{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,28]],"date-time":"2026-09-28T10:56:09Z","timestamp":1790592969343,"version":"4.1.0"},"reference-count":28,"publisher":"MDPI AG","issue":"19","license":[{"start":{"date-parts":[[2026,9,26]],"date-time":"2026-09-26T00:00:00Z","timestamp":1790380800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100017192","name":"USC Norris Comprehensive Cancer Center","doi-asserted-by":"publisher","award":["P30CA014089"],"award-info":[{"award-number":["P30CA014089"]}],"id":[{"id":"10.13039\/100017192","id-type":"DOI","asserted-by":"publisher"}]},{"award":["P30CA014089"],"award-info":[{"award-number":["P30CA014089"]}],"id":[{"id":"https:\/\/ror.org\/01nmyfr60","id-type":"ROR","asserted-by":"publisher"}]},{"name":"USC Michelson Convergent Science Institute in Cancer"},{"name":"Vassiliadis Research Fund"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Cancers"],"abstract":"<jats:p>Background: Outcomes for patients with osteosarcoma (OS) presenting with metastases at diagnosis or relapsed remain poor with five-year survival rate of only 20\u201330%. Risk stratification at diagnosis remains limited, and no validated prognostic liquid biopsy analyte currently exists for this disease. OS lacks the recurring mutational hotspots and epithelial surface markers that underpin liquid biopsy strategies developed for carcinomas, necessitating fundamentally different approaches to circulating biomarker detection. Methods: Peripheral blood samples from 25 pediatric and young adult OS patients at clinically apparent disease timepoints (n = 29 samples) and 76 adult healthy donors (ND) were processed without cell enrichment using fluorescent whole-slide imaging. An outlier detection pipeline employing deep contrastive learned features classified rare cells across eight immunophenotypic categories defined by DAPI, vimentin (V), cytokeratin (CK), and CD45\/CD31 (CD) expression. From the same patient cohort, plasma cell-free DNA (cfDNA) was analyzed by ichorCNA-based tumor fraction estimation across 42 samples (10 clinically apparent disease timepoints shared with the rare cell cohort and 32 alternative timepoints) from ten patients, and single-cell copy number alteration (scCNA) profiling was performed on 80 rare cells from five patients. Results: Rare cells were detected at a median of approximately 127 cells\/mL (range: 29\u20131114) in OS patients at clinically apparent disease timepoints versus 34 cells\/mL (range: 4\u2013225) in healthy donors. DAPI-only (D) cells, identified using a data-driven optimal cutpoint (38.32 cells\/mL) that is subject to optimization bias and requires independent validation, were associated with relapse-free survival in this exploratory, single-center OS cohort (log-rank p = 0.0003), with high D cell burden associated with markedly shorter relapse-free survival (median 393 days, 95% CI: 106\u2013572) compared to patients with low D cell counts (median 835 days, 95% CI: 494\u20131671). D cells also showed the broadest clinical associations, correlating with metastatic burden and disease subgroup. The Vimentin+\/CD45\u2212 (D|V) population was the strongest discriminator between OS patients and healthy donors (p = 4.64 \u00d7 10\u221212) but did not distinguish relapse-free survival groups within the OS cohort (log-rank p = 0.8367). Genomic analyses at the plasma, single-cell, and tumor tissue levels were largely uninformative. Conclusions: Enrichment-free rare cell profiling identifies a DAPI-only circulating rare cell population whose abundance is associated with relapse-free survival in this exploratory, single-center OS cohort, accessible by nuclear morphology alone without biomarker enrichment. This is a novel, hypothesis-generating observation that, together with the mesenchymal-forward D|V population characterized here, motivates a larger, independent, prospective validation cohort and dedicated single-cell molecular (proteomic) characterization of the D-cell population, rather than establishing DAPI-only rare cell enumeration as a validated liquid biopsy stratification tool at this stage.