Introduction
Glioma as the most common primary malignant brain tumor in adults, is regarded as one of the leading causes of cancer death worldwide [
1,
2]. Despite notable advances in therapy, patients with glioma, particularly those with high-grade glioma, persistently experience an unfavorable prognosis. According to a multicentric data, the median overall survival of glioblastoma (GBM) patients is approximately 15 months, with a 5-year survival rate of < 10% [
1,
3].
The resected tumor or biopsy tissue allows direct access to genetic information or immunohistochemical biomarkers in glioma. Multiple molecular biomarkers have been identified from the tumor, including isocitrate dehydrogenase 1 and 2 (IDH1/2), codeletion of chromosome arms 1p and 19q (1p/19q codeletion), and O-6-methylguanine-DNA methyltransferase (MGMT), which play important roles in patient stratification, delineation of risk groups, and prognostication of treatment response, among other aspects [
4]. Tissue specimens acquired via a highly invasive procedure present substantial clinical risk. Furthermore, the implementation of repeated tumor tissue sampling in clinical practice is deemed entirely impractical. The assessment following treatment is currently predicated exclusively on the consecutive analysis of magnetic resonance imaging (MRI), which is assessed using the modified RANO criteria [
5,
6]. Therefore, in contrast to other types of tumors, the incorporation of verified circulating biomarkers into the diagnosis and treatment of glioma has not yet been achieved. MRI serves as a conventional modality for glioma imaging and demonstrates effectiveness; however, it possesses the potential to yield misleading results due to hysteresis and pseudoprogression. The monitoring of early-stage glioma relapse through exclusive reliance on MRI-based detection is challenging. Therefore, there is an urgent need to develop a widely accessible and minimally invasive method for tracking glioma. The monitoring of glioma progression should incorporate the utilization of tumor-based circulating biomarkers as an adjunctive parameter. In certain circumstances, when the likelihood of tumor recurrence is uncertain, the inclusion of supplementary detection would be highly advantageous in facilitating clinical decision-making.
FAP is a membrane protease in cancer-associated stromal fibroblasts and contributes to tumor progression but is absent or insignificant in most normal tissues [
7‐
9]. The findings from immunohistochemical analyses conducted on extracranial tumor tissues indicate that elevated FAP expression is indicative of an unfavorable prognosis, suggesting a significant involvement of FAP in tumorigenesis [
10]. Histopathological studies revealed that FAP expression was elevated in gliomas, particularly in mesenchymal subtypes [
11]. Although FAP has been extensively investigated as a biomarker in various cancer types, there is currently a lack of studies reporting on the longitudinal monitoring of glioma progression using sequential serum FAP.
In the present study, we conducted an investigation to identify the presence of the serum marker FAP and assess its viability as a means of monitoring the progression of glioma. Based on our findings, it can be inferred that the integration of serum autoantibody relative FAP level and MRI examination has the potential to enhance the precision of tumor progression monitoring in a clinical setting.
Materials and methods
Patients
From February 2020 to May 2021, 87 glioma patients (47 males and 40 females, median age 48.2 years, range 18–74 years) were recruited for this study at the Affiliated Tumor Hospital of Xinjiang Medical University, the Third Affiliated Hospital of Zhengzhou University and the Sun Yat-Sen University Cancer Center. These patients met the following inclusion and exclusion criteria: no combined tumors other than gliomas, age > 18 years, no cancer history, and no prior anti-cancer therapy before blood collection. Following tumor removal, all patients received standard care. Glioma was classified using the current World Health Organization (WHO) Classification Criteria. All patients were diagnosed using pathological examination and histological specimens were confirmed by two independent experienced pathologists. Clinical characteristics were collected from the medical records. All research methods and experiments were conducted under the applicable regulations. The study was authorized by the medical ethics committee (SL-B2022-567-01). Individual participants or their family members provided written informed consent.
