MD-PhD Antoine Galmiche
email: galmiche.antoine@chu-amiens.fr

Zuzana Saidak

Jebrane Bouaoud

Haitham Mirghani

Cyril Page

Stéphanie Dakpé

Florian Clatot

Genomics and Precision Surgery for Head and Neck Squamous Cell Carcinoma

Keywords: elective neck dissection, HNSCC: Head and neck squamous cell carcinoma, HPV: Human papilloma virus, ICB: immune-checkpoint blockade, ITH: intratumor heterogeneity, lncRNA: Long non coding RNA, miRNA: microRNA, PNI: perineural invasion, SNB: Sentinel node biopsy, SM: Surgical margins, TCGA: The Cancer Genome Atlas, TME: Tumor microenvironment, HNSCC, precision surgery, genomics, surgical margins, post-surgical recurrence, circulating DNA

Introduction

With over 500,000 new cases worldwide every year, head and neck squamous cell carcinoma (HNSCC) is ranked as the 8 th most common cancer worldwide [START_REF] Bray | Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries[END_REF]. Typically, HNSCC carcinogenesis is related to alcohol and tobacco use, but the independent role of the human papilloma virus (HPV) in HNSCC carcinogenesis has also recently been proven [START_REF] Cramer | The changing therapeutic landscape of head and neck cancer[END_REF]. The incidence of HPV-induced HNSCC is increasing, mostly in western countries, while alcohol/tobacco-induced HNSCC is decreasing. HPVrelated HNSCCs are usually restricted to the oropharynx, and they are associated with a better outcome, due to a different biology and natural history [START_REF] Cramer | The changing therapeutic landscape of head and neck cancer[END_REF][START_REF] Ang | Human papillomavirus and survival of patients with oropharyngeal cancer[END_REF]. Besides this important distinction in terms of etiology, HNSCC is a heterogeneous disease. Depending on the primary location of the HNSCC (mainly oral cavity, larynx, oropharynx and hypopharynx), tumors meet anatomical constraints that dictate their progression, the associated symptoms and the conditions of their diagnosis and staging. Because of its complexity, the clinical handling of HNSCC is increasingly carried out at comprehensive cancer centers by multidisciplinary teams, permitting an integrated management [START_REF] Cramer | The changing therapeutic landscape of head and neck cancer[END_REF]. In this context, the emergence of HPV has greatly changed the therapeutic landscape of HNSCC [START_REF] Cramer | The changing therapeutic landscape of head and neck cancer[END_REF]. Indeed, it is now increasingly recognised that a better understanding of tumor biology is crucial in order to select the best treatment strategy. The present review proposes to explore the concept of "precision surgery" applied to HNSCC. "Precision surgery" is a transposition of the concept of "precision medicine", i.e. a move toward personalized therapies based on disease biology, applied to head and neck surgery.

Analyses centred on the tumor genome or transcriptome, using either DNA or RNA at the level of the whole genome/exome or targeted analyses, have recently emerged as an irreplaceable tool for comprehensive studies of cancer biology. Large scale genome sequencing has established a landmark in HNSCC biology [START_REF] Stransky | The mutational landscape of head and neck squamous cell carcinoma[END_REF][START_REF] Cancer | Comprehensive genomic characterization of head and neck squamous cell carcinomas[END_REF][START_REF] Hedberg | Genetic landscape of metastatic and recurrent head and neck squamous cell carcinoma[END_REF][START_REF] Leemans | The molecular landscape of head and neck cancer[END_REF]. The comprehensive genomic characterization reported within the frame of The Cancer Genome Atlas (TCGA) has laid the foundation for a new molecular taxonomy reflecting the presence of molecular alterations and distinct gene expression profiles [START_REF] Cancer | Comprehensive genomic characterization of head and neck squamous cell carcinomas[END_REF][START_REF] Leemans | The molecular landscape of head and neck cancer[END_REF]. An extensive description of the genetic alterations and the identification of driver genes in HNSCC, that have confirmed the existence of two separate pathways (HPV-negative vs positive), can be found in a recent review [START_REF] Leemans | The molecular landscape of head and neck cancer[END_REF]. Briefly, HPV-negative tumors are characterized by the accumulation of numerous mutations, as well as chromosomal gains and losses, leading to alterations in some well-known driver genes that regulate the cell cycle, apoptosis, WNT signaling and epigenetics, such as PIK3CA, TP53, FAT1 and NSD1 [START_REF] Leemans | The molecular landscape of head and neck cancer[END_REF]. On the other hand, HPV-driven carcinogenesis is highly dependent on the transforming potential of the viral oncogenes E6 and E7 [START_REF] Leemans | The molecular landscape of head and neck cancer[END_REF]. Overall, directly actionable genomic alterations are found at a low frequency in HNSCC [START_REF] Bailey | Comprehensive Characterization of Cancer Driver Genes and Mutations[END_REF]. More recently, sequencing was also applied to recurrent HNSCC and metastatic lesions of HNSCC [START_REF] Hedberg | Genetic landscape of metastatic and recurrent head and neck squamous cell carcinoma[END_REF][START_REF] Huang | Molecular Classification of Lymph Node Metastases Subtypes Predict for Survival in Head and Neck Cancer[END_REF]. Chromosomal instability is associated with the progression of HNSCC to metastasis, through mechanisms that are now better understood [START_REF] Bakhoum | Chromosomal instability drives metastasis through a cytosolic DNA response[END_REF].

In addition to the improved understanding of the genomic alterations, sequencing has proven invaluable for the analysis of complex facets of tumor biology, ranging from fundamental aspects of gene regulation [START_REF] Xiang | Maximizing the Utility of Cancer Transcriptomic Data[END_REF] to the exploration of the tumor microenvironment and the tumor immune infiltrate [START_REF] Thorsson | The Immune Landscape of Cancer[END_REF][START_REF] Lau | RNA Sequencing of the Tumor Microenvironment in Precision Cancer Immunotherapy[END_REF], the presence of viruses and microorganisms within the tumor tissue [START_REF] Guerrero-Preston | 16S rRNA amplicon sequencing identifies microbiota associated with oral cancer, human papilloma virus infection and surgical treatment[END_REF][START_REF] Zhang | Compositional and Functional Analysis of the Microbiome in Tissue and Saliva of Oral Squamous Cell Carcinoma[END_REF], or even the intratumor heterogeneity (ITH) found within the tumor lineage itself (Fig. 1). Interestingly, the analyzis of ITH has been the focus of many recent studies and multiple strategies have been proposed to extrapolate ITH and the clonal structure of HNSCC from the data of bulk tumor DNA sequencing, for example by measuring mutant-allele tumor heterogeneity [START_REF] Mroz | Intra-tumor genetic heterogeneity and mortality in head and neck cancer: analysis of data from the Cancer Genome Atlas[END_REF][START_REF] Andor | Pan-cancer analysis of the extent and consequences of intratumor heterogeneity[END_REF][START_REF] Morris | Pan-cancer analysis of intratumor heterogeneity as a prognostic determinant of survival[END_REF][START_REF] Mroz | Intratumor heterogeneity could inform the use and type of postoperative adjuvant therapy in patients with head and neck squamous cell carcinoma[END_REF]. Importantly, the phenotypic plasticity of cancer cells, under epigenetic regulation is also a major contributor to ITH [START_REF] Peitzsch | Cancer Stem Cells in Head and Neck Squamous Cell Carcinoma: Identification, Characterization and Clinical Implications[END_REF]. Some biologically important issues, such as the existence of a population of cancer stem cells [START_REF] Peitzsch | Cancer Stem Cells in Head and Neck Squamous Cell Carcinoma: Identification, Characterization and Clinical Implications[END_REF] or the occurrence of an Epithelial Mesenchymal Transition (EMT) within the tumor lineage [START_REF] Dongre | New insights into the mechanisms of epithelial-mesenchymal transition and implications for cancer[END_REF] are difficult to address using bulk tumor sequencing [START_REF] Mccorry | Epithelial-to-mesenchymal transition signature assessment in colorectal cancer quantifies tumour stromal content rather than true transition[END_REF]. The advent of single-cell sequencing permits a further characterization of tumor composition at the cellular level. The first study to perform single-cell RNA sequencing of HNSCC was reported by Puram et al. (5902 cells from 18 tumors), leading to the classification of HNSCC based on transcriptomic analyses into three major subtypes: basal, classical, atypical [START_REF] Puram | Single-Cell Transcriptomic Analysis of Primary and Metastatic Tumor Ecosystems in Head and Neck Cancer[END_REF]. With technical advances that permit single-cell sequencing, it is now possible to further characterize the ITH, the tumor microenvironment (TME), to identify cancer stem cells and malignant cells undergoing EMT, and to chart the genomic events along tumor progression at a cellular resolution [START_REF] Qi | Single-cell sequencing and its applications in head and neck cancer[END_REF][START_REF] Yofei | single-cell genomic approaches for developing the next generation of immunotherapies[END_REF]. These advances and the corresponding landmark studies show how genomics permits the study of virtually all aspects of tumor biology from clinical material. Despite this striking progress, genomic sequencing technologies have not yet contributed to the development of strategies for precision medicine for HNSCC. We argue that sequencing offers new perspectives for the integrative study of HNSCC and the clinical transposition of new biomarkers.

