Cryoablation Combined with Programmed Cell Death Protein 1 Inhibitor Pembrolizumab for Advanced Non-small Cell Lung Cancer

Cryoablation Combined with Programmed Cell Death Protein 1 Inhibitor Pembrolizumab for Advanced Non-small Cell Lung Cancer | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Cryoablation Combined with Programmed Cell Death Protein 1 Inhibitor Pembrolizumab for Advanced Non-small Cell Lung Cancer Li Wang, Guotai Wang, Hubao Yuan, Zhongqiang Cao, Xiaodong Yang, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4831983/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Introduction: To evaluate the efficacy and safety of cryoablation combined with pembrolizumab treatment versus cryoablation alone in patients with advanced non-small cell lung cancer (NSCLC). Methods This retrospective study was conducted from February 2018 and October 2021. A total of 90 patients with NSCLC (AJCC stage IIIB/IV) were included, with 36 patients receiving cryoablation combined with pembrolizumab (Group A) and 54 patients receiving cryoablation alone (Group B). The primary outcome measures included objective response rate (ORR), overall survival (OS), and progression-free survival (PFS), immune responses and adverse events serving as secondary endpoints. Risk factors for OS and PFS were identified using univariate and multivariate analyses. Results No treatment related deaths were observed. Group A demonstrated a higher ORR (75.0% vs. 61.1%), longer median OS (28.1 months vs. 24.2 months), and longer median PFS (12.8 months vs. 8.4 months) compared to Group B. Additionally, Group A showed significant increases in CD3+, CD4+, and CD8 + T cells, and elevated levels of IL-2, IL-6, TNF-β, and IFN-γ. The multivariate analysis showed the combination of cryoablation and pembrolizumab was an independent prognostic factor for OS and PFS. Conclusions : Cryoablation combined with pembrolizumab significantly improves clinical outcomes in advanced NSCLC patients compared to cryoablation alone, highlighting the potential of this combination therapy in enhancing anti-tumor immunity and prolonging survival. Cryoablation Programmed cell death protein 1 Inhibitor Pembrolizumab Advanced non-small cell lung cancer Combination Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Non-small cell lung cancer (NSCLC) represents approximately 85% of all lung cancer cases and remains a leading cause of cancer-related mortality worldwide ( 1 ). Despite advances in targeted therapies and immunotherapies, the prognosis for patients with advanced NSCLC remains poor, with a 5-year survival rate of less than 5% for stage IV disease ( 2 – 4 ). Traditional treatment modalities, including chemotherapy and radiotherapy, have limited efficacy and are often associated with significant toxicity ( 5 – 7 ). Therefore, there is an urgent need for novel therapeutic strategies that can improve outcomes for these patients. Cryoablation, a minimally invasive procedure that uses argon to destroy cancer cells, has emerged as a promising local treatment for various malignancies, including NSCLC ( 8 – 10 ). This technique not only induces direct tumor cell death but also has the potential to elicit systemic anti-tumor immune responses ( 11 – 13 ). However, the efficacy of cryoablation as a monotherapy is often limited by incomplete tumor eradication and the potential for residual disease. Combining cryoablation with immunotherapy, such as immune checkpoint inhibitors, may enhance the anti-tumor immune response and improve clinical outcomes. Programmed death-1 (PD-1) inhibitors, such as pembrolizumab, have revolutionized the treatment landscape for advanced NSCLC by blocking the interaction between PD-1 and its ligands, PD-L1 and PD-L2, thereby restoring T-cell activity and promoting anti-tumor immunity ( 14 , 15 ). Pembrolizumab has demonstrated significant clinical benefits in patients with PD-L1-positive NSCLC, leading to its approval as a first-line treatment for this population ( 16 , 17 ). However, the response rates to pembrolizumab monotherapy remain suboptimal, particularly in patients with low or negative PD-L1 expression ( 18 ). Given the complementary mechanisms of action of cryoablation and pembrolizumab, we hypothesize that the combination of these two modalities may synergistically enhance anti-tumor immunity and improve clinical outcomes in patients with advanced NSCLC. This study aims to evaluate the efficacy and safety of cryoablation combined with pembrolizumab compared to cryoablation alone in patients with advanced NSCLC. We will assess tumor response, progression-free survival (PFS), overall survival (OS), and immune-related biomarkers to elucidate the potential benefits of this combination therapy. In summary, this study addresses a critical clinical need by exploring a novel combination therapy for advanced NSCLC. By integrating cryoablation with pembrolizumab, we aim to enhance the anti-tumor immune response and improve patient outcomes. The findings from this study could provide valuable insights into the potential of combining local and systemic therapies for the treatment of advanced NSCLC. Methods Patients This retrospective study was approved from the Institutional Ethic Committee of Affiliated Hospital of Shaanxi University of Chinese Medicine and conducted in accordance with the Declaration of Helsinki and the Declaration of Good Clinical Practice. Written informed consent was obtained from each participant. From February 2018 and October 2021, all patients with advanced NSCLC underwent either a combination of cryoablation + pembrolizumab (group A) or cryoablation alone (group B). The inclusion criteria were as follows: ( 1 ) Histologically and radiologically verified advanced NSCLC; ( 2 ) Stage IIIB/IV, according to the American Joint Committee on Cancer (AJCC) criteria; ( 3 ) Patients over 18 years; ( 4 ) Sufficient bone marrow, laboratory examination of hepatic, renal and ardio-pulmonary function; ( 5 ) Relapsed after multimodal treatment (surgical resection, radiotherapy, or disease progressed after chemotherapy); ( 7 ) Life expectancy of at least 3 months; ( 6 ) Patients received pembrolizumab therapy for at least 1month after cryoablation in this study. The exclusion criteria were as follows: ( 1 ) Patients with distant metastases; ( 2 ) previous received anti-PD-1/PD-L1 therapy; ( 3 ) platelet count of less than 50×10 9 /L; ( 4 ) Severe impairment of pulmonary functions with the maximum voluntary ventilation less than 40%. Treatment Procedure Cryoablation Preoperative imaging was performed to select supine, lateral or oblique position. Intraoperative real-time monitoring of the patient's blood pressure, oxygen saturation, heart rate, and electrocardiogram. Preoperative intravenous access was established for preoperative and intraoperative medications. Under the guidance of CT combined with ultrasound, we determined the location and path of the puncture needle, and planned the level, angle and depth of the puncture into the tumor. Thus, important tissue structures such as the heart, large blood vessels, and trachea were avoided to ensure surgical safety. Local aesthesia was applied at the puncture point with 1% lidocaine injection. The cryoprobe was inserted into the target according to the needle plan designed, and then confirmed by CT/MR scanning. The type and number of cryoprobes are selected according to the morphology, size and location of the lesion, with the aim of obtaining full coverage of the tumor with the "ice ball". Rapid freezing with argon for 15–20 min, rapid rewarming with helium for 2–5 min, and repeated freezing and rewarming were performed. The decision to increase the freezing time was made according to the coverage of the lesion by the "ice ball" on imaging. When the edge of the "ice ball" exceeds the lesion by more than 1cm, it is heated and the cryoprobe is withdrawn. CT or MR scanning was performed for complications such as pneumothorax and hemorrhage. Pembrolizumab Patients in group A received pembrolizumab therapy (100 mg/3w). The first dose of pembrolizumab was administered within 2 weeks after cryoablation. Patients received continuous pembrolizumab treatment until intolerance toxicity or progressive disease. The treatment protocol is illustrated in Fig. 1 B. PD-L1 expression analysis PD- L1 expression was assessed using a proprietary immunohistochemistry assay. We classified PD-L1 expression into three levels: negative PD-L1 expression (TPS < 1%), low PD-L1 expression (TPS 1–49%), and high PD-L1 expression (TPS ≥ 50%). Flow cytometric analysis Blood samples of each patient’s blood (2 ml) were collected just before the cryoablation procedure (Pretreatment) and 30 days (Posttreatment) after cryoablation procedure. Flow cytometry techniques (FACS caliber, four-color system, BD Bioscience, CA, US) were used to analyze the absolute number of CD3 + T cells (CD3 + ), CD4 + T cell (CD3 + CD4 + ), CD8 + T cell (CD3 + CD8 + ), Treg cell (CD4 + CD25 + CD127dim). A similar method was used to measure serum cytokines, including interleukin-2 (IL-2), interleukin-6 (IL-6), tumor-necrosis factor-beta (TNF-β), and interferon-γ (IFN-γ). Collection of clinical data The tumor response was evaluated based on the mRECIST ( 28 ). The objective response rate (ORR) was calculated as follows: ORR = (CR + PR cases) / total cases × 100%. Follow-up and endpoints assessment All patients in this study were followed up at 1-, 3-, and 6-months posttreatment and then every three months. Two radiologists independently interpreted all follow-up scan results. Adverse events were evaluated and classified according to the National