Heterogeneity of fibroblast activation protein expression in the microenvironment of an intracranial tumor cohort: head-to-head comparison of gallium-68 FAP inhibitor-04 (⁶⁸Ga-FAPi-04) and fluoride-18 fluoroethyl-L-tyrosine (¹⁸F-FET) in positron emission tomography-computed tomography imaging

Introduction

Intracranial tumors exhibit substantial heterogeneity, which could be attributed to both tumor parenchymal cells and benign cells in the tumor microenvironment (TME). Gliomas, the most common central nervous system tumors, exemplify this, demonstrating pronounced intratumoral and intertumoral heterogeneity (1-3). Interactions between glioma cells and adjacent TME cells in the tumor stroma occur through various mechanisms, promoting tumor pathophysiological behaviors such as proliferation, migration, and angiogenesis (4,5).

Positron emission tomography (PET) imaging with radio-labeled amino acid tracers such as fluoride-18 fluoroethyl-L-tyrosine (¹⁸F-FET) can contribute to brain tumor grading, differential diagnosis, prognostication, treatment planning, and monitoring (6-8). The Response Assessment in Neuro-Oncology (RANO) working group endorses this imaging modality as a valuable complement to magnetic resonance imaging (MRI) in all stages of glioma management (9). Although these radio-labeled amino acid tracers have appreciable efficacy, they primarily target L-type amino acid transporters (LATs), which are transmembrane proteins expressed in tumor cells (10,11).

Cancer-associated fibroblasts (CAFs), capable of secreting growth factors and inflammatory cytokines, are critically involved in the interactions between tumor cells and stromal cells (12,13). Fibroblast activation protein (FAP), a transmembrane glycoprotein, may be overexpressed by CAFs in the TME (14,15). Radio-labeled fibroblast activation protein inhibitors (FAPis) have demonstrated efficacy in imaging CAFs activities across a range of solid tumors with satisfactory results.

As there is significant FAP accumulation in the stroma of malignant tumors and satisfactory tissue contrast, FAP-targeted imaging has efficacy in malignant tumor detection, tumor delineation, metastatic lymph node recognition, tumor staging and restaging, and radiotherapy planning (16-18). Research suggests that FAP-targeted imaging can influence the treatment decisions after the detection of extra-lymph node metastasis in breast carcinomas (19). FAPi imaging also aids in the differentiation of malignant transformation of pancreatic intraductal papillary mucinous neoplasms (20). While there is some evidence indicating that certain types of gliomas overexpress FAP (21), data on the FAP expression in other intracranial tumors have not been well established in the literature and should be pursued further. Moreover, the correlation between the volume of FAP in the TME and tumor malignancy degree in various intracranial tumors is of considerable interest.

This prospective, head-to-head study applied gallium-68 FAPi-04 (⁶⁸Ga-FAPi-04) and ¹⁸F-FET PET-computed tomography (CT) imaging to patients with intracranial tumors before surgery to investigate the spectrum of FAP expression. Immunohistochemical staining was used to examine the patterns of FAP expression. A quantification analysis of PET parameters was employed to characterize the relationship between FAP expression volume in tumor stroma and the degree of malignancy.

Methods

Ethical approval

This study was conducted according to the Declaration of Helsinki (as revised in 2013). Ethical approval of our previously written study protocol and consequent analytical design was obtained from Ethics Committee of Huashan Hospital, Fudan University (No. 2021-891), and informed consent was obtained from all individual participants. The study protocol was not registered on a public platform and did not involve any interventional procedures.

Study design

Adult preoperative patients with intracranial mass lesions were enrolled. Anatomic MRI after symptom occurrence was collected and reviewed for initial diagnosis by outpatient neurosurgeons in Huashan Hospital before admission for further investigation and treatment. Those patients who had already received treatment including surgery, radiosurgery, radiotherapy, or chemotherapy were excluded from the study. Enrolled adult patients with intracranial tumors received ⁶⁸Ga-FAPi-04 and ¹⁸F-FET PET/CT brain imaging before surgery from October 2022 to March 2023 in the Neurosurgery Department of Huashan Hospital, Fudan University. Semiquantitative imaging parameters and immunohistochemical staining were obtained for diagnostic evaluation of this head-to-head study. Written informed consent from patients for dual-tracer PET/CT imaging and follow-up analysis was obtained.