Primary surgery in HNSCC: current practice, trends and open issues

HNSCC are traditionally considered as good indications for primary surgery [START_REF] Cramer | The changing therapeutic landscape of head and neck cancer[END_REF][START_REF] Bozec | Current role of primary surgical treatment in patients with head and neck squamous cell carcinoma[END_REF]. The main goal of primary surgery is to resect the entire tumor, but an important challenge is to preserve the function of the organs involved after resection to maintain a decent quality of life for HNSCC patients [START_REF] Orosco | Positive Surgical Margins in the 10 Most Common Solid Cancers[END_REF]. The surgical indication and the type of procedure used are chosen after initial tumor staging that essentially relies on physical and endoscopic examination of the patient combined with tumor imaging (using CT scan, magnetic resonance imaging (MRI) and PET scan, among others). The microscopic examination of the resected tumor sample is a major element of prognosis. Achieving R0 clear surgical margins (SM) and removing all potentially involved neck lymph nodes are the primary aims of oncology surgery. The main determinants that guide whether or not to apply post-operative radiation therapy are the SM status, the presence of Perineural invasion (PNI), vascular invasion, the number and size of metastatic lymph nodes and the presence of extracapsular spread (ECS).

Cisplatin-based chemotherapy is added to post-operative radiation therapy in the case of positive SM or ECS [START_REF] Bernier | Defining risk levels in locally advanced head and neck cancers: a comparative analysis of concurrent postoperative radiation plus chemotherapy trials of the EORTC (#22931) and RTOG (# 9501)[END_REF], but can also be proposed when a combination of factors of poor prognosis is present.

The recognition that HPV-driven oropharynx tumors have specific properties compared to those diagnosed in the context of chronic exposure to alcohol and tobacco has put into question the estimation of the post-operative risk [START_REF] Sinha | High metastatic node number, not extracapsular spread or N-classification is a node-related prognosticator in transorallyresected, neck-dissected p16-positive oropharynx cancer[END_REF]. Several retrospective studies suggest that the classical histological criteria used to assess the prognosis and the therapeutic indications of HNSCC might not be optimal for HPV-driven tumors. For example, the prognosis of HPV-driven oropharynx tumors might depend on the number of metastatic lymph nodes rather than the presence of ECS [START_REF] Sinha | High metastatic node number, not extracapsular spread or N-classification is a node-related prognosticator in transorallyresected, neck-dissected p16-positive oropharynx cancer[END_REF].

Meanwhile, locally advanced tumors of the oropharynx that are induced by HPV were found to respond better to radio-chemotherapy than those induced by alcohol and tobacco [START_REF] Ang | Human papillomavirus and survival of patients with oropharyngeal cancer[END_REF][START_REF] Göttgens | HPV, hypoxia and radiation response in head and neck cancer[END_REF]. These two lines of findings led to the emergence of a revised staging system for HNSCC taking into account their HPV status [START_REF] O'sullivan | Development and validation of a staging system for HPV-related oropharyngeal cancer by the International Collaboration on Oropharyngeal cancer Network for Staging (ICON-S): a multicentre cohort study[END_REF][START_REF] Colevas | NCCN Guidelines Insights: Head and Neck Cancers, Version 1[END_REF]. The inclusion of HPV among the criteria that are useful for clinical staging of HNSCC illustrates the importance of tumor biology for the prognosis of these tumors. To date however, no specific treatment can be proposed for HNSCC based on the HPV status, and evidence is lacking regarding the possibility of proposing a general therapeutic de-escalation for HPV-positive oropharynx HNSCC [START_REF] Cramer | The changing therapeutic landscape of head and neck cancer[END_REF]. Recently, two randomized studies examining this possibility for locally advanced HPV-induced HNSCC even reported worse overall survival when the standard radiochemotherapy treatment was modified by replacing cisplatine with cetuximab [START_REF] Mehanna | Radiotherapy plus cisplatin or cetuximab in low-risk human papillomavirus-positive oropharyngeal cancer (De-ESCALaTE HPV): an open-label randomised controlled phase 3 trial[END_REF][START_REF] Gillison | Radiotherapy plus cetuximab or cisplatin in human papillomavirus-positive oropharyngeal cancer (NRG Oncology RTOG 1016): a randomised, multicentre, non-inferiority trial[END_REF]. A discussion about the ongoing de-escalation trials is beyond the scope of the present review [START_REF] Cramer | The changing therapeutic landscape of head and neck cancer[END_REF].

However, applying tailored de-intensification, based on the characteristics of each tumor (tumor stage, risk of locoregional recurrence and metastatic dissemination), including its predicted sensitivity to radiotherapy, chemotherapy or immunotherapy is a promising perspective [START_REF] Cramer | The changing therapeutic landscape of head and neck cancer[END_REF].

The need to preserve the vital functions and quality of life of HNSCC patients has been an important criterion for the introduction of minimally-invasive surgical procedures, especially Trans-Oral Surgery (TOS) (including Trans-Oral Robotic and Trans-Oral Laser Microsurgery) for oropharyngeal tumors [START_REF] Bozec | Current role of primary surgical treatment in patients with head and neck squamous cell carcinoma[END_REF][START_REF] Remacle | Laser-assisted surgery of the upper aero-digestive tract: a clarification of nomenclature. A consensus statement of the European Laryngological Society[END_REF][START_REF] Golusiński | Functional Organ Preservation Surgery in Head and Neck Cancer: Transoral Robotic Surgery and Beyond[END_REF]. TOS is widely considered to be a safe primary surgical modality, provided that it is applied to small tumors with little local risk (T1T2 cN0) [START_REF] Achim | Long-term Functional and Quality-of-Life Outcomes After Transoral Robotic Surgery in Patients With Oropharyngeal Cancer[END_REF]. An interesting controversy has arisen as to whether TOS should be favored over radiotherapy for OSCC, especially those that are HPV-positive [START_REF] Huang | The interplay of IMRT and transoral surgery in HPV-mediated oropharyngeal cancer: Getting the balance right[END_REF][START_REF] Nichols | Radiotherapy versus transoral robotic surgery and neck dissection for oropharyngeal squamous cell carcinoma (ORATOR): an openlabel, phase 2, randomised trial[END_REF]. Favoring TOS as a first line therapy comes with the risk of having to post-operatively revise tumor stage and prognosis (the pTNM status replacing cTNM). In this situation, patients that are initially refered to TOS may subsequently receive post-operative radiation therapy +/-chemotherapy, in addition to surgery. New mini-invasive procedures therefore hold the potential to reduce patient morbidity and improve the functional outcome of surgery, but they require an optimal initial tumor staging. Analyzing depth of invasion (DOI) of a tumor has been introduced among the criteria that define the risk of local extension, although its predictive value depends on the pathologist's expertise and the cut-off applied [START_REF] Wu | Can pattern and depth of invasion predict lymph node relapse and prognosis in tongue squamous cell carcinoma[END_REF][START_REF] Caldeira | Tumor depth of invasion and prognosis of early-stage oral squamous cell carcinoma: A meta-analysis[END_REF]. Besides the choice of the therapy used as first line against HNSCC, the choice of surgical procedure at the level of tumor lymphatic drainage is also a topic of hot debate. It is yet not established whether the staging of T1T2 cN0 OSCC should rely on elective neck dissection (END), simple clinical surveillance or the sentinel node biopsy (SNB) procedure [START_REF] Ren | Elective versus therapeutic neck dissection in nodenegative oral cancer: Evidence from five randomized controlled trials[END_REF]. The SNB procedure, typically based on the injection of the radiopharmaceutical agent [(99m)Tc]-Tilmanocept, targeting the CD206 receptor, can predict the pathologic nodal status of oral HNSCC with high accuracy [43] and might be considered an appropriate mini-invasive approach in this setting. Collectively, with the expanding number of procedures available and the increasing complexicty of clinical situations, standardization of the surgical treatment of HNSCC is lacking. This is best shown by the number of controversial issues identified in recent surveys of surgical practices [START_REF] Dulguerov | A Review of Controversial Issues in the Management of Head and Neck Cancer: A Swiss Multidisciplinary and Multi-Institutional Patterns of Care Study-Part 1 (Head and Neck Surgery)[END_REF], such as the decision to perform END, surveillance or SNB for cN0 tumors of the oral cavity, the use of up-front neck dissection for advanced neck stages, or the adequate resection margin for tumors of the oral cavity with bone invasion [START_REF] Dulguerov | A Review of Controversial Issues in the Management of Head and Neck Cancer: A Swiss Multidisciplinary and Multi-Institutional Patterns of Care Study-Part 1 (Head and Neck Surgery)[END_REF]. We argue, as discussed later in this review, that progress in addressing HNSCC biology, driven by genomics, will help to improve HNSCC pathological diagnosis and staging and could contribute to refine the indications for surgery, the type of surgical procedure used and also perhaps to adapt the follow up of operated patients.

