Synthesis and Preliminary Study of 99mTc-Labeled HYNIC-FAPi for Imaging of Fibroblast Activation Proteins in Tumors.

Fibroblast activation protein (FAP) is an emerging target for cancer diagnosis. Different types of FAP inhibitor (FAPI)-based radiotracers have been developed and applied for tumor imaging. However, few FAPI tracers for single photon emission computed tomography (SPECT) imaging have been reported. SPECT imaging is less expensive and more widely distributed than positron emission tomography (PET), and thus, 99mTc-labeled FAPIs would be more available to patients in developing regions. Herein, we developed a FAPI-04-derived radiotracer, HYNIC-FAPi-04 (HFAPi), for SPECT imaging. 99mTc-HFAPi, with a radiochemical purity of >98%, was prepared using a kit formula within 30 min. The specificity of 99mTc-HFAPi for FAP was validated by a cell binding assay in vitro and SPECT/CT imaging in vivo. The binding affinity (Kd value) of 99mTc-HFAPi for human FAP and murine FAP was 4.49 and 2.07 nmol/L, respectively. SPECT/CT imaging in HT1080-hFAP tumor-bearing mice showed the specific FAP targeting ability of 99mTc-HFAPi in vivo. In U87MG tumor-bearing mice, 99mTc-HFAPi had a higher tumor uptake compared with that of HT1080-hFAP and 4T1-mFAP tumor models. Interestingly, 99mTc-HFAPi showed a relatively high uptake in some murine joints. 99mTc-HFAPi accumulated in tumor lesions with a high tumor-to-background ratio. A preliminary clinical study was also performed in breast cancer patients. Additionally, 99mTc-HFAPi exhibited an advantage over 18F-FDG in the detection of lymph node metastatic lesions in breast cancer patients, which is helpful in improving treatment strategies. In short, 99mTc-HFAPi showed excellent affinity and specificity for FAP and is a promising SPECT radiotracer for (re)staging and treatment planning of breast cancers.

FAP imaging in rare cancer entities-first clinical experience in a broad spectrum of malignancies.

PURPOSE: 68 Ga-FAPI (fibroblast activation protein inhibitor) is a rapidly evolving and highly promising radiotracer for PET/CT imaging, presenting excellent results in a variety of tumor entities, particularly in epithelial carcinomas. This retrospective analysis sought to evaluate the potential and impact of FAPI-PET/CT in rare cancer diseases with respect to improvement in staging and therapy, based on tracer uptake in normal organs and tumors. MATERIAL AND METHODS: Fifty-five patients with rare tumor entities, defined by a prevalence of 1 person out of 2000 or less, received a 68 Ga-FAPI-PET/CT scan. Fourteen women and 41 men (median age 60) were included within the following subgroups: cancer of unknown primary (n = 10), head and neck cancer (n = 13), gastrointestinal and biliary-pancreatic cancer (n = 17), urinary tract cancer (n = 4), neuroendocrine cancer (n = 4), and others (n = 7). Tracer uptake was quantified by standardized uptake values SUVmax and SUVmean and the tumor-to-background ratio (TBR) was determined (SUVmax tumor/SUVmean organ). RESULTS: In 20 out of 55 patients, the primary tumor was identified and 31 patients presented metastases (n = 88), characterized by a high mean SUVmax in primary (10.1) and metastatic lesions (7.6). The highest uptake was observed in liver metastases (n = 6) with a mean SUVmax of 9.8 and a high TBR of 8.7, closely followed by peritoneal carcinomatosis (n = 16) presenting a mean SUVmax of 9.8 and an excellent TBR of 29.6. In terms of the included subgroups, the highest uptake regarding mean SUVmax was determined in gastrointestinal and biliary-pancreatic cancer with 9.8 followed closely by urinary tract cancer with 9.5 and head and neck cancer (9.1). CONCLUSION: Due to excellent tumor visualization and, thereby, sharp contrasts in terms of high TBRs in primary and metastatic lesions in different rare malignancies, 68 Ga-FAPI-PET/CT crystallizes as a powerful and valuable imaging tool, particularly with respect to epithelial carcinomas, and therefore an enhancement to standard diagnostics imaging methodologies. The realization of further and prospective studies is of large importance to confirm the potential of FAP imaging in oncology.

68Ga-DOTA-D-Alanine-BoroPro Radiotracer for Imaging of the Fibroblast Activation Protein in Malignant and Non-Malignant Diseases.

