Discrimination between Inflammatory and Fibrotic Activity in Crohn’s Disease-Associated Ileal-Colonic Anastomotic Strictures by Combined Ga-68-FAPI-46 and F-18-FDG-PET/CT Imaging Open Access

Crohn’s disease (CD), which is one of the main entities of inflammatory bowel diseases (IBD), is a chronic, debilitating, immune-mediated disorder of the gastrointestinal tract, which is characterized by a relapsing-remitting course [1]. Its clinical symptoms comprise abdominal pain, diarrhoea, weight loss, nausea, general malaise and fatigue [2]. The progressive nature of the disease is exemplified by the frequent development of intestinal strictures, which are commonly accompanied by obstructive symptoms in affected patients [3]. In a prospective population-based inception cohort study, up to 21% of patients present with stricturing CD at diagnosis, and 10% of patients without baseline findings of strictures may develop symptomatic strictures over a 5‐year follow‐up [4]. In paediatric CD patients, nearly 20% of patients are found to have strictures at the time of diagnosis, increasing to 40% of patients by 10 years [5]. The mechanisms by which strictures develop in CD are complex and may be due to either excessive inflammation or exaggerated fibrotic responses. Recent data indicated that early initiation of intensified anti-inflammatory medical therapy might be associated with decreased rates of stricturing complications [6, 7], but there are currently no therapeutic agents available that primarily target intestinal fibrosis. It could be shown that intestinal fibrosis is the result of the chronic inflammatory processes in CD, which leads to the activation of myofibroblasts and the uncontrolled deposition of connective tissue in the extracellular matrix, mainly in the submucosa and subserosa [8]. Additionally, due to the described increase of smooth muscle cells and expansion of the muscle layers, there is fibrotic wall thickening and formation of stricturing disease. This interaction would explain the reported overlap between both inflammatory and fibrotic components [9]. Correspondingly, histopathological analyses in advanced strictures also detect both inflammatory and fibrotic components [10]. Their respective impact on disease prognosis and the approaches to treating them differ fundamentally and therefore need to be assessed before initiation of appropriate treatment. Predominantly inflammatory strictures necessitate intensified medical therapy, while strictures with a predominantly fibrotic component would rather benefit from balloon dilatation or surgery [11‒15]. Simultaneous assessments and quantification of inflammatory and fibrotic processes in CD associated intestinal strictures are therefore very much required in order to gauge disease prognosis accurately and tailor therapies appropriately toward one process or the other [16]. The segregation of fibrostenotic predominant from inflammatory‐predominant strictures is a prerequsite to enable optimal patient care in CD. This differentiation could also help us to understand the role of fibrosis in occurring clinical symptoms in the absence of inflammation. Furthermore, the accurate measurement of fibrosis is also essential for the development of drugs to prevent and treat fibrotic strictures [17].

However, fibrosis assessment in CD is challenging and evaluation of fibrosis remains elusive.

There are several imaging modalities, mainly intestinal ultrasound (IUS), computed tomography (CT), and magnetic resonance enterography (MRE) that are currently used in clinical practice to assess the intestinal wall [18]. Here, MRE is currently the most established and used technique in CD, allowing both the evaluation of bowel inflammation and the identification of strictures [18, 19]. Given its accuracy and its lack of ionizing radiation exposure, the CD anti‐fibrotic STRICTure therapies (CONSTRICT) group recommended MRE as the ideal test for assessing and following‐up on CD strictures [20]. However, currently used imaging methods are not able to quantify inflammatory and fibrotic activity in the intestinal strictures [19, 21]. Therefore, there is a direct clinical need to develop novel imaging methods that enable differentiation between fibrosis and inflammation in stricturing CD. Here, positron emission tomography/computed tomography (PET) as a hybrid imaging method, combines anatomical and molecular information. The most common tracer, 18F-fluorodeoxyglucose (FDG) can be used to locate inflammatory processes within a patient and was also found to correctly identify inflamed bowel segments in CD [22, 23]. However, the use in fibrotic disease is limited [20].