Refining the indications of surgery

In general, biological analyses and tumor genomics offer perspectives for optimal staging and therapeutic stratification. Early gene expression studies, performed using microarrays, proved to accurately reflect the main aggressive histopathological features of these tumors, such as angiolymphatic invasion and ECS [START_REF] Roepman | Multiple robust signatures for detecting lymph node metastasis in head and neck cancer[END_REF][START_REF] Van Hooff | Validation of a gene expression signature for assessment of lymph node metastasis in oral squamous cell carcinoma[END_REF]. A transcriptomic analysis of ECS was for example reported by Wang et al., who constructed an 11 gene expression signature that correlated with ECS and presented a prognostic value for early stage SCC of the oral cavity [START_REF] Wang | An eleven gene molecular signature for extra-capsular spread in oral squamous cell carcinoma serves as a prognosticator of outcome in patients without nodal metastases[END_REF]. Importantly, the studies reported to date identify different sets of genes with little or no ovelap between the sets [START_REF] Tonella | Gene Expression Signatures for Head and Neck Cancer Patient Stratification: Are Results Ready for Clinical Application?[END_REF]. While the cellular pathways involved are often common and involve for example cell cycle control, hypoxia, the extracellular matrix and signal transduction, the issue of the variability has until now constituted a real challenge to clinical transposition of gene expression analysis [START_REF] Tonella | Gene Expression Signatures for Head and Neck Cancer Patient Stratification: Are Results Ready for Clinical Application?[END_REF]. One explanation for the relative lack of reproducibility resides paradoxically in the huge progress made in gene expression analyses over the past decade. An increasing number of analytical platforms and protocols are available, creating a first source of variability between studies [START_REF] Byron | Translating RNA sequencing into clinical diagnostics: opportunities and challenges[END_REF]. Perhaps more importantly, quality control and standardization of the preanalytical phase is lagging behind (peri-operative hypoxia, use of fresh vs fixed samples, etc.) [START_REF] Byron | Translating RNA sequencing into clinical diagnostics: opportunities and challenges[END_REF]. The use of computer algorithms that track transcriptional noise, systematic bias and "batch effects" in gene expression analysis could offer practical solutions to derive consolidated data sets with a better chance of clinical transposition [START_REF] Riegman | How standardization of the pre-analytical phase of both research and diagnostic biomaterials can increase reproducibility of biomedical research and diagnostics[END_REF][START_REF] Hunter | The "industrial" revolution in biomedical research: Data explosion and reproducibility crisis drive changes in lab workflows[END_REF]. Besides the establishment of gene expression profiles and RNA-seq analyses, progress has also been made in the description of the genomic features that define aggressive tumors at the time of diagnosis. The CCND1 locus, encoding Cyclin D1, is for example frequently amplified in OSCC presenting with small metastases [START_REF] Noorlag | Amplification and Protein Overexpression of Cyclin D1: Predictor of Occult Nodal Metastasis in Early Oral Cancer[END_REF]. Recently, Biswas et al. reported that a small number of genomic characteristics analysed by exome sequencing (specific hotspot somatic mutations in the genes TP53 and CASP8, various alterations of the genes involved in DNA repair by homologous recombination or the nonhomologous end joining (NHEJ) pathways, chromosomal instability) correlated with the occurrence of lymph node metastasis in OSCC [START_REF] Biswas | Lymph node metastasis in oral cancer is strongly associated with chromosomal instability and DNA repair defects[END_REF].

While the literature regarding the biomarkers that could assist in the choice of surgical indications remains scarse, the prospect of better tumor staging at diagnosis offers interesting perspectives [START_REF] Budach | Novel prognostic clinical factors and biomarkers for outcome prediction in head and neck cancer: a systematic review[END_REF]. As mentioned previously, SCC of the oral cavity carry a significant risk of occult lymph node metastases at the time of diagnosis. In this context, the negative predictive value of transcriptomic signatures reflecting nodal metastasis might be an important element to validate the indication for a minimally invasive surgical resection, as suggested by Van Hooff et al. for T1-T2N0 tumors of the oral cavity [START_REF] Van Hooff | Validation of a gene expression signature for assessment of lymph node metastasis in oral squamous cell carcinoma[END_REF]. Genomic alterations, such as the amplification of the chromosomal region 11q13.3 carrying the gene CCND1 are also associated with occult nodal metastases and their detection could equally be useful in this respect [START_REF] Noorlag | Amplification and Protein Overexpression of Cyclin D1: Predictor of Occult Nodal Metastasis in Early Oral Cancer[END_REF]. A more advanced exploration of the genome of oral SCC, as reported by Biswas et al., provides a proof of principle that a limited number of genomic characteristics have a high predictive value for the diagnosis of nodal metastasis, permiting an accurate classification of almost 90% of positive-node vs negative-node tumors [START_REF] Biswas | Lymph node metastasis in oral cancer is strongly associated with chromosomal instability and DNA repair defects[END_REF]. Interestingly, a recent study shows that it is also possible to address the existence of copy number alterations by low-coverage sequencing of cell-free tumor DNA from the plasma of HNSCC patients [START_REF] Schirmer | Cell-Free Plasma DNA for Disease Stratification and Prognosis in Head and Neck Cancer[END_REF].

Importantly, such an approach offers perspectives for a non-invasive molecular staging of HNSCC around the time of diagnosis [START_REF] Schirmer | Cell-Free Plasma DNA for Disease Stratification and Prognosis in Head and Neck Cancer[END_REF]. We discuss later in more detail the issue of circulating DNA (ctDNA) analyses, in the section devoted to the follow-up of cancer patients after surgery.

Additionally, genomics might be helpful in the context of premalignant lesions and, at the other end of the clinical spectrum, for oligometastatic disease. Oral pre-malignant lesions carry a significant risk of transformation, but their current pathological diagnosis is subjective and poorly standardized. Dysplastic lesions are usually removed by surgery, but it is unclear whether resection reduces the risk of subsequent oral SCC [START_REF] Balasundaram | Is there any benefit in surgery for potentially malignant disorders of the oral cavity?[END_REF]. Molecular genomics studies offer the promising perspective of better identification of pre-malignant lesions at risk of progression to oral SCC [START_REF] Zhang | Loss of heterozygosity (LOH) profiles--validated risk predictors for progression to oral cancer[END_REF][START_REF] Foy | Immunological and classical subtypes of oral premalignant lesions[END_REF].

Molecular analyses might also be interesting for metastatic HNSCC. Systemic therapy has until now been the standard of care for patients with metastatic disease [START_REF] Sun | Approach to oligometastatic disease in head and neck cancer, on behalf of the GORTEC[END_REF][START_REF] Pitroda | Integrated molecular and clinical staging defines the spectrum of metastatic cancer[END_REF]. It is however increasingly clear that a subset of HNSCC present characteristics of indolent tumors, and they might constitute possible indications for surgical metastasectomy [START_REF] Sun | Approach to oligometastatic disease in head and neck cancer, on behalf of the GORTEC[END_REF]. Exploring the biology of oligometastatic lesions with high-throughput molecular profiling offers the attractive perspective of broadening the indication for surgical resection to more advanced stages of HNSCC. Recent fundamental studies that have addressed tumor dormancy point to a complex intersection of intrinsic characteristics of cancer cells with the effects of the immune system and specific niches [START_REF] Goddard | Dormant tumour cells, their niches and the influence of immunity[END_REF]. The identification of some transcriptional regulators of HNSCC cell quiescence, such as the transcription factor NR2F1 (Nuclear Receptor subfamily 2 group F member 1) [START_REF] Sosa | NR2F1 controls tumour cell dormancy via SOX9-and RARβ-driven quiescence programmes[END_REF], paves the way to the detection of the corresponding changes in the tumor transcriptome, for an optimized stratification of advanced stages of HNSCC based on functional genomics.

