Human Papillomaviruses in Head and Neck Squamous cell carcinomas : the relevance of HPV16 variants

1 HUMAN PAPILLOMAVIRUSES IN HEAD AND NECK SQUAMOUS CELL CARCINOMAS THE RELEVANCE OF HPV16 VARIANTS DANIELA COCHICHO TAVARES DE SOUSA Tese para obtenção do grau de Doutor em Biomedicina Doutoramento em associação entre: Faculdade de Ciências Médicas | NOVA Medical School - Universidade NOVA de Lisboa Universidade de Aveiro Janeiro, 2023 2 HUMAN PAPILLOMAVIRUSES IN HEAD AND NECK SQUAMOUS CELL CARCINOMAS: THE RELEVANCE OF HPV16 VARIANTS Daniela Cochicho Tavares de Sousa Supervisor: Ana Félix MD PhD Faculdade de Ciências Médicas | NOVA Medical School da Universidade NOVA de Lisboa, Serviço de Anatomia Patológica do Instituto Português de Oncologia de Lisboa Francisco Gentil, (IPOLFG) Co-Supervisor: Rui M. Gil da Costa PhD LEPABE, Laboratory for Process Engineering, Environment, Biotechnology and Energy, Faculty of Engineering, University of Porto ALiCE - Associate Laboratory in Chemical Engineering, Faculty of Engineering, University of Porto Molecular Oncology and Viral Pathology Group, Research Center of IPO Porto (CIIPOP) / RISE@CI-IPOP (Health Research Network), Portuguese Oncology Institute of Porto (IPO Porto), Porto Comprehensive Cancer Center (Porto.CCC) Post-graduate Programme in Adult Health (PPGSAD), University Hospital (HUUFMA) and Morphology Department, Federal University of Maranhão, Brazil TESE PARA OBTENÇÃO DO GRAU DE DOUTOR NA ESPECIALIDADE EM BIOMEDICINA Janeiro, 2023 3 HOST INSTITUTIONS 4 FINANCIAL SUPPORT This research was not directly financed but some experimental tasks and personal included in some articles were part of projects with funds and the cost were partially covered, and for that we briefly mention here: • Fundação para a Ciência e a Tecnologia (FCT) para a Ciência e Tecnologia/Ministério da Ciência, Tecnologia e Ensino Superior (FCT/MCTES, Portugal) through national funds to iNOVA4Health. (UIDB/04462/2020 and UIDP/04462/2020) – IPO Lisboa • GenomePT project supported by COMPETE 2020 Operational Programme for Competitiveness and Internationalisation (POCI), Lisboa Portugal Regional Operational Programme (Lisboa2020), Algarve Portugal Regional Operational Programme (CRESC Algarve2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF) and by Fundação para a Ciência e a Tecnologia (FCT). POCI-01-0145-FEDER-022184) - INSA • FEDER funds through the Programa Operacional Factores de Competitividade – COMPETE and by National funds through the Fundação para a Ciência e a Tecnologia within the scope of the project Centre for Toxicogenomics and Human Health -ToxOmics). (UID/BIM/00009/2019) – INSA • National funds through FCT/MCTES (PIDDAC) to 2SMART (NORTE-01-0145FEDER-000054) supported by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF). (LA/P/0045/2020 ALICE) (UIDB/00511/2020 and UIDP/00511/2020 LEPABE) – IPO Porto 5 LIST OF SCIENTIFIC PUBLICATIONS These are the main scientific articles published by the author whose content was totally or partially used in the elaboration of the thesis: I. Detecção e Genotipagem do HPV em esfregaços e biópsias buco‐ faringeos. Daniela Cochicho, Luís Martins, Pedro Montalvão, Lígia Ferreira, Luís Oliveira, Rui Fino, Hugo Estibeiro, Ricardo Pacheco, Ana Hebe, Mário Cunha, Miguel Magalhães. Revista Portuguesa de Otorrinolaringologia e Cirurgia Cervicofacial, vol.55, nº3 Setembro 2017.https://www.journalsporl.com/index.php/sporl/article/view/414 The author contributed to the study's idea and design, performed all laboratory technical procedures. Performed data collection and compilation. Directly contribute to analysing and interpreting data. Prepare all the writing, editing, and rewriting the final manuscript. II. Exploring the roles of HPV16 variants in head and neck squamous cell carcinoma: Current challenges and opportunities. Daniela Cochicho; Rui Gil da Costa; Ana Félix. Virol J 18, 217 (2021). https://doi.org/10.1186/s12985-021-01688-9 The author contributed to the study´s concepts and study design, including the literature search using three databases (meta-analysis), the data collection, analysis, and interpretation. Also conducted the preparation of the manuscript, in its editing and reviewed entirely. III. Distribution and clinical significance of HPV16 Variants in Head and Neck Squamous Cell Carcinomas: Data from a Portuguese cohort and systematic review. Daniela Cochicho, Alexandra Nunes, João Paulo, Susana Esteves, Joana Mendonça, Luís Vieira, Luís Martins, Mário Cunha, Pedro Montalvão, Miguel Magalhães, Rui Gil da Costa, Ana Félix. Pathobiology - DOI: 10.1159/000529723 The author contributed to the study´s concepts and study design, 6 including the systematic review of the literature using two databases. Also performed the data collection, analysis, and interpretation. Design and developed the entire NGS workflow, directly contribute to the analysing and interpreting of the molecular data. Conducted the preparation and editing the final manuscript. IV. Characterization of the Human papillomavirus 16 oncogenes in K14HPV16 mice: Sublineage A1 drives multi-organ carcinogenesis. Daniela Cochicho, Alexandra Nunes, João Paulo Gomes, Luís Martins, Mário Cunha, Beatriz Medeiros-Fonseca, Paula Oliveira, Margarida Bastos, Rui Medeiros, Joana Mendonça, Luís Vieira, Rui M. Gil da Costa, Ana Félix. Int. J. Mol. Sci. 2022, 23, 12371. https://doi.org/10.3390/ijms232012371 The author contributed on the study's design, performed all laboratory technical procedures for DNA HPV molecular analysis. Performed data collection and compilation. Directly contribute to molecular analysing and interpreting data. Prepare all the writing, editing, and rewriting the final manuscript. V. PIK3CA Gene Mutations in HNSCC: Systematic Review and Correlations with HPV Status and Patient Survival. Cochicho D, Esteves S, Rito M, Silva F, Martins L, Montalvão P, Cunha M, Magalhães M, Gil da Costa RM, Félix A. Cancers. 2022; 14(5):1286. https://doi.org/10.3390/cancers14051286 The author contributed to the study´s concepts and study design, including the literature search using three databases (meta-analysis), the data collection, analysis, and interpretation. Performed all laboratory technical optimization and procedures. Conducted the preparation of the manuscript, in its editing and reviewed entirely. 7 Fulfilling the Portuguese Law, the Candidate declares to have actively participated in the collection and study of all material and data included in this Thesis, having written all papers. This Thesis and the all the work included in it have been approved by the: • Ethics Research Committee of NOVA Medical School (nº 70/2019/CEFCM) • Scientific board of Instituto Português de Oncologia de Lisboa and the Ethics Committee (UIC/1168) • The animal model experiments were performed and approved by the University of Trás-os-Montes and Alto Douro Ethics Committee (Approval Number 10/2013) and by the Portuguese Veterinary Directorate (Approval Number 0421000/000/2014). 8 9 TABLE OF CONTENTS THESIS OUTLINE ........................................................................................................................................ 14 SUMÁRIO ...................................................................................................................................................... 15 ABSTRACT .................................................................................................................................................... 17 ACRONYMS AND ABBREVIATIONS ..................................................................................................... 19 FOREWORD ................................................................................................................................................ 21 CHAPTER 1 ........................................................................................................................................................ 22 Background ..................................................................................................................................................... 22 1. Human Papillomaviruses .................................................................................................................... 23 1.1 Structure and regulation of the viral genome ........................................................................ 23 1.2 Infection and Replication cycle ................................................................................................... 24 1.3 Carcinogenesis ................................................................................................................................. 28 1.4 Classification ..................................................................................................................................... 30 1.5 HPV Variants ...................................................................................................................................... 32 2. Head and Neck Squamous cell carcinoma .................................................................................. 37 2.1 Epidemiology .................................................................................................................................... 37 2.2 Risk factors ........................................................................................................................................ 38 2.3 Clinical-pathological characterization ..................................................................................... 39 2.4 Molecular characterization .......................................................................................................... 40 2.4.1 Non-HPV associated HNSCC ............................................................................................... 42 2.4.2 HPV associated HNSCC ......................................................................................................... 43 2.5 HNSCC carcinogenesis, HPV infection and PI3K/AKT/mTOR pathway ........................ 45 2.5 Treatment .......................................................................................................................................... 49 3. K14HPV16 Animal Model ..................................................................................................................... 51 CHAPTER 2 ....................................................................................................................................................... 54 Aim .................................................................................................................................................................. 55 General hypothesis ................................................................................................................................... 55 Specific objectives ..................................................................................................................................... 55 CHAPTER 3 ....................................................................................................................................................... 56 16 No estudo exploratório das respectivas linhagens não foi possível estabelecer nenhuma associação de confiança com as variáveis clinico-patológicas, incluindo as variáveis de sobrevivência, devido ao reduzido número de casos incluídos na análise (n=35). Na análise comparativa do genoma dos 35 pacientes foram reportadas 243 variações nucleotídicas, 84 das quais a alteração resulta num aminoácido diferente. O perfil mutacional apresentado foi concordante com o previamente descrito, em 41% (100/243) das variações nucleotídicas foram publicadas por diferentes autores na patologia do colo e/ou na patologia região de cabeça-pescoço. As restantes SNV contribuem com novos dados em diferentes genes do genoma viral, incluindo os genes que até à data se encontram menos explorados, nomeadamente o gene E4, E5 e L2. Da análise exploratória do significado biológico o total de 23% (23/100) variantes foram descritas, a maioria já estudada no carcinoma do colo do útero. No geral, os nossos resultados revelam existir uma maior variabilidade, precisamente nos genes E5 e L2, que não estavam tão estudados, e confirmaram que o gene mais conservado é o E7, o que está de acordo com o reportado previamente por outros autores. Os nossos resultados no estudo da frequência de mutações do gene PIK3CA sustentam a elevada ocorrência destas mutações nos tumores de cabeça-pescoço. Na nossa coorte identificamos a presença de pelo menos uma mutação no gene PIK3CA em 39% dos pacientes incluídos e a mutação E545D foi a mais frequentemente detectada. A análise do genoma do modelo murino que efetuámos permitiu pela primeira vez identificar qual a é linhagem do HPV16 presente neste modelo que é a sublinhagem A1 (Europeia). Este dado novo é muito interessante porque esta é a mais prevalente em doentes e que reforça a hipótese de que esta é uma linhagem com elevado potencial oncogénico na orofaringe, tornando este modelo ainda mais interessante. Globalmente, os nossos dados corroboram os previamente reportados por outros grupos, destacando a importância da análise molecular para melhor compreender a distribuição das variantes do HPV16 e das mutações do PIK3CA e a sua possível influência na carcinogénese, diagnóstico e abordagem clínica no cancro de cabeça e pescoço. PALAVRAS-CHAVE: CARCINOMA, OROFARINGE, HPV16 VARIANTES, PIK3CA, GENOMA, NGS 17 ABSTRACT Head and Neck Carcinoma is considered the sixth most common cancer worldwide and is responsible for 450,000 deaths/year. These tumours appear in various locations of the upper aero-digestive tract. Most of these neoplasms originate in the squamous epithelium and correspond to 90 to 95% of all squamous cell carcinomas of the head and neck region. Despite their common origin in the squamous epithelium, tumours from different locations within the head and neck region show high heterogeneous phenotypic, etiological, and biological characteristics. The registered risk factors are tobacco and alcohol consumption, described as the most common causes for the appearance of these tumours, and Human Papillomavirus (HPV) infection as a risk factor associated with tumours that arise in the oropharynx. The developed project aimed to obtain information about some specific aspects of squamous cell carcinoma of the head and neck region through a detailed molecular analysis of a case series. The study design was developed in distinct and structured tasks to: 1obtain data related to clinical and pathological variables, 2identify the different HPV16 variants present and 3determine the frequency of PIK3CA gene mutations. The cohort included in the study was selected from a universe of patients diagnosed with squamous cell carcinoma of the head and neck region at the Instituto Português de Oncologia Francisco Gentil de Lisboa IPOLFG, being composed mostly of male patients with oropharyngeal cancer, HPV-positive and with active consumption habits. Additionally, a series of samples from a murine model of oropharyngeal carcinogenesis induced by HPV16 was studied to identify the virus variant present in this experimental model. The sequencing of the complete genome of HPV16 was performed using Next Generation Sequencing methodology, developed from previously described primers, which were adapted to optimize a laboratory workflow that allows the identification of HPV16 variants from multiple tissues. Finally, the frequency of the PIK3CA mutation strongly associated with squamous cell carcinoma of the head and neck region was calculated based on the detection of the presence of four different mutations (E545D, E545K, E542K and H1047L) by real-time PCR methodology. The main data obtained express the clinical characterization of the pathology in the series of patients, describe the different HPV16 strains identified in the total 18 cohort of patients, as well as the mutational profile for each HPV16 gene. Phylogenetic analysis shows lineage A as the most prevalent in our cohort. In the exploratory study of the respective strains, it was not possible to establish any reliable association with clinicopathological variables, including survival variables, due to the small number of cases included in the analysis (n=35). Regarding the genomic comparative analysis of the 35 patients, 243 nucleotide variations were reported, 84 of which integrate an amino acid alteration. The mutational profile agrees with that previously described; a total of 41% (100/243) single nucleotide variation (SNV) were published by other authors based on cervix pathology and/or in the pathology of the head-neck region. The remaining SNV that were not described in the systematic review contribute with new data in different genes of the viral genome, including those genes that until now are less explored, such as the E4, E5 and L2 gene. From the exploratory analysis of biological significance, a total of 23 variants were described, the majority studied in cervical carcinoma. Overall, the greatest variability was observed in the E5 and L2 genes that were not previously studied. Oppositely, the most conserved gene was E7, as previously reported by other authors. The frequency of PIK3CA gene mutations found in our cohort support the high occurrence of these mutations among head and neck tumours. In our cohort, we identified the presence of at least one mutation in the PIK3CA gene in 39% of the patients included, and the E545D mutation was the most frequently detected. It should also be noted that the data from the analysis of the murine model genome allowed for the first time to identify the HPV16 lineage present as A1 (European) sublineage, which was the most prevalent in patients, supporting the hypothesis that this is a lineage with high oncogenic potential in the oropharynx and making this model even more interesting. Overall, our data corroborate those previously reported by other groups, highlighting the importance of molecular analysis to better understand the distribution of HPV16 variants and PIK3CA mutations and their potential influence on carcinogenesis, diagnosis and clinical approach in head and neck cancer. KEYWORDS: HNSCC, HPV16 VARIANTS, PIK3CA, WHOLE-GENOME, NGS, SNVS 19 ACRONYMS AND ABBREVIATIONS AA - Asia-American HPV16 Variant AF - African HPV16 Variant AJCC - American Joint Committee on Cancer AS - Asian HPV16 Variant A260Absorbance at 260 nm A280 - Absorbance at 280 nm bp - Base pair cDNA - Complementary DNA CDKN2A - Cyclin-dependent kinase inhibitor 2A CT - Chemotherapy CRT - Chemoradiotherapy DFS - Disease-free survival DNA - Deoxyribonucleic acid E - European HPV16 variant E HPV - Early protein EU - European Union FFPE - Formalin-fixed paraffin-embedded H&E - Hematoxylin and eosin staining HN – Head and Neck HNSCC - Head and Neck Squamous cell carcinoma HPV - Human Papillomavirus HNSCC-HPV - Head and Neck Squamous cell carcinoma associated to Human Papillomavirus HNSCC-nonHPV - Head and Neck Squamous cell carcinoma without Human Papillomavirus association IARC - International Agency for Research on cancer ICTV - International Committee on Taxonomy of Viruses ISO - International Organization for Standardization K14HPV16 - Early HPV16 genes under the control of the cytokeratin 14 LCR - Long control Region MUT - Mutated mRNA - Messenger RNA NA - North American HPV16 Variant 20 NGS - Next-generation sequencing NTC – No Target Control O.C.T. - Compound Optimal cutting temperature compound OD - Odds ratio ORF - Open reading frame OS - Overall survival PCR - Polymerase chain reaction PIK3CA - Phosphatidylinositol-4,5-biphosphate 3-kinase PI3K - Phosphoinositide 3-kinases PI3-K-AKT - Phosphatidylinositol-3-kinase-AK PIP2 - Phosphatidylinositol-(4,5)-bisphosphate PIP3 - Phosphatidylinositol-(3,4,5)-trisphosphate PRB - Retinoblastoma protein PTEN - Phosphatase and tensing homolog PV - Papilloma virus P16 - p16INK4a protein, cyclin-dependent kinase inhibitor 2A, or CDKN2A P53 – Tumour suppressor protein 53 qPCR - Real-time PCR RNA - Ribonucleic acid RT – Radiotherapy SR – Systematic review TNM - Tumour, lymph nodes, metastasis TCGA - Tumour Cancer Genome Atlas UICC - Union for International Cancer Control's USA - United States of America WT - Wild type WHO - World health organization 21 FOREWORD Head and neck carcinoma is the sixth leading cancer by incidence worldwide and is responsible for more than 800,000 new cases yearly. HPV is present in approximately 35% of HNSCC, emerges in the lingual and palatine tonsils, and HPV16 is the most detected type. The epidemiological, etiological, and molecular data suggest that intra-type HPV variants are biologically distinct and may be associated with different risks in uterine cervical cancer progression. Nonetheless, HPV16 variants may also play an important role in head and neck carcinogenesis, but few have established their relevance. The genomic profile of HNSCC published by the Cancer Genome Atlas in 2015 broadened the development of mutation studies and highlighted a high frequency of changes. The identification of biomarkers to predict response to therapy will allow the selection of patients more likely to respond to new therapeutic combinations making a valuable contribution to clinical treatment strategies. Essentially, it is critical to refine the mutational profile of HPV-positive and HPV-negative within HNSCC patients. This project addresses a deep molecular analysis of a Portuguese cohort of HNSCC patients and explores its correlation with clinicopathological variables and their potential contribution to clinical practice. 22 CHAPTER 1 BACKGROUND 23 1. HUMAN PAPILLOMAVIRUSES The Papilloma Virus (PV) a member of the Papillomaviridae family (De Villiers et al., 2004), are ubiquitous viruses that infect the epidermis and mucosa of several mammals, including humans. PV infections are usually transient and asymptomatic, with no relevant clinical manifestations. However, they can cause benign tumours (warts, papilloma) that usually regress or establish persistent infections that can lead to tumours (zur Hausen, 1996). There are over 300 distinct types of PVs isolated. About 200 can infect humans – human papillomavirus (HPV), where 40 types infect the anogenital region (World Health Organization, 2010). Only a small group is associated with the development of neoplastic pathologies. Although the wide number of HPV types isolated and tropism differences, they all share the same genome arrangement (Bernard, Calleja-Macias and Dunn, 2006). 