Recently, small molecule inhibitors of the fibroblast activating protein (FAPI) linked to Gallium-68 (Ga-68) and used as PET tracers have emerged for the imaging of fibrotic disorders. Fibroblast activating protein (FAP) is a surface glycoprotein overexpressed on the stroma of epithelial cancer but also on activated fibroblasts in several chronic inflammatory diseases, such as idiopathic pulmonary fibrosis or IgG₄-related diseases [24, 25]. Activated myofibroblasts showing a FAP overexpression play a crucial role in the development of intestinal fibrosis in CD [26]. First studies have shown that FAPI-PET can identify and measure fibrosis in CD patients and furthermore allows the discrimination of fibrotic and inflamed non-fibrotic strictures [27, 28]. However, stenosis with both inflammatory and fibrotic components could not be distinguished from non-inflamed fibrotic strictures [27]. Yet, given the different therapeutic approaches of anti-inflammatory and anti-fibrotic drugs, possible side-effects and the growing knowledge on the development on intestinal fibrosis, a further discrimination is highly sought after. The aim of this study was to combine the evaluation of sequentially acquired FDG-PET and FAPI-PET and to explore its potential for further differentiation of both fibrotic and inflammatory components within ileocecal strictures of patients with CD.

In this study, 3 consecutive patients with established CD diagnosis and endoscopically confirmed strictures in the anastomotic region after previous ileo-caecal resection were recruited at the First Medical Department of the University Hospital Erlangen (Erlangen, Germany). Demographic and disease-specific characteristics of the patients are summarized in Table 1. Two patients were male and one female. The mean age of the patients was 54 years and the mean disease duration 26 years. All patients had previously undergone one CD-related surgery due to stenosis of the ileo-caecal region and obstructive symptoms. All included patients had active disease with a mean Harvey-Bradshaw Index of 7. All patients were treated with a biological therapy (2 anti-TNF antibodies and 1 ustekinumab) and underwent MRE and endoscopy as part of our clinical routine proceedings to assess characteristics and extent of the stenoses. Patients underwent both FDG-PET and FAPI-PET examination between September 2022 and February 2023. All patients underwent this examination as part of the clinical workup in order to accumulate diagnostic evidence and potentially optimize their individual treatment. The Harvey-Bradshaw Index was used to assess the clinical disease activity for each patient at the time of the PET scan [29]. Two patients (patient 2 and 3) subsequently had to undergo surgical resection of the strictured anastomosis and part of the neoterminal ileum due to therapy-refractory obstructive symptoms. Surgery took place 5 months (patient 2) and 4 months (patient 3) after FDG-PET and FAPI-PET examination. There was no change in medical therapy between the time of imaging and bowel wall resection. Written informed consent was obtained from all patients prior to FDG-PET and FAPI-PET examination. The Institutional Review Board of the Friedrich Alexander Universität Erlangen-Nürnberg approved the analysis of study results (Identification Number: 23-455-Br).

Ga-68-FAPI-46 is an investigational radiopharmaceutical and not yet approved by the Food and Drug Administration or the European Medicines Agency. It was, therefore, administered under the conditions outlined in §13 (2b) of the Arzneimittelgesetz (German Medicinal Products Act) and in compliance with the Declaration of Helsinki. The fully automated GMP-compliant radiosynthesis of Ga-68-FAPI-46 started from the elution of Ga-68³⁺, which was obtained from the Ge-68/Ga-68 radionuclide pharmacy grade generator (1,850 MBq, GalliaPharmTM, Eckert and Ziegler AG, Berlin, Germany). The elution volume of Ga-68³⁺ (5.5 mL, 0.1 m HCl) was added to a solution of FAPI-46 (30 μg in 1.5 m Hepes buffer, 3 L) and the pH value was adjusted by the addition of acetic acid (0.13 mL). The reactor vial was heated to 100°C for 12 min. After trapping of 68Ga-FAPI-46 on a solid phase cartridge (Sep-Pak C18 Plus Light 130 mg, Waters, Eschborn, Germany), the solid phase was washed with water (22 mL). The elution of Ga-68-FAPI-46 was performed by the use of ethanol/water (50:50, v/v, 2 mL) and the final formulation of Ga-68-FAPI-46 was done using PBS (14 mL) and sterile filtration. The radioactivity yield of Ga-86-FAPI-46 was 47% (referred to the eluted Ga-68³⁺) after a total synthesis time of 34 min and the radiochemical purity was at least 98% as determined by radio-instant thin-layer chromatography (ITLC [SGI001, Agilent], ammoniumacetate [1 m]: methanol, 50:50, v/v, Rf = 0.7–1.0) and radio-high-performance liquid chromatography (HPLC; Chromolith Performance RP-18e, 100 × 4.6 mm, 10% acetonitrile (0.1% trifluoroacetic acid) [0–6 min], 4 mL/min, 254 nm, tR = 1.22 min).