The prospect that HPV-positive tumors might in general be sensitive to radiotherapy has opened discussion regarding the possibility of favoring radiotherapy over surgery in specific situations [START_REF] Huang | The interplay of IMRT and transoral surgery in HPV-mediated oropharyngeal cancer: Getting the balance right[END_REF][START_REF] Sinha | Survival for HPVpositive oropharyngeal squamous cell carcinoma with surgical versus non-surgical treatment approach: A systematic review and meta-analysis[END_REF]. There is currently no evidence that HPV-positivity, typically assessed by p16 immunohistochemistry and the detection of HPV DNA, can be used for this purpose [START_REF] Sinha | Survival for HPVpositive oropharyngeal squamous cell carcinoma with surgical versus non-surgical treatment approach: A systematic review and meta-analysis[END_REF]. Arguably however, genomic analyses that more closely address the impact of HPV infection on tumor biology could be useful in this context. A recent study examining HPV RNA levels in oropharyngeal HNSCC tumors from TCGA suggests the interest of considering HPV infection as a continuous variable rather than a binary status [START_REF] Gleber-Netto | Variations in HPV function are associated with survival in squamous cell carcinoma[END_REF]. By more accurately reflecting active viral replication and its impact on tumor biology, this type of approach will likely help in the understanding of why HPV-positive HNSCC tumors are more sensitive to chemotherapy and radiation therapy [START_REF] Gleber-Netto | Variations in HPV function are associated with survival in squamous cell carcinoma[END_REF]. Beyond the classical knowledge that the E6 and E7 proteins of HPV neutralize p53 and Rb, many questions regarding how HPV interferes with the DNA damage response still remain [START_REF] Göttgens | HPV, hypoxia and radiation response in head and neck cancer[END_REF]. Recent studies have explored the possibility of predicting the response of HNSCC to radiotherapy from gene analyses [START_REF] Toustrup | Development of a hypoxia gene expression classifier with predictive impact for hypoxic modification of radiotherapy in head and neck cancer[END_REF][START_REF] Tawk | Comparative analysis of transcriptomics based hypoxia signatures in head-and neck squamous cell carcinoma[END_REF][START_REF] Hess | Genomic amplification of Fanconi anemia complementation group A (FancA) in head and neck squamous cell carcinoma (HNSCC): Cellular mechanisms of radioresistance and clinical relevance[END_REF][START_REF] Foy | A 13-gene expressionbased radioresistance score highlights the heterogeneity in the response to radiation therapy across HPV-negative HNSCC molecular subtypes[END_REF][START_REF] Schmidt | Development and Validation of a Gene Signature for Patients with Head and Neck Carcinomas Treated by Postoperative Radio(chemo)therapy[END_REF][START_REF] You | Prognostic signature associated with radioresistance in head and neck cancer via transcriptomic and bioinformatic analyses[END_REF]. The different gene signatures reflect various tumor characteristics, ranging from the intrinsic radioresistance of cancer cells (for example accounted for by DNA repair deficiency) to the physical conditions encountered by the tumor tissue, such as hypoxia. They vary greatly in their ability to predict patient survival and tumor recurrence, reflecting the heterogeneity of the populations analyzed (some excluding HPV-positive HNSCC patients, others aiming to identify a signature independent of HPV status). There are also differences in the main aims of these studies (patient stratification or validation of basic research). Other than the necessity of prospective validation of these findings, an important remaining challenge is the identification of the key genes whose expression will contribute to robust signatures. The intricate regulation of the various aspects of tumor biology also represents a challenge, as was shown for example in a recent study showing an inverse correlation between the expression of hypoxic genes and immune T cell infiltration in HNSCC [START_REF] Brooks | Development and Validation of a Combined Hypoxia and Immune Prognostic Classifier for Head and Neck Cancer[END_REF]. Combining the various sources of genomic information with biomarkers obtained for example by immunohistochemistry or tumor imaging, will require the use of powerful and integrative computational approaches [START_REF] Budach | Novel prognostic clinical factors and biomarkers for outcome prediction in head and neck cancer: a systematic review[END_REF]. Despite the absence of validated biomarkers to guide the choice of surgery over radiotherapy, progress toward prediction of tumor aggressiveness and radiosensitivity could contribute to the definition of surgical indications for subgroups of HNSCC.

Adapting the surgical procedure

The definition of surgical resectability is an essential initial step for the choice of surgical procedure. Complete resection of a tumor, leading to R0 SM is classically considered as the single most important goal of surgery in HNSCC [START_REF] Clark | Understanding the surgical margin: a molecular assessment[END_REF][START_REF] Backes | Therapeutic implications of tumor free margins in head and neck squamous cell carcinoma[END_REF]. Often, the emphasis is put on the fact that invaded SM are explained by the need to balance the extent of resection with the need to preserve the vital functions and quality of life of the HNSCC patients that are operated on [START_REF] Orosco | Positive Surgical Margins in the 10 Most Common Solid Cancers[END_REF]. In a recent study, we addressed the possibility that the outcome of surgery might in some instances be linked to tumor biology in HNSCC from different primary locations [START_REF] Saidak | A three-gene expression signature associated with positive surgical margins in tongue squamous cell carcinomas: Predicting surgical resectability from tumour biology?[END_REF]. We identified a three gene-expression signature that was strikingly associated with the presence of positive SM in tongue tumors among TCGA patients (85% sensitivity, 76% specificity) [START_REF] Saidak | A three-gene expression signature associated with positive surgical margins in tongue squamous cell carcinomas: Predicting surgical resectability from tumour biology?[END_REF]. The corresponding signature was not associated with the TNM status of the tumors, but it correlated with higher grade (i.e. less differentiated tumor cells), elevated proliferation levels, a tumor stroma with fewer fibroblasts and endothelial cells and a diffusely invasive growth pattern that could mislead the surgeon during tongue tumor resection [START_REF] Saidak | A three-gene expression signature associated with positive surgical margins in tongue squamous cell carcinomas: Predicting surgical resectability from tumour biology?[END_REF].

These findings suggest for the first time that tumor biology, analyzed by functional genomics, could help to predict surgical resectability for a subset of HNSCC [START_REF] Saidak | A three-gene expression signature associated with positive surgical margins in tongue squamous cell carcinomas: Predicting surgical resectability from tumour biology?[END_REF]. They also illustrate how genomics can be used to explore the most practical aspects of oncological surgery without any former a priori.

Other aspects of HNSCC tumor biology that can be explored by studying the transcriptome of HNSCC tumors could be of potential interest in this respect, such as the study of the tropism of HNSCC cells for bone tissue [START_REF] Park | Loss of RUNX3 expression inhibits bone invasion of oral squamous cell carcinoma[END_REF]. The corresponding knowledge could be used for example to anticipate the ability of HNSCC tumors to invade the mandible. Combined with optimal tumor imaging, information about biological characteristics of tumors could potentially help the surgeon to choose between a minimally invasive procedure and a large resection, consider the performance of a mandibulectomy, or modulate the extent of neck dissection.

In addition to its predictive use, the molecular diagnosis of SM offers interesting clinical perspectives. Local recurrence of HNSCC is observed in 10 to 15% of patients with clear R0 SM. The pionneering study of Brennan et al. revealed that some of the molecular alterations of the TP53 gene that are present in the primary tumor can be detected in a fraction of SM that were considered negative at the conventional histological examination [START_REF] Brennan | Molecular assessment of histopathological staging in squamous-cell carcinoma of the head and neck[END_REF]. Since then, the possibility of augmenting the classical pathological assessment of SM to better evaluate the SM status post-operatively has been actively investigated in several studies. It is unclear what the optimal strategy could be for the molecular diagnosis of SM, considering the variety of applicable biomarkers: mutations of TP53 [START_REF] Brennan | Molecular assessment of histopathological staging in squamous-cell carcinoma of the head and neck[END_REF][START_REF] Murillo | The utility of molecular diagnostics to predict recurrence of head and neck carcinoma[END_REF], changes in DNA methylation [START_REF] Hayashi | Correlation of gene methylation in surgical margin imprints with locoregional recurrence in head and neck squamous cell carcinoma[END_REF], microsatellite instability [START_REF] Liu | Genetic analysis of surgical margins in oral cavity cancer[END_REF] or increased expression levels of specific genes [START_REF] Ogbureke | Expressions of matrix metalloproteinase-9 (MMP-9), dentin sialophosphoprotein (DSPP), and osteopontin (OPN) at histologically negative surgical margins may predict recurrence of oral squamous cell carcinoma[END_REF]. Molecular analysis might render SM analysis more accurate and could be a useful element in the assessment of surgical resection of HNSCC [START_REF] Clark | Understanding the surgical margin: a molecular assessment[END_REF].

