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68Ga-DOTA.SA.FAPi as a Versatile Diagnostic Probe for Various Epithelial Malignancies: A Head-to- Head Comparison with 18F-FDG
Sejal Chopra, Yamini Mathur, Frank Roesch, Euy Sung Moon, Nivedita Rana, Santhosh Irrinki, Rama Walia, Ajay Duseja, Harmandeep Singh, Rajender Kumar, Jaya Shukla, Bhagwant Rai Mittal
Rationale and Objectives: Fibroblast Activation Protein (FAP) expressing cancer-associated fibroblasts has been a major break- through causing a paradigm shift in targeted theranostics focusing on the tumor microenvironment. In this study, a squaric acid deri- vative DOTA.SA.FAPi (SA.FAPi) has been evaluated as a potential diagnostic probe in diverse epithelial cancers and compared to the standard-of-care 18F-FDG.
Methods: 25 patients enrolled in this prospective study underwent 18F-FDG and 68Ga-SA.FAPi PET scans on two different days. For biodis- tribution, standardized uptake values (SUV) were computed by delineating region-of-interest on various body organs. For comparative analysis in disease identification, lesion tracer uptake was quantified using SUVs corrected for lean body mass (SUL), SUVmax, tumor-to-background ratio (TBR) with liver and blood pool as the reference, total lesion glycolysis (TLG for 18F-FDG) and total lesion FAP expression (TLF for 68Ga-SA.FAPi).
Results: 25 patients (mean age: 58 ± 8 years) with four types of cancers including hepatocellular carcinoma (HCC, 56% of cohort), gall bladder carcinoma (GB Ca, 12%), adrenocortical carcinoma (ACC, 16%), and breast carcinoma (breast Ca, 16%) were prospectively evaluated. Physiological tracer uptake of 68Ga-SA.FAPi was noted in the salivary glands, thyroid, liver, pancreas, muscles and kidneys with variable uptake in the lacrimal glands, extra-ocular muscles, oral mucosa and uterus. Lesion-based comparative analysis between both the radiotracers demonstrated complete concordant findings in detection of all primary lesions and distant metastases in liver, bones, adrenals and peritoneum whereas discordant findings were noted in lung nodules (20%) and lymph nodes (13%). In overall analysis, 68Ga-SA.FAPi exhibited significantly higher SUVmax (10.3 vs 8.8, p-0.019), SULpeak (6.8 vs 4.9, p-0.000) and SULavg (5.4 vs 4.1, p-0.019) in comparison to 18F-FDG whereas TBR was comparable for both the tracers [TBRLiver: median 1.9 (IQR: 2.6-1.4) vs 1.8 (2.6-1.1), p-0.275; TBRBloodpool: 2.1 (3.7-1.4) vs 2.0 (2.7-1.4), p-0.207]. In subcategorical analysis, 68Ga-SA.FAPi demonstrated higher SUVmax, SULpeak and SULavg values for primary disease (SUVmax: 14.8 (18.7-9.7) vs (12.9-6.6), p-0.087; SULpeak: 8.2 (11.2-6.8) vs 6.3 (8.5-4.4), p-0.037; SULavg: 6.9 ± 2.5 vs 5.1 ± 2.2, p-0.023] and distant metastases (8.8 vs 7.2, p-0.038); 6.3 (8.8-4.4) vs 3.6 (4.4-2.0), p- 0.000; 5.4 vs 3.5, p-0.000] whereas comparable values were noted for both the tracers in nodal metastases [9 (13.5-4.1) vs 8 (12.7-4.7), p-0.726; 4.5 (6.2-1.8) vs 4.3 (5.7-2.2), p-0.727; 4.1 ± 2.3 vs 3.7 ± 1.8, p-0.129]. In primary disease, highest 68Ga-SA.FAPi avidity was noted in ACC followed by GB Ca and HCC. In distant metastases, gall bladder, lung and skeletal lesions demonstrated higher 68Ga- SA.FAPi avidity. Moreover, 68Ga-SA.FAPi identified five additional lung lesions which were missed by 18F-FDG in one case of ACC.
Conclusion: 68Ga-SA.FAPi emerged as an effective, versatile diagnostic probe for imaging various epithelial malignancies similar to 18F-FDG.
Key Words: 68Ga-DOTA.SA.FAPi; Broad-spectrum; 18F-FDG; HCC; ACC.
@ 2024 The Association of University Radiologists. Published by Elsevier Inc. All rights are reserved, including those for text and data mining, AI training, and similar technologies.
Acad Radiol 2024; 31:2521-2535
From the Department of Nuclear Medicine, Postgraduate Institute of Medical Education and Research, Chandigarh, India (S.C., Y.M., N.R., H.S., R.K., J.S., B.R.M.); Department of Chemistry, Johannes Gutenberg University, Mainz, Germany (F.R., E.S.M.); Department of General Surgery, Postgraduate Institute of Medical Education and Research, Chandigarh, India (S.I.); Department of Endocrinology, Postgraduate Institute of Medical Education and Research, Chandigarh, India (R.W.); Department of Hepatology, Postgraduate Institute of Medical Education and Research, Chandigarh, India (A.D.). Received August 2, 2023; revised November 28, 2023; accepted December 1, 2023. Address correspondence to: J.S. e-mail: shuklajaya@gmail.com
@ 2024 The Association of University Radiologists. Published by Elsevier Inc. All rights are reserved, including those for text and data mining, Al training, and similar technologies.
INTRODUCTION
18
F -FDG PET-CT is the standard-of-care in the diagnosis, staging and response assess- ment of many malignancies. The glucose analog is an excellent metabolic marker that assists the ima- ging of over-expression of GLUT receptors within the tumor, which is a well-acknowledged hallmark of cancer (1). However, in view of it’s limitations in certain malignancies, there’s a growing emphasis on radiopharmaceuticals targeting molecular pathways such as tumor cell proliferation, angio- genesis, hypoxia and receptors expressed on tumor cells. This has led to the development of more specific tracers for re- ceptor-based imaging, which have gained increasing clinical acceptance over time (2). One of the most promising tumor component which can be targeted for drug and tracer de- velopment is the tumor microenvironment (TME).
The TME is a complex framework of non-cellular com- ponents such as extracellular matrix (ECM) as well as myo- fibroblasts and cellular components, such as fibroblasts, adipose cells, neuroendocrine (NE) cells, immune-in- flammatory cells, blood and the lymphatic system (3). Fi- broblasts are present ubiquitously in the body and are characterized by dipeptidyl peptidase 4 (DPP4) expression with minimal or no Fibroblast Activation Protein (FAP) expression (4). One of the most promising stromal targets in the TME are the cancer associated fibroblasts (CAFs) which play a crucial role in tumorigenesis by ECM remodeling and promoting cancer cell proliferation (5-9). CAFs are a sub- population of fibroblasts having myofibroblastic phenotype specifically characterized by increased FAP expression (5,6,8). They are associated with more than 90% of epithelial carcinomas, including colon, pancreatic, lung, ovarian and breast cancer constituting a major component (> 90%) of the gross tumor mass (10-13). Hence, targeting FAP+-CAF’s using appropriate FAP inhibitors (FAPI) is a potentially re- warding strategy for the development of suitable theranostic molecular probe.
