{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,18]],"date-time":"2026-09-18T00:27:16Z","timestamp":1789691236706,"version":"4.0.1"},"reference-count":34,"publisher":"MDPI AG","issue":"16","license":[{"start":{"date-parts":[[2023,8,18]],"date-time":"2023-08-18T00:00:00Z","timestamp":1692316800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Israel Science Foundation","award":["2279\/22"],"award-info":[{"award-number":["2279\/22"]}]},{"name":"Israel Science Foundation","award":["3099\/22"],"award-info":[{"award-number":["3099\/22"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Cancers"],"abstract":"<jats:p>Sarcoma classification is challenging and can lead to treatment delays. Previous studies used DNA aberrations and machine-learning classifiers based on methylation profiles for diagnosis. We aimed to classify sarcomas by analyzing methylation signatures obtained from low-coverage whole-genome sequencing, which also identifies copy-number alterations. DNA was extracted from 23 suspected sarcoma samples and sequenced on an Oxford Nanopore sequencer. The methylation-based classifier, applied in the nanoDx pipeline, was customized using a reference set based on processed Illumina-based methylation data. Classification analysis utilized the Random Forest algorithm and t-distributed stochastic neighbor embedding, while copy-number alterations were detected using a designated R package. Out of the 23 samples encompassing a restricted range of sarcoma types, 20 were successfully sequenced, but two did not contain tumor tissue, according to the pathologist. Among the 18 tumor samples, 14 were classified as reported in the pathology results. Four classifications were discordant with the pathological report, with one compatible and three showing discrepancies. Improving tissue handling, DNA extraction methods, and detecting point mutations and translocations could enhance accuracy. We envision that rapid, accurate, point-of-care sarcoma classification using nanopore sequencing could be achieved through additional validation in a diverse tumor cohort and the integration of methylation-based classification and other DNA aberrations.<\/jats:p>","DOI":"10.3390\/cancers15164168","type":"journal-article","created":{"date-parts":[[2023,8,18]],"date-time":"2023-08-18T09:13:44Z","timestamp":1692350024000},"page":"4168","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":6,"title":["Rapid Classification of Sarcomas Using Methylation Fingerprint: A Pilot Study"],"prefix":"10.3390","volume":"15","author":[{"given":"Aviel","family":"Iluz","sequence":"first","affiliation":[{"name":"Leslie and Michael Gaffin Center for Neuro-Oncology, Hadassah Medical Center and Faculty of Medicine, The Hebrew University of Jerusalem, Jerusalem 9190501, Israel"},{"name":"Agnes Ginges Center for Human Neurogenetics, Department of Neurology, Hadassah Medical Center and Faculty of Medicine, The Hebrew University of Jerusalem, Jerusalem 9190501, Israel"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Myriam","family":"Maoz","sequence":"additional","affiliation":[{"name":"Oncology Department, Sharett Institute of Oncology, Hadassah Medical Organization and Faculty of Medicine, The Hebrew University of Jerusalem, Jerusalem 9190501, Israel"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nir","family":"Lavi","sequence":"additional","affiliation":[{"name":"Leslie and Michael Gaffin Center for Neuro-Oncology, Hadassah Medical Center and Faculty of Medicine, The Hebrew University of Jerusalem, Jerusalem 9190501, Israel"},{"name":"Agnes Ginges Center for Human Neurogenetics, Department of Neurology, Hadassah Medical Center and Faculty of Medicine, The Hebrew University of Jerusalem, Jerusalem 9190501, Israel"},{"name":"Department