Dynamic Changes of Cytokine Profiles and Their Correlation With Tumor Recurrence Following Thermal Ablation in Hepatocellular Carcinoma

Abstract

The 5-year recurrence rate of thermal ablation for hepatocellular carcinoma (HCC) is high, and whether this treatment strategy induces systemic immune response remains elusive. This study aimed to investigate the effects of thermal ablation on HCC patients’ cytokine profiles and to explore the correlation of cytokine profiles with tumor recurrence after ablation. A total of 22 HCC patients were included in this prospective study. The levels of 27 cytokines in the peripheral blood of HCC patients were measured before ablation (baseline), week 1, and week 4 after ablation using a Bio-Plex Pro Human Cytokine 27-plex Assay kit. Cytokines showed different dynamic changing trends after ablation treatment. It was found that the level of IL-6 was significantly elevated at week 1 and returned to the baseline level at week 4 after ablation. The level of IL-10 was slightly reduced at week 1 and significantly decreased at week 4. The levels of MCP-1, macrophage inflammatory protein-1β (MIP-1β), and TNF-α were similarly reduced at week 1 and increased at week 4. The levels of IL-17, platelet-derived growth factor-BB (PDGF-BB), and regulated upon activation, normal T cell expressed and secreted (RANTES) showed little to no change at week 1 while an observable increase at week 4. Patients with a high IL-10 level (2.99 pg/ml) at baseline and low levels of TNF-α (20.4 pg/ml), PDGF-BB (107.78 pg/ml), and RANTES (2303.94 pg/ml) at week 4 were at risk of tumor recurrence during 1-year follow-up. The results suggested that thermal ablation activated systemic immune responses by changing the levels of cytokines. The results also demonstrated that measurement of IL-10 at baseline, TNF-α, PDGF-BB, and RANTES at week 4 after ablation might predict the risk of tumor recurrence.

Keywords

hepatocellular carcinoma (HCC)thermal ablation cytokine profiles tumor recurrence

Introduction

Hepatocellular carcinoma (HCC) is the sixth most common type of cancer and ranks third in mortality globally.¹ In China, HCC ranks second in terms of the mortality rate of malignancies.² Surgical resection and liver transplantation are curative therapies for HCC, however, due to the complex features (eg, cirrhosis, underlying liver function, and organ shortage), most patients are ineligible for these treatments.^3,4 In recent decades, local thermal ablation therapies, such as radiofrequency ablation (RFA) and microwave ablation (MWA), have become potentially curative strategies for early-stage HCC in most of clinical practice guidelines due to their excellent curative effect and minimal invasiveness.^5–7 However, the 5-year recurrence rate of HCC with thermal ablation is as high as 70%.⁸ It has been indicated that thermal ablation may induce a systemic antitumor immune response by the release of cytokines,⁹ such as interleukin-10 (IL-10) and transforming growth factor-β (TGF-β), which may not only directly suppress cytotoxic T cells and natural killer (NK) cells, but also promote tumor progression and a poor prognosis.¹⁰ A recent study found that the levels of IL-10 and TGF-β significantly decreased at 1 week after RFA; nevertheless, the serum interferon-gamma (IFN-γ) level, which is an important proinflammatory factor stimulating antitumor immunity, was significantly elevated at 1 week after RFA.¹¹ However, few studies have concentrated on the dynamic changes of immune cytokine/chemokine profiles induced by thermal ablation, and only a limited number of plasma biomarkers were proposed to predict prognosis after thermal ablation.

The study aimed to investigate the effects of thermal ablation on 27 cytokine profiles at 3 different time points (baseline, week 1, and week 4), and to explore the correlation of dynamic changes of cytokine profiles with 1-year tumor recurrence after ablation.

Materials and Methods

Study Design and Patients

The reporting in this study conformed to STROBE guidelines.¹²

In this prospective study, a total of 22 HCC patients who were admitted to the Beijing You’an Hospital (Beijing, China) from January 2019 to December 2020 were included. The diagnosis of HCC was based on the guideline of the American Association for the Study of Liver Diseases,⁴ and HCC stages were classified as China liver cancer (CNLC) staging. The inclusion criteria were as follows: (1) age between 18 and 75 years; (2) transcatheter arterial chemoembolization combined with complete thermal ablation (RFA/MWA) therapy; (3) Child‒Pugh class A or B; and (4) CNLC staging I/II. The exclusion criteria were as follows: (1) Child‒Pugh class C, (2) incomplete ablation, (3) the presence of infection, (4) secondary liver cancer, (5) the presence of immune-related diseases, and (6) history of undergoing combined immunotherapy. The study protocol conformed to the Declaration of Helsinki and was approved by the Ethics Committee of Beijing Youan Hospital, Capital Medical University. Written informed consent was obtained from all subjects. In this study, 10 mL of blood sample was collected from each patient at 3 time points: baseline, week 1, and week 4 after ablation. The study deidentified all patient details.