Imaging protocols

⁶⁸Ga-FAPi-04 and ¹⁸F-FET tracers were synthesized in the Department of Nuclear Medicine & PET Center of Huashan Hospital, Fudan University.

For ¹⁸F-FET PET/CT imaging, patients fasted for a minimum of 4 hours prior to imaging. A 20-minute static scan was conducted in 3-dimensional (3D) mode with a Biograph mCT Flow Edge 128 PET/CT system (Siemens Healthineers, Erlangen, Germany) 20 minutes after intravenous bolus injection of ¹⁸F-FET (185±17.0 MBq). Attenuation correction was performed using low-dose CT (tube current =150 mAs, voltage =120 kV, acquisition =64×0.6 mm, convolution kernel = H30s, slice thickness =5 mm, interslice gap =1.5 mm) prior to the emission scan. Postacquisition, PET images were reconstructed using the ordered subset expectation maximization (OSEM) algorithm with a Gaussian filter and a full width at half maximum of 3.5 mm at the center of the field of view.

In ⁶⁸Ga-FAPi-04 PET/CT imaging, 30 minutes after intravenous bolus injection of ⁶⁸Ga-FAPi-04 (185±29.2 MBq), a 30-minute static scan was conducted in 3D mode with a uMI510 PET/CT (United Imaging, Shanghai, China). Attenuation correction was similarly performed using low-dose CT prior to the emission scan. PET images were also reconstructed using the OSEM algorithm with a Gaussian filter and the same full width at half maximum after acquisition.

Image analysis

PET/CT images were analyzed with a syngo.via workstation (Siemens Healthineers). Two experienced nuclear medicine physicians (W.Z. and T.H., with over 6 and 13 years of experience, respectively) performed blinded ⁶⁸Ga-FAPi-04 and ¹⁸F-FET PET/CT positive lesion judgement and lesion delineation before surgical treatment.

Structural MRI was initially read before PET/CT lesion delineation. For ¹⁸F-FET PET/CT imaging, mean standardized uptake value (SUVmean) of the brain background was measured in a crescent-shaped area, encompassing both gray and white matter on the lesion’s contralateral hemisphere. Subsequently 1.6 times of background SUVmean was used for lesion delineation, and the maximal standardized uptake value (SUVmax), metabolic tumor volume (MTV), and total lesion tracer uptake (TLU) were obtained. Maximal tumor-to-brain ratio (TBRmax) was calculated by dividing the intracranial lesion SUVmax with the background SUVmean.

For ⁶⁸Ga-FAPi-04 imaging, the background SUVmean was measured similarly to that of ¹⁸F-FET PET. Lesion SUVmax and TBRmax were measured. Due to the lack of guidelines for a FAPi-positive lesion delineation and extremely low uptake of the brain background, a series of isocontour volumetric thresholds including 20%, 30%, 40%, and 50% of lesion SUVmax were measured for MTV_FAPi and TLU_FAPi measurements. Different MTV_FAPi:MTV_FET and TLU_FAPi:TLU_FET ratios were calculated for further analysis.

Histological analysis

Pathological diagnosis was completed in the Pathology Department of Huashan Hospital, Fudan University. Characteristic surgical resection slices were selected for FAP immunohistochemistry staining as per the FAP kit instruction (Abcam, Cambridge, UK). Briefly, heat-mediated antigen retrieval with buffer was applied before FAP immunohistochemical staining. Tissue sections were incubated at 4 ℃ overnight with a 1:250 anti-fibroblast activation antibody (RRID: AB_207178; Abcam, Cambridge, UK). Streptavidin-biotin complex was used for incubation before staining and visualization.

FAP immunohistochemical staining scores were assigned by two independent pathologists who were blinded to patients’ clinical and PET/CT imaging analysis results. The scoring method was applied as previously described (22), with 0 indicating complete absence or very minimal FAP staining in less than 1% of the evaluation area, 1 indicating weak FAP immunohistochemical staining from 1% to 10% of the evaluation area, 2 indicating moderate FAP immunohistochemical staining from 11% to 50% of the evaluation area, and 3 indicating strong FAP immunohistochemical staining over 50% of the evaluation area.