Numerous FAPI molecules have been formulated over time, commencing with the first-generation compounds consisting of a boronic acid warhead which utilized boronic acid to interact with the target. However, they were even- tually replaced by carbonyl warheads owing to greater sta- bility and enzyme specificity (14,15). Recent FAPI derivatives were further conjugated with the widely used bifunctional chelator (BFC) DOTA for allowing use in theranostic applications by radiolabelling with suitable iso- topes (68Ga,177Lu, 225 Ac, etc.). FAPI-02, the first DOTA- FAPI combination created employing piperidine as a linker molecule, exhibited excellent tumor uptake and great image contrast. However, shorter tumor retention time warranted the need for further improvement for therapeutic applica- tions (16). Further structural variations, especially in the linker region paved the pathway towards development of more potent, analogous FAPI compounds viz. FAPI-04,
FAPI-21 and FAPI-46 which showed promising results in both preclinical and clinical trials (17,18). Various preclinical and clinical studies have been conducted so far for evaluating the therapeutic efficacy of diverse FAPI derivatives (16,18-21). Although introduction of 68Ga-FAPI may have been one of the major breakthrough in both oncological and non-oncological imaging, the quest for an ideal theranostic FAPI probe is still going on.
More recent advancements have led to the addition of squaric acid motif as a linker moiety in DOTA.SA.FAPI (SA.FAPi) because of it’s facile coupling to the chelator and target vector without the requirement of any protecting groups. The preclinical study conducted using 68Ga- SA.FAPi in HT-29 human colorectal cancer xenograft mouse model demonstrated promising results with high tumor uptake and good image contrast (19). Moreover, it may further lead to improved pharmacokinetics as observed in 68Ga-TRAM.SA.PSMA, 68Ga-DOTAGA.SA.PSMA and 68Ga-NODAGA.SA.PSMA with good tumor uptake and overall high tumor-to-background ratio (22). A single- center, clinical study conducted on 54 patients with diverse malignancies demonstrated rapid uptake of 68Ga-SA.FAPi with high contrast images and favorable pharmacokinetics (23). However, to validate the diagnostic accuracy of this molecule, elaborative research focusing on particular malig- nancies is required due to the paucity of literature.
To the best of our knowledge, the role of SA.FAPi in hepatocellular carcinoma (HCC) and adrenocortical carci- noma (ACC) has not been explored yet. 18F-FDG (FDG) suffers from a well-known limitation in HCC owing to the limited activity of the hexokinase enzyme. Moreover, ex- ploring SA.FAPi could be pivotal, especially in treating challenging conditions like metastatic ACC where current therapies fall short. This prospective study aims to evaluate the diagnostic efficacy of 68Ga-SA.FAPi in four malignancies i.e. HCC, ACC, gall bladder (GB ca) and breast (BC) car- cinomas by comparative analysis with FDG PET. The radiosynthesis, quality control and stability studies of this radiopharmaceutical have also been described.
MATERIALS AND METHODS
Materials
SA.FAPi, Hydrochloric acid (HCl) (Merck), Sodium Acetate (Sigma Aldrich), Sodium Citrate (Advanced Biochemical Compounds), Ethanol (Laboratory grade, Honeywell, Riedel- de-Haen™M), 0.9% Saline (Otsuka, India), C18 catridge (Sep- Pak), 0.22 um filter (Millex ® -GV), silica-coated Instant Thin Layer Chromatography (ITLC) strips (Sigma Aldrich), Whatmann Paper III strips (Sigma Aldrich), Acetonitrile (Sigma Aldrich), High Purity Liquid Chromatography (HPLC) Water (Merck), Fluid Thioglycollate Medium (FTM) (Sigma Aldrich), Soybean-Casein Digest Medium (Sigma Aldrich), Endpoint Chromogenic Limulus Amebocyte Lysate (LAL) test kit (Charles River, U.S.). 1-Octanol (Sigma Aldrich).
68GA-SA.FAPI: PREPARATION AND QUALITY CONTROL
Radiosynthesis
SA.FAPi was obtained from Dr. Frank Roesch and Dr. Moon Euy Sung, Mainz, Germany. Radiolabelling of SA.FAPi was performed in a semi-automated iQS ® (ITG, isotope technolo- gies Garching GmbH, Germany) Fluidic Labelling Module. 68 GaCl3 was eluted using 4 mL 0.05 M HCI from 68Ge-68Ga radionuclide generator (ITM Medical Isotopes, Munich, Germany). 20 µgm of SA.FAPi was dissolved in 0.25 M Sodium Acetate (NaOAc) buffer (pH 8). The reaction mixture was he- ated at 90 ℃ for 10 min (pH ~ 4) followed by purification using solid-phase extraction (Sep-Pak C-18 column). The radiolabelled product was obtained using 1 mL 50% ethanol as an eluent after passing through 0.22 um Millipore filter.
Quality Control (QC)
For QC testing, radionuclidic purity (RNP), radiochemical purity (RCP), sterility and apyrogenicity of the labeled product were assessed. A batch record of three consecutive 68
68Ga-SA.FAPi productions was maintained to ensure re- producibility of results.
Shelf Life, In-vitro Stability and Lipophilicity
The shelf-life of 68Ga-SA.FAPi was estimated at room temperature (RT) and 4 ℃ up to 4 h. The RCP of the radiolabelled product was assessed using ITLC technique at 1, 2, 3 and 4 h post-production. The stability of radiolabelled product was assessed in human serum and saline at 37 ℃ for 4 h. Lipophilicity of 68Ga-SA.FAPi was estimated by cal- culating the Log P value obtained by partitioning between 1- Octanol (organic phase) and 0.9% Isotonic saline (aqueous phase). Batch records of three 68Ga-SA.FAPi productions was obtained to ensure reproducibility of the results for all the previously mentioned parameters.
CLINICAL STUDY
Study Design
This prospective study was duly approved by the Institutional Ethics Committee (IEC INT/IEC/2021/SPL-500). 25 diag- nosed/suspected cases of localized or metastatic ACC, GB Ca and Breast Ca were recruited for FDG and 68Ga-SA.FAPi PET for disease evaluation and comparative analysis. All the patients were above 18 years of age. Patients who refused to provide a written informed consent, pregnant and lactating females, or facing difficulty to undergo two scans due to other serious illness were excluded from the study.
Acquisition Parameters and Scan Protocol
All patients underwent both FDG and 68Ga-SA.FAPi PET scans on two different days, spaced within a week, for disease
evaluation and comparative analysis. For FDG, whole-body (WB) images (Discovery 710, GE Healthcare) were acquired from vertex to mid-thigh, one hour post tracer administra- tion with mean injected activity 298.9 ± 48.1 MBq. Computed Tomography (CT) Images were acquired fol- lowing oral and intravenous contrast media with following parameters: Matrix-size 512 * 512; kV 120; mA 100-350 (auto mA); pitch 0.984:1; helical thickness 3.75 mm and rotation time 0.75 s. This was followed by emission PET acquisition for same axial coverage (one minute per bed frame; matrix size 192 *192). For 68Ga-SA.FAPi, WB images (Discovery 710, GE Healthcare) were acquired from vertex to mid-thigh, 30 minutes post tracer administration with mean injected activity 82.5 ± 19.6 MBq. A low-dose, non- contrast CT (mA 40; kV 120) was acquired followed by PET acquisition for same axial coverage with the previously mentioned parameters (two minutes per bed frame).
