Clinical application of Al18F-NOTA-FAPI PET/CT in diagnosis and TNM staging of pancreatic adenocarcinoma, compared to 18F-FDG

Purpose This study aimed to investigate the ability of Al18F-NOTA-FAPI PET/CT to diagnose pancreatic carcinoma and tumor-associated inflammation with the comparison of 18F-FDG PET/CT. Methods Prospective analysis of Al18F-NOTA-FAPI PET/CT and 18F-FDG PET/CT scans of 31 patients from 05/2021 to 05/2022 were analyzed. Al18F-NOTA-FAPI imaging was performed in patients who had Ce-CT and FDG PET/CT and the diagnosis was still unclear. Follow-up histopathology or radiographic examination confirmed the findings. Radiotracer uptake, diagnostic performance, and TNM (tumor-node-metastasis) classifications were compared. Results A total of 31 patients with pancreatic carcinoma (all were adenocarcinoma) underwent Al18F-NOTA-FAPI-04 PET/CT, including 20 male and 11 female patients, with a mean age of 58.2 ± 8.5 years. FAPI-04 PET/CT imaging showed a higher value of SUVmax-15min/30min/60min, SUVmean-15min/30min/60min, TBR1, and TBR2 in pancreatic carcinoma than FDG (all P < 0.01). The mean level of Al18F-NOTA FAPI-04 uptake values of the pancreatic ductal adenocarcinoma was higher than that of pancreatitis in both SUVmax-30min (P < 0.01), SUVmean-30min (P < 0.05), SUVmax-60min (P < 0.01), and SUVmean-60min (P < 0.01). The FAPI △SUVmax-1, △SUVmax-2, and △SUVmean-2 uptake values of pancreatic carcinoma were higher than tumor-associated inflammation (all P < 0.01). TNM staging of 16/31 patients changed after Al18F-NOTA FAPI-04 PET/CT examination with all upstaging changes. Conclusion Al18F-NOTA-FAPI-04 PET/CT at 15 and 30 min also demonstrated an equivalent detection ability of pancreatic lesion to 18F-FDG PET/CT. Delayed-phase Al18F-NOTA-FAPI-04 PET/CT can help differentiate pancreatic carcinoma and tumor-associated inflammation. Al18F-NOTA FAPI-04 PET/CT also performed better than FDG PET/CT in TNM staging. Trial registration Chinese Clinical Trial Registry, ChiCTR2100051406. Registered 23 September 2021, https://www.chictr.org.cn/showproj.html?proj=133033


Introduction
A 5-year survival rate of 10% for pancreatic ductal adenocarcinoma (PDAC) is a leading cause of cancer mortality worldwide [1].A comprehensive imaging study of PDAC is essential for accurate initial staging, selection of treatment, and follow-up examinations to detect recurrence and/or metastatic spread.The utilization of 18 F-FDG PET/CT and PET/MR imaging in clinical contexts is on the rise, however, their usage is limited to the diagnosis of inflammatory and malignant pancreatic ailments [2][3][4].A novel PET tracer, fibroblast activation protein inhibitor (FAPI), targets cancer-associated fibroblasts (CAFs) [5].A CAF in PDAC is derived from pancreatic stellate cells and transforms its tumor-promoting properties by crosstalking with neoplastic cells [6].The CAF is thought to promote tumor growth, invasion, metastasis, and therapy resistance in PDAC [7,8].This study investigated whether Al 18 F-NOTA-FAPI could improve screening efficiency and differentiate between PDAC and tumorassociated inflammation.