1.1 STRUCTURE AND REGULATION OF THE VIRAL GENOME The virus particle is small (55nm-diameter), non-enveloped, suitable only for a protein capsid of icosahedral symmetry that encloses the viral DNA genome (Münger et al., 2004). The viral DNA, with a length of 7,906 base pairs (bp), is circular, double-stranded and structured into three functional regions: Early Region, Late Region, and Long Control Region. These three regions are separated by two polyadenylation sites (pA) as shown in Figure 1 (Zheng and Baker, 2006). The genetic information is distributed among the eight coding sequences for proteins classified as early (E) and late (L) according to their temporal expression. The Early region occupies more than 50% of the virus genome and encodes six open reading frames (ORFs): E1, E2, E4, E5, E6 and E7. The late region contains the L1 and L2 ORFs that encode two structural proteins. In addition, there is a long control region (LCR) situated between the E6 and L1 genes, which does not contain ORFs but includes the origin of replication (ori) as well as multiple elements (enhancers) required for transcription (Münger et al., 2004). 24 Figure 1. HPV16 complete genome representation adapted from (Zheng and Baker, 2006). The coloured line illustrates the linear form of the virus genome (≈8000 pb), presenting the three functional regions LCR (blue), Early (red) and Late (grey); the promoters P (arrows) and polyadenylation sites (pA); the early (AE) and late (AL). Above the line, white boxes illustrate the ORFs for each coding region (red and grey): the Early E6, E7, E1, E2, E4 and E5 genes; and the late genes L2 and L1. The numbers above represent the first nucleotide in the start codon and the last nucleotide position in the stop codon for each ORF. The transcription of the genome occurs from a single strand of DNA considered to be polycistronic, with multiple introns that undergo extensive alternative RNA splicing producing multiple RNAs with several open reading frames. The transcription of the Early and Late genes occurs unidirectionally from primers and promoters. It is initiated at p97 promoter located upstream of the ORF of the E6 gene and is responsible for almost all early gene expression (Smotkin and Wettstein, 1986). Next is followed by the p670 promoter located within the ORF of the E7 gene, which is responsible for the transcription of late genes. This mechanism gives to HPV the ability to express different proteins at a given time and in the differentiation stage of the infected epithelium (Zheng and Baker, 2006). It also ensures the smooth progression of key events for the virus, such as the replication cycle, along with immune evasion. 1.2 INFECTION AND REPLICATION CYCLE HPV accesses to the basal layer through micro abrasions in the epithelium and binds to host cells using cell surface molecules (Shafti-Keramat et al., 2003; Patterson, Smith and Ozbun, 2005). The first events are induced by the interaction between the L1 structural protein and the cell surface receptor (heparin sulphate 25 proteoglycan) (Giroglou et al., 2001). Followed by the interaction of the L2 protein with the cell surface recruiting a second receptor that allows the entrance of the virus (Kawana et al., 1999), which as a non-enveloped particle, adopts mechanisms through clathrinid-mediated endocytosis (Day, Lowy and Schiller, 2003). Once inside the cell, the viral particle is transported to the nucleus, not before losing the capsid first. This process allows the uncoating of the circular genome protected in the endosome (Merle et al., 1999; Nelson, Rose and Moroianu, 2002) and is mediated by the L2 protein, which can disrupt the endosome membrane, and the viral genome is released into the cytosol. Then the viral genome (8000 bp) diffuses through the cytosol, mediated thereby L2 protein, which interacts with both actin and tubulin (Yang et al., 2003). Upon reaching the nucleus, the viral DNA enters through the nuclear pores. The PVs, as DNA viruses, replicate and are assembled exclusively in the nucleus. In tissues, the replication begins in undifferentiated cells, located in the basement layer, involving host cell factors that interact with the LCR region and thus initiate the transcription of viral genes (Burd, 2003). The first proteins to be expressed are E1 and E2, necessary for viral DNA replication and to establish genomes with low copy numbers of 20-100 copies per cell (Ruesch, Stubenrauch and Laimins, 1998; Ozbun, 2002). The viral genomes replicate simultaneously with the replication of cellular DNA (You et al., 2004). The infected cell, when it divides, originates new cells that remain in the basal layer, thus serving as a reservoir for the virus. Just the same these infected cells proceed with expression and replication in a tightly controlled and regulated manner by keratinocyte differentiation (zur Hausen, 1996). The complete viral genome encodes only 8 to 10 viral proteins, which require host cell factors to regulate viral transcription and replication. The E1 and E2 proteins accompany this process, by recruiting the cellular machinery involved in DNA replication. The E1 protein is an ATP-dependent helicase which opens the replication origin, allowing viral genome replication to begin (Wilson et al., 2002), while E2 interact as a transcription factor through its functions to regulate the expression of early ORF promoters (Ham et al., 1991). With the epithelial differentiation, cells stop dividing and the differentiated keratinocytes do not express the crucial factors involved in DNA replication. The virus solves this problem by using viral oncoproteins E6 and E7 that act on the regulation of the cell cycle progress and blockage of apoptosis of those cells that enter the cell cycle 32 Figure 4. Comparison of Epidemiological and Phylogenetic classification. The colour diagram presents the Taxonomy by phylogenetic classification and epidemiological classification, with the distribution of the HPV Types into high- (red) and low-risk (green) groups. Above all, except (yellow) HPV 70 and HPV73, observe good agreement between the two classifications. This correlation is helpful to predict whether a new genotype may be of a high-risk sequence (Muñoz et al., 2003). 1.5 HPV VARIANTS There is also clear evidence of a wide sequence diversity within each HPV type (De Villiers et al., 2004; Burk, Harari and Chen, 2013), where the genome sequencing revealed intra-type variants with genetic differences ranging between 0.5 and 1% (Ho et al., 1991; Mirabello et al., 2016). HPVs, being a double-stranded DNA virus, uses the efficient revision of the host's DNA polymerase for its replication, which avoids high mutation rates; therefore, changes in HPV genomes are acquired slowly, the random mutations that eventually occur within viral types generate the nucleotide polymorphisms contained in intratype variants (Gillison et al., 2000; Muñoz et al., 2003). Initially, intratype variants were essentially studied in HPV16 and HPV18 and were named according to their geographical distribution depending on the origin and ethnicity of the infected population (Muñoz et al., 2003). Thus, six distinct phylogenetic clusters were defined for HPV16: European (E), Asian (As), AsianAmerican (AA), African 1 (Af1), African 2 (Af2) (Bernard et al., 1993; Ho et al., 1993; 33 Yamada et al., 1995) and North American 1 (NA1) (Yamada et al., 1997; Burk, Harari and Chen, 2013; Jackson et al., 2016). Posteriorly, it was proposed a more precise nomenclature, establishing lineages and sub-lineages (Burk, Harari and Chen, 2013) as shown in Figure 5. Regarding HPV16 genotype, whole-genome analysis allowed the characterization of four main variant lineages (A, B, C, D) and sixteen sublineages (A1, A2, A3, A4, B1, B2, B3, B4, C1, C2, C3, C4, D1, D2, D3, D4). According to these findings, lineage A grouped European cluster with sublineage A1, A2 and A3, and Asiatic cluster with sublineage A4. The lineage B encloses the African (Af1) cluster with sublineage B1 and the Lineage C encloses the African (Af2) cluster with appropriate sublineage (C1 to C4); Lineage D encloses the American cluster with sublineage D1, to North American cluster sublineage D2 and to Asian-American cluster sublineage D3 (Burk, Harari and Chen, 2013). 34 Figure 5. Phylogenetic tree from HPV16 intra-type variants. The phylogeny tree from (Burk, Harari and Chen, 2013; Mirabello et al., 2018) illustrates human papillomavirus 16 (HPV16) lineages (A-D) and sublineage (A1-4, B1-4, C1-4, D1-4) relationships, highlighted on different colours, indicating the main lineage branches. 35 The emerging epidemiological, etiological, and molecular data suggest that the intra-type variants of HPV are biologically distinct and may be associated with different risk of cancer progression. As shown by in vitro studies using 3D organotypic epithelial cell cultures, the HPV16 E6 variants differ in their ability to abolish keratinocyte differentiation and to induce p53 degradation (Hoffmann et al., 2004; Agrawal et al., 2008; Cancer Genome Atlas Network, 2015). Therefore, it was postulated that the occurrence of nucleotide variations between intratype HPV variants reflects in their functional differences and pathogenicity (De la CruzHernández et al., 2005; Hochmann et al., 2016; Zhao et al., 2020; Hadami et al., 2021). In addition, several isolates of intratype variants of HPV, previously described by different clinical studies from cohorts of carcinoma of the cervix, anal canal, oropharynx and oral cavity, contribute to a careful classification of variants of the most frequent HPV types in cancer (Bernard et al., 2010; Burk, Harari and Chen, 2013; Chen et al., 2015). The HPV16 T350G (L83V) variant is the most frequently found among invasive cervical cancers (Hassani et al., 2015; Betiol et al., 2016) and has been associated with an increased oncogenic potential than the prototype (Pakdel et al., 2021), possibly by facilitating persistent viral infection (Boscolo-Rizzo et al., 2009; Joseph et al., 2013; Zhang et al., 2015). Following this, several studies have reported that intra-type variants of HPV16 and HPV18 alter host cells in a differently way, which translates in different risks of persistence and progression to cancer (Yamada et al., 1995; Sichero et al., 2007; Cornet et al., 2013). Further functional studies explored differences in biological behaviour concerning the clinical evolution of patients with HPV-related cancers. Decoding the impact that these variations may be important to know the impact in the patient's prognosis and in the development of therapeutic strategies. All these variants’ impact were established for cervical cancer as the focus of most studies, compared to studies performed on other HPV-induced pathologies. In fact, in HNSCC cancer, what is known about distribution HPV16 variants resemble the knowledge acquired in cervical cancer. Indeed, authors described both share common biological features. A presenting data from a cohort study that compares genomic characteristics of HPV associated with cervical versus oropharyngeal tumours using DNA sequence analysis showed no significant differences between distribution in HPV16 variants (LeConte et al., 2018) where both presenting major prevalence of the European variant. 36 Unlike the HPV E6 gene amplified from oropharyngeal samples reported over more nonsynonymous mutations, also for the E7 gene, no differences were found in mutation rates between the two anatomical locations (LeConte et al., 2018; Zhu et al., 2020). Notably, the E7 gene is conserved in both locations corroborating that the apparent restriction on E7 mutations seems to be present in the oropharynx, as well. Otherwise it is still poorly understood the potential contribution of those specific variants in cancer risk increasing, or prognosis (Gillison et al., 2000) or the clinicopathological relevance of the identified variants (Hoffmann et al., 2004; Badaracco et al., 2007). The main reason behind this lack of conclusions in this field of knowledge may be the relatively low number of studies performed and the small size of the HNSCC HPV-associated cohorts (Hassani et al., 2015). Therefore, recently this picture is starting to change, and some more studies on HPV16 variants have explored the HN region carcinomas. In 2022, a large study was published using HNSCC cases from two tertiary cancer centres. The study performed a NGS analysis of nearly 400 patients and found that specific variations have different clinical implications, namely eight HPV16 SNPs were significantly associated with worse survival (E1 gene position 1053, L2 gene positions 4410, 4539, 5050, 5254, L1 gene positions 5962, 6025, and 7173 in the upstream regulatory region) in a US population (Lang Kuhs et al., 2022). Although there is still data limitation, crucial steps have been achieved, and hopefully new studies with larger cohorts of patients and other HPV-associated tumours, leveraged due to the introduction of NGS techniques that have optimized workflows that faster and more robust sequencing results (Bernard et al., 1993; Yamada et al., 1995; De la Cruz-Hernández et al., 2005). 37 2. HEAD AND NECK SQUAMOUS CELL CARCINOMA Head and neck squamous cell carcinoma (HNSCC) are a group of tumours located in several distinct structures in the oral cavity, pharynx, and larynx subsites of the upper aerodigestive tract (Figure 6). Figure 6. Illustration of the Head and Neck anatomic region adapted from (Johnson et al., 2020). The cross-section (left) and front section (right) classification by each sublocation. 2.1 EPIDEMIOLOGY Worldwide HNSCC is considered to be the sixth leading cancer regarding incidence and estimated to have an impact of ~890,000 new patients and responsible for 450,000 deaths/year (Global cancer Observatory, 2021). The incidences are rising and expected to continue to increase over the next decade (Global cancer Observatory, 2021). The age at diagnosis usually ranges between 50 and 70 years of age, and men are significantly more likely to develop the disease than women (up to a 4:1 proportion) (Bray et al., 2018). Although this disease burden rate varies across countries, over the past decade, several epidemiologic studies reported higher prevalence in regions such as Southeast Asia and Australia, the majority associated with specific carcinogen substances such as chronic 38 consumption of alcohol and tobacco (Mork et al., 2010; Chaturvedi et al., 2013; Hwang et al., 2015; McDermott and Bowles, 2019). Contrasting to this, the increase trending incidence, in high-income countries such as the USA and Western Europe, of HNSCC is associated with oropharyngeal HPV related cancers (Chaturvedi et al., 2011; Centers for Disease Control and Prevention, 2018). In Portugal, according to data from the National Cancer Registry, the oral cavity and pharynx are considered to be the seventh most diagnosed malignant tumour (Registo Oncológico Regional do Norte, 2010; Miranda et al., 2018). Every year approximately 3,000 new cancer cases in the aerodigestive tract (Miranda et al., 2018) are diagnosed, but the mortality rate from these malignant tumours is low compared to the EU average. In conclusion, this is a relatively common human cancer, characterized by high morbidity, high mortality, and few therapeutic options, outside of standard schemes that may include surgery, chemotherapy, and radiation are available. 2.2 RISK FACTORS The wide geographic variability in the incidence of these tumours reflects the prevalence of certain risk factors in specific regions (Johnson et al., 2020). In addition, HNSCC can also occur in young patients without association with known risk factors (Rothenberg and Ellisen, 2012). In general, the most represented risk factors are the tobacco use and alcohol consumption, as well as infection with oncogenic virus such the HPV (Katabi and Lewis, 2017). The association established between risk factors and Head and Neck subsites reported in epidemiological studies, have linked the oropharynx tumours to a previous infection with human papillomavirus (HPV) and the other HNSCC subsites, which majority are a consequence of the harmful effects of smoking and alcohol consumption and collectively referred to as HNSCC-nonHPV (Katabi and Lewis, 2017). In these two groups (HPV and nonHPV) positive HNSCC, genetic and prognostic differences (Hwang et al., 2015) reflect the critical role of the molecular profile triggered by each risk factors that contribute to its carcinogenesis (Mork et al., 2010). Currently, these two groups are recognised as major carcinogenic pathways leading to HNSCC (Katabi and Lewis, 2017). 39 2.3 CLINICAL-PATHOLOGICAL CHARACTERIZATION Head and neck cancer comprises very different anatomical areas and involves wide histological tissues. However, most of these tumours originate from the squamous epithelium, corresponding to 90 to 95% of all lesions (Katabi and Lewis, 2017). This cancer is characterized by its multifocal development, coupled with slow and silent growth. Despite evidence of histological progression from cellular atypia through various degrees of dysplasia, most patients are diagnosed in advanced stage disease without clinical evidence of the antecedent premalignant lesion (Nelson, Rose and Moroianu, 2002). The main clinical manifestations of the disease vary according to the site of origin of the tumour, often characterized by mucosal ulcers or tumour, dysphagia, odynophagia or otalgia, chronic cough, and neck nodes tumefaction (Johnson et al., 2020). The diagnosis is usually established based on the clinical otorhinolaryngological exam that is complemented with imaging exams to evaluate the locoregional and distant extension of the disease classified using the TNM (Tumour, Node, Metastases staging system) classification of the American Joint Committee on Cancer and the International Union for Cancer Control (Katabi and Lewis, 2017; Johnson et al., 2020), and histological evaluation from tumour tissue (El-Naggar AK et al., 2017). The histopathological spectrum of squamous cell carcinoma is characterized by the evaluation of cellular atypia and squamous differentiation. It defines a well-differentiated tumour, a stratified epithelium that contains mature cells organized in layers with irregular keratinization, called a “keratin pearl”. On the contrary, the poorly differentiated tumour presents mostly immature cells with nuclear pleomorphism and atypical mitoses, with a poorly defined stratification and without keratinization (Johnson et al., 2020) Figure 7. 40 Figure 7. Squamous cell carcinoma of the oropharynx, with HPV16 DNA identified. Invasive cell nests with very irregular borders with stromal reaction (A). High power area showing poorly differentiated cells with mitotic figures (arrow) and focal necrosis (*), infiltrating striated muscle cell tissue (B); neoplastic cells show intense p16 nuclear (and also cytoplasmic) staining, a surrogate marker of HPV. 2.4 MOLECULAR CHARACTERIZATION The HNSCC molecular analysis reveals substantial genetic instability with frequent loss or gain of chromosomal regions as noted by copy number alterations and chromosomal fusions (Cancer Genome Atlas Network, 2015). Cho and colleagues describe the comprehensive genetic profile of these tumours, characterising TP53, CDKN2A, PIK3CA, HRAS, PTEN, and NOTCH1 as being changed differentially across all Head and Neck locations in contrast to CASP8, which is also confined within oral cavity (Loyo et al., 2013; Farah, 2021). Mutations in RAS oncogenes are less frequent, than in other solid tumour malignancies, characterizing HNSCC as neoplasms predominately associated with loss of tumour suppressors genes (Cancer Genome Atlas Network, 2015). 41 Genes have been grouped into categories including cell survival and proliferation - TP53, EGFR, PIK3CA, and HRAS, cell-cycle control genes - CDKN2A and CCND1, cellular differentiation genes - NOTCH1, and cellular adhesion and invasion signalling - FAT1, have been classified (Agrawal et al., 2011; Stransky et al., 2011; Cancer Genome Atlas Network, 2015). The overview of the genetic profile of HNSCC tumours (Figure 8) grouped according to Copy Number Alterations, gene mutations and gene expression profiles intent classify distinct classes of tumours based on patterns of protein expression that correlate with clinical behaviour (Rothenberg and Ellisen, 2012). The biological variability shown within HNSCC translates the division of the two main carcinogenesis pathways established for HNSCC-HPV and HNSCC-nonHPV (Farah, 2021). Figure 8. Molecular characterization of the HNSCC. Scheme adapted from (Farah, 2021) presenting the main profiles grouped according to copy number alteration (CNA), genetic mutations and gene expression profiles. Accordingly, the mutational analysis divided the HNSCC-nonHPV into CNA-silent and CNA-high tumours. Normally the TP53 and RB pathways are abrogated in HNSCC-nonHPV tumours but remain active in CNA-silent tumours. These CNA-silent are considered a subgroup of tumours that, although their aetiology is not well established, age apparently is the most risk factor associated and exhibiting mutations in the HRAS and CASP8 genes and often found in tumours of the oral cavity. On the other hand, HNSCC-nonHPV tumours (CNA-high), in which tobacco is the associated risk factor, exhibited changes in oncogenes such FAT1 and NOTCH1 pathway (WNT-Bcatenin). 48 Particularly, in HPV-positive HNSCC tumours, oncoproteins E6 and E7 interact directly in signalling pathways such PI3K/Akt/mTOR. This is thought to play a very important role in HPV-induced carcinogenesis by acting through multiple cellular and molecular events (Pim et al., 2005; Contreras-Paredes et al., 2009). These viral oncoproteins increase PI3K/AKT/mTOR signalling in several ways: the E7 was shown to significantly upregulate AKT activity not only through degradation of RB but also in an RB and PI3K independent manner (Figure 10). It interacts with protein phosphatase 2A (PP2A) and prevents dephosphorylation of AKT that causes significantly higher levels of phosphorylated Bcl-2 antagonist of cell death (BAD) (Marquard and Jücker, 2020). The E6 can activate Akt as well, or bind TSC2, leading to its degradation and resulting in stimulation of mTORC1. The PI3K pathway is unique, in that all the major components of this pathway are frequently found amplified or mutated in HPV-induced cancers (Zhang et al., 2017). The mutation frequency among HNSCC-HPV tumours was reported to be approximately half of that found in HNSCC-nonHPV (Stransky et al., 2011). Even so, the PIK3CA gene remains one of the most mutated in HNSCC-HPV (Sewell et al., 2014; Cancer Genome Atlas Network, 2015). Gene mutations targeting the p110α catalytic subunit were found in 56% of HPV HNSCC tumours and in 34% of HPVnegative HNSCC tumours (Cancer Genome Atlas Network, 2015). Among the PIK3CA mutations observed in HNSCC, 63% occur at three specific locations encoding the p110α subunit, namely E542, E545, and H1047, known as canonical mutations (Henderson et al., 2014; Miao et al., 2018). Among these three activating mutations the E542 and E545 affects the helical domain of p110α, inducing PI3K hyperactivity by disrupting the regulatory activity of p85 on p110α (Miled et al., 2007), and the H1047 affects the p110α kinase domain and is thought to cause a conformational change allowing easier access to the phospholipid substrate (Mandelker et al., 2009). Also, mutations targeting the p110α subunit have been associated with adverse outcomes in solid tumours although the prognostic significance in oropharyngeal SCC is still unclear. To be precise, the specific survival data did not differ between PIK3CA wild-type (WT) and mutated (MUT) lesions, however, the WT-PIK3CA patients had a significantly higher 3-year disease-free survival (DFS) compared to PIK3CA mutated patients. So, the prognostic value of PIK3CA mutations is still a matter of debate (Alexandrov and Stratton, 2014). 