The time interval between FDG-PET and FAPI-PET was 8 to 11 days. Both investigations were carried out on the same dedicated PET/CT system (Biograph VISION 600 Edge, Siemens Healthineers, Erlangen, Germany). The covered field-of-view was from the cervical region to mid-thigs. FDG-PET was acquired 59 to 64 min after injection of 2–3 Megabequerel (MBq) F-18-FDG/per kg of bodyweight and 20 mg of Hyoscine butylbromide intravenously. Patients were fasting at least 6 h prior to the investigation. Blood sugar levels prior to injection were in between 98 and 100 mg/dL. FAPI-PET was acquired dynamically starting with the injection of 1.5–3 MBq Ga-68-FAPI-46/per kg of bodyweight intravenously. Overall, 6 passes of PET scanning over the field-of-view were conducted, resulting in a scanning time of 40 min per patient. PET data were corrected for random and scattered coincidences, as well as for decay during scanning. PET attenuation correction was carried out by the CT portion of the multimodal acquisition using a low dose CT protocol (120 kV tube voltage, 40 mAs Ref. of tube current with CAREDOSE dose modulation (Siemens Healthineers, Erlangen, Germany). All corrections and reconstructions were obtained using the PET manufacturer’s software.

Two physicians specialized in nuclear medicine and skilled in interpreting PET scans, blinded for all clinical information of the patients, reviewed all studies, both visually and quantitatively. All reviews took place on dedicated workstations using certified software (syngo.via; Siemens Healthineers). For the evaluation, the sections of the intestine were divided as follows: stomach, small intestine, terminal ileum, caecum and ascending colon, transverse colon, descending and sigmoid colon, and rectum. For both tracers, lesions with questionable tracer uptake with uptake levels higher than the tracer uptake of the liver were rated positive by consensus. Quantitative assessment was performed based on the static PET reconstructions, using iso-contour VOIs with a threshold of 41 percent. Maximum and mean radiotracer activity as bodyweight corrected standardized uptake values (SUVs) were calculated, additionally the metabolic active lesion volume and the total lesion FAP expression quotient for FAPI-PET and the total lesion glycolysis in FDG-PET were measured. For FAPI-PET, time-activity-curves of the tracer accumulation in the suspected stenosis were calculated.

MRE was performed due to routine clinical care in all patients prior to the FAPI-PET and FDG-PET examination. The time intervals between MRE and PET scans were 7 months for patient 1, 1 month for patient 2 and 3 days for patient 3. One board certified radiologist interpreted the MRE and performed visual analysis concordantly to PET analysis.

Colonoscopy was performed due to routine clinical care in all patients within 4 months prior to the FAPI-PET and FDG-PET examination. The Simple Endoscopic Score (SES-CD) was recorded for each endoscopy [30]. Two of the strictures in the anastomotic region were not passable upon endoscopy (patients 2 and 3), while one was endoscopically just passable (patient 1).

Histopathological examination was performed in the surgically resected gut specimen of the strictures in the anastomotic region by an experienced pathologist, blinded to all clinical data of the respective patients, to evaluate the level of inflammation and fibrosis, respectively.

Statistical analysis and the creation of the graphs were performed via GraphPad Prism version 10.2.0 for Windows, GraphPad Software, Boston, Massachusetts USA, www.graphpad.com.

