Clinical applications of fibroblast activation protein inhibitor positron emission tomography (FAPI-PET)

Clinical applications of fibroblast activation protein inhibitor positron emission tomography (FAPI-PET)

Introduction

The importance of the tumor microenvironment (TME) and its influence on tumor immunity has been a dominant theme of cancer research for the last decade. Multiple immune-modulating therapies have been introduced including checkpoint inhibitors, chimeric antigen receptor T cells (CAR-T), cancer vaccines and T cell transfer therapy, among others, which depend on activating the anti-tumor immune response. These therapies can lead to dramatic clinical responses1. Cancer-associated fibroblasts (CAFs) are stromal cells found in the TME of some tumors. They represent the “activated state” of tissue fibroblasts and are known to aid cancer growth2 by interfering with immune surveillance while producing stromal proteins that physically isolate cancer cells from exposure to the immune response and drug therapy. Indeed, the full range of pro-tumor effects of activated fibroblasts is still not completely understood. CAFs exhibit a specific membrane expression profile including the expression of fibroblast activation protein (FAP). Recent advances in cell biology and radiochemistry have enabled the development of radiolabeled FAP inhibitors that bind to membrane-bound FAP with high affinity. The original compounds have now grown into a class of FAP-targeting ligands and permit the non-invasive imaging of FAP expression in vivo.

In this review we provide an overview of the current status of FAPI-PET, beginning with a brief description of FAP and FAP tracers, the importance of tumor stroma, and the potential clinical applications of this new class of PET tracers in comparison to FDG-PET. Here, we summarize the current understanding of FAPI-PET in selected diseases.

Stromal targeting with FAP

Fibroblast activation protein (FAP)

Fibroblast activation protein (FAP) was initially described by Wolfgang Rettig in 1988 as a cell surface antigen expressed on reactive stromal fibroblasts within many epithelial cancers and in granulation tissue of wound healing, as well as on tumor cells of many sarcomas3. Conversely, FAP expression is absent in most normal tissue, including normal fibroblasts, non-malignant epithelial cells, or the stroma of benign epithelial tumors3.

FAP was subsequently identified as a membrane-bound type II serine protease4,5, which is chemically classified as a member of the dipeptidyl peptidase IV (DPPIV) family of proteins. FAP consists of 760 amino acids with a short intra- (6 amino acids), trans- (20 amino acids), and a large extracellular membrane domain (734 amino acids)6, the latter offering a favorable docking site for ligands7,8. FAP possesses both endo- and exopeptidase activities and, thus, enables matrix remodeling through the cleavage of matrix proteins9,10. FAP is overexpressed in CAFs, which can be found in over 90% of epithelial cancers to varying degrees10. For instance, CAFs are commonly found in abundance in neoplasms with strong desmoplastic reactions such as pancreatic11,12, colorectal13,14, and breast15,16,17 cancer. The possible association of FAP expression with tumor aggressiveness was noted even in the early literature13,18,19 and subsequently confirmed in various cancer types3,11,12,13,14,15,16,17,18,19,20,21,22,23. Moreover, FAP can also be expressed directly in a limited number of cancer cells, such as some ovarian20,21,22, breast15,16, an pancreatic cancers12,24 as well as in sarcomas3,23, suggesting possible theranostic applications in the future.

FAP-targeting tracer

Small enzyme inhibitors specific for FAP were initially proposed by Jansen et al, who designed several compounds including UAMC-1110, which showed specificity for FAP25,26. Through the chemical modification of the quinoline group of UAMC-1110, researchers at the University of Heidelberg successfully synthesized a family of FAPI tracers beginning in 201827. These compounds demonstrated inhibition of the endopeptidase activity of FAP. Moreover, the attachment of 1,4,7,10-tetraazacylclododecane-1,4,7,10-tetrayl tetraacetic acid (DOTA)-chelator enabled binding of Ga-68 for PET imaging, as well as other therapeutic radioisotopes such as Lu-17727. In the initial production of FAPI tracers, FAPI-04 revealed the most favorable pharmacokinetic profile with higher uptake and more tumor retention up to 3 h post injection28. Dosimetric analysis showed a suitable equivalent dose of approximately 3–4 mSv for 200 MBq, which is comparable to 18F-FDG or 68Ga-DOTA-(Tyr3)-octreotate (DOTATATE)28. In the subsequent series of tracer development, 68Ga-FAPI-46 showed even higher tumor retention and tumor-to-background ratio (TBR)29. Currently, these tracers namely 68Ga-FAPI-04 and -46, are the most widely used FAP tracers around the world30. While 68Ga-labeling has inherent limitations due to its shorter half-life and limited synthesis capacity, 18F- or 99mTc-labeled FAPI compounds (18F-FAPI-74 for PET, 99mTc-FAPI-34 for SPECT imaging) have also been synthesized31,32,33. These compounds provide potential advantages for wider clinical use because of the longer half-life of these isotopes. For instance, labeling with F-18 enables a larger production batch which can then be distributed around a metropolitan area rather than requiring labeling at each imaging site. 99mTc with its 6 h half-life utilizes local labeling but enables the application of SPECT cameras instead of PET, which could reduce costs34.

Other promising approaches to targeting FAP include targeting peptides or peptidomimetic compounds35,36. OncoFAP is a small organic FAP ligand with ultrahigh affinity35,37. Baum and colleagues presented a new cyclic peptide FAP-2286 that selectively binds to FAP with low off-target activity36,38. Further modification of the quinoline-based structure has been performed by Ballal and colleagues, who developed 68Ga-DOTA.SA.FAPi as a quinoline-based monomer which offers the possibility of wider theranostic use through dimerization (DOTAGA.(SA.FAPi)239,40. The chemical structures of the selected FAP tracers are shown in Fig. 1.

Fig. 1: Chemical structures of the selected FAP tracers.
Clinical applications of fibroblast activation protein inhibitor positron emission tomography (FAPI-PET)

Chemical structures of the selected FAP tracers for diagnostic use, except (DOTAGA.(SA.FAPi)2, which is the dimer of the diagnostic tracer DOTA.SA.FAPi and more suitable for therapeutic purposes due to the longer tumor retention.

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Oncological indications

Overview

18F-FDG is currently the most widely used radiotracer in oncological imaging41, however, it has well known limitations42,43,44. For instance, FDG accumulates only in glucose-consuming cells and many cancers are either metabolically inactive or rely on energy sources other than glucose42. Moreover, FDG is also taken up in inflammation, which can lead to uncertainty over whether residual uptake is due to persistent tumor or inflammatory response to therapy45. FDG is also subject to variability due to patient preparation which includes fasting and resting prior to scanning42. In contrast, FAPI-PET is independent of glucose metabolism and there is no need for patient preparation. Whereas an incubation period of 60 min is recommended for FDG-PET45, FAPI-PET can be initiated as early as 10 min post injection (p.i.) with an acceptable imaging quality up to 3 h p.i28,46. Whereas FDG can be retained in background tissues, biodistribution studies of 68Ga-FAPI-PET show fast renal clearance and low tracer uptake in normal organs28. This results in significantly lower background signal compared to FDG, especially in FDG-avid organs such as brain, liver, or gastrointestinal tract28. Lower background results in higher target-to-background ratios (TBRs) with improved image contrast, contributing to the higher sensitivity for detecting malignant lesions that has been observed in many comparisons studies43,44,47,48.

Cancer-associated fibroblasts (CAFs) and stroma

To be an effective diagnostic biomarker, FAPI-PET requires that tumors contain FAP-expressing CAFs in the tumor stroma or on the tumor cells themselves. Stroma typically consists of various immune cells, fibroblasts, endothelial cells, and extracellular matrix (ECM), surrounding neoplastic cells19,49,50 and is present even in small tumors of 1–2 mm in diameter51. Cancer-associated fibroblasts (CAFs) are often the most abundant cell type in the TME19,50 and arise mainly from normal, resting fibroblasts19,52,53. However, other precursors of CAFs include hematopoietic stem cells, as well as epithelial and endothelial cells54,55,56,57,58. CAFs interact with other key immunomodulatory cells including tumor‐associated macrophages (TAMs), regulatory T cells (Tregs), and myeloid‐derived suppressor cells (MDSCs) by releasing growth factors and proinflammatory cytokines, such as transforming growth factor β (TGF-β), vascular endothelial growth factor (VEGF), and interleukin-6 (IL-6)50,59,60,61,62,63,64,65. For example, in some breast cancers CAFs play an immunosuppressive role by promoting monocyte migration and transforming macrophages to the M2-subtype using monocyte chemotactic protein‐1 (MCP‐1) and stromal cell‐derived factor‐1 (SDF‐1)59. Hence, CAFs emerge as a potential target for immune modulation59,66. The interactions of FAP with various immune cells in TME are schematically illustrated in Fig. 264.

