Abstract

Objectives

This study aimed to evaluate the potential efficacy of ⁶⁸Ga-fibroblast activation protein inhibitor (FAPi) positron emission tomography/computed tomography (PET/CT) for detecting, staging, and restaging digestive system malignancies that are ¹⁸F-fluorodeoxyglucose (¹⁸F-FDG) negative or show equivocal ¹⁸F-FDG uptake.

Methods

We conducted a prospective analysis of 30 patients with pathologically confirmed primary tumors or metastases of the digestive system. Participants underwent ⁶⁸Ga-FAPi PET/CT and ¹⁸F-FDG PET/CT imaging for staging or restaging purposes within the same week. The efficacy of ⁶⁸Ga-FAPi PET/CT was assessed by comparing its ability to detect lesions and influence disease staging with that of ¹⁸F-FDG PET/CT.

Results

⁶⁸Ga-FAPi PET/CT imaging was performed in 30 patients with ¹⁸F-FDG-negative or indeterminate lesions. Of the 30 patients, 23 had gastric cancer and 7 had colorectal cancer. Among all patients, histopathological diagnosis of signet ring cell carcinoma was present in 15 (50%) patients. Primary tumor or local recurrence was detected in 19 (63%) patients, lymph node metastasis in 8 (27%) patients, visceral metastasis in 4 (13%) patients, peritoneal metastasis in 14 (47%) patients, and bone metastasis in 3 (10%) patients on ⁶⁸Ga-FAPi PET/CT images. All patients underwent histopathological confirmation on ⁶⁸Ga-FAPi PET/CT images. The disease stage was upgraded in 20 patients (67%) after ⁶⁸Ga-FAPi PET/CT imaging. Of the 20 patients, 12 had no evidence of recurrence or metastasis on ¹⁸F-FDG PET/CT.

Conclusion

Based on our study, ⁶⁸Ga-FAPi PET/CT alters the disease stage in the majority of gastrointestinal malignancies with negative or equivocal ¹⁸F-FDG PET/CT findings. ⁶⁸Ga-FAPi PET/CT appears to be effective in both staging and restaging of gastrointestinal malignancies, such as signet-ring cell carcinomas of the stomach that frequently show low ¹⁸F-FDG -avidity.

Introduction

The World Health Organization (WHO) reclassified digestive system tumors in 2020 and emphasized the importance of molecular pathology in clinical practice (1). According to the WHO, approximately 5 million new cases and 3.6 million deaths from digestive system cancers will occur worldwide in 2020, and the incidence of various types of digestive system cancers is gradually increasing (2, 3). Most cancers of the digestive tract have a poor prognosis and differ in clinical presentation because of the involvement of multiple organs (4, 5). Therefore, early diagnosis and accurate evaluation are of great clinical importance in the treatment of these tumors.

Malignancies in the digestive system are investigated using standard imaging techniques, such as biomarkers, ultrasound, and endoscopic procedures (6, 7). However, these techniques have numerous drawbacks, including the inability to accurately determine the stage and metastasis of cancers of the digestive system. ¹⁸F-FDG positron emission tomography/computed tomography (PET/CT) is currently used as a standard imaging method in the clinical applications of oncology, for preoperative systemic evaluation, and for determining tumor stage. However, it may be inadequate for imaging certain types of cancer, such as signet-ring cell cancers, mucinous-serous adenocarcinomas, and peritoneal tumors, which have low glucose metabolism (8, 9, 10). Another important factor affecting the sensitivity to ¹⁸F-FDG PET/CT is the size of the tumor (11). In addition, the physiological uptake of ¹⁸F-FDG through the gastrointestinal tract may lead to false-positive results, limiting the use of ¹⁸F-FDG-PET/CT (12). Therefore, the search for new tumor diagnostic methods has always been an important issue.

PET/CT imaging methods based on fibroblast activation protein (FAP) expressed by cancer-associated fibroblasts (CAFs) in cancer tissues have recently been developed. FAP was first demonstrated in malignant sarcoma cells in 1988 (13). FAP is a type 2 transmembrane serine protease consisting of 760 amino acids with endopeptidase and dipeptidyl peptidase activities (14). It is expressed on the surface of CAFs, which are also found in many tumor tissues. The current FAP inhibitors (FAPi) are peptidomimetic quinoline derivatives that bind to FAP with high affinity and can be used for PET imaging by binding to the ⁶⁸Ga (15).