Compared to other types of tumors, HNSCC have high levels of infiltration with inflammatory and immune cells [START_REF] Thorsson | The Immune Landscape of Cancer[END_REF][START_REF] Mandal | The head and neck cancer immune landscape and its immunotherapeutic implications[END_REF][START_REF] Horton | Immune Evasion by Head and Neck Cancer: Foundations for Combination Therapy[END_REF]. The prospect of using neoadjuvant immunotherapy, i.e. one or two administrations of immune-checkpoint blockers (ICB) before surgical resection is currently an exciting topic of discussion in surgical oncology [START_REF] O'donnell | The Promise of Neoadjuvant Immunotherapy and Surgery for Cancer Treatment[END_REF]. A number of preclinical and clinical studies in melanoma, glioblastoma and non-small-cell lung cancer suggest the practical feasability and the efficacy of neoadjuvant anti-tumor immunotherapy [START_REF] O'donnell | The Promise of Neoadjuvant Immunotherapy and Surgery for Cancer Treatment[END_REF]. A few experimental studies also provide a rationale for the interest of this approach in HNSCC [START_REF] Horton | Immune Evasion by Head and Neck Cancer: Foundations for Combination Therapy[END_REF][START_REF] Liu | Improved Efficacy of Neoadjuvant Compared to Adjuvant Immunotherapy to Eradicate Metastatic Disease[END_REF][START_REF] Friedman | Neoadjuvant PD-1 immune checkpoint blockade reverses functional immunodominance among tumor-antigen specific T cells[END_REF]. The first trial assessing the safety of a single-dose neoadjuvant pembrolizumab, directed against PD1 was reported in 2017 as an oral communication by Uppaluri et al. [START_REF] Uppaluri | Neoadjuvant pembrolizumab in surgically resectable, locally advanced HPV negative head and neck squamous cell carcinoma (HNSCC)[END_REF]. Several clinical trials are currently ongoing with other protocols, either using other ICB, such as nivolumab [START_REF] Horton | Neoadjuvant presurgical PD-1 inhibition in oral cavity squamous cell carcinoma[END_REF] or combinations of anti-PD1 and anti-CTLA4 antibodies [START_REF] Ferrarotto | Checkpoint inhibitors assessment in oropharynx cancer (CIAO): Safety and interim results[END_REF][START_REF] Zuur | Feasibility and toxicity of neoadjuvant nivolumab with or without ipilimumab prior to extensive (salvage) surgery in patients with advanced head and neck cancer (the IMCISION trial, NCT03003637)[END_REF].

Neoadjuvant immunotherapy may lead to partial or complete pathological responses in around one third of patients with HNSCC [START_REF] Horton | Neoadjuvant presurgical PD-1 inhibition in oral cavity squamous cell carcinoma[END_REF][START_REF] Ferrarotto | Checkpoint inhibitors assessment in oropharynx cancer (CIAO): Safety and interim results[END_REF][START_REF] Zuur | Feasibility and toxicity of neoadjuvant nivolumab with or without ipilimumab prior to extensive (salvage) surgery in patients with advanced head and neck cancer (the IMCISION trial, NCT03003637)[END_REF]. From a surgical point of view, neoadjuvant ICB may therefore lead to tumor down-staging, thus eventually converting an unresectable tumor to a resectable one or allowing a reduction in adjuvant therapy. Another potential benefit of neoadjuvant ICB might be the reduction of the risk of metastatic spread [START_REF] Hanna | Rationale for neoadjuvant immunotherapy in head and neck squamous cell carcinoma[END_REF]. Finally, comparison between baseline biopsies and samples resected after neoadjuvant ICB will provide valuable material to identify biomarkers of response / resistance to these treatments. It is not yet clear whether neoadjuvant ICB will be beneficial in controlling tumor recurrence and extending patient survival. Nevertheless, the corresponding trials do challenge the current surgical practice of proposing a rapid upfront resection for HNSCC tumors, and shed light on the importance of genomic information as a source of clinicallyapplicable biomarkers in HNSCC.

Immunotherapy with pembrolizumab is currently approved for recurrent/metastatic HNSCC [START_REF] Cohen | Pembrolizumab versus methotrexate, docetaxel, or cetuximab for recurrent or metastatic head-and-neck squamous cell carcinoma (KEYNOTE-040): a randomised, open-label, phase 3 study[END_REF][START_REF] Burtness | Pembrolizumab alone or with chemotherapy versus cetuximab with chemotherapy for recurrent or metastatic squamous cell carcinoma of the head and neck (KEYNOTE-048): a randomised, open-label, phase 3 study[END_REF][START_REF] Cramer | Immunotherapy for head and neck cancer: Recent advances and future directions[END_REF]. In this context, the only approved predictive biomarker is the histological detection of PD-L1 (Programmed death-ligand 1, CD274), analyzed using a combined proportion score (CPS) that takes into account the presence of cancer and immune cells. This companion assay is of limited predictive interest, since most patients with a high CPS (>20) do not benefit from pembrolizumab. Moreover, determination of the PD-L1 status is not easily standardizable [START_REF] Rasmussen | Intratumor heterogeneity of PD-L1 expression in head and neck squamous cell carcinoma[END_REF]. The other immunotherapy approved in this setting is nivolumab [START_REF] Ferris | Nivolumab for Recurrent Squamous-Cell Carcinoma of the Head and Neck[END_REF], for which no predictive biomarkers are currently clinicallyapproved. Information regarding the HPV status is not considered to be useful for HPV-driven tumors, despite the presence of a more active immunological environment in HPV-positive tumors [START_REF] Mandal | The head and neck cancer immune landscape and its immunotherapeutic implications[END_REF][START_REF] Gameiro | Treatment-naïve HPV+ head and neck cancers display a T-cell-inflamed phenotype distinct from their HPVcounterparts that has implications for immunotherapy[END_REF]. In Table 1, we list a number of molecular markers based on DNA or RNA analyses that might be of interest for the stratification of HNSCC patients and the identification of those that are the most likely to respond to adjuvant PD1 treatment (Table 1) [START_REF] Straub | CD274/PD-L1 gene amplification and PD-L1 protein expression are common events in squamous cell carcinoma of the oral cavity[END_REF][START_REF] Ayers | IFN-γ-related mRNA profile predicts clinical response to PD-1 blockade[END_REF][START_REF] Cristescu | Pan-tumor genomic biomarkers for PD-1 checkpoint blockade-based immunotherapy[END_REF][START_REF] Goodman | Prevalence of PDL1 Amplification and Preliminary Response to Immune Checkpoint Blockade in Solid Tumors[END_REF][START_REF] Lecerf | Immune gene expression in head and neck squamous cell carcinoma patients[END_REF][START_REF] Mandal | Genetic diversity of tumors with mismatch repair deficiency influences anti-PD-1 immunotherapy response[END_REF]. A major progress of the past years lies in the interpretation of the composition of the tumor microenvironment (TME) from transcriptomic data [START_REF] Lau | RNA Sequencing of the Tumor Microenvironment in Precision Cancer Immunotherapy[END_REF]. The TME consists of different cell types whose symbiotic relationship determines the response of individual tumors to treatment [START_REF] Binnewies | Understanding the tumor immune microenvironment (TIME) for effective therapy[END_REF]. Analyzing tumor biology to predict the sensitivity of locally advanced tumors to ICB may lead to a more rational and standardized choice of treatment in the neoadjuvant setting. Combining information extracted from tumor genomics, such as the tumor mutational burden (TMB), the abundance of CD8-positive T cells and the expression levels of checkpoint molecules, seems to be the most promising predictive strategy [START_REF] Mandal | Genetic diversity of tumors with mismatch repair deficiency influences anti-PD-1 immunotherapy response[END_REF][START_REF] Lee | Multiomics Prediction of Response Rates to Therapies to Inhibit Programmed Cell Death 1 and Programmed Cell Death 1 Ligand 1[END_REF]. The list is not exhaustive and the ongoing studies addressing neoadjuvant immunotherapy in HNSCC offer interesting perspectives to identify new molecular markers, such as the analysis of the immune repertoire and the expansion of clones of tumor-resident T cells [START_REF] Lau | RNA Sequencing of the Tumor Microenvironment in Precision Cancer Immunotherapy[END_REF]. Interestingly, the information gained from TME analysis might not only be of interest to predict tumor response to ICB, but also to better understand the response of HNSCC to surgery itself. A number of experimental studies point to invasive surgical procedures as a source of systemic and local inflammation, potentially fostering tumor growth [START_REF] Horowitz | Exploiting the critical perioperative period to improve long-term cancer outcomes[END_REF][START_REF] Tohme | Surgery for Cancer: A Trigger for Metastases[END_REF][START_REF] Krall | The systemic response to surgery triggers the outgrowth of distant immune-controlled tumors in mouse models of dormancy[END_REF]. To the best of our knowledge, there is currently no study linking the TME and the immune activation status of HNSCC tumors to the outcome of oncological surgery. This is however an interesting possibility, that will be more easily addressed in the clinical trials that include molecular analyses to examine the TME. The neoadjuvant trials with ICB are therefore not only challenging some practical aspects of HNSCC surgery, but also offer new opportunities to better understand the biology of HNSCC in the perioperative period.

