Fibroblast Activation Protein Expression Research Articles

Safety and Efficacy of 90Y-FAPI-46 Radioligand Therapy in Patients with Advanced Sarcoma and Other Cancer Entities.

We report efficacy and safety of 90Y-labeled FAPI-46 (90Y-FAPI-46-RLT) in patients with advanced sarcoma, pancreatic cancer, and other cancer entities. Up to four cycles of radioligand therapy (RLT) were offered to patients with (i) progressive metastatic malignancy, (ii) exhaustion of approved therapies, and (iii) high fibroblast activation protein (FAP) expression, defined as SUVmax ≥ 10 in more than 50% of tumor. Primary endpoint was RECIST response after RLT. Secondary endpoints included PET response (PERCIST), overall survival (OS), dosimetry, and safety of FAP-RLT. Among 119 screened patients, 21 (18%) were found eligible [n = 16/3/1/1 sarcoma/pancreatic cancer/prostate/gastric cancer; 38% Eastern Cooperative Oncology Group (ECOG) ≥ 2] and received 47 90Y-FAPI-46-RLT cycles; 16 of 21 (76%) patients underwent repeat RLT. By RECIST, disease control was confirmed in 8 of 21 patients [38%; 8/16 (50%) of evaluable patients). There was one partial response (PR) and seven stable diseases after RLT. Disease control was associated with prolonged OS (P = 0.013). PERCIST response was noted in 8 of 21 patients [38%; 8/15 (53%) of evaluable patients]. Dosimetry was acquired in 19 (90%) patients. Mean absorbed dose was 0.53 Gy/GBq in kidney, 0.04 Gy/GBq in bone marrow, and <0.14 Gy/GBq in liver and lung. Treatment-related grade 3 or 4 adverse events were observed in 8 (38%) patients with thrombocytopenia (n = 6) and anemia (n = 6) being most prevalent. 90Y-FAPI-46-RLT was safe and led to RECIST PR in one case as well as stable disease in about one third of patients with initially progressive sarcomas, pancreatic cancer, and other cancers. Discontinuation after the first cycle and a low rate of PR requires future improvement of FAP-RLT.

Non-oncologic incidental uptake on FAPI PET/CT imaging.

Fibroblast-activation protein (FAP) is a serine protease classified in the dipeptidyl peptidase 4 (DPP4) family. FAP is predominantly expressed in activated fibroblasts such as the cancer-associated fibroblasts (CAFs). FAP expression in CAFs is associated with tumor progression and poor prognosis in solid cancers. Recently, radiolabeled FAP inhibitors (FAPI) has been developed, which enables positron emission tomography (PET) imaging of FAP. FAPI PET/CT can provide a higher tumor-to-background ratio (TBR) than 18F-fludeoxyglucose PET/CT in various cancers, and thus has attracted substantial attention. As studies on FAPI PET grow in number and size, incidental findings related to non-oncologic conditions have been increasingly reported. FAPI PET uptake has been reported in various conditions such as benign tumors, fibrotic, granulomatosis, scarring/wound, degenerative diseases, and inflammatory diseases.The knowledge of physiological and non-oncologic causes of FAPI uptake is indispensable for accurate FAPI PET/CT interpretation and can help appropriate management of incidental findings on FAPI PET/CT in patients referred for cancer staging indications. In this review article, we describe for each organ system (Brain, Oral mucosa, Salivary Glands, Thyroid, Lung, Myocardium, Breast, Esophagus, Stomach, Intestine, Liver, Gallbladder, Pancreas, Spleen, Kidney, , Uterus, Bone marrow, Joints, Muscle, Vessels, Lymph nodes), the patterns of physiological FAPI uptake and the main causes of non-oncological uptake reported from the literature with FAPI-02, FAPI-04 and FAPI-46. We also illustrate some examples from our institutional database at UCLA.

Fibroblast activation protein (FAP) expression in CK5/6 expressed (Basal subtype) & CK20 expressed (Luminal subtype) urothelial bladder carcinoma: an immunohistochemical study

ABSTRACT Urinary bladder cancer incidence varies all over the world. Egypt displays high incidence rates. Molecular subtyping helps risk stratification and personalized treatment. Cancer-associated fibroblasts (CAFs) in the tumor microenvironment may provoke tumor-promotion or tumor suppression. Fibroblast activation protein (FAP) is a marker of CAFs, suggested to accelerate tumor progression in various cancers. In urothelial carcinoma, investigations regarding impact of FAP expression on prognosis are needed. This work aims to study impact of FAP expression in urothelial carcinoma and find its relation to CK 5/6 (basal) expressed and CK 20 (luminal) expressed immunohistochemical markers. This retrospective study included 70 urothelial carcinoma specimens. Immunohistochemistry was performed and results were analyzed. FAP was expressed in 67.1% of cases and showed significant association with advanced tumor stage, muscle invasion, mitoses in tumor cells and stratified groups; as 73.9% of FAP positive cases were of Ck5/6+/Ck20- (basal subtype). All studied parameters did not show significant association with patient’s overall survival. In conclusion, FAP could have a role in modulating tumor microenvironment and promoting tumor invasion. FAP is correlated with basal subtype of urothelial carcinoma, which may be an indicator of tumor aggressiveness. FAP antagonists may be helpful in preventing tumor progression.