Treatment

All eligible patients underwent locoregional ablation (RFA or MWA). A safety margin of 0.5–1.0 cm of the adjacent nonneoplastic tissue was ablated to ensure complete coverage.¹³ Abdominal dynamic contrast-enhanced computed tomography (CECT) or magnetic resonance imaging (MRI) was used to evaluate the ablation effect.

Measurement of Cytokines/Chemokines

The serum levels of cytokines and chemokines were measured using a Bio-Plex Pro Human Cytokine 27-plex Assay kit (Bio-Rad Laboratories, Hercules, CA, USA) with Bio-Plex Manager 6.0 software in a Bio-Plex TM 200 system (Bio-Rad Laboratories). This system was applied to quantitatively measure levels of 27 different chemokines and cytokines, including IL-1β, IL-1ra, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12 (p70), IL-13, IL-15, IL-17, eotaxin, basic fibroblast growth factor (bFGF), granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage CSF (GM-CSF), IFN-γ, IP-10, monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory protein-1α (MIP-1α), platelet-derived growth factor-BB (PDGF-BB), MIP-1β, regulated upon activation, normal T cell expressed and secreted (RANTES), tumor necrosis factor-α (TNF-α), and vascular endothelial growth factor.

Follow-up

Patients were followed up at 1 month after ablation and then every 3 months, including alpha-fetoprotein/protein induced by vitamin K absence or antagonist II (AFP/PIVKA-II) and CECT or MRI. Tumor recurrence was defined in the presence of new lesions which showed arterial contrast enhancement and portal phase washout on contrast-enhanced images. The recurrence modality was classified as local tumor recurrence or intrahepatic distant/extrahepatic recurrence. Local tumor recurrence was designated as tumor recurrence within or adjacent to the original lesion (<2 cm from the edge of the ablation site). Intrahepatic distant/extrahepatic recurrence was designated as a new tumor with typical HCC enhancement features within different liver subsegments distinct from the original ablation site or metastases outside the liver.

Statistical Analysis

Normally distributed continuous variables were expressed as the mean ± standard deviation (SD), and abnormally distributed continuous variables were presented as the median (interquartile range). Comparisons between two groups were performed using the independent-samples t-test, Mann‒Whitney Wilcoxon test, or Pearson Chi-squared tests. Repeated measures data were analyzed by repeated-measures analysis of variance (ANOVA) or generalized estimating equations using the Bonferroni posthoc test, comparing all pairs of time points (baseline, week 1, and week 4). Differences were considered significant at P < .05. Spearman's rank correlation coefficient was used for linear correlation analysis between plasma levels of cytokines and WBC count. A receiver operating characteristic (ROC) curve was used to determine the cut-off value. Statistical analysis was performed using SPSS 25.0 software (IBM, Armonk, NY, USA) and graphs were constructed with GraphPad Prism 8.0 (GraphPad software Inc).

Results

Patient Characteristics

A total of 22 HCC patients were involved in this study. The present study included 18 males (81.8%) and 4 females (18.2%), their average age was 54.86 ± 9.37 years, and the average tumor size was 26.50 (17.00-44.00) mm. For etiology, all the patients were HBV-related HCC, and all patients with cirrhosis. Based on whether tumor recurrence occurred within 1 year or not, all patients were divided into recurrence and nonrecurrence group. During 1-year follow-up, 10 patients were diagnosed with tumor recurrence, of which 7 patients were local tumor recurrence and 3 patients were intrahepatic distant/extrahepatic recurrence. And, 12 patients were without tumor recurrence. The baseline characteristics of these two groups were similar, and no significant differences were found in several important variables, such as age, gender, CNLC staging, tumor number, tumor size, and AFP levels (Table 1).

Table 1.