Adapting the follow up of cancer patients after surgery

In addition to refining the indications and the type of surgical procedure, the use of functional genomics holds great potential for the optimization of the follow up of HNSCC patients after surgery [START_REF] Budach | Novel prognostic clinical factors and biomarkers for outcome prediction in head and neck cancer: a systematic review[END_REF]. In Table 2, we list some of the molecular markers that could be helpful in the identification of tumors at risk of post-surgical recurrence, either analysing DNA [START_REF] Peng | Somatic copy number alterations detected by ultra-deep targeted sequencing predict prognosis in oral cavity squamous cell carcinoma[END_REF][START_REF] Lapke | Missense mutations in the TP53 DNAbinding domain predict outcomes in patients with advanced oral cavity squamous cell carcinoma[END_REF][START_REF] Liu | Somatic Mutations and Genetic Variants of NOTCH1 in Head and Neck Squamous Cell Carcinoma Occurrence and Development[END_REF][START_REF] Hedberg | Use of nonsteroidal anti-inflammatory drugs predicts improved patient survival for PIK3CA-altered head and neck cancer[END_REF] or RNA [START_REF] Wang | An eleven gene molecular signature for extra-capsular spread in oral squamous cell carcinoma serves as a prognosticator of outcome in patients without nodal metastases[END_REF][START_REF] Schmidt | Development and Validation of a Gene Signature for Patients with Head and Neck Carcinomas Treated by Postoperative Radio(chemo)therapy[END_REF][START_REF] Saidak | A gene expression profile associated with perineural invasion identifies a subset of HNSCC at risk of post-surgical recurrence[END_REF][START_REF] Hess | A Five-MicroRNA Signature Predicts Survival and Disease Control of Patients with Head and Neck Cancer Negative for HPV Infection[END_REF] (Table 2). In a recent study, we have addressed the issue of post-surgical recurrence of HNSCC by addressing the basic biology of perineural invasion (PNI) [START_REF] Saidak | A gene expression profile associated with perineural invasion identifies a subset of HNSCC at risk of post-surgical recurrence[END_REF]. Perineural growth and tumor extension along nerve tracts and small nerve fibers is often observed upon pathological examination of HNSCC [START_REF] Roh | Perineural growth in head and neck squamous cell carcinoma: a review[END_REF]. It is generally considered to be an aggressive hallmark of HNSCC, but the analysis of PNI relies on histology and poses difficulties in standardization. PNI results from active molecular mechanisms that lead to a cellular cross-talk between the malignant cells and nerve cells [START_REF] Saidak | Perineural invasion in head and neck squamous cell carcinoma: background, mechanisms, and prognostic implications[END_REF]. Based on gene expression analysis of data from TCGA, we established a PNI gene expression profile that was found to be distinct from the profiles associated with angiolymphatic invasion or ECS [START_REF] Saidak | A gene expression profile associated with perineural invasion identifies a subset of HNSCC at risk of post-surgical recurrence[END_REF]. In a multivariate analysis conducted on patients treated by primary surgery with tumors considered to be of low to intermediate risk of recurrence (i.e. those with neither ECS nor positive SM), the PNI gene expression profile turned out to be the main determinant associated with tumor recurrence and shorter overall survival [START_REF] Saidak | A gene expression profile associated with perineural invasion identifies a subset of HNSCC at risk of post-surgical recurrence[END_REF]. In contrast to the PNI gene expression profile, the histological diagnosis of PNI did not reach significance as a predictor of post-surgical recurrence [START_REF] Saidak | A gene expression profile associated with perineural invasion identifies a subset of HNSCC at risk of post-surgical recurrence[END_REF]. These findings suggest that tumor biology is a key determinant of recurrence after surgery, and suggest the contribution of PNI to post-surgical recurrence for HNSCC tumors in the absence of ECS or positive SM. In addition, the study highlights the fact that molecular genomic analyses could potentially outperform pathological diagnosis. Interestingly, another recent study provided evidence that analysing bulk tumor gene expression can predict post-surgical locoregional recurrence in patients with locally advanced HNSCC [START_REF] Schmidt | Development and Validation of a Gene Signature for Patients with Head and Neck Carcinomas Treated by Postoperative Radio(chemo)therapy[END_REF]. Using a selection of genes previously reported to be associated with radio(chemo)sensitivity and machine-learning algorithms, the authors designed a 7gene expression signature that predicted locoregional recurrence of HNSCC [START_REF] Schmidt | Development and Validation of a Gene Signature for Patients with Head and Neck Carcinomas Treated by Postoperative Radio(chemo)therapy[END_REF]. The conclusions of this important study, designed with an independent, multicentric validation cohort, support the applicability of bulk tumor RNA profiling to improve the prediction of tumor recurrence in locally advanced HNSCC [START_REF] Schmidt | Development and Validation of a Gene Signature for Patients with Head and Neck Carcinomas Treated by Postoperative Radio(chemo)therapy[END_REF]. Exploring ITH also illustrates the potential of sequencing for the discovery of biomarkers predictive of post-surgical recurrence, as was for example shown in another study based on TCGA [START_REF] Mroz | Intratumor heterogeneity could inform the use and type of postoperative adjuvant therapy in patients with head and neck squamous cell carcinoma[END_REF]. In this study, whole exome sequencing retrieved from surgical HNSCC were used to calculate a score reflecting mutant-allele tumor heterogeneity. The results of this interesting study, although still preliminary, point to the interest of sequencing as a source of new biomarkers that could independently predict which patients benefits the most from adjuvant radiotherapy / chemotherapy, and those at higher risk of post-surgical recurrence [START_REF] Mroz | Intratumor heterogeneity could inform the use and type of postoperative adjuvant therapy in patients with head and neck squamous cell carcinoma[END_REF]. Importantly, single-cell sequencing could further expand the information gained from bulk sequencing [START_REF] Qi | Single-cell sequencing and its applications in head and neck cancer[END_REF][START_REF] Yofei | single-cell genomic approaches for developing the next generation of immunotherapies[END_REF]. Single-cell sequencing of DNA / RNA offers a direct access to the ITH and the composition of the tumor microenvironment [START_REF] Qi | Single-cell sequencing and its applications in head and neck cancer[END_REF][START_REF] Yofei | single-cell genomic approaches for developing the next generation of immunotherapies[END_REF]. Compared to bulk DNA / RNA sequencing, single cell analyses pose a number of practical problems and their interpretation is challenging [START_REF] Qi | Single-cell sequencing and its applications in head and neck cancer[END_REF]. They nevertheless open the exciting perspectives of examining the clonal structure of malignant cells within a primary HNSCC tumor, eventually compared to metastatic / recurrent lesions. As briefly discussed earlier in this review, the single-cell approach promises to be useful for examining the role of phenotypic plasticity of cancer cells in the clinical setting (Cancer Stem cells, EMT) in HNSCC [START_REF] Qi | Single-cell sequencing and its applications in head and neck cancer[END_REF]. Interestingly, the prospect of analyzing single tumor cells will eventually be facilitated by the possibility of noninvasively recovering Circulating Tumor Cells (CTC), for example from the blood of HNSCC patients.

