PD-L1 status on Circulating Tumor Cells: A Promising Predictor in Advanced Lung Cancer with PD-1/PD-L1 immunotherapies

PD-L1 status on Circulating Tumor Cells: A Promising Predictor in Advanced Lung Cancer with PD-1/PD-L1 immunotherapies | 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 PD-L1 status on Circulating Tumor Cells: A Promising Predictor in Advanced Lung Cancer with PD-1/PD-L1 immunotherapies Xiaoying Wei, Lin Chen, Zhonglin Yang, Daxiong Zeng, Dongjiang Tang, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7920255/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 31 Jan, 2026 Read the published version in European Journal of Medical Research → Version 1 posted 11 You are reading this latest preprint version Abstract Background Tumor heterogeneity and invasive sampling limit traditional tissue-based PD-L1 testing in advanced lung cancer. We explored a non-invasive alternative by assessing PD-L1 expression on circulating tumor cells (CTC PD-L1) via liquid biopsy to predict immunotherapy outcomes. Methods Fifty-two advanced lung cancer patients were recruited and 38 individuals received combination of chemotherapy and PD-1/PD-L1 inhibitor therapy, with serial blood samples (baseline, during treatment, and at progression) analyzed using the LiquidBiopsy™ platform and CTC PD-L1 assay. Primary endpoints included objective response rate (ORR), progression-free survival (PFS), and overall survival (OS). Results CTC PD-L1 was identified on special phenotype with ratio of CK/CD45 > 1.7 and PD-L1/CD45 > 3.2. At baseline, 71.2% (37/52) of patients had detectable CTCs, with 50.0% (26/52) being CTC PD-L1 + . CTC PD-L1 + patients exhibited significantly higher ORR (84.2% vs. 36.8%), longer median PFS (16 months vs 4 months; HR = 0.28, 95% CI: 0.096–0.78, p = 0.0041), and superior OS (median survival, undefined; HR = 0.19, 95% CI: 0.05–0.74, p = 0.017). Multivariate analysis confirmed CTC PD-L1 + as an independent predictor of response (p = 0.003) and potential as a complementary biomarker to tPD-L1. Conclusions We developed a novel procedure to detect and characterize PD-L1 expression on CTCs, which was a feasible, non-invasive biomarker for predicting combination of chemotherapy and immunotherapy efficacy in advanced lung cancer, addressing tissue sampling limitations and enhancing patient stratification and monitoring. Trial registration The study was approved by the Medical Ethics Committee of Fourth Affiliated Hospital of Soochow University, with ethics approval number 220154 on 23th January, 2022 and retrospectively registered under clinical trial number ChiCTR2500096312 on 21th January, 2025. circulating tumor cells CTC PD-L1 objective response rate (ORR) progression-free survival (PFS) and overall survival (OS) Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction PD-1/PD-L1 blockade immunotherapy has established itself as a cornerstone in the first-line and second-line treatment of advanced lung cancer ( 1 ), significantly enhancing patients' survival and quality of life ( 2 ). Various PD-1 or PD-L1 monoclonal antibodies have been developed and evaluated in clinical trials, demonstrating efficacy as monotherapy or in combination with cytotoxic chemotherapy ( 3 ), radiotherapy ( 4 ), or other immune checkpoint inhibitors ( 5 , 6 ). Pivotal studies including Keynote-001 ( 7 ), Keynote-010 ( 8 ), and Keynote-024 ( 9 ), and CheckMate 057 ( 10 ). have shown that non-small cell lung cancer (NSCLC) patients with PD-L1-positive (PD-L1+) tumors, particularly those with high PD-L1 expression, achieve higher objective response rates (ORR), longer progression-free survival (PFS), and improved overall survival (OS) when treated with immunotherapies. Despite these significant clinical advantages, durable responses are only observed in a minority of patients, with just 20%-30% showing lasting benefits from PD-1/PD-L1 blockade immune-therapies ( 11 ). Furthermore, immune-related adverse events, including skin reactions and liver abnormalities, and potentially fatal conditions like pneumonitis, hepatitis, and myocarditis, underscore the critical importance of early identifying of patients likely to benefit from PD-1/PD-L1 blockade therapy early in the treatment process ( 12 ). Currently, tissue PD-L1 (tPD-L1), alongside tumor mutational burden (TMB) ( 13 ) and microsatellite instability (MSI) ( 14 ), stands as one of the most established predictive biomarkers for response to PD-1/PD-L1 blockade therapy ( 15 ). However, several factors limit the clinical utility of these biomarkers, including tumor tissue scarcity, the invasiveness and metastatic risks associated with tissue biopsy ( 16 ), and variability in PD-L1 detection methodologies ( 17 ). Notably, 10% to 20% of patients with negative tPD-L1 status may still respond positively to immune checkpoint inhibitors (ICIs), while certain tPD-L1-positive tumors, especially those with high PD-L1 expression, can be refractory to immunotherapy ( 18 ). The assessment of tPD-L1 often relies on archived paraffin-embedded tissues, which may account for some of the observed inconsistencies ( 19 ). This variability likely stems from the dynamic regulation of PD-L1 expression, which can be modulated by treatments such as targeted therapy, chemotherapy ( 20 ), or radiation therapy ( 21 ). Furthermore, the limited tumor tissue size obtained from biopsies may not adequately represent the heterogeneous tumor tissue, potentially compromising the accuracy of PD-L1 analysis ( 22 , 23 ). Therefore, identifying novel predictive biomarkers is essential for optimizing the efficacy and safety of PD-1/PD-L1 inhibitors and for identifying patients most likely to derive sustained benefits from this class of immunotherapies. Circulating tumor cells (CTCs) play a pivotal role in metastasis, which is the primary cause of mortality in cancer patients ( 24 ). These cells can spread through the bloodstream as individual cells, clusters ( 25 ), or in conjunction with immune cells ( 26 ) after detaching from the primary tumor ( 27 ). Several studies have reported that the presence of PD-L1-positive CTCs is associated with poorer progression-free survival (PFS) and overall survival (OS) ( 28 , 29 ), suggesting that these cells might facilitate immune evasion in cancer. Conversely, other investigations have demonstrated that patients with PD-L1-positive CTCs tend to experience improved PFS and OS following PD-1/PD-L1 immune checkpoint therapy ( 30 ), suggesting that CTC PD-L1 could be a significant prognostic and predictive biomarker in immunotherapy. However, some studies have found no significant association with better PFS and OS in NSCLC patients treated with immune checkpoint inhibitors (ICIs) ( 31 ). Consequently, the precise role of PD-L1 in CTCs regarding its potential as a potential biomarker to predict response to combination of chemotherapy and immunotherapy remains unclear. To investigate the correlation between CTC PD-L1 expression and response to combination of chemotherapy and immunotherapy, we have developed a novel assay for detecting CTC PD-L1 using the LiquidBiopsy™ system. This approach assesses PD-L1 expression in blood samples from patients with advanced lung cancer at multiple time points: pre-treatment (baseline, T0), during treatment (after the second cycle and before the third cycle (T1)), and after the fourth cycle and before the fifth cycle (T2)). This research aims to investigate the correlation between CTC PD-L1 levels and treatment outcomes, specifically focusing on the overall response rate (ORR) and survival outcomes. Materials and Method 1. Study design This study was reported in accordance with the Consolidated Standards of Reporting Trials (CONSORT) guidelines. It was a non-interventional, double-blind, prospective, real-world investigation conducted at the Fourth Affiliated Hospital of Soochow University (Ethics review board approval number 220003), enrolling advanced lung cancer patients without autoimmune diseases. Participants received at least two cycles of combination of chemotherapy and PD-1/PD-L1 blockade immunotherapy starting from April 2022, with informed consent and under a registered clinical trial number (ChiCTR2500096312). To maintain blind, peripheral blood samples (10mL) were collected at baseline (T0), after two cycles (T1), after four cycles (T2), and at disease progression (PD) for CTC detection and PD-L1 expression analysis at Zhuhai Sanmedbio (Fig. 1 and Supplementary Fig. 1), the effects of response to combination of chemotherapy and immune checkpoint inhibitor (ICI) therapy and follow-up care are being studied at the Fourth Affiliated Hospital of Soochow University, all the data was sent to the third party for storage and analysis immediately. The primary objective was to assess the predictive value of baseline CTC PD-L1 for objective response rate (ORR), progression-free survival (PFS), and overall survival (OS). Secondary objectives included monitoring changes in CTC counts and their relationship to therapeutic response. All the patients were followed-up with more than 3 months for ORR evaluation. 2. CTC isolation and detection by using LiquidBiopsy™ CTCs were isolated and counted using the LiquidBiopsy™ system (Zhuhai Sanmed Biotech LTD, Zhuhai, China), as detailed elsewhere( 32 ). PBMCs were processed using Ficoll from 10 mL blood, fixed with PFA, and incubated with capture cocktail kits and streptavidin beads for CTC capture. Anti-pan CK antibody, anti-PD-L1 antibody, and anti-CD45 antibody, along with DAPI, were used for detection. CTCs were identified based on specific phenotypes with threshold of CK mean of fluorescence intensity (MFI) and ratio of CK/CD45 ( 33 ). CTC PD-L1 was analyzed on HRP labeled anti-PD-L1 antibody with anti HRP IgG alexa Fluor594 secondary antibody (Supplementary Fig. 2) and classified with threshold of PD-L1 MFI and ratio of PD-L1/CD45. Patients with one or more PD-L1 positive CTCs were classified as CTC PD-L1 + , while those with one or more CK positive CTCs were classified as CTC positive. 22C3 pharmDx (Dako) was used for evaluation of PD-L1 expression in tumor tissues according to previous study( 34 ). 3. Treatment response and disease progression assessment Tumor responses were radiologically assessed after two treatment cycles using the RECIST 1.1 criteria. ORR was defined as the ratio of patients with complete response (CR) or partial response (PR) to all patients. Disease control rate (DCR) included patients with CR, PR, or stable disease (SD). PFS was measured from the start of combination of chemotherapy and PD-1/PD-L1 immunotherapy to first documented disease progression, and OS was measured from the start of PD-1/PD-L1 immunotherapy in combination with chemotherapy to death after recruitment. PR patients were defined as responders at combination of chemotherapy and PD-1/PD-L1 immunotherapy. 4. Statistical analysis The study analyzed the total number of CTCs and CTC PD-L1 to characterize patient disease features. Patients were categorized into two groups: those with at least one PD-L1-positive CTC (CTC PD-L1 + ) or none (CTC PD-L1 − ). A receiver operating characteristic (ROC) curve determined the optimal cutoff value to distinguish between treatment responders and non-responders. A univariate logistic regression model was then used to calculate odds ratios (ORs) and 95% confidence intervals (CIs) at this cutoff. Differences in quantities of CTC PD-L1 + between the partial response (PR) group and the stable disease/progressive disease (SD/PD) group were assessed using a two-tailed, unpaired t-test for variables with normal distribution. Fisher's exact tests evaluated the association between CTC PD-L1 status and treatment response, as well as the link between changes in total CTC counts and treatment outcomes. These tests also assessed variations in the objective response rate (ORR) based on categorical variables like age, gender, cancer subtype, tissue PD-L1 (tPD-L1) status, type of immunotherapy, CEA, CYFRA21-1, NSE, SCC, and other clinical factors. Survival analysis was conducted using the Kaplan-Meier method to plot curves and the Mantel-Cox test to calculate hazard ratios. Both univariate and multivariate Cox proportional hazards regression models assessed the correlation between CTC PD-L1 status and progression-free survival (PFS) and overall survival (OS), considering factors like age, gender, cancer subtype, treatment type, CEA, CYFRA21-1, NSE, SCC, smoking status, hypertension, diabetes, and ECOG score. Statistical analyses were performed using GraphPad Prism 8.2.0 and IBM SPSS Statistics version 25, with p-values of less than 0.05 considered statistically significant. Result 1. Baseline Characteristics and CTC Detection From April 2022 to February 2024, we prospectively enrolled 52 patients with advanced lung cancer (stages III-IV) in the Department of Respiratory and Critical Care Medicine at the Fourth Affiliated Hospital of Soochow University (Fig 1). The cohort comprised 44 males (84.6%) and 8 females (15.4%), with a median age of 67.8 years (range: 45-82 years). Histological subtypes included adenocarcinoma (n=15, 28.8%), squamous cell carcinoma (n=23, 44.2%), and small cell lung cancer (n=14, 27.0%). All patients were driver gene-negative and received PD-1/PD-L1 inhibitor therapy in combination with chemotherapy. At baseline assessment, CTCs, which were identified based on specific phenotypes, with CK+/DAPI+/CD45-, or CK+/PD-L1+/DAPI+/CD45- (Fig 2A) at ratio of CK/CD45 >1.7 (Sensitivity and specificity was 100% (95% CI 99.52% to 100.0%) and 100% (95% CI 99.27% to 100.0%) respectively, with AUC 1.0 (95% CI 1.0 to 1.0, p3.2 (Sensitivity and specificity was 99.90% (95% CI 99.81% to 99.95%) and 99.62% (95% CI 98.61% to 99.93%) respectively, with AUC 0.9999 (95% CI 1.0 to 1.0, p<0.0001)) with CK+/PD-L1+/DAPI+/CD45- (Fig 2Bd-2Bf). CTC were successfully detected in 37 patients (71.2%) out of the 52 patients recruited, with varying detection rates across histological subtypes: adenocarcinoma (8/15, 53.3%), squamous cell carcinoma (19/23, 82.6%), and small cell lung cancer (10/14, 71.4%). CTC PD-L1 + were identified in 26 patients (50.0%), with subtype-specific positivity rates of 40.0% (6/15) in adenocarcinoma, 56.5% (13/23) in squamous cell carcinoma, and 50.0% (7/14) in small cell lung cancer (Table 1). 2. Association Between CTC PD-L1 Status and Clinical Response Of the 52 patients enrolled, 38 who completed at least two cycles of combination of chemotherapy and immunotherapy were assessed for treatment response per RECIST 1.1 (follow-up through 24 February 2024; Fig 1); the other 14 were excluded because they received chemotherapy only. CTCs were detected in 27 patients (71.1%), with 19 (50.0%) individuals exhibiting CTC PD-L1 + . The accordance of CTC PD-L1 and tPD-L1 was 52.6% (20/38). In the CTC PD-L1 + group (n=19), 16 patients achieved partial response (PR), 2 maintained stable disease (SD), and 1 developed progressive disease (PD), resulting in an objective response rate (ORR) of 84.2% (Fig 3A) and disease control rate (DCR) of 94.7% (Fig 3C). In contrast, the CTC PD-L1 - group (n=19) demonstrated significantly lower efficacy measures, with an ORR of 36.8% and DCR of 68.4%, which was similar to the results of ORR (Fig 3C; 76.0% vs 41.2%) and DCR (Fig 3D; 95.2% vs 64.7%) to tPD-L1. The positive and negative predictive values (PPV and NPV) of CTC PD-L1 for ORR were 84.2% and 63.2%, respectively. Quantitative analysis revealed significantly higher quantities of CTC PD-L1 + in responders (mean ± SD: 2.9 ± 1.14) compared to non-responders (mean ± SD: 0.2 ± 0.11, p = 0.046) (Fig 3E), but not in tPD-L1 (Fig 3F). ROC curve analysis established an optimal CTC PD-L1 cutoff value of 1 (AUC = 0.7165, 95% CI 0.5418 to 0.8792, p = 0.0265; sensitivity = 69.5%, specificity = 80%, p < 0.001), yielding a sensitivity of 69.5% and specificity of 80.0% (Fig 3G), which was similar to tPD-L1 with cutoff value of 1% (AUC=0.6751, 95% CI 0.4987 to 0.8515, p = 0.0665; sensitivity = 69.5%, specificity = 80%) (Fig 3H). Furthermore, the sensitivity and the specificity would be improved to 86.9% and 78% respectively when combined CTC PD-L1 and tPD-L1, which meant that CTC PD-L1 was potential act as a complementary biomarker for tPD-L1 in prediction of benefit from combination of chemotherapy and immunotherapy. Additionally, a multivariate logistic regression analysis evaluated the prognostic value of various factors including age, gender, cancer subtype, type of ICI drugs (PD-1 or PD-L1 antibody), CEA, CYFRA21-1, NSE, and SCC. Only CTC PD-L1 and tPD-L1 showed significant difference between the PR and SD/PD groups ( p = 0.003 and p = 0.028) (Table 2), confirming its role as an independent predictor of ORR in ICI therapy and potential act as a complementary biomarker for tPD-L1. 