Statistical analysis

Descriptive statistics are expressed as the mean and standard deviation or median and range. The t-test and one way analysis of variance was used to compare continuous variables. The Wilcoxon signed rank or Kruskal-Wallis test was performed if a normal distribution of variables was not met. Linear regression analysis was applied to investigate the relationship of pathological diagnosis and PET parameters. The variance inflation factor was used to control multicollinearity. Intraclass correlation coefficients (ICCs) for PET parameter measurements and FAP immunohistochemical scoring were assessed, and the results were classified as poor (less than 0.2), fair (0.21–0.4), moderate (0.41–0.6), good (0.61–0.8), and very good (0.8–1.0). All statistical analyses were performed with Stata version 17 (College Station, TX, USA). In all analyses, P<0.05 was considered to indicate a statistically significant difference.

Results

Patients characteristics

This study enrolled 22 patients, including 14 men and 8 women, with a mean age of 50±13 years (range, 27–69 years). Consecutive ⁶⁸Ga-FAPi-04 and ¹⁸F-FET PET/CT imaging scans were applied with an at least a 24-hour interval for each patient in our cohort. Surgical treatment was performed after dual-tracer PET/CT within 8 days and included 20 surgical resections and 2 stereotactic biopsies. Primary brain tumor, brain metastasis, and noncancerous disease were observed. Eight specific histological diagnoses were identified according to the 2021 World Health Organization (WHO) Classification of Tumors of the Central Nervous System, which included WHO grade 4 glioblastoma isocitrate dehydrogenase wild type (IDH-wt), metastatic carcinoma, oligodendroglioma, WHO grade 2 IDH mutation (IDH-mu) and 1p/19q codeleted, noncancerous lesions, WHO grade 4 astrocytoma IDH-mu, WHO grade 3 astrocytoma IDH-mu, WHO grade 4 medulloblastoma not otherwise specified (NOS), and WHO grade 1 ganglioglioma. Demographic details of the cohort are provided in Table 1.

Comparison of two tracers in PET imaging of lesions

A total of 32 intracranial lesions were observed in the 22 ¹⁸F-FET PET images, including in 5 patients with multiple lesions; meanwhile, 18 lesions were observed in 16 ⁶⁸Ga-FAPi-04 PET images. In 1 case of glioblastoma (No. 6), ¹⁸F-FET PET showed 4 lesions while ⁶⁸Ga-FAPi-04 PET showed 2. In 1 case of WHO grade 4 astrocytoma IDH-mu (No. 5), ¹⁸F-FET PET showed 4 lesions while ⁶⁸Ga-FAPi-04 PET showed 1. In one case of brain metastasis (No. 2), ¹⁸F-FET PET showed 2 lesions while ⁶⁸Ga-FAPi-04 PET showed 1. In another case of brain metastasis (No. 16), both PET tracers showed 2 lesions. In 1 case of noncancerous disease (No. 10), ¹⁸F-FET PET showed 3 lesions but ⁶⁸Ga-FAPi-04 PET showed none. The difference in detection between the tracers in PET was significant (P=0.046).

Analysis of patient-based PET semiquantitative parameters

ICCs showed very good agreement between the different tracers for ¹⁸F-FET and ⁶⁸Ga-FAPi-04 PET/CT parameter measurements (ICC >0.95, P<0.001; ICC >0.87, P<0.001), and the results of reader one (W.Z.) were used for analysis.