<\/jats:p>","DOI":"10.3390\/cancers18193123","type":"journal-article","created":{"date-parts":[[2026,9,28]],"date-time":"2026-09-28T10:00:34Z","timestamp":1790589634000},"page":"3123","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Enrichment-Free Rare Cell Profiling in Pediatric Osteosarcoma Reveals a Circulating Cell Population Associated with Relapse-Free Survival"],"prefix":"10.3390","volume":"18","author":[{"given":"Stephanie N.","family":"Shishido","sequence":"first","affiliation":[{"name":"Convergent Science Institute in Cancer (CSI-Cancer), Michelson Center for Convergent Bioscience, University of Southern California, Los Angeles, CA 90089, USA"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"methodology","vocabulary":"credit"},{"role":"validation","vocabulary":"credit"},{"role":"investigation","vocabulary":"credit"},{"role":"data-curation","vocabulary":"credit"},{"role":"supervision","vocabulary":"credit"},{"role":"writing-review-editing","vocabulary":"credit"}]},{"given":"Siddhant","family":"Chaudhary","sequence":"additional","affiliation":[{"name":"Convergent Science Institute in Cancer (CSI-Cancer), Michelson Center for Convergent Bioscience, University of Southern California, Los Angeles, CA 90089, USA"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"software","vocabulary":"credit"},{"role":"validation","vocabulary":"credit"},{"role":"formal-analysis","vocabulary":"credit"},{"role":"investigation","vocabulary":"credit"},{"role":"data-curation","vocabulary":"credit"},{"role":"writing-original-draft","vocabulary":"credit"},{"role":"writing-review-editing","vocabulary":"credit"},{"role":"visualization","vocabulary":"credit"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8720-1264","authenticated-orcid":false,"given":"Ian","family":"Ward","sequence":"additional","affiliation":[{"name":"Convergent Science Institute in Cancer (CSI-Cancer), Michelson Center for Convergent Bioscience, University of Southern California, Los Angeles, CA 90089, USA"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"methodology","vocabulary":"credit"},{"role":"software","vocabulary":"credit"},{"role":"formal-analysis","vocabulary":"credit"},{"role":"investigation","vocabulary":"credit"},{"role":"data-curation","vocabulary":"credit"},{"role":"visualization","vocabulary":"credit"},{"role":"writing-review-editing","vocabulary":"credit"}]},{"ORCID":"https:\/\/orcid.org\/0009-0009-7825-4450","authenticated-orcid":false,"given":"Camilla","family":"Plascencia-Laija","sequence":"additional","affiliation":[{"name":"Convergent Science Institute in Cancer (CSI-Cancer), Michelson Center for Convergent Bioscience, University of Southern California, Los Angeles, CA 90089, USA"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"validation","vocabulary":"credit"},{"role":"investigation","vocabulary":"credit"},{"role":"data-curation","vocabulary":"credit"},{"role":"writing-review-editing","vocabulary":"credit"}]},{"given":"Anya","family":"Zdanowicz","sequence":"additional","affiliation":[{"name":"Cancer and Blood Disease Institute, Children\u2019s Hospital Los Angeles, Los Angeles, CA 90027, USA"},{"name":"Department of Pediatrics, Keck School of Medicine, University of Southern California, Los Angeles, CA 90027, USA"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"data-curation","vocabulary":"credit"},{"role":"writing-review-editing","vocabulary":"credit"},{"role":"resources","vocabulary":"credit"}]},{"given":"Venkata","family":"Yellapantula","sequence":"additional","affiliation":[{"name":"Cancer and Blood Disease Institute, Children\u2019s Hospital Los Angeles, Los Angeles, CA 90027, USA"},{"name":"Department of Pediatrics, Keck School of Medicine, University of Southern California, Los Angeles, CA 90027, USA"},{"name":"Division of Genomic Medicine, Department of Pathology and Laboratory Medicine, Children\u2019s Hospital Los Angeles, Los Angeles, CA 90027, USA"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"writing-review-editing","vocabulary":"credit"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5933-5003","authenticated-orcid":false,"given":"Jeremy","family":"Mason","sequence":"additional","affiliation":[{"name":"Convergent