The Ki-67 Proliferation Index in Tertiary Lymphoid Structures as a Predictor of Survival Outcomes in Patients with Resectable Non-Small Cell Lung Cancer

The Ki-67 Proliferation Index in Tertiary Lymphoid Structures as a Predictor of Survival Outcomes in Patients with Resectable 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 The Ki-67 Proliferation Index in Tertiary Lymphoid Structures as a Predictor of Survival Outcomes in Patients with Resectable Non-Small Cell Lung Cancer Xinyu Bao, Mei Xie, Xuwen Lin, Yiming Liu, Yinguang Zhang, Xidong Ma, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9249156/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 12 You are reading this latest preprint version Abstract Background & Objectives : Although the presence of tertiary lymphoid structures (TLS) has been associated with improved immunotherapy efficacy across multiple solid tumors, the prognostic significance of proliferative activity within TLS in patients with resectable non-small cell lung cancer (NSCLC) remains unclear. This study aims to evaluate the predictive value of Ki-67 expression within TLS for survival outcomes in patients with NSCLC and to explore the association between TLS maturity and the cellular proliferation index within these structures. Methods : Postoperative paraffin-embedded pathological specimens from 88 patients with NSCLC were analyzed. Hematoxylin-eosin (H&E) staining and immunohistochemistry (IHC) were performed to evaluate the presence, spatial distribution, and number of TLS. Multiplex immunofluorescence (mIF) staining was used to assess TLS maturity. Kaplan-Meier survival analysis was conducted to determine the prognostic significance of TLS maturity and the cellular proliferation index within TLS. Results : Tumor-associated TLS were identified in the tumor microenvironment of 74 (84.1%) of the 88 patients. The mature TLS was significantly associated with improved recurrence-free survival (RFS) in patients with NSCLC ( P = 0.0068). The cellular proliferation index within TLS demonstrated a significant positive correlation with TLS maturity. Compared with early-stage TLS and primary follicular-like TLS, secondary follicular-like TLS exhibited a significantly higher proliferation index ( P < 0.0001). Furthermore, a high TLS cellular proliferation index was predictive of favorable RFS in patients with NSCLC ( P = 0.0231). Multivariate analysis further indicated that a low TLS cellular proliferation index was an independent adverse prognostic factor for RFS (HR= 1.9040, 95% CI: 1.054-3.440; P = 0.0330). Conclusion : This study demonstrates the prognostic relevance of the cellular proliferation index within TLS in the tumor microenvironment of NSCLC. A high TLS cellular proliferation index was significantly associated with prolonged RFS. These findings suggest that an elevated TLS cellular proliferation index may contribute to improved clinical outcomes by promoting TLS maturation and enhancing antitumor immune responses. Non-small cell lung cancer Tertiary lymphoid structures Ki-67(+) immune cells Surgery Prognosis Full Text Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Revision Version 1 posted Editorial decision: Revision requested 13 May, 2026 Reviews received at journal 10 May, 2026 Reviews received at journal 17 Apr, 2026 Reviews received at journal 17 Apr, 2026 Reviewers agreed at journal 17 Apr, 2026 Reviewers agreed at journal 17 Apr, 2026 Reviewers agreed at journal 01 Apr, 2026 Reviewers invited by journal 01 Apr, 2026 Editor invited by journal 30 Mar, 2026 Editor assigned by journal 30 Mar, 2026 Submission checks completed at journal 30 Mar, 2026 First submitted to journal 27 Mar, 2026 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-9249156","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":617844409,"identity":"711d0549-a508-4267-8c1f-f7a577c2666c","order_by":0,"name":"Xinyu Bao","email":"","orcid":"","institution":"Affiliated Hospital of Shandong Second Medical University, Shandong Second Medical University","correspondingAuthor":false,"prefix":"","firstName":"Xinyu","middleName":"","lastName":"Bao","suffix":""},{"id":617844410,"identity":"072ed509-d656-41b3-9646-b0a36d2cc733","order_by":1,"name":"Mei Xie","email":"","orcid":"","institution":"Capital Medical