For ⁶⁸Ga-FAPi-04 PET images, brain metastasis showed highest uptake. The lesion median SUVmax and TBRmax were 4.8 (range, 4.5–19.1) and 161.0 (range, 148.7–382.2), respectively. In primary brain tumors, FAPi uptake in WHO grade 4 glioblastoma IDH-wt was the most obvious, and the lesion median SUVmax and TBRmax were 4.2 (range, 0.2–11.7) and 126.8 (range, 5.3–389.0), respectively. In noncancerous lesions, the median SUVmax and TBRmax were 2.2 (range, 0.3–4.0) and 57.4 (range, 14.5–100.3), respectively. The brain background median SUVmean was 0.03 (range, 0.02–0.06). There were no significant differences in intracranial lesion types, including brain metastasis, primary brain tumor, and noncancerous disease, in terms of SUVmax-FAPi (P=0.092) or TBRmax-FAPi (P=0.189); there were similarly no significant differences among specific histological diagnoses (SUVmax-FAPi: P=0.212; TBRmax-FAPi: P=0.132).

For ¹⁸F-FET PET imaging, the highest tracer uptake was present in primary brain tumors, and the median SUVmax and TBRmax were 3.5 (range, 1.6–6.3) and 4.4 (range, 2.1–5.9), respectively. In brain metastasis, the median SUVmax and TBRmax were 2.7 (range, 2.5–3.7) and 3.6 (range, 2.8–4.2), respectively. In noncancerous patients, the median SUVmax and TBRmax were 3.0 (range, 2.2–3.8) and 4.4 (range, 2.7–6.1), respectively. There were no significant differences between the three intracranial lesion types in terms of SUVmax-FET (P=0.648) or TBRmax-FET (P=0.709); there were similarly no significant differences among specific histological diagnoses (SUVmax-FET: P=0.644; TBRmax-FET: P=0.553).

We found no evidence of differences in patient-based SUVmax between the PET images of the different tracers (FAPi: 4.15±4.66; FET 3.42±1.19; P=0.935), while the TBRmax of FAPi was significantly higher than that of FET (FAPi: 120.83±117.47; FET 4.09±1.21; P=0.0001). The comparisons are provided in Figure 1, the scatter plot of the TBRmax uptake in lesions with both tracers is shown in Figure 2, and illustrative cases are provided in Figure 3.

Figure 1 Comparison of the median SUVmax and TBRmax of ⁶⁸Ga-FAPi-04 and ¹⁸F-FET uptake in intracranial lesions. 1: ganglioglioma; 2: oligodendroglioma; 3: grade 3 astrocytoma IDH-mu; 4: grade 4 astrocytoma IDH-mu; 5: grade 4 glioblastoma IDH-wt; 6: metastatic carcinoma; 7: noncancerous disease; 8: medulloblastoma. SUVmax, maximal standardized uptake value; FAPi, fibroblast activation protein inhibitor; FET, fluoroethyl-L-tyrosine; TBRmax, maximal tumor-to-brain ratio; ⁶⁸Ga-FAPi-04, gallium-68 fibroblast activation protein inhibitor 04; ¹⁸F-FET, fluoride-18 fluoroethyl-L-tyrosine; IDH, isocitrate dehydrogenase; wt, wild type; mu, mutant.

Figure 2 Scatter plot and regression plot of lesion tracer uptake and immunohistochemical score. (A) Scatter plot and regression plot of participants’ dual-tracer TBRmax with ⁶⁸Ga-FAPi-04 and ¹⁸F-FET PET images. (B) Scatter plot and regression plot of participants’ ⁶⁸Ga-FAPi-04 uptake SUVmax and FAP immunohistochemical staining score. TBRmax, maximal tumor-to-brain ratio; FAPi, fibroblast activation protein inhibitor; FET, fluoroethyl-L-tyrosine; CI, confidence interval; FAP, fibroblast activation protein; SUVmax, maximal standardized uptake value; ⁶⁸Ga-FAPi-04, gallium-68 fibroblast activation protein inhibitor 04; ¹⁸F-FET, fluoride-18 fluoroethyl-L-tyrosine; PET, positron emission tomography.

Figure 3 Typical MRI, ¹⁸F-FET, and ⁶⁸Ga-FAPi-04 PET imaging of representative cases. (A-C) A 56-year-old male (No. 1) with WHO grade 4 left temporo-parietal glioblastoma IDH-wt. (D-F) A 58-year-old male (No. 15) with right fronto-parietal metastatic carcinoma. (G-I) A 35-year-old male (No. 21) with right cerebellar medulloblastoma. (J-L) A 46-year-old male (No. 13) with right frontal multifocal encephalomalacia. MRI, magnetic resonance image; ¹⁸F-FET, fluoride-18 fluoroethyl-L-tyrosine; ⁶⁸Ga-FAPi-04, gallium-68 fibroblast activation protein inhibitor 04; PET, positron emission tomography; WHO, World Health Organization; IDH, isocitrate dehydrogenase; wt, wild type.