The Comparison of Clinical Data Between Recurrence and Nonrecurrence Group at Baseline

Variables	Total n = 22	Recurrence group	Nonrecurrence group	P-value ^#
Variables	Total n = 22	n = 10	n = 12	P-value ^#
Age (years)	54.86 ± 9.37	54.40 ± 7.46	55.25 ± 11.04	0.838
Gender (male/female)	18/4	8/2	10/2	0.840
Etiology (HBV/HCV)	22/0	10/0	12/0	1
Cirrhosis (yes/no)	22/0	10/0	12/0	1
Child-Pugh class (A/B)	19/3	9/1	10/2	0.650
CNLC staging (I/II)	17/5	7/3	10/2	0.457
Tumor number (single/multiple)	13/9	5/5	8/4	0.429
Tumor size (mm)	26.50 (17.00–44.00)	34.00 (25.25–47.75)	20.50(13.25–36.00)	0.093
Ablation method (RFA/MWA)	12/10	6/4	6/6	0.639
White blood cell (10^9/L)	5.15 ± 1.70	5.44 ± 2.05	4.90 ± 1.39	0.467
Platelet count (10^9/L)	130.82 ± 70.78	127.00 ± 77.07	134.00 ± 68.42	0.824
ALT (U/L)	23.50 (19.00-41.00)	21.50 (18.75-48.75)	31.00 (20.75-41.00)	0.418
AST (U/L)	26.00 (22.75-39.00)	25.50 (21.00-41.25)	26.00 (23.75-36.00)	0.923
AFP (ng/ml)	27.83 (3.21-196.6)	51.15 (2.18-196.63)	21.02 (3.58-194.9)	0.771
HBsAg (IU/ml)	850.50 (318.50-1123.00)	566.15 (318.64-1073.95)	1102.50 (399.50-1231.00)	0.381
HBV DNA (negative/positive)	15/7	6/4	9/3	0.452

# Compare between the recurrence group and the nonrecurrence group.

Abbreviations: HBV, hepatitis B virus; HCV, hepatitis C virus; RFA, radiofrequency ablation; MMA, microwave ablation; ALT, alanine aminotransferase; AST, aspartate aminotransferase; AFP, α-fetoprotein.

Dynamic Changes in Cytokines/Chemokines After Thermal Ablation

The variations in the levels of cytokines/chemokines in 22 HCC patients at baseline and at week 1 and week 4 after thermal ablation were compared. It was revealed that the level of IL-6 was significantly elevated at week 1 and returned to the baseline level at week 4. (Figure 1a). Compared with the baseline level, the level of IL-10 was slightly reduced at week 1 (no significant difference), and then decreased at week 4 with a statistical difference (P = .012, Figure 1b). The dynamic changes in levels of MCP-1, MIP-1β, and TNF-α were similar, which decreased at week 1 and increased at week 4 (Figures 1c-e). The levels of IL-17, PDGF-BB, and RANTES showed little to no change at week 1 while an observable increase at week 4. (Figures 1f-h).

Figure 1.

Dynamic changes in levels of cytokines/chemokines after ablation treatment (a-h). The levels of IL-6 (a), IL-10 (b), MCP-1 (c), MIP-1β (d), TNF-α (e), IL-17 (f), PDGF-BB (g), and RANTES (h) were compared at baseline and at week 1 and week 4 after ablation. Repeated measures data were subjected to repeated measures ANOVA or generalized estimating equations with the Bonferroni post-hoc test.

Correlation Between IL-6 Level and WBC Count at Week 1 After Ablation

It was attempted to measure ALT level, AST level, WBC count, and neutrophil count as indicators of hepatic necrosis and systemic inflammation. As shown in Figure 2, there was a significantly positive correlation between serum IL-6 level and WBC count at week 1 (rs = 0.555, P = .007). The changing characteristic of IL-6 release following thermal ablation suggests that IL-6 is involved in the acute-phase response against tissue damage and inflammation caused by ablation.

Figure 2.

There was a positive correlation between the IL-6 level and WBC count at week 1. Spearman's rank correlation analysis was used for linear correlation analysis.

The Correlation Between Levels of Cytokines/Chemokines and 1-Year Tumor Recurrence

The correlation between levels of cytokines/chemokines and 1-year tumor recurrence was assessed. It was revealed that the recurrence group had significantly higher levels of IL-10 at baseline (P = .005; Figure 3a). Univariate and multivariate analyses included CNLC staging, tumor number, tumor size, and IL-10 (baseline). Results showed that only high IL-10 (baseline) (OR, 0.190; 95% CI, 0.047-0.772; P = .020) was a significant predictor of 1-year tumor recurrence (Table 2).