Cancer Institute Common Terminology Criteria for Adverse Events, version 4.0. The last follow-up was completed on June 16, 2023. The tumor response was assessed using Response Evaluation Criteria in Solid Tumors, version 1.1 (RECIST v1.1)( 19 ). The objective response rate (ORR) after 3months posttreatment was calculated which is defined as the percentage of patients with a best overall response of complete response (CR) or partial response (PR). Overall survival (OS) was defined as the time from diagnosis to death or until the last follow-up. progression-free survival (PFS) was defined as the time from diagnosis to disease progression until the last follow-up. Adverse events, including immune-mediated adverse events, were collected throughout the treatment until 90 days after the last infusion of pembrolizumab. All Adverse events were graded based on the NCI Common Terminology Criteria for Adverse Events v5.0. Statistical analysis All statistical analyses were performed and compiled using GraphPad Prism (version 8.01, GraphPad Software, San Diego, CA, USA) and SPSS software (version 20.0, SPSS Inc., Chicago, IL, USA). Categorical variables were compared using the chi-square or Fisher exact test. The continuous data, categorical data, and survival curves of the two groups were compared using the Mann-Whitney test, Fisher's exact test, and log-rank (Mantel-Cox) tests individually. The Cox regression model was used in survival analysis to assess the influence factor of OS and PFS. All statistical tests were two-sided; a p -value < 0.05 was considered statistically significant. Results Patient Characteristics Between February 2018 and October 2021, a total of 387 advanced NSCLC patients were included in this study (Fig. 1 ). Based on the exclusion criteria, totally 297 patients were excluded due to receiving other treatment ( n = 138), tumor metastasis ( n = 106), incomplete data, or missing ( n = 48). A total of 90 advanced NSCLC patients were finally evaluated in this study: Group A ( n = 36) and Group B ( n = 54) (Fig. 1 A). The baseline characteristics were well balanced between the two groups (Table 1 ). The median ages were 58 (29–80) and 54 (33–78) in Group A and B, respectively. Male patients had a higher risk than female patients between the two groups. The median tumor sizes were 4.6 cm (range 2.9–8.5) and 4.8 cm (range 3.1–8.7) for patients in groups A and B, respectively. The majority of patients were nonsquamous histology (81.1%) and had former or current smokers (82.2%). In the 90 patients who underwent a PD-L1 expression assay before treatment, 12 (13.3%) patients had PD-L1 negative expression, and 56 (62.2%) had PD-L1 positive expression, including 29 (32.2%) patients more than 50% (≥ 50%) PD-L1 expression (Table 1 ). Table 1 Patients characteristics. Characteristic Group A ( n = 36) Group B ( n = 54) P Value Age, year 0.326 ≤ 60 25 32 > 60 11 22 Sex Male 26 35 0.461 Female 10 19 Tumor size (cm) 0.666 2 ~ 5 14 24 ≥5 22 30 Tumor pattern 0.660 Squamous 6 11 Nonsquamous 30 43 TNM stage 0.648 IIIA 11 19 IV 25 35 Smoking status 0.736 Former or current 29 45 Never 7 9 PD-L1 TPS 0.371 ≥50% 9 20 ≥ 1% and < 50% 11 16 < 1% 4 8 Unknown 12 10 Preoperative therapy 0.702 Surgery 14 21 Chemotherapy 10 10 Radiotherapy 9 16 Others 3 7 TNM, tumor node metastasis; PD-L1, programmed death-ligand 1; TPS, tumor proportion score Adverse Events As shown in Table 2 , there was no treatment-related mortality in either group in our study. Overall, the frequency of adverse events was similar between the two groups. The most common cryoablation treatment-related adverse events were pain (61.1%), nausea (63.9%), vomiting (58.3%) and pneumothorax (33.3%) in group A. The major (grade 3 ~ 4) cryoablation-related adverse events were pneumothorax (5.6%) and hemorrhage (2.7%) in Group A (Table 2 ). All adverse events were relieved or improved after symptomatic treatment. The most common immune-related adverse events were fatigue (19.4%), pruritus (13.8%), diarrhea (11.1%), nausea (11.1%). The major (grade 3 ~ 4) immune-related adverse events were fatigue (2.7%), pruritus (2.7%), AST increased (2.7%), lipase increased (2.7%), ALT increased (2.7%) in group A, which was resolved after the treatment with a corticosteroid. There were no immune-related adverse events in group B (Table 3 ). Table 2 Adverse events related to cryoablation treatment. Characteristic Group A (n = 36) Group B (n = 54) All Grade Grade 3/4 All Grade Grade 3/4 Pain 22 (61.1%) 0 36 (66.7%) 0 Nausea 23 (63.9%) 0 28 (51.9%) 0 Vomiting 21 (58.3%) 0 32 (59.3%) 0 Pneumothorax 12 (33.3%) 2 (5.6%) 17 (31.5%) 4 (7.4%) Pleural effusion 7 (19.4%) 0 15 (27.8%) 0 Hemorrhage 9 (25.0%) 1 (2.7%) 22 (40.7%) 2 (3.7%) Infection 1 (2.7%) 0 2 (3.7%) 0 Table 3 Adverse events related to immune treatment. Adverse event Group A (n = 36) All Grade Grade 3/4 Fatigue 7 (19.4%) 1 (2.7%) Pruritus 5 (13.8%) 1 (2.7%) Diarrhea 4 (11.1%) 0 Nausea 4 (11.1%) 0 Decreased appetite 2 (5.6%) 0 Hypothyroidism 2 (5.6%) 0 Cough 2 (5.6%) 0 Fever 2 (5.6%) 0 AST increased 2 (5.6%) 1 (2.7%) Lipase increased 1 (2.7%) 1 (2.7%) ALT increased 1 (2.7%) 1 (2.7%) Pneumonitis 1 (2.7%) 0 AST, aspartate transaminase; ALT, alanine aminotransferase Response Overall, 11 (30.6%) patients achieved CR, 16 (44.4%) patients received PR, 6 (16.7%) patients developed SD, and 3 (8.3%) patients suffered PD in group A. The tumor response in group B was 10 (18.5%) patients with CR, 23 (42.6%) patients with PR, 9 (16.7%) patients with SD and 12 (22.2%) with PD. The ORR was 75.0% vs. 61.1% ( p = 0.045, Table 4 ) in groups A and B, respectively. Table 4 Tumor response 3 months posttreatment Characteristic Group A (n = 36) Group B (n = 54) P Value CR 11(30.6%) 10(18.5%) PR 16(44.4%) 23(42.6%) SD 6(16.7%) 9(16.7%) PD 3(8.3%) 12(22.2%) ORR 27(75%) 33(61.1%) 0.045 CR, complete response; PR, partial response; SD, stable disease; PD, progressive disease; ORR, objective response rate Immune parameters We compared the absolute number of immune cells after treatment to investigate possible effects on the tumor immune microenvironment of each treatment. The results demonstrated that the absolute number of CD3 + T cells ( p = 0.033), CD4 + T cells ( p = 0.012), and CD8 + T cells ( p = 0.003) steadily increased after treatment in group A comparing to group B (Figs. 2 A–C), while the proportion of Tregs cells ( p = 0.035) decreased (Figs. 2 D). To further investigate the variations of the immune microenvironment, we compared the levels of cytokines in both groups. The data suggested that the IL2 ( p = 0.009), IL-6 ( p = 0.000), TNF-β ( p = 0.001), and IFN-γ ( p = 0.014) levels significantly increased after treatment in group A comparing to group B (Fig. 3 ). Survival The median follow-up time was 24.6 months (9.9–41.4 months). The median OS from diagnosis was significantly longer in group A than in group B, respectively (28.1 months vs. 24.2 months, p = 0.009, Fig. 4 A). Similarly, the median PFS from diagnosis was significantly longer in group A than in group B, respectively (12.8 months vs. 8.4 months, p = 0.011, Fig. 4 B). Univariate and Multivariate Analyses of OS and PFS Univariate Cox regression analysis and multivariate Cox regression analysis were used to evaluate the predictors of OS and PFS. As shown in Table 5 , tumor size (HR: 1.024, 1.045–1.038, p = 0.025), tumor pattern (HR: 0.645, 0.328–1.525, p = 0.048), PD-L1 TPS (HR: 0.975, 0.415–2.045, p = 0.038), cryoablation plus pembrolizumab (HR: 0.447, 0.238–0.765, p = 0.015) were significantly correlated with OS in the univariate Cox regression analysis. Multivariate analysis showed that the tumor size (HR: 2.056, 1.010–4.256, p = 0.046), PD-L1 TPS (HR: 0.172, 0.042–0.746, p = 0.028) and cryoablation plus pembrolizumab (HR: 0.472, 0.285–0.778, p = 0.006) were predictors of OS (Table 5 ). Furthermore, PD-L1 TPS (HR: 0.488, 0.214–1.425, p = 0.045) and cryoablation plus pembrolizumab (HR: 1.757, 0.965–3.287, p = 0.047) were also significant prognostic factors for PFS (Table 6 ). Table 5 Univariate and multivariate analyses of OS in patients Characteristic Univariate analysis Multivariate analysis HR (95%CI) P value HR (95%CI) P value Age (> 60/≤60) 0.724 (0.146–0.786) 0.356 Sex (female/male) 0.354 (0.278–0.628) 0.758 Tumor size (≥ 5/2 ~ 5) 1.024 (1.045–1.038) 0.025 2.056 (1.010–4.256) 0.046 Tumor pattern (squamous/nonsquamous) 0.645 (0.328–1.525) 0.048 0.024 (0.014–0.524) 0.067 TNM stage 0.987 (0.648–1.546) 0.983 Smoking status (Current/former/Never) 1.012 (0.985–1.41) 0.225 PD-L1 TPS (≥ 50%/1–49%) 0.975 (0.415–2.045) {Novello, 2023 #8} 0.172 (0.042–0.746) 0.028 Preoperative therapy (surgery/chemotherapy/ radiotherapy/others) 1.345 (0.789–2.458) 0.178 Cryoablation (with pembrolizumab/without pembrolizumab) 0.447 (0.238–0.765) 0.015 0.472 (0.285–0.778) 0.006 OS, overall survival; PD-L1, programmed death-ligand 1; HR, hazard ratio; CI, confidence interval; Table 6 Univariate and multivariate analyses of PFS in patients Characteristic Univariate analysis Multivariate analysis HR (95%CI) P value HR (95%CI) P value Age (> 60/≤60) 1.145 (0.561–2.110) 0.656 Sex (female/male) 0.351 (0.212–0.653) 0.751 Tumor size (≥ 5/2 ~ 5) 0.714 (0.484–1.564) 0.428 Tumor pattern (squamous/nonsquamous) 1.124 (0.748–1.658) 0.572 TNM stage 0.942 (0.631–1.412) 0.752 Smoking status (Current/former/Never) 0.946 (0.645–1.412) 0.746 PD-L1 TPS (≥ 50%/1–49%) 0.625 (0.258–1.445) 0.021 0.488 (0.214–1.425) 0.045 Preoperative therapy (surgery/chemotherapy/ radiotherapy/others) 0.615 (0.378–1.224) 0.208 Cryoablation (with pembrolizumab/without pembrolizumab) 1.825 (1.045–3.275) 0.013 1.757 (0.965–3.287) 0.047 PFS, progression free