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Research Article

Early enlarging cavitation after percutaneous microwave ablation of primary lung cancer

, , , , , , , , , , & ORCID Icon show all
Article: 2210269 | Received 20 Feb 2023, Accepted 29 Apr 2023, Published online: 16 May 2023

Abstract

Purpose

This retrospective study assessed the incidence rate, risk factors, and clinical course of early enlarging cavitation after percutaneous microwave ablation (MWA) of primary lung cancer (PLC).

Methods

This study included 557 lesions of 514 patients with PLC who underwent CT-guided percutaneous MWA between 1 January 2018 and 31 December 2021. Of these patients, 29 developed early enlarging cavitation and were enrolled in the cavity group, and 173 were randomly enrolled in the control group. Early enlarging cavitation of the lung was defined as the development of a cavity ≥30 mm within 7 days after MWA.

Results

Overall, 31 (5.57%, 31/557 tumors) early enlarging cavitations occurred at an average of 5.83 ± 1.55 d after MWA. The risk factors were lesion contact with a large vessel (diameter ≥3 mm), lesion contact with the bronchus (diameter ≥2 mm), and a large ablated parenchymal volume. The cavity group had a higher incidence rate of delayed hydropneumothorax (12.9%) and bronchopleural fistula (9.68%) than the control group, resulting in a longer hospitalization (9.09 ± 5.26 days). Until Dec 31, 2022, 27 cavities disappeared after a mean of 217.88 ± 78.57 d (range, 111–510 d), two persisted, and two were lost to follow-up.

Conclusions

Early enlarging cavitation occurred in 5.57% PLC cases that underwent MWA, causing serve complications and longer hospitalization. The risk factors were ablated lesion contact with large vessels and bronchi, as well as a larger ablated parenchymal volume.

Introduction

Primary lung cancer (PLC) remains one of the most common malignancies and leading causes of cancer‑associated mortality worldwide [Citation1]. Surgical excision is widely recognized as a first-line treatment for early-stage PLC. However, about 60% of PLCs cannot be surgically removed for various reasons, including poor cardiopulmonary function or advanced stage [Citation2]. Stereotactic ablative therapy improves local efficacy over standard radiotherapy. It is a good option for most patients with lung cancer that cannot be surgically resected, but it has limitations [Citation3,Citation4]. As an alternative technique, thermal ablation is increasingly used in the local treatment of patients with PLCs who cannot tolerate surgery or SBRT [Citation5,Citation6]. Despite that cryoablation has produced encouraging results for the treatment of lung tumors in recent years, radiofrequency ablation (RFA) and microwave ablation (MWA) is the most widely used techniques [Citation7–9]. Although thermal ablation is a safe and effective treatment for lung tumors, complications should not be ignored [Citation10,Citation11]. Among previous reports, cavity formation is an additional imaging finding after lung MWA without being clinically symptomatic [Citation12–14]. A recent study has shed new light on cavity formation, with early enlarging and persistent cavities after RFA in lung tumors. The formation mechanism and evolution of this type of cavity are quite different from those previously reported [Citation15]. MWA has a greater ablation power than RFA, but whether it is more likely to cause early enlarging cavitation has not been reported. The objective of this study was to retrospectively determine the incidence rate of early enlarging cavitation after MWA sessions in patients with PLC, identify risk factors, and follow the clinical course of patients.

Materials and methods

Written informed consent was obtained from all patients before performing MWA and CT examinations. This study used a retrospectivecase–cohort design. The cavity group included patients with early enlarging cavitations. The control group included randomly selected patients from the total study samples. This study was approved by the relevant ethics committee and performed in accordance with the ethical standards as laid down in the 1964 Declaration of Helsinki and its later amendments or comparable ethical standards. The approved protocol number is YXLL-KY-2018 (011).