of Military Medicine and \u201cTzameret\u201d, Faculty of Medicine, The Hebrew University of Jerusalem, Jerusalem 9190501, Israel"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hanna","family":"Charbit","sequence":"additional","affiliation":[{"name":"Leslie and Michael Gaffin Center for Neuro-Oncology, Hadassah Medical Center and Faculty of Medicine, The Hebrew University of Jerusalem, Jerusalem 9190501, Israel"},{"name":"Agnes Ginges Center for Human Neurogenetics, Department of Neurology, Hadassah Medical Center and Faculty of Medicine, The Hebrew University of Jerusalem, Jerusalem 9190501, Israel"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7045-680X","authenticated-orcid":false,"given":"Omer","family":"Or","sequence":"additional","affiliation":[{"name":"Orthopedic Department, Hadassah Medical Organization and Faculty of Medicine, The Hebrew University of Jerusalem, Jerusalem 9190501, Israel"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0509-8222","authenticated-orcid":false,"given":"Noam","family":"Olshinka","sequence":"additional","affiliation":[{"name":"Orthopedic Department, Hadassah Medical Organization and Faculty of Medicine, The Hebrew University of Jerusalem, Jerusalem 9190501, Israel"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3604-127X","authenticated-orcid":false,"given":"Jonathan Abraham","family":"Demma","sequence":"additional","affiliation":[{"name":"Surgical Department, Hadassah Medical Organization and Faculty of Medicine, The Hebrew University of Jerusalem, Jerusalem 9190501, Israel"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mohammad","family":"Adileh","sequence":"additional","affiliation":[{"name":"Surgical Department, Hadassah Medical Organization and Faculty of Medicine, The Hebrew University of Jerusalem, Jerusalem 9190501, Israel"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Marc","family":"Wygoda","sequence":"additional","affiliation":[{"name":"Radiotherapy Institute, Sharett Institute of Oncology, Hadassah Medical Organization and Faculty of Medicine, The Hebrew University of Jerusalem, Jerusalem 9190501, Israel"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Philip","family":"Blumenfeld","sequence":"additional","affiliation":[{"name":"Radiotherapy Institute, Sharett Institute of Oncology, Hadassah Medical Organization and Faculty of Medicine, The Hebrew University of Jerusalem, Jerusalem 9190501, Israel"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Masha","family":"Gliner-Ron","sequence":"additional","affiliation":[{"name":"Radiology Department, Hadassah Medical Organization and Faculty of Medicine, The Hebrew University of Jerusalem, Jerusalem 9190501, Israel"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2239-8203","authenticated-orcid":false,"given":"Yusef","family":"Azraq","sequence":"additional","affiliation":[{"name":"Radiology Department, Hadassah Medical Organization and Faculty of Medicine, The Hebrew University of Jerusalem, Jerusalem 9190501, Israel"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Joshua","family":"Moss","sequence":"additional","affiliation":[{"name":"Oncology Department, Sharett Institute of Oncology, Hadassah Medical Organization and Faculty of Medicine, The Hebrew University of Jerusalem, Jerusalem 9190501, Israel"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tamar","family":"Peretz","sequence":"additional","affiliation":[{"name":"Oncology Department, Sharett Institute of Oncology, Hadassah Medical Organization and Faculty of Medicine, The Hebrew University of Jerusalem, Jerusalem 9190501, Israel"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Amir","family":"Eden","sequence":"additional","affiliation":[{"name":"Department of Genetics, The Hebrew University