Immunohistochemical expression of FAP and PCNA in neoplastic epithelial colonic lesions

Background Colorectal carcinoma is one of the leading causes of morbidity and mortality worldwide. Fibroblast-activation protein is expressed in cancer-associated fibroblasts and its expression is upregulated during the transformation of benign colorectal tissue into cancer. Proliferating-cell nuclear antigen expression was used as a marker of cell proliferation and is considered as a marker able to identify aggressive adenomas that may in turn become malignant. Aim To study the expression of fibroblast-activation protein and proliferating-cell nuclear antigen in neoplastic epithelial colonic lesions and their role in adenoma–carcinoma sequence. Patients and methods This retrospective study aimed to evaluate the expression of fibroblast-activation protein and proliferating-cell nuclear antigen in 80 cases of neoplastic epithelial colonic lesions. Results In malignant colorectal epithelial tumors, high fibroblast-activation protein expression was detected with an increase in histological grade, depth of invasion, vascular invasion, and lymph-node metastasis. No fibroblast-activation protein expression was detected in all benign cases. In included benign cases, there was a significant relation between proliferating-cell nuclear antigen labeling index and both histological type and grade of dysplasia. In the included malignant cases, there was a significant relation between proliferating-cell nuclear antigen labeling index with grade of tumor and the depth of invasion. Conclusions Both fibroblast-activation protein and proliferating-cell nuclear antigen labeling index suggested to have a role in early detection of malignant transformation and had a prognostic role in malignant cases.

Preclinical evaluation and pilot clinical study of [18F]AlF-NOTA-FAPI-04 for PET imaging of rheumatoid arthritis.

Fibroblast-like synoviocytes (FLSs) are key effector cells in the inflamed joints of patients with rheumatoid arthritis (RA). Previous studies have suggested that fibroblast activation protein (FAP) is highly expressed in RA-derived FLSs and is a specific marker of activated RA FLSs. In this study, we developed aluminum-[18F]-labeled 1,4,7-triazacyclononane-N,N',N″-triacetic acid-conjugated FAP inhibitor 04 ([18F]AlF-NOTA-FAPI-04) to image RA-FLSs in vitro and arthritic joints in collagen-induced arthritis (CIA) mice and RA patients. RA FLSs and NIH3T3 cells transfected with FAP were used to perform in vitro-binding studies. Biodistribution was conducted in normal DBA1 mice. Collagen-induced arthritis (CIA) models with different arthritis scores were subjected to [18F]AlF-NOTA-FAPI-04 and 18F-FDG PET imaging. Histological examinations were performed to evaluate FAP expression and Cy3 dye-labeled FAPI-04(Cy3-FAPI-04) uptake. Blocking studies with excess unlabeled FAPI-04 in CIA mice and NIH3T3 xenografts in immunocompromised mice were used to evaluate the binding specificity of [18F]AlF-NOTA-FAPI-04. Additionally, [18F]AlF-NOTA-FAPI-04 PET imaging was performed on two RA patients. The binding of [18F]AlF-NOTA-FAPI-04 increased significantly in RA FLSs and NIH3T3 cells overexpressing FAP compared to their parental controls (FAP-GFP-NIH3T3 vs. GFP-NIH3T3, 2.40 ± 0.078 vs. 0.297 ± 0.05% AD/105 cells; RA FLSs vs. OA FLSs, 1.54 ± 0.064 vs. 0.343 ± 0.056% AD/105 cells). Compared to 18F-FDG imaging, [18F]AlF-NOTA-FAPI-04 showed high uptake in inflamed joints in the early stage of arthritis, which was positively correlated with the arthritic scores (Pearson r=0.834, P<0.001). In addition, the binding of [18F]AlF-NOTA-FAPI-04 to cells with high FAP expression and the uptake of [18F]AlF-NOTA-FAPI-04 in arthritic joints both could be blocked by excessive unlabeled FAPI-04. Fluorescent staining showed that the intensity of Cy3-FAPI-04 binding to FAP increased accordingly as the expression of FAP protein increased in cells and tissue sections. Furthermore, the uptake of [18F]AlF-NOTA-FAPI-04 in FAP-GFP-NIH3T3 xenografts was significantly higher than that in GFP-NIH3T3 xenograft (35.44 ± 4.27 vs 7.92 ± 1.83% ID/mL). Finally, [18F]AlF-NOTA-FAPI-04 PET/CT imaging in RA patients revealed nonphysiologically high tracer uptake in the synovium of arthritic joints. [18F]AlF-NOTA-FAPI-04 is a promising radiotracer for imaging RA FLSs and could potentially complement the current noninvasive diagnostic parameters.