Image Interpretation
The image reconstruction was performed using Ordered Subset Expectation Maximum (OSEM) employing three iterations and 24 Subsets with Gaussian filter having full width at half maximum (FWHM) of 5 mm. Both FDG and 68 Ga-SA.FAPi scans were concurrently accessed and co- registered using carina as a reference anatomical point. The scan interpretations were done by independently by two Nuclear Medicine experts with more than 10 years experi- ence. The lesions identified by both the interpreters were included in comparative analysis. The physiological uptake of 68Ga-SA.FAPi was noted by drawing region of interests (ROI’s) on various body organs and calculating maximum and mean standardized uptake values (SUVmax, SUVmean). To determine SUV in reference organs, spherical regions of interest (ROIs) with a volume of 1.22 cm3 were delineated on the thyroid, salivary glands and pancreas. Conversely, spherical ROIs with a volume of 3.52 cm3 were defined on normal brain tissue, the mediastinal blood pool (BP), psoas major muscle, liver, uterus and kidneys. For disease identi- fication, 68Ga-SA.FAPi/FDG avid lesions were distributed into three anatomical categories: local disease (primary/re- sidual/recurrent lesion), nodal lesions and distant metastases. The ROI metrics were represented as SUV adjusted for lean body mass (SUL), SULpeak and SULavg. For comparing tracer uptake, a 3D auto-contour ROI, set at a 40% threshold of SULpeak, was meticulously drawn around tracer-avid lesions to determine the total lesion glycolysis (TLG; SULXcm3) for FDG and total lesion FAP expression (TLF; SULXcm3) for 68 Ga-SA.FAPi derived from the product of FAP expressing tumor volume (FTV; cm3) and the average (avg.) SUL (Advantage Workstation 4.7, GE Healthcare, USA). The tumor-to-background ratios (TBR) for both tracers were also determined using liver and BP as reference backgrounds (24). Non-specific uptake of each tracer, if any, was further quantitatively compared using the previously mentioned metrics.
Statistical Analysis
All continuous variables were presented in the form of mean ± standard deviation (SD) for normal distribution and median (Inter-quartile range, IQR) for skewed distribution. The nor- mality of the data was tested using Kolmogorov-Smirnov test (no of observations (n) > 50) and Shapiro-Wilk test (n < 50) (25). Cohn’s weighted kappa statistics were employed to evaluate the inter-observer agreement for scan interpretation. Kappa va- lues below 0.4 signify weak agreement, values ranging from 0.4 to 0.75 suggest fair to good concordance, and values of 0.75 and above denote outstanding agreement (26). The statistical sig- nificance of the difference in the lesion uptake for both the tracers was determined using Student t-test (normal distribution) and Wilcoxon Signed-Rank test (skewed distribution). The p-value of < 0.05 was considered to be statistically significant.
RESULTS
Quality Control (QC) of the Radiopharmaceutical
The radiochemical yield (RCY) for 68Ga-SA.FAPi was 98.06 ± 1.4%. The half-life measured using the dose cali- brator was 69 ± 1.2 min. The gamma ray spectrum of the radiolabelled product showed a prominent peak corre- sponding to photon energy of 511 keV and a small sum peak corresponding to 1022 keV. No peak was observed in the gamma ray spectrum at 24 and 48 h. Based upon gamma spectrum and half-life measurement, the RNP of the radi- olabelled product was > 99%. The RCP of the labeled product obtained using ITLC was more than 99%. The product was found to be sterile as no microbial growth was observed upto 14 days. The radiolabelled product was non- pyrogenic as the calculated endotoxin values were less than 175 EU/V.
Shelf Life, In-vitro Stability and Lipophilicity of the Radiolabelled Product
68 Ga-SA.FAPi was found to be stable up to 4 h at RT and 4 ℃ with an RCP of 98% and > 99% respectively. In in- vitro stability study, 68Ga-SA.FAPi was found to be stable in saline for up to 4 h with a RCP of > 99%. In serum, 68Ga- SA.FAPi had an RCP of > 99% for up to 2 h which further decreased to 80% at 4 h. The product was hydrophilic in nature with a log P value - 1.51 ± 0.47.
Patient Demographics
Twenty-five patients [HCC, n = 14 (56%); GB Ca, n = 3 (12%); ACC, n = 4 (16%); BC, n = 4 (16%)] were enrolled in the study. The mean age of the cohort was 58 ± 8 years. Out of the cohort, 10 had only primary disease, five had primary disease with nodal metastases, three had primary disease with distant metastases, four had primary disease with both lymph nodal and distant metastases, one had residual
disease and two had recurrent disease with nodal and distant metastases (Table 1).
Biodistribution Analysis: 68Ga-SA.FAPi vs FDG
Physiological tracer uptake of 68Ga-SA.FAPi was noted in the salivary glands, thyroid, liver, pancreas, muscles and kidneys with variable uptake in the lacrimal glands, extra- ocular muscles, oral mucosa and uterus. Higher SUV max and SUV mean values were noted for 68Ga-SA.FAPi in the salivary glands, thyroid and liver (Table 2, Fig 1). Intense 68Ga- SA.FAPi tracer uptake was noted in the pancreas [SUV max: 15.4 + 3.9] in comparison to minimal FDG avidity [SUV max: 2.8 ± 0.8. A very low 68Ga-SA.FAPi uptake was noted in the brain [SUVmax: 0.7 ± 0.4] in comparison to intense FDG uptake [SUVmax:13.6 ± 4.8]. Additionally, muscles showed physiological tracer uptake in 68Ga-SA.FAPi scans whereas minimal tracer avidity was noted in FDG scans (Table 2).
Disease Identification: 68Ga-SA.FAPi vs FDG
In 25 patients enrolled for the study, 88 lesions spanning local disease, nodal and distant metastases were identified. In local disease detection, a perfect agreement (k = 1) was observed between both the observers. For distant metastases, a near perfect agreement was observed (k=0.87), with good agreement in detection of lung lesions (k = 0.78). For nodal disease, a fair inter-observer agreement was noted (k = 0.72). Lesion-based comparative analysis between both radiotracers demonstrated complete concordant findings in detection of all primary lesions and distant metastases in liver, bones, adrenals and peritoneum whereas discordant findings were noted in lung nodules (22%) and lymph nodes (13%). In overall analysis, 68Ga-SA.FAPi exhibited significantly higher SUVmax (10.3 ± 5.7 vs 8.8 ± 6.0, p-0.019), SULpeak (6.8 ± 3.8 vs 4.9 ± 3.0, p-0.000) and SULavg (5.4 ± 2.7 vs 4.1 ± 2.4, p-0.019) in comparison to FDG whereas TBR was comparable for both the tracers [TBR Liver: median 1.9 (IQR: 2.6-1.4) vs 1.8 (2.6-1.1), p-0.275; TBRBp: 2.1 (3.7-1.4) vs 2.0 (2.7-1.4), p-0.207]. Additionally, the mean TLF for the cohort was significantly higher than the mean TLG [38.8 (108.6-8.1) vs 16.7 (49.8-4.8)] (Table 3).