survival; PD-L1, programmed death-ligand 1; HR, hazard ratio; CI, confidence interval; Discussion In this study, we evaluated the efficacy and safety of combining cryoablation with pembrolizumab treatment compared to cryoablation alone in patients with advanced NSCLC. Our main findings indicate that the combination therapy significantly improved ORR and prolonged OS and PFS compared to cryoablation alone. Specifically, patients receiving the combination therapy exhibited a more favorable immune profile, as evidenced by increased levels of CD3 + T cells, CD4 + T cells, and CD8 + T cells, along with elevated serum cytokines such as IL-2 and IFN-γ. These results align with our initial hypothesis that the addition of pembrolizumab, an anti-PD-1 monoclonal antibody, would enhance the anti-tumor immune response initiated by cryoablation. Furthermore, the safety profile of the combination therapy was manageable, with adverse events consistent with those previously reported for pembrolizumab and cryoablation treatments ( 16 , 20 ). These findings suggest that the integration of cryoablation and pembrolizumab could offer a promising therapeutic strategy for patients with advanced NSCLC. The primary novelty of this study lies in its comprehensive evaluation of the combined treatment of cryoablation and pembrolizumab in NSCLC patients, a therapeutic approach that has not been extensively explored in previous research. This study uniquely integrates cryoablation, a minimally invasive procedure that induces tumor cell death through argon, with pembrolizumab, an immune checkpoint inhibitor targeting PD-1. The combination aims to enhance anti-tumor immunity by not only directly reducing tumor burden but also by potentially increasing the immunogenicity of the tumor microenvironment. Our findings demonstrate a significant improvement in both OS and PFS in the combination therapy group compared to cryoablation alone, with median OS of 28.1 months versus 24.2 months and median PFS of 12.8 months versus 8.4 months, respectively. This is consistent with the hypothesis that cryoablation can act as an in situ vaccine, enhancing the efficacy of subsequent immunotherapy ( 21 – 23 ). Furthermore, the observed increase in CD3+, CD4+, and CD8 + T cells, along with elevated levels of cytokines such as IL-2, IL-6, TNF-β, and IFN-γ, supports the notion of an augmented immune response post-treatment. These results align with previous studies that have suggested the potential of combining local ablative therapies with systemic immunotherapies to achieve synergistic effects ( 11 , 24 , 25 ). However, our study is among the first to provide robust clinical evidence in a human cohort, thereby filling a critical gap in the current understanding of multimodal treatment strategies for advanced NSCLC ( 16 , 26 ). The clinical implications of our study are significant, particularly in the context of treatment strategies for advanced NSCLC. These findings suggest that integrating immunotherapy with local ablative techniques can enhance the anti-tumor immune response, potentially leading to better clinical outcomes. This is particularly relevant given the limited treatment options and poor prognosis associated with advanced NSCLC. The observed increase in CD3 + T cells, CD4 + T cells, and CD8 + T cells, along with elevated levels of cytokines such as IL-2, IL-6, TNF-β, and IFN-γ, underscores the immunomodulatory effects of this combination therapy. These results align with previous studies that have highlighted the synergistic effects of combining local therapies with immune checkpoint inhibitors ( 14 , 27 , 28 ). However, our study is limited by its retrospective nature and the relatively small sample size. Future research should focus on conducting large-scale, multicenter randomized controlled trials to validate our findings and explore the underlying mechanisms of this combined treatment approach. Additionally, longer follow-up periods are necessary to assess the long-term efficacy and safety of this therapeutic strategy. Understanding the molecular and immunological changes induced by this combination therapy could pave the way for more personalized and effective treatment regimens for NSCLC patients. In conclusion, our study demonstrates that the combination of cryoablation and pembrolizumab therapy significantly improves the clinical outcomes in patients with advanced non-small cell lung cancer (NSCLC) compared to cryoablation alone. These findings suggest that the synergistic effect of cryoablation and immunotherapy could potentially overcome the limitations of monotherapy in treating advanced NSCLC. Declarations Conflicts of interest All authors report no conflicts of interest in this work. FUNDING This study was supported by the Project of Shaanxi Provincial Department of Science and Technology (No. 2022SF-513). Author Contribution SW and PW designed the project and edited the manuscript. LW, GW, HY, ZC and XY conducted study selection, dataextraction, statistical analyses, and drafted the main manuscript. All authors contributed to the article and approved thesubmitted version. Acknowledgements We thank all patients and their families for their help and support. We thank all my colleagues for their assistance. Availability of data and material The dataset generated during and/or analyzed during this study are available from the corresponding author on reasonable request. References Siegel RL, Giaquinto AN, Jemal A, Cancer Statistics. 2024. CA Cancer J Clin (2024) 74(1):12–49. 10.3322/caac.21820 Herbst RS, Morgensztern D, Boshoff C. The Biology and Management of Non-Small Cell Lung Cancer. Nature. 2018;553(7689):446–54. 10.1038/nature25183 . Jonna S, Subramaniam DS. Molecular Diagnostics and Targeted Therapies in Non-Small Cell Lung Cancer (Nsclc): An Update. Discov Med. 2019;27(148):167–70. 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Rate of Freeze Alters the Immunologic Response after Cryoablation of Breast Cancer. Ann Surg Oncol. 2010;17(4):1187–93. 10.1245/s10434-009-0846-1 . Yu Z, Wang D, Qi Y, Liu J, Zhou T, Rao W, et al. Autologous-Cancer-Cryoablation-Mediated Nanovaccine Augments Systematic Immunotherapy. Mater Horiz. 2023;10(5):1661–77. 10.1039/d3mh00092c . den Brok MHMGM, Sutmuller RPM, Nierkens S, Bennink EJ, Toonen LWJ, Figdor CG, et al. Synergy between in Situ Cryoablation and Tlr9 Stimulation Results in a Highly Effective in Vivo Dendritic Cell Vaccine. Cancer Res. 2006;66(14):7285–92. Seguin J, El Hajjam M, Legagneux J, Diakhaby S, Mignet N, Boudy V, et al. Radiofrequency Combined with Intratumoral Immunotherapy: Preclinical Results and Safety in Metastatic Colorectal Carcinoma. Pharmaceutics. 2024;16(3). 10.3390/pharmaceutics16030315 . Wang J, Zong D, Dong S, Gao S, Yang Y, Zhang P, et al. Argon-Helium Knife Cryoablation Plus Programmed Cell Death Protein 1 Inhibitor in the Treatment of Advanced Soft Tissue Sarcomas: There Is No Evidence of the Synergistic Effects of This Combination Therapy. Front Oncol. 2023;13:1185291. 10.3389/fonc.2023.1185291 . Brahmer J, Reckamp KL, Baas P, Crinò L, Eberhardt WEE, Poddubskaya E, et al. Nivolumab Versus Docetaxel in Advanced Squamous-Cell Non-Small-Cell Lung Cancer. N Engl J Med. 2015;373(2):123–35. 10.1056/NEJMoa1504627 . Brahmer JR, Tykodi SS, Chow LQM, Hwu W-J, Topalian SL, Hwu P, et al. Safety and Activity of Anti-Pd-L1 Antibody in Patients with Advanced Cancer. N Engl J Med. 2012;366(26):2455–65. 10.1056/NEJMoa1200694 . Wang J, Ge H, Tian Z. Immunotherapy Plus Radiotherapy for the Treatment of Sarcomas: Is There a Potential for Synergism? Onco Targets Ther. 2023;16:385–97. 10.2147/OTT.S410693 . Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4831983","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":347939386,"identity":"f28dfd84-c0f4-46a5-bcca-6e87d0b816dc","order_by":0,"name":"Li Wang","email":"","orcid":"","institution":"Shaanxi University of Chinese Medicine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Li","middleName":"","lastName":"Wang","suffix":""},{"id":347939387,"identity":"5fd5f216-8bd6-476c-8032-6227337484a0","order_by":1,"name":"Guotai Wang","email":"","orcid":"","institution":"Affiliated Hospital of Shaanxi University of Chinese Medicine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Guotai","middleName":"","lastName":"Wang","suffix":""},{"id":347939388,"identity":"8c04203e-7757-4bcc-9dfa-540661155263","order_by":2,"name":"Hubao Yuan","email":"","orcid":"","institution":"Affiliated Hospital of Shaanxi University of Chinese Medicine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Hubao","middleName":"","lastName":"Yuan","suffix":""},{"id":347939389,"identity":"8fe8b548-e287-4699-bbbf-69d081d25ac8","order_by":3,"name":"Zhongqiang Cao","email":"","orcid":"","institution":"Affiliated Hospital of Shaanxi University of Chinese Medicine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Zhongqiang","middleName":"","lastName":"Cao","suffix":""},{"id":347939390,"identity":"3e937286-f8b7-472d-9cfa-d27fc1ffee9f","order_by":4,"name":"Xiaodong Yang","email":"","orcid":"","institution":"Affiliated Hospital of Shaanxi University of Chinese Medicine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Xiaodong","middleName":"","lastName":"Yang","suffix":""},{"id":347939391,"identity":"3316a139-4d0c-43d1-8ef5-bb2c112c25be","order_by":5,"name":"Pengli Wang","email":"","orcid":"","institution":"Affiliated Hospital of Shaanxi University of Chinese Medicine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Pengli","middleName":"","lastName":"Wang","suffix":""},{"id":347939392,"identity":"c6b815e7-21ba-43de-9ab3-676830fe5b59","order_by":6,"name":"Shaohui