Abstract 4142: Fibroblast activation protein transmembrane receptor targeted near-infrared fluorochrome specifically identifies patient derived lung cancer associated fibroblasts in pre-clinical models

Abstract Objective: Cancer-associated fibroblasts (CAFs) are a key component of the tumor microenvironment and affect neoplastic progression by stromal remodeling, paracrine signaling, potentiating cancer cell proliferation, and immune system evasion. As such, they are a potential target for optimizing therapeutic and diagnostic strategies. The purpose of our study was to evaluate if Fibroblast Activation Protein (FAP) can be a suitable target for molecular imaging guided cancer localization during surgery because FAP is overwhelmingly expressed by CAFs. Methods: Patient derived lung adenocarcinoma FAP+/alpha SMA+ myofibroblasts (CAFs) were isolated by flow cytometry and cultured according to institutional best practices. Normal fibroblasts from 3T3 cell line were used as negative control whereas normal lung fibroblasts transfected with human FAP cDNA were used as positive control. Cell expression of FAP was compared using SDS Page analysis and flow cytometry. FAP ligand coupled to NIR S0456 fluorochrome (FAP-S0456) was then evaluated with in-vitro dose escalation, cell-toxicity study, fluorescence parameters, and immunofluorescence microscopy. Results: Patient derived lung cancer (LC) CAFs had lower FAP-S0456 dye binding expression compared to transfected fibroblasts (TF), while normal fibroblasts (FAP-, aSMA-) had no FAP-S0456 dye binding detected on FACS direct antibody analysis (MFI 1033 vs 244, p&amp;lt;0.05). SDS page immunofluorescence protein analysis with FAP-S0456 showed 5.33x relative expression of FAP in LC CAFs and transfected fibroblasts compared to normal controls on densitometric analysis (p&amp;lt;0.05). Both the LC CAFs and FAP transfected fibroblasts demonstrated targeted fluorochrome endosomal internalization at 798 nm wavelength with mean MFI of 418 (SD 41) compared to minimal uptake by normal fibroblasts (MFI 49 (SD 5.12)p&amp;lt;0.05). FAP-S0456 was internalized by FAP-expressing fibroblasts in a dose- and time-dependent manner, as compared to normal fibroblasts, which did not internalize the dye. Matrix co-culturing FAP+/GFP+ fibroblasts with A549 cells showed FAP-S0456 uptake in the GFP+ fibroblasts with no fluorescence observed in neoplastic cells. Evaluation of FAP-S0456 specificity for FAP transmembrane protein by competitive inhibition via 1000x free FAP ligand showed no uptake in the FAP expressing fibroblasts (LC CAF, FAP+ TF) with MFI similar to negative controls (normal fibroblasts, (39 vs 48, p=0.77). Conclusion: FAP transmembrane receptor is highly expressed in cancer associated fibroblasts and can serve as a potential target for FAP ligand conjugated near-infrared tracers. This study demonstrates proof of principle of utilizing FAP-S0456 in CAF in-vitro models and paves a path for pre-clinical diagnostic and therapeutic validation for surgical applications. Citation Format: Feredun Azari, Gregory Kennedy, Ashley Chang, Bilal Nadeem, Neil Sullivan, Emanuel Encarnado, Steven Albelda, Evgeniy Eruslanov, Sunil Singhal. Fibroblast activation protein transmembrane receptor targeted near-infrared fluorochrome specifically identifies patient derived lung cancer associated fibroblasts in pre-clinical models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4142.

Abstract CT251: LuMIERE: A phase 1/2 study investigating safety, pharmacokinetics, dosimetry, and preliminary antitumor activity of 177Lu-FAP-2286 in patients with advanced or metastatic solid tumors

Abstract Background: Fibroblast activation protein (FAP) is a membrane-bound protease that is highly expressed on the surface of cancer-associated fibroblasts (CAFs) present in the tumor microenvironment of most epithelial cancers. With limited expression observed in normal tissues, FAP is a promising therapeutic and imaging target for delivery of radionuclides to cancer tissues. FAP-2286, a low-molecular-weight cyclic peptide that potently and selectively binds to FAP, is attached to a linker and tetraazacyclododecane tetraacetic acid (DOTA) cage that can be used to conjugate radionuclides such as lutetium-177 (177Lu) or gallium-68 (68Ga) for therapeutic or imaging applications, respectively. 177Lu-FAP-2286 has demonstrated antitumor activity in preclinical studies. The LuMIERE study (NCT04939610) is the first phase 1/2 clinical trial of an FAP-targeted radionuclide therapy evaluating 177Lu-FAP-2286 in patients with FAP-expressing solid tumors that are identified with the imaging agent 68Ga-FAP-2286. Methods: Eligible adult patients with advanced or metastatic solid tumors and measurable disease (per RECIST v1.1) will be selected for treatment with 177Lu-FAP-2286 on the basis of FAP expression using 68Ga-FAP-2286. In phase 1, tumors must be refractory to or have progressed following prior treatment, with no satisfactory alternative treatment options. Phase 1 includes dose escalation of 177Lu-FAP-2286, starting at 3.7 GBq to a maximum of 9.25 GBq. Patients may receive up to 6 cycles of 177Lu-FAP-2286 administered on day 1 of each 6-week cycle. Three to 12 patients will be treated at each dose level in the dose-escalation portion. Tolerability will be assessed across multiple doses for determination of a recommended phase 2 dose (RP2D), which may be further evaluated in a phase 1 dose-expansion portion. FDG-PET/CT scans will be collected prior to treatment, and patients will be imaged by planar and SPECT/CT scans at multiple time points to calculate organ and tumor dosimetry to determine absorbed radiation dose. Disease status will be assessed per RECIST v1.1 every 6 weeks. The primary objective of phase 1 is to evaluate the safety of 177Lu-FAP-2286 and determine the RP2D. Secondary objectives include the assessment of radiation dosimetry, pharmacokinetics, and preliminary efficacy of 177Lu-FAP-2286 and the evaluation of safety and tumor uptake of 68Ga-FAP-2286. Phase 2 will evaluate the efficacy, safety, and dosimetry of 177Lu-FAP-2286 in patients with select FAP-expressing solid tumors. Citation Format: Jonathan McConathy, Mallika Dhawan, Ajit H. Goenka, Emerson A. Lim, Yusuf Menda, Beth Chasen, Moh'd Khushman, Akiva Mintz, Yousef Zakharia, John J. Sunderland, Owen Bowles, Jim Xiao, Andrew D. Simmons, Kenton Wride, Aaron Enke, Thomas A. Hope. LuMIERE: A phase 1/2 study investigating safety, pharmacokinetics, dosimetry, and preliminary antitumor activity of 177Lu-FAP-2286 in patients with advanced or metastatic solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr CT251.

