{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,31]],"date-time":"2026-07-31T19:40:13Z","timestamp":1785526813361,"version":"3.56.0"},"reference-count":42,"publisher":"MDPI AG","issue":"7","license":[{"start":{"date-parts":[[2026,3,24]],"date-time":"2026-03-24T00:00:00Z","timestamp":1774310400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Programmed death-ligand 1 (PD-L1) expression is routinely used to guide immune checkpoint inhibitor (ICI) therapy in advanced non-small cell lung cancer (NSCLC), yet clinical benefit remains heterogeneous even among PD-L1\u2013high tumors. Liquid biopsy based on cell-free DNA (cfDNA) enables minimally invasive, real-time monitoring of tumor evolution. We report four cases of metastatic lung adenocarcinoma treated with atezolizumab, integrating longitudinal whole-exome sequencing (WES) of cfDNA with radiological assessment. Four patients with PD-L1\u2013positive (\u226560%) metastatic NSCLC received atezolizumab monotherapy. Serial cfDNA samples (1\u20133 per patient) were analyzed by high-depth WES. Distinct molecular trajectories paralleled divergent clinical outcomes. One patient achieved a complete molecular response, characterized by progressive clearance of KRAS, ATM, and NF1 mutant clones, which was concordant with radiological remission. A second patient showed an initial molecular response, followed by clonal rebound of TP53, NF1, and NOTCH2 mutant populations and the emergence of PTEN and KIF1A variants, suggesting clinical progression. Two patients exhibited primary resistance despite high PD-L1 expression, with persistent or expanding clones and early subclonal diversification; in one case, new EGFR and BRAF alterations emerged under treatment pressure. Notably, switching to platinum-based chemotherapy in a non-responder induced a measurable molecular response, highlighting discordance between PD-L1 status and immunotherapy efficacy. Longitudinal cfDNA WES captured dynamic clonal remodeling under immunotherapy and anticipated radiological outcomes. These findings underscore the clinical necessity of integrating dynamic molecular monitoring by liquid biopsy to overcome the limitations of static PD-L1 assessment, refine therapeutic stratification, and identify early resistance mechanisms in advanced NSCLC.<\/jats:p>","DOI":"10.3390\/ijms27072947","type":"journal-article","created":{"date-parts":[[2026,3,25]],"date-time":"2026-03-25T09:11:14Z","timestamp":1774429874000},"page":"2947","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Longitudinal Whole-Exome Sequencing of Cell-Free DNA Reveals Molecular Evolution and Heterogeneous Clinical Outcomes in PD-L1 Stratified Advanced NSCLC Adenocarcinoma Patients Treated with Atezolizumab"],"prefix":"10.3390","volume":"27","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-4566-6124","authenticated-orcid":false,"given":"Viola Bianca","family":"Serio","sequence":"first","affiliation":[{"name":"Cancer Genomics & Systems Biology Lab, University of Siena, 53100 Siena, Italy"},{"name":"Med Biotech Hub and Competence Centre, Department of Medical Biotechnologies, University of Siena, 53100 Siena, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tommaso","family":"Regoli","sequence":"additional","affiliation":[{"name":"Cancer Genomics & Systems Biology Lab, University of Siena, 53100 Siena, Italy"},{"name":"Med Biotech Hub and Competence Centre, Department of Medical Biotechnologies, University of Siena, 53100 Siena, Italy"},{"name":"Department of Medical, Surgical and Neurological Sciences, University of Siena, 53100 Siena, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0006-0549-4953","authenticated-orcid":false,"given":"Debora","family":"Maffeo","sequence":"additional","affiliation":[{"name":"Cancer Genomics & Systems Biology Lab, University of Siena, 53100 Siena, Italy"},{"name":"Med Biotech Hub and Competence Centre, Department of Medical Biotechnologies, University of Siena, 53100 Siena, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ignazio","family":"Martellucci","sequence":"additional","affiliation":[{"name":"Oncology Unit, Azienda Ospedaliero Universitaria Senese, 53100 Siena, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Diletta","family":"Rosati","sequence":"additional","affiliation":[{"name":"Cancer Genomics & Systems Biology Lab, University of Siena, 53100 Siena, Italy"},{"name":"Med Biotech Hub and Competence Centre, Department of Medical Biotechnologies, University of Siena, 53100 Siena, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Marco","family":"Ghisalberti","sequence":"additional","affiliation":[{"name":"Thoracic Surgery Unit, Azienda Ospedaliera Universitaria Senese, 53100 