CAFs differ from other fibroblasts in that they express higher levels of FAP in the tumor microenvironment. Therefore, FAP is expressed at a very low level in healthy tissues, which allows ⁶⁸Ga-FAPi PET/CT to provide low background uptake. The low background uptake of ⁶⁸Ga-FAPi PET/CT provides technical advantages, such as higher tumor detection sensitivity. The requirement for supportive stroma in tumor tissue larger than 1-2 mm in size and the fact that the stromal volume is higher than the cancer cell volume provide an advantage for ⁶⁸Ga-FAPi PET/CT (16).

Studies comparing ¹⁸F-FDG PET/CT with ⁶⁸Ga-FAPi PET/CT have demonstrated the contribution of ⁶⁸Ga-FAPi PET/CT in the staging of digestive system tumors (17). However, there is limited information on the success of ⁶⁸Ga-FAPi PET/CT in ¹⁸F-FDG-negative patients. The aim of this study was to detect ¹⁸F-FDG-negative or equivocal ¹⁸F-F-FDG lesions using ⁶⁸Ga-FAPi PET/CT and to evaluate the contribution of ⁶⁸Ga-FAPi PET/CT to the clinical staging or restaging of digestive tumors.

Materials and methods

Patients

This single-center prospective clinical trial was conducted between September 2020 and March 2024, a total of 30 patients with digestive tumors enrolled for ¹⁸F-FDG PET/CT with the indication of staging or restaging who met the following inclusion criteria were offered a ⁶⁸Ga-FAPi PET/CT: (a) low ¹⁸F-F-FDG affinity in the metastasis sites of tumor on ¹⁸F-FDG PET/CT; (b) an elevation in tumor markers without any focal findings on ¹⁸F-FDG PET/CT; (c) presence of an indeterminate finding on ¹⁸F-FDG PET/CT; (d) the presence of a lesion in the CT component of ¹⁸F-FDG PET/CT that does not exhibit ¹⁸F-FDG avidity; (e) patients with stage 1-3 disease diagnosed on ¹⁸F-FDG PET/CT. The term indeterminate finding was assigned to areas exhibiting uptake indistinguishable from the background that could not be identified as abnormal.

The exclusion criteria were as follows: (a) aged 18 years; (b) having two or more primary diseases; (c) patients identified as stage 4 on ¹⁸F-FDG -PET/CT; (d) pregnant or suspected of being pregnant; (e) inability to remain still during the scan (20-30 minutes).

Informed consent was obtained from all patients. This prospective study was approved by the Yeditepe University Clinical Research Ethics Committee (decision no: 1576, date: 02.03.2022).

Preparation and Quality Control of ⁶⁸Ga-FAPi

⁶⁸Ga-DOTA-FAPi-04 was prepared using a modular-based fully automated synthesizer (GRP V4, Scintomics GmbH, Germany). Briefly, the ⁶⁸Ga obtained from the ⁶⁸Ge/⁶⁸Ga generator (iThemba LABS) was sent to the reaction vial containing DOTA-FAPi-04. After completion of the labeling process, the reaction solution was purified with an extraction cartridge and subjected to sterile filtration to prepare the final patient dose. The total synthesis time was 20-25 minutes. The radiochemical purity and radiolabeling efficiency ⁶⁸Ga-FAPi were determined by combining a radioactive detector with reversed-phase high-pressure liquid chromatography (retinitis pigmentosa- high-performance liquid chromatography). ⁶⁸Ga-FAPi with a radiochemical purity of ≥ 95% was administered to patients.

⁶⁸Ga-FAPi and ¹⁸F-FDG PET/CT Imaging

Whole-body imaging of ⁶⁸Ga-FAPi and ¹⁸F-FDG PET/CT was performed using a PET scanner (Discovery PET/CT 710, General Electric Medical Systems, Milwaukee, WI, USA) with integrated 64-slice CT, high resolution, time-of-flight function, and LYSO crystal. After intravenous injection of radiopharmaceuticals with an average activity of 240±60 MBq (range: 122-312 MBq), patients were fixed supine on the bed of the PET scanner 60 minutes after injection. CT and PET images were acquired from the vertex region to mid-thigh. ⁶⁸Ga-FAPi PET/CT was performed within 7 days after ¹⁸F-FDG PET/CT.