3. Survival Analysis Based on CTC PD-L1 Status Kaplan-Meier survival analysis demonstrated significant differences between CTC PD-L1 + and CTC PD-L1 - groups. The median progression-free survival (PFS) was markedly longer in CTC PD-L1 + patients (16 months vs 4 months; HR = 0.28, 95% CI: 0.096-0.78, p = 0.0041) (Fig 4A) but not in tPD-L1 (median survival, 12 months vs undefined; HR = 0.83, 95% CI: 0.28-2.5, p = 0.74) (Fig 4C). Similarly, overall survival (OS) showed significant improvement in the CTC PD-L1 + group, with median OS not reached compared to the CTC PD-L1 - group (HR = 0.19, 95% CI: 0.05-0.74, p = 0.017) (Fig 4B), also not in tPD-L1 (HR = 0.88, 95% CI: 0.23-3.4, p = 0.85) (Fig 4D). Multivariate analysis incorporating clinical variables (age, gender, histology, treatment modality) and laboratory parameters (CEA, CYFRA21-1, NSE, SCC) identified CTC PD-L1 as the sole independent predictor of both PFS ( p = 0.039) and OS ( p = 0.033) (Table 3). 4. Dynamic CTC Monitoring During Treatment Serial blood sampling was performed in 21 patients at day 42 (T1) to evaluate CTC dynamics. The overall CTC positivity rate decreased from 71.2% at baseline to 33.3% at T1. In the PR patients (16/21), 14 patients (87.5%) exhibited CTC decline or stable, correlating with clinical response. Extended monitoring at day 84 (T2) was obtained in 8 patients, of which 7 patients were PR and 1 patient was SD. CTC analysis revealed consistent patterns: 7 PR patients showed either decreased or stable CTC counts, while the SD single patient presented increased CTC counts. Notably, NSE levels showed better correlation with treatment response compared to other conventional markers (CEA, CYFRA21-1, SCC), suggesting its potential complementary role in monitoring treatment efficacy. These comprehensive findings demonstrate the robust predictive and prognostic value of CTC PD-L1 assessment in advanced lung cancer patients receiving combination of chemotherapy and immunotherapy, offering a promising liquid biopsy approach for treatment stratification and monitoring (Fig 5). Discussion This study prospectively enrolled 52 patients with advanced lung cancer (stages III-IV) and examined the association between PD-L1 expression in CTCs and the efficacy of combination of chemotherapy and immunotherapy. A novel procedure to detect and characterize PD-L1 expression on CTCs was developed. CTC PD-L1 was identified on special phenotype with MFI ratio of CK/CD45 > 1.7 and PD-L1/CD45 > 3.2. The core findings revealed that CTC PD-L1 was associated with significantly higher objective response rate (ORR) (84.2%) and disease control rate (DCR) (94.7%) compared to CTC PD-L1 − (ORR: 36.8%, DCR: 68.4%), which was similar to the results of tPD-L1, though the accordance of CTC PD-L1 status and tPD-L1 was only 52.6%. Moreover, CTC PD-L1 + emerged as an independent predictor of combining chemotherapy and PD-1/PD-L1 immunotherapy efficacy with significant prognostic value for progression-free survival (PFS) (HR = 0.28, 95% CI: 0.096–0.78, p = 0.0041) and overall survival (OS) (HR = 0.19, 95% CI: 0.05–0.74, p = 0.017). Furthermore, detailed dynamic monitoring data confirmed that the change of CTCs served as a prognostic indicator for therapy efficacy. The majority of PR patients (87.2%) showed a decrease in CTC counts from baseline (T0) to the first follow-up (T1). Compared to traditional tissue biopsies, CTC testing in patients with advanced lung cancer was previously shown to provide higher feasibility in real-time assessment of tumor changes during PD-1/PD-L1 antibody immunotherapy( 35 ). CTC dynamic monitoring suggested that change in CTC counts might act as a prognostic indicator for therapy efficacy( 36 ). Moreover, CTC PD-L1 was associated with poor PFS and OS indicated that its potential prognostic value in prediction for lung cancer( 37 ). Although prior investigations have provided preliminary insights into the potential utility of CTC PD-L1 assessment during immunotherapy, comprehensive evaluations delineating the quantitative and qualitative concordance between CTC PD-L1 and tPD-L1 expression remain conspicuously absent. Moreover, the clinical relevance of CTC PD-L1 as a predictive biomarker for combining chemotherapy and PD-1/PD-L1 immunotherapy remains contentious, with conflicting data regarding its association with therapeutic benefit( 38 , 39 ). In the current study, we investigated not only the correlation between CTC PD-L1 and tPD-L1, but also that clinical benefit of short-term treatment outcome (ORR and DCR) and long-term survival (PFS and OS), providing valuable insights for clinical practice. Moreover, we also explored dynamic changes of CTC before and after therapy, and the role of CTC testing in combination with tumor markers in predicting the efficacy of immunotherapy. In summary, the study employed a prospective cohort design, enrolling 52 patients with advanced lung cancer who were intended to treat with immunotherapy and followed for up to two years, ensuring data integrity and reliability. Multivariate analysis adjusted for multiple clinically relevant covariates (e.g., age, gender, histology, treatment modality, CEA, CYFRA21-1, NSE, SCC), allowing for a more accurate and comprehensive assessment of independent predictive value of CTC PD-L1. Additionally, the study evaluated PD-L1 status in CTCs not only at baseline but also dynamically during treatment, providing a comprehensive perspective on treatment response. Furthermore, monitoring the changes of CTC counts in combination with tumor markers would be better in tracking the disease progression of patients. This integrated study design and data analysis strategy offer new insights into the role of CTC monitoring in immunotherapy for advanced lung cancer, which may help identify patients with poor treatment response early, thereby optimizing therapeutic strategies. However, several limitations exist in this study. A limitation of the study was its small sample size of 38 patients, which may have affected the observed correlation between CTC PD-L1/tPD-L1 and clinical outcomes. To overcome this and to better understand the relationship between CTC PD-L1/tPD-L1 and combination of chemotherapy and immunotherapy outcomes, future studies should aim for larger and more diverse patient populations. This approach will enable a more accurate assessment of these biomarkers' impact on treatment response and survival, potentially leading to more personalized and effective immunotherapy strategies. Additionally, as a single center study, the generalizability of our findings also requires validation through further studies. In conclusion, the LiquidBiopsy™ detection system for evaluating CTC PD-L1 proved feasible, effective, and non-invasive. The baseline CTC PD-L1 was significantly correlated with clinical outcomes in combination of chemotherapy and PD-1/PD-L1 blockade therapy, including objective response rate (ORR), progression-free survival (PFS), and overall survival (OS). The study supports the potential use of CTC PD-L1 as a reliable predictive biomarker that could be integrated into therapeutic decision-making algorithms for combination of chemotherapy and PD-1/PD-L1 inhibitor therapy. Abbreviations AUC area under curve CD45 leukocyte common antigen,LCA CEA carcinoembryonic antigen CI confidence interval CK Cytokeratin CTCs circulating tumor cells CYRFRA19 cytokeratin fragment antigen 19 DAPI 4',6-diamidino-2-phenylindole DCR disease control rate HR hazard ratio HRP horseradish peroxidase ORR objective response rate NSE neuron-specific enolase OS overall survival PD progression disease PD-1 programmed death 1 PD-L1 programmed death ligand 1 PFS progression free survival PR partial response ROC receiver operating characteristic curve SCC Squamous Cell Carcinoma Antigen SD stable disease WBC white blood cell. Declarations Data availability statement All data relevant to the study are included in the article. Consent for publication statement Written informed consent for publication was obtained from the patient or their legal guardian. Funding The study was supported by the Science and Technology Project of Suzhou Municipal Health Commission (LCZX202234) and Medical and Health Innovation Research Project of Suzhou Municipal Health Commission (CXYJ2024A07). Conflict of interest disclosure Authors declare no competing interests. Ethics statement This study was approved by the Fourth Affiliated Hospital of Soochow University (Approval number 220154) and under a registered clinical trial number (ChiCTR2500096312: Prediction value of PD-L1 statue on circulating tumor cells to PD-1/PD-L1 immunotherapy of NSCLC). Written informed consent was obtained from all participants. Authors’ contributions Conceptualization: Junhong Jiang, Dongjiang Tang, and Daxiong Zeng. Data curation: Xiaoying Wei. Formal analysis: Lin Chen. Funding acquisition: Junhong Jiang. Investigation: Lin Chen. Methodology: Lin Chen. Project administration: Dongjiang Tang. Resources: Xiaoying Wei. Supervision: Dongjiang Tang. Validation: Zhonglin Yang. Visualization: Lin Chen. Writing: Lin Chen. Review and editing: All the authors. References Brito ABC CM, de Lima VCC. Anti-PD1 versus anti-PD-L1 immunotherapy in first-line therapy for advanced non-small cell lung cancer: A systematic review and meta-analysis. Thorac Cancer. 2021;12(7):1058–66. Wang, Jing, Miaomiao, Li, Yan, Zhang, et al. PD-1/PD-L1 blockades in non-small-cell lung cancer therapy. Onco Targets Ther. 2016;9:489–502. Grasselly C DM, Bourguignon A, Talhi N, Mathe D, Tourette A, Serre L, Jordheim LP, Matera EL, Dumontet C. The Antitumor Activity of Combinations of Cytotoxic Chemotherapy and Immune Checkpoint Inhibitors Is Model-Dependent. Front Immunol. 2018;9:2100. Takamori S, Toyokawa G, Takada K, Shoji F, Maehara Y. Combination Therapy of Radiotherapy and Anti-PD-1/PD-L1 Treatment in Non-Small-cell Lung Cancer: A Mini-review. Clinical Lung Cancer. 2017;19(1):12–6. Johnson CB, Win SY. Combination therapy with PD-1/PD-L1 blockade: An overview of ongoing clinical trials. Oncoimmunology. 2018;7(4):e1408744. Hayashi H, Nakagawa K. Combination therapy with PD-1 or PD-L1 inhibitors for cancer. Int J Clin Oncol. 2020;25(5):818–30. Gandhi L R-AD, Gadgeel S, Esteban E, Felip E, De Angelis F, Domine M, Clingan P, Hochmair MJ, Powell SF, Cheng SY, Bischoff HG, Peled N, Grossi F, Jennens RR, Reck M, Hui R, Garon EB, Boyer M, Rubio-Viqueira B, Novello S, Kurata T, Gray JE, Vida J, Wei Z, Yang J, Raftopoulos H, Pietanza MC, Garassino MC. Pembrolizumab plus Chemotherapy in Metastatic Non–Small-Cell Lung Cancer. N Engl J Med. 2018;378(22):2078–92. Baas P, Garon EB, Herbst RS, Felip E, Kim DW. Relationship between level of PD-L1 expression and outcomes in the KEYNOTE-010 study of pembrolizumab vs docetaxel for previously treated, PD-L1-Positive NSCLC. Journal of Clinical Oncology. 2016;34(15):9015-. Reck M R-AD, Robinson AG, Hui R, Csőszi T, Fülöp A, Gottfried M, Peled N, Tafreshi A, Cuffe S, O'Brien M, Rao S, Hotta K, Vandormael K, Riccio A, Yang J, Pietanza MC, Brahmer JR. Updated Analysis of KEYNOTE-024: Pembrolizumab Versus Platinum-Based Chemotherapy for Advanced Non-Small-Cell Lung Cancer With PD-L1 Tumor Proportion Score of 50% or Greater. J Clin Oncol. 2019;37(7):537–46. Paz-Ares L, Horn L, Borghaei H, Spigel DR, Brahmer JR. Phase III, randomized trial (CheckMate 057) of nivolumab (NIVO) versus docetaxel (DOC) in advanced non-squamous cell (non-SQ) non-small cell lung cancer (NSCLC). Journal of Clinical Oncology. 2015;33(18):LBA109. Shukuya T, Carbone DP. Predictive Markers for the Efficacy of Anti-PD-1/PD-L1 Antibodies in Lung Cancer. J Thorac Oncol. 2016;11(7):976–88. Darnell EP, Mooradian MJ, Baruch EN, Yilmaz M, Reynolds KL. Immune-Related Adverse Events (irAEs): Diagnosis, Management, and Clinical Pearls. Curr Oncol Rep. 2020;22(4):39. Gandara DR, Paul SM, Kowanetz M, Schleifman E, Shames DS. Blood-based tumor mutational burden as a predictor of clinical benefit in non-small-cell lung cancer patients treated with atezolizumab. Nat Med. 2018;24(9):1441–8. Overman MJ, Mcdermott R, Leach JL, Lonardi S, André T. Nivolumab in patients with metastatic DNA mismatch repair-deficient or microsatellite instability-high colorectal cancer (CheckMate 142): an open-label, multicentre, phase 2 study. Lancet Oncol. 2017;18(9):1182–91. Gao G, Zhang XD, Qu H, Yao B, Zhou Y, Xiang J, et al. A comprehensive pan-cancer analysis of CD274 gene amplification, tumor mutation burden, microsatellite instability, and PD-L1 expression in Chinese cancer patients. Ann Transl Med. 2021;9(8):677. Kara M AG, Sak SD, Kavukçu S. Implantation metastasis caused by fine needle aspiration biopsy following curative resection of stage IB non-small cell lung cancer. Eur J Cardiothorac Surg. 2001;20(4):868–70. Oh KS, Mahalingam M. PD-L1 Detection—Pearls and Pitfalls Associated With Current Methodologies Focusing on Entities Relevant to Dermatopathology. Am J Dermatopathol. 2019;41(8):539–65. Zhu J, Armstrong AJ, Friedlander TW, Kim W, Pal SK, George DJ, et al. Biomarkers of immunotherapy in urothelial and renal cell carcinoma: PD-L1, tumor mutational burden, and beyond. J Immunother Cancer. 2018;6(1):4. Chen YL, Huang WC, Lin FM, Hsieh HB, Hsieh CH, Hsieh RK, et al. Novel circulating tumor cell-based blood test for the assessment of PD-L1 protein expression in treatment-nave, newly diagnosed patients with non-small cell lung cancer. Cancer Immunol. 2019;68(7):1087–94. Chi Z PL, Karamchandani DM, Xu J.. PD-L1 (22C3) expression and prognostic implications in esophageal squamous cell carcinoma. Ann Diagn Pathol. 2025;74:152394. Azad A, Lim SY, D'Costa Z, Jones K, Diana A, Sanso OJ, et al. PD-L1 blockade enhances response of pancreatic ductal adenocarcinoma to radiotherapy. EMBO Mol Med. 2016;9(2):167–80. Hong L, Negrao MV, Dibaj SS, Chen R, Zhang J. Programmed Death Ligand 1 Heterogeneity and its Impact on Benefit from Immune Checkpoint Inhibitors in Non-Small-Cell Lung Cancer. J Thorac Oncol. 2020;15(9):1449–59. Ilie M, Long-Mira E, Bence C, Butori C, Lassalle S, Bouhlel L, et al. Comparative study of the PD-L1 status between surgically resected specimens and matched biopsies of NSCLC patients reveal major discordances: a potential issue for anti-PD-L1 therapeutic strategies. Ann Oncol. 2015;27(1):147–53. Thawani R, Fakhoury K, Becker KD. Cause of mortality in patients with lung cancer and brain metastasis. Journal of Clinical Oncology. 2020;38(15):e21743-e. Yu JJ, Shu C, Yang HY, Xiao W. The Presence of Circulating Tumor Cell Cluster Characterizes an Aggressive Hepatocellular Carcinoma Subtype. Front Oncol. 2021;11:734564. Szczerba BM, Castro-Giner F, Vetter M, Krol I, Gkountela S, Landin J, et al. Neutrophils escort circulating tumour cells to enable cell cycle progression. Nature. 2019;566(7745):553–7. Lin D SL, Luo M, Zhang K, Li J, Yang Q, Zhu F, Zhou D, Zheng S, Chen Y, Zhou J. Circulating tumor cells: biology and clinical significance. Signal Transduct Target Ther. 2021;6(1):404. Sinoquet L, Jacot W, Gauthier L, Pouderoux S, Alix-Panabières C. Programmed Cell Death Ligand 1-Expressing Circulating Tumor Cells: A New Prognostic Biomarker in Non-Small Cell Lung Cancer. Clin Chem. 2021;66(11):1503–12. Cheng Y WT, Lv X, Li R, Yuan L, Shen J, Li Y, Yan T, Liu B, Wang L. Detection of PD-L1 Expression and Its Clinical Significance in Circulating Tumor Cells from Patients with Non-Small-Cell Lung Cancer. Cancer Manag Res. 2020;12:2069–78. Ikeda M, Koh Y, Teraoka S, Sato K, Oyanagi J, Hayata A, et al. Longitudinal Evaluation of PD-L1 Expression on Circulating Tumor Cells in Non-Small Cell Lung Cancer Patients Treated with Nivolumab. Cancers (Basel). 