Head-to-head Intra-individual Comparison of [68Ga]-FAPI and [18F]-FDG PET/CT in Patients with Bladder Cancer

Aim/Purpose Fibroblast activation protein-(FAP)-ligands, a novel class of tracers for PET/CT imaging, demonstrated promising results in previous studies in various malignancies compared to standard [18F]FDG PET/CT. 68Ga-labeled fibroblast activation protein inhibitor-([68Ga]Ga-DOTA-FAPI)-PET/CT impresses with sharp contrasts in terms of high tumor uptake and low background noise leading to clear delineation. [18F]FDG PET/CT has limited accuracy in bladder cancer due to high background signal. Therefore, we sought to evaluate the diagnostic potential of [68Ga]FAPI in patients with bladder cancer. Material and Methods This retrospective analysis consisted of 8 patients (median age 66), 7 of whom underwent both [68Ga]FAPI and [18F]FDG PET/CT scans with a median time interval of 5 days (range 1–20 days). Quantification of tracer uptake was determined with SUVmax and SUVmean. Furthermore, the tumor-to-background ratio (TBR) was derived by dividing the SUVmax of tumor lesions by the SUVmax of adipose tissue, skeletal muscle, and blood pool. Results Overall, 31 metastases were detected in five patients including lymph node metastases (n = 23), bone metastases (n = 4), lung metastases (n = 3), and a peritoneal metastasis (n = 1). In one patient, [68Ga]FAPI demonstrated significant uptake in the primary tumor located in the bladder wall. [68Ga]FAPI-PET/CT demonstrated significantly higher uptake compared to [18F]FDG PET/CT with higher mean SUVmax (8.2 vs. 4.6; p = 0.01). Furthermore, [68Ga]FAPI detected additional 30% (n = 9) lesions, missed by [18F]FDG. TBR demonstrated favorable uptake for [68Ga]FAPI in comparison to [18F]FDG. Significant differences were determined with regard to metastasis/blood pool ([68Ga]FAPI 5.3 vs [18F]FDG 1.9; p = 0.001). Conclusion [68Ga]FAPI-PET/CT is a promising diagnostic radioligand for patients with bladder cancer. This first described analysis of FAP-ligand in bladder cancer revealed superiority over [18F]FDG in a small patient cohort. Thus, this so far assumed potential has to be confirmed and extended by larger and prospective studies. Supplementary Information The online version contains supplementary material available at 10.1007/s11307-022-01715-3.

platelet derived growth factor receptor-alpha and -beta (PDGFR-α/-β) and especially in advanced stages significantly increased FAP-expression [12,13]. FAP is a type II transmembrane serine protease with post proline dipeptidyl peptidase as well as endopeptidase activity and its increased expression appears to be an independent adverse prognostic factor in urothelial bladder cancer [13]. Similar correlations are observed in breast cancer, colorectal cancer, ovarian cancer, and pancreatic ductal adenocarcinoma. FAP-expression is also observed in chronic inflammation and fibrotic diseases [14,15].
This multicenter, retrospective pilot analysis aimed to evaluate the potential role of [ 68 Ga]FAPI-PET/CT in the assessment of bladder cancer in comparison with [ 18 F]FDG in patients with bladder cancer regarding biodistribution and tumor uptake.

Patient cohort
Eight male patients were recruited from three centers (University Hospital Pretoria n = 4, Azerbaijan National Centre of Oncology n = 3, and University Hospital Heidelberg n = 1) with histopathologically confirmed therapy-naïve or pretreated bladder cancer (early and advanced stages). These patients underwent both [ 18

PET image acquisition
Imaging data was acquired 60 min after tracer application and whole body images encompassing from the head to midthigh for both [ 18 F]FDG PET/CT and [ 68 Ga]FAPI PET/CT. All PET scans were performed in 3D mode with an acquisition time of 3-5 min/bed position at all sites. All patients were monitored for adverse events up to 30 min after the end of the examination. The median time interval between [ 18 F]