Patients
A total of 31 patients who had undergone Al 18 F-NOTA-FAPI-04 PET were prospectively analyzed.All patients gave written informed consent to undergo FAPI PET/CT following the German Pharmaceuticals Act §13(2b) regulations.The clinical translational study of Al 18 F-NOTA-FAPI-04 was approved by the Ethics Committee (approval No. 2021XJSS01) and registered in the Chinese Clinical Trial Registry (ChiCTR2100051406).All patients enrolled in this study signed written informed consent forms.We assessed clinical features, including clinical manifestation, imaging parameters from contrast-enhanced CT (CE-CT), and laboratory panels such as carbohydrate antigen (CA)19-9 and serum amylase.FAPI imaging was performed in patients who had CE-CT and FDG PET/CT and the diagnosis was still unclear.Only patients who had a contraindication (e.g., pregnant women, the start of treatment before FAPI PET/CT examination or patients who refused the procedure) did not undergo Al 18 F-NOTA-FAPI-04 PET/CT imaging.All reported investigations were conducted by the Declaration of Helsinki and with the national regulations.All patients suspected of solid tumors underwent surgery or biopsy to confirm the pathological diagnoses.As defined in previous study [9], it was considered a positive lymph node if the uptake of Al 18 F-NOTA-FAPI-04 or 18 F-FDG exceeded that of surrounding tissue.For patients suspected of pancreatitis, complementary treatment and imaging follow-up were performed.The minimum follow-up period was three months.

Radiotracer synthesis
18 F-FDG was routinely synthesized at the department of Nuclear Medicine of the First Affiliated Hospital of Harbin Medical University, following standard methodology [10].The synthesis and labeling of Al 18 F-NOTA-FAPI-04 have already been described previously [11].Following the German Pharmaceuticals Act §13(2, b) regulations, indication and labeling of the FAPI-tracers were conducted under the physician's direct responsibility.Injected activities were dependent on labeling yields.According to a previous dosimetry estimate -an effective dose of 1.6 mSv / 100 MBq (2.7 mCi)-an upper limit of 370 MBq (10.0 mCi) regarding radiation exposure, and a lower limit of 100 MBq (2.7 mCi) per exam to achieve a sufficient count rate have been considered [5].The radiochemical purity was > 95% for both 18 F-FDG and Al 18 F-NOTA-FAPI-04.Al 18 F-NOTA-FAPI-04 and 18 F-FDG tracers met all standard criteria before human administration.

PET/CT image acquisition
All patients underwent sequential 18 F-FDG and Al 18 F-NOTA-FAPI-04 PET/CT scanning within 1 week.The patients underwent 18 F-FDG PET/CT examination first. 18F-FDG PET/CT was conducted after > 6 h of fasting and among patients with normal blood glucose levels (3.9-11.1mmol/L).The 18 F-FDG PET/CT was conducted after urinating in quiet, light-avoidance conditions (60min).Al 18 F-NOTA-FAPI-04 PET/CT scan was conducted without fasting at 15 min, 30 min, and 1 h, respectively.No patients were required to fast, and venous blood glucose levels were not controlled for Al 18 F-NOTA-FAPI-04 PET/CT.Radioactivity ranging from 129.5 to 148 MBq (3.5 to 4.0 mCi) of Al 18 F-NOTA-FAPI-04 isotope (Jiangyuan Industrial technology trade Co., LTD, Jiangsu, China, radiochemical purity > 95%) was intravenously injected.The Al 18 F-NOTA-FAPI-04 PET/CT were conducted at 15min, 30min and 60min after injection.Both 18 F-FDG and Al 18 F-NOTA-FAPI-04 PET/CT images were acquired using a 16-slice Gemini GXL PET/CT scanner (Philips Medical System).A low-dose CT scan (tube voltage: 120 kV, tube current: 50 mAs, slice thickness: 5.0 mm, pitch: 1.0) was acquired for attenuation correction, and then the PET images were acquired (1.5 min per bed position, 6-7 PET bed positions).According to the agency's standard clinical protocols, the scan range was from the head to the mid-thigh.The line of response reconstruction algorithm was used to reconstruct the image without post-reconstruction filtering after automatic random and scattering correction.