Science Institute in Cancer (CSI-Cancer), Michelson Center for Convergent Bioscience, University of Southern California, Los Angeles, CA 90089, USA"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"methodology","vocabulary":"credit"},{"role":"software","vocabulary":"credit"},{"role":"formal-analysis","vocabulary":"credit"},{"role":"data-curation","vocabulary":"credit"},{"role":"writing-review-editing","vocabulary":"credit"},{"role":"visualization","vocabulary":"credit"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5353-4338","authenticated-orcid":false,"given":"James","family":"Hicks","sequence":"additional","affiliation":[{"name":"Convergent Science Institute in Cancer (CSI-Cancer), Michelson Center for Convergent Bioscience, University of Southern California, Los Angeles, CA 90089, USA"},{"name":"USC Norris Comprehensive Cancer Center, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033, USA"},{"name":"Department of Biological Sciences, Dornsife College of Letters, Arts and Sciences, University of Southern California, Los Angeles, CA 90089, USA"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"writing-review-editing","vocabulary":"credit"}]},{"given":"Fariba","family":"Navid","sequence":"additional","affiliation":[{"name":"Cancer and Blood Disease Institute, Children\u2019s Hospital Los Angeles, Los Angeles, CA 90027, USA"},{"name":"Department of Pediatrics, Keck School of Medicine, University of Southern California, Los Angeles, CA 90027, USA"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"conceptualization","vocabulary":"credit"},{"role":"resources","vocabulary":"credit"},{"role":"writing-review-editing","vocabulary":"credit"},{"role":"project-administration","vocabulary":"credit"},{"role":"funding-acquisition","vocabulary":"credit"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2629-4505","authenticated-orcid":false,"given":"Peter","family":"Kuhn","sequence":"additional","affiliation":[{"name":"Convergent Science Institute in Cancer (CSI-Cancer), Michelson Center for Convergent Bioscience, University of Southern California, Los Angeles, CA 90089, USA"},{"name":"USC Norris Comprehensive Cancer Center, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033, USA"},{"name":"Department of Biological Sciences, Dornsife College of Letters, Arts and Sciences, University of Southern California, Los Angeles, CA 90089, USA"},{"name":"Catherine and Joseph Aresty Department of Urology, Institute of Urology, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033, USA"},{"name":"Department of Aerospace and Mechanical Engineering, Viterbi School of Engineering, University of Southern California, Los Angeles, CA 90089, USA"},{"name":"Department of Biomedical Engineering, Viterbi School of Engineering, University of Southern California, Los Angeles, CA 90089, USA"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"conceptualization","vocabulary":"credit"},{"role":"resources","vocabulary":"credit"},{"role":"writing-review-editing","vocabulary":"credit"},{"role":"supervision","vocabulary":"credit"},{"role":"project-administration","vocabulary":"credit"},{"role":"funding-acquisition","vocabulary":"credit"}]}],"member":"1968","published-online":{"date-parts":[[2026,9,26]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"1531","DOI":"10.1002\/cncr.24121","article-title":"Osteosarcoma incidence and survival rates from 1973 to 2004: Data from the Surveillance, Epidemiology, and End Results Program","volume":"115","author":"Mirabello","year":"2009","journal-title":"Cancer"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"423","DOI":"10.1016\/j.ctrv.2006.05.005","article-title":"Primary bone osteosarcoma in the pediatric age: State of the art","volume":"32","author":"Longhi","year":"2006","journal-title":"Cancer Treat. Rev."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"72","DOI":"10.3892\/or.2026.9077","article-title":"Liquid biopsy in malignant primary bone tumors: Clinical applications of circulating tumor DNA and circulating tumor cells for diagnosis, prognosis, and treatment monitoring (Review)","volume":"55","author":"Tian","year":"2026","journal-title":"Oncol. Rep."