Fig. 2: Effects of cancer-associated fibroblast (CAF) on immune cells.
figure 2

Major effects of cancer-associated fibroblast (CAF) on immune cells in the tumor microenvironment. TGF-β transforming growth factor beta, VEGF vascular endothelial growth factor, IL interleukin 6, GM-CSF granulocyte-macrophage colony-stimulating factor, M-CSF macrophage colony-stimulating factor, CCL C-C motif chemokine ligand, CXCL C-X-C motif ligand. BMDSC bone marrow-derived suppressor cells, Tregs regulatory T cells. [From ref. 244].

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When resident tissue fibroblasts convert to CAFs they morph from spindle- to stellate-shape and begin to express new surface markers, including α-smooth muscle actin (α-SMA), platelet-derived growth factor receptor (PDGFRα/β), vimentin, and FAP15,67,68,69, among which FAP is the most specific49,67 (Table 1). This activation can be caused by growth factors, such as TGF-β70,71. CAFs are actually quite heterogeneous with diverse functions, leading to several subclassifications68,72,73. Two commonly identified subpopulations of CAFs, ‘”myoCAFs” and “iCAFs”, are based on whether the CAF exhibits a matrix-producing contractile phenotype (myoCAF) or an immunomodulating phenotype (iCAF), respectively67,74. In pancreatic cancer, CAFs near the cancer cells exhibit a myoCAF phenotype with high TGF-β-driven α-SMA expression and lower levels of IL-6 (α‐SMAhighIL‐6low), while the more peripheral CAFs have lower αSMA expression with higher levels of IL-6 (α‐SMAlowIL‐6high) consistent with iCAFs74. The potential impact of these CAF subpopulations on tumor growth and on FAP imaging is still unknown and needs further elucidation.

Table 1 Markers of CAF
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Liver cancer

About half of hepatocellular carcinomas (HCC) are hypo- or isometabolic compared to normal liver and thus, show poor FDG uptake75. Diminished FDG uptake is due to high FDG-6 phosphatase activity and low glucose transporter expression, generally found in moderate to well-differentiated HCC76. Thus, FDG-PET is of limited value in HCC management.

Multiple studies comparing FDG-PET to FAPI-PET in HCC have documented the superior sensitivity and TBR of FAPI-PET77,78,79,80,81,82. For instance, the sensitivity of FAPI-PET for HCC varies in studies from 96 to 100% compared to 50–80% for FDG imaging77,78,79,80. The difference in sensitivity is especially pronounced for small lesions (≤2 cm in diameter) where the sensitivity of FAPI-PET vs. FDG-PET was 69% vs. 19% respectively and for well- or moderately differentiated vs. poorly differentiated HCCs where the difference was 83% vs. 33% respectively81. Higher SUVmax and TBR values for HCC have been found with 18F-FAPI-74 PET compared to 18F-FDG-PET (SUVmax: 6.7 vs. 4.3, TBR: 3.9 vs. 1.7, both P < 0.0001)82. This study also documented a higher detection rate for intrahepatic lesions (92.2% vs 41.1%, P < 0.0001) and lymph node metastases (97.9% vs 89.1%; P = 0.01), while the detection of distant metastases was more comparable (63.6% (42/66) vs 69.7% (46/66), P > 0.05)82. FAPI-PET resulted in upstaging, leading to changes in therapy planning in 48% of patients82. FAPI-PET sensitivity (96%) has also been favorably compared to other modalities such as MRI (100%) or contrast-enhanced CT for detection of HCC (96%) in comparison with FDG-PET (65%)77. The specificity of FAPI-PET varies from 90–100%.

Thus, FAPI-PET appears to be a highly sensitive and specific method of identifying HCC and is superior in sensitivity and specificity to FDG-PET. It will likely prove to be very helpful in identifying recurrences after focal therapy of HCC and thus, could become an important modality in the management of patients with HCC.

Biliary tract cancer

Cholangiocarcinoma (CCC) is characterized by a strong desmoplastic reaction similar to that seen in pancreatic cancer83, making FAPI-PET a promising modality for detecting this cancer. Studies have documented that FAPI-PET has superior sensitivity to FDG-PET for primary CCC (98% vs. 86%), lymph node metastases (90% vs. 87%), and distant metastases (100% vs. 84%)84. Higher tracer uptake was observed for FAPI-PET compared to FDG-PET for intrahepatic lesions, pelvic nodal metastases, and distant metastases such as in the pleura or omentum84,85. FAPI-PET led to changes in tumor staging in many patients85.

A possible correlation between FAPI uptake and the grade of malignancy has been suggested by several authors84,85,86. For instance, Pabst et al. demonstrated that Grade 3 CCC tumors showed a significantly higher 68Ga-FAPI-46 uptake than grade 2 tumors (SUVmax 12.6 vs. 6.4; P = 0.009)86. FAP expression was high in the tumor stroma of CCC (90% of cells were positive)86. Furthermore, FAPI-PET uptake correlated with carcinoembryonic antigen (CEA) and carbohydrate antigen19-9 (CA19-9), which are prognostic biomarkers84.

Thus, FAPI-PET demonstrates superior sensitivity to FDG-PET for CCC including at the localized, locally advanced, and metastatic stages of the disease.

Gastric cancer

In general, FDG-PET has low uptake in many gastric cancers including signet-ring cell carcinoma, mucinous adenocarcinoma, and non-interstitial diffuse type cancer87. In such patients, FAPI-PET has shown superior sensitivity to FDG-PET88,89,90,91,92,93,94,95 (Fig. 3). For instance, in gastric signet-ring cell carcinoma (GSRCC), FAPI-PET showed a higher detection rate of primary lesions (73% vs. 18%), lymph nodes (77% vs. 23%), and distant metastasis (93% vs. 39%)91. SUVmax and TBR values of 68Ga-FAPI-PET were significantly higher than 18F-FDG-PET in primary tumors (SUVmax: 5.2 vs. 2.2; TBR: 7.6 vs. 1.3, P < 0.001), lymph nodes (SUVmax: 6.8 vs. 2.5; TBR: 5.8 vs. 1.3, both P < 0.001), and bone and visceral metastases (SUVmax: 6.5 vs. 2.4; TBR: 6.3 vs. 1.3, both P < 0.001)91. In a cohort with mixed gastric cancer subtypes, three studies confirmed superior sensitivity of FAPI-PET over FDG-PET (88% vs. 60%)88,89,90.

Fig. 3: Head-to-head comparison of 18F-FDG and 18F-FAPI-74 PET imaging in selected oncologic patients.
figure 3

Nine representative oncologic patients who underwent 18F-FDG and 18F-FAPI-74 PET imaging. 18F-FAPI-74 PET outperforms 18F-FDG PET in detecting primary tumors (patients 11, 39, 50, 58, 79, and 101; solid black arrows), local recurrences (patient 4; blue arrows), abdomen lymph node metastases (patients 4 and 50; green arrows), intrahepatic metastases (patient 50; red arrows), bone metastases (patient 85; arrowheads), and peritoneal metastases (patients 4, 11, 85, 97, and 101; dotted arrows). [From ref. 88].

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Despite these favorable results, other studies have reported limitations of FAPI-PET in assessing lymph node status in gastric cancer92,95, probably due to the heterogeneous nature of these tumors. For example, one study found no difference in sensitivity and specificity in lymph node staging between the FDG and FAPI groups (P > 0.05)95. However, the median age of the patients in this study was relatively high (68 years), which might have led to a higher incidence of pathological abdominal conditions such as preceding surgery or co-existing infection, that could influence the tracer accumulation of both FAPI and FDG95.

The prognostic value of FAPI-PET in gastric cancer has been demonstrated by several authors. One study demonstrated that SUVmax and TBR of 68Ga-FAPI-04 correlated with clinical outcomes (T) and lymph nodal status (N), which suggested FAPI-PET is a prognostic surrogate92. Another study suggested that FAP expression of the primary tumor was significantly higher in patients who did not benefit from immune checkpoint blockade (ICB) therapy, making FAPI-PET a potential predictor of immunotherapy response74. The data suggest that high expression of FAP might indicate a poor prognosis in metastatic GC and FAPI-PET may serve as a non-invasive biomarker to select patients who are likely to benefit from the ICB therapy74.

Thus, FAPI-PET has a sensitivity advantage over FDG-PET for gastric cancer and may provide prognostic information as well, which should be evaluated in further studies.

Pancreatic cancer

In pancreatic ductal adenocarcinoma (PDAC), FAP overexpression is found not only in tumor stroma but also in the PDAC cells and may be associated with metastatic spread and worse clinical outcome12,24.

FAPI-PET outperforms FDG-PET in sensitivity and detection rate in PDAC96,97,98,99,100,101,102. In one study, FAPI-PET was superior to FDG-PET in detecting the primary tumor (100% vs. 95.0%), metastatic lymph nodes (96.2% vs. 61.5%), and distant metastases (100% vs. 84.0%) (all P < 0.0001)96,97. Consequently, disease management was altered in many cases96,97,98,101,102. Other studies suggest that FAPI-PET can alter tumor stage in 43% compared with conventional imaging99 (Fig. 4).