Patients

A total of 775 patients with 838 lung lesions underwent CT-guided percutaneous MWA between 1 January 2018 and 31 December 2021. Among these patients, 417 lesions underwent synchronous MWA and biopsy, and were diagnosed with highly suspicious malignant lesions after discussing the findings with a multidisciplinary team [Citation16]. Among these lesions, 257 were pathologically diagnosed as adenocarcinoma and 30 were diagnosed as squamous cell carcinoma. This study included 287 lesions. In addition, 358 patients with 391 preprocedural pathologically diagnosed PLC underwent MWA. Of these cases, 221 adenocarcinomas, 47 squamous cell carcinomas, and 2 adeno-squamous carcinomas were enrolled in the final sample. Overall, 514 patients with 557 PLC were enrolled in the total study samples. Early enlarging cavitation was defined as the development of a cavity having a diameter ≥30 mm within 7 d after the procedure. Ninety-three cavities were detected in the total sample; of which, 38 cavities were excluded because the maximum diameter was <30 mm and 24 cavities were excluded because the detection time was >7 d. Finally, 29 patients (mean age: 63.87 ± 15.46 years, range: 48–84 years) with 31 early enlarging cavitations were enrolled in the cavity group. The control group was randomly selected using a random number table from the total sample, and 173 patients (mean age 66.34 ± 10.64 years, range: 39–88 years) in 186 sessions were enrolled. shows the flowchart of patient selection. The primary endpoint was to evaluate the incidence of early enlarging cavitation after MWA for PLC. The secondary endpoints were: (i) the risk of early enlarging cavitation; (ii) Characteristics of concomitant complications; and (iii) the clinical course during cavitation.

Figure 1. The flowchart of patient selection. Reference- CT: computerized tomography; MWA: Microwave ablation; PLC: primary lung cancer.

Figure 1. The flowchart of patient selection. Reference- CT: computerized tomography; MWA: Microwave ablation; PLC: primary lung cancer.

MWA procedure

The MWA procedure has been described previously [Citation14,Citation17]. To relieve the pain experienced by the patient during the procedure, local anaesthesia (1% lidocaine) and intravenous anaesthesia (sufentanil, 0.25 μg/kg) were administered. Moreover, the patient was kept under moderate sedation and provided oxygen via a nasal tube [Citation18]. CT (Lightspeed 64 V, GE General Electric or NeuViz 64, Neusoft Co. Ltd. or uCT 760, United Imaging Healthcare Co. Ltd.) was used to guide the procedures. MWA was performed using the MTC-3C MWA system (Vison-China Medical Devices R&D Center), the ECO-100A1 MWA system (ECO Medical Instrument Co. Ltd.), or the KY-2450B MWA system (CANYOU Medical Inc.), with a frequency of 2450 ± 50 MHz and an adjustable continuous wave output power of 0–100 W. The microwave antenna had an effective length of 100–180 mm, an outer diameter of 19-G (19-G antennas have high puncture accuracy and few complications), and a radiating tip of 1.5 cm (tapered end). The surface temperature of the antenna was cooled using a water circulation cooling system. The procedure was performed as follows: (1) a treatment plan was designed according to the pre-procedural CT findings, including appropriate body placement, puncture site on the body surface, optimal puncture trajectory, target skin distance, and antenna number; (2) the ablation antenna was placed at the position determined using the treatment plan; (3) after confirming the proper positioning of antennae using CT, MWA was performed at the predetermined power and time; (4) the procedure aimed to ablate the tumor with at least 5-mm margin; (5) after completion of the procedure, whole-lung CT scan was performed to assess the ablation area and immediate complications; and (6) the patient’s electrocardiographic tracing, heart rate, respiratory rate, oxygen saturation, and blood pressure was continuously monitored throughout the MWA session and for an additional 6 h after they returned to the ward. Steroid hormones and antibiotics were not routinely used during MWA.

Follow-up examination and evaluation of early enlarging cavitation

All patients were routinely followed up with chest CT within 24 h after MWA to evaluate post-procedural complications. The severity of the complications in the patients was classified as major or minor according to the Cirse Classification System [Citation19]. Major complications were defined as events that led to substantial morbidity and disability, an increased level of care, hospital admission, or a substantially prolonged hospital stay (classification levels 3–5). Intubation drainage was required for severe pneumothorax, pleural effusion, and pleural hemorrhage. Typically, the patient was hospitalized for 3–4 d [Citation20,Citation21]. If persistent fever, severe pain, severe cough, or dyspnea occurred, an emergency chest CT examination was required to rule out delayed complications (complications occurred >72 h after the procedure) [Citation22]. A chest CT examination was performed 1, 3, 6, 9, and 12 months after the procedure and every 6 months thereafter. The CT images were retrospectively reviewed by two board-certified diagnostic specialists with more than 10 years of experience. The evaluation criteria for early enlarging cavitation are mentioned above, and the detection time, evolution, and clinical course were recorded.