For further information on this rapidly evolving research topic, we refer the reader to recent reviews on single-cell analyses [START_REF] Qi | Single-cell sequencing and its applications in head and neck cancer[END_REF][START_REF] Yofei | single-cell genomic approaches for developing the next generation of immunotherapies[END_REF][START_REF] Payne | Circulating Tumour Cell Biomarkers in Head and Neck Cancer: Current Progress and Future Prospects[END_REF]. Tumor genomics therefore offers exciting perspectives for translational research on cancer cell stemness, the presence of immune or nerve cells within the TME, ITH, etc… and their contribution to post-surgical recurrence. Besides the interest of genomics as a research tool, sequencing also appears to be a source of new biomarkers that could be of prognostic use.

Analysing nucleic acids shed from tumors, and in particular circulating tumor DNA (ctDNA) is the focus of considerable interest in oral oncology. A detailed overview of the strategies used to detect and quantify ctDNA and their application for the treatment of solid tumors can be found in a recent review [START_REF] Corcoran | Application of Cell-free DNA Analysis to Cancer Treatment[END_REF]. Briefly, ctDNA consists of small DNA fragments <200 base pairs that are shed by malignant cells and that can be detected in biological fluids. Analyzing ctDNA offers the attractive possibility of performing « liquid biopies », i.e. to address the genomic landscape of most HNSCC in a non-invasive manner, even at an early, surgically curable stage [START_REF] Wang | Detection of somatic mutations and HPV in the saliva and plasma of patients with head and neck squamous cell carcinomas[END_REF] (Fig. 2). This analysis presents some technical challenges that are common to all solid tumors, such as the need to perform sensitive DNA sequencing analyses. In addition, HNSCC analyses pose specific problems, such as the design of an optimal gene panel, considering the variability of genomic alterations found in different HNSCC tumors [START_REF] Corcoran | Application of Cell-free DNA Analysis to Cancer Treatment[END_REF]. While ctDNA could potentially be of diagnostic and prognostic interest, its most evident application arguably resides in the follow-up of HNSCC treatment. The direct detection of HPV DNA, in the subset of patients with virally-induced HNSCC tumors, provides a demonstration of the applicability of ctDNA analysis to the follow up of HPV-associated oropharyngeal tumors [START_REF] Chera | Rapid Clearance Profile of Plasma Circulating Tumor HPV Type 16 DNA during Chemoradiotherapy Correlates with Disease Control in HPV-Associated Oropharyngeal Cancer[END_REF]. In a recent prospective trial based on digital PCR analysis for the detection of the most prevalent HPV strains in 103 patients treated by radio(chemo)therapy, Chera et al. detected ctHPVDNA in the plasma of almost 90% of HPV-related oropharyngeal tumors. A rapid clearance of ctHPVDNA (>95% by week 4 after radio(chemo)therapy) was associated with better local and regional disease control [START_REF] Chera | Rapid Clearance Profile of Plasma Circulating Tumor HPV Type 16 DNA during Chemoradiotherapy Correlates with Disease Control in HPV-Associated Oropharyngeal Cancer[END_REF]. This interesting study provides a proof of feasability that ctDNA brings clinically useful information for the post-surgical follow up of HNSCC. This molecular follow up could for example constitute the basis for a tailored therapeutic de-intensification [START_REF] Chera | Rapid Clearance Profile of Plasma Circulating Tumor HPV Type 16 DNA during Chemoradiotherapy Correlates with Disease Control in HPV-Associated Oropharyngeal Cancer[END_REF]. Besides HNSCC, a recent study exploring variations in the copy number of cell free-EBV in the plasma of patients with nasopharyngeal carcinoma illustrates the power of liquid biopsies to guide risk-adapted therapeutic adjustment in head and neck oncology [START_REF] Lv | Liquid biopsy tracking during sequential chemo-radiotherapy identifies distinct prognostic phenotypes in nasopharyngeal carcinoma[END_REF]. Further studies are required to explore the application of ctDNA to the follow-up of surgical patients and its extension to non HPV-related HNSCC [START_REF] Lauritano | Liquid biopsy in head and neck squamous cell carcinoma : prognostic significance of circulating tumour cells and circulating tumour DNA. A systematic review[END_REF].

Importantly, ctDNA may potentially serve as a reflection of tumor burden and its response to therapy, considering especially the fact that currently there are no satisfactory biological markers for the detection of residual cancer or progression of HNSCC after curative treatment. Besides this relatively straightforward application of ctDNA, other uses still need to be explored. For example, ctDNA sequencing could be of interest for the analysis of tumor mutational burden and the emergence of mutations associated with resistance to adjuvant / neoadjuvant therapies in HNSCC patients (Table 1 and2

).
Finally, genomic analysis of circulating nucleic acid material originating from HNSCC can also be extended to RNA, even though RNA is usually considered to be an unstable molecule. The past decade has witnessed the characterisation of an expanding number of RNA species, each with specific cellular functions [START_REF] Fang | Comprehensive analysis of competitive endogenous RNAs network associated with head and neck squamous cell carcinoma[END_REF]. Long non-coding RNA (lncRNA) and microRNA (miRNA) form an extensive RNA network whose contribution to the regulation of gene expression and carcinogenesis can be investigated with RNAseq [START_REF] Fang | Comprehensive analysis of competitive endogenous RNAs network associated with head and neck squamous cell carcinoma[END_REF]. Interestingly, miRNA can also be readily detected and analyzed in membrane encapsulated vesicles that are released into the extracellular space by cancer cells [START_REF] Xie | The role of extracellular vesicles from different origin in the microenvironment of head and neck cancers[END_REF]. Exosomal miRNA can for example be analyzed from the saliva of HNSCC patients [START_REF] Langevin | Comprehensive microRNA-sequencing of exosomes derived from head and neck carcinoma cells in vitro reveals common secretion profiles and potential utility as salivary biomarkers[END_REF].

Circular RNA (circRNA) is another type of promising clinical biomarkers. CircRNAs are the product of the splicing of a single mRNA and, because of their closed structure, are resistant to enzymatic degradation [START_REF] Slack | The Role of Non-coding RNAs in Oncology[END_REF]. This property might confer more stability to cirRNA in body fluids, making them potential cancer biomarkers in HNSCC [START_REF] Lu | RNA-Seq profiling of circular RNAs in human laryngeal squamous cell carcinomas[END_REF]. The practical validity of exploring different elements of the circulating transcriptome still needs to be demonstrated in the clinical context [START_REF] Sole | The circulating transcriptome as a source of cancer liquid biopsy biomarkers[END_REF].

Conclusion

The past decade has witnessed several technical improvements in the field of surgery of HNSCC, such as new mini-invasive surgical procedures that can potentially reduce the morbidity of surgery [START_REF] Cramer | The changing therapeutic landscape of head and neck cancer[END_REF][START_REF] Bozec | Current role of primary surgical treatment in patients with head and neck squamous cell carcinoma[END_REF] and improved perioperative care of HNSCC patients [START_REF] Dort | Optimal Perioperative Care in Major Head and Neck Cancer Surgery With Free Flap Reconstruction: A Consensus Review and Recommendations From the Enhanced Recovery After Surgery Society[END_REF][START_REF] Aeberhard | Effect of preoperative immunonutrition on postoperative short-term outcomes of patients with head and neck squamous cell carcinoma[END_REF]. While the number of available therapeutic options and surgical procedures is growing, there is an unmet need regarding the personalization of therapeutic approaches based on disease biology in HNSCC [START_REF] Cramer | The changing therapeutic landscape of head and neck cancer[END_REF]. Genomics has the major advantage of providing an integrative platform to study almost any aspect of tumor biology. The past years have shown how this single general approach can be instrumental in the transposition of the most fundamental knowledge on immune or nerve cells within the TME, cancer cell stemness, ITH to the surgical setting [START_REF] Mroz | Intratumor heterogeneity could inform the use and type of postoperative adjuvant therapy in patients with head and neck squamous cell carcinoma[END_REF][START_REF] Saidak | A gene expression profile associated with perineural invasion identifies a subset of HNSCC at risk of post-surgical recurrence[END_REF]. Genomics facilitates the conduct of systems biology studies without any biological a priori, and has therefore proven to be a powerful approach for surgical research [START_REF] Saidak | A three-gene expression signature associated with positive surgical margins in tongue squamous cell carcinomas: Predicting surgical resectability from tumour biology?[END_REF]. It is becoming increasingly clear that tumor biology at least partially determines the outcome of surgery and the post-surgical recurrence of HNSCC. Besides the interest of genomics as a research tool, sequencing will likely also be a source of new biomarkers that could be of prognostic use. Nucleic acid sequencing may «enhance» the pathological analysis of HNSCC by addressing new facets of tumor biology, by achieving a more objective and reliable tumor staging, and arguably because it offers perspectives of better standardization than the current routine pathological examinations. Of note, an important aim of future research should be to promote high quality translational research programs associated with large phase 3 trials. This might help in the personalization of cancer therapeutics and management of HNSCC patients.