Evaluation of ⁶⁸Ga-FAPİ PET/CT and ¹⁸F-FDG PET/CT Images

Activity uptake in the tumor was measured by maximum standard uptake value (SUV_max) using circular regions of interest drawn around the lesions with focal uptake in transaxial slices and automatically adapted to a 3D voxel area within the 60% iso-contour. All images were reviewed by three senior nuclear medicine physicians who reached consensus for confirmation.

Statistical Analysis

Statistical analysis were performed using SPSS software (version 25.0; IBM Inc.). Descriptive analyses were conducted to assess the characteristics of the patients and their tumors. The mean and standard deviation were calculated for normally distributed measurements, and the median and range were calculated for non-normal measurements. Diagnostic parameters were calculated using a simple matrix method. Using the sample size, 95% Confidence Intervals were also calculated. Pearson’s chi-square test was used to compare ⁶⁸Ga-FAPi PET/CT and ¹⁸F-FDG PET/CT. McNemar’s test was used to evaluate the staging accuracy of ⁶⁸Ga-FAPi PET/CT and ¹⁸F-FDG PET/CT. A P-value of less than 0.05 was considered statistically significant.

Results

A total of 30 patients were included in the study. Of all patient group for 14 (47%) patients, ⁶⁸Ga-FAPi PET/CT was performed for restaging because of suspected progressive disease, whereas the 16 (53%) patients with new diagnoses underwent PET imaging for primary staging. The mean age of the patients was 52.7±12.0 (range: 35-77 years). Of the 30 patients, 23 had gastric (77%), and 7 had colorectal (23%). Among all patients, histopathological diagnosis of signet ring cell carcinoma was present in 15 (50%) patients. The demographic characteristics of patients are presented in Table 1.

In half of the patients (n=15), ¹⁸F-FDG PET/CT findings were completely negative, while others had equivocal findings. ⁶⁸Ga-FAPi PET/CT was performed in these patients due to suspicion of potential oversight in staging and evaluation, prompted by clinical progression and/or elevated tumor markers, such as CA 19-9 and CEA. Primary tumor or local recurrence was detected in 19 (63%) patients, lymph node metastasis in 8 (27%) patients, visceral metastasis in 4 (13%) patients, peritoneal metastasis in 14 (47%) patients, and bone metastasis in 3 (10%) patients on ⁶⁸Ga-FAPi PET/CT images. For ¹⁸F-FDG PET/CT, primary tumor or local recurrence was detected in 13 (43%) patients and lymph node metastasis in 3 (10%) patients. Otherwise, visceral, peritoneal, and bone metastases could not be detected on ¹⁸F-FDG PET/CT (Table 1). ⁶⁸Ga-FAPi PET/CT demonstrated a higher detection rate for primary lesions at 96%, compared to 71% with ¹⁸F-FDG PET/CT.

At least one lesion in all patients was confirmed histopathologically after ⁶⁸Ga-FAPi PET/CT imaging. In one patient, although peritoneal fluid sampling was negative, ⁶⁸Ga-FAPi PET/CT revealed signs consistent with peritoneal carcinomatosis. Since the findings of peritoneal carcinomatosis were confirmed radiologically in the patient’s subsequent follow-up visits, clinical follow-up confirmed that the cytology result was false negative. In one patient, despite the presence of a primary tumor on CT and/or magnetic resonance imaging (MRI), both ⁶⁸Ga-FAPi and ¹⁸F-FDG PET/CT findings were negative.

Although no findings were detected in 15 patients on ¹⁸F-FDG PET/CT, ⁶⁸Ga-FAPi PET/CT identified 2 patients (7%) as stage 2, 1 patient (3%) as stage 3, and 9 patients (30%) as stage 4 (Table 2). While there was clinical suspicion in 2 patients, no evidence of recurrence or metastasis was found in ¹⁸F-FDG and ⁶⁸Ga-FAPi PET/CT as well as CT and/or MRI. A patient diagnosed with signet ring cell carcinoma of the stomach who presented for staging showed false-negative results on both ¹⁸F-FDG and ⁶⁸Ga-FAPi PET/CT. We found that 12 of 15 patients with negative ¹⁸F-FDG PET/CT results (80%) experienced an increase in disease stage after undergoing ⁶⁸Ga-FAPi PET/CT. Additionally, 8 of 15 patients with initially staged 1-3 on ¹⁸F-FDG PET/CT (53%) showed an elevation in their primary disease stage when assessed with ⁶⁸GaFAPi PET/CT. Notably, a discrepancy in staging between ⁶⁸Ga-FAPi and ¹⁸F-FDG PET/CT was observed in 67% of patients, leading to significant alterations in their oncologic treatment plans (Table 2). Our findings indicate that staging with ⁶⁸Ga-FAPi PET/CT is statistically more effective than ¹⁸F-FDG PET/CT (Pearson chi-square value of 27.18; p=0.007).

