Superior Tumor Detection for 68Ga-FAPI-46 Versus 18F-FDG PET/CT and Conventional CT in Patients with Cholangiocarcinoma

Visual Abstract

Management of cholangiocarcinoma is among other factors critically determined by accurate staging. Here, we aimed to assess the accuracy of PET/CT with the novel cancer fibroblast-directed 68 Gafibroblast activation protein (FAP) inhibitor (FAPI)-46 tracer for cholangiocarcinoma staging and management guidance. Methods: Patients with cholangiocarcinoma from a prospective observational trial were analyzed. 68 Ga-FAPI-46 PET/CT detection efficacy was compared with 18 F-FDG PET/CT and conventional CT. SUV max /tumor-to-background ratio (Wilcoxon test) and separately uptake for tumor grade and location (Mann-Whitney U test) were compared. Immunohistochemical FAP and glucose transporter 1 (GLUT1) expression of stromal and cancer cells was analyzed. The impact on therapy management was investigated by pre-and post-PET/CT questionnaires sent to the treating physicians. Results: In total, 10 patients (6 with intrahepatic cholangiocarcinoma and 4 with extrahepatic cholangiocarcinoma; 6 with grade 2 tumor and 4 with grade 3 tumor) underwent 68 Ga-FAPI-46 PET/CT and conventional CT; 9 patients underwent additional 18 F-FDG PET/CT. Immunohistochemical analysis was performed on the entire central tumor plain in 6 patients. Completed questionnaires were returned in 8 cases. Detection rates for 68 Ga-FAPI-46 PET/CT, 18 F-FDG PET/CT, and CT were 5, 5, and 5, respectively, for primary tumor; 11, 10, and 3, respectively, for lymph nodes; and 6, 4, and 2, respectively, for distant metastases. 68 Ga-FAPI-46 versus 18 F-FDG PET/CT SUV max for primary tumor, lymph nodes, and distant metastases was 14.5 versus 5.2 (P 5 0.043), 4.7 versus 6.7 (P 5 0.05), and 9.5 versus 5.3 (P 5 0.046), respectively, and tumor-tobackground ratio (liver) was 12.1 versus 1.9 (P 5 0.043) for primary tumor. Grade 3 tumors demonstrated a significantly higher 68 Ga-FAPI-46 uptake than grade 2 tumors (SUV max , 12.6 vs. 6.4; P 5 0.009). Immunohistochemical FAP expression was high on tumor stroma (90% of cells positive), whereas GLUT1 expression was high on tumor cells (80% of cells positive). Overall, average expression intensity was estimated as grade 3 for FAP and grade 2 for GLUT1. Positive 68 Ga-FAPI-46 PET findings led to a consequent biopsy workup and diagnosis of cholangiocarcinoma in 1 patient. However, patient treatment was not adjusted on the basis of 68 Ga-FAPI-46 PET. Conclusion: 68 Ga-FAPI-46 demonstrated superior radiotracer uptake, especially in grade 3 tumors, and lesion detection in patients with cholangiocarcinoma. In line with this result, immunohistochemistry demonstrated high FAP expression on tumor stroma. Accuracy is under investigation in an ongoing investigator-initiated trial. Chol angiocarcinomas originate from intra-and extrahepatic locations of the biliary tract (1). They are the second most common liver malignancy (2), are rising in incidence (3) and are often diagnosed late, frequently leading to a fatal outcome (4). Primary tumors are typically diagnosed by contrast-enhanced and diffusionweighted MRI with MR cholangiopancreatography (5). Additional imaging by whole-body CT is offered for the detection of distant metastases and vascular involvement (6).
Current guidelines do not routinely recommend PET/CT for the diagnosis and staging of biliary tract malignancies. These recommendations refer to imaging using the radioactive tracer 18 F-FDG (6). The accuracy of 18 F-FDG is limited by intertumoral heterogeneous uptake, that is, a high glycolytic rate for high-grade cholangiocarcinoma and a low glycolytic rate for low-grade cholangiocarcinoma (7,8).
In recent years, quinoline-based fibroblast activation protein (FAP)specific inhibitors (9) coupled to 68 Ga have been developed for PET imaging. FAP is expressed by predominantly cancer-associated fibroblasts in the stroma of various tumor entities, leading to highly tumor-specific expression (10).
Because of an abundant tumor stroma whose main cellular components are cancer-associated fibroblasts, cholangiocarcinoma is a promising tumor entity for 68 Ga-FAP inhibitor (FAPI)-46 PET imaging (11).
Previous publications without a systematic histopathologic workup indicated FAP-directed PET to be highly accurate for the imaging of cholangiocarcinoma (12,13). Here, we performed a head-to-head comparison of 68 Ga-FAPI-46 PET, 18 F-FDG PET, and contrastenhanced CT and compared the efficacy of these 3 modalities for cholangiocarcinoma detection. Furthermore, we investigated immunohistochemical FAP and glucose transporter 1 (GLUT1) expression from tumor samples of our patient cohort and assessed the impact of 68 Ga-FAPI-46 PET/CT on cholangiocarcinoma management.