While there are great differences between countries, regions, and even between cancer centers in their access to genomics analyses, the use of sequencing is bound to expand in the coming years [START_REF] Nangalia | Genome Sequencing during a Patient's Journey through Cancer[END_REF]. Sequencing already constitutes a part of the routine management of many cancer patients, for example in order to identify some of the simple genetic defects that can be exploited for therapeutic targeting [START_REF] Nangalia | Genome Sequencing during a Patient's Journey through Cancer[END_REF]. Great effort toward standardization of genomic protocols has already been initiated, but national/international quality-assurance programs still need to be installed, for example before RNA-seq data can be used as an element in clinical routine [START_REF] Nangalia | Genome Sequencing during a Patient's Journey through Cancer[END_REF]. The success of TCGA, an international consortium that juxtaposes molecular analyses performed in different cancer centres, represents one the first attempts to introduce a new molecular taxonomy for HNSCC, and it has turned out to be a milestone in HNSCC research [START_REF] Cancer | Comprehensive genomic characterization of head and neck squamous cell carcinomas[END_REF]. Precision surgery, i.e. biology-based surgery, has become a stimulating field of research for various tumors in addition to HNSCC [START_REF] Evrard | Parenchymal sparing surgery brings treatment of colorectal liver metastases into the precision medicine era[END_REF][START_REF] Willaert | Biology-Based Surgery: The Extent of Lymphadenectomy in Cancer of the Colon[END_REF]. The coming decade promises to bring more insights regarding the contribution of tumor biology to the outcome of HNSCC surgery and to identify new biomarkers that could help in the personalization of surgical care. highly specific facets of the immune response (expression of immune checkpoint regulatory molecules, tumor mutational burden and the immune repertoire for example). Tumor mutational burden (TMB), defined as the sum of non-synonymous mutations per megabase [START_REF] Cristescu | Pan-tumor genomic biomarkers for PD-1 checkpoint blockade-based immunotherapy[END_REF] in the whole exome, predicts optimal response to PD1 Mismatch repair deficiency / microsatellite instability and indels mutational load [START_REF] Mandal | Genetic diversity of tumors with mismatch repair deficiency influences anti-PD-1 immunotherapy response[END_REF] correlates with increased immune infiltration and cytolytic activity in HNSCC RNA

T cell inflammation gene expression profile

An 18 gene-expression signature predicts the response of HNSCC to anti-PD1, [START_REF] Ayers | IFN-γ-related mRNA profile predicts clinical response to PD-1 blockade[END_REF] associated with PD-L1 mRNA expression and TMB for an optimal prediction of PD1 response [START_REF] Cristescu | Pan-tumor genomic biomarkers for PD-1 checkpoint blockade-based immunotherapy[END_REF] Immune checkpoint gene expression analysis [START_REF] Lecerf | Immune gene expression in head and neck squamous cell carcinoma patients[END_REF] One third of HNSCC tumors coexpress PD1/PD-L1 with other potentially important immune checkpoint regulators (OX40/OX40L, CTLA4, TIGIT) [START_REF] Peng | Somatic copy number alterations detected by ultra-deep targeted sequencing predict prognosis in oral cavity squamous cell carcinoma[END_REF] in OSCC referred for radical tumor excision and neck dissection PIK3CA mutations + amplifications associated with longer OS in patients treated with [START_REF] Hedberg | Use of nonsteroidal anti-inflammatory drugs predicts improved patient survival for PIK3CA-altered head and neck cancer[END_REF] non-steroidal anti-inflammatory drugs (NSAID)

TP53 mutations [START_REF] Lapke | Missense mutations in the TP53 DNAbinding domain predict outcomes in patients with advanced oral cavity squamous cell carcinoma[END_REF] Analyzed with a scoring system (missense, stop, splice site, frameshift and inframe deletions)

Predictive of post-operative recurrence / overall survival in advanced OSCC NOTCH1 somatic mutations / genetic variants [START_REF] Liu | Somatic Mutations and Genetic Variants of NOTCH1 in Head and Neck Squamous Cell Carcinoma Occurrence and Development[END_REF] Confer a higher risk of recurrence / worse overall survival in HNSCC (odds ratio 4.5)

Intratumor heterogeneity, based on Mutant allele tumor heterogeneity (MATH) analysis [START_REF] Mroz | Intratumor heterogeneity could inform the use and type of postoperative adjuvant therapy in patients with head and neck squamous cell carcinoma[END_REF] Adjuvant Radiotherapy only increases the OS of HNSCC patients with high MATH scores RNA

ECS gene expression profile

An 11 gene-expression signature predicts a worse overall survival in node-negative oral SCC [START_REF] Wang | An eleven gene molecular signature for extra-capsular spread in oral squamous cell carcinoma serves as a prognosticator of outcome in patients without nodal metastases[END_REF]

PNI gene expression profile

A 26 gene-expression profile predicts recurrence and a worse overall survival in early stages of [START_REF] Saidak | A gene expression profile associated with perineural invasion identifies a subset of HNSCC at risk of post-surgical recurrence[END_REF] HNSCC (R0 SM and no ECS)

Stemness markers / hypoxia / tumor biology related gene expression profile

A 7 gene-expression signature (cancer stem cell markers, tumor hypoxia-associated genes, DNA repair, [START_REF] Schmidt | Development and Validation of a Gene Signature for Patients with Head and Neck Carcinomas Treated by Postoperative Radio(chemo)therapy[END_REF] cell-cycle regulation, epithelial-mesenchymal transition, proliferation or invasion) predicts postoperative recurrence for HPV-negative locally advanced HNSCC receiving radio(chemo)therapy microRNA A 5 microRNA signature predicts postoperative recurrence for HPV-negative locally advanced [START_REF] Hess | A Five-MicroRNA Signature Predicts Survival and Disease Control of Patients with Head and Neck Cancer Negative for HPV Infection[END_REF] HNSCC receiving radio(chemo)therapy.

Fig. 2 .

 2 Fig. 2. A possible frame for future practice and personalized surgery for patients with HNSCC.

Table 1 : A list of candidate molecular tumor markers for the stratification of HNSCC patients treated with ICB directed against PD1

 1

	Molecule analyzed	Biomarker and main findings

Table 2 : A list of potentially useful molecular tumor markers for the identification of HNSCC patients at risk of post-surgical recurrence

 2 Confer a higher risk of post-operative local recurrence (hazard ratio of 2.1 and 2.6, respectively)

	Molecule analyzed	Biomarker and main findings

This work was funded by La Ligue Nationale contre le Cancer, Comité du Pas de Calais and CHU Amiens. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Nucleic acid sequencing holds the potential for exploring many important parameters of tumor biology with a single approach. We present some of the biological parameters that can be explored with DNA/RNAseq and could become clinically-relevant for precision surgery of HNSCC. The exome, i.e. the protein-coding portion of the eukaryotic genome has received the most attention to date, although it only represents approximately 2% of the total cell DNA. Mutations or structural rearrangements are known to potentially alter the tumor cell proteome. Genomic mutations may also reveal the presence of a defect in the DNA repair machinery, for example in the nonhomologous end joining (NHEJ) or mismatch repair (MMR) pathways, potentially predicting the response of tumor cells to agents that damage DNA, such as radiotherapy. Some structural rearrangements, such as those that lead to gene fusions can also be found in a small percentage of solid tumours and offer potential therapeutic targets. Intra-tumor heterogeneity and the tumor clonal structure can be extrapolated, for example by measuring mutant-allele tumor heterogeneity. Nucleic acid sequencing also allows for the detection of viral integration and the identification of the corresponding genomic site. More generally, sequencing is well suited for exploring the presence of viruses and microorganisms within the tumor, i.e. the tumor virome and microbiome. Finally, sequencing offers the opportunity to gain some understanding of the different levels of regulation of gene expression: mRNA expression, differential splicing, micro-RNA (miRNA) and long non-coding RNA (lncRNA). This information can be further interpreted to examine the cellular composition of the tumor (endothelial cells, fibroblasts and immune cells), the activation status of the tumor immune microenvironment or