Wang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAvklEQVRIiWNgGAWjYDACCRBhwMDAz8x8+AFpWiTb2dIMSNAC0nWeR0ECr0oYkJ/d/PAzT8HhxM2HeYCW1dhEE9RicOeYseQMg7TEbYd5DzxgOJaW20BQi0SCgcQHAxugFr4EA8aGw4S1yM9I//wDqCtxczOPgQRRWhhu5JiBbdnATKwWgxs5ZZZAvxjPOAwM5ARi/AJ02ObbPH8Oy/b3Hz784EONDREOgwJHsMoEYpWDgD0pikfBKBgFo2CEAQAP4z6M4tPP1QAAAABJRU5ErkJggg==","orcid":"","institution":"Affiliated Hospital of Shaanxi University of Chinese Medicine","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Shaohui","middleName":"","lastName":"Wang","suffix":""}],"badges":[],"createdAt":"2024-07-31 03:08:02","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4831983/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4831983/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":64569062,"identity":"290cac27-102b-48ba-934e-306cdeef2dfd","added_by":"auto","created_at":"2024-09-16 00:45:09","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":72814,"visible":true,"origin":"","legend":"\u003cp\u003eFlowchart of the included patients and treatment protocol. (\u003cstrong\u003eA\u003c/strong\u003e) Flowchart of the included patients. A total of 387 patients with advanced NSCLC were included, and 297 participants were excluded. Finally, 36 patients were analyzed in group A and 54 in group B. (\u003cstrong\u003eB\u003c/strong\u003e) Treatment protocol. Group A treated with a combination of cryoablation with pembrolizumab; Patients received pembrolizumab every 3 weeks until disease progression or intolerable toxicity post cryoablation, and group B was treated withcryoablation alone.\u003c/p\u003e","description":"","filename":"OnlineFigure1.png","url":"https://assets-eu.researchsquare.com/files/rs-4831983/v1/cb2e5cd032a74e71901a67ef.png"},{"id":64569724,"identity":"5f656da0-fab7-45e3-a3a6-3cb983d93093","added_by":"auto","created_at":"2024-09-16 00:53:09","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":102808,"visible":true,"origin":"","legend":"\u003cp\u003eThe number of lymphocytes in the blood was measured before (Pretreatment) and after 30 days (Posttreatment) of cryoablation. \u003cstrong\u003e(A) \u003c/strong\u003eThe number of CD3+ T cells, \u003cstrong\u003e(B) \u003c/strong\u003eCD4+ T cells, \u003cstrong\u003e(C) \u003c/strong\u003eCD8+ T cells, \u003cstrong\u003e(D) \u003c/strong\u003eTregs cells were analyzed by multicolor flow cytometry. Comparison within groups: *\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05; **\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.01; ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001; comparison between groups: #\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05; ##\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.01; ###\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"OnlineFigure2.png","url":"https://assets-eu.researchsquare.com/files/rs-4831983/v1/e8d84ad7b87dcdc67b5edfb1.png"},{"id":64567807,"identity":"107216b4-e062-4d59-b7b1-b4bc480aa667","added_by":"auto","created_at":"2024-09-16 00:37:09","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":98921,"visible":true,"origin":"","legend":"\u003cp\u003eCytokine expression measured before (Pretreatment) and after 30 days (Posttreatment) of cryoablation. \u003cstrong\u003e(A) \u003c/strong\u003eThe numbers of IL-2, \u003cstrong\u003e(B) \u003c/strong\u003eIL-6\u003cstrong\u003e,\u003c/strong\u003e \u003cstrong\u003e(C) \u003c/strong\u003eTNF-β, and \u003cstrong\u003e(D)\u003c/strong\u003e INF-γ were analyzed by multicolor flow cytometry. Comparison within groups: *\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05; *\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.01; ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001; comparison between groups: #\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05; ##\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.01; ###\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"OnlineFigure3.png","url":"https://assets-eu.researchsquare.com/files/rs-4831983/v1/f97b405d92736ae4ec384ea6.png"},{"id":64567809,"identity":"16855d79-9c40-4430-95cb-1dfa29a30c92","added_by":"auto","created_at":"2024-09-16 00:37:09","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":28372,"visible":true,"origin":"","legend":"\u003cp\u003eGraph showing Kaplan-Meier survival curves. \u003cstrong\u003e(A) \u003c/strong\u003eMedian overall survival (OS) from diagnosis. \u003cstrong\u003e(B)\u003c/strong\u003e Median progression-free survival (PFS) from diagnosis.\u003c/p\u003e","description":"","filename":"OnlineFigure4.png","url":"https://assets-eu.researchsquare.com/files/rs-4831983/v1/c910fdbfc95ce36e4637351b.png"},{"id":65924884,"identity":"51c091d5-5938-4e6b-b8fd-e6f767ae0698","added_by":"auto","created_at":"2024-10-04 12:39:10","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1251589,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4831983/v1/dc9767b3-4de8-42e5-8f48-9262743e2338.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Cryoablation Combined with Programmed Cell Death Protein 1 Inhibitor Pembrolizumab for Advanced Non-small Cell Lung Cancer","fulltext":[{"header":"Introduction","content":"\u003cp\u003eNon-small cell lung cancer (NSCLC) represents approximately 85% of all lung cancer cases and remains a leading cause of cancer-related mortality worldwide (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). Despite advances in targeted therapies and immunotherapies, the prognosis for patients with advanced NSCLC remains poor, with a 5-year survival rate of less than 5% for stage IV disease (\u003cspan additionalcitationids=\"CR3\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). Traditional treatment modalities, including chemotherapy and radiotherapy, have limited efficacy and are often associated with significant toxicity (\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). Therefore, there is an urgent need for novel therapeutic strategies that can improve outcomes for these patients.\u003c/p\u003e \u003cp\u003eCryoablation, a minimally invasive procedure that uses argon to destroy cancer cells, has emerged as a promising local treatment for various malignancies, including NSCLC (\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). This technique not only induces direct tumor cell death but also has the potential to elicit systemic anti-tumor immune responses (\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). However, the efficacy of cryoablation as a monotherapy is often limited by incomplete tumor eradication and the potential for residual disease. Combining cryoablation with immunotherapy, such as immune checkpoint inhibitors, may enhance the anti-tumor immune response and improve clinical outcomes.\u003c/p\u003e \u003cp\u003eProgrammed death-1 (PD-1) inhibitors, such as pembrolizumab, have revolutionized the treatment landscape for advanced NSCLC by blocking the interaction between PD-1 and its ligands, PD-L1 and PD-L2, thereby restoring T-cell activity and promoting anti-tumor immunity (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). Pembrolizumab has demonstrated significant clinical benefits in patients with PD-L1-positive NSCLC, leading to its approval as a first-line treatment for this population (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). However, the response rates to pembrolizumab monotherapy remain suboptimal, particularly in patients with low or negative PD-L1 expression (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eGiven the complementary mechanisms of action of cryoablation and pembrolizumab, we hypothesize that the combination of these two modalities may synergistically enhance anti-tumor immunity and improve clinical outcomes in patients with advanced NSCLC. This study aims to evaluate the efficacy and safety of cryoablation combined with pembrolizumab compared to cryoablation alone in patients with advanced NSCLC. We will assess tumor response, progression-free survival (PFS), overall survival (OS), and immune-related biomarkers to elucidate the potential benefits of this combination therapy.\u003c/p\u003e \u003cp\u003eIn summary, this study addresses a critical clinical need by exploring a novel combination therapy for advanced NSCLC. By integrating cryoablation with pembrolizumab, we aim to enhance the anti-tumor immune response and improve patient outcomes. The findings from this study could provide valuable insights into the potential of combining local and systemic therapies for the treatment of advanced NSCLC.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePatients\u003c/h2\u003e \u003cp\u003e This retrospective study was approved from the Institutional Ethic Committee of Affiliated Hospital of Shaanxi University of Chinese Medicine and conducted in accordance with the Declaration of Helsinki and the Declaration of Good Clinical Practice. Written informed consent was obtained from each participant. From February 2018 and October 2021, all patients with advanced NSCLC underwent either a combination of cryoablation\u0026thinsp;+\u0026thinsp;pembrolizumab (group A) or cryoablation alone (group B). The inclusion criteria were as follows: (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e) Histologically and radiologically verified advanced NSCLC; (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e) Stage IIIB/IV, according to the American Joint Committee on Cancer (AJCC) criteria; (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e) Patients over 18 years; (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e) Sufficient bone marrow, laboratory examination of hepatic, renal and ardio-pulmonary function; (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e) Relapsed after multimodal treatment (surgical resection, radiotherapy, or disease progressed after chemotherapy); (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e) Life expectancy of at least 3 months; (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e) Patients received pembrolizumab therapy for at least 1month after cryoablation in this study. The exclusion criteria were as follows: (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e) Patients with distant metastases; (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e) previous received anti-PD-1/PD-L1 therapy; (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e) platelet count of less than 50\u0026times;10\u003csup\u003e9\u003c/sup\u003e/L; (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e) Severe impairment of pulmonary functions with the maximum voluntary ventilation less than 40%.