Data collection

The patient characteristics, preprocedural examination, tumor characteristics, procedural characteristics, and post-procedural follow-up were recorded. The patient characteristics included age, sex, emphysema, diabetes, interstitial pneumonia, chronic obstructive pulmonary disease, lung function, and history of antitumor therapy. The tumor characteristics included tumor size, density, location, and distance from the interlobular fissure, chest wall, blood vessels (diameter ≥3 mm), and bronchi (diameter ≥2 mm) (). The tumor and ablation zone volumes were calculated using the following formula: π/6 × maximum diameter × maximum diameter in the perpendicular direction × maximum diameter in the craniocaudal direction. The ablation zone parameters were measured on CT images obtained 24 h after MWA. The ablated parenchymal volume was calculated by subtracting the tumor volume from the ablation zone volume. Follow-up features were recorded for patients with early enlarging cavitation, including hospitalization days, cavity detection days, the maximum diameter of the cavity, the number of days the cavity was at the maximum diameter, and the days to cavitation disappearance. MWA-related major complications were recorded, including delayed complications.

Figure 2. ( A) Some large bronchi are surrounded by a GGO located in the left lower lobe of the lung (black triangle). (B) A GGO in contact with a large bronchus (black triangle). (C) A pure GGO surrounding a few bronchi and vessels (white arrowheads).

Figure 2. ( A) Some large bronchi are surrounded by a GGO located in the left lower lobe of the lung (black triangle). (B) A GGO in contact with a large bronchus (black triangle). (C) A pure GGO surrounding a few bronchi and vessels (white arrowheads).

Statistical analysis

Logistic regression analysis was performed to calculate the RRs and 95% confidence intervals. The exponent of the regression coefficient in the logistic regression analysis was calculated as an estimate of the RR. The analyses were performed by an experienced epidemiologist/biostatistician using Stata 16.1/MP4 (Stata Corporation, College Station, TX, USA). A P-value < 0.05 was considered significant. RRs were calculated for significant categorical values.

Results

Between 1 January 2018 and 31 December 2021, 93 cavities were detected in 557 MWA sessions; of which, 24 cavities were detected after >7 d and 38 cavities were developed with a maximum diameter of <30 mm. Thus, 31 early enlargement cavitations (5.57%, 31/557) of 29 patients were enrolled in the cavity group (). A total of 186 patients were randomly selected from the total study sample and enrolled in the control group. Among them, cavities formed after <7 and >7 days in 6 and 34 patients, respectively. The diameter of the cavity was <30 mm and >30 mm in 32 and 8 patients, respectively.

Figure 3. A 59-year-old woman with lung cancer in the right lower lobe of the lung was treated with percutaneous microwave ablation (MWA). (A) subsolid GGO (white arrowhead) located in the lower right lobe of the lung, in contact with a large bronchus (white triangle). (B) A CT image obtained 24 h after MWA showing a larger fried-egg sign (white arrowhead) on the ablation zone. (C) A CT image obtained four days after MWA shows an enlarging cavitation (white arrowhead) with a maximum diameter of 58 mm. (D) A CT image obtained 3 months after MWA showing a shrinking cavitation (white arrowhead). (E) A CT image obtained 12 months after MWA showing the disappearance of cavitation and shrinking as a scar (white arrowhead).

Figure 3. A 59-year-old woman with lung cancer in the right lower lobe of the lung was treated with percutaneous microwave ablation (MWA). (A) subsolid GGO (white arrowhead) located in the lower right lobe of the lung, in contact with a large bronchus (white triangle). (B) A CT image obtained 24 h after MWA showing a larger fried-egg sign (white arrowhead) on the ablation zone. (C) A CT image obtained four days after MWA shows an enlarging cavitation (white arrowhead) with a maximum diameter of 58 mm. (D) A CT image obtained 3 months after MWA showing a shrinking cavitation (white arrowhead). (E) A CT image obtained 12 months after MWA showing the disappearance of cavitation and shrinking as a scar (white arrowhead).