of Jerusalem, Jerusalem 9190501, Israel"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5077-9249","authenticated-orcid":false,"given":"Aviad","family":"Zick","sequence":"additional","affiliation":[{"name":"Oncology Department, Sharett Institute of Oncology, Hadassah Medical Organization and Faculty of Medicine, The Hebrew University of Jerusalem, Jerusalem 9190501, Israel"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6526-1573","authenticated-orcid":false,"given":"Iris","family":"Lavon","sequence":"additional","affiliation":[{"name":"Leslie and Michael Gaffin Center for Neuro-Oncology, Hadassah Medical Center and Faculty of Medicine, The Hebrew University of Jerusalem, Jerusalem 9190501, Israel"},{"name":"Agnes Ginges Center for Human Neurogenetics, Department of Neurology, Hadassah Medical Center and Faculty of Medicine, The Hebrew University of Jerusalem, Jerusalem 9190501, Israel"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2023,8,18]]},"reference":[{"key":"ref_1","doi-asserted-by":"publisher","first-page":"18","DOI":"10.3978\/j.issn.2306-9759.2014.10.01","article-title":"The role of mesenchymal stem\/progenitor cells in sarcoma: Update and dispute","volume":"1","author":"Yang","year":"2014","journal-title":"Stem Cell Investig."},{"key":"ref_2","doi-asserted-by":"publisher","first-page":"93","DOI":"10.3322\/caac.21388","article-title":"The Eighth Edition AJCC Cancer Staging Manual: Continuing to build a bridge from a population-based to a more \u201cpersonalized\u201d approach to cancer staging","volume":"67","author":"Amin","year":"2017","journal-title":"CA Cancer J. Clin."},{"key":"ref_3","doi-asserted-by":"publisher","first-page":"e000914","DOI":"10.1136\/esmoopen-2020-000914","article-title":"Unravelling the heterogeneity of soft tissue and bone sarcoma patients\u2019 health-related quality of life: A systematic literature review with focus on tumour location","volume":"5","author":"Fiore","year":"2020","journal-title":"ESMO Open"},{"key":"ref_4","doi-asserted-by":"publisher","first-page":"102259","DOI":"10.1016\/j.ctrv.2021.102259","article-title":"Uncommon and peculiar soft tissue sarcomas: Multidisciplinary review and practical recommendations for diagnosis and treatment. Spanish group for Sarcoma research (GEIS\u2014GROUP). Part I","volume":"99","author":"Bernabeu","year":"2021","journal-title":"Cancer Treat. Rev."},{"key":"ref_5","doi-asserted-by":"publisher","first-page":"55","DOI":"10.1002\/mas.21570","article-title":"DNA adducts: Formation, biological effects, and new biospecimens for mass spectrometric measurements in humans","volume":"39","author":"Guo","year":"2020","journal-title":"Mass Spectrom. Rev."},{"key":"ref_6","doi-asserted-by":"publisher","first-page":"3406","DOI":"10.1038\/s41467-022-30496-0","article-title":"Clinical genomic profiling in the management of patients with soft tissue and bone sarcoma","volume":"13","author":"Gounder","year":"2022","journal-title":"Nat. Commun."},{"key":"ref_7","doi-asserted-by":"publisher","first-page":"3405","DOI":"10.1038\/s41467-022-30453-x","article-title":"Clinical sequencing of soft tissue and bone sarcomas delineates diverse genomic landscapes and potential therapeutic targets","volume":"13","author":"Nacev","year":"2022","journal-title":"Nat. Commun."},{"key":"ref_8","doi-asserted-by":"publisher","first-page":"498","DOI":"10.1038\/s41467-020-20603-4","article-title":"Sarcoma classification by DNA methylation profiling","volume":"12","author":"Koelsche","year":"2021","journal-title":"Nat. Commun."},{"key":"ref_9","first-page":"1065","article-title":"Mapping of amplification units in the q13-14 region of chromosome 12 in human sarcomas: Some amplica do not include MDM2","volume":"4","author":"Forus","year":"1993","journal-title":"Cell Growth Differ."