{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,12,11]],"date-time":"2025-12-11T05:36:16Z","timestamp":1765431376497,"version":"3.46.0"},"reference-count":34,"publisher":"MDPI AG","issue":"24","license":[{"start":{"date-parts":[[2025,12,9]],"date-time":"2025-12-09T00:00:00Z","timestamp":1765238400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Cancers"],"abstract":"<jats:p>Background: Human papillomavirus (HPV) plays a crucial role in the pathogenesis of oropharyngeal squamous cell carcinomas (OPSCC). Accurate HPV status classification is essential for therapeutic stratification. While p16 immunohistochemistry (IHC) is the clinical surrogate marker, it has limited specificity. Methods: In this study, we implemented a weakly supervised deep learning approach using the Clustering-constrained Attention Multiple-Instance Learning (CLAM) framework to directly predict HPV status from hematoxylin and eosin (H&amp;E)-stained whole-slide images (WSIs) of OPSCC. A total of 123 WSIs from two cohorts (The Cancer Genome Atlas (TCGA) cohort and OPSCC cohort from the University of Naples Federico II (OPSCC-UNINA)) were used. Results: Attention heatmaps revealed that the model predominantly focused on tumor-rich regions. Errors were primarily observed in slides with conflicting p16\/in situ hybridization (ISH) status or suboptimal quality. Morphological analysis of high-attention patches confirmed that cellular features extracted from correctly classified slides align with HPV status, with a Random Forest classifier achieving 83% accuracy at the cell level. Conclusions: This work supports the feasibility of deep learning-based HPV prediction from routine H&amp;E slides, with potential clinical implications for streamlined, cost-effective diagnostics.<\/jats:p>","DOI":"10.3390\/cancers17243938","type":"journal-article","created":{"date-parts":[[2025,12,9]],"date-time":"2025-12-09T12:42:43Z","timestamp":1765284163000},"page":"3938","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["A Weakly Supervised Approach for HPV Status Prediction in Oropharyngeal Carcinoma from H&amp;E-Stained Slides"],"prefix":"10.3390","volume":"17","author":[{"ORCID":"https:\/\/orcid.org\/0009-0003-6344-7942","authenticated-orcid":false,"given":"Angela","family":"Crispino","sequence":"first","affiliation":[{"name":"Pathology Unit, Department of Advanced Biomedical Sciences, University of Naples Federico II, Via S. Pansini, 5, 80131 Napoli, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4984-0046","authenticated-orcid":false,"given":"Silvia","family":"Varricchio","sequence":"additional","affiliation":[{"name":"Pathology Unit, Department of Advanced Biomedical Sciences, University of Naples Federico II, Via S. Pansini, 5, 80131 Napoli, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0009-0004-4671-3310","authenticated-orcid":false,"given":"Alessandra","family":"Marfella","sequence":"additional","affiliation":[{"name":"Pathology Unit, Department of Advanced Biomedical Sciences, University of Naples Federico II, Via S. Pansini, 5, 80131 Napoli, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0009-0007-7394-3754","authenticated-orcid":false,"given":"Dora","family":"Cerbone","sequence":"additional","affiliation":[{"name":"Pathology Unit, Department of Advanced Biomedical Sciences, University of Naples Federico II, Via S. Pansini, 5, 80131 Napoli, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6961-3695","authenticated-orcid":false,"given":"Daniela","family":"Russo","sequence":"additional","affiliation":[{"name":"Pathology Unit, Department of Advanced Biomedical Sciences, University of Naples Federico II, Via S. Pansini, 5, 80131 Napoli, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0404-0141","authenticated-orcid":false,"given":"Rosa Maria","family":"Di Crescenzo","sequence":"additional","affiliation":[{"name":"Pathology Unit, Department of Advanced Biomedical Sciences, University of Naples Federico II, Via S. Pansini, 5, 80131 Napoli, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4993-9388","authenticated-orcid":false,"given":"Stefania","family":"Staibano","sequence":"additional","affiliation":[{"name":"Pathology