Serum platelet-derived growth factor-BB levels as a potential biomarker in assessing the metabolic activity of lesions in alveolar echinococcosis patients

Objective Alveolar echinococcosis (AE) is a chronic disease caused by the larval stage of Echinococcus multilocularis. Assessing the metabolic activity of AE lesions is critical to evaluate disease progression and survey treatment options. There is an urgent need to identify more rapid, convenient, and noninvasive clinical detection methods to substitute the current techniques. Herein, we evaluated the viability of platelet-derived growth factor-B B (PDGF-BB) as a biomarker for detecting the metabolic activity of AE patients. Concentration of serum PDGF-BB homodimers (sPDGF-BB) was assessed via ELISA. Correlations of PDGF-BB expression levels with clinicopathological features of AE patients were analyzed using SPSS. Results The concentrations of sPDGF-BB were signicantly lower in AE patients (p<0.0001), particularly in High Metabolically Active AE patients (HMAE) patients (p<0.05). The expression levels of PDGF-BB were signicantly higher in close liver tissue (CLT) in AE patients (p<0.0001). We found that metabolically active AE and sPDGF-BB are signicantly negatively correlated (r=-0.624, p=0.0004). Besides, the local expression levels of PDGF-BB was positively correlated with metabolic activity, PNM stage, and lesion size. Notably, the sPDGF-BB levels were proposed as a potential biomarker for assessing metabolic activity of AE, with 81% sensitivity and 85.7% specicity (95% condence interval, p = 0.003).


Introduction
Alveolar echinococcosis (AE) is a gradual and invasive form of echinococcosis caused by Echinococcus multilocularis infection that gradually invades the liver. It is considered a signi cant global public health concern, highly prevalent in Northwest China, Central Asia, Western Europe, and Middle East (1). The lesions of AE damage the hepatic tissue and obstructs bile ducts and vessels (2). This causes severe or even lethal complications (2).
The status of the parasitic lesion in the patient which can be classi ed as calci ed, highly active, and or general is used to determine the degree of disease progression. Therefore, they are important parameters that affect the decision-making process in clinical treatment and can be used to determine the appropriate time to discontinue albendazole therapy (2,3). The F-labeled uoro-2-deoxyglucose 18 (F-FDG), positron emission tomography and computed tomography (PET-CT) techniques have been integrated and currently provide a superior non-invasive tool for assessing the metabolic activity of AE lesions (4). However, several limitations of PET-CT such as the high cost of the procedure and the need for professionals and reagents implies that there is an urgent need to develop more economical, convenient, and rapid methods for in vivo analysis of the parasitic activity.
Platelet-derived growth factors (PDGFs) are alkaline proteins stored in platelet alpha granules (5). The isoform PDGF-BB participates in the maturity and reconstructing of the vascular network by recruiting parietal cells (pericytes and vascular smooth muscle cells) around new blood vessels (6). Notably, platelets have a central function in hepatic dynamic balance and pathobiology (7). PDGF-BB as one of the platelet-related biomarkers, was released from many different cell types, and played pivotal roles in cell proliferation and transformation, chemotaxis, angiogenesis, wound healing, and the regulation of apoptosis (8). Furthermore, PDGF-BB has demonstrated several implications in the eld of oncology and other diseases including screening, diagnostic, and prognostic relevance of diseases. (9)(10)(11). For instance, higher serum levels of PDGF-BB in hepatocellular carcinoma (HCC) patients undergoing sorafenib treatment are indicators for extended survival bene ts (12). Patients with low levels of serum plateletderived growth factor-BB (sPDGF-BB) during the perioperative period of HCC are more likely to exhibit post-operation relapse (13).
In this study, we investigated the expression of PDGF-BB in peripheral and local tissues in AE patients and determine the prognostic value of sPDGF-BB in assessing the metabolic activity of lesions in AE patients.

Human Subjects
A total of 28 AE patients admitted at the First A liated Hospital of Xinjiang Medical University from February 2016 to October 2019 were included in the study cohort. Patients were diagnosed with AE following international recommendations (14). The exclusion criteria included: (1) Patients with cystic echinococcosis. (2) Patients with viral hepatitis, autoimmune diseases, and tumors. We grouped 28 AE patients following the WHO Informal Working Group on Echinococcosis PNM (P = parasitic mass in the liver, N = involvement of neighboring organs, and M = metastasis) classi cation and staging of alveolar echinococcosis. 28 healthy volunteers from the same demographic origin who received annual health examinations were included as the control group. Detailed baseline information for all subjects is summarized in Table S1.The handling of human subjects followed the principles of the Helsinki Declaration and was approved by the Clinical Research Ethics Committee of the First A liated Hospital of Xinjiang Medical University. We obtained informed consent from all participants or their legal custody before commencing the study.