49 Figure 10. The PI3K pathway and HPV HNSCC. Representative scheme of the PI3K–AKT–mTOR pathway adaptation from (Medda, Duca and Chiocca, 2021). The HPV16 E7 protein binds to protein phosphatase 2A (PP2A) subunits, preventing their interaction with p-Akt and keeping it active. The E6 can activate Akt, or bind TSC2, leading to its degradation and resulting in the stimulation of mTORC1. 2.5 TREATMENT The description of current treatment management of these patients is complex due to the variety of tumour origin sites, particularly in the definition of the surgical options, with the intention of preservation of the integrity of the surrounding anatomical structures and to ensure that the involved organs still maintain is functions. Nowadays, therapeutic decisions are made in a multidisciplinary approach, that includes several specialities and medical areas such as otorhinolaryngologists, oncologists, radiation oncologists, dentists, dieticians, psychologists, and various therapists involved in the rehabilitation area. The varying presentations defined for specific subsite profiles evolve a range of therapies including surgery, radiotherapy, chemotherapy and more recently immunotherapy (Farah, 2021). The therapeutic scheme must consider the tumour features in terms of its size, growth pattern, depth of invasion, presence and 50 location of invaded lymph nodes, presence of extracapsular nodal extension, and perineural and lymphatic infiltration: all these characteristics are important for the tumour staging and prognosis, which help to determine surgical margins and the need for postoperative adjuvant therapy (Machiels et al., 2020). Therefore, in general, carcinomas diagnosed at an early stage of the disease (I and II) are usually treated with isolated therapy (surgery or radiotherapy). However, for carcinomas diagnosed in more advanced stages (III, IVA and IVB), combined therapy (surgery, radiotherapy and/or chemotherapy or others) is usually used. A good therapeutic response in low stages of disease occurs and the majority of patients have a good prognosis with a 5-year survival rate of 70 to 90%, in contrast to high stage disease where the 5-year survival is between 30 to 60% (Johnson et al., 2020). All patients, at the end of active treatment phase, require regular surveillance and follow-up, as the rate of recurrence and appearance of a new primary neoplasm is frequent within tumours with the worst prognosis (Farah, 2021). Recurrences occur in 80 to 90% of cases within the first 2 to 4 years of follow-up. For that, this period of follow-up must be more frequent and thorough. Disease’s recurrence has a dismal prognosis, since therapeutic options are limited by the previous treatment and tumour biological aggressiveness resulting that most of patients have a low mean survival, around 6 to 9 months. It should be noted, that despite all treatments like surgery, radiation, and chemotherapy, approximately half of the patients will die of the disease. The risk stratification for HNSCC by its anatomic site, stage, and histologic characteristics of the tumour, has been continuously investigated aiming to improve strategies regarding better treatment of these patients. However, it should be noted that the inputs of the current knowledge relative to genomic alterations or gene expression profiles previously described have limited clinical utility, and management of most patients is still predominantly the “classic” (Cancer Genome Atlas Network, 2015). In the last decades research is being actively explored on the mutational, genomic, and transcriptomic landscape and most recent available data show that HPV positivity is an indicator of better overall survival and has a substantially better prognosis after therapy compared with non-HPV+ HNSCC (Mehanna et al., 2019). The initial data from HPV-positive H&N cancer cell lines have shown that cancer cells have an increased sensitivity to radiation, which may explain the enhanced 51 survival of HPV-positive patients (Nulton et al., 2017). However, data obtained in several clinical trials provide evidence to consider de-intensified therapy for these patients (Argiris et al., 2008; Smith et al., 2010), but until now, data do not support any modification in contemporary treatment protocols, reducing the associated morbidity while maintaining tumour control (Hoffmann and Tribius, 2019). A large proportion of HNSCCs is still left with limited treatment options, underscoring the urgent need to identify novel clinical strategies and more importantly, biomarkers that could help to select patients for current and future therapies. A better understanding of the mutational, genomic, and transcriptomic landscape of HNSCC has become the promise leveraged to explore new therapeutic approaches to manage this group of diseases, and it is hoped that these additional insights will further drive the correct stratification of patients enabling them to apply more specific treatments for each group of tumours (Farah, 2021). 3. K14HPV16 ANIMAL MODEL The association of HPV with human cancer has been the subject of extensive research and considerable effort has been expended to model the diseases they cause (Doorbar, 2016). Currently, there are several useful in vitro and in vivo models that allow studying the process of cancer progression and the role of viral proteins in this process. Overall, in vitro models of epithelial cell differentiation can support the entire productive lifecycle of high-risk HPV types (Doorbar, 2016), and in vivo models are useful for studying and modulating the function of different cell populations, which serve to promote antitumor activity and reduce protumour signs (Santos et al., 2017). Despite these various efforts, the current models of HPV-associated disease have a few limitations that are linked to the fact that these viruses only replicate and complete their life cycle in humans, and cause cancers at discrete epithelial sites that are not straightforward to the model (Doorbar, 2016). Nevertheless, each model has its own potential and limitations, and adequate models should be chosen carefully (Santos et al., 2017). The K14-HPV16 in vivo model is valuable to explore components of the immune response against HPV-induced lesions and is promising in the development of novel, preventive and therapeutic, strategies (Santos et al., 2017). This model for 52 HPV induced cancer relies on the cytokeratin 14 (CK14) gene promoter to drive the expression of all HPV16 early oncogenes and reproduces the multi-stage carcinogenic process occurring in human disease, sharing histological features with the human lesions (Du et al., 2012). K14-HPV16 transgenic mice were first developed in the 1990s using standard techniques by microinjection of B6D2/F2 embryos. A K14 expression cassette developed by E. Fuchs at the University of Chicago was prepared, which contains 2 kb of the K14 promoter/enhancer and 500 bp of a 3' flanking sequence that includes the K14 polyadenylation signal. Instead of the endogenous long control region, the viral genes are regulated by this human K14 enhancer/promoter (Arbeit, Howley and Hanahan, 1996). Three forms of HPV16 DNA of the early region were used by constructed plasmids p1203, p16Nt and p16Pt, which contain respectively a genome of wild-type HPV16 (p1203), a genome with a translation termination ligand (TTL) in ORF E1 at nucleotide 1311 (p16Nt) and a genome that contains a TTL in ORF E2 at nucleotide 2922 (p16Pt). The fragments were then excised and cloned into the BamHI site of the K14 expression plasmid to generate plasmids pK14-1203 (K14-wt), pK14-16Nt (K14-Elttl) and pK14-16Pt (K14-E2ttl). Therefore, the K14-HPV16 transgenic mice contain, within their genomes, the entire HPV16 early region (Arbeit, Howley and Hanahan, 1996), however, since the two early-region variants contain specific mutations (E1ttl and E2ttl), the functions of the E1 and E2 genes are abolished and the E6 and E7 genes remained intact in these mutants (Arbeit et al., 1994). For the creation of the following transgenic lines several inbred backgrounds were carried out for several generations (minimum of five) and maintained in heterozygotes state (Arbeit et al., 1994), including C57BL/6, BALB/c, and SSIN/SENCAR genetic backgrounds, however, only mice backcrossed into the FVB/n background progress to malignant squamous cell carcinomas (Coussens, Hanahan and Arbeit, 1996). The successful targeting of HPV DNA to basal keratinocytes (Arbeit et al., 1994) generated lesions in the same sites observed in cancer patients allowed a better understanding of the functions of oncoproteins in vivo (Arbeit et al., 1993; Yang, Liu and Iannaccone, 1995). For instance, when female E5+/− transgenic mice were treated with oestrogen, this gene was capable of inducing cervical cancer (SmithMcCune et al., 1997). But, on the other hand, the absence of the late viral coding region (L1 and L2) and the fact that gene expression is regulated by the cytokeratin 14 promoter, introduce significant differences compared to human keratinocytes 53 infected by HPV and constitute significant limitations of these models (Santos et al., 2017). Furthermore, this model has proved to be very profitable in several epithelial cancers, and pre-malignant lesions. Most of the early studies were predominantly performed on uterine cervix carcinomas. But later other reports have also used in vivo animal models in the study of other HPV-associated malignancies, such as head and neck (Jabbar et al., 2010), penile (Dias et al., 2022) and anus cancers (Arbeit et al., 1994). In Head and Neck tumours, Jabbar (Jabbar et al., 2010) demonstrated the potential importance of studying cancer growth associated with HPV at various stages. They used a chemical carcinogen 4-nitroquinoline-1-oxide (4-NQO) to induce head and neck cancer in mice K14E6 and K14E7 transgenic models and demonstrate the E7 gene as a dominant oncogene, synergising with 4-NQO. In contrast, under the condition used in the previous studies, E6 transgenic mice treated similarly did not develop HNSCC (Strati and Lambert, 2007). These results are consistent with the dominant oncogenic properties of E7 gene in the head and neck region. Recent data studied the distribution of oral and pharyngeal lesions in K14HPV16 transgenic mice (Mestre et al., 2020). These findings provided experimental evidence that HPV16 is sufficient to drive HNSCC in the absence of chemical carcinogens and is observed a difference in their distribution. The HPV-driven lesions in the model preferentially target a clinically relevant area at the base of the tongue namely a squamocolumnar junction in the circumvallate papilla (Mestre et al., 2020). Additionally, a different incidence of tongue base cancer in male and female HPV16-transgenic mice was found being these tumours more common in female mice (Neto et al., 2021). The K14HPV16 animal model can be quite useful to the study of oral carcinogenesis, However, it should be pointed out, that this model lacks a deeper molecular study, a valuable issue to explore the biological significance of the HPV16 variant based on clinical and pathological observations. The characterization of the mutational profile with the identification of the specific lineage and sublineage involved in K14HPV16 mice (phylogenetic analysis) would be useful to broaden clinicopathologic data association, in addition to improving basic and translational studies in this field. 54 CHAPTER 2 AIM 55 AIM The main purpose of this study is to explore the hypothesis that specific HPV16 E6E7 variants influence the occurrence, the morphological and molecular phenotypes of HPV-induced HNSCC in a select population and to validate this molecular setting in a mouse model of HPV-induced HNSCC. GENERAL HYPOTHESIS Specific HPV16 E6 E7 variants identified in the carcinomas of the Head and Neck region may be associated with different morphological and molecular phenotypes and with clinical-pathologic settings and molecular markers of prognosis and response to therapy. SPECIFIC OBJECTIVES 1To identify and to study the frequency of HPV16 variants in squamous cell carcinomas of the head and neck region in a selected population, and to describe the clinical-pathological characteristics of HPV associated carcinomas, including tumour location, histological type, biological behaviour, and response to therapy. 2To study gene mutations typically associated with HNSCC-HPV positive, as well as the expression of potential prognostic and therapeutic markers, regarding HPV variants identified in each patient sample. 3Characterization of oral cancer in an animal model to develop a tool for experimental research: • To identify the HPV16 variant (E6/E7) present in the K14HPV16 animal model under CK14 regulation to elucidate for a more accurate use of the animal model. 56 CHAPTER 3 MATERIALS AND METHODS 57 1. CHARACTERIZATION OF THE BIOLOGICAL SAMPLES 1.1 SQUAMOUS CELL CARCINOMA OF HEAD AND NECK SAMPLES A detailed characterization of this material was done in the different articles that were published or submitted to publication and are presented in Chapter 4Results. The tumour samples are from a universe of patients diagnosed with HNSCC, treated in the Instituto Português de Oncologia de Lisboa Francisco Gentil (IPOLFG) between 2008 and 2019 and were included within the scope of the research project (ref. IPOLFG's UIC/1092), to study the prevalence of HPV in these tumour types. The patients were clinically followed up by the medical staff of the otorhinolaryngology and head and neck surgery departments of IPOLFG. The diagnosis was established by histological analysis of tumour tissue in the Pathology department. The molecular analysis and HPV DNA detection was performed in the twin tumour sample. A total of 457 cases were selected using following selection criteria: ● Histological confirmation of squamous cell carcinoma in the Head and Neck region (HNSCC), namely the oropharynx (base of the tongue, soft palate, tonsils, wall) and oral cavity (a lip, oral mucosa, mobile tongue, floor, jaw, and hard palate) ● HPV DNA status identified ● Patients without a previous diagnosis of a malignant tumour (relapse) or synchronous diagnosis of a malignant neoplasm in another location besides the upper respiratory/digestive airway ● Only cases of patients aged over 18 years were included 64 2.4.1.1 LABORATORY PROTOCOLS Product Nature: Sample Preparation All biological samples used for the identification of HPV by molecular methodologies can be used for NGS. The pre-analytical sample processing is applied with the same specific procedures for the various biospecimen types (product nature) such as fresh biopsy samples conserved: liquid-based cytology, dry frozen swab samples (-80ºC), or tissue derived from formalin-fixed paraffinembedded (FFPE). In general, all samples were fresh biopsies from tumours in the Head and Neck region. The DNA extraction was performed in Automatic equipment or Manual extraction, following the manufactures specificities. The quality and suitability of DNA extracted was spectrophotometrically controlled (A260/A280). This checkpoint allows to determine the suitability of the samples: only those with an acceptable range of quantity, purity and integrity values are validated for the next step of the NGS workflow. Target enrichment: PCR design and optimization Our approach was the amplification of the complete HPV16 DNA genome (~8000pb) and the primer design was based on 47 primers set previously described by Cullen et al 2015, which makes possible to cover the entire genome based on fragmented amplification producing amplicons with a size that ranged between 120-359 nucleotides. This strategy contributes to a better sensitivity of the assay, as opposed to opting for a long-fragment strategy that lowers the sensitivity values. The sensibility of the assay is an important parameter that we must overcome since the main target (viral DNA) is in a small quantity within the abundant host genomic DNA. An extensively evaluation from primer characteristics and in silico validation were performed. For the 103 primers that composes the 47 primers set, it was described the respective sequence, size, melt temperature, target region, and fragment size produced and evaluate secondary amplifications to other organisms with the Blast tool from PubMed (Supplement Table 2 in silico validation). For the pool primer conception was consider the in silico validation and intention to promote the best balance between specificity and sensibility of the assay. The best of 3 combination tested was an approach with 4 pools with 7 to 10 primer each. 65 (Supplement Table 2 Pool Primer Design): these were structured to amplify the viral whole-genome, avoiding the development of primer-dimer. For that, each primer was single amplified and verify within each primer pool to validate specificity. The PCR conditions were optimized for a real-time PCR system (60ºC hybridization temperature) in the Applied Biosystems QuantStudio 5 Real-Time PCR System, for a reaction volume of 25uL. The mixed compounds were accurate to work with High-Fidelity DNA Polymerase, a proofreading hot-start DNA polymerase for high yield sensitivity and specificity, being over 300x more accurate than Taq-enzyme, resulting in ultra-low error rates. Validation was carried out for each pool primer with input concentration set to 1 pmol and normalised each DNA template into ~100-250ng. The reaction is performed in 3 stages for complete amplification: (1) 95ºC Denaturation; following 40 cycles of segment (2) 60ºC hybridization; and (3) 72ºC elongation. The detection method by agarose gel was used to identify the PCR products. The 4 independent PCR products of each sample were pooled together in equal molar amounts based on the signal intensity. Library Preparation and Sequencing Library preparation protocols were based on Amplification-based NGS. There are several different reaction reagents available for specific protocols due to the diversity of platforms. At present, there is not a recommend protocol for any HPV whole-genome applications. Our in-house assay has been optimized for Illumina technology with the collaboration of the Core NGS from INSA (National Institute of Health). The optimization of the quality, quantity, and fragment length includes confirmation of expected results from positive control and validates the measurements data for the specific analytical purpose. The pooled amplicons were purified from small fragments and primers using the AMPure XP beads (Beckman Coulter) according to the manufacturer´s instructions before library preparation. The Nextera XT DNA library prep kit was used to prepare bead-based normalized dual-indexed libraries for sequencing. The validation considered fragments size to cluster efficiency and concentration of the prepared libraries since low concentration may result into a low number of sequenced reads. The library pool was sequenced on a MiSeq benchtop sequencer using a reagent kit V3 (illumina MS-102-3001). A sequencing strategy with 250-bp paired-end reads was used. 66 2.4.1.2 BIOINFORMATIC DATA ANALYSIS NGS bioinformatics pipelines consists in a specific platform to analyse sequence consensus single nucleotide variant (SNV) and may be customizable based on laboratory needs. The Bioinformatics Analysis of NGS Data were developed with Core bioinformatic INSA collaboration. Primary analyses were performed on INSaFLU (https://insaflu.insa.pt/) an online platform for amplicon-based nextgeneration sequencing data analysis (Borges et al., 2018) and used for reads’ quality control, variant detection/inspection and sequence consensus generation. The genome sequence of a representative of HPV16 sublineage A1 (GenBank accession number NC001526.4) was used as a reference for mapping and SNV annotation. Regions with a depth of coverage below 10-fold were automatically masked in the INSaFLU pipeline by placing undefined bases “N” in the consensus sequence. Lowcoverage regions were visually inspected using Integrative Genomics Viewer (IGV). SNVs were assumed in consensus when they displayed more than 50% intrasample frequency. MEGA11 software (http://www.megasoftware.net) (Tamura, Nei and Kumar, 2004) was applied to calculate matrices of nucleotide distances and to perform phylogenetic reconstructions. 2.4.2 ANALYTICAL VALIDATION The performance characteristics of this method were validated based on data measurements from Positive Control, Negative Control, and Internal Control. The cell line SiHa, positive for HPV16, was used as positive control with 100% concordance and were included in each run to evaluate PCR efficiency (HPV16 A lineage (GenBank:AF001600.1). The negative control (No Target Control) provides insight regarding chemical reagents contamination; Albumin that was also used as an internal control to distinguish true target negatives from PCR failure or inhibition. To evaluate the assay performance: the precision of the method was established by performing positive reference control for 10 repeats and the assay performance results were analysed by agarose gel. To estimate efficiency sensibility the positive control was quantified and diluted 10x series into 4 points between 5 to 5000 copies. The assays demonstrated a wide dynamic range efficiency and can 67 analytically detect at 5 HPV16 copies/PCR. The specificity was accurate for each pool primer, which the specific target was analysed through fragment size in agarose gel with Biorad geldocX tool. The reproducibility was based on 10 replicates of the positive control and has demonstrated excellent reproducibility inter and intra-assay (Supplement Table 3). 