Expression of the Prostate Specific Membrane Antigen (PSMA) and Programmed Death-Ligand 1 (PD-L1) on Disseminated Tumor Cells from Early Triple Negative Breast Cancer Patients and their Clinical Impact

Expression of the Prostate Specific Membrane Antigen (PSMA) and Programmed Death-Ligand 1 (PD-L1) on Disseminated Tumor Cells from Early Triple Negative Breast Cancer Patients and their Clinical Impact | 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 Article Expression of the Prostate Specific Membrane Antigen (PSMA) and Programmed Death-Ligand 1 (PD-L1) on Disseminated Tumor Cells from Early Triple Negative Breast Cancer Patients and their Clinical Impact Anastasios Tolios, Vasileios Vardas, Ann-Kathrin Bittner, Oliver Hoffmann, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6975457/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 Triple-negative breast cancer (TNBC) is the most aggressive breast cancer (BC) subtype, with a worse outcome, highlighting the need for novel biomarkers to treat patients accordingly. This study aimed to evaluate the presence of prostate-specific membrane antigen (PSMA) and programmed death-ligand 1 (PD-L1) on DTCs in the bone marrow of TNBC patients before and after neo-adjuvant chemotherapy, to provide critical insights into their prognostic and therapeutic potential. Methods We investigated the expression of PSMA and PD-L1 in disseminated, cytokeratin (CK)-positive tumor cells (DTCs) from the bone marrow of TNBC patients before (55 patients) and after (47 patients) neo-adjuvant treatment using triple immunofluorescence stainings and VyCAP platform analysis. A weighted scoring system was developed to quantify PSMA and PD-L1 expression on DTCs, categorizing patients into negative, low/intermediate, and high expression groups. Statistical analyses included Spearman’s correlation, Wilcoxon signed-rank, chi-square, and McNemar’s tests, with significance set at p < 0.05. Results At baseline, 69% of patients exhibited CK + PSMAhighCD45- DTCs, which significantly decreased to 9% post-therapy ( p < 0.001). Similarly, CK + PD-L1highCD45- DTCs declined from 43–27% after therapy ( p = 0.045). Based on our scoring system, most patients initially classified as PSMA-high transitioned to PSMA-negative (n = 20, p < 0.001), while only one patient remained PSMA-high post-therapy. Post-NACT, a strong inverse correlation emerged between CK + PD-L1highCD45- and CK + PSMA-CD45- cells ( p = 0.010), and dual-positive phenotypes decreased to 4% ( p < 0.001). Presence of PSMA + DTCs at diagnosis was associated with shorter progression-free ( p = 0.002, HR = 16.1) and overall survival ( p = 0.016, HR = 2.1). Patients transitioning from PSMA-high to PSMA-negative post-therapy had improved overall survival compared to those remaining PSMA-high ( p < 0.001, HR = 1). Conclusions PSMA and PD-L1 are frequently overexpressed on DTCs of TNBC patients, predominantly before the administration of NACT. PSMA-positive DTCs emerged as a significantly poor prognostic factor, holding promise as a biomarker for identifying individuals at higher risk of relapse. It also provides a potential therapeutic target. Similarly, the presence and persistence of PD-L1-positive DTCs suggests their utility as biomarkers to stratify patients for ICI therapies. Biological sciences/Cancer Health sciences/Biomarkers Triple Negative Breast Cancer (TNBC) Prostate - Specific Membrane Antigen (PSMA) programmed death-ligand 1 (PD-L1) Disseminated Tumor Cells (DTCs) Bone marrow (BM) Full Text Additional Declarations No competing interests reported. Supplementary Files SupplementaryFigures.pptx Table1Patientcharacteristics.docx 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-6975457","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":489695656,"identity":"6f2b2a0b-a2d5-4a09-a5b6-e61d58c1cf91","order_by":0,"name":"Anastasios Tolios","email":"","orcid":"","institution":"University of Patras","correspondingAuthor":false,"prefix":"","firstName":"Anastasios","middleName":"","lastName":"Tolios","suffix":""},{"id":489695663,"identity":"4ac14567-732d-4e48-a520-73c8b61ce742","order_by":1,"name":"Vasileios Vardas","email":"","orcid":"","institution":"University of 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Clinical Impact","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":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Triple Negative Breast Cancer (TNBC), Prostate - Specific Membrane Antigen (PSMA), programmed death-ligand 1 (PD-L1), Disseminated Tumor Cells (DTCs), Bone marrow (BM)","lastPublishedDoi":"10.21203/rs.3.rs-6975457/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6975457/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003eTriple-negative breast cancer (TNBC) is the most aggressive breast cancer (BC) subtype, with a worse outcome, highlighting the need for novel biomarkers to treat patients accordingly. This study aimed to evaluate the presence of prostate-specific membrane antigen (PSMA) and programmed death-ligand 1 (PD-L1) on DTCs in the bone marrow of TNBC patients before and after neo-adjuvant chemotherapy, to provide critical insights into their prognostic and therapeutic potential.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e\u003cp\u003eWe investigated the expression of PSMA and PD-L1 in disseminated, cytokeratin (CK)-positive tumor cells (DTCs) from the bone marrow of TNBC patients before (55 patients) and after (47 patients) neo-adjuvant treatment using triple immunofluorescence stainings and VyCAP platform analysis. A weighted scoring system was developed to quantify PSMA and PD-L1 expression on DTCs, categorizing patients into negative, low/intermediate, and high expression groups. Statistical analyses included Spearman\u0026rsquo;s correlation, Wilcoxon signed-rank, chi-square, and McNemar\u0026rsquo;s tests, with significance set at \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eAt baseline, 69% of patients exhibited CK\u0026thinsp;+\u0026thinsp;PSMAhighCD45- DTCs, which significantly decreased to 9% post-therapy (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Similarly, CK\u0026thinsp;+\u0026thinsp;PD-L1highCD45- DTCs declined from 43\u0026ndash;27% after therapy (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.045). Based on our scoring system, most patients initially classified as PSMA-high transitioned to PSMA-negative (n\u0026thinsp;=\u0026thinsp;20, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), while only one patient remained PSMA-high post-therapy. Post-NACT, a strong inverse correlation emerged between CK\u0026thinsp;+\u0026thinsp;PD-L1highCD45- and CK\u0026thinsp;+\u0026thinsp;PSMA-CD45- cells (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.010), and dual-positive phenotypes decreased to 4% (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Presence of PSMA\u0026thinsp;+\u0026thinsp;DTCs at diagnosis was associated with shorter progression-free (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.002, HR\u0026thinsp;=\u0026thinsp;16.1) and overall survival (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.016, HR\u0026thinsp;=\u0026thinsp;2.1). Patients transitioning from PSMA-high to PSMA-negative post-therapy had improved overall survival compared to those remaining PSMA-high (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, HR\u0026thinsp;=\u0026thinsp;1).\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e\u003cp\u003ePSMA and PD-L1 are frequently overexpressed on DTCs of TNBC patients, predominantly before the administration of NACT. PSMA-positive DTCs emerged as a significantly poor prognostic factor, holding promise as a biomarker for identifying individuals at higher risk of relapse. It also provides a potential therapeutic target. Similarly, the presence and persistence of PD-L1-positive DTCs suggests their utility as biomarkers to stratify patients for ICI therapies.\u003c/p\u003e","manuscriptTitle":"Expression of the Prostate Specific Membrane Antigen (PSMA) and Programmed Death-Ligand 1 (PD-L1) on Disseminated Tumor Cells from Early Triple Negative Breast Cancer Patients and their Clinical Impact","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-24 18:58:51","doi":"10.21203/rs.3.rs-6975457/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"59985e4d-b6d1-4e4d-ae74-c7e6eb489437","owner":[],"postedDate":"July 24th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":51990344,"name":"Biological sciences/Cancer"},{"id":51990345,"name":"Health sciences/Biomarkers"}],"tags":[],"updatedAt":"2025-12-19T10:09:02+00:00","versionOfRecord":[],"versionCreatedAt":"2025-07-24 18:58:51","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6975457","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6975457","identity":"rs-6975457","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}