{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,3,19]],"date-time":"2026-03-19T22:43:15Z","timestamp":1773960195299,"version":"3.50.1"},"reference-count":47,"publisher":"MDPI AG","issue":"9","license":[{"start":{"date-parts":[[2019,9,19]],"date-time":"2019-09-19T00:00:00Z","timestamp":1568851200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Research Center of Portuguese Oncology Institute of Porto","award":["PI 74-CI-IPOP-19-2016"],"award-info":[{"award-number":["PI 74-CI-IPOP-19-2016"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["JCM"],"abstract":"<jats:p>Background: Lung cancer (LCa) is the most frequently diagnosed and lethal cancer worldwide. Histopathological subtyping, which has important therapeutic and prognostic implications, requires material collection through invasive procedures, which might be insufficient to enable definitive diagnosis. Aberrant DNA methylation is an early event in carcinogenesis, detectable in circulating cell-free DNA (ccfDNA). Herein, we aimed to assess methylation of selected genes in ccfDNA from LCa patients and determine its accuracy for tumor subtyping. Methods: Methylation levels of APC, HOXA9, RAR\u03b22, and RASSF1A were assessed in three independent study groups (study group #1: 152 tissue samples; study group #2: 129 plasma samples; study group #3: 28 benign lesions of lung) using quantitative methylation-specific PCR. Associations between gene promoter methylation levels and LCa subtypes were evaluated using non-parametric tests. Receiver operating characteristic (ROC) curve analysis was performed. Results: In study group #2, HOXA9 and RASSF1A displayed higher methylation levels in small-cell lung cancer (SCLC) than in non-small-cell lung cancer (NSCLC). HOXA9 displayed high sensitivity (63.8%), whereas RASSF1A disclosed high specificity (96.2%) for SCLC detection in ccfDNA. Furthermore, HOXA9 methylation levels showed to be higher in squamous cell carcinoma in comparison with adenocarcinoma in study group #1. Conclusions: Methylation level assessments in ccfDNA may provide a minimally invasive procedure for LCa subtyping, complementing standard diagnostic procedures.<\/jats:p>","DOI":"10.3390\/jcm8091500","type":"journal-article","created":{"date-parts":[[2019,9,19]],"date-time":"2019-09-19T11:02:01Z","timestamp":1568890921000},"page":"1500","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":52,"title":["Subtyping Lung Cancer Using DNA Methylation in Liquid Biopsies"],"prefix":"10.3390","volume":"8","author":[{"given":"Sandra P.","family":"Nunes","sequence":"first","affiliation":[{"name":"Cancer Biology & Epigenetics Group \u2013 Research Center, Portuguese Oncology Institute of Porto (CI-IPOP), 4200-072 Porto, Portugal"}]},{"given":"Francisca","family":"Diniz","sequence":"additional","affiliation":[{"name":"Cancer Biology & Epigenetics Group \u2013 Research Center, Portuguese Oncology Institute of Porto (CI-IPOP), 4200-072 Porto, Portugal"}]},{"given":"Catarina","family":"Moreira-Barbosa","sequence":"additional","affiliation":[{"name":"Cancer Biology & Epigenetics Group \u2013 Research Center, Portuguese Oncology Institute of Porto (CI-IPOP), 4200-072 Porto, Portugal"}]},{"given":"Vera","family":"Const\u00e2ncio","sequence":"additional","affiliation":[{"name":"Cancer Biology & Epigenetics Group \u2013 Research Center, Portuguese Oncology Institute of Porto (CI-IPOP), 4200-072 Porto, Portugal"}]},{"given":"Ana Victor","family":"Silva","sequence":"additional","affiliation":[{"name":"Lung Cancer Clinic and Department of Medical Oncology, Portuguese Oncology Institute of Porto, 4200-072 Porto, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9576-3486","authenticated-orcid":false,"given":"J\u00falio","family":"Oliveira","sequence":"additional","affiliation":[{"name":"Lung Cancer Clinic and Department of Medical Oncology, Portuguese Oncology Institute of Porto, 4200-072 Porto, Portugal"}]},{"given":"Marta","family":"Soares","sequence":"additional","affiliation":[{"name":"Lung Cancer Clinic and Department of Medical Oncology, Portuguese Oncology Institute of Porto, 4200-072 Porto, Portugal"}]},{"given":"Sofia","family":"Paulino","sequence":"additional","affiliation":[{"name":"Cancer Biology & Epigenetics Group \u2013 Research Center, Portuguese Oncology Institute of Porto (CI-IPOP), 4200-072 Porto, Portugal"},{"name":"Department of Pathology, Portuguese Oncology Institute of Porto, 4200-072 Porto, Portugal"}]},{"given":"Ana Lu\u00edsa","family":"Cunha","sequence":"additional","affiliation":[{"name":"Cancer Biology & Epigenetics Group \u2013 Research Center, Portuguese Oncology Institute of Porto (CI-IPOP), 4200-072 Porto, Portugal"},{"name":"Department of Pathology, Portuguese Oncology Institute of Porto, 4200-072 Porto, Portugal"}]},{"given":"J\u00e9ssica","family":"Rodrigues","sequence":"additional","affiliation":[{"name":"Department of Epidemiology, Portuguese Oncology Institute of Porto, 4200-072 Porto, Portugal"}]},{"given":"Lu\u00eds","family":"Antunes","sequence":"additional","affiliation":[{"name":"Department of Epidemiology, Portuguese Oncology Institute of Porto, 4200-072 Porto, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3171-4666","authenticated-orcid":false,"given":"Rui","family":"Henrique","sequence":"additional","affiliation":[{"name":"Cancer Biology & Epigenetics Group \u2013 Research Center, Portuguese Oncology Institute of Porto (CI-IPOP), 4200-072 Porto, Portugal"},{"name":"Department of Pathology, Portuguese Oncology Institute of Porto, 4200-072 Porto, Portugal"},{"name":"Department of Pathology and Molecular Immunology, Institute of Biomedical Sciences Abel Salazar, University of Porto (ICBAS-UP), 4050-313 Porto, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4186-5345","authenticated-orcid":false,"given":"Carmen","family":"Jer\u00f3nimo","sequence":"additional","affiliation":[{"name":"Cancer Biology & Epigenetics Group \u2013 Research Center, Portuguese Oncology Institute of Porto (CI-IPOP), 4200-072 Porto, Portugal"},{"name":"Department of Pathology and Molecular Immunology, Institute of Biomedical Sciences Abel Salazar, University of Porto (ICBAS-UP), 4050-313 Porto, Portugal"}]}],"member":"1968","published-online":{"date-parts":[[2019,9,19]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","unstructured":"Bray, F., Ferlay, J., Soerjomataram, I., Siegel, R.L., Torre, L.A., and Jemal, A. 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