Unit, Department of Advanced Biomedical Sciences, University of Naples Federico II, Via S. Pansini, 5, 80131 Napoli, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0711-6738","authenticated-orcid":false,"given":"Francesco","family":"Merolla","sequence":"additional","affiliation":[{"name":"Department of Medicine and Health Sciences \u201cV. Tiberio\u201d, University of Molise, Via De Sanctis, 86100 Campobasso, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9354-4087","authenticated-orcid":false,"given":"Gennaro","family":"Ilardi","sequence":"additional","affiliation":[{"name":"Pathology Unit, Department of Advanced Biomedical Sciences, University of Naples Federico II, Via S. Pansini, 5, 80131 Napoli, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2025,12,9]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"306","DOI":"10.1038\/s41571-022-00603-7","article-title":"HPV-associated oropharyngeal cancer: Epidemiology, molecular biology and clinical management","volume":"19","author":"Lechner","year":"2022","journal-title":"Nat. Rev. Clin. Oncol."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"24","DOI":"10.1056\/NEJMoa0912217","article-title":"Human papillomavirus and survival of patients with oropharyngeal cancer","volume":"363","author":"Ang","year":"2010","journal-title":"N. Engl. J. Med."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"261","DOI":"10.1093\/jnci\/djn011","article-title":"Improved survival of patients with human papillomavirus\u2013positive head and neck squamous cell carcinoma in a prospective clinical trial","volume":"100","author":"Fakhry","year":"2008","journal-title":"J. Natl. Cancer Inst."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"4142","DOI":"10.1200\/JCO.2010.29.2904","article-title":"Prognostic significance of p16INK4A and human papillomavirus in patients with oropharyngeal cancer treated on TROG 02.02 phase III trial","volume":"28","author":"Rischin","year":"2010","journal-title":"J. Clin. Oncol."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"1071","DOI":"10.1093\/annonc\/mdr006","article-title":"Survival and human papillomavirus in oropharynx cancer in TAX 324: A subset analysis from an international phase III trial","volume":"22","author":"Posner","year":"2011","journal-title":"Ann. Oncol."},{"key":"ref_6","doi-asserted-by":"crossref","unstructured":"Perri, F., Longo, F., Caponigro, F., Sandomenico, F., Guida, A., Della Vittoria Scarpati, G., Ottaiano, A., Muto, P., and Ionna, F. (2020). Management of HPV-related squamous cell carcinoma of the head and neck: Pitfalls and caveat. Cancers, 12.","DOI":"10.3390\/cancers12040975"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"40","DOI":"10.1634\/theoncologist.2020-0303","article-title":"Optimizing treatment de-escalation in head and neck cancer: Current and future perspectives","volume":"26","author":"Rosenberg","year":"2021","journal-title":"Oncologist"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"LBA6006","DOI":"10.1200\/jco.2014.32.18_suppl.lba6006","article-title":"E1308: Reduced-dose IMRT in human papilloma virus (HPV)-associated resectable oropharyngeal squamous carcinomas (OPSCC) after clinical complete response (cCR) to induction chemotherapy (IC)","volume":"32","author":"Cmelak","year":"2014","journal-title":"J. Clin. Oncol."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"2347","DOI":"10.1002\/cncr.31338","article-title":"Mature results of a prospective study of deintensified chemoradiotherapy for low-risk human papillomavirus-associated oropharyngeal squamous cell carcinoma","volume":"124","author":"Chera","year":"2018","journal-title":"Cancer"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"S2","DOI":"10.1016\/j.ijrobp.2024.08.014","article-title":"Interim futility results of NRG-HN005, A randomized, phase II\/III non-inferiority trial for non-smoking p16+ oropharyngeal cancer patients","volume":"120","author":"Yom","year":"2024","journal-title":"Int. J. Radiat. Oncol. Biol. Phys."