Image evaluation
PET data were analyzed by two experienced nuclear medicine specialists (P.F. and C.Z.) on a consensus decision.The readers were blind to the results of 18 F-FDG PET/CT when reporting Al 18 F-NOTA-FAPI-04 PET/ CT.Al 18 F-NOTA-FAPI-04 tracer uptake was quantified as SUV max (15min/30min/60min), SUV mean (15min/30min/60min), and tumor to background ratio (TBR) from static images 15 min after tracer injection.The TBR is classified as TBR 1 , TBR 2 , and TBR 3 depending on the background of the mediastinal blood pool, the liver blood pool, and the muscle.△SUV max-1 and △SUV mean-1 have been defined as the SUV max-30min values minus the SUV max-15min values, and the SUV mean-30min values minus SUV mean-15min .△SUV max-2 and △SUV mean-2 have been defined as the SUV max-60min values minus the SUV max-30min values, and the SUV mean- 60min values minus SUV mean-30min . 18F-FDG tracer uptake was quantified as SUV max and SUV mean .The discrepancies were discussed and a consensus was reached.

Statistical analysis
Statistical analyses were performed using SPSS software version 23.0 (SPSS, Chicago, IL, USA), GraphPad Prism (version 8.4.2;GraphPad Software, San Diego, Calif ), and the R language (version 3.6.3,http:// www.rproje ct.org).Quantitative values were expressed as mean ± SD or median and appropriate range, and categorical variables were presented as a rate or percentage.Shapiro-Wilk test for continuous variables shows that all continuous variable data do not meet normal distribution.A comparison of nonparametric data was performed using a Wilcoxon test.For correlation analyses, Kendall's TaU-B test was used to test the correlation between categorical and continuous variables.The Spearman test was used to test the correlation between continuous and non-continuous variables.All statistical tests were performed 2-sided, and P < 0.05 indicated statistical significance.

Patients' characteristics
A total of 31 patients with pancreatic carcinoma (all were adenocarcinoma) have been enrolled and underwent Al 18 F-NOTA-FAPI-04 PET/CT from 05/2021-05/2022, including twenty male and eleven female patients were recruited in the prospective study, with an overall mean age of 58.2 ± 8.5 years at the time of the PET scan (Table 1).The study flow diagram is presented in Fig. 1.The median values of serum CA19-9 and amylase level were 89.2 U/mL (10.6-1000U/ml, 25 th -75 th percentile: 35.9-144.8U/mL) and 60.7 U/L (26.3-61089.8U/L 25 th -75 th percentile 33.8-83.8U/L), respectively.Among the 31 patients, twelve (38.7%) had jaundice.Most of the patients (n = 20, 64.5%) underwent PET/CT examination to clarify the nature of the lesion.Other clinical indications for PET/CT were initial staging (n = 9, 29.0%) and restaging (n = 2, 6.5%).CE-CT was used in all patients as the most commonly used imaging modality for pancreatic cancer.All cases finally underwent both Al 18 F-NOTA-FAPI-04 and 18 F-FDG PET/CT.

Adverse events
All participants tolerated the Al 18 F-NOTA-FAPI-04 PET/ CT scan.No Al 18 F-NOTA-FAPI-04-related pharmacological effects or physiological responses occurred.None of the participants reported any abnormal symptoms.

Changes in TNM staging after Al 18 F-NOTA-FAPI-04 PET/CT and clinical management
Compared to 18 F-FDG PET/CT, Al 18 F-NOTA-FAPI-04 PET/CT improved the N stage in 16/31 patients (51.6%).A total of 16 patients had their TNM staging upstaged as a result of the new lesions detected by Al 18 F-NOTA-FAPI-04 PET/CT, and all changes are upstaging (Table 2).Seven cases (22.6%) were upstaged by detecting Al 18 F-NOTA-FAPI-04 uptake in abdominal lymph nodes, leading to an upstaging to stage IIB (Fig. 5A-D), and nine cases (29.0%) were upstaged to stage III.Because of newly detected lymph node metastases, the therapeutic regimen was changed in 12 patients (38.7%, surgically resectable to unresectable).

