This monocentric prospective study was performed at our institution from January 2020 to December 2020. This study was approved by the local Institutional Review Board and informed consent was obtained from all eligible patients. After a multidisciplinary meeting that included surgeons, interventional radiologists, pathologists and oncologists, 104 consecutive patients who were recommended to receive MWA for pulmonary malignancies were assessed. Patients aged > 18 years, with a diagnosis of primary and metastatic lung malignancies by pathology, with less than five lesions, and with tumors that had a maximum diameter ≤5cm were included. Exclusion criteria included: 1) those with contraindications of MWA; 2) received chemotherapy, immunotherapy, radiotherapy or MWA within three months before enrollment in the study or within one month after MWA; 3) experienced severe complications from MWA such as infections or massive bleeding; 4) received glucocorticoid treatment or immunosuppressive therapy; 5) declined to participate in the study.

The day when MWA was performed was defined as D1. Patients’ peripheral blood samples were collected on the day before (D0) and one month (M1) after MWA, and were used to measure changes in immune cell subsets and cytokines. All patients received a clinical follow-up (Figure 1).

Percutaneous MWA was performed by an experienced interventional radiologist under the guidance of a dual-source computed tomography (CT) system (SOMATOM Force, Siemens Healthcare, Forchheim, Germany) with local anesthesia (lidocaine). The MWA system (MTC-3C, Vison-China Medical Devices R&D Center, Nanjing, China) consisted of a 2450 ± 50MHz generator generating a maximum of 100 W and a water-cooled antenna (outer diameter was 16G or 18G and effective length was 150 mm or 180 mm).

Tumors with a maximal diameter less than 3 cm were treated with a single applicator that was placed in the center of the tumor. To ensure the ablation range completely covered the tumors and 0.5 cm around each tumor, tumors with a diameter larger than 3 cm were treated using two to three different sites based on tumor size and shape. A second or even third ablation was performed for those with a progression in ablation lesions.

A tumor treated according to the protocol and with a patient who was completely recovered as determined at the time of the procedure was deemed a “technical success”.

All patients received a chest CT scan the next day after MWA to assess for pneumothorax or hemorrhage. Analgesics and antipyretics were prescribed if necessary.

Peripheral blood samples were collected from patients on D0 and M1. T cells (CD3+), CD4+ T cells (CD3+CD4+), CD8+ T cells (CD3+CD8+), B cells (CD3-CD19+), natural killer (NK) cells (CD3-CD16+CD56+), CD20+ B cells (CD45+CD3-CD20+), and Treg cells (CD3+CD4+CD25+CD127-) were analyzed by flow cytometry (BD FACSCalibur, BD Biosciences, Sparks, MD, USA) using BD Multi-TEST IMK Kits (BD Biosciences, Shanghai, China). Levels of cytokines: interleukin-2 (IL-2), IL-4, IL-6, IL-10, IL-17A, tumor necrosis factor-alpha (TNF-α), and IFN-γ were determined by a Luminex 200 analyzer (Shanghai Tellgen Life Science Co. Ltd, Shanghai, China) using cytokine detection kits (Bio-Rad, Hercules, CA, USA).

For primary lung malignancies, patients with sensitive mutations were treated with epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) or anaplastic lymphoma (ALK) kinase inhibitors. For those without mutations, platinum-based chemotherapy was administered. For patients with metastatic lung malignancies, systematic chemotherapy was administered according to the pathology of the primary tumor. For all patients, if local therapy (such as radiotherapy, a second thermal ablation, transhepatic arterial chemotherapy, and embolization) or immunotherapy was necessary, such treatments were administered one month after MWA.

Treatment-related complications were noted within 30 days after ablation, and were classified in accordance with the Common Terminology Criteria for Adverse Events version 4.0 (24). Patients with major complications were excluded from the study.

Patient follow-up was conducted at 1 month and every 3 months thereafter until death or the last follow-up. Follow-up evaluation included physical examination, laboratory testing, and contrast-enhanced imaging (ultrasound, CT, magnetic resonance imaging, or positron emission tomography).

