Protocol TitleDevelopment of Patient-Derived Xenografts (PDXs) and analysis of tyrosine kinase receptor expression in patients with Head and Neck Squamous Cell Carcinoma (HNSCC) to study resistance mechanisms induced by standard therapy (SPAR).Brief SummaryThe SPAR study aims to better understand why some patients with head and neck squamous cell carcinoma (HNSCC) stop responding to standard treatments. Researchers will create a specialized set of "Patient-Derived Xenografts" (PDXs)-biological models developed by grafting fresh human tumor samples into immunocompromised mice.By analyzing these models alongside historical patient samples, the study will investigate the expression of various protein receptors (such as the ERBB family, MET, and AXL) and their signaling pathways. The goal is to identify alternative therapeutic targets that could allow the use of existing drugs approved for other cancers, providing new hope for patients with drug-resistant HNSCC.Study DesignStudy Type: Combined Prospective and Retrospective observational/translational study.Prospective Phase: Over 5 years, researchers will collect fresh tumor and blood samples from patients undergoing surgery. These samples will be used to develop the PDX models and undergo genetic and molecular characterization (NGS, IHC, WB).Retrospective Phase: Researchers will analyze archived biological materials (FFPE samples) from patients treated over the last 10 years to evaluate molecular markers and resistance pathways.Duration: 6.5 years total (5 years for enrollment, 1.5 years for data analysis).Participant PopulationTotal Enrollment: 120 patients.Retrospective Group (approx. 40 cases): Patients with HNSCC treated with Cetuximab in the last 10 years.Prospective Group (80 cases): Patients with HNSCC scheduled for surgery at the IRCCS Policlino S. Orsola in Bologna.Eligibility CriteriaInclusion Criteria:Age $\ge$ 18 years at the time of tissue collection.Signed informed consent.Confirmed histological diagnosis of squamous cell carcinoma of the head and neck.For the retrospective group: Prior treatment with Cetuximab.For the prospective group: Candidates for surgery within the Otorhinolaryngology or Maxillofacial units.Exclusion Criteria:Retrospective: Biological material of insufficient quality or quantity for immunohistochemical/molecular analysis.Prospective: Any diagnosis other than squamous cell carcinoma.Outcome Measures / MethodologyThe study will utilize advanced molecular techniques to map the tumor landscape, including:Genomic/Transcriptomic Analysis: Next-Generation Sequencing (NGS), qPCR, and ddPCR to identify mutations and gene rearrangements.Protein Expression: ELISA, Immunohistochemistry (IHC), Immunoblotting, and QF-Pro/PLA technology to evaluate receptor activity.Virology: Identification of HPV presence and genotypes.