Site of Malignancy
Local (primary/residual/recurrent) disease: 21 local lesions including two GB, four breast, one adrenal and 14 hepatic lesions were identified in the study cohort. Both radiotracers detected all primary lesions, yielding 100% concordant scan findings. Higher 68Ga-SA.FAPi avidity was noted in com- parison to FDG with avg. SUVmax values of 14.8 (18.7-9.7) [vs 10.0 (12.9-6.6); p-0.087], SULpeak 8.2 (11.2-6.8) [vs 6.3 (8.5-4.4); p-0.037] and SULavg 6.9 ± 2.5 (vs 5.1 ± 2.2, p- 0.023) with comparable TBR [TBR Liver: 2.0 (2.5-1.7) vs 2.4 (3.5-1.4), p-0.818; TBRBP: 2.3 (3.3-1.3) vs 3.1 (4.2-1.7), p-0.052]. For local disease, the lesions demonstrated higher
| Patient | Age (years) | Sex | Diagnosis | Extent of Disease |
|---|---|---|---|---|
| 1 | 70 | M | Hepatocellular Carcinoma | Primary |
| 2 | 54 | M | Gall Bladder Carcinoma | Recurrent + LN's + Distant Mets |
| 3 | 51 | F | Breast Carcinoma | Primary + LN's |
| 4 | 54 | F | Breast Carcinoma | Primary + LN's + Distant Mets |
| 5 | 65 | F | Breast Carcinoma | Primary + LN's |
| 6 | 61 | M | Hepatocellular Carcinoma | Primary + LN's |
| 7 | 71 | M | Hepatocellular Carcinoma | Primary + LN's + Distant Mets |
| 8 | 50 | F | Adrenocortical Carcinoma | Primary + LN's + Distant Mets |
| 9 | 39 | F | Adrenocortical Carcinoma | Recurrent + LN's + Distant Mets |
| 10 | 55 | F | Gall Bladder Carcinoma | Primary + Distant Mets |
| 11 | 60 | M | Hepatocellular Carcinoma | Primary + Distant Mets |
| 12 | 58 | M | Hepatocellular Carcinoma | Primary |
| 13 | 65 | M | Hepatocellular Carcinoma | Primary |
| 14 | 65 | M | Hepatocellular Carcinoma | Primary |
| 15 | 50 | M | Hepatocellular Carcinoma | Primary |
| 16 | 53 | M | Hepatocellular Carcinoma | Primary |
| 17 | 62 | M | Hepatocellular Carcinoma | Primary + LN's |
| 18 | 65 | M | Hepatocellular Carcinoma | Primary |
| 19 | 61 | F | Hepatocellular Carcinoma | Primary |
| 20 | 62 | M | Hepatocellular Carcinoma | Primary + LN's + Distant Mets |
| 21 | 50 | F | Adrenocortical Carcinoma | Residual |
| 22 | 43 | M | Adrenocortical Carcinoma | Primary + Distant Mets |
| 23 | 75 | M | Gall Bladder Carcinoma | Primary |
| 24 | 57 | F | Breast Carcinoma | Primary + LN's |
| 25 | 62 | M | Hepatocellular Carcinoma | Primary |
mean TLF [104.4 (276.8-40.8)] in comparison to mean TLG [77.6 (227.4-21.7)], although the difference was not statis- tically significant (p-0.280). In sub-categorical analysis, higher 68Ga-SA.FAPi avidity was noted in ACC followed by GB Ca (Fig 2) and HCC (Fig 3) in contrast to Breast Ca (Fig 4) exhibiting higher FDG avidity as depicted in Table 4.
Additionally, in 28% (4/14 patients) of the HCC cohort, a pronounced 68Ga-SA.FAPi uptake was observed in the tu- mor’s periphery compared to its core [periphery SUV max: 9.8 (13.4-7.9); vs inside lesion SUVmax: 5 (7.9-3); SULpeak: 7.8
vs 6.8; SULavg: 4.9 vs 4.4]. Conversely, FDG uptake was noted primarily within the lesion, with avg. SUVmax: 9.5 (10.2-6.2) SULpeak: 8.5 and SULavg: 7.3. A similar case has been depicted in Figure 5. The TLF measurement in such cases was overestimated and hence were excluded. In the context of portal vein thrombosis (PVT), out of two cases, 68 Ga-SA.FAPi was able to identify only one case in contrast to FDG which identified both cases with higher SUVmax, SULavg and SULpeak values of 6.6 vs. 8.8, 3.0 vs 5.8 and 4.3 vs 7.4 respectively. Furthermore, for patients diagnosed with
| TABLE 2. SUVmax and SUVmean Values for Various Body Organs in 68Ga-SA.FAPi vs FDG PET | |||||
|---|---|---|---|---|---|
| S.No | Organs | 68Ga-SA.FAPi | FDG | ||
| SUVmax (Max ± SD) | SUVmean (Mean ± SD) | SUVmax (Max ± SD) | SUVmean (Mean ± SD) | ||
| 1 | Brain | 0.7 ± 0.4 | 0.2 ± 0.1 | 13.6 ± 4.8 | 8.6 ± 2.8 |
| 2 | Salivary glands | 6.9 ± 4.0 | 5.4 ± 3.0 | 2.4 ± 0.8 | 1.9 ± 0.7 |
| 3 | Thyroid | 12.6 ± 5.1 | 8.2 ± 3.8 | 2.2 ± 0.8 | 1.5 ± 0.6 |
| 4 | Liver | 6.2 ± 2.7 | 4.0 ± 1.9 | 5.1 ± 2.3 | 3.3 ± 1.3 |
| 5 | Pancreas | 15.4 ± 3.9 | 10.9 ± 2.3 | 2.8 ± 0.5 | 1.8 ± 0.3 |
| 6 | Blood Pool | 5.9 ± 1.5 | 3.8 ± 1.0 | 3.7 ± 1.3 | 2.6 ± 1.0 |
| 7 | Muscles | 2.7 ± 0.8 | 1.7 ± 0.6 | 1.4 ± 0.3 | 0.8 ± 0.1 |
| 8 | Kidneys | 8.2 ± 2.9 | 5.1 ± 1.8 | 5.4 ± 1.3 | 3.3 ± 0.8 |
| 9 | Uterus | 7.7 ± 2.7 | 4.4 ± 0.7 | 3.3 ± 1.8 | 1.9 ± 0.9 |
a
30
68Ga-SA.FAPİ
FDG
25
20
SUVmax
15
10
5
L
1
I
1
1
1
T
1
0
I
Brain
Salivary Glands
Thyroid
Liver
Pancreas
Uterus
Muscles
Kidney
Bloodpool
b
20
68Ga-SA.FAPi
17
FDG
13
SUVmean
10
1
7
3
1
T
L
L
T
1
1
1
L
1
0
2
Brain
Salivary Glands
Thyroid
Liver
Pancreas
Uterus
Muscles
Kidney
Bloodpool
hepatobiliary malignancies (n = 17), there was a notably high liver background (SUVmax: 8.1 ± 2.9). On contrary, in the cohort with other malignancies (n= 8), a diminished liver background activity was observed (SUVmax: 4.0 ± 0.7).
Nodal Metastases
Twenty-three nodal lesions were detected in the cohort with a scan concordance of 87%. While FDG identified all 23 nodal sites, 68Ga-SA.FAPi pinpointed 20 of these lesions. Comparable nodal avidity was noted for both the tracers with avg. SUVmax, SULavg and SULpeak values of 9 (13.5-4.1) vs 8 (12.7-4.7); p-0.726, 3.7 ± 1.8 vs 4.1 ± 2.3; p-0.129 and 4.5 (6.2-1.8) vs 4.3 (5.7-2.2); p-0.727 respectively. The TBRBP was significantly higher for 68Ga-SA.FAPi [2.1 (4.2-2.1) vs 2.0 (2.3-1.4), p-0.001] in comparison to TBRliver which was higher for FDG [1.5 ± 0.8 vs 2.0 ± 1.2; p-0.011]. The mean TLF was significantly higher than the mean TLG [9.1 (31.6-4.6) vs 4.4 (5.4-2.4), p-0.000] (Table 3).