The first patient had an established diagnosis of ileal CD and had undergone ileocecal resection due to a stenosis and obstructive symptoms 24 years ago. Postoperative therapy included methotrexate and then adalimumab, which was stopped due to secondary loss of response after 6 years. Therapy was then switched to infliximab, which was continued in an intensified dose (10 mg/kg bodyweight every 4 weeks). Nevertheless, 5 years after infliximab initiation the patient presented himself with moderate disease activity and endoscopic examination showed heightened inflammatory activity and a strictured neoterminal ileum, which was just passable by the endoscope. The MRE examination demonstrated a moderately strictured ileo-colonic anastomosis of 3.4 cm length. The FAPI-PET showed a minimally increased, focal tracer uptake at the beginning and end of the known stenosis in the neoterminal ileum. Tracer uptake was stable over time. The rest of the gastrointestinal tract exhibited an unremarkable tracer distribution. Altogether, the tracer uptake was only minimally increased. The FDG-PET revealed a more intense and more extensive uptake in the known stenosis in the neoterminal ileum. However, there was also a noticeable and extended radionuclide accumulation in the rectosigmoid, which remained without correlation in the other examinations (Colonoscopy, MRE). This is most likely a false-positive finding, possibly due to intestinal motility. The imaging findings are demonstrated in Figure 1. In summary, the combined evaluation of FAPI-PET and FDG-PET in this case suggested a predominantly inflammatory activity with only minimal fibrotic activity. The patient remained clinically stable without obstructive symptoms for another 1.5 years after the PET scans were performed, without a need for surgical intervention.

The second patient had an established diagnosis of ileal and perianal fistulizing CD and had undergone ileocecal resection due to a stenosis and obstructive symptoms 5 years ago. Postoperative therapy consisted of adalimumab, which was continued in an intensified dose (40 mg every week). After 5 years, the patient showed moderate clinical disease activity with obstructive symptoms. Endoscopic examination showed heightened inflammatory activity and an endoscopically non-passable anastomotic stricture. The MRE examination demonstrated a strictured ileo-colonic anastomosis of 10 cm length. The patient had to undergo surgical resection of the strictured anastomosis due to significant abdominal symptoms 5 months after the PET examinations. Postoperatively, ustekinumab therapy was initiated.

In the FAPI-PET scan, a strong and extensive tracer uptake was observed in the anastomotic transition area extending into the preterminal parts of the ileum. Additionally, there were other, focal, intense tracer accumulations in the small intestine. In contrast, the FDG-PET showed a more circumscribed, focal tracer uptake exclusively in the ileo-ascending transition area, with otherwise only extended functional uptake in the transverse colon, lacking correlation in other imaging methods. The imaging findings are demonstrated in Figure 2. In summary, the distribution pattern, when considering both examinations, suggested a predominantly and extensive active fibrotic process with only a minor inflammatory component.

The third patient had an established diagnosis of ileo-colonic disease and upper gastrointestinal tract involvement and had undergone ileocecal resection due to a stenosis and obstructive symptoms 3 years ago. Postoperative therapy initially consisted of infliximab, which had to be discontinued due to progredient arthralgia and development of exanthema. Therapy was then switched to ustekinumab, which had to be applied in a shortened interval of 6 weeks during maintenance therapy due to clinical signs of secondary loss of response. After 3 years, the patient showed moderate clinical disease activity with obstructive symptoms and endoscopic examination demonstrated heightened inflammatory activity and an endoscopically non-passable anastomotic stricture. The MRE examination demonstrated a strictured ileo-colonic anastomosis of 12 cm length with signs of heightened inflammatory activity. The patient had to undergo surgical resection of the strictured anastomosis due to significant abdominal symptoms, 4 months after the PET examinations. Post-operatively, vedolizumab therapy was initiated. The FAPI-PET and the FDG-PET scans both demonstrated a clear, extensive tracer accumulation along the entire stenosis in the neoterminal ileum, as seen in the MRE. No local differences in the strength or extent of the uptake were observed. The imaging findings are demonstrated in Figure 3. Considering both the FAPI-PET and the FDG-PET findings, a high level of both fibrotic and inflammatory activity in the area of the stenosis in the neoterminal ileum can be diagnosed.