Abstract 4134: Stromal FAP expression is associated with MRI visibility and patient survival in prostate cancer

Abstract Some clinically significant prostate cancers (PCa) are missed by magnetic resonance imaging (MRI). We asked if the tumor stroma in surgically treated primary PCa lesions positive or negative with MRI are different in their cellular and molecular properties, and whether the differences are reflected to the clinical course of the disease. We profiled the stromal and immune cell composition of MRI-classified tumor lesions by applying multiplexed fluorescence immunohistochemistry (mfIHC) and automated image analysis in a clinical cohort of 343 patients (Cohort I). We compared stromal variables between MRI-visible, invisible lesions, and benign tissue and assessed the predictive significance for biochemical recurrence (BCR) and disease-specific survival (DSS) using Cox regression and Log rank analysis. Subsequently, we carried out a prognostic validation of the identified biomarkers in a population-based cohort of 319 patients (Cohort II). MRI true-positive lesions are different from benign tissue and MRI false-negative lesions in their stromal composition. CD163-positive cells and fibroblast activation protein (FAP)-positive cells were more abundant in MRI true-positive than in MRI false-negative lesions or benign areas. In MRI true-visible lesions, a high proportion of stromal FAP-positive cells was associated with PTEN status and increased immune infiltration (CD8+, CD163+), and predicted elevated risk for BCR. High FAP phenotype was confirmed to be a strong indicator of poor prognosis in two independent patient cohorts using also conventional IHC. The molecular composition of the tumor stroma may determine whether early prostate lesions are detectable by MRI and associates with survival after surgical treatment. Citation Format: Teijo Pellinen, Kevin Smith, Sami Blom, Riku Turkki, Annabrita Hemmes, Katja Välimäki, Juho Eineluoto, Anu Kenttämies, Stig Nordling, Olli Kallioniemi, Antti Rannikko, Tuomas Mirtti. Stromal FAP expression is associated with MRI visibility and patient survival in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4134.

Influence of Wound Fluid on the Transdifferentiation of Human Mesenchymal Bone Marrow Stem Cells into Cancer-Associated Fibroblasts

Cancer-associated fibroblasts (CAF) in the tumor microenvironment have a decisive influence on tumor growth and metastatic behavior. The cellular origins as well as the stimuli leading to CAF formation are heterogenous, impeding a precise characterization. Aim of this study was to analyze the influence of cytokines secreted in the process of wound healing, tumor cell-associated paracrine-secreted factors, and direct cell-cell contact on the expression of the CAF-associated markers fibroblast activation protein (FAP), α-smooth muscle actin (α-SMA), thrombospondin-1 (THBS1), and tenascin-c (TNC) by RT-PCR in mesenchymal stem cells (MSC). Cells developed different morphological characteristics after incubation with wound fluid (WF). Moreover, expression of FAP and α-SMA in MSC was significantly reduced after WF compared to tumor-conditioned medium and in co-culture with tumor cells; THBS1 and TNC were not significantly altered after any of the different incubation methods. There were no alterations of expression patterns of FAP and α-SMA in the immunohistochemical analysis. Differ­ences in the cytokine composition of the media were found in the dot blot. The heterogeneity of the results emphasizes the complexity of the interactions of tumor cells and cells of the microenvironment, particularly through the addition of human-derived WF.

