistently considered progressive according to the RANO criteria. We were able to carry out a follow-up PET/MR in another two cases (patients #9 and #16, both SD at the PET/MR examination after two cycles of regorafenib): a) patient # 9 presented minimal variations in FET vol/pat and ADC vol/pat after two cycles of regorafenib, and remained stable (presenting a decrease in FET vol/pat and ADC vol/pat ) at the follow-up PET/MR (TP3 in Fig 4); b) patient #16 showed a signi cant increase in both FET vol/pat and ADC vol/pat between the PET/MR performed after two cycles of regorafenib (Fig 4) and the follow-up PET/MR, and was considered progressive according to the RANO criteria.
These four cases, although insu cient to draw de nitive conclusions, seem to show consistent variations in FET and ADC pathological volumes in followup examinations performed after six cycles of regorafenib, and con rm the greater predictive value of these parameters compared with the standard RANO criteria. In fact, the variations in FET and ADC predicted the follow-up in three out of four cases.
Moreover, it is notable that an increase in both ADC and FET pathological volumes (in the PET/MR after two cycles of regorafenib) predicted disease progression in subjects #5 and #8 (who were categorized as stable according to the RANO criteria). The so identi ed 18 F-FET and ADC areas and values, which are correlated but were obtained from completely different measures, could serve as independent biomarkers of treatment response. It is worth bearing in mind, however, that the RANO criteria include cutoffs for the percentage change in contrast-enhancing tumor volume, and small increases in contrast-enhanced volumes are not su cient to classify a patient as progressive. Therefore, appropriate thresholds should be established for the newly proposed DWI-and FET-derived parameters in prospective case series with adequate outcome measures.
Our study has some limitations, including that regorafenib was introduced only recently: 1. It was retrospective in nature and included only a relatively small number of patients; 2. Adequate follow up was not available for all patients due to the novelty of the treatment; 3. The current RANO criteria were assumed as gold standard.
Despite these limitations, we focused on a highly homogeneous patient population comprising GBM subjects at their rst disease relapse, and all patients were treated with a recently approved chemotherapeutic agent (regorafenib). Moreover, all imaging studies were acquired at the same institution with an integrated PET/MR system and a standardized protocol.

Conclusions
In the present study, we have proposed a method to identify the pathological ADC volume based on the corresponding 18 F-FET positive region in intrinsically co-registered 18 F-FET PET/MR images. We found a correlation between the percentage changes in pathological FET and DWI-ADC volumes in glioblastoma patients treated with regorafenib at their rst disease relapse. In 4/16 cases followed up with a third PET/MR, the results seemed encouraging compared to the RANO criteria. The 18 F-FET and ADC metrics identi ed could, given they were correlated but obtained from completely different measures, serve as semiquantitative independent biomarkers of response to regorafenib treatment.

Declarations
Compliance with Ethical Standards: Funding: This research received no external funding.
Disclosure of potential con icts of interest: All the authors declare no con ict of interest.
Research involving human participants and/or animals: All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. This was a single-center, retrospective, observational study conducted after formal approval by our local Ethics Committee (protocol number: AOP1673 -4831/AO/20).
Informed consent: All patients gave written informed consent before undergoing the 18F-FET PET/MR, including access to their data for research purposes.
Data availability: Due to the retrospective nature of this research, participants of this study did not explicitly agree for their data to be shared publicly, so supporting data is not available.   Figure 1 Variations in the ADC-derived parameters after regorafenib in study patients sorted into response groups according to RANO criteria. Left, the variation in pathological ADC volume (ADCvol/pat); center, the variation in the mean ADC values in the pathological ADC volume (mean ADCvol/pat); right, the variation in pathological FET volume (FETvol/pat). SD = Stable Disease; PD = Progressive Disease Figure 2 Scatter plots of the variation in pathological ADC volume (ADCvol/pat) versus the corresponding pathological FET volume (FETvol/pat) after 2 cycles of treatment with regorafenib. Red and green dots represent the patients presenting with progressive and stable disease after regorafenib, respectively. The regression line is shown in blue, and the gray area represents the 95% con dence interval. SD = Stable Disease; PD = Progressive Disease.

Figure 3
Box plots of the variation in the pathological ADC volume (ADCvol/pat) after two cycles of regorafenib. Study patients were subdivided into two groups: one including patients with at least a duplication in FETvol/pat after treatment with regorafenib (in yellow), the other including subjects with a less than twofold increase in FETvol/pat (in green).

Figure 4
Variations in the pathological FET volume (FETvol/pat) and pathological ADC volume (ADCvol/pat) between timepoints (TP1, TP2 and TP3 representing, respectively, the rst baseline PET/MR before regorafenib, the second PET/MR after two cycles of regorafenib, and the third PET/MR after six cycles of regorafenib). Subjects 5, 8, 9 and 16 were classi ed (at the latest PET/MR) as progressive disease (8,16) or stable disease (5,9) according to RANO criteria. Figure 5