Distant Metastases
37 distant metastatic sites were identified in the cohort in- cluding one GB, one breast, three liver, three adrenal, five
peritoneal, six skeletal, and 18 lung lesions with 84% con- cordant scan findings. 68Ga-SA.FAPi was able to identify four additional lung lesions which were missed by FDG. For dis- tant metastases, higher 68Ga-SA.FAPi avidity was noted compared to FDG with avg. SUVmax, SULavg and SULpeak values of 8.8 ± 5.1 vs 7.2 ± 5.6; p-0.038, 5.4 ± 2.6 vs 3.5 ± 2.4; p-0.000 and 6.3 (8.8-4.4) vs 3.6 (4.4-2.0); p- 0.000, respectively. 68Ga-SA.FAPi demonstrated significantly higher TBRliver [2.2 (3.5-1.3) vs 1.6 (2.1-1.0), p-0.005] whereas TBRBp was comparable to FDG [2.3 ± 1.2 vs 2.0 ± 1.3, p-0.367]. The mean TLF was significantly higher than the mean TLG [36.6 (90.9-8.1) vs 21.6 (37.1-6.3); p- 0.015] (Table 3). In sub-categorical analysis, gall bladder, lung and skeletal lesions demonstrated higher 68Ga-SA.FAPi avidity in comparison to the adrenal, liver and peritoneal lesions ex- hibiting higher FDG avidity. Breast lesion demonstrated si- milar avidity for both the tracers as depicted in Table 5.
Non-specific Tracer Uptake
Seven sites with non-specific tracer uptake were noted in FDG PET out of which five sites showed minimal
| Category of Analysis (n, no of lesions) | Overall (n = 88) | Primary (n =21) | Nodal Mets (n = 23) | Distant Mets (n = 37) | Non-Specific (n = 7) | |
|---|---|---|---|---|---|---|
| SUVmax [Mean ± S.D./Median (IQR)] | 68Ga-SA.FAPi | 10.3 ± 5.7 | 14.8 (18.7-9.7) | 9.0 (13.5-4.1) | 8.8 ± 5.1 | 4.6 ± 1.9 |
| FDG | 8.8 ± 6.2 | 10.0 (12.9-6.6) | 8 (12.7-4.7) | 7.2 ± 5.6 | 6.0 ± 2.4 | |
| p-value | 0.019 | 0.087 | 0.726 | 0.038 | NA* | |
| SULavg [Mean ± S.D./Median (IQR)] | 68Ga-SA.FAPi | 5.4 ± 2.7 | 6.9 ± 2.5 | 3.7 ± 1.8 | 5.4 ± 2.6 | 2.0 ± 0.5 |
| FDG | 4.1 ± 2.4 | 5.1 ± 2.2 | 4.1 ± 2.3 | 3.5 ± 2.4 | 2.3 ± 1.4 | |
| p-value | 0.000 | 0.023 | 0.129 | 0.000 | NA* | |
| SULpeak [Mean ± S.D./Median (IQR)] | 68Ga-SA.FAPi | 6.8 ± 3.8 | 8.2 (11.2-6.8) | 4.5 (6.2-1.8) | 6.3 (8.8-4.4) | 2.9 ± 1.0 |
| FDG | 4.9 ± 3.0 | 6.3 (8.4-4.4) | 4.3 (5.7-2.2) | 3.6 (4.4-2.0) | 3.9 ± 3.5 | |
| p-value | 0.000 | 0.037 | 0.727 | 0.000 | NA* | |
| TBRBP [Mean ± S.D./Median (IQR)] | 68Ga-SA.FAPi | 2.1 (3.7-1.4) | 2.3 (3.3-1.3) | 2.1 (4.2-2.1) | 2.3 ± 1.2 | 0.8 ± 0.6 |
| FDG | 2.0 (2.7-1.4) | 1.3 (4.2-1.7) | 2.0 (2.3-1.4) | 2.0 ± 1.3 | 1.2 ± 0.3 | |
| p-value | 0.207 | 0.052 | 0.001 | 0.367 | NA* | |
| TBRLiver [Mean ± S.D./Median (IQR)] | 68Ga-SA.FAPi | 1.9 (2.6-1.4) | 2.0 (2.5-1.7) | 1.5 ± 0.8 | 2.2 (3.5-1.3) | 1.0 ± 0.2 |
| FDG | 1.8 (2.6-1.1) | 2.4 (3.5-1.4) | 2.0 ± 1.2 | 1.6 (2.1-1.0) | 1.3 ± 1.2 | |
| p-value | 0.275 | 0.818 | 0.011 | 0.005 | NA* | |
| Volumetric Analysis [Mean ± S.D./ Median (IQR)] | TLF (SULxcm3) | 38.8 (108.6-8.1) | 104.4 (276.8-40.4) | 9.1 (31.6-4.6) | 36.6 (90.9-8.1) | 55.0 ± 86.2 |
| TLG (SULxcm3) | 16.7 (49.8-4.8) | 77.6 (227.4-21.7) | 4.4 (5.2-2.4) | 21.6 (37.1-6.3) | 29.0 ± 27.5 | |
| p-value | 0.000 | 0.280 | 0.000 | 0.015 | NA* |
* Statistical analysis not applicable (NA) due to small sample size.
68Ga-SA.FAPi
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68 8Ga-SA.FAPi avidity (site 1, dental carries: SA.FAPi SUV max: 3.8 vs FDG SUV max: 5.6 vs; site 2, non-specific uptake in rectum: 3.5 vs 10.9 vs, site 3, uptake in right hilar LN: 4.5 vs 7.4; site 4 non-specific nodal uptake: 3.4 vs 5.1;
site 5 atelectatic changes: 4.2 vs 5.9) whereas two sites had more 68Ga-SA.FAPi uptake (site 6 mediastinal LN uptake: 6.2 vs 4.9; site 7, post-op bed: 4.1 vs 1.0). 68Ga-SA.FAPi demonstrated lower non-specific tracer uptake in contrast to
68Ga-SA.FAPi
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FDG with an avg. SUV max, SULavg and SULpeak values of 5.5 ± 1.9 (vs 5.8±2.6; FDG), 2.0 ± 0.5 (vs 2.3± 1.4) and 5.5 ± 1.9 (vs 3.9 ± 3.0) respectively. The TBR was also lower for 68Ga-SA.FAPi in comparison to FDG (TBRliver: 1.0 ± 0.2 vs 1.3 ± 1.2; TBRBp: 0.8 ± 0.6 vs 2.0 ± 0.2 although the mean TLF was higher than the mean TLG (55.1 ± 86 vs 29.0 ±27.5).
DISCUSSION
FDG plays a pivotal role in numerous oncological and non- oncological applications. The era of personalized medicine has caused a paradigm shift focusing towards CAF’s in the TME characterized by increased FAP expression (3-10,13). Qui- noline-based FAP inhibitors have shown promise in clinical studies (27,28). While 68Ga-SA.FAPi displayed strong pre- clinical results, multi-centric studies are essential for global clinical validation. This prospective study assessed the diag- nostic performance of 68Ga-SA.FAPi across different cancer types, comparing it to the current standard-of-care FDG.
Radiosynthesis of 68Ga-SA.FAPi was facile using semi- automated module and good RCY of > 98%. The pre- paration was sterile and nonpyrogenic with RNP and RCP of > 99%. The radiopharmaceutical was hydrophilic in nature (Lop P - 1.51) with a shelf life of 4 h at RT and 4 ℃. 68 Ga-SA.FAPi demonstrated good stability in human serum and 0.9% saline for two hours, consistent with the results of an in-vitro study conducted by Moon et al (19).