2.4.3 IMPLEMENTATION AS ROUTINE PROCEDURE For the assay implementation as a routine procedure, several actions were established based on specific requirements face to quality control metric as described in Table 2 for each section of the workflow. Table 2. Implementation and Quality Control Metrics NGS Workflow sections Requirements Action Sample Preparation Biological Specimen Pre-analytical sample processing DNA Monitoring quantity and purity PCR enrichment Reaction performance Positive control amplification HPV16 infected cell line Siha or CaSki Pool Primer Efficiency Monitoring amplicons concentration (signal intensity) HPV16 Whole-genome Specificity Monitoring Fragment size (migration position) Target integrity Internal control amplification albumin gene Library Preparation and Sequencing Library Qualification and Quantification Monitoring number of reads Data analysis Bioinformatic pipeline Standardization of analytical parameters Biologic significance Data systematization into a database 68 2.5 DETECTION OF MUTATIONS OF THE PIK3CA GENE Assessment of the PIK3CA gene mutations were performed by real-time PCR assay (Amoy Dx) for the qualitative detection of four mutations (H1047L; E542K, E545K and E545D) target in exon nine and exon twenty from the human genomic DNA extracted from tumour tissue. The assay comprises specific primers and fluorescent probes. During the nucleic acid amplification, the targeted mutant DNA is detected by FAM-labelled probes. It used an internal control system containing primers and a HEX-labelled probe for a region of genomic DNA without known mutations and polymorphisms. All the procedures of pre-analytical, analytical and data interpretation were performed according to the manufacturer's specifications. For the PCR conditions, the preparation of the reaction components (master mix) was optimized for the eluted DNA evaluated. This amount was normalized to 15 ng of template DNA for all samples. The different compounds PIK3CA Reaction Mixes, Mixed Standard and Taq DNA polymerase were combined at different concentrations (ng/µL) for a final volume of 20µl. Therefore amplification were performed on qPCR BioRad CFX96 equipment with Cycling Parameters programmed for 3 stages defined by Temperature, Time and Cycles: Stage 1 defined by 1 cycle at 95 ℃ 5 min 1; Stage (2) defined in 15 cycles at 95 ℃ 25 s, follow decrease to 64 ℃ 20 s 15 and increase to 72 ℃ 20 s; and Stage (3) defined in 31 cycles at 93 ℃ 25 s, follow decrease 60 ℃ 35 s (Data collection of FAM and HEX/VIC) and increase to 72 ℃ 20 s. Analysis data run automatically in software to check the FAM and VIC Ct value for each sample. Based on different mutant Ct values, the detection results are divided into strong positive, weak positive or negative. 69 CHAPTER 4 RESULTS 70 1. DESCRIPTIVE ANALYSIS OF THE STUDY SERIES 1.1. IDENTIFICATION OF HPV DNA IN HNSCC SAMPLES From a total of 457 patient´s with the diagnosis of HNSCC, HPV was found in 34.4% (n=157) of the cases. The HPV genotype was achieved for 101 HNSCC-HPV despite 56 cases where genotyping was inconclusive. Single infection was present in 93 cases which the most frequent type was HPV16 (n=68), followed by HPV18 (n=4), HPV35 (n=4), HPV31 (n=2), HPV53 (n=3), HPV33 (n=2), HPV51 (n=2), HPV56 (n=2), HPV59 (n=2), HPV6 (n=2), HPV52 (n=2) and HPV26 (n=1) (Graphic 1). In 5.7% (n=8) of the cases, more than one type of HPV was detected - coinfection, where HPV16 was present with other HPV types both low-risk and/or high-risk types. Graphic 1. Frequency HPV types of single-infection cases (n=93). Legend: coloured red - High-Risk Types; coloured grey - Low-Risk Types 71 1.1.1 DEMOGRAPHIC CHARACTERISTICS The age at diagnosis of HNSCC-HPV patients range between 36 and 91 years with a median of 61 years. Both genders are present with 76.4% (120/157) males and 23.6% (37/157) females (Graphic.2-A). In our cohort, the gender distribution is similar in both groups HNSCC-HPV and HNSCC-nonHPV with a male to female ratio of 3:1. The median age of 61 years was also recorded for HNSCC-nonHPV group, which ranged from 27 to 93 in HNSCC-nonHPV, with 76.3% (229/300) of males and 23.7% (71/300) females (Graphic.2-B). Graphic 2. Age at diagnosis per group HNSCC-HPV (A) and HNSCC-nonHPV (B) stratified by patients' gender. The data refers to the total number of patients diagnosed with HNSCC in last decades (2008 and 2019) in IPOLFG. 1.1.2 CONSUMPTION HABITS The consumption habits were evaluated according to self-reported in the clinical files. Data analysis was based on stratification by never drank, ex-drinker, sporadic, moderate, and severe drinker for Alcohol exposure. Tobacco consumption was defined in units as one pack/year (equal to one pack of cigarettes/day/year, with 20 cigarettes/pack) and classified as current or heavy smokers: current tobacco users included those who used tobacco ≤ 30 pack/years and heavy users were those who smoked > 30 pack/year. Patients without consumption habits were defined as not having consumed none of these substances prior to cancer diagnosis. Of the total of the 457 patients included, 41 patients had missing information in files. The analysis from the remaining 416 patients, considered two groups active consumption vs. no consumption. Most patients have an active consumption (82%; 72 n=341/416) with a consumption practice of both tobacco and alcohol. On the other hand, 18% (n=75/416) of the patients self-reported no consumption habits. The analysis of the distribution of HPV by consumption habits (Graphic 3) shows that a higher active consumption among HNSCC-nonHPV patients [65% (n=223/341)] compared to the consumption observed among HNSCC-HPV patients [35% (n=118/341)]. The analysis of consumption by gender reveals that the noconsumption group is mostly represented by females (Table 3). Graphic 3. Characterization of HNSCC patients' consumption habits by HPV detection Table 3. Characterization of HNSCC patients’ consumption habits by HPV and by gender HNSCC-HPV HNSCC-nonHPV Consumption n male female male female no consumption 75 7 20 21 27 active 341 105 13 190 33 no data 41 8 4 18 11 Total 457 120 37 229 71 73 1.1.3 TUMOUR SITES The Table 4 shows the distribution of the different tumour locations. Tumours were in different subsites of the oropharynx [45.3% (207/457)] and oral cavity [54.7% (250/457)]. The site of the tumour from HNSCC-HPV patients is more frequent in the oropharynx with 55% cases (86/157), unlike HNSCC-nonHPV patients which the tumour site is more frequent in the oral cavity with 60% cases (179/300). Table 4. Distribution of tumour site by HPV detection Tumour site HNSCC-HPV HNSCC-nonHPV oropharynx 207 86 55% 121 40% base tongue 25 15 17% 10 8% soft palate 46 10 12% 36 30% tonsil 118 56 65% 62 51% oropharynx wall 18 5 6% 13 11% oral cavity 250 71 45% 179 60% hard palate 5 2 3% 3 2% oral mucosa 42 11 15% 31 17% oral floor 52 13 18% 39 22% tongue 151 45 63% 106 59% Total 457 157 300 1.1.4 TUMOUR HISTOLOGY All cases included in this series were confirmed as invasive squamous cell carcinoma in the histological analysis. Tumour were diagnosed in different stages of disease at time of diagnosis (Table 5). The distribution regarding stage of disease revealed that tumours were predominately in stage II and I, followed by stage III and few cases were diagnosed at stage IV. 80 Cochichoetal. Virol J (2021) 18:217 https://doi.org/10.1186/s12985-021-01688-9 REVIEW Exploring theroles ofHPV16 variants inhead andneck squamous cell carcinoma: current challenges andopportunities Daniela Cochicho1,2†, Rui Gil da Costa3,4,5† and Ana Felix1,6*† Abstract The incidence of squamous cell carcinomas of the head and neck (HNSCC) is consistently increasing, in association with human papillomavirus (HPV) infection, especially HPV16. HPV variants show heterogeneity in the pathogenicity of cervical cancer, but little has been established about their relevance on HNSCC. This review addresses the distribution of HPV16 variants in HNSCC and their potential contribution to clinical practice. A search was performed in PubMed using the keywords HNSCC HPV16 variants. Sixty articles were identified between 2000 and 2020 and 9 articles were selected for a systematic analysis. Clinical cohorts comprised 4 to 253 patients aged between 17 and 91 years with confirmed HPV16-positive HNSCC. Samples were collected from fresh biopsies of the tumour, oral rinse or formol fixed/paraffin embedded tissue, from the oral cavity, oropharynx, hypopharynx, larynx and Waldeyer’s tonsillar ring. HPV16 variants were identified using Sanger sequencing techniques. Seven studies addressed the HPV16 E6 gene, one studied E6 and E7, another studied L1 and one focused on the long control region. European variants represent 25–95%, Asian-American 5–57% and African 2–4% of the total isolates, suggesting a marked predominance of European strains. No correlations could be drawn with patient prognosis, partly because many studies relied on small patient cohorts. Additional studies are needed, particularly those employing next generation sequencing techniques (NGS), which will allow faster and accurate analysis of large numbers of samples. Keywords: HNSCC, HPV16, HPV16 variants, Next generation sequencing © The Author(s) 2021. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. The Creative Commons Public Domain Dedication waiver (http:// creat iveco mmons. org/ publi cdoma in/ zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Background Head and neck cancer is the sixth leading cancer by incidence worldwide [1] and comprises many different pathological entities. The diagnosis is often characterized by multifocal development and presentation at an advanced stage. [2] The age at diagnosis usually ranges between 50 and 70years of age and men are significantly more likely to develop the disease than women (up to a 4:1 proportion, depending on geographical localization) [3]. Currently, two major carcinogenic pathways leading to head and neck squamous cell carcinomas are recognized: the first is associated with risk factors like smoking and abusive alcohol consumption while the second is associated with human papillomavirus (HPV) infection [4, 5]. Over the past decade, several epidemiologic studies reported a 36.5% increase in the incidence of head and neck squamous cell carcinomas (HNSCC), particularly in high-income countries and among men < 60years [4, 6–8]. Available data indicates that these changes specifically involve oropharyngeal cancers [9, 10]. Although tobacco consumption has decreased, the incidence of HPV-positive oropharyngeal cancers has increased [11], indicating that HPV infection is the underlying cause for the overall increase in HNSCC incidence [12–14]. Open Access *Correspondence: [email protected] †Daniela Cochicho, Rui Gil da Costa and Ana Felix contributed equally to this work 1 NOVA Medical School, NOVA University of Lisbon, CEDOC, Campo Mártires da Pátria 130, 1169-056 Lisbon, Portugal Full list of author information is available at the end of the article 81 Page 2 of 9 Cochichoetal. Virol J (2021) 18:217 Patients showing oral HPV infection are 53 times more likely to develop HNSCC [15]. Presently, HPV is present in approximately 35% of HNSCC and most HPV-positive cases emerge in lingual and palatine tonsils [16]. The presence of HPV DNA in tumour cells defines a specific pathologic entity within HNSCC with specific epidemiology, molecular characteristics, and biological behaviour [17]. HPV interferes with key signaling pathways to promote carcinogenesis via its viral oncoproteins E6 and E7, which lead to the inactivation of tumour protein 53 (p53) and the retinoblastoma protein (pRB), respectively [18]. HPV is a group of small, double-stranded DNA viruses that infect the epidermis and keratinizing mucosae. More than 100 HPV types have been identified [19, 20]. Presently, 40 different HPV types are known to infect mucosal epithelia and are categorized into low-risk and high-risk HPV types according to their epidemiologic association with cervical cancer [21]. In HNSCC, the majority of HPV types belong to this group including HPV16, HPV18, HPV39 and HPV45 [22]. HPV16 is by far, the most detected type, accounting for 90% of all HPV-positive HNSCC cases [23], a significantly greater proportion than in cervical cancer where it accounts for little over 50% of cases [24]. Sequencing of the HPV genome also revealed intra-type variants with genetic differences ranging between 0.5 and 1% [25, 26]. HPV variants arise mainly from nucleotide substitutions in some restricted positions in the genome coding region or in the noncoding region [27, 28]. The prevalence of variants for each HPV type varies significantly in different geographical areas [27]. Regarding HPV16, whole-genome analysis allowed the characterization of five distinct phylogenetic clusters named according to their original geographical distribution: European (E), Asian (As), Asian-American (AA), African 1 (Af1) and African 2 (Af2) [29–31]. Subsequently, a new branch, North American 1 (NA1) was identified [32, 33]. Currently, emerging epidemiological, etiological, and molecular data suggest that intra-type HPV variants are biologically distinct and may be associated with different risk of cervical cancer progression [28]. In vitro studies using 3D organotypic epithelial cell cultures showed that HPV16 E6 variants differ in their ability to abolish keratinocyte differentiation and to induce p53 degradation [34–36]. Those experimental results are corroborated by clinical studies on cervical cancer. The T350G (L83V) HPV16 variant is the most frequently found among invasive cervical cancers [21, 37] and has been linked to a higher oncogenic potential than the prototype [38], possibly by facilitating persistent viral infection, a critical factor for cancer development [39–41]. Despite the growing number of studies concerning HPV16 variants on cervical cancer, there are currently very few reports describing their distribution in HNSCC and nothing is established about their impact on the development, treatment response and impact on disease outcome. Nonetheless, HPV16 variants may also play an important role in head and neck carcinogenesis and studying their impact is needed due to the rising incidence of HPV-positive HNSCC [18, 23, 25, 42]. The purpose of this work is to systematically review the distribution of HPV16 variants in HNSCC and to assess the available knowledge concerning their potential contribution for HNSCC pathologic heterogeneity in published studies. Main text Review compilation data A systematic review was performed on the PubMed electronic database (https:// pubmed. ncbi. nlm. nih. gov) using the following keywords search criteria: HNSCC HPV16 variants. Observational studies reporting the distribution of HPV16 variants in HNSCC and published between 2000 and 2020 were included. Review articles, case reports and articles dealing with other types of cancer were excluded (Fig.1). The selection search criteria were based on tumour localization, nature of sample and background of the variant study field. The studies were independently assessed to identify the prevalence of HPV16 variants in HNSCC and evaluate in respective cohorts for correlation with clinicopathological parameters. HNSCC Fig. 1 Analytical flow for systematically reviewing published articles dealing with HPV16 variants in HNSCC 82 Page 3 of 9 Cochichoetal. Virol J (2021) 18:217 patients were categorized into different sites (oral cavity and oropharynx) and further into sub cohorts based on the demographic and clinical information provided. The published data were summarized using frequencies and percentages for HPV16 variants, stratified by tumour location, types of tumour samples, patient cohort size, sequencing methodology, and clinical variables such as age and gender (Fig.2.) General findings From 61 records found using the search criteria HNSCC HPV16 DNA cell carcinoma variants frequency patients’ cohorts, we selected 9 studies while 52 studies were excluded from further analysis, including review articles and papers which fit the exclusion criteria (Fig.1). Selected articles report data from a total of 945 patients in the period between 2000 and 2020, distributed by 6 countries. (Table1) Three studies were performed in Europe, threein the USA, two in Asia and Middle East (Japan and Iran) and one in South America (Brazil). HPV16 variants were evaluated in nine different clinical cohorts. All studies were different and did not contain the evaluation of the same cases. Overall, the study population consisted of patients whose age at diagnosis ranged between 17 and 91years. The gender ratio was 1:3 or 1:4 (female to male), and all cases were histologically confirmed as HNSCC. The specimens used to evaluate HPV16 were primary tumours located in the oral cavity, oropharynx, hypopharynx, larynx and Waldeyer’s tonsillar ring. Regional lymph node metastases were also included in one study. The samples used to analyze were diverse and consisted of fresh tissue, oral rinse, or formalin fixed/paraffin embedded tissue. The use of the p16INK4a marker is to clinically detect an oncogenically active HPV infection and is considered as a surrogate marker for HPV infection [43, 44]. From the 9 studies evaluated three described the expression pattern of p16INK4a among negative HNSCC HPV(−) and HNSCC HPV16 cases. For all studies there was a significant difference in p16INK4a expression between HNSCC HPV(−) and HNSCC HPV16(+). Pakdel etal. 2020 observed the expression of p16INK4a among HPV variants and showed that a strong p16INK4a expression was present in poorly differentiated tumour tissues infected with HPV16 sublineage A2 [45]. HPV detection andgenotyping HPV was detected using PCR-based techniques employing consensus degenerate primers: MY09/11 (4 studies) MY09/11 and/or GP05/06 (2 studies), PGMY (one study) and SPF1/2 (one study); all of which are complementary to the conserved L1 region. One study detected HPV using insitu hybridization. HPV genotyping was performed using different methods. Two studies using a commercial kit (INNO LIPA) which identifies 28 different HPV types (6, 11, 16, 18, 26, 31, 33, 35, 39, 40, 43, 44, 45, 51, 52, 53, 54, 56, 58, 59, 66, 68, 69, 70, 71, 73, 74, 82) by reverse blot hybridization. Two other studies used TaqMan PCR methods targeting the E6 or E6/LCR. One study used restriction fragment length polymorphism (RFLP) analysis. Two studies performed direct Sanger sequencing of the PCR products. The HPV frequency in the different cohorts ranged from 10 to 100% of the cases, and the HPV16 distribution ranged between 67 and 100% of all HPV positive cases (Table1). Except the study done in 2020 in Fig. 2 Analytical variables flow for included articles dealing with HPV16 variants in HNSCC search criteria 83 Page 4 of 9 Cochichoetal. Virol J (2021) 18:217 Table 1 Identification of HPV16 variants in HNSCC in the literature (PubMed 2000–2020) E6 Early gene 6; E7 early gene 7; LCR long control region; L1 late gene 1; FF/PE Formalin fixed Paraffin embedded Year publication Reference Country Mean age patients (yrs) Gender (% male) Consumption (% active) Samples Total cases (n) Total cases HPV detected %(n) Total cases HPV16 detected %(n) Total isolates for HPV sequencing Genome region location Amplicon size (pb) Sequencing 2000 [48] USA 63 no data available 87% Fresh tissue 253 24% (62) 90% (56) 52 E6 455 Sanger 2004 [49] Germany no data available no data available no data available FF/PE 24 100% (24) 100% (24) 21 E6 and E7 793 Sanger 2007 [50] Italy 63 74% 67% Fresh tissue FF/PE 115 18% (21) 67% (14/21) 13 L1 150 Sanger 2008 [51] USA 57 77% 76% Oral rinse FF/PE 135 32% (44) 100% (44/44) 19 E6 609 Sanger 2009 [52] Japan 64 86% 79% Fresh tissue 77 10% (8) 100% (8/8) 8 E6 323 Sanger 2013 [53] Brazil 59 100% 50% FF/PE 4 100% (4) 100% (4/4) 4 E6 no data available Sanger 2015 [54] Italy 65 88% 63% FF/PE 24 100% (10) 100% (10/10) 10 E6 no data available Sanger 2016 [55] USA 60 86% No data available FF/PE 205 18% (36) 70% (25) 21 LCR 193 Sanger 2020 [56] Iran 56 58% No data available FF/PE 108 23% (25) 16% (17) 13 E6 no data available Sanger 84 Page 5 of 9 Cochichoetal. Virol J (2021) 18:217 Iran, where just 16% of the HPV detected were HPV16. The most common HPV in their series was HPV16 but also followed by HPV18 and HPV11 and the cases were almost located in Oral cavity and Larynx; only 6 tonsils in all 108 cases were evaluated, explaining the low HPV frequency overall. Identification HPV16 variants Of the 9 studies, seven studies identified HPV16 variants by sequencing the E6 gene, one addressed variants in the L1 gene and another the long control region. In one study the E7 gene was also sequenced along with E6 (Table1). In all studies, the identification of HPV16 variants was performed by Sanger sequencing techniques and by comparison to the reference sequence (prototype). Next generation sequencing (NGS) was not used in any of the studies evaluated. However, the reliance on conventional Sanger sequencing is likely to limit the number of cases that can be analyzed and the reliability of results. [26, 46, 47] Sanger sequencing techniques show limitations concerning the size of amplicons, which may compromise the assay sensitivity. Additionally, to overcome polymerase errors and guarantee the integrity and robustness of the results, it is necessary to perform replicates for each case or vector molecular assays [48]. Such replicates add significantly to the workload and costs and constitute a limitation to analyze large patient cohorts. In fact, only two out of nine studies included over 200 HNSCC patients and none analyzed more than 52 HPV-positive patients for HPV16 variants. Future studies aiming to study larger patient cohorts are likely to benefit from NGS techniques, which provide faster and reliable sequencing results of larger amplicons [25, 26, 46]. Prevalence ofHPV16 variants The frequency of each HPV16 variant (E, NA, AF, AS and AA) was estimated for each of the 9 studies included in the systematic review (Table2). Gillison etal. [37] first reported data concerning the distribution of HPV16 E6 variants in HNSCC. The authors evaluated 52 HPV16positive patients from an overall HNSCC cohort comprising 259 patients. The age at diagnosis ranged between 17 and 91years-old (median, 63years-old) and the majority were smokers, with or