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"827","DOI":"10.1038\/s41416-019-0414-9","article-title":"Recommendations for determining HPV status in patients with oropharyngeal cancers under TNM8 guidelines: A two-tier approach","volume":"120","author":"Craig","year":"2019","journal-title":"Br. J. Cancer"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"945","DOI":"10.1097\/PAS.0b013e318253a2d1","article-title":"Validation of methods for oropharyngeal cancer HPV status determination in US cooperative group trials","volume":"36","author":"Jordan","year":"2012","journal-title":"Am. J. Surg. Pathol."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"2166","DOI":"10.1002\/cncr.25033","article-title":"Comparison of human papillomavirus in situ hybridization and p16 immunohistochemistry in the detection of human papillomavirus-associated head and neck cancer based on a prospective clinical experience","volume":"116","author":"Singhi","year":"2010","journal-title":"Cancer"},{"key":"ref_14","doi-asserted-by":"crossref","unstructured":"Wang, H., Zhang, Y., Bai, W., Wang, B., Wei, J., Ji, R., Xin, Y., Dong, L., and Jiang, X. (2020). Feasibility of immunohistochemical p16 staining in the diagnosis of human papillomavirus infection in patients with squamous cell carcinoma of the head and neck: A systematic review and meta-analysis. Front. Oncol., 10.","DOI":"10.3389\/fonc.2020.524928"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"412","DOI":"10.1038\/s41374-020-00514-0","article-title":"Artificial intelligence and computational pathology","volume":"101","author":"Cui","year":"2021","journal-title":"Lab. Investig."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"38","DOI":"10.4103\/jpi.jpi_53_18","article-title":"Artificial intelligence and digital pathology: Challenges and opportunities","volume":"9","author":"Tizhoosh","year":"2018","journal-title":"J. Pathol. Inform."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"e29080","DOI":"10.1002\/jmv.29080","article-title":"Artificial intelligence for HPV status prediction based on disease-specific images in head and neck cancer: A systematic review and meta-analysis","volume":"95","author":"Song","year":"2023","journal-title":"J. Med. Virol."},{"key":"ref_18","doi-asserted-by":"crossref","unstructured":"Broggi, G., Maniaci, A., Lentini, M., Palicelli, A., Zanelli, M., Zizzo, M., Koufopoulos, N., Salzano, S., Mazzucchelli, M., and Caltabiano, R. (2024). Artificial intelligence in head and neck cancer diagnosis: A comprehensive review with emphasis on radiomics, histopathological, and molecular applications. Cancers, 16.","DOI":"10.3390\/cancers16213623"},{"key":"ref_19","doi-asserted-by":"crossref","unstructured":"Migliorelli, A., Manuelli, M., Ciorba, A., Stomeo, F., Pelucchi, S., and Bianchini, C. (2024). Role of Artificial Intelligence in Human Papillomavirus Status Prediction for Oropharyngeal Cancer: A Scoping Review. Cancers, 16.","DOI":"10.3390\/cancers16234040"},{"key":"ref_20","doi-asserted-by":"crossref","unstructured":"Yao, H., and Zhang, X. (2023). A comprehensive review for machine learning based human papillomavirus detection in forensic identification with multiple medical samples. Front. Microbiol., 14.","DOI":"10.3389\/fmicb.2023.1232295"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"1131","DOI":"10.1158\/1078-0432.CCR-20-3596","article-title":"Deep learning predicts HPV association in oropharyngeal squamous cell carcinomas and identifies patients with a favorable prognosis using regular H&E stains","volume":"27","author":"Klein","year":"2021","journal-title":"Clin. Cancer Res."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"100320","DOI":"10.1016\/j.modpat.2023.100320","article-title":"A novel deep learning algorithm for human papillomavirus infection prediction in head and neck cancers using routine histology images","volume":"36","author":"Wang","year":"2023","journal-title":"Mod. Pathol."},{"key":"ref_23","doi-asserted-by":"crossref","unstructured":"Varricchio, S., Ilardi, G., Crispino, A., D\u2019Angelo, M.P., Russo, D., Di Crescenzo, R.M., Staibano, S., and Merolla, F. (2024). A machine learning approach to predict HPV positivity of oropharyngeal squamous cell carcinoma. Pathol.