shows the patient characteristics, preprocedural lung function, and history of antitumor therapy of 31 early enlarging cavitations and 186 control sessions. The tumor characteristics, procedural characteristics, and post-procedural characteristics of the two groups are shown in . also shows the results of univariable analyses performed to determine the risk factors for early enlarging cavitation. The following risk factors were significantly associated with cavitation: contact with vessels that were ≥3 mm in diameter (p = 0.0004; RR 3.35-fold higher than that of vessels that were <3 mm in diameter; 95% CI: 1.72 − 5.55) and bronchus ≥2 mm in diameter encompassed in the ablation zone (p < 0.0001; RR 6.15-fold higher than that of <2 mm in diameter; 95% CI: 3.13 − 12.08). In addition, the univariable analyses also showed that the ablated parenchymal volume of the two groups were significantly different (46.53 ± 12.36 cm³ vs, 29.66 ± 8.57 cm³; p < 0.0001). The clinical course of cavitation is presented in . The mean hospitalization in the cavitation group and control group was 9.09 ± 5.26 d (range: 3–38 d) and 6.34 ± 2.69 d (range: 2–22 d), respectively. There was a significant statistical difference in hospitalization between the two groups (p = 0.0028). Of 31 early enlarging cavitations, follow-up CT images showed the largest size (mean, 42.9 ± 11.71 [SD] mm; range: 30–73 mm) appeared at an average of 14.77 ± 4.65 [SD] d (range: 8–25 d) after the procedure. Finally, 27 cavitations disappeared at an average of 217.88 ± 78.57 [SD] d (range: 111–510 d) after the procedure. Until 31 December 2022, two cavities persisted and two were lost to follow-up. The major complications of the cavity group are shown in . Briefly, 11 of 31 sessions had ≥ Grade 3 pneumothorax, four of which presented as delayed hydropneumothorax. Three cases had persistent subcutaneous emphysema, which was clinically considered a bronchopleural fistula. After continuous negative pressure closed drainage, two cases were effectively controlled within 14 d, while the other case presented with aggravating subcutaneous emphysema. After bronchoscopic treatment, the bronchopleural fistula was under control 38 d after the procedure ().

Figure 4. A 74-year-old man with lung cancer in the right upper lobe of the lung was treated with percutaneous microwave ablation (MWA). (A) solid GGO (white arrowhead) located in the right upper lobe of the lung. (B) A CT image obtained 24 h after MWA showing a larger fried-egg sign (white arrowhead) on the ablation zone. (C) A CT image obtained six days after MWA showing an enlarged cavitation (white arrowhead) with a maximum diameter of 71 mm, accompanied by subcutaneous emphysema. (D) A CT image obtained 11 days after MWA showing a cavitation continuing to enlarge and increased symptoms of subcutaneous emphysema. (E) A sagittal CT showing that the cavity was directly connected with the large bronchus. (F) A CT image obtained 37 days after MWA showing shrinking cavitation (white arrowhead), with subcutaneous emphysema being controlled. (G) A CT image obtained 3 months after MWA showing shrinking cavitation (white arrowhead). (H–I) CT images obtained 1 and 2 years after MWA showing the disappearance of cavitation and shrinking as a scar (white arrowhead).

Figure 4. A 74-year-old man with lung cancer in the right upper lobe of the lung was treated with percutaneous microwave ablation (MWA). (A) solid GGO (white arrowhead) located in the right upper lobe of the lung. (B) A CT image obtained 24 h after MWA showing a larger fried-egg sign (white arrowhead) on the ablation zone. (C) A CT image obtained six days after MWA showing an enlarged cavitation (white arrowhead) with a maximum diameter of 71 mm, accompanied by subcutaneous emphysema. (D) A CT image obtained 11 days after MWA showing a cavitation continuing to enlarge and increased symptoms of subcutaneous emphysema. (E) A sagittal CT showing that the cavity was directly connected with the large bronchus. (F) A CT image obtained 37 days after MWA showing shrinking cavitation (white arrowhead), with subcutaneous emphysema being controlled. (G) A CT image obtained 3 months after MWA showing shrinking cavitation (white arrowhead). (H–I) CT images obtained 1 and 2 years after MWA showing the disappearance of cavitation and shrinking as a scar (white arrowhead).

Table 1. Patient baseline characteristics.

Table 2. Results of univariable analyses to determine risk factors for early enlarging cavitation.

Table 3. Clinical course of the patients with early enlarging cavitation.

Table 4. Major adverse events after procedure (According to The Cirse Classification System).