Out of the 16 ⁶⁸Ga-FAPi-04-positive patients, 15 surgically treated intracranial tumors were used to determine the efficacy of the PET parameters, with 1 noncancerous patient being excluded due to not meeting our research purpose. At a 20% FAPi lesion SUVmax threshold, the P value of the MTV_FAPi:MTV_FET ratio was 0.027 [95% confidence interval (CI): 0.572–7.712], the P value of the TLU_FAPi:TLU_FET ratio was 0.032 (95% CI: −5.057 to −0.288), and the P value of TBRmax-FAPi was 0.044 (95% CI: 0.000–0.016). At a 30% FAPi lesion SUVmax threshold, the P value of TBRmax-FET was 0.047 (95% CI: −1.482 to −0.012), while the P values of the MTV_FAPi:MTV_FET ratio, TLU_FAPi:TLU_FET ratio, and TBRmax-FAPi were 0.052 (95% CI: −0.045 to 11.013), 0.067 (95% CI: −6.447 to 0.257), and 0.081 (95% CI: −0.001 to 0.015), respectively. A 40% FAPi lesion SUVmax threshold, the P value of the MTV_FAPi:MTV_FET ratio was 0.049 (95% CI: 0.044–17.903), while the P values of TBRmax-FAPi and TLU_FAPi:TLU_FET were 0.082 (95% CI: −0.001 to 0.015) and 0.098 (95% CI: −8.174 to 0.818), respectively. At a 50% FAPi lesion SUVmax threshold, the P value of the MTV_FAPi:MTV_FET ratio was 0.065 (95% CI: −1.131 to 30.596). The detailed regression results details can be seen in Table 2 and Figure 4 and the scatter plot matrix of PET parameters and pathological diagnosis is provided in Figure 5.

Figure 4 Linear regression results of the semiquantitative PET image parameters from 15 ⁶⁸Ga-FAPi-04-positive participants with intracranial tumor at 20% (A), 30% (B), 40% (C), and 50% (D) lesion SUVmax thresholds. TBRmax, maximal tumor-to-brain ratio; FAPi, fibroblast activation protein inhibitor; FET, fluoroethyl-L-tyrosine; MTV ratio, MTV_FAPi:MTV_FET ratio; MTV, metabolic tumor volume; TLU ratio, TLU_FAPi:TLU_FET ratio; TLU, total lesion tracer uptake; PET, positron emission tomography; ⁶⁸Ga-FAPi-04, gallium-68 fibroblast activation protein inhibitor 04; SUVmax, maximal standardized uptake value.

Figure 5 Scatter plot matrix from the MTV_FAPi:MTV_FET ratio, TLU_FAPi:TLU_FET ratio, TBRmax-FAPi, TBRmax-FET, and pathological diagnosis with 20% (A), 30% (B), 40% (C), and 50% (D) lesion SUVmax thresholds. MTV ratio, MTV_FAPi:MTV_FET ratio; MTV, metabolic tumor volume; TLU ratio, TLU_FAPi:TLU_FET ratio; TLU, total lesion tracer uptake; TBRmax, maximal tumor-to-brain ratio; FAPi, fibroblast activation protein inhibitor; FET, fluoroethyl-L-tyrosine; SUVmax, maximal standardized uptake value.