Siena, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7349-9725","authenticated-orcid":false,"given":"Alberto","family":"Balistreri","sequence":"additional","affiliation":[{"name":"Bioengineering Laboratory, University of Siena, 53100 Siena, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9284-7492","authenticated-orcid":false,"given":"Gianluca","family":"Santamaria","sequence":"additional","affiliation":[{"name":"Department of Experimental and Clinical Medicine, University \u201cMagna Graecia\u201d of Catanzaro, Campus \u201cS. Venuta\u201d, Germaneto, 88100 Catanzaro, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1759-0313","authenticated-orcid":false,"given":"Niccol\u00f2","family":"Vono","sequence":"additional","affiliation":[{"name":"Department of Experimental and Clinical Medicine, University \u201cMagna Graecia\u201d of Catanzaro, Campus \u201cS. Venuta\u201d, Germaneto, 88100 Catanzaro, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1992-1654","authenticated-orcid":false,"given":"Francesca","family":"Mari","sequence":"additional","affiliation":[{"name":"Department of Medical, Surgical and Neurological Sciences, University of Siena, 53100 Siena, Italy"},{"name":"Clinical Pathology Unit, Azienda Ospedaliero-Universitaria Senese, 53100 Siena, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2015-4317","authenticated-orcid":false,"given":"Francesca","family":"Colombo","sequence":"additional","affiliation":[{"name":"Institute for Biomedical Technologies, National Research Council, Segrate, 20054 Milan, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5634-031X","authenticated-orcid":false,"given":"Elisa","family":"Frullanti","sequence":"additional","affiliation":[{"name":"Cancer Genomics & Systems Biology Lab, University of Siena, 53100 Siena, Italy"},{"name":"Med Biotech Hub and Competence Centre, Department of Medical Biotechnologies, University of Siena, 53100 Siena, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3014-1552","authenticated-orcid":false,"given":"Maria","family":"Palmieri","sequence":"additional","affiliation":[{"name":"Cancer Genomics & Systems Biology Lab, University of Siena, 53100 Siena, Italy"},{"name":"Med Biotech Hub and Competence Centre, Department of Medical Biotechnologies, University of Siena, 53100 Siena, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,3,24]]},"reference":[{"key":"ref_1","first-page":"209","article-title":"Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries","volume":"71","author":"Sung","year":"2021","journal-title":"CA Cancer J. Clin."},{"key":"ref_2","first-page":"7","article-title":"Cancer statistics, 2020","volume":"70","author":"Siegel","year":"2020","journal-title":"CA Cancer J. Clin."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"551","DOI":"10.5732\/cjc.011.10059","article-title":"Lung carcinoma signaling pathways activated by smoking","volume":"30","author":"Wen","year":"2011","journal-title":"Chin. J. Cancer"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"1447","DOI":"10.1101\/gad.263145.115","article-title":"Origins, genetic landscape, and emerging therapies of small cell lung cancer","volume":"29","author":"Semenova","year":"2015","journal-title":"Genes Dev."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"1601734","DOI":"10.1183\/13993003.01734-2016","article-title":"Molecular biomarkers for lung adenocarcinoma","volume":"49","author":"Calvayrac","year":"2017","journal-title":"Eur. Respir. J."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"1","DOI":"10.18632\/genesandcancer.3","article-title":"ALK-rearrangements and testing methods in non-small cell lung cancer: A review","volume":"5","author":"Shackelford","year":"2014","journal-title":"Genes Cancer"},{"key":"ref_7","doi-asserted-by":"crossref","unstructured":"Yan, N., Guo, S., Zhang, H., Zhang, Z., Shen, S., and Li, X. (2022). BRAF-Mutated Non-Small Cell Lung Cancer: Current Treatment Status and Future Perspective. Front. Oncol., 12.","DOI":"10.3389\/fonc.2022.863043"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"1277","DOI":"10.1007\/s40265-019-01164-3","article-title":"Targeting ROS1 Rearrangements in Non-small Cell Lung Cancer: Crizotinib and Newer Generation Tyrosine Kinase Inhibitors","volume":"79","author":"Morris","year":"2019","journal-title":"Drugs"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"112470","DOI":"10.1016\/j.biopha.2021.112470","article-title":"Biomarkers related to immune checkpoint inhibitors therapy","volume":"147","author":"Li","year":"2022","journal-title":"Biomed. Pharmacother."