Grouping patients based on lesion 'metabolic activity'
We performed the [18F] Fluorodeoxyglucose positron emission tomography/computed tomography ([18F] FDG PET-CT) scan of the patients immediately after admission or before the operation. The PET/CT acquisitions were conducted 3 hours after 18F-FDG injection (15). The images were analyzed by two experienced nuclear physicians blinded to the clinical, biological, and pathological characteristics of the patients. Subsequently, following the maximum standardized uptake value (SUV MAX ) of [18F]FDG PET-CT, patients were grouped into High Metabolically Active AE (HMAE SUV MAX >4.0) and Low Metabolically Active AE (LMAE SUV MAX ≤4.0) categories (15). The demographic characteristics of subgroups (HMAE vs. LMAE) did not exhibit any signi cant difference (Table S1).

Sample acquisition
Blood samples were collected from the subjects on the admission day. Serum was extracted from the samples through centrifugation at 2,500 × g for 10 minutes and stored at -80°C awaiting further analysis. Besides, a similar procedure was used to process samples from the control subjects.
Sections of the liver tissue were obtained from the 28 AE patients previously underwent surgical resection. One specimen was obtained from a region close to the parasitic lesion including the metacestode (close liver tissue [CLT], about 0.5 cm from lesion). Another specimen was obtained from the macroscopically normal liver distant from the lesion (distant liver tissue [DLT], at least 2 cm distant from lesion) as previously described (16).

Quantifying sPDGF-BB
The quantities of sPDGF-BB were estimated via enzyme-linked immunosorbent assay (ELISA) tests (Quantizing; R&D Systems, Minneapolis, MN, USA). The concentrations of sPDGF-BB were adjusted to the platelet count levels which were obtained from a complete blood count.

Immunohistochemistry IHC
Liver tissue samples were prepared for immunohistochemical analysis. We analyzed the PDGF-BB staining results using IPWIN (version 4.5.0.29 Image-Pro Plus, California, USA). IHC staining was conducted as described previously by Zhang et al (16). Stained sections were visualized by two senior pathologists blinded to each other's results. Brie y, we incubated tissue sections with primary antibodies for mouse PDGF-BB (Abcam, 1:200 Polyclonal) overnight at 4°C. Subsequent incubation with corresponding secondary antibodies was performed at room temperature for 1 hour in the dark.
Immunoreactivity was observed using a horseradish peroxidase-streptavidin detection system (Abcam).

Statistics
Statistical analyses were performed using the SPSS statistical software package version 20.0 (SPSS, Inc, Chicago, IL, USA) and GraphPad Prism (version 7.0d for MacOS X, USA, GraphPad Software, San Diego, California, USA). Non-parametric data were analyzed via the Mann-Whitney U test, and categorical data were subjected to the Chi-square test. The data of PNM grade was analyed by Spearman correlation. In between the state of "no correlation at all" (r = 0) and "perfect correlation" (r = 1), interim values of the correlation coe cient are interpreted by convention. Thus, any value <0.30 would be poor correlation and a p-value <0.05 was considered signi cant (17). The results of PDGF-BB-positive area Lesion volume and PLT were normalized using the following formula (18).
We mapped the range of values of a set of data X to an interval , where Y is the mapped value, Xmax is the maximum value, and Xmin is the minimum score in this data set. Data that did not conform to the normal distribution such as ALT, AST, WBC,TBIL,DBIL and hospitalization days were log-transformed prior to Pearson correlation analysis.

Results
Low sPDGF-BB concentration in AE patients ELISA results indicated signi cantly low sPDGF-BB levels in AE patients compared to the healthy control subjects (p<0.0001; Fig. 1A). However, sPDGF-BB levels in HMAE patients were signi cantly lower compared to the LMAE patients (p<0.01; Fig. 1B).
PDGF-BB is highly expressed in CLT from AE patients IHC results showed signi cantly higher expression of PDGF-BB in the CLT compared to DLT (Fig. 2).