Patient Population
The patient flowchart is shown in Figure 1. This is a subgroup analysis of the ongoing observational trial (NCT04571086) at the University Hospital Essen. Until August 2021, 10 patients with cholangiocarcinoma were included (1.8% of the entire trial). Before enrollment, patients gave written informed consent to undergo 68 Ga-FAPI-46 PET for a clinical indication. Inclusion criteria were scheduling a 68 Ga-FAPI PET examination for staging or restaging of cholangiocarcinoma in routine clinical practice and being at least 18 y old. Pregnant, lactating, or breastfeeding women, as well as patients unable to tolerate PET scanning, were excluded. This study was approved by the local Ethics Committee (permits 19-8991-BO and 20-9485-BO).
Patients were not required to be fasting at the time of application and did not require specific preparation. The median intravenously administered activity was 89 MBq (interquartile range [IQR], 79-128 MBq). The median uptake time was 15 min after injection (IQR, 10-38 min). Lowdose CT was performed without application of intravenous contrast medium. Clinical PET/CT scans were obtained in the craniocaudal direction on a Biograph mCT Vision scanner (Siemens Healthineers) (15). 18 F-FDG PET/CT. 18 F-FDG PET/CT was performed in 8 of 10 (80%) patients and 18 F-FDG PET/MRI in 1 of 10 (10%). One patient did not undergo additional 18 F-FDG PET/CT. The median injected activity was 317 MBq (IQR, 266-344 MBq). The median uptake time was 63 min after injection (IQR, 54-80 min after injection). Diagnostic CT was performed, and intravenous contrast medium was given to 6 of 9 (66.7%) patients. The PET protocol was in accordance with the European Association of Nuclear Medicine procedure guidelines for tumor imaging, version 2.0 (16).
Conventional CT. Conventional CT was performed on all patients either as part of 18 F-FDG PET/CT (n 5 5) or as a stand-alone examination before PET/CT (n 5 5); the median interval between 68 Ga-FAPI-46 PET/CT and CT was 17 d (range, 0-36 d). In all patients, diagnostic CT was acquired after application of intravenous contrast medium in the arterial and portal venous phases.

Image Evaluation
For comparison of 68 Ga-FAPI-46 and 18 F-FDG PET/CT, a lesion-based analysis of SUV max , SUV mean , SUV peak , and metabolic tumor volume was performed in consensus by 2 independent, masked readers. For calculation of SUV mean and metabolic tumor volume, volumes of interest were determined by an isocontour threshold of 41% of SUV max . Syngo.via software (Siemens Healthineers) was used for measurements of SUV and metabolic tumor volume (16). Lesions visible on only one PET modality were compared with the background of the other PET modality in the same region for statistical reasons. Three regions were selected for evaluation of tumorto-background ratios (TBRs) using a spheric region of interest: mediastinal blood pool (center of the aortic arch), liver (noninvolved area of the right lobe), and left gluteal muscle (center of the left gluteus). Diagnostic CT was analyzed in consensus by 2 independent, masked radiologists.

Detection Efficacy
Detection efficacy was assessed through lesion-based evaluation of 68 Ga-FAPI-46 PET/ CT, 18 F-FDG PET/CT, and conventional CT in 9 of 10 patients. Each detected lesion was considered positive, regardless of the imaging modality. On PET, areas with focal uptake above the background level, not attributable to physiologic findings, were rated positive. On CT, lymph nodes larger than 1 cm in short diameter with suggestive features (contrast enhancement and a round shape, among others) were considered positive. Furthermore, on CT, morphologically delineated or hyperarterialized organ lesions were considered suggestive of malignancy. Follow-up imaging (CT or PET/CT), clinical data, or histologic confirmation were used as the standard of truth.

Immunohistochemical Analysis of FAP and GLUT1 Expression
Immunohistochemistry was performed on formalin-fixed paraffinembedded human tissue samples according to the standard laboratory procedures (17). The following antibodies were used: anti-GLUT1 Abcam ab652 (RRID:AB 305540), diluted 1:5,000; anti-FAP a-antibody (SP325); and Abcam ab227703, diluted 1:100. Immunohistochemical expression was evaluated on tumor cells and tumor stroma, and the percentage of intratumoral necrosis related to the tumor areas was also assessed. A simplified visual FAP/GLUT1 grading was applied for stromal and tumor cells, as well as for necrosis. A FAP/GLUT1 grading legend is shown in Table 1. For larger neoplasms, a central slice of the tumor was stained completely. Immunohistochemical analyses were performed on a resection of bioptic samples of the primary or localrecurrence tumors before 68 Ga-FAPI-46 or 18 F-FDG PET/CT and consequently do not correspond to visible PET lesions. Two pathologists and 2 biologists performed masked immunohistochemical analysis in consensus.

