{"paper_id":"0c61c6f3-d5b6-4100-9ac0-30046bfbb23c","body_text":"Tissue and Blood Immune Status Predicts Risk of Recurrence in Resected Non-small Cell Lung Cancer | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Tissue and Blood Immune Status Predicts Risk of Recurrence in Resected Non-small Cell Lung Cancer Giovanni Bocchialini, Simona D’Agnelli, Giulia Mazzaschi, Federico Quaini, and 11 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7273225/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract BACKGROUND The integration of Immune checkpoint inhibitors(ICIs) into perioperative strategies for early-stage non-small-cell lung cancer(NSCLC) has shown promising survival benefits; however, reliable biomarkers to predict response and recurrence risk remain limited. The aim of the study was to determine whether a comprehensive immune profiling, including tumor immune microenvironment(TIME) at tissue level and peripheral blood(PB), might unveil immune-biological grounds and improve prognostic stratification in resected NSCLC. METHODS We prospectively analyzed 115 patients with resected stage I–IIIA NSCLC. Comprehensive immune profiling was performed on tumor tissue using digital microscopy, and on paired PB samples by Fluorescence-Activated-Cell-Sorting(FACS). The microarchitecture of tumor-infiltrating lymphocytes(TILs) subtypes, and levels of circulating lymphocytes were correlated with clinicopathological features and recurrence-free survival(RFS). RESULTS Immune profiling of patients with node-negative disease showed a TIME with high density of CD4+( p =0.01) and CD8+( p =0.05) TILs. Stage I disease compared to stage II-III, had increased densities of CD3+( p =0.004), stromal -CD4+( p <0.001), and intratumoral -CD8+ TILs( p =0.002) and, in PB higher levels of CD3+( p =0.01) and CD4+( p =0.03) circulating lymphocytes. At log-rank test, improved RFS was documented in patients carrying high stromal- CD4+(p<0.001; HR 0.24, 95%CI 0.11-0.52) and intratumoral- CD8+(p<0.001; HR 0.24, 95%CI 0.11-0.53) TILs. TIME with PD-1-to-CD8 ratio 0.5 was associated with a high risk of relapse (p<0.001; HR 3.62, 95%CI 1.77-7.41). Multivariate analysis confirmed histology, intratumoral CD8+ TILs and PD-1-to-CD8 ratio as independent prognostic factors of RFS. CONCLUSIONS Our findings suggest that both tissue and blood immune profiles reflect tumor immune status and may serve as prognostic markers in resectable NSCLC. Integrated immune profiling could enhance patient stratification and optimize perioperative immunotherapy strategies. NSCLC Biomarkers Tumor Immune Microenvironment Tumor Infiltrating Lymphocytes Peripheral Blood Immunotherapy Full Text Additional Declarations No competing interests reported. Supplementary Files Supplements.pdf Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {\"props\":{\"pageProps\":{\"initialData\":{\"identity\":\"rs-7273225\",\"acceptedTermsAndConditions\":true,\"allowDirectSubmit\":true,\"archivedVersions\":[],\"articleType\":\"Research Article\",\"associatedPublications\":[],\"authors\":[{\"id\":531743953,\"identity\":\"956ba66c-1e17-4839-a36d-1ac8ab241a40\",\"order_by\":0,\"name\":\"Giovanni 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in Resected Non-small Cell Lung Cancer\\u003c/p\\u003e\",\"fulltext\":[],\"fulltextSource\":\"\",\"fullText\":\"\",\"funders\":[],\"hasAdminPriorityOnWorkflow\":false,\"hasManuscriptDocX\":false,\"hasOptedInToPreprint\":true,\"hasPassedJournalQc\":\"\",\"hasAnyPriority\":false,\"hideJournal\":true,\"highlight\":\"\",\"institution\":\"\",\"isAcceptedByJournal\":false,\"isAuthorSuppliedPdf\":true,\"isDeskRejected\":\"\",\"isHiddenFromSearch\":false,\"isInQc\":false,\"isInWorkflow\":false,\"isPdf\":true,\"isPdfUpToDate\":true,\"isWithdrawnOrRetracted\":false,\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"researchsquare\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":true,\"externalIdentity\":\"\",\"sideBox\":\"\",\"snPcode\":\"\",\"submissionUrl\":\"/submission\",\"title\":\"Research Square\",\"twitterHandle\":\"researchsquare\",\"acdcEnabled\":true,\"dfaEnabled\":false,\"editorialSystem\":\"\",\"reportingPortfolio\":\"\",\"inReviewEnabled\":false,\"inReviewRevisionsEnabled\":true},\"keywords\":\"NSCLC, Biomarkers, Tumor Immune Microenvironment, Tumor Infiltrating Lymphocytes, Peripheral Blood, Immunotherapy\",\"lastPublishedDoi\":\"10.21203/rs.3.rs-7273225/v1\",\"lastPublishedDoiUrl\":\"https://doi.org/10.21203/rs.3.rs-7273225/v1\",\"license\":{\"name\":\"CC BY 4.0\",\"url\":\"https://creativecommons.org/licenses/by/4.0/\"},\"manuscriptAbstract\":\"\\u003cp\\u003e\\u003cstrong\\u003eBACKGROUND\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe integration of Immune checkpoint inhibitors(ICIs) into perioperative strategies for early-stage non-small-cell lung cancer(NSCLC) has shown promising survival benefits; however, reliable biomarkers to predict response and recurrence risk remain limited.