The results of the quantitative analysis of both FDG PET and FAPI-PET can be found in Table 2. Compared to the FAP expression, visually larger portions of the gastrointestinal tract exhibited increased glucose metabolism (affected segments 3 on FAPI-PET vs. 9 affected segments on FDG PET). Concerning the known strictures, we observed three different tracer distributions in the 3 patients examined. First, unremarkable to low FAPI uptake with concurrently moderate FDG retention, suggesting predominantly inflammatory activity with only minor or no fibrosis (patient 1); second, high FAPI uptake with low FDG uptake, possibly indicating predominantly fibrotic activity with only minor inflammatory involvement (patient 2); and third, significant and matching uptake in both examinations, most likely indicating both high inflammatory and fibrotic activity in patient 3 (Fig. 4a). Dynamic analysis revealed two different patterns of tracer uptake with 2 patients showing a stable FAPI uptake, while 1 patient (P2) showed a significantly increasing tracer uptake over time (Fig. 4b, c, slope = 0.123, p = 0.0045). All imaging procedures were well tolerated by the patients.

The results of the analysis can be found in Table 2. In summary, MRE revealed a pathological wall thickening in the stenotic bowel segments. Furthermore, abnormalities in contrast agent enhancement (CE) were found in these segments. A wall oedema was identified in 2 out of 3 patients. MRE was able to correctly identify and rate the extent of inflammation; however, it failed to differentiate between inflammation and fibrosis. MRE identified one additional affected bowel segment compared to FAPI-PET. In comparison, only 4 out of 9 FDG-PET positive areas showed a correlation in the MRE procedure, suggesting that these are most likely nonspecific functional radiopharmaceutical accumulations.

Resection of the stenotic bowel segment was performed in patient 2 and patient 3. In patient 2, histological examination revealed severe fibrotic activity in the resected segment of the neoterminal ileum. The inflammatory activity, however, was scored as mild with basal lymphocytic infiltration. Patient 3 on the other hand, showed signs of highly active inflammation with rather moderate fibrotic activity. Histological examination correlated well with the PET and MRE findings, as patient 2 showed both a high FAPI uptake as well as severe fibrotic changes in the histological examination. Furthermore, concordantly to a high inflammatory activity in the resected segment, patient 3 also showed high uptake in the FDG-PET and signs of acute inflammation, e.g., wall oedema and local lymphadenopathy in MRE.

To the best of our knowledge, this is the first study reporting on the results of the combined use of FAPI-PET and FDG-PET scans to assess areas of fibrotic tissue remodelling in ileo-colonic anastomotic strictures in patients with CD. We could show that it is possible to identify different uptake patterns of Ga-68-FAPI-46 in Crohn’s associated strictures. In regard to FAPI-PET, we also observed a varying uptake behaviour over time in the strictures. Our results indicate that high FAPI uptake with minor FDG uptake indicate predominantly fibrotic activity with only minor inflammatory involvement, while a high FDG uptake with lower FAPI uptake might primarily represent inflammatory changes. A low or locally limited uptake in both examinations might be indicative for patients exhibiting a stable course of disease, with no need for surgical intervention.

Accurate grading of fibrosis and differentiation from inflammation in intestinal strictures is imperative for effective clinical management of patients with CD, yet remains an unmet necessity in current clinical practice. At the moment, the mainstay for evaluation of intestinal strictures in CD is still represented by MRE, which can depict signs of acute intestinal inflammation via mural oedema, CE of the bowel wall, and increased T2 signal intensity [20, 31]. Fibrotic strictures, however, rather demonstrate a decreased T2 mural signal intensity. However, intestinal wall enhancement in fibrotic strictures is variably present and therefore not a reliable indicator for the presence of fibrosis [31]. This clearly underlines that current conventional imaging procedures are insufficient to differentiate the fibro-stenotic structures from the inflammatory ones in CD associated stenosis reliably. Imaging of fibrotic lesions has important implications for therapeutic decisions, as it may guide early surgical or endoscopic balloon dilation over initiation of advanced medical treatments. Furthermore, multiple potential anti-fibrotic medications are currently evaluated for the treatment of CD associated intestinal strictures [17, 32, 33]. Here, a quantitative imaging modality to depict fibrotic bowel segments is a prerequisite for conducting a clinical trial and define potential response criteria [17].