2021;13(10):2290. Acheampong E AR, Page K, Wadsley MK, Beasley AB, Coombes RC, Shaw JA, Gray ES. Meta-Analysis of Circulating Tumor Cell PD-L1 Expression and the Association with Clinical Outcomes in Non-Small Cell Lung Cancer. Clin Chem. 2024;70(1):234–49. Winer-Jones JP VB, Arquilevich N, Fang C, Ferguson S, Harkins D, Hill C, Klem E, Pagano PC, Peasley C, Romero J, Shartle R, Vasko RC, Strauss WM, Dempsey PW. Circulating Tumor Cells: Clinically Relevant Molecular Access Based on a Novel CTC Flow Cell. PLoS One. 2014;9(1):e86717. Chen W ZJ, Huang L, Chen L, Zhou Y, Tang D, Xie Y, Wang H, Huang C. Detection of HER2-positive Circulating Tumor Cells Using the LiquidBiopsy System in Breast Cancer. Clin Breast Cancer. 2019;19(1):e239-e46. Ilie M JJ, Huang L, Hofman V, Khambata-Ford S, Hofman P. Use of the 22C3 anti-programmed death ligand 1 antibody to determine programmed death ligand 1 expression in cytology samples obtained from non-small cell lung cancer patients. Cancer Cytopathol. 2018;126(4):264–74. Cao L YW, Zhao X, Chen Z. Diagnostic and prognostic value of circulating tumor cells in renal cell cancer: A systematic review and meta-analysis. Asian J Surg. 2024;47(8):3425–34. Su X ZC, Chen S, Ma Q, Xiao H, Chen Q, Zou H. Prognosis value of circulating tumor cell PDL1 and baseline characteristics in patients with NSCLC treated with immune checkpoint inhibitors plus platinumcontaining drugs. Oncol Lett. 2024;27(3):131. Zhang B LY, Zhou S, Jiang H, Zhu K, Wang R. Predictive effect of PD-L1 expression for immune checkpoint inhibitor (PD-1/PD-L1 inhibitors) treatment for non-small cell lung cancer: A meta-analysis. Int Immunopharmacol. 2020;80:106214. Kloten V LR, Krahn T, Schlange T. Circulating Tumor Cell PD-L1 Expression as Biomarker for Therapeutic Efficacy of Immune Checkpoint Inhibition in NSCLC. Cells. 2019;8(8):809. Strati A EP, Lianidou E, Psyrri A. Clinical Significance of PD-L1 Status in Circulating Tumor Cells for Cancer Management during Immunotherapy. Biomedicines. 2023;11(6):1768. Tables Table 1: Characteristics of recruited patients and CTC detection Characteristic N CTC positive (%) CTC PD-L1 (%) Total 52 37 (71.2%) 26 (50.0%) Gender Female 8 6 (75.0%) 4 (50.0%) Male 44 31 (70.5%) 22 (50.0%) Cancer type Adenocarcinoma 15 8 (53.3%) 6 (40.0%) Squamous cell carcinoma 23 19 (82.6%) 13 (56.5%) Small cell lung cancer 14 10 (71.4%) 7 (50.0%) ICI therapy type PD-1 30 21 (70.0%) 15 (50.0%) PD-L1 8 6 (75.0%) 4 (50.0%) Table 2: Comparison of clinical and biological factors between patients with PD/SD and PR responses PD/SD PR p Age 66.5±13.4 69.3±8.9 0.439 Gender 0.28 Male 12 22 Female 3 1 Cancer subtype 0.44 NSCLC 13 17 SCLC 2 6 PD-1 or PD-L1 monotherapy 0.66 PD-1 14 21 PD-L1 1 2 CTC PD-L1 0.003* Positive 3 16 Negative 12 7 tPD-L1 positive cells 0.028* >=1% 5 16 <1% 10 7 CEA 10.9(4.86, 65.7) 3.63(1.93, 7.65) 0.091 CYRFRA19 6.07(3.12, 11.4) 4.41(3.29, 10.3) 0.153 NSE 15.7(12.7, 23.0) 16.2(12.5, 19.8) 0.153 SCC 1.97(0.82, 4.34) 1.81(1.26, 3.8) 0.062 *Significant relevance at p <0.05. Table 3: Univariate analysis of potential factors affecting progression-free survival (PFS) and overall survival (OS) Factors PFS OS Age 0.746 0.554 Gender 0.063 0.137 Cancer subtype 0.502 0.934 PD-1 or PD-L1 monotherapy 0.684 0.847 Smoking or not 0.144 0.074 Hypertension 0.059 0.106 Diabetes 0.765 0.748 ECOG score 0.072 0.203 CTC PD-L1 0.039* 0.033* tPD-L1 0.405 0.844 CEA 0.051 0.658 CYRFRA19 0.361 0.688 NSE 0.567 0.122 SCC 0.777 0.556 *Significant relevance at p <0.05. Additional Declarations No competing interests reported. Supplementary Files Supplementaryfigures.doc CONSORT2025expandedchecklist.pdf Cite Share Download PDF Status: Published Journal Publication published 31 Jan, 2026 Read the published version in European Journal of Medical Research → Version 1 posted Editorial decision: Revision requested 12 Dec, 2025 Reviews received at journal 12 Dec, 2025 Reviews received at journal 24 Nov, 2025 Reviewers agreed at journal 23 Nov, 2025 Reviewers agreed at journal 23 Nov, 2025 Reviewers agreed at journal 21 Nov, 2025 Reviewers agreed at journal 10 Nov, 2025 Reviewers invited by journal 06 Nov, 2025 Editor assigned by journal 03 Nov, 2025 Submission checks completed at journal 01 Nov, 2025 First submitted to journal 31 Oct, 2025 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-7920255","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":545793641,"identity":"87c150dc-6595-4d18-9f97-5b4c3e790885","order_by":0,"name":"Xiaoying Wei","email":"","orcid":"","institution":"The Fourth Affiliated Hospital of Soochow University","correspondingAuthor":false,"prefix":"","firstName":"Xiaoying","middleName":"","lastName":"Wei","suffix":""},{"id":545793642,"identity":"0a37360d-2889-46e6-bf95-65e607ccef9e","order_by":1,"name":"Lin Chen","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA2klEQVRIiWNgGAWjYDACCRBRIMHAD+EyJ7ARp8VAgkGygUQtQHQAqoWgDv7ZzcceMBhY5BmfP/zs0Y0K6zw+9gbGDx9z8Fhy51i6AdBhxWY30syNc86kF7PxHGCWnLkNtxYDiRwzCSCZuO0Gg5l0btvhxDaJBDZmXrxa8r+BtWzuP/5NOvcfUVpy2MBaNjDkAG1pIEKLxI00M4kEoJYZN3LKpHOOgfxysBmvX/hnJD+T+FBRl9jff3ybdE6NdZ58e/PBDx/xaAGDBFQuYwMB9aNgFIyCUTAKCAEAJ21JTCLeHecAAAAASUVORK5CYII=","orcid":"","institution":"Zhuhai Sanmed Biotech Ltd.","correspondingAuthor":true,"prefix":"","firstName":"Lin","middleName":"","lastName":"Chen","suffix":""},{"id":545793643,"identity":"14413d9d-604c-4a75-a564-331a17b3e7e7","order_by":2,"name":"Zhonglin Yang","email":"","orcid":"","institution":"Zhuhai Sanmed Biotech Ltd.","correspondingAuthor":false,"prefix":"","firstName":"Zhonglin","middleName":"","lastName":"Yang","suffix":""},{"id":545793644,"identity":"ec221343-8741-4661-b9c1-da64a71e53a2","order_by":3,"name":"Daxiong Zeng","email":"","orcid":"","institution":"The Fourth Affiliated Hospital of Soochow University","correspondingAuthor":false,"prefix":"","firstName":"Daxiong","middleName":"","lastName":"Zeng","suffix":""},{"id":545793645,"identity":"8eba8a7f-ec0e-4f0d-ab9e-af9d88178884","order_by":4,"name":"Dongjiang Tang","email":"","orcid":"","institution":"Zhuhai Sanmed Biotech Ltd.","correspondingAuthor":false,"prefix":"","firstName":"Dongjiang","middleName":"","lastName":"Tang","suffix":""},{"id":545793646,"identity":"abb227c1-960d-42c7-9c35-5cfe33303ab5","order_by":5,"name":"Junhong Jiang","email":"","orcid":"","institution":"The Fourth Affiliated Hospital of Soochow University","correspondingAuthor":false,"prefix":"","firstName":"Junhong","middleName":"","lastName":"Jiang","suffix":""}],"badges":[],"createdAt":"2025-10-22 08:41:32","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7920255/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7920255/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s40001-026-03945-5","type":"published","date":"2026-01-31T15:58:37+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":96159852,"identity":"27c07ec8-fac5-4dc5-a026-3f543f122ec4","added_by":"auto","created_at":"2025-11-18 08:45:58","extension":"doc","order_by":0,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":168448,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.doc","url":"https://assets-eu.researchsquare.com/files/rs-7920255/v1/9423df8c07290f3ca234e595.doc"},{"id":96159873,"identity":"510443f9-efba-4d7a-97aa-d4911292e32f","added_by":"auto","created_at":"2025-11-18 08:45:58","extension":"doc","order_by":1,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":24050688,"visible":true,"origin":"","legend":"","description":"","filename":"Figures.doc","url":"https://assets-eu.researchsquare.com/files/rs-7920255/v1/b3eb08af1ffa694375b352ff.doc"},{"id":96159854,"identity":"7cc7cfa9-6190-482d-b56b-c04c64119592","added_by":"auto","created_at":"2025-11-18 08:45:58","extension":"doc","order_by":2,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":58368,"visible":true,"origin":"","legend":"","description":"","filename":"tables.doc","url":"https://assets-eu.researchsquare.com/files/rs-7920255/v1/7a7ca834fb95ae5b2e2fe1bc.doc"},{"id":96251130,"identity":"cdb2c719-0916-4062-8b0c-f82700bde2a7","added_by":"auto","created_at":"2025-11-19 07:39:23","extension":"json","order_by":3,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":7799,"visible":true,"origin":"","legend":"","description":"","filename":"8d20ded123d34f12a0662bb8a37f6d0b.json","url":"https://assets-eu.researchsquare.com/files/rs-7920255/v1/5af6671c3518a98aac60d3d4.json"},{"id":96251886,"identity":"2b9b1b30-d42f-4379-9223-930496509809","added_by":"auto","created_at":"2025-11-19 07:40:09","extension":"pdf","order_by":4,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":273257,"visible":true,"origin":"","legend":"","description":"","filename":"CONSORT2025expandedchecklist.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7920255/v1/0f47af1f8078e8c1454c0634.pdf"},{"id":96250035,"identity":"4ba44eb5-4166-4195-a16b-7f69e77e3e0c","added_by":"auto","created_at":"2025-11-19 07:37:11","extension":"doc","order_by":5,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":658944,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryfigures.doc","url":"https://assets-eu.researchsquare.com/files/rs-7920255/v1/5eb3decdea24c176d8ed61f0.doc"},{"id":96252115,"identity":"dc209a80-4fff-41e0-9a2a-49507b05d150","added_by":"auto","created_at":"2025-11-19 07:40:28","extension":"xml","order_by":6,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":133938,"visible":true,"origin":"","legend":"","description":"","filename":"8d20ded123d34f12a0662bb8a37f6d0b1enriched.xml","url":"https://assets-eu.researchsquare.com/files/rs-7920255/v1/dd8c0db9b99d8aa67691bf22.xml"},{"id":96249025,"identity":"1af964d7-11e0-489d-b84f-9686c35cad50","added_by":"auto","created_at":"2025-11-19 07:29:57","extension":"eps","order_by":8,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":51492,"visible":true,"origin":"","legend":"","description":"","filename":"drawingimage10.eps","url":"https://assets-eu.researchsquare.com/files/rs-7920255/v1/dd93afccc3032e12e0e9cbb8.eps"},{"id":96251147,"identity":"5559f558-e63d-414a-8604-a6c403aaccbf","added_by":"auto","created_at":"2025-11-19 07:39:24","extension":"eps","order_by":9,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":462,"visible":true,"origin":"","legend":"","description":"","filename":"drawingimage2.eps","url":"https://assets-eu.researchsquare.com/files/rs-7920255/v1/4c576c9bf85ffb089dfcdb6a.eps"},{"id":96159862,"identity":"0d539e33-a223-4244-a372-d57d288abb07","added_by":"auto","created_at":"2025-11-18 08:45:58","extension":"eps","order_by":10,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":220,"visible":true,"origin":"","legend":"","description":"","filename":"drawingimage3.eps","url":"https://assets-eu.researchsquare.com/files/rs-7920255/v1/20ab2c50267cdcbee3836709.eps"},{"id":96159866,"identity":"d19c1620-52f1-4c8b-8c95-485955548bda","added_by":"auto","created_at":"2025-11-18 08:45:58","extension":"eps","order_by":11,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":51308,"visible":true,"origin":"","legend":"","description":"","filename":"drawingimage4.eps","url":"https://assets-eu.researchsquare.com/files/rs-7920255/v1/9844f85014cf1241044f111b.eps"},{"id":96159867,"identity":"e2354d3b-3d06-40de-88c5-170f9b707793","added_by":"auto","created_at":"2025-11-18 08:45:58","extension":"eps","order_by":12,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":52024,"visible":true,"origin":"","legend":"","description":"","filename":"drawingimage5.eps","url":"https://assets-eu.researchsquare.com/files/rs-7920255/v1/6be1b661b53a2197d31f33af.eps"},{"id":96251911,"identity":"d146facb-0c52-45d7-a3bd-438b07ff7e4f","added_by":"auto","created_at":"2025-11-19 07:40:11","extension":"eps","order_by":13,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":51432,"visible":true,"origin":"","legend":"","description":"","filename":"drawingimage6.eps","url":"https://assets-eu.researchsquare.com/files/rs-7920255/v1/8b36c3380e8ce61ad0bd41ef.eps"},{"id":96250061,"identity":"c874287b-0aeb-40f6-87a1-7e3e1859056c","added_by":"auto","created_at":"2025-11-19 07:37:15","extension":"eps","order_by":14,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":51351,"visible":true,"origin":"","legend":"","description":"","filename":"drawingimage7.eps","url":"https://assets-eu.researchsquare.com/files/rs-7920255/v1/8e468792ff8f2395fff79894.eps"},{"id":96159871,"identity":"143dc64e-0d8d-4c4a-baa3-43335c2c9de5","added_by":"auto","created_at":"2025-11-18 08:45:58","extension":"eps","order_by":15,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":51212,"visible":true,"origin":"","legend":"","description":"","filename":"drawingimage8.eps","url":"https://assets-eu.researchsquare.com/files/rs-7920255/v1/f40ea82f02237af51c12a4ce.eps"},{"id":96249987,"identity":"b11674e8-9215-42c9-a6b3-f02c25c75d1b","added_by":"auto","created_at":"2025-11-19 07:36:59","extension":"png","order_by":17,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":74892,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7920255/v1/38a4d068a12c00a1de8d9c4b.png"},{"id":96159878,"identity":"69a301e5-2df7-419f-929e-9ba31a4bde0a","added_by":"auto","created_at":"2025-11-18 08:45:59","extension":"emf","order_by":18,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":31524,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage10.emf","url":"https://assets-eu.researchsquare.com/files/rs-7920255/v1/aa7be371961bcff3d9b60e3f.emf"},{"id":96251075,"identity":"4aad46e8-394b-4ca6-9450-bef64c26d2e9","added_by":"auto","created_at":"2025-11-19 07:39:17","extension":"emf","order_by":19,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":29128,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage11.emf","url":"https://assets-eu.researchsquare.com/files/rs-7920255/v1/a18ced17bd47600f722cd337.emf"},{"id":96252293,"identity":"7a1e4e4c-4ed6-447f-8291-3a0d42831e26","added_by":"auto","created_at":"2025-11-19 07:40:45","extension":"emf","order_by":20,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":31388,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage12.emf","url":"https://assets-eu.researchsquare.com/files/rs-7920255/v1/b16ccdbcff313c863832b76a.emf"},{"id":96251957,"identity":"5a89fbb0-8e0e-418f-9438-3700aa5dc423","added_by":"auto","created_at":"2025-11-19 07:40:14","extension":"jpeg","order_by":21,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":131401,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage13.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7920255/v1/90f48ac866f018137d097160.jpeg"},{"id":96159884,"identity":"ceb48470-eabb-448c-8d17-0a1ef86b7a28","added_by":"auto","created_at":"2025-11-18 08:45:59","extension":"jpeg","order_by":22,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":209755,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage14.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7920255/v1/430c9b2babce8d74f3a32eb1.jpeg"},{"id":96159882,"identity":"fe39507a-9c9a-4b28-8ae6-baefbdca6609","added_by":"auto","created_at":"2025-11-18 08:45:59","extension":"jpeg","order_by":23,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":203252,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage15.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7920255/v1/58850a660ce2503dbabe4c5c.jpeg"},{"id":96159895,"identity":"2defe21c-b346-46f1-8883-9fe4bff45f67","added_by":"auto","created_at":"2025-11-18 08:45:59","extension":"jpeg","order_by":24,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":206278,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage16.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7920255/v1/892fdde83d70343a3c6a972a.jpeg"},{"id":96159874,"identity":"0029e050-b073-45a4-a1d8-db44c6599a68","added_by":"auto","created_at":"2025-11-18 08:45:58","extension":"jpeg","order_by":25,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":244241,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage17.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7920255/v1/c0b3d775627188fa7e76e228.jpeg"},{"id":96251042,"identity":"d5684c52-b391-49a1-966c-af787a064888","added_by":"auto","created_at":"2025-11-19 07:39:15","extension":"jpeg","order_by":26,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":215676,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage18.