Recently, we reported a new fibroblast activation protein (FAP) inhibitor radiopharmaceutical based on the 99mTc-((R)-1-((6-hydrazinylnicotinoyl)-D-alanyl) pyrrolidin-2-yl) boronic acid (99mTc-HYNIC-D-Alanine-BoroPro)(99mTc-HYNIC-iFAP) structure for tumor microenvironment SPECT imaging. This research aimed to synthesize 68Ga-[2,2',2″,2‴-(2-(4-(2-(5-(((S)-1-((S)-2-boronopyrrolidin-1-yl)-1-oxopropan-2-yl)carbamoyl)pyridin-2-yl)hydrazine-1-carbothioamido)benzyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrayl)tetraacetic acid] (68Ga-DOTA-D-Alanine-BoroPro)(68Ga-iFAP) as a novel radiotracer for PET imaging and evaluate its usefulness for FAP expression in malignant and non-malignant tissues. The coupling of p-SCN-benzene DOTA with HYNIC-iFAP was used for the chemical synthesis and further labeling with 68Ga. Radiochemical purity was verified by radio-HPLC. The specificity of 68Ga-iFAP was evaluated in HCT116 cells, in which FAP expression was verified by immunofluorescence and Western blot. Biodistribution and biokinetic studies were performed in murine models. 68Ga-iFAP uptake at the myocardial level was assessed in mice with induced infarction. First-in-human images of 68Ga-iFAP in healthy subjects and patients with myocardial infarction, glioblastoma, prostate cancer, and breast cancer were also obtained. DOTA-D-Alanine BoroPro was prepared with a chemical purity of 98% and was characterized by UPLC mass spectroscopy, FT-IR, and UV-vis. The 68Ga-iFAP was obtained with a radiochemical purity of >95%. In vitro and in vivo studies demonstrated 68Ga-iFAP-specific recognition for FAP, rapid renal elimination, and adequate visualization of the glioblastoma, breast tumor, prostate cancer, and myocardial infarction sites. The results of this research justify further dosimetry and clinical trials to establish the specificity and sensitivity of 68Ga-iFAP PET for FAP expression imaging.

The Diagnostic Value of Fibroblast Activation Protein Imaging in Hepatocellular Carcinoma and Cholangiocellular Carcinoma.

Hepatocellular carcinoma (HCC) is the most common primary liver malignancy with worldwide high incidence and mortality. In more than 90% of cases, HCC arise from a cirrhotic liver that is mostly induced by viral diseases and especially in developed countries alcoholic steatohepatitis and non-alcoholic steatohepatitis. In contrast, cholangiocellular carcinoma (CCC) is a very rare cancer entity with a high mortality due to insidious onset. The only curative option for both cancer entities is a timely and definitive surgical therapy, which mandates an accurate early diagnosis. To this end, [18F]FDG PET/CT scan could demonstrate only little benefit, as there is an unmet clinical need for an alternative, pan-cancer agent for initial diagnostic work-up of CCC or evaluation of Milan criteria for HCC patients.

[99mTc]Tc-iFAP Radioligand for SPECT/CT Imaging of the Tumor Microenvironment: Kinetics, Radiation Dosimetry, and Imaging in Patients.

Tumor microenvironment fibroblasts overexpress the fibroblast activation protein (FAP). We recently reported the preclinical evaluation of [99mTc]Tc-iFAP as a new SPECT radioligand capable of detecting FAP. This research aimed to evaluate the kinetic and dosimetric profile of [99mTc]Tc-iFAP in healthy volunteers, and to assess the radioligand uptake by different solid tumors in three cancer patients. [99mTc]Tc-iFAP was obtained from lyophilized formulations prepared under GMP conditions with >98% radiochemical purity. Whole-body scans of six healthy subjects were obtained at 0.5, 2, 4, and 24 h after [99mTc]Tc-iFAP (740 MBq) administration. A 2D-planar/3D-SPECT hybrid activity quantitation method was used to fit the biokinetic models of the source organs (volume of interest: VOI) as exponential functions (A(t)VOI). The total nuclear transformations (N) that occurred in the source organs were calculated from the mathematical integration (0,∞) of A(t)VOI. The OLINDA code was used to estimate the radiation doses. Three treatment-naive patients (breast, lung, and cervical cancer) with a prior [18F]FDG PET/CT scan underwent whole-body, chest, and abdominal SPECT/CT scanning after [99mTc]Tc-iFAP (740 MBq) administration. Both imaging methods were compared visually and quantitatively. Oncological diagnoses were performed histopathologically. The results showed favorable [99mTc]Tc-iFAP biodistribution and kinetics due to rapid blood activity removal (t1/2α = 2.22 min and t1/2ß = 90 min) and mainly renal clearance. The mean radiation equivalent doses were 5.2 ± 0.8 mSv for the kidney and 1.7 ± 0.3 mSv for the liver after administration of 740 MBq. The effective dose was 2.3 ± 0.4 mSv/740 MBq. [99mTc]Tc-iFAP demonstrated high and reliable uptake in the primary tumor lesions and lymph node metastases in patients with breast, cervical, and lung cancer, which correlated with that detected by [18F]FDG PET/CT. The tumor microenvironment molecular imaging from cancer patients obtained in this research validates the performance of additional clinical studies to determine the utility of [99mTc]Tc-iFAP in the diagnosis and prognosis of different types of solid tumors.