Fig. 4: Example images of 64-year-old woman with recurrent pancreatic ductal adenocarcinoma.
figure 4

A Maximum-intensity projection (MIP) of 68Ga-FAPI PET. B Axial 68Ga-FAPI PET/CT images and contrast-enhanced CT (ceCT) images of lesions (arrows: lesions 1 and 2, pulmonary metastasis and mediastinal lymph node metastasis; lesion 3, paraaortic lymph node metastasis) detected by 68Ga-FAPI PET. HU: Hounsfield units. [From ref. 99].

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In addition to detecting the extent of pancreatic cancer, FAPI-PET may also provide prognostic information96,102,103. For instance, one study established that tumors with 68Ga-DOTA-FAPI-04 PET/CT SUVmax > 14.9 before chemotherapy were more likely to progress96. Others have suggested that higher baseline metabolic tumor volumes (MTV) of 18F-AlF-NOTA-FAPI-04 were associated with poorer overall survival, although the hazard ratio was minimally elevated (hazard ratio HR = 1.016, P = 0.016)102. In treatment-naïve patients, 68Ga-FAPI-04 uptake correlates with both ex-vivo FAP expression and aggressive pathological features103. For instance, FAP expression was higher in poorly differentiated PDAC than in well- to moderately differentiated neoplasms103. Tumor uptake was also significantly correlated with tumor size, differentiation, and perineural invasion103. In this study, SUVmax was a significant independent prognostic predictor of recurrence-free survival (HR = 2.46, P < 0.05)103.

False positive results due to inflammatory conditions, such as pancreatitis, are an ongoing concern98,104. For the differentiation of inflammation from malignancy, the kinetics of uptake may be important. One group suggested that uptake values be obtained at 10 min, 1 h, and 3 h post-injection. Decreasing uptake was observed in pancreatitis (SUVmax 7.24, 6.55, and 5.63 after 10, 60, and 180 min, respectively), whereas the uptake of PDACs was stable or slightly increased (SUVmax 11.48, 12.66, and 13.23 after 10, 60, and 180 min, respectively)98. The earlier washout of FAPI-PET agents in inflammation was further confirmed in a separate study in patients with pancreatitis, where reduced SUVmax on 3 h delayed static scans were observed, while malignant lesions showed no significant tracer washout104.

FAPI-PET has a very promising role to play in PDAC diagnosis and staging. It is more sensitive than FDG-PET and with the use of washout kinetic studies at three hours, FAPI-PET may be able to distinguish benign and malignant lesions.

Colorectal cancer

Much like other GI tract tumors, increased FAP expression is associated with higher tumor aggressiveness and poor prognosis in colorectal cancer (CRC)13,14,105,106. While the majority of CRCs are FDG avid, the diagnostic performance of FDG imaging in CRC is substantially limited by high physiological bowel uptake41. Accordingly, head-to-head comparisons of FAPI and FDG generally show superior performance in sensitivity, detection rate, TBR, and higher SUVmax for FAPI-PET.

Multiple studies have reported a higher sensitivity of FAPI-PET compared to FDG-PET for the detection of primary CRC tumors (100% vs. 53%)107, lymph node metastases (90% vs. 80%)108, distant metastases (89% vs. 57%), and peritoneal metastases (100% vs. 55%)107,108. The specificity of FAPI- vs. FDG-PET for nodal metastases was also higher (100% vs. 81.8%)108. This is especially true of specific subtypes of CRC such as signet-ring cancer that tends to be non-FDG avid108,109,110. TBR of most CRC lesions was significantly higher on FAPI-PET108,109,110. SUVmax of primary lesions, peritoneal, and liver metastases was higher than in FDG-PET108,109, except for lymph node metastases107. FAPI-PET led to an overall change of TNM stage in 50% of the treatment-naïve patients108 or led to a change of treatment options in 21% of patients with confirmed primary CRC109,110. Another study demonstrated that higher TBR was obtained from 68Ga-FAPI-04 vs. 18F-FDG-PET scans (13.3 vs. 8.2) in primary tumors109. Both SUVmax in peritoneal metastases and TBR in liver metastases of 68Ga-FAPI-04 were higher than those of 18F-FDG (5.2 vs. 3.8 and 3.7 vs. 1.9, both P < 0.001)109. Clinical TNM staging based on 68Ga-FAPI-04 PET/CT led to upstaging and downstaging in 16% and 8.2% respectively109.

Peritoneal carcinomatosis

Peritoneal carcinomatosis occurs in multiple cancer types, especially in ovarian cancer, gastric, and colorectal cancer111. FDG has poor sensitivity for identifying peritoneal carcinomatosis, due to the physiological FDG uptake in the GI tract that masks carcinomatosis, and the low FDG avidity in some cancer subtypes42. Overall, FAPI-PET exhibits higher sensitivity and TBR112 for peritoneal carcinomatosis than FDG90,108.

Many studies confirm the higher sensitivity of FAPI-PET for peritoneal carcinomatosis. In colorectal carcinomatosis, 68Ga-FAPI-04 PET/CT exhibited a higher sensitivity (100% vs. 40%) with a comparable specificity to FDG (both 100%) for the detection of peritoneal involvement108, although not all studies reach the same conclusion90. In peritoneal gastric cancer, 68Ga-FAPI-04 had a sensitivity 100% vs. 40% for 18F-FDG, while both scans had a high specificity of 100%90,92. 68Ga-DOTA-FAPI-04 also showed higher TBR in peritoneal metastases (8.1 vs. 3.2, P < 0.001) compared to 18F-FDG-PET/CT92.

In peritoneal ovarian cancer, FAPI-PET demonstrated superior sensitivity compared to FDG112,113,114. In one study, 68Ga-FAPI-04 PET/CT showed higher sensitivity for detecting peritoneal metastases (97% vs. 76%; P < 0.001)113 (Fig. 5) and higher SUVmax (17.31 vs. 13.68; P = 0.026), leading to upstaging in many patients113. TBRs for FAPI-PET were also consistently higher than FDG-PET (median TBR 5.8 vs. 2.7, respectively; P < 0.001)112. Thus, FAPI-PET, across a broad range of cell types, demonstrates substantially better sensitivity for peritoneal carcinomatosis with higher specificity compared to FDG-PET.

Fig. 5: A 66-year-old woman underwent preoperative staging after being diagnosed with left ovarian high-grade serous carcinoma.
figure 5

68Ga-FAPI-04 PET/CT demonstrated mild focal uptake (SUVmax, 4.5) in the left primary tumor ((a); dotted arrow head) and distinctive uptake (SUVmax, 9.7) in widespread peritoneal metastasis ((a); solid arrowhead). 18F-FDG PET/CT revealed high focal uptake (SUVmax, 8.8) in this cystic-solid ovary tumor ((b); dotted arrowhead) and slight and diffuse uptake (SUVmax, 2.5) in the peritoneal metastases ((b); solid arrowhead). For this representative participant, 68Ga-FAPI-04 PET/CT detected more metastatic lesions compared with 18F-FDG PET/CT regarding the peritoneal metastases. [From ref. 113].

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Lung cancer

Since lung cancer (LC) is the most common malignancy, the performance of FAPI-PET compared to FDG-PET has generated multiple studies112,113,114,115,116. Overall, FAPI-PET surpasses FDG-PET for detecting primary cancer and metastases in the brain, pleura, bone, and lymph nodes115,116,117,118. In one study comparing patients with all forms of LC, 18F-FAPI-74 PET/CT demonstrated higher sensitivity (99% vs. 87%; P < 0.001), specificity (93% vs. 79%; P = 0.004), and accuracy (97% d vs. 85%; P < 0.001)118. In a similar study of 68Ga-FAPI, a higher detection rate of primary tumors and metastases including lymph nodes, brain, bone, and pleura was found when compared to 18F-FDG-PET/CT117. A standard limitation of such studies is that histologic confirmation is usually not possible and confirmation often relies on other methods such as conventional imaging and clinical history117.

Several subtypes of LC are less avid for FDG-PET and therefore, might be expected to yield even better results for FAPI-PET. For instance, in patients with lung adenocarcinoma 18F-AlF-NOTA-FAPI-42 PET/CT outperformed 18F-FDG, demonstrating higher SUVmax in lymph nodes, pleura, bones, and other distant metastases115. Moreover, 18F-FAPI-PET detected more lesions than 18F-FDG (total lesions 554 vs.464, P = 0.003), lymph nodes (258 vs. 229, P = 0.039), brain metastases (34 vs. 9, P = 0.002), and pleural metastases (56 vs. 30, P = 0.041)115. A comparison between different subtypes of LC including adenocarcinoma (ADC), squamous cell carcinoma (SCC), small cell lung cancer (SCLC) revealed no difference between 18F-AlF-NOTA-FAPI-04 and 18F-FDG119 with the minor exception that bone metastases of SCC and ADC exhibited higher uptake than those of SCLC119.

False positive uptake is a limitation of FAPI-PET in staging lung cancer. Various benign conditions e.g., co-existing post-radiation injury, surgery, or inflammation may also lead to positive FAP accumulation, thus making the precise differentiation between malignant and benign pulmonary findings challenging120,121,122,123,124,125,126,127. Inflammatory lesions tend to exhibit statistically lower uptake compared to cancers. Despite statistically different results among the groups, the degree of overlap between the two categories makes distinguishing benign and malignant disease in a specific patient, problematic128. The role of tracer washout in distinguishing inflammation from cancer is yet to be investigated. Thus, consensus has not yet been reached on the utility of FAPI-PET vis a vis FDG-PET in lung cancer128.