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7920255/v1/8b1c4162fa1158bb1ee8e84a.jpeg"},{"id":96159891,"identity":"63e9e9dc-a97c-4140-9317-d201a549d636","added_by":"auto","created_at":"2025-11-18 08:45:59","extension":"jpeg","order_by":27,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":252810,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage19.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7920255/v1/416bd2a1989f8c3c139fc945.jpeg"},{"id":96159876,"identity":"f4c34c92-abc3-4387-acc3-09f2bef77e54","added_by":"auto","created_at":"2025-11-18 08:45:58","extension":"jpeg","order_by":28,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":361768,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7920255/v1/d3bf4c84ad02fea83ea30979.jpeg"},{"id":96250984,"identity":"e849669e-8507-4d58-912f-bcde7c87b25c","added_by":"auto","created_at":"2025-11-19 07:39:11","extension":"jpeg","order_by":29,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":200997,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage20.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7920255/v1/c27dcfc683830859ea6f784f.jpeg"},{"id":96159877,"identity":"35ff94af-3e09-4d0f-98c7-ca3526fb1576","added_by":"auto","created_at":"2025-11-18 08:45:59","extension":"jpeg","order_by":30,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":255151,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage21.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7920255/v1/10772e9bf44e8d9f8c34c8b7.jpeg"},{"id":96250149,"identity":"d1329fac-5d34-4acd-8082-877c3eb8a6e1","added_by":"auto","created_at":"2025-11-19 07:37:38","extension":"jpeg","order_by":31,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":143221,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7920255/v1/030b063bec8da64849f9b8a8.jpeg"},{"id":96159883,"identity":"56279f11-239f-4f7d-aba4-71e494da7c78","added_by":"auto","created_at":"2025-11-18 08:45:59","extension":"emf","order_by":32,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":5565872,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage4.emf","url":"https://assets-eu.researchsquare.com/files/rs-7920255/v1/77aa5002c3935c545621362e.emf"},{"id":96159896,"identity":"46309418-e741-4829-8f72-555ce857032d","added_by":"auto","created_at":"2025-11-18 08:45:59","extension":"emf","order_by":33,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":7140792,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage5.emf","url":"https://assets-eu.researchsquare.com/files/rs-7920255/v1/a601ccc3a665483a232c2f3e.emf"},{"id":96248974,"identity":"52b9b131-7bda-41bb-9f1d-b9d7af587217","added_by":"auto","created_at":"2025-11-19 07:29:50","extension":"emf","order_by":34,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":632084,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage6.emf","url":"https://assets-eu.researchsquare.com/files/rs-7920255/v1/1759420fcf04ee036b964a5c.emf"},{"id":96252239,"identity":"1e2bae35-acba-4068-980d-ba6cd5fce1cf","added_by":"auto","created_at":"2025-11-19 07:40:41","extension":"jpeg","order_by":35,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":193625,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7920255/v1/3e1a57560ed7d13b77353ae8.jpeg"},{"id":96250853,"identity":"5ea7cbf3-cfd6-42ab-b66c-e8d69894ccdb","added_by":"auto","created_at":"2025-11-19 07:39:03","extension":"jpeg","order_by":36,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":94128,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage8.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7920255/v1/d92defba7328bd913433d2b5.jpeg"},{"id":96159886,"identity":"0a4b6dc6-4e14-486c-8190-abc3f0779a77","added_by":"auto","created_at":"2025-11-18 08:45:59","extension":"emf","order_by":37,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":31524,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage9.emf","url":"https://assets-eu.researchsquare.com/files/rs-7920255/v1/304adb831823b0bd2df1bb63.emf"},{"id":96250930,"identity":"d113ae41-b422-4b79-85e7-63003fc46eee","added_by":"auto","created_at":"2025-11-19 07:39:08","extension":"png","order_by":38,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":32397,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7920255/v1/5bc73e4338bb33b0d9fc2774.png"},{"id":96251133,"identity":"0dcc6aa6-e01a-4776-8e9f-cd45f2a69695","added_by":"auto","created_at":"2025-11-19 07:39:23","extension":"png","order_by":39,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":7405,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage10.png","url":"https://assets-eu.researchsquare.com/files/rs-7920255/v1/cbbf02e2f4c278450cef46e2.png"},{"id":96250143,"identity":"b03570f3-17bb-4a83-9f9c-f53c2f0ac51e","added_by":"auto","created_at":"2025-11-19 07:37:37","extension":"png","order_by":40,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":6281,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage11.png","url":"https://assets-eu.researchsquare.com/files/rs-7920255/v1/8dbece59ff2dff0478c78b33.png"},{"id":96159909,"identity":"a66e8efb-2aaf-4f3e-af9b-b8b963250149","added_by":"auto","created_at":"2025-11-18 08:45:59","extension":"png","order_by":41,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":6284,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage12.png","url":"https://assets-eu.researchsquare.com/files/rs-7920255/v1/3191fe244ad0b43f319e28a4.png"},{"id":96249155,"identity":"e3f4db56-951d-4c84-9974-b4c0268fe992","added_by":"auto","created_at":"2025-11-19 07:30:28","extension":"png","order_by":42,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":21196,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage13.png","url":"https://assets-eu.researchsquare.com/files/rs-7920255/v1/9775a95e7ffb1aca09ed6bca.png"},{"id":96159908,"identity":"8a5c9511-a401-4e26-a659-6677af299f65","added_by":"auto","created_at":"2025-11-18 08:45:59","extension":"png","order_by":43,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":46659,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage14.png","url":"https://assets-eu.researchsquare.com/files/rs-7920255/v1/597b17468ae03070152bf2fc.png"},{"id":96251169,"identity":"88f346eb-a6bf-401d-ae75-aad9242eecc6","added_by":"auto","created_at":"2025-11-19 07:39:27","extension":"png","order_by":44,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":47470,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage15.png","url":"https://assets-eu.researchsquare.com/files/rs-7920255/v1/6fd13009345a4e430b000770.png"},{"id":96159879,"identity":"6a7e8181-9319-40dc-ad52-662a361d7068","added_by":"auto","created_at":"2025-11-18 08:45:59","extension":"png","order_by":45,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":44832,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage16.png","url":"https://assets-eu.researchsquare.com/files/rs-7920255/v1/1dad51560f906b60833d9910.png"},{"id":96251973,"identity":"aedbe6a9-590f-4063-828a-199a1d6a2590","added_by":"auto","created_at":"2025-11-19 07:40:15","extension":"png","order_by":46,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":58941,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage17.png","url":"https://assets-eu.researchsquare.com/files/rs-7920255/v1/7ae4e5e8a02ca65aedb9196a.png"},{"id":96249130,"identity":"1ff1af7c-0081-4fcc-9d28-ab57f49a7808","added_by":"auto","created_at":"2025-11-19 07:30:23","extension":"png","order_by":47,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":48122,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage18.png","url":"https://assets-eu.researchsquare.com/files/rs-7920255/v1/9b97cd903a9db493219331ed.png"},{"id":96159880,"identity":"b487fc1f-08a8-4a22-a1e3-0076f9907026","added_by":"auto","created_at":"2025-11-18 08:45:59","extension":"png","order_by":48,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":63414,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage19.png","url":"https://assets-eu.researchsquare.com/files/rs-7920255/v1/5f73d8dd70a330bd4fde3526.png"},{"id":96159903,"identity":"82d19cc5-a50f-4685-a5bd-49a1a7b848cc","added_by":"auto","created_at":"2025-11-18 08:45:59","extension":"png","order_by":49,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":64122,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7920255/v1/5b701675d8b7829274f52ba6.png"},{"id":96252207,"identity":"34802f23-0190-4396-b2c7-bf9e3e95225e","added_by":"auto","created_at":"2025-11-19 07:40:40","extension":"png","order_by":50,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":43918,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage20.png","url":"https://assets-eu.researchsquare.com/files/rs-7920255/v1/0087a0b019ace2160d621246.png"},{"id":96252006,"identity":"5a97394e-af6c-423a-a8b0-3e82c91d8bff","added_by":"auto","created_at":"2025-11-19 07:40:18","extension":"png","order_by":51,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":65501,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage21.png","url":"https://assets-eu.researchsquare.com/files/rs-7920255/v1/a6e314f82d236b43bcd15c4a.png"},{"id":96159910,"identity":"02acc5f3-425f-4d1a-bfee-5d907a8f65f4","added_by":"auto","created_at":"2025-11-18 08:45:59","extension":"png","order_by":52,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":24939,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7920255/v1/c2cee3a2df525123e3f27db0.png"},{"id":96250997,"identity":"1724bb90-7a17-4c51-bd8c-098020281eaa","added_by":"auto","created_at":"2025-11-19 07:39:12","extension":"png","order_by":53,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":4579,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7920255/v1/aa4c401142b333987ff501e6.png"},{"id":96159900,"identity":"f62a190f-16eb-4337-9e4e-156579d8aac3","added_by":"auto","created_at":"2025-11-18 08:45:59","extension":"png","order_by":54,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":5227,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-7920255/v1/c3eff3030a490cbfb1ad9950.png"},{"id":96159913,"identity":"72d477df-a8ed-4c23-bfef-d11985c03573","added_by":"auto","created_at":"2025-11-18 08:46:00","extension":"png","order_by":55,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":5631,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-7920255/v1/7816e2ba837251ab6ccafc8a.png"},{"id":96252003,"identity":"64a3c541-87a6-4b7e-838c-5b8bae732540","added_by":"auto","created_at":"2025-11-19 07:40:18","extension":"png","order_by":56,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":40376,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-7920255/v1/c4f3178c7346df8c0f17e3c4.png"},{"id":96159888,"identity":"54b2c43c-e9f6-4c56-9eff-9e4d1421f484","added_by":"auto","created_at":"2025-11-18 08:45:59","extension":"png","order_by":57,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":24272,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-7920255/v1/7bd50669f41a167e97b69b0f.png"},{"id":96159912,"identity":"c9c794a5-2dab-4879-b698-41ba6b79a837","added_by":"auto","created_at":"2025-11-18 08:46:00","extension":"png","order_by":58,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":7227,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-7920255/v1/3d5ae0e79723492f467bd64e.png"},{"id":96250936,"identity":"4836d9b4-54be-439f-9012-81a7fdf1ec8b","added_by":"auto","created_at":"2025-11-19 07:39:08","extension":"xml","order_by":59,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":133975,"visible":true,"origin":"","legend":"","description":"","filename":"8d20ded123d34f12a0662bb8a37f6d0b1structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7920255/v1/4b0654b83b72835664b72e00.xml"},{"id":96159904,"identity":"62c37659-61cb-4fcd-ab67-c84ccb0d1e3f","added_by":"auto","created_at":"2025-11-18 08:45:59","extension":"html","order_by":60,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":146603,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7920255/v1/af119c8dbda9a6082150fa65.html"},{"id":96251028,"identity":"97598b00-4c4e-45c1-bd6c-0de2a8dd6788","added_by":"auto","created_at":"2025-11-19 07:39:14","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":92020,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eWorkflow of the study. \u003c/strong\u003eA total of 52 advanced lung cancer patients were enrolled in this study, among whom 38 received at least 2 cycles of combination chemotherapy and PD-1/PD-L1 immunotherapy. The relationship between baseline CTC PD-L1 expression levels and ORR, PFS, and OS was evaluated.\u003c/p\u003e","description":"","filename":"Figures1.png","url":"https://assets-eu.researchsquare.com/files/rs-7920255/v1/a910b7eb9fcfb86e145f1da3.png"},{"id":96159851,"identity":"99b98af3-8bdc-4b0a-8e75-7b47cb050abf","added_by":"auto","created_at":"2025-11-18 08:45:58","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":258461,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCharacterization of CTC in immunofluorescence staining.\u003c/strong\u003eCTCs were identified as CK+/PD-L1+/DAPI+/CD45-, or CK+/PD-L1-/DAPI+/CD45-; WBCs were detected by using CD45 antibody with CK-/PD-L1-/DAPI+/CD45+. To establish an interpretive method for CK and PD-L1, we compared the CK staining fluorescence intensity between the NCI-H820 tumor cell line and leukocytes WBC, as well as the PD-L1 staining fluorescence intensity between NCI-H820 and leukocytes. Due to the fact that some leukocytes also express PD-L1, there is partial overlap in the PD-L1 fluorescence signals between tumor cells and leukocytes. Therefore, we further calculated the ratio of PD-L1 to CD45 staining signal intensity and performed an analysis using the ROC curve. A:\u003cstrong\u003e \u003c/strong\u003eIdentification of CTCs and their PD-L1 expression. Ba, Bb: CK fluorescence intensity and CK/CD45 ratio to distinguish tumor cells from leukocytes. Bc: ROC analysis evaluating the CK/CD45 ratio for tumor cell discrimination (Sensitivity and specificity was 100% (95% CI 99.52% to 100.0%) and 100% (95% CI 99.27% to 100.0%) respectively at threshold of 1.7 with AUC 1.0 (95% CI 1.0 to 1.0, p\u0026lt;0.0001)). Bd, Be: PD-L1 fluorescence intensity and PD-L1/CD45 ratio to distinguish tumor-specific PD-L1 expression. Bf: ROC analysis evaluating the PD-L1/CD45 ratio for specific PD-L1 expression assessment (Sensitivity and specificity was 99.90% (95% CI 99.81% to 99.95%) and 99.62% (95% CI 98.61% to 99.93%) respectively at threshold of 3.2 with AUC 0.9999 (95% CI 1.0 to 1.0, p\u0026lt;0.0001)).\u003c/p\u003e","description":"","filename":"Figures2.png","url":"https://assets-eu.researchsquare.com/files/rs-7920255/v1/b7c8e82c560ea137c3e7b641.png"},{"id":96159849,"identity":"4520613c-d2a0-4f65-942b-82f6b9dd87f2","added_by":"auto","created_at":"2025-11-18 08:45:58","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":36326,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCorrelation between disease status and the counts of CTC PD-L1.\u003c/strong\u003e PD-L1 expression on CTC was evaluated before combining chemotherapy and PD-1/PD-L1 immunotherapy (at baseline) from 38 patients. ORR and DCR were recorded after 2 cycles therapy. Association of CTC PD-L1 to ORR, DCR had been analyzed. Difference of CTC PD-L1\u003csup\u003e+\u003c/sup\u003e counts was analyzed on PR and SD/PD by using student’s t-test. Sensitivity and specificity of CTC PD-L1 were analyzed by using ROC curve. A: Objective response rate of patients with or with-out CTC PD-L1 at baseline (84.2% vs 36.8%). B: Objective response rate of patients with or with-out tPD-L1 at baseline (76.0% vs 41.2%). C: Disease control rate of patients with or with-out CTC PD-L1 at baseline (94.7% vs 68.4%). D: Disease control rate of patients with or with-out tPD-L1 at baseline (95.2% vs 64.7%). E: Count distribution of PD-L1 positive CTCs in the PR, SD, and PD groups at baseline (p=0.046). F: Percentage of tPD-L1 positive in the PR, SD, and PD groups at baseline (p=0.9124). G: Analyzed the cutoff of CTC PD-L1 to ORR by ROC curve (AUC=0.7105, 95% CI 0.5418 to 0.8792, p=0.0265; cutoff=1, sensitivity=69.5%, specificity=80%). H: Analyzed the cutoff of tPD-L1 to ORR by ROC curve (AUC=0.6751, 95% CI 0.4987 to 0.8515, p=0.0665; cutoff =1%, sensitivity=69.5%, specificity=80%).\u003c/p\u003e","description":"","filename":"Figures3.png","url":"https://assets-eu.researchsquare.com/files/rs-7920255/v1/28576496a05e4cb14c9824fa.png"},{"id":96252280,"identity":"cb295c55-7c85-4157-91eb-e9cda35dda29","added_by":"auto","created_at":"2025-11-19 07:40:44","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":23120,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAssociation between clinical outcome and CTC PD-L1 in the study.