Introduction: Cell-free tumor DNA (ctDNA) is an emerging biomarker in head and neck squamous cell carcinoma (HNSCC) for disease staging, patients’ recurrence risk stratification and early detection of relapse. We aimed to compare variants identified in ctDNA versus surgical tumor specimen, and to study the evolution of the mutational landscape of ctDNA over time in HNSCC. Patients and Method: Forty-one HNSCC patients treated with curative-intent primary surgery from SCANDARE cohort (NCT03017573) were evaluated for longitudinal ctDNA-based NGS. Overall, 28 patients were treated with adjuvant (chemo)radiotherapy, and 31 experienced recurrence. Formalin-fixed paraffin-embedded tumor tissues at surgery were available for 41 patients. Serial contributive ctDNA were retrieved from all 41 patients at the date of surgery, 36 patients within 19 weeks after surgery, 20 patients at six months after surgery, and 22 patients at recurrence. Tissue DNA was personalized detected with a custom NGS panel of 571 genes (DRAGON) and ctDNA was sequenced using another personalized dedicated NGS panel including up to 15 genes (OncoFOLLOW). Results: Most frequently mutated genes in tissue included TP53 (15.9%), FAT1 (6.7%), NOTCH1 (5.5%) and PIK3CA (4.3%) with similar allelic ratio to ctDNA at baseline surgery. Higher prevalence of KRAS and TP53 mutations was found in ctDNA at recurrence in comparison with ctDNA and tissue, respectively, at baseline surgery (KRAS: 6.3% versus 1.6% and 0.6%; TP53: 31.2% versus 21.1% and 15.9%). Additional variants in NRAS, HRAS, TP53, JAK2 and SDHA were detected in 6 patients in ctDNA at surgery and were not found in tissue, suggesting spatial intratumor heterogeneity. Twenty-three/36 patients (64%) had detected ctDNA within 19 weeks after surgery among whom, 17/23 patients (74%) had disease recurrence. Eleven/20 patients (including 10 with adjuvant treatment) had detected ctDNA at six months after surgery among whom 6 patients (55%) had disease recurrence. Fifteen/22 patients (68%) had detected ctDNA at recurrence. Emerging pathogenic variants were found in patients with detected ctDNA after surgery (n=7/23; 30%), at six months after surgery (n=1/11; 9%) and at recurrence (n=4/15; 27%). Conclusion: Our study suggests spatial and longitudinal tumor heterogeneity and reports emerging mutations in ctDNA over time in HNSCC. Prognostic significance characterization of the ctDNA dominant clone allele frequency is ongoing. Citation Format: Grégoire Marret, Constance Lamy, Sophie Vacher, Mathieu Séné, Ladidi Ahmanache, Laura Courtois, Zakhia El Beiano, Jerzy Klijanienko, Charlotte Martinat, Nicolas Servant, Choumouss Kamoun, Linda Larbi Chérif, Thierry Bronzini, Cédric Balsat, Jean-François Laes, Aubray Prévot, Sébastien Sauvage, Maxime Lienard, Emmanuel Martin, Bérengère Genin, Nathalie Badois, Maria Lesnik, Antoine Dubray-Vautrin, Olivier Choussy, Wahib Ghanem, Rabah Taouachi, Julien Masliah Planchon, Ivan Bièche, Maud Kamal, Christophe Le Tourneau. Spatial and longitudinal tumor heterogeneity in head and neck squamous cell carcinoma patients treated with primary surgery [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3363.

Background: Epidermal growth factor receptor (EGFR) inhibitors, including monoclonal antibodies and tyrosine kinase inhibitors, have limited efficacy in head and neck squamous cell carcinoma (HNSCC) patients. In the randomized phase II PREDICTOR trial, we aimed at identifying predictive and pharmacodynamics biomarkers of 2-4 weeks afatinib (an irreversible pan-HER inhibitor) versus no treatment in the pre-operative setting (NCT01415674). We previously reported a 59% metabolic response rate on PET imaging in the afatinib arm. We report here the evaluation of predictive genomic and transcriptomic biomarkers of afatinib efficacy. Patients and Methods: All patients (41 in the afatinib arm and 20 in the no treatment arm) underwent a pre-treatment biopsy. We performed targeted DNA sequencing using an in-house NGS panel of 571 genes on baseline biopsies from 56 patients, and RNA-sequencing (RNAseq) in 54 patients. In the afatinib arm, 26 patients had paired pre- and post-treatment tumor samples. DNA and RNA alterations were correlated with metabolic response to afatinib using PET imaging, as well as overall survival (OS). Results: Most frequent molecular alterations, including known activating mutations and/or focal amplifications for oncogenes or homozygous deletions and inactivating mutations for tumor suppressor genes, involved genome integrity (TP53 [70%]), cell cycle (CCND1 [38%], CDKN2A [32%], CDKN2B [14%]), senescence (TERT [23%]), Wnt signaling (NOTCH1 [16%]), and the PI3K pathway (PIK3CA [14%]). In the afatinib arm, metabolic response was observed in 1 out of 7 patients (14%) and in 19 out of 28 patients (68%) in the Wnt altered and unaltered groups (p = 0.03, fisher exact test), respectively. In the whole cohort of patients, homozygous deletions of both CDKN2A and CDKN2B correlated with shorter OS, with 6-year survival of 22% in the CDKN2A/B altered group and 70% in the CDKN2A/B wild-type group (p = 0.004; log-rank test). In the afatinib treated patients, using a generalized linear mixed model with a patient as random effect and a quasi-binomial family, the ratio of B cells expression levels in the post-treated versus pre-treated samples was significantly higher in responder as compared to non-responder patients (p = 0.001). Conclusions: Wnt signaling pathway alterations and treatment-related dynamic changes in B cells proportions were identified as predictive and pharmacodynamics biomarkers of afatinib efficacy. CDKN2A/B homozygous deletions were associated with a poor prognosis in HNSCC patients treated with upfront surgery. Citation Format: Grégoire Marret, Maud Kamal, Jocelyn Gal, Stéphane Temam, Jerzy Klijianenko, Jean-Pierre Delord, Caroline Hoffmann, Gilles Dolivet, Olivier Malard, Jerôme Fayette, Olivier Capitain, Caroline Even, Sébastien Vergez, Lionel Geoffrois, Frédéric Rolland, Philippe Zrounba, Laurent Laccourreye, Joël Guigay, Nicolas Aide, Valérie Bénavent, Constance Lamy, Elodie Girard, Marta Jimenez, Ivan Bièche, Christophe Le Tourneau. Randomized phase II trial of pre-operative afatinib in non-metastatic head and neck squamous cell carcinoma patients: Identification of predictive biomarkers of response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1237.