Introduction
Urothelial carcinoma is the most common (> 90%) cell type of bladder cancer associated with worldwide highly varying incidence and prevalence rates mainly depending on environmental factors [1][2][3][4]. While bladder cancer in its early clinical stages (non-muscle-invasive bladder cancer, NMIBC ≤ T1) has an overall good prognosis, it nevertheless is associated with high recurrence rates (40-70% rate within 5 years). The prognosis in advanced clinical stage disease (muscle-invasive bladder cancer; MIBC ≥ T2) is poor due to the early development of distant metastases. Accurate staging plays a crucial role for proper patient stratification and therapy management [5,6].
Besides T-stage, nodal status is the most important prognostic factor that correlates with 5-year disease-free survival. Furthermore, the extent of nodal metastasis shows a direct correlation with T-status at the time of initial presentation which reveals a lymph node involvement of approximately 30% in T2 and up to 60% in ≥ T3 cancers [7]. The imaging modalities of computed tomography urography (CTU) and multiparametric magnetic resonance imaging (mpMRI) along with the urological examination methods provide a clinically acceptable diagnostic performance in patients with early clinical stages (NMIBC), whereas the diagnostic performance of the conventional imaging modalities for initial tumor staging has been disappointing due to their low sensitivity for lymph node involvement (≤ 50%) in patients with advanced clinical stages (MIBC) (7)(8)(9). Although 2-deoxy-2-[ 18 F] fluoro-D-glucose positron emission tomography/computed tomography ([ 18 F]FDG PET/CT) is substantially better than conventional imaging modalities in tumor surveillance and therapy response monitoring, its diagnostic performance in detection of lymph node involvement for initial tumor staging in advanced clinical stages is only slightly better than conventional imaging (up to 56%) [7,8]. The renal clearance and high tracer accumulation in the urinary bladder are further limiting factors for the use of [ 18 F]FDG PET/CT in primary tumor detection despite techniques such as urinary catheterization and administration of diuretics to reduce bladder activity [9][10][11].
Cancer-associated fibroblasts (CAFs) in the tumor microenvironment enhance pro-tumorigenic effects in many cancers including bladder cancer, which influences as a part of supportive tumor stroma various aspects of tumor development and progression as well as therapeutic response. CAFs promote tumorigenesis in urothelial bladder carcinoma via multiple markers including alpha smooth muscle actin (ASMA), CD90/Thy-1, fibroblast activation protein (FAP), 5 days (range 1-20 days). There was no change in therapy between the scans.

Image analysis
Tracer uptake was quantified by mean and maximum standardized uptake values (SUV mean and SUV max ) for both [ 68 Ga] FAPI and [ 18 F]FDG PET/CT scans. SUV values were determined by drawing volumes of interest (VOIs) on metastatic lesions observed with [ 18 F]FDG and [ 68 Ga]FAPI. Volumes of interest (VOI) were placed over the normal organs by one UKD investigator (EN; supervised by FLG) with a diameter of 1 cm for small organs (thyroid, parotid gland, myocardium, oral mucosa, and spinal cord) or 2 cm for other organs (brain, muscle, liver, spleen, kidney, fat, aortic lumen, and lung). Circular regions of interest (ROI) were placed on axial slices around lesions with focally increased tracer uptake and were automatically incorporated into a 3-dimensional volume of interest (ESoft; Siemens). A 40% iso-contouring approach was used for organs as well as lesions. Only unequivocally positive lesions on the [ 18 F]FDG or [ 68 Ga]FAPI scan, whether primary or metastatic, were considered to be suitable for further analysis. The lesions are classified as unequivocally positive, when the SUV max is more than 3 times that of the blood pool. Tumor-to-background ratios (TBRs) were determined to quantify the image contrast. TBR was calculated by dividing the SUV max of the metastases by background values (blood pool, fat tissue and skeletal muscle).

Statistical analysis
We used descriptive analyses for demographics, tumor characteristics and tracer uptake. Comparison between [ 68 Ga] FAPI-and [ 18 F]FDG PET/CT-SUV metrics in tumor and normal tissue as well as TBRs was performed with the paired t-test and Wilcoxon-Mann-Whitney-Test. A p value of < 0.05 was considered statistically significant. All statistical analyses were performed using SigmaStat Version 3.5 (Systat Software, Inc., San Jose, CA, USA) and SigmaPlot Version 11.0 (Systat Software, Inc., San Jose, CA, USA) for graphical visualization.