The mean SUV_max values of the primary tumors in gastric and colorectal cancer were 14.8±5.8 (range: 5.5-21.8) and 9.5±4.2 (range: 5.3-13.6), respectively. Notably, all peritoneal metastases were negative on ¹⁸F-FDG PET/CT, whereas they exhibited significant uptake on ⁶⁸Ga-FAPi PET/CT, with a mean SUV_max value of 10.5±4.9 (range: 3.6-21.8) (Figure 1).

A histopathological diagnosis of signet ring cell cancer was made in 15 patients (50%). Among these, 14 patients (47%) had gastric signet ring cell cancer, and 1 patient (3%) had colonic signet ring cell malignancy (Table 1). In patients with signet ring cell cancer, 45% of primary lesions (5/11) and 80% of lymph node metastases (4/5) were negative on ¹⁸F-FDG PET/CT, whereas all these lesions demonstrated increased uptake on ⁶⁸Ga-FAPi PET/CT. Additionally, peritoneal metastases were identified in 4 patients using ⁶⁸Ga-FAPi PET/CT, whereas these lesions were negative on ¹⁸F-FDG PET/CT. The mean SUV_max for primary lesions was 13.4±5.2 (range: 5.5-17.0) on ⁶⁸Ga-FAPi PET/CT and 6.8±2.1 (range: 3.8-10.1) on ¹⁸F-FDG PET/CT. For peritoneal metastases, the mean SUV_max on ⁶⁸Ga-FAPi PET/CT was 10.6±3.6 (range: 5.9-14.5) (Figure 2).

Discussion

In this study, ¹⁸F-FDG-positive cases were excluded because they were already well documented. Our study highlights the diagnostic superiority of ⁶⁸Ga-FAPi PET/CT over ¹⁸F-FDG PET/CT in staging digestive system malignancies, particularly when ¹⁸F-FDG PET/CT results are equivocal or negative. The pivotal role of ⁶⁸Ga-FAPi PET/CT is attributed to its targeted imaging of CAFs, which are prominently expressed in the stromal components of gastrointestinal tumors. This expression pattern significantly enhances tumor detection sensitivity, highlighting the critical role of stromal involvement in gastrointestinal cancer pathology.

¹⁸F-FDG PET/CT and CT demonstrate suboptimal lesion detectability, primarily due to the mucinous types of gastric cancer and signet ring cell carcinoma, which constitute the majority of cases in this study and typically manifest as small, diffusely growing patterns characterized by a scarcity of tumor cells (12). Some lesions exhibit low expression of tumor glucose transporters but high levels of dephosphorylation, resulting in lessened accumulation of ¹⁸F-FDG PET/CT (18, 19). Furthermore, in contrast to the relatively high physiological uptake of ¹⁸F-FDG PET/CT in the gastrointestinal tract, the low background uptake of ⁶⁸Ga-FAPi PET/CT in the abdominopelvic cavity contributes to the superior performance of ⁶⁸Ga-FAPi PET/CT (20, 21).

As with the clinical presentation of gastrointestinal cancers, imaging characteristics and workflows can exhibit substantial variability, resulting in unequal diagnostic efficacy among different imaging modalities (22). The findings of this study indicate that ⁶⁸Ga-FAPi PET/CT holds considerable promise for detecting disease extent in gastrointestinal cancer, a conclusion that is consistent with other published reports (23, 24, 25). Many other studies have shown that ⁶⁸Ga-FAPi PET/CT is superior to other modalities, such as MRI, CT, and ¹⁸F-FDG PET/CT, in digestive tract malignancies. However, very few studies have investigated the effect of ⁶⁸Ga-FAPi PET/CT. The current study is one of the few studies that emphasizes stage changes after ⁶⁸Ga-FAPi PET/CT performed for staging or restaging of gastrointestinal tumors correlated with biopsy. The results demonstrated that ⁶⁸Ga-FAPi PET/CT changed disease staging in approximately 67% of cases with ¹⁸F-FDG-negative or equivocal lesions. This is a significant finding for accurate staging and the application of correct treatment algorithms.