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"100846","DOI":"10.1016\/j.tranon.2020.100846","article-title":"Pediatric sarcomas display a variable EpCAM expression in a histology-dependent manner","volume":"13","author":"Tombolan","year":"2020","journal-title":"Transl. Oncol."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"352","DOI":"10.1016\/j.cell.2024.12.005","article-title":"Ongoing chromothripsis underpins osteosarcoma genome complexity and clonal evolution","volume":"188","author":"Trevers","year":"2025","journal-title":"Cell"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"615","DOI":"10.1038\/s41416-018-0212-9","article-title":"Detection of circulating tumour DNA is associated with inferior outcomes in Ewing sarcoma and osteosarcoma","volume":"119","author":"Shulman","year":"2018","journal-title":"Br. J. Cancer"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"21","DOI":"10.1038\/s41698-023-00357-0","article-title":"Combined low-pass whole genome and targeted sequencing in liquid biopsies for pediatric solid tumors","volume":"7","author":"Christodoulou","year":"2023","journal-title":"npj Precis. Oncol."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"3333","DOI":"10.2147\/CMAR.S176515","article-title":"Significance of circulating tumor cells in osteosarcoma patients treated by neoadjuvant chemotherapy and surgery","volume":"10","author":"Wu","year":"2018","journal-title":"Cancer Manag. Res."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"15838","DOI":"10.1038\/s41598-026-47094-5","article-title":"Decoding CTCs in osteosarcoma: The molecular journey from initial tumor to metastasis","volume":"16","author":"Gangi","year":"2026","journal-title":"Sci. Rep."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"78965","DOI":"10.18632\/oncotarget.20697","article-title":"Size-based detection of sarcoma circulating tumor cells and cell clusters","volume":"8","author":"Hayashi","year":"2017","journal-title":"Oncotarget"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"1645","DOI":"10.1158\/0008-5472.CAN-13-1739","article-title":"Universal marker and detection tool for human sarcoma circulating tumor cells","volume":"74","author":"Satelli","year":"2014","journal-title":"Cancer Res."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"899","DOI":"10.1158\/1078-0432.CCR-14-0894","article-title":"Epithelial-mesenchymal transitioned circulating tumor cells capture for detecting tumor progression","volume":"21","author":"Satelli","year":"2015","journal-title":"Clin. Cancer Res."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"100357","DOI":"10.1016\/j.jbo.2021.100357","article-title":"Using a combination of gangliosides and cell surface vimentin as surface biomarkers for isolating osteosarcoma cells in microfluidic devices","volume":"28","author":"Fasanya","year":"2021","journal-title":"J. Bone Oncol."},{"key":"ref_14","doi-asserted-by":"crossref","unstructured":"Batth, I.S., Meng, Q., Wang, Q., Torres, K.E., Burks, J., Wang, J., Gorlick, R., and Li, S. (2020). Rare osteosarcoma cell subpopulation protein array and profiling using imaging mass cytometry and bioinformatics analysis. BMC Cancer, 20.","DOI":"10.1186\/s12885-020-07203-7"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"100236","DOI":"10.1016\/j.jbo.2019.100236","article-title":"Prognostic and clinicopathological significance of circulating tumor cells in osteosarcoma","volume":"16","author":"Li","year":"2019","journal-title":"J. Bone Oncol."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"1397","DOI":"10.20892\/j.issn.2095-3941.2022.0279","article-title":"Detection and surveillance of circulating tumor cells in osteosarcoma for predicting therapy response and prognosis","volume":"19","author":"Mu","year":"2022","journal-title":"Cancer Biol. Med."