University","correspondingAuthor":false,"prefix":"","firstName":"Mei","middleName":"","lastName":"Xie","suffix":""},{"id":617844411,"identity":"9edc16b2-1b7d-4246-9e65-ff6caa076b4d","order_by":2,"name":"Xuwen Lin","email":"","orcid":"","institution":"Capital Medical University","correspondingAuthor":false,"prefix":"","firstName":"Xuwen","middleName":"","lastName":"Lin","suffix":""},{"id":617844419,"identity":"91d985a4-750b-43ec-b435-09941a05541f","order_by":3,"name":"Yiming Liu","email":"","orcid":"","institution":"Chinese PLA General Hospital","correspondingAuthor":false,"prefix":"","firstName":"Yiming","middleName":"","lastName":"Liu","suffix":""},{"id":617844423,"identity":"fe0d73f9-8e9b-40e8-a736-4c90490cb1e8","order_by":4,"name":"Yinguang Zhang","email":"","orcid":"","institution":"Capital Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yinguang","middleName":"","lastName":"Zhang","suffix":""},{"id":617844426,"identity":"645f07a0-72b0-46f1-98ee-2c4d5e05414e","order_by":5,"name":"Xidong Ma","email":"","orcid":"","institution":"Capital Medical University","correspondingAuthor":false,"prefix":"","firstName":"Xidong","middleName":"","lastName":"Ma","suffix":""},{"id":617844436,"identity":"b6a75c2d-d866-4e0d-a784-26cee222e3a8","order_by":6,"name":"Xin Zhang","email":"","orcid":"","institution":"Affiliated Hospital of Shandong Second Medical University, Shandong Second Medical University","correspondingAuthor":false,"prefix":"","firstName":"Xin","middleName":"","lastName":"Zhang","suffix":""},{"id":617844437,"identity":"9e3cd1d3-7e36-4843-9862-e3437ad2d03c","order_by":7,"name":"Yiran Liang","email":"","orcid":"","institution":"Capital Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yiran","middleName":"","lastName":"Liang","suffix":""},{"id":617844449,"identity":"3b2e1d07-2111-4681-860f-c2501e374e0f","order_by":8,"name":"Xinying Xue","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA8UlEQVRIiWNgGAWjYLCCBAYGHgYG5gMMjA2kaWFLIEELBPAYEKfF4EbyMYmHOw7LmPOv+SbNu8PG3pyB+djHL3i0SM5ISzZIPHOYx3LG283GvGfSEnc2sCXPlsGjhV8ix/BBYtthHoMbZzc+zm07nGBwgMeYWQKPFjaJ/A8HIFrOPDgM1GJPUAvQFkaILed7GEG2MG4AamH8gM8vPc+MDRLb0oG2sBkb/20D+qWZLZkZjw4Gg+PJzyR/tlnbG5w//ExyZhswxNibDzP+wKcHApoZGCQSoIYArWDmIaylDuirA1AtQEyMLaNgFIyCUTByAAAi/k800C3VAQAAAABJRU5ErkJggg==","orcid":"","institution":"Affiliated Hospital of Shandong Second Medical University, Shandong Second Medical University","correspondingAuthor":true,"prefix":"","firstName":"Xinying","middleName":"","lastName":"Xue","suffix":""}],"badges":[],"createdAt":"2026-03-28 02:38:20","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9249156/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9249156/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":106404179,"identity":"f9e2a82a-8956-425d-81b8-a3a318de56ad","added_by":"auto","created_at":"2026-04-08 09:15:36","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1457841,"visible":true,"origin":"","legend":"","description":"","filename":"Manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9249156/v1_covered_912d594a-0f10-47f2-b97e-f2075b743336.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"The Ki-67 Proliferation Index in Tertiary Lymphoid Structures as a Predictor of Survival Outcomes in Patients with Resectable Non-Small Cell Lung Cancer","fulltext":[],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":true,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":true,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-cancer","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bcan","sideBox":"Learn more about [BMC Cancer](http://bmccancer.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/bcan/default.aspx","title":"BMC Cancer","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Non-small cell lung cancer, Tertiary lymphoid structures, Ki-67(+) immune cells, Surgery, Prognosis","lastPublishedDoi":"10.21203/rs.3.rs-9249156/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9249156/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground \u0026amp; Objectives\u003c/strong\u003e: Although the presence of tertiary lymphoid structures (TLS) has been associated with improved immunotherapy efficacy across multiple solid tumors, the prognostic significance of proliferative activity within TLS in patients with resectable non-small cell lung cancer (NSCLC) remains unclear. This study aims to evaluate the predictive value of Ki-67 expression within TLS for survival outcomes in patients with NSCLC and to explore the association between TLS maturity and the cellular proliferation index within these structures.