Figure 3.

Levels of cytokines/chemokines were associated with tumor recurrence. IL-10 level at baseline (a), TNF-α level at week 4 (b), PDGF-BB level at week 4 (c), and RANTES level at week 4 (d) were associated with tumor recurrence. Statistical analysis of data was performed using repeated measures ANOVA or generalized estimating equations with the Bonferroni posthoc test.

Table 2.

Univariate and Multivariate Analysis of Factors at Baseline Associated With 1-Year Tumor Recurrence

Variables	Univariate analysis		Multivariate analysis
Variables	OR (95% CI)	P-value	OR (95% CI)	P-value
CNLC staging	0.467 (0.061-3.565)	0.463	-
Tumor number	0.750 (0.131-4.290)	0.746	-
Tumor size	0.958 (0.902-1.018)	0.167
IL-10 (baseline)	0.340 (0.129-0.895)	0.029	0.190 (0.047-0.772)	0.020

Abbreviations: CNLC, China liver cancer staging; IL-10, interleukin-10.

We also found that lower levels of TNF-α, PDGF-BB, and RANTES at week 4 were significantly associated with tumor recurrence (P = .026, .025, and .013, respectively; Figure 3b-d). Other cytokines did not show significant differences between recurrence and nonrecurrence groups at the 3 different time points.

Predictive Values of IL-10 (Baseline), TNF-α (Week 4), PDGF-BB (Week 4), and RANTES (Week 4) Levels for Tumor Recurrence

According to the analysis of the relationship between the levels of cytokines/chemokines and tumor recurrence, the predictive values of IL-10 (baseline), TNF-α (week 4), PDGF-BB (week 4), and RANTES (week 4) levels for tumor recurrence were further analyzed (Figures 4a, b). The ROC curves for predicting recurrence were plotted. It was found that the area under the curve (AUC) of the IL-10 level at baseline was 0.783 (95% confidence level (CI): 0.570–0.997, P = .025), and the cut-off value was 2.99 pg/ml. Low levels of TNF-α, PDGF-BB, and RANTES at week 4 were associated with tumor recurrence; the AUC values were 0.792 (95% CI: 0.575-1, P = .021), 0.750 (95% CI: 0.533-0.967, P = .048), and 0.783 (95% CI: 0.577-0.989, P = .025), respectively. Notably, tumor recurrence predicted by the three combinations had a higher AUC (0.9, 95% CI: 0.750-1, P = .002), with a sensitivity of 83.3% and a specificity of 100% (Table 3). Therefore, these cytokines/chemokines can be used as predictors of tumor recurrence.

Figure 4.

ROC curves of IL-10, TNF-α, PDGF-BB, and RANTES levels to predict tumor recurrence. The ROC curve of IL-10 at baseline to predict tumor recurrence (a). The ROC curves of TNF-α, PDGF-BB, and RANTES and their combination at week 4 (b). The AUC values were all more than 0.75, which indicated that these cytokines could predict tumor recurrence well, especially when combined.

Table 3.

Predictive Values of IL-10, TNF-α, PDGF-BB, and RANTES Levels for Tumor Recurrence

Variable (s)	AUC	P-value	Cut-off (pg/ml)	Sensitivity	Specificity
IL-10 (baseline)	0.783 (0.570-0.997)	0.025	2.99	70%	91.7%
TNF-α (week 4)	0.792 (0.575-1.000）	0.021	20.4	83.3%	90%
PDGF-BB (week 4)	0.750 (0.533-0.967)	0.048	107.78	83.3%	70%
RANTES (week 4)	0.783 (0.577-0.989)	0.025	2303.94	66.7%	90%
Combined (week 4)	0.900 (0.750-1)	0.002	0.702	83.3%	100%

Abbreviations: IL-10, interleukin-10; TNF-α, tumor necrosis factor-α; PDGF-BB, platelet-derived growth factor-BB; RANTES, regulated upon activation, normal T cell expressed and secreted; AUC, area under the curve.

Discussion

In this prospective cohort, it was found that thermal ablation induced a systemic immune response by changing the levels of cytokines. The IL-10 level at baseline and levels of TNF-α, PDGF-BB, and RANTES at week 4 were correlated with tumor recurrence.