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eTreatment Procedure\u003c/h2\u003e \u003cdiv id=\"Sec5\" class=\"Section3\"\u003e \u003ch2\u003eCryoablation\u003c/h2\u003e \u003cp\u003ePreoperative imaging was performed to select supine, lateral or oblique position. Intraoperative real-time monitoring of the patient's blood pressure, oxygen saturation, heart rate, and electrocardiogram. Preoperative intravenous access was established for preoperative and intraoperative medications. Under the guidance of CT combined with ultrasound, we determined the location and path of the puncture needle, and planned the level, angle and depth of the puncture into the tumor. Thus, important tissue structures such as the heart, large blood vessels, and trachea were avoided to ensure surgical safety. Local aesthesia was applied at the puncture point with 1% lidocaine injection. The cryoprobe was inserted into the target according to the needle plan designed, and then confirmed by CT/MR scanning. The type and number of cryoprobes are selected according to the morphology, size and location of the lesion, with the aim of obtaining full coverage of the tumor with the \"ice ball\". Rapid freezing with argon for 15\u0026ndash;20 min, rapid rewarming with helium for 2\u0026ndash;5 min, and repeated freezing and rewarming were performed. The decision to increase the freezing time was made according to the coverage of the lesion by the \"ice ball\" on imaging. When the edge of the \"ice ball\" exceeds the lesion by more than 1cm, it is heated and the cryoprobe is withdrawn. CT or MR scanning was performed for complications such as pneumothorax and hemorrhage.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003ePembrolizumab\u003c/h2\u003e \u003cp\u003ePatients in group A received pembrolizumab therapy (100 mg/3w). The first dose of pembrolizumab was administered within 2 weeks after cryoablation. Patients received continuous pembrolizumab treatment until intolerance toxicity or progressive disease. The treatment protocol is illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003ePD-L1 expression analysis\u003c/h2\u003e \u003cp\u003ePD- L1 expression was assessed using a proprietary immunohistochemistry assay. We classified PD-L1 expression into three levels: negative PD-L1 expression (TPS\u0026thinsp;\u0026lt;\u0026thinsp;1%), low PD-L1 expression (TPS 1\u0026ndash;49%), and high PD-L1 expression (TPS\u0026thinsp;\u0026ge;\u0026thinsp;50%).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eFlow cytometric analysis\u003c/h2\u003e \u003cp\u003eBlood samples of each patient\u0026rsquo;s blood (2 ml) were collected just before the cryoablation procedure (Pretreatment) and 30 days (Posttreatment) after cryoablation procedure. Flow cytometry techniques (FACS caliber, four-color system, BD Bioscience, CA, US) were used to analyze the absolute number of CD3\u003csup\u003e+\u003c/sup\u003e T cells (CD3\u003csup\u003e+\u003c/sup\u003e), CD4\u003csup\u003e+\u003c/sup\u003eT cell (CD3\u003csup\u003e+\u003c/sup\u003eCD4\u003csup\u003e+\u003c/sup\u003e), CD8\u003csup\u003e+\u003c/sup\u003eT cell (CD3\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003e), Treg cell (CD4\u003csup\u003e+\u003c/sup\u003eCD25\u003csup\u003e+\u003c/sup\u003eCD127dim). A similar method was used to measure serum cytokines, including interleukin-2 (IL-2), interleukin-6 (IL-6), tumor-necrosis factor-beta (TNF-β), and interferon-γ (IFN-γ).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eCollection of clinical data\u003c/h2\u003e \u003cp\u003eThe tumor response was evaluated based on the mRECIST (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). The objective response rate (ORR) was calculated as follows: ORR = (CR\u0026thinsp;+\u0026thinsp;PR cases) / total cases \u0026times; 100%.\u003c/p\u003e \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003ch2\u003eFollow-up and endpoints assessment\u003c/h2\u003e \u003cp\u003eAll patients in this study were followed up at 1-, 3-, and 6-months posttreatment and then every three months. Two radiologists independently interpreted all follow-up scan results. Adverse events were evaluated and classified according to the National Cancer Institute Common Terminology Criteria for Adverse Events, version 4.0. The last follow-up was completed on June 16, 2023. The tumor response was assessed using Response Evaluation Criteria in Solid Tumors, version 1.1 (RECIST v1.1)(\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). The objective response rate (ORR) after 3months posttreatment was calculated which is defined as the percentage of patients with a best overall response of complete response (CR) or partial response (PR). Overall survival (OS) was defined as the time from diagnosis to death or until the last follow-up. progression-free survival (PFS) was defined as the time from diagnosis to disease progression until the last follow-up.\u003c/p\u003e \u003cp\u003eAdverse events, including immune-mediated adverse events, were collected throughout the treatment until 90 days after the last infusion of pembrolizumab. All Adverse events were graded based on the NCI Common Terminology Criteria for Adverse Events v5.0.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAll statistical analyses were performed and compiled using GraphPad Prism (version 8.01, GraphPad Software, San Diego, CA, USA) and SPSS software (version 20.0, SPSS Inc., Chicago, IL, USA). Categorical variables were compared using the chi-square or Fisher exact test. The continuous data, categorical data, and survival curves of the two groups were compared using the Mann-Whitney test, Fisher's exact test, and log-rank (Mantel-Cox) tests individually. The Cox regression model was used in survival analysis to assess the influence factor of OS and PFS. All statistical tests were two-sided; a \u003cem\u003ep\u003c/em\u003e-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003ePatient Characteristics\u003c/h2\u003e \u003cp\u003eBetween February 2018 and October 2021, a total of 387 advanced NSCLC patients were included in this study (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Based on the exclusion criteria, totally 297 patients were excluded due to receiving other treatment (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;138), tumor metastasis (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;106), incomplete data, or missing (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;48). A total of 90 advanced NSCLC patients were finally evaluated in this study: Group A (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;36) and Group B (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;54) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). The baseline characteristics were well balanced between the two groups (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The median ages were 58 (29\u0026ndash;80) and 54 (33\u0026ndash;78) in Group A and B, respectively. Male patients had a higher risk than female patients between the two groups. The median tumor sizes were 4.6 cm (range 2.9\u0026ndash;8.5) and 4.8 cm (range 3.1\u0026ndash;8.7) for patients in groups A and B, respectively. The majority of patients were nonsquamous histology (81.1%) and had former or current smokers (82.2%). In the 90 patients who underwent a PD-L1 expression assay before treatment, 12 (13.3%) patients had PD-L1 negative expression, and 56 (62.2%) had PD-L1 positive expression, including 29 (32.2%) patients more than 50% (\u0026ge;\u0026thinsp;50%) PD-L1 expression (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePatients characteristics.