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"1252","DOI":"10.1007\/s12094-022-03046-9","article-title":"New update to the guidelines on testing predictive biomarkers in non-small-cell lung cancer: A National Consensus of the Spanish Society of Pathology and the Spanish Society of Medical Oncology","volume":"25","author":"Isla","year":"2023","journal-title":"Clin. Transl. Oncol."},{"key":"ref_11","doi-asserted-by":"crossref","unstructured":"Lim, M., Kim, C.J., Sunkara, V., Kim, M.-H., and Cho, Y.-K. (2018). Liquid Biopsy in Lung Cancer: Clinical Applications of Circulating Biomarkers (CTCs and ctDNA). Micromachines, 9.","DOI":"10.3390\/mi9030100"},{"key":"ref_12","first-page":"3911","article-title":"Applications of liquid biopsy in lung cancer diagnosis, prognosis prediction, and disease monitoring","volume":"10","author":"Luo","year":"2018","journal-title":"Am. J. Transl. Res."},{"key":"ref_13","doi-asserted-by":"crossref","unstructured":"Miyanaga, A., Masuda, M., and Yamada, T. (2019). Biomarkers of Lung Cancer: Liquid Biopsy Comes of Age. Biomarkers in Cancer Therapy: Liquid Biopsy Comes of Age, Springer.","DOI":"10.1007\/978-981-13-7295-7_10"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"579","DOI":"10.1200\/JCO.2012.45.2011","article-title":"Liquid biopsies: Genotyping circulating tumor DNA","volume":"32","author":"Diaz","year":"2014","journal-title":"J. Clin. Oncol."},{"key":"ref_15","doi-asserted-by":"crossref","unstructured":"Serio, V.B., Rosati, D., Maffeo, D., Rina, A., Ghisalberti, M., Bellan, C., Spiga, O., Mari, F., Palmieri, M., and Frullanti, E. (2024). The Personalized Inherited Signature Predisposing to Non-Small-Cell Lung Cancer in Non-Smokers. Cancers, 16.","DOI":"10.20944\/preprints202407.1249.v1"},{"key":"ref_16","doi-asserted-by":"crossref","unstructured":"Phan, L.M., and Rezaeian, A.H. (2021). ATM: Main Features, Signaling Pathways, and Its Diverse Roles in DNA Damage Response, Tumor Suppression, and Cancer Development. Genes, 12.","DOI":"10.3390\/genes12060845"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"654","DOI":"10.1158\/2159-8290.CD-21-0450","article-title":"Atezolizumab Treatment of Tumors with High Tumor Mutational Burden from MyPathway, a Multicenter, Open-Label, Phase IIa Multiple Basket Study","volume":"12","author":"Friedman","year":"2022","journal-title":"Cancer Discov."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"202","DOI":"10.1158\/2159-8290.CD-15-0283","article-title":"Loss of PTEN Promotes Resistance to T Cell\u2013Mediated Immunotherapy","volume":"6","author":"Peng","year":"2016","journal-title":"Cancer Discov."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"1732","DOI":"10.1038\/s41416-020-0834-6","article-title":"Emerging role of PTEN loss in evasion of the immune response to tumours","volume":"122","author":"Vidotto","year":"2020","journal-title":"Br. J. Cancer"},{"key":"ref_20","first-page":"535","article-title":"The role of PTEN in the tumor microenvironment and its implications for immunotherapy","volume":"18","author":"Sahin","year":"2021","journal-title":"Nat. Rev. Clin. Oncol."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"dmm049362","DOI":"10.1242\/dmm.049362","article-title":"RAS and beyond: The many faces of the neurofibromatosis type 1 protein","volume":"15","author":"Anastasaki","year":"2022","journal-title":"Dis. Model. Mech."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"290","DOI":"10.1038\/nrc3911","article-title":"A RASopathy gene commonly mutated in cancer: The neurofibromatosis type 1 tumour suppressor","volume":"15","author":"Ratner","year":"2015","journal-title":"Nat. Rev. Cancer"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"942","DOI":"10.1002\/hep.27566","article-title":"Differential effects of targeting Notch receptors in a mouse model of liver cancer","volume":"61","author":"Huntzicker","year":"2014","journal-title":"Hepatology"},{"key":"ref_24","doi-asserted-by":"crossref","unstructured":"Xing, L., Xu, L., Zhang, Y., Che, Y., Wang, M., Shao, Y., Qiu, D., Yu, H., Zhao, F., and Zhang, J. (2022). Recent Insight on Regulations of FBXW7 and Its Role in Immunotherapy. Front. Oncol., 12.","DOI":"10.3389\/fonc.2022.925041"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"24394","DOI":"10.18632\/aging.202293","article-title":"E3 ligase FBXW7 restricts M2-like tumor-associated macrophage polarization by targeting c-Myc","volume":"12","author":"Zhong","year":"2020","journal-title":"Aging"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"151","DOI":"10.1038\/s41571-018-0142-8","article-title":"Cancer immunoediting and resistance to T cell-based immunotherapy","volume":"16","author":"Teng","year":"2019","journal-title":"Nat. Rev. Clin. Oncol."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"227","DOI":"10.1158\/0008-5472.CAN-14-3362","article-title":"Control of PD-L1 Expression by Oncogenic Activation of the AKT\u2013mTOR Pathway in Non\u2013Small Cell Lung Cancer","volume":"76","author":"Lastwika","year":"2016","journal-title":"Cancer Res."