-J. Ital. Soc. Anat. Pathol. Diagn. Cytopathol., 116.","DOI":"10.32074\/1591-951X-1027"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"555","DOI":"10.1038\/s41551-020-00682-w","article-title":"Data-efficient and weakly supervised computational pathology on whole-slide images","volume":"5","author":"Lu","year":"2021","journal-title":"Nat. Biomed. Eng."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"16878","DOI":"10.1038\/s41598-017-17204-5","article-title":"QuPath: Open source software for digital pathology image analysis","volume":"7","author":"Bankhead","year":"2017","journal-title":"Sci. Rep."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"152","DOI":"10.1038\/s41746-023-00901-z","article-title":"Predicting HPV association using deep learning and regular H&E stains allows granular stratification of oropharyngeal cancer patients","volume":"6","author":"Klein","year":"2023","journal-title":"npj Digit. Med."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"4506","DOI":"10.1038\/s41598-024-55288-y","article-title":"Extracting interpretable features for pathologists using weakly supervised learning to predict p16 expression in oropharyngeal cancer","volume":"14","author":"Adachi","year":"2024","journal-title":"Sci. Rep."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"S44","DOI":"10.1038\/modpathol.2016.152","article-title":"Morphologic diversity in human papillomavirus-related oropharyngeal squamous cell carcinoma: Catch Me If You Can!","volume":"30","author":"Lewis","year":"2017","journal-title":"Mod. Pathol."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"186","DOI":"10.1007\/s12105-009-0126-1","article-title":"HPV-Related Nonkeratinizing Squamous Cell Carcinoma of the Oropharynx: Utility of Microscopic Features in Predicting Patient Outcome","volume":"3","author":"Chernock","year":"2009","journal-title":"Head Neck Pathol."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"2939","DOI":"10.1038\/s41388-023-02819-y","article-title":"Clinical, morphologic and molecular heterogeneity of HPV-associated oropharyngeal cancer","volume":"42","author":"Lim","year":"2023","journal-title":"Oncogene"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"104670","DOI":"10.1016\/j.oraloncology.2020.104670","article-title":"The role of tumour morphology in assigning HPV status in oropharyngeal squamous cell carcinoma","volume":"105","author":"Molony","year":"2020","journal-title":"Oral Oncol."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"45","DOI":"10.1186\/s13000-022-01225-4","article-title":"Visual and digital assessment of Ki-67 in breast cancer tissue\u2014A comparison of methods","volume":"17","author":"Skjervold","year":"2022","journal-title":"Diagn. Pathol."},{"key":"ref_33","doi-asserted-by":"crossref","unstructured":"Martino, F., Varricchio, S., Russo, D., Merolla, F., Ilardi, G., Mascolo, M., dell\u2019Aversana, G.O., Califano, L., Toscano, G., and De Pietro, G. (2020). A Machine-learning Approach for the Assessment of the Proliferative Compartment of Solid Tumors on Hematoxylin-Eosin-Stained Sections. Cancers, 12.","DOI":"10.3390\/cancers12051344"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"60","DOI":"10.1186\/s13073-025-01484-y","article-title":"Closing the gap in the clinical adoption of computational pathology: A standardized, open-source framework to integrate deep-learning models into the laboratory information system","volume":"17","author":"Angeloni","year":"2025","journal-title":"Genome Med."}],"container-title":["Cancers"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2072-6694\/17\/24\/3938\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,12,11]],"date-time":"2025-12-11T05:31:19Z","timestamp":1765431079000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2072-6694\/17\/24\/3938"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,12,9]]},"references-count":34,"journal-issue":{"issue":"24","published-online":{"date-parts":[[2025,12]]}},"alternative-id":["cancers17243938"],"URL":"https:\/\/doi.org\/10.3390\/cancers17243938","relation":{},"ISSN":["2072-6694"],"issn-type":[{"type":"electronic","value":"2072-6694"}],"subject":[],"published":{"date-parts":[[2025,12,9]]}}}