Breast cancer

The reported presence of abundant stroma in most primary breast cancers17,129 and the reported FAP expression in breast cancer cells16 suggest a highly promising role for FAPI-PET. FDG-PET has known limitations in some forms of breast cancer, particularly in invasive lobular breast cancers (ILC), which are characterized by lower tumor cell density17 and lower FDG uptake130,131. In this setting, FAPI-PET may provide a valuable alternative for diagnosis and staging, although specific analysis of different histological types is still lacking.

Numerous studies have demonstrated the effectiveness of FAPI-PET in detecting primary and metastatic breast cancer lesions with high sensitivity and specificity132. 68Ga-FAPI-04 PET/CT was superior to 18F-FDG PET/CT in detecting primary tumors demonstrating higher sensitivity, SUVmax, and TBR133. Additionally, 68Ga-FAPI-04 PET/CT was superior to 18F-FDG PET/CT in detecting lymph nodes, hepatic, bone, and cerebral metastases133. 68Ga-FAPI PET/CT also detected a higher number of primary breast lesions due to higher tracer uptake compared to 18FDG-PET/CT134. In one study, FAPI was effective in the major histologic subtypes (invasive ductal carcinoma: IDC 89.6%) and ILC (10.4%), while 18F-FDG showed lower uptake, especially in the ILC subtype134. In a selected cohort of patients with low FDG uptake, FAPI-PET demonstrated higher sensitivity and uptake135. In another study, FAPI-PET consistently demonstrated higher SUVmax (11.06 ± 5.48 vs. 8.33 ± 6.07, P = 0.02) and TBR (15.32 ± 10.33 vs. 8.25 ± 5.51, P < 0.001) compared to 18F-FDG in primary tumors, confirming the results of previous studies136.

Despite these promising results, there are a number of limitations of FAPI-PET in the diagnostic imaging of breast cancer. Hormonal status can influence uptake with pre-menopausal patients, showing higher uptake in the normal breast than post-menopausal women. Uptake is significantly increased with lactation137. A higher background in pre-menopausal women can reduce TBR and consequently reduce accuracy137. Additional limitations of FAPI-PET, which may potentially compromise diagnostic accuracy will be discussed in the “Limitations” section.

Ovarian cancer

FAP is expressed in most primary epithelial ovarian cancers with most of the expression arising from the tumor stroma, but some arising from the tumor cells themselves138. Mhawech-Fauceglia et al. evaluated ex vivo the expression of FAP in 338 primary epithelial ovarian cancer specimens and found positive FAP expression in 70 cases (21%) in neoplastic cells and 207 cases (61%) in the tumor stroma138. Notably, almost all cases (66/70), which expressed FAP in the neoplastic cells also expressed FAP in the stroma, while 42% showed FAP expression only in the stroma138. Since FAP expression in ovarian cancers was shown to be associated with cancer promotion, invasion, chemoresistance, and worse clinical outcomes, it is not surprising that FAP theranostics have been suggested as potential adjuvants to existing treatment paradigms20,138,139.

There have been several head-to-head comparative studies of FAPI- vs. FDG-PET in ovarian cancer112,113,114,140. Overall, FAPI-PET has demonstrated higher sensitivity for ovarian cancer compared to FDG-PET, especially in the detection of primary tumors, nodal, and distant metastases including peritoneal involvement and peridiaphragmatic metastases114. Although the SUVmax was comparable to FDG in some studies112,140, FAPI-PET scans generally showed higher TBR and higher detection rates of disease. For example, FAPI demonstrated improved sensitivity for detecting peritoneal metastases (96.8% vs. 83.0%), retroperitoneal (99.5% vs. 91.4%), and supradiaphragmatic lymph node metastases (100% vs. 80.4%) (all P < 0.001)113. 68Ga-FAPI-04 also showed higher SUVmax for peritoneal metastases (17.31 vs. 13.68; P = 0.026), retroperitoneal (8.72 vs. 6.56; P < 0.001) and supradiaphragmatic lymph node metastases (6.39 vs. 4.20; P < 0.001) compared to 18F-FDG113.

The hormone status of the patient influences background activity in the uterus and breasts137. There is significantly higher SUVmax in the endometrium (11.7 vs. 3.0; P < 0.001) and breast (1.8 vs. 1.0; P = 0.004) of pre-menopausal women than in post-menopausal women137. In contrast, FAPI accumulation in the ovaries showed no statistically significant differences between pre- and post-menopausal women (SUVmax 2.8 vs 1.6; p = 0.14)137. Overall, FAPI uptake in 167 female probands showed a mean SUVmax of 4.0 (±3.2) in endometrium (n = 128), 1.7 (±0.8) in ovary (n = 64), and 1.1 (±0.5) in breast tissue (n = 147)137. Although future validation is needed, lower physiological FAP accumulation in ovaries both in pre- and post-menopausal women, underscores the diagnostic potential of FAPI-PET in ovarian cancer.

Sarcoma

While undifferentiated sarcoma and rhabdomyosarcoma are strongly FDG avid141,142, low-grade sarcomas such as myxoinflammatory fibroblastic sarcoma, low-grade leiomyosarcoma, low-grade liposarcoma, solitary fibrous tumor, myxoid liposarcoma, and synovial sarcoma are known to be less FDG avid and thus, it is more likely that FAPI-PET will be advantageous141,143.

The expression of FAP in sarcoma was recently evaluated ex vivo by Crane et al., who demonstrated that 78% of stromal cells and 51% of tumor cells exhibited positive FAP staining144. The highest stromal FAP expression was observed in desmoid fibromatosis, myxofibrosarcoma, solitary fibrous tumor, and undifferentiated pleomorphic sarcoma144. The FAP expression in sarcoma cells was initially described by Rettig et al. in 1988, who evaluated the binding of FAP antibody (F19) in 12 known sarcoma cell lines. The study found FAP expression in the cell lines of fibrosarcoma, malignant fibrous histiocytoma (MFH), leiomyosarcoma, osteosarcoma, chondrosarcoma, liposarcoma, synovial sarcoma, and undifferentiated sarcoma, although the number of samples in each category was small (n ≤ 10)3. Conversely, no FAP expression was found in embryonal rhabdomyosarcoma, Ewing sarcoma, mesenchymal chondrosarcoma, and rhabdomyosarcoma, again with small numbers in each category3. In subsequent studies, these early findings appear to have been confirmed17,23.

Clinical studies have shown a significant correlation between FAPI-PET uptake and histopathologic FAP expression145. The positive predictive value and sensitivity were 100% and 96%, respectively. However, detection rates of 68Ga-FAPI and 18F-FDG were comparable in this cohort with no clear advantage for either PET agent. The subgroup analysis was limited due to the small number of patients in each sarcoma subtype145. In another study, FAPI-PET/CT detected more lesions compared to FDG-PET/CT and outperformed FDG significantly in sensitivity, specificity, positive and negative predictive values, and accuracy (P < 0.001)146. Moreover, 68Ga-DOTA-FAPI-04 demonstrated significantly higher values of SUVmax and TBR compared to 18F-FDG-PET/CT in liposarcoma, malignant solitary fibrous tumor (MSFT), and interdigitating dendritic cell sarcoma (IDCS)146. However, mean SUVmax and TBR suggested that 18F-FDG was more sensitive than 68Ga-DOTA-FAPI-04 in undifferentiated pleomorphic sarcoma (UPS) (P = 0.003 and P < 0.001, respectively) and rhabdomyosarcoma (RMS) (P < 0.001)146. This finding agrees with Koerber et al., who reported high SUVmax and TBR in a cohort of 15 patients with various sarcomas147. The highest uptake was found in liposarcomas and high-grade disease. Moreover, a high SUVmax (>10) was observed for more aggressive disease, indicating the higher uptake of FAPI correlates with a higher grade of malignancy147.

Thus, a general statement regarding the efficacy of FAPI-PET cannot be made due to the variable nature of tumors that fall under the “sarcoma” rubric. Among sarcomas, undifferentiated pleomorphic sarcoma, high-grade osteosarcoma, high-grade liposarcomas, and the rare solitary fibrous tumors (SFT) are currently the sarcoma subtypes with the highest likelihood of FAPI uptake, which should be validated in a future study.

Non-oncological indications

Overview

Multiple benign processes result in fibroblast activation, e.g., fibrosis, inflammation, benign tumors, or scar formation which may be amenable to imaging with FAPI-PET148 (Fig. 6). It was observed that in many FAPI-PET studies obtained for cancer evaluation, non-malignant findings were very common149.