The key immune cytokines/chemokines in HCC patients (n = 22) before and after thermal ablation were analyzed. The present study provided evidence that thermal ablation could induce a systemic antitumor immune response. It was revealed that IL-6 level was markedly elevated, and that there was a significantly positive correlation between IL-6 level and WBC count at week 1 after thermal ablation. IL-6 is a pleiotropic cytokine involved not only in the immune response, but also in inflammation by stimulating signal transducer and activator of transcription 3 (STAT3).^14,15 Some studies have reported that IL-6 level is significantly elevated at 3–15 days postablation.^16–18 Therefore, it was hypothesized that IL-6 is a biomarker reflecting the degree of hepatic trauma and inflammation caused by thermal ablation. It was also found that IL-10 level, an anti-inflammatory Th2 cytokine that promotes immunosuppression, was significantly reduced from baseline to week 4 after ablation, while the Th1 cytokine TNF-α level was first reduced at week 1 and then elevated at week 4, however, there was no significant difference between baseline and week 4. These findings indicated that thermal ablation could offset the Th1/Th2 balance, which was reshaped and polarized to the Th1 status, thereby alleviating HCC tumor-induced immune suppression. In addition, the dynamic changes in levels of MCP-1, MIP-1β, and TNF-α were similar, which decreased at week 1 and increased at week 4. The levels of IL-17, PDGF-BB, and RANTES showed little to no change at week 1 while an observable increase at week 4. Taken together, these data showed the different dynamic changing trends of cytokines/chemokines after ablation, which could be caused by different mechanisms. First, thermal ablation could cause trauma to the liver, and the wound healing process could result in alterations in levels of some cytokines/chemokines. Second, heat-induced injury could promote acute thermal coagulative necrosis and apoptosis in liver and tumor tissues. The ablated tissue or tumor cells release cytokines/chemokines. Third, importantly, immunogenic signals released by dying tumor cells can induce antigen uptake, as well as antigen processing and presentation, further stimulating nonspecific and specific immune responses. Therefore, cytokine expression affected by wound healing, ablated tissue, and nonspecific immune responses lasts for a shorter duration than that affected by specific immune responses,¹⁹ which may justify why changes in the levels of cytokines/chemokines lasted for different durations. Therefore, it could be concluded that thermal ablation induced changes in levels of specific immune cytokines/chemokines at week 4.

In our study, we did not find significant differences in the baseline demographic and clinicopathological data between recurrence and nonrecurrence groups, such as age, gender, CNLC staging, tumor number, tumor size and AFP levels. We further analyzed and found that the levels of IL-10, TNF-α, PDGF-BB, and RANTES were associated with tumor recurrence. IL-10 may contribute to a tumor microenvironment, promoting HCC carcinogenesis and progression.²⁰ In our study showed that a high IL-10 level at baseline rather than after thermal ablation, was easily associated with tumor recurrence (Figure 3a). Multivariate analysis showed that IL-10 (baseline) was an independent prognostic factor for 1-year tumor recurrence in HCC patients (Table 2). This is consistent with the finding of a previous study describing IL-10 as a major biomarker for the poor prognosis of HCC.²¹ The present study also indicated that the levels of TNF-α, PDGF-BB, and RANTES were significantly lower at week 4 and were closely associated with an unfavorable prognosis. The results further indicated that thermal ablation induced antitumor immune function at week 4. TNF-α is one of the most important proinflammatory cytokines and has been demonstrated to be an antitumor cytokine that activates the nuclear factor-κB (NF-κB) pathway. Some recent studies also demonstrated that TNF-α level was higher after ablation and was associated with a good prognosis of HCC patients.^22–24

PDGF-BB is a pluripotent angiogenic ligand that is present in platelets and released upon degranulation, and it plays a biological function by activating PDGFR-α and PDGFR-β.²⁵ PDGF-BB plays roles in tumor growth, invasion, and metastasis.^26,27 A previous study revealed that in HCC patients undergoing curative resection, PDGF-BB level was lower in the recurrence group than that in the nonrecurrence group both before surgery and at 4 weeks postoperation.²⁸ In another study,²⁹ after sorafenib treatment, PDGF-BB level was significantly lower in nonlong survivors than that in long survivors (overall survival ≥2 years). The present study, for the first time, showed that depleted serum concentration of PDGF-BB could be associated with tumor recurrence after ablation. In patients with recurrence, the molecular mechanism involved in the exhaustion of serum PDGF-BB concentration has remained elusive. The reason could be that platelet dysfunction is already known to contribute to cancer progression,^30,31 and therefore, our hypothesis was that the depletion of serum PDGF-BB concentration might be attributed to platelet exhaustion.