Compared to other FAPI derivatives, 68Ga-SA.FAPi had a higher physiological uptake in the liver (SUVmax: 6.2 vs. 1.7 for FAPI-02) and pancreas (SUVmax: 15.4 vs. ~ 2 for FAPI- 02) (27). However, the increased pancreatic uptake may not hinder it’s therapeutic translation due to its rapid clearance over time (23). Consequently, delayed imaging (at 2-3 h) due to prolonged tumor retention may offer better lesion delineation in pancreatic malignancies. In comparison to another study by Meyer et al on FAPI-46, a comparable physiological liver uptake (SUVmax: 6.2 vs. ~ 6.3 for FAPI- 46) was noted (18). Compared to FDG, 68Ga-SA.FAPi showed minimal brain uptake, making it advantageous for imaging primary diseases or brain metastases. The physiolo- gical uptake of 68Ga-SA.FAPi in salivary glands, pancreas and muscles may be attributed to its half-maximal inhibitory concentration (IC50 8.7 ± 0.9) for FAP related prolyl oli- gopeptidase (PREP) (19). Although selective for FAP (IC50 1.4 ± 0.2) (19), the presence of PREP or related dipeptidyl peptidase 4 (DPP4), may be responsible for the uptake (29,30). Uterine uptake may be linked to FAP expression variation with the menstrual cycle (31). However, the un- derlying uptake mechanisms in thyroid, salivary glands and extra-ocular muscles are still unclear (31-33).
For disease detection, 68Ga-SA.FAPi demonstrated sig- nificantly higher tracer avidity compared to FDG while TBR was similar for both tracers. The mean TLF was no- tably higher than the mean TLG for the cohort. Sub-cate- gorical analysis revealed significantly higher 68Ga-SA.FAPi uptake in local disease and distant metastases. Chen et al also
| TABLE 4. Average SUVmax, SULavg, SULpeak, TBRBP, TBRliver, TLG and TLF Values for Various Sites of Local Disease in 68Ga-SA.FAPi vs FDG PET | |||||
|---|---|---|---|---|---|
| Site of local disease | Gall Bladder (n =2) | Breast (n = 4) | Adrenal Gland (n =1) | Liver | |
| (n = 21) | (n = 14) | ||||
| SUVmax [Mean ± S.D.] | 68Ga-SA.FAPi | 13.4 ± 2.5 | 14.6 ± 4.9 | 22.0 | 13.3 ± 6.0 |
| FDG | 9.3 ± 1.1 | 19.5 ± 9.0 | 10.0 | 9.3 ± 3.4 | |
| Paired lesion | 4.1 ± 1.4 | 4.9 ± 4.1 | 12.0 | 4.0 ± 6.0 | |
| subtraction | |||||
| p-value | 0.158 | 0.209 | NA* | 0.040 | |
| SULavg [Mean ± S.D.] | 68Ga-SA.FAPi | 5.9 ± 1.9 | 5.7 ± 0.3 | 7.6 | 7.2 ± 2.9 |
| FDG | 4.0 ± 1.1 | 7.2 ± 2.3 | 5.5 | 4.8 ± 2.3 | |
| Paired lesion | 1.9 ± 0.8 | 1.5 ± 2.0 | 2.1 | 2.4 ± 3.5 | |
| subtraction | |||||
| p-value | 0.194 | 0.329 | NA* | 0.019 | |
| SULpeak [Mean ± S.D.] | 68Ga-SA.FAPi | 8.1 ± 2.3 | 7.9 ± 0.3 | 11.9 | 9.8 ± 4.4 |
| FDG | 5.4 ± 1.3 | 8.9 ± 2.5 | 7.8 | 6.1 ± 2.5 | |
| Paired lesion | 2.7 ± 1.0 | 1.0 ± 2.2 | 4.1 | 3.7 ± 4.8 | |
| subtraction | |||||
| p-value | 0.161 | 0.532 | NA* | 0.008 | |
| TBRBP [Mean ± S.D.] | 68Ga-SA.FAPi | 2.2 ± 0.7 | 1.8 ± 0.8 | 4.1 | 2.5 ± 1.1 |
| FDG | 4.3 ± 2.3 | 3.5 ± 1.3 | 3.1 | 2.9 ± 1.5 | |
| Paired lesion | 2.1 ± 1.6 | 1.7 ± 0.5 | 1.0 | 0.4 ± 1.8 | |
| subtraction | |||||
| p-value | 0.307 | 0.142 | NA* | 0.411 | |
| TBRLiver [Mean ± S.D.] | 68Ga-SA.FAPi | 2.8 ± 0.6 | 3.3 ± 1.4 | 2.7 | 2.0 ± 0.8 |
| FDG | 2.7 ± 1.2 | 3.3 ± 1.0 | 2.4 | 2.2 ± 1.0 | |
| Paired lesion | 0.1 ± 0.6 | 0.0 ± 0.4 | 0.3 | 0.2 ± 1.1 | |
| subtraction | |||||
| p-value | 0.360 | 0.970 | NA* | 0.429 | |
| Volumetric Analysis [Mean ± S.D.] | TLF (SULxcm3) | 86.0 ± 52.5 | 126.6 ± 56.2 | 396.8 | 746.4 ± 1402.4 |
| TLG (SULxcm3) | 45.9 ± 33.8 | 172.7 ± 24.4 | 279.3 | 571.1 ± 846.5 | |
| Paired lesion* | 40.1 ± 18.7 | 46.1 ± 31.8 | 117.5 | 175.3 ± 1119.5 | |
| subtraction | |||||
| p-value | 0.203 | 0.280 | NA* | 0.553 | |
* Statistical analysis not applicable (NA) due to small sample size.