\u003c/strong\u003e A total of 38 patients who received combined chemotherapy and PD-1/PD-L1 immunotherapy were followed up for more than 3 months. PFS and OS were evaluated. Kaplan-Meier survival curves were used to analyze the differences in PFS and OS between patients with baseline CTC PD-L1\u003csup\u003e+\u003c/sup\u003e and baseline CTC PD-L1\u003csup\u003e−\u003c/sup\u003e status, and baseline tPD-L1\u003csup\u003e+\u003c/sup\u003e and baseline tPD-L1\u003csup\u003e−\u003c/sup\u003e status. A: Kaplan-Meier evaluates of PFS for patients in the subgroups of CTC PD-L1\u003csup\u003e+\u003c/sup\u003e and CTC PD-L1\u003csup\u003e− \u003c/sup\u003eat baseline (median survival, 16 months vs 4 months; HR=0.28, 95% CI: 0.096-0.78, \u003cem\u003ep\u003c/em\u003e=0.0041). B: Kaplan-Meier evaluates of OS for patients in the subgroups of CTC PD-L1\u003csup\u003e+\u003c/sup\u003e and CTC PD-L1\u003csup\u003e−\u003c/sup\u003e at baseline (median survival, undefined; HR=0.19, 95% CI: 0.05-0.74, \u003cem\u003ep\u003c/em\u003e=0.017). C: Kaplan-Meier evaluates of PFS for patients in the subgroups of tPD-L1\u003csup\u003e+\u003c/sup\u003e and tPD-L1\u003csup\u003e− \u003c/sup\u003eat baseline (median survival, 12 months vs undefined; HR=0.83, 95% CI: 0.28-2.5, \u003cem\u003ep\u003c/em\u003e=0.74). D: Kaplan-Meier evaluates of OS for patients in the subgroups of tPD-L1\u003csup\u003e+\u003c/sup\u003e and tPD-L1\u003csup\u003e−\u003c/sup\u003e at baseline (median survival, undefined; HR=0.88, 95% CI: 0.23-3.4, \u003cem\u003ep\u003c/em\u003e=0.85).\u003c/p\u003e","description":"","filename":"Figures4.png","url":"https://assets-eu.researchsquare.com/files/rs-7920255/v1/8da510c218518946994b2b14.png"},{"id":96159859,"identity":"56f3dbfb-9144-472d-bca0-1c0bd1f8bbf2","added_by":"auto","created_at":"2025-11-18 08:45:58","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1377797,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eComparing circulating biomarkers with CTCs for monitoring tumor dynamics.\u003c/strong\u003e CTC and tumor biomarkers in blood samples were evaluated at multiple time points for 8 patients: pre-treatment (baseline, T0), during treatment (after the second cycle and before the third cycle, T1), and after the fourth cycle and before the fifth cycle (T2). The association between the dynamic changes of CTC and tumor biomarkers and ORR was analyzed. Each case represents data from a different patient, and the red boxes indicate the corresponding tumor tissue. Case 1: A patient with lung squamous cell carcinoma who received combination therapy of Gemcitabine, Carboplatin, and Tislelizumab had PR after 2 cycles and PR after 4 cycles therapy (A); Case 2: A patient with lung adenocarcinoma who received combination therapy of Pemetrexed, Pembrolizumab had SD after 2 cycles and PR after 4 cycles therapy (B); Case 3: A patient with SCLC who received combination therapy of Etoposide, Carboplatin, Serplulimabhad PR after 2 cycles and PR after 4 cycles therapy (C); Case 4: A patient with SCLC who received combination therapy of Etoposide, Carboplatin, Serplulimab had PR after 2 cycles and PR after 4 cycles therapy (D); Case 5: A patient with lung squamous cell carcinoma who received combination therapy of Paclitaxel, Cisplatin, Tislelizumab had SD after 2 cycles and PR after 4 cycles therapy (E); Case 6: A patient with lung squamous cell carcinoma who received combination therapy of Albumin paclitaxel, Sintilimab had PR after 2 cycles and PR after 4 cycles therapy (F); Case 7: A patient with lung squamous cell carcinoma who received combination therapy of Paclitaxel, Cisplatin, Sintilimab had SD after 2 cycles and SD after 4 cycles therapy (G); Case 8: A patient with lung adenocarcinoma who received combination therapy of Carboplatin, Pemetrexed, Pembrolizumab had PR after 2 cycles and PR after 4 cycles therapy (H).\u003c/p\u003e","description":"","filename":"Figures5.png","url":"https://assets-eu.researchsquare.com/files/rs-7920255/v1/cafddb38de3c25de4f0b3c7e.png"},{"id":101691362,"identity":"35fcb8c1-fbd9-44d1-9357-47062e4cc23b","added_by":"auto","created_at":"2026-02-02 16:13:40","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2655214,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7920255/v1/99d897ed-ccfa-462b-8887-17434b99fad4.pdf"},{"id":96251003,"identity":"3a34b194-5b08-4ae9-b3cb-b707c0e31baf","added_by":"auto","created_at":"2025-11-19 07:39:12","extension":"doc","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":658944,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryfigures.doc","url":"https://assets-eu.researchsquare.com/files/rs-7920255/v1/4d89580b1fbd6bd88d159855.doc"},{"id":96159857,"identity":"e056f859-8495-4ff2-85ee-1b500a17b875","added_by":"auto","created_at":"2025-11-18 08:45:58","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":273257,"visible":true,"origin":"","legend":"","description":"","filename":"CONSORT2025expandedchecklist.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7920255/v1/3afcaeb0a120b6491c6b07bc.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"PD-L1 status on Circulating Tumor Cells: A Promising Predictor in Advanced Lung Cancer with PD-1/PD-L1 immunotherapies","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePD-1/PD-L1 blockade immunotherapy has established itself as a cornerstone in the first-line and second-line treatment of advanced lung cancer (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e), significantly enhancing patients' survival and quality of life (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). Various PD-1 or PD-L1 monoclonal antibodies have been developed and evaluated in clinical trials, demonstrating efficacy as monotherapy or in combination with cytotoxic chemotherapy (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e), radiotherapy (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e), or other immune checkpoint inhibitors (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). Pivotal studies including Keynote-001 (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e), Keynote-010 (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e), and Keynote-024 (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e), and CheckMate 057 (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). have shown that non-small cell lung cancer (NSCLC) patients with PD-L1-positive (PD-L1+) tumors, particularly those with high PD-L1 expression, achieve higher objective response rates (ORR), longer progression-free survival (PFS), and improved overall survival (OS) when treated with immunotherapies. Despite these significant clinical advantages, durable responses are only observed in a minority of patients, with just 20%-30% showing lasting benefits from PD-1/PD-L1 blockade immune-therapies (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). Furthermore, immune-related adverse events, including skin reactions and liver abnormalities, and potentially fatal conditions like pneumonitis, hepatitis, and myocarditis, underscore the critical importance of early identifying of patients likely to benefit from PD-1/PD-L1 blockade therapy early in the treatment process (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eCurrently, tissue PD-L1 (tPD-L1), alongside tumor mutational burden (TMB) (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e) and microsatellite instability (MSI) (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e), stands as one of the most established predictive biomarkers for response to PD-1/PD-L1 blockade therapy (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). However, several factors limit the clinical utility of these biomarkers, including tumor tissue scarcity, the invasiveness and metastatic risks associated with tissue biopsy (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e), and variability in PD-L1 detection methodologies (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). Notably, 10% to 20% of patients with negative tPD-L1 status may still respond positively to immune checkpoint inhibitors (ICIs), while certain tPD-L1-positive tumors, especially those with high PD-L1 expression, can be refractory to immunotherapy (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). The assessment of tPD-L1 often relies on archived paraffin-embedded tissues, which may account for some of the observed inconsistencies (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). This variability likely stems from the dynamic regulation of PD-L1 expression, which can be modulated by treatments such as targeted therapy, chemotherapy (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e), or radiation therapy (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). Furthermore, the limited tumor tissue size obtained from biopsies may not adequately represent the heterogeneous tumor tissue, potentially compromising the accuracy of PD-L1 analysis (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). Therefore, identifying novel predictive biomarkers is essential for optimizing the efficacy and safety of PD-1/PD-L1 inhibitors and for identifying patients most likely to derive sustained benefits from this class of immunotherapies.\u003c/p\u003e\u003cp\u003eCirculating tumor cells (CTCs) play a pivotal role in metastasis, which is the primary cause of mortality in cancer patients (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). These cells can spread through the bloodstream as individual cells, clusters (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e), or in conjunction with immune cells (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e) after detaching from the primary tumor (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). Several studies have reported that the presence of PD-L1-positive CTCs is associated with poorer progression-free survival (PFS) and overall survival (OS) (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e), suggesting that these cells might facilitate immune evasion in cancer. Conversely, other investigations have demonstrated that patients with PD-L1-positive CTCs tend to experience improved PFS and OS following PD-1/PD-L1 immune checkpoint therapy (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e), suggesting that CTC PD-L1 could be a significant prognostic and predictive biomarker in immunotherapy. However, some studies have found no significant association with better PFS and OS in NSCLC patients treated with immune checkpoint inhibitors (ICIs) (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e). Consequently, the precise role of PD-L1 in CTCs regarding its potential as a potential biomarker to predict response to combination of chemotherapy and immunotherapy remains unclear.\u003c/p\u003e\u003cp\u003eTo investigate the correlation between CTC PD-L1 expression and response to combination of chemotherapy and immunotherapy, we have developed a novel assay for detecting CTC PD-L1 using the LiquidBiopsy\u0026trade; system. This approach assesses PD-L1 expression in blood samples from patients with advanced lung cancer at multiple time points: pre-treatment (baseline, T0), during treatment (after the second cycle and before the third cycle (T1)), and after the fourth cycle and before the fifth cycle (T2)). This research aims to investigate the correlation between CTC PD-L1 levels and treatment outcomes, specifically focusing on the overall response rate (ORR) and survival outcomes.\u003c/p\u003e"},{"header":"Materials and Method","content":"\u003ch3\u003e1. Study design\u003c/h3\u003e\n\u003cp\u003eThis study was reported in accordance with the Consolidated Standards of Reporting Trials (CONSORT) guidelines. It was a non-interventional, double-blind, prospective, real-world investigation conducted at the Fourth Affiliated Hospital of Soochow University (Ethics review board approval number 220003), enrolling advanced lung cancer patients without autoimmune diseases. Participants received at least two cycles of combination of chemotherapy and PD-1/PD-L1 blockade immunotherapy starting from April 2022, with informed consent and under a registered clinical trial number (ChiCTR2500096312). To maintain blind, peripheral blood samples (10mL) were collected at baseline (T0), after two cycles (T1), after four cycles (T2), and at disease progression (PD) for CTC detection and PD-L1 expression analysis at Zhuhai Sanmedbio (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and Supplementary Fig.\u0026nbsp;1), the effects of response to combination of chemotherapy and immune checkpoint inhibitor (ICI) therapy and follow-up care are being studied at the Fourth Affiliated Hospital of Soochow University, all the data was sent to the third party for storage and analysis immediately. The primary objective was to assess the predictive value of baseline CTC PD-L1 for objective response rate (ORR), progression-free survival (PFS), and overall survival (OS). Secondary objectives included monitoring changes in CTC counts and their relationship to therapeutic response. All the patients were followed-up with more than 3 months for ORR evaluation.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\n\u003ch3\u003e2. CTC isolation and detection by using LiquidBiopsy™\u003c/h3\u003e\n\u003cp\u003eCTCs were isolated and counted using the LiquidBiopsy™ system (Zhuhai Sanmed Biotech LTD, Zhuhai, China), as detailed elsewhere(\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e). PBMCs were processed using Ficoll from 10 mL blood, fixed with PFA, and incubated with capture cocktail kits and streptavidin beads for CTC capture. Anti-pan CK antibody, anti-PD-L1 antibody, and anti-CD45 antibody, along with DAPI, were used for detection. CTCs were identified based on specific phenotypes with threshold of CK mean of fluorescence intensity (MFI) and ratio of CK/CD45 (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e). CTC PD-L1 was analyzed on HRP labeled anti-PD-L1 antibody with anti HRP IgG alexa Fluor594 secondary antibody (Supplementary Fig.\u0026nbsp;2) and classified with threshold of PD-L1 MFI and ratio of PD-L1/CD45. Patients with one or more PD-L1 positive CTCs were classified as CTC PD-L1\u003csup\u003e+\u003c/sup\u003e, while those with one or more CK positive CTCs were classified as CTC positive. 22C3 pharmDx (Dako) was used for evaluation of PD-L1 expression in tumor tissues according to previous study(\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003e3. Treatment response and disease progression assessment\u003c/h3\u003e\n\u003cp\u003eTumor responses were radiologically assessed after two treatment cycles using the RECIST 1.1 criteria. ORR was defined as the ratio of patients with complete response (CR) or partial response (PR) to all patients. Disease control rate (DCR) included patients with CR, PR, or stable disease (SD). PFS was measured from the start of combination of chemotherapy and PD-1/PD-L1 immunotherapy to first documented disease progression, and OS was measured from the start of PD-1/PD-L1 immunotherapy in combination with chemotherapy to death after recruitment. PR patients were defined as responders at combination of chemotherapy and PD-1/PD-L1 immunotherapy.\u003c/p\u003e\n\u003ch3\u003e4. Statistical analysis\u003c/h3\u003e\n\u003cp\u003eThe study analyzed the total number of CTCs and CTC PD-L1 to characterize patient disease features. Patients were categorized into two groups: those with at least one PD-L1-positive CTC (CTC PD-L1\u003csup\u003e+\u003c/sup\u003e) or none (CTC PD-L1\u003csup\u003e−\u003c/sup\u003e). A receiver operating characteristic (ROC) curve determined the optimal cutoff value to distinguish between treatment responders and non-responders. A univariate logistic regression model was then used to calculate odds ratios (ORs) and 95% confidence intervals (CIs) at this cutoff.