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e: Postoperative paraffin-embedded pathological specimens from 88 patients with NSCLC were analyzed. Hematoxylin-eosin (H\u0026amp;E) staining and immunohistochemistry (IHC) were performed to evaluate the presence, spatial distribution, and number of TLS. Multiplex immunofluorescence (mIF) staining was used to assess TLS maturity. Kaplan-Meier survival analysis was conducted to determine the prognostic significance of TLS maturity and the cellular proliferation index within TLS.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e: Tumor-associated TLS were identified in the tumor microenvironment of 74 (84.1%) of the 88 patients. The mature TLS was significantly associated with improved recurrence-free survival (RFS) in patients with NSCLC (\u003cem\u003eP =\u003c/em\u003e 0.0068). The cellular proliferation index within TLS demonstrated a significant positive correlation with TLS maturity. Compared with early-stage TLS and primary follicular-like TLS, secondary follicular-like TLS exhibited a significantly higher proliferation index (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001). Furthermore, a high TLS cellular proliferation index was predictive of favorable RFS in patients with NSCLC (\u003cem\u003eP =\u003c/em\u003e 0.0231). Multivariate analysis further indicated that a low TLS cellular proliferation index was an independent adverse prognostic factor for RFS (HR= 1.9040, 95% CI: 1.054-3.440; \u003cem\u003eP =\u003c/em\u003e 0.0330).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion\u003c/strong\u003e: This study demonstrates the prognostic relevance of the cellular proliferation index within TLS in the tumor microenvironment of NSCLC. A high TLS cellular proliferation index was significantly associated with prolonged RFS. These findings suggest that an elevated TLS cellular proliferation index may contribute to improved clinical outcomes by promoting TLS maturation and enhancing antitumor immune responses.\u003c/p\u003e","manuscriptTitle":"The Ki-67 Proliferation Index in Tertiary Lymphoid Structures as a Predictor of Survival Outcomes in Patients with Resectable Non-Small Cell Lung Cancer","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-07 19:12:01","doi":"10.21203/rs.3.rs-9249156/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-05-13T09:38:32+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-10T21:58:55+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-17T18:22:49+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-17T13:08:59+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"207909860480779238851060656834066870516","date":"2026-04-17T12:09:57+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"210328813875657720924434310229483741929","date":"2026-04-17T11:54:45+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"159489336965521947657027670235366774187","date":"2026-04-02T01:51:09+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-02T01:42:14+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2026-03-30T14:42:50+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-03-30T11:32:48+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-03-30T11:32:09+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Cancer","date":"2026-03-28T02:27:34+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-cancer","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bcan","sideBox":"Learn more about [BMC Cancer](http://bmccancer.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/bcan/default.aspx","title":"BMC Cancer","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"33077b76-ce25-464b-9ac3-f58ffe902b14","owner":[],"postedDate":"April 7th, 2026","published":true,"recentEditorialEvents":[{"type":"decision","content":"Revision requested","date":"2026-05-13T09:38:32+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-10T21:58:55+00:00","index":46,"fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[],"tags":[],"updatedAt":"2026-05-13T09:56:59+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-07 19:12:01","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9249156","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9249156","identity":"rs-9249156","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}