In our study, all patients had histopathological confirmation after ⁶⁸Ga-FAPi PET/CT images. ⁶⁸Ga-FAPi PET/CT findings of one patient were compatible with peritoneal carcinomatosis, although the peritoneal fluid sample was negative. However, on clinical radiological follow-up, these findings confirmed that the pathology result was false negative. In another patient, despite the presence of a tumor, the ⁶⁸Ga-FAPi and ¹⁸F-FDG PET/CT findings were negative.

⁶⁸Ga-FAPi PET/CT showed more peritoneal implants and lymph node metastases than ¹⁸F-FDG PET/CT, which led to upstaging based on the tumor-node-metastasis system. In addition, ⁶⁸Ga-FAPi PET/CT detected more primary lesions than ¹⁸F-FDG PET/CT in individuals diagnosed with digestive system malignancy, with detection rates of 96% and 71%, respectively. These results are consistent with literature (17, 21). Recent research has also highlighted the promising potential of ⁶⁸Ga-FAPi PET/CT in guiding the clinical management of pancreatic and gastric cancer (26, 27). Koerber et al. (25) have shown that ⁶⁸Ga-FAPi PET/CT resulted in changes in treatment classified as high, intermediate, and low in 19%, 33%, and 29% of patients, respectively. In our study, we observed that 80% (12/15) of patients with no detectable uptake in the primary tumor and/or metastasis sites on ¹⁸F-FDG PET/CT showed increased uptake on ⁶⁸Ga-FAPi PET/CT. In addition, we demonstrated that of the patients identified by ¹⁸F-FDG PET/CT at any stage (stage 1, 2, 3), 53% showed an increase in the primary disease stage when examined by ⁶⁸Ga-FAPi PET/CT. In summary, 67% of patients showed a difference in staging between ⁶⁸Ga-FAPi PET/CT and ¹⁸F-FDG PET/CT, leading to significant changes in their oncologic treatment strategies.

In agreement with the literature, we found a higher SUV_max on ⁶⁸Ga-FAPi PET/CT than on ¹⁸F-FDG PET/CT in primary tumors (17). However, it is worth noting that we did not include patients with high ¹⁸F-FDG uptake in our study. The lesions of the patients in our study had either no ¹⁸F-FDG uptake or very low uptake outside the primary lesion, which could not be distinguished from the background. Therefore, our patients were expected to have a higher SUV_max on ⁶⁸Ga-FAPi PET/CT. In our study, higher uptake was observed in primary lesions of gastric cancer (SUV_max: 14.8) than in colorectal cancer (SUV_max: 9.5) on ⁶⁸Ga-FAPi PET/CT.

The role of ¹⁸F-FDG-PET/CT in signet ring cell carcinoma is of limited diagnostic value in terms of both primary lesions and metastases. Peritoneal metastases are often overlooked in ¹⁸F-FDG PET/CT. This is because these tumors are mucin-rich, do not consume glucose, and express low levels of glucose transporters (28, 29). In addition, the peritoneal implants are usually small and can be missed even with diagnostic tools such as CT and/or MRI. In this context, previous studies have shown that ⁶⁸Ga-FAPi PET/CT is extremely sensitive in signet ring cell carcinomas (30, 31). In our study, 45% of primary lesions (5/11) and 80% of lymph node metastases (4/5) of patients with signet ring cell carcinoma were negative on ¹⁸F-FDG PET/CT. Of these patients, 4 had peritoneal metastasis, which could not be detected by ¹⁸F-FDG PET/CT. The average SUV_max was 13.4 for the primary tumor and 10.6 for the peritoneal metastasis.

Conclusion

In conclusion, ⁶⁸Ga-FAPi PET/CT is superior for staging and restaging indications in digestive system tumors, especially in patients with ¹⁸F-FDG-negative or equivocal lesions, such as signet ring cell carcinoma. The strength of our study lies in its ability to stage cases in which ¹⁸F-FDG PET/CT fails to resolve using ⁶⁸Ga-FAPi PET/CT and in corroborating these findings with biopsy results in all patients. The ⁶⁸Ga-FAPi PET/CT modality is a promising imaging modality for the diagnosis and management of FDG-negative GI tumors. The use of this method has the potential to introduce new applications for tumor staging or restaging. Future studies should explore the longitudinal impact of ⁶⁸Ga-FAPi PET/CT-guided treatment decisions on patient outcomes, potentially establishing this modality as a standard component of gastrointestinal cancer management protocols.