POS0249 FIBROBLAST ACTIVATION PROTEIN AS A LINK BETWEEN INFLAMMATION AND VASCULAR REMODELING IN GIANT CELL ARTERITIS

BackgroundGiant cell arteritis (GCA) can present with serious complications such as blindness, stroke and aortic aneurysm that are related to both inflammation and remodeling of the vessel wall. GCA frequently overlaps with polymyalgia rheumatica (PMR). The pathogenesis of GCA starts in the adventitia where fibroblasts are the major stromal cell population. A preliminary study [1] reported the migration of adventitial fibroblasts to the intima, contributing to intimal hyperplasia in GCA. Fibroblast activation protein (FAP) is a non-classical serine protease which can be present in both a membrane-bound form and a soluble form and has been demonstrated to promote inflammation and fibrosis in coronary artery disease and rheumatoid arthritis [2-3]. We hypothesize that FAP is involved in GCA vasculopathy due to its pro-inflammatory and pro-fibrotic effects.ObjectivesAs a first step to unravel the contribution of FAP to the vasculopathy in GCA, we determined FAP plasma levels and FAP protein expression at the site of vascular inflammation in GCA.MethodsIn our prospective cohort of GCA, PMR and healthy elderly (GPS), we measured the plasma FAP levels with ELISA in new-onset, treatment-naïve GCA (N=62), and PMR (N=63) patients and 42 age-matched healthy controls (HC). In addition, we measured the plasma FAP levels at follow-up (3-months, 1-year, 1.5-year and treatment-free remission (TFR)). Temporal artery biopsies (TAB) from treatment-naïve GCA patients (n=9) and non-GCA patients (n=9), aorta samples from GCA-related aneurysm (n=9) and atherosclerosis (n=11) were stained for FAP using immunohistochemistry. Immunofluorescence staining for CD90, CD68, αSMA and FAP was performed to detect FAP expression in fibroblasts, macrophages and vascular smooth muscle cells (VSMC), respectively.ResultsBaseline plasma FAP levels were significantly lower in GCA patients (52.72±2.93 ng/ml) than in PMR patients (66.42±2.86 ng/ml) and HC (80.47±3.38 ng/ml). FAP levels at baseline correlated inversely with C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), interleukin (IL)-6, macrophage-associated protein YKL-40 (Chitinase 3-like 1/CHI3L1) levels and monocyte counts. Plasma FAP levels in GCA patients decreased even further at 3 months (37.23±2.10 ng/ml) upon glucocorticoid-induced remission, and gradually increased to the level of HC (76.84±5.43 ng/ml) when patients were in TFR. Increased FAP expression in GCA TAB (adventitia, media, intima) and aorta (media) compared with tissues from non-GCA was documented. FAP was abundantly expressed in fibroblast- and macrophage-rich areas but not in VSMC-rich areas in TAB and aorta of GCA patients.ConclusionFAP expression is clearly modulated both in plasma and at the site of vascular inflammation in GCA and may represent a pathogenic link between the inflammatory and remodeling processes in GCA. As such, FAP may have utility as a biomarker and should be further investigated as target for therapeutic intervention.

Dichotomous Roles of Men1 in Macrophages and Fibroblasts in Bleomycin-Induced Pulmonary Fibrosis.

Pulmonary fibrosis therapy is limited by the unclear mechanism of its pathogenesis. C57BL/6 mice were used to construct the pulmonary fibrosis model in this study. The results showed that Men1, which encodes menin protein, was significantly downregulated in bleomycin (BLM)—induced pulmonary fibrosis. Mice were made to overexpress or had Men1 knockdown with adeno-associated virus (AAV) infection and then induced with pulmonary fibrosis. BLM—induced pulmonary fibrosis was attenuated by Men1 overexpression and exacerbated by Men1 knockdown. Further analysis revealed the distinct roles of Men1 in fibroblasts and macrophages. Men1 inhibited fibroblast activation and extracellular matrix (ECM) protein expression while promoting macrophages to be profibrotic (M2) phenotype and enhancing their migration. Accordingly, pyroptosis was potentiated by Men1 in mouse peritoneal macrophages (PMCs) and lung tissues upon BLM stimulation. Furthermore, the expression of profibrotic factor OPN was positively regulated by menin in Raw264.7 cells and lung tissues by binding to the OPN promoter region. Taken together, although Men1 showed antifibrotic properties in BLM—induced pulmonary fibrosis mice, conflictive roles of Men1 were displayed in fibroblasts and macrophages. The profibrotic role of Men1 in macrophages may occur via the regulation of macrophage pyroptosis and OPN expression. This study extends the current pathogenic understanding of pulmonary fibrosis.

Assaying fibroblast activation protein (FAP) expression in vivo and in vitro for possible targeting with chimeric antigen receptor (CAR) T cells