Statistical Analysis
Descriptive statistics and individual patient data are reported. For continuous data, the median, IQR, and range were used. SUV max , SUV mean , and TBR were compared using the Wilcoxon test. The Mann-Whitney U test was performed to compare subgroups for tumor grade and location. To demonstrate the results, visualization with scatterplots was used, with a P value of less than 0.05 being considered statistically significant. All analyses were performed using SPSS Statistics (version 27.0; IBM).

Patient Characteristics
Overall, 10 patients (6 men and 4 women) were reviewed. The median age was 55.5 y (range, 40-79 y). Included were 6 patients with intrahepatic cholangiocarcinoma and 4 patients with extrahepatic cholangiocarcinoma.
We performed initial staging in 2 patients and restaging in 8. The median interval between diagnosis and initial staging or restaging was 1 mo or 22 mo (range, 5-56 mo), respectively, whereas the median interval was 17 d (range, 0-36 d) between 68 Ga-FAPI-46 PET/CT and conventional CT and 0 d (range, 0-35 d) between 68 Ga-FAPI-46 PET/CT and 18 F-FDG PET/CT. Further clinical information can be found in Table 2.

Detection Efficacy
Detection efficacy is summarized in Table 3. Figure 2 shows maximum-intensity projections of all 10 patients. Overall, 22 lesions were detected across all modalities, including primary tumors (n 5 5), lymph node metastases (n 5 11), and distant metastases (n 5 6). All primary tumors were detected by all 3 imaging modalities.    Figure 3A.

Change in Management
Eight of 10 questionnaire pairs were completed by the referring physicians. According to the survey, diagnostic tests were not avoided or triggered, and intended therapy did not change in any patient. In 1 patient with an unknown primary, 68 Ga-FAPI-46 PET/CT localized the tumor. Subsequent biopsy with immunohistochemical analysis led to a cholangiocarcinoma diagnosis.   Table 2. N.A. 5 not applicable.