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"83","DOI":"10.1016\/j.jbo.2018.07.002","article-title":"Isolation of circulating tumor cells in a preclinical model of osteosarcoma: Effect of chemotherapy","volume":"12","author":"Chalopin","year":"2018","journal-title":"J. Bone Oncol."},{"key":"ref_18","doi-asserted-by":"crossref","unstructured":"Dai, S., Shao, X., Wei, Q., Du, S., Hou, C., Li, H., and Jin, D. (2023). Association of circulating tumor cells and IMP3 expression with metastasis of osteosarcoma. Front. Oncol., 13.","DOI":"10.3389\/fonc.2023.819357"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"1075","DOI":"10.3892\/ijo.2017.3905","article-title":"A liquid biopsy-based method for the detection and quantification of circulating tumor cells in surgical osteosarcoma patients","volume":"50","author":"Zhang","year":"2017","journal-title":"Int. J. Oncol."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"016003","DOI":"10.1088\/1478-3975\/9\/1\/016003","article-title":"Fluid biopsy in patients with metastatic prostate, pancreatic and breast cancers","volume":"9","author":"Marrinucci","year":"2012","journal-title":"Phys. Biol."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"22","DOI":"10.1038\/s41523-022-00390-5","article-title":"Disease characterization in liquid biopsy from HER2-mutated, non-amplified metastatic breast cancer patients treated with neratinib","volume":"8","author":"Shishido","year":"2022","journal-title":"npj Breast Cancer"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"36589","DOI":"10.1038\/s41598-025-20514-8","article-title":"Representation learning enables robust single cell phenotyping in whole slide liquid biopsy imaging","volume":"15","author":"Naghdloo","year":"2025","journal-title":"Sci. Rep."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"11775","DOI":"10.1038\/s41598-023-38881-5","article-title":"Plasticity of circulating tumor cells in small cell lung cancer","volume":"13","author":"Seo","year":"2023","journal-title":"Sci. Rep."},{"key":"ref_24","doi-asserted-by":"crossref","unstructured":"Narayan, S., Courcoubetis, G., Mason, J., Naghdloo, A., Kolen\u010d\u00edk, D., Patterson, S.D., Kuhn, P., and Shishido, S.N. (2022). Defining a liquid biopsy profile of circulating tumor cells and oncosomes in metastatic colorectal cancer. Cancers, 14.","DOI":"10.3390\/cancers14194891"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"36","DOI":"10.1038\/s41698-024-00521-0","article-title":"Cancer-related cells and oncosomes in the liquid biopsy of pancreatic cancer patients undergoing surgery","volume":"8","author":"Shishido","year":"2024","journal-title":"npj Precis. Oncol."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"2203","DOI":"10.1002\/(SICI)1097-0258(19961030)15:20<2203::AID-SIM357>3.0.CO;2-G","article-title":"A simulation study of cross-validation for selecting an optimal cutpoint in univariate survival analysis","volume":"15","author":"Faraggi","year":"1996","journal-title":"Stat. Med."},{"key":"ref_27","doi-asserted-by":"crossref","unstructured":"Rota, M., Antolini, L., and Valsecchi, M.G. (2015). Optimal cut-point definition in biomarkers: The case of censored failure time outcome. BMC Med. Res. Methodol., 15.","DOI":"10.1186\/s12874-015-0009-y"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"e66","DOI":"10.1093\/nar\/gks1352","article-title":"Genome-wide copy number profiling of single cells in S-phase reveals DNA-replication domains","volume":"41","author":"Cheng","year":"2013","journal-title":"Nucleic Acids Res."}],"container-title":["Cancers"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2072-6694\/18\/19\/3123\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,9,28]],"date-time":"2026-09-28T10:02:20Z","timestamp":1790589740000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2072-6694\/18\/19\/3123"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,9,26]]},"references-count":28,"journal-issue":{"issue":"19","published-online":{"date-parts":[[2026,10]]}},"alternative-id":["cancers18193123"],"URL":"https:\/\/doi.org\/10.3390\/cancers18193123","relation":{},"ISSN":["2072-6694"],"issn-type":[{"value":"2072-6694","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,9,26]]}}}