Fibroblast activation protein (FAP) is a marker of activated fibroblasts in various human cardiovascular diseases. FAP is of special interest because it is expressed exclusively in activated, pathogenic fibroblasts and has been proposed as a target for chimeric antigen receptor (CAR) T cell ablation therapy. It has been shown previously that fibroblasts from patients with hypertrophic and dilated cardiomyopathy express FAP, while fibroblasts from non‐failing hearts did not. The aim of the project was to examine FAP expression in other human cardiac diseases by obtaining heart samples from patients who suffered diseases characterized by cardiac fibrosis so that we could determine which cardiac diseases might be amenable to FAP CAR T therapy. In addition, we sought to characterize an in vitro model of FAP expression to aid in the ability to assess the effectiveness of engineered T cells to kill FAP‐expressing cells. Heart tissue from patients diagnosed with various cardiac disorders, including idiopathic giant cell myocarditis, COVID‐19, and sarcoidosis were stained for FAP expression and compared to non‐failing, control hearts. The results showed significant FAP expression in all cardiac specimens with interstitial fibrosis compared to non‐failing hearts. Notably, samples from patients who died from COVID‐19 exhibited significant fibrosis and FAP expression. This suggests that there may be a large and growing population that may be amenable to anti‐fibrotic therapy. In order to develop an in vitro model of FAP expression to assay anti‐FAP CAR T cell activity, we treated IMR‐90 human fibroblasts with TGF‐B, a known activator of fibrosis, and assayed for FAP expression. TGF‐B exposure resulted in an increase in FAP expression 24‐hours after treatment. Our observation of fibroblast restricted FAP expression in multiple different cardiovascular diseases supports the potential use of FAP CAR T cell therapy as an anti‐fibrotic treatment in multiple heart diseases.

CT radiomics signature: a potential biomarker for fibroblast activation protein expression in patients with pancreatic ductal adenocarcinoma.

To develop and validate a radiomics model to predict fibroblast activation protein (FAP) expression in patients with pancreatic ductal adenocarcinoma (PDAC). This retrospective study included consecutive 152 patients with PDAC who underwent MDCT scan and surgical resection from January 2017 to December 2017 (training set) and from January 2018 to April 2018 (validation set). In the training set, 1409 portal radiomic features were extracted from each patient's preoperative imaging. Optimal features were selected using the least absolute shrinkage and selection operator (LASSO) logistic regression algorithm, whereupon the extreme gradient boosting (XGBoost) was developed using the radiomics features. The performance of the XGBoost classifier performance was assessed by its calibration, discrimination, and clinical usefulness. The patients were divided into FAP-low (n = 91; 59.87%) and FAP-high (n = 61; 40.13%) groups according to the optimal FAP cutoff (45.71%). Patients in the FAP-low group showed longer survival. The XGBoost classifier comprised 13 selected radiomics features and showed good discrimination in the training set [area under the curve (AUC), 0.97] and the validation set (AUC, 0.75). It also performed well in the calibration test and decision-curve analysis, demonstrating its potential clinical value. The XGBoost classifier based on CT radiomics in the portal venous phase can non-invasively predict FAP expression and may help to improve clinical decision-making in patients with PDAC.

Feasibility of [68Ga]Ga-FAPI-46 PET/CT for detection of nodal and hematogenous spread in high-grade urothelial carcinoma

Background[68Ga]Ga-FAPI-46 is a novel positron emission tomography (PET) ligand that targets fibroblast activation protein (FAP) expression as FAP inhibitor (FAPI) and could already show promising results in several tumor entities. It could be demonstrated that an increased FAP expression correlates with tumor aggressivity in urothelial carcinoma (UC). Given the limited value of [18F]FDG in UC, [68Ga]Ga-FAPI-46 could add diagnostic information in staging and response assessment in UC. We present the first data of [68Ga]Ga-FAPI-46 PET imaging in a pilot cohort of UC patients evaluating uptake characteristics in metastases and primary tumors.MethodsFifteen patients with UC prior to or after local treatment underwent [68Ga]Ga-FAPI-46 PET/CT imaging for detection of metastatic spread. We compared the biodistribution in non-affected organs and tumor uptake of UC lesions by standard uptake value measurements (SUVmean and SUVmax). Additionally, metastatic sites on PET were compared to its morphological correlate on contrast-enhanced computed tomography (CT).ResultsOverall, 64 tumor sites were detected on PET and/or CT. The highest uptake intensity was noted at the primary site (SUVmax 20.8 (range, 8.1–27.8)) followed by lymph node metastases (SUVmax 10.6 (range, 4.7–29.1)).In 4/15 (26.7%) patients there were [68Ga]Ga-FAPI-46-positive lesions that were missed on standard routine CT imaging. On the other hand, 2/15 patients had suspicious prominent bipulmonary nodules as well as pelvic lymph nodes previously rated as suspicious for metastatic spread on CT, but without increased FAPI expression; here histopathology excluded malignancy.Conclusion[68Ga]Ga-FAPI-46 PET shows distinctly elevated uptake in UC lesions. Therefore, the tracer has potential as a promising new biomarker in metastatic UC patients, as [68Ga]Ga-FAPI-46 PET might improve detection of metastatic sites compared to CT alone. These findings highly emphasize larger studies investigating FAPI imaging in UC patients.

Expression of fibroblast activation protein in lung cancer and its correlation with tumor glucose metabolism and histopathology.