68Ga-SA.FAPi 68Ga-SA.FAPi
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FDG
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| TABLE 5. Average SUVmax, SULavg, SULpeak, TBRBP, TBRliver, TLG and TLF Values for Various Sites of Distant Metastases in 68Ga-SA.FAPi vs FDG PET | ||||||||
|---|---|---|---|---|---|---|---|---|
| Site of Distant Metastases (n = 37) | Lung (n = 18) | Skeleton (n = 6) | Peritoneum (n = 5) | Liver (n =3) | Adrenal Gland (n = 3) | Gall Bladder (n =1) | Breast (n = 1) | |
| SUVmax [Mean ± S.D.] | 68Ga-SA.FAPi | 6.5 ± 5.9 | 14.7 ± 7.0 | 9.1 ± 3.0 | 12.6 ± 1.8 | 11.2 ± 0.9 | 8.4 | 11.0 |
| FDG | 3.3 ± 2.9 | 10.0 ± 3.3 | 11.9 ± 7.1 | 10.8 ± 0.9. | 17.0 ± 1.5 | 3.9 | 11.1 | |
| Paired lesion | 3.2 ± 3.9 | 5.9 ± 4.4 | 2.8 ± 4.2 | 1.8 ± 1.3 | 10.2 ± 13.1 | 4.5 | 0.1 | |
| subtraction | ||||||||
| p-value | 0.003 | 0.023 | 0.216 | 0.142 | 0.216 | NA* | NA* | |
| SULavg [Mean ± S.D.] | 68Ga-SA.FAPi | 4.6 ± 2.6 | 5.7 ± 1.6 | 3.5 ± 1.0 | 6.3 ± 3.5 | 7.5 ± 4.3 | 4.7 | 5.3 |
| FDG | 1.8 ± 1.2 | 2.9 ± 0.8 | 6.9 ± 3.2 | 6.5 ± 2.4 | 9.5 ± 0.6 | 1.8 | 6.0 | |
| Paired lesion | 2.5 ± 1.7 | 2.8 ± 1.6 | 1.4 ± 0.7 | 0.2 ± 3.2 | 1.3 ± 9.5 | 3.0 | 0.7 | |
| subtraction | ||||||||
| p-value | 0.000 | 0.001 | 0.031 | 0.937 | 0.886 | NA* | NA* | |
| SULpeak [Mean ± S.D.] | 68Ga-SA.FAPi | 5.8 ± 3.7 | 6.9 ± 2.0 | 6.6 ± 2.3 | 7.7 ± 3.5 | 9.6 ± 5.8 | 7.1 | 9.2 |
| FDG | 2.2 ± 1.6 | 3.5 ± 1.0 | 4.7 ± 1.5 | 6.1 ± 1.4 | 12.7 ± 2.5 | 3.2 | 10.5 | |
| Paired lesion | 3.2 ± 2.5 | 3.4 ± 1.9 | 1.8 ± 1.0 | 1.6 ± 2.1 | 1.6 ± 2.1 | 3.9 | 1.3 | |
| subtraction | ||||||||
| p-value | 0.000 | 0.001 | 0.033 | 0.333 | 0.874 | NA* | NA* | |
| TBRBP [Mean ± S.D.] | 68Ga-SA.FAPi | 1.8 ± 0.2 | 1.7 ± 0.8 | 3.2 ± 1.6 | 2.9 ± 0.7 | 1.2 ± 0.6 | 3.1 | 2.8 |
| FDG | 1.6 ± 1.0 | 1.4 ± 0.3 | 1.4 ± 0.3 | 1.8 ± 0.3 | 4.1 ± 0.2 | 2.9 | 3.2 | |
| Paired lesion | 0.8 ± 0.6 | 0.3 ± 0.9 | 1.5 ± 1.3 | 1.1 ± 2.3 | 3.2 ± 0.3 | 0.2 | 0.4 | |
| subtraction | ||||||||
| p-value | 0.001 | 0.421 | 0.119 | 0.490 | 0.039 | NA* | NA* | |
| TBRLiver [Mean ± S.D.] | 68Ga-SA.FAPi | 4.6 ± 2.0 | 1.9 ± 0.7 | 2.3 ± 1.0 | 1.6 ± 0.4 | 2.3 ± 1.1 | 2.7 | 2.5 |
| FDG | 1.3 ± 0.8 | 1.2 ± 0.4 | 1.5 ± 0.3 | 3.0 ± 1.3 | 3.4 ± 0.2 | 2.5 | 2.8 | |
| Paired lesion | 2.7 ± 1.8 | 0.7 ± 0.5 | 0.9 ± 0.7 | 1.4 ± 1.6 | 1.6 ± 0.7 | 0.2 | 0.3 | |
| subtraction | ||||||||
| p-value | 0.004 | 0.016 | 0.101 | 0.283 | 0.204 | NA* | NA* | |
| Volumetric Analysis [Mean * ± S.D.] | TLF (SULxcm3) | 39.6 ± 46.3 | 74.9 ± 60.2 | 142.9 ± 164.1 | 526.7 ± 865.6 | 29.2 ± 23.6 | 59 | 30 |
| TLG (SULxcm3) | 13.4 ± 6.6 | 42.3 ± 49.0 | 258.2 ± 308.2 | 310.3 ± 504.3 | 14.1 ± 11.0 | 48 | 42 | |
| Paired lesion | 25.3 ± 30.6 | 32.6 ± 37.3 | 115.3 ± 143.8 | 216.4 ± 361.3 | 16.1 ± 22.2 | 11 | 12 | |
| subtraction | ||||||||
| p-value | 0.015 | 0.042 | 0.207 | 0.408 | 0.492 | NA* | NA* | |
* Statistical analysis not applicable (NA) due to small sample size.
reported significantly higher uptake of 68Ga-FAPI-04 in primary and metastatic lesions compared to FDG, con- cordant with our findings (34).
Various studies have documented that TLG and Metabolic Tumor Volume (MTV) may serve as superior prognostic indicators for overall survival compared to SUVs in various cancers, including lung, ovarian, head and neck cancers, as well as pleural mesothelioma (35-38). Hong et al emphasized that TLG can independently serve as a prognostic factor for overall survival, surpassing MTV and SUV as predictors of survival in patients undergoing radiotherapy for locally ad- vanced esophageal cancer (39). In this study, a markedly higher mean TLF was noted in nodal disease and metastatic lung and skeletal lesions, as opposed to mean TLG value. However, in local disease, mean TLG and TLF were com- parable. Hence, in the realm of FAPi aided theranostics, the integration of volumetric parameters FTV and TLF with conventional clinicopathological parameters may offer a novel approach for prognostic stratification, enabling more refined treatment planning and response assessment.
This study demonstrated highest 68Ga-SA.FAPi tracer uptake was noted in ACC followed by HCC in local disease detection. Ballal et al reported highest 68Ga-FAPi uptake in the head and neck cancers followed by gall bladder carci- noma (23). Kratochwil et al. also reported highest 68Ga- FAPI-04 uptake in lung cancer followed by breast, esopha- geal cancer, cholangiocellular carcinoma and sarcoma (28). Chen et al. reported the highest tracer avidity of 68Ga-FAPI- 04 in pancreatic cancer, liver cancer, sarcoma, esopha- geal and gastric cancer (34). The variation in study results may stem from diverse patient groups and the lack of ACC cohorts in these studies.
In BC, similar tracer avidity was observed for both 68Ga- SA.FAPi and FDG with concordant scans and these findings align with the results reported by Kratochwil et al. and Ballal et al. In our study, 68Ga-SA.FAPi SULpeak ranged from 6.0 to 10.7, while Ballal et al. reported a SULpeak range of 3.3-12.5 for in patients. Kratochwil et al. noted an average SUVmax of > 12, aligning with our findings (SUVmax 14.6) in breast cancer patients. Despite similar study outcomes, the varying uptake of 68Ga-SA.FAPi could be due to changes in FAPa expression during different metastatic stages. Ding et al. highlighted the shifting FAPa expression across meta- static stages in a breast cancer animal model. Early tumor metastases showed greater 68Ga-FAPI-04 sensitivity than advanced stages, which had higher FDG sensitivity (40). In this study, all four breast cancer patients had primary disease with node involvement, and one had distant metastases, possibly leading to lower 68Ga-SA.FAPi avidity.
In HCC, 68Ga-SA.FAPi displayed a higher avidity than FDG but exhibited a distinct uptake pattern. Interestingly, in 44% of the HCC cohort with local disease (4/9 patients), 68 Ga-SA.FAPi uptake was more pronounced in the peritu- moral region rather than within the tumor itself (avg. SUV max: 9.8 vs 5.0). The presence of FDG in the tumor negated the possibility of tumor necrosis. Variations in FAP
expression based on cellular differentiation have been pre- viously reported (34,40). Gua et al observed a positive cor- relation between 68Ga-FAPI-04 tracer avidity and the pathological grade of primary tumor (41). Shi et al noted a relatively lower 68Ga-FAPI-04 uptake in less-aggressive, well-differentiated HCC’s in comparison to the poorly dif- ferentiated lesions (42). In a study on clinicopathologic fea- tures of 26 cases of HCC, Okuda et al identified a clear fibrous capsule in well-differentiated tumors, varying in thickness from a few millimeters to 1 cm resulting from hepatocyte compression, reticulin fiber condensation and collagenization, and portal area inclusion (43). Yuki et al also reported a fibrous capsule in their autopsy study of 240 HCC cases (44). Yin et al performed immunohistochemical staining (IHC) of 57 HCC tissues, identifying CAFs in the fibrous septum, fibrous capsule, and hepatic blood sinusoids (45). In the study cohort, patients with peritumoral 68Ga- SA.FAPi uptake had localized primary disease, potentially linked to low FAP expression within the tumor due to cellular differentiation. The strong peritumoral uptake might be related to the presence of a fibrous capsule around well- differentiated lesions. However, for further validation, future studies with larger, more consistent sample sizes and histo- pathological correlations are necessary. Additionally, HCC FAP expression could signify an epithelial-mesenchymal transition (EMT), a factor in disease aggressiveness (46). Therefore, 68Ga-SA.FAPi imaging in HCC may offer va- luable insights into disease severity, prognosis and patient management.