Fig. 6: Pathological characteristics of fibrosis in different tissues. In tissues such as liver, kidney, lung, heart, and tumor, common events lead to fibrosis progression (and regression).
figure 6

If the pathological stimulus is persistent and the healing process is dysregulated, the continuous recruitment and activation of inflammatory cells and myofibroblasts can result in fibrosis. Core features of fibrotic processes that are shared by all of these organs include overproduction of cytokines, growth factors, ECM proteins, and ultimately the loss of tissue architecture as well as function. [From ref. 148].

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The mechanisms by which fibroblasts are activated are multifaceted, complex, and beyond the scope of this review. The most common pathway is thought to arise from transforming growth factor (TGF)-β signaling149,150,151,152,153. TGF-β modulates multiple cellular processes including synthesis of ECM150,154,155,156. and regulates the differentiation of fibroblasts into myofibroblasts157, thus playing an essential role in initiating and sustaining fibrosis150,151,152. Additionally, TGF-β takes part in the pathological wound-healing process and is considered to play an essential role in tissue inflammation158,159,160,161, suggesting a close relationship between inflammatory and fibrotic processes.

Myocardial infarction

Not surprisingly, myocardial infarction demonstrates FAPI uptake due to ischemia-induced fibroblast activation162. In a preclinical study, it has been shown that after the left coronary artery was ligated, 68Ga-FAPI-04 uptake in the injured myocardium peaked on day 6162. The tracer accumulation in the myocardial corresponded with the region of decreased 18F-FDG uptake162. Histopathologic evaluation revealed that 68Ga-FAPI-04 accumulated mainly at the border zone of the infarcted myocardium, suggesting this area is the most fibrotically active due to ongoing tissue remodeling and reparatory processes in response to acute ischemia162.

In humans, Diekmann et al. demonstrated the prognostic significance of early cardiac fibroblast activation after the acute myocardium infarction (AMI)163. In this study, 35 patients underwent cardiac MRI, perfusion SPECT, and 68Ga-FAPI-46 PET/CT after AMI (68Ga-FAPI-46 PET/CT was performed on day 7.5 (±1.3 d) after AMI)163. The FAP-positive region was significantly larger than the area of SPECT perfusion defect (P < 0.001) or the infarct area evaluated by cardiac MRI (P < 0.001)163 (Fig. 7). These areas include viable tissue in the border zone, where the injury may contribute to the development of interstitial fibrosis163. The FAPI-PET-based myocardial injury volume was predictive of outcome after AMI163,164. Additionally, the PET signal was predictive of subsequent development of left ventricular (LV) dysfunction at follow-up (r = −0.58, P = 0.007)114. These results suggest that FAPI-PET might be a useful imaging tool in the setting of AMI, and serve as a predictor of ventricular remodeling after MI163. This conclusion is further supported by a study that assessed the predictive value of 68Ga-DOTA-FAPI-04-PET/MR for late LV remodeling after AMI165. FAPI uptake volume at baseline was a significant predictor (OR = 1.048, P = 0.011) for LV remodeling at 12 months after myocardial infarction165,166.

Fig. 7: Myocardial perfusion images using 99mTc-tetrofosmin at rest, 68Ga-FAPI PET, LGE from CMR, and schematic drawings of LV.
figure 7

Area of fibroblast activation as indicated by 68Ga-FAPI-46 PET signal exceeds infarct area and LGE signal, the most common type of myocardial FAP distribution. HLA 5 horizontal long axis; SA 5 short axis; VLA 5 vertical long axis. [From ref. 163].

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Cardiac fibrosis

Approximately, 15–20% of cells in the adult heart are identified as cardiac fibroblasts163. Cardiac damage triggers fibroblast activation167, leading to adverse remodeling of cardiac tissue, characterized by replacement or reactive fibrosis168. Therefore, activated cardiac fibroblasts might serve as an attractive therapeutic target to modulate the remodeling process and improve functional outcomes169,170. Some preclinical studies using mouse models of cardiac fibrosis suggest the possibility of selectively targeting activated fibroblasts to arrest or even reverse reactive fibrosis171,172,173. In a pressure overload heart failure model in Sprague-Dawley rats, FAPI uptake both in the heart and liver correlated with the early stages of fibrosis174, confirming the well-known connection between chronic heart failure and congestive liver fibrosis175. FAPI uptake in the heart muscle also correlated with right ventricular pressure overload, leading to cardiac hypertrophy and fibrosis after pulmonary artery banding176.

Cardiac fibrosis is currently difficult to assess and FAPI-PET may thus, be a welcome tool to assist cardiologists177,178. In a study of hypertrophic cardiomyopathy (HCM), FAPI-PET demonstrated intense and inhomogeneous myocardial uptake across the LV myocardium (median target-to-background ratio, 8.8 vs. 2.1 in healthy controls; P < 0.001)179. In HCM, more segments with elevated FAPI uptake were detected than the number of hypertrophic segments (median; 14 vs. 5; P < 0.001), possibly indicating functionally active regions of disease179. The degree of FAPI accumulation was shown to correlate with the 5-year sudden cardiac death (SCD) risk score (r = 0.32; P = 0.03)179. FAPI uptake in the left ventricle was also associated with obesity, diabetes mellitus, and radiation exposure to the chest180. However, the possible prognostic value of FAPI-PET in cardiac fibrosis will require further research before it is used as a routine clinical tool.

Liver fibrosis

Liver fibrosis is a precursor of liver malignancies and hepatic dysfunction, with 80%–90% of hepatic malignancies developing in fibrotic or cirrhotic liver parenchyma181. Activated hepatic stellate cells are believed to be the source of hepatic fibrosis and are known to express FAP, thereby promoting fibrosis in the liver182. A quantitative, non-invasive measure of fibrosis would be useful clinically. In a swine model, hepatic fibrosis as measured by the collagen proportionate area (CPA) correlated with 68Ga-FAPI-46 uptake (r = 0.89, P < 0.001)183. 68Ga-FAPI-46 uptake increased progressively with increasing hepatic fibrosis183. The strong correlation between liver 68Ga-FAPI-46 uptake and the histologic stage of liver fibrosis suggests that 68Ga-FAPI-PET can play an impactful role in non-invasive staging of liver fibrosis183.

Because tumors arise in a background of fibrosis, theoretically, this could interfere with the detection of cancer77, on the other hand, simply identifying sites of significant fibrosis could help facilitate the early detection of liver malignancies. In actual practice, SUVmax and lesion-to-background ratio (LBR) of 18F-FAPI-74 PET were significantly higher in hepatocellular carcinoma than in benign fibrosis (HCC: SUVmax: 6.4 vs. 4.5, P = 0.017; LBR: 5.1 vs. 1.5, P = 0.003)184. These findings suggest that FAPI-PET imaging could differentiate malignant from non-malignant and non-inflammatory fibrosis in the liver, potentially making it a useful screening test in high-risk individuals.

Lung fibrosis/inflammation

Pulmonary fibrosis is a common disorder and is largely detected by pulmonary function testing followed by computed tomography. Unchecked, it can lead to severe disability and death, however, early intervention can prevent many later sequelae. Interstitial lung diseases (ILD) can arise from multiple causes, e.g., idiopathic pulmonary fibrosis (IPF), systemic sclerosis, or radiation among others185,186,187,188.

Pulmonary fibrosis is usually the result of acute lung inflammation that fails to resolve over time, causing the deposition of fibrosis in the lungs150,161,185. Thus, acute inflammation can lead to chronic inflammation and finally, to fibrosis, a pattern replicated in many organs150. Once it begins, pulmonary fibrosis can destroy lung architecture and lead to respiratory failure150,161. It is thought that wound healing dysregulation is the initiating step in fibrosis development150,158. Myofibroblasts are one of the key cells in wound healing but if they fail to regress after healing can eventually cause pulmonary fibrosis150,189. The differentiation of fibroblasts into myofibroblasts is regulated by the secretion of TGF-β and mechanical stress150,190. Myofibroblasts actively synthesize ECM components during lung tissue repair. During chronic inflammation, myofibroblasts evade apoptosis, forming hyperproduction of ECM and finally, pulmonary fibrosis150,190. In vitro data suggest that FAP expression is significantly increased, even in the early phase of profibrosis191.

FAPI-PET/CT uptake in patients with systemic sclerosis-associated ILD was higher in patients with progressive disease, compared to those with stable or inactive disease192. This study also found that increased 68Ga-FAPI-04 uptake at baseline was associated with ILD progression within 6–10 months, independently of the extent of involvement on initial high-resolution CT (HRCT) scan and the forced vital capacity at baseline192. The potential predictive value of FAPI-PET has been demonstrated in several studies191,193,194. In one study of ILD, baseline SUVtotal of 68Ga-FAPI-04 was significantly related to the pulmonary functional decline (decrease of vital capacity) (r = −0.5257, P = 0.0017)191.

Unfortunately, pulmonary inflammation alone or in combination with ILD can lead to FAPI-PET uptake191. Thus, increased activity in the lung can be due to inflammation or fibrosis, and teasing out the component of each can be challenging124,126,195,127. For example, patients with SARS-CoV-2 infection and suspected pulmonary fibrosis, have increased uptake of 68Ga-FAPI-46-PET/CT compared to the control group196. However, it remains unclear, whether such uptake represents inflammation, or early fibrotic change, not visible on CT. Inflammation and fibrosis share a common TGF-β activation pathway and distinguishing between the two entities is predicted to be difficult.