The CC-chemokine RANTES (CCL5) is a T-cell chemoattractant and an immunoregulatory molecule. RANTES can be expressed by a number of cell types, including T lymphocytes, macrophages, platelets, and tubular epithelium.³² Nevertheless, the role of RANTES in tumor development remains controversial. Some studies have shown that RANTES overexpression facilitates tumor progression and metastasis via the RANTES/CCR5 axis.^33,34 In addition, one recent study has reported that the increased RANTES expression, which chemotactically attracts a substantial number of immunocytes to tumor tissues to exert antitumor efficacy, may be associated with favorable outcomes in some diseases.³⁵ In the present study, elevated serum levels of RANTES were found in nonrecurrent patients compared with recurrent patients at week 4 after ablation. This could indicate a protective role of RANTES in antitumor immune responses to prevent tumor recurrence.

The present study suggests that thermal ablation induced changes of cytokines which affect tumor recurrence. Over the last decade, we have witnessed the emergence and advent of immune checkpoint inhibitors (ICIs) in solid tumors, such as programmed cell death-1 (PD-1), cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4).³⁶ ICIs seem to have found their role in advanced HCC patients, and have shown impressive benefits in progress-free survival and overall survival.³⁷ The combination of thermal ablation therapies with ICIs presents a more recent promising approach for the treatment of HCC, as it can lead to a decrease in recurrence rate and improve survival rates in HCC patients. Therefore, two therapeutic strategies should be explored in the future.^38,39

The present study revealed that immune mediators were associated with tumor recurrence, and 27 cytokines/chemokines, immune mediators, and growth factors at 3 time points before and after ablation were compared, which have rarely been reported previously. Nevertheless, several limitations of this study should be pointed out. First of all, the study is limited to very small sample size, and larger cohorts are needed to validate the findings. Second, this research merely evaluated circulating cytokines/chemokines, and additional studies on their association with the immune response in the ablation zone should be conducted.

Conclusions

In conclusion, the results suggested that thermal ablation could be an effective modality to overcome the immunosuppressive tumor microenvironment and induce a systemic immune response. The results also indicated that the levels of cytokines/chemokines after 4 weeks of thermal ablation were associated with tumor recurrence and could be used as noninvasive and useful biomarkers to predict the postablation prognosis. In the future, thermal ablation combined with immune checkpoint inhibitors may enhance the antitumor immune response and improve the prognosis of patients.

Footnotes

Abbreviations

Acknowledgements

The authors highly appreciate all participants who were involved in the present study and our team from Beijing You’an Hospital.

Author Contributions

Yonghong Zhang, Dandan Guo, and Yan Zhao carried out the studies, participated in collecting data, and drafted the manuscript. Ling Qin, Jianping Sun, and Kang Li performed the statistical analysis and participated in its design. Chaoran Zang, Qi Wang, Wenying Qiao, and Biyu Liu participated in the acquisition, analysis, and interpretation of data and drafted the manuscript. All authors read and approved the final manuscript.

Availability of data and materials

The data used to support the findings are available from the corresponding author upon request.

Consent for publication

Not applicable.

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Ethics Approval and Consent to Participate

The study protocol conformed to the Declaration of Helsinki, and was approved by the Ethics Committee of Beijing You’An Hospital Affiliated to Capital Medical University (Jingyou Kelun Zi [2022]017). Written informed consent was obtained from all subjects.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This study was financially supported by the Beijing Municipal Natural Science Foundation (Grant No. 7191004), the Capital health development project (Grant Nos. 2020-1-2182 and 2020-2-1153), the Beijing Key Laboratory (Grant No. BZ0373), the Beijing Municipal Administration of Hospitals’ Ascent Plan (Grant No. DFL20181701), the Key medical professional development plan of Beijing municipal administration of hospitals (Grant No. ZYLX201711), the Beijing You’an Hospital plan (Grant No. YNKTQN2021014), and the Beijing Municipal Administration of Hospitals’ Incubating Program (Grant No. PX2018059).

ORCID iD

Yonghong Zhang

Notes

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