without alcohol consumption (87%). Tumours were localized in the nasopharynx (n = 2), oral cavity (n = 84), oropharynx (n = 60), hypopharynx (n = 21), larynx (n = 86). HPV-positive patients showed significantly improved disease outcomes compared with HPV-negative patients. All samples used were HNSCC fresh tumour specimens. The HPV16 variants were classified into the same phylogenetic group as the European prototype in 75% of cases, Asian in 17%, North American in 4.0% and African 1 in 4.0% of cases. Six novel variants not previously reported (E-G315T, E-G315G, E-C395G, E-A478T, E-A132T, Af1-C311, Af1-A389) were also identified. The authors remarked that the distribution of variants in HNSCC resembled that observed in cervical cancer. However, no conclusions were drawn concerning the potential contribution of specific HPV16 E6 variants for increasing cancer risk or modifying tumour biopathology and prognosis. Four years later, Hoffmann etal. [38] identified HPV16 variants in 7 out of 21 tumour specimens of HNSCC. The authors analyzed the E6 and E7 ORFs. Altogether, the DNA samples carried HPV16 prototype European Table 2 Frequency of HPV16 variants (–) not evaluated; AA Asia-American HPV16 Variant; AF African HPV16 Variant; AS Asian HPV16 variant; E European HPV16 variant; E6 HPV Early protein 6; E7 HPV Early protein 7; LCR long control region; L1 late gene; NA North American HPV16 Variant Reference Total cases HPV16 detected (n) Total isolates HPV sequencing (n) HPV16 variant region location European (E) North American African Asian Asia American All lineages (n) E-350-G (n) (NA) (n) (AF1|AF2) (n) (AS) (n) (AA) (n) [48] 56 52 E6 39 6 2 2 9 0 [49] 24 21 E6 E7 15 8 0 0 0 0 [50] 14 13 L1 9 0 1 2 0 1 [51] 44 19 E6 18 4 0 0 1 0 [52] 8 8 E6 5 5 0 0 0 0 [53] 4 4 E6 2 1 0 0 0 2 [54] 10 10 E6 8 8 0 0 0 0 [55] 25 21 LCR 9 – 0 0 0 12 [56] 17 13 E6 11 – 0 0 0 2 85 Page 6 of 9 Cochichoetal. Virol J (2021) 18:217 variant (29%), the European variant T350G (38%) and 33% Euro-German variant (A131G + C712A). Again, no conclusions could be drawn concerning the clinicalpathological relevance of the HPV16 variants identified, partly because of the cohort’s small size. Badaracco etal. [49], published molecular analysis data on the HPV16 L1 ORF, from an Italian cohort (total n = 115, of which only 13 were tested for HPV variants), composed of 86 men and 29 women, with a mean age of 63.21years old. A high percentage of patients were smokers (67%) and alcohol drinkers 43%. Tumours were localized mostly in the oral cavity (n = 60), followed by the larynx (n = 30), the oropharynx (n = 10), the tonsil area (n = 8), the hypopharynx (n = 5) and the sinus/nose (n = 2). In this study, the presence of HPV was not significantly associated with disease-free survival at 2years. Sixty nine percent of the cases (13 cases) analyzed showed European-German variants (9 cases), 15% were African type 2 (2 cases), 8% Asian-American (1 case), and the remaining 8% (1 case) had an unclassified variant. The predominance of the European variant was unsurprising considering the Italian origin of the patients. No correlations were drawn between the presence of HPV16 variants and any epidemiological, pathological, or clinical data. Agrwal etal. [50], studied 19 HPV16 isolates from a universe of 135 HNSCC samples. The median age at diagnosis was 57years-old, 77% of the patients were men, most patients had a history of smoking (n = 62 smokers versus 51 nonsmokers) but were non-drinkers (73 non-drinkers versus 41 drinkers). All analyzed cases showed European variants and a single case carried an Asian variant. The most common European variant was E-350T (n = 6), followed by E-350G (n = 4) and E-T131G (n = 2). Importantly, eight of the 19 isolates contained European variants with sequences unique to a single individual. BoscoloRizzo etal. [51] analyzed HPV E6 variants in a short (n = 8) case series. The authors showed the presence of the T350G mutation in 5 cases located in the oral cavity and oropharynx, while three tumours located in the larynx and hypopharynx contained HPV 16 prototype sequences. Joseph etal. [52] compared HPV16 variants present in four patients with bilateral tonsillar HNSCC. Two cases carried European variants while two others carried Asian-American variants. The results show that, in all 4 patients, the same HPV16 variant was present in the bilateral tumours, supporting the hypothesis that a single HPV infection, rather than independent infections with distinct agents, is responsible for those bilateral tumours. Hassani etal. [53] studied the HPV16 variants present in 10 cases of tonsillar HNSCC. The authors found that the E-350G-variant was present in 80% of cases while the European prototype was identified in the other 20%. Again, this short case series did not provide data concerning the pathobiological relevance of HPV16 variants. In the following year, Betiol etal. [54] reported the distribution of HPV16 variants in a Brazilian cohort of 21 HNSCC patients. The authors analyzed the HPV16 LCR and found that 12 (57.1%) patients carried European and 9 carried Asian-American (42.9%) variants. The authors remarked that the slight predominance of European variants accompanied observation from their normal cervical samples, suggesting that the distribution of HPV16 variants reflects the overall frequency in each studied population. The most recent study was published by Pakdel etal. [45]. Thirteen HNSCC tissue specimens tested positive for HPV16 using overlapping PCR assays and were analyzed for the presence of E6 variants. There was a marked predominance of European variants (84.6%) followed by Asian-American (15.4%) variants. Discussion We reviewed the published literature on HPV16 variants in HNSCC, aiming to evaluate the biological meaning of those variants in this particular location. The clinical management of HNSCC improved greatly since the recognition of HPV-positive and HPV-negative lesions. Although tumour recurrence still occurs in 10–20% of HPV-positive HNSCC patients, the majority of these patients clearly benefit from therapeutic de-escalation [55]. The lack of adequate biomarkers to define more tailored approaches is still necessary in SCC HPV associated tumours [56]. The large majority of these tumours are associated with HPV16 and a deeper understanding of the bio pathological implications of distinct HPV16 variants would contribute to the molecular characterization of HPV-positive HNSCC and may help to define patient’s subgroups that would benefit from specific therapeutic approaches. Data from cervical cancer patients showed an association between the presence of nonEuropean HPV16 lineages, a longer viral persistence [57, 58] and an increased risk of developing high-grade cervical intraepithelial neoplasia [59]. Apparently, within individuals, HPV genomes harbour high levels of variability in HPV16 genome sequence upon normal to pre-cancer/ cancer [60] revealing several fundamental discoveries and suggesting a paradigm shift from HPV16 as a single viral entity to theorize each HPV16 isolated to be a separate virus with distinct carcinogenic potential [61]. This would imply that within HPV16, the genetic variation partly predicts the risk of pre-cancer and cancer [62]. In particular, specific sublineages (A4, C, D2, and D3) have shown a significantly increased risk compared to the most common A1/A2 sublineages [26] and assured D2, for the strongest risk of cancer within glandular epithelium (adenocarcinomas) [26]. 86 Page 7 of 9 Cochichoetal. Virol J (2021) 18:217 The major data from whole-sequences obtained from individual clinical specimens agrees that the genetic variation occurs more commonly on low-grade or benign HPV16 infections [61] and corrected explained In case–control analyses describing the highest amino acid changing variants HPV16 in the controls throughout the genome with cervix cancer. [62] Therefore, E7 oncogene lacks nonsynonymous (amino acid changing) variants in cervical cancers, suggesting the E7 conservation is admitted for carcinogenicity. [61] The specific conservation of the 98 amino acids of E7, that directly disrupts Rb function, was shown to be crucial for trigger carcinogenesis, owing to be commented as a highly specific target for etiologic and therapeutic research [26]. Presenting data from a cohort study that compares genomic characteristics of HPV associated with cervical versus oropharyngeal tumours using DNA sequence analysis showed no significant differences between distribution in HPV16 variants [24], both presenting major prevalence of the European variant. Instead, the HPV E6 gene amplified from oropharyngeal samples reported over more nonsynonymous mutations, but also for the E7 gene, no differences were found in mutation rates between the two anatomical locations [24, 62]. Notably, the E7 gene is conserved in both locations corroborating the recent findings on cervical cancer studies. The apparent restriction on E7 mutations seems to be present in the oropharynx, as well. Indeed, described all both share common biological features, but the important differences present in HPV-genome may explain their distinct pathophysiological mechanisms and susceptibility to treatment. Nevertheless, the invariability of E7 presents an attractive potential target for therapy at both locations. [24] In all the studies evaluated in this analysis, the most prevalent variant was the European Variant, which belongs to the European lineage and within this lineage the E-350-G HPV16 variant was the most frequent in majority of the studies sequencing based E6 and/or E7 regions with 29% and 53% respectively, although the cohorts had or not an European origin, (cohorts were from Brazil, United States of America, Japan, Italy and Germany (Table3). As it was established from molecular analysis studies in cervical cancer, that the European variant T350G, was the variant frequently found in cervical intraepithelial neoplasms and cancers, and has been associated with progression to cervical cancer particularly in North European women. The detection of the T350G variant in a large proportion of HNSCC patients, therefore, indicates that this variant might also play an important role in HN carcinogenesis. Hassani etal.in 2015 supported this hypothesis, elucidating that the HPV-16 E-350G variant has a polymorphism in residue 83, a leucine for valine (L83V), probably responsible for the increased cancer risk [53]. Conclusions In HNSCC different lineages of HPV16 variants can be identified and differ geographically. Although most reviews described only the distribution of HPV16 variants in HNSCC, LeConte in a more detailed analysis of those studies found important differences. They found the distribution of two HPV16 variant groups differ significantly in oropharyngeal cancer and cervical cancer. The European + South America (E + AS) variant groups showed a higher prevalence in the oropharyngeal samples, representing 90.2%, than in cervical carcinomas (71.4%). Moreover, the Asia-American (AA1 + AA2) variant groups were present in 22.5% of cervical cancers in contrast to 4.4% in the oropharyngeal cancers [24]. The number of cases studied does not allow us to explore the importance of those differences nor to understand the potential role determining the clinical behaviour and potential use to select treatment and prognosis. Hopefully, the new sequencing era will enrich the study of HPV and related cancers. The advances in HPV wholegenome sequencing [46] provided technically achievable large-scale longitudinal studies on HPV whole-genomic sequences and promotes an exhaustive understanding of Table 3 Frequency HPV16 Variants by region genome location (–) not evaluated; AA Asia-American HPV16 Variant; AF African HPV16 Variant; AS Asian HPV16 Variant; E European HPV16 variant; E6 HPV Early protein 6; E7 HPV Early protein 7; LCR long control region; L1 late gene; NA North American HPV16 variant Reference Total number of cases Total isolates for HPV sequencing HPV16 variant region location European North American (NA) African (AF1|AF2) Asian (AS) Asia American (AA) All lineages E-350-G [50] 115 115 L1 69% Not Detected - 15% - 8% [48, 51–54, 56] 621 106 E6 78% 29% 2% 2% 9% 4% [49] 24 21 E6E7 71% 53% - - - - [55] 205 21 LCR 42.8% - - - - 57.2% 87 Page 8 of 9 Cochichoetal. Virol J (2021) 18:217 the viral genetic diversity within and between infected individuals and will make the link between variants and cancer risk, comprehensively [46, 61]. Abbreviations AA: Asia-American HPV16 variant; AF: African HPV16 variant; AS: Asian HPV16 variant; E: European HPV16 variant; E6: HPV Early protein 6; E7: HPV Early protein 7; HNSCC: Head and neck squamous cell carcinoma; HPV: Human papillomavirus; NA: North American HPV16 VARIANT; NGS: Next generation sequencing. Acknowledgements Not applicable. Authors’ contributions These authors contributed equally to this work. Study concepts: AF, DC, RGC. Study design: AF, DC, RGC. Data acquisition: DC, AF, RGC. Data analysis and interpretation: RGC, DC, AF. Manuscript preparation: DC. Manuscript editing: AF, DC, RGC. Manuscript review: AF, DC, RGC. All authors read and approved the final manuscript. Funding Not applicable. Availability of data and materials Not applicable. Declarations Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Competing interests The authors declare that they have no competing interests. Author details 1 NOVA Medical School, NOVA University of Lisbon, CEDOC, Campo Mártires da Pátria 130, 1169-056 Lisbon, Portugal. 2 Virology Laboratory IPOLFG, Rua Professor Lima Bastos, 1099-023 Lisbon, Portugal. 3 LEPABE, Laboratory for Process Engineering, Environment, Biotechnology and Energy, University of Porto, Rua Dr. Roberto Frias, 4200-465 Porto, Portugal. 4 Post-graduate Programme in Adult Health (PPGSAD), University Hospital (HUUFMA) and Morphology Department, Federal University of Maranhão, Av. dos Portugueses 1966 - Vila Bacanga, São Luís, MA 65080-805, Brazil. 5 Molecular Oncology and Viral Pathology Group, Research Center of IPO Porto (CI-IPOP) / RISE@CI-IPOP (Health Research Network), Portuguese Oncology Institute of Porto (IPO Porto) / Porto Comprehensive Cancer Center (Porto.CCC), Rua Dr. António Bernardino de Almeida, 4200-072 Porto, Portugal. 6 Pathology Department IPOLFG, Rua Professor Lima Bastos, 1099-023 Lisbon, Portugal. 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Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 89 2.2 THE RELEVANCE OF HPV16 VARIANTS IN HEAD AND NECK SQUAMOUS CELL CARCINOMAS Accepted for publication in Pathobiology. Abstract The variants of the human papillomavirus (HPV) are believed to play an important role in the carcinogenesis process of head and neck squamous cell carcinomas (HNSCC). This study aims to establish the prevalence of HPV16 variants in a Portuguese HNSCC cohort, describe clinical-pathological characteristics and determine the association with patient survival. We retrieved 68 HNSCC patients from the Institute of Oncology in Lisboa. DNA samples were available from tumour biopsy at the time of the primary diagnosis. Targeted NGS was used to analyse whole-genome sequences and variants were established based on phylogenetics classification. The majority of the HPV16 samples clustered in lineage A and the remaining clustering within sublineages B, C and D. Comparative genome analysis revealed a total of 1356 SNV fall within the coding regions E1, E2, E4, E5, E6, E7 early genes and L1 and L2 late genes. Our integrated approach reports a deep molecular HNSCC HPV16 mutational profile characterization, intended to describe molecular characteristics of HPV16 variants and their biological significance in HN cancer. Keywords: K14HPV16, Variant 96 excluded due to persistence). In the remaining 27 records with HPV16 lineage A (n=20), Lineage B (n=2) and Lineage D (n=5) at 3 years of follow-up there were 4 events (relapse or death). Patients with lineage A and D had overlapping DFS with 100% at 3 years (no events). The Lineage B patients with complete response (n=2) died without relapse at median time 1.37-years. HPV16 variants identified and clinical presentation of HNSCC A recent publication identified eight SNVs as markers of poor prognosis [23]. Our study reports four of them individually present in thirteen patients (genome position 4410 n=1; position 4539 n=9; position 5962 n=1 and position 7173 n=2). Among these, we have ten men and three women, with an average age ≥65 and tumours in stages II-III (n=11). Most cases presented a complete response, while two cases showed disease persistence. In total, four death events were registered. The remaining patients are well without evidence of disease. Discussion The different oncogenic potential of HPV lineages in the development of cervical cancer has been recognized [37]. Conversely, in head-and-neck pathology, the mutational profile of HPV16 is still little explored and few studies present a wholegenome analysis of HPV-associated cancers [31]. In this context, our study of 35 cases provides a valuable contribution to a better characterization of the molecular profile of HPV-driven SCC in the head-and-neck region of a European country. The clinical-pathological characteristics of our cohort are in line with published literature. Male gender and oropharynx location are the predominant clinicalpathological aspects of this HPV16 positive squamous cell carcinoma series. Regarding HPV16 analysis most samples clustered in lineage A (n=26), the most common lineage described in Europe. This observation agrees with previous studies [38–41] showing a preponderance of lineage A strains, as recently reviewed [17]. This is also the case with cervical cancer [11,13,18], revealing an interesting similarity between the HPV16 strains involved in carcinogenesis at these two anatomical sites. In the present study, we analysed and compared the frequency of different HPV16 variants. The mutation profile describes 243 variable sites, of which 100 have been previously reported in cervical pathology, nine in SCC head-and-neck pathology, and 143 are possible new changes. The largest study on HNSCC HPV16 variants and SNV was very recently published and eight HPV16 SNVs were significantly associated with worse survival in a US population [23]. This was now compared with our Portuguese series, and we found four variants (one missense and 3 synonymous) in common (positions 4410, 4539, 5962 and 7173). However, our data do not allow us to draw conclusions regarding the potential of these variants to influence the tumour's biological behaviour. Altogether, 25 patients were alive at the last follow-up, and the remaining ten had death events. Several studies have shown that in the E6 coding region of HPV16 in the European lineage the E31G, L83V and D25E transitions were significantly associated with the development of cervical carcinoma [22,38–40]. Strikingly, none of these three substitutions was found in our data, highlighting the need for a more detailed 97 molecular characterization of HPV-associated cancers in different anatomic locations. In this context, it is also remarkable that the E7 N29S variant, associated with a higher oncogenic risk to cervical cancer [41], was only detected in a single HNSCC case, further highlighting the molecular differences associated with cervical and head-and-neck carcinogenesis. Similarly, previous observations from the HPV16-transgenic mouse model show differences in cancer penetrance and microRNA expression profiles between different anatomical sites [42–46], this is in the same line as what is observed in HPV-driven cancers. Taken together, these clinical and experimental data suggest that the interplay between different HPV16 strains and tissue-specific host factors differentially modulates carcinogenesis in different organs. The E7 gene was shown to be the most conserved, corroborating recent findings on the apparent restriction on E7 gene mutations in cervical cancer and in the oropharynx, sustaining the hypothesis that carcinogenicity depends on a highly conserved E7 protein [11,23]. Interestingly, the indicator of selective pressure revealed that none of the genes reached the positive pressure cut-off, and this was especially noticeable for the E7 gene, owing to purifying selection against nonsynonymous changes. We acknowledge that one of the limitations of the present study is the low number of cases. However, we provide high-quality whole-genome data on the HNSCC HPV16 mutational profile in 35 new cases of a Portuguese cohort, adding to the available knowledge of HPV16 variants and mutation site and amino acid changes that allow the understanding of the biological significance of each variant. In conclusion, the present data provide an in-depth genomic characterization of HPV16 present in HNSCC samples, revealing a marked predominance of lineage A strains. A total of 243 genome variable sites were identified, of which 143 are potentially new variations. Major differences were observed concerning the distribution of SNVs in our HSNCC cohort and in another HPV-associated malignancy, cervical cancer. Overall, these data contribute for a deeper understanding of the influence of these mutations on carcinogenesis, diagnostics, and clinical management. Statements Acknowledgement (optional) In the Acknowledgement section, authors must include individuals and organizations that have made substantive contributions to the research or the manuscript. An exception is where funding was provided, which should be included in Funding Sources. Please refer to the Guidelines issued by the ICMJE to determine nonauthor contributors that should be included in the Acknowledgement section. Statement of Ethics All the data were anonymized, and the study was conducted in accordance with the Helsinki Declaration and approved by the Ethics Committee of the Portuguese Oncologic Institute Francisco Gentil, Lisbon (Ref. UIC/2019/1168). A written informed consent was obtained from all participants of the study. Conflict of Interest Statement 98 The authors have no conflicts of interest to declare. Funding Sources This study was financially supported by the Virology Laboratory Pathology Department from the Portuguese Oncology Institute of Lisboa IUIC/1168, with contributions by the GenomePT project (POCI-01-0145-FEDER-022184), supported by COMPETE 2020 - Operational Programme for Competitiveness and Internationalisation (POCI), Lisboa Portugal Regional Operational Programme (Lisboa2020), Algarve Portugal Regional Operational Programme (CRESC Algarve2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF), and by Fundação para a Ciência e a Tecnologia (FCT). Author Contributions Contribution authors AF, DC and RGC for study concepts and study design; the authors AN, DC, DS, JPG, JM, LM, LV, MC, MM, and PM performed and data acquisition; the author's AN, DC, LV and JPG analyse the genomic data; the authors AF, AN, DC, LM, MC, RGC and SE were responsible for analysis and interpretation; the author DC for manuscript preparation; the authors AF, AN, DC, DS, LM, MC, RGC and SE for manuscript editing and review. Critical revision of the article for important intellectual content was performed by AF, AN, DC, JPG, LV, MC, MM, PM and RGC. All the authors reviewed and approved the final version of the manuscript. Data Availability Statement The authors confirm that the data supporting the findings of this study are available within the article and its supplementary materials. Further enquiries can be directed to the corresponding author. References 1 Ferlay J, Soerjomataram I, Rajesh Dikshit, Sultan Eser, Mathers C, Rebelo M, et al. 