Study population
Eight male patients were included in the study with a median age of 66 years (range 57-78). Four of them had been newly diagnosed with bladder cancer, while four patients were under evaluation for recurrent disease with advanced tumor stage. Two of the patients with newly diagnosed bladder cancer were advanced stage (IIIA and IVB), while the other two patients had localized disease (Stage I). Both of the latter two patients demonstrated a local, intracystic lesion of NMIBC, which is consistent with the previous studies that patients with NMIBC are much less likely to exhibit metastatic lesions (5,6). The patients with recurrent bladder cancer had received either a local BCG-Therapy (intravesical Bacillus Calmette-Guérin immunotherapy) following a TUR-B-(Transurethral resection of bladder tumor)-Resection or a radical cystoprostatectomy with a neobladder reconstruction. In total, we detected 31 metastatic lesions in five patients (median metastatic lesion 7, range 2-11).
Staging of bladder cancer currently employs several conventional imaging tools. CT Urography (CTU) is the method of choice for primary bladder cancer staging but has relatively poor soft tissue resolution for muscular invasion and also a very low sensitivity for lymph node involvement. In light of the limitations of CTU, mpMRI is considered to be an alternative method for local staging of bladder cancer because of its higher soft-tissue contrast resolution. Although it can assess muscular invasion more successfully, it also has very limited sensitivity and specificity for nodal involvement. Bladder cancer can be detected with [ 18 F]FDG PET/CT scans; however, it has been of limited use for primary staging of bladder cancer due to high renal clearance and tracer accumulation in the urinary bladder. However, [ 18 F]FDG PET/ CT imaging is relatively insensitive for nodal involvement [7]. Since lymph node involvement directly correlates with T-stage, differentiation of T2 and ≥ T3 tumor is essential for accurate interpretation of [ 18 F]FDG PET/CT. Thus, conventional imaging modalities such as mpMRI, CTU, and [ 18 F] FDG PET/CT are relatively poor at establishing nodal status in primary tumor staging which has implications for therapy planning and prognosis in patients with bladder cancer [7,8].
In the primary setting, improved tumor delineation by [ 68 Ga] FAPI would facilitate planning of treatment in cases with high grade carcinoma in situ or locally advanced disease. However, [ 68 Ga]FAPI, like [ 18 F]FDG, is excreted via urinary tract leading to high background within the bladder, thereby masking uptake in the primary tumor and requiring additional measures such as forced diuresis and/or voiding before scanning. Therefore, it is expected that the one patient in our series in whom it was possible to identify the primary tumor, is an unusual event, however, this needs to be confirmed in a larger cohort. However, with regard to N staging, this study suggests more promising results and may find a role in initial staging or recurrence.
Although different equipment and imaging protocols were utilized at the three centers involved, the findings were consistent across sites (Supplementary Table 1), particularly in terms of detected metastatic lesions based on increased tumor uptake as well as a significantly higher tumor-to-background ratio (SUV max tumor/blood pool). Since the most frequently used FAPI-ligands [ 68 Ga]FAPI-04 and [ 68 Ga]FAPI-46 are considered to be interchangeable due to comparable biodistribution at 1 h p.i. and similar diagnostic performance so that the use of both agents in this study is not thought to be a problem [19,23].
[ 68 Ga]FAPI-based tumor assessment may improve treatment monitoring, especially after immune checkpoint inhibitors. It is well known that [ 18 F]FDG may result in false positives due to pseudoprogression. As [ 68 Ga]FAPI PET reflects the population of CAFs in the tumor microenvironment and CAFs are thought to decrease in number during response to therapy, it may be a better method of assessing patients thought to have pseudoprogression.
There are several limitations to this study, particularly its small size. Additionally, no histopathological verification was possible for lesions other than primary bladder lesions. In light of the important role of the histopathological subtypes of bladder cancer for the prognosis, we cannot perform a correlation between tumor grading and [ 68 Ga]FAPI uptake due to the small patient cohort. Furthermore, an analysis of TBR and specific tracer uptake of the various metastatic lesions could not be performed.

Conclusion
To our knowledge, this is the first investigation to evaluate the potential of FAP-ligands in bladder cancer.  Acknowledgements The authors gratefully acknowledge all participating patients.
Funding Open Access funding enabled and organized by Projekt DEAL.
Data availability The data used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Declarations
Ethics approval and consent to participate All procedures performed in studies involving human participants were approved by ethics committee and carried out in accordance with the ethical standards of the institutional and/or national research committees and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. Informed consent was obtained from all individual participants included in the study.
Conflict of interest UH and FLG have a patent application for quinolone based FAP-targeting agents for imaging and therapy in nuclear medicine. UH and FLG also have shares of a consultancy group for iTheranostics. FLG is also advisor at ABX, Telix, and SOFIE Biosciences. SAK reports grants from Viewray Inc. and honoraria from IBA Dosimetry, outside the submitted work. The other authors declare no conflict of interest regarding this manuscript.
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Fig. 5
The depiction of overall metastatic lesions in a box-plot, FAPI vs. FDG