\\u003c/p\\u003e\\n\\u003cp\\u003eThe aim of the study was to determine whether a comprehensive immune profiling, including tumor immune microenvironment(TIME) at tissue level and peripheral blood(PB), might unveil immune-biological grounds and improve prognostic stratification in resected NSCLC.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eMETHODS\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eWe prospectively analyzed 115 patients with resected stage I–IIIA NSCLC. Comprehensive immune profiling was performed on tumor tissue using digital microscopy, and on paired PB samples by Fluorescence-Activated-Cell-Sorting(FACS). The microarchitecture of tumor-infiltrating lymphocytes(TILs) subtypes, and levels of circulating lymphocytes were correlated with clinicopathological features and recurrence-free survival(RFS).\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eRESULTS\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eImmune profiling of patients with node-negative disease showed a TIME with high density of CD4+(\\u003cem\\u003ep\\u003c/em\\u003e=0.01) and CD8+(\\u003cem\\u003ep\\u003c/em\\u003e=0.05) TILs. Stage I disease compared to stage II-III, had increased densities of CD3+(\\u003cem\\u003ep\\u003c/em\\u003e=0.004), \\u003cem\\u003estromal\\u003c/em\\u003e-CD4+(\\u003cem\\u003ep\\u003c/em\\u003e\\u0026lt;0.001), and \\u003cem\\u003eintratumoral\\u003c/em\\u003e-CD8+ TILs(\\u003cem\\u003ep\\u003c/em\\u003e=0.002) and, in PB higher levels of CD3+(\\u003cem\\u003ep\\u003c/em\\u003e=0.01) and CD4+(\\u003cem\\u003ep\\u003c/em\\u003e=0.03) circulating lymphocytes.\\u003c/p\\u003e\\n\\u003cp\\u003eAt log-rank test, improved RFS was documented in patients carrying high \\u003cem\\u003estromal-\\u003c/em\\u003eCD4+(p\\u0026lt;0.001; HR 0.24, 95%CI 0.11-0.52) and \\u003cem\\u003eintratumoral-\\u003c/em\\u003eCD8+(p\\u0026lt;0.001; HR 0.24, 95%CI 0.11-0.53) TILs. TIME with PD-1-to-CD8 ratio \\u0026nbsp;\\u0026nbsp;\\u0026nbsp;\\u0026nbsp;\\u0026nbsp;\\u0026nbsp;\\u0026nbsp;\\u0026nbsp;\\u0026nbsp;\\u0026nbsp;\\u0026nbsp;\\u0026nbsp;\\u0026nbsp;\\u0026nbsp;\\u0026nbsp;\\u0026nbsp;\\u0026nbsp;\\u0026nbsp;\\u0026nbsp;0.5 was associated with a high risk of relapse (p\\u0026lt;0.001; HR 3.62, 95%CI 1.77-7.41). Multivariate analysis confirmed histology, intratumoral CD8+ TILs and PD-1-to-CD8 ratio as independent prognostic factors of RFS.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eCONCLUSIONS\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eOur findings suggest that both tissue and blood immune profiles reflect tumor immune status and may serve as prognostic markers in resectable NSCLC. Integrated immune profiling could enhance patient stratification and optimize perioperative immunotherapy strategies.\\u003c/p\\u003e\",\"manuscriptTitle\":\"Tissue and Blood Immune Status Predicts Risk of Recurrence in Resected Non-small Cell Lung Cancer\",\"msid\":\"\",\"msnumber\":\"\",\"nonDraftVersions\":[{\"code\":1,\"date\":\"2025-10-20 19:16:53\",\"doi\":\"10.21203/rs.3.rs-7273225/v1\",\"editorialEvents\":[{\"type\":\"communityComments\",\"content\":0}],\"status\":\"published\",\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"researchsquare\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":true,\"externalIdentity\":\"\",\"sideBox\":\"\",\"snPcode\":\"\",\"submissionUrl\":\"/submission\",\"title\":\"Research Square\",\"twitterHandle\":\"researchsquare\",\"acdcEnabled\":true,\"dfaEnabled\":false,\"editorialSystem\":\"\",\"reportingPortfolio\":\"\",\"inReviewEnabled\":false,\"inReviewRevisionsEnabled\":true}}],\"origin\":\"\",\"ownerIdentity\":\"0a031f17-72f8-47fe-a4d4-d141583082ea\",\"owner\":[],\"postedDate\":\"October 20th, 2025\",\"published\":true,\"recentEditorialEvents\":[],\"rejectedJournal\":[],\"revision\":\"\",\"amendment\":\"\",\"status\":\"posted\",\"subjectAreas\":[],\"tags\":[],\"updatedAt\":\"2026-02-25T09:27:23+00:00\",\"versionOfRecord\":[],\"versionCreatedAt\":\"2025-10-20 19:16:53\",\"video\":\"\",\"vorDoi\":\"\",\"vorDoiUrl\":\"\",\"workflowStages\":[]},\"version\":\"v1\",\"identity\":\"rs-7273225\",\"journalConfig\":\"researchsquare\"},\"__N_SSP\":true},\"page\":\"/article/[identity]/[[...version]]\",\"query\":{\"redirect\":\"/article/rs-7273225\",\"identity\":\"rs-7273225\",\"version\":[\"v1\"]},\"buildId\":\"8U1c8b4HqxoKbykW_rLl7\",\"isFallback\":false,\"isExperimentalCompile\":false,\"dynamicIds\":[84888],\"gssp\":true,\"scriptLoader\":[]}","source_license":"CC-BY-4.0","license_restricted":false}