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Braz J Med Biol Res. 2006 Jun;39(6):707–17. 41 Song YS, Kee SH, Kim JW, Park NH, Kang SB, Chang WH, et al. Major sequence variants in E7 gene of human papillomavirus type 16 from cervical cancerous and noncancerous lesions of Korean women. Gynecol Oncol. 1997 Aug;66(2):275–81. 42 Santos C, Vilanova M, Medeiros R, Gil da Costa RM. HPV-transgenic mouse models: Tools for studying the cancer-associated immune response. Virus Research. 2017 May;235:49– 57. 43 Costa AC, Santos JMO, Medeiros-Fonseca B, Oliveira PA, Bastos MMSM, Brito HO, et al. Characterizing the Inflammatory Microenvironment in K14-HPV16 Transgenic Mice: Mast Cell Infiltration and MicroRNA Expression. Cancers (Basel). 2022 Apr;14(9):2216. 44 Araújo R, Santos JMO, Fernandes M, Dias F, Sousa H, Ribeiro J, et al. Expression profile of microRNA-146a along HPV-induced multistep carcinogenesis: a study in HPV16 transgenic mice. J Cancer Res Clin Oncol. 2018 Feb;144(2):241–8. 102 45 Paiva I, Gil da Costa RM, Ribeiro J, Sousa H, Bastos MMSM, Faustino-Rocha A, et al. MicroRNA-21 expression and susceptibility to HPV-induced carcinogenesis - role of microenvironment in K14-HPV16 mice model. Life Sci. 2015 May;128:8–14. 46 Paiva I, Gil da Costa RM, Ribeiro J, Sousa H, Bastos M, Faustino-Rocha A, et al. A role for microRNA-155 expression in microenvironment associated to HPV-induced carcinogenesis in K14-HPV16 transgenic mice. PLoS One. 2015;10(1):e0116868. Figure Legends Fig.1 HPV16 phylogeny in HNSCC samples. The phylogeny tree was reconstructed using the Neighbor-Joining method [38] with the Maximum Composite Likelihood model [37]. Numbers next to the branch nodes indicate the bootstrap values (1000 replicates). 103 Fig.2 Non-synonymous variant sites in HPV16 genome. For each genome position of the HPV16 A1 sublineage representative (NC_001526.4), all non-synonymous mutations displayed by the 35 HNSCC tumour samples are shown. Viral coding regions are represented on the top of the figure, while on the left, the phylogenetic tree with the patient samples shown in Figure 1. Nucleotide variants are colouredcoded, with nucleotides C, G, T, and A represented by yellow, red, blue and green (coloured-coded), respectively. Fig.3 Nonsynonymous-to-synonymous mutation ratio for each HPV16 gene. For each gene, estimates were based on the p-distance model. Minimum and maximum values represent lower and upper limits of the standard error (SE) of the estimate (vertical bar). For better visualization, the positive pressure threshold is shown (horizontal red line). 104 Fig.4 Kaplan–Meier plots for overall survival by each HPV lineage. 105 2.3 CHARACTERIZATION OF THE HPV16 E6 AND E7 ONCOGENES IN K14HPV16 MICE: SUBLINEAGE A1 DRIVES MULTI-ORGAN CARCINOGENESIS. Int. J. Mol. Sci. DOI: https://doi.org/10.3390/ijms232012371 Abstract The study of HPV-induced carcinogenesis relies on multiple in vivo mouse models, one of which relies on the cytokeratin 14 gene (CK14) promoter to drive the expression of all HPV early oncogenes. Our study evaluated HPV DNA from 17 samples from 4 animals, wild-type (WT, n=2) and HPV16-transgenic mice (MUT, n=2). Total DNA was extracted and detection of HPV16 was performed using a qPCR multiplex. HPV16-positive samples were subsequently whole-genome sequenced by NGS techniques. Comparative genome analysis of K14HPV16 samples revealed a total of 12 mutations to the HPV16 sublineage A1 representative strain. Most of the mutations fall within the coding regions of E1, E2, E4 and E5 early genes and of L1 and L2 late genes. The phylogenetic positioning clearly shows K14HPV16 samples clustering together in the sub-lineage A1 (NC001526.4). The knowledge of the variant is very important, and these findings will allow the rational use of this animal model to explore the role of the A1 sublineage in HPV-driven cancer. Keywords: K14HPV16, Variant 112 Int. J. Mol. Sci. 2022,23, 12371 5 of 10 exhibiting differential intermediate frequencies across the nine K14HPV16 samples (from 12.3% for K14HPV16_15 to 22.1% for K14HPV16_11). 3. Discussion HPV16 variants show a differential prevalence in diverse geographical locations and are associated with differential oncogenic potential [ 5 – 8 ]. Caucasian women infected with the A1/A2 variant are at a higher risk of CIN3+ compared to women of other genetic backgrounds [ 19 ]. This is also the most common variant found in head and neck oral cancers in Caucasians and cervical carcinomas [ 20 ], suggesting a heightened oncogenic potential compared with other HPV16 variants. For the first time, the present study determined the variant lineage present in the K14HPV16 transgenic mouse model, based on the relatedness of reference sequences including ten HPV16 A, B, C, and D variant lineages. The K14HPV16 mouse model was shown to carry the HPV16 A1 sublineage. Despite their isolation from distinct tissues, the K14HPV16 samples were found to be highly genetically related among them, only differing by a mean of 1.0 ± 0.8 nucleotides and showing a mean distance of 3.1 ± 1.7 nucleotides from the phylogenetically closest sublineage A1 representative strain. On the other hand, K14HPV16 samples exhibited a mean distance to other sublineages that ranged from 22.0 ± 4.5 (sublineage A3) to 112.0 ± 10.3 (sublineage D2). Considering that variant HPV sublineages are empirically defined as exhibiting genome differences in the 0.5–1.0% range [ 21 ], respectively, these results point to a clonal origin of K14HPV16 samples, in agreement with the congenic nature of this mouse strain [12]. Having determined the variant lineage and sublineage present in K14HPV16 mice, it is now possible to discuss its association with the various kinds of neoplastic lesions observed in this mouse strain. K14HPV16 mice have been used to mimic the development of cervical cancer in the 1990s [ 13 ]. In this study, the HPV16 oncogenes were necessary but insufficient to induce cervical cancer and chronic estrogen supplementation was required for carcinogenesis. Based on the present results, it is interesting to speculate that, although the A1 sublineage is clinically associated with a heightened risk of cervical cancer, it may require hormonal co-factors for efficient carcinogenesis. Recently, our group employed K14HPV16 mice for producing the first mouse model of HPV-related penile cancer [ 22 ]. Again, the A1 lineage was necessary but insufficient to induce invasive squamous cell carcinoma and a tobacco-related co-carcinogen was needed. However, the HPV16 A1 lineage was able to induce a range of penile intraepithelial lesions in this model without additional co-factors. Interestingly, K14HPV16 mice develop squamous cell carcinomas specifically located at the tongue base, without the need for any chemical or hormonal co-carcinogens—although tumors were more frequent in female mice [ 15 ]. Oropharyngeal cancer incidence could be increased in this model by exposure to ptaquiloside, a bracken fern carcinogen, suggesting a synergistic effect [ 23 ]. Based on these observations, we speculate that the oncogenic potential of the HPV16 A1 variant lineage in this mouse model is dependent on the anatomic site: cervical and penile carcinogenesis require the presence of co-factors while oropharyngeal cancer may be solely induced by the viral oncogenes, although the predominant incidence in female mice suggests a role of the higher physiological state of estrogen. Further studies are warranted to examine the interplay between different HPV16 lineages and hormonal co-factors. Remarkably, another study using mice carrying only the HPV16 E6 and E7 transgenes without variant assignment found that oropharyngeal carcinogenesis depended on a chemical co-carcinogen [24]. The K14HPV16 mouse model was also used to test new therapies and cancer preventive strategies by our group and others [ 14 , 16 , 25 , 26 ]. In general, the development of lesions in this animal model is strongly associated with modulation of the immune response within the tumor microenvironment, and combined immune therapy approaches are required to prevent lesion development [ 26 ]. Now, it will be possible to assess the specific impact of those approaches on the HPV16 A1 sublineage, and it would be desirable to develop animal models that represented other HPV16 strains as well. 113 Int. J. Mol. Sci. 2022,23, 12371 4 of 10 samples were not included in the phylogenetic analysis. Nevertheless, they share the same major mutations as the remaining K14HPV16 samples against the HPV16 sublineage A1 representative strain (see details below). The phylogenetic analysis (Figure 3) clearly shows K14HPV16 samples clustering together in the sub-lineage A1 (NC001526.4). Int. J. Mol. Sci. 2022, 23, x FOR PEER REVIEW 4 of 11 2.3. K14HPV16 Lineage Classification HPV16 variants have been classified into four major lineages (A–D) and sublineages (A1–A4, B1, B2, and D1–D3), based on their genome sequence diversity [17]. To determine the phylogenetic positioning of the HPV16 transgenes in K14HPV16 samples, the respective consensus sequences were aligned against the genomes of lineage/sublineage representative sequences (Supplementary Table S1). For K14HPV16-3 and K14HPV16-6, the generation of a consensus sequence failed (% of genome covered <70%), and those two samples were not included in the phylogenetic analysis. Nevertheless, they share the same major mutations as the remaining K14HPV16 samples against the HPV16 sublineage A1 representative strain (see details below). The phylogenetic analysis (Figure 3) clearly shows K14HPV16 samples clustering together in the sub-lineage A1 (NC001526.4). Figure 3. Phylogenetic positioning of K14HPV16 samples within the major HPV16 lineages (A–D). Legend: the phylogenetic tree was generated using the neighbor-joining method [18] with the maximum composite likelihood model [17] and depicts the genetic relationships of the obtained K14HPV16 genome consensus sequences to each representative genome. Numbers next to the branch nodes indicate the bootstrap values (1000 replicates). HPV16 lineages are shown in grey Figure 3. Phylogenetic positioning of K14HPV16 samples within the major HPV16 lineages (A–D) . Legend: the phylogenetic tree was generated using the neighbor-joining method [ 18 ] with the maximum composite likelihood model [ 17 ] and depicts the genetic relationships of the obtained K14HPV16 genome consensus sequences to each representative genome. Numbers next to the branch nodes indicate the bootstrap values (1000 replicates). HPV16 lineages are shown in grey boxes next to the main tree branches, while sublineages (A1–A4, B1, B2, and D1–D3) are illustrated in black boxes. 2.4. HPV16 Sequencing Comparative genome analysis of K14HPV16 samples revealed a total of three mutations to the HPV16 sublineage A1 representative strain. All mutations fall within the coding region of the E1 early gene, with more than half yielding amino acid changes. Two of these (330A > G|Leu110Leu and 978A > G|Ile326Met) have become evolutionarily fixed (frequency of 100%) based on the observed deep coverage supporting each alteration. Indeed, 100% of the reads mapping each position (for pos 330: ranging from 1580x for sample 6 to 15456x for sample 8; and for pos. 978; ranging from 636x for sample 6 to 11951x for sample 8) support the two variants found in all K14HPV16 samples. The last mutation is a non-synonymous minor intra-patient single nucleotide variant (166A > T|Asn56Tyr) 114 Int. J. Mol. Sci. 2022,23, 12371 3 of 10 2. Results 2.1. Sample Characterization Histological samples from wild-type (WT) animals showed normal histology and HPV16-transgenic (MUT) mice samples showed typical proliferative epithelial lesions of the skin and tongue, while the liver and lymph node samples from WT and HPV16trangenic mice showed mild inflammatory changes or none at all. The oral tumor found in the HPV16-trangenic mice was identified as a squamous cell carcinoma, with minimal invasion of the tongue (Figure 2). Int. J. Mol. Sci. 2022, 23, x FOR PEER REVIEW 3 of 11 Figure 1. Schematic of the HPV-16R genome. The genome organization of 7906 base pairs (bp) arranged in a circular form represents the three functional regions, early, late, and long control (LCR); and a non-coding region (NCR). These are separated by two polyadenylation sites, described as early (pAE) and late (pAL). In total, the genome encodes eight open reading frames (ORFs), of which E1, E2, E4, E5, E6, and E7 are from the early region; and L1 and L2 are from the late region. The LCR, located between the E6 and L1 gene, is a regulatory region that includes the origin of replication (ori) and the p97 promoter. 2. Results 2.1. Sample Characterization Histological samples from wild-type (WT) animals showed normal histology and HPV16-transgenic (MUT) mice samples showed typical proliferative epithelial lesions of the skin and tongue, while the liver and lymph node samples from WT and HPV16trangenic mice showed mild inflammatory changes or none at all. The oral tumor found in the HPV16-trangenic mice was identified as a squamous cell carcinoma, with minimal invasion of the tongue (Figure 2). Figure 2. Histological findings of WT and MUT tongue and oral cancer. Legend: Histological samples from wild-type and transgenic K14HPV16 mice stained with H&E. (A) low power view of normal squamous cell epithelium of the tongue from a wild-type mouse; (B) low power view of mild dysplastic squamous cell epithelium with enlarged hyperchromatic nuclei in the upper layers of the epithelium from a transgenic K14HPV16 mouse and (C) low power view of a focally invasive squamous cell carcinoma with thickened rete ridges and dysplastic cells with hyperchromatic nuclei, irregular basement membrane and focally disrupted associated with inflammatory cells in the stroma from a transgenic K14HPV16 mouse; the inset shows the invasive front. (D) low power view of the liver of one transgenic K14HPV16 mouse showing normal structure and cytology. 2.2. HPV16 Genome Coverage and Quality Among the nine HPV16-positive samples, the mean depth of coverage was 2080-fold, ranging between 870-fold for the node sample and 3726-fold for the tumor sample. On average, samples had 75% of the HPV16 genome covered, with 95% of it covered by at least 10-fold. The only exception was a ~1900 bp region comprising most of the L1 gene, the upstream regulatory region (URR) and the beginning of the E6 gene for which no amplification signal was observed for all samples. This is in agreement with the model design [12] which encompasses the entire HPV16 early coding region from bp 97 to 6152, although the gap between the L1 and E6 region (~1945 pb) compromises the hybridization efficiency of specific primers within the primer pool for that region. Figure 2. Histological findings of WT and MUT tongue and oral cancer. Legend: Histological samples from wild-type and transgenic K14HPV16 mice stained with H&E. ( A ) low power view of normal squamous cell epithelium of the tongue from a wild-type mouse; ( B ) low power view of mild dysplastic squamous cell epithelium with enlarged hyperchromatic nuclei in the upper layers of the epithelium from a transgenic K14HPV16 mouse and ( C ) low power view of a focally invasive squamous cell carcinoma with thickened rete ridges and dysplastic cells with hyperchromatic nuclei, irregular basement membrane and focally disrupted associated with inflammatory cells in the stroma from a transgenic K14HPV16 mouse; the inset shows the invasive front. ( D ) low power view of the liver of one transgenic K14HPV16 mouse showing normal structure and cytology. 2.2. HPV16 Genome Coverage and Quality Among the nine HPV16-positive samples, the mean depth of coverage was 2080-fold, ranging between 870-fold for the node sample and 3726-fold for the tumor sample. On average, samples had 75% of the HPV16 genome covered, with 95% of it covered by at least 10-fold. The only exception was a ~1900 bp region comprising most of the L1 gene, the upstream regulatory region (URR) and the beginning of the E6 gene for which no amplification signal was observed for all samples. This is in agreement with the model design [ 12 ] which encompasses the entire HPV16 early coding region from bp 97 to 6152, although the gap between the L1 and E6 region (~1945 pb) compromises the hybridization efficiency of specific primers within the primer pool for that region. 2.3. K14HPV16 Lineage Classification HPV16 variants have been classified into four major lineages (A–D) and sublineages (A1–A4, B1, B2, and D1–D3), based on their genome sequence diversity [ 17 ]. To determine the phylogenetic positioning of the HPV16 transgenes in K14HPV16 samples, the respective consensus sequences were aligned against the genomes of lineage/sublineage representative sequences (Supplementary Table S1). For K14HPV16-3 and K14HPV16-6, the generation of a consensus sequence failed (% of genome covered <70%), and those two 115 Int. J. Mol. Sci. 2022,23, 12371 2 of 10 1. Introduction Papillomaviruses are species-specific double-stranded DNA viruses of 8000 base pairs (bp) length (Figure 1) that have preferential tropism for epithelial cells. Infection with human papillomavirus (HPV) is the most common sexually transmissible infection and induces a range of benign (e.g., condylomas) and malignant lesions, such as cervical cancer and other anogenital squamous cell carcinomas and a growing subset of oropharyngeal squamous cell carcinomas [ 1 ]. HPV can be classified into genotypes defined by a greater than 10.0% variation in their L1 gene sequence [ 2 ]. Currently, over 200 HPV types are recognized and grouped as high-risk (e.g., HPV16 and HPV18, associated with malignant cervical lesions) or low-risk (HPV6 and HPV11, associated with benign lesions) [ 2 – 4 ]. Accumulating data indicate that HPV intra-type variants, lineages (defined by a 1.0–10.0% genomic variation), and sub-lineages (0.5–1.0% variation) may differ in their carcinogenic potential [ 5 – 8 ], as recently reviewed [ 9 , 10 ]. The study of HPV-induced carcinogenesis uses multiple in vivo mouse models [ 11 ], one of which relies on the Krt14 (cytokeratin 14) gene promoter to drive the expression of all HPV16 early oncogenes and specifically target basal keratinocytes (known as K14HPV16 mice) [ 12 ]. This widely used model was first developed in the 1990s and has since been used to model cervical cancer [ 13 ] and other HPV16-associated malignancies [ 14 – 16 ]. This animal model has also proved useful to elucidate the immune-modulatory mechanisms involved in HPV16-induced cancers and to test potential new therapies. However, the specific variant and sub-lineage involved in K14HPV16 mice remain unknown [ 12 ]. Determining which variant and sublineage are present in this animal model would help test their potential associations with the development of cancer at specific locations. Additionally, this information would help researchers. This study aimed to determine the HPV16 variant and sublineage present in the K14HPV16 mouse model, further characterizing this important research tool and potentiating its use for basic and translational studies. Int. J. Mol. Sci. 2022, 23, x FOR PEER REVIEW 2 of 11 Keywords: K14HPV16; carcinogenesis; HPV16; variant; lineage 1. Introduction Papillomaviruses are species-specific double-stranded DNA viruses of 8000 base pairs (bp) length (Figure 1) that have preferential tropism for epithelial cells. Infection with human papillomavirus (HPV) is the most common sexually transmissible infection and induces a range of benign (e.g., condylomas) and malignant lesions, such as cervical cancer and other anogenital squamous cell carcinomas and a growing subset of oropharyngeal squamous cell carcinomas [1]. HPV can be classified into genotypes defined by a greater than 10.0% variation in their L1 gene sequence [2]. Currently, over 200 HPV types are recognized and grouped as high-risk (e.g., HPV16 and HPV18, associated with malignant cervical lesions) or low-risk (HPV6 and HPV11, associated with benign lesions) [2– 4]. Accumulating data indicate that HPV intra-type variants, lineages (defined by a 1.0– 10.0% genomic variation), and sub-lineages (0.5–1.0% variation) may differ in their carcinogenic potential [5–8], as recently reviewed [9,10]. The study of HPV-induced carcinogenesis uses multiple in vivo mouse models [11], one of which relies on the Krt14 (cytokeratin 14) gene promoter to drive the expression of all HPV16 early oncogenes and specifically target basal keratinocytes (known as K14HPV16 mice) [12]. This widely used model was first developed in the 1990s and has since been used to model cervical cancer [13] and other HPV16-associated malignancies [14–16]. This animal model has also proved useful to elucidate the immune-modulatory mechanisms involved in HPV16-induced cancers and to test potential new therapies. However, the specific variant and sub-lineage involved in K14HPV16 mice remain unknown [12]. Determining which variant and sublineage are present in this animal model would help test their potential associations with the development of cancer at specific locations. Additionally, this information would help researchers. This study aimed to determine the HPV16 variant and sublineage present in the K14HPV16 mouse model, further characterizing this important research tool and potentiating its use for basic and translational studies. Figure 1. Schematic of the HPV-16R genome. The genome organization of 7906 base pairs (bp) arranged in a circular form represents the three functional regions, early, late, and long control (LCR); and a non-coding region (NCR). These are separated by two polyadenylation sites, described as early (pAE) and late (pAL). In total, the genome encodes eight open reading frames (ORFs), of which E1, E2, E4, E5, E6, and E7 are from the early region; and L1 and L2 are from the late region. The LCR, located between the E6 and L1 gene, is a regulatory region that includes the origin of replication (ori) and the p97 promoter. 