Blood sample collection
Blood samples were obtained from 87 patients diagnosed with glioma through the collection of cubital vein specimens, which were subsequently incubated in 3 ml tubes containing ethylene-diamine tetra acetic acid dipotassium salt. Among them, 33 patients diagnosed with high-grade gliomas were enrolled in the study, with the objective of obtaining a minimum of two consecutive peripheral venous blood samples during the course of adjuvant chemotherapy. All these samples were rested for 30 min at room temperature before being centrifuged at 3500 rpm at 4℃ for 10 min to separate the serum. The supernatants were collected and aliquoted into 250 µl cryo-tubes, which were then stored in liquid nitrogen until the ELISA technique was employed to ascertain the relative level of the FAP antigen.
ELISA for blood samples
The plates were coated with 100 ng of recombinant FAP extracellular protein per well and incubated overnight at 4℃. After blocking the plates for 2 h at room temperature with 300 µl blocking buffer (10 mM pH 7.4 PBS with 15% goat serum), the serum specimens were added to the wells at 37℃ for 1 h and washed 5 times. Another 1 h at 37 °C was spent incubating 100 µl of diluted HRP-labeled goat anti-human IgG antibody (Promega, USA). The plates were washed 3 times with the washing buffer before being treated with 100 µl TBM per well for 15 min at 37℃ and 50 µl 2 M H2SO4 to block the response. The optical density of each well within 5 min was determined using a microplate reader set to 450 nm.
Immunohistochemistry (IHC) on human glioma tissue
IHC was conducted according to the manufacturer’s instructions to detect FAP expression in glioma tissue. Briefly, following the segmentation of formalin-fixed paraffin-embedded (FFPE) tissue specimens with a thickness of 0.4 μm, the slides underwent a deparaffinization and rehydration process utilizing progressive ethanol gradients. High-temperature antigen extraction was conducted in 10 mM/ml citrate buffer (pH 6.0) for 30 min. Endogenous peroxidase was quenched twice for 15 min with 3% hydrogen peroxide. Primary FAP-specific antibody (Promega, USA) was incubated for FAP staining in glioma slides. Finally, two neuropathologists conducted counterstaining, observation, and photography of the slides.
MR image examination
All patients in the study underwent preoperative and postoperative MRI scans on a regular basis. Following the completion of each postoperative FAP test, a corresponding multimodal MRI examination was accessible. The same multiplex scanner model, the protocol for acquisition, and reconstruction software were used for these patients. All MRI examinations were conducted using contrast agents and subsequently analyzed in a retrospective manner. The radiologists were aware of the pathological diagnosis of gliomas for the tumors, yet they did not know knowledge regarding the treatment details of the patients. Two imaging specialists, each possessing over a decade of experience, conducted an autonomous analysis. The resolution of disputed results was achieved through expert discussion in accordance with pertinent guidelines and consensuses. Tumor volumes were automatically calculated using computer-aided planimetric software.
Data analyses
The statistical analyses were conducted utilizing R (version 4.3.1). The Shapiro-Wilk test was employed to assess normality, while Bartlett’s statistic was utilized to evaluate homogeneity of variances. If the data met the criteria of normal distribution and homogeneity of variance, the statistical tests employed were Student’s t-test for two groups or ANOVA for more than two groups. In instances where the data did not exhibit conformity to a normal distribution and homogeneity of variance, the Mann-Whitney U test was employed for two groups, while the Kruskal-Wallis test was utilized for more than two groups, in order to conduct data analysis (*p < 0.05, **p < 0.01, ***p < 0.001).
Discussion
Precise diagnosis, to an extreme, can improve the efficacy of existing treatment modalities for patients. The accurate identification of progressive glioma continues to present a significant challenge in the present time. Monitoring serum tumor markers is a highly advantageous approach for patients diagnosed with glioma due to its minimal invasiveness and safety. According to our findings, it has been observed that the utilization of serum-derived FAP obtained from patients diagnosed with glioma can serve as a viable method for disease detection. The monitoring of tumor response to treatment is of utmost importance, however, it presents significant challenges, particularly in glioma, which is recognized as the most aggressive variant of primary brain tumor. Neuroimaging predominantly serves as the primary means for assessing the diagnosis and therapeutic response of gliomas. Nevertheless, it is often observed that tumors subjected to surgical intervention and chemoradiotherapy tend to exhibit an increase in size during subsequent MRI examinations, thereby suggesting the occurrence of either progression or radiation necrosis (also known as pseudoprogression). The utilization of MR imaging or CT examinations is a commonly employed method, although it can occasionally lead to misleading outcomes and less than optimal accuracy.