\u003c/p\u003e\u003cp\u003eDifferences in quantities of CTC PD-L1\u003csup\u003e+\u003c/sup\u003e between the partial response (PR) group and the stable disease/progressive disease (SD/PD) group were assessed using a two-tailed, unpaired t-test for variables with normal distribution. Fisher's exact tests evaluated the association between CTC PD-L1 status and treatment response, as well as the link between changes in total CTC counts and treatment outcomes. These tests also assessed variations in the objective response rate (ORR) based on categorical variables like age, gender, cancer subtype, tissue PD-L1 (tPD-L1) status, type of immunotherapy, CEA, CYFRA21-1, NSE, SCC, and other clinical factors.\u003c/p\u003e\u003cp\u003eSurvival analysis was conducted using the Kaplan-Meier method to plot curves and the Mantel-Cox test to calculate hazard ratios. Both univariate and multivariate Cox proportional hazards regression models assessed the correlation between CTC PD-L1 status and progression-free survival (PFS) and overall survival (OS), considering factors like age, gender, cancer subtype, treatment type, CEA, CYFRA21-1, NSE, SCC, smoking status, hypertension, diabetes, and ECOG score. Statistical analyses were performed using GraphPad Prism 8.2.0 and IBM SPSS Statistics version 25, with p-values of less than 0.05 considered statistically significant.\u003c/p\u003e"},{"header":"Result","content":"\u003cp\u003e\u003cstrong\u003e1. Baseline Characteristics and CTC Detection\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFrom April 2022 to February 2024, we prospectively enrolled 52 patients with advanced lung cancer (stages III-IV) in the Department of Respiratory and Critical Care Medicine at the Fourth Affiliated Hospital of Soochow University (Fig 1). The cohort comprised 44 males (84.6%) and 8 females (15.4%), with a median age of 67.8 years (range: 45-82 years). Histological subtypes included adenocarcinoma (n=15, 28.8%), squamous cell carcinoma (n=23, 44.2%), and small cell lung cancer (n=14, 27.0%). All patients were driver gene-negative and received PD-1/PD-L1 inhibitor therapy in combination with chemotherapy.\u003c/p\u003e\n\u003cp\u003eAt baseline assessment, CTCs, which were identified based on specific phenotypes, with\u0026nbsp;CK+/DAPI+/CD45-, or CK+/PD-L1+/DAPI+/CD45-\u0026nbsp;(Fig 2A) at ratio of CK/CD45 \u0026gt;1.7\u0026nbsp;(Sensitivity and specificity was 100% (95% CI 99.52% to 100.0%) and 100% (95% CI 99.27% to 100.0%) respectively, with AUC 1.0 (95% CI 1.0 to 1.0, p\u0026lt;0.0001)) (Fig 2Ba-2Bc), while CTC PD-L1 was classified as ratio of PD-L1/CD45 \u0026gt;3.2 (Sensitivity and specificity was 99.90% (95% CI 99.81% to 99.95%) and 99.62% (95% CI 98.61% to 99.93%) respectively, with AUC 0.9999 (95% CI 1.0 to 1.0, p\u0026lt;0.0001)) with\u0026nbsp;CK+/PD-L1+/DAPI+/CD45- (Fig 2Bd-2Bf). CTC\u0026nbsp;were successfully detected in 37 patients (71.2%) out of the 52 patients recruited, with varying detection rates across histological subtypes: adenocarcinoma (8/15, 53.3%), squamous cell carcinoma (19/23, 82.6%), and small cell lung cancer (10/14, 71.4%).\u0026nbsp;CTC PD-L1\u003csup\u003e+\u003c/sup\u003e were identified in 26 patients (50.0%), with subtype-specific positivity rates of 40.0% (6/15) in adenocarcinoma, 56.5% (13/23) in squamous cell carcinoma, and 50.0% (7/14) in small cell lung cancer (Table 1).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2. Association Between CTC PD-L1 Status and Clinical Response\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOf the 52 patients enrolled, 38 who completed at least two cycles of combination of chemotherapy and immunotherapy were assessed for treatment response per RECIST 1.1 (follow-up through 24 February 2024; Fig 1); the other 14 were excluded because they received chemotherapy only. CTCs were detected in 27 patients (71.1%), with 19 (50.0%) individuals exhibiting CTC PD-L1\u003csup\u003e+\u003c/sup\u003e. The accordance of\u0026nbsp;CTC PD-L1\u0026nbsp;and tPD-L1 was 52.6% (20/38). In the\u0026nbsp;CTC PD-L1\u003csup\u003e+\u003c/sup\u003e group (n=19), 16 patients achieved partial response (PR), 2 maintained stable disease (SD), and 1 developed progressive disease (PD), resulting in an objective response rate (ORR) of 84.2% (Fig 3A) and disease control rate (DCR) of 94.7% (Fig 3C). In contrast, the\u0026nbsp;CTC PD-L1\u003csup\u003e-\u003c/sup\u003e group (n=19) demonstrated significantly lower efficacy measures, with an ORR of 36.8% and DCR of 68.4%, which was similar to the results of ORR (Fig 3C; 76.0% vs 41.2%) and DCR (Fig 3D; 95.2% vs 64.7%) to tPD-L1. The positive and negative predictive values (PPV and NPV) of\u0026nbsp;CTC PD-L1\u0026nbsp;for ORR were 84.2% and 63.2%, respectively. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eQuantitative analysis revealed significantly higher quantities of\u0026nbsp;CTC PD-L1\u003csup\u003e+\u003c/sup\u003e in responders (mean \u0026plusmn; SD: 2.9 \u0026plusmn; 1.14) compared to non-responders (mean \u0026plusmn; SD: 0.2 \u0026plusmn; 0.11, \u003cem\u003ep\u003c/em\u003e = 0.046) (Fig 3E), but not in tPD-L1 (Fig 3F). ROC curve analysis established an optimal\u0026nbsp;CTC PD-L1\u0026nbsp;cutoff value of 1 (AUC = 0.7165, 95% CI 0.5418 to 0.8792, \u003cem\u003ep\u0026nbsp;\u003c/em\u003e= 0.0265; sensitivity = 69.5%, specificity = 80%, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001), yielding a sensitivity of 69.5% and specificity of 80.0% (Fig 3G), which was similar to tPD-L1 with cutoff value of 1% (AUC=0.6751, 95% CI 0.4987 to 0.8515, \u003cem\u003ep\u0026nbsp;\u003c/em\u003e= 0.0665; sensitivity = 69.5%, specificity = 80%) (Fig 3H). Furthermore, the sensitivity and the specificity would be improved to 86.9% and 78% respectively when combined\u0026nbsp;CTC PD-L1\u0026nbsp;and tPD-L1, which meant that\u0026nbsp;CTC PD-L1\u0026nbsp;was potential act as a complementary biomarker for tPD-L1 in prediction of benefit from combination of chemotherapy and immunotherapy.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp;Additionally, a multivariate logistic regression analysis evaluated the prognostic value of various factors including age, gender, cancer subtype, type of ICI drugs (PD-1 or PD-L1 antibody), CEA, CYFRA21-1, NSE, and SCC. Only\u0026nbsp;CTC PD-L1\u0026nbsp;and tPD-L1 showed significant difference between the PR and SD/PD groups (\u003cem\u003ep\u0026nbsp;\u003c/em\u003e= 0.003 and \u003cem\u003ep\u0026nbsp;\u003c/em\u003e= 0.028) (Table 2), confirming its role as an independent predictor of ORR in ICI therapy and potential act as a complementary biomarker for tPD-L1.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3. Survival Analysis Based on CTC PD-L1 Status\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eKaplan-Meier survival analysis demonstrated significant differences between CTC PD-L1\u003csup\u003e+\u003c/sup\u003e and CTC PD-L1\u003csup\u003e-\u003c/sup\u003e groups. The median progression-free survival (PFS) was markedly longer in CTC PD-L1\u003csup\u003e+\u003c/sup\u003e patients (16 months vs 4 months; HR = 0.28, 95% CI: 0.096-0.78, \u003cem\u003ep\u0026nbsp;\u003c/em\u003e= 0.0041) (Fig 4A) but not in tPD-L1 (median survival, 12 months vs undefined; HR = 0.83, 95% CI: 0.28-2.5, \u003cem\u003ep\u0026nbsp;\u003c/em\u003e= 0.74) (Fig 4C). Similarly, overall survival (OS) showed significant improvement in the\u0026nbsp;CTC PD-L1\u003csup\u003e+\u003c/sup\u003e group, with median OS not reached compared to the CTC PD-L1\u003csup\u003e-\u003c/sup\u003e group (HR = 0.19, 95% CI: 0.05-0.74, \u003cem\u003ep\u0026nbsp;\u003c/em\u003e= 0.017) (Fig 4B), also not in tPD-L1 (HR = 0.88, 95% CI: 0.23-3.4, \u003cem\u003ep\u0026nbsp;\u003c/em\u003e= 0.85) (Fig 4D). \u0026nbsp;Multivariate analysis incorporating clinical variables (age, gender, histology, treatment modality) and laboratory parameters (CEA, CYFRA21-1, NSE, SCC) identified CTC PD-L1 as the sole independent predictor of both PFS (\u003cem\u003ep\u0026nbsp;\u003c/em\u003e= 0.039) and OS (\u003cem\u003ep\u0026nbsp;\u003c/em\u003e= 0.033) (Table 3).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4. Dynamic CTC Monitoring During Treatment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSerial blood sampling was performed in 21 patients at day 42 (T1) to evaluate CTC dynamics. The overall CTC positivity rate decreased from 71.2% at baseline to 33.3% at T1. In the PR patients (16/21), 14 patients (87.5%) exhibited CTC decline or stable, correlating with clinical response.\u003c/p\u003e\n\u003cp\u003eExtended monitoring at day 84 (T2) was obtained in 8 patients, of which 7 patients were PR and 1 patient was SD. CTC analysis revealed consistent patterns: 7 PR patients showed either decreased or stable CTC counts, while the SD single patient presented increased CTC counts. Notably, NSE levels showed better correlation with treatment response compared to other conventional markers (CEA, CYFRA21-1, SCC), suggesting its potential complementary role in monitoring treatment efficacy.\u003c/p\u003e\n\u003cp\u003eThese comprehensive findings demonstrate the robust predictive and prognostic value of CTC PD-L1 assessment in advanced lung cancer patients receiving combination of chemotherapy and immunotherapy, offering a promising liquid biopsy approach for treatment stratification and monitoring (Fig 5). \u0026nbsp;\u0026nbsp;\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study prospectively enrolled 52 patients with advanced lung cancer (stages III-IV) and examined the association between PD-L1 expression in CTCs and the efficacy of combination of chemotherapy and immunotherapy. A novel procedure to detect and characterize PD-L1 expression on CTCs was developed. CTC PD-L1 was identified on special phenotype with MFI ratio of CK/CD45\u0026thinsp;\u0026gt;\u0026thinsp;1.7 and PD-L1/CD45\u0026thinsp;\u0026gt;\u0026thinsp;3.2. The core findings revealed that CTC PD-L1 was associated with significantly higher objective response rate (ORR) (84.2%) and disease control rate (DCR) (94.7%) compared to CTC PD-L1\u003csup\u003e\u0026minus;\u003c/sup\u003e (ORR: 36.8%, DCR: 68.4%), which was similar to the results of tPD-L1, though the accordance of CTC PD-L1 status and tPD-L1 was only 52.6%. Moreover, CTC PD-L1\u003csup\u003e+\u003c/sup\u003e emerged as an independent predictor of combining chemotherapy and PD-1/PD-L1 immunotherapy efficacy with significant prognostic value for progression-free survival (PFS) (HR\u0026thinsp;=\u0026thinsp;0.28, 95% CI: 0.096\u0026ndash;0.78, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0041) and overall survival (OS) (HR\u0026thinsp;=\u0026thinsp;0.19, 95% CI: 0.05\u0026ndash;0.74, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.017). Furthermore, detailed dynamic monitoring data confirmed that the change of CTCs served as a prognostic indicator for therapy efficacy. The majority of PR patients (87.2%) showed a decrease in CTC counts from baseline (T0) to the first follow-up (T1).\u003c/p\u003e\u003cp\u003eCompared to traditional tissue biopsies, CTC testing in patients with advanced lung cancer was previously shown to provide higher feasibility in real-time assessment of tumor changes during PD-1/PD-L1 antibody immunotherapy(\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e). CTC dynamic monitoring suggested that change in CTC counts might act as a prognostic indicator for therapy efficacy(\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e). Moreover, CTC PD-L1 was associated with poor PFS and OS indicated that its potential prognostic value in prediction for lung cancer(\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e). Although prior investigations have provided preliminary insights into the potential utility of CTC PD-L1 assessment during immunotherapy, comprehensive evaluations delineating the quantitative and qualitative concordance between CTC PD-L1 and tPD-L1 expression remain conspicuously absent. Moreover, the clinical relevance of CTC PD-L1 as a predictive biomarker for combining chemotherapy and PD-1/PD-L1 immunotherapy remains contentious, with conflicting data regarding its association with therapeutic benefit(\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e). In the current study, we investigated not only the correlation between CTC PD-L1 and tPD-L1, but also that clinical benefit of short-term treatment outcome (ORR and DCR) and long-term survival (PFS and OS), providing valuable insights for clinical practice. Moreover, we also explored dynamic changes of CTC before and after therapy, and the role of CTC testing in combination with tumor markers in predicting the efficacy of immunotherapy.\u003c/p\u003e\u003cp\u003eIn summary, the study employed a prospective cohort design, enrolling 52 patients with advanced lung cancer who were intended to treat with immunotherapy and followed for up to two years, ensuring data integrity and reliability. Multivariate analysis adjusted for multiple clinically relevant covariates (e.g., age, gender, histology, treatment modality, CEA, CYFRA21-1, NSE, SCC), allowing for a more accurate and comprehensive assessment of independent predictive value of CTC PD-L1. Additionally, the study evaluated PD-L1 status in CTCs not only at baseline but also dynamically during treatment, providing a comprehensive perspective on treatment response. Furthermore, monitoring the changes of CTC counts in combination with tumor markers would be better in tracking the disease progression of patients. This integrated study design and data analysis strategy offer new insights into the role of CTC monitoring in immunotherapy for advanced lung cancer, which may help identify patients with poor treatment response early, thereby optimizing therapeutic strategies.\u003c/p\u003e\u003cp\u003eHowever, several limitations exist in this study. A limitation of the study was its small sample size of 38 patients, which may have affected the observed correlation between CTC PD-L1/tPD-L1 and clinical outcomes. To overcome this and to better understand the relationship between CTC PD-L1/tPD-L1 and combination of chemotherapy and immunotherapy outcomes, future studies should aim for larger and more diverse patient populations. This approach will enable a more accurate assessment of these biomarkers' impact on treatment response and survival, potentially leading to more personalized and effective immunotherapy strategies. Additionally, as a single center study, the generalizability of our findings also requires validation through further studies.\u003c/p\u003e\u003cp\u003eIn conclusion, the LiquidBiopsy\u0026trade; detection system for evaluating CTC PD-L1 proved feasible, effective, and non-invasive. The baseline CTC PD-L1 was significantly correlated with clinical outcomes in combination of chemotherapy and PD-1/PD-L1 blockade therapy, including objective response rate (ORR), progression-free survival (PFS), and overall survival (OS). The study supports the potential use of CTC PD-L1 as a reliable predictive biomarker that could be integrated into therapeutic decision-making algorithms for combination of chemotherapy and PD-1/PD-L1 inhibitor therapy.