68 Ga-FAPI-04 PET Distinguishes Malignancy from Inammatory Lesions

Background The 68 Ga-labelled FAPI provides new oncology imaging option other than 18 F-FDG-PET. However, it's unclear about whether the FAPI-PET distinguishes malignancy from benign lesions. Methods We established an AOM/DSS-induced rat colorectal tumor model. A double PET/CT tracer of 68 Ga-FAPI-04 and 18 F-FDG was used in the rat colorectal tumor model. Histological examination, immunohistochemistry staining, and radioautography were performed in this study.

Introduction 18 F-uorodeoxyglucose (FDG) positron emission tomography/computer tomography (PET/CT) is frequently applied in cancer diagnosis and therapeutic response monitoring. However, FDG high-uptake lesions indicate carcinoma and benign lesions, such as in ammatory diseases [1,2]. Colorectal cancer (CRC) is one of the most common malignancies worldwide. The increased uptake of FDG in the benign lesions of the colon/rectum has been described in several studies [3] [4]. Identifying malignant colorectal tumors from high FDG uptake lesions is a clinical challenge.
In this study, we further evaluate the capacity of FAPI PET in distinguishing colorectal cancer diagnosis from in ammatory lesions in a rat colorectal tumor model.

AOM/DSS-induced colon cancer
Six-week-old male F344 rats were used for the AOM/DSS-induced colon cancer model. All animals were administered AOM (Sigma) by intraperitoneal injection at a 10 mg/kg dose on the rst day of the rst week. After one week, rats were given 2% (w/v) DSS (MP Biomedicals, Solon, OH, USA) dissolved in drinking water on the second and fourth weeks. AOM/DSS-induced colon lesions were veri ed by microenteroscopy, 18 F-FDG / 68 Ga-FAPI-04 micro-PET/CT, and pathological examination.
In Vivo PET/CT imaging on rats AOM/DSS-induced rats were subjected to 18  Germany) and frozen after animal sacri ce. Five contiguous tissue sections of 5/10 µm thick were carried out by a 3050S cryostat microtome (Leica) for autoradiography and immunohistochemistry analysis. The tumor sections were placed in a cassette against an imaging lm (Fuji, Tokyo, Japan). A Typhoon 5 (GE Healthcare, CA, USA) read the exposed plate with a Phosphor reader scanning system. The same tumor section was used for autoradiography, and the adjacent sections were used for histological assays.

Immunohistochemistry
Immunohistochemistry of FAP and HK2 was performed using the tumor sections obtained from induced rats' colorectal lesions. Slides containing the sections were stained with antibodies against HK2 (2867S, endogenous peroxidase was neutralized with 3% hydrogen peroxidase in methanol for 15 mins at room temperature, and primary antibodies were applied at 4°C overnight. Anti-rabbit IgG biotinylated secondary antibody was applied for 30 mins at 37°C, followed by SA-HRP for 30 mins at 37°C. The sections were visualized using DAB for 5 minutes and counterstained in Mayer's hematoxylin for 20 minutes.

Statistical analysis
Data were presented as mean ± SD. The student t-test was used to compare two variables. A p-value of 0.05 or less was considered to be signi cant. Statistical analysis was performed using SPSS Statistics software (version 24.0.0; IBM).  (Fig. 3A). After the dissection, some of the rats were nodular hyperplasia and bowel wall invasion, and the others were only congestion and edema of the bowel (Fig. 3B).
Eight rats were divided into two groups, the neoplasia group and the in ammation group without neoplasia (Fig. 2B), which were veri ed by pathological examination (Fig. 3C). The expression of FAP and hexokinase2 (HK2) was detected by IHC in the neoplasia or the in ammatory tissue. HK2 is a key glycolysis enzyme. As shown in Fig. 3C, the HK2 expression was upregulated in both the in ammatory lesion and the neoplasia. In contrast, the FAP expression was increased merely in neoplasia. This observation was highly consistent with the double tracer imaging of FDG-PET and FAPI-PET (Fig. 3D).
The SUVmax of 18 F-FDG was 2.325 ± 0.64 and 2.250 ± 1.74 in the neoplasia group and in the in ammation group (p = 0.9380), while the SUVmax of 68 Ga-FAPI-04 was 3.475 ± 1.07 in the neoplasia group and 0.675 ± 0.33 in the in ammation group (p = 0.0024), suggesting that 68 Ga-FAPI PET imaging distinguishes malignancy from in ammation. 68 Ga-FAPI PET distinguishes malignancy from in ammatory lesions polyp to avoid background interference. As shown in Fig. 3E, the invasive neoplasia was 18 F-FDG-positive and 68 Ga-FAPI-04 positive, while the polyp was only 18 F-FDG positive. Two lesions were located in different parts of the colorectum (Fig. 3F). Consistent with the report, the AOM/DSS-induced tumors were more often in the distal colorectum [13]. After dissection, the slices from two segments were analyzed by the autoradiography of 68 Ga-FAPI-04 and the FAP staining. As shown in the lower panel of Fig. 3F, the radiation intensity of the distal slice was much higher than the proximal one. Consistent with the autoradiography, only the distal sections were FAP-positive. This malignant lesion was further con rmed by pathological examination (Fig. 3G). in the sixth month. However, FDG SUVmax was still 4.63 ± 1.06 at the same time point (Fig. 4B). The change fold of the SUVmax of 68 Ga-FAPI-04 was 8.67 ± 2.68 at the sixth month compared with the third month (p < 0.05), while the change fold of the SUVmax of 18 F-FDG was 1.32 ± 0.13. There were no signi cant changes in rat weight, which was 1.09 ± 0.14 ( Fig. 4B). This observation further supported the above nding that non-malignant in ammation does not affect 68 Ga-FAPI PET imaging, distinguishing neoplasia from in ammation.

Discussion
High uptake of FDG in the intestine is nonspeci c as the 18 F-FDG is known to accumulate in acute in ammatory lesions. Therefore, high uptake of FDG could present in various colon and rectum diseases, including tumor, polyp, Crohn's disease, or colitis [14,15]. However, no optimal management algorithm is helpful for patients with incidental colorectal 18 F-FDG PET-avid lesions. Thus, it's challenging to distinguish a malignant tumor from in ammatory diseases based on the FDG uptake [3].
This study found that FAPI-04 was uptaken by the AOM/DSS-induced rat CRC tumor, where the washout was slower than the other organs except the kidney and bladder. Also, it has a high tumor-to-background contrast ratio in vivo. Moreover, the increased accumulation of FAPI was only in colorectal carcinomas.
The 68 Ga-FAPI-04 signal was negative or low at the in ammatory lesions, where 18 F-FDG uptake was markedly increased. Therefore, the speci city of 68 Ga-FAPI PET is more than 18 F-FDG PET, at least to colorectal carcinomas.