},{"key":"ref_10","doi-asserted-by":"publisher","first-page":"17","DOI":"10.1080\/13577149778434","article-title":"Amplification and Over-Expression of the MDM2 Gene in Human Soft Tissue Tumours","volume":"1","author":"Patterson","year":"1997","journal-title":"Sarcoma"},{"key":"ref_11","doi-asserted-by":"publisher","first-page":"342","DOI":"10.1002\/ijc.2910640511","article-title":"MDM2 gene amplification in bone and soft-tissue tumors: Association with tumor progression in differentiated adipose-tissue tumors","volume":"64","author":"Nakayama","year":"1995","journal-title":"Int. J. Cancer"},{"key":"ref_12","first-page":"37","article-title":"Cytogenetics and molecular biology of soft tissue tumors","volume":"38","author":"Fletcher","year":"1996","journal-title":"Monogr. Pathol."},{"key":"ref_13","doi-asserted-by":"publisher","first-page":"777","DOI":"10.1016\/S0140-6736(18)31268-6","article-title":"Principles of DNA methylation and their implications for biology and medicine","volume":"392","author":"Dor","year":"2018","journal-title":"Lancet"},{"key":"ref_14","doi-asserted-by":"publisher","first-page":"469","DOI":"10.1038\/nature26000","article-title":"DNA methylation-based classification of central nervous system tumours","volume":"555","author":"Capper","year":"2018","journal-title":"Nature"},{"key":"ref_15","doi-asserted-by":"publisher","first-page":"253","DOI":"10.1038\/nrc1045","article-title":"The power and the promise of DNA methylation markers","volume":"3","author":"Laird","year":"2003","journal-title":"Nat. Rev. Cancer"},{"key":"ref_16","doi-asserted-by":"publisher","first-page":"316","DOI":"10.1111\/dgd.12608","article-title":"Nanopore sequencing: Review of potential applications in functional genomics","volume":"61","author":"Kono","year":"2019","journal-title":"Dev. Growth Differ."},{"key":"ref_17","doi-asserted-by":"publisher","first-page":"672804","DOI":"10.3389\/fgene.2021.672804","article-title":"Cancer Biomarkers Discovery of Methylation Modification With Direct High-Throughput Nanopore Sequencing","volume":"12","author":"Zhang","year":"2021","journal-title":"Front. Genet."},{"key":"ref_18","doi-asserted-by":"publisher","first-page":"407","DOI":"10.1038\/nmeth.4184","article-title":"Detecting DNA cytosine methylation using nanopore sequencing","volume":"14","author":"Simpson","year":"2017","journal-title":"Nat. Methods"},{"key":"ref_19","doi-asserted-by":"publisher","first-page":"691","DOI":"10.1007\/s00401-017-1743-5","article-title":"Same-day genomic and epigenomic diagnosis of brain tumors using real-time nanopore sequencing","volume":"134","author":"Euskirchen","year":"2017","journal-title":"Acta Neuropathol."},{"key":"ref_20","doi-asserted-by":"publisher","first-page":"e12856","DOI":"10.1111\/nan.12856","article-title":"Robust methylation-based classification of brain tumors using nanopore sequencing","volume":"49","author":"Kuschel","year":"2023","journal-title":"Neuropathol. Appl. Neurobiol."},{"key":"ref_21","first-page":"2825","article-title":"Scikit-learn: Machine Learning in Python","volume":"12","author":"Pedregosa","year":"2011","journal-title":"J. Mach. Learn. Res."},{"key":"ref_22","unstructured":"Fischer, B., Smith, M., Pau, G., Morgan, M., and van Twisk, D. (2023, January 29). rhdf5: R Interface to HDF5, R Package version 2.42.0. Available online: https:\/\/github.com\/grimbough\/rhdf5."},{"key":"ref_23","unstructured":"Krijthe, R., van der Maaten, L.R., and Rtsne, L. (2023, January 29). Rtsne: T-Distributed Stochastic Neighbor Embedding using a Barnes-Hut Implementation. Available online: https:\/\/github.com\/jkrijthe\/Rtsne."