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCharacteristic\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGroup A (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;36)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGroup B (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;54)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e\u0026nbsp;Value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAge, year\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.326\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u0026le;\u0026thinsp;60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSex\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.461\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFemale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTumor size (cm)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.666\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2\u0026thinsp;~\u0026thinsp;5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u0026ge;5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTumor pattern\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.660\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSquamous\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNonsquamous\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTNM stage\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.648\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIIIA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIV\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSmoking status\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.736\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFormer or current\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNever\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePD-L1 TPS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.371\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u0026ge;50%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u0026ge;\u0026thinsp;1% and \u0026lt;\u0026thinsp;50%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;1%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUnknown\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePreoperative therapy\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.702\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSurgery\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eChemotherapy\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRadiotherapy\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOthers\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"4\"\u003eTNM, tumor node metastasis; PD-L1, programmed death-ligand 1; TPS, tumor proportion score\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eAdverse Events\u003c/h2\u003e \u003cp\u003eAs shown in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, there was no treatment-related mortality in either group in our study. Overall, the frequency of adverse events was similar between the two groups. The most common cryoablation treatment-related adverse events were pain (61.1%), nausea (63.9%), vomiting (58.3%) and pneumothorax (33.3%) in group A. The major (grade 3\u0026thinsp;~\u0026thinsp;4) cryoablation-related adverse events were pneumothorax (5.6%) and hemorrhage (2.7%) in Group A (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). All adverse events were relieved or improved after symptomatic treatment. The most common immune-related adverse events were fatigue (19.4%), pruritus (13.8%), diarrhea (11.1%), nausea (11.1%). The major (grade 3\u0026thinsp;~\u0026thinsp;4) immune-related adverse events were fatigue (2.7%), pruritus (2.7%), AST increased (2.7%), lipase increased (2.7%), ALT increased (2.7%) in group A, which was resolved after the treatment with a corticosteroid. There were no immune-related adverse events in group B (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eAdverse events related to cryoablation treatment.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCharacteristic\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eGroup A (n\u0026thinsp;=\u0026thinsp;36)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003eGroup B (n\u0026thinsp;=\u0026thinsp;54)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAll Grade\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGrade 3/4\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eAll Grade\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eGrade 3/4\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePain\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e22 (61.1%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e36 (66.7%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNausea\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e23 (63.9%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e28 (51.9%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVomiting\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e21 (58.3%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e32 (59.3%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePneumothorax\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e12 (33.3%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2 (5.6%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e17 (31.5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e4 (7.4%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePleural effusion\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e7 (19.4%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e15 (27.8%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHemorrhage\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e9 (25.0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1 (2.7%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e22 (40.7%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2 (3.7%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eInfection\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1 (2.7%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e2 (3.7%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eAdverse events related to immune treatment.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAdverse event\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eGroup A (n\u0026thinsp;=\u0026thinsp;36)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAll Grade\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGrade 3/4\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFatigue\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e7 (19.4%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1 (2.7%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePruritus\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e5 (13.8%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1 (2.7%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDiarrhea\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4 (11.1%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNausea\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4 (11.1%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDecreased appetite\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2 (5.6%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHypothyroidism\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2 (5.6%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCough\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2 (5.6%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFever\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2 (5.6%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAST increased\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2 (5.6%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1 (2.7%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLipase increased\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1 (2.7%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1 (2.7%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eALT increased\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1 (2.7%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1 (2.7%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePneumonitis\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1 (2.7%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"3\"\u003eAST, aspartate transaminase; ALT, alanine aminotransferase\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eResponse\u003c/h2\u003e \u003cp\u003eOverall, 11 (30.6%) patients achieved CR, 16 (44.4%) patients received PR, 6 (16.7%) patients developed SD, and 3 (8.3%) patients suffered PD in group A. The tumor response in group B was 10 (18.5%) patients with CR, 23 (42.6%) patients with PR, 9 (16.7%) patients with SD and 12 (22.2%) with PD. The ORR was 75.0% \u003cem\u003evs.\u003c/em\u003e 61.1% (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.045, Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e) in groups A and B, respectively.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eTumor response 3 months posttreatment\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCharacteristic\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGroup A (n\u0026thinsp;=\u0026thinsp;36)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGroup B (n\u0026thinsp;=\u0026thinsp;54)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e\u0026nbsp;Value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e11(30.6%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e10(18.5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e16(44.4%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e23(42.6%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6(16.7%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e9(16.7%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3(8.3%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e12(22.2%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eORR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e27(75%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e33(61.1%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.045\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"4\"\u003eCR, complete response; PR, partial response; SD, stable disease; PD, progressive disease; ORR, objective response rate\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eImmune parameters\u003c/h2\u003e \u003cp\u003eWe compared the absolute number of immune cells after treatment to investigate possible effects on the tumor immune microenvironment of each treatment. The results demonstrated that the absolute number of CD3\u003csup\u003e+\u003c/sup\u003eT cells (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.033), CD4\u003csup\u003e+\u003c/sup\u003eT cells (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.012), and CD8\u003csup\u003e+\u003c/sup\u003e T cells (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.003) steadily increased after treatment in group A comparing to group B (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA\u0026ndash;C), while the proportion of Tregs cells (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.035) decreased (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo further investigate the variations of the immune microenvironment, we compared the levels of cytokines in both groups. The data suggested that the IL2 (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.009), IL-6 (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.000), TNF-β (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.001), and IFN-γ (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.014) levels significantly increased after treatment in group A comparing to group B (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eSurvival\u003c/h2\u003e \u003cp\u003eThe median follow-up time was 24.6 months (9.9\u0026ndash;41.4 months). The median OS from diagnosis was significantly longer in group A than in group B, respectively (28.1 months \u003cem\u003evs.