Activation and local accumulation of myofibroblasts are a common denominator of fibrotic diseases [17]. Here, prolyl endopeptidase FAP (alternatively known as fibroblast activation protein-α) is a type II transmembrane protease with dipeptidyl peptidase and endopeptidase activity [34], which is induced in fibroblasts upon activation [24, 35]. FAPI uptake is associated with mesenchymal cell stimulation during the course of tissue fibrosis rather than immune cell metabolic activity of inflammation [16, 36, 37]. Fittingly, our study results, using FAPI-PET and FDG-PET scans show that it is feasible to dissect inflammatory from fibrotic activity in intestinal strictures.

The accuracy of FDG-PET in identifying inflamed segments of the intestine in CD has been examined in various studies [22, 23]. These studies have demonstrated a good correlation between disrupted intestinal glucose metabolism and ileocolonoscopy [23] or CT enterography [22]. Additionally, the level of tracer uptake appears to correlate with disease activity. Epelboym et al. [28] also utilized FDG-PET in this context to assess the therapeutic response to systemic therapy with anti-TNF antibodies. Although the reported detection rates of intestinal strictures for FDG-PET imaging are very high, the examination cannot be used for the identification of intestinal fibrosis [38]. Additionally, there is a high rate of false-positive enrichments, which has also been confirmed in our analysis [22]. Various causes of non-inflammatory increased intestinal FDG uptake are discussed [39], with peristaltic bowel movements [40] and certain medications, such as metformin [41] appearing to be the main causative factors. This limits the clinical utility of standalone FDG-PET scans in patients with CD.

The evidence for FAPI-PET application in CD is limited, but current studies suggest high accuracy in identifying intestinal fibrosis. Chen et al. [28] reported a detection accuracy of 93.3% compared to endoscopy. A significantly better performance was achieved when compared to CT enterography. In a recently published comparative analysis of an animal model and additionally of ten CD patients, FAPI-PET uptake was better associated with the histological degree of fibrosis than FDG-PET uptake [42]. FAPI-PET/MRE, the combination of FAPI-PET and MRE, was applied in a single examination using a dedicated PET/MR device. The study aimed to assess the level of radionuclide accumulation in intestinal fibrosis. Histological examination of surgical specimen served as a comparator. In this proof-of concept study that evaluated 14 CD patients with clinical signs and symptoms of intestinal strictures, a correlation was found between the extent and grade of histologically confirmed fibrosis and the level of FAPI uptake [27]. However, it was observed that using FAPI-PET does not provide information about the extent of inflammation in significantly fibrotic strictures. Additionally, there was no significant FAPI uptake in intestinal segments that were only inflamed [27].

The data from our analysis suggest that this issue might be addressed through the combination of FDG-PET and FAPI-PET. Alternatively, the combination of FAPI-PET and MRE could be used to further discriminate between inflammation and fibrosis. In our analysis, known inflammatory MRE parameters such as wall oedema or additional findings adjacent to the stenotic ileal segments were associated with increased inflammatory activity. This is also supported by the results of another study [27]. However, due to the time gap between the PET scans and the surgery, spanning 5 and 4 months for patient 2 and patient 3, respectively, there may have been interim changes in the degree of fibrosis and local inflammation, hampering comparability to the histological examination. In the case of patient 1, there is a 7-month gap between the MRE and the PET scans. Given the overall low level of inflammatory activity and the stable disease condition, no significant changes in the degree of inflammation or fibrosis are expected in this case. Combined with the limited number of investigated patients, this is a major limitation to our study, which precludes definite conclusions regarding the capacity of combined use of FAPI-PET and FDG-PET scans to assess areas of fibrotic tissue in CD. Due to the novelty of the procedure, there is currently limited data regarding FAPI-PET in CD, despite promising initial publications. This makes it difficult to estimate a valid time interval between the PET scan and surgery. Here, there is a need for larger, preferably multicentre collaborations including a larger patient cohort and more standardized time intervals in between the single investigations to establish quantitative cut-off values of both FAPI-PET and FDG-PET and to determine the interobserver agreement [21].

It would be interesting to evaluate, if this novel diagnostic method could also be applied in other diseases to differ between inflammatory and fibrotic disease. For example, elevated FAPI uptake has been demonstrated in IgG4-associated sclerosing cholangitis [43]. However, these findings are currently limited to small patient populations. To what extent further differentiation, for example, from malignant diseases can be achieved remains unclear at this point in time. Some FAPI compounds show relatively high hepatobiliary excretion, which can lead to physiological accumulation in the bile ducts and significantly affect their assessment regarding fibrosis.