116 2.4 PIK3CA GENE MUTATIONS IN HNSCC: SYSTEMATIC REVIEW AND CORRELATIONS WITH HPV STATUS AND PATIENT SURVIVAL Cancers. DOI: 10.3390/cancers14051286 Abstract PIK3CA mutations are believed to contribute to the pathogenesis of human papillomavirus (HPV)-associated head and neck squamous cell carcinomas (HNSCC). This study aims to establish the frequency of PIK3CA mutations in a Portuguese HNSCC cohort and to determine their association with HPV status and patient survival. A meta-analysis of scientific literature also done revealed widely different mutation rates in cohorts from different world regions and a trend towards improved prognosis among patients with PIK3CA mutations. DNA samples were available from 95 patients diagnosed with HNSCC at the Portuguese Institute of Oncology in Lisbon, between 2010 and 2019. HPV status was established based on viral DNA detected using real-time PCR. Evaluation of PIK3CA gene mutations was performed by real-time PCR for 4 mutations (H1047L; E542K, E545K, E545D). 37 cases were found to harbour PIK3CA mutations (39%) with the E545D mutation (73%) more frequent detected. There were no significant associations between mutational status and HPV status (74% WT and 68% MUT were HPV (+); p=0.489) or overall survival (OS) (3-yr OS: WT 54% and MUT 65%; p= 0.090). HPV status was the only factor significantly associated with both OS and disease-free survival (DFS), with HNSCC-HPV patients having consistently better outcomes (3yr OS: HPV (+) 65% and HPV (-) 36%; p=0.007; DFS HPV (+) 83% and HPV (-) 43%; p=0.001). There was a statistically significant interaction effect between HPV status and PIK3CA mutation regarding DFS (Interaction test: p=0.026). In HNSCC-HPV patients, PIK3CA wild type is associated with a significant 4.64 times increase in the hazard of recurrence or death (HR=4.64; 95% CI 1.02-20.99; p=0.047). Overall, PIK3CA gene mutations are present in many patients and may help define patient subsets who can benefit from therapies targeting the PI3K pathway. The systematic assessment of PIK3CA gene mutations in HNSCC patients will require further methodological standardization. Keywords: HNSCC; HPV; p16 INK4a; PIK3CA 117 !"#!$%&'(! !"#$%&' Citation: Cochicho, D.; Esteves, S.; Rito, M.; Silva, F.; Martins, L.; Montalvão, P.; Cunha, M.; Magalhães, M.; Gil da Costa, R.M.; Felix, A. PIK3CA Gene Mutations in HNSCC: Systematic Review and Correlations with HPV Status and Patient Survival. Cancers 2022,14, 1286. https:// doi.org/10.3390/cancers14051286 Academic Editor: David Wong Received: 5 February 2022 Accepted: 25 February 2022 Published: 2 March 2022 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). cancers Article PIK3CA Gene Mutations in HNSCC: Systematic Review and Correlations with HPV Status and Patient Survival Daniela Cochicho 1,2, Susana Esteves 3, Miguel Rito 4, Fernanda Silva 1, Luís Martins 2, Pedro Montalvão5, Mário Cunha 2, Miguel Magalhães 5, Rui M. Gil da Costa 6,7,8,9,† and Ana Felix 1,4,*,† 1NOVA Medical School, NOVA University of Lisbon, 1099-085 Lisbon, Portugal; [email protected] (D.C.); [email protected] (F.S.) 2Virology Laboratory from Clinical Pathology Department, IPOLFG, 1099-023 Lisbon, Portugal; [email protected] (L.M.); [email protected] (M.C.) 3Clinical Research Unit, IPOLFG, 1099-023 Lisbon, Portugal; [email protected] 4Pathology Department, IPOLFG, 1099-023 Lisbon, Portugal; [email protected] 5Otorhinolaryngology Department, IPOLFG, 1099-023 Lisbon, Portugal; [email protected] (P.M.); [email protected] (M.M.) 6Post-Graduate Programme in Adult Health (PPGSAD), Morphology Department, University Hospital (HUUFMA), Federal University of Maranhão, São Luís 65080-805, Brazil; [email protected] 7LEPABE, Laboratory for Process Engineering, Environment, Biotechnology and Energy, Faculty of Engineering, University of Porto, Rua Roberto Frias, 4200-465 Porto, Portugal 8Centre for the Research and Technology of Agro-Environmental and Biological Sciences (CITAB), Inov4Agro, University of Trás-os-Montes e Alto Douro (UTAD), Quinta de Prados, 5000-801 Vila Real, Portugal 9Molecular Oncology and Viral Pathology Group, Research Center of IPO Porto (CI-IPOP)/RISE@CI-IPOP (Health Research Network), Portuguese Oncology Institute of Porto (IPO Porto)/Porto Comprehensive Cancer Center (Porto.CCC), 4200-162 Porto, Portugal *Correspondence: [email protected] † These authors contributed equally to this work. Simple Summary: Mutations of the PIK3CA gene are thought to contribute to the development of head and neck squamous cell carcinomas (HNSCC), especially those associated with human papillomavirus infection. Furthermore, these mutations may help identify patients who can benefit from specific targeted therapies. This study presents a systematic review of the PIK3CA mutations profile in HNSCC. The results are compared with a cohort of Portuguese patients to study the possible associations with HPV status and patient survival. The Portuguese cohort harboured PIK3CA mutations in 39% of patients, and there were no significant associations with the HPV status or with the OS. In this original case series, there was a statistically significant interaction effect between HPV status and PIK3CA mutation regarding disease-free survival. In HPV-positive patients, the PIK3CA wild-type is associated with a significant 4.64 times increase in the hazard of recurrence or death. Additional studies are needed to clarify the implications of PIK3CA mutations for patient prognosis. Abstract: PIK3CA mutations are believed to contribute to the pathogenesis of human papillomavirus (HPV)-associated head and neck squamous cell carcinomas (HNSCC). This study aims to establish the frequency of PIK3CA mutations in a Portuguese HNSCC cohort and to determine their association with the HPV status and patient survival. A meta-analysis of scientific literature also revealed widely different mutation rates in cohorts from different world regions and a trend towards improved prognosis among patients with PIK3CA mutations. DNA samples were available from 95 patients diagnosed with HNSCC at the Portuguese Institute of Oncology in Lisbon between 2010 and 2019. HPV status was established based on viral DNA detected using real-time PCR. The evaluation of PIK3CA gene mutations was performed by real-time PCR for four mutations (H1047L; E542K, E545K, and E545D). Thirty-seven cases were found to harbour PIK3CA mutations (39%), with the E545D mutation (73%) more frequently detected. There were no significant associations between the mutational status and HPV status (74% WT and 68% MUT were HPV (+); p= 0.489) or overall survival Cancers 2022,14, 1286. https://doi.org/10.3390/cancers14051286 https://www.mdpi.com/journal/cancers 118 Cancers 2022,14, 1286 19 of 19 44. Shigaki, H.; Baba, Y.; Watanabe, M.; Murata, A.; Ishimoto, T.; Iwatsuki, M.; Iwagami, S.; Nosho, K.; Baba, H. PIK3CA mutation is associated with a favorable prognosis among patients with curatively resected esophageal squamous cell carcinoma. Clin. Cancer Res. 2013,19, 2451–2459. [CrossRef][PubMed] 45. Zi˛eba, S.; Chechli´nska, M.; Kowalik, A.; Kowalewska, M. Genes, pathways and vulvar carcinoma-New insights from nextgeneration sequencing studies. Gynecol. Oncol. 2020,158, 498–506. [CrossRef][PubMed] 46. Qiu, W.; Tong, G.X.; Manolidis, S.; Close, L.G.; Assaad, A.M.; Su, G.H. 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[CrossRef][PubMed] 120 Cancers 2022,14, 1286 17 of 19 Institutional Review Board Statement: The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Institutional Ethics Committee of IPOLFG (protocol code UIC/1168, approved on 29-10-2019) and Ethics Committee of NMS|FCM-UNL (CEFCM) (protocol code 70/2019/CEFCM, approved on 23-01-2020). Informed Consent Statement: Informed consent was obtained from all subjects involved in the study. Data Availability Statement: The data presented in this study are available on request from the corresponding author. The data are not publicly available due to privacy. Conflicts of Interest: The authors declare no conflict of interest. References 1. Ferlay, J.; Soerjomataram, I.; Dikshit, R.; Eser, S.; Mathers, C.; Rebelo, M.; Parkin, D.M.; Forman, D.; Bray, F. Cancer incidence and mortality-Major patterns in GLOBOCAN 2012, worldwide and Georgia. Int. J. Cancer 2015 ,136, E359–E386. [CrossRef][PubMed] 2. 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We evaluated the prognostic impact of PIK3CA mutations and p16 overexpression on the overall survival (OS) and disease-free survival (DFS). Overall survival was defined as the period of time (in years) from the date of diagnosis to the date of death from any cause, patients alive were censored at the date of last follow-up assessment. Disease-free survival was evaluated in the patients with a complete response after the first treatment and was defined as the time from the end of the first treatment to disease relapse or death from any cause; patients alive without disease recurrence were censored at the date of the last follow-up assessment. We used Kaplan–Meier curves to visualize the differences in survival between subgroups defined by PIK3CA mutation, p16 overexpression, and HPV status and the log-rank test for group comparisons. Cox proportional hazards regression analysis was used to compute the hazard ratios (HRs) and 95% Confidence Intervals based on Wald statistics, with OS and DFS as the outcome variables and adjusting for age, HPV status, and drinking/smoking habits as potential confounding factors. Age and HPV status were chosen a priori as the most important confounding factors based on clinical criteria, and drinking/smoking habits were also included due to the imbalances observed between the PIK3CA MUT and WT groups in our cohort. The proportional hazards assumption was checked using statistical tests and graphical diagnosis based on Schoenfeld residuals. We also graphically assessed the functional form of the age variable in the models using Martingale residuals. As this analysis showed that the linearity assumption was not acceptable in the OS model, we adjusted the nonlinear effect of age with a smoothing spline using the “pspline” function of the R package “survival”. The potential interaction between HPV status and PIK3CA mutations on survival was tested using the log-likelihood ratio test between the fitted Cox regression models with and without the interaction term. The analyses were conducted in the complete case dataset. All statistical tests were two-sided, and we considered a significance level of 5%. As this was an exploratory study, no p-value correction for multiple testing was done. We used the software R package version 4.1.0 (http://www.R-project.org, accessed on 1 October 2021) [47]. 5. Conclusions Overall, these results confirmed a high frequency of canonical PIK3CA mutations (substitutions H1047L and E542K, E545K, and E545D) in HNSCC, including in HPV (  ) cases, suggesting that screening for PIK3CA mutations should not be restricted to HPV (+) patients. Additional studies are needed to clarify the implications of PIK3CA mutations for HNSCC patient prognosis. Supplementary Materials: The following supporting information can be downloaded at https: //www.mdpi.com/article/10.3390/cancers14051286/s1: Figure S1: Participant flow chart. Figure S2 : Kaplan–Meier overall survival curves for the included and excluded groups. Figure S3: Flow chart from the meta-analysis inclusion criteria. Table S1: Systematic Review HNSCC PIK3CA qPCR. Table S2: Overall, the 17 articles selected for HNSCC PIK3CA mutation. Table S3: HNSCC PIK3CA mutational profile. Table S4: Clinical and demographic characterisation of the 295 excluded cases. Author Contributions: Conceptualisation, A.F. and R.M.G.d.C.; Data curation: D.C., M.M., P.M., M.R., F.S., M.C. and L.M.; Formal analysis, D.C., S.E., M.R. and A.F.; Writing—original draft preparation, D.C., R.M.G.d.C. and A.F.; and Writing—review and editing, R.M.G.d.C. and A.F. All authors have read and agreed to the published version of the manuscript. Funding: This study was financially supported by the Virology Laboratory from the Pathology Department of the Portuguese Oncology Institute of Lisboa IUIC/1168, with contributions by the Research Center of the Portuguese Oncology Institute of Porto (project no. PI86-CI-IPOP-66-2017), by Base Funding-UIDB/00511/2020 of the Laboratory for Process Engineering, Environment, Biotechnology, and Energy—LEPABE—funded by national funds through the FCT/MCTES (PIDDAC), and Project 2SMART-engineered Smart materials for Smart citizens, with reference NORTE-010145-FEDER-000054, supported by Norte Portugal Regional Operational Programme (NORTE 2020) under the PORTUGAL 2020 Partnership Agreement through the European Regional Development Fund (ERDF). 128 Cancers 2022,14, 1286 9 of 19 Figure 1. Kaplan–Meier curves comparing patients with PIK3CA-mutated vs. the wild-type at diagnosis ( A ). Overall survival (Log-rank test chi-square = 2.9 with 1 degree of freedom, p= 0.090). (B). Disease-free survival (Log-rank test chi-square = 1.7 with 1 degree of freedom, p= 0.198). Figure 2. Cont. 129 Cancers 2022,14, 1286 8 of 19 Analysis PIK3CA, HPV Status, and p16 Immunohistochemistry OS and DFS were determined separately for patients grouped by the PIK3CA mutational status, HPV DNA, and p16 INK4a (Table 4and Figures 1and 2). During the univariable analysis, we could not demonstrate a significant association between the PIK3CA mutation status and OS or DFS (Table 4and Figure 1A,B). The HPV status was the only factor significantly associated with both OS and DFS, with patients with positive HPV having consistently better outcomes compared with the ones with non-detected HPV (Table 4and Figure 2A,B). Patients with p16 overexpression had significantly longer DFS than patients with no p16 overexpression, but no significant difference could be demonstrated concerning OS (Table 4). Table 4. PIK3CA mutation, HPV, and p16 status association with the overall and disease-free survival by univariable analysis. Overall Survival Disease-Free Survival Median (Years) 3-Year % (95% CI) HR (95%CI) p*Median, (Years) 3-Year % (95% CI) HR (95% CI) p* PIK3CA MUT 6.2 65 (50–83) 1 0.090 NR 77 (63–95) 1 0.198 WT 4.0 54 (41–70) 1.71 (0.91–3.18) 4.8 72 (58–90) 1.80 (0.73–4.42) HPV status Positive 5.8 65 (54–78) 1 0.007 NR 83 (72–94) 1 0.001 Not detected 2.4 36 (19–69) 2.40 (1.15–4.61) 2.2 43 (21–90) 4.81 (1.74–13.29) p16 overexpression No 2.6 46 (33–64) 1 0.089 4.8 59 (44–80) 1 0.029 Yes 6.9 73 (60–90) 0.58 (0.31–1.10) NR 91 (81–100) 0.34 (0.13–0.94) * Log-rank test; HR = Hazard Ratio; 95% CI = 95% Confidence Interval; NR = Not Reached; MUT = mutated; Wt = wild-type. Figure 1. Cont. 130 Cancers 2022,14, 1286 7 of 19 2.2.2. Prevalence of PIK3CA Mutations The PIK3CA mutations were present in 39% of HNSCC cases (n= 37, 95% CI: 29–49% ). The prevalence of PIK3CA mutations in HNSCC HPV (+) (n= 68) and HNSCC HPV (  )(n= 27) was, respectively, 36.8% (n= 25) and 44.4% (n= 12). Next, we analysed the distribution of PIK3CA mutations in patient subgroups defined by both the HPV DNA and p16 INK4a status, as summarised in Table 2. We could not demonstrate a statistically significant association between the presence of PIK3CA mutations and HPV DNA detection (p= 0.489, Table 1). When evaluating a possible association between PIK3CA mutation and the p16 INK4a status without stratification by the HPV DNA status, no statistically significant association was found (p= 0.163, Table 1). Similarly, there was no evidence of an association between the p16 INK4a status and PIK3CA mutation when considering HPV status stratification (p= 0.245). Table 2. Frequency of PIK3CA mutations in HNSCC with and without HPV DNA and p16 INK4a overexpression (n= 89; p16 missing data n= 6). HNSCC PIK3CA Gene WT n(%) MUT n(%) HPV (+) p16(+) 22 (68.8%) 10 (31.3%) p16() 17 (56.7%) 13 (43.3%) HPV ()p16(+) 3 (75%) 1 (25%) p16() 12 (52.2%) 11 (47.8%) 2.2.3. Classification of PIK3CA Gene Mutations: H1047L, E542K, E545K, and E545D HNSCC cases were screened for four different single substitutions, known as canonical PIK3CA mutations. Among the cases harbouring PIK3CA mutations (n= 37), the majority carried E545D (73%; n= 27), followed by E545K (5%; n= 2), E542K (3%; n= 1), and H1047R (3%; n= 1). We observed the occurrence of combined substitutions E545D|E542K (5.4%; n= 2), E545D|E545K (8.1%; n= 3), and H1047R|E545D (2.7%; n= 1). The distribution of PIK3CA mutations in the HPV-positive and -negative subgroups is summarised in Table 3. Table 3. Classification of PIK3CA gene mutations (substitutions H1047L and E542K, E545K, and E545D). HPV Status PIK3CA Gene MUT One Substitution Two Substitutions E545D %(n) E545K %(n) E542K %(n) H1047R %(n) E545D|E545K %(n) E545D|E542K %(n) E545D|H1047R %(n) HPV (+) 76% (19) 8% (2) 4% (1) 4% (1) 8% (2) ND ND HPV () 67% (8) ND ND ND 8% (1) 17% (2) 8% (1) Total 73% (27) 5% (2) 3% (1) 3% (1) 8% (3) 5% (2) 3% (1) Legend: ND = not detected. 2.2.4. PIK3CA Mutations and Patient Prognosis The median follow-up determined using the reverse Kaplan–Meier method was 4.12 years (95% CI 3.1–5.5 years). A total of 46 deaths were reported during the follow-up period. The median overall survival (OS) in the whole sample (n= 95) was 4.75 years (95% CI 2.6–6.9 years). The 3-year OS was 58% (95% CI 48–70%). Disease-free survival (DFS) was evaluated in the 66 patients with a complete response to the first-line treatment ( 17 patients with persistent disease and 12 with missing information concerning response to treatment were excluded from the DFS analysis). Overall, the median DFS was 6.16 years (95% CI 4.6–NA), and the 3-year DFS was 75% (64–87%). 131 Cancers 2022,14, 1286 6 of 19 Table 1. Clinical, pathological, and demographic characteristics and association with PIK3CA mutation. Variable Categories PIK3CA Evaluation Total Cases n= 95 p-Value WT (n= 58) MUT (n= 37) n(%) n(%) n(%) Gender Male 45 (77.6%) 25 (67.6%) 70 (73.7%) 0.2795 Female 13 (22.4%) 12 (32.4%) 25 (26.3%) Age at diagnosis Mean (Standard Deviation) 62 (10.7) 62 (12.7) 62 (11.5) 0.9740 65 Years 24 (41.4%) 12 (32.4%) 36 (37.9%) 0.3807 <65 Years 34 (58.6%) 25 (67.6%) 59 (62.1%) Consumption habits (tobacco and/or alcohol) Active consumption 44 (75.9%) 22 (59.5%) 66 (69.5%) 0.0325 * Alcohol active 18 (31.0%) 10 (27%) 28 (29.5%) Tobacco active 7 (12.1%) 1 (2.7%) 8 (8.4%) Both active 19 (32.8%) 11 (29.7%) 30 (39.6%) No consumption 9 (15.5%) 13 (35.1%) 22 (23.2%) Never 5 (8.6%) 10 (27%) 15 (15.8%) Nonactive 4 (6.9%) 3 (8.1%) 7 (7.4%) No data 5 (8.6%) 2 (5.4%) 7 (7.4%) Tumour Anatomic region Oropharynx 46 (79.3%) 29 (78.4%) 75 (78.9%) 0.9135 Oral cavity 12 (20.7%) 8 (21.6%) 20 (21.1%) Tumour stage I 4 (6.9%) 4 (10.8%) 8 (8.4%) 0.9342 II 11 (19%) 8 (21.6%) 19 (20.0%) III 16 (27.6%) 10 (27.0%) 26 (27.4%) IV 25 (43.1%) 15 (40.5%) 40 (42.1%) No data 2 (3.4%) 0 2 (2.1%) p16 IHQ HNSCC p16() 29 (50.0%) 24 (64.9%) 53 (55.8%) 0.1627 HNSCC p16(+) 25 (43.1%) 11 (29.7%) 36 (37.9%) No data 4 (6.9%) 2 (5.4%) 6 (6.3%) HPV infection HPV16 DNA() 15 (25.9%) 12 (32.4%) 27(28.4%) 0.4887 HPV16 DNA(+) 43 (74.1%) 25 (67.6%) 68 (71.6%) Single infection 39 (90.7%) 23 (92.0%) 62 (91.2%) Co infection with other HR/LR HPV 4 (9.3%) 2 (8%) 6 (8.8%) Primary Treatment Surgery 1 (1.7%) 2 (5.4%) 3 (3.2%) 0.7376 Radiotherapy (RT) 10 (17.2%) 6 (16.2%) 16 (16.8%) Chemotherapy (CTX) 46 (79.3%) 29 (78.4%) 75 (78.9%) CTX + RT 26 (56.5%) 12 (41.4%) 38 (50.7%) Surgery + RT 9 (19.6%) 11 (37.9%) 20 (26.7%) Surgery + CTX 11 (23.9%) 6 (20.7%) 17(22.7%) No data 1 (1.7%) 0 1 (1.1%) Treatment response Complete 38 (65.5%) 28 (75.7%) 66 (69.5%) 0.5190 Persistence 11 (19.0%) 6 (16.2%) 17 (17.9%) No data 9 (15.5%) 3 (8.1%) 12 (12.6%) Legend: No data = no information available; RT = Radiotherapy; CTX = Chemotherapy; * p-value calculate for active vs. no consumption group. 132 Cancers 2022,14, 1286 5 of 19 marker (n= 208) and all cases associated with HPV infection other than HPV16 (n= 79); the remaining 103 cases were included in the study and analysed for PIK3CA status (Figure S1). Eight cases were further excluded from the study due to technical failure in the PIK3CA mutational status evaluation. As such, our study sample comprised 95 patients. The demographic and clinical features of the excluded patients were similar to the included patients (Table S4), and both groups showed overlapping survival curves (Figure S2), which suggests the absence of selection bias. The demographic and clinical–pathological characteristics of the 95 HNSCC patients included in the analysis are summarized in Table 1. The average age at diagnosis was 62 years old and ranged between 37 and 90 years old. Most patients were men (73.7%, n= 70) and, according to self-reported consumer habits, the majority had active tobacco and/or alcohol consumption (70%, n= 66) . Stratification was performed for each consumption habit. Alcohol exposure was characterised according to qualitative data in clinical records: never drank, ex-drinker, sporadic, moderate, and severe drinkers. The classification for tobacco consumption was defined as one pack/year (equal to one pack of cigarettes/day/year, with 20 cigarettes in a pack) distributed among current and heavy smokers. Current tobacco users included those who used tobacco  30 pack/years. Heavy users were those who smoked > 30 pack/years. The ex-smoker group included smokers who stopped until the date of diagnosis. Cases where the pack-a-year unit information was not available were classified as unrated smokers, and the data only reported consumption in a qualitatively way. Never users of tobacco or alcohol were defined as not having consumed either of these substances prior to cancer diagnosis. The analysis considered the two groups of active consumption vs. no consumption and did not consider the missing values. In 75 cases (79%), the primary tumour was located in the oropharynx, including the palatine tonsil (n= 47), the base of the tongue (n= 12), uvula (n= 2), soft palate ( n= 10 ), trigone retromolar (n= 1), and oropharynx wall (n= 3). In the remaining 20 cases, the primary tumour was located in the oral cavity, including the tongue body (n= 13), buccal floor (n= 4), oral mucosa (n= 2), and mandible (n= 1). HPV infection was detected in 68 HNSCC cases , including 62 cases of HPV16 single-infection and six cases of coinfection with HPV16 and other HR or LR HPV types (HPV6, HPV18, HPV53, and HPV58). Data from p16 INK4a immunohistochemical assays were available for 89 patients. Patients were treated with different schemes involving surgery and radiotherapy (RT) as single therapies or in association with systemic antineoplastic treatment (Table 1). This treatment heterogeneity was expected, taking into consideration the clinical characteristics and tumour stage distribution observed in our cohort. Most patients showed complete response to the applied treatment (n= 66), but cases with disease persistence (n= 17) and relapse ( n=6 ) were also reported. In 12 cases, the treatment response could not be retrospectively evaluated (Table 1). There were no differences between the wild-type (WT) and mutant (MUT) PIK3CA groups regarding age, primary tumour location, tumour stage at diagnosis, HPV16 infection status, and primary treatment administered (Table 1). The PIK3CA WT group had a numerically higher proportion of males (78% vs. 68%), p16 overexpression (43% vs. 30%), and complete responses to treatment (65% vs. 76%) compared to MUT PIK3CA, although none of these differences was statistically significant. There was a statistically significantly higher proportion of patients with active tobacco/alcoholic consumption habits among the WT compared to the MUT PIK3CA (76% vs. 60%) group; as such, this variable was also considered in the multivariable analysis of the prognostic impact of PIK3CA mutations. 