\u003c/em\u003e 24.2 months, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.009, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Similarly, the median PFS from diagnosis was significantly longer in group A than in group B, respectively (12.8 months \u003cem\u003evs.\u003c/em\u003e 8.4 months, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.011, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eUnivariate and Multivariate Analyses of OS and PFS\u003c/h2\u003e \u003cp\u003eUnivariate Cox regression analysis and multivariate Cox regression analysis were used to evaluate the predictors of OS and PFS. As shown in Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, tumor size (HR: 1.024, 1.045\u0026ndash;1.038, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.025), tumor pattern (HR: 0.645, 0.328\u0026ndash;1.525, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.048), PD-L1 TPS (HR: 0.975, 0.415\u0026ndash;2.045, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.038), cryoablation plus pembrolizumab (HR: 0.447, 0.238\u0026ndash;0.765, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.015) were significantly correlated with OS in the univariate Cox regression analysis. Multivariate analysis showed that the tumor size (HR: 2.056, 1.010\u0026ndash;4.256, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.046), PD-L1 TPS (HR: 0.172, 0.042\u0026ndash;0.746, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.028) and cryoablation plus pembrolizumab (HR: 0.472, 0.285\u0026ndash;0.778, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.006) were predictors of OS (Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Furthermore, PD-L1 TPS (HR: 0.488, 0.214\u0026ndash;1.425, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.045) and cryoablation plus pembrolizumab (HR: 1.757, 0.965\u0026ndash;3.287, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.047) were also significant prognostic factors for PFS (Table\u0026nbsp;\u003cspan refid=\"Tab6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab5\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eUnivariate and multivariate analyses of OS in patients\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eCharacteristic\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eUnivariate analysis\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003eMultivariate analysis\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHR (95%CI)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e value\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eHR (95%CI)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAge (\u0026gt;\u0026thinsp;60/\u0026le;60)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.724 (0.146\u0026ndash;0.786)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.356\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSex (female/male)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.354 (0.278\u0026ndash;0.628)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.758\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTumor size (\u0026ge;\u0026thinsp;5/2\u0026thinsp;~\u0026thinsp;5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.024 (1.045\u0026ndash;1.038)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.025\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e2.056 (1.010\u0026ndash;4.256)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.046\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTumor pattern (squamous/nonsquamous)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.645 (0.328\u0026ndash;1.525)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.048\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.024 (0.014\u0026ndash;0.524)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.067\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTNM stage\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.987 (0.648\u0026ndash;1.546)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.983\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSmoking status (Current/former/Never)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.012 (0.985\u0026ndash;1.41)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.225\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePD-L1 TPS (\u0026ge;\u0026thinsp;50%/1\u0026ndash;49%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.975 (0.415\u0026ndash;2.045)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e{Novello, 2023 #8}\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.172 (0.042\u0026ndash;0.746)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.028\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePreoperative therapy\u0026nbsp;(surgery/chemotherapy/ radiotherapy/others)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.345 (0.789\u0026ndash;2.458)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.178\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCryoablation (with pembrolizumab/without pembrolizumab)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.447 (0.238\u0026ndash;0.765)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.015\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.472 (0.285\u0026ndash;0.778)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.006\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"6\"\u003eOS, overall survival; PD-L1, programmed death-ligand 1; HR, hazard ratio; CI, confidence interval;\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab6\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 6\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eUnivariate and multivariate analyses of PFS in patients\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eCharacteristic\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eUnivariate analysis\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003eMultivariate analysis\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHR (95%CI)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e value\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eHR (95%CI)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAge (\u0026gt;\u0026thinsp;60/\u0026le;60)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.145 (0.561\u0026ndash;2.110)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.656\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSex (female/male)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.351 (0.212\u0026ndash;0.653)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.751\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTumor size (\u0026ge;\u0026thinsp;5/2\u0026thinsp;~\u0026thinsp;5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.714 (0.484\u0026ndash;1.564)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.428\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTumor pattern (squamous/nonsquamous)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.124 (0.748\u0026ndash;1.658)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.572\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTNM stage\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.942 (0.631\u0026ndash;1.412)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.752\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSmoking status (Current/former/Never)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.946 (0.645\u0026ndash;1.412)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.746\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePD-L1 TPS (\u0026ge;\u0026thinsp;50%/1\u0026ndash;49%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.625 (0.258\u0026ndash;1.445)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.021\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.488 (0.214\u0026ndash;1.425)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.045\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePreoperative therapy\u0026nbsp;(surgery/chemotherapy/ radiotherapy/others)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.615 (0.378\u0026ndash;1.224)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.208\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCryoablation (with pembrolizumab/without pembrolizumab)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.825 (1.045\u0026ndash;3.275)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.013\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.757 (0.965\u0026ndash;3.287)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.047\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"6\"\u003ePFS, progression free survival; PD-L1, programmed death-ligand 1; HR, hazard ratio; CI, confidence interval;\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we evaluated the efficacy and safety of combining cryoablation with pembrolizumab treatment compared to cryoablation alone in patients with advanced NSCLC. Our main findings indicate that the combination therapy significantly improved ORR and prolonged OS and PFS compared to cryoablation alone. Specifically, patients receiving the combination therapy exhibited a more favorable immune profile, as evidenced by increased levels of CD3\u0026thinsp;+\u0026thinsp;T cells, CD4\u0026thinsp;+\u0026thinsp;T cells, and CD8\u0026thinsp;+\u0026thinsp;T cells, along with elevated serum cytokines such as IL-2 and IFN-γ. These results align with our initial hypothesis that the addition of pembrolizumab, an anti-PD-1 monoclonal antibody, would enhance the anti-tumor immune response initiated by cryoablation. Furthermore, the safety profile of the combination therapy was manageable, with adverse events consistent with those previously reported for pembrolizumab and cryoablation treatments (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). These findings suggest that the integration of cryoablation and pembrolizumab could offer a promising therapeutic strategy for patients with advanced NSCLC.