FAP immunohistochemistry and ⁶⁸Ga-FAPi-04 PET imaging analysis

The FAP immunohistochemical scores from the two pathologists were in satisfactory agreement (ICC >0.92; P<0.001). In primary brain tumors, moderate FAP expression in the tumor stroma and peri-vascular areas could be observed in most of the patients with WHO grade 4 glioblastoma IDH-wt. Mild FAP expression was observed in the tumor stroma and perivascular areas in patients with WHO grade 4 and grade 3 astrocytoma IDH-mu. Scant-to-mild FAP expression was found in the tumor stromal areas of patients with low-grade gliomas including oligodendroglioma and ganglioglioma. Mild FAP expression was found in the tumor stroma of the patient with medulloblastoma but not in the perivascular region. In brain metastasis, significant tumor stroma, perivascular, and small vessel epithelial expression were found. In patient 13, who had a noncancerous lesion, moderate FAP expression was present in the stromal cells in inflamed regions, the perivascular region, the small-vessel epithelium, and the gliotic region; meanwhile, mild FAP expression was observed in the small vessel epithelium and gliotic region in patient 10 (Figure 6).

Figure 6 Typical case illustrations of FAP immunohistochemical staining imaging (original magnification, 200×). (A) WHO grade 4 glioblastoma IDH-wt (No. 1) with moderate FAP expression in tumor stroma and perivascular region. (B) Metastatic carcinoma (No. 15) with significant FAP expression in tumor stroma, perivascular region, and small-vessel epithelial tissue. (C) Medulloblastoma (No. 21) with mild FAP expression in the tumor stroma. (D) Multifocal encephalomalacia (No. 13) with moderate FAP expression in stromal cells in inflamed regions, perivascular region, small-vessel epithelial tissue, and gliotic region. FAP, fibroblast activation protein; WHO, World Health Organization; IDH, isocitrate dehydrogenase; wt, wild type.

The lesion median SUVmax was 0.4 (range, 0.2–1.3), 4.0 (range, 3.2–5.8), and 10.4 (range, 8.9–19.1) in patients with an FAP immunohistochemical score from 1 to 3, respectively. The lesion FAPi SUVmax was significantly correlated with FAP immunohistochemical score (r²=0.70; P<0.001) (Figure 2).

Discussion

Despite there being distinct characteristics of FAP expression across a wide range of carcinomas, limited evidence exists regarding its expression patterns in intracranial tumors. Our study found there to be heterogeneous uptake of ⁶⁸Ga-FAPi-04 across our cohort, with generally significant uptake noted in patients with brain metastasis, glioblastoma, and medulloblastoma. The immunohistochemical results showed varied FAP expression patterns in primary tumors, brain metastasis, and noncancerous disease. In FAPi-positive intracranial tumors, the MTV_FAPi:MTV_FET ratio, a semiquantitative parameter which can be used to reflect the quantification results of CAFs activity in tumor parenchyma, has shown promising differential potential in intracranial tumors.

Limited research in the FAP of gliomas suggests there to be increased FAPi uptake in high-grade gliomas (21). Other analysis indicates that FAPi uptake in high-grade gliomas partly correlates with the tumor perfusion but not with the cell density of these gliomas (23). Our study expanded the scope to an intracranial tumor cohort, and FAP expression status was generally associated with tumor malignancy degree, in accordance with previous investigations. Immunohistochemical analysis indicated that the differences of FAP expression were mainly present in the areas of the TME, including perivascular region. These patchy- and spot-like FAP expressions in intracranial tumor stroma correlated with ⁶⁸Ga-FAPi-04 uptake. Based on this, we hypothesized that FAP expression in perivascular stromal region is associated with tumor lesion perfusion, while the cellular density status of the intracranial tumor is not correlated with its FAP expression status, which results in the absence of relationship between FAPi uptake and the apparent diffusion coefficient of the tumor.

Radio-labeled amino acid tracers have satisfactory diagnostic efficacy in brain tumors (9,24). ¹⁸F-FET PET imaging can well delineate a wide range of intracranial tumors in our cohort, even some small lesions. However, TME cell-targeting tracers can offer new possibilities in evaluating the heterogeneities of intracranial tumors in addition to tumor cell-targeted imaging. The TME consists of a range of benign cells that interact with tumor cells, influencing growth, migration, and recurrence (25-27). FAP expression has been observed in most epithelial neoplasms (28). Due to unique classification and characteristics of central nervous system (29), moderate FAP expression was present in most of the patients with WHO grade 4 glioblastoma IDH-wt in our cohort. These patients typically have a very poor prognosis, and the expression pattern of FAP could provide valuable data for future FAP-targeted theranostic methods, serving as a complement to classical treatment procedures.