Correlation analysis of PDGF-BB status with other clinical variables in AE patients
In this section, we analyzed different clinical variables to understand the correlation between PDGF-BB expression, lesion activity, and severity of AE. Of note, the levels of sPDGF-BB negatively correlated with lesion metabolic activity(r=-0.624, p=0.0004) which was expressed by SUV MAX , a numerical value that indicated the uorodeoxyglucose F 18 ([18F]-FDG) uptake by the liver lesions in AE patients, the expression of PDGF-BB (r=-0.540, p=0.006) in the tissues around AE lesions ( Fig. 3A;), but not with PNM stages, volume of lesion, number of lesions, inpatient days, and ALT, and AST levels ( Table S2). The expressions of PDGF-BB around the lesion were positively correlated with lesion metabolic activity (SUVmax), the PNM stage, and the lesion size but not with other clinical variables ( Fig. 3B and Table S2).

Diagnostic value of serum PDGF-BB levels in predicting the metabolic activities
The level of sPDGF-BB was assessed as a potential biomarker for assessing metabolic activity, exhibiting 81% sensitivity and 85.7% speci city with AUC > 0.8 (95% con dence interval, p= 0.003) and a cut-off point of 4.66 pg/10 9 platelets. Detailed results on the analysis of the receiver operating characteristic (ROC) curves are presented in Fig. S2.

Discussion
We present the rst report indicating a decrease in serum PDGF-BB levels and increase expression of PDGF-BB in local liver tissues from AE patients. Our results showed that AE patients, particularly those with high-activity lesions have signi cantly lower levels of serum PDGF-BB compared to healthy subjects, and a higher expression level of PDGF-BB around the lesion, a bigger lesion size, a worse disease grade compared to those AE patients with low-activity lesions.
Notably, AE patients require lifelong follow-up after surgery and chemotherapy. A previous study reported recurrence cases nearly 20 years post-surgery (19). The lesion progression has been reported to be extremely slow in immunocompetent and early diagnosed AE patients (2). However, prolonged follow-up must be conducted before ruling on the activity of lesions and regression or progression to determine the need for surgery. Currently, [18F]FDG PET/CT is almost the only available noninvasive tool for detecting metabolic activity in AE patients, however, it is scarce and associated with high cost thereby limiting its usage(4). In Africa, for example, many countries have limited access to [18F]FDG PET/CT noninvasive strategy for managing AE patients due to scarce medical resources. In our study, PDGF-BB was found to potentially predict metabolically active lesions with 81% sensitivity, 85.7% speci city, and AUC > 0.8. The techniques used in assessing the PDGF-BB level in serum are more readily available, and affordable compared with PET. Therefore, it can be applied in most regions globally including the developing nations. Many studies have reported that the angiogenic cytokine PDGF-BB is associated with the prognosis and severity of several diseases of the liver such as hepatocellular carcinoma, liver brosis, and chronic viral hepatitis. Besides, a previous report indicated that serum levels of PDGF-BB are signi cantly correlated with the degree of liver damage and brosis in chronic hepatitis B (CHB) patients (20). Additionally, serum PDGF-BB levels have been suggested to decrease with increased brosis, indicating that the depleted serum PDGF-BB is a potential biomarker for assessing the brosis stage in patients with CHB (21).
Previously, the underlying mechanism associated with reduced PDGF-BB levels in peripheral blood and increased levels of PDGF-BB in the tissue enclosing the lesion has been obscure. We speculated that this phenomenon could be attributed to the high levels of PDGF-BB secreted in the local tissues. Also, the PDGF-BB generated from platelet α-granules responded chemotactically to some chemotactic factor and arrived around the local tissues may cause the relative decrease at the periphery. In addition, several studies have shown that damaged liver exhibits an in ux of many platelets (7,22). White blood cells recruit platelets during in ammation which interacts with the hepatic sinusoidal endothelial cells. These cellular interactions can induce the release of a range of up to 300 bioactive proteins (including cytokines, chemokines, growth factors, hemostatic proteins, bioactive lipids, bactericidal agents6, and PDGF-BB) from platelet α-granules (23). Increasing evidence highlights the role of PDGF-BB at each stage during the progression of liver injury, repair, and brosis (20,21).

Conclusion
These ndings suggest that sPDGF-BB could provide a simple, non-invasive, and rapid strategy for monitoring the status of different stages of AE.

Limitations
This pilot study has some limitations that need to be addressed. First, we used a relatively small sample size, therefore, the results should further be validated using larger patient groups. Second, this study did not examine the molecular mechanisms by which sPDGF-BB is eliminated in AE patients, which would be vital in con rming the actual role of PDGF-BB as a biomarker for AE lesion metabolic activity. Third, we did not characterize the molecular events underling sPDGF-BB depletion in AE patients. This could provide novel strategies for monitoring the diagnosis and treatment of AE. The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.