\u003c/p\u003e \u003cp\u003eThe primary novelty of this study lies in its comprehensive evaluation of the combined treatment of cryoablation and pembrolizumab in NSCLC patients, a therapeutic approach that has not been extensively explored in previous research. This study uniquely integrates cryoablation, a minimally invasive procedure that induces tumor cell death through argon, with pembrolizumab, an immune checkpoint inhibitor targeting PD-1. The combination aims to enhance anti-tumor immunity by not only directly reducing tumor burden but also by potentially increasing the immunogenicity of the tumor microenvironment. Our findings demonstrate a significant improvement in both OS and PFS in the combination therapy group compared to cryoablation alone, with median OS of 28.1 months versus 24.2 months and median PFS of 12.8 months versus 8.4 months, respectively. This is consistent with the hypothesis that cryoablation can act as an in situ vaccine, enhancing the efficacy of subsequent immunotherapy (\u003cspan additionalcitationids=\"CR22\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). Furthermore, the observed increase in CD3+, CD4+, and CD8\u0026thinsp;+\u0026thinsp;T cells, along with elevated levels of cytokines such as IL-2, IL-6, TNF-β, and IFN-γ, supports the notion of an augmented immune response post-treatment. These results align with previous studies that have suggested the potential of combining local ablative therapies with systemic immunotherapies to achieve synergistic effects (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). However, our study is among the first to provide robust clinical evidence in a human cohort, thereby filling a critical gap in the current understanding of multimodal treatment strategies for advanced NSCLC (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe clinical implications of our study are significant, particularly in the context of treatment strategies for advanced NSCLC. These findings suggest that integrating immunotherapy with local ablative techniques can enhance the anti-tumor immune response, potentially leading to better clinical outcomes. This is particularly relevant given the limited treatment options and poor prognosis associated with advanced NSCLC. The observed increase in CD3\u0026thinsp;+\u0026thinsp;T cells, CD4\u0026thinsp;+\u0026thinsp;T cells, and CD8\u0026thinsp;+\u0026thinsp;T cells, along with elevated levels of cytokines such as IL-2, IL-6, TNF-β, and IFN-γ, underscores the immunomodulatory effects of this combination therapy. These results align with previous studies that have highlighted the synergistic effects of combining local therapies with immune checkpoint inhibitors (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eHowever, our study is limited by its retrospective nature and the relatively small sample size. Future research should focus on conducting large-scale, multicenter randomized controlled trials to validate our findings and explore the underlying mechanisms of this combined treatment approach. Additionally, longer follow-up periods are necessary to assess the long-term efficacy and safety of this therapeutic strategy. Understanding the molecular and immunological changes induced by this combination therapy could pave the way for more personalized and effective treatment regimens for NSCLC patients.\u003c/p\u003e \u003cp\u003eIn conclusion, our study demonstrates that the combination of cryoablation and pembrolizumab therapy significantly improves the clinical outcomes in patients with advanced non-small cell lung cancer (NSCLC) compared to cryoablation alone. These findings suggest that the synergistic effect of cryoablation and immunotherapy could potentially overcome the limitations of monotherapy in treating advanced NSCLC.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eConflicts of interest\u003c/h2\u003e \u003cp\u003eAll authors report no conflicts of interest in this work.\u003c/p\u003e\u003ch2\u003eFUNDING\u003c/h2\u003e \u003cp\u003eThis study was supported by the Project of Shaanxi Provincial Department of Science and Technology (No. 2022SF-513).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eSW and PW designed the project and edited the manuscript. LW, GW, HY, ZC and XY conducted study selection, dataextraction, statistical analyses, and drafted the main manuscript. All authors contributed to the article and approved thesubmitted version.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eWe thank all patients and their families for their help and support. We thank all my colleagues for their assistance.\u003c/p\u003e\u003ch2\u003eAvailability of data and material\u003c/h2\u003e \u003cp\u003eThe dataset generated during and/or analyzed during this study are available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSiegel RL, Giaquinto AN, Jemal A, Cancer Statistics. 2024. \u003cem\u003eCA Cancer J Clin\u003c/em\u003e (2024) 74(1):12\u0026ndash;49. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3322/caac.21820\u003c/span\u003e\u003cspan address=\"10.3322/caac.21820\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHerbst RS, Morgensztern D, Boshoff C. The Biology and Management of Non-Small Cell Lung Cancer. 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Onco Targets Ther. 2023;16:385\u0026ndash;97. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.2147/OTT.S410693\u003c/span\u003e\u003cspan address=\"10.2147/OTT.S410693\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Cryoablation, Programmed cell death protein 1 Inhibitor, Pembrolizumab, Advanced non-small cell lung cancer, Combination","lastPublishedDoi":"10.21203/rs.3.rs-4831983/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4831983/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eIntroduction:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo evaluate the efficacy and safety of cryoablation combined with pembrolizumab treatment versus cryoablation alone in patients with advanced non-small cell lung cancer (NSCLC).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis retrospective study was conducted from February 2018 and October 2021. A total of 90 patients with NSCLC (AJCC stage IIIB/IV) were included, with 36 patients receiving cryoablation combined with pembrolizumab (Group A) and 54 patients receiving cryoablation alone (Group B). The primary outcome measures included objective response rate (ORR), overall survival (OS), and progression-free survival (PFS), immune responses and adverse events serving as secondary endpoints. Risk factors for OS and PFS were identified using univariate and multivariate analyses.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo treatment related deaths were observed. Group A demonstrated a higher ORR (75.0% vs. 61.1%), longer median OS (28.1 months vs. 24.2 months), and longer median PFS (12.8 months vs. 8.4 months) compared to Group B. Additionally, Group A showed significant increases in CD3+, CD4+, and CD8 + T cells, and elevated levels of IL-2, IL-6, TNF-β, and IFN-γ. The multivariate analysis showed the combination of cryoablation and pembrolizumab was an independent prognostic factor for OS and PFS.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions\u003c/strong\u003e: Cryoablation combined with pembrolizumab significantly improves clinical outcomes in advanced NSCLC patients compared to cryoablation alone, highlighting the potential of this combination therapy in enhancing anti-tumor immunity and prolonging survival.\u003c/p\u003e","manuscriptTitle":"Cryoablation Combined with Programmed Cell Death Protein 1 Inhibitor Pembrolizumab for Advanced Non-small Cell Lung Cancer","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-09-16 00:37:04","doi":"10.21203/rs.3.rs-4831983/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"9f7661df-a4c5-4091-af67-6c51ffbe3c85","owner":[],"postedDate":"September 16th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-10-04T12:38:48+00:00","versionOfRecord":[],"versionCreatedAt":"2024-09-16 00:37:04","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4831983","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4831983","identity":"rs-4831983","version":["v1"]},"buildId":"7rjqhiLT3MXkJMwkYKINL","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}