The identification of serum tumor markers has recently gained prominence as a potential diagnostic tool for glioma [
12]. The accuracy of image examination in assessing gliomas post-therapy is generally limited, whereas serum tumor markers have the potential to effectively compensate for this limitation, thereby alleviating the need for reoperation and reducing patient suffering [
13,
14]. Tumor biomarkers present in circulating bodily fluids have the potential to serve as valuable tracers, facilitating the diagnosis process and enhancing the assessment of therapeutic outcomes in patients. Recently, there has been identification of circulating proteins released by tumor cells or cancer-associated cells as potential biomarkers for glioma. However, the current markers exhibit inadequate sensitivity and patient coverage, limiting their broad clinical applicability. [
15] So far, little progress has been made in developing effective blood-based methods for tracking glioma. Though molecular and histological pathology based on tissue samples could provide accurate diagnosis and distinguish tumor markers for prognostic prediction, fluid-based tumor markers provide a minimally invasive approach for monitoring glioma without sampling tumors, despite its heterogeneity and evolution [
16,
17]. In the current study, the potential diagnostic value of serum FAP detection as a marker was investigated in conjunction with tumor images. FAP is produced by human cancer-associated fibroblasts (CAFs) in tumors such as glioma. It, a transmembrane serine protease, is highly expressed in many tumors but completely absent in normal tissues [
18,
19]. FAP has been identified as an independent biomarker associated with a poor prognosis in a growing number of cancers [
20‐
23]. The presence of proangiogenic FAP in CAFs has been reported which is consistent with our findings [
9,
12,
24].
CAFs cause the accumulation of FAP within tumors, consequently leading to an elevation in the level of FAP in the bloodstream. A group of researchers have identified a notable elevation in FAP levels among patients diagnosed with glioma [
25]. According to the literature, the FAP expression in grade 2 gliomas is generally lower than that of patients with grade 3 and grade 4 gliomas, indicating that high-grade gliomas are associated with a high level of FAP expression [
26]. To the best of our best knowledge, the utilization of the dynamic serum FAP test as a diagnostic tool for glioma detection has not been documented in existing literature. Due to the limited amount of research conducted on blood FAP for glioma trace, it is imperative to explore the potential of dynamic monitoring of tumor markers for clinical utilization. In comparison to an MRI examination, blood FAP test is less invasive, more accessible, inexpensive, and more convenient. In this regard, it would be extremely interesting for future studies to continuously track gliomas using serum FAP.
In order to explore the correlation between serum FAP expression and tumor characteristics, we conducted an analysis of serum FAP levels and their association with imaging observations. Our study demonstrates serum FAP levels in preoperative gliomas are significantly higher than those in postoperative patients, suggesting a positive correlation between serum FAP levels and tumor burden. The findings suggest that patients with tumor progression exhibit significantly elevated FAP levels compared to those without recurrent glioma, thereby highlighting the potential of blood tumor markers for glioma as a sensitive tool for early diagnosis. FAP expression in gliomas promotes tumor progression [
24], though serum FAP levels vary. FAP-positive cells in immunohistochemical tests are spindle-shaped, fibroblast-like cells, which is consistent with our findings in gliomas [
27]. Multiple studies conducted on stromal cells, specifically CAFs, within gliomas have revealed the presence of FAP expression in neoplastic glial cells [
28]. In the majority of human solid cancers, the expression of FAP is observed in a selective manner among cancer-associated fibroblasts (CAFs) and pericytes, while tumor cells do not exhibit this expression [
29]. The direct observation revealed the presence of prominent FAP staining in fibroblasts surrounding the tumor cells, while minimal or absent expression was observed in adjacent normal tissue. Due to its highly selective distribution in tumors, FAP served as a biomarker of reactive CAFs [
29,
30]. Based on our research findings, there exists a positive correlation between the level of serum FAP and both the grade and molecular state of glioma. Multiple studies conducted on different types of cancers have revealed a strong correlation between elevated levels of FAP and the presence of cancer [
31,
32]. The observed phenomenon was construed as a systemic reaction to the progression of the tumor [
17,
33]. The occurrence of the homologous phenomenon was not documented in glioma.