},{"key":"ref_28","doi-asserted-by":"crossref","unstructured":"Caforio, M., de Billy, E., De Angelis, B., Iacovelli, S., Quintarelli, C., Paganelli, V., and Folgiero, V. (2021). PI3K\/Akt Pathway: The Indestructible Role of a Vintage Target as a Support to the Most Recent Immunotherapeutic Approaches. Cancers, 13.","DOI":"10.3390\/cancers13164040"},{"key":"ref_29","doi-asserted-by":"crossref","unstructured":"Cartwright, D., Kidd, A.C., Ansel, S., Ascierto, M.L., and Spiliopoulou, P. (2025). Oncogenic Signalling Pathways in Cancer Immunotherapy: Leader or Follower in This Delicate Dance?. Int. J. Mol. Sci., 26.","DOI":"10.3390\/ijms26094393"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"e003402","DOI":"10.1136\/jitc-2021-003402","article-title":"PI3K activation allows immune evasion by promoting an inhibitory myeloid tumor microenvironment","volume":"10","author":"Collins","year":"2022","journal-title":"J. Immunother. Cancer"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"516","DOI":"10.1038\/s41568-020-0273-y","article-title":"Metabolism of immune cells in cancer","volume":"20","author":"Leone","year":"2020","journal-title":"Nat. Rev. Cancer"},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"202","DOI":"10.1038\/s41588-018-0312-8","article-title":"Tumor mutational load predicts survival after immunotherapy","volume":"51","author":"Samstein","year":"2019","journal-title":"Nat. Genet."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"390","DOI":"10.14336\/AD.2015.0827","article-title":"Phenylketonuria Pathophysiology: On the Role of Metabolic Alterations","volume":"6","author":"Schuck","year":"2015","journal-title":"Aging Dis."},{"key":"ref_34","unstructured":"Vaupel, P., and Multhoff, G. (2021). Overcoming the Hostile Tumor Microenvironment: Obstacles to Successful Immunotherapy. Front. Oncol., 11."},{"key":"ref_35","first-page":"e173501","article-title":"Association of Cell-Free DNA Tumor Fraction and Somatic Copy Number Alterations with Survival in Metastatic Triple-Negative Breast Cancer","volume":"4","author":"Stover","year":"2018","journal-title":"JAMA Oncol."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"1463","DOI":"10.1126\/science.aaf1490","article-title":"Clonal neoantigens elicit T cell immunoreactivity and sensitivity to immune checkpoint blockade","volume":"351","author":"McGranahan","year":"2016","journal-title":"Science"},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"212","DOI":"10.1038\/s41591-021-01233-9","article-title":"Intratumoral heterogeneity in cancer progression and response to immunotherapy","volume":"27","author":"Vitale","year":"2021","journal-title":"Nat. Med."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"83","DOI":"10.1038\/nrc2290","article-title":"FBW7 ubiquitin ligase: A tumour suppressor at the crossroads of cell division, growth and differentiation","volume":"8","author":"Welcker","year":"2008","journal-title":"Nat. Rev. Cancer"},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"455","DOI":"10.1016\/j.ccell.2014.09.013","article-title":"Tumor Suppression by the Fbw7 Ubiquitin Ligase: Mechanisms and Opportunities","volume":"26","author":"Davis","year":"2014","journal-title":"Cancer Cell"},{"key":"ref_40","first-page":"263","article-title":"The Fbxw7 Tumor Suppressor: Mechanisms and Opportunities for Therapeutics","volume":"2","author":"Yeh","year":"2018","journal-title":"Annu. Rev. Cancer Biol."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"1499","DOI":"10.1126\/science.1162981","article-title":"Fbxw7 targets mTOR for degradation and cooperates with PTEN in tumor suppression","volume":"321","author":"Mao","year":"2008","journal-title":"Science"},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"1441","DOI":"10.1038\/s41591-018-0134-3","article-title":"Blood-based tumor mutational burden as a predictor of clinical benefit in non-small-cell lung cancer patients treated with atezolizumab","volume":"24","author":"Gandara","year":"2018","journal-title":"Nat. Med."}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/27\/7\/2947\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,4,8]],"date-time":"2026-04-08T04:12:34Z","timestamp":1775621554000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/27\/7\/2947"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,3,24]]},"references-count":42,"journal-issue":{"issue":"7","published-online":{"date-parts":[[2026,4]]}},"alternative-id":["ijms27072947"],"URL":"https:\/\/doi.org\/10.3390\/ijms27072947","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,3,24]]}}}