1. Protocol Identification & Administrative StatusStudy Acronym: SPAR Protocol Date: 04-02-2026 Version: 3 (Superseding version 1 from 26/11/2025) Study Duration: 6.5 years total (5 years enrollment/selection, 8-12 months for PDX expansion, 3 months molecular analysis, 2 months data analysis) Principal Investigator: Mattia Lauriola Coordinating Institution: IRCCS Azienda Ospedaliero-Universitaria di Bologna (Policlinico S. Orsola) 2. Scientific Background & RationaleHead and Neck Squamous Cell Carcinoma (HNSCC) remains a global health challenge, representing the seventh most common cancer with approximately 890,000 new cases and 450,000 deaths annually.The Resistance ProblemWhile standard therapies include surgery, chemotherapy, and radiation, recent innovations like PD-1 inhibitors (pembrolizumab, nivolumab) benefit only 20-30% of patients. Cetuximab (CTX), an anti-EGFR monoclonal antibody, is a standard treatment, but acquired resistance is a frequent barrier.The Biological HypothesisResistance is often driven by:RTK Bypass: Activation of alternative receptor tyrosine kinases (RTKs) such as MET, AXL, IGFR1, and MERTK.ERBB Family Interactions: The ERBB family (EGFR, HER2, HER3, HER4) utilizes complex heterodimerization to maintain signaling despite EGFR inhibition.Signaling Pathways: Downstream activation of RAS and PI3K/AKT pathways, as well as alterations in the tumor microenvironment (e.g., cytokines, EMT markers).The SPAR project aims to address this by developing unique Italian Patient-Derived Xenograft (PDX) models to study these specific resistance mechanisms.3. Study Objectives & OutcomesPrimary ObjectiveGoal: To develop a set of HNSCC PDX models.Method: Implantation of surgical tumor samples into immunocompromised mice.Assessment: Success is evaluated 2-3 months post-implantation.Secondary ObjectivesMolecular Characterization: Comprehensive analysis of RTK expression (EGFR, HER2, HER3, HER4, MET, AXL, IGFR1, MERTK) and their ligands (e.g., NRG1-4, EGF, HGF).Technological Scope: Utilize NGS (genomic and transcriptomic), RT-qPCR, ddPCR, ELISA, Western Blot, and QF-Pro Technology to map the tumor landscape.Virology: Detection of HPV genotypes and presence of viral oncogene transcripts (E6).4. Study Design & MethodologyThis is a non-profit, observational, cross-sectional study using both retrospective and prospective cohorts.Retrospective Phase (n = 40)Sample Source: Archived FFPE (formalin-fixed paraffin-embedded) materials from the last 10 years at IRCCS AOU Bologna.Population: Patients treated with Cetuximab (both locally advanced and R/M settings).Inclusion: Patients must have adequate biological material for immunohistochemical/molecular analysis.Prospective Phase (n = 80)Duration: Enrollment over 5 years.Source: Patients undergoing surgery for HNSCC at IRCCS S. Orsola.PDX Development: Fresh tumor/blood samples collected during standard care.Feasibility: Researchers estimate a 30% success rate for PDX development, targeting sufficient models for a robust biobank.5. Clinical Procedures & Data ManagementClinical EvaluationThis study does not introduce experimental pharmacological treatments; it aligns with the patient's "usual care".Patients in therapy are seen every 2-3 weeks; patients not in active therapy are followed up every 3 months for the first 2 years, then every 6 months for the next 3 years.Data GovernancePlatform: Data is recorded in a pseudonymized electronic Case Report Form (eCRF) using the REDCap platform.Personnel: The Principal Investigator maintains a delegation log for data management duties.Statistical Analysis: Conducted using GraphPad Prism (v.9.5.0). Demographics are reported as frequencies (categorical) or mean/median (continuous).6. Ethics & Regulatory ComplianceInformed ConsentWritten informed consent is mandatory for participation.Special Case (Deceased/Unreachable Patients): For the retrospective cohort, if patients are deceased or unreachable despite reasonable efforts, researchers may proceed without explicit consent under Art. 110, D.Lgs 196/2003, provided they comply with the Italian Data Protection Authority's requirements.Insurance & AmendmentsAs an observational study, no study-specific insurance is required.Any protocol changes must be submitted to the Ethics Committee as formal amendments.7. Dissemination & ReproducibilityOpen Science: The study will be registered on platforms such as clinicaltrials.gov or osf.io.Publication: The investigator commits to submitting findings to a peer-reviewed journal within 12 months of study conclusion, regardless of the results.Data Sharing: Anonymous data will be made available only after publication

Inclusion Criteria:

1. Patients aged 18 years or older at the time of tissue collection
2. Obtaining written informed consent

For patients enrolled retrospectively:

1. Patients with squamous cell carcinoma of the head and neck treated by the Medical Oncology Unit because they underwent medical treatment with cetuximab in the last 10 years following histological diagnosis carried out in the Otolaryngology or Maxillofacial Surgery Units

For prospectively enrolled patients:

1. Patients with squamous cell carcinoma of the head and neck who will be recruited over the next 5 years

Exclusion Criteria:

• For patients enrolled retrospectively:

Patients with material that is qualitatively and/or quantitatively unsuitable for the necessary immunohistochemical and molecular investigations.

- For patients enrolled prospectively: None, except for diagnoses other than squamous cell carcinoma.

The SPAR study will involve approximately 120 patients with head and neck squamous cell carcinoma (HNSCC). The retrospective phase will analyze tissue samples (FFPE) from about 40 patients treated with cetuximab over the last 10 years at the IRCCS in Bologna, regardless of their disease stage. The prospective phase will enroll 80 surgical candidates over the next 5 years, collecting samples during standard clinical care to develop PDX models and perform molecular analysis. For both groups, clinical, demographic, and outcome data will be collected following the acquisition of informed consent.