Following surgical intervention and/or in conjunction with subsequent chemoradiotherapy, the evaluation of the disease predominantly relies on MRI, posing challenges in accurately discerning tumor progression from radiation necrosis within specific timeframes. While tissue biopsies are essential for precise diagnosis and molecular profiling, their limitations lie in their ability to solely capture a fixed moment in time, unable to consistently depict changes in the mutational spectrum, microenvironment, and heterogeneity evolution. The correlation between tumor volume and blood FAP levels suggests potential utility in guiding treatment strategy selection. The promotion of posttreatment glioma invasive growth by FAP suggests the existence of actively proliferating tumor cells [
10]. Even if no obvious mass is visible on the MR image, blood FAP of glioma patient may serve as a tumor tracer.
Until now, the utilization of craniocerebral MRI scans has been suggested as a conventional diagnostic approach for post-treatment evaluation of gliomas. The utilization of MRI examination aids in the confirmation of the underlying cause responsible for the upregulation of FAP expression in gliomas. Investigating the origin of FAP detected in blood samples will contribute to a more comprehensive comprehension of its role as a protein biomarker. The dynamic serum FAP effectively addresses the limitations of MRI in differentiating between radiation necrosis and tumor progression. The integration of serial serum FAP test results with neuroimaging enhances the precision of early glioma recurrence detection, underscoring the potential of combining tumor markers with imaging as a viable approach in the clinical diagnosis of glioma. The early detection of glioma recurrence still remains challenging. In the present study, it was observed that serum FAP exhibited a progressive elevation in conjunction with the augmentation of tumor volume. This finding suggests that various cellular components implicated in glioma progression, including parenchymal cells, mesenchymal cells, and endothelial cells, might contribute to the synthesis of this protein.
In the present study, we conducted an analysis to determine the levels of FAP in the serum of patients diagnosed with glioma, and subsequently compared these levels with the assessments of tumor burden obtained through MRI imaging. The results of our study indicate a significant elevation in serum FAP levels as tumor progression occurs, suggesting that serum FAP has potential as a valuable tool for disease monitoring and as a marker for tumor progression. Additionally, our research reveals substantial variations in serum FAP levels among gliomas, with a notable elevation observed in a considerable proportion of high-grade gliomas compared to low-grade gliomas. Concurrently, a notable reduction in serum FAP level was observed in patients who did not experience tumor recurrence subsequent to successful treatment. The serum levels of FAP exhibited a significant increase in the presence of recurrent tumor, whereas the serum levels of FAP displayed fluctuations in accordance with the condition of the tumor. The utilization of longitudinal variations in serum FAP level in our analyses has led to the confirmation that serum FAP is a reliable indicator for evaluating the status of the disease. Another intriguing finding is that serum FAP level has a suggestive role in the molecular pathological subtypes of glioma. However, the association between serum FAP levels and MGMT promoter methylation status appears to be less definitive compared to other molecular statuses such as IDH and 1p/19q. We did not search for a direct link between MGMT status and blood FAP level in the literature. Our study found that serum FAP levels fluctuated in some cases while MRI assessments were stable. This phenomenon may be attributed to the constrained sensitivity of MRI in discerning minute masses within tumor dimensions, thereby requiring additional validation within a more extensive sample group.
Collectively, we conducted an investigation into the potential of serum-derived FAP obtained from patients with glioma to function as a biomarker for the disease. The results of our study unequivocally demonstrate that the dynamic detection of serum FAP serves as a straightforward approach to ascertain treatment response and evaluate tumor status. These findings suggest that serum FAP may be a potentially reliable biomarker for disease monitoring in the context of glioma, which is critical for the timely and accurate assessment of therapeutic effects. The incorporation of serum-derived FAP obtained from glioma patients, in conjunction with MRI evaluation, substantially enhances the precision of disease diagnosis.
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