Lesion delineation is vital for devising a treatment plan. Radio-labeled amino acid tracer imaging could be used to outline lesions (30). There is evidence supporting the potential of FAP-targeted imaging in the differentiation and delineation of intracranial malignant tumors. It is reasonable to suggest that FAPi imaging can complement current imaging protocols for intracranial malignancies. Besides FAPi imaging, comprehensive analysis of anatomic MRI and amino acid PET imaging are necessary to exclude occasional benign lesions with FAP expression. The costs of FAP-targeted imaging are similar to those of widely clinically used tracers, and the potential benefits of the noninvasive malignant degree recognition and evidence-guided treatment planning via FAP-targeted imaging should be considered.

There is currently no standard for brain lesion delineation in FAP-targeted imaging. Considering the characteristics of brain tumors and FAP expression status in normal brain background, we applied 20% lesion SUVmax as the threshold for the initial observation in ⁶⁸Ga-FAPi-04 PET analysis. Further observations were investigated with 30%, 40%, and 50% of the lesion SUVmax being used as thresholds. This strategy was employed to control the influence of delineation fluctuation owing to the extremely low uptake of the brain background. Given the significant tumor-to-brain ratio of FAPi uptake, a 20% isocontour volumetric threshold is reasonable for lesion delineation.

There are growing amount of studies on ⁶⁸Ga-FAPi uptake in noncancerous diseases including autoimmune disease, cardiovascular disease, wound healing (31-34). The results from this varied research indicates the potential of FAPi in the molecular imaging of inflammation. In our cohort, the participants with noncancerous lesions also had ⁶⁸Ga-FAPi-04 uptake, suggesting that caution should be taken in the diagnosis of positive ⁶⁸Ga-FAPi-04 lesions. In classical semiquantitative parameters, characteristic time-activity curve patterns from dynamic ¹⁸F-FET PET imaging could help to differentiate between glioma subtypes and exclude noncancerous lesions. Other imaging parameters should be considered for participants for whom diagnosis is unclear.

Our study involved certain limitations which should be addressed. First, our head-to-head study included a relatively small size, and the results of this exploratory investigation need to be further confirmed with a more robust patient cohort. Second, this initial study mainly concentrated on cohort ⁶⁸Ga-FAPi-04 and ¹⁸F-FET PET/CT imaging analysis, the combination of anatomic MRI parameters with those examined in our study will complement these results. More investigations including histopathological validation of the different FAPi imaging threshold-based lesion delineations and imaging quantification analysis combined with anatomic MRI features will doubtlessly contribute to furthering our understanding of the effects of CAFs in the intracranial TME.

Conclusions

Our study found heterogeneities in ⁶⁸Ga-FAPi-04 uptake in this intracranial tumor cohort, with tumors with greater malignancy generally having an elevated FAP expression, although the difference between the primary brain tumor, brain metastasis, and noncancerous disease were statistically nonsignificant. Immunohistochemical analysis revealed a diversity of FAP expression patterns. For intracranial tumor patients with positive ⁶⁸Ga-FAPi-04 uptake, the MTV_FAPi:MTV_FET ratio demonstrated potential in differentiating between various intracranial tumors, suggesting that the interactions between intracranial tumor parenchyma and CAF cells in the TME are distinct across various types of tumor genesis.

Acknowledgments

We appreciate the kind support from pharmacists, nurses, and technicians of the Department of Nuclear Medicine & PET Center; and pathologists Haixia Cheng and Jingjing Zhu from the Department of Pathology, Huashan Hospital, Fudan University.

Funding: This work was supported by the Science and Technology Commission of Shanghai Municipality (grant Nos. 18411952100 and 2018SHZDZX01) and Zhang Jiang Lab.

Footnote

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://qims.amegroups.com/article/view/10.21037/qims-24-82/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. This study was conducted according to the Declaration of Helsinki (as revised in 2013). Ethical approval of our previously written study protocol and consequent analytical design was obtained from Ethics Committee of Huashan Hospital, Fudan University (No. 2021-891), and informed consent was obtained from all individual participants.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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