To explore the expression of fibroblast activation protein (FAP) in lung cancer (LC) and its correlation with tumor glucose metabolism and histopathology. From June 2018 to November 2020, 73 patients with newly diagnosed LC were included. Immunohistochemical staining was used to quantify FAP expression in tumors. The histopathological type and tumor grade were determined via histopathological examination. The tumor glucose metabolism parameters and tumor maximal diameter were measured via [18F] F-FDG PET/CT. Univariate and multivariate analysis were performed to study the correlation of FAP expression levels with glucose metabolism variables and tumor histopathology. Positive FAP expression was observed in 97.3% (71/73) LC lesions, which was significantly higher than 87.7% (64/73) of [18F] F-FDG positivity observed on PET/CT (χ2 = 4.818, P = 0.028). In 12 early adenocarcinomas (ADCs), only three lesions (25%) were positive for [18F] F-FDG on PET/CT; however, 10 lesions (83.3%) were positive for FAP. When FAP expression was classified into low level (scores ≤ 3) and high level (scores > 4), high FAP level was found in 80.8% tumors and low FAP level in the other 19.2% tumors. High FAP level was identified in 100.0% of squamous cell carcinomas (SCCs), 85.7% of ADCs, 66.7% (4/6) of large cell neuroendocrine carcinomas (LCNCs), and 40.0% (4/10) of small cell lung cancers (SCLCs) (P < 0.05). In non-mucinous ADC lesions, on univariate analysis, FAP expression level showed a close relationship with tumor metabolism parameters (maximal standard uptake value (SUVmax), mean standard uptake value (SUVmean), and total lesion glycolysis (TLG)), tumor diameter, tumor grade, and lesion attenuation (P < 0.05). The present study demonstrates that FAP is widely expressed in LC and shows great variation in different histopathological types. A high positive rate of FAP expression implies that FAP-targeted imaging may be a sensitive modality for diagnosing LC, especially in early ADCs. Further validation with such probes is warranted.

Serum Exosomal lncRNA AC007099.1 Regulates the Expression of Neuropeptide-Related FAP, as a Potential Biomarker for Hepatocarcinogenesis.

Neuropeptide-associated fibroblast activation protein (FAP) may be an important risk factor for neurovascular metastasis in hepatocellular carcinoma. Analysis of The Cancer Genome Atlas (TCGA) database showed that FAP mRNA was highly expressed in most human tumor tissues. The HPA database then verified that FAP was highly expressed in tumor tissues following protein translation. Survival analysis then showed that the level of FAP expression significantly affected the overall survival (OS), progress free interval (PFI), and disease specific survival (DSS) of patients with hepatocellular carcinoma. A high expression of FAP in tumor tissue is associated with poor patient prognosis. According to the results of spearman correlation, AC009099 and FAP were negatively correlated with miR-7152 expression, while AC009099 and FAP expression were positively correlated. The lncRNA AC007099.1, which may serve as a potential target for the treatment of hepatocellular carcinoma, was associated with liver cancer. AC007099.1/miR-7152/FAP was found to be associated with immune infiltration in patients with hepatocellular carcinoma. Enrichment analysis suggests that the AC009099/miR-7152/FAP ceRNA regulatory network is associated with neuropeptide functional pathways. In conclusion, a neuropeptide-related AC009099/miR-7152/FAP ceRNA regulatory network was constructed in this study.

Expression of Potential Biomarker Targets by Immunohistochemistry in Cervical Carcinomas.

There have been few clinically useful targetable biomarkers in uterine cervical carcinomas. Estrogen receptor (ER), HER2, and fibroblast activation protein (FAP) are potential therapeutic or theranostic targets in other gynecologic and genitourinary carcinoma types. We determined the immunohistochemical expression patterns of these markers in treatment-naive cervical carcinoma, and whether expression correlated with clinical outcomes after definitive chemoradiation therapy. Tissue microarrays were created from 71 patient samples taken before therapy (57 squamous cell carcinomas and 14 nonsquamous cell carcinomas) and stained for ER, HER2, and FAP. ER was positive in 25/70 cases (36%). Of 66 tumors with evaluable HER2 staining, only 1 had positive (3+) staining (3%, positive for HER2 amplification by fluorescence in situ hybridization), and 1 had equivocal (2+) staining (negative for amplification by fluorescence in situ hybridization). The remainder were negative for HER2 overexpression. FAP expression was widely variably in the tumor stroma. ER positivity and FAP expression did not correlate with cervical recurrence, pelvic recurrence, distant recurrence, or cancer death. In conclusion, HER2 amplification is very rare in nonmetastatic treatment-naive cervical carcinomas, but if present, could represent a target for antibody therapy. ER and FAP were expressed in a subset of tumors, but expression did not correlate with clinical outcomes. These immunohistochemical markers do not demonstrate prognostic significance in treatment-naive cervical cancer, but they may have utility in targeted therapy or imaging.

P082 The profibrogenic role of neutrophil extracellular traps in stenotic Crohn’s disease: a new antifibrotic target?