Discussion

The present study reports an incidence of 5.57% (31/557) for early enlarging cavitation after MWA treatment for PLC lesions, which were more likely to incorporate delayed hydropneumothorax and bronchopleural fistula. This result suggests that early enlarging cavitation should be given more attention and research. However, previous studies show that cavities after ablation are a natural change with limited clinical significance, and no special treatment is required. Okuma et al. studied 100 lung tumours after RFA ablation and showed that cavitation occurred at a frequency of 14% at 1.5 ± 0.8 months, but the majority of the patients were asymptomatic. Lesions near the chest wall, lung cancer as the primary lesion, and pulmonary emphysema were the risk factors [Citation23]. It is traditionally considered that after the ablation of the cavity necrotic tissues are discharged along the large tracheae within 1–3 months. As there were significant differences in the occurrence time and imaging findings, we believe that the cavitation in previous studies was not similar to the early enlarging cavitation reported in the present study. A recent study on RFA for lung tumors reported early enlarging cavitation, which led to the development of more complications, including pneumothorax, rupture of cavitation, pleural effusion, bronchopleural, acute interstitial pneumonia, and intractable haemorrhage, leading to deterioration to a severe condition or even death. addition, this study has analyzed the significant risk factors for early enlarging cavitation, including a distance from tumors to the pleura of ≥20 mm, tumors contact with a vessel ≥3 mm in diameter, tumors contact with a bronchus ≥2 mm in diameter and using a multi-tined expandable electrode [Citation15]. The authors hypothesized two mechanisms for lung early enlarging cavitation after RFA. One hypothesis was the “check valve” theory: (1) RFA damages the bronchi in the ablation zone. (2) The necrotic tissue and inflammatory exudate obstruct the injured bronchi. (3) A “check valve” structure and cavitation are created and enlarged. Another hypothesis is the “shear stress” theory: (1) The flexibility between the ablation zone and the normal lung tissue is different. (2) The boundary between these two structures is subjected to constant shear stress during respiration. (3) Air flows into the cracks created by shear stress, forming a cavity.

MWA has different characteristics from RFA, including greater heat generation, larger regions of active heating, and less dependence on thermal conduction, which reduced the sensitivity to a heat sink. This indicates that MWA can more thoroughly destroy the structure of the great vessels and bronchus adjacent to the lesions than RFA. In addition, unlike RFA, MWA is not limited by heat and tissue electric conduction and can reach a larger ablation region, leading to a larger ablated parenchymal volume [Citation5]. This study showed that lesion contact with vessels that are ≥3 mm in diameter, lesion contact with bronchus ≥2 mm in diameter, and a larger ablated parenchymal volume were risk factors for early enlarging cavitation. A larger ablated volume might result in deeper structural damage to the large vessels and bronchi. We also observed a relatively large ablative volume and severe exudation in cases with early enlarging cavitation. Masaoka et al. reported that excessive lung ablation and continuous aggravated exudation are closely related to fever after ablation [Citation24], which may indicate the rapid progression of local inflammation. Therefore, we think that the severe structural damage of large vessels and bronchi was an important factor in this kind of cavity formation. Thus, the probable underlying mechanism of early enlarging cavitation after MWA is described as follows. (1) The elasticity of the large vessels and bronchus is different from normal lung tissue, which remained in a relatively balanced pulling mode when the structure was intact. (2) After ablation, the structures were destroyed, and the local traction balance was broken, resulting in blood vessels and bronchus with a higher elasticity coefficient being pulled away from the ablation area, which was passively inflated to form a cavity. This speculation could explain the following phenomena: (1) The cavity formed within 2–7 d after the procedure, and there was not enough time for necrotic tissues to be discharged along the large tracheae. (2) Most patients showed a larger ablation area but almost normal 24 h CT findings after the procedure until experiencing sudden aggravation pain at 2–7 d. Subsequently, enlarging cavity and severe hydropneumothorax were detected after intubation drainage. This was probably because the blood vessels and bronchus had not been broken at 24 h. Severe pulling might force the pleural tearing, causing pain, and delayed hydropneumothorax. This also explains why the ablation of tumors near the interlobular fissure would be more likely to form cavities [Citation25]. These theories are speculative, so additional experiments or pathology are necessary to identify the exact mechanisms of cavity formation after MWA.