Kidney fibrosis

The final common pathway of chronic kidney disease (CKD) is renal fibrosis. The severity of fibrosis correlates with the degree of renal dysfunction and its reversibility. In preclinical studies, high FAP expression was observed in animals with CKD and the expression increased with the progression of renal fibrosis. In another preclinical study, higher SUVmax and TBR in 68Ga-FAPI-04-PET/CT were found in animals with CKD, but not in controls197. These results indicate the potential usefulness of FAPI-PET for non-invasive evaluation of renal fibrosis with the potential to reduce the need for renal biopsy197.

In a clinical study, three different radiotracers, 68Ga-FAPI, 68Ga-PSMA, and 68Ga-DOTATOC were compared in CKD patients198. The authors found a negative correlation between the glomerular filtration rate (GFR) and 68Ga-FAPI uptake, indicating the functional decline of kidney is associated with higher FAPI accumulation in the parenchyma198. Meanwhile, neither 68Ga-DOTATOC nor 68Ga-PSMA uptake correlated with CKD stage198. In another study in patients with confirmed renal fibrosis, 68Ga-FAPI-04-PET/CT SUVmax measurements in mild, moderate, and severe fibrosis were 3.92 ± 1.50, 5.98 ± 1.6, and 7.67 ± 2.23, respectively, demonstrating an increase in uptake with increased fibrosis199.

Overall, FAPI-PET may be a reasonable surrogate of renal fibrosis and therefore, may be able to predict the severity of fibrosis, monitor response to anti-fibrotic therapies, and help determine patient outcomes.

Rheumatoid arthritis

Rheumatoid arthritis, along with other auto-immune arthritides, are systemic illnesses that are often treated with systemic therapies. While the mainstay of assessment is self-reported patient symptoms, imaging can play an important role in the quantitative assessment of disease burden and response to therapy. Fibroblast-like synoviocyte cells (FLSs) are central to the formation of joint inflammation and are known to overexpress FAP200.

Preclinical models of rheumatoid arthritis (RA), including the collagen-induced arthritis (CIA) mouse model, have demonstrated that the activated FLSs are detectable with FAPI-PET200,201. For instance, 18F-FAPI-04 uptake has been shown to correlate with inflammation and response to treatment201. In a clinical study comparing 18F-FDG-PET with 18F-AlF-NOTA-FAPI-04, the latter demonstrated higher uptake in inflamed joints even at an early stage of arthritis and showed a positive correlation with clinical arthritis scores (r = 0.834, P < 0.001)200. 68Ga-FAPI-04 has substantially higher sensitivity for affected joints than 18F-FDG-PET/CT imaging202. The SUVmax value of the most affected joint in each participant was higher with 68Ga-FAPI compared to 18F-FDG PET/CT (9.54 vs. 5.85; P = 0.001)202. The 68Ga-FAPI-04 uptake in the joints correlated significantly with the clinical and radiographic grade of joint damage202, showing the therapeutic effect after anti-rheumatic treatment (Fig. 8).

Fig. 8: Pre- and post-treatment dual-tracer PET/CT in the three participants with different responses undergoing tight control treatment.
figure 8

Participant 12 is a 55-year-old woman with a 1-month history of rheumatoid arthritis (RA) who was treated with methotrexate, etoricoxib, tripterygium wilfordii, and iguratimod. Participant 17 is a 53-year-old woman with a 1-year history of RA who was treated with methotrexate and etanercept. Participant 18 is a 55-year-old woman with a 19-month history of RA who was treated with methotrexate, etanercept, and tripterygium wilfordii. There is residual active uptake of 68Ga-FAPI and 18F-FDG in three major joints 6 months after treatment in participant 18 (black arrows). Major joints with rheumatoid affection are marked with blue arrows. [Adapted, from ref. 202].

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These results suggest that FAPI-PET may be a promising radiotracer for aiding diagnosis and monitoring disease progression or therapy response, especially in difficult cases such as seronegative rheumatoid arthritis, which exhibits no classical immunological markers and can be difficult to diagnose and monitor203.

IgG4-related disease

Immunoglobulin G4-related disease (IgG4-RD) is a relatively recently identified systemic fibroinflammatory disease, characterized by lymphoplasmacytic infiltration and variable degrees of fibrosis204. The disease can occur in virtually any organ, but most commonly is found in lymph nodes, liver, pancreas, retroperitoneum, lacrimal, and salivary glands205,206.

Here again, 68Ga-FAPI-PET/CT is more sensitive for organ involvement, particularly in the pancreas, bile duct, liver, and salivary glands, than FDG-PET204. However, FDG avid lymph nodes did not accumulate 68Ga-FAPI, which was attributed to the fact that these lymph nodes lacked the characteristic storiform fibrosis207, which is one of the defining histopathologic features of IgG4-RD208,209. As with other similar diseases, a distinction between inflammation and fibrosis remains important, as the latter is less likely to be reversible. One study showed the possibility of distinguishing inflammatory from fibrotic activities, demonstrating that 68Ga-FAPI-04-PET uptake in IgG4-RD was associated with abundant FAP-positive activated fibroblasts, while 18F-FDG-PET positive lesions showed dense lymphoplasmacytic infiltration of IgG4 positive cells210.

Therefore, FAPI-PET may be useful in assessing IgG4-RD organ involvement and is likely more sensitive and specific than FDG-PET.

Crohn´s disease

Crohn’s disease (CD) is a chronic disease that causes inflammation throughout the digestive tract, particularly in the small and large bowel. Although fibrosis and inflammation are hallmarks of CD and differentiating between them has clinical relevance for treatment, current imaging modalities are not predictive211,212.

MR and CT enterography have become standard imaging methods for assessing bowel narrowing and fistula formation in CD212. FAPI-PET/MR enterography is a promising diagnostic tool for differentiating bowel wall fibrosis and inflammation212. In addition to showing anatomic regions of bowel lumen narrowing on MRI, SUVmax of FAPI-PET was significantly higher in segments with fibrosis compared to inflammation (7.6 vs. 2.0; P < 0.001)212. In severe fibrosis, SUVmax was higher than in mild to moderate fibrosis (8.9 vs. 6.2; P = 0.045)212. In addition, bowel segments with isolated active inflammation had lower FAPI uptake than segments with combined active inflammation and fibrosis (SUVmax, 3.2 vs. 8.1; P = 0.005)212. The sensitivity and specificity of FAPI-PET/MR enterography were 93% and 83%, respectively212. In another study211, FAPI-PET/CT showed superior performance compared to CT enterography (CTE) with a higher sensitivity for involved bowel segments: 93.3% vs. 86.7%)211. 68Ga-FAPI-04 PET/CT correlated well with endoscopy, CTE, and clinical biomarkers of CD211. Higher signal-to-background ratios were associated with clinical severity determined by the CTE score (r = 0.81; P < 0.0001)211. Further, the global FAPI-PET/CT score (defined as the sum of TBR in the affected segments, divided by the number of studied intestinal segments213) correlated with biomarkers such as C-reactive protein (CRP) or clinical indices for disease severity such as Crohn’s disease activity index (CDAI) or Crohn’s disease endoscopy index of severity (CDEIS). These findings indicate that FAPI-PET/CT might be a reliable surrogate for non-invasive assessment of disease activity in CD211. Further, FAPI-PET may also be useful in differentiating Crohn’s disease from ulcerative colitis (UC)214, because FAPI uptake was strongly increased only in CD but not in UC214. This result supports other in vitro studies that demonstrate the overexpression of FAP is found in the intestinal myofibroblasts in CD, but not in the corresponding areas of UC215.

Erdheim–Chester disease

Erdheim–Chester disease (ECD) is a rare disease characterized by the abnormal accumulation of histiocytes, or tissue macrophages. ECD involves multiple organs and tissues and has diverse manifestations, which makes it difficult to distinguish ECD from other diseases216.

ECD is characterized by varying degrees of organ fibrosis217. The most common manifestations of ECD are in the bone, heart, brain, skin, lung, kidney, peritoneum, or omentum218. 68Ga-FAPI-PET/CT is more sensitive than 18F-FDG-PET/CT for detecting lesions218. Moreover, the SUVmax for 68Ga-FAPI-PET/CT was significantly higher than 18F-FDG-PET/CT in the heart (4.9 vs. 2.8; P = 0.050), lung or pleura (6.8 vs. 3.1; P = 0.025), peritoneum or omentum (5.7 vs. 2.8 ± 1.7; P = 0.032), and kidney or perinephric infiltration (4.9 vs. 2.9; P = 0.009)218. The utility of FAPI-PET in ECD is still limited and further work is needed to better understand its role in the management of these patients.