Comparisons of Al 18 F-NOTA-FAPI-04 and 18 F-FDG between the detection of abnormal uptake in the liver and bones
In this cohort, Al 18 F-NOTA-FAPI-04 PET/CT examination found extra uptake in two liver lesions with the SUV max of 10.6, and one bone lesion of the cervical spine with the SUV max of 3.2.While 18 F-FDG PET/CT examination found two liver lesions with the SUV max of 6.0, no extra 18 F-FDG uptake was found in the skeleton (Fig. 6 A-D).Two liver lesions were confirmed as liver metastasis by biopsy.Besides, 2 cases of diffused liver fibrosis resulted in overall FAPI uptake in the liver, affecting the interpretation of PET examination.Combined with the imaging of the patient in the previous two years, the lesion in the cervical spine was diagnosed as a benign lesion caused by skeletal degeneration after consultation with two experienced nuclear medicine physicians and two experienced radiologists.

Discussion
A single-center prospective analysis was conducted to compare Al 18 F-NOTA-FAPI-04 PET/CT and 18 F-FDG PET/CT for pancreatic lesions diagnosis and malignant tumor staging.PDAC is clinically challenging and is associated with an extremely high mortality rate.  1F-FDG uptake values in PDAC.Compared with FDG, FAPI PET imaging showed the lesions in the head of the pancreas clearly with higher TBR.The image quality of 15-minute, 30-minute, and 1-hour imaging can satisfy the diagnostic requirements (SUV max : 12.9, 12.7, 10.1), where no uptake of FDG showed(SUV max : 1.8).b. c.The SUV max , SUV mean , TBR 1 , TBR 2 , and TBR 3 values of Al 18 F-NOTA-FAPI-04 PET / CT imaging were significantly higher than those of FDG 18 F-FDG PET has proven superior to other anatomical imaging modalities such as CT, MRI, and ultrasound.Improving survival can only be achieved by selecting treatment approaches tailored to the individual patient's condition.In a study comparing FDG PET and FAPI PET/CT in various tumor types, four pancreatic carcinomas showed an upstaging tendency following FAPI PET [12].Therefore, FAPI-tracer may open new opportunities for staging and restaging PDAC [13].
It is becoming increasingly recognized that CT may be limited in representing pancreatic tumors.There is a correlation between tumor size and sensitivity (83% for lesions less than 2 cm) [14].Additionally, about 10% of pancreatic adenocarcinomas and metastatic lesions are iso-attenuating on CT and, thus, cannot be detected, even if larger than 2 cm [15].A CT combined with FDG PET could provide more information than can be obtained by CT alone [16].Adenocarcinomas of the pancreas typically exhibit an increased FDG uptake area [17].A pancreatic tumor is characterized by vascular deficiency and abundant desmoplastic stroma, which comprise 90% of its volume.The stroma is composed of extracellular matrix proteins and CAFs [18].A high TBR and a clear tumor boundary were observed with 68 Ga-FAPI PET/CT in pancreatic cancer [19].
Our study showed that the Al 18 F-NOTA-FAPI-04 SUV max , SUV mean , TBR 1 and TBR 2 uptake of pancreatic adenocarcinoma were higher than those of 18 F-FDG with statistical significance.Abnormal diffused uptake of FAPI uptake in the liver was observed in 2 cases, possibly due to hepatic fibrosis [20,21].Therefore, although TBR 3 was higher than FDG, it did not show statistical significance.This suggests that though the background FAPI of normal liver tissue is low, physicians should pay attention to its sensitivity to liver fibrosis when reviewing Al 18 F-NOTA-FAPI-04 PET/CT images.In contrast, choosing the mediastinal blood pool as TBR is less likely to be misinterpreted.Due to the uptake of FDG in pancreatic adenocarcinoma lesions is not as strong as FAPI, FDG PET imaging often fails to accurately identify the boundary of the lesion, which results in an incorrect measurement of the lesion volume.Our research found that strong uptake of FAPI in displaying pancreatic adenocarcinoma lesions leads to clear boundaries, which may affect the T-staging of the tumor, selection of surgical scope, and delineation of radiotherapy target.Another study also showed how automatic contouring based on FAPIs could improve radiation oncology target definitions for pancreatic cancer [22].