This study also observed an elevated 68Ga-SA.FAPi liver background in patients with hepatobiliary malignancies (4 HCC and 1 GB Ca with liver infiltration), possibly due to cirrhosis or fibrosis linked with these conditions (47). Similar findings of increased 68Ga-FAPI-04 uptake in cirrhotic livers were reported by Shi and Gua et al (36,37) (41,42). Wu et al found 68Ga-FAPI less useful for detecting primary HCC in cirrhotic livers than FDG due to high background uptake (48). Cirrhosis shows heightened intrahepatic FAP expres- sion, linked to liver fibrosis severity (46,49). Thus, 68Ga- SA.FAPi could illuminate underlying molecular pathways and enhance cirrhosis diagnosis.
Both tracers exhibited 87% agreement in nodal disease detection with comparable tracer avidity in this study. Serfling et al. also found FDG superior to 68Ga-FAPI for nodal mets (50). Ballal et al. presented a case of NSCLC with no discernible 68Ga-SA.FAPi avidity in lymph nodes (23). Conversely, Chen et al observed better performance with 68Ga-FAPI-04 compared to FDG in identifying nodal me- tastases (34). A meta-analysis by Sollini et al on various FAPI derivatives revealed variable performance for 68Ga-FAPI PET in detecting nodal metastases, with sensitivities ranging from 59% to 100%. This variability may be linked to cancer biology and the lymph node’s cellular composition (51). Inflammatory lymph nodes could also impact the differential uptake of FDG and 68Ga-SA.FAPi. However, assessing each lymph node can be challenging. Additionally, the choice of
radioisotope (68Ga/18F) may influence spatial resolution and hence, the detection of smaller tumor clusters within lymph nodes (52).
In distant metastases, 68Ga-SA.FAPi outperformed FDG, identifying four more lung lesions. This is in line with the observations made by Chen et al., who also reported higher number of metastatic lesions detected with 68Ga-FAPI-04. In a subcategory analysis, the SUV values from 68Ga- SA.FAPi were 2-3 times higher than FDG (skeletal: 12.8 vs 6.9; GB: 8.4 vs 3.9; lung: 6.5 vs 3.3). Chen et al. also found similar disparities between 68Ga-FAPI-04 and FDG derived SUV values for lung and skeletal metastases (34).
68 Ga-SA.FAPi also demonstrated a reduced non-specific tracer uptake compared to FDG. However, physiological uptake of the radiotracer, especially in wound healing, de- generative lesions, muscles, thyroid, salivary glands and uterus uterus may act as potential pitfalls in 68Ga-SA.FAPi PET imaging. Additionally, the peri-tumoral uptake owing to a fibrous capsule or the increased liver background in cirrhotic/fibrotic livers seen in HCC may require meticulous examination for precise disease identification.
To the best of our knowledge, the utility of68Ga-SA.FAPi in ACC and HCC is being assessed for the first time in this study. FDG has a recognized shortcoming in HCC due to the subdued activity of the hexokinase enzyme, suggesting that 68Ga-SA.FAPi might be a superior diagnostic probe (27,52). Shi et al. also reported higher sensitivity of 68Ga- FAPI compared to FDG PET in patients with suspected primary hepatic tumors (41). Given the challenges in dis- cerning 68Ga-SA.FAPi uptake across varied HCC cellular differentiation stages, comprehensive, consistent studies with histopathological validation are essential to better understand the diagnostic value of this molecule. Our team previously explored the diagnostic capabilities of 68Ga-SA.FAPi in metastatic and recurrent ACC (53).
Several FAPi derivatives have been reported so far with comparable diagnostic affinity. However, the clinical appli- cations of FAPi with therapeutic utility are limited. The recently introduced DOTA(SA.FAPi)2 dimer shows promise for therapeutic applications due to its prolonged tumor re- tention (20). Consequently, 68Ga-SA.FAPi and 177 Lu/225 Ac- DOTA(SA.FAPi)2 present a promising theranostic duo for precision cancer treatment, particularly for challenging can- cers like metastatic ACC and HCC. Moreover, no fasting requirement and shorter uptake period in 68Ga scans may be an additional benefit for diabetic and elderly patients.
LIMITATIONS
· Our study features a small sample size, heterogenous co- hort with both FDG-avid and non-avid malignancies. Multi-centric studies with homogenous patient popula- tion are required to establish the diagnostic performance of 68Ga-SA.FAPi for incorporation into routine clinical practice.
· Not all nodal and metastatic lesions in the comparative analysis have confirmed metastasis through biopsy. Ethically, obtaining pathological confirmation for all analyzed lesions solely to validate the PET/CT results may not be justifiable.
· The observed peritumoral uptake in the HCC group may be due to a fibrous capsule surrounding well-differ- entiated lesions. Yet, more comprehensive studies with consistent sample sizes and correlations to histopatholo- gical FAP expression are needed for further confirmation.
CONCLUSION
68Ga-SA.FAPi consistently matched the standard-of-care FDG in detection of local disease and distant metastases in liver, bones, adrenals and peritoneum with discordant findings were noted in lung nodules and lymph nodes. Significantly higher 68Ga-SA.FAPi tracer avidity was noted in local disease and distant metastases with comparable TBR. These findings emphasize the selective expression of 68Ga-SA.FAPi in various cancers and add value to existing knowledge on FAP inhibitor molecular imaging. This highlights an additional role for FAPi in the realm of theranostics.
LEADERSHIP ROLES
All authors actively contributed in the study conception and design. The DOTA.SA.FAPi molecule was provided by Prof. Frank Roesch and Dr. Euy Sung Moon. The patients were referred by Prof. Ajay Duseja, Dr. Rama Walia, and Dr. Santhosh Irrinki. The study was performed by Ms. Sejal Chopra under the supervision of Dr. Jaya Shukla, Prof. Bhagwant Rai Mittal and Dr. Harmandeep Singh. The ac- quisition was performed by Dr. Nivedita Rana. The patient images were interpreted by Dr. Yamini Mathur and Dr. Rajender Kumar. The first draft of manuscript was prepared by Ms. Sejal Chopra. All the authors have read and approved the final manuscript.
DATA STATEMENT
The author(s) declare(s) that they had full access to all of the data in this study and the author(s) take(s) complete re- sponsibility for the integrity of the data and the accuracy of the data analysis.
FUNDING
The authors would like to acknowledge Postgraduate Institute of Medical Education and Research, Chandigarh (Project ID:9719-126) for providing financial support to conduct the study.
DECLARATION OF COMPETING INTEREST
The authors declare the following financial interests/personal relationships which may be considered as potential com- peting interests: Dr. Jaya Shukla reports financial support was provided by Post Graduate Institute of Medical Education and Research.
APPENDIX A. SUPPORTING INFORMATION
Supplementary data associated with this article can be found in the online version at doi:10.1016/j.acra.2023.12.002.
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