Limitations

While we have focused on some of the opportunities afforded by FAPI-PET, there are also several inherent limitations44,120,219. CAFs are intrinsically heterogeneous in origin, phenotype, and function68, which impact the amount of FAP expression. The heterogeneity of FAP expression, especially in non-oncological conditions may lead to false positive findings, as reported in numerous studies and case reports. Most of the false positives are associated with fibrous or sclerotic foci, including post-radiation injury or scar formation, causing activation of quiescent fibroblasts149,220. In a systematic search for studies with non-malignant FAPI PET/CT findings, a total of 1178 papers comprising a total of 2372 FAPI avid non-malignant findings were reported, with wide-ranging spectrum of benign diseases149 including inflammation. The most frequent incidental finding was the uptake of FAPI-PET in atherosclerosis, followed by degenerative and traumatic bone and joint disease or arthritis149. FAPI avid lymph nodes and tuberculosis are potential false positives in cancer staging149. These findings and their respective detection thresholds are depicted in Fig. 9.

Fig. 9: FAP tracer uptake in various non-oncologic diseases.
figure 9

Reported SUVs of non-oncologic diseases (*SUVmax, **SUVmean, ***SUVpeak, † median, ‡ average). SUV standardized uptake value. [From ref. 120].

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It may also be difficult to distinguish between malignant and benign conditions on FAPI-PET. For instance, previous extended surgery near the region of interest may lead to false positive results for recurrence due to wound healing. In ovarian cancer patients, postoperative findings in the omentum and in the abdominal wall (at the surgical incision site) may also be a source of false positives140. Upon histologic evaluation, these areas are typically characterized microscopically by fibrous tissue hyperplasia and calcium salt deposition, that can cause false positive findings on FAPI-PET140.

Some investigators have suggested that the use of delayed static scans can differentiate inflammation from malignancy in the pancreas98. In pancreatitis, 68Ga-FAPI-PET/CT scan 3 h p.i. showed a significant decrease in uptake, while the uptake in pancreatic ductal adenocarcinoma was stable or slightly increasing98,104,221 (Fig. 10), indicating that this might provide a valuable, practical tool to non-invasively distinguish inflammatory vs. malignant conditions. Whether this method will prove useful in in other situations and whether such additional scans can be integrated into the clinical workflow without excessive disruption, remains to be determined.

Fig. 10: 68Ga-FAPI tracer uptake in PDAC and in accompaniying pancreatitis.
figure 10

A, B Average SUVmax and SUVmean 1 h after injection of 68Ga-labeled FAPI tracers in 8 PDAC and in accompanying pancreatitis in rest of pancreas. C Exemplary images of tumor-related (red arrow) and pancreatitis-related (yellow arrow) 68Ga-FAPI uptake 10, 60, and 180 min after application. D 68Ga-FAPI uptake 10, 60, and 180 min after application (SUVmax and SUVmean values) in PDAC lesions of 6 patients. [From ref. 98].

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FAP theranostics

Overview

Theranostic agents are those that can be used both for imaging and therapy by substituting therapeutic isotopes for diagnostic isotopes. The theranostic potential of FAP-targeted pharmaceuticals, based on the excellent demarcation of primary and metastatic lesions in multiple cancers, has been recognized since the early phase of FAP compound development. The first-in-human theranostic application of FAPI was the administration of Y-90-FAPI-04 in a female patient with metastasized breast cancer after a preceding diagnostic FAP-PET, showing a substantial intratumoral uptake27. This treatment led to a significant reduction in pain medication in this patient with minimal toxicity27. Currently, FAPI-04 and FAPI-46, containing DOTA-chelators, which have the capability of binding Ga-68, Y-90, or Lu-177, are the compounds most widely used as theranostic FAPI ligands34. Major innovations include peptide compounds (Ga-68/Lu-177-FAP 2286)38 and dimeric FAPI derivates (Ga-68/Lu-177-DOTAGA.(SA.FAPi)2)40 as theranostic pairs. Currently published data on FAP theranostics is summarized in Table 2. Although these results are promising, they are only proof-of-concept studies performed in single institutions, and the larger validation studies are needed.

Table 2 Overview of selected therapeutic applications of FAP ligands
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Challenges in the development of a theranostic tracer

The main challenge in the development of a theranostic tracer is to create a compound with prolonged tumor retention, matching that of the half-life of the radioisotope222. This maximizes dose to the tumor while minimizing toxicity and biosafety hazards. The ideal theranostic agent is characterized by high initial tumor uptake with minimal off-target uptake and rapid clearance, thus minimizing toxicity to major organs222. For this reason, some compounds may be suitable for diagnostic purposes only in combination with a shorter-lived diagnostic emitter such as Ga-68222. For example, 68Ga-FAPI-02, one of the first FAPI compounds developed in 2018, showed a desirable, high internalization rate into the target cells, but due to the relatively rapid efflux and shorter intra-tumoral retention compared to other compounds e.g., FAPI-04 and FAPI-46, FAPI-02 has not been further considered for therapeutic use27. Given that the tumor retention time remains short, short-lived isotopes such as α-emitter Bi-213 or the β-emitter Re-188 may ultimately be more suitable to deliver higher radiation dose to the tumor than current longer-lived isotopes like Lu-17734. Precise dosimetric analysis with larger number of patients is also pivotal for the accurate estimation of delivered radiation dose to tumors and normal tissues.

New trends

Covalent targeting

Covalent targeted radioligand (CTR) is a novel strategy for the prolonged binding of radioligands to their targets223,224. In this strategy, a ligand “warhead” which binds covalently to the target molecule, is conjugated to a linker and the chelated radioisotope225. When CTRs reach the tumor, the molecule first non-covalently binds to the target through a conventional ligand-target interaction but rapidly becomes irreversibly bound due to covalent interactions of the warhead. Other CTRs that do not bind to the target undergo rapid renal excretion, preventing off-target binding226, thus limiting the risk of systemic toxicity. Recently, Cui et al. developed a CTR, using FAPI-04 with an attached sulfur fluoride-based warhead225. This warhead reacts with the nucleophilic sites of FAP in the side chain to create covalent binding. Instead of sulfone fluoride (SF), fluorosulfate (FS) has been introduced as an alternative warhead, because it exhibits higher stability with increased cellular uptake and retention225. Thus, covalent binding strategies might be able to enhance the tumor uptake and retention of radioligands considerably. However, an important design criterion for CTRs is that they avoid uncontrollable ligation reactivity during circulation or in healthy tissues, which will require more development.

FAPI theranostics in combination with immunotherapy

It is increasingly evident that targeted radionuclide therapy might affect the immunogenicity of tumors through direct and indirect immunostimulatory effects227,228,229. In vitro studies have shown that radiation-induced DNA damage can cause the accumulation of nucleic acid fragments in the cytosol of cancer cells, leading to the stimulation of pro-inflammatory cytokines230,231. Increased levels of pro-inflammatory cytokines attract immune cells that may boost the immune response against the tumor231. Thus, radionuclide therapy has the potential to convert immunologically inactive (so-called “cold”) tumors into immunologically active (“hot”) lesions more likely to respond to immunotherapy227,232. Numerous preclinical studies suggest the synergistic effect of radiation and immunotherapy233,234,235,236,237,238. The majority of these studies assess the effect of combined radionuclide therapy with immune checkpoint inhibitors (ICIs) in melanoma, prostate, colon, or breast cancer233,234,235,236,237,238. So far, however, there is sparse evidence this can be extended to humans with cancer. Zboralski et al. have recently demonstrated that Lu-177-FAP-2287, a murine-targeted-FAP agent, enhanced anti-PD-1 mediated tumor growth inhibition239. This result offers hope that FAP-targeted radionuclide therapy in combination with ICI may be more effective than either strategy alone.

Summary and future perspectives

The current state of the art of FAPI-PET reveals wide-ranging potential applications in oncology and non-oncologic diseases. While the exploration of many of the non-oncological indications is currently just emerging, evidence that FAPI-PET surpasses FDG-PET in many cancer types has been steadily accumulating and suggests a promising role for FAPI-PET, especially in tumors where FDG-PET has proven insensitive or non-specific. This includes hepatic, gastric, pancreatic, colorectal, lung, breast, and ovarian cancer, where the superior sensitivity of FAPI-PET compared with FDG-PET is evident. While FAPI-PET consistently demonstrates higher sensitivity than FDG in multiple malignancies, there are limitations to FAPI due its uptake in inflammation and benign fibrosis.

Currently, there are several gaps in knowledge on the performance of FAPI-PET. One such gap is how well the tracer can differentiate between benign and malignant lesions given the avidity for both. The differentiation of inflammatory from malignant lesions is one of the major issues, which needs further analysis. Some hope is offered by the observation that multi-time point imaging obtained over several hours, can demonstrate washout of activity with inflammation but plateauing or increases in cancer, however, some overlap between the two is inevitable98,221. Detailed histopathological and immunological evaluation of FAP expression in various inflammatory or infectious diseases might be a way to clarify this issue.

It is also now clear that FAPI uptake can vary with specific histological subtypes, particularly in diseases with low FDG avidity. Several comparative studies have shown that tumors with known low FDG avidity such as lung adenocarcinoma or signet-ring gastric carcinoma, are most benefitted by the use of FAPI-PET91,115. Larger clinical studies stratified by histologic subtype and FDG uptake, e.g., invasive lobular breast cancers130,131 or multiple sarcoma subtypes, are needed to fully characterize the role of FAPI-PET more precisely.