For cases with only pancreatic cancer lesions, a single time point Al 18 F-NOTA-FAPI-04 scan is sufficient for diagnosis.However, 15min time point imaging is not able to differentiate PDAC from tumor-associated inflammation.We recommend multi-time point Al 18 F-NOTA-FAPI-04 scans in the differential diagnosis of PDAC and tumor-associated pancreatitis.This cohort showed statistically significant differences in FAPI SUV max (15min/30min/60min) uptake between PDAC and tumor-associated inflammation.We also found a statistical difference in △SUV max-1, △SUV max-2 and △SUV mean-2 uptake values in Al 18 F-NOTA-FAPI-04 PET/CT examination between PDAC and tumor-associated inflammation.The tumor-associated inflammation decreased as the uptake time lengthened, while primary pancreatic malignant lesions increased the FAPI uptake value.In other words, even though the pancreas may show diffused FAPI uptake on the same scan, the SUV value at the cancer range is higher than the inflammation.Such changes mentioned above in SUV max values were not observed in 18 F-FDG PET/CT examination.The SUV max values of 18 F-FDG were generally low, and the visual assessment might not be quite Fig. 5 An evaluation of a patient with a local PDAC.Al 18 F-NOTA-FAPI-04 PET/CT showed increased FAPI uptake in lymph nodes (a, white arrow, SUV max-15min =4.8), which all showed no uptake in 18 F-FDG PET/CT (b, white arrow).Meanwhile, the contrast-enhanced CT showed little enhancement in both arteries (c, white arrow) or the venous phase (d, white arrow).Thus, the N stage was upstaging to the IIB clear.Al 18 F-NOTA-FAPI-04 PET/CT imaging can be a promising imaging supplement.
Pancreatic adenocarcinoma is staged based on its local and distant spread.Our study showed TNM upstaging caused by Al 18 F-NOTA-FAPI-04 PET/CT imaging over half of the patients (16/31, 51.6%) compared to FDG PET/CT imaging.The most impressive observation was that FAPI PET/CT imaging tended to show a clearer and broader lesion contour.This can affect the assessment of tumor-vessel relationship.However, a CE-CT examination is always recommended if vascular invasions is suspected, regardless of whether FAPI was the racer or FDG.Our study showed that more lymph nodes were involved with Al 18 F-NOTA-FAPI-04 PET/CT than with 18 F-FDG PET/CT, which would affect N-staging.Sensitivity, specificity, and accuracy for the diagnosis of metastatic lymph nodes for Al 18 F-NOTA-FAPI-04 PET/CT were higher than that of 18 F-FDG PET/CT.Al 18 F-NOTA-FAPI-04 PET/CT may improve pancreatic adenocarcinoma prognosis by overcoming existing challenges in N staging and rationalizing surgical planning.However, whether FAPI PET/CT imaging can accurately identify lymph node metastasis requires further research on large samples and pathological diagnosis.
A lower liver background has long been an advantage of FAPI PET/CT imaging, revealing more lesions that FDG PET/CT cannot identify.In this cohort, Al 18 F-NOTA-FAPI-04 PET/CT examination found extra uptake in two liver lesions with the SUV max higher than that of 18 F-FDG.We also found FAPI uptake in the cervical spine in one case.FAPI uptake in bones and joints is a common incidental finding on PET/CT [23].The FAPI uptake in bone occurs often with degenerative changes where osteophytes are typically visible on CT scans [24].There have been reports of increased FAPI uptake in cases of bone fractures [25], Schmorl's nodes [25], fibrous dysplasia [26], avascular necrosis [27], mastoiditis [25], bone tuberculosis [28], and myositis ossificans [29] as well.Thus, we sought a discussion with experienced radiologists and nuclear medicine physicians.By comparing the patient's imaging studies over the previous two years, it was concluded that FAPI uptake in this lesion was caused by a local inflammatory response induced by skeletal degeneration.These findings suggest that skeletal FAPI uptake should be carefully interpreted.Comparison with past Fig.6 Comparisons between Detection of Extra Abnormal Al 18 F-NOTA-FAPI-04 and 18 F-FDG Uptake.a and b both showed Al 18 F-NOTA-FAPI-04 and 18 F-FDG uptake in the liver metastases (SUV max : 10.6 vs. 6.0), demonstrating a better TBR of FAPI than FDG.c showed Al 18 F-NOTA-FAPI-04 uptake in cervical bone hyperplasia, while d showed no 18 F-FDG uptake (SUVmax: 3.2 vs. 1.7), suggesting that skeletal lesions should be carefully analyzed imaging examination or regular follow-up is necessary and diagnosis valuable.
The equivocal/benign findings which could be present in both scans.Al 18 F-NOTA-FAPI-04 PET/CT may improve the diagnostic rate of PDAC, but it also shows more uptake of benign bone lesions.In addition, FAPI PET can accurately detect more metastatic lymph nodes.In this cohort, FAPI PET did reduce the number of equivocal/benign findings.However, considering the intense uptake of Al 18 F-NOTA-FAPI-04 in liver fibrosis, benign skeletal lesions, etc., we agreed that it is not yet certain that Al 18 F-NOTA-FAPI-04 can replace 18 F-FDG.Larger sample size, multi-center studies are the focus of future work.