133 Cancers 2022,14, 1286 4 of 19 (two cases) in exon 20). The highest reported frequency (32%) was observed in a Japanese study (Suda 2012) with a cohort of 115 cases, equally distributed across different subsites of the HN region (oral cavity n= 31, oropharynx n= 25, larynx n= 23, hypopharynx n= 25, and nasopharynx n= 11). Five mutations spots were evaluated in exon 9 and exon 20. The distribution of mutations by location revealed a predominance of oropharyngeal sites (oral cavity n= 9 (24%), oropharynx n= 10 (27%), larynx n= 8 (22%), hypopharynx n= 9 (24%), and nasopharynx n= 1 (3%) data from T Suda 2012). In addition, we performed a second analysis to explore the HNSCC PIK3CA mutation profile in greater depth. For this second analysis, we assessed 17 selected articles comprising data from 1286 HNSCC patients published between 2006 and 2021 where mutations were detected using DNA sequencing techniques instead of PCR-based methods ( Supplemental Data Table S2 ). The most used sequencing methodology was the classic Sanger assay ( n= 10 studies ), followed by cutting-edge NGS technology (n= 7). All studies evaluated tumour tissue, and the majority of samples were collected at the time of diagnostic (i.e., were treatment-naive samples). Primary tumours were located at different subsites from the HN region stratified by the oral cavity, oropharynx, larynx, hypopharynx, and nasopharynx; all data that did not specifically describe these locations were considered as not stratified. Three out of 17 studies evaluated SCC exclusively from the oral cavity, three others from the oropharynx, and one study from the hypopharynx. The remaining eight studies include data from different subsites. Overall, the most represented subsite was the oral cavity (n= 678 cases, reported by 11 articles), followed by the oropharynx ( n= 340 , reported by seven articles), larynx (n= 101, reported by five articles), hypopharynx (n= 94, reported by six articles), nasopharynx (n= 40, reported by five articles), and 32 non-stratified cases (reported by four articles). The HPV status was described in nine out of 17 studies: three studies evaluated single HPV status cohorts HPV (+) or HPV (  ), and at least six studies presented both HPV (+) and HPV (  ) cases. HPV (  ) HNSCC was the most represented subgroup (n= 330 cases), followed by HNSCC HPV (+) (n= 244 cases). Overall, the frequency of PIK3CA mutations ranged between 3% and 36%. Only two studies reported the lowest frequency (3%) (Bruckman 2010 and Cortelazzi 2015), at least five studies presented frequencies above 20%, and three studies above 32%. Overall, 45 mutations were reported, distributed by both exon 9 and exon 20, and novel mutations were reported in a few studies. All mutations were stratified by exon, except for 30 cases reported in three studies. The analysis based on general data for the mutational profile was supported by data reported in each of the studies. The three most frequently encountered mutations were the E545K mutation (n= 31 cases; data from 15 studies), followed by E542K (n= 21; nine studies) on exon 9 and H1047R (n= 16; nine studies), T1025T (n= 16; two studies), M10431 (n= 8; two studies), and G1049 (n= 2; three studies) on exon 20 (Supplementary Table S3). The PIK3CA-mutated cases per HN subsite were accurate; the highest number was in the oral cavity (n= 115, reported by seven studies), followed by oropharynx (n= 32, three studies), hypopharynx (n= 10, three studies), larynx (n= 8, three studies), and nasopharynx with no cases reported. We also evaluated the PIK3CA mutation (data reported from six studies) for subgroups of the HPV status. A total of 33 cases of HNSCC HPV (+) and 19 cases of HNSCC HPV (  ) harboured PIK3CA mutations. In five of the 17 studies, the presence frequency of PIK3CA mutations was associated with the clinical parameters and correlated with the respective outcome (OS and DFS). Four studies estimated a better prognosis associated with the presence of the mutation (Alsofyania 2020, Lim 2019, García-Carracedo 2016, and Cohen 2011), and in one study, no correlation was found (Chau 2016). 2.2. Portuguese HNSCC Study Population 2.2.1. Clinical and Demographic Characteristics Cases were selected from a universe of 390 consecutive primary HNSCC cases located in the oropharynx or oral cavity at our tertiary cancer centre between 2010 and 2019. We excluded all cases with HPV-negative and missing information concerning the p16INK4a 134 Cancers 2022,14, 1286 3 of 19 patients had significantly higher 3-year disease-free survival (DFS) compared to PIK3CA MUT patients, and a multivariable analysis for age, sex, smoking, TNM stage, and treatment showed associations between PIK3CA status and disease recurrence [ 31 ]. A greater understanding of therapies targeting the PI3K pathway has been achieved, developing new therapeutic combinations to improve the survival of HNSCC patients, but their efficacy is variable [ 32 , 33 ]. The identification of biomarkers to predict the response to therapy will allow the selection of patients who are more likely to respond to new therapeutic combinations [ 32 ], suggesting that different treatment strategies need to be considered based on the molecular phenotype of each tumour [ 34 , 35 ]. In this context, it is critical to refine the profile of PIK3CA mutations in HPV-positive and HPV-negative HNSCC patients and its association with prognosis. To address these issues, the present study includes a systematic review of the frequency of PIK3CA mutations in HNSCC and the impact of the different detection techniques. The study also reports the prevalence of canonical PIK3CA gene mutations (substitutions for H1047L and E542K, E545K, and E545D) within an HNSCC Portuguese patient series at the time of diagnosis and conducted an exploratory analysis to test the hypotheses: (1) whether PIK3CA (canonical) mutations are associated with HPV-positive HNSCC, as evaluated by HPV DNA and p16INK4a and (2) whether PIK3CA mutations are independently associated with overall and disease-free survival. 2. Results 2.1. Systematic Review We performed a systematic review to study the PIK3CA mutation frequency in different HNSCC cohorts (total case number range between 25 and 115 cases; total number of cases was 479) in scientific articles published from 2012 to 2021. The PIK3CA gene mutation analysis was performed by qPCR designed for specific targets in the regions known as hotspots of the PIK3CA gene exon 9 and exon 20, such E542K, E545K, E545D, H1047R, and H1047L, respectively. This analysis covers three different geographical regions: Europe (three studies), Asia (two studies), and North America (one study) (Supplemental Data Table S1). The majority of the studies performed a mutational analysis using untreated tumour biopsy tissue, the male gender was the most represented in all studies, always representing more than half of the sample (55–93%), the mean age ranged between 63 and 65 years, and all the cohorts presented the active consumption of tobacco and/or alcohol. Almost all the studies reported a stratification by HN subsites (oral cavity, oropharynx, larynx, hypopharynx, and nasopharynx), and the majority of the cases originated from the oropharynx (five out of six studies, a total of 138 cases), followed by the oral cavity (four studies, a total of 97 cases), larynx (four studies, a total of 80 cases), hypopharynx (four studies, a total of 80 cases), and nasopharynx (three studies, a total 16 cases). All patients presented a histological diagnosis of SCC, most of them at advanced stages (III and IV). Only three of the six studies reported the HPV DNA status of the cohort. In total, 54 HNSCC HPV (+) and 219 HNSCC HPV (  ) were evaluated. In three studies, the p16INK4 status was determined by immunohistochemistry with a total of 45 positive cases. The mutation frequency was estimated in a qualitative analysis of the number of cases with a mutation present. All studies reported PIK3CA gene mutations, and their frequency ranged from 8 to 32%. The lowest frequency (8%) was reported in a cohort of 113 cases (Borkowska 2021), mostly consisting of laryngeal tumours (43%) and, largely, HPV (  ) lesions (77%). This low frequency of mutation detection can be related to the study’s design targeting only H1047R. Four other studies presented frequencies ranging between 16% and 18% and mainly consisted of lesions from the oropharynx and oral cavity. Among these four studies, HPV (  ) HNSCC cases prevailed in the two studies where the HPV status was determined (García-Escudero 2018; SB Pattle 2017). All but one of these studies evaluated at least four mutations distributed both in the exon 9 and exon 20. The highest frequency was found (taking into account only (three cases) E545D (two cases) in exon 9 and H1047R 135 Cancers 2022,14, 1286 2 of 19 (OS) (3-year OS: WT 54% and MUT 65%; p= 0.090). HPV status was the only factor significantly associated with both OS and disease-free survival (DFS), with HPV (+) patients having consistently better outcomes (3-year OS: HPV (+) 65% and HPV (  ) 36%; p= 0.007; DFS HPV (+) 83% and HPV (  ) 43%; p= 0.001). There was a statistically significant interaction effect between HPV status and PIK3CA mutation regarding DFS (Interaction test: p= 0.026). In HPV (+) patients, PIK3CA wild-type is associated with a significant 4.64 times increase in the hazard of recurrence or death (HR = 4.64; 95% CI 1.02–20.99; p= 0.047). Overall, PIK3CA gene mutations are present in a large number of patients and may help define patient subsets who can benefit from therapies targeting the PI3K pathway. The systematic assessment of PIK3CA gene mutations in HNSCC patients will require further methodological standardisation. Keywords: HNSCC; HPV; p16 INK4a; PIK3CA 1. Introduction Head and neck carcinoma (HN) is the sixth leading cancer by incidence worldwide [ 1 ], according to data published in 2018, and is responsible for more than 800,000 new cases yearly and 450,000 deaths/year worldwide [ 2 ]. It comprises many different common and rare entities, the large majority (90%) being squamous cell carcinomas (SCC) [ 3 ]. There is a wide variation in disease progression and overall patient survival at different anatomic subsites [ 4 , 5 ], which may be explained by its multifactorial aetiology, in which environmental factors have a strong contribution, such as smoking and alcohol consumption. Infection with human papillomavirus (HPV), mainly the high-risk (HR) type HPV16, also plays an important role in a subgroup of these tumours, particularly in oropharyngeal cancers [ 6 ]. HPV-positive and HPV-negative tumours are clinically distinct [ 7 ], and two separate carcinogenesis routes are recognized [ 8 ]. Over the past decades, the incidence of HPV-associated oropharyngeal SCC has increased, as reported by the United States Center for Disease Control (CDC) in 2018 [ 9 ]. HPV-associated HNSCC has a substantially better prognosis after therapy compared with HPV-negative cases [ 10 ]. De-intensified therapy for these patients is being actively explored to reduce the associated morbidity while maintaining tumour control [11,12]. The genomic profile of HNSCC published by The Cancer Genome Atlas (TCGA) in 2015 allowed the development of new mutation studies and highlighted a high frequency of changes in components of the phosphatidylinositol-3-kinase (PI3K) signalling pathway, pointing out PIK3CA as the most frequently altered gene [ 13 ]. Activating PIK3CA gene mutations upregulate intracellular signalling via the PI3K–protein kinase T (Akt)–mammalian target of rapamycin (mTOR) pathway, contributing to multiple hallmarks of cancer, such as resisting cell death and uncontrolled proliferation [ 14 – 16 ]. As described previously, HPV genome integration leads to keratinocyte immortalization and transformation by inhibiting the tumour suppressor p53 and retinoblastoma protein (pRb) but also by interacting with other pathways, including PI3K/Akt/mTOR [ 17 – 20 ]. Several studies have addressed the role of the PIK3CA pathway in cervical cancer and other types of HPV-associated cancers [ 21 – 25 ]. In the evaluation of 151 HNSCC whole-exome sequences, the PI3K pathway was found to be the most commonly altered mitogenic pathway (30.5% of tumours) compared to the JAK/STAT pathway (9.3%) and MAPK pathway (8.0%) [ 26 ]. Among the PIK3CA mutations observed in HNSCC, 63% occur at three specific locations encoding the p110 ↵ subunit, namely E542, E545, and H1047, known as canonical mutations [ 27 , 28 ]. The PIK3CA mutation frequency among HPV-positive tumours was reported to be approximately half of that found in HPV-negative cancers [ 29 ]. Even so, the PIK3CA gene remains one of the most mutated in HPV-associated HNSCC [ 7 , 30 ]. Additionally, mutations have been associated with adverse outcomes in solid tumours, but their prognostic significance in oropharyngeal SCC is still unclear. In previous studies, the specific survival data did not differ between PIK3CA wild-type (WT) and mutated (MUT) lesions. However, WT-PIK3CA 136 CHAPTER 5 GENERAL DISCUSSION 137 HNSCC and HPV type, lineage and NGS The HNSCC associated with HPV typically arises in the oropharynx and is considered a distinct entity compared with other HN tumours named HNSCCnonHPV. The recognition of this difference incited the development of studies in this field, to gain insights into patient risk stratification and more personalized treatments. In the early 2000s with the introduction of new molecular tools aided several research groups to explore molecular mechanism-based approaches to define genetic signatures that may play a prime role in patient stratification (Alexandrov and Stratton, 2014; Chai, Lim and Cheong, 2020). In this context, our research project led us to explore if specific HPV16 variants could influence the occurrence, the morphology, and the molecular phenotype of HNSCC-HPV. Apparently, within each host, HPV genomes harbour high variability levels in the evolution from low-grade lesions to high-grade lesions and cancer, which may reveal several genetic signatures (Nacher et al., 2020). The growing number of studies on HPV16 variants, was mainly done in uterine cervical cancer and has exposed a very large number of different papillomavirus genomes. Moreover, the research on HPV phylogenetic lineages with clinical host specificity reports several differences within its oncogenic potential (Sichero et al., 2007; Zhang et al., 2015). Our general hypothesis was that the specific morphological and molecular phenotype HPV16 variants identified in HNSCC tumour could be associated with different clinical pathologic settings. In this manner, our study was conceived to identify the frequency of HPV16 variants in squamous cell carcinomas of the head and neck region and to evaluate their correlation with clinical variables. At the onset, we carried out a systematic review (SR) of the literature to scrutinize which sequencing methodologies were performed. We concluded that in most studies in the HN region, a limited number of samples were examined, mainly because of the labour-intensive techniques used (PCR). Sanger sequencing techniques were applied to identify HPV16 variants in all nine HN studies included in the analysis; no Next Generation Sequencing (NGS) was used, most likely due to difficulties on the access to the technique and the limitation on supporting costs to applying this new sequencing methodology. The use of conventional Sanger sequencing likely limited the number of cases analysed and introduced limitations in a whole genome analysis with respect to the size of the amplicons, which may compromise the sensitivity of the assay (Hoffmann et al., 2004; Park et al., 2016; 144 and survival of cancer cells and is associated with a poor patient prognosis. Determining which patients carry PIK3CA mutations is of great clinical importance since it can help to define subgroups of patients with specific needs and who may benefit from promising targeted therapies against the PI3K-Akt-mTOR signalling pathway. Our study proposed a qualitive analysis of the mutations commonly associated with HNSCC tumours. The present results contribute to better defining this complex scenario, determining the frequencies of canonical PIK3CA mutations in a Portuguese cohort of patients with HNSCC and testing the putative associations between PIK3CA mutations and multiple clinicopathological parameters. In our dataset, most patients with HNSCC carrying canonical PIK3CA mutations had advanced disease stages (III, IV), especially among HNSCC-HPV, suggesting a negative impact on patient prognosis and corroborating previously published data (Zieba et al., 2020). However, there was no significant association between mutation frequency and HPV status, and no correlation between PIK3CA and overall survival between univariate or multivariate correlations. Importantly, our data evidence a high frequency of canonical PIK3CA mutations in HNSCC, including in HNSCC-nonHPV cases, suggesting that screening for PIK3CA mutations should not be restricted to the HNSCC-HPV group but can also be relevant to HNSCC-nonHPV patients, which contrasts with previous studies that indicated a higher frequency in HNSCC-HPV tumours. Overall, our results contribute with new data in the field of molecular analysis in oncological pathology applied to HPV16-positive oropharyngeal tumours. Currently, it is valuable to consider that tumour recurrence still occurs in 10-20% of HPV-positive HNSCC patients. Although the clinical management of HNSCC has improved since the recognition of HPV-positive and HPV-negative lesions, further therapeutic advances will require effective clinical validation of all these promising biomarkers in large-scale population studies. Currently, in response to the clinical diagnosis, the HPV status is determined in suspicious lesions of the oropharynx by qualitative PCR methodologies that translate its presence or not. These methodologies designed to detect viral DNA only identify the genotype present. The new molecular tools developed in the present work magnify the information on this diagnosis and enrich the information on the Virus-Host dynamics, promoting better patient risk stratification and more 145 targeted treatments in clinical practice. Particularly, one of the strengths of this project was the development of the NGS workflow, validated against the ISO standard 15189 to certify its implementation in a clinical laboratory, thus contributing with new molecular inputs for the clinical practice among head and neck tumours. Finally, our future perspective is to apply this workflow to clinically referred casuistic associated to HPV tumours pretending to expand, sustain and validate biomarkers to answer the clinical practice needs. Hopefully, the new sequencing era will innovate the study of HPV and related cancers. 146 CHAPTER 6 CONCLUDING REMARKS 147 Molecular analysis using next-generation sequencing methodology represents a valuable tool to consolidate genetic markers with their respective clinicopathological characteristics. The studies published in this field reinforce its application on a larger scale to improve the clinical implications of early diagnosis, prognosis, and therapeutics. The NGS methodologies implementation at the routine laboratory within the scope of HPV-associated pathology potentially increases the studies of these cohorts, provide a detailed description of mutational profiles associated with the respective clinicopathological characteristics and assesses greater robustness as a complementary of diagnosis. A strong point of this project was the development of an NGS workflow for the identification of HPV16 variants in HNSCC tumours, optimized to analyse DNA from different types of products, and therefore also promising for its application in the context of other associated pathologies, such as the cervix and anal canal. The analytical validation defines the characteristics of the assay based on studies the sensitivity and specificity of cell lines infect HPV16 (SiHa cell), tissue sample lesions from the K14HPV16 model and tissue tumours samples from a cohort of patients diagnosed with HNSCC. The weaknesses are highlighted mainly by the unsatisfactory results of 33 samples to determine HPV16 variants, directly compromising the expected cohort for the phylogenetic analysis and association with the clinicopathological characteristics and survival. However, the vulnerable points identified in the global process and their resolution make it possible to refine technical flaws and contribute to increase the success of the NGS workflow. Finally, in general, it is a technically consistent study that reveals original data from genomic analysis that directly contribute to deepening the study of the role of variants in the pathology of HNSCC and reinforcing its promissory purpose as a complementary tool of diagnosis. In our future perspectives, we intend to expand our study, increasing the number of cases included, and invest in the improvement of bioinformatics analysis. 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