Abstract Background Neutrophil extracellular traps (NETs) are structures constituted of DNA filaments and protein granules, extruded by neutrophils after various insults, such as chronic inflammation. Their formation is dependent on the citrullination of histone H3 (H3cit) in the nucleus by peptidylarginine deiminase 4 (PAD4). The pathogenic effect of NETs in Crohn’s disease (CD) has been poorly addressed so far. Recently, a profibrogenic role of NETs has been proposed in the lung. The aim of the present study was to assess whether NETs may trigger the fibrotic process in patients with CD. Methods Multiplex immunofluorescence (M-IF) for PAD4, myeloperoxidase, neutrophil elastase, H3cit, vimentin and alpha-SMA were performed in unaffected, inflamed and fibrotic ileum of patients operated for stricturing CD. From the same patients, primary intestinal fibroblasts (PIF) were cultured from unaffected ileum. NETs were obtained from healthy volunteers’ neutrophils, incubated with 50 nM phorbol myristate acetate for 3.5 hours. Subconfluent PIF were stimulated in vitro with NETs for 6 hours and RNA extracted for sequencing (RNAseq). Cell culture medium was collected for soluble collagen analysis, using SIRCOL®. Experiments were replicated in 5 patients. CCD-18Co cell line was used to test the migratory activity of fibroblasts with a scratch test and to confirm the expression of profibrogenic proteins, such as fibroblast activation protein (FAP) and collagen 3A1. M-IF was interpreted using computational analysis. Statistics was performed with Kruskal-Wallis Test for collagen quantification, with p &amp;lt; 0.05. For RNAseq, Poisson distribution and DESeq2 were used, with significance set at Q &amp;lt; 0.05; the resulting gene list was then analysed with ClueGo® software. Results M-IF showed that NETs were primarily found in inflamed areas, with clusters of these traps located close to intestinal myofibroblasts in the deeper submucosa layer (Figure 1). The gene ontology of RNAseq (Figure 2) showed significant upregulation of profibrogenic pathways of TGFB1/SMAD and p38/MAPK in the group of PIF co-cultured with NETs. NETs stimulation determined a higher release of collagen in the medium (p = 0.003), and an increase in the protein expression of FAP and collagen 3A1 in PIF. Accordingly, NETs profibrotic effect resulted also in a slower migratory ability of activated fibroblasts. Conclusion Our data show that NETs are produced during active inflammation of CD and their colocalisation with intestinal myofibroblasts suggests that they may represent an early trigger of fibroblast activation. Clinically, the inhibition of PAD4 might result in both contrasting inflammation and fibrogenesis. An in vivo chronic model of colitis on PAD4-KO mice is ongoing to confirm these findings.

Expression of Fibroblast Activation Protein Is Enriched in Neuroendocrine Prostate Cancer and Predicts Worse Survival.

Background: Advanced prostate cancer (PC) may accumulate genomic alterations that hallmark lineage plasticity and transdifferentiation to a neuroendocrine (NE) phenotype. Fibroblast activation protein (FAP) is a key player in epithelial-to-mesenchymal transition (EMT). However, its clinical value and role in NE differentiation in advanced PC has not been fully investigated. Methods: Two hundred and eight patients from a multicenter, prospective cohort of patients with metastatic castration-resistant prostate cancer (CRPC) with available RNA sequencing data were analyzed for tumor FAP mRNA expression, and its association with overall survival (OS) and NE tumor features was investigated. Results: Twenty-one patients (10%) were found to have high FAP mRNA expression. Compared to the rest, this subset had a proportionally higher exposure to taxanes and AR signaling inhibitors (abiraterone or enzalutamide) and was characterized by active NE signaling, evidenced by high NEPC- and low AR-gene expression scores. These patients with high tumor mRNA FAP expression had a more aggressive clinical course and significantly shorter survival (12 months) compared to those without altered FAP expression (28 months, log-rank p = 0.016). Conclusions: FAP expression may serve as a valuable NE marker indicating a worse prognosis in patients with metastatic CRPC.

Circulating Dipeptidyl Peptidase Activity Is a Potential Biomarker for Inflammatory Bowel Disease.

INTRODUCTION:Dipeptidyl peptidase (DPP)-4 is part of a larger family of proteases referred to as DPPs. DPP4 has been suggested as a possible biomarker for inflammatory bowel disease (IBD). Circulating DPP4 (cDPP4) enzyme activity was investigated as a potential biomarker for IBD. In addition, DPP enzyme activity and gene expression were quantified in colonic tissue of patients with IBD and non-IBD.METHODS:In study 1, DPP enzyme activity was quantified in plasma samples from 220 patients with IBD (Crohn's disease [CD] n = 130 and ulcerative colitis [UC] n = 90) and non-IBD controls (n = 26) using a colorimetric assay. In study 2, tissue and plasma samples were collected from 26 patients with IBD and 20 non-IBD controls. Plasma C-reactive protein (CRP) was quantified in all patients. Colonic DPP4, DPP8, DPP9, and fibroblast activation protein (FAP) gene expression was determined by quantitative polymerase chain reaction. cDPP and cFAP enzyme activity was also measured. Sensitivity and specificity were determined by receiver operating characteristic curve analysis.RESULTS:In study 1, total cDPP activity was found to differentiate patients with CD with active disease (n = 18) from those in remission (n = 19; sensitivity 78% and specificity 63%). In study 2, total cDPP and cFAP activity was 28% and 48% lower in patients with elevated CRP (>10 mg/L), respectively, compared with patients with normal CRP. Gene expression of DPP4, FAP, and DPP8 was also significantly higher in colonic biopsies from patients with IBD compared with non-IBD patients (P < 0.05).DISCUSSION:Our findings implicate the DPP enzyme family in intestinal inflammation and suggest future biomarker applications to differentiate the pathophysiological aspects of IBD.