},{"key":"ref_24","doi-asserted-by":"publisher","first-page":"3094","DOI":"10.1093\/bioinformatics\/bty191","article-title":"Minimap2: Pairwise alignment for nucleotide sequences","volume":"34","author":"Li","year":"2018","journal-title":"Bioinformatics"},{"key":"ref_25","doi-asserted-by":"publisher","first-page":"7","DOI":"10.1186\/1477-5751-10-7","article-title":"Histopathological grading of pediatric ependymoma: Reproducibility and clinical relevance in European trial cohorts","volume":"10","author":"Ellison","year":"2011","journal-title":"J. Negat. Results BioMed."},{"key":"ref_26","doi-asserted-by":"publisher","first-page":"373","DOI":"10.1023\/A:1006527517576","article-title":"Aging trends\u2014Puerto Rico","volume":"12","author":"Muschkin","year":"1997","journal-title":"J. Cross Cult. Gerontol."},{"key":"ref_27","doi-asserted-by":"publisher","first-page":"459","DOI":"10.1016\/S1470-2045(09)70025-7","article-title":"Effects of radiotherapy with concomitant and adjuvant temozolomide versus radiotherapy alone on survival in glioblastoma in a randomised phase III study: 5-year analysis of the EORTC-NCIC trial","volume":"10","author":"Stupp","year":"2009","journal-title":"Lancet Oncol."},{"key":"ref_28","doi-asserted-by":"crossref","unstructured":"Pembaur, A., Sallard, E., Weil, P.P., Ortelt, J., Ahmad-Nejad, P., and Postberg, J. (2021). Simplified Point-of-Care Full SARS-CoV-2 Genome Sequencing Using Nanopore Technology. Microorganisms, 9.","DOI":"10.1101\/2021.07.08.21260171"},{"key":"ref_29","doi-asserted-by":"publisher","first-page":"4170","DOI":"10.1172\/JCI67315","article-title":"Retinal angiogenesis suppression through small molecule activation of p53","volume":"123","author":"Chavala","year":"2013","journal-title":"J. Clin. Invest."},{"key":"ref_30","doi-asserted-by":"publisher","first-page":"346","DOI":"10.1002\/gcc.23041","article-title":"Methylation classifiers: Brain tumors, sarcomas, and what\u2019s next","volume":"61","author":"Koelsche","year":"2022","journal-title":"Genes Chromosomes Cancer"},{"key":"ref_31","doi-asserted-by":"publisher","first-page":"109","DOI":"10.1007\/s00428-019-02720-8","article-title":"Ewing sarcoma and Ewing-like tumors","volume":"476","author":"Sbaraglia","year":"2020","journal-title":"Virchows Arch."},{"key":"ref_32","doi-asserted-by":"publisher","first-page":"941","DOI":"10.1097\/PAS.0000000000000846","article-title":"Sarcomas With CIC-rearrangements Are a Distinct Pathologic Entity With Aggressive Outcome: A Clinicopathologic and Molecular Study of 115 Cases","volume":"41","author":"Antonescu","year":"2017","journal-title":"Am. J. Surg. Pathol."},{"key":"ref_33","doi-asserted-by":"publisher","first-page":"313","DOI":"10.1097\/PAS.0000000000000570","article-title":"CIC-rearranged Sarcomas: A Study of 20 Cases and Comparisons With Ewing Sarcomas","volume":"40","author":"Yoshida","year":"2016","journal-title":"Am. J. Surg. Pathol."},{"key":"ref_34","doi-asserted-by":"publisher","first-page":"70","DOI":"10.32074\/1591-951X-213","article-title":"The 2020 WHO Classification of Soft Tissue Tumours: News and perspectives","volume":"113","author":"Sbaraglia","year":"2021","journal-title":"Pathologica"}],"container-title":["Cancers"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2072-6694\/15\/16\/4168\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T20:36:46Z","timestamp":1760128606000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2072-6694\/15\/16\/4168"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2023,8,18]]},"references-count":34,"journal-issue":{"issue":"16","published-online":{"date-parts":[[2023,8]]}},"alternative-id":["cancers15164168"],"URL":"https:\/\/doi.org\/10.3390\/cancers15164168","relation":{},"ISSN":["2072-6694"],"issn-type":[{"value":"2072-6694","type":"electronic"}],"subject":[],"published":{"date-parts":[[2023,8,18]]}}}