Previous studies have found that some severe complications may develop from cavities, such as hemoptysis, infection, and bronchopleural fistula. In a multicenter study, Huang et al. reported that 23 of 1596 patients developed invasive pulmonary aspergillosis secondary to MWA. Among them, 20/23 (86.96%) were secondary to a persistent cavity, and 6/23 (26.1%) patients died [Citation12]. Other studies have reported that bronchopleural fistula is often secondary to cavities, which is considered a rare but severe complication and should be treated actively using surgical pleurodesis, thoracic drainage, chemical pleurodesis, percutaneous injection of synthetic hydrogel surgical sealant, and endobronchial valve therapy [Citation26–28]. The detection of cavitation, largest diameter, and days to reach the maximum diameter of the present study almost consistent with previous studies. However, the confirmation of cavity disappearance was 217.88 ± 78.57 d (range: 111–510 d), significantly longer than that of RFA ablation (111.9 ± 64.9 d; range: 41 − 274 d). This is probably due to MWA having a larger ablation range than other thermal ablation techniques [Citation29–32]. The incidence of complications requiring catheter drainage was 17/31 (54.84%), including 7/31 (22.58%) cases of pneumothorax, 3/31 (9.68%) cases of pleural effusion, 4/31 (12.9%) cases of hydropneumothorax, and 3/31 (9.68%) cases of bronchopleural fistula. Among these, four cases presented as delayed and severe hydropneumothorax, possibly because early enlarging cavities are more likely to cause extensive exudation and rupture of the pleura [Citation20,Citation33]. Two cases had a bacterial infection, and three cases had a fungal infection, all of which occurred several months after the cavity, accompanied by high fever, cough, and sputum [Citation34]. Patients with bacterial and fungal infections were cured with sensitive antibiotics and voriconazole. In addition, three patients developed extensive subcutaneous emphysema immediately after early enlarging cavitation with pneumothorax, which was considered a bronchopleural fistula [Citation35–38]. Two patients were cured with continuous negative pressure closed drainage with 10 F drainage tube plus a chest area skin incision (a 2-cm incision) for decompression for 14 days. Another case underwent treatment with a 10 F drainage tube and surgical “mushroom head” drainage, while the subcutaneous emphysema continued to worsen. This case was not successfully treated until bronchoscopy treatment [Citation39], with hospitalization for 38 days. These served complications developed a longer hospitalization in the cavity group. Until 31 December 2022, 27 cavities disappeared; in addition, there were two cavities persisted and two cavities were lost to follow-up.

There are some limitations of this study. First, this is a single-center retrospective study with a small number of cases, and two patients were lost to follow-up. Second, because a CT examination is not routinely performed on a postprocedural 7th day, we could not accurately determine whether the patients with early enlarging cavitation without symptoms were enrolled in the study or incorrectly grouped. Third, the analysis did not include medical treatment during the cavity, such as antibiotics and corticosteroids.

In conclusion, early enlarging cavitation occurred in 5.57% of PLC after the MWA procedure, which is more likely to develop into delayed hydropneumothorax and bronchopleural fistula, causing a longer hospitalization. Therefore, watchful follow-up and proper management should be taken to reduce the risk of these severe complications. The significant risk factors of early enlarging cavitation include lesion contact with a vessel of ≥3 mm in diameter and lesion contact with a bronchus of ≥2 mm in diameter, as well as a larger ablated parenchymal volume.

Author contributions

Wang Nan and Jingwen Xu contributed equally to this article. Nan Wang was responsible for acquiring the data and drafting the manuscript. Nan Wang and Jingwen Xu were responsible for the analysis of the data. Pikun Cao, Zhichao Li, Guoliang Xue, Yanting Hu, Cuiping Han, Haitao Zhang, and Wenhua Zhao provided and collected the clinical data. Yang Xia, Zhigang Wei, and Xin Ye were responsible for designing the experiments and supervising the study. All the listed authors have made a substantial, direct, and intellectual contribution to the work and have approved it for publication.

Disclosure statement

The authors report there are no competing interests to declare.

Additional information

Funding

This work was supported by National Natural Science Foundation of China (81502610 and 82072028) and Shandong Provincial Natural Science Foundation, China (ZR2020MH294 and ZR2021MH143). This study has also received funding from the Project funded by China Postdoctoral Science Foundation 2022M711979 and academic promotion program of Shandong First Medical University (2019LJ005).

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