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eAUC\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003earea under curve\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eCD45\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eleukocyte common antigen,LCA\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eCEA\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003ecarcinoembryonic antigen\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eCI\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003econfidence interval\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eCK\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eCytokeratin\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eCTCs\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003ecirculating tumor cells\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eCYRFRA19\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003ecytokeratin fragment antigen 19\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eDAPI\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003e4',6-diamidino-2-phenylindole\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eDCR\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003edisease control rate\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eHR\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003ehazard ratio\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eHRP\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003ehorseradish peroxidase\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eORR\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eobjective response rate\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eNSE\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eneuron-specific enolase\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eOS\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eoverall survival\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003ePD\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eprogression disease\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003ePD-1\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eprogrammed death 1\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003ePD-L1\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eprogrammed death ligand 1\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003ePFS\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eprogression free survival\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003ePR\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003epartial response\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eROC\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003ereceiver operating characteristic curve\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eSCC\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eSquamous Cell Carcinoma Antigen\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eSD\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003estable disease\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eWBC\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003ewhite blood cell.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability statement\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data relevant to the study are included in the article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWritten informed consent for publication was obtained from the patient or their legal guardian.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study was supported by the Science and Technology Project of Suzhou Municipal Health Commission (LCZX202234) and Medical and Health Innovation Research Project of Suzhou Municipal Health Commission (CXYJ2024A07).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest disclosure\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAuthors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was approved by the\u0026nbsp;Fourth Affiliated Hospital of Soochow University (Approval number 220154) and under a registered clinical trial number (ChiCTR2500096312: Prediction value of PD-L1 statue on circulating tumor cells to PD-1/PD-L1 immunotherapy of NSCLC). Written informed consent was obtained from all participants.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConceptualization:\u003c/strong\u003e Junhong Jiang, Dongjiang Tang, and Daxiong Zeng. \u003cstrong\u003eData curation:\u0026nbsp;\u003c/strong\u003eXiaoying Wei. \u003cstrong\u003eFormal analysis:\u003c/strong\u003e Lin Chen. Funding acquisition: Junhong Jiang. \u003cstrong\u003eInvestigation:\u0026nbsp;\u003c/strong\u003eLin Chen. \u003cstrong\u003eMethodology:\u0026nbsp;\u003c/strong\u003eLin Chen. \u003cstrong\u003eProject administration:\u003c/strong\u003e Dongjiang Tang. \u003cstrong\u003eResources:\u003c/strong\u003e Xiaoying Wei. \u003cstrong\u003eSupervision:\u003c/strong\u003e Dongjiang Tang. \u003cstrong\u003eValidation:\u003c/strong\u003e Zhonglin Yang. \u003cstrong\u003eVisualization:\u003c/strong\u003e Lin Chen. \u003cstrong\u003eWriting:\u0026nbsp;\u003c/strong\u003eLin Chen.\u003cstrong\u003e\u0026nbsp;Review and editing:\u003c/strong\u003e All the authors.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBrito ABC CM, de Lima VCC. Anti-PD1 versus anti-PD-L1 immunotherapy in first-line therapy for advanced non-small cell lung cancer: A systematic review and meta-analysis. Thorac Cancer. 2021;12(7):1058\u0026ndash;66.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWang, Jing, Miaomiao, Li, Yan, Zhang, et al. PD-1/PD-L1 blockades in non-small-cell lung cancer therapy. Onco Targets Ther. 2016;9:489\u0026ndash;502.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGrasselly C DM, Bourguignon A, Talhi N, Mathe D, Tourette A, Serre L, Jordheim LP, Matera EL, Dumontet C. The Antitumor Activity of Combinations of Cytotoxic Chemotherapy and Immune Checkpoint Inhibitors Is Model-Dependent. Front Immunol. 2018;9:2100.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTakamori S, Toyokawa G, Takada K, Shoji F, Maehara Y. Combination Therapy of Radiotherapy and Anti-PD-1/PD-L1 Treatment in Non-Small-cell Lung Cancer: A Mini-review. Clinical Lung Cancer. 2017;19(1):12\u0026ndash;6.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eJohnson CB, Win SY. Combination therapy with PD-1/PD-L1 blockade: An overview of ongoing clinical trials. Oncoimmunology. 2018;7(4):e1408744.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHayashi H, Nakagawa K. Combination therapy with PD-1 or PD-L1 inhibitors for cancer. Int J Clin Oncol. 2020;25(5):818\u0026ndash;30.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGandhi L R-AD, Gadgeel S, Esteban E, Felip E, De Angelis F, Domine M, Clingan P, Hochmair MJ, Powell SF, Cheng SY, Bischoff HG, Peled N, Grossi F, Jennens RR, Reck M, Hui R, Garon EB, Boyer M, Rubio-Viqueira B, Novello S, Kurata T, Gray JE, Vida J, Wei Z, Yang J, Raftopoulos H, Pietanza MC, Garassino MC. Pembrolizumab plus Chemotherapy in Metastatic Non\u0026ndash;Small-Cell Lung Cancer. N Engl J Med. 2018;378(22):2078\u0026ndash;92.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBaas P, Garon EB, Herbst RS, Felip E, Kim DW. Relationship between level of PD-L1 expression and outcomes in the KEYNOTE-010 study of pembrolizumab vs docetaxel for previously treated, PD-L1-Positive NSCLC. Journal of Clinical Oncology. 2016;34(15):9015-.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eReck M R-AD, Robinson AG, Hui R, Csőszi T, F\u0026uuml;l\u0026ouml;p A, Gottfried M, Peled N, Tafreshi A, Cuffe S, O'Brien M, Rao S, Hotta K, Vandormael K, Riccio A, Yang J, Pietanza MC, Brahmer JR. Updated Analysis of KEYNOTE-024: Pembrolizumab Versus Platinum-Based Chemotherapy for Advanced Non-Small-Cell Lung Cancer With PD-L1 Tumor Proportion Score of 50% or Greater. J Clin Oncol. 2019;37(7):537\u0026ndash;46.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePaz-Ares L, Horn L, Borghaei H, Spigel DR, Brahmer JR. Phase III, randomized trial (CheckMate 057) of nivolumab (NIVO) versus docetaxel (DOC) in advanced non-squamous cell (non-SQ) non-small cell lung cancer (NSCLC). Journal of Clinical Oncology. 2015;33(18):LBA109.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eShukuya T, Carbone DP. Predictive Markers for the Efficacy of Anti-PD-1/PD-L1 Antibodies in Lung Cancer. J Thorac Oncol. 2016;11(7):976\u0026ndash;88.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDarnell EP, Mooradian MJ, Baruch EN, Yilmaz M, Reynolds KL. Immune-Related Adverse Events (irAEs): Diagnosis, Management, and Clinical Pearls. Curr Oncol Rep. 2020;22(4):39.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGandara DR, Paul SM, Kowanetz M, Schleifman E, Shames DS. Blood-based tumor mutational burden as a predictor of clinical benefit in non-small-cell lung cancer patients treated with atezolizumab. Nat Med. 2018;24(9):1441\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eOverman MJ, Mcdermott R, Leach JL, Lonardi S, Andr\u0026eacute; T. Nivolumab in patients with metastatic DNA mismatch repair-deficient or microsatellite instability-high colorectal cancer (CheckMate 142): an open-label, multicentre, phase 2 study. Lancet Oncol. 2017;18(9):1182\u0026ndash;91.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGao G, Zhang XD, Qu H, Yao B, Zhou Y, Xiang J, et al. A comprehensive pan-cancer analysis of CD274 gene amplification, tumor mutation burden, microsatellite instability, and PD-L1 expression in Chinese cancer patients. Ann Transl Med. 2021;9(8):677.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKara M AG, Sak SD, Kavuk\u0026ccedil;u S. Implantation metastasis caused by fine needle aspiration biopsy following curative resection of stage IB non-small cell lung cancer. Eur J Cardiothorac Surg. 2001;20(4):868\u0026ndash;70.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eOh KS, Mahalingam M. PD-L1 Detection\u0026mdash;Pearls and Pitfalls Associated With Current Methodologies Focusing on Entities Relevant to Dermatopathology. Am J Dermatopathol. 2019;41(8):539\u0026ndash;65.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZhu J, Armstrong AJ, Friedlander TW, Kim W, Pal SK, George DJ, et al. Biomarkers of immunotherapy in urothelial and renal cell carcinoma: PD-L1, tumor mutational burden, and beyond. J Immunother Cancer. 2018;6(1):4.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChen YL, Huang WC, Lin FM, Hsieh HB, Hsieh CH, Hsieh RK, et al. Novel circulating tumor cell-based blood test for the assessment of PD-L1 protein expression in treatment-nave, newly diagnosed patients with non-small cell lung cancer. Cancer Immunol. 2019;68(7):1087\u0026ndash;94.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChi Z PL, Karamchandani DM, Xu J.. PD-L1 (22C3) expression and prognostic implications in esophageal squamous cell carcinoma. Ann Diagn Pathol. 2025;74:152394.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAzad A, Lim SY, D'Costa Z, Jones K, Diana A, Sanso OJ, et al. PD-L1 blockade enhances response of pancreatic ductal adenocarcinoma to radiotherapy. EMBO Mol Med. 2016;9(2):167\u0026ndash;80.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHong L, Negrao MV, Dibaj SS, Chen R, Zhang J. Programmed Death Ligand 1 Heterogeneity and its Impact on Benefit from Immune Checkpoint Inhibitors in Non-Small-Cell Lung Cancer. J Thorac Oncol. 2020;15(9):1449\u0026ndash;59.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eIlie M, Long-Mira E, Bence C, Butori C, Lassalle S, Bouhlel L, et al. Comparative study of the PD-L1 status between surgically resected specimens and matched biopsies of NSCLC patients reveal major discordances: a potential issue for anti-PD-L1 therapeutic strategies. Ann Oncol. 2015;27(1):147\u0026ndash;53.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eThawani R, Fakhoury K, Becker KD. Cause of mortality in patients with lung cancer and brain metastasis. Journal of Clinical Oncology. 2020;38(15):e21743-e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYu JJ, Shu C, Yang HY, Xiao W. The Presence of Circulating Tumor Cell Cluster Characterizes an Aggressive Hepatocellular Carcinoma Subtype. Front Oncol. 2021;11:734564.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSzczerba BM, Castro-Giner F, Vetter M, Krol I, Gkountela S, Landin J, et al. Neutrophils escort circulating tumour cells to enable cell cycle progression. Nature. 2019;566(7745):553\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLin D SL, Luo M, Zhang K, Li J, Yang Q, Zhu F, Zhou D, Zheng S, Chen Y, Zhou J. Circulating tumor cells: biology and clinical significance. Signal Transduct Target Ther. 2021;6(1):404.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSinoquet L, Jacot W, Gauthier L, Pouderoux S, Alix-Panabi\u0026egrave;res C. Programmed Cell Death Ligand 1-Expressing Circulating Tumor Cells: A New Prognostic Biomarker in Non-Small Cell Lung Cancer. Clin Chem. 2021;66(11):1503\u0026ndash;12.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCheng Y WT, Lv X, Li R, Yuan L, Shen J, Li Y, Yan T, Liu B, Wang L. Detection of PD-L1 Expression and Its Clinical Significance in Circulating Tumor Cells from Patients with Non-Small-Cell Lung Cancer. Cancer Manag Res. 2020;12:2069\u0026ndash;78.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eIkeda M, Koh Y, Teraoka S, Sato K, Oyanagi J, Hayata A, et al. Longitudinal Evaluation of PD-L1 Expression on Circulating Tumor Cells in Non-Small Cell Lung Cancer Patients Treated with Nivolumab. Cancers (Basel). 2021;13(10):2290.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAcheampong E AR, Page K, Wadsley MK, Beasley AB, Coombes RC, Shaw JA, Gray ES. Meta-Analysis of Circulating Tumor Cell PD-L1 Expression and the Association with Clinical Outcomes in Non-Small Cell Lung Cancer. Clin Chem. 2024;70(1):234\u0026ndash;49.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWiner-Jones JP VB, Arquilevich N, Fang C, Ferguson S, Harkins D, Hill C, Klem E, Pagano PC, Peasley C, Romero J, Shartle R, Vasko RC, Strauss WM, Dempsey PW. Circulating Tumor Cells: Clinically Relevant Molecular Access Based on a Novel CTC Flow Cell. PLoS One. 2014;9(1):e86717.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChen W ZJ, Huang L, Chen L, Zhou Y, Tang D, Xie Y, Wang H, Huang C. Detection of HER2-positive Circulating Tumor Cells Using the LiquidBiopsy System in Breast Cancer. Clin Breast Cancer. 2019;19(1):e239-e46.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eIlie M JJ, Huang L, Hofman V, Khambata-Ford S, Hofman P. Use of the 22C3 anti-programmed death ligand 1 antibody to determine programmed death ligand 1 expression in cytology samples obtained from non-small cell lung cancer patients. Cancer Cytopathol. 2018;126(4):264\u0026ndash;74.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCao L YW, Zhao X, Chen Z. Diagnostic and prognostic value of circulating tumor cells in renal cell cancer: A systematic review and meta-analysis. Asian J Surg. 2024;47(8):3425\u0026ndash;34.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSu X ZC, Chen S, Ma Q, Xiao H, Chen Q, Zou H. Prognosis value of circulating tumor cell PDL1 and baseline characteristics in patients with NSCLC treated with immune checkpoint inhibitors plus platinumcontaining drugs. Oncol Lett. 2024;27(3):131.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZhang B LY, Zhou S, Jiang H, Zhu K, Wang R. Predictive effect of PD-L1 expression for immune checkpoint inhibitor (PD-1/PD-L1 inhibitors) treatment for non-small cell lung cancer: A meta-analysis. Int Immunopharmacol. 2020;80:106214.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKloten V LR, Krahn T, Schlange T. Circulating Tumor Cell PD-L1 Expression as Biomarker for Therapeutic Efficacy of Immune Checkpoint Inhibition in NSCLC. Cells. 2019;8(8):809.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eStrati A EP, Lianidou E, Psyrri A. Clinical Significance of PD-L1 Status in Circulating Tumor Cells for Cancer Management during Immunotherapy. Biomedicines. 2023;11(6):1768.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTable 1: Characteristics of recruited patients and CTC detection\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003eCharacteristic\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003eN\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003eCTC positive (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003eCTC PD-L1 (%)\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003eTotal\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003e52\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003e37 (71.2%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003e26 (50.0%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003eGender\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003eFemale\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003e6 (75.0%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003e4 (50.0%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003eMale\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003e44\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003e31 (70.5%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003e22 (50.0%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003eCancer type\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003eAdenocarcinoma\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003e8 (53.3%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003e6 (40.0%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003eSquamous cell carcinoma\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003e23\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003e19 (82.6%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003e13 (56.5%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003eSmall cell lung cancer\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003e14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003e10 (71.4%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003e7 (50.0%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003eICI therapy type\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003ePD-1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003e30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003e21 (70.0%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003e15 (50.0%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003ePD-L1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003e6 (75.0%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003e4 (50.0%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTable 2:\u0026nbsp;\u003cstrong\u003eComparison of clinical and biological factors between patients with PD/SD and PR responses\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 174px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 110px;\"\u003e\n \u003cp\u003ePD/SD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003ePR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003e\u003cem\u003ep\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 174px;\"\u003e\n \u003cp\u003eAge\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 110px;\"\u003e\n \u003cp\u003e66.5\u0026plusmn;13.