Next, although an expert IBD pathologist scored the histopathological samples, a validated scoring system could not be applied, but instead visualized assessment was performed using a 3-point scale (low, moderate, high), which is used in clinical routine. Patient 1 did not undergo surgery due to a clinical stable course of disease since the patient suffered only from minor symptoms. The stenotic bowel segment was not passable by colonoscopy; hence, there was no possibility of gathering adequate histological material and histological assessment was limited to only the 2 patients who had to undergo a surgical procedure. In our study, anastomotic ileo-colonic strictures were the only ones investigated, limiting potential applicability of our imaging procedure results in de novo small bowl or colonic strictures. Endoscopic evaluation, which directly visualizes lesions in the intestinal mucosa, could not be used in our study for correlation analyses due to presence of impassable strictures in 2 patients, precluding macroscopic assessment of inflammation. The availability of FAPI-PET is mostly limited to larger tertiary centres with their own Radiopharmacy Department. As a result, this method is not yet suitable for routine diagnostics. Nevertheless, we hope that our pilot study has provided initial insights that will encourage further research into the potential role of nuclear medicine imaging techniques, particularly in relation to therapy management in CD.

The potential for dynamic FAPI-PET studies in CD has not been addressed in studies thus far. In our analysis, we identified different enrichment patterns over time. The patient with the highest fibrotic burden showed an increasing uptake over time. Dynamic FAPI-PET might therefore further help characterize CD associated stenosis. In conclusion, this pilot study demonstrates promising results and warrants further studies with combined use of Ga-68-FAPI-46-PET/CT and F-18-FDG-PET/CT imaging in CD for the characterization of fibrotic components in intestinal strictures, which probably remains the biggest diagnostic challenge in stricturing CD.

The authors thank all patients for participating in this study and all physicians and nurses contributing to an effective patient recruitment.

The study has been granted an exemption from requiring ethics approval by the Ethics Committee of the Friedrich-Alexander-University Erlangen-Nürnberg (Identification Number: 23-455-Br). All patients gave written informed consent prior to study inclusion.

M.F.N. has served as a speaker, or consultant, or received research grants from AbbVie, MSD, Takeda Pharma, Boehringer, Roche, Pfizer, Janssen, Pentax, PPD. R.A. has served as a speaker, or consultant, or received research grants from AbbVie, Abivax, AstraZeneca, Bristol-Myers Squibb, Celltrion Healthcare, Galapagos, Janssen-Cilag, Lilly, MSD Sharp and Dohme, Pfizer, Takeda Pharma. DFG-SFB/TRR241 Project No. C02 and IBDome (R.A.) are funded by the German Research Council (DFG). A.A. is supported by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) – 493624887 (Clinician Scientist Program NOTICE). All other authors declare no conflict of interest.

This work was supported by the Friedrich-Alexander-University Erlangen-Nürnberg and the Erlangen University Hospital (IIT – Investigator-initiated study). This work was also funded by the FIBROTARGET project. The FIBROTARGET project has received funding from the European Union’s Horizon Europe Research and Innovation programme under grant agreement No. 101080523. Funded by the European Union. Views and opinions expressed are, however, those of the author(s) only and do not necessarily reflect those of the European Union or European Health and Digital Executive Agency (HADEA). Neither the European Union nor the granting authority can be held responsible for them.

M.B., T.K., T.H., A.A., M.G., A.H., M.S., M.U., M.F.N., and R.A. oversaw the study, analysed and interpreted the data, and contributed to the writing and editing of the manuscript. M.B., T.K., M.S., M.U., M.F.N., and R.A. planned the study protocol. M.B., T.K., T.H., A.A., M.G., A.H., M.S., M.U., M.F.N., and R.A. provided clinical and scientific input, recruited patients, and conducted the trial and reviewed the manuscript. A.H. performed histological assessment.

Individual participant data collected during the trials after de-identification are available for the present study. All data provided are anonymized to respect the privacy of patients who have participated in the trial, in line with applicable laws and regulations. Proposals should be directed to corresponding author at [email protected] to gain access, data requestors will need to sign a data access agreement.