## 2 Materials and Methods  
The patients assessed in this study belong to two previous study cohorts, the HPV-AHEAD study realized in collaboration with IARC-Lyon, and a collaborative study between the University of Medicine and Pharmacy \"[PERSON]\", lasi, Romania and Karolinska Institute-Stockholm. Fifty-four HNSCC patients were selected from the Departments of Oral and Maxillofacial Surgery, Otorhinolaryngology, \"[PERSON]\" University of Medicine and Pharmacy (Iasi, Romania), from January 2010 to October 2017. The formalin-fixed, paraffin-embedded (FFPE) HNSCC blocks included squamous cell carcinoma of the orophynx (International Classification of Diseases for Oncology (ICD-O) C01--base of tongue, C02.4--lingual tonsil, C09--tonsil, C10--oropharynx) and were retrieved from the hospital archives. The OPSCC samples were collected from patients with a median age of 54 years (limits: 41-79), from which 9% (5/54) were women, 54% (29/54) declared being smokers, and 21% (11/54) declared being chronic alcohol users. The education of the patients was high school or professional school. All patients were diagnosed with keratinizing or non-keratinizing squamous cell carcinomas. One patient with base of tongue of tongue cancer was treated previously (2014) with radiotherapy for larynx carcinoma. Ethical clearance for the investigations reported in this study was obtained from the Institutional Ethical Committee of the \"Grigore T. Popa\" University of Medicine and Pharmacy, Iasi, Romania, reference number 7150/2014 and 3953/2018 respectively. The study implied the use of archival material only, and it did not envisage any contact with the patients or to disclosure any personal data files. Adequate measures to ensure data protection, confidentiality, patients' privacy, and anonymization were considered. No informed consent was available. All data were fully anonymized before access. Preparation of paraffin sections and DNA extraction: The FFPE blocks were each sectioned into 31 sections, following the HPV-AHEAD protocol; three were used for histology analysis (sections S1, S10, and S31), two for p16 immunohistochemistry (S2 and S9), and S11 \(\pm\) S30 were archived for future independent studies. For DNA and RNA and RNA analysis, sections S3 \(\pm\) S5 and S6 \(\pm\) S8 were collected in two separate vials. In order to ensure the risk of cross-contamination was minimal, each FFPE block was sectioned using a different blade. Ethanol 70% and DNA Away (Dutscher, Brumath, France) were used to clean the microtome after each block. After every 10 th tissue FFPE block, an empty paraffin block was sectioned and processed to control for DNA cross-contamination. For DNA extraction, the paraffin tissue sections were incubated overnight in a digestion buffer (10 mM Tris/HCl pH 7.4, 0.5 mg/mL proteinase K, and 0.4% Tween 20). Nucleic acids extraction was performed from FFPE OPSCC sections according to QIAamp DNA FFPE Tissue Procedure: remove paraffin from sample, lyse with proteinase K, heat, bind DNA, wash, and elute the ready to use DNA in 50 \(\upmu\)L buffer. HPV DNA genotyping: Type-specific multiplex genotyping (TS-MPG) assay, a method combining multiplex PCR and bead-based Luminex technology (Luminex Corporation, Austin, TX, USA), was used to determine HPV DNA positivity [21]. This assay is able to detect 19 HR (HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 59) or probable high-risk(HPV26, 53, 66, 68a and b, 70, 73, 82) and two low-risk HPV types (HPV6 and 11). Cellular beta-globin gene was used to control for DNA quality. Following PCR amplification, a 10 \(\mu\)L sample of each reaction mixture was used for multiplex HPV genotyping by Luminex technology (Luminex Corporation, Austin, TX, USA) [22]. HPV RNA analysis: Total RNA was purified using the Pure Link FFPE Total RNA Isolation Kit (Invitrogen, Carlsbad, CA, USA) from three pooled sections of the same tissue block.