FAP and GLUT1 Immunohistochemistry
FAP and GLUT1 immunohistochemistry findings are shown in Figures 6A-6C. Surgical samples of primary tumors (n 5 5) or local recurrences (n 5 1) from 6 of 10 patients were examined. Figure 6D demonstrates FAP and GLUT1 expression within a tumor sample. According to visual assessment (Table 1) Currently, the only curative treatment for cholangiocarcinoma is radical surgery of the primary tumor, including lymphadenectomy (6). Patients with unresectable intrahepatic cholangiocarcinoma may benefit from local ablative interventions, such as radioembolization with 90 Y-microspheres or transarterial chemoembolization (18). In the presence of distant metastases, systemic chemotherapy is the therapy of choice (6). Accurate staging is therefore crucial for management of cholangiocarcinoma.
MRI in combination with MR cholangiopancreatography is the clinical standard for local detection of cholangiocarcinoma (6). According to the guidelines of the European Society for Medical Oncology, additional contrast-enhanced CT determines the relationship between tumor and vasculature (6). Contrast-enhanced CT is currently the imaging modality of choice for staging lymph nodes and distant metastases, although sensitivity and specificity vary significantly across studies (lymph node metastases: sensitivity, 67% [95% CI, 28%-86%]; specificity, 88% [95% CI, 74%-95%]) (19). 18 F-FDG PET/CT shows advantages in detecting small cholangiocarcinomas as well as lymph node and distant metastases (20)(21)(22).
However, extrahepatic cholangiocarcinomas and low-grade tumors are difficult to detect because of reduced 18 F-FDG or a high background signal (8). Here, we show the lowest detection rates for contrast-enhanced CT: we attribute this in particular to the size, exemplified by lymph node metastases, which partly presented at 10 mm or smaller in the investigated cohort. 68 Ga-FAPI-46 is a novel radioligand that binds to FAP in the tumor stroma and has shown high detection rates for stroma-rich tumors (23). FAP is selectively expressed at high levels by cancer-associated fibroblasts (24,25) in more than 90% of human epithelial cancers (26).
Recently, Kratochwil et al. reported a high 68 Ga-FAPI PET SUV max for cholangiocarcinoma (12). In addition, Lan et al. compared detection efficacy for biliary tract   cancer of primary tumors, lymph nodes, and distant metastases between 68 Ga-FAPI and 18 F-FDG PET/CT and showed 68 Ga-FAPI to be superior in all 3 subgroups (13). Here, we confirm that 68 Ga-FAPI-46 PET/CT is superior to 18 F-FDG PET/CT, and also to conventional CT, for detection of primary tumor but especially for detection efficacy for lymph node and distant metastases.
Here we, for the first time to our knowledge, present a systematic immunohistochemistry assessment of the imaging cohort. Immunohistochemistry showed high and very specific FAP expression in tumor stroma whereas GLUT1 was expressed mainly on cholangiocarcinoma tumor cells. A high expression level of FAP in tumor stroma was reported previously (23,27). Cholangiocarcinoma typically presents with a pronounced stromal compartment, which consists mainly of cancer-associated fibroblasts (28,29). The tumorspecific FAP expression, high stromal content in cholangiocarcinoma and good specificity and retention properties of 68 Ga-FAPI-46 radioligand probably led to the observed superior TBR and detection rate. In contrast, GLUT1 is a universal glucose transporter that is expressed in many healthy cells in the body, contributing to a higher background level in liver and blood pool that leads to lower TBR ratios and a lower detection specificity for 18 F-FDG PET.
We could not detect major changes in tumor treatment, mainly because most patients presented for restaging and metastatic stage was already known. With limited therapeutic options for cholangiocarcinoma, the treatment of choice was mostly already performed or planned.
Efficacious treatment options for cholangiocarcinoma are limited (6). In the past decade, target-directed radioligand therapy (RLT) combined with PET, so-called radiotheranostics, has seen unprecedented expansion (30). Theranostic ligands are carrierbound small molecules that provide diagnostic imaging or therapy depending on the type of radiolabel. Novel RLT has led to prolonged survival in patients with metastatic neuroendocrine tumors ( 177 Lu-DOTATOC) (31) and prostate cancer ( 177 Lu-PSMA) (32). RLT is characterized by favorable safety and improvement of health-related quality of life (33).
Our study comes with limitations. 18 F-FDG PET was mostly combined with contrast-enhanced CT, whereas 68 Ga-FAPI-46 PET/CT was performed as low-dose CT without a contrast agent. This may affect attenuation correction and SUV quantification. However, Schoen et al. (38) did not find a significant difference with respect to the SUV max of the liver or muscle, for PET/CT with or without contrast enhancement. Other limitations are a small number of patients and the retrospective design. An ongoing prospective interventional investigator-initiated trial (NCT 05160051) aims to assess diagnostic accuracy and target expression in a larger cohort of patients.

CONCLUSION
In patients with cholangiocarcinoma, 68 Ga-FAPI-46 demonstrates superior radiotracer uptake, especially in grade 3 tumors, and improved lesion detection when compared with 18   Pharma15. Katharina Lueckerath is a consultant for SOFIE Bioscience. Stefan Kasper receives honoraria from Merck Serono, MSD, Novartis, BMS, Amgen, Roche, Sanofi-Aventis, Servier, Incyte, and Lilly and research funding from Merck Serono, Lilly, BMS, and Roche. Ken Herrmann receives personal fees from Bayer, Sofie Biosciences, SIRTEX, Adacap, Curium, Endocyte, IPSEN, Siemens Healthineers, GE Healthcare, Amgen, Novartis, ymabs, Aktis Oncology, and Pharma15; nonfinancial support from ABX; and grants or personal fees from BTG. Rainer Hamacher is supported by the Clinician Scientist Program of the University Medicine Essen Clinician Scientist Academy (UMEA) sponsored by the faculty of medicine and Deutsche Forschungsgemeinschaft (DFG); has received travel grants from Lilly, Novartis, and PharmaMar; and has received fees from Lilly and PharmaMar. Wolfgang Fendler receives research funding from SOFIE Bioscience and Bayer; is a consultant to Janssen, Calyx, and Bayer; is on the speakers bureau for Janssen, Bayer, Novartis, and Telix; and does image review for Parexel. No other potential conflict of interest relevant to this article was reported.

KEY POINTS
QUESTION: Is 68 Ga-FAPI-46 PET/CT superior to 18 F-FDG PET/CT and conventional CT in a head-to-head comparison for staging cholangiocarcinoma?
PERTINENT FINDINGS: 68 Ga-FAPI-46 PET/CT is superior to both other imaging modalities for detection efficacy, uptake intensity, and TBR. In line with these findings, immunohistochemistry demonstrates high FAP expression of the tumor samples.
IMPLICATIONS FOR PATIENTS CARE: 68 Ga-FAPI-46 is a promising novel diagnostic test for staging cholangiocarcinoma. In the future, FAP-directed RLT may represent a new treatment option.