A final gap in knowledge is to what extent FAPI-PET uptake reflects prognostic information and whether it is a possible surrogate for disease outcomes. Several studies have addressed the prognostic impact of FAPI-PET, showing that the FAP signal intensity correlated with the clinical severity and disease extent, which in some cases is predictive of disease outcome74,92,95,102,103,163,192, however, more work is needed.

The future of FAPI-PET is quite promising. However, we also need to consider other radionuclidic forms of FAPI imaging including 99mTc-labeled FAPI-SPECT imaging, which although lower in resolution, will be more flexible and cost-effective and perhaps more useful in environments with fewer PET scanners31,33,240. At the other end of the cost spectrum, FAPI-PET/MRI may also lead to increased flexibility in clinical choice, as MRI is regarded as the standard radiographic method in several cancers (e.g., liver, brain, breast cancer, or soft tissue sarcomas).

To summarize, well-conducted clinical trials with sufficiently large cohorts of patients to allow for valid subgroup analysis, detailed analysis of histological subtypes as well as the correlation with longitudinal clinical outcome, will be important in defining the future of FAPI-PET. New developments in super-sensitive whole-body PET technology will also, no doubt, advance the utility of FAPI imaging. Finally, the potential to directly target FAP expression either by FAP-targeted drugs or radioligand therapy, opens further opportunities for image-directed therapy in cancer and non-cancer applications. FAPI-PET will continue to be an exciting area of research in the coming years.

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Stromal architecture and fibroblast subpopulations with opposing effects on outcomes in hepatocellular carcinoma

Dissecting the spatial heterogeneity of cancer-associated fibroblasts (CAFs) is vital for understanding tumor biology and therapeutic design. By combining pathological image analysis with spatial proteomics, we revealed two stromal archetypes in hepatocellular carcinoma (HCC) with different biological functions and extracellular matrix compositions. Using paired single-cell RNA and epigenomic sequencing with Stereo-seq, we revealed two fibroblast subsets CAF-FAP and CAF-C7, whose spatial enrichment strongly correlated with the two stromal archetypes and opposing patient prognosis. We discovered two functional units, one is the intratumor inflammatory hub featured by CAF-FAP plus CD8_PDCD1 proximity and the other is the marginal wound-healing hub with CAF-C7 plus Macrophage_SPP1 co-localization. Inhibiting CAF-FAP combined with anti-PD-1 in orthotopic HCC models led to improved tumor regression than either monotherapy. Collectively, our findings suggest stroma-targeted strategies for HCC based on defined stromal archetypes, raising the concept that CAFs change their transcriptional program and intercellular crosstalk according to the spatial context.

Targeting of TAMs: can we be more clever than cancer cells?

With increasing incidence and geography, cancer is one of the leading causes of death, reduced quality of life and disability worldwide. Principal progress in the development of new anticancer therapies, in improving the efficiency of immunotherapeutic tools, and in the personification of conventional therapies needs to consider cancer-specific and patient-specific programming of innate immunity. Intratumoral TAMs and their precursors, resident macrophages and monocytes, are principal regulators of tumor progression and therapy resistance. Our review summarizes the accumulated evidence for the subpopulations of TAMs and their increasing number of biomarkers, indicating their predictive value for the clinical parameters of carcinogenesis and therapy resistance, with a focus on solid cancers of non-infectious etiology. We present the state-of-the-art knowledge about the tumor-supporting functions of TAMs at all stages of tumor progression and highlight biomarkers, recently identified by single-cell and spatial analytical methods, that discriminate between tumor-promoting and tumor-inhibiting TAMs, where both subtypes express a combination of prototype M1 and M2 genes. Our review focuses on novel mechanisms involved in the crosstalk among epigenetic, signaling, transcriptional and metabolic pathways in TAMs. Particular attention has been given to the recently identified link between cancer cell metabolism and the epigenetic programming of TAMs by histone lactylation, which can be responsible for the unlimited protumoral programming of TAMs. Finally, we explain how TAMs interfere with currently used anticancer therapeutics and summarize the most advanced data from clinical trials, which we divide into four categories: inhibition of TAM survival and differentiation, inhibition of monocyte/TAM recruitment into tumors, functional reprogramming of TAMs, and genetic enhancement of macrophages.

CRISPR knock-in of a chimeric antigen receptor into GAPDH 3’UTR locus generates potent B7H3-specific NK-92MI cells

CAR-NK therapy is becoming a promising approach to treat solid tumors. However, the random insertion of the CAR gene and inflexible CAR expression caused by common preparation methods significantly impact its efficacy and safety. Here we successfully established a novel type of CAR-NK cells by integrating CAR sequences into the GAPDH 3’UTR locus of NK-92MI cells (CRISPR-CAR-NK), achieving site-specific integration of the CAR gene and allowing endogenous regulatory components to govern CAR expression. CRISPR-CAR-NK cells had comparable growth capacity but displayed superior anti-tumor activity compared with their lentiviral counterparts. They activated and degranulated more effectively when co-cultured with tumor cells, due to increased expression of activating receptors and decreased expression of inhibitory molecules. They also enhanced the production of Granzyme B and IFN-γ, and more effectively triggered the IFN-γ pathway. Moreover, CRISPR-CAR-NK cells demonstrated distinct properties from conventional CAR-NK concerning metabolic features and signal dependence. Notably, CRISPR-CAR-NK cells exhibited lower metabolic levels without compromising antitumor activity, and their function was less reliant on the PI3K-AKT pathway, implying that the CRISPR-CAR-NK cells have significant potential for enhanced synergy with AKT inhibitors and adaptation to nutrient stress within the tumor microenvironment. These findings provide a novel potential strategy for cancer immunotherapy and an experimental foundation and paradigm for optimizing CAR-NK cells utilizing CRISPR technology, highlighting the potential of CRISPR to advance immunotherapies.

Age-dependent differences in breast tumor microenvironment: challenges and opportunities for efficacy studies in preclinical models

Immunity suffers a function deficit during aging, and the incidence of cancer is increased in the elderly. However, most cancer models employ young mice, which are poorly representative of adult cancer patients. We have previously reported that Triple-Therapy (TT), involving antigen-presenting-cell activation by vinorelbine and generation of TCF1+-stem-cell-like T cells (scTs) by cyclophosphamide significantly improved anti-PD-1 efficacy in anti-PD1-resistant models like Triple-Negative Breast Cancer (TNBC) and Non-Hodgkin’s Lymphoma (NHL), due to T-cell-mediated tumor killing. Here, we describe the effect of TT on TNBC growth and on tumor-microenvironment (TME) of young (6–8w, representative of human puberty) versus adult (12 m, representative of 40y-humans) mice. TT-efficacy was similar in young and adults, as CD8+ scTs were only marginally reduced in adults. However, single-cell analyses revealed major differences in the TME: adults had fewer CD4+ scTs, B-naïve and NK-cells, and more memory-B-cells. Cancer-associated-fibroblasts (CAF) with an Extracellular Matrix (ECM) deposition-signature (Matrix-CAFs) were more common in young mice, while pro-inflammatory stromal populations and myofibroblasts were more represented in adults. Matrix-CAFs in adult mice displayed decreased ECM-remodeling abilities, reduced collagen deposition, and a different pattern of interactions with the other cells of the TME. Taken together, our results suggest that age-dependent differences in the TME should be considered when designing preclinical studies.

Type 2 immunity in allergic diseases

Significant advancements have been made in understanding the cellular and molecular mechanisms of type 2 immunity in allergic diseases such as asthma, allergic rhinitis, chronic rhinosinusitis, eosinophilic esophagitis (EoE), food and drug allergies, and atopic dermatitis (AD). Type 2 immunity has evolved to protect against parasitic diseases and toxins, plays a role in the expulsion of parasites and larvae from inner tissues to the lumen and outside the body, maintains microbe-rich skin and mucosal epithelial barriers and counterbalances the type 1 immune response and its destructive effects. During the development of a type 2 immune response, an innate immune response initiates starting from epithelial cells and innate lymphoid cells (ILCs), including dendritic cells and macrophages, and translates to adaptive T and B-cell immunity, particularly IgE antibody production. Eosinophils, mast cells and basophils have effects on effector functions. Cytokines from ILC2s and CD4+ helper type 2 (Th2) cells, CD8 + T cells, and NK-T cells, along with myeloid cells, including IL-4, IL-5, IL-9, and IL-13, initiate and sustain allergic inflammation via T cell cells, eosinophils, and ILC2s; promote IgE class switching; and open the epithelial barrier. Epithelial cell activation, alarmin release and barrier dysfunction are key in the development of not only allergic diseases but also many other systemic diseases. Recent biologics targeting the pathways and effector functions of IL4/IL13, IL-5, and IgE have shown promising results for almost all ages, although some patients with severe allergic diseases do not respond to these therapies, highlighting the unmet need for a more detailed and personalized approach.

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