Limitations
The present study had several limitations.First, this is a single-center study with a small sample size, making it difficult to draw any conclusions about the diagnostic value of the Al 18 F-NOTA-FAPI-04 PET/CT.Second, not all histopathology results were available for lymph node metastasis.There were times when a biopsy by needle or surgery could not be done due to their aggressive nature.Further studies with larger patient cohorts are needed to confirm these results.

Conclusion
In this prospective study, 15/30-min Al 18 F-NOTA-FAPI-04 PET/CT demonstrated an equivalent detection ability of pancreatic lesions to 18 F-FDG PET/CT, which may improve the efficiency of clinical application.Multi-time point Al 18 F-NOTA-FAPI-04 PET/CT can help differentiate PDAC, tumor-associated inflammation, and inflammatory pancreatic lesions.In terms of TNM staging, Al 18 F-NOTA-FAPI-04 PET/CT performed better than 18 F-FDG PET/CT.
List of all quantitative measurements used 1.Al 18 F-NOTA-FAPI-04 tracer uptake was quantified as: SUV max (15min/30min/60min) SUV mean (15min/30min/60min) tumor to background ratio (TBR): from static images 15 minutes after tracer injection.• thorough peer review by experienced researchers in your field • rapid publication on acceptance • support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year

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Fig. 2
Fig. 2 Biodistribution analysis (SUV max and SUV mean ) of 31 patients with PDAC based on PET/CT imaging 60min after an injection of Al 18 F-NOTA-FAPI-04 tracer molecules (PDAC data included 15 min, 30 min, and 60min after injection)

Fig. 3 a
Fig. 3 a.Female, 62 years old, comparison of Al 18 F-NOTA-FAPI-04 and 18 F-FDG uptake values in PDAC.Compared with FDG, FAPI PET imaging showed the lesions in the head of the pancreas clearly with higher TBR.The image quality of 15-minute, 30-minute, and 1-hour imaging can satisfy the diagnostic requirements (SUV max : 12.9, 12.7, 10.1), where no uptake of FDG showed(SUV max : 1.8).b. c.The SUV max , SUV mean , TBR 1 , TBR 2 , and TBR 3 values of Al 18 F-NOTA-FAPI-04 PET / CT imaging were significantly higher than those of FDG

TBR 1 :
Tumour to meditational blood pool background TBR 2 : Tumour to liver background TBR 3 : Tumour to muscle background △SUV max-1 : the SUV max-30min values minus the SUV max-15min values △SUV max-2 : the SUV max-60min values minus the SUV max-30min values △SUV mean-1 : the SUV mean-30min values minus SUV mean-15min values △SUV mean-2 : the SUV mean-60min values minus SUV mean-30min values 2. 18 F-FDG tracer uptake was quantified as SUV max and SUV mean • fast, convenient online submission

Table 2
Comparison of Al 18 F-NOTA-FAPI-04 and 18 F-FDG PET/CT based TNM staging of 16/31 patients with primary and recurrent/ progressive PDAC