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 142px;\"\u003e\n \u003cp\u003e69.3\u0026plusmn;8.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 142px;\"\u003e\n \u003cp\u003e0.439\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 174px;\"\u003e\n \u003cp\u003eGender\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 110px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003e0.28\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 174px;\"\u003e\n \u003cp\u003eMale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 110px;\"\u003e\n \u003cp\u003e12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003e22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 174px;\"\u003e\n \u003cp\u003eFemale\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 110px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 174px;\"\u003e\n \u003cp\u003eCancer subtype\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 110px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003e0.44\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 174px;\"\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; NSCLC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 110px;\"\u003e\n \u003cp\u003e13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003e17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 174px;\"\u003e\n \u003cp\u003eSCLC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 110px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 174px;\"\u003e\n \u003cp\u003ePD-1 or PD-L1 monotherapy\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 110px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003e0.66\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 174px;\"\u003e\n \u003cp\u003ePD-1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 110px;\"\u003e\n \u003cp\u003e14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003e21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 174px;\"\u003e\n \u003cp\u003ePD-L1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 110px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 174px;\"\u003e\n \u003cp\u003eCTC PD-L1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 110px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003e0.003*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 174px;\"\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; Positive\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 110px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 174px;\"\u003e\n \u003cp\u003eNegative\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 110px;\"\u003e\n \u003cp\u003e12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 174px;\"\u003e\n \u003cp\u003etPD-L1 positive cells\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 110px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003e0.028*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 174px;\"\u003e\n \u003cp\u003e\u0026gt;=1%\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 110px;\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 174px;\"\u003e\n \u003cp\u003e\u0026lt;1%\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 110px;\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 174px;\"\u003e\n \u003cp\u003eCEA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 110px;\"\u003e\n \u003cp\u003e10.9(4.86, 65.7)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 142px;\"\u003e\n \u003cp\u003e3.63(1.93, 7.65)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 142px;\"\u003e\n \u003cp\u003e0.091\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 174px;\"\u003e\n \u003cp\u003eCYRFRA19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 110px;\"\u003e\n \u003cp\u003e6.07(3.12, 11.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 142px;\"\u003e\n \u003cp\u003e4.41(3.29, 10.3)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 142px;\"\u003e\n \u003cp\u003e0.153\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 174px;\"\u003e\n \u003cp\u003eNSE\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 110px;\"\u003e\n \u003cp\u003e15.7(12.7, 23.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 142px;\"\u003e\n \u003cp\u003e16.2(12.5, 19.8)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 142px;\"\u003e\n \u003cp\u003e0.153\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 174px;\"\u003e\n \u003cp\u003eSCC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 110px;\"\u003e\n \u003cp\u003e1.97(0.82, 4.34)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 142px;\"\u003e\n \u003cp\u003e1.81(1.26, 3.8)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 142px;\"\u003e\n \u003cp\u003e0.062\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e*Significant relevance at \u003cem\u003ep\u003c/em\u003e\u0026lt;0.05.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTable 3: Univariate analysis of potential factors affecting progression-free survival (PFS) and overall survival (OS)\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 261px;\"\u003e\n \u003cp\u003eFactors\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 137px;\"\u003e\n \u003cp\u003ePFS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 137px;\"\u003e\n \u003cp\u003eOS\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 261px;\"\u003e\n \u003cp\u003eAge\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 137px;\"\u003e\n \u003cp\u003e0.746\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 137px;\"\u003e\n \u003cp\u003e0.554\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 261px;\"\u003e\n \u003cp\u003eGender\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 137px;\"\u003e\n \u003cp\u003e0.063\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 137px;\"\u003e\n \u003cp\u003e0.137\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 261px;\"\u003e\n \u003cp\u003eCancer subtype\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 137px;\"\u003e\n \u003cp\u003e0.502\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 137px;\"\u003e\n \u003cp\u003e0.934\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 261px;\"\u003e\n \u003cp\u003ePD-1 or PD-L1 monotherapy\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 137px;\"\u003e\n \u003cp\u003e0.684\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 137px;\"\u003e\n \u003cp\u003e0.847\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 261px;\"\u003e\n \u003cp\u003eSmoking or not\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 137px;\"\u003e\n \u003cp\u003e0.144\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 137px;\"\u003e\n \u003cp\u003e0.074\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 261px;\"\u003e\n \u003cp\u003eHypertension\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 137px;\"\u003e\n \u003cp\u003e0.059\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 137px;\"\u003e\n \u003cp\u003e0.106\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 261px;\"\u003e\n \u003cp\u003eDiabetes\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 137px;\"\u003e\n \u003cp\u003e0.765\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 137px;\"\u003e\n \u003cp\u003e0.748\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 261px;\"\u003e\n \u003cp\u003eECOG score\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 137px;\"\u003e\n \u003cp\u003e0.072\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 137px;\"\u003e\n \u003cp\u003e0.203\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 261px;\"\u003e\n \u003cp\u003eCTC PD-L1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 137px;\"\u003e\n \u003cp\u003e0.039*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 137px;\"\u003e\n \u003cp\u003e0.033*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 261px;\"\u003e\n \u003cp\u003etPD-L1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 137px;\"\u003e\n \u003cp\u003e0.405\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 137px;\"\u003e\n \u003cp\u003e0.844\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 261px;\"\u003e\n \u003cp\u003eCEA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 137px;\"\u003e\n \u003cp\u003e0.051\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 137px;\"\u003e\n \u003cp\u003e0.658\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 261px;\"\u003e\n \u003cp\u003eCYRFRA19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 137px;\"\u003e\n \u003cp\u003e0.361\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 137px;\"\u003e\n \u003cp\u003e0.688\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 261px;\"\u003e\n \u003cp\u003eNSE\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 137px;\"\u003e\n \u003cp\u003e0.567\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 137px;\"\u003e\n \u003cp\u003e0.122\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 261px;\"\u003e\n \u003cp\u003eSCC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 137px;\"\u003e\n \u003cp\u003e0.777\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 137px;\"\u003e\n \u003cp\u003e0.556\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e*Significant relevance at \u003cem\u003ep\u003c/em\u003e\u0026lt;0.05.\u0026nbsp;\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"european-journal-of-medical-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ejmr","sideBox":"Learn more about [European Journal of Medical Research](http://eurjmedres.biomedcentral.com)","snPcode":"40001","submissionUrl":"https://submission.nature.com/new-submission/40001/3","title":"European Journal of Medical Research","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"circulating tumor cells, CTC PD-L1, objective response rate (ORR), progression-free survival (PFS), and overall survival (OS)","lastPublishedDoi":"10.21203/rs.3.rs-7920255/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7920255/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003eTumor heterogeneity and invasive sampling limit traditional tissue-based PD-L1 testing in advanced lung cancer. We explored a non-invasive alternative by assessing PD-L1 expression on circulating tumor cells (CTC PD-L1) via liquid biopsy to predict immunotherapy outcomes.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e\u003cp\u003eFifty-two advanced lung cancer patients were recruited and 38 individuals received combination of chemotherapy and PD-1/PD-L1 inhibitor therapy, with serial blood samples (baseline, during treatment, and at progression) analyzed using the LiquidBiopsy\u0026trade; platform and CTC PD-L1 assay. Primary endpoints included objective response rate (ORR), progression-free survival (PFS), and overall survival (OS).\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eCTC PD-L1 was identified on special phenotype with ratio of CK/CD45\u0026thinsp;\u0026gt;\u0026thinsp;1.7 and PD-L1/CD45\u0026thinsp;\u0026gt;\u0026thinsp;3.2. At baseline, 71.2% (37/52) of patients had detectable CTCs, with 50.0% (26/52) being CTC PD-L1\u003csup\u003e+\u003c/sup\u003e. CTC PD-L1\u003csup\u003e+\u003c/sup\u003e patients exhibited significantly higher ORR (84.2% vs. 36.8%), longer median PFS (16 months vs 4 months; HR\u0026thinsp;=\u0026thinsp;0.28, 95% CI: 0.096\u0026ndash;0.78, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0041), and superior OS (median survival, undefined; HR\u0026thinsp;=\u0026thinsp;0.19, 95% CI: 0.05\u0026ndash;0.74, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.017). Multivariate analysis confirmed CTC PD-L1\u003csup\u003e+\u003c/sup\u003e as an independent predictor of response (p\u0026thinsp;=\u0026thinsp;0.003) and potential as a complementary biomarker to tPD-L1.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e\u003cp\u003eWe developed a novel procedure to detect and characterize PD-L1 expression on CTCs, which was a feasible, non-invasive biomarker for predicting combination of chemotherapy and immunotherapy efficacy in advanced lung cancer, addressing tissue sampling limitations and enhancing patient stratification and monitoring.\u003c/p\u003e\u003ch2\u003eTrial registration\u003c/h2\u003e\u003cp\u003eThe study was approved by the Medical Ethics Committee of Fourth Affiliated Hospital of Soochow University, with ethics approval number 220154 on 23th January, 2022 and retrospectively registered under clinical trial number ChiCTR2500096312 on 21th January, 2025.\u003c/p\u003e","manuscriptTitle":"PD-L1 status on Circulating Tumor Cells: A Promising Predictor in Advanced Lung Cancer with PD-1/PD-L1 immunotherapies","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-18 08:45:53","doi":"10.21203/rs.3.rs-7920255/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-12-12T20:21:46+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-12T20:00:45+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-24T08:16:06+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"80595842315251052630907927935590357080","date":"2025-11-23T13:44:55+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"28036502381619547753284298185862999814","date":"2025-11-23T08:00:04+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"43985886987228696172747715336514006444","date":"2025-11-21T12:46:26+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"254541578684275681261099431222961974612","date":"2025-11-10T20:02:02+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-11-06T13:52:40+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-11-03T05:44:18+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-11-01T11:49:25+00:00","index":"","fulltext":""},{"type":"submitted","content":"European Journal of Medical Research","date":"2025-10-31T06:37:59+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"european-journal-of-medical-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ejmr","sideBox":"Learn more about [European Journal of Medical Research](http://eurjmedres.biomedcentral.com)","snPcode":"40001","submissionUrl":"https://submission.nature.com/new-submission/40001/3","title":"European Journal of Medical Research","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"9faf2825-780f-4ab9-87d5-ce5a7c09344d","owner":[],"postedDate":"November 18th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-02-02T16:07:07+00:00","versionOfRecord":{"articleIdentity":"rs-7920255","link":"https://doi.org/10.1186/s40001-026-03945-5","journal":{"identity":"european-journal-of-medical-research","isVorOnly":false,"title":"European Journal of Medical Research"},"publishedOn":"2026-01-31 15:58:37","publishedOnDateReadable":"January 31st, 2026"},"versionCreatedAt":"2025-11-18 08:45:53","video":"","vorDoi":"10.1186/s40001-026-03945-5","vorDoiUrl":"https://doi.org/10.1186/s40001-026-03945-5","workflowStages":[]},"version":"v1","identity":"rs-7920255","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7920255","identity":"rs-7920255","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}