Technique efficacy referred to a defined prospective time point when “complete ablation” of the macroscopic tumor was achieved as noted on follow-up imaging. Complete ablation was defined as lesion disappearance, complete cavitation formation, fibrotic progression or scar formation, solid nodule involution or no change, and/or atelectasis presenting as no contrast-enhanced signs on follow-up CT images. Incomplete ablation was indicated by incomplete cavernous formation with several remaining solid or liquid components; partial fibrosis or fibrotic lesions with solid residues; and/or solid nodules with unchanged or increased size displaying irregular peripheral or internal enhancement signs on follow-up CT images.

Treatment response was evaluated according to the Response Evaluation of Criteria in Solid Tumors version 4.0 (25). Progression-free survival (PFS), was defined as the interval between initial treatment and disease progression or death.

Statistical software (SPSS version 19.0; SPSS, Chicago, IL, USA) was used for analysis. Continuous variables were presented as mean ± standard deviation and compared by paired sample t test. Categorical variables were presented as frequencies and compared using the chi-square test. Univariate and multivariate analyses were used to identify factors associated with PFS. Progression-free survival was analyzed using Kaplan–Meier curves and a log-rank test. A Cox proportional hazards model was used to examine risk factors associated with PFS. Variables with a p value less than 0.1 were entered into the multivariate model. The relationship among those variables was analyzed by using spearman correlation coefficients. A p value less than 0.05 indicated a significant difference.

Between January 2020 and December 2020, 104 consecutive patients were assessed. A total of 24 eligible patients were enrolled in the study. Technical success was achieved in all 24 patients. One patient was excluded due to experiencing pneumonia after MWA. Another patient was excluded due to a lack of M1 blood sample. Finally, 22 patients finished the study. Table 1 summarizes the main characteristics of the studied population.

The variations in immune cell subsets and cytokines after MWA are shown in Table 2 and Figures 2A , B. The proportion of CD8+ T cells increased from 22.95 ± 7.38% (D0) to 25.95 ± 9.16% (M1) (p = 0.031). An increase in the proportion of CD8+ T cells was observed in 15 (68.2%) patients. The proportion of Treg cells decreased from 10.82 ± 4.52% (D0) to 8.77 ± 2.05% (M1) (p = 0.049). A decrease in proportion of Treg cells was observed in 15 (68.2%) patients. The IL-2 concentration at M1 was significantly lower than that at D0 (1.26 ± 0.60 pg/mL vs. 1.58 ± 0.46 pg/mL, p = 0.028). A decrease in the IL-2 concentration was observed in 13 (59.1%) patients. Other immune cell subsets and cytokines showed no significant difference between D0 and M1.

No major post-treatment complications occurred in all patients after MWA. The average follow-up interval was 12.68 ± 7.56 (3 – 24) months. Technical efficacy was achieved in 16 patients (72.7%) during follow-up. One patient was lost to follow-up 15 months after MWA without disease progression. During the follow-up, a total of 16 patients (72.7%) showed disease progression.

In a Cox regression analysis of variables potentially associated with PFS (Table 3), gender (hazard ratio [HR] = 0.22, 95% confidence interval [CI]: 0.06–0.82), stage (HR = 4.26, 95% CI: 0.88–20.71), PS (HR = 1.98, 95% CI: 1.15–3.42), technical efficacy (HR = 0.31, 95% CI: 0.10–0.97), and a reduction in the proportion of Treg cells (HR = 3.30, 95% CI: 1.06–10.25) were significantly associated with PFS in univariate analysis. No linear correlation was found among the variables that entered into the multivariate model according to spearman correlation analysis (all p > 0.05). In multivariate analysis, a reduction in the proportion of Treg cells (HR = 4.97, 95% CI: 1.32–18.67, p = 0.018) was independently associated with PFS.

Patients with an absolute percentage decrease in the proportion of Treg cells over the average showed longer PFS (median PFS: 16.0 months) than patients with an absolute percentage decrease below the average (median PFS: 8.5